Documentos de Académico
Documentos de Profesional
Documentos de Cultura
Editors
Subhash Bhore,
K. Marimuthu &
M. Ravichandran
Biotechnology for Sustainability
Achievements, Challenges and Perspectives
Editors
2017
1
Biotechnology for Sustainability
Achievements, Challenges and Perspectives
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AIMST University
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2017 by the authors; Licensee, Editors; AIMST University,
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Edited by
Dr. Subhash J. Bhore (Senior Associate Professor)1,
Dr. K. Marimuthu (Professor)1, 2, and
M. Ravichandran (Senior Professor)1, 2
Edition
First; July 18, 2017
3
Dedication
4
Preface
World Environment Day (WED) is a biggest global annual event celebrated each
year on June 5 to create the positive awareness to preserve the environment and planet
earth. This year, the theme for WED-2017 was Connecting people to nature. Our
environment should be healthy for our growth, development and to achieve the sustainable
development goals (SDGs) adopted by the international community to transform the world.
Most recently, Antnio Guterres (United Nations Secretary General) precisely
highlighted that Without a healthy environment we cannot end poverty or build prosperity.
We all have a role to play in protecting our only home: we can use less plastic, drive less,
waste less food and teach each other to care. In fact, to achieve the SDGs by protecting
environment, everyone needs to do their part.
We strongly believe that biotechnology can play an important role directly or
indirectly in achieving various SDGs. Hence, we had decided to publish a book,
Biotechnology for Sustainability to commemorate the WED and to highlight the
achievements, challenges and perspectives in various domains of the biotechnology. In
response to our call for articles, we had received 50 manuscripts. The selected articles
published in this book are highlighting various issues, achievements, challenges and
perspectives for the viable development and sustainability. The World Commission on the
Environment and Development defined sustainability as the development that meets the
needs of the present without compromising the ability of future generations to meet their
own needs. The United Nations recent estimate suggest that the worlds food supply needs
to be doubled by the year 2050 to keep up with the growing demand. To achieve this is a
huge challenge; because, the amount of arable land is continuously decreasing as a result of
rising urbanization, saline soils and desertification. Biotechnologists (and plant breeders)
around the world are working persistently to produce crops which will boost the food
production to meet the growing demand. Genetically engineered crop varieties do offer
many promising possibilities to boost nutritive value of the food, sustain farming on
marginal lands, and to minimize the loss by creating pests and disease resistant varieties.
The articles published in this book are going to be useful in creating awareness
about the environmental issues, natural resources, biodiversity conservation, sustainable
development and various biotechnological approaches that could be used to alleviate the
respective challenges.
We would like to express our sincere gratitude and thanks to Dato' Seri Utama Dr.
S. Samy Vellu, Chancellor and Chairman, AIMST University for his support in publishing
this book.
We wish to thank all contributing authors for making a common cause with us. This
book publication project could not have been completed without the courteous cooperation
of the authors to highlight achievements, challenges and or perspectives in using
biotechnological approaches for the sustainability.
We are confident that this book will serve as a reference to various researchers,
scientists, academicians and graduate students involved in biodiversity conservation,
environmental protection and various fields of biology and biotechnology.
It is hoped that a prudent use of biotechnology in the biodiversity conservation,
environmental protection, and production of more and better quality of food, fiber, fuel and
drugs will contribute in accomplishing SDGs and to promote peace in the world.
Subhash J. Bhore
K. Marimuthu
M. Ravichandran
Preface ................................................................................................................................ i
Contents ............................................................................................................................ ii
Plant Tissue Culture for Sustainability
C. K. John ....................................................................................................................... 1
Traditional Medicine of the Tribes in Tamil Nadu and Its Sustainable
Use through Biotechnology
Valli Gurusamy, Kavitha Valampuri John, Usha Raja Nanthini
Ayyakkanu, Ramani Bai Ravichandran ......................................................................... 14
Vermitechnology An Eco-Biological Tool for Sustainable
Environment
Mahaly Moorthi, Koilpathu Senthil Kumar Abbiramy, Arumugam Senthil
Kumar and Karupannan Nagarajan................................................................................ 41
Role of Biotechnology in Food Authentication
Shobana Manoharan, Raghavan Kuppu and Ramesh Uthandakalaipandian ................... 51
Management Strategies against Tiny Tigers for Sustainable
Development of Agriculture
Viswa Venkat Gantait ................................................................................................... 58
Designing Greener Pharmaceuticals and Practicing Green Health Is
Required for Sustainability
Sridevi Chigurupati, Jahidul Islam Mohammad, Kesavanarayanan
Krishnan Selvarajan, Saraswati Simansalam, Shantini Vijayabalan and
Subhash Janardhan Bhore ............................................................................................. 68
Clonal Propagation of a High Value Multipurpose Timberline Tree
Species Quercus semecarpifolia Sm. of West Himalaya, India
Aseesh Pandey and Sushma Tamta ............................................................................... 79
Spent Mushroom Substrate of Hypsizygus ulmarius: A Novel
Multifunctional Constituent for Mycorestoration and Mycoremediation
Padmavathi Tallapragada and Ranjini Ramesh .............................................................. 88
Biotechnology for Sustainability of Forests
Kumud Dubey and Kesheo Prasad Dubey ................................................................... 104
Biotechnological Approaches for Conservation and Sustainable Supply
of Medicinal Plants
Sagar Satish Datir and Subhash Janardhan Bhore ........................................................ 117
Plant Tissue Culture Division, CSIR-National Chemical Laboratory, Dr. Homi Bhabha
Road, Pune 411008, India;*Correspondence: ck.john@ncl.res.in; Tel.: +91-9822531551
Abstract: The United Nations has placed great emphasis on sustainability. Three of the
most important requirements of sustainable development are: eradicating extreme pov-
erty and hunger, protecting the environment, and conserving biodiversity. Because of hu-
man activities the stable functioning of earths life support system which includes the at-
mosphere, oceans, forests, waterways, biodiversity and biogeochemical cycles, is at risk.
One of the major contributing factors is the large scale destruction of natural forests. Defor-
estation had many adverse effects; most importantly, the effects on climate, environment,
and biodiversity. The three pillars of sustainable development are: sustainable agriculture,
conserving biodiversity, and protecting the environment through reversing the effects of
deforestation by large scale afforestation. Plant Tissue Culture can greatly contribute in all
the three.
lar the essential needs of the worlds poor, the principles of sustainable develop-
to which overriding priority should be ment into country policies and programs,
given, and the limitations imposed by the reversing loss of environmental resources,
State of Technology and Social organiza- and reducing biodiversity loss.
tion on the Environments ability to meet In 2012, the United Nations
present and future needs should be ad- Rio+20 summit in Brazil committed gov-
dressed. The Brundtland Commission Re- ernments to create a set of Sustainable
port emphasized the need to integrate Development Goals (SDGs). On Sep-
economic and ecological factors in deci- tember 25th 2015, countries adopted a set
sion-making at all levels for sustainable of goals to end poverty, protect the planet,
development. These factors include, re- and ensure prosperity for all as part of
viving growth, changing quality of a 2030 Sustainable Development Agenda.
growth, meeting essential needs for jobs, Each goal has specific targets to be
food, energy, water and sanitation, ensur- achieved in 15 years. The 17 Sustainable
ing the resource base, reorienting tech- Development Goals (SDGs), otherwise
nology and managing risks. In its broadest known as the Global Goals, are a uni-
sense, the strategy for sustainable devel- versal call for action to end poverty,
opment aims to promote harmony among protect the planet and ensure that all
people and between human beings and people enjoy peace and prosperity al-
environment. ways. The goals are interconnected.
In 1992, at the Earth Summit (Rio, The key to success on one will involve
1992) there was consensus that environ- tackling issues associated with anoth-
ment, and economic and social develop- er. The SDGs work in the spirit of
ment cannot be considered in isolation, partnership and pragmatism, to make
and in addition to treaties and agreements the right choices now to improve life,
on climate change, biological diversity, in a sustainable way, for future genera-
deforestation, and desertification, the Rio tions. They provide clear guidelines
Declaration contains fundamental princi- and targets for all countries to adopt in
ples on which nations can base their fu- accordance with their own priorities
ture decisions and policies, considering and the environmental challenges of
the environmental implications of socio- the world at large. The SDGs are an
economic development. inclusive agenda. They tackle the root
In 2000 the Millennium Sum- causes of poverty and unite all nations
mit of the United Nations, following the together to make a positive change for
adoption of the United Nations Millenni- both people and planet (UNDP).The
um Declaration, established the eight Mil- 15th SDG of UN relates to Life on land,
lennium Development Goals (MDGs) to and involves protecting, restoring and
be achieved by the year 2015. The MDGs promoting sustainable use of terrestri-
are: 1. to eradicate extreme poverty and al ecosystems, sustainably managing for-
hunger, 2. to achieve universal primary ests, combating desertification, and halt-
education, 3. to promote gender equali- ing and reversing land degradation and
ty and empower women, 4. to re- halting biodiversity loss.
duce child mortality, 5. to im- The stable functioning of Earths
prove maternal health, 6. to com- life support system which includes the
bat HIV/AIDS, malaria, and other diseas- atmosphere, oceans, forests, waterways,
es, 7. to ensure environmental sustainabil- biodiversity and biogeochemical cycles, is
ity, and 8. to develop a global partnership a prerequisite for future human develop-
for development. In the present context ment. However, as per recent research
Goal 7: Ensuring environmental sustaina- findings this functioning is at risk (Rock-
bility is very important. Two of the im- strm et al., 2009). Further human pres-
portant targets of MDG 7 are: Integrating sure may lead to large-scale, abrupt, and
undifferentiated tissue, regenerate into gregates of few cells. These cells/ cell ag-
whole plants. gregates grow/ divide/ separate as a result
of agitation, and can be continually main-
4.1. Types of cultures tained in this state. Growth of cells in
Different types of cultures are suspension culture can be more easily
possible: (i) culture of whole plants, (ii) manipulated in liquid medium than on
embryo culture (embryo rescue), (iii) or- semi-solid medium. Slowly agitating the
gan culture (shoot tip culture, root culture, liquid medium on a rotary shaker is nec-
leaf culture, anther culture etc.), (iv) cal- essary for the growth of the cultures,
lus culture, (v) cell suspension and single which can be sub-cultured. Growth in
cell culture, (vi) protoplast culture. single isolated cells can be induced by
culturing them in hanging drops in micro-
4.2. Callus culture chambers. Suspension cultures are useful
Callus is an amorphous mass pro- in plant production by somatic embryo-
duced by cell proliferation, occurring in genesis form single cells. In regeneration
an unorganized manner. In nature it is a of plants from callus established on semi-
wound response, or a plant reaction to the solid media from small cell aggregates,
presence of micro-organisms, insects, or and for the production of secondary me-
to some kind of stress. Under in vitro tabolites. Suspension cultures can also be
conditions callusing is a response to en- initiated from tissue other than callus
dogenous or exogenous growth regula- (Geile and Wagner, 1980).
tors. The potential for callus formation is
dependent on the tissue (explant) type. 4.5. Protoplast cultures
Meristematic tissues are more suitable for Protoplasts are plant cells without
callus induction than mature tissues. Cal- cell walls. In 1882, Klercker isolated pro-
lus cultures can be maintained for long by toplasts mechanically for the first time.
sub-culturing the primary callus (callus The yield of protoplasts was very low. In
established originally from the explant), 1960, Cocking using enzymes for the first
at periodic intervals. time could isolate protoplasts in large
numbers. Protoplasts can be isolated from
4.3. Somaclonal variations different plant parts, or from tissues al-
Long term callus cultures can ready in culture. Enzymatic isolation is
however, suffer from spontaneously aris- now the most commonly used method. A
ing genetic variations, reflected in the combination of these two can also be
phenotype of plants regenerated from used.
such calli. These variations are known as One of the important applications
somaclonal variations. Somaclonal varia- of protoplasts is in somatic hybridization.
tions are reported in many species. The Many agents like NaNO3 (Power et al.,
basis of somaclonal variations is not well 1990), a higher pH, and a higher concen-
understood. Chromosomal rearrange- tration of calcium ions in the medium
ments, activation of endogenous trans- (Melchers and Labib, 1974), polyethylene
posons, and changes in the status of DNA glycol (Kao and Michayuluk, 1974;
methylation, are considered to be the con- Wallin et al., 1974), and a high strength
tributing factors. electric field (Zimmermann and Scheu-
rich, 1981), are used for obtaining fusion
4.4. Suspension cultures between protoplasts. Protoplasts, when
Culture of unorganized plant cells, placed in appropriate media regenerate
as single cells/ cell aggregates, in liquid cell walls and form calli, from which
medium. Friable callus when cultured in plants can be regenerated. Protoplasts are
agitated liquid medium, the cells separate used for producing somatic hybrids (para-
and form a suspension of single cells/ ag- sexual hybrids), for genetic manipulation,
and for basic studies on a variety of as- shoot tips of virus infected plants are also
pects. Regenerating plants from proto- virus-free. Morel and Martin (1950; 1955)
plasts is difficult in some species. Con- could produce healthy plants from virus-
ventional hybridization depends on affini- free plants through shoot-tip culture from
ty of gametes. Wide crosses are not pos- infected mother plants. This is possible
sible because of well-established cross because the pathogen concentration is not
breeding barriers. Protoplast fusion makes uniform in the infected plants, and apical
such hybridizations possible. buds of rapidly growing shoots are often
not invaded by the virus. Morel (1960)
4.6. Anther (isolated microspore) cultures used shoot apices of orchids to obtain
Guha and Maheshwari (1964) ob- their rapid clonal multiplication. Shoot tip
tained haploid embryos, directly from an- culture has two important practical appli-
ther cultures of Datura innoxia. The cations: (i) virus eradication and (ii) mi-
origin of these embryos was traced to the cro-propogation. These developments
pollen grains. The potential of anther cul- were followed by in vitro propagation of
ture for obtaining haploid plants, and plants from shoot tip culture. Initially
from them by chromosome doubling of most of the species micropropogated were
homozygous diploid plants was apparent. herbaceous (Morel, 1964; Murashige,
In 1974, Nitsch had reported regeneration 1974). Now methods are available for the
of haploids and homozygous diploids by micropropagation of a large number of
chromosome doubling, from isolated mi- species belonging to a wide range of plant
crospore culture (Nitsch, 1974a; 1974b). groups.
Culturing the microspores along with an-
ther wall is essential for success. In iso- 4.8. Embryo culture
lated microspores, pollen embryogenesis Very young to mature embryos
is induced only rarely. can be cultured in vitro. Embryo culture is
This technique has great potential one of the oldest applications of plant tis-
in plant breeding. Normally it takes self- sue culture in plant breeding. It has many
ing for many generations to obtain homo- practical applications, and very useful in
zygosity in parental lines required in obtaining hybrid plants from crosses in
breeding programmes. This time can be which post-zygotic incompatibility exists.
considerably reduced by haploid culture In post zygotic incompatibility, fertiliza-
techniques. tion and zygote formation occur on cross
pollination. The zygote grows, but is not
4.7. Meristem culture and shoot tip cul- accepted by the endosperm. This results
ture in embryo abortion at some stage of de-
When growing points (meristems) velopment before maturing of the seed. In
of shoots are cultured they continue their such instances when the ovary/ ovule/
organized growth. The shoots/multiple embryo with a part of the maternal tissue
shoots produced can be rooted to produce is excised and cultured on a suitable me-
plantlets. This capacity has practical ap- dium and under optimum culture condi-
plication and economic significance for tions, it matures to produce a seedling.
plant propagation. This procedure is hence called embryo
Culture of the meristemic zones rescue. Sharma et al. (1980) obtained few
/extreme shoot tip is known as meristem hybrids between Solanum melongena and
culture, and culture of small segments (5- S. khasianum by this method. Embryo
10 mm in size) from the shoot tip is culture is useful also in overcoming seed
known as shoot tip culture. It was known dormancy and for obtaining seed germi-
that meristems of virus infected roots are nation in some vegetatively propagating
free of the pathogen (White, 1933; 1934). species in which seeds are produced but
Limasset and Cornuet (1949) found that
normally do not germinate (e.g. some shoots or leaves. These organs may arise
wild bananas). out of pre-existing meristems or out of
differentiated cells. Indirect pathway in-
4.9. Invitro pollination and fertilization cludes a callus stage.
Pre-zygotic incompatibility is one Direct pathway bypasses a callus
of the major limitations in obtaining hy- stage. The cells in the explant act as direct
bridization between many plant species precursors of a new primordium, an organ
and varieties. In pre-zygotic incompatibil- or a part in its most rudimentary form or
ity, the zygote is not formed on cross pol- stage of development.
lination. The pollen either do not germi-
nate on the stigma of the female parent or 4.12. Somatic embryogenesis
the pollen tube gets arrested at some point In plants, embryo-like structures
of its growth on the stigma/ in the style. can be generated from non-germ cells
A variety of methods used in vivo to (somatic cells), by circumventing the
overcome this barrier. process of normal fertilization. As somat-
Kanta et al. (1962) developed an ic embryos are formed without fertiliza-
in vitro technique for overcoming pre- tion event, they are genetically identical
zygotic incompatibility. In this method, to the parent tissue, and are therefore
the mature/nearly mature ovaries/ovules clones.
are cultured on suitable media and polli- Somatic embryogenesis may be
nated in vitro with cross pollen to obtain direct or indirect. Indirect somatic embry-
hybrids (Zenktler, 1980; Raghavan, ogenesis involves a callus phase prior to
1990). embryo production. Direct somatic em-
bryogenesis involves production of em-
4.10. Root cultures bryos from organized tissue without an
Tip portions from primary and intervening callus phase. Irrespective of
secondary roots of many plants can be the mode of production, anatomical and
cultured. In 1922, Kotte and Robbins in- physiological features of somatic embryos
dependently postulated that true in vitro are highly comparable to zygotic embry-
cultures could be raised from meristemat- os. The morphological and temporal de-
ic cells from root tips and shoot tips. velopments of somatic embryos are very
Kotte (1922) could cultivate root tips of similar to that of zygotic embryos. They
pea and maize in nutrient media for long, both proceed through a series of distinct
but no sub-culturing were done. Robbins stages, namely, globular, heart, torpedo
(1922) could subculture his maize root and cotyledon or plantlet stages for dicot-
cultures. White (1934) obtained unlim- yledons, and globular, elongated, scutellar
ited growth of tomato roots, using the and coleoptilar stages for monocotyle-
same medium as Robbins (1922), with dons. These stages typically span a period
yeast extract. Root cultures are useful in: of several days. In dicots initially small
(i) secondary metabolite production, and globular embryos form which undergo
(ii) in basic studies on nematode infec- isodiametric growth and establish bilat-
tions, mycorrhizal associations, and root eral symmetry. In monocots, especially in
nodulation by Rhizobium bacteria. grasses, the transition from globular stage
follows a series of events occurring sim-
4.11. Organogenesis ultaneously; such as the development of
Organogenesis is the process of scutellum, initiation of the coleoptilar
initiation and development of a structure notch, tissue differentiation with the de-
that shows natural organ form and/or velopment of embryogenic vascular sys-
function. It is the ability of non- tem and accumulation of intracellular
meristematic plant tissues to form various storage substances. Somatic embryogene-
organs de novo; the production of roots, sis is used for: large-scale clonal propaga-
storage, simple crop improvement meth- jor driver of loss of biodiversity. This puts
ods such as selection and bulking by rapid in jeopardy the sustainability of agricul-
and large scale cloning by micropropaga- ture and ecosystem services and their
tion can be of great use. Plant tissue cul- ability to adapt to changing conditions.
ture techniques such as: anther culture, This also poses serious threat to food and
dihaploid production, embryo rescue, and livelihood security.
in vitro pollination and fertilization can be
very useful in developing crop varieties 6.4. Plant tissue culture methods for con-
through hybridization. Callus culture, cell serving biodiversity
suspension culture, organogenesis and Plant Tissue Culture offers novel
somatic embryogenesis are essential for options for collection, multiplication and
crop improvement through transgenics medium/ long-term ex situ conservation
(various genetic engineering techniques). of plant biodiversity. By plant cell, tissue,
and organ culture techniques, rapid and
6. Role of plant tissue culture in con- large scale multiplication and season in-
serving biodiversity dependent production of planting material
is possible. This has helped in the conser-
6.1. Biodiversity vation of many endangered species. Me-
"Biological diversity means the dium-term conservation is achieved by
variability among living organisms from slow growing cultures. Cryopreservation
all sources including, inter alia, terrestrial, (at 196 C, in liquid nitrogen) allows the
marine and other aquatic ecosystems and safe and cost-effective long-term conser-
the ecological complexes of which they vation.
are part; this includes diversity within
species, between species and of ecosys- 6.5. In vitro collection
tems. - Definition of Biodiversity by Potential advantages of in vitro
CBD. methods are: (i) Less space requirement,
(ii) Pathogen-fee plants, (iii) No need for
6.2. Importance of biodiversity transfer (under storage conditions), (iv)
Biodiversity is essential to: (i) en- Stored cultures can be used as stock for
sure the production of food, fibre, fuel, vegetative preservation, and (v) Interna-
fodder, etc., (ii) maintain other ecosystem tional exchange of plant material made
services, (iii) allow adaptation to chang- easy because, no use of soil, and no path-
ing conditions - including climate change, ogens.
and (iv) sustain rural peoples' livelihoods Basic goals of an in vitro storage
(Convention of Biological Diversity). system are: to maintain genetic stability,
to keep in indefinite storage without loss
6.3. Threats to biodiversity of viability, and most importantly, to be
Biodiversity is under seri- economical.
ous threat as a result of human activities. Three types of Plant Tissue Culture sys-
The main dangers worldwide are: (i) In- tems are available. They are: (i) Normal
vasion by alien species, (ii) Environmen- growth, (ii) Slow growth, and (iii) Cryo-
tal degradation, (iii) Climate change and preservation.
global warming, (iv) Urbanization and
habitat conversion, (v) Population growth 6.5.1. Normal growth
and ever-increasing demand for resources, Normal Growth is achieved either
(vi) Unsustainable over-exploitation of on semi solid media or in liquid media.
natural resources. Normal growth is similar to multiplication
Agriculture contributes signifi- stage in micro-propagation, and requires
cantly to conservation and sustainable use frequent sub-culture. Considered as genet-
of biodiversity. However, it is also a ma- ically stabile when achieved through di-
al, and (iii) industrial raw material. About due to conversion to agriculture land and
2 billion people in the developing world urbanization. Deforestation has severe
are dependent on forests for their basic consequences for the environment and
energy needs (fuel for cooking food). Un- climate. More than 2000 times the total
til recently wood has been the chief con- energy consumption of the world popula-
struction material. High strength to tion, of solar energy reaches the earths
weight ratio and availability in many surface. Because of deforestation, not on-
kinds, ease of cutting and shaping with ly this natural source of energy is wasted,
simple tools, and insulation to heat, make but also has a serious negative impact on
wood an ideal construction material for the environment, by way of surface heat-
many purposes. Major industrial uses of ing, and desertification.
wood are: (i) paper and pulp, (ii) rayon, In the tropical and sub-tropical re-
and (iii) plywood. Besides wood, forests gions of the world, receiving about 600
are a source of a variety of non-wood for- mm rainfall and above, plantation forestry
est products (NWFPs). Thus forests con- of economically important tree species
tribute greatly to the economy. Around (say teak for timber and eucalypts for
1.6 billion people depend on forests for pulp) can take away pressure for forestry
their livelihood. This includes some 70 resources from natural forests and can add
million indigenous people. to the forest cover. For this, large num-
Forests play an important role in bers (in millions) of plating material of
maintaining ecological balance. Forests superior varieties (fast growing, better
are atmospheric filters. They are the ma- adapted, disease and insect-pest resistant
jor suppliers of oxygen. In photosynthesis etc.) are necessary. But forest tree species
they fix atmospheric carbon dioxide into are difficult to breed, because of their
carbohydrates, sugars, proteins, and many long generation cycles, highly heterozy-
forms of biomass, thus playing a signifi- gous natural populations, openly cross
cant part in the global carbon cycle. For- pollinated nature, and lack of knowledge
ests contribute large quantities of mois- about their genetics.
ture to the atmosphere, thus regulating Clonal propagation of superior
climate. Forests also conserve soil and genotypes (identified for desirable traits)
water resources. through tissue culture has been used very
The term forest implies natural profitably in case of many tree species.
vegetation of the area, existing from Eucalypts have been multiplied and used
thousands of years and supporting a varie- for plantation forestry for a long time
ty of biodiversity. More than half of the (FAO Report, 1981). Eucalyptus wood
known terrestrial plant and animal species from plantation forestry has been used as
live in forests (Millennium Ecosystem timber, industrial raw material and fuel.
Assessment, 2005). The forest ecosystem Plantation forestry using eucalypts may
has two components - biotic and abiotic. not be suitable for some places because of
The living component includes plants high water demand. Teak (Tectona gran-
(trees, shrubs, herbs etc.), animals and dis Linn. f.) is another success story. In
microorganisms. Forests are home to early 1980s, scientists from CSIR-
more than 80 per cent of all terrestrial National Chemical Laboratory, Pune for
species of animals, plants and insects. the first time regenerated complete plant-
Globally, deforestation is the major cause lets from an 80 year old elite tree (Gupta
of loss of biological diversity, and is a et al., 1983). Poplars (Populus spp.) are
matter of great concern (Laurance, 2007). another tree species which clonally prop-
Worldwide, the area of natural agated through plant tissue culture tech-
forests decreases by some 13 million ha niques, and widely cultivated in planta-
annually (this is about 3% of the total for- tion forestry in many parts of the world.
est area). This loss of forest area is mostly In India, poplars (Populus spp.) are the
Abstract: India is a land of mega biodiversity representing about 7% of the worlds flora
and 6.5 per cent of worlds fauna. Tamil Nadu, one of the southern most states of India, is
rich in forest cover and cultural diversity. Genomic evidence supports the peopling of Tamil
Nadu from the 1st wave of migration of humans from the Out of Africa exodus and points
out that the tribes of state were among the earliest settlers in the region. The tribal popula-
tion of Tamil Nadu represents 1.02% of the total population of the state. Living in close as-
sociation with the forest, they have accumulated a treasure trove of ethno botanical
knowledge in the form of traditional medicine. The future of sustainable use of renewable
forest product lies with the molecular tools of Biotechnology. We present here an analysis
of the documented literature of the medicinal plants used by the tribes of Tamil Nadu for
treatment of common disorders. We also present the challenges and prospects within the
scope of Biotechnology to ensure sustainable use of traditional medicine for the betterment
of mankind and environment.
process of evolution through trial and er- cient lineage from the 1st wave of the out
ror which makes documentation and rec- of Africa exodus and thus in vast ethno-
ord-keeping almost impossible. This pro- botanical knowledge. According to the
cess of exchange and assimilation is con- 2011 census, 104 million tribal people
tinuous, and today there is a growing speaking over 227 linguistic groups in-
awareness among the medical community habit varied geographic and climatic
about the intrinsic value of traditional zones of the Indian subcontinent. Ethno-
medicine, and as a result in India Ayurve- medicine includes plants, animal products
da, Unani and Siddha have entered the and minerals used by tribal communities
mainstream to compliment biomedicine. of a particular region or country for me-
Contemporary Indian society faces the dicinal purposes other than those men-
challenge of integrating the best of the tioned in classical streams of the respec-
different healing traditions to provide a tive cultures. Tribal people have been us-
holistic health care. ing a large number of wild plants as doc-
umented by ethnobotanical investigations.
2. Traditional knowledge The application of most of the plants rec-
orded are either lesser known or hitherto
Even before classical medical unknown to the outside world. India has
knowledge of ancient India was codified been the country most concerned about
into the canonical texts of Ayurveda in the conservation of its medicinal plants.
the 6th century BC, there were abundant There are over 45,000 species of vascular
sources of medical knowhow in the sub- plants reported from India of which as
continent from prehistoric times. Tradi- many as 15,000 may be used medicinally.
tional healers can be either folk or tribal The folk medicine system of India use
healers and have worked in intimate rela- about 5,000 plant species with about
tion with their environment. Traditional 25,000 formulation, whereas the tribal
healing ranges from simple home reme- medicine involves the use of over 8,000
dies related to nutrition and treatment for plant species with about 1,75,000 specific
minor illnesses, to more sophisticated preparations (Pushpangadan and George,
procedures such as midwifery, bone set- 2010). More than 90% of the raw material
ting, blood-letting (therapeutic phleboto- for traditional medicine comes from wild
my) and treatment of snake bites and harvesting as this the common method
mental disorders. Some healing practices used for collecting them (Tandon, 1996;
were considered to be sacred and were Gupta 1998; Ved et al., 1998). About 71
associated with rituals that helped safe- medicinal plant species are classified as
guard them for there is a substantial over- rare, and of this 92% are in active trade,
lap between healing plants and sacred and 74% are traded nationally. It has been
plants. Categories of traditional healers estimated that between 4,000 and 10,000
are traditionally trained healers, old indi- medicinal plant species in India face ex-
viduals of the community, educated indi- tinction in the local, regional and national
viduals acquiring certain knowledge from levels (Hamilton, 2004). In an effort to
their predecessors, ancient inscriptions in create leadership in affordable and holis-
the form of copper plate/palm leaf writ- tic health care, India is committed to
ings, old and recent publications in re- promoting traditional medicines like
gional language. Ayurveda which remained untapped due
to inadequate scientific scrutiny. Steps
3. Indian ethnobotany are being taken to bring in regulatory
amendments in research and effective en-
India is one of the richest coun- forcement for integration of quality prod-
tries in the world not only in biodiversity ucts, practices and practitioners into the
but also in different ethnic groups of an- AYUSH (Ayurveda, Yoga and Naturopa-
thy, Unani, Siddha and Homoeopathy) in moderate altitude of the district name-
system at central and state level. ly: New Kotagiri (Aggal), Kil-Kotagiri,
Kundah, Kallimalai, Gudalur, Trichigadi
4. Traditional knowledge of the tribes and Sholur Kokal.Their settlement is
of Tamil Nadu called the Kokkal with linear row of
houses in streets, Keri. Each village has
According to the 2001 census, three Keri known as Kizhkeri, Nadukeri
tribal population in Tamil Nadu is 6, and Melkeri. Keri, clan, exogamy is note-
51,321 which constitute 1.02% of the to- worthy among Kotas (Kavitha V.J.,
tal population. There are 36 tribes and sub 2008). Their chief diety is Kambattrayan.
tribes in Tamil Nadu. Out of the 36 Their population was 1,894 in 2001 cen-
Scheduled Tribe communities in the sus.
state, about six tribal populations Todas, Irulas: They are also called as Iu-
Kotas, Krumbas, Paniyas, Irulas, Kat- van,Villiar. The Irulas are distributed in
tunayakas have been classified as primi- the lower altitudes of the Nilgiri hills (dis-
tive tribes with incredibly high anthropo- trict). They are negrotoid in appearance
logical significance. The primitive tribes whose chief occupation is as plantation
occupy the length and breadth of the Nil- labourers in the estates. Their settlements
giri district in the Western Ghats of Tamil are called Aral. Their dialect is Tamil
Nadu. One sixth of the land mass of Tam- mixed with Malayalam. Their community
il Nadu is covered by forests. The tribes is divided into seven exogamous clans
of Tamil Nadu live in and around the re- (Kuems): Kupper, Sambe, Kalkatti, Ku-
served forests and have gained immense runagar, Devanan, Peradar and Punger
knowledge on the use of forest produce to (Rajan and Sethuraman, 1991). They are
treat common disorders. basically hunter gatherers. Their popula-
Todas: They are called by other tion was 6,700 in 2001 census.
names like Tudas, Thuduvans, and Todar Kurumbas: The Kurumbas prac-
(Kavitha V.J., 2008). They are profes- tice hunting food gathering economy,
sional pastoralists and dairy men, a purely well-versed in honey collection tech-
pastoral economy in India today, living in niques. They are plain dwelling people
the higher altitudes in the traditional living in the interior forests of the district.
houses called Munds that are half barrel Their staple foods are wild tubers (Di-
shaped and are vegetarians. Their dialect oscorea bulbosa), wild fruits and other
is independent form of Dravidian Tamil- minor forests produces. Their settlements
Malayalam. They are fair skinned and are called Mottam. They are dark
wear ornaments and their dress is akin to skinned and speak the Kurumba dialect.
the Roman toga. They have two exoga- Kurumbas are a heterogenous population
mous divisions called Tarthar and Teivali. having divisions such as Halu Kurumbas,
There are five socially distinguishable Betta Kurumbas, Mullu Kurumbas, Jess
sects (clans) such as Pelki, Pekkan, kurumbas and Urali Kurumbas. Their
Kuttan, Kenna and Jodi (Rajan and Sethu- population was 6,872 in the 2001 census.
raman, 1993). Their population was 1,600 Paniyas:The Paniyas are negro-
in the 2001 census. toid people living in bamboo huts at the
Kotas: Their other names are Ko- junction of Kerala and Tamil Nadu bor-
ter, Kothewars, and Kohatur. The Kotas der. They work as labourers with Waya-
are musicians and excellent craftsmen nad Chettis though they were basically
having mastery over ironworking. They hunter gatherers. Their settlements are
are light skinned, with copper hair. They called Paddi. They possess excellent
speak the Kota a Dravidain language. skills in the art of fishing by employing
Their distribution in the Nilgiri district is certain plant parts like bark of Eugenia sp.
confined only to seven villages inhabiting and leaves of Aibizzia sp. as stupefying
agents. They speak the Paniya dialect, dicinal knowledge of the tribes of Tamil
practice Illam, patilineage, and their in- Nadu.
heritance is by Marumakkathayam.
They numbered 6,700 in 2001 census. 5. Ethnomedical wealth of tribes of
Kattunayakas: They are also Tamil Nadu
called as Shola Nayakans, Jenu or Teen
Kurumbas. They are another group of for- A total of 229 medicinal plants
est dwellers who are nomadic in nature, used by the tribes of Tamil Nadu belong-
their staple foods are honey, wild fruits ing to 79 families for the treatment of
and tubers. Their settlements are called more than 40 disorders were documented.
Paadi. They are short and black with The percent representation of the families
protruding forehead. They have curly hair of plants used as medicine by the tribes of
and speak Kannada language. Eating bi- Tamil Nadu is represented in Figure 1.
son flesh is a cultural taboo with them. Euphorbiaceae is the largest family repre-
The social customs and religious practices sented by 18 species at 12% followed by
of Kattunayakas are akin to Kurumbas in Fabaceae by 14 species (9%), Lamaceae
many respects. They population was by 13 species (8%), Asteraceae by 11
1,425 in the 2001 census. species (7%), Solaneaceae and Rutaceae
Paliyars: They are found in the by 10 species each (6%) and Ascelpiada-
hilly regions of Madurai, Dindigul, Theni, cea by 8 species(5%). This data marks a
Tirunelveli and Virudhunagar districts. It direction for scientific researchers as to
is believed that Paliyars are indigenous which of the families to search for to
people of Palani hills of Kodaikanal and identify bio active compounds.
speak Tamil. Physically they are similar Of the primary parts of the plant
to the Semong of Malaya and other Indian used the leaves formed 40% of usage in
tribal communities. They can be grouped traditional medicine followed by the root
into three categories based on their life and bark at 11%, whole plant and fruit at
styles, namely, nomadic, semi nomadic 8%, stem at 7% and seed at 5% (Figure
and settled. Nomadic Paliyars don't build 2). It is of utmost importance to see this
houses; they live temporarily in rock data in the light of the major families rep-
caves called 'Pudai'. resented for medicinal use by the tribes of
Ethnobotanical traditional Tamil Nadu. It is also important to test
knowledge for the tribes of Tamil Nadu these bioactive compounds from different
was retrieved using Pubmed and Google parts of the plant as a combination and to
using the keywords ethnomedicne, tribes, use bioactive compounds different fami-
Tamil Nadu. The ethnobotanical data pre- lies in conjunction for any particular dis-
sented here included knowledge form order.
seven tribal groups of Irula, Pani- The data when analysed for major
ya,Kurumba, Kota, Thoda, Kattunayak- remedies against diseases it revealed that
kans and Paliyars (Table 1). These pub- the tribes of Tamil Nadu had traditional
lished research articles were then ana- remedies for wounds (31) and skin prob-
lysed manually to ascertain the traditional lems (29), followed by stomach aches
knowledge of these communities related (17), diahorrea and headache (13), Cold,
to the study area and the usage pattern of cough, fever (12) and rheumatic diseases,
the medicinal plant species. The data was gastric disorders and toothache (9). It is
further analysed using graphical represen- interesting to note that the ethnomedicine
tations for summarizing and interpreting of the tribals has 9 remedies for women to
for the major families of plant species ease labour pain and 3 to induce lactation
represented, part of the plant used and (Figure 3). Tribal ethnomedicine also has
cure for disorder from the traditional me- remedies for diabetes and jaundice (5).
Figure 1: Percent representation of major plant families used by Tamil Nadu tribes as medi-
cine.
Branches
Heartwood Nuts Resin
Fruits Flowers Rhizome
0% 0% 0% 0%1% 1% 2%
Tuber
2%
Latex
3%
Seed
5%
Stem
Leaves 7%
40%
Fruit
8%
Whole Plant
8%
Bark
Root
11%
11%
Figure 2: Parts of the Plant used in traditional healing among the tribes of Tamil Nadu.
No. Botanical Name Local name Part Used Medicinal uses Tribe Reference
Acanthaceae
1 Adhatoda zeylanica Medicus Adathodai Leaves Asthma, Cold, fever PL e
Adhatoda zeylanica Medicus Auduthoda Root, bark, Cough, asthma ,eye pain IR g
flowers
2 Andrographis lineata Wallich ex Siriyanangai Leaves Cough, diabetes, scorpion and PL e
Nees snake bite
3 Andrographis paniculata (Burm.f.) Periyanangai or Leaves Scorpion sting PL e
Wall. ex Nees Nilavembu and snakebites, menorrhagia
4 Asystasia gangetica Valukai keerai Leaves Appetite PL e
5 Blepharis maderaspatensis (L.) Roth. Vettukaaya pachilai Leaves Fractured bones, Cuts PL e
6 Phlebophyllus kunthianum Nees Kurinji chedi Leaves Nervous disorder PL e
Alangiaceae
7 Alangium salvifolium (L.f.) Wangerin Alinji Fruit Eye infections PL e
Amaranthaceae
8 Achyaranthes aspera L. Uthrunk Leaves Cuts, wounds and sores KO b
Achyranthes aspea L. Cherukadalai Whole Plant Sprain ached in the Joints KT b
Achyranthes aspera L Nayuruvi Leaves Diverticulosis & Diverticulitis KU i
Achyranthes aspera L. Nayurvi Geeda Whole Plant Ease child birth and labour KU b
pain
Achyranthes aspera L. leaves New born babies, lactation IR g
9 Achyranthes bidentata Blume, Kithoop Leaves Rapid healing of wounds KU i
10 Achyranthus bidentata Blume Naiyur Leaves Skin disorders including sca- KO a
bies
11 Aerva lanata (L.) Juss.ex Schult Kannupila & Pan- leaves New born babies, lactation IR g
Celosia argentea L. naipoo
12 Alternanthera sessilis (L.) Nilakirai Leaves Diarrhoea KU i
Alternanthera sessilis (L.) Nilakirai Leaves Roughage KU i
13 Amaranthus gangeticus L. Mulai keerai Whole plant Good digestion,Constipation, KU i
Table 1: Continued
Anacardiaceae
14 Anacardium occidentale Bark, Fruit Fever, warts IR, c
latex PA
15 Mangifera indica Maavae pattae Bark Stomach pain IR, c
PA
16 Spondias pinnata Kattu Maa Bark Acute diarrhoea IR, c
PA
Annonaceae
17 Annona squanmosa L. Seetha mara Seeds Vermifuge PA b
Apiaceae
18 Centella asiatica (L) Urban Vallarai Whole Plant Refrigerant TH b
Centella asiatica (L) Urban Gottala Whole Plant Toothache KT b
Centella asiatica (Linn.) Urban. Kidth Kot Leaves Stomach problems Cools the KO a
body
19 Coriandrum sativum Linn. Kothumull Leaves Refrigerant and diuretic KO a
Aplaceae
20 Buplerum wightii P.K. Mukherjee, Malai seragam Root Easy delivery IR-S d
21 Centella asiatica (L) Urban, Kutheraikokku Leaves Digestive agent, blood circula- IR-S d
tion
22 Heracleum ceylanicum Gardner ex C.B. Poonaikal sedi Leaves Insect allergy IR-S d
Clarke
Apocymeeae
23 Rauvolfia serpentine (L.) Benth. ex Kurz Chivanamelpodi Root Stomachache KT f
24 Alstonia scholaris (L.) R.Br. Paalooram pattai Stem Lactation PL e
Araceae
25 Acorus calamus L. Vasambu Rhizome Speech PL e
Table 1: Continued
27 Phoenix sylvestris (L.) Roxb. Injai Stem Easy Delivery IR g
Aristolochiaceae
28 Aristolochia indica L. Perumanthikodi Root Fever IR g
29 Aristolochia tagala Cham. Modhalaikodi. Leaves Diarrhea and vomiting IR-S d
Asclepiadaceae
30 Ceropegia candelabrum L. Perun kodi Leaves Headache PL e
31 Cryptolepis buchananii Roem & Paalkodi/Karunkodi Latex Wound PL e
Schul
32 Gymnema hirsutum W&A Sakarasedi Leaves Diabetes KU i
33 Gymnema sylvestre (Retz.) R. Br. Ex Sirukurinjan Leaves Diabetes and nervous disorder PL e
Gymnema sylvestre R.Br. Sirukurinjan & ha- Leaves Diabetes IR g
karikolli
34 Hemidesmus indicus H.f. Nannari Whole Fever, menorrhagia, stomach- PL e
Plant, Root, ache
leaves
35 Holostemma ada-kodien Schult. Ada kizhangu Tuber Fever KT f
36 Pergularia daemia (Fors.) Chio Veli parutthi Leaves Headache PL e
37 Tylophora indica (Burm. f.) Merr. Nangilai Leaves, Snakebite PL e
Root
Asparagaceae
38 Asparagus racemosus Willd. Ammaikodi Tuber Stomachache KT f
Asteraceae
39 Adenostemma lavenia (L.) Kuntze Kasirukai Leaves Skin diseases IR-S d
40 Ageratum conyzoides L. Nasar soppu Leaves Cough and cold KU b
Ageratum conyzoides Linn. Pugudu thalai Leaves Wound KO a
41 Artemisia nilagarica (C. B Clarke) Manikoland Leaves & Removing worms from KU i
Pamp. stem wounds both in humans and
animals
42 Artemisia parviflora Buch ham. Ex Railpundu Leaves Headache IR-S d
Roxb.
43 Bidens pilosa L. Katu kunni Leaves White patches on the legs KU h
Table 1: Continued
44 Blumea lacera (Burm.f.) DC. Navakkarandai Leaves Improving vision KT f
45 Chromolaena odorata (L.) King & Rob- Vettukkaya pacha Leaves Cuts and wounds KT f
ins.
46 Elephantopus scaber L. Anashovadi Root Stomach ache KT f
47 Siegesbeckia orientalis Linn. Potaz Leaves Skin rashes, insect bites and KO a
allergies
Sigesbeckia orientalis L. Nadukadachi Leaves Wounds and parasitic skin KU h
problems
48 Sonchus oleraceus L. Kaalaadi pachilai Leaves Wound PL e
49 Tridax procumbens L. Vettukayapoondu leaves Wounds to stop bleeding IR g
Tridax procumbens L. Vettukkaya thalai Leaves Sores KT f
Balanophoraceae
50 Balanophora fungosa Fors and Fors. Vaer chedi Whole Plant Skin disease PL e
Berberidaceae
51 Mahonia leschenaultii (Wight & Mullu kadambu Bark Skin disease PL e
Arn.) Tak. ex Gamble
52 Berberis tinctoria Lesch Jakkala Leaves & Dysentery, Bloating of stom- KU i
stem ach
Bignoniaceae
53 Radermackera xylocarpa (Roxb.) K. Vadencarni Stem Fever KT f
Schum.
Bischofiaceae
54 Bischofia javanica Blume. Romaviruksha patta Bark Nervous disorder, hair growth PL e
Boraginaceae
55 Carmona retusa (Vahl) Masam. Kurangu vetthilai Leaves Fertility PL e
56 Nasturtium indicum (L.) DC. Kadge Root Por- Ear diseases KU i
tion
57 Trichodesma zeylanica R.Br Jalke maram Root Round patches appearing on KU h
the skin
Burseraceae
58 Boswellia serrata Roxb. Ex Colebr. Kungiliyam Resin Cold PL e
Table 1: Continued
Caesalpiniacea
59 Caesalipinia bonduc (L) Roxb. Porumaielai Root, seeds Gastric disorders Increase IR-S d
body weight
60 Bauhinia racemosa Mara avara Bark Boils IR, c
PA
61 Cassia auriculata L. Avarai Fruit, Skin and scalp IR g
leaves,
flowers
62 Cassia fistula Gaggai pattai, Bark Sudden sicknesses , diar- IR, c
Konnai mara rhoea PA
and stomach pain
Cassia fistula L. Konnei Stem bark Stomach ache KT f
63 Pterolobium hexapetalum Kari indu Leaves Ease delivery pain PL e
64 Tamarindus indica Puli Fruit Nursing mothers, eczema IR, c
PA
Caparidaceae
65 Capparis sepiaria L. Thoratti Root Wounds and scratches IR g
Capparis zeylanica L. Adandai leaves Increase appetite IR g
66 Celome monophylla L. Kadugu sedi Leaves Earache IR-S d
Caricaceae
67 Carica papaya L. Poppilli Poppilli mara Fruit Indigestion and Constipation KU i
Caryophyllaceae
68 Drymaria cordata (L.) Roem. & Kodi charai Leaves Heel cracks PL e
Schult.
Celastraceae
69 Celastrus paniculatus Willd. Valulurai Root Body pain KT f
Chenopodiaceae
70 Chenopodium ambrosioides L. Jaregida Whole plant Intestinal cramps KU h
Colchicaceae
71 Gloriosa superba L. Kodanki kizhangu. Tuber Sleeping tablet IR-S d
Combretaceae
Table 1: Continued
72 Pterocarpus marsupium Roxb. Vengai Stem Rheumatic pain KT f
73 Terminalia bellirica (Gaertn.) Roxb. Tani Bark Body pain KT f
74 Terminalia chebula Retz. Kadukai Fruit Muscular dislocation KT f
Terminalia chebula Retz. Kadukkai maram Leaves Cold, Cough, Fever, stomach- PL e
ache
75 Terminalia crenulata Heyne ex Roth Karimathi Bark Internal bleeding KT f
Compositae
76 Bidens pilosa L., Katu Katu kunni Leaves White patches on the legs KU i
Convolvulaceae
77 Aroyreia hirsute Wight & Arn. Meenidal Leaves Male Child KO a
78 Ipomoea alba L. Velutha Leaves Skin diseases KU h
79 Melothria maderaspatana Cogn. Solapushni kai Stem Prolonged cough KU i
80 Trichosanthes cucumerina L. Peyppadal Fruit Headaches IR g
Dioscoreaceae
81 Dioscorea oppositifolia L. var. Nurulai/Valli ki- Rhizome Stomacache PL e
tomentosa. langu
Ebenaceae
82 Diospyros ferrea (Wild.) Bahk. Var. bux- Veeraii Fruit Bood circulation IR g
ifolia
Ericaceae
83 Gaultheria fragrantissima Wall. Ameerpan Leaves Headache Relieve body KO a
sprains and pains
Erthoroxylaceae
84 Erythroxyium monogynum Roxb. Jeevadalli maram Stem Bark Acute skin disease KU b
Erythroxylum monogynum Jeevathalimara Bark Scabies IR, c
PA
Euphorbiaceae
85 Acalypha fruticosa Forsskal. Chinni chedi Leaves Dysentery, skin disease PL e
86 Acalypha indica L. Kuppaimeni leaves Ear pain, snake bite and sca- IR g
bies
Table 1: Continued
88 Breynia rhamnoides, Muell Poolan Root & White patches on the skin all KU h
Leaves over the body
89 Euphorbia antiquorum L. Sathura kalli Latex Body pain PL e
90 Euphorbia hirta L. Ammanpatcharisi Leaves & Pimples KU h
Latex
91 Euphorbia rothiana Spreng, Kopot Latex Sores, grow hairs, insect repel- KU i
lant
92 Excoecaria agallocha L. Thillai Latex Antiseptic IR g
93 Excoecaria crenulata L. Vellai thillai Stem Skin disease PL e
94 Jatropha curcas L. Kaatu amanku Leaves Headache KT f
95 Jatropha tanjorensis Ellis & Saroja Katamanukku Latex Antiseptic IR g
96 Mallotus philippensis Chaneri mara Bark Stomach pain and diarrhoea IR, c
PA
97 Phyllanthus amarus Schum. & Thonn. Kila nelli Whole plant Jaundice KT f
Phyllanthus amarus Schum. & Thonn. Kizhanelli leaves Jaundice IR g
98 Phyllanthus emblica L. Nelli Fruit Stomachache KT f
99 Ricinus communis Kottamuthu Bark Quick delivery, sprains, IR, c
breathing PA
problems
100 Ricinus communis Linn. Amanaku Leaves, Headache KO a
Seeds
101 Securinega virosa (Willd.) Baill. Pula Root Joint pain KT f
102 Euphorbia rothiana Spreng. Kapsi Leaves Sudden sickness and giddiness KO a
Fabaceae
103 Acacia nilotica (L.) Willd. ex. Del. Karuvelam leaves Dysentery, burns or scalds IR g
104 Albizia lebbeck (L.) Benth. Vagai seeds Lesions of lepers IR g
105 Caesalpinia bonduc (L.) Roxb. Kazhchikai seeds Hydrocele IR g
106 Canavalia lineata (Thunb.) DC. Kozhiavarai seeds Several Disorders, General IR g
health
Table 1: Continued
Table 1: Continued
126 Leucas biflora (Vahl.)R.Br. Kaduthumbae Whole plant Skin irritations KU h
127 Leucas indica (L.) R. Br. ex Vatke Mosappullu Leaves Cough KT f
128 Ocimum basilicum Var purpurasens Katu thulasi Whole plant Skin inflammations after the KU h
insect bites
129 Plectranthus coleoides Benth. Mudupattan or Leaves Cold, delivery pain, hair PL e
Omavalli chedi growth, wounds
130 Plectranthus malabaricus (Benth.) R.H. Ellamabai Leaves Heart attack IR-S d
Willemse
131 Prunella vulgaris Linn. Kadthur Root Refrigerant Haematanic KO a
132 Aloe vera (L.) Burm.f Sotru Kattrazhai Leaves Hair and skin Diseases KU h
133 Asparagus racemosus Willd. Thanneer vittan Leaves Heel cracks PL e
kilangu
Lobeliaceae
134 Lobelia heyneana Roem. & Schult. Upperi chedi Leaves, Skin disease PL e
Flowers
135 Lobelia leschenaultiana (Presl) Skottsb. Bombari thalai. Whole Plant Sickness in cattle KO a
Loganiaceae
136 Strychnos nux-vomica Yetti Bark Acute stomach pain IR, c
PA
Lythraceae
137 Lagestroemia microcarpa Wight Tindiyam Bark Burns KT f
Malvaceae
138 Hibiscus rosa sinensis L. Chembarathi Flowers Strenthening hair KU h
139 Malvatrum coromandelianum (L) Garke Kalakenikai Root Stomach pain IR-S d
140 Sida acuta Burm f. Pilla valatthi chedi. Leaves Dandruffs , strengthening hair PL e
141 Sida rhombifolia L. Kal gadale Leaves Wounds KU i
Sida rhombifolia L. Chitra mutti Root Rheumatic pain KT f
142 Side cordifolia L. Arathae Leaves Snakebite PA b
Meliaceae
143 Azadirachta indica Veppamaram Stem Toothache, post-natal compli- IR, c
cations PA
Table 1: Continued
144 Azadirachta indica A. Juss. Veppam leaves Stomach worms IR g
145 Cipadessa baccifera (Roth.) Miq. Pulipancheddi Leaves Unconsciousness and anaemia KT f
Cipadessa baccifera (Roth.) Miq. Pulippan chedi Leaves Diarrhoea PL e
Cipadessa baccifera (Roth.) Miq. Marundha soppu Leaves Rheumatism IR-S d
Menispermaceae
146 Cissampelos pareira L. var. hirsuta Urikkakodi Tuber Snakebite KT f
(Ham. ex DC.) Forman
147 Cissampelos pureira, L. Koodibatale Leaves Headache, fever, burning sen- KU i
sation in chest
148 Cyclea peltata (Lam.) Hook. f. & Thom- Para Tuber Body pain KT f
son
Cyclea peltata (Lam.) Hook.f. Sethari Kodi Leaves Cough, cold and body pain IR-S d
149 Tinospora cordifolia (Wild) Miers ex Amrithavalli Leaves White rashes appearing on the KU h
Hook.F. & Thoms body
Mimosaceae
150 Acacia caesia (L.) Willd. Nanjupattai Bark Wound PL e
Acacia caesia (L.) Willd. Kari Indu Stem bark Body pain KT f
151 Acacia leucophloea (Roxb.) Willd. Sarayapattai maram Bark Cuts PL e
152 Mimosa pudica L. (Mimosaceae) Thotalvadi Whole plant Body pain KT f
Moraceae
153 Ficus infectoria Roxb Selakai Fruit Food KU i
154 Ficus racemosa L Athikai Fruit Eye sight KU i
Moringaceae
155 Moringa concanensis Nimmo Kattu murukka Bark Abortifacient IR, c
PA
156 Moringa oleifera Murunga Bark Dog or scorpion bite IR, c
PA
Musaceae
157 Musa paradisiaca Vazhai Stem Acute diarrhoea IR, c
PA
Table 1: Continued
Myrataceae
158 Eucalyptus polybractea R. T Baker Karpura mara Leaves & Round patches between fin- KU h
bark gers
159 Psidium guajava L. Koyyapazham Fruit Gastric troubles and ulcers in PA b
stomach
Psidium guajava L. Koyyapazham Fruit & Anti-dysenteric and Antidiar- KU i
Leaves rhoeal
160 Syzygium cumini (L.) Skeels Naval Pa- Stem Bark, Sore throat, dysentery, ulcers, KU i
zham, Fruit & purifying blood, antidiarrhoeal
Seeds and anti diabetic
Syzygium cumini (L.) Skeels Naval bark Diarrhoea IR g
Nyctaginaceae
161 Mirabilis jalapa L. Thottanembi Root , Cuts and wounds KO b
Leaves
Orchidaceae
162 Cymbidium aloifolium (L.) Sw. Ottai Root Ear pain IR g
163 Malaxis densiflora (A. Rich.) Kuntze Kuntze, Nelnethch Leaves Wounds KU i
Oxalidaceae
164 Oxalis corniculata L. Pulichen segae Whole Plant Febrifuge PA b
Oxalis corniculata L. Puliyankeerai Leaves Vomiting and headache IR-S d
Oxalis corniculata Linn. Pulch Leaves Anti-emetic Restorative tonic KO a
after child birth
Pandanaceae
165 Pandanus odoratissimus Kaithae Stem Fracture IR, c
PA
166 Passiflora calcarata Mast Potul Leaves Skin diseases KU h
167 Passiflora foetida L. Narati chedi Whole Plant Arthritic problems KU b
Periplocaceae
168 Hemidesmus indicus (L.) R. Br. Nannari Root Mouth ulcers KT f
Piperaceae
169 Piper betle L. Thabulam Whole plant Cuts and wounds KT f
Table 1: Continued
170 Piper brachystachyum Wall., Kadu kurumulaku Fruit Cough KU i
171 Piper nigrum L. Milagu Seeds Throat infection PL e
Plantaginaceae
172 Plantago erosa Wall. Kalthal Leaves Wounds as a antiseptic KO a
173 Plantago lanceolata L. Neela kare Leaves Boils on the legs KU h
Plumbaginaceae
174 Plumbago zeylanica L. Chitthira moolam Root Stomachache PL e
Plumbago zeylanica L. Cithiramalliver. Root Insect bite IR-S d
Poaceae
175 Cymbopogon citratus L. Karppura pul Root Pimples KU h
176 Cynodon dactylon (L.) Pers. Arugampul Branches Body coolant IR g
Polygonaceae
177 Rumex nepalensis Spreng. Gundott or Sukkutu Root Refrigerant and laxative KO a
Keerai
Rumex nepalensis spreng. Kekal Ott, Gund Root Jaundice KO b
Ott
Proaceae
178 Cynodon dactylon (Linn.) Pers. Nagirki Leaves Relief from sudden sickness KO a
Ranunculaceae
179 Clematis gauriana Roxb. Meenae Leaves & Wounds & skin Diseases KU h
stem
Clematis gouriana Roxb. Ex. DC. Attumeesai chedi Leaves Skin disease PL e
Rhamnaceae
180 Ziziphus mauritiana Yelluchi maram Bark Gastric disturbance IR, c
PA
Ziziphus mauritiana Lam. Elanthai Bark Old wounds IR g
Ziziphus mauritiana Lam. Ilantha Whole Plant Mouth freshener KT f
Rubiaceae
181 Catunaregam spinosa (Thunb.) Madukarei Root Ulcers KT f
Tirvengadum
182 Rubia cordifolia L. Sappli Koth Stem Restorative, jaundice KO b
Table 1: Continued
Rubia cordifolia L. Kalutharupan chedi Leaves Heel cracks PL e
Rubia cordifolia L. Periya nangai. Leaves Cough, cold and nervous dis- IR-S d
orders
Rubia cordifolia Linn. Maral Leaves Injuries caused by fire KU i
Rubia cordifolla L. Muthang Root Dysmenorrhoea KT b
Rutaceae
183 Citrus aurantium L. Eravae kai Fruit Digestion, Hemorrhoids KU i
184 Clausena dentata (Willd.) Roem. Anai thazhai Leaves Wound PL e
185 Glycosmis mauritiana (Lam.) Yaich. Panasedi Leaves Headache IR-S d
186 Glycosmis pentaphylla (retz.) DC. Eruputtal Whole Plant Stomach ache and abdominal KT b
discomfort
187 Murraya koenigii L. Karivepilla Leaves Skin inflammations KU h
188 Murraya paniculata Chedichi Bark Toothache IR, c
PA
189 Naringi crenulata (Roxb) Nicolson Naivalampattai Bark General health IR-S d
190 Ruta chalepensi L. Aruvatha Geeda Leaves Infant convulsions KO b
191 Ruta graveolens L. Aruvadam Leaves Skin diseases KU h
Ruta graveolens L. Arubathansedi Leaves Diarrhea, stomach pain and IR-S d
vomiting
192 Toddalia asiatica (L.) Lam. Surai leaves, Fuit Fever, headache IR g
Toddalia asiatica (L.) Lam. Kindu mullu Leaves, Stomachache, toothache PL e
stem, Root
bark
Toddalia asiatica (Linn). Lam. Vaseri Leaves , Vermifuge KO a
Seeds
Salvadoraceae
193 Azima tetracantha Lam. Sankan leaves Fever IR g
194 Salvadora persica L. Vagai Fruit, Root Rheumatic pains, tooth brush IR g
Santalaceae
195 Santalum album Santhana mara Heartwood Refrigerant, skin diseases IR, c
PA
Table 1: Continued
Santalum album L. Sanddanamara Seed Skin troubles, refrigerant KU i
196 Thesium wightianum Wall. ex Wight Anaikchi Whole Plant Cheek to prevent bulging IR-S d
Sapindaceae
197 Cardiospermum halicacabum L. Mudakkathan whole plant Rheumatoid arthritis IR g
Cardiospermum halicacabum L. Poovanthi Nuts Body wash IR g
198 Dodonaea angustifolia L.f. Marundha soppu Leaves Rheumatism IR-S d
199 Dodonaea viscose (Linn.) Jacq. Vilari thalai Leaves Wounds and injuries, joint KO a
sprains and bone fracture
200 Dodonea viscosa Linn. Manantha Leaves Fracture KU i
201 Schleichera oleosa Jagada mara Bark Abortifacient IR, c
PA
Sapotaceae
202 Manilkara hexandra (Roxb.) Dubard Pala maram Latex Toothaches IR g
203 Tinospora cordifolia (Willd.) Miers Seenthil stem Many Disorders IR g
Simaroubaceae
204 Ailanthus excelsa Roxb. Pekalathi Bark, leaves After child birth IR g
Solanaceae
205 Datura stramnium L. Umbathi Leaves Inflamed wound and sores KO b
Datura stramonium L. Yemmuth Leaves & Piles KU i
Fruit
206 Physalis peruviana L. Urechithuvar Leaves Wound KU i
207 Solanum anguivi Lam. Kandan kathiri Fruit, leaves
Colds, coughs IR g
and fever, intestinal worms
208 Solanum denticulatum Periya midinje Whole Plant Migraine KU i
209 Solanum erianthum D.Don Malai sundai Fruit Toothache PL e
210 Solanum indicum Linn. Sunda maram Root & Toothache and snakebite KU i
Leaves
211 Solanum nigrum L. Mana thakkali Leaves Ulcer, wound PL e
Solanum nigrum Linn. Ikki sop Leaves Stomach disorders Skin rashes KO a
212 Solanum sisymbrifolium Limk. Vadadana Seeds Vermifuge KO a
213 Solanum surattrense Burm. F Kandankathiri Fruit Toothache PL e
Table 1: Continued
214 Solanum trilobatum L. Thoodhuvalai Leaves Asthma, Cold PL e
Tamaricaceae
215 Tamarix indica willd Kattuchaukku leaves Laxative IR g
Thunberigiacea
216 Thunbergia fragrans Roxb. Kakka Valli Root Snake-bite KT b
Tiliaceae
217 Grewia aspera Roxb Dadchi maram Bark Diarrhoea KU i
218 Grewia tiliifolia Vahl. Unu Root bark Swellings KT f
Ulmaceae
219 Holoptelea integrifolia Vellaya Bark Swellings IR, c
PA
Umbelliferae
220 Centella asiatica (L.) Urban. Vallarai Leaves Jaundice PL e
Verbenaceae
221 Gmelina arborea Roxb Perungilai/ Root bark Piles PL e
Kumilamaram
222 Gmelina asiatica L. Kumalai Nochi Fruit Bathing IR g
223 Lantana camara L. Unni chedi Whole plant Cuts and wounds KT f
Lantana camara L. Thusik Leaves Gum bleeding and tooth-ache KO b
Lantana camera L. Parale gida Flowers Skin inflammations KU h
224 Tectona grandis Thekku Bark Constipation IR, c
PA
Tectona grandis F. Thekku Bark Ease child birth and labour KU b
pain
Tectona grandis L. Thekku Bark Ease child birth and labour PA b
pain
Tectona grandis L.f. Thekku Stem Stomach ache and dysentery KT f
225 Vitex negundo L. Nochhi Leaves Rejunvating skin KU h
Vitex negundo L. Kumalai Nochi leaves Repel mosquitoes, body pain IR g
Vitex negundo L. Notchi Fruit Cold, Cough, Fever, headache PL e
Table 1: Continued
Zingiberaceae
226 Alpinia calcarata Rosc. Arathi poo Rhizome Immunity PL e
227 Costus speciosus (J. Koen.) Smith. Koshtam Leaves Diabetes PL e
228 Curcuma longa L. Manjal Rhizome Itching, glowing skin KU h
Curcuma longa L. Manjal Rhizome Scorpion bite KT f
229 Elatteria cardamomum (L.) Maton. Yelakkai Fruit Stomachache PL e
KU = Kurumba, IR = Irula, KT = Kota, TH = Thoda, PA = Paniya, PL = Paliyar, KT = Kattunayakas. a, (Rajan and Sethuraman, 1991); b, (Ra-
jan et al., 1997); c, (Rajan et al., 2001); d, (Murugesan et al., 2005); e, (Ignacimuthu et al., 2006); f, (Udayan et al., 2007); g, (Ragupathy
and Newmaster, 2009); h, (Deepak et al., 2014a); I, (Deepak et al., 2014b)
Thus, the medicinal uses of the tribal traditional medicines depict The tropical countries are gifted with vast resources of medicinal
their medical history through the ages. They have tried and found plants and the recent global renaissance in traditional medicines has
cure to their major health problems such as wounds and skin prob- created a large market for herbal products that can be exploited by
lems. This data also throws light on a cultural aspect of their life these countries they meet up to quality and safety specifications.
style, mainly that they held their women in high esteem for they Population explosion, incidence of side effects of synthetic medi-
have painstakingly developed remedies form medicinal plants to al- cines and our inability to provide modern medicines to a vast section
leviate labour pains and post-partum medical care. of the population living in rural and remote areas of the country due
to non-availability, in accessibility and unaffordability have been the
6. Towards sustainable local production of traditional medicines prime reasons for the growing popularity of alternative medicines
amongst rural and remote population, and neo-rich people in the de-
Sustainable development denotes a development that meets the veloped countries. Access to quality health care is an enormous pub-
needs of the present without compromising the ability of future gen- lic health global issue at the scientific, clinical, economic, political
erations to meet their own needs. It encompasses two key concepts, and policy levels. It is one aspect of the "great divide" that exists
the concept of needs, in particular meeting the essential needs of the between and within every country in the world; it is the difference in
poor and the idea of limitations that is the environments ability to access to health care between the rich and the poor. The very basis
meet these needs. Thus sustainable development seeks to relieve for promoting the local production of herbal remedies is to provide
poverty, create equitable standards of living, satisfy the basic needs cost effective medicines to populations who cannot afford costly
of all peoples, and establish sustainable political practices, while en- medicines. The World Health Organization (WHO) has been repeat-
suring that there are no irreversible damages to natural resources and edly stressing that the goal of health for all cannot be accom-
nature. plished without herbal medicines.
Nervous disorder
Hair
Burns
Antiseptic
Lactation
Heel cracks
Fracture
Eye infections
Boils
Asthma
Vermifuge
Ear pain
Abortifacient
Snake bite
Jaundice
Diabetes
Body pain
Refrigerant
Fever
Toothache
Gastric disorders
Labour pain
Rheumatic disease
Cold, Cough, Fever
Headache
Diahorrea
Stomacache
Skin Problems
Wound
0 5 10 15 20 25 30 35
Figure 3: Traditional Medicine of the Tribes of Tamil Nadu in the treatment of common ail-
ments.
7. Strategies for promoting sustainable ered and collected from the wild and rela-
production of traditional medicines tively few are cultivated in farmlands.
World Wildlife fund report (2004) report-
The first step in promoting sustainable ed that 20% of the medical plants world-
production of traditional medicine is de- wide are in the treat of disappearing (Pan
veloping a standardized mode of produc- et al., 2013). A paradigm shift from wild
tion which will meet the standards of qual- collection to structured cultivation of me-
ity, efficacy and safety as defined in the dicinally important plants will further en-
WHO guidelines. Thus bringing these sure the purity, authenticity and sustaina-
plants into large scale cultivation ensures ble supply of raw drugs. Suitable infra-
that endangered plant are protected by cul- structure like production equipment, pota-
tivation and by involving the indigenous ble water, storage facilities and post-
people over-exploitation will be avoided harvest quality monitoring and marketing
while adding to income for the indigenous are very critical. Trained man power with
tribes. Majority of the plants are still gath- an appropriate background in pharmaceu-
flux, seasonal variation in enzyme activi- There are three main types of
ties, and the biosynthetic genes which are search strategies: biorational, chemo-
present, but not fully functional. Method- rational and random approaches. An anti-
ologies that are able to characterise the HIV bioactive compound Conocurvone
majority of the constituents without indi- was discovered as a result of random ap-
vidual isolation of active constituents need proach of screening strategies. Drugs dis-
to be developed to help validation and covered using bio-rational approaches
standardization. India has a vast and di- were artemisinin, morphine, quinine, and
verse wealth of traditional medicinal ephedrine. Bio-rational approach is mostly
knowledge that can be used for bio- guided by the ethnomedical information
prospecting to benefit both the country and generated from the traditional medicines
the indigenous people. and the most effective approach to date.
These medicinal plants contain reservoir of
9. Bioprospecting of traditional medi- etho-medical and ethno-botanical tradi-
cine to combat disease tional knowledge, which is an important
guide to discovery of many new drug lead
The search for new drugs is the molecules (Table 2). As there are many
vast opportunity in the ambit of Biotech- existing and emerging diseases that cannot
nology given the vast majority of diseases be treated by the current plethora of drugs
encountered today and improved health and the additional burden of increasing
care services.
Table 2: Bioactive compounds from medicinal plants and their clinical uses
SN Bioactive compound Species Clinical Uses
Cholesterol
1 Mevastatin & lovastatin Penicillin spp.
lowering
Anthelmintic and antipar-
2 Ivermectins Streptomycetes spp.
asitic
3 Reserpine Rauwolfia serpentine Antihypertensive
4 Ephedrine Ephedra sinca Antiasthma
5 Atropine Belladonna Anticholinergic
6 Teprotide Bothrops jaracaca Cardiovascular diseases
Vincristine and
7 Catharanthus roseus Anti-cancer drug
vinblastine
Ovarian
11 Bryostatin-1 Bugula neritina carcinoma and non-
Hodgkins lymphoma
drug resistant pathogens, the need for the drugs, the costs escalate and these drugs
hour is the development of new arsenal of will not be available to the local people
drugs to combat them based on the tradi- because of the costs which is also a critical
tional knowledge. an ethical issue (Pan et al., 2013).
10. Biotechnological tools to be exploited 11.1 The Kani model of benefit sharing of
for sustainable production of tradi- traditional medicine
tional medicines The Kanis inhabit the forests of the
Thiruvananthapuram district of Kerala and
Biotechnology produces a vast array of Thirunelveli district of Tamil Nadu in
tools for the successful discovery and vali- southwestern India. In 1987, scientists
dation of traditional medical drugs. These from Tropical Botanic Garden and Re-
tools have been developed in the 80s and search Institute (TBGRI) while collecting
90s and today there is vast improvement ethnomedical data form Kani tribal people
in their technological aspects after subse- discovered that the tender fruits of Arog-
quent modification of the initial technolo- yappacha (Trichopus zeylanicus subsp.
gy. Each technique has its own pros and travancoricus) have anti-ageing, anti-
cons and care needs to exercise in the em- depressant and anti-fatigue property. This
ployment of a suitable technology to pro- paved a way for the scientists at TBGRI to
vide an appropriate traditional medicine develop a scientifically validated and
based drug. Techniques like in vitro regen- standardised herbal drug called Jeevani, a
eration through mciropropagation, callus formulation consisting of four ingredients
mediated organogenesis, somatic embryo- and Arogyappacha was one of the constit-
genesis, cryopreservation, production of uents. Jeevani has been found to be thera-
secondary metabolites and genetic trans- peutically effective having anti-fatigue and
formation holds a tremendous potential for immuno-enhancing properties and it has
the production of high quality plant based also shown good hepato-protective and
medicines mainly because of the multipli- anti-stress properties. Subsequently,
cation rate, pathogen free material, plant TBGRI decided to share 50% of the li-
preservation and regeneration success to cence fee and royalty with the Kani people
yield bioactive compound that ensures re- to encourage an equitable sharing of the
duced costs compared to the natural syn- benefits arising from the utilisation of such
thesis by the plants. knowledge, innovations and practices as
stated in the mandate of Article 8(j) of the
11. Intellectual property rights (IPR) for Convention on Biological Diversity
sustainable use of traditional medi- (CBD). This is considered to be one of the
cine first models for benefit sharing in the
world, which is popularly known as the
It is very vital to consider intellec- TBGRI Model for Benefit Sharing.
tual property rights of the local people
when the ethnomedical documentation is 12. Conclusion
done for traditional medical knowledge.
The economic implication of the eventual Sustainable local production of tra-
commercial production of standardized ditional medicines requires an enabling
traditional medicinal drug should include environment and effective partnerships
the welfare of the people from whom the between traditional health practitioners,
traditional knowledge was documented. researchers, public and the private sector.
Biopiracy becomes an ever looming issue There is a strong need for indexing eco and
when large pharmaceutical companies con- ethno information of medicinal plants, sus-
fiscate medicinal plants to make new tainable cultivation of the traditional medi-
yats, zilla panchayats, which organize cessed potato; wastes from supermarkets
seminars and teach farmers the methods and restaurants; wastes from poultry,
of Vermitechnology. Government agricul- pigs, cattles, sheeps, goats, horses (Ed-
tural departments also buy earthworms in wards and Bater, 1992) as well as horti-
bulk from these SHGs to conduct their cultural residues from dead plants and
own projects on vermiculture. In fact, the spent wastes from mushroom industry
Supreme Courts ruling criteria is that to (Edwards,1988).
decide on metro status would be the par- The degradable organic matter
ticular districts level of participation in from these wastes when dumped in open
organic farming. undergoes either aerobic or anaerobic
But the low sense of awareness degradation. These un-engineered
still remains a problem even within the dumpsites permit fine organic matter to
municipalities that have undergone semi- become mixed with percolating water to
nars on Vermitechnology. Neither the form leachate. The potential for this
Boards nor the local bodies have ideas on leachate to pollute adjoining water and
the perspectives of Vermitechnology. soil is high. India where a lot of solid or-
Technical expertise alone does not make ganic waste is available in different sec-
organic farming. Unless there is a uni- tors with no dearth of manpower, the en-
formity of procedure, the advantages of vironmentally acceptable Vermitechnolo-
organic farming low cost, more soil fer- gy using earthworms can very well be
tility and eco-friendliness - will not come adopted for converting waste into wealth.
through. Considerable work has been carried out
After all, by preparing Vermitech on vermicomposting of various organic
products, the farmer is making the soil materials and it has been established that
healthy. In turn, hes supplying healthy epigeic forms of earth-worms can hasten
crops into the market. The content of or- the composting process to a significant
ganic carbon, the index for the presence extent, with production of a better quality
of humus in the soil, is in high Ranges. So of composts as compared with those pre-
the farmers must contribute considerably pared through traditional methods. The
more towards ecology and food produc- viability of using earthworms as a treat-
tion. He deserves all the support he can ment or management technique for nu-
get. With the right financial support from merous organic waste streams has been
the Government and a more organized investigated by a number of workers
network of cultural units, Vermitech (Logsdon, 1994; Madan, 1988; Singh,
products, as a form of enrichment can 2002). Similarly a number of industrial
generate a steady source of income for the wastes have been vermicomposted and
impoverished folk of agricultural areas. turned into nutrient rich manure
There are numerous sources of (Sundaravadivel, 1995). Hand et al.
waste produced in India where degradable (1988) defined vermicomposting as a low
organic matter is either partially or fully cost technology system for the processing
generated. Solid waste consists of the dis- or treatment of organic wastes.
carded portion of the households, dead A growing awareness of some of
animals, trade, commercial, agricultural the adverse economic and environmental
and industrial waste and other large waste impacts of agrochemicals in crop produc-
like debris from construction site, furni- tion has stimulated greater interest in the
ture etc. Solid wastes are generally cate- utilization of organic amendments such as
gorized as domestic, industrial and haz- compost or vermicompost for crop pro-
ardous or biomedical waste. Studies were duction (Follet, 1981). Therefore, the sus-
made on some solid wastes like sewage tainability has to be restored by some
sledges and solids from waste water means of regular food security. Utiliza-
(Mitchell et al., 1980); wastes from pro- tion of earthworms may be an answer as
viable or can be placed in spaces availa- organic matter for partial decomposition,
ble inside the house itself. Thus depend- care must be taken that, both the organic
ing on the economical status, the place matter and the cow dung are shad dried.
can be selected. The conditions in main- This is because; wet organic matter and
taining the vermibeds are, direct sunlight cow dung may contain the cyst and larvae
must be avoided and it must be safe from of other microorganisms and insects. So if
insects and rodents. implemented wet, they may grow along
The second method of culturing with earthworms and do hindrance.
earthworms is by tank method. By this The alternate layers of organic
way multiplication of earthworms can be matter and cow dung are arranged in the
done in large scale. For this method, ce- form of heap and 50% moisture must be
ment tanks of dimensions one meter maintained. Cover the heap with thatched
width one meter height 3-5 meter coconut leaves. This set up must be kept
length can be constructed. These tanks for atleast 30 days. The organic matter,
must be constructed inside a thatched roof along with cow dung mixture must be
shed so that direct sunlight and rain can turned over with a spade once in a week.
be avoided. The shed can be of any di- The microorganisms which are present in
mensions according to the land available. the cow dung slowly degrade the organic
Thus the length of the tank and the no of matter. After 40 days the process of par-
tanks to be constructed can also be done tial decomposition will be finished and
accordingly. The shed must be construct- now it can be used as a medium for ver-
ed in East-West direction length wise to mibed.
avoid direct sunlight and preferably open In the containers, the vermibed
from all sides with unpaved floor with must be prepared by mixing the partially
raised ground (atleast 6 inches) to protect decomposed organic waste and cow dung
the area from flooding during the rains. (shade dried and powdered) in 1: 1 ratio.
This ratio is recommended by many sci-
2.1.1. Vermibed preparation entists and has been proved in many liter-
The vermibed for both the meth- atures also. Nearly 50% of moisture must
ods can be prepared using any kind of de- be maintained in the bed. Earthworms can
gradable, non-toxic organic waste like be introduced after two days. Leave the
leaf litters, agricultural wastes (Suthar, setup for 45 days. The worms eat the de-
2010), shredded newspaper (Updegraff, graded organic matter and convert them
1971), cardboard, etc... can be utilized as into vermicastings. At the same time they
medium. Wastes from fruit and vegetable also reproduce and increase in number.
market such as potato peels, onion peels, Vermibed for tank method can al-
cabbage leaves, carrot and radish leaves, so be prepared in the same way but the
lettuce leaves, moldy bread can also be partially degraded waste and the cow
used (Suthar, 2009). Though organic dung must be added in large quantities
wastes serve as food for earthworms, they into the tank. Earthworms can also be re-
cant be directly implemented for ver- leased in large quantities. Water must be
miculture. In other words, the worms sprinkled on the vermibed once or twice
cant eat the organic matter directly. Thus in a week according to the humidity pre-
it must be subjected to partial decomposi- vailing in the atmosphere. Temperature of
tion and then used as a medium for ver- about 37 degree Celsius must be main-
mibed. tained. If the climate is hot, wet jute bags
The organic matters must be can be screened on the sides of the tank.
shredded and alternate layers of organic By the tank method, earthworms can the
matter and mixture of cow dung is spread reproduced 300 times greater within 45
on the floor of shed constructed for ver- days or 60 days (depending upon the
miculture. Before the implementation of worms cultured). The reproducing capaci-
ty of exotic worms is greater than the in- small worms (juveniles) present segregate
digenous worms. It is said that eight red in the centre of the heap and can be col-
worms multiply into 1,500 redworms (Ei- lected.
senia fetida) in six months. These earth-
worms can be used to prepare vermicom- 2.2. Application of earthworms
post or for other purposes or can be sold. Adult earthworms can be sold as
The vermibeds must be covered fish bait and the young ones can be used
with a Jute mat to protect earthworms as inoculums for fresh bedding. Many
from birds and insects. Water is sprinkled industries like aquaculture and Poultry
on the vermibeds daily according to re- will buy earthworms for using it as live
quirement and season to keep them moist. feed. Business people who are interested
The waste is turned upside down once in in constructing vermicomposting units
a week. The appearance of black granular (house hold, small scale or large scale
crumbly powder on top of vermibeds in- unit) will be purchasing the earthworms.
dicates the casts of earthworm. Addition Earthworms can be sold to new business
of more amount of cow dung ensures the people who are about to start their ver-
fecundity rate of earthworms. miculture or composting unit. Worms are
also bought by academic institutions for
2.1.2. Harvesting of earthworms research purposes. Earthworms can be
The vermibed after 45 days is sold to wholesalers who then resell the
converted into vermicastings. This indi- worms to bait shops, home and organic
cates that all the organic matter along nurseries, and other users.
with cow dung is eaten up by the worms
and converted into vermicastings. Now 3. Vermicomposting
there is no food available for the worms.
The content in the container or in the tank Earthworms facilitate the stabili-
must be removed and new beds must be zation of organic wastes because their ac-
replaced. Before the replacement, the tivity maintains aerobic conditions and
worms must be harvested. The very re- ingested solids are converted into discrete
cent and new technique followed to har- odourless casts (Edwards, 1988). Thus the
vest earthworms is using fresh cow dung end product of vermicomposting is re-
balls. Fresh cow dung attracts the earth- ferred to as "vermicasting" or vermicom-
worms very much. Thus balls of fresh post. This is a nutrient rich organic sub-
cow dung, of 15 to 20 cm in diameter are stance that can be added to soil to in-
placed inside the vermibeds. For contain- crease its organic matter content and
er method, one ball is enough while for available nutrients. Vermicomposting is
tank method, four to six balls can be used getting enormous importance in the ame-
to harvest earthworms. lioration of severe problems associated
As soon as the cow dung balls are with the disposal of large quantities of
placed, the earthworms start migrating organic wastes (John Paul, 2005). Earth-
into the fresh cow dung balls. The cow worms feed on organic matter, in which
dung balls were kept for 6 to 8 hours, af- 5-10% is taken as food intake for their
ter which are removed. Thus the earth- growth and the rest is excreted. Exotic
worms can be harvested and used as in- earthworms like Red worms (Eisenia feti-
oculums for the new vermibed. The ver- da) and African worm (Eudrilus eugeni-
micastings in the container or the tank is ae) and Indigenous species like Lampito
removed and made into heap. This heap is mauritti and Perianyx excavatus are
left undisturbed for a day. Next day, the proved to be effectively in vermicompost-
vermicastings are removed slowly from ing (Karmegam and Daniel, 2009, Kaur,
the outer side with the help of spade, et al., 2010). Vermicomposting can be
sieved, and packed as biofertilizer. The done in three ways.
in getting the pure form of vermicompost, It neutralizes the soil acidity or alka-
i.e. the vermicasts, which are collected on linity.
the top of vermibed. At that case, ver- Vermicompost is free from patho-
micasts can be collected with a spatula gens, toxic elements, weed seeds
once in a week and can be packed and etc.
sold. The rate of pure form of vermi- Vermicompost minimizes the inci-
castings is also higher when compared to dence of pest and diseases.
the vermicompost. When large amount of It contains valuable vitamins, en-
compost are to be harvested, for example zymes and hormones like auxins,
the compost obtained from tank method gibberellins etc.
or from pit method, first the worms are The nutrients available in ver-
harvested with the help of fresh cow dung micompost (in general) are Organic
balls, then the whole material is moved to carbon (9.5 17.98%), Nitrogen
a plain area or on a plastic sheet and made (0.5 1.50%), Phosphorous (0.1
into a single heap and is exposed to light, 0.30%), Potassium (0.15 0.56%),
scooped, sieved and packed for sales. Sodium (0.06 0.30%), Calcium
Earthworms or undecomposed materials and Magnesium (22.67 to 47.60
if seen any, are collected and returned to meq/100g), Copper (2 9.50 mg
the compost pile. kg-1), Iron (2 9.30 mg kg-1), Zinc
For faster rate of harvesting ver- (5.70 11.50 mg kg-1) and Sulphur
micompost, the original heap is better di- (128 548 mg kg-1).
vided into several smaller pyramids. To
enhance the earthworm movement to- 3.7. Storing and packing of vermicompost
wards the centre of the heap, ball of raw Watering is stopped for atleast 5
or fresh cow dung can be placed at the days at this stage. The first lot of Ver-
centre. Always earthworms have high af- micompost is ready for harvesting after 2-
finity towards fresh cow dung. Thus after 2 months and the subsequent lots can
few hours the worms can be easily sepa- be harvested after every 6 weeks of load-
rated and the compost can be harvested ing. The vermibed is loaded for the next
and packed. treatment cycle. The harvested ver-
micompost should be stored in dark, cool
3.6. Advantages of vermicompost place. It should have minimum 40%
Vermicompost is rich in all essential moisture. Sunlight should not fall over the
plant nutrients, hence provide excel- composted material. It will lead to loss of
lent effect on overall plant growth, moisture and nutrient content. It is advo-
encourages the growth of new cated that the harvested composted mate-
Shoots / leaves rial is openly stored rather than packed in
Vermicompost is free flowing, easy over sac. Packing can be done at the time
to apply, handle and store and does of selling. If it is stored in open place, pe-
not have bad odour. riodical sprinkling of water may be done
It improves soil structure, texture, to maintain moisture level and also to
aeration, and water holding capaci- maintain beneficial microbial population.
ty. If the necessity comes to store the materi-
Vermicompost is rich in beneficial al, laminated over sac is used for packing.
microorganisms which fix the Ni- This will minimize the moisture evapora-
trogen and Phosphorous in the soil. tion loss. Vermicompost can be stored for
Vermicompost may contain earth- one year without loss of its quality, if the
worm cocoons from which juveniles moisture is maintained at 40% level.
may come and increases in popula-
tion in the soil where applied. 4. Vermiwash
Abstract: Food is the most essential component of life for survival. The quality of food
products is essentially to be authenticated as they are closely related to the health of human
beings. Foodomics, is a concept to utilize technology for improvement of food and nutri-
tion. Food products are authenticated in benefit of both consumers as well as commercial
traders. Food authentication is done by analytical techniques like chromatography, Fourier
Transform Infrared Spectroscopy (FTIR), Nuclear Magnetic Resonance Spectroscopy
(NMR), Gas Chromatography - Mass Spectroscopy (GC-MS) and Liquid Chromatography
- Mass Spectroscopy (LC-MS). Plant or animal based food products are widely authenticat-
ed for their origin, species nomenclature using DNA barcodes - a genomics based approach.
The protein molecules of food products serve as key molecules for authentication using
proteomics approach such as 2-dimensional gel electrophoresis and 2-dimensional differ-
ence gel electrophoresis (DIGE). Thus, food authentication is mandatory to substantiate the
geographical origin, species nomenclature, food composition, genetic modification of a
food product that reaches to the hands of consumers in the food markets. Thus, the devel-
opment of rapid, novel food authentication methods to validate sea food products, Genet-
ically Modified (GM) food products based on biotechnological approaches helps to provide
quality assured food products to the human community. This review briefs about the vari-
ous techniques employed in food authentication, their advantages and applications in food
biology.
Food Labelling
Food Authentication
Food
Food Product
Authentication
Chromatographic
techniques
Adulteration
DNA Barcodes
Species
Confusion Proteome
analysis
HPLC
Barcode FTIR
Food
Authentication
DGGE NMR
GC-MS
genetically modified products, soy pro- databases will be fruitful for high
teins in foods and dairy products are done throughput validation of food products.
based on proteomic analysis (Gallardo et
al., 2013). This proteomics based ap- Acknowledgement
proach is of wide importance and applica-
tions in food industry as they accurately Authors are thankful to the Educa-
pin point the difference between original tional fellowship, UGC-NON-NET
and adulterated food products. Scheme. Authors are also thankful to the
State-of-art infrastructure facility provid-
6. Immunological methods ed by CEGS, School of Biological Sci-
ences, Madurai Kamaraj University, In-
Immunological methods rely on dia.
the binding specificity of the antibodies
designed to an atypical antigen i.e., aller- References
gens, toxins etc. in food by use of En-
zyme Linked Immuno Sorbent Assay Ali, M. E., Razzak, M. A., and Hamid,
(ELISA) (Asensio et al., 2008). For in- S. B. A. (2014). Multiplex PCR in
stance, they are used to spot the presence species authentication: probability
of potential fish allergen parvalbumin and prospects - a review. Food Ana-
(Gajewski and Hsieh, 2009) in seafood lytical Methods, 7(10), 1933-1949.
industry. These techniques rely on speci- Arcuri, E. F., El Sheikha, A. F., Rych-
ficity and also have an added advantage lik, T., Piro Metayer, I., and Mon-
of validating a large number of samples tet, D. (2013). Determination of
with high precision in a short span of cheese origin by using 16S rDNA
time. fingerprinting of bacteria communi-
ties by PCRDGGE: Preliminary
7. Perspectives application to traditional Minas
cheese. Food Control, 30(1), 1-6.
Food authentication not only en- Asensio, L., Gonzalez, I., Garcia, T.,
sures food quality but also assures human and Martin, R. (2008). Determina-
health. Emergence of new research fields tion of food authenticity by enzyme-
such as transgenics, foodomics, nutri- linked immunosorbent assay (ELI-
genomics has resulted in wide range of SA). Food Control, 19(1), 1-8.
neoteric products that need to be validated But P. P. H. (1994). Herbal poisoning
for substitutes prior to their distribution in caused by adulterants or erroneous
commercial markets for edible purpose. substitutes. Journal of Tropical
Some of the practical applications of food Medicine and Hygiene, 97,371374.
authentication based on the above- Capozzi, F., and Bordoni, A. (2013).
mentioned techniques are tabulated in Ta- Foodomics: a new comprehensive
ble 1. Construction of food authentication approach to food and nutri-
tion. Genes and nutrition, 8(1), 1-4.
Capuano, E., Lommen, A., Heenan, S., trometry (ICP-MS). Food Chemis-
de la Dura, A., Rozijn, M., and try, 114(2), 706-711.
van Ruth, S. (2012). Wild salmon Hebert, P. D., Cywinska, A., and Ball,
authenticity can be predicted by 1H S. L. (2003). Biological identifica-
NMR spectroscopy. Lipid Technol- tions through DNA bar-
ogy, 24(11), 251-253. codes. Proceedings of the Royal So-
Cozzolino, D. (2012). Recent trends on ciety of London B: Biological Sci-
the use of infrared spectroscopy to ences, 270(1512), 313-321.
trace and authenticate natural and Hohmann, M., Monakhova, Y., Erich,
agricultural food products. Applied S., Christoph, N., Wachter, H.,
Spectroscopy Reviews, 47(7), 518- and Holzgrabe, U. (2015). Differ-
530. entiation of organically and conven-
Cserhati, T., Forgacs, E., Deyl, Z., and tionally grown tomatoes by chemo-
Miksik, I. (2005). Chromatography metric analysis of combined data
in authenticity and traceability tests from proton nuclear magnetic reso-
of vegetable oils and dairy products: nance and mid-infrared spectrosco-
a review. Biomedical Chromatog- py and stable isotope analy-
raphy, 19(3), 183-190. sis. Journal of Agricultural and
Danezis, G. P., Tsagkaris, A. S., Camin, Food Chemistry, 63(43), 9666-9675.
F., Brusic, V., and Georgiou, C. A. Ibanez, C., Garcia Canas, V., Valdes,
(2016). Food authentication: tech- A., and Simo, C. (2013). Novel
niques, trends and emerging ap- MS-based approaches and applica-
proaches. TrAC Trends in Analytical tions in food metabolomics. Trends
Chemistry, 85, 123-132. in Analytical Chemistry, 52, 100-
Drivelos, S. A., and Georgiou, C. A. 111.
(2012). Multi-element and multi- Janzen, D. H., Hallwachs, W., Blandin,
isotope-ratio analysis to determine P., Burns, J. M., Cadiou, J., Cha-
the geographical origin of foods in con, I., and Franclemont, J. G.
the European Union. Trends in Ana- (2009). Integration of DNA barcod-
lytical Chemistry, 40, 38-51. ing into an ongoing inventory of
Gajewski, K. G., and Hsieh, Y. H. P. complex tropical biodiversi-
(2009). Monoclonal antibody specif- ty. Molecular Ecology Re-
ic to a major fish allergen: parval- sources, 9(s1), 1-26.
bumin. Journal of Food Protec- Longobardi, F., Ventrella, A., Bianco,
tion, 72(4), 818-825. A., Catucci, L., Cafagna, I., Gallo,
Gallardo, J. M., Ortea, I., and Carrera, V., and Agostiano, A. (2013). Non-
M. (2013). Proteomics and its appli- targeted 1 H NMR fingerprinting
cations for food authentication and and multivariate statistical analyses
food-technology research. Trends in for the characterisation of the geo-
Analytical Chemistry, 52, 135-141. graphical origin of Italian sweet
Georgiou, C. A., and Danezis, G. P. cherries. Food Chemistry, 141(3),
(2015). Elemental and isotopic mass 3028-3033.
spectrometry. Comprehensive Ana- Moore, J. C., Spink, J., and Lipp, M.
lytical Chemistry, 131-143. (2012). Development and applica-
Giannenas, I., Nisianakis, P., Gavriil, tion of a database of food ingredient
A., Kontopidis, G., and Kyria- fraud and economically motivated
zakis, I. (2009). Trace mineral con- adulteration from 1980 to
tent of conventional, organic and 2010. Journal of Food Sci-
courtyard eggs analysed by induc- ence, 77(4), R118-R126.
tively coupled plasma mass spec- Nurrulhidayah, A. F., Man, Y. B., Al-
Kahtani, H. A., and Rohman, A.
Zoological Survey of India, M-Block, New Alipore, Kolkata-700053, West Bengal, India;
*Correspondence: v.gantait@rediffmail.com; Tel: 09433463555
Abstract: Amongst different pests of agriculture, the plant parasitic nematodes are consid-
ered the worst one because of devastations they cause to the crops. Due to their microscopic
nature, they are the hidden enemies of soil and crop damages caused by them have not been
fully formulated. For sustainable development of agriculture and to stop the yield loses
causes by these tiny pests of crops, potential control measures should be explored and
adopted against them. By following various physical, chemical, biological and botanical
methods, some of cultural practices and also by regulatory methods nematode infestation in
agricultural fields must be stopped or checked at certain level. This will help to enhance
crop production, which will ultimately be helpful to improve the gross national product
(GNP) of country and agricultural sustainability. This article provides an overview of man-
agement strategies which could be used against plant parasitic nematodes to boost the sus-
tainable development of agriculture.
zoan invertebrate group, because they are where fine plant roots are concentrated;
found in all types of habitats (Bohra and adequate soil moisture and oxygen also
Baqri, 1997). They are so numerous that favours dense populations of nematodes.
if everything on the earth were to disap-
pear except the nematodes, the outlines of 1.3. What they feed?
habitats would still be dimly visible; the Nematodes are microphagous,
mountains, lakes and oceans, the plants mycetophagous, saprophytic, phytopara-
and the animals would all be outlined by sitic, predatory, carnivorous and even
the nematodes present (Cobb, 1914). cannibalistic in nature. Most of them feed
Based on the habitat, the total on bacteria, fungi, other microorganisms
nematode population can be categorized and decaying matter. They can parasitize
into three groups: marine, animal- plants, different animals including man
parasitic, soil and freshwater nematodes and even other nematodes also. Depend-
(Ayoub, 1980). The marine nematodes ing on the diverse diets Yeates et al.
constitute about 50% of the total nema (1993) categorized nematodes into eight
population, where as animal-parasitic feeding groups: plant feeders, algal feed-
nematodes make up only 15% of the ers, hyphal feeders, substrate ingesters,
known nematode species. The soil and unicellular eukaryote feeders, bacterial
freshwater nematodes can be spitted into feeders, predators and omnivorous.
two finer divisions: free-living and plant-
parasitic. Free living nematodes compris- 2. Important roles in agricultural as-
ing about 25% of the total nematode pop- pects
ulation. The plant-parasitic nematodes
constitute only 10% of the total nema- Plant parasitic and soil-inhabiting
todes (Figure 1). nematodes have been known for their vir-
According to Crofton (1966) most ulence causing significant loss to agricul-
of the soil holds about 90% nematodes at tural and horticultural crops (Bohra and
top six inches. (Nicholas, 1984) opined Baqri, 2004). Several species have come
that the soil nematodes are usually most to be recognized as useful predators in the
abundant near the soil surface, with the control of different insects and nematodes
majority within the top 10 cm, though also. The possibility of using nematodes
some may be found much deeper. He also for control of different plant parasitic
stated that, populations are much denser nematodes was first suggested by Cobb
in the zones, rich in organic matter or (1917). The role of predatory nematodes
rect and indirect benefits to control these Fallowing is a very common prac-
tiny tigers of agricultural fields improved tice and cheaper way to minimize the
plant health, thereby reducing their nematode problems by keeping the lands
changes of suffering from nematode dis- from all vegetations for a certain period.
eases and also increased the plants ability Complete fallow without allowing any
to withstand adverse growing conditions, plant or weed to grow invariably ensures
resulted improvement of crop production. the parasitic nematodes will have no host
Principal strategies for nematode man- to feed. Thus, those are deprived of food
agement are cultural practices, physical, and killed by the solar heat and soil desic-
biological, botanical, chemical and regu- cation.
latory methods. Deep summer ploughing involves
exposure of soils to solar heat and desic-
6. Cultural Practices cation, helps to kill the nematode pests
along with other pathogens. For en-
To control nematode pests, cultur- hancement of the efficacy of solar heat-
al practices are the most effective and ing, polyethylene mulching of moist soil
economical means which can be achieved during hottest period is being advocated.
by crop rotation, fallowing, deep summer It considered as a very effective and good
plaughing, water flooding, growing an- control measure against nematode pests
tagonistic or trap crops, removal of in- and can be used with other cultural prac-
fected plant debris, selection of healthy tices like crop rotation, green manuring,
planting materials, application of organic inter cropping as well as with nematicides
manures and fertilizers etc. etc. (Mathur et al., 1987).
Some species of nematodes are In the field where there is enor-
able to feed and multiply on certain crops, mous availability of water and nematode
but not on others. The crop plants on infested area is uniformly leveled, flood-
which they cannot feed and abundantly ing can be adopted as a routine practice
reproduce are called non-host. By crop and very effective measure. Under sub-
rotation system, the crops to be grown in merged condition, chemicals lethal like
between the susceptible host crops should hydrogen sulphide and ammonia to these
be immune or resistant to nematodes or at noxious pests are released. Asphyxiation
least non-host plants which help to eradi- and microbial decomposition products
cate or minimize the nematodes problems.
due to anaerobic condition help to kill the terials like banana corns, onion bulbs, tu-
nematodes. ber seeds and roots of seeding can be
The crops like mustard, marigold, dipped in 5-55C hot water for about 10
neem etc. are treated as antagonistic minutes.
crops, those have chemicals or alkaloids The most primitive way of heating
as root exudates which repeal or suppress the soil on large scale is by putting a fire
the plant parasitic nematodes. These over it. Burning a layer of dry leaves on
plants can be grown along with main crop soil killed nematode pests. Rabbing of
or may be included in crop rotation. bajara husk, paddy husk, wheat straw etc.
The basic concept with trap crop- become most effective against nematodes.
ping system is that two crops are grown in Burning materials outside and incorporat-
the field, out of which one crop is highly ing ash into the soil surface satisfaction
susceptible to nematode. When nema- nematode kill.
todes attack such crop, very carefully it Soil solarization is the most recent
should be remove and destroyed or burnt method for control of plant parasitic nem-
totally. Thus, the main crop escapes from atodes (Sharma & Trivedi, 1991). The
nematode infestation. Cowpea is a very technique consists of covering the moist
good example of such susceptible crop for soil with a good quantity, clear and trans-
destroying root-knot nematodes. parent plastic film during the period of
Early detection of infected plants, intense sunshine and increasing substan-
immediate removal and destruction of tial soil temperature over the non-
those helps to reduce the spreading of solarized soil. Increased soil temperature
nematodes in the field. Selection of nema- coupled with soil moisture invariably re-
tode-free healthy planting materials or sult the significant reduction in popula-
plantation of nematode-resistant varieties tion densities of phytoparasitic nema-
is also a very effective method to avoid todes. The nematode pests of crops are
nematode infestation in field. effectively controlled by this method but
Soil treatments with organic ma- it is restricted to summer season and only
nures, green crop residues or green leaf in the tropical and subtropical region of
manures, farm yard or poultry manures, the world.
oil cakes, and oils of neem, karanja, cas- Careful washing of tubers, bulb
tor etc. significantly checked nematode and other planting materials prevent this
population. The use of such materials also nematode infestation in new planting
encourages the development of nematode- fields. Modern mechanical seed cleaning
antagonistic microbes and predacious methods have been developed to remove
nematodes also those help to control the seed galls to form normal healthy
nematode infestation in agricultural seeds. Sanitation, the use of clean tools
fields. and equipments in field also prevent nem-
atodes infestation. Soil amendments and
6.1. Physical methods frequent irrigation can also help to reduce
The hot water treatment, hot water nematode-damage of crops.
drenching, rabbing with slow burning ma-
terials, soil solarization, electrical soil 6.2. Biological methods
heating, washing and cleaning of seed etc. Biological control method of
are the most effective physical methods to nematodes include the use of predaceous
control nematodes infestations to crops. and parasitic organisms such as fungi,
For denamatization, rhizomes, bacteria, protozoans, viruses, nematodes,
bulbs, corns, tubers and fleshy roots of tardigrades, collembolans, mites etc, even
plantations and also other planting mate- antagonistic higher plants also. This
rials are submerged into hot water for cer- method, in fact, should be considered a
tain periods. Prior to plating the seed ma- skillful manipulation of the biosphere
ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 62
Biotech Sustainability (2017)
Management Strategies against Tiny Tigers Gantait
Table 2: Continued
13. Ditylenchus angustus Rice nematode
14. Ditylenchus destructor Potato root nematode, Potato tuber nematode,
Iris nematode
15. Ditylenchus dipsaci Stem nematode, Tulip root nematode, Bulb
nematode
16. Ditylenchus myceliophagus Mushroom spawn nematode
17. Dolichodorus spp. Awl nematodes
18. Dorylaimus spp. Spear nematodes
19. Globodera rostochiensis Golden nematode of potato
20. Globodera spp. Cyst nematode
21. Helicotylenchus spp. Spiral nematodes
22. Hemicriconemoides spp. Sheathoid nematodes
23. Hemicycliophora spp. Sheath nematodes
24. Heterodera avenae Great root nematode, Cereal nematodes
25. Heterodera cruciferae Cabbage cyst nematode
26. Heterodera glycines Soybean cyst nematode
27. Heterodera goettingiana Pea cyst nematode, Pea root nematode, Alfalfa
root nematode
28. Heterodera schachtii Sugar beet nematode
29. Heterodera spp. Cyst-forming nematodes
30. Hirschmanniella oryzae Rice root nematode
31. Hoplolaimus spp. Lance nematode, Spear nematode
32. Longidorus spp. Needle nematode
33. Meloidodera spp. Cystoid nematode
34. Meloidogyne arenaria Peanut root knot nematode
35. Meloidogyne brevicauda Indian root knot nematode
36. Meloidogyne exigua Coffee root knot nematode, Brazilian root knot
nematode
37. Meloidogyne incognita Southern root knot nematode
38. Meloidogyne javanica Javanese root knot nematode
39. Meloidogyne spp. Root knot nematodes, Root-gall nematodes
40. Nacobbus spp. False root knot nematodes
41. Paratylenchus spp. Pin nematodes
42. Pratylenchus spp. Root lesions nematodes, Meadow nematodes
43. Radopholus similis Burrowing nematode
44. Rhadinaphelenchus cocophilus Coconut palm nematode, Red ring nematode
45. Rotylenchulus reniformis Reniform nematode
46. Rotylenchus spp. Spiral nematodes
47. Trichodorus spp. Stubby root nematodes
48. Tylenchorhynchus claytoni Stunt nematode, Teaselate stylet nematode
49. Tylenchorhynchus martini Sugarcane stylet nematode
50. Tylenchorhynchus spp. Stunt nematode, Stylet nematode.
51. Tylenchulus semipenetrans Citrus root nematode
52. Xiphenema spp. Dagger nematodes
Certain botanicals in the form of aqueous charantia L.; leaves of Ageratum coni-
extracts of various parts have great poten- zoides L., Anacardium occidentale L.,
tial against nematodes, the aqueous ex- Argemone mexicana L., Datura stramo-
tracts of fresh neem leaves; fruit skin of nium L. etc.; aqueous root extract of Oci-
Citrus reticulata Blanco and Momordica mum sanctum L.; seed extracts of
ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 64
Biotech Sustainability (2017)
Management Strategies against Tiny Tigers Gantait
University, Manipur, India. pp. 159- Yeates, G. W., Bongers, T., De Goede,
177. R. G. M., Freckman, D. W. and
Wardle, D. A. and Yeates, G. W. Georgieva, S. S. (1993). Feeding
(1993). The dual importance of habits in soil nematode families and
competition and predation as regula- genera: An outline for soil ecol-
tory forces in terrestrial ecosystem: ogists. Journal of Nematology 25,
evidence from decomposer food 315-331.
webs. Oecologia 93, 303-306.
Abstract: The global demand for drugs is increasing day-to-day and in the same fashion
pharmaceutical effluents released from industry adversely affect the environment and hu-
man health. Discharge of pharmaceutical effluents either directly or indirectly into the envi-
ronment results in substantial pollution. Disposal of such toxic effluents into the environ-
ment also affects the ecosystems either by direct or indirect pathway. Several researchers
from environmental biotechnology domain have reported the presence of pharmaceuticals
in water and soil. To minimize the environmental pollution and to develop the sustainable
solutions, various methods to convert pharmaceutical effluents into non-toxic and biode-
gradable organic matter has been proposed and discussed by the scientific community. This
chapter discusses the fate of pharmaceutical waste in the environment, its impact on human
health, methods adopted to treat effluents before its disposal into the environment, QSAR
studies in the design of biodegradable agents and future research directions to protect the
environment for sustainability.
Figure 1: A diagrammatic sketch showing imaginable sources and paths for the pharma-
ceutical deposits to reach into the water bodies including our drinking water.
and transformation products if not eradi- teams in few countries. However, the
cated during sewage handling then they presence of approximately 160 various
may come in the aquatic environment and drugs and or various pharmaceuticals
in the long run can reach the drinking wa- chemical associates is reported in STPs
ter bodies and may come to our homes effluent and in ground and surface water
through water supply (see Figure 1). The bodies (WHO, 2011). Even in drinking
active amalgams pharmaceuticals or its water samples, some Active Pharmaceuti-
derivatives could enter into environment cal Ingredients (APIs) were found (WHO,
by various routes or non-point sources; 2011). They are additionally distin-
for instance waste, sewage treatment guished in the freezing environment. Con-
plants (STPs) and landfill effluent or from trasted with the free water, phase analysis
animals treatment facilities (Calamari et of APIs is difficult in bio solids and sew-
al., 2003). age sludge for proper comprehension
Pharmaceuticals, particularly (Daughton and Ternes, 1999).
hormones were the primary focus of re- Medications are useful in aquacul-
search and public consciousness in the ture, livestock farming, and veterinary
1970s. The hormones do not (bio) de- and in other sectors of agriculture. How-
grade completely. However, the concept ever, improper practices results in con-
produced enthusiasm in 1980s. Other tamination of environment; for instance,
constituents like heavy metals, aromatic seepage of growth promoters are found in
polycyclic hydrocarbons, pesticides and contaminated soil. Various types of chem-
detergents were also the issue of wide- icals used in agriculture and those become
spread examination during 1980s (Jones source of environmental contamination or
et al., 2008). biowaste can have an adverse impact on
Deliberate research on contamina- human health (Table 1). Veterinary anti-
tion of environment by various pharma- biotics could end up in the groundwater
ceuticals is undertaken by some research or in the soil. By runoff, it may be cleared
Table 1: Different types of wastes and their impact on human health (Ferrari et al., 2003;
Tauxe-Wuersch et al., 2005; Isidori et al., 2006; Khanal et al., 2006)
No Type of waste Effect on humans
1. Nuclear waste Hair: The losing of hair occurs with radiation exposure at 200
rems.
Brain: Brain cells do not replicate, they damaged directly at
the exposure is 5,000 rems.
Thyroid: The thyroid gland is vulnerable to radioactive iodine.
In adequate amounts, radioactive iodine can destroy all or part
of the thyroid.
Blood System: The blood's lymphocyte cell count will be re-
duced once a person is exposed to around 100 rems, leaving
the victim more susceptible to infection. This is often referred
to as mild radiation sickness.
Heart: Immediate damage to small blood vessels and perhaps
cause heart failure and death directly by intense exposure to
radioactive material at 1,000 to 5,000 rems.
Gastrointestinal Tract: Exposure to 200 rems or more radia-
tion harm to the intestinal tract lining will cause nausea,
bloody vomiting and diarrhea. The radiation will begin to de-
stroy the cells in the body that goes division rapidly. These in-
cluding blood, GI tract, reproductive and hair cells, and harms
their DNA and RNA of living cells.
Reproductive Tract: Since reproductive tract cells multiply
rapidly, these areas of the body can be damaged at rem levels
as low as 200. Long-term, some radiation sickness victims will
become sterile.
2. Environmental Interaction of humans to agrichemicals is common and results
waste in acute and chronic health threats, together with acute and
chronic neurotoxicity (fumigants, insecticides and fungi-
cides,), lung chemical burns (anhydrous ammonia), newborn
methemoglobinemia and also impairment (paraquat).
3. Agrichemicals Soil pollution effects causes leukemia and it is danger for
young children as it can cause developmental damage to the
brain furthermore it illustrated that mercury in soil increases
the risk of neuromuscular blockage, causes headaches, kidney
failure, depression of the central nervous system, eye irritation
and skin rash, nausea and fatigue. Soil contamination closely
related to air and water pollution, so numerous things come out
as similar as caused by water and air pollution.
into surface water bodies. Wide variety of namics in soils. The profusion of these
indications of various active ingredients natural antibiotics was less and appeared
in liquid state and in the soil has been es- as restricted to the adjacent surroundings
tablished. The primary reviews that have (Kmmerer, 2004). The composition of
researched the transfer and the related the soil-lodging species is found to be in-
risks have been published only recently fluenced by antimicrobial agents. Antibi-
(Corcoran et al., 2015). otics in the soil appear to support fungal
Antibiotics transpire naturally into development. The circumstances in the
the soils and the resistance against these areas beneath fish farms are critical due to
antibiotics influences the inhabitants dy- more concentrations of antimicrobial
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Biotech Sustainability (2017)
Designing Greener Pharmaceuticals and Practicing Green Chigurupati et al.
agents as a result of seepage. Some anti- well as affects the flora and fauna at vari-
microbial agents are responsible to dimin- ous extents. Hence, the dumping of phar-
ish the number of microscopic organisms maceuticals into the environment is now
around aquaculture facilities or where the recognized as a serious issue (Boxall,
seepage of antimicrobial agents was no- 2004).
ticeable (Oliveira et al., 1995). The de- There are many techniques and or
gree of the impact of ciprofloxacin on mi- processes utilized to overcome this type
crobial salt marsh groups was contrarily of pollution problem. Few of them are -
related to the level of sorption to the sed- (i) Thermal process; (ii) Chemical pro-
iments. Even though ciprofloxacin is a cess; (iii) Irradiation process; (iv) Biolog-
wide-spectrum antibiotic, its influence on ical process (biodegradation); and (v)
sediment microbial organisms was selec- Mechanical process.
tive and seemed to favor Gram-negative Among the five above stated pro-
and sulfate-reducing bacteria. (Sengupta cesses used to minimize the waste or pol-
et al., 2013). This type of the environ- lution, biodegradation techniques (biolog-
mental contamination can cause the im- ical process) are considered as greener
balance in ecosystems. technique/technology that involves the
The impacts of oxytetracycline in use of various biological processes to
environmentally applicable concentra- overcome the adverse effects of pharma-
tions on enchytraeids, springtails and ceutical effluents into the environment.
earthworms have been analyzed. Neither Biodegradation is a complex and natural
one of the antibiotics demonstrated any decomposition process of organic sub-
harmfulness against the organisms under stances with the help of biochemical reac-
scrutiny. However, the capability of a pol- tions of microbes (OECD Statistics
lutant to accumulate in organisms must be Directorate, 2002). Although, pharmaceu-
considered seriously. Antibiotics that are ticals signify a minor fraction of all chem-
inadequately water soluble, particularly if icals that are dumped into the environ-
the bio-concentration factor is between ment, exceptional care must be taken
500 and 1000 or if the octanol/water dis- about their presence in the environment.
tribution coefficient crosses the value of Because, they are ubiquitous and dissem-
1000, have a tendency to get accumulated inate easily; they act on biological sys-
in organisms. The substance enrichment tems; they show a lot of side effects in
in organisms has been proved for a few non-targeted bodies; and they are known
antibiotics, e.g., sulphadimethoxine (Call to cause chronic toxicity even at low con-
et al., 2013). centrations (Enick and Moore, 2007).
As stated by Jones et al. (2005),
3. Biodegradation of pharmaceutical the occurrence of pharmaceutical contam-
products inants in the environment was reported
first time in late 1970s (Jones et al,
Globally, the production of phar- 2005). The major source of pharmaceuti-
maceuticals continues to grow year by cals in the environment is due to their im-
year, and with its environmental concerns proper disposal and leaching from landfill
pertaining to not just production, but also sites to natural water (Jones et al., 2004)
consumer waste and disposal (Wu et al., and most often the drugs enter into sew-
2010). The drugs (pharmaceuticals) con- ages and culminate in water sources.
sumed are eventually either excreted by There is no any guarantee that the phar-
humans and animals and or disposed maceutical drugs and their effluents are
which causes environment pollution. removed by merely dumping in sewage
Most of the pharmaceuticals produced, (Boxall, 2004). Moreover, many times, it
finally make their way to the ecosystems is proven that the water waste treatment
and causes environmental pollution as
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Biotech Sustainability (2017)
Designing Greener Pharmaceuticals and Practicing Green Chigurupati et al.
Figure 2: A diagrammatic sketch showing degradation of natural aromatic and some xeno-
biotic compounds.
of year 2016, the Ministry of Health long time in the environment. The organic
(MoH) had disposed nearly RM 2 million carcinogenic solvents should be replaced
worth of medicines, most of which were with green solvents wherever it is possi-
obtained through the Return Your Medi- ble. Industries should more focus on the
cines Programme. The most common minimum utilization of atoms for the syn-
medicines returned under this programme thesis of pharmaceutical products.
were the ones used for treatment of diabe- Biological based products are naturally
tes, hypertension, hyperlipidemia and gas- less poisonous and promote the principles
tritis. The common reason for the patients of green chemistry by expanding pro-
to incomplete the course of medicines spects to develop expected processes ex-
was a change or discontinuation of a ploiting renewable resources. Ultimately,
treatment (Ministry destroys RM 2 million renewable resources do have a potential
worth of expired meds - Nation | The Star to produce a significant amount of phar-
Online, 2016). maceutical materials which are currently
produced using either hazardous and or
7. Concluding remarks nonrenewable materials. Green chemical
synthetic procedures should replace the
The pharmaceuticals should be conventional procedures of producing
designed by using green chemistry tech- pharmaceuticals. It is not only essential to
niques; so that at the end, products can be promote the health of people but also for
easily broken down into innocuous deg- the sustainability of the industry and
radation products and do not persist for planet.
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Biotech Sustainability (2017)
Designing Greener Pharmaceuticals and Practicing Green Chigurupati et al.
2017 by the authors. Licensee, Editors and AIMST University, Malaysia. This arti-
cle is an open access article distributed under the terms and conditions of the Creative
Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
Abstract: Tree species across the alpine timberline are most vulnerable to climate change
and requires immediate attention for their conservation. Quercus semecarpifolia Sm. forms
the extensive forests in alpine timberline region and it is among the highly exploited tree
species of western Himalaya. Attempts were made to develop in vitro clonal propagation
procedure for Q. semecarpifolia. Nodal explants, derived from a single mature tree growing
in natural stands, were cultured on different nutrient media for the optimization of nutrient
medium. Woody plant (WP) medium supplemented with 8.88 M 6-benzylaminopurine
(BA) and 0.72 M Gibberellic acid (GA3) has yield significant shoot multiplication re-
sponse. For root induction a two- step method was applied. Microshoots treated with 100
M indole-3-butyric acid (IBA) for 24h, showed the significantly higher rooting response.
Present study leads a way forward to conservation and sustainable utilization of this high
value tree species. Moreover, further strengthening and optimization efforts are required to
develop a low cost procedure for the development of the nursery of clonally propagated Q.
semecarpifolia plants. Thus, the threat-mitigation strategies can be developed through the
introduction of these in vitro raised plants into the natural forest.
vided by cool fluorescent tubes (40 W; (w/v) and the media was solidified with
Philips from Saveer Biotech Limited, 0.8% agar (w/v) and/or 0.24% clarigel
New Delhi, India). (w/v). Besides, casein hydrolysate (CH,
w/v, 500mg l-1) and activated charcoal
2.3. Explant preparation (AC, w/v, 20mg l-1) were also incorpo-
rated in to each shoot multiplication me-
Nodal explant were initially dium. Each experiment consisted of 12
washed in running tap water for 10 min explants and repeated twice. Subculturing
and then immersed in detergent solution was carried out at 6 week interval and da-
(labolene, 0.1%, v/v; 20 min), and rinsed ta on shoot number and shoot length were
thoroughly with distilled water. Washed recorded during subculture.
explants were treated with fungicide (ba- Explant selection experiment was
vistin, 0.5%, w/v; 30 min) with gentle carried out in culture vessels (50 ml vol-
shaking. Those treated explants were in- ume, 20 ml medium per tube). The con-
troduced to laminar air flow cabinet, tamination-free explants were transferred
where after 5 rinses of autoclaved double to conical flasks (250 ml volume, 100 ml
distilled water, nodal segments were sur- medium per flask) for further growth and
face disinfected with mercuric chloride development. WP basal media devoid of
(HgCl2, 0.1%, w/v; 10 min). This treat- growth regulators served as control in all
ment was followed by subsequent 5 rinses experiments. All culture vessels and
of autoclaved double distilled water (2 flasks were plugged with non-absorbent
min each). The exposed ends of nodal cotton plugs and sterilized at 121C for
segments were excised out prior to inocu- 15 min. The pH was adjusted to 5.7- 5.8
lation. To check the contamination nodal by 0.1M NaOH or HCl before autoclaving
explants were cultured in WA: water agar at 1.06 kg cm-2 (121C) for 20 min.
medium for one week (Figure 1a).
2.6. Rooting of microshoots
2.4. Shoot induction and nutrient medium Microshoots (2.0-3.0 cm, with
selection well-developed leaves) were introduced
To select the nutrient medium for to a two step-rooting procedure; as de-
shoot induction and multiplication, con- scribed by Pandey and Tamta et al (2012).
tamination-free nodal segments were in- Briefly, during the first step excised mi-
oculated either in full strength MS: Mu- croshoots were cultured in full strength
rashige and Skoog (1962) or WP: Lloyd WP media supplemented with different
and McCown (1980) nutrient medium. concentrations of indole-3-butyric acid
Each medium was supplemented with 6- (IBA 50.0 or 100.0 M) for 24 or 48 h
benzyleamenopurene (BA, 4.44 M) and and cultures were placed in a dark during
activated charcoal (AC, w/v, 20mg l-1) to this step (Table 2). In the second step the-
monitor the shoot induction responses. se treated shoots were transferred to PGR-
free half-strength WP medium, solidified
2.5. Shoot multiplication with clarigel (0.25%) and exposed to 16 h
After 30 day on shoot induction photoperiod. The percentage of root for-
media, explants with positive shoot induc- mation, lengths of the formed roots and
tion response were transferred to shoot length of longest root were recorded after
multiplication media (Figure 1). Different 4 weeks of incubation in PGR-free half-
concentrations of cytokinin (BA, 4.44- strength WP medium.
22.20 M) alone or in combination with
IAA (1.43 M) or GA3 (0.73-1.44 M) 2.7. Acclimatization
were tested for shoot multiplication re- After 4 week of transfer to PGR-
sponses in WP full strength nutrient me- free medium, these shoots with roots were
dium. The sucrose concentration was 3.0% taken out from the culture flasks and wa-
-shed gently with distilled water to re- col pots (8 cm width and 10 cm height)
move traces of clarigel. After recording of containing soil and farmyard manure (3:1,
data, plantlets were transferred to therma- w/w). These plants were placed inside
used; BA reported to be the best plant yan Environment and Sustainable Devel-
growth regulator in Quercus shumardii opment, Almora, Uttarakhand, India for
for shoot multiplication (Bennett and Da- providing financial assistance through
vies, 1986). 20 M BA was found to be integrated eco-development research pro-
best for adventitious shoot induction and gramme (IERP) for Indian Himalayan re-
multiplication of individual shoots of Q. gion (IHR) during 2009-2011. The head
semecarpifolia (Tamta et al., 2008). In department of botany, D.S.B. Campus and
contrast to multiplication from petiolar department of biotechnology, Bhimtal
tube of Q. semecarpifolia the higher con- Campus of Kumaun University, Nainital,
centrations of BA were not found to be Uttarakhand India are highly acknowl-
the best concentration although it induced edged for providing necessary facilities
highest number of shoots/bud but the for experimentation.
length of shoots was reduced. Shoot mul-
tiplication was followed by rooting. Earli- References
er study in Q. semecarpifolia suggests
that IBA is the better responded auxin Bennet, L.K. and Davies, F.T. (1986). In
(root inducer) than NAA and a two step- vitro propagation of Quercus shu-
rooting procedure resulted 100.0% rooting mardii seedlings.Horticulture Sci-
response, without the formation of basal ence 21(4), 1045-1047.
callus (Tamta et al., 2008). In present Bisht, H., Prakash, V. and Nautiyal,
study two step method was used and A.R. (2012). Factors Affecting Re-
about 88.89percent rooting was recorded generation Potential of Quercus
within 20 days of culture. Similar method semecarpifolia, Smith: A Poor Re-
of rooting has also been reported earlier in generated Oak of Himalayan Tim-
other Quercus species such as Q. suber berline.Research Journal of Seed
(Manzanera and Pardos, 1990) and Q. Science 5, 63-70. doi:
leucotrichophora and Q. glauca (Purohit 10.3923/rjss.2012.63.70.
et al., 2002b). During acclimatization the Bonga, J. and Aderkas, P. (1992).In
semi-rooted microshoots were not able to vitro culture in trees, Kluwer Aca-
survive. The survival rate of microshoots demic Publ, Dordrecht-Boston-
was poor due to undeveloped rooting sys- London. pp. 207.
tem to survive ex vitro conditions. Brijwal, L., Pandey, A. and Tamta, S.
(2015). In vitro propagation of the
5. Conclusion endangered species Berberis aris-
tata DC. via leaf-derived callus. In
Results of present study are en- Vitro Cellular and Developmental
couraging and could be useful in develop- Biology-Plant 51(6), 637-647.
ing a rapid clonal propagation method for Chalupa, V. (1987). Effect of benzyla-
this important multipurpose tree species. minopurine and thidiazuron on in
However, the low rate of multiplication vitro shoot proliferation of Tilia
and poor survival rate of plantlets in the cordata Mill., Sorbus aucuparia L.
soil are the main challenges at the present. and Robinia pseudoacacia L. Biolo-
Therefore, to address the low regeneration gia Plantarum 29, 425-429.
and to improve survival rate of Q. Harsch, M.A., Hulme, P.
semecarpifolia further research is re- E., McGlone, M. S. and Duncan,
quired. R. P. (2009). Are tree lines advanc-
ing? A global meta-analysis of tree-
Acknowledgements line response to climate warm-
ing.Ecology Letters 12,10401049.
The authors express their gratitude IPCC, (2013). Climate Change 2013: The
to G.B. Pant National Institute of Himala- Physical Science Basis. Contribu-
safe in the Western Himalaya? Cur- Vyas, S.C. (1984). Systemic fungicides,
rent Science 98, 1420-1421. Tata McGraw hill Publishing Co.
Singh, J.S. and Singh, S.P. (1992). For- Ltd. New Delhi.
ests of Himalaya: Structure, func- Walck, J. L., Hidayati, S. N., Dixon, K.
tioning and impact of man. W., Thompson, K.E.N. and
Gyanodaya Prakashan, Nainital, Poschlod, P. (2011). Climate
pp.294. change and plant regeneration from
Smith, W. K., Germino, M. J., Johnson, seed. Global Change Biology 17,
D. M. and Reinhardt, K. (2009). 21452161.
The altitude of alpine treeline: a Wang, B., Chen, T., Xu, G., Liu, X.,
bellwether of climate change ef- Wang, W., Wu, G. and Zhang, Y.
fects. The Botanical Review 75(2), (2016). Alpine timberline popula-
163-190. tion dynamics under climate change:
Tamta, S., Palni, L.M.S., Purohit, V.K. a comparison between Qilian juni-
and Nandi, S.K. (2008). In vitro per and Qinghai spruce tree species
propagation of brown oak (Quercus in the middle Qilian Mountains of
semecarpifolia Sm.) from seedling northeast Tibetan Plateau. Boreas.
explants.In Vitro Cellular and De- 10.1111/bor.12161. ISSN 0300-
velopment Biology-Plant44(2),136- 9483.
141. Wilhelm, E. (2000). Somatic embryogen-
Vengadesan, G. and Pijut, P.M. esis in oak (Quercus spp.). In Vitro
(2009).In vitro propagation of Cellular and Developmental Biolo-
northern red oak (Quercus rubra gy-Plant 36(5), 349-357.
L.). In Vitro Cellular and Develop- Yam, T.Y., Ernst, R., Arditti, J., Nair,
ment Biology-Plant45, 474-482. H. and Weatherhead, M.A.
Vieitez, A. M., Pintos, F., San-Jose, M. (1990). Charcoal in orchid seed
and Ballester, A. (1993). In vitro- germination and tissue culture me-
shoot proliferation determined by dia: a review. Lindleyana5, 256
explant orientation of juvenile and 265.
mature Quercus rubra L. Tree Phys-
iology 12, 107-117.
Abstract: Spent Mushroom Substrate (SMS) is a composted growing medium that results
from the mushroom growing process. The spent substrate remains after harvesting the
mushrooms, which is entangled with innumerable mushroom threads (collectively referred
as mycelia), would have been biochemically modified by the mushroom enzymes into a
simpler and more readily digestible form, which could then be used in mycorestoration
and mycoremediation. Mushroom mycelia can produce a group of complex extracellular
enzymes that can degrade and utilize the lignocellulosic wastes found in nature, which also
reduces their potential for pollution. It has been revealed recently that mushroom mycelia
can play a significant role in the restoration of damaged environments. Saprotrophic, endo-
phytic, mycorrhizal and even parasitic fungi or mushrooms can be used in mycorestora-
tion, which can be performed in four different ways: mycofiltration (using mycelia to fil-
ter contaminated water), mycoforestry (using mycelia to restore degraded forests), my-
coremediation (using mycelia to eliminate toxic wastes from soil and water) and my-
copesticides (using mycelia to control insect pests). These methods represent the potential
to create a clean ecosystem, where no damage will be left after fungal implementation. Ap-
plied Mushroom Biology can not only convert this huge amount of lignocellulosic wastes
into human food but also can produce notable nutraceutical products, which have several
health benefits and it is discussed in this chapter.
steady reduction of food grains. In addi- being utilized extensively in industry, ag-
tion, the increase in industrialization has riculture, medicine, food and textile in-
polluted our environment with chemicals dustries (Prabhakaran et al., 2011).
and toxins of various kinds (Singh et al., Hypsizygus ulmarius, the Elm
2011) Most contaminated sites usually Oyster Mushroom (Figure 1), is a new
contain a mixture of non biodegradable variety of edible mushroom, developed by
persistent compounds, which increase the the Indian Institute of Horticultural Re-
difficulties of remediation. This is due to search (IIHR), Bangalore -
the intensification of agriculture, range of www.iihr.res.in. It is a type of basidiomy-
crops grown and the diversity of manu- cete, also known as white rot fungi, of
facturing industries. The excess usage of which there are about 1,400 known spe-
chemical fertilizers has also contributed cies. It can be commercially cultivated by
to the deterioration of the environment, solid-state fermentation method, using
with soil degradation, loss of soil fertility agricultural wastes such as paddy straw,
and agricultural productivity being the coconut husk, tea and saw dust, among
main consequences (Khan and Ishaq, others. Mushroom cultivation is environ-
2011). ment-friendly, in addition to providing a
For improving the long-term sus- cost-effective source of food protein for
tainability of industry and agriculture, vegetarians and a source of income for
emphasis should be on the holistic man- rural women (Ahmed et al., 2009). Mush-
agement of natural resources. Microor- rooms are a good source of vitamins and
ganisms can control pollution and pests, minerals, while having low content of
maintain the fertility of soil and enhance fats, carbohydrates and dietary fiber. With
plant growth, with no major adverse ef- their nutritional value, mushrooms can
fects on the environment or other non- reduce malnutrition in the rural poor to a
target organisms (Gomathi and Ambika- large extent, and are also effective in re-
pathy, 2011). These types of mechanisms ducing the occurrence of life-style diseas-
rely on stimulating the growth of specific es like hypercholesterolemia, hyperten-
species of micro-organisms or mixtures of sion, diabetes and cancer (Alam et al.,
microflora native to the contaminated 2007).
sites and are thus, able to remediate the
area more easily and efficiently (Kumar et
al., 2010).
Recent research has favored the
techniques of bioremediation for clean-
ing up the above types of sites, as it is
both environment-friendly and of relative-
ly low-cost (Sasek, 2003). Bioremediation
is the addition of biological agents, main-
ly microbes like yeast cells, fungi or bac-
teria to detoxify the contaminated soil and
water. When fungi are specifically used, it
is known as mycoremediation. Lignino-
Figure 1: Hypsizygus ulmarius: The Elm
lytic basidiomycete fungi such as Phan-
Oytser Mushroom, growing naturally on
erochaete chrysosporium, Pleurotus os-
the bark of Elm trees
treatus, Lentinula edodes, etc. are well
(http://www.mushroomexpert.com/hypsiz
known mycoremediation agents. These
ygus_ulmarius.html)
microbes use the xenobiotics to be de-
graded as nutrients or as sources of ener-
Spent mushroom substrate
gy (Tang et al., 2007). Fungi play an im-
(SMS) is the by-product of mushroom
portant role in bioremediation, besides
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Biotech Sustainability (2017)
Spent Mushroom Substrate of Hypsizygus ulmarius Tallapragada and Ramesh
cultivation, and contains the fungal myce- lic acid, 2,6-dimethoxyphenol, etc (Ike-
lium, fermented substrate, residues of in- hata et al., 2004).
organic nutrients and secreted enzymes Phenol, also known as carbolic ac-
such as ligno-cellulases, proteases and id, is a highly toxic element that is added
peroxidases (Medina et al., 2009). SMS in the manufacture of resins, herbicides
is also a rich source of carbon, nitrogen and various other industrial processes
and other nutrients, and can be added to (Amara and Salem, 2010). It is one of the
enhance crop growth and maintain soil most persistent chemicals, with high tox-
fertility. It contains a consortium of bacte- icity even at low concentrations, and is
ria and fungi which can mediate the for- considered a priority pollutant under the
mation and weathering of soil, nutrient Environment Protection Act, 1986
and water mobilization, nitrogen fixation (Chandrakant et al., 2006). Phenols can
and denitrification processes. The fungal be degraded by various white rot fungi
mycelium on the spent substrate is similar like P.florida, L.edodes and H.ulmarius
to Arbuscular mycorrhizal fungi AMF (Ranjini and Padmavathi, 2013; 2012).
(Jonathan et al., 2013). AM fungi espe- Pleurotus florida, Pleurotus os-
cially function to mobilize water and treatus, Pleurotus flabellatus and Pleuro-
phosphorus for plants (Manimegalai et tus sajor-caju have also been studied for
al., 2011). ). Pleurotus florida is a known their potential in dye decolourization
mycorestoration agent, as it improves (Faraco et al., 2009). Azo dyes are added
soil fertility by phosphate solubilization, in textile, pharmaceutical, cosmetic and
increases aeration and water movements food industries. After processing, almost
through soil and enhances plant growth forty percent of the dye is released into
(Kumar et al., 2010). wastewater. This affects the aesthetics,
White rot fungi are some of na- transparency and oxygen levels of the re-
tures most efficient lignin degraders from ceiving water, making it toxic (Ali et al.,
the microbial world, due to their ability to 2008). Even at very low concentrations
produce several kinds of lignin and phe- (<1 mg/L) in the effluent, they are visible
nol-degrading enzymes such as laccases and cause turbidity, especially red colour
and peroxidases. Laccases are copper- (Forgacs and Oros, 2004). Reactive dyes
containing, glycosylated polyphenol oxi- are significant because of their bright col-
dases. Their broad substrate specificity our and low energy usage during applica-
increases their significance in industrial tion (Aksu, 2005). In exhausted dye baths
and biotechnological applications such as and rinsing water, they are not recyclable
biomechanical pulping of cellulosic mat- or biodegradable.
ter, bleaching of pulp and degradation of Dyes can cause allergies, skin irri-
dyes, chloro-phenols and a variety of xe- tation and skin cancers, in addition to ge-
nobiotic and aromatic compounds, mainly netic mutations (Inbaraj et al., 2002).
by reduction of oxygen to water (Patel et Bacterial degradation of azo and reactive
al., 2008). Extracellular laccase is usually dyes usually occurs anaerobically, trans-
secreted into the medium in small quanti- forming them into carcinogenic interme-
ties. Its production is affected by typical diates; thus it is not considered suitable
fermentation factors such as media com- on a large scale (Manikandan et al.,
position, carbon-nitrogen ratio of growth 2012). Fungi, in aerobic conditions, can
media, pH, temperature and diffusion of uptake and remove dyes without creating
oxygen into the media (Revankar and the above carcinogens. Physical adsorp-
Lele, 2006). Their production is stimulat- tion onto the surface of spent mycelium,
ed by the addition of inducers such as followed by enzymatic breakdown is the
phenolic and aromatic compounds like usual mechanism of fungal remediation of
catechol, guaiacol, veratryl alcohol, ferru- dyes (Zumriye and Karabayir, 2008). The
effectiveness of fungi in bioremediation
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Biotech Sustainability (2017)
Spent Mushroom Substrate of Hypsizygus ulmarius Tallapragada and Ramesh
fungal enzymes present in the above bacteria (PGPR). The nitrogen content of
generated spent substrate (Figure 2). SMS varies from 0.4-13.7% with a C: N
ratio of 9 to 15: 1. It contains cations like
2. Mycorestoration of soil using spent K+, Na+, Ca2+, Mg2+; and anions like Cl-,
mushroom substrate from mush- NO3-, SO42-; all essential for optimal plant
room cultivation growth. It improves physical soil proper-
ties by decreasing its density, surface
Spent mushroom substrate (SMS) crust formation and diurnal temperature
also known as spent mushroom compost changes; in addition to increasing infiltra-
(SMC), is generated as a by-product after tion, aeration and water-retaining capaci-
the harvest of mushroom crop. Recently, ties. It maintains a high organic content of
it has been proposed to re-name it as post soil. It can be added singly or as a sup-
mushroom substrate because it is not re- plement to conventional bio-fertilizers,
ally spent and has many uses remaining. though it functions better in combination
The composition of spent mushroom sub- with other bio-fertilizers (Rinker and Kan
strate varies depending on the type of Zeri, 2004).
mushroom cultivated and the agricultural Restoring a degraded or stressed
waste material used as substrate. It is an soil using mycorrhizae and myco-bio-
excellent source of humus, although much fertilizers is known as mycorestoration.
of its nitrogen content is used up by the Humans have the ability to synergize my-
growing mushrooms. Overall, it is a good corrhizae and use them for healing forest
source of the macro nutrients viz., nitro- habitats that have suffered from stress,
gen, phosphorus and potassium, in addi- toxic waste or poor nutrition, making
tion to having trace elements. This makes them critical to our mutual evolutionary
it suitable for supporting plant growth survival (Stamets, 2006). Most plants also
(Kulshreshtha and Sharma, 2014). This is have associated with them diverse groups
also because it behaves similar to arbus- of plant-growth-promoting-fungi (PGPF)
cular mycorrhizal fungi (AMF), associ- and AMF.
ated with plant-growth-promoting rhizo-
Mycoremediation Mycorestoration
Nutraceuticals
2.1. The mushroom industry and mush- For obtaining the spent mushroom
room cultivation substrate (SMS) of H.ulmarius, it is culti-
The ever-increasing demand for vated on agricultural wastes such as pad-
protein-rich vegetarian food and the inef- dy straw and coconut husk (Khan et al.,
ficiency of conventional methods have 2008), by solid-state fermentation meth-
resulted in the need to explore alternatives od, as prescribed by Chang (1999).
for low cost production of protein-rich
food like mushrooms (Mukherjee and 2.2.1. Substrate preparation and sterili-
Nandi, 2004). The mushroom industry zation
has a world production greater than 25 Crushed rice straw is used for cul-
million tonnes. The largest producer is tivation. Straw is cut to 2-6 cm pieces,
China, which cultivates more than 20 mil- soaked overnight and autoclaved, at
lion tonnes and account for over 80% of 121oC and 15 psi, while coconut husk is
the world's mushroom production (Li, separated and soaked for 5-6 hours, then
2012). Research has proved that produc- autoclaved as above.
tion of 1 kg mushrooms will generate 5
kg of spent residual material called spent 2.2.2. Spawn rate
mushroom substrate or SMS. An average The quantity of spawn used for
farm discards about 24 tonnes of SMS per inoculation is 5% of its total weight (50
month (Singh et al., 2011). In Ireland, gm spawn for 1 kg substrate).
approximately 2,54,000 tonnes of SMS is
generated each year (Barry et al., 2012) 2.2.3. Spawning of substrate bag
and in The Netherlands, more than The pasteurized substrate is filled
8,00,000 tonnes (Oei and Albert, 2012). into transparent perforated polyethylene
In some countries, waste management of bags; incubated at 23-25oC for 12 to 14
SMS is a major problem faced by farmers days. Mushrooms form around the edges
and the government. The obvious solution of bag perforations and are harvested ap-
is to increase the demand for SMS proximately 3 to 4 weeks later.
through exploration of new applications,
being recycled and reused. 2.2.4. Spawn broadcasting
The spent substrate is a composted After spawning, the bags are
organic medium, made from renewable moved to a room where temperature is
agricultural residues such as paddy straw, around 1820oC and relative humidity is
wheat straw, sawdust, sugarcane bagasse, close to 95-98%. The first 12-21 days are
hay, poultry manure, ground corncobs, completed without artificial lighting. At
cottonseed meal, cocoa shells, gypsum the end of this period, 4 hours light is
and other substances (Jordan et al., 2008). provided daily using fluorescent bulbs. At
Generally, each cultivation cycle lasts for the time of formation of mushrooms,
5 to 6 months, after which the spent sub- fresh air is let in to lower CO2 levels.
strate would be disposed. In Malaysia, an Studies have shown that paddy
average farm producing 100 tonnes of straw is the preferred substrate for culti-
fresh mushrooms per annum generates vation of H.ulmarius. However, coconut
approximately 438 tonnes of SMS. The husk could be used as a supplement to
current disposal strategy of SMS in Ma- enhance stipe length. Increasing stipe
laysia is by burning, spreading on land, length can make picking the fruit during
burying, composting with animal manure harvest much easier and thereby, increase
or land-filling. its competitiveness in the commercial
market. Rice straw, cotton waste, coir,
2.2. Cultivation process for hypsizygus baggase and banana leaves are all also
ulmarius considered suitable substrates for growing
oyster mushrooms (Belewu and Belewu,
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Biotech Sustainability (2017)
Spent Mushroom Substrate of Hypsizygus ulmarius Tallapragada and Ramesh
2005). The yield and quality depends on There is a reduction or complete ab-
the C: N ratio, composition of vitamins, sence in the growth of weeds- when SMS
phytohormones, macro, and micro ele- is added to soil.
ments present in the substrate (Ad- SMS is already supplemented with
enipekun and Gbolagade, 2006). The use nitrogen- along with its innate ability to
of these agricultural wastes in mushroom release phosphorus and potassium, it
cultivation provides a solution for their makes the soil enriched in all the three
disposal, and effective solid waste man- major plant nutrients nitrogen, phospho-
agement. rus and potassium.
The cultivation of edible mush- There is absence of heavy metals and
rooms offers one of the most feasible and toxins- in soil supplemented with SMS.
economic methods for environment-
friendly bio-conversion and disposal of 2.4. Use of spent mushroom substrate
agro-lignocellulose waste (Cohen et al., (SMS) of Hypsizygus ulmarius as a
2002). The spent substrate after two or mycorestoration agent
three harvests of mushrooms can be used The potential of the SMS of H.
for mycorestoration and mycoremediation ulmarius has been studied for improving
studies. soil fertility and plant growth by the re-
lease of soil phosphorous, improving aer-
2.3. Applications of spent mushroom sub- ation and disease resistance. Phosphorous
strate is one of the limiting factors for plant
The most significant applications of growth in most soils. SMS added singly
spent mushroom substrate (SMS) is its increased soil phosphorus; with conven-
ability to increase and retain the organic tional bacterial biofertilizers such as Azo-
content of soil or the potting medium (by tobacter sp. and fungal biofertilizers like
increasing the release of major plant nu- G.intraradices, it enhances soil porosity,
trients such as phosphorus and potassi- production of leaves, auxiliary buds and
um); it also increases the porosity of soil flowers; also root biomass, soil carbon
by creating air spaces (due to the thread- and nitrogen. Hence, it is more beneficial
like nature of fungal mycelia) when the SMS of H.ulmarius was used as
www.mushroom-sms.com. a supplement to conventional fertilizers,
Some of the other applications of rather than as a stand-alone bio-fertilizer.
SMS are as follows: A lot of literature has indicated that there
Consistency of quantity and quality is a stimulatory effect of plant growth-
A consistent amount of SMS can be pro- promoting Rhizobacteria (PGPR) on the
duced annually as mushrooms can be cul- growth of plants, due to the presence of
tivated throughout the year with regulated beneficial micro-organisms. However the
temperature and humidity conditions. mechanisms of this stimulation have not
Consistent high quality can also be pro- been discussed in detail in most of the
duced by standardizing the process and literature. The modes of action that have
materials used for cultivation. been studied, are, however, as follows:
High water and nutrient retention Increased supply of nitrogen to the host
capacity - Water and nutrient retention by microbial nitrogen-fixation; increase in
capacity of the soil increases by addition supply of phosphorus, sulphur and iron;
of SMS due to the filamentous nature of increase in surface area of roots due to
the mycelia that creates a network, similar production of phytohormones and stimu-
to mycorrhizal fungi, which traps the lation of mutualistic relationships be-
water molecules and also the released nu- tween the host plant and other algae and
trients. fungi (Banerjee, 2006).
and acute toxicity. They are being contin- the formation of dihydroxy benzene. Fur-
uously introduced into ponds, lakes and ther degradation proceeds through the
rivers through from chemical and textile cleavage of dihydroxy benzene to give
industries of different scale and category. pent 2-enedioic acid and formaldehyde, in
The major sources of phenol pol- addition to benzoquinone and maleic acid.
lution are waste water coming from paint The decarboxylation of maleic acid and
industries, pesticides, resin production ring opening of hydroquinone result in the
and petrochemical industries. Phenols are formation of oxalic acid. Decarboxylation
considered as primary pollutants under of oxalic acid leads to the formation of
directive 80/778/EC, since they are harm- carbon dioxide and water.
ful to organisms even at very low concen-
trations (Calace et al., 2002). The maxi- 3.2.1. Application of spent mushroom
mum concentration has been set at 0.5 substrate of white rot fungi in my-
mg/l for total phenols in drinking water, coremediation of phenolic com-
and individual concentration should be pounds
under 0.1 mg/l. The enzyme systems of white rot
The main routes of exposure to fungi contain laccase, lignin peroxidase
phenol include breathing contaminated and manganese-dependent peroxidases
air; inhaling cigarette smoke, drinking which catalyses metabolism of many lig-
water from contaminated surface or nin-like structures, for example, PAHs
groundwater supplies, swallowing or in- and phenols (Eggen and Sasek, 2002).
haling products containing phenol or Phenol oxidation using SMS from
coming into contact with contaminated A.bisporus was reported and laccase was
water and products containing phenol identified as the main enzyme responsible
through bathing (Ahmaruzzaman, 2008). (Trejo-Hernandez et al., 2001). Studies
The common method to detect in- have also supported the idea of de-
termediate products of phenol breakdown contaminating phenolic compounds using
is extraction by organic solvent after es- SMS from cultivation of edible mush-
terification or acetylation, followed by room.
gas chromatography and mass spectros-
copy (GC-MS). However, this method 3.2.2. Phenol tolerance and degradation
has two disadvantages: (1) as the inter- by spent mushroom substrate of
mediates are mostly polar compounds, Hypsizygus ulmarius
they dissolve easily in water and the non- H.ulmarius tolerated and degraded
or low-polar solvents dont get extracted phenol better under carbon and nitrogen
completely (2) Esterification is suitable limiting conditions of the growth medi-
only for derivatizing acids, while acetyla- um. The optimum carbon sources for its
tion only for hydroxylated compounds. In growth were glucose, mannitol and cellu-
addition, it is not possible to detect these lose, while ammonium nitrate, ammoni-
intermediates by high-performance liquid um chloride and sodium nitrate were the
chromatography (HPLC). This method optimum nitrogen sources. Peroxidase
needs not only many calibration standards and manganese peroxidase were the en-
but takes a lot of time as there are several zymes secreted in maximum quantity dur-
unknown compounds in the intermedi- ing phenol degradation, followed by lac-
ates, and some compounds would have case (Ranjini and Padmavathi, 2012;
the same retention time (Guo et al., 2013).
2006).
A possible degradation mecha- 3.3 Mycoremediation of dyes using spent
nism for phenol was given by Devi and mushroom substrate
Rajashekhar (2011). The hydroxyl radi- The majority of natural dyes are
cals attack the phenol molecule leading to produced from plant sources like roots,
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Biotech Sustainability (2017)
Spent Mushroom Substrate of Hypsizygus ulmarius Tallapragada and Ramesh
berries, bark, leaves, wood and also fungi Chen (2002 a, b) also reported the use of
and lichens. Azo compounds have aryl or biomass for the removal of basic dyes.
alkyl functional groups, with vivid colors Decolourization by living and
like reds, oranges and yellows. About dead microbial cells involves mechanisms
50% of dyes produced in the world are such as surface adsorption, ion-exchange,
azo dyes (Perumal et al., 2007). They are complexation (coordination), complexa-
extremely recalcitrant and when released tionchelation and micro-precipitation.
with the wastewater, remains in the water Cell walls consist of polysaccharides, pro-
or soil and adversely impacts the photo- teins and lipids and offer many functional
synthetic ability of phytoplanktons in wa- groups for the above reactions. Dyes can
ter. They interfere with the functioning of interact with these active groups on the
chlorophyll in the plankton, as they color surface of the cell. The accumulation of
the water and prevent the proper absorp- dyes by biomass may involve a combina-
tion of light (Duran and Esposito, 2000). tion of active, metabolism-dependent and
Many methods have been tried for achiev- passive transport mechanisms. It starts
ing decolorization of azo dyes in with diffusion of the adsorbed solute to
wastewater, but most of them like nano- the surface of the microbial cell
filtration, specific coagulation, use of ac- (OMahony et al., 2002).
tivated carbon and multiple effect evapo- Laccase oxidizes the phenolic
rators are very expensive. Bio-treatment group of the azo dye with the participa-
is a cheaper and environmentally better tion of one electron, generating a phenoxy
alternative (Olukanni et al., 2006). Deg- radical and then oxidizes it to a carboni-
radation by micro-organisms utilizes their um ion. A nucleophilic attack on the phe-
enzymes, mainly laccases and peroxidas- nolic ring carbon bearing the azo linkage
es produced by few bacteria and white to produce 3-diazenyl-benzenesulfonic
rot, brown rot and soft fungi (Duran and acid (III) and 1, 2-naphthoquinone then
Esposito, 2000). Only these enzymes are takes place (Camarero et al., 2005). Phe-
found to be effective in breaking down nolic radicals get oxidized further to yield
the high structural integrity and variety of oligomers. Under certain conditions, the
azo dyes. Decolourization and/or bio- C-C-formed dimers take part in coupling
adsorption of dye-containing wastewater reactions to form extended quinines (Zille
by dead or living biological matter (bio- et al., 2005).
mass), white-rot fungi and other microbial In reactive dyes, the chromophore
cultures are the subject of many studies contains a substitute that is activated and
reviewed in several recent papers (Aksu, allows the dye to directly react to the sub-
2005). In particular, these studies demon- strate surface. They are added for dyeing
strate that bio-sorbents from suitable of cotton or flax.
microbial biomass can be used for dye The spent mushroom substrate
decolourization; this is because certain (SMS) of P.sajor-caju offer an economi-
dyes have an affinity for binding with cal source of industrially important en-
some microbial species. The use of bio- zymes decolourize dyes (Singh et al.,
mass is becoming popular due to its 2002). Singh et al. (2011) has shown the
availability in large quantities and cost- decolorization of eight dyes (viz.) trypan
effectiveness. It is produced in fermenta- blue, amido black, remazol brilliant blue
tion processes to synthesize antibiotics R, bromophenol blue, crystal violet, me-
and enzymes. Here, a large amount of by- thyl green, congo red and methylene blue
products are generated, which can be used using lignin peroxidase extracted from 5-
in bio-sorption of pollutants. Aksu and month-aged SMS of P. sajor-caju cou-
Tezer (2005) have shown the uptake of pled with veratryl alcohol as a redox me-
588.2 mg of reactive black 5 per g of bi- diator. Further, three azo group dyes, re-
omass of Rhizopus arrhizus. Chu and active black 5, reactive orange 16 and
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Biotech Sustainability (2017)
Spent Mushroom Substrate of Hypsizygus ulmarius Tallapragada and Ramesh
disperse blue 79; two anthraquinone corestoration. Using the spent mushroom
group dyes, disperse red 60 and disperse substrate (SMS), a by-product of its culti-
blue 56 and textile wastewater from all vation, as a bio-fertilizer, either singly or
the five reactive and disperse dyes were in combination with conventional bio-
also successfully decolourized by crude fertilizers improves soil fertility, especial-
enzymes from SMS of P.sajor-caju. The ly phosphorus, which is mostly insoluble
mechanisms of enzymatic dye decolouri- and unavailable to plants. Phosphorus is
zation were most probably due to laccase one of the three macro nutrients of soil
and manganese peroxidase, as a recent that can determine the yield of crops and
study showed that under stimulation by agricultural income to farmers, and re-
malachite green, a triphenylmethane dye, duce their dependence on chemical ferti-
the laccase and manganese peroxidase lizers.
levels in the enzyme extracts of Soil contamination by phenolic
P.ostreatus were increased by 1.4 and compounds and dyes can be remedied us-
2.1-fold, respectively (Papinutti and For- ing the spent mushroom substrate (SMS)
chiassin, 2010). Thus, dyes can be re- of H.ulmarius. Phenolic waste is an inte-
moved, degraded and detoxified by en- gral part of biomedical and industrial
zymatic biological processes and also waste. Present treatment methods are
physical adsorption using SMS (Gao et chemical-intensive and further pollute the
al., 2011). Use of SMS in bioremediation environment.
of dyes is both time saving and cost- The spent mushroom substrate
effective. (SMS) of H. ulmarius can also be used to
decolorize dyes. Dyes color and pollute
3.3.1. Dye degradation using spent mush- water bodies, kill aquatic organisms by
room substrate (sms) of hypsizygus increasing toxicity, and known toreduce
ulmarius of light and dissolved oxygen in water.
The SMS of H.ulmarius was ef- The present treatment methods are mostly
fective in degrading three categories of physico-chemical in nature.
dyes (viz.) azo (Congo red), heterocyclic
(Methylene blue) and reactive (Solo- References
chrome black), with Methylene blue be-
ing most effectively decolourized, fol- Adenipekun, C.O. and Gbolagade, J.S.
lowed by Solochrome black and Congo (2006). Nutritional requirements of
red. Here, laccase was the enzyme secret- Pleurotus florida (Mont.) Singer, a
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ganese peroxidase. Maximum secretion of nal of Nutrition 6, 597-600.
both enzymes was observed during de- Ahmaruzzaman, Md. (2008). Adsorp-
colourization of Congo red (Ranjini and tion of phenolic compounds on low-
Padmavathi, 2015). cost adsorbents: a review. Advances
in Colloid and Interface Science
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Ahmed, S.A., Kadam, J.A., Mane, V.P.,
To conclude the effective man- Patil, S.S. and Baig, M.M.V.
agement of agricultural wastes as a part of (2009). Biological efficiency and
solid waste management is mushroom nutritional contents of Pleurotus
cultivation which utilizes agricultural florida (Mont.) Singer cultivated on
wastes in an environment-friendly manner different agro-wastes. Nature and
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are generated in large amounts each year. Aksu, Z. (2005). Application of biosorp-
The fertility of marginal soils increases in tion for the removal of organic pol-
an environment-friendly manner - my-
ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 98
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posed for the degradation of phenol sorbent for the removal of Reactive
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Spent Mushroom Substrate of Hypsizygus ulmarius Tallapragada and Ramesh
International Society for Mushroom and reactive dyes using spent myce-
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Biotech Sustainability (2017)
Spent Mushroom Substrate of Hypsizygus ulmarius Tallapragada and Ramesh
Abstract: Biotechnology has various applications which could be utilized to enhance the
sustainability in various sectors. Forest Biotechnology was started only during 1990s in
the earnest sense. It encompasses structural and functional studies of genes and genomes
(including development and application of genetic molecular markers); various methods
of vegetative reproduction, and asexual insertion of genes into forest plant species. Tra-
ditionally, productivity of forests has been improved by introduction of new germplasm
developed through tree genetics and breeding, as trees are strategically and efficiently
able to utilize both horizontal as well as vertical space in an optimum way. The applica-
tion of modern biotechnological tools and techniques that span the diverse fields of plant
biology, genetic transformation and discovery of genes associated with complex multi-
genic traits. Modern innovations have added an exclusively new dimension to forest tree
improvement programs. However, forest biotechnology is still lagging behind because of
longer time periods required for planning, investigation and field research, poor juvenile-
mature correlations and multiplicity of selection criteria. But in the present scenario,
with growing population, economic development, environmental degradation and Cli-
mate Change, the demand for more biomass production from trees for production of
miscellaneous forest products and services, renewable energy alternatives to fossil fuels
and carbon sequestration etc. is ever increasing. Currently, the major challenge is to
maintain the original characteristics of pristine virgin forests for in situ biodiversity con-
servation for a sound natural genetic foundation. Genetic trees for pest and disease re-
sistance will help in enhancing plantation survival, productivity and yield that would
lead to eco-restoration of native tree species. This chapter highlights various application
of biotechnology in forestry which could help in boosting sustainability.
needs. Three billion people depend on such newly developed planting stocks as
wood for fuel. So we must harvest wood. new varieties generated through clonal
But forests also are an essential compo- propagation and advanced breeding pro-
nent of our ecology. It is essential to en- grams or as transgenic trees with high-
hance the productivity of plantation for- value traits, is expected in the near future,
ests in order to meet the ever increasing and these trees will enhance the quality
future world demand for wood and wood- and productivity of our plantation forests.
based products in a sustainable manner The application of plant biotechnology
that preserves natural forests and biodi- techniques promises potentially signifi-
versity (Scholes and Noble, 2001). The cant genetic improvement of forestry spe-
major challenge of foresters today is to cies and may become very attractive in
maintain the natural characteristics of for- view of several constraints traditionally
ests while meeting societys need for imposed by the long life cycles and phys-
products produced from trees. Implemen- ical size of trees (Schuch,1991). The po-
tation of improved silvicultural tech- tential applications of biotechnology in
niques and forest management practices forestry may be classified in three broad
are being used to manage the forest in categories: Plant Culture, Plant Protec-
sustainable manner. Productivity of the tion, and Plant Utilization.
forest has also been improved by the in-
troduction of new germplasm developed 2.1. Plant tissue culture
through genetics and breeding efforts for Genetic improvement in planta-
tree species. The application of new bio- tion forestry relies significantly on con-
technological tools and techniques that ventional breeding techniques which have
span the fields of plant biology, genetic been extensively used to improve various
transformation and discovery of genes characteristics in forest trees such as
associated with complex multigenic traits growth and form, volume yield, resistance
along with genetic engineering have add- to pathogens and quality of the end prod-
ed a new dimension to forest tree im- uct (Walter et al., 1998). Traditional
provement programs. Significant progress breeding techniques involve identification
has been made during the past few years of superior trees with desired traits and
in the area of plant regeneration via or- selection of the offspring having desired
ganogenesis and somatic embryogenesis traits. It was the major technique used for
(SE) for economically important tree spe- the planting stock improvement in 1970s.
cies. These advances have not only In the 1990s, biotechnology was intro-
helped the development of efficient gene duced in forestry in earnest (Sedjo, 2001).
transfer techniques but also have opened Forest biotechnology offers new perspec-
up avenues for using new high growth tives in the genetic improvements of for-
performance clonally replicated planting est trees through tissue culture and genet-
stocks in forest plantations. Advance- ic engineering. Plant tissue culture broad-
ments in gene cloning and genomics ly refers to the techniques of growing
technology in forest trees have enabled plant tissues or parts on a nutrient medi-
the discovery and introduction of value- um containing minerals, sugars, vitamins
added traits for wood quality and re- and plant hormones, all under sterilized
sistance to biotic and abiotic stresses into conditions. It is a basic technique to be
improved genotypes. One of the greatest used for multiplying elite clonal
challenges today is the ability to extend germplasm or genetically engineered
this technology to the most elite plants of forestry species. Plant tissue cul-
germplasm, such that it becomes an eco- ture of forestry species are aimed to fulfill
nomically feasible means for large-scale following objectives:
production and delivery of improved i. Micro-Propagation: To develop pro-
planting stock. Commercialization of tocol for micro propagation of im-
some undifferentiated cells of these calli it more reliable, especially when using
can evolve into somatic embryos charac- mature selected genotypes.
terized similarly to zygotic embryos, by a Organogenesis or the creation of
shootroot bipolar structure. This basical- plantlets from tissues such as cotyledons
ly distinguishes somatic embryogenesis is not common and rarely used in forestry.
from microcuttings consisting first of a Organogenesis and somatic embryogene-
shoot from which an adventitious root sis have been achieved in a large number
must subsequently develop. Somatic em- of woody plant species. However, in a
bryogenesis offers the advantage of rapid majority of tree species, micro-
embryo multiplication in a small space. propagation has been carried out by em-
Somatic embryogenesis is of specific in- ploying juvenile explants, for example
terest in the long term because it has the embryos, cotyledons and shoots tips from
potential for producing, inexpensively, seedlings. Clonal propagation from ma-
large numbers of clones that can be prop- ture trees, in particular conifers, is still
agated as artificial seeds. In the case of very difficult by tissue culture and re-
conifers, somatic embryogenesis, espe- mains a challenging biotechnological
cially when derived from a single cell, problem. Micro propagation of commer-
seems the most suitable regeneration and cial forest tree species is a major research
propagation technique. In broad-leaved and development goal.
species, vegetative propagation of genet-
ically engineered materials is likely to use 2.1.2. Genetic engineering
a combination of micropropagation and Future prospects for genetic engi-
rooted stem cuttings, at least in the begin- neering of forest trees are high. Through
ning. The advantages of somatic embryo- the use of genetic engineering techniques,
genesis in comparison with micropropa- individual genes of interest may be isolat-
gation through microcuttings are especial- ed from the donor organism and trans-
ly with regard to multiplication rate and ferred to a target microbe, animal, or
genetic modification applications. Somat- plant cell. Forest trees have generally
ic embryogenesis is preferred to micro been more difficult to work with mainly
propagate conifers (Sutton, 2002; Lelu- due to their long generation times and life
Walter and Harvengt, 2004). However, cycles. Through genetic engineering, for-
there are still serious obstacles to large- eign genes can be transferred to a forest
scale operational application of somatic tree resulting in faster tree improvement
embryogenesis to forest trees, for exam- and unique gene combinations which
ple: cannot be achieved by traditional tree
Only few species and within these breeding. The successful genetic engi-
species, only few genotypes can pro- neering of a forest tree requires four fac-
duce somatic embryos. tors:
Success has been obtained with few i. A desirable gene must be identi-
exceptions, mainly with juvenile tis- fied and isolated from a donor or-
sues coming for instance from imma- ganism.
ture zygotic embryos. ii. A plant regeneration protocol
There are risks that somaclonal varia- from single cells or a small group
tion may decrease the value of the of cells.
genotypes produced by somatic em- iii. A mechanism for inserting or
bryogenesis, resulting in a considera- transferring foreign DNA into a
ble waste of time, material and mon- target cell is required.
ey. True-to-typeness, particularly, iv. Additional DNA sequences for
may remain a problem for certain regulating the target gene, e.g. a
genotypes and efforts are still needed promoter is necessary to cause the
for optimizing this technique to make target gene to function in the
proper tissue when and where de- troporation and polyethylene glycol. Most
sired. successful work on genetic transformation
of forest tree species genomes so far has
Considerable research is focused been obtained by using juvenile material,
on identifying, characterizing and isolat- for example from an explants produced
ing genes in trees that are responsible for from juvenile tissues which have much
traits of particular interest, including higher regeneration capacities than older
stress responses viz. drought, frost and material. Successful genome modification
water-logging etc., disease tolerance and reports of adult selected plant material are
or resistance growth characteristics, wood very rare, except in poplars precisely be-
quality and flowering. The examples of cause of its greater capacity to regenerate.
traits for which genes are being sought in Transformation of a number of
commercial forest trees include lignin tree species by genetically engineering
composition, disease and insect re- trees has been reported. Populus spp.
sistance, dormancy, cold hardiness and serves as the model for much of the re-
growth rate etc. Genes of interest can be search because techniques for genetic en-
divided into two categories. First, genes gineering and micropropagating are rela-
those are responsible in governing agro- tively advanced. However, research in
nomic traits or growth of trees. These this area is still modest because several
genes are expected to lower the cost of technical problems have to be solved.
wood. This category includes genes for Limitations to the broad use of
disease and pest resistance or tolerance of genetic engineering to improve forest
environmental stresses. Genes that help a trees include primarily the following
crop to grow more efficiently, such as facts:
herbicide tolerance, also fall into this i. Only a few genes for specific
group. A second category includes genes traits of commercial interest have
for value-added traits that improve pro- been identified,
duction efficiency, product quality, or ii. Culturing cells of many tree spe-
product value and are expected to in- cies is difficult and
crease the value of wood or quantity and iii. Regenerating whole plants from
quality of active bio-chemicals. For in- cultured cells of commercial tree
stance, genes for reduced lignin content species has met with only limited
or lignin type which are more easily re- success.
moved during pulping fall into this cate- Those biochemical activi-
gory. Genes responsible for improved fi- ties that are specific to woody plants or
ber characteristics would also be included even to single species or varieties of trees,
here. Several value-added traits are being such as wood fiber formation and re-
actively studied in order to isolate valua- sistance (or susceptibility) to specific in-
ble genes. sects or diseases often must be studied in
Several gene transfer systems are the tree species of interest. It is important
available for movement of foreign DNA to note, however, that much of the re-
into target plants. The most common search on non-forest plants is relevant to
method uses Agrobacterium tumefaciens. commercial forest trees. For example, the
This soil-borne bacterium is able to natu- research underway on cellulose biosyn-
rally genetically engineer plants to create thesis in cotton plants is focused on un-
an environment in which the bacterium covering enzymes and genes for which
can thrive. Molecular biologists have al- similar counterparts can be anticipated in
tered this organism to insert target genes trees. The same is true for photosynthesis
into plants without causing plant disease. research on spinach. Major research ef-
Other methods for gene transfer in forest forts are underway in many laboratories
trees include particle bombardment, elec- on the small plant Arabidopsis thaliana
because it completes its life cycle in 6 like drought and water logging add to it,
weeks. Funds for much basic biochemical quite substantially. Shoot borer in Shorea
research are more effectively spent on robusta and Fusarium wilt of Dalbergia
plants other than forest trees. sissoo greatly damage these important
species of timber. Through the application
2.1.3. Molecular markers of Biotechnology threats to tree health
A serious problem today is in can be reduced. Research is showing
identifying desired progeny before they promise in the introduction of traits that
get too old to propagate vegetatively, by confer resistance to pests and pathogens
root cuttings, for example. Marker- that weaken or cause heavy mortality of
assisted selection (MAS) has given fur- trees. Improvements through tree bio-
ther impetus to tree breeding and selec- technology may also improve weed con-
tion. Molecular markers allow rapid iden- trol enabling young trees to get a head
tification of the gene or genes of interest start over nutrient-robbing competitors.
in minute samples. Molecular markers are The research falls into two categories: (a)
genetically linked to a given allele on a direct protection through control of in-
given locus and can therefore, be used to sects and diseases and (b) indirect protec-
predict the presence of the allele with tion through development of resistant tree
great accuracy (FAO, 2004). Biochemical varieties.
and molecular markers play a significant
role in many forest biotechnology activi- 3.1. Direct protection
ties for the selection of desired proge- The biotechnology product Bt
ny.Research aimed at developing molecu- toxin is already being used commercially
lar markers for screening is accelerating. in agriculture and forestry. Produced by
Marker applications for fingerprinting and the bacterium Bacillus thuringiensis, this
paternity analysis been used for character- toxin is effective against lepidopterous
ization of genetic diversity. Isozymes, insects. One biological approach is to use
randomly amplified polymorphic DNAs pheromones to attract the insects to a cen-
(RAPDs) and restriction fragment length tral point for control. Techniques of mo-
polymorphisms (RFLPs) have been wide- lecular biology apparently have not yet
ly used for genetic diversity and mapping been applied to these pests. Direct bio-
studies, though the current trend favours control of rusts and other fungal, bacterial
microsatellites (nuclear and cytoplasmic) and viral diseases of trees is labour inten-
and AFLPs (amplified fragment length sive and costly at best.
polymorphisms). Currently, ESTs (ex-
pressed sequence tags) and SNPs (single 3.2. Indirect protection
nucleotide polymorphisms) represent the Trees have a wide variety of natu-
most active area of marker development ral defenses against insects and diseases,
(FAO, 2004). which is why most microbes, insects and
viruses do not cause tree damage. Con-
3. Plant protection siderable research is underway to identify
genes that confer resistance to fusiform
Forest trees are affected by a rust and white pine blister rust in resistant
large number of different insect pests and varieties and species of pines. When these
diseases, the latter caused by fungi, bacte- genes have been identified, they can theo-
ria and viruses. Biotechnology offers a retically be inserted into susceptible trees
powerful approach to mitigating the dam- to provide resistance. This is fairly long-
age caused by insects and diseases as well range research. In the near term, the iden-
as by environmental stressors. Insects and tification of markers for these resistance
diseases probably cause great damage to genes will allow marker-assisted tree
Indian forests; environmental stressors breeding and asexual progeny selection to
biotechnology driven tree plantations may desirable traits would be the easy break-
free huge chunks of natural or primary down of wood fibers and the removal of
forest areas from ever increasing pres- lignin during chemical processing.
sures to supply industrial wood and thus Through Biotechnology, the raw material
improve their ability to maintain and pre- for paper production can be customized to
serve biodiversity. And as trees are genet- meet the requirements of producers there-
ically modified to be able to grow in pre- by wood values increase to that extent.
viously unsuitable areas such as arid or
Usar lands and saline soils the newly cre- 5.1.2. Environmental benefits
ated forests could not only produce more Forestry Biotechnology may also
wood but also enhance watershed protec- be used to create a number of desirable
tion and sequester carbon for climate environmental outputs (Table 1).
change mitigation.
Table 1: Utilization of biotechnology for
5.1. Benefits of biotechnology environmental benefits
5.1.1. Economic benefits No. Biotechnological Environmental
Introduction of any technology innovation output
for the consumer simply means that rela- 1 Cheaper planta- Pressure to log
tive prices of the desired goods fall com- tion wood substi- primary forests
pared to that which would have been in tutes for wood can be reduced
the absence of the particular technological from natural for-
innovation. In other words, technology ests
features increased productivity, that is, 2 Trees are genet- Ecological forests
enhanced output per unit of input. Alter- ically modified to can be estab-
natively, technology can be either cost grow in arid, usar lished on de-
(input) reducing or yield (output) enhanc- or saline condi- graded lands
ing. For society, more output for the tions
equivalent expenditure of inputs means 3 Trees are genet- Carbon-
societal increase in efficiency. Incidental- ically modified to sequestrating for-
ly, Plantation Forestry has received some adapt to tradi- ests can be estab-
success in recent decades because of its tionally unsuita- lished on sites
associated cost-reducing technology that ble sites previously not
has given wood from planted forests a suitable for for-
competitive price advantage over that estry
harvested from natural forests. The poten- 4 Cold-tolerant The altitudinal
tial applications of immediate interest of species of a de- and geographical
cost-reducing biotechnology to forestry sired genus are range of desirable
are increased wood production, improved developed tree species can
tree form and wood quality, enhanced be extended
survival, quicker growth rates and en-
hanced resistance to insects, diseases and Trees genetically engineered for
herbicides. In addition, production and pest and disease resistance may promote
processing costs of wood or chips could plantation survival and yield and also lead
be reduced as well as financial and envi- to eco-restoration of native tree species
ronmental costs for pulping. like Sal. Biotechnology has the potential
Paper production, for instance, to enhance the ability of trees to tolerate
requires fiber with adequate strength to abiotic stresses; restoration of contami-
allow sheets to be produced on high- nated sites through phytoremediation; fa-
speed machines, an attribute determined cilitation of weed control using more en-
by the wood fiber characteristics. There- vironmentally safe treatments; modifica-
fore, in pulp making for paper production, tion of biomass chemistry to improve
pulp and biofuels production; and im- ment. Thus, the presence of that modified
proving carbon sequestration to mitigate gene in the natural environment appears
greenhouse gas emissions. Biotechnol- unlikely to constitute any serious envi-
ogy also offers the potential to assist in ronmental problem, either short- or long-
ecosystem restoration and repair. Similar- term.
ly, biotechnology may help deal with in- For qualitative genes that affect
vasive exotics, which have in many plac- tree form or wood fibre characteristics,
es threatened locally and easily available release into the natural environment is
indigenous species. Modified tree species unlikely to provide a competitive ad-
also prove useful in providing ecological vantage in survival and therefore, unlikely
and environmental services in areas to have significant or adverse conse-
where trees now have difficulty in surviv- quences on the ecology. However, the
ing, such as arid or drought-prone areas consequences could be different if a sur-
areas and areas with saline soil or frost vival gene is involved. For example, the
zones and industrial waste effluents viz. introduction of Bacillus thuringiensis (Bt)
Dairy waste disposal sites, Alumunium gene makes a plant toxic to certain pests.
waste effluents-Red Mud, Fly Ash Ponds The release into the wild of such a gene
in Thermal Power Plants etc.. Another could constitute a major problem if it al-
contemporary and very important applica- tered the comparative competitive posi-
tion of biotechnology from the present tion of wild vegetation vis-a-vis those
Climate Change scenario involves crea- pests. However, the seriousness of this
tion of biological sinks, a potential tool to problem depends on the probability of the
mitigate the build-up of greenhouse gases transfer of a survival gene into the wild,
associated with global warming. Land the scale of the transfer and the compara-
Areas and Wetlands not currently forested tive change in the competitive ecological
could grow carbon-sequestering planta- balance within the natural habitat. Since
tions of Genetically Modified transgenic pests adapt via natural selection to modi-
trees (GM Trees). fied genes, the long-term impact of the
The most threatening cost of Bio- release of the modified gene into the natu-
technology is the after-effect of transgen- ral environment will be mitigated. Subse-
ic plants on the natural ecosystem, when quently, wild populations would gradual-
there would be genetic exchange(s) be- ly become resistant to the Bacillus thurin-
tween domestic and wild populations. In giensis (Bt) gene, thereby undermining its
cases where plantation tree species are long-term effectiveness against those
exotic, genetic outcrossing to the natu- pests.
ral environment would not be a factor. Transgenic biotechnology has be-
Where genetic exchange could be a prob- come controversial in agriculture. Some
lem, planting sterile trees or varieties with of those controversies appear to be spill-
reduced or delayed flowering would less- ing over to forestry too. The controversy
en the likelihood of their escape to the revolves around a number of issues. One
natural environment. In the case of the such issue involves the effects of biotech-
herbicide-tolerant gene, the consequences nology, particularly the introduction of
of release into the wild are probably transgenic plants on human health. The
small. Herbicides are unlikely to be ap- food safety issue is not generally raised
plied to most of the natural environment, for plants such as forest trees, which are
and where necessary, other types could be not usually a food source. However, cel-
used to which the escaped genes do not lulose is increasingly being used as filler
confer tolerance. In the long term, the in food products, and the food safety issue
herbicide in question will almost surely could become a concern, subsequently to
be replaced periodically in the normal be encountered by the forest biotechnolo-
course of product change and develop- gists.
FAO. (2004). Preliminary review of bio- Teaknet Publication No. 3, pp. 161-
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biotechnology and their implication
Abstract: Food and medicines are integral part of human life. Continuously increasing
global population and food demand has created an alarm about sustainable use of natural
resources. Due to adverse environmental conditions such as drought, salinity, temperature
and pathogens, it is very challenging to achieve high yield with current agricultural practic-
es. Due to deterioration of food quality and unpredictable environmental conditions, there is
a major public health concern about various diseases. Plant-derived compounds are playing
significant role in combating various human diseases since prehistoric times and therefore,
there is an increasing demand for production of plant-derived secondary metabolites. How-
ever, due to mismanagement of natural resources and faulty agricultural practices, several
medicinal plant species have become rare, vulnerable and endangered. Hence, alternative
strategies are needed to protect medicinally important plant species. Biotechnology has be-
come a center of attraction due to its innumerable advantages in agriculture, pharmaceuti-
cals, forestry and food sectors. In recent years, plant-derived compounds (also called as
natural compounds) are widely studied and biotechnological tools such as, in vitro propaga-
tion, transgenic for secondary metabolite production and cryopreservation not only provid-
ed alternative but also offer sustainable approaches towards conservation of medicinally
important plant species. This brief review highlights various biotechnological approaches
for conservation and sustainable supply of medicinal plants. Achievements, challenges and
perspectives on in vitro propagation for the conservation of medicinal plants are also high-
lighted.
become endangered mainly due to the though they are facing the threat of be-
human activity (European Commission, coming endangered and or extinct (Ma-
2008; Ibrahim et al., 2013). Whereas, nohar, 2012).
over-utilization of natural resources, pol- One third of the global plant spe-
lution of the soil, water and the atmos- cies are threatened at different level ac-
phere, and introduction of invasive spe- cording to International Union of Conser-
cies have resulted into reduced biodiversi- vation of Nature (IUCN, 2013). Further-
ty (Hunde, 2007). more, the habitat destruction and loss also
Medicinal plants are important for leads to the fragmentation of the remain-
the wellbeing of human population and ing habitat which eventually results in
there is an increasing demand for the pro- further isolation of the respective plant
duction of plant-derived secondary me- species population. The destructive har-
tabolites/ novel drug leads (Atanasov et vest of underground parts of slow repro-
al., 2015). Currently, there is constant ducing, slow growing and habitat-specific
demand for plants and plant parts in plant species are the crucial factors in
pharmaceutical industries as well as from making them vulnerable and rare
Ayurveda professionals. Furthermore, due (Ghimire et al., 2005; Kala, 2005).
to major public concerns about dreadful Providing high quality planting material
diseases such as cancer, HIV etc., phar- for sustainable use and thereby saving the
maceutical industries are actively engaged genetic diversity of plants in the wild is
in production of plant-derived drugs. Due important (Krishnan et al., 2011). How-
to the toxicity and side effects of synthet- ever, due to the human intervention there
ic drugs, the plant-derived drugs are be- is rapid dwindling of plant resources for
coming more popular and as a result there medicines; hence, alternative strategies
is increase in the number of herbal drug and or innovative approaches are needed
manufacturers (Verma and Singh, 2008; for their conservation. Bukuluki et al.
Agrawal, 2005; Lahlou, 2013). (2014) had scrutinized the harvesting
The projected escalating demand practices of medicinal plants in Uganda
for medicinal plants is increasing which and identified harvesting methods for sus-
leads to unscrupulous collection from the tainable supply of medicinal plants. The
wild and adulteration of raw material good harvesting practices suggested in-
supplied to the manufacturers. Ultimately, clude, careful harvesting of roots without
this practice has resulted into the over- affecting tap root, careful removal of stem
harvesting of many plants from wild and bark to avoid damaging the innermost
disturbed the population of various me- layer that contributes to drying of the
dicinal plant species and several species plant, plucking of leaves without breaking
even became endangered (Kala et al., the shoots, picking flowers those are fall-
2006; Rao et al., 2004). It has been re- en down or selecting only a few in order
vealed that more than 50,000 plant spe- to allow the plant to bear fruits and repro-
cies are used in phytotherapy and medi- duce. Recently, Hishe et al. (2016) re-
cine of which 2/3 are harvested from na- viewed the value chain of medicinal
ture leading to local extinction of many plants and the associated challenges. They
species or degradation of their habitats have conducted detailed studies of modes
(Tasheva and Kosturkova, 2012). Due to of harvesting, storage, packaging, supply
the constant expansion of herbs trade, the and distribution of medicinal plants. They
insufficient cultivation fields, and the highlighted that the medicinal plants sup-
weak management of harvesting and ply chains have varying requirements for
overharvesting of medicinal plants have their cultivation, resource management in
led to exhaustion of the natural resources the wild, harvesting, processing and mar-
and reduction in the biodiversity. Medici- keting. Considering these facts, they had
nal plants are always in demand even concluded that in order to become com-
petitive in the medicinal plants global for food, pharmaceutical and cosmetic
market place, value chain must become industries (Nalawade et al., 2003). A sys-
more flexible, innovative and efficient, so tematic concept of sustainability was pro-
it can bring to market new products in a posed by Prescott-Allen and Prescott-
timely fashion. Allen (1996). According to them, both
As medicinal plants represent con- humans and ecosystem are interrelated
sistent part of biodiversity, their utiliza- and dependent on each other. Hence, in
tion and conservation strategies needs conceptual terms, the essence of sustaina-
planned management for sustainability. ble development is expressed by the rela-
Therefore, systematic efforts should not tionship between people and the ecosys-
only be directed towards preservation of tem around them. They further stated that
the plant populations but also elevating the society is thought to be sustainable
the level of knowledge for sustainable when both the human condition and the
utilization of these plants in medicine condition of the ecosystem are satisfacto-
(WHO 2010). Developing strategies for ry or improving. They concluded that the
long-term sustainable supply of medicinal system improves only when both the con-
plants is challenging; therefore, it has dition of the ecosystem and the human
been suggested that to meet future public condition improve (Prescott-Allen and
food and healthcare demand, integration Prescott-Allen, 1996).
of conventional methods and biotechnol- In order to supply medicinal
ogy are essential. Biotechnological meth- plants or medicinal plant-based raw mate-
ods not only offer faster cloning and con- rial in a sustainable manner, various in
servation of the genotype of the plants; situ and ex situ strategies (which includes
but also enable genetic modification, gene in vitro techniques, botanical gardens,
regulation and expression for an efficient plant banks, GenBank, gene sanctuaries
production of valuable natural substances and seed banks) have been suggested for
in higher amounts or with better proper- the conservation of critically endangered
ties (Tasheva and Kosturkova, 2012). Be- plant species (Khan et al., 2012). Genetic
cause of innumerable advantages of bio- diversity preservation is of prime im-
technology in agriculture, pharmaceuti- portance while conserving plant genetic
cals, forestry, food industry and other sec- resources. For the conservation of plant
tors, the field of biotechnology has be- and or their germplasm, ex situ and in situ
come a center of attraction for conserva- strategies are used. The in situ approach
tion and sustainable supply of medicinal includes the maintenance of plant species
plants. and or their populations in their habitats,
where they can naturally occur, grow and
2. Biotechnological approaches for con- reproduce. Whereas, ex situ approach of
servation of medicinal plants conservation focuses on the maintenance
of plant species germplasm under con-
It appears that biotechnology is trolled conditions (Pathak and Abido,
emerging dramatically as a key enabling 2014; Rai et al., 2010). The multiplication
technology for environmental protection of plants by classical methods such as
and stewardship in a sustainable manner cuttings, budding, layering, and or graft-
(Cantor, 2000; Gavrilescu, 2004; Arai, ing in nurseries produces enormous num-
2006). Biotechnological advances have ber of plants. However, biotechnological
encompassed almost every aspect of hu- methods such as micropropagation, meta-
man life including food, fuel, cosmetics, bolic engineering and genetic manipula-
medicines and beverages. Most im- tions are especially appropriate for spe-
portantly,, biotechnology based-methods cies which are difficult to propagate in
are reliable and provides continuous sup- vivo (Tasheva and Kosturkova, 2012).
ply of raw material and natural products Hence, in situ approach of conservation
alone would not be efficient and effective (Sreekumar and Renuka, 2006). Biotech-
strategy for conservation and multiplica- nological approaches for sustainable sup-
tion of medicinal plants. Krishnan et al. ply and conservation of medicinal plants
(2011) suggested that prudent application include micropropagation, mycorrhiza-
of propagating biotechnology tools in tion, genetic transformation and devel-
plant conservation program is a prerequi- opment of the DNA banks (Sheikhpour et
site to succeed (in sustainable use of me- al., 2014; Rai et al., 2010). Figure 1 de-
dicinal plants) and to complement the ex- picts the biotechnological approaches use-
isting ex situ measures. The systematic ful in sustainable supply of medicinal
study on genetic diversity of rare and en- plants. Biotechnological approaches can
dangered plant species is very important be used to conserve plants from any
mainly because, it will be helpful in for- group. Table 1 shows some examples of
mulating plans for management and pre- successfully conserved plant species us-
serving their genetic diversity as well as ing biotechnology approaches.
ensuring their long term survival
Table 1: Names of some endangered medicinal plants those are propagated using plant tis-
sue culture techniques
Name Family Common Explant used Reference
name
Aerva lanata (L.) Amaranthaceae Chaya Node Shekhawat and
Juss. Revathi, 2017
ex Schult.
Bacopa monnieri L. Scrophulariaceae Bramhi Leaves, node Rathore and
&shoot apex Singh, 2013
Commiphora wightii Burseraceae Guggul node, shoot tip, Tejovathi et al.,
(Ar.) Bhandari & axillary bud 2011
Gentiana kurroo L. Gentianaceae Indian Node Verma et al.,
gentian 2012
Paris polyphylla sm. Trilliaceae Bulb Verma et al.,
2012
Picrorhiza kurroa Scrophulairaceae Kaur Nodal sector Jan et al., 2010
Royle ex. Benth
Picrorrhiza kurroa Scrophulariaceae Satuva Leaf/ node Verma et al.,
Royle ex Benth. 2012
Psoralea corylifolia Fabaceae Indian Apical meristem Pandey et al.,
Linn bread root 2013
Psoralea corylifolia Fabaceae Kutki cotyledons, hypo- Sehrawat et al.,
Linn cotyls 2013
Rheum emodii L. Polygonaceae Himalayan Basal disc Verma et al.,
rhubarb 2012
Salvia sclarea L. Labiateae Clary sage Node Verma et al.,
2012
Saussurea esthonica Asteraceae -- Seeds Gailte et al.,
Baer ex Rupr. 2010
Stevia rebaudiana Asteraceae Candy leaf Leaf/Node Verma et al.,
(Bertoni) Bertoni 2012
Table 2: Some examples of successfully conserved plant species using biotechnology ap-
proaches
Plant species Approach Explant used Reference
Calophyllum apetalum Micropropagation Lakshmi and Seeni,
2003
Cineraria maritima L. Cryopreservation- Shoot tips and Srivastava et al., 2009
Encapsulation nodal seg-
ments
Chlorophytum borivilianum Micropropagation Floral buds Sharma and Mohan,
Sant. Et Fernand 2006
Decalepis arayalpathra Micropropagation Gangaprasad et al.,
2005
Dioscorea floribunda Cryopreservation Shoot tip Ahuja et al., 2002
Gomortega keule (Mol.) Micropropagation Zygotic em- Muoz-Concha and
Baillon bryos Dave, 2011
Psoralea corylifolia L. Micropropagation Apical meri- Pandey et al., 2013
stem
preservation is considered as one of the traditional applications and for the devel-
most important conservation techniques opment of novel drugs and or supplemen-
and has been a successfully approach for tary products. However, plant-derived
the conservation of number of medicinal drugs as medicines have been based on
plants. Conservation of Atropa belladon- the assumption that the plants will be
na, Digitalis lanata, Hyoscyamus sp. and available on a continuing basis. As much
Rauvolfia serpentine are some examples more attention is given for discovery of
to state (Jain et al., 2012). Initiatives for new drugs from medicinal plants, the de-
conservation of medicinally important mand for raw material is steadly growing.
endangered plants have already been tak- However, there are no enough meticulous
en by several institutions. For instance, efforts to ensure the availability of target-
more than 110 accessions of rare or ed medicinal plants for harvesting to fulfil
threatened plant species are stored using the industrys demand. Moreover, there
cryopreservation approach of conserva- are no sustainable approaches developed
tion at the Kings Park and Botanic Gar- for harvesting as well as for conservation
den in Perth, Australia (Touchell and of in-demand medicinal plants. Due to
Dixon, 1994). Likewise, The National this awful situation, many medicinal plant
Bureau for Plant Genetic Resources species are becoming rare, vulnerable,
(NBPGR, New Delhi, India) has stored endangered and or extinct. Unfortunately,
more than 1,200 accessions from 50 dif- very meager efforts have been undertaken
ferent plant species (Mandal, 2000). for their conservation and sustainable
Shoot tips of endangered and medicinally supply. Therefore, there is a need to in-
important plant, Picrorhiza kurroa have tensify the efforts for not only for the
been preserved using same approach conservation but also to ensure sustaina-
(Sharma and Sharma, 2003). Cryo- ble supply of medicinal plants. Biotech-
preserved embryogenic cultures of Di- nological tools such as micropropagation,
oscorea bulbifera was performed using an cryopreservation and transgenic approach
encapsulation-dehydration procedure. Af- should be used efficiently for the conser-
ter cryopreservation, the sub-culturing vation of medicinal plants for the sustain-
showed 53.3% recovery of growth of em- able growth and development of the in-
bryogenic culture (Mandal et al., 2009). dustry, people and the planet.
These studies clearly provide the insights
about the usefulness of cryo-techniques in References
conservation of valuable medicinal plants
(Tashdeva and Kosturkova, 2012). How- Agrawal, A. (2005). Current issues in
ever, research on cryopreservation tech- quality control of natural products.
niques is relatively limited; hence, further Pharma Times 37, 9-11.
research and development is essential in Ahuja, S. Mandal, B.B. Dixit, S. and
this area to utilize this technique/approach Srivastava, P. S. (2002). Molecular
efficiently for the conservation of medici- phenotypic and biosynthetic stabil-
nal plants. ity of plants recovered from cryo-
preserved shoot-tips of Dioscorea
3. Perspectives floribunda. Plant Science3, 971-
977.
Understanding of the challenges Arai, K. (2006). Toward biotechnology:
and opportunities in conservation of me- The mission of IUBMB in the 21st
dicinal plants and to develop the biotech- century. IUBMB Life 58, 267-268.
nological approaches to deal with it is Atanasov, A. G. Waltenberger, B.
important for the sustainable supply of Pferschy-Wenzig, E. Linder, T.
medicinal plants. Medicinally important Wawrosch, C. Uhrin, P. Temml,
plants are continuously harvested for their V. Wang, L. Schwaiger, S. Heiss,
Abstract: Himalayas are among the most vulnerable ecosystems of the world. They harbor
unique geology/geography, ecosystem, biodiversity, and several other life sustaining biotic
and abiotic resources. Sustainability of Himalayas is being challenged by increased tourism
activities, deforestation, pollution, unmanaged exploitation of bio-resources, climate change
and unplanned developmental activities. Besides, the region has several resources of aes-
thetic and economic interest that can be harnessed for generation of income and employ-
ment to millions of the people residing in the region. Understanding the specific problems
of Himalaya and carving out the prospects for its sustainable development is a difficult task.
Looking at the present scenario, the chapter provides an overview of the opportunities and
challenges for achieving sustainability in Indian Himalayan Region. The present trends
suggest that the existing interventions in the region are unsustainable. Further, the unscien-
tific exploitation of natural resources is increasing the environmental degradation in the re-
gion. Proper policies and their implementation for harnessing the potential of the natural
resources are urgently needed for the sustainable development of the region.
rum, Western Himalaya and the Kashmir. degradation (Singh, 2002; Blaikie and
This situation is particularly damaging to Muldavin, 2004). Several scientific and
fragile ecosystem whose recovery is par- political forums have emphasized the
ticularly slow (Bawa et al., 2010). The uniqueness, environmental challenges and
political situation thus prevail prevent the political legacies of the Himalayan region
proper policy implementation for devel- so that sustainable planning and manage-
opmental programs causing the unfair ment in the region can be worked out.
treatment of the people residing in these Global change and Worlds Mountains
areas. These synergistic effects seem to conference held at Perth, Scotland in 2010
make Himalayas the hotspot for the phys- also identified several research gaps in
ical, economic and social vulnerability. sustainable mountain development. Look-
The studies carried out on the ing at the current scenario, the present
mountain ecosystems throughout the chapter provides the overview of the op-
world concluded that mountains are in portunities and challenges of sustainabil-
dire need of relief from anthropogenic ity in the Himalayas with special refer-
activities (Jodha, 2005). Sustainability of ence to Indian Himalayan Region.
Himalayas is being challenged by in-
creased tourism activities, deforestation, 2. Indian Himalayan region (IHR)
pollution, climate change and unplanned
development. Besides all this, the region IHR occupies a special place in
has several resources of symbolic and the mountain ecosystem of the world
economic values whose harnessing can (Singh, 2006). The region extends be-
provide income and employment to mil- tween latitude 26o20 and 35o40 North,
lions of the people residing in the region. and between longitudes 74o50 and 95o40
Understanding the specific Himalayan East covering 530, 795 sq. km of geo-
problems and prospects of the sustainable graphic area. It spreads across the states
development is not an easy task. Interna- of Jammu and Kashmir, Himachal Pra-
tional failure to recognize the economic desh, Uttarakhand, Sikkim, Arunachal
value of the issues of sustainability using Pradesh, Meghalaya, Nagaland, Manipur,
policy tools in the Himalayas has been Mizoram, Tripura and the hill regions of
cited as the major cause of this continued Assam and West Bengal (Figure 1). It co-
nstitutes about 16.2% of Indias adminis- veloped and 9.09% is under construction
tered geographical area. Most of the area (CEA, 2009). Accordingly the Prime
in the region is covered with the snow Minister of India launched a 50,000 MW
clad mountain peaks, glaciers in the high- hydroelectric initiative program, formu-
er Himalayas and dense forest in the mid- lated by CEA for preparation of Prelimi-
Himalayas thus harbouring a rich variety nary Feasibility reports of 162 new hy-
of flora, fauna and cultural diversity. dropower schemes (47,930MW) and out
of these 133 are in IHR (Agarwal et al.,
3. Opportunities and challenges for 2010).
sustainability in the IHR The development of hydropower
offers several advantages to the economy
3.1. Water potential of the nation. It is the source of clean re-
Himalayas has been rightly newable source of energy offering the
acknowledged as the Water Tower of mitigation of climate change issues and
Asia. Approximately 10-20 % of the area achieving the sustainability goals. It pro-
is covered by glaciers while 30-40% re- vides the inexpensive power, especially
mains under seasonal snow cover (Baha- when the project achieves financial
dur, 2004). Being the source of Asias 10 breakeven. The development of the hy-
largest rivers (Amu Darya, Indus, Ganges, dropower project is expected to improve
Brahmaputra, Irrawaddy, Salween, Me- the infrastructure of the remote areas as
kong, Yangtze, Yellow and Tarim) (Xu et well as it helps in flood moderation, irri-
al., 2007) they provide drinking water, gation, navigation and providing drinking
irrigation, fisheries, hydropower, and water all the year around.
supports several terrestrial and aquatic Unfortunately several challenges
ecosystems. The Ganges River system is are associated with the development of
the main source of fresh water to more the hydropower in IHR. It has been real-
than half the population of India and ized that the development of the hydro-
Bangladesh and nearly entire population power projects has significant environ-
of Nepal (Rasul, 2014). About 60% of the mental and social impact (Goldsmith and
Indias irrigated area of 546,820 Km2 is in Hildyard, 1984). These projects alter the
the Ganges basin (National Ganga River vital ecological process such as flow of
Basin Authority, 2011). Indus irrigation water, sediments, nutrients, energy and
system irrigates about 14.3 million hec- biota (Franklin et al., 1995). The IHR is
tares of farmland constituting the worlds among the most seismically active zone
largest contiguous irrigation system; ena- of the world; the construction of the hy-
bling the production of more than 80% dropower project increases the chances of
food grains of Pakistan (GoP, 2010). Amu the earthquakes which may shatter the
Darya irrigates 385,000 ha of farmland in lives of millions of people. Proper Envi-
Afghanistan contributing a reliable source ronment Impact Assessment appears to be
of Afghanistans food and water security the biggest challenge for the implementa-
(NAS, 2012). The ground water flow tion of the hydropower project in IHR. In
through bedrocks is approximately six a nutshell it may cause multi-dimensional
times the annual contribution from glacial unpredictable ecological disturbances and
ice melt and snow melt to central Himala- loss of biodiversity, productive land, so-
yan Rivers (Andermann et al., 2012). cial and cultural heritage. Hence, the de-
The government of India had rec- velopment interventions related to hydro-
ognized the hydropower potential of the power in the IHR should have different
IHR. The countrys hydropower potential approach. As per the reports of Chopra
is 148,701 MW out of which more than committee, formulated by the order of
75% (117,139 MW) resides in IHR; how- Supreme Court of India in the year 2014
ever only 22.37 % potential has been de- to assess the role of hydroelectric projects
biodiversity. However, biodiversity loss for the biodiversity loss in IHR. At the
from the region had become a matter of current rate of deforestation in IHR, the
great concern. There are several factors total forest cover (84.9 % in 2000) and
such as anthropogenic pressure and the coverage of dense forest (75.4% in 2000)
climate change which contributes to the is expected to be reduced to 52.8% and
loss of biodiversity in the region. 34% respectively by the year 2100 (Pan-
Due to the collation of several ge- dit et al. 2007). This could have serious
ographic and climatic features IHR pro- implications on the diversity of the flora
vides very suitable environment for flour- and fauna of the region. Looking at the
ishing the huge biodiversity. IHR sup- ever-increasing threats to the biological
ports nearly 50% of the total flowering diversity in the region; there is an urgent
plants in India of which 30% are endemic need of the proper actions or otherwise it
to the region (Singh et al., 2006). It har- may bring huge setback to the economic
bours 816 tree species, 675 edibles and benefits of the local populations.
1748 species of medicinal value (Samant
et al., 1998). The forests of the region 3.3. Medicinal plants
have phenomenal diversity that meets the Among the biodiversity elements,
diverse needs of the people (Singh and the roles of medicinal plants is remarka-
Singh, 1992). The forest of the region acts ble in the health care of the Himalayan
as the sink of the carbon dioxide and pro- people as most of the them resides in the
vides timber, wild edibles, gums, resins remote locations where the allopathic sys-
and other numerous products of immense tem of medicines is not practiced to such
value. an extent. Besides the health care, medic-
Massive deforestation, extensive inal plants have high socio-cultural, sym-
shifting cultivation and the demands for bolic and economic value, providing in-
agricultural land are the primary drivers come and employment to millions of peo-
ple living in the region (Ghimire, 2008). (2001) estimated 3200 medicinal plants
Himalayan medicinal plants meet most of from Indian Himalayas including 700
the international demand. Over- from trans-Himalaya (Table 2). India is
exploitation seems the biggest challenge ranked 2nd after China in terms of medici-
for the survival of these plants though nal plants export and exports about
they also have slow growth rates, low 32,600 tons of medicinal raw material
population densities and narrow geo- worth about US $46 million annually
graphical ranges (Kala et al., 2006). (Lange, 1997). High nativity and ende-
IHR host 1748 plants species of mism of medicinal plants is associated
medicinal value including 1685 angio- with the IHR. Out of 1748 medicinal
sperms, 12 gymnosperms and 51 pterido- plants, about 548 species are identified in
phytes (Samant et al., 1998). Ved et al. HP, 707 in Sikkim and Darjeeling and
Table 2: Medicinal plants, species diversity and representative species of different biogeo-
graphic zones of India#
Biogeographic re- Estimated no. of Examples of some typical medicinal species
gion medicinal plants
Trans-Himalayas 700 Ephedra geradiana Wall., Hippophae rham-
noides L., Arnebia euchroma (Royle) John
Himalayan 2500 Aconitum heterophyllum Wall. ex Royle,.Ferula
jaeshkeana Vatke and Saussurea costus (Balc).
Lipsch., Nardostachys grandiflora D.C. Taxus
wallichiana Zucc,. Rhododendron anthopo-
gon D.Dun and Ponax pseudoginseng Wall.
Desert 500 Convolvulus microphyllus Seib ex Spreng.,
Tecomella undulata (Sm.) Seem., Citrulus colo-
cynthis (L.), Schraderand Cressa crertica L.
Semi-Arid 1000 Commiphora wightii (Arn.)
Bhandari, Caesalpinia bonduc (L.)
Roxb, Balanites aegyptiaca (L.), Delilie
and Tribulus rajasthanensis Bhandari & Sharma.
Western Ghats 2000 Myristica malabarica Lam., Garcinia indi-
ca (Thou.) Choisy, Utleria salicifolia Bedd
and Vateria indica L.
Deccan Peninsula 3000 Pterocarpus santalinus L.f., Decalepis hamilto-
nii Wigh & Arn, Terminalia pallida Brandis
and Shorea tumbuggaia Roxb
Gangetic Plain 1000 Holarrhenaq pubescens (Buch-Ham.) Wall. ex
DC., Mallotus philippensis (Lam.) Muell
Arg., Pluchea lanceolata C.B. Clarke
and Peganum harmala L.
North-East India 2000 Aquilaria malaccensis Lam., Smilax gla-
bra Roxb., Ambroma augusts (L.) L.f.
and Hydnocarpus hurzii (King) Warb.
Islands 1000 Claophyllum inophyllum L. Adnanthera pavoni-
na L., Barringtonia asiatica (L.), Kurz
and Aisandra butyracea (Roxb.), Baehni.
Coasts 500 Rhizophora mucronata Lam., Acanthus ilicifoli-
us L., Avicennia marina Vierth and Sonneratia
caseolaris (L.) engl.
#
Source: Ved et al., 2001; http://www.fao.org/docrep/007/ad871e/ad871e09.htm
701 in the Uttarakhand (Badola and Ait- medicines (Fuentes et al., 1993). Using in
ken, 2003). The economic potential of the vitro propagation methods several rare
medicinal plants has been recognized in and endangered plant species can be
the IHR. It possesses various forest types quickly and successfully propagated with
with diverse habitats, slopes, aspects and low impact on wild population (Cuenca et
altitudinal ranges which offers congenial al., 1999). Plant cell cultures represent a
environment for the natural and artificial potential source of valuable secondary
propagation of the diverse medicinal metabolites which can be used as food
plants. Such diversity of the medicinal additives, nutraceuticals, and pharmaceu-
plants would be helpful for further scien- ticals. The major advantage of the synthe-
tific research on exploring their medical sis of phytochemicals by the cell cultures
efficacy (Kala et al., 2006). The aim set is that they are independent of environ-
by India for establishing golden triangle mental conditions and quality fluctua-
between traditional medicine, modern tions. Therefore, this sector undoubtedly
medicine and modern science will be a offers several opportunities for the sus-
boon for development of traditional herb- tainable development of the region how-
al medicine and medicinal plant sector ever there are lots of challenges associat-
(Mashelkar et al., 2005). ed with the sound development of the sec-
However, there is a paucity of re- tor in the region.
search on the biology, habitat and adapta-
tion mechanism of the majority of the 3.4. Agriculture assets and food security
threatened trade taxa of the medicinal Agriculture assets and food securi-
plants (Dhar et al., 2000). Further, proper ty is a critical issue of Himalayas due to
agro techniques for the cultivation of the complete dependency on rain besides
medicinal plant are lacking in the Hima- general characteristics of remoteness, low
layas. It is noteworthy to mention here market integration and underdeveloped
that < 90% of the plant raw material for agrarian resources. However, agriculture
herbal and industries in India and for ex- and allied sector forms the pivotal part of
port is drawn from natural habitats (Tan- the people living in the Himalayas. The
don, 1996, Ved et al., 1998; Dhar et al., huge diversity in the Himalayas has been
2000). Several challenges are associated maintained through a variety of crop
with the sustainable development of the composition, indigenous methods of
medicinal plant sector in IHR. Most im- maintaining soil fertility, socio-cultural
portant among these are low population and religious rituals (Negi and Maikhuri,
size, habitat specificity, narrow distribu- 2013).
tion ranges, unscientific collection for IHR is the storehouse for the di-
commercial purposes, land use disturb- verse genetic stocks. For instance, farmers
ances, introduction of non-native species, of the Central Himalaya grow about 100
habitat loss and alteration, climate chang- varieties of paddy, 170 varieties of kidney
es, heavy livestock grazing, unregulated beans, 8 varieties of wheat,4 varieties of
tourism, construction of dams and roads, barley and about a dozen varieties of
explosion of human population, popula- pulses and oil seeds each year and farmers
tion bottlenecks and genetic drift (Kala, of Uttarakhand Himalaya are known for
2005). cultivating 34 crop species comprising of
Recent advancement in the field of 6 types of cereals, 5 types of pseudo cere-
Plant Biotechnology especially Plant tis- als, 6 types of millets, 16 types of pulses,
sue culture has emerged as the promising 4 types of oilseeds, 5 types of condiments
technique for the conservation of these and 8 types of vegetables (Negi and
medicinal plants. In-vitro propagation of Maikhuri, 2013). The wild fruits of IHR
plants holds tremendous potential for the have significantly attracted the attention
production of high-quality plant-based of the entire world from the Nutraceutical
point of view. However, at present the tion and social equality. In order to utilize
decline in the interest of farming has been the tourism industry market, uncontrolled
observed as a result of climatic uncertain- number of tourist there should be proper
ty and cultural transformation. The con- infrastructural facilities. The ecological
sequences of this may be disastrous be- pressures are threatening land, water and
cause of genetic erosion of some unique wildlife resources through direct and indi-
and diverse gene bank. rect environmental impacts together with
generation of solid and liquid wastes
3.5. Tourism /aesthetic/ ecological ser- (Singh, 2002). So, ecotourism or green
vices tourism should be promoted for the sus-
Tourism industry typically de- tainable development of the region.
pends on the quality of the natural re-
sources. Himalayas are blessed with natu- 3.6. Other challenges (disasters, climate
ral beauty and pilgrim centers which at- change, population increase, pov-
tracts the millions of International tourists erty and waste management)
throughout the year. They have abundant People living in IHR, are facing the prob-
of natural resources like spectacular land- lems and damages due to some sudden
scapes, mountains, glaciers, rivers, lakes, events such as forest fire, avalanches,
fountains, snow clad peaks, forests having cloud bursting, land and mudslides, earth-
high floral and faunal diversity, which quakes, debris flows, flash floods, paraly-
offers strong resonance with tourism. ses the life and property of the people
These bioresources make Himalayas suit- (Nyaupane and Chhetri, 2009). There are
able for establishing Sanatoria and reju- several evidences of Climate change and
venating centers. Thus, they have huge its impact in Himalayas (Beniston, 2003;
potential for promoting tourism in the Cruz et al., 2007; Xu et al., 2009).
form of natural and cultural heritage. Among these impacts, the most widely
Economic potential of tourism that reported is the receding of glaciers which
could promote to sustainability in IHR is could have disastrous impact on the sur-
well recognized.IHR host several tourist vival of the millions of people. The ongo-
destinations which provides livelihood to ing climate change over succeeding dec-
millions of people residing in the region. ades will likely to have additional nega-
With the arrival of Britishers in 19th cen- tive impacts across these mountains, in-
tury, several hill stations like Nainital, cluding significant cascading effects on
Darjelling, Mussoorie, Shimla etc. were the river flows, ground water recharge,
established. They are the tourist hotspots natural hazards, and biodiversity; ecosys-
for thousands of national and international tem composition, structure and human
tourist. In India, both the state and central livelihoods (Xu et al., 2009). The popula-
government have declared tourism to be tion in Himalayas is rapidly increasing;
an industry and provides same conces- however there are finite resources to en-
sions and incentivizes of the industrial hance the production ultimately promot-
sector (Cole and Sinclair, 2002). ing the poverty. Recently proper waste
However, several challenges are management appears as the critical prob-
associated with the sustainable develop- lem in the Himalayas. Reducing forest
ment of the tourism industry in IHR. Hol- cover, accelerated soil erosion, drying
len,(2010) had identified lack of confi- springs, biodiversity loss etc. seems the
dence in the economic certainty of tour- ever increasing challenges for the sustain-
ism as the major challenge in the sustain- ability in the Himalayas.
able development of the tourism in Hima-
layas. He also advocates the philosophy 4. Concluding remarks
of sustainable development constructed
upon conservation, community participa-
Himalayas have undoubtedly huge Bawa, K. S., Koh, L. P., Lee, T. M.,
ecological, life sustaining, recreational, Liu, J., Ramakrishnan, P., Yu,
educational and scientific values. Several D. W., Zhang, Y.P. and Raven,
challenges are associated for the sustaina- P. H. (2010). China, India and the
bility in the region. The present trends Environment. Science 327, 1457-
suggest that the existing interventions in 1459.
the region are unsustainable; further un- Beniston, M. (2003). Climatic change in
scientific exploitation of natural resources mountain regions: a review of
is increasing the environmental degrada- possible impacts. Climatic Change
tion in the region (Singh, 2006). Proper 59, 531.
policies and their implementation for har- Blaikie, P. M. and Muldavin, J. S.
nessing the potential of the natural re- (2004). Upstream, downstream,
sources is the need of the hour for the sus- China, India: the politics of envi-
tainable development in the region. ronment in the Himalayan re-
gion. Annals of the Association of
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Abstract: Green bioactive polyphenol from Mother Nature especially from medicinal
plants are a rich source of novel therapeutics. Therefore, the search for bioactive molecules
from nature continues to play an important role in the invention of new medicinal agents.
Most plants do provide an array of phytochemicals that may contribute to reduction of dis-
ease and slowing of aging. Oxidative stress which is continuously produced in vivo by oxy-
gen-centred free radicals and other reactive oxygen species may leads to various diseases.
Since the oxidative stress has a great impact on the human health, it is appropriate to exam-
ine the role of natural antioxidant as a defence system. In this line, medicinal plant based
natural antioxidants such as polyphenols with free radical scavenging activity are emerging
as the primary components of holistic approaches in impeding adverse effect of oxidative
stress. This chapter focuses on biological effects of natural polyphenols on oxidative stress
and related diseases as an aalternative green nutraceutical approach.
Keywords: Free radicals; medicinal plant; natural antioxidants; oxidative stress; polyphenol
Figure 1: Free radicals are generated due to factors such as stress, alcohol, tobacco, harm-
ful chemicals including pesticides and drugs, fried food and radiation. Found abundantly in
natural food, antioxidants are free radical scavengers that react with free radicals to neutral-
ize them. Antioxidants function by donating an electron to the free radical before the latter
oxidizes other components within the cell. The free radical is stabilized and becomes non-
damaging to cells once it receives a free electron from the antioxidant.
antioxidants. The human body manufac- was first introduced by Selye (1955). A
tures endogenous antioxidant enzymes in continuously activity of human body re-
order to control these destructive free rad- acts with oxygen when breathing resulted
ical chain reactions (Valko et al., 2007). in energy production by cells and gener-
However, the body also relies on dietary ate highly reactive molecules within the
antioxidants to fulfil the needs of other cells known as free radicals. The effect
antioxidants it cannot produce (Diplock et exerted by free radicals in the body is
al., 1998), such as those found abundantly called oxidative stress (Finaud et al.,
in bioactive food components including 2006).
fruits, vegetables, grains and mushrooms. Since overwhelming research has
Examples of dietary antioxidants include been developed, the contemporary con-
lycopene, beta-carotene, lutein, zeaxan- cept of oxidative stress was updated and
thin, alpha-tocopherol, vitamin A and vit- briefly redefined as disturbance in the
amin C. balance between oxidants production and
antioxidants defences (Sies and Jones,
3. Oxidative stress 2007; Sies, 2015) which are associated
with electron transfer influencing the re-
The origin of term oxidative stress dox state of cells and organism. Subse-
from its very nature defined as the combi- quently, the imbalance redox mechanism
nation of electron transfer, free radicals, leads to the production of reactive oxygen
oxygen metabolites such as the superox- species (ROS) that includes free radicals
ide anion radical, hydrogen peroxide, hy- (superoxide, hydroxyl radical, peroxyl,
droxyl radical and singlet molecular oxy- alkoxyl, and hydroperoxyl) and non-free
gen with a biological concept of stress radicals (hydrogen peroxide, hypo-
ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 143
Biotech Sustainability (2017)
Natural Polyphenols and Its Potential in Sasidharan et al.
chlorous acid, ozone, singlet oxygen) are 4. Oxidative stress and immune re-
mainly involved in growth, differentia- sponse
tion, progression and death of the cells.
(Rahman et al., 2012; Rajendran et al., Immune system is important for our
2014). body to protect from the invasive patho-
Free radicals known as small mol- gens. The human defence system has been
ecules and unpaired electron amid highly well developed and can be divided into
reactive proliferation characteristic which two immunity reactions, namely innate
differ from most biological molecules that immunity and adaptive immunity. As its
tend to involve in initiate chain reactions name suggests, the innate immunity is a
from single free radical until propagate to non-specific first barrier of defence which
damage multiple molecules. Oxidative is able to act fast towards microbial inva-
stress occurs when excessive free radical sion. The adaptive immunity, on the other
formation within a cell or organism as a hand, is a highly specific barrier of de-
form of ROS due to aerobic metabolism fence due to possess immunological
and immune activation (Delmastro- memory function that can rapidly and ef-
Greenwood and Piganelli, 2013), UV ra- ficiently remove the pathogens that in-
diation (Halliwell and Gutteridge, 2015), vaded into body (Poland et al., 2013).
heme-oxygenase accumulation (Vanella Components of the adaptive immune sys-
et al., 2013), and hypoxia (Yang et al., tem are normally silent; however, when
2011). Failure of the cells defence mech- activated, these components adapt to the
anisms to neutralize or balance the accu- presence of infectious agents by activat-
mulation of free radicals may leads to mi- ing, proliferating, and creating potent
tochondrial dysfunction, DNA damage mechanisms for neutralizing or eliminat-
and lipid peroxidation can trigger pro- ing the microbes. In general, there are two
grammed cell death pathways (Dixon and types of adaptive immune responses
Stockwell, 2014). The findings could ex- where humoral immunity, mediated by
plain that low concentration of ROS pro- antibodies produced by B lymphocytes
ductions play role in intracellular signal- (B-cells), and cell-mediated immunity,
ling and defence against pathogens, while mediated by T lymphocytes (T-cells). Un-
the higher concentration of ROS has been like B-cells, T-cells recognize circulating
linked to clinically relevant diseases in- antigens of many chemical structures, the
cluding cancer, cardiovascular disease, vast majority of T cells (> 95%) are only
asthma, ischemia, diabetes and neuro- able to recognize peptide fragments that
degenerative diseases (Reynolds et al., are displayed by specialized molecules,
2007; Halliwell and Gutteridge, 2015; MHC molecules, on the surfaces of anti-
Phaniendra et al., 2015). Mitochondria is gen presenting cells. Therefore, this sys-
a major part of cellular sources of ROS tem ensures that T-cells are able to recog-
which consume oxygen with the process nize antigens that might be floating in the
of oxidative phosphorylation. Excessive cytosol or contained within ingested vesi-
formation of free radicals is known to cles of various cells (Thomas et al.,
weaken defence mechanisms against oxi- 2013).
dation and it results in more cell damages An imbalance between reactive
(Zorov, 2014). Consequently, the increas- oxygen species (ROS) and reducing
ing level of oxidative stress involved in agents (antioxidants) towards a pro-
pathological redox reaction, process of oxidant state can always result to oxida-
ageing which can initiate tissue damage tive stress (Sies, 1997). The damaging of
via apoptosis and necrosis (Cui et al., macromolecular in the form of protein
2012). carbonylation, lipid peroxidation and
DNA oxidation has historically has been
proven as harmful to particular functional
cells and being part of the main factors to are greatly debated in healthcare man-
hypertension and aging problems (Annor agement.
et al., 2015; Zhao et al., 2017). Up to
date, ROS is widely recognised as signal- 5. Oxidative stress and cancer
ling molecular substances (Suzuki and
Sevanian, 1997) that enable to trigger a Oxidative stress is closely related to
wide range of biological effects. With re- all aspects of cancer, from carcinogenesis
gard to the immune system, high levels of to the tumor-bearing state, from treatment
ROS can also be beneficial. For instances, to prevention. Cancer cells generally dis-
neutrophils generate ROS and release play elevated ROS level compared to
them intracellularly and extracellularly in normal cells that give them a proliferative
the form of an oxidative burst to defend advantage and promote malignant pro-
against and destroy pathogens, thus gression. The excess level of ROS typi-
providing antimicrobial protection cally observed in cancer cells are the re-
(Dahlgren and Karlsson, 1999). However, sult of accumulation of intrinsic and envi-
excessive ROS are generated in the pres- ronmental factors. In cancer cells, high
ence of immune complexes with auto- levels of ROS can result from hypoxia,
antigens where further macromolecular mitochondrial dysfunction, peroxisome
damage is induced. Prolonged exposure to activity, enhanced cellular metabolic ac-
high ROS concentrations can inhibit T- tivity, increased cellular receptor signal-
cell proliferation and lead to apoptosis ing, oncogene activity, increased activity
(Thoren et al., 2007), and incubation of of oxidases, cyclooxygenases, lipoxigen-
T-cells with the reactive nitrogen species ases and thymidine phosphorylase, and
(RNS) peroxynitrite can also inhibit their the crosstalk between cancer cells and
proliferation (Kasic et al., 2011). Previous immune cells recruited to the tumor site
study has revealed that different T-cell (Holmstrm and Finkel, 2014). Environ-
responses to ROS production may be due mental sources of ROS that can signifi-
to the extent of change to the cellular re- cantly contribute to tumorigenesis include
dox environment (Griffiths, 2005). In ionizing radiation, xenobiotics, tobacco
some cases such as absence of antigen components, chlorinated compounds, bar-
presenting cell, reactive carbonyls includ- biturates, metal ions and phorbol esters
ing 4-hydroxy-2-nonenal and malondial- etc. Figure 2 illustrates the potential out-
dehyde (MDA), which are generated on comes when such ROS level exceeds the
proteins and lipids randomly in the pres- capacity of the oxidation-reduction sys-
ence of ROS, will promote differentiation tem of the cell, may cause DNA, protein
towards a Th2 phenotype (Moghaddam et and lipid damage, leading to cellular re-
al., 2011). These data emphasize that the sponses such as chromosomal instability,
importance of ROS homeostasis and flux genetic mutation, alterations in cellular
in governing cell maturation and that the metabolism and modulation of cell
balance between oxidising and reducing growth that may lead to malignant trans-
agents is a delicate process which must be formation by affecting crucial hallmarks
tightly regulated and well managed, de- of cancer.
pending on whether the requirement is for When present at high and sus-
protecting against bacteria, in an immune tained levels, ROS can cause severe dele-
response, or requirements for T-cell sig- terious modifications to DNA, protein,
nalling, activation and regulation of func- and lipids. During the initiation stage,
tion. Owing to their pivotal role, the effect ROS may produce DNA damage by in-
of the oxidative stress on T-cell biochem- troducing gene mutations and structural
istry and its implications in autoimmune alterations into the DNA. ROS-induced
disease such as rheumatoid arthritis (RA) DNA damage can result in single- or dou-
ble-strand breakage, base modifications,
Inciting factors
Intrinsic source of ROS Extrinsic source of ROS
Increased metabolic activity Ultraviolet rays
Mitochondrial dysfunction Environmental agents
Peroxisome activity Pharmaceuticals
Oncogene activity Industrial chemicals
Increased cellular receptor signalling
Increased activity of enzymes
Carcinogenesis
Initiation Promotion Progression Invasion Metastasis
with protein and DNA and have been im- Kamendulis, 2007). Most initiating muta-
plicated in the pathogenesis of cancer tions affect proto-oncogenes or tumor
(Tuma, 2002). ROS also contribute to suppressor genes. Proto-oncogenes code
chromosomal instability and mutation for a variety of growth factors, growth
through its role in increasing the rates of factor receptors, enzymes or transcription
mutation, enhancing sensitivity to muta- factors that promote cell growth and cell
genic agents and compromising the sur- division while oncogenes are mutated
veillance systems. In addition to inducing versions of proto-oncogenes that promote
DNA, lipid and protein damage, oxidative abnormal cell proliferation. Activation of
damage to protein-coding or non coding oncogenes and loss of tumor suppressors
RNA may potentially cause errors in pro- cause alterations to multiple intracellular
tein synthesis or dysregulation of gene signaling pathways that promote metabol-
expression. ROS-induced alteration of ic reprogramming in cancer, resulting in
gene expression can occur through modu- enhanced nutrient uptake to supply ener-
lation of a host of signaling pathways in- getic and biosynthetic pathways for en-
cluding cAMP-mediated cascades, calci- hanced growth and survival (Zhang et al.,
um-calmodulin pathways and intracellular 2013).
signal transducers such as nitric oxide Oxidative stress may participate in
(Bertin and Averbeck, 2006). ROS stimu- the progression stage of the cancer pro-
lation on signal transduction pathways cess by adding further DNA alterations to
can lead to activation of key transcription the initiated cell population (Qanungo et
factors such as Nrf2 and NF-B. al., 2005). It can also promote many as-
ROS interact with the multistage pects of tumor development and progres-
processes in carcinogenesis including ini- sion through various biological processes.
tiation, promotion, proliferation, invasion, It acts on cellular proliferation through
angiogenesis and metastasis. ROS act as extracellular-regulated kinase 1/2
essential signalling molecules and modu- (ERK1/2) activation and ligand independ-
late a number of redox-sensitive signal- ent receptor tyrosine kinases (RTK) acti-
ling pathways in initiation of carcinogen- vation. ROS were shown as positive regu-
esis. Well-characterized targets include lators of tumor cell proliferation by
ROS-mediated regulation of the mitogen- modulating key proteins in cell cycle pro-
activated protein (MAP) kinase/Erk cas- gression such as cyclin, ATM (ataxia tel-
cade, phosphoinositide-3-kinase angiectasia mutated) and antioxidant en-
(PI3K)/Akt-regulated signaling cascades, zymes like MnSOD, catalase and gluta-
as well as the IB kinase (IKK)/nuclear thione peroxidase (Browne et al., 2004;
factor -B (NF-B)-activating pathways Lewis et al., 2005). ROS are involved in
(Ray et al., 2012). Oxidative stress- Anoikis resistance, PTEN inactivation,
mediated signaling pathways are persis- activation of Src, NF-B, CREB and
tently elevated in many types of cancers phosphatidylinositol-3 kinase (PI3K)/Akt,
and affect all characters of cancer cell be- enabling the tumor cells to escape from
havior, where they participate in cell apoptosis (Giannoni et al., 2009; Zhu et
growth/proliferation, differentiation, pro- al., 2011). In invasion and metastasis,
tein synthesis, glucose metabolism, cell ROS play a role in Met over-expression,
survival and inflammation (Storz, 2005). matrix metalloproteinase secretion into
In promotion stage, ROS can con- the extracellular matrix (ECM), invado-
tribute to abnormal gene expression, inhi- podia formation, Rho-Rac interaction,
bition of intercellular communication and plasticity in cell motility and epithelial
modification of second-messenger sys- mesenchymal transition (EMT). These
tems, thus resulting in an increase in cell help cancer cells to escape the primary
proliferation or a decrease in apoptosis of tumor, invade matrix of different organs,
the initiated cell population (Klaunig and find a suitable metastatic niche and then
lee, 1995; Lee et al., 1995; Ganz and 1988; Li et al., 1996). These results in
Seftel, 2000; Brownlee, 2005). The effect auto-oxidation of glucose to glyoxals, de-
of oxidative stress damage is aggravated composition of the Amadori product (glu-
by inactivation of anti-atherosclerotic en- cose-derived 1-amino 1-deoxyfructose
zymes (endothelial nitric oxide synthase lysine adducts, to 3-deoxyglucosone, and
(eNOS) and prostacyclin synthase. In ad- fragmentation of glyceraldehyde-3-
dition, oxidative stress also activates phosphate and dihydroxyacetone phos-
stress-sensitive signaling pathways, such phate to methylglyoxal (Brownlee, 2001).
as nuclear redox sensitive transcription In addition, increased AGEs production
factor (NF-B), p38 MAPK, and NH2- promotes the binding of AGEs to its re-
terminal Jun kinases/stress-activated pro- ceptors (RAGE). Binding of AGEs to
tein kinases (JNK/SAPK) leads to both RAGE induces overproduction of ROS
insulin resistance and impaired insulin and activation of NF-kB signaling and
secretion (Folli et al., 2011; Brownlee, upregulation of intracellular adhesion
2001). molecule-1(ICAM-1), vascular adhesion
cell molecule-1 (VCAM-1), monocyte
7. Molecular mechanisms of hypergly- chemotactic protein-1 (MCP-1), PAI-1,
caemia-induced oxidative stress in tissue factor, and VEGF (Yamagishi et
diabetes al., 1997; Bierhaus et al., 2001).
Previous studies demonstrated that
Hyperglycaemia-induced activation of PCK activity was increased in the retina,
polyol pathway was the first mechanism kidney and microvasculature of diabetic
discovered (Gabbay et al., 1966). This rats (Craven, P.A. and F.R. DeRubertis,
pathway has been therapeutic target for 1989; Lee et al., 1989), suggested that the
diabetes neuropathy (Oates and Mylari, lipolytic pathway and production of di-
1999). Recent human genetic study has acylglycerol induces PKC activation
implicated polymorphisms of the aldose (Ishii et al., 1998). Hyperglycaemia in-
reductase gene associated with increased creases diacylglycerol synthesis, which is
risk for diabetes complications (Oates and a critical activating co-factor for PKC
Mylari, 1999). Excess glucose activates isoforms (Derubertis and Craven, 1994;
polyol pathway. Aldose reductase (de- Xia et al., 1994; Koya et al., 1997; Koya
pendent upon NADPH as co-factor) in- and King, 1998). PKC activation has been
creases conversion of glucose to polyal- shown to have diverse effects on gene ex-
cohol sorbitol. Excessive activation of pression in different cell types. PKC acti-
polyol pathway results in depletion of in- vation inhibits insulin-stimulated endothe-
tracellular NADPH and GSH which is an lial Nitric Oxide Synthase (eNOS) ex-
important intracellular antioxidant (Lee pression in the endothelial cells and de-
and Chung, 1999). Accumulation of sor- creases nitric oxide production in the
bitol forms cellular osmotic stress (Ste- smooth muscle cells (Vlassara et al.,
vens et al., 1993). 1995). In vascular smooth muscle cells,
Excess glucose induces auto- PKC activation induces over-expression
oxidation through activation of the AGEs of fibrinolytic inhibitor, plasminogen ac-
pathway. tivator inhibitor (PAI-1) and activation of
The AGE precursor damages cells by NF-kB (Abordo and Thornalley, 1997).
three mechanisms: modification of pro- PKC enhances accumulation of microvas-
teins involve in gene transcription cular matrix protein by up-regulation of
(Giardino et al., 1994; Shinohara et al., transforming growth factor (TGF-), fi-
1998), modification of extracellular ma- bronectin and type 4 collagen in both cul-
trix molecules (McLellan et al., 1994), ture mesangial cells and glomeruli of dia-
and modification of circulating protein in betic rats (Doi et al., 1992). PKC also en-
the blood (e. g. albumin) (Vlassara et al., hances vascular permeability by increas-
ing the expression of vascular endothelial creatic -cells triggers oxidative stress
growth factor (VEGF) (Skolnik et al., and ER stress, exacerbated by high circu-
1991). lating glucose and lipids (non-esterified
Lastly, hyperglycaemia causes fatty acid). Oxidative stress and ER stress
damage to the blood vessel through acti- induce chemokine production and acti-
vation of hexosamine pathway. The end vates inflammatory cells in the pancreatic
product of this pathway, uridine diphos- islet. In turn, the activated inflammatory
phate (UDP)-N-acetyl glucosamine regu- cells produce cytokines that further exac-
lates gene expression implicated in vascu- erbate oxidative and ER stress and disrupt
lar complications (such as PAI -, TGF-, -cell secretory pathway function. In ad-
TGF-1). In addition, activation of hex- dition, oxidative stress induces unfolded
osamine pathway impairs Insulin Recep- protein response (UPR) and NF-B acti-
tor Substrate (IRS)/phosphatidylinositol vation. In early diabetes (manifested by
3-kinase (PI3-K)/Akt pathway, resulting chronic ER stress and inflammation), in-
in deregulation of eNOS activity (Bucala creased proinsulin:insulin ratio impairs
et al., 1991; Kolm-Litty et al., 1998). insulin signaling further aggravates hy-
perglycaemia. Overall, this vicious cycle
8. Oxidative stress and -cell dysfunc- leads to -cell apoptosis and progression
tion in diabetes to diabetes (summarized schematically in
Figure 5). Several mechanisms have been
Diabetes mellitus (DM) is character- implicated in -cell apoptosis (Nakagawa
ized by failure of the pancreatic -cells to et al., 2000; Oyadomari et al., 2002;
maintain glucose homeostasis. Physiolog- Puthalakath et al., 2007; Song et al.,
ically, the pancreatic -cells secrete hor- 2008; Mahdi et al., 2012; Supale et al.,
mone insulin and regulate glucose home- 2012; Uruno et al., 2015). The
ostasis. Insulin drives glucose uptake in PERK/ATF4-mediated activation of
the liver (reducing hepatic gluconeogene- CHOP and IRE1a/TRAF2/ASK1-
sis both directly and in conjunction with mediated activation of JNK are important
suppression of glucagon secretion), mus- molecular mechanisms (reviewed in Papa
cle and fat (Knner, 2007). Because of FR 2012) (Papa, 2012). A growing body
their high biosynthetic load and require- of evidence suggests that ER stress induc-
ment for oxygen, pancreatic -cells are es autophagy (an important mechanism
very vulnerable to oxidative stress (Len- for removal of terminally misfolded pro-
zen, 2008; Newsholme et al., 2012; Cao tein from the endoplasmic reticulum (ER)
and Kaufman, 2014; Kaneto and Mat- leads to induction of apoptosis (Wang et
suoka, 2015). al., 2014; Li et al., 2012; Quan et al.,
Oxidative stress and endoplasmic 2012). Another mechanism involves NF-
reticulum stress (ER) are key pathological kB and interleukin 1 beta (IL1b) activa-
features in particular type 2 diabetes tion (reviewed in Hasnain SZ et al., 2012;
mellitus (T2DM), contribute to pancreatic Hasnain et al., 2014).
-cell dysfunction, inducing inflammation
(immune activation) and -cell apoptosis. 9. Current therapeutics in diabetes
Previous studies have suggested the oxi-
dative stress is able to suppress insulin Good glycaemic control is the most effec-
transcription and associated with accumu- tive mean of mitigating diabetes compli-
lation of -amyloid in the human pancre- cations in particular type 1 diabetes
atic islet (Kaneto and Matsuoka, 2015). In (Greene et al., 1992; Molitch et al.,
obesity and early stage of diabetes, nutri- 1993). In general, drug available for dia-
ent overload leads to development of mild betes work by reducing stress on -cells
insulin resistance and hyperglycaemia. biosynthesis pathway. In diabetes pa-
Increased insulin production by the pan- tients, the use of drug that promotes insul-
Figure 5: Schematic representation of the cycle of oxidative and ER stress and its effects
on glucose homeostasis in diabetes (Hasnain et al., 2015).
pids, proteins and nucleic acid in the cells Polyphenols are a large group of
(Rock et al., 1996; McCord, 2000). natural antioxidants found mostly in
The concept of oxidative stress fruits, vegetables, cereals and beverages
was first hypothesised in the 1950s by (Arts and Hollman, 2005; Pandey and
researchers that investigated the toxic ef- Rizvi, 2009). There are more than 8,000
fects of ionizing radiation, free radicals, polyphenolic compounds that have been
and the similar toxic effects of molecular identified in various plant species, which
oxygen (Gerschman et al., 1954), as well arise from a common intermediate, phe-
as its possible contribution to the aging nylalanine or an immediate precursor, and
process (Harman, 1956). Interest in this shikimic acid. Polyphenols contain phenol
field of research grew (Hybertson et al., rings in the basic structure. Based on the
2011) when studies reported that the bio- number of phenol rings and the basis of
logical systems are capable of producing the structural elements that binds to these
substantial amounts of superoxide free rings, polyphenols can be classified as
radical, O2- through the natural metabolic phenolic acids, flavonoids, stilbenes and
pathways (McCord & Fridovich, 1968) lignans (Spencer et al., 2008; Pandey and
and the activity of antioxidant enzymes, Rizvi, 2009). Epidemiological studies
superoxide dismutases (SODs) is neces- have shown that high consumption of
sary for the survival of aerobic organisms polyphenols lowered the risk of chronic
(McCord and Fridovich, 1969; McCord et human diseases (Scalbert et al., 2005;
al., 1971). Besides the aging process, oxi- Arts and Hollman, 2005). Besides that,
dative stress has been postulated to play a the consumption of polyphenols have also
role in many other conditions, as well. been linked with cardio-protective effect,
These conditions includes inflammatory anti-cancer effect, anti-diabetic effect,
diseases (arthritis, vasculitis, glomerulo- anti-aging effect and neuroprotective ef-
nephritis, lupus erythematous, adult res- fect (Pandey and Rizvi, 2009). Several
piratory diseases syndrome), ischemic types of research have also concluded that
diseases (heart diseases, stroke, intestinal high phenolic content correlates with a
ischemia), hemochromatosis, acquired good antioxidant capability and lower ox-
immunodeficiency syndrome, emphyse- idative stress-related chronic diseases
ma, carcinogenesis, gastric ulcers, hyper- (Aruoma, 1998; Kohen and Nyska, 2002).
tension and preeclampsia, neurological Clinacanthus nutans is an example of a
disorders (Alzheimer's disease, Parkin- traditional herb that is rich in polyphenols
son's disease, muscular dystrophy), alco- and has potent antioxidant capabilities
holism and smoking-related diseases (Aromdee et al., 2007; Yong et al., 2013).
(Pham-Huy et al., 2008; Lobo et al., It is traditionally used to treat oxidative
2010). stress-related diseases, such as diabetes
To protect cellular components and various kinds of cancers (Ching et al.,
from the free radical-induced damage, the 2013; Png et al., 2013).
body possess several mechanisms to The antioxidants protect cellular
counteract oxidative stress by producing components by acting as radical scaven-
an extensive range of antioxidant, both gers, hydrogen and electron donors, per-
endogenous (generated in situ) and exog- oxide decomposer, singlet oxygen
enous (supplied through food, e.g. poly- quencher, enzyme inhibitors, synergist,
phenols) in the cells. The antioxidants can and metal-chelating agents or by gene ex-
be divided into three main categories pression regulation (Frei et al., 1988; Lo-
which are antioxidant enzymes, chain bo et al., 2010). The antioxidant process
breaking antioxidants and transition metal has been proposed to have two principle
binding proteins (Young and Woodside, mechanisms of action, a chain-breaking
2001; Pham-Huy et al., 2008). mechanism and a prevention mechanism.
The first mechanism donates an electron
from the primary antioxidant to the free by Yim et al. (1990), CuZnSOD catalyzes
radical. The free radical is unstable and hydroxyl radical (OH) from H2O2. Dele-
highly reactive. Therefore, it either tion of sod1 gene in knock-out mice was
releases or steals an electron from another reported to show high level of oxidative
molecule in order to stabilise itself. This damage to proteins, lipids, and DNA in
activity causes the molecule to become skeletal muscle tissue due to lack of
unstable, thus forming a second radical. CiZnSOD (Muller et al., 2006). Compre-
The second radical then releases or steals hensive oxidative damage was also ob-
an electron from another molecule, thus served in liver tissues of sod1-/- mice
forming the following radical.This (Elchuri et al., 2005). Hence, CuZnSOD
process is continued until a chain- is a potential antioxidant enzyme.
breaking antioxidant helps to stabilise the Another enzymatic antioxidant de-
free radical. On the other hand, the second fence mechanism is glutathione perox-
mechanism involves the removal of idise (GSHPx) activity where free radicals
ROS/RNS initiators by inhibiting chain- are reduced by glutathione into water and
initiation catalyst in the cells (Pham-Huy its analogous alcohols (Wendel, 1980).
et al., 2008; Lobo et al., 2010). Both The selenieum-containing GSHPx is
mechanisms play a significant role in pre- mainly localized in cytoplasm and mito-
venting oxidative stress-related diseases. chondria (Zarowski and Tappel, 1978;
Epp et al., 1983; Tappel, 1984; Timcen-
11. The defences against free radical ko-Youssef, 1985). The free radical de-
attack and oxidative stress fence mechanism through GSHPx is evi-
dent through the study conducted by
Human body is naturally adapted with Hawker et al. (1993) who demonstrated
several defence mechanisms to counter- an excessive ROS generation in selenium-
balance the excessive free radicals pro- deficient mice. Although GSHPx-1 was
duced through the oxidative phosphoryla- reported to be insignificantly contribute
tion. Enzymatic antioxidant mechanism is against oxidative damage by evaluating
the most important defence system carbonyl content, lipid peroxidation activ-
against free radical. One such major free ity and rate of extracellular H2O2 con-
radical scavenging enzyme is the superox- sumption in several tissues of GPSH-1-/-
ide dismutase (SOD). Manganese SOD knock-out mice samples (Ho et al., 1997),
(MnSOD) was reported to be directly in- studies conducted by Zhang et al. (2009)
volved in the suspension of superoxide revealed an elevation of free radicals in
anions which was evident by the death of Sod2+/Gpx1/ mice through evaluation
sod2 gene-knocked out mice within 10 of DNA and protein oxidation in several
days after birth due to dilated cardiomyo- tissues of the transgenic mice. In addition,
pathy, lipid amassing in liver and skeletal Gpx4+/ mice was reported to be highly
muscle and metabolic acidosis caused by sensitive to oxidative stress (Yant et al.,
the deficiency of MnSOD (Li et al., 2003; Ran et al., 2003).
1995). Furthermore, tissues of those mice Furthermore, catalase is also a po-
lacking MnSOD exhibited an elevated tential free radical defence enzyme which
degree of oxidative damage DNA with eliminates hydrogen peroxide by catalys-
higher rate cancer prevalence (Remmen et ing it into water and oxygen in which one
al., 2003). Another type of SOD is the molecule of catalase decomposes approx-
Copper, Zinc SOD (CuZnSOD) which is imately six million of H2O2 molecules per
mainly localized in cytoplasm, nucleus, minute (Kellin and Hartree, 1938; Mates
lysosome and intermembrane space of et al., 1999). Several studies have demon-
mitochondria (Chang et al., 1988; Keller strated the defence mechanism of free
et al., 1991; Crapo et al., 1992; Sturtz et radicals by catalyse. Transgenic mice
al., 2001). Based on the study conducted over expressing catalase (hCatTg+/0) was
shown to decrease the generation of H2O2 antioxidants that quench free radicals, a
which eventually resulted in reduced physiological response usually not present
blood pressure in contrast to the wild-type in lower elevation plants (Alonso-Amelot,
mice (Yang et al., 2002). In addition, 2008).
knockout mice with under expression of
catalase by disruption of cat or cas1 gene 12.1. Verbascum sinaiticum Benth.
was reported to show decreased rate of (Scrophulariaceae)
decomposition of hydrogen peroxide with The plant, Verbascum sinaiticum
an increased tendency to oxidative stress is a biennial rosette plant (a taproot with a
(Ho et al., 2004). cluster of leaves on the soil surface), upon
On the other hand, there are sever- where this species is listed as rare in
al other non-enzymatic defence mecha- Egypt and wide-reaching to Sinai Penin-
nisms against free radicals such as vita- sula (Hegazy, 2000). The leaves and
min E, vitamin C and Thiol antioxidants. flowers from Verbascum species had been
Vitamin E is a fat soluble vitamin which utilized in the treatment of bronchitis, dry
was reported to act against lipid peroxida- coughs, tuberculosis and asthma while
tion (Pryor, 2000). Vitamin C is a water studies have confirmed expectorant, mu-
soluble vitamin which acts in correspond- colytic and demulcent properties (Kozan
ence with vitamin E. -tocopherol is an et al., 2011). Extraordinary results were
active form of vitamin E which eliminates obtained from V. sinaiticum when it was
lipid peroxyl radical by donating a hydro- observed for selectivity in anti-
gen atom and itself becoming an - proliferation response against carcinoma
tocopherol radical while vitamin C reduc- and normal cells. The effect of anti-
es -tocopherol radical into its original proliferate was evaluated in hepatocellu-
form (Carr and Frei, 1999; Kojo, 2004). lar carcinoma (Hep-G2) and normal
Thiol antioxidants include the tripeptide (MRC-5) cells after exposing the cells to
glutathione (GSH), thioredoxin (TRX), the extract for 48 hours (Tauchen et al.,
and -lipoic acid (ALA) also acts against 2015). The extract comprises composi-
free radical through redox reactions tions of luteolin, chrysoeriol, hydrocarpin
(Rahman, 2007). and sinaiticin which were earlier tested
for cytotoxicity reaction towards leukae-
12. Medicinal plants as green approach mia P-388 cells, providing a positive
defences against free radical attack feedback (Afifi et al., 1993). These com-
pounds falls under the classification of
In recent times, substantial evidence flavonoids and flavonolignans (Afifi et
emerged indicating a mutual relationship al., 1993 and Mahmoud et al., 2007) to
between the consumption of antioxidant- which they will not cause possible haz-
rich foods and the episodes of human ards to consumer and consequently an
health deterioration (Sies, 1997). Con- attribution to the selective anti-
versely, when synthetic antioxidants proliferative activity of V. sinaiticum
namely butylated hydroxytoluene (BHT) against Hep-G2 and MRC-5 cell line
and butylated hydroxyanisole (BHA) (Tauchen et al., 2015).
were extensively employed as additives in
food manufacturing business, a multitude 12.2. Ugni myricoides (Kunth) O.Berg
of cases were reported on liver injuries Ugni myricoides are inhabitants of
and carcinogenesis (Grice, 1988; Wichi, western Latin America (WCSP, 2016),
1986). Considering this fact, attentions possessing the properties for anti-
were shifted into utilizing natural antioxi- hypernociceptive effect (Quinto et al.,
dants. Plants, given the fact that can grow 2010) and anti-nociceptive effects in mice
successfully adapting to the exposure of (Camplo, 2011). Brovo et al., had car-
ultraviolet, are also reservoir for potent ried out an investigation between U. my-
ricoides fruits antioxidant properties and indigenous medicine for the administra-
their inhibitory interaction against skin tion of many ailments. It also has measur-
aging-related enzymes. U. myricoides has ably multitude biological activities asso-
been measured for high values in mitigat- ciated to antioxidant, anti-inflammatory
ing free radical damages of UV light in and cancer preventive properties (Teiten
skin, by both the ORAC (Oxygen radical et al., 2010; Goel et al., 2008; Hatcher et
absorbance capacity) and TEAC assays al., 2008).
(The Trolox Equivalent Antioxidant Ca- In a paper reported by Dall'Acqua
pacity). As a result, U. myricoides were et al., a pattern of significant changes
determine to retain high total phenolic were observed in the urinary metabolome
content, with high correlation coefficient of healthy rats, orally treated with curcu-
above 0.80 and its aptitude in hindering min, compared to controls in data sets ob-
collagenase, elastase, hyaluronidase and tained both by NMR and HPLCMS
tyrosinase enzymes propose that the pres- (Dall'Acqua et al., 2016). The effect of
ence of high values of polyphenols are Curcuma extract on urinary composition
potential conducive for these responses in healthy rats disclosed evidence for in
(Bravo et al., 2016). vivo reduction of the amount of urinary
biomarkers of oxidative stress namely al-
12.3. Black highland barley (BHLPE) lantoin, 3-nitrotyrosine, m-tyrosine, 8-
The black highland barley known OHdG. The m-Tyrosine is examined as a
in Tibet for a crop of high importance is biomarker for oxidative damage to pro-
referred as Qing Ke in Chinese. The po- teins while urinary 8-OHdG (Orhan et al.,
tential of BHLPE was measured and the 2004) is measured as a biomarker of
findings exhibited potent superoxide radi- comprehensive cellular oxidative stress
cal, hydroxyl radical and 2,2-diphenyl-1- due to its prevalent generation of oxidized
picrylhydrazyl radical-scavenging activi- DNA repair (Tsikas et al., 2005).
ty, ferric reducing antioxidant power and
moderate metal ion-chelating activity 12.5. Beta vulgaris L.
(Shen et al., 2016). Two groups of mice Beetroot (Beta vulgaris) is well
were prepared; a high fat diet (HFD) known for its high total phenolic content
group and a group that has been adminis- (5060 mol/g dry weight) (Khknen et
trated with 600 mg BHLPE/kg body al., 1999; Vinson et al., 1998), and the
weight. The treated group of mice dis- presence of considerable amount of phe-
played remarkable reduction in total cho- nolic acids: ferulic, protocatechuic, vanil-
lesterol, low-density lipoprotein choles- lic, p-coumaric, p-hydroxybenzoic and
terol and the atherosclerosis index. The in syringic acids (Kujala, et al., 2000). It al-
vivo test further provides information on so a source of betalains, water-soluble
antioxidant defence system and antioxi- nitrogenous pigments that feed on reac-
dant gene expression where significant tive oxygen species (ROS) to protect its
amelioration had occurred within BHLPE plant from wound injuries and pathogenic
treated group as compared to HFD mice penetration as studied in red beet
group. Conclusively, the study highlight- (Seplveda-Jimnez et al., 2004). Five
ed the tendency of natural antioxidant of beetroot pomace extracts from different
BHLPE in generating good health by re- cultivar were studied upon where Detroit
ducing the incident of disease (Shen et al., beetroot pomace extract (DBPE) exhibit-
2016). ed the highest antiradical properties in
vitro, by effectively scavenging DPPH
12.4. Curcuma longa L. radicals (EC50=2.060.10 g/ml) and high
Curcuma longa L is a spice that reducing power (EC50=123.3906.05
has considerable usage in the practise of g/ml). The effect of in vivo antioxidant
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Abstract: The most important aspect of nanotechnology is the synthesising and characteriz-
ing the nanoparticles (NPs). Nano particles are manufactured in large quantities because of
their wide range of applications. All Physical, Chemical and Biological methods have been
used in the metal nanoparticles production. The major problems in the production are the
stability, aggregation, morphology, crystal growth, size and size distribution. In recent
years, the green synthesis of metal NPs has become more attractive because of cost effec-
tiveness and its various applications in developing new technologies. It is considered as an
eco-friendly technology for the production of well characterized NPs. The metal nanoparti-
cles produced by the plant are more stable and the rate of synthesis is faster as compared to
other methods. Currently, researchers are mainly focused on searching new antimicrobial
agents against various pathogenic bacteria which cause infectious diseases. Moreover, the
NPs have effective antimicrobial properties against infectious pathogens. In this review, we
have discussed different kind of plants which are used in synthesising NPs and highlighted
their antimicrobial applications. We also discussed the basic mechanism by which nanopar-
ticles interact with microbes.
cus carica (Singh and Bhakat, 2012), be synthesised using stem latex of Eu-
Sphaeranthus amaranthoides (Nellore et phorbia nivulia (Common milk hedge).
al., 2012) and Putranjiva roxburghii Some green methods for synthesis of
(Badole and Dighe, 2012), plant extract of copper nanoparticles are reported using
Mango (Yang et al., 2014); Gymnocladus plant leaf extracts such as Capparis
assamicus (Tamuly et al., 2013); Cacu- zeylanica Linn (Saranyaadevi et al.,
men platycladi (Wu et al., 2013); Po- 2014), tamarind, lemon juice (Sastry et
gestemon benghalensis (Paul et al., al., 2013); Ocimum sanctum as capping
2015); Nerium oleander (Tahir et al., agents (Kulkarni and Kulkarni 2013);
2015); Butea monosperma (Patra et al., Magnolia kobus leaf extract (Lee et al.,
2015); Pea nut (Raju et al., 2014); Hibis- 2013); Syzygium aromaticum (cloves)
cus cannabinus (Bindhu et al., 2014); aqueous extract (Subhankari et al., 2013)
Sesbania grandiflora (Das and Velusamy, and Nerium oleander (Gopinath et al.,
2014). 2014). Brassica juncea, Medicago sativa
and Helianthus annus and Tridax pro-
3.2. Silver nanoparticle cumbens (Abdul and Samarrai, 2012;
Normaly silver (Ag) nanoparticles Asim et al., 2012), Lantana camara (Ma-
ranged in size from 15 to 65 nm with an jumder, 2012), Zingiber officinale (Ipsa
average size of 34 nm and cuboidal, rec- and Nayak, 2013) and Ocimum sanctum
tangular in shape. The medicinally im- (Vasudev and Pramod, 2013). Copper na-
portant plants like Boerhaavia diffusa noparticles could be directly used for ad-
(Vijaykumar et al., 2014), Aloe vera ministration/in vivo delivery of nanoparti-
(Chandran et al., 2006), Terminalia cles for cancer therapy (Valodkar et al.,
chebula (Edison and Sethuraman 2012), 2011).
Catharanthus roseus (Mukunthan et al.,
2011), Ocimum tenuiflorum (Patil et al., 3.4. Copper oxide nanoparticles
2012) Azadirachta indica (Tripathi et al., According to Padile et al. (2013)
2009), Emblica officinalis (Ankamwar et Sterculia urens (Karaya gum) synthesizes
al., 2005) Cocos nucifera (Roopan et al., Cuprous Oxide (CuO at a range of 4.8 nm
2013), common spices Piper nigrum size). Jayalakshmi and Yogamoorthi,
(Shukla et al., 2010), Cinnamon zeylan- (2014) tried out the copper oxide nano-
icum (Satishkumar et al., 2009a) have al- particles synthesis using flower extract of
so been used for Ag NPs synthesis. Cassia alata. Rinkesh et al. (2016) use
Au-Ag bimetallic nanoparticles also syn- Floral extract of Caesalpinia pulcherrima
thesize successfully by plants include for the synthesis of CuO in an eco-
Azadirachta indica (neem) (Shankar et friendly method.
al., 2004), Anacardium occidentale (cash-
ew nut) (Sheny et al., 2011), Swietenia 3.5. Palladium nanoparticles
mahagony (West Indies mahogany) Palladium nanoparticles were syn-
(Mondal et al., 2011) and cruciferous thesised using an extract of C. Zeylanicum
vegetable extracts (Jacob et al., 2012). taken from (cinnamon) bark Satishkumar
et al. (2009b) and also using Annona
3.3. Copper nanoparticles squamosa (Custard apple) peel extract
Many variety of plant extracts with the size ranging from 75 to 85 nm
have been used for the synthesize of Cop- (Roopan et al., 2011). The leaf extract of
per (Cu) and copper oxide (CuO) nano- soybean (Glycine max) have been able to
particles. Magnolia leaf extract and synthesise nanoparticles with a mean size
Syzygium aromaticum (Clove) extracts is of 15 nm (Kumar et al., 2012). According
used to Cu nanoparticles with the size to Nadagouda et al. (2008) Coffea arabi-
ranging from 40 to 100 nm (Subhankari ca (Coffee) and Camellia sinensis (Tea)
and Nayak, 2013). Cu nanoparticles can extracts have been utilised to synthesise
ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 173
Biotech Sustainability (2017)
A Review on Green Synthesis of Nanoparticles Arumugam and Sharma
The ionic silver released by the silver na- sised green NPs have a good potential as
noparticles inactivates bacterial enzymes an antimicrobial agent against different
by interacting with thiol group. It inhibits microorganism. However, further re-
bacterial DNA replication and damage the search is required to tap the potential.
bacterial cytoplasm membranes thereby
depleting the level of ATP and inhibit References
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ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 176
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Abstract: Secondary metabolites produced by Medicinal plants are useful for the pro-
duction of life saving drugs. The use of natural phytochemicals has its own advantage
as it has no side effects. Because of sustainability, attention on in vitro plant materials
as potential factories for secondary phytochemical products is increasing. More than
80% of the worlds population relies on traditional medicine as it has negligible side
effects for their primary health care needs. Annually, about 95% of the medicinal
plants used as a raw material is growing at the rate of more than 40%. However, ma-
jority of medicinal plants are collected from their wild habitats, especially from forests.
Repeated use of these medicinal plants has resulted in depletion and extinction of many
important medicinal plant species from their natural habitats. The other most common
reason is poor regeneration capacity of plant under natural habitats due to improper en-
vironmental factors like weather conditions, seasonal change, soil erosion etc. Due to
these reasons, the need of hour is to protect and conserve valuable important medicinal
plants otherwise many of these plants will be lost from natural vegetation forever. In
this article, biotechnological approaches such as plant cell culture and hairy root culture
is described as alternative methods for the production of secondary metabolites. The
advantages of these methods and enhancements of secondary metabolites production by
different methods are also discussed.
Over one quarter of new drugs that most secondary metabolites. The recent
have been approved in the last 30 years are advances, new directions, and opportuni-
based on a lead from a molecule from ties in plant cell-based processes are being
plant origin. Moreover, 9 of the top 20 critically examined. Such as biotransfor-
selling drugs are derived from knowledge mation using an exogenous supply of bio-
of plant secondary metabolites (Harvey, synthetic precursors, genetic manipulation,
2000; Tulp and Bohlin, 2002). Higher and metabolic engineering may improve
plants are rich source of bioactive constit- the accumulation of compounds. Use of
uents or phyto-pharmaceuticals used in biotic and a biotic elicitors, can also be
pharmaceutical industry. Some of the plant used for triggering the formation of sec-
derived natural products include drugs ondary metabolites. The possible use of
such as morphine, codeine, cocaine, qui- plant cell cultures for the specific biotrans-
nine etc; anti-cancer Catharanthus alka- formation of natural compounds has been
loids, belladonna alkaloids, colchicines, demonstrated (Cheetham, 1995; Scragg,
phytostigminine, pilocarpine, reserpine 1997; Krings and Berger, 1998; Ravishan-
and steroids like diosgenin, digoxin and kar and Ramachandra Rao, 2000). In the
digitoxin. Many of these pharmaceuticals search for alternatives for production of
are still in use today and often no useful desirable medicinal compounds from
synthetic substitutes have been found that plants, biotechnological approaches, spe-
possess the same efficacy and pharmaco- cifically, plant tissue cultures, are found
logical specificity. Currently one fourth of to have potential as a supplement to tradi-
all prescribed pharmaceuticals in industri- tional agriculture and also in the industri-
alized countries contain compounds that al production of bioactive plant metabo-
are directly or indirectly, via semi- lites (Ramachandra Rao and Ravishankar,
synthesis, derived from plants. Studies on 2000). Cell suspension culture systems
plant secondary metabolites have been in- could be used for large scale culturing of
creasing over the last 50 years. plant cells from which secondary me-
There are three potential pathways tabolites could be extracted. Due to these
for primary metabolism: the Embden advances, research in the area of tissue
Meyerhof-Parnas Pathway (EMP), the culture technology for production of plant
Entner-Dourdorof pathway, and the hexose chemicals has bloomed beyond expecta-
monophosphate (HMP) pathway. Due to tions. The oncogenic strains of Agrobacte-
rapid deforestation and depletion of genet- rium rhizogenes is used to transform a
ic stocks, various efforts were made to range of plant species, which induces hairy
implement new methods for several plant roots. The hairy roots induced from Agro-
species conservation and also yielding in bacterium rhizogenes transformation has
high amounts of secondary metabolites, shown to have attractive properties for
with photosynthetic efficiency, pest re- secondary metabolite production as com-
sistant and disease resistant plants. To pared to differential cell cultures (Kim et
overcome these problem new improved al., 2002). While research to date has suc-
methods of plant tissue culture and trans- ceeded in producing a wide range of valu-
formation through molecular approaches able secondary photochemical in unor-
presents an alternative option for multipli- ganized callus or suspension cultures, in
cation, development and conservation of other cases production requires more dif-
elite plant species. Biotechnological ap- ferentiated micro plant or organ cultures
proaches such as plant tissue culture hold (Davioud et al., 1989). In this chapter we
great promise for controlled production of will be describing the popular types of bio-
useful secondary metabolites on demand. technological approaches for production of
The current yield and productivity cannot secondary metabolites.
fulfill the commercial goal of plant cell-
based bioprocess for the production of
2. Biotechnological approaches for pro- Plant cell and tissue cultures can be
duction of secondary metabolites established routinely under sterile condi-
tions from explants, such as plant leaves,
The increased appeal of natural stems, roots, and meristems for multiplica-
products for medicinal purposes coupled tion and extraction of secondary metabo-
with the low product yields and supply lites. The explants selected from high
concerns of plant harvesting has renewed yielding mother plants are usually selected
interest in large-scale plant cell and hairy as the starting material ((Figure 1)). Fol-
root culture technology. Secondary metab- lowing the standard sterilization proce-
olites can be produced by using different dures the cultures are inoculated on a suit-
biotechnological approaches (Table1). In able plant tissue culture medium for induc-
this chapter, some techniques have been tion of callus which is the base for all fur-
described. ther production work (Figure 2). Callus
cultures, can be divided into one of two
2.1. Plant cell culture
ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 189
Biotech Sustainability (2017)
Production of Secondary Metabolites Using a Biotechnological Approach Shankar and Rajagopal
Table 1: Bioactive secondary metabolites produced using plant tissue cultures (Table cour-
tesy: Vanisree and Tsay, 2004)
Plant name Active ingredient Culture type Reference
Agave amaniensis Saponins Callus Andrijany et al., 1999
Ailanthus altissima Alkaloids Suspension Anderson et al., 1987
Ailanthus altissima Canthinone alkaloids Suspension Anderson et al., 1986
Allium sativum L. Alliin callus Malpathak and David,
1986
Aloe saponaria Tetrahydroanthracene glucosides suspension Yagi et al., 1983
Ambrosia tenuifolia Altamisine Callus Goleniowski and Trip-
pi, 1999
Anchusa officinalis Rosmarinic acid Suspension De-Eknamkul and
Ellis, 1985
Brucea javanica (L.) Merr. Canthinone alkaloids Suspension Liu et al., 1990
Bupleurum falcatum Saikosaponins Callus Wang and Huang,
1982
Bupleurum falcatum L. Saikosaponins Root Kusakari et al., 2000
Camellia sinensis Theamine, -glutamyl derivatives suspension Orihara and Furuya,
1990
Canavalia ensiformis L-Canavanine Callus Ramirez et al., 1992
Capsicum annuum L. Capsaicin Suspension Johnson et al., 1990
Cassia acutifolia Anthraquinones Suspension Nazif et al., 2000
Catharanthus roseus Indole alkaloids Suspension Moreno et al., 1993
Catharanthus roseus Catharanthine Suspension Zhao et al., 2001b
Cephaelis ipecacuanha A. Emetic alkaloids Root Teshima et al., 1988
Richard
Chrysanthemum cinerari- Pyrethrins Callus Rajasekaran et al.,
aefolium 1991
Chrysanthemum cinerari- Chrysanthemic acid and pyethrins Suspension Kueh et al., 1985
aefolium
Cinchona L. Alkaloids Suspension Koblitz et al., 1983
Cinchona robusta Robustaquinones Suspension Schripsema et al.,
1999
Cinchona spec. Anthraquinones Suspension Wijnsma et al., 1985
Cinchona succirubra Anthraquinones Suspension Khouri et al., 1986
Citrus sp. Naringin, Limonin Callus Barthe et al., 1987
Coffea arabica L. Caffeine Callus Waller et al., 1983
Cruciata glabra Anthraquinones Suspension Dornenburg and
Knorr, 1996
Cryptolepis buchanani Cryptosin Callus Venkateswara et al.,
Roem. & shult 1987
Digitalis purpurea L. Cardenolides Suspension Hagimori et al., 1982
Dioscorea deltoidea Diosgenin Suspension Heble and Staba, 1980
Dioscorea doryophora Diosgenin Suspension Huang et al., 1993
Hance
Duboisia leichhardtii Tropane alkaloids Callus Yamada and Endo,
1984
Ephedra spp. L- Ephedrine, D Pseudoephidrin Suspension ODowd et al., 1993
Eriobotrya japonica Triterpenes Callus Taniguchi et al., 2002
Eucalyptus tereticornis SM. Sterols and Phenolic compounds callus Venkateswara et al.,
1986
Fumaria capreolata Isoquinoline alkaloids Suspension Tanahashi and Zenk,
1985
Table 1: Continued
Plant name Active ingredient Culture type Reference
Gentiana sp. Secoiridoid glucosides Callus Skrzypczak et al.,
1993
Ginkgo biloba Ginkgolide A Suspension Carrier et al., 1991
Glehnia littoralis Furanocoumarin Suspension Kitamura et al., 1998
Glycyrrhiza echinata Flavanoids Callus Ayabe et al., 1986
Glycyrrhiza glabra var. Triterpenes callus Ayabe et al., 1990
glandulifera
Hyoscyamus niger Tropane alkaloids Callus Yamada and Hashimo-
to, 1982
Isoplexis isabellina Anthraquinones Suspension Arrebola et al., 1999
Linum flavum L. 5-Methoxypodophyllotoxin Suspension Uden et., al 1990
Lithospermum erythrorhizon Shikonin derivatives Suspension Fujita et al., 1981
Lithospermum erythrorhizon Shikonin derivatives Suspension Fukui et al., 1990
Lycium chinense Cerebroside Suspension Jang et al., 1998
Mentha arvensis Terpenoid Shoot Phatak and Heble,
2002
Morinda citrifolia Anthraquinones Suspension Zenk et al., 1975
Morinda citrifolia Anthraquinones Suspension Bassetti et al., 1995
Mucuna pruriens L-DOPA Suspension Wichers et al., 1993
Mucuna pruriens L-DOPA Callus Brain, 1976
Nandina domestica Alkaloids Callus Ikuta and Itokawa,
1988
Nicotiana rustica Alkaloids Callus Tabata and Hiraoka,
1976
Nicotiana tabacum L. Nicotine Suspension Mantell et al., 1983
Ophiorrhiza pumila Camptothecin related alkaloids Callus Kitajima et al., 1998
Panax ginseng Saponins and Sapogenins Callus Furuya et al., 1973
Panax notoginseng Ginsenosides Suspension Zhong and Zhu, 1995
Papaver bracteatum Thebaine Callus Day et al., 1986
Papaver somniferum L. Alkaloids Callus Furuya et al., 1972
Papaver somniferum Morphine, Codeine Suspension Siah and Doran, 1991
Peganum harmala L. -Carboline alkaloids Suspension Sasse et al., 1982
Phytolacca americana Betacyanin Suspension Sakuta et al., 1987
Picrasma quassioides Ben- Quassin Suspension Scragg and Allan,
nett 1986
Podophyllum hexandrum Podophyllotoxin Suspension Uden et al., 1989
royle
Polygala amarella Saponins Callus Desbene et al., 1999
Polygonum hydropiper Flavanoids Suspension Nakao et al., 1999
Portulaca grandiflora Betacyanin Callus Schroder and Bohm,
1984
Ptelea trifoliata L. Dihydrofuro [2,3-b] quinolinium Callus Petit-Paly et al., 1987
Rauwolfia sellowii Alkaloids Suspension Rech et al., 1998
Rauwolfia serpentina Benth. Reserpine Suspension Yamamoto and Yama-
da, 1986
Rauvolfia serpentina x Rha- 3-Oxo-rhazinilam Callus Gerasimenko et al.,
zya stricta 2001
Rhus javanica Gallotannins Root Taniguchi et al., 2000
Ruta sp. Acridone and Furoquinoline alka- Callus Baumert et al., 1992
loids and cumarins
Table 1: Continued
Plant name Active ingredient Culture type Reference
Salvia miltiorrhiza Lithospermic acid B and rosma- Callus Morimoto et al., 1994
rinic acid
Salvia miltiorrhiza Cryptotanshinone Suspension Miyasaka et al., 1989
Scopolia parviflora Alkaloids Callus Tabata et al., 1972
Scutellaria columnae Phenolics Callus Stojakowska and
Kisiel, 1999
Solanum chrysotrichum Spirostanol saponin Suspension Villarreal et al., 1997
(Schldl.)
Solanum laciniatum Ait Solasodine Suspension Chandler and Dodds,
1983a
Silybum marianum Flavonolignan Root Alikaridis et al., 2000
Solanum paludosum Solamargine Suspension Badaoui et al., 1996
Tabernaemontana divarica- Alkaloids Suspension Sierra et al., 1992
ta
Taxus spp. Taxol Suspension Wu et al., 2001
Taxus baccata Taxol baccatin III Suspension Cusido et al., 1999
Thalictrum minus Berberin Suspension Kobayashi et al., 1987
Thalictrum minus Berberin Suspension Nakagawa et al., 1986
Torreya nucifera var. radi- Diterpenoids Suspension Orihara et al., 2002
cans
Trigonella foenumgraecum Saponins Suspension Brain and Williams,
1983
Withaina somnifera Withaferin A Shoot Ray and Jha, 2001
.
um. This results in dilution of the suspen- lites. The secondary metabolites produced
sion and the initiation of another batch by hairy roots arising from the infection of
growth cycle. The degree of dilution dur- plant material by A. rhizogenes are the
ing subculture should be determined em- same as those usually synthesized in intact
pirically for each culture. Too great a de- parent roots, with similar or higher yields.
gree of dilution will result in a greatly ex- This feature, together with genetic stability
tended lag period or, in extreme cases, and generally rapid growth in simple me-
death of the transferred cells. After subcul- dia lacking phytohormones, makes them
ture, the cells divide and the biomass of especially suitable for biochemical studies
the culture increases in a characteristic not easily undertaken with root cultures of
fashion, until nutrients in the medium are an intact plant. The hairy roots are normal-
exhausted and/or toxic byproducts build up ly induced on aseptic, wounded parts of
to inhibitory levels this is called the sta- plants by inoculating them with A. rhi-
tionary phase. If cells are left in the sta- zogenes.
tionary phase for too long, they will die
and the culture will be lost. Therefore, 2.3. Biotransformation using precursors
cells should be transferred as they enter the Commercial production of second-
stationary phase. It is therefore important ary metabolites requires a reproducible and
that the batch growth-cycle parameters are standardized protocol for cultivation of
determined for each cell-suspension cul- plant cells/organs on a large scale. Em-
ture. Strain improvement, methods for the ploying precursor feeding, transformation
selection of high-producing cell lines, and methods, and immobilization techniques.
medium optimizations can lead to an en- The treatment of plant cells with biotic
hancement in secondary metabolite pro- and/or abiotic elicitors has been a useful
duction. Compared to whole plant cultiva- strategy to enhance secondary metabolite
tion system cell culture system has the ad- production in cell cultures (Karuppusamy,
vantages as follows: 2009). The most frequently used elicitors
i. Useful compounds can be pro- are fungal carbohydrates, yeast extract,
duced under controlled conditions Methyl Jasmonate (MJ) and chitosan. MJ,
independent of climatic change or a proven signal compound, is the most ef-
soil conditions; fective elicitor of taxol production in Tax-
ii. Cultured cells will be free of path- us chinensis Roxb (Wink et al., 2008) and
ogens and pests; gonsenoside production in P. ginseng C.A.
iii. Less space is required and produc- Meyer cell/organ culture (Xu et al., 2008;
tion is uniform. Yamanaka et al., 1996).
ly active enzyme or to a cell within a reac- iv. Mixing with suitable materials,
tor system and prevents its entry into the changing their consistency with
mobile phase, which carries the substrate temperature (embedding).
and product. The first successful immobi- v. Physical retention within the
lization of plant cells was reported by framework of diverse pore size and
Brodelius et al., (1979) and they entrapped permeability (entrapment, micro
Catharathus roseus and Daucus carota encapsulation).
cells in alginate beds. Following success
with enzymatic and microbial process, 2.4.1.1. Selection of immobilization system
immobilization has been suggested as a The choice of a suitable immobili-
strategy to enhance the overall productivi- zation system is determined by the follow-
ty of secondary metabolite in plant cell ing requirements.
culture. The ability to immobilize plant i. The polymer material used for im-
cells has been reported for a large number mobilization must be available in
of plant cells and protoplasts by using a large quantities; it must be inert,
variety of polymers. Immobilization of non-toxic and cheap.
plant cells has been used for a wide range ii. It must be able to carry large quan-
of reactions, which can be divided into tities of biomass and its fixing po-
three groups. (1) Biotransformation or bio- tential must be high.
conversion, (2) synthesis from precursors iii. The immobilization process must
and (3) the De Novo synthesis of com- not diminish enzymatic activity of
pounds. Some of the advantages of immo- biological catalyst.
bilization are, retention of biomass enables iv. Manipulation of the biological cat-
its continuous reutilization as a production alyst must be as simple as possible.
system. The immobilization of cells allows
the use of a higher biomass level compared 2.4.2. Methods for immobilization
to cell suspension culture, Separation of
cells from medium and the product is extra 2.4.2.1. Gel entrapment by polymerization
cellular, which will simplify downstream A monomer or a mixture of mon-
processing compared to extract from tis- omers is polymerized in the presence of a
sue. Immobilization allows a continuous cell suspension, which is entrapped inside
process, which increase volumetric the lattice of the polymer.
productivity and allows the removal of
metabolic inhibitors. Reduces problems 2.4.2.2. Gel entrapment by ionic net work
such as aggregate, growth and foaming formation
.Some of the disadvantage is the microen- In this method, polymerization of
vironment favoring optimal production can polyelectrolyte is achieved by addition of
be unfavorable for released secondary me- multivalent ions. The most common meth-
tabolites and cause their degradation or od is the entrapment in calcium alginate.
metabolization. This is a non-toxic process in which sodi-
um alginate solution containing the cell
2.4.1. Different types of immobilization suspension is dropped into a mixture of
i. Direct intracellular binding due to counter ion solution such as calcium chlo-
natural affinity (adsorption, adhe- ride. A uniform, spherical and highly mi-
sion and agglutination). crospores structure results, which retains
ii. Covalent coupling on otherwise in- the cell.
ert matrices.
iii. Intracellular connection via bi or 2.4.2.3. Gel entrapment formation by pre-
poly functional reagent (cross- cipitation
linking). Gels may be formed by precipita-
tion of some natural and synthetic poly-
Abstract: Photobioreactor (PBR) is a reactor which exploits a light supply to farm photo-
trophic microorganisms which can generate biomass using light, water and carbon dioxide.
Macro- and microalgae are developed naturally in fresh water as well as marine water
(highly economical) or brackish water. Universal production of marine algal monocultures
is basically limited to a selective species such as Scenedesmus spp., Nannochloropsis spp.,
and Chlorella spp., and some extremophiles like Arthrospira spp., and Dunaliella spp.,.
Owing to their inborn resistance to predators and competitors, algal species can be cultivat-
ed naturally in open systems akin to ponds, stirred tanks and bubble columns. Nowadays,
numerous research groups are focusing on PBRs (viz., selective closed type or open sys-
tems) to produce marine algae (cyanobacteria or seaweeds) through utilizing a light source
with lavish biomass accumulation. The cultivation of marine algae has distinguished a new
extension in numerous fields crucial for humanity, especially energy, food, health and envi-
ronment. Currently, marine algae and algae-based extracts remain predominantly unex-
ploited despite their huge potential applications. This chapter highlights the present and fu-
ture prospects of marine plants and their derived aqueous extracts for crop improvement
and enhancement of plant biomass production.
case of filamentous micro algae, a variety population over the foregoing few dec-
of strains are recognized to make intra- or ades. In the year 2012, an incredible 90.4
extracellular metabolites by dissimilar million tonnes of marine based food and
biological activities (Glombitza and beverages were farmed and this amount is
Koch, 1989). However, numerous re- expected to increase until about 2030, at
search groups focusing on photobioreac- which moment it is indefinite that capture
tors (PBRs) for making of prominent al- fisheries and aquaculture will distribute
gal biomass production, is not nearly as equal quantities (FAO, 2013; Taelman et
superior as requisite by the worlds de- al., 2015). Nevertheless, an entire control
sires, particularly in the pharmaceutical, on culture conditions is sufficient only
for human utilization as well as fish and through closed systems, only PBR.
animal feed, and for field level agricultur- Commonly, PBR is a bioreactor
al applications. Still, there is inert ques- which uses a light source to grow photo-
tion as to whether algal based cultures are trophic microorganisms which can pro-
able to make plant growth stimulants and duce biomass through light and CO2 and
concerning their mechanisms of action include plants, macro and micro algae,
towards elevated yielding, biotic and abi- cyanobacteria, purple bacteria and moss-
otic stress resistance and high biomass es. The first step was taken by Richmond
production of crops in agriculture through and group in 1978 as the introduction of
regulation of growth succession such as external algal biotechnology in Israel
cell division and in the sensitivity of envi- (Richmond and Vonshak, 1978;
ronmental changes. Masojidek et al., 2003, 2009). Compared
to open culture systems like ponds and
2. Recent developments for cultivation raceways, PBR systems have several ad-
of marine algae using photobioreac- vantages viz., reproducible farming with
tors regard to environmental changes, mainte-
nance of dissolved oxygen concentration,
The majority of marine algae use sufficient mixing of the culture with the
photosynthesis to confine light energy to appropriate flow rate, the opportunity of
change inorganic substances into useful temperature regulation with low CO2
sugars and then other molecules which is losses and making it feasible to focus and
similar to plants. The limitations of de- regulate the irradiance for augmentation
velopment forced by the constraint and of algal cultures with reduced risk of cul-
saturation of light and results have im- ture contamination (Masojidek et al.,
pelled algal biotechnologists to come up- 2009). Rorrer et al. (2004) showed the
on for resolution to convince this outcome bioprocess engineering method, especial-
in outdoor cultures (Masojidek et al., ly for the marine algae process through
2003). Due to the significance of light the development of cell and tissue culture
saturation, information on the behavior of systems and his group described a diversi-
cyanobacteria cultures showed to very ty of techniques to grow phototropic sus-
high illumination is comparatively sparse pension cultures appropriate for the culti-
and most of it has been acquired in labor- vation in PBR systems. Also, the same
atory experiments. The possible devel- group compared three foremost PBR con-
opment of algal biotechnology is moving figurations for macroalgal suspension cul-
towards the field of high charge commod- ture systems viz., bubble-column or air-
ities grown in well controlled cultivation lift, tubular recycle and stirred tank, and
conditions, such as temperature, nutrient considered the major factors that limit
supply, pH, light, and CO2 (Masojidek et their development and cultivation perfor-
al., 2009). Aquaculture has turned into mance (Rorrer et al., 2004). Later on, a
extremely important in the salvation of novel tubular PBR with linear Fresnel
food and nutrition for the growing world lenses depends on solar concentrators dis-
covered and studied the correlation be- energy and environment (Muller-Feuga et
tween changes in physiological and pho- al., 2012). Greater attention to several
tochemical characters in central Europe enterprises and businesses has to be the
(Masojidek et al., 2009). Nevertheless, a reality that there is plenty of other value
number of guideline ideologies about the added product opportunities that exist
best possible design of PBRs have been based on algae. The recombinant pharma-
established (refer for recent reviews, Pulz, ceutical protein production began to be
2001; Carvalho et al., 2006). But, still the improved more than 25 years before and
worldwide monoculture of algae is large- today above 300 protein commodities are
ly limited to some unique species, includ- in the market or in late clinical stages
ing extremophiles, easy and quick grow- (Decker and Reski, 2008). Though, a few
ers and this slow growing spp., have to be years back, the moss Physcomitrella pat-
cultivated using PBR systems in order to ens were suggested and merchandised as
certify their control, although the cultiva- substitute production host that convene
tion of subtle spp., needs unusual protec- this concern through providing a special
tion to keep away from forces which amenability for accurate genetic engineer-
could give stress to the cells, centrifugal ing together with economic cultivation
forces, mainly surface and shear stress (Decker and Reski, 2008). However, the
(Masojidek et al., 2009). production cost of algae in large scale is
not yet competitive, predominantly be-
3. A long history of algal use cause of the prevalence of the PBR tech-
nology worldwide. As well the major
Over the past two decades a great progress in classic PBR design, a number
covenant of literature has been focused on of novel configurations have been antici-
the PBR potential of algal commercial pated in the last two decades to advance
applications, due to space limitations we their management expressed in terms of
account a few of them in this chapter. biomass productivity, photosynthetic effi-
Kelps are cost-effectively precious and ciency, light absorption and light to bio-
primary producers, as a result, numerous mass acquiesce.
studies on algal cultivation through breed-
ing have considered enhancing its quality 4. Advantages of PBRs in algal culti-
and productivity (Sato et al., 2017). How- vation and exploring the power of
ever, most cultivation tests have been per- algae
formed in the ocean, thereby limiting the
development of new cultivars. Macroal- The novel advantages of PBR
gae was being eaten at least 1,500 years have been recommended in the last two
ago in Japan and it remains an important decades as a substitute to the open type
food in many cultures where it is valued cultures and they are widely used in food,
for its high mineral content (i.e., Nori and cosmetics and pharmaceutical industries
Laverbread). Closer to home in Europe, to produce algal biomass in large scale. In
kelp was farmed extensively from the 17th another way, the cost of nutrient media
to 19th centuries for processing into soda for algal (cyanobacteria) cultivation is
for the linen industry and into iodine for greatly cheaper than to heterotrophic bac-
medicinal purposes. Micro algae have teria and the inorganic nutrients at low
been used for decades as a food supple- concentration limit the contamination
ment (i.e., spirulina) and as a feedstock through other microorganisms (Chetsu-
for farmed shellfish and finfish. Com- mon et al., 1994). The harvesting cost can
pounds extracted from both micro- and be controlled through the production of
macroalgae today find their way into eve- bioactive substances from the marine al-
ryday foods, health, cosmetics and phar- gae cultivated by PBRs. An evidence of
maceutical industries, agricultural fields, the rising interest in PBR applications is
the large number of research and review consumption (Ngo et al., 2011; Taelman
articles published over the past few years et al., 2015).
showing novel PBR developments
(Olivieri et al., 2014; reviewed in this 5. Extraction technology of bioactive
chapter). An interesting application of compounds from marine algae
micro algal cultures in PBRs concerns the
welfare of space mission teams. A PBR Extraction technology is critical
intended to produce algae protein along for the complete use of marine algae
with oxygen and to remediate devastate (seaweeds and cyanobacteria) raw materi-
and CO2 can form a closed ecological life als to reducing time, with high yields at
support system where it is elemental for low costs and with low consumption of
astronauts concerned in long term inves- solvents (Caamal-Fuentes et al., 2013).
tigation task and on space stations on oth- Natural bioactive composite consists of
er planets like the Moon and Mars an extensive range of functionalities and
(Olivieri et al., 2014). Recently, Cao et al. structures which give an outstanding pool
(2012) patented a method for micro algal of molecules for the assembly of func-
biomass cultivation by 100% CO2 to pro- tional foods, nutraceuticals and food addi-
long the life support system in such a tives. A few of those natural compounds
harsh environment. can be originated in nature at an elevated
Algal products are again being level eg., polyphenols but other com-
commercially explored and developed pounds can only be available at very
with the help of a growing global industry small concentrations, so that enormous
using the latest algal biotechnologies. harvesting is essential to acquire adequate
This involves the mass cultivation of mi- amounts. The structural complexity of the
cro- and macroalgae and conversion of compounds makes chemical synthesis un-
the harvested biomass into a range of val- successful and the intrinsic complications
ue-added products. The EnAlgae project in producing and screening of these com-
aims to develop technologies that will be pounds include led to the improvement of
both economically-viable and environ- advanced technologies (Gil-Chavez et al.,
mentally-friendly ways so that the pro- 2012). Marine algae derived bioactive
duction of algal biomass can be rolled out compound production may be regulated
on industrial scales. Since macroalgae by the assortment of suitable cultivation
generates energy through photosynthesis, conditions and making these algae accu-
algae biomasses are situated in the aquatic rate natural bioreactors (Ibanez et al.,
euphotic zone upper layers. Algal photo- 2012). For extraction of novel compounds
synthetic systems are comparable to that from algae, it is essential to estimate how
of plants growing on terrestrial lands, efficient ingredients are acquired. In this
however, usually, they are highly capable view, there is a need to unite relevant,
of converting sunlight into biomass since cost-effective, choosy and environmental-
less complex cellular structure and their ly friendly extraction methods with the
direct access to water, nutrients and CO2 permitting requirements about the use of
(Kilinc et al., 2013; Taelman et al., 2015). food grade reagents/solvents and progres-
Chetsumon et al. (1994) produced antibi- sions. The application of nature friendly,
otic through the immobilized cyanobacte- advanced and clean extraction methods
rium, Scytonema spp., in a seaweed type allows for the accomplishment of the
PBR. European Union imports of aimed compounds of significance with
macroalgae have conventionally been high efficient extraction techniques,
used as the products for agriculture or in whereas, at the similar moment in time,
the food, pharmaceutical and cosmetic reducing the utilize of organic toxic sol-
industries for their useful extracts and are vents.
very less frequently used for direct human
The Green Chemistry associa- synthesized from marine algae have been
tion has been investigating traditions to industriously illustrated in literature for
ease the risk of harmful chemical contact their biological and physico-chemical
to the environment and humans. Ibanez et characteristics in various models (Caillot
al. (2012) showed a schematic diagram et al., 2012). To give an instance, a linear
which stated about the changes in the -1,3-glucan polymer known as laminarin
waste prevention hierarchy. The innova- derived from brown algae provoked the
tion and progress of marine algae based development of antifungal compounds in
bioactive compounds is a quite new area alfalfa (Medicago sativa) cotyledons
when compared to the invention of bioac- (Kobayashi et al., 1993) and a number of
tive compounds from other global re- defense responses in rice (Oryza sativa)
sources. As a result, in the growth of this (Inui et al., 1997), tobacco (Nicotiana
field, novel, eco-friendly and sustainable tabacum) cell suspension cultures
trends should be followed (reviewed by (Klarzynski et al., 2000), and grapevine
Ibanez et al., 2012). (Vitis vinifera) (Aziz et al., 2003). The
effectiveness of two different algal sac-
6. Application of algal extracts for charides, glucuronan and oligoglucu-
plant growth and development ronans against postharvest gray mold
caused by Botrytis cinerea and blue mold
Worldwide, the policy drivers caused by Penicillium expansum on apple
supporting the application of agricultural fruit, and the associated defense responses
biostimulants derived marine algae in ag- implicated were assessed (Abouraicha et
riculture and are also emphasised in crop al., 2015, 2016). Ulvan (algae derived
management through soil drenches, seed glyco product) activated a large set of re-
priming, hydroponic treatments and foliar lated defense enzymes and accumulated a
sprays (Sharma et al., 2014). The majori- variety of resistance substances induced
ty of the research works associated to the resistance to anthracnose caused by
crop improvement and protection has at- Colletotrichum lindemuthianum in beans
tracted in recent times an extraordinary (Phaseolus vulgaris) (Paulert et al., 2009;
awareness in order to widen safer and de Freitas and Stadnik, 2012), and pro-
new control methods as an alternative of tected from Fusarium wilt in seedlings of
these methods depending on chemical tomato (Lycopersicon esculentum) (El
pesticides. One approach might be the Modafar et al., 2012). Pre-treatment of
generation of the self-defenses in plants wheat (Triticum aestivum) and barley
through natural extracts (Terry and Joyce, (Hordeum vulgare) plants with ulvan ob-
2004; Walters et al., 2005). Marine mi- tained from green macroalgae Ulvan fas-
cro- and macroalgae are the abundant for ciata significantly reduced the symptom
several bioactive compounds, including severity of Blumeria graminis infection
algal polysaccharides, and the composi- (Paulert et al., 2010).
tion and structure of these compounds The application of algae derived
have greatly contributed to their potential agricultural biostimulants on crop plants
on activating signaling pathways and en- produced numerous benefits in the midst
hancing defense mechanisms in a variety of reported effects including enhanced
of plants (El Modafar et al., 2012; Arman seed germination, increased shooting pro-
and Qader, 2012; Abouraicha et al., 2015; liferation, enhanced rooting (Hernandez-
de Freitas and Stadnik, 2015). In the late Herrera et al., 2013; Satish et al., 2015,
1940s, the first seaweed liquid extract for 2016), higher crop and fruit yields, en-
agricultural utilize has been urbanized hanced photosynthetic activity, salinity,
and sold as Maxicrop (Satish et al., 2015). drought and freezing tolerance, and re-
All through the past two decades, -(1,4)- sistance to various bacteria, fungi and vi-
d-polyglucuronic acids (glucuronans) ruses (Sharma et al., 2014). We examined
the various marine macroalgae extracts in spp., through increased chlorophyll con-
bioassays of Solanum trilobatum and El- tent, enhanced nutrient uptake, augmented
eusine coracana seed germination, flower and fruit set leading to elevated
growth assays and in vitro regeneration yields, delayed senescence and longer
including Bacopa monnieri (Satish et al., shelf life of fruits (Briceno-Dominguez et
2015, 2016; Rency et al., 2017). In the al., 2014). In addition, the chemical
past, marine algae extracts have been suc- breakdown of marine algae and their bio-
cessfully affianced as biostimulatns in active compounds has made known the
numerous plants, such as Arabidopsis tha- standpoint of a wide range of substances
liana (Khan et al., 2011) and Nicotiana such as gibberellins, auxins and cytokin-
tobaccum (Sanderson and Jameson, 1986) ins which are stimulating plant shoot and
and Glycine max (Stirk and Van Staden, root growth, maturation and production
1997) for the detection of cytokinin-like (Hernandez-Herrera et al., 2013; Satish et
activity. Marine algae or their substances al., 2015). The supplementation of algae
are applied successfully for the improve- based extracts showed positive response
ment of vegetables like Brassica oleracea to the growth of various vegetables, fruits
(Abetz and Young, 1983), Lactuca sativa and other food crops since micro (Cu, Zn,
(Abetz and Young, 1983; Crouch et al., B, Mn, Co and Mo), macro (Ca, K and P)
1990), Beta vulgaris (Featonby-Smith and nutrients, amino acids, organic acids, vit-
Van Staden, 1983b), Cucumis sativus amins, antioxidants and complex minerals
(Nelson and Van Staden, 1984; Jayara- are available in their extracts (Hernandez-
man et al., 2011), Daucus carota (Jayaraj Herrera et al., 2013; Briceno-Dominguez
et al., 2008), Lycopersicon esculentum et al., 2014; Satish et al., 2015, 2016;
(Featonby-Smith and Van Staden, 1983a; Rency et al., 2017). More or less, around
Finnie and Van Staden, 1985; Kumari et 15 million metric tons of marine plant
al., 2011; Zadope et al., 2011; Basher et products are emerging every year (FAO,
al., 2012; Vinoth et al., 2012, 2014; Her- 2006), a substantial percentage of which
nandez-Herrera et al., 2013; Briceno- distributed for nutrient supplementation
Dominguez et al., 2014), Brassica rapa and as biostimulants or biofertilizers in
(Chinese cabbage) (Sharma et al., 2012), worldwide agriculture. At present, the
Abelmoschus esculentus (Papenfus et al., advanced crop growing is paying atten-
2013) and Brassica napus plants (Ferreira tion for existing biotechnologies where it
and Lourens, 2002). Other than this, puls- would consent for a concession in the ap-
es such as Phaseolus vulgaris (Featonby- plication of chemicals without toxic ef-
Smith and Van Staden, 1984; Beckett et fects on crop growth and yield as well as
al., 1994), Phaseolus acutifolius (Beckett the farmers' earnings (Hernandez-Herrera
el al., 1994), Vigna sinensis (Sivasankari et al., 2013). These biostimulants can be
et al., 2006), Glycine max (Rathore et al., useful as an alternative to herbs and pesti-
2009) and Vigno mungo (Sharma et al., cides, or used in conjunction through syn-
2012; Selvam and Sivakumar, 2013; Bri- thetic/artificial crop protection goods and
ceno-Dominguez et al., 2014), in cereals as plant growth stimulants (Satish et al.,
like Zea mays (Jeannin, 1991), Hordeum 2015, 2016), and they play a role in sus-
vulgare (Steveni et al., 1992) and Triti- taining the crop production levels, high
cum aestivum (Beckett and Van Staden, yield, health and quality (Sharma et al.,
1989; Kumar and Sahoo, 2011), horticul- 2014), and marine algae or their deriva-
tural crops like Fragaria ananassa (Spi- tives are remaining largely unexploited
nelli et al., 2010) and in Malus domestica universally.
(Spinelli et al., 2009) seaweed extracts
applied successfully in different forms. 7. Future prospects
Marine algae extracts elicit a wide
range of responses in the different plants
growth and yield of Triticum aes- tions, operation strategies and appli-
tivum var. Pusa Gold. Journal of cations. Journal of Chemical Tech-
Applied Phycology 23, 251255. nology and Biotechnology 89, 178
Kumari, R., Kaur, I. and Bhatnagar, 195.
A. K. (2011). Effect of aqueous ex- Papenfus, H. B., Kulkarni, M. G.,
tract of Sargassum johnstonii Setch- Stirk, W. A., Finnie, J. F. and Van
ell & Gardner on growth, yield and Staden, J. (2013). Effect of a com-
quality of Lycopersicon esculentum mercial seaweed extract (Kelpak)
Mill. Journal of Applied Phycology and polyamines on nutrient-
23, 623633. deprived (N, P and K) okra seed-
Masojidek, J., Papacek, S., Sergejevo- lings. Scientia Horticulturae 151,
va, M., Jirka, V., Cerveny, J., 142146.
Kunc, J., Korecko, J., Verboviko- Paulert, R., Talamini, V., Cassolato, J.,
va, O., Kopecky, J., Stys, D. and Duarte, M., Noseda, M., Smania,
Torzillo, G. (2003). A closed solar A. J. and Stadnik, M. (2009). Ef-
photobioreactor for cultivation of fects of sulfated polysaccharide and
microalgae under supra-high irradi- alcoholic extracts from green sea-
ance: basic design and performance. weed Ulva fasciata on anthracnose
Journal of Applied Phycology 15, severity and growth of common
239248. bean (Phaseolus vulgaris L.). Jour-
Masojidek, J., Sergejevova, M., Rott- nal of Plant Diseases and Protection
nerova, K., Jirka, V., Korecko, J., 116, 263270.
Kopecky, J., Zatkova, V., Torzillo, Pulz, O. (2001). Photobioreactors: pro-
G. and Stys, D. (2009). A two-stage duction systems for phototrophic
solar photobioreactor for cultivation microorganisms. Applied Microbi-
of microalgae based on solar con- ology and Biotechnology 57, 287
centrators. Journal of Applied Phy- 293.
cology 21, 5563. Rathore, S. S., Chaudhary, D. R.,
Muller-Feuga, A., Lemar, M., Vermel, Boricha, G. N. and Ghosh, A.
E., Pradelles, R., Rimbaud, L. and (2009). Effect of seaweed extract on
Valiorgue, P. (2012). Appraisal of a the growth, yield and nutrient up-
horizontal two-phase flow photobio- take of soybean (Glycine max) un-
reactor for industrial production of der rainfed conditions. South Afri-
delicate microalgae species. Journal can Journal of Botany 75, 351355.
of Applied Phycology 24, 349355. Rency, A. S., Satish, L., Pandian, S.,
Nelson, W. R. and Van Staden, J. Rathinapriya, P. and Ramesh, M.
(1984). The effect of seaweed con- (2017). In vitro propagation and ge-
centrate on growth of nutrient- netic fidelity analysis of alginate-
stressed greenhouse cucumbers. encapsulated Bacopa monnieri
Hortscience 19, 8182. shoot tips using Gracilaria salicor-
Ngo, D. H., Wijesekaraa, I., Voa, T. S., nia extracts. Journal of Applied
Van Taa, Q. and Kima, S. K. Phycology 29, 481494.
(2011). Marine food-derived func- Richmond, A. and Vonshak, A. (1978).
tional ingredients as potential anti- Spirulina culture in Israel. Biologi-
oxidants in the food industry: an cal constraints in algal biotechnolo-
overview. Food Research Interna- gy 11, 274279.
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Olivieri, G., Salatino, P. and Mar- (2004). Bioprocess engineering of
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photobioreactors for intensive mi- seaweeds. Aquacultural Engineering
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Abstract: Environmental degradation is one of the main threats confronting the world and
the widespread use of chemical fertilizers contributes essentially to the degeneration of the
environment through exhaustion of fossil fuels, generation of carbon dioxide (CO2) and
contamination of water bodies. Excessive use of fertilizers has adversely affected agricul-
tural productivity causing soil degradation. Biotransformation through vermicomposting
implies the production of nutrient-rich excreta of worms. After earthworms digest organic
matter, they excrete a high-nutrient product known as Castings. Sustainable development
can be achieved by providing adequate quantity of food for which agricultural land man-
agement is an important aspect. Earthworms are known to consume all types of organic
wastes including vegetable waste, wastes generated by pulse and rice processing industries
and other organic wastes. The food passes through the digestive tract and the worms secrete
chemicals that break down organic matter into sustainable nutrition. Vermicompost is a
peat like material consisting of excellent porosity, structure, aeration and moisture holding
capacity that makes it a good organic manure for growing plants. Another important proper-
ty of vermicompost is its vast surface area that provides strong absorbability and nutrient
retention ability. Vermicompost increases soil fertility, enhance plant growth and suppress
the population of plant pathogens and pests. As a soil conditioner, vermicompost is healthi-
er to traditional compost for its capability to improve and enhance soil configuration and its
water-holding capacity. Thus vermicomposting can be proposed as a cost effective and en-
vironment friendly method for efficient utilization of various organic wastes. This will
promote healthy plant growth and aid in sustainable management of agricultural lands with
cleaner environment.
municipal wastes. The disposal of bio- of the total waste produced in countries. It
degradable solid wastes from domestic, has also been noted that 60% of food pro-
agricultural and industrial sources has cessing industry waste belongs to organic
caused ever-increasing environmental and matter.
economic problems (Garg et al., 2006). Global urbanization has led to in-
Moreover, an additional key threat crease in the volume of solid wastes. Ac-
is the environmental degradation due to cording to a survey conducted in 1990,
the extensive use of chemical fertilizers about 1.3 billion metric tons of municipal
that has led to deterioration of the envi- solid waste was generated globally
ronment through generation of carbon (Beede and Bloom, 1995). In todays sce-
dioxide (CO2), exhaustion of fossil fuels, nario, the generation of solid waste per
and water resources being contaminated. year equals to 1.6 billion metric tons ap-
Agricultural productivity also has taken a proximately. A significant amount of cap-
heavy toll due to disproportionate use of ital is being invested into managing such
fertilizers leading to soil degradation. huge volumes of solid waste suggesting
Recycling of wastes through bio- that solid waste management (SWM) has
transformation of various biodegradable become a large, complex and costly ser-
wastes can reduce the problem of non- vice.
utilization of wastes. Locally available The solid waste and its manage-
organic wastes of anthropogenic nature, ment at various stages are chiefly affected
domestic waste and agricultural lignocel- by the ever growing population. The
lulosic waste products can be used as bio- numbers of households owing to the in-
fertilizer as an alternative to chemical fer- crease in population also has an important
tilizers. Vermicomposting employing role in generation and collection of the
earthworm as decomposers, for degrada- solid waste. Solid waste is mainly collect-
tion and recycling, may be used to en- ed by municipalities and the uncollected
hance the production of crops which are waste, approx. 31% to 49%, is left on
free from pollution and health hazard street or road corners, open spaces like
(Bakthvathsalam and Ramakrishnan, vacant plots that pollute the environment
2004). Preserving the quantity and quality on continuous basis. The collected waste
of soil is one of the main objectives of is usually disposed of within or outside
current efforts to make agriculture more the municipal limits into low lying areas
"sustainable". like ponds etc, without any treatment ex-
cept separation of recyclable waste by
2. Current waste disposal practices & scavengers.
its impact Extensive research around the
world today is being carried out to find a
Currently, waste is a major con- solution for utilization of various agricul-
cern worldwide becoming exceedingly tural residues as energy sources. A simple
significant in developing countries like example is the sugarcane processing in-
India, China as well as in Europe. Waste dustries. Sugarcane bagasse and sugar-
can be classified into industrial, agricul- cane agriculture residues are the two main
tural, sanitary and solid urban residues on residues of sugar and ethanol production
the basis of their origin. There might be a process. Sugarcane bagasse is the fibrous
significant change in their distribution waste that remains after recovery of sugar
depending on the country. Waste generat- juice via crushing and extraction. It is one
ed by various food processing industries of the principal fuels used around the
is a good example of globally generated world in the sugarcane agro-industry be-
waste on a large scale. Waste of this type cause of its well-known energy proper-
has become a great source of concern as ties. However, the bagasse management
in certain cases it is found to be over 50% and disposal practices employed by the
sugar agro-industry have, in most cases, of pesticides, heavy metals, and patho-
remained the same as those used back in gens polluting the soil via poultry wastes
the early 19th century leading to enor- are the central environmental issues at the
mous amount of bagasse being disposed present time.
(Faria et al., 2012). This bagasse can be
an excellent material for biotransfor- 3. Role of chemical fertilizers in envi-
mation by vermicomposting. A similar ronmental degradation
scenario is also found in industries deal-
ing with pulses and grains. The amount of 3.1. Nutrient requirements for plant
waste generated as husk and bran is growth
enormous. Although the husk and bran For the survivability and growth
are being widely used in various industri- of a plant, 16 essential nutrients are re-
al applications like industrial fuel, acti- quired including carbon, hydrogen, oxy-
vated carbon, as pet food fiber, substrate gen, nitrogen, phosphorous, potassium,
for various fermentation processes etc, magnesium, calcium and sulphur, iron,
there is still lot of waste that is left un- zinc, copper, manganese, boron, chlorine
used. and molybdenum. Air, water and sunlight
Consumption of fruits and vegeta- provide the necessary oxygen, carbon,
bles has radically increased in the various hydrogen and energy.
nations by more than 30% during the past
few years. It is also projected that approx- 3.2. Chemical fertilizers a threat
imately 20% of all the fruits and vegeta- Current practices mainly use the
bles produced is lost each year due to chemical fertilizer to accomplish the nu-
spoilage. Also, vegetable markets in vari- trients requirement of a growing plant
ous cities & towns are known to produce which has a serious impact on the soil and
significant amount of non-edible vegeta- water bodies. Excessive utilization of
ble wastes. As mentioned earlier, solid chemical fertilizer leads to increased sa-
waste management of spoilt fruits and linity of soil which is one of the major
vegetables is one of the biggest problems threats causing lower productivity in the
faced today by all the cities including col- soil. Some of the serious impacts of using
lection, transportation and disposal of the chemical fertilizers are:
waste. These wastes are usually discarded Pollution of ground & surface wa-
in the market itself and allowed to rot. ter
However, this discarding leads to produc- Soil fertility is reduced leading to
tion of hazardous ecological impacts reduced food production
(Kumari, 2013). Depletion in the soil microbial
The poultry industry is one of the ecosystem
biggest and fastest growing livestock pro- Ground water pollution and de-
duction systems in the world (Edwards struction of the aquatic life
and Daniel, 1992). As per the reports of Loss of terrestrial and aquatic bio-
Foreign Agricultural Service in 1992, in- diversity
ternationally, approximately 40 million Depletion of the Ozone leading to
metric tons of poultry meat and 600 bil- global warming
lion eggs were produced. Although rea-
sonably flourishing, the poultry industry Exhaustive cropping involving
is at present facing a challenging envi- continuous use of high levels of chemical
ronmental problem. From an agricultural fertilizers (CF) often leads to nutritional
standpoint, the role of poultry wastes in disparity in soil and decline in crop
the contamination of soil and groundwa- productivity (Nambiar, 1994). Numerous
ter, the eutrophication of surface waters properties characterizing the status of soil
by nitrogen and phosphorus, and the fate microbial biomass, activity and nutrient
and valuable organic fertilizer that is rich cess is the conversion of important plant
in humus. nutrients into a more soluble state (Nair et
C6H12O6 + 6O2 6CO2 + 6H2O + 2840kJ al., 2006).
2 Several parameters can be used
Compared to chemical fertilizers, for the evaluation of the vermicomposting
compost is an excellent product that re- process, like, survival of the worms, bio-
tains most of the original nutrients benefi- mass growth, and increase in worm popu-
cial for the increase of soils organic and lation. Since earthworms are known to
nutrient constituents. Application of com- feed on the pathogens present in the waste
post can improve structure and fertility of used, vermicomposting process guaran-
the soil. There are basically four im- tees pathogen removal. Hence, as per
portant parameters that must be consid- USEPA, Vermicomposting has been rec-
ered for the evaluation of quality of com- ognized as Class A stabilization process
post and process performance. They are: (Eastman et al., 2001). Another advantage
volatile solids, respiration rate, germina- of biotransformation of waste through
tion tests and pathogen indicators. vermicomposting is the loss of moisture
to yield a drier product. High concentra-
5. Vermicomposting tion of moisture in the compost can lead
to process failure. In vermicomposting,
Vermicomposting is an ecological the worm burrows act as channels for air
stabilization process involving the break- passage, hence, this can support higher
down of organic waste by the joint action humidity.
of earthworms and mesophilic microor- The main problems encountered
ganisms. Earthworms require an envi- with composting of many organic wastes
ronment conducive for microbial degrada- are their high moisture content, need of
tion and maintenance of biochemical pro- bulking substrate and components unde-
cesses for enhanced microbial decomposi- sirable for worm consumption. As a re-
tion. Various intestinal microflora of sult, composting of raw organic wastes
earthworms are transferred to the compost requires constant monitoring of moisture
matrix along with their gut enzymes that level, composition of the waste, C/N ratio
play an important role (Whiston and Seal, and the composting period. This has been
1988). Furthermore, earthworms are also overcome by the process of pre-digestion
known to augment the microbial activities of organic waste before it can be utilized
by improvement of the environment nec- for vermicomposting. Geetha et al.,
essary for their growth (Syers et al., 1979; (2016) and Nair et al., (2006) have car-
Mulongoy and Bedoret, 1989). Through ried out pre-digestion of the non-edible
the process of vermicomposting, wastes vegetables waste and kitchen wastes re-
are converted to a better homogenized, spectively, in order to achieve a better
nutrient rich and well stabilized product. quality of vermicompost. Pre-digestion
Several studies reveal that vermicompost- prior to vermicomposting was helpful in
ing can be used as an effective technique pH, moisture and waste stabilization. Pre-
for the treatment of wastes rich in patho- digestion was also found to effectively
gens (Eastman et al., 2001). Vermicom- reduce the pathogen load from the ver-
posting gives a better end product than micompost.
composting due to the enzymatic and mi-
crobial activity that takes place during the 5.1. Earthworms
process (Bajsa et al., 2003). Various stud- Earthworms belong to kingdom
ies indicate that vermicomposting can at- Animalia, phylum Annelida, class Clite-
tain harmless pathogen levels which may latta and subclass Oligochaetae. They are
be aided by the microbial and enzymatic known as soil forming organisms inhabit-
activity. An added advantage of this pro- ing almost all part of the earth. They can
be found in different groups of varying excrete. The following steps are involved
size, shape, colouration, life span, feeding in vermicomposting process (Abbasi et
habit and depth in the soil where they are al., 2008):
found. The shape of the worm is cylindri- i. Ingestion and breaking down of the in-
cal with segmented body tapering off at gested organic waste by the action of
both ends. The segments are separated by earthworms gizzard, located next to
fluid-filled compartment that surrounds a the mouth of the worm.
central digestive tract. ii. Digestion of the broken down particles
More than 1800 known species of by the action of enzymes and microbes
earthworm have been found all over the while it passes through the earth-
world (Minnich, 1977) and they can be worms body.
subdivided in three basic groups: iii. Exit of the digested matter as Ver-
Epigeics, Endogeics and Anecics. Alt- micast after few hours of ingestion.
hough there are probably many species of
earthworms available, only a few have Vermicomposting bins (Figure 3)
been utilized for biotransformation of or- are set up using Vermitech pattern
ganic waste processing. The most com- (Geetha et al., 2016). The plastic or ceme-
monly used species of earthworms in-
clude Eisenia foetida (Red wiggler),
Lumbricus rubellus (Red worm), Eisenia
Andrei (Red tiger), Perionyx excavates
(Blue worm), Eudrilus eugeniae (African
night crawler), Enchytraeids (White
worm), Dendroba enaveneta and Peri-
onyx hawayana. Although all the above
mentioned species may be used for ver-
micomposting, Eisenia foetida and Lum-
bricus rubellus are the most commonly
used as it is easy to replicate the suitable
environmental condition for their growth
and regeneration. Among the two species,
Eisenia foetida has been proved best for
vermicomposting of any organic waste
(Edwards and Bater, 1992) because the
growth and reproduction of these worms
is quite rapid. Eisenia foetida, commonly Figure 3: Schematic representation of
known as Red wiggler, is an epigenic design of vermicompost bin (Geetha et
worm that favours living in organic ma- al., 2016).
nure or compost. It has the ability to pro-
cess large quantities of organic matter as -nt bins are placed in a shaded elevated
under ideal conditions; it is known to area on a pedestal of bricks for effective
consume food as much as its body weight water drainage. The basal layer of the
each day. vermibed comprises of broken bricks and
stones above which a layer of sand up to
5.2. Vermicomposting process the height of 10 cm is set up to ensure
The process of vermicomposting proper filtration and drainage. This is fol-
can be carried out either in batch or in lowed by first a 10 cm high layer of cow
continuous modes. The time taken for the dung (3kg) and 5cm high layer of pre-
completion of decomposition process may digested organic waste. A 6~8 cm layer of
vary depending on the time taken by the straw is added to cover the bedding mate-
earthworms to ingest the feed, digest and rial that helps in retaining moisture. Ap-
proximately 60-75 earthworms are inocu- in various enzymes, high quality organics
lated in the composting bins. The ver- (humus) and plant growth regulators.
micomposting units are kept in shade and
covered with a mesh. Water is sprinkled 6. Application of biotransformed waste
to maintain sufficient moisture. For op- for better agricultural productivity
erative collection of wormy wash, a small
hole is drilled near the base of compost- One of the biggest environmental
ing bin and a tube is attached to it. Turn- challenges that the world is facing today
ing of the vermicomposting pile must be is solid waste management. A sustainable
carried out periodically to ensure proper approach towards management of this
aeration. problem may be the use of biotransfor-
mation technique to treat and convert or-
5.3. Advantages of waste management by ganic waste into vermicompost. Compost-
vermicomposting ing is the most cost-effective and ecologi-
Composting is known as an effec- cal option for management of various or-
tive biotransformation technique for treat- ganic wastes since it is easily operable
ing organic wastes, following natures and can be carried out in a small scale
way of recycling. Vermicomposting level with proper management of the pro-
requires very less resources like water, cess to obtain a good quality product. A
energy and land/space required for treat- list of various wastes converted to ver-
ment of per unit of bio-waste as compared micompost is given in Table 1. Although,
to aerobic composting. It is a rapid, cost husk is abundantly available as lignocel-
effective and sustainable alternative for lulosic waste, the problem with using
transformation of organic waste, carried husks as vermicomposting feedstock is its
out by earthworms, leading to formation high initial carbon (lignin and cellulose)
of a product rich in plant nutrients and contents, which impedes the composting
humic acids. The compost thus generated process (Kumar et al., 2013). Hence, the
has the ability to hold nutrients for a substrate requires amendments to achieve
longer period without unfavorably im- an optimum range of C/N ratio to attain
pacting the environment. No curing is optimal process efficiency (Goyal et al.,
necessary for vermicompost as it is highly 2005). This can be achieved with the help
rich in beneficial microorganisms. The of pre-digestion of husks along with other
overall time required for processing of organic wastes (such as non-edible vege-
waste is therefore reduced to a great ex- table waste) as amendment, prior to ver-
tent, resulting in non-toxic by-products. micomposting. This will help in conver-
Gardeners, all over the world, highly pre- sion of husk into biofertilizer within few
fer the application of vermicompost over months.
chemical fertilizers; hence, vermicompost Integrated use of fly ash along
is finding a significant market value with organic wastes has been effectively
(Sudhakar et al., 2002). used in increasing the yield of crops when
Application of vermicompost has compared to continuous use of chemical
been proved to improve the texture, struc- fertilizers alone (Rautaray et al., 2003).
ture, aeration, fertility and water holding This may be due to beneficial effects on
capacity of soil. This way most of the rice and residual effects on mustard. Ac-
valuable nutrients that are taken out of the cording to Rautaray et al. (2003) greater
soil during crop cultivation are replen- crop yield was related to higher uptake of
ished. Also, vermicompost addition is nutrient. Further to the yield advantage, a
known to enhance plant-root develop- better soil chemical properties were also
ments that help in control of soil erosion. noted namely pH, organic carbon and
Vermicompost are also known to be rich available N, P and K as compared to the
soils where chemical fertilizers were con-
tinuously used. When fly ash was used in ability of macro and micronutrients from
combination with organic waste during vermicompost and wormy wash (Erich et
vermicomposting, the end product ob- al., 2002). Vermicompost and wormy
tained gave better plant growth than with wash augments crop growth and yield
chemical fertilizers alone. when added to soil (Lalitha et al., 2000).
Vigna radiata (Green gram) when According to the studies conducted by
grown in soil amended with a combina- Atiyeh et al. (2001), and Suthar (2009),
tion of vermicompost and wormy wash addition of vermicompost in bedding me-
were found to develop nodes and new dia enhanced seed germination and
leaves at a significantly higher rate when growth leading to overall increase in plant
compared to control (Geetha et al., 2016). productivity.
This might be due to increase in bioavail-
Abstract: Plant health and development promoted through microbial interactions have been
the main motif for sustainable agriculture. To find new and beneficial endophytic microor-
ganisms from plants of different ecosystems is highly considerable because endophytic bac-
teria are not restricted to a specific species but showed a wide range of host diversity. En-
dophytes colonize an ecological niche similar to that of phytopathogens and baffle disease
development through endophyte-mediated de novo synthesis of novel compounds and anti-
fungal metabolites. Seedling emergence, plant growth and plants establishment under ad-
verse conditions can be accelerated by endophytes. Endophytic Pseudomonas sp. and Bacil-
lus sp. recorded a significant improvement in morphological characters and ISR in the
plantlets. Endophyte fortifies plant cell wall strength, alters host physiology and metabolic
responses thereby enhance the defense mechanism by the synthesis of different metabo-
lites such as phenolic compounds, pathogenicity related protein (PR-1, PR-2, PR-5), oxida-
tive stress enzymes (chitinases, peroxidases, polyphenyoxidase, phenyl alanine ammonia
lyase, Oxidase and/or chalcone synthase, phytoalexins etc. The contribution of endophytes
as biological fertilizers is highly significant because of their metabolic acclimatization in
the host plant with mutualism. Endophytic microbes must be properly selected, combined
and formulated with respect to the environmental conditions for development of efficient
endophytic biofertilizers that can very much contribute to the enhanced food production in
the world. This review aims to provide an overview on bacterial endophytes and their po-
tential application for sustainable agriculture.
(Figure 1) and often phyllosphere bacteria Siciliano, 2001; Zinniel et al., 2002; Ses-
may also be a source of endophytes sitsch et al., 2002; Dent et al., 2004; Sun
(Hallmann et al., 1997; Germaine et al., et al., 2008). More than 200 bacterial
2004). Though each individual plant ex- genera from 16 phyla have been reported
ists on the earth is a host to one or more as endophytes since the first report of en-
endophytes (Strobel et al., 2004), only a dophytic bacteria (Samish et al., 1963a)
few of these plants have ever been com- and include both culturable and uncul-
pletely studied to their endophytic biolo- turable bacteria (Sun et al., 2008; Berg
gy and to find novel beneficial endophytic and Hallmann, 2006; Mengoni et al.,
microorganisms. 2009; Manter et al., 2010; Sessitsch et al.,
2012). Most predominantly studied endo-
2. Endophytic bacterial diversity in the phytes belong to three major phyla (Ac-
host plants tinobacteria, Proteobacteria and Firmicu-
tes) and include members of Streptomyces
Endophytic bacteria were isolated (Suzuki et al., 2005), Azoarcus (Krause et
from both monocotyledonous and dicoty- al., 2006), Acetobacter (renamed as Glu-
ledonous plants (Table 1) ranging from conobacter) (Bertalan et al., 2009), Pseu-
woody trees to herbaceous crops such as domonas, Serratia, Stenotrophomonas
prairie plants, agronomic crops, tuberous (Ryan et al., 2009), Enterobacter (Ped-
crops and grasses (McInroy and rosa et al., 2011). Bacteria which are
Kloepper, 1995; Gutirrez-Zamora and ubiquitous in the soil/ rhizosphere repre-
Martnez-Romero, 2001; Germida and sent the main source of endophytic
Table 1: Continued
Grapevine (Vitis spp.) Bacillus, Clavibacter, Coma- Bell et al., 1995a; 1995b
monas, Curtobacterium, Enter-
obacter, Klebsiella, Moraxella,
Pantoea, Pseudomonas, Rah-
nella, Rhodococcus, Staphylo-
coccus,
Xanthomonas
Soybean Enterobacter sakazakii Kuklinsky-Sobral et al.,
Enterobacter agglomerans 2004
Erwinia sp.
Klebsiella oxytoca
Klebsiella pneumoniae
Pseudomonas citronellolis
Maize Burkholderia pickettii McInroy and Kloepper,
Enterobacter spp. 1995
Arthrobacter globiformis Chelius and Triplett, 2000a
Microbacterium testaceum Zinniel et al., 2002
Citrus plants Bacillus spp. Araujo et al., 2001, 2002
Curtobacterium flaccumfaciens
Nocardia sp.
Methylobacterium mesophili-
cum
Carrot Pseudomonas putida Surette et al., 2003
Pseudomonas fluorescens
Staphylococcus saprophyticus
Klebsiella terrigena
Sugar cane (Saccharum Herbaspirillum rubrisulbalbi- Olivares et al., 1996
officinarum L.) cans, Acetobacter, Herbaspiril- Cavalcante & Dbereiner,
lum 1988;
Gillis et al., 1989;
Boddey et al., 1991;
Dong et al., 1994;
Ohvares et al., 1997
Corn (Zea mays L.) Bacillus, Burkholderia, Lalande et al., 1989;
Corynebacterium, Enterobac- Fisher et al., 1992;
ter, Klebsiella, Pseudomonas Mclnroy & Kloepper, 1995;
Palus et al., 1996
Wheat Streptomyces Coombs and Franco, 2003a
Alfalfa (Medicago sativa Erwinia-likc, Pseudomonas Gagne et al., 1987
L.)
Coffee (Coffea arabica Acetobacter Jimenez-Salgado et al.,
L.); Cameroon grass 1997; Reis et al., 1994
(Pennisetum purpureum
Schumach)
Cotton (Gossypium hirsu- Agrobacterium, Bacillus, Misaghi & Donndelinger,
tum L.) Burkholderia, Clavibacter 1990;
Erwinia, Serratia, Mclnroy & Kloepper,
Xanthomonas 1995
Cucumber (Cucumis sa- Agrobacterium, Arthrobacter, Mclnroy & Kloepper,
tivis L.) Bacillus, Burkholderia, 1995
Table 1: Continued
Chryseobacterium, Enterobac-
ter,
Pseudomonas, Stenotrophomo-
nas
Hybrid spruce {Picea Bacillus, Pseudomonas, O'Neill et al., 1992;
glauca x Engelmannii) Phyllobacterium, actinomy- Chanway et al., 1994
cetes,
Staphylococcus
Kallar grass (Leptochloa Azoarcus Reinhold et al., 1986;
fusca [L.] Kunth) root Reinhold-Hurek et al.,
1993
Lodgepole pine (Pinus Bacillus Shishido et al., 1995
contorta Dougl. Ex Loud)
root
Sorghum bicolor L. Herbaspirillum James et al., 1997
Moench shoot
Sugar beet (Beta vulgaris Bacillus, Corynebacterium, Er- Jacobs et al., 1985
L.) winia,
Lactobacillus; Pseudomonas,
Xanthomonas
Teosinte (Zea luxurians Klebsiella Palus et al., 1996
Itins
and Doebley) stem
Banana Azospirillum brasilense Weber et al., 1999
Citrobacter sp. Martnez et al., 2003
Marigold Kocuria varians Sturz and Kimpinski, 2004
Microbacterium esteraromati-
cum
Banana, pineapple Azospirillum amazonense Weber et al., 1999
Sugarcane, coffee Gluconacetobacter diazo- Cavalcante and Dbereiner,
trophicus 1988; Jimnez-Salgado et
al., 1997
Scots pine, citrus plants Methylobacterium extorquens Araujo et al., 2002; Pirttil
et al., 2004
Carrot, rice Rhizobium (Agrobacterium) Surette et al., 2003
radiobacter
Kallar grass, rice Azoarcus sp. Reinhold-Hurek et al., 1993,
Engelhard et al., 2000;
Yellow lupine, citrus Burkholderia cepacia Araujo et al., 2001; Barac et
plants al., 2004
Banana, pineapple, rice Burkholderia sp. Araujo et al., 2001; Barac et
al., 2004
Sugarcane, rice, maize, Herbaspirillum seropedicae Olivares et al., 1996; Weber
sorghum, banana et al., 1999
Banana, rice, maize, sug- Klebsiella variicola Rosenblueth et al., 2004.
arcane
Citrus plants, sweet pota- Pantoea agglomerans Araujo et al., 2001, 2002;
to Asis and Adachi 2003
Rice, soybean Pantoea sp. Kuklinsky-Sobral et al.,
2004; Verma et al., 2004
Table 1: Continued
Marigold (Tagetes spp.), Pseudomonas chlororaphis Sturz and Kimpinski, 2004;
carrot Surette et al., 2003
Alfalfa, carrot, radish, Salmonella enterica Cooley et al., 2003; Guo et
tomato al., 2002; Islam et al., 2004
Dune grasses (Ammophila Stenotrophomonas Dalton et al., 2004
arenaria and
Elymus mollis)
Maize, carrot, citrus Bacillus megaterium Araujo et al., 2001; McInroy
plants and Kloepper, 1995; Surette
et al., 2003
Grass Miscanthus sinen- Clostridium Miyamoto et al., 2004
sis
Wheat, Scots pine Mycobacterium sp. Conn and Franco 2004; Prit-
til et al., 2005
Citrus plants, maize Enterobacter cloacae Araujo et al. 2002; Hinton et
al., 1995
Lettuce Escherichia coli Ingham et al., 2005
Wheat, sweet potato, rice Klebsiella sp. Engelhard et al., 2000;
Iniguez et al., 2004;
colonizers (Hallmann and Berg, 2006). et al., (2008) isolated 77 endophytic bac-
Earlier studies reported that endophytic teria from roots, stems and leaves of So-
population of Bacillus polymyxa inside lanum nigrum grown in two different na-
the root is highly specific and less diverse tive habitats of Jena and Germany. Ara-
than the root surface population, though vind et al., (2009) isolated 80 endophytic
there are different populations of Bacillus bacteria from different varieties of Piper
polymyxa in soil, rhizosphere, and rhi- nigrum L. cultivated in different regions
zoplane of wheat field. It clearly indicates of India. Bhore and Sathisha (2010) iso-
that endophytes appear to be originated lated 115 putative cultivable endophytic
from rhizosphere or rhizoplane (Mavingui bacteria from leaves of 72 different plant
et al., 1992; Germida et al., 1998). species collected from northern part of
Endophytic genera isolated from Peninsular Malaysia. Magnani et al.,
the interior of ginseng roots cultivated in (2010) isolated 32 endophytic bacteria
three different areas showed marked dif- from Brazilian sugarcane. A total of 264
ferences in microbial community (Cho et colonies of endophytic bacteria were re-
al. 2007). Ryan et al., (2008) reported ported from leaves and roots of young
that endophytic bacteria in a single plant radish (Seo et al., 2010).
host not restricted to a single species but Pereira et al., (2011) investigated
comprise several genera and species. En- endophytic bacterial diversity associated
dophytic bacteria belong to one genera with the roots of maize through culture-
was not only restricted a specific host but dependent and culture-independent meth-
also showed wide range of host diversity. ods. Enterobacter, Erwinia, Klebsiella,
Bacteria belonging to the genera Bacillus Pseudomonas, and Stenotrophomonas
and Pseudomonas were identified as pre- genera belong to -Proteobacteria were
dominantly occurring endophytes (Segh- reported as predominant group. Based on
ers et al., 2004). culturable component of the bacterial
Jha and Kumar (2007) isolated community genus Bacillus belong to Fir-
and characterized 10 endophytic diazo- micutes was identified as another pre-
trophic bacteria from surface-sterilized dominant group, while Achromobacter,
roots and culm of Typha australis. Long Lysinibacillus, and Paenibacillus genera
were rarely found in association with the Internal colonization of plant tissues
roots. by bacteria is primarily intercellular and
Yang et al. (2011) isolated 45 xylem vessels act as reservoirs of internal
strains from stems and 27 strains from populations of bacteria (Gardner et al.,
leaves of tomato as endophytic bacteria 1982; Jacobs et al., 1985; Sumner, 1990;
and reported that endophytic efficiency of Frommel et al., 1991; Kloepper et al.,
bacteria in stem was higher than leaves. 1992; Bell et al., 1995; Sprent and James
Out of 72 endophytic bacteria isolated 1995; Reinhold-Hurek and Hurek 1998a).
from tomato on of the W4 was identified Intracellular endophytic bacteria have al-
as Brevibacillus brevis W4 on the bases so been found in the cytoplasm and vacu-
of 16S rDNA gene analysis and Biolog oles of epidermal cells (Quadt - Hallman
systemic analysis. Patel et al. (2012) iso- and Kloepper, 1996), root hairs (Vance,
lated and characterized bacterial endo- 1983), and parenchyma cells (Jacobs et
phytes from root and stem of Lycopersi- al., 1985).
con esculentum. Out of 18 endophytic
bacteria selected from tomato only one 3. Endophytes in agriculture
isolate HR7 was identified as Pseudomo-
nas aeruginosa by 16S rDNA analysis. The endophyte-plant interaction is one
Fisher et al. (1992) found that endo- of the least studied biochemical systems
phytic bacteria appear to be preferentially in nature. Plants host to one or more en-
located in the lower part of the stems of dophytic microorganisms include fungi,
corn, with a declining gradient running bacteria and actinomycetes. Endophytes
from the base to the top of the plant. reside in the tissues beneath the epidermal
Roots and other below ground tissues cell layers (Stone et al., 2000) are transi-
tend to yield the highest numbers of CFU ently symptomless and inconspicuous
of bacteria compared with above-ground with several beneficial effects on plants
tissues is an indicative of the upward path (Hallmann et al., 1997). More than thou-
of bacteria (Table 2) from the roots into sand endophytic bacteria were reported in
the stem during plant development the last decade (Table 3). Like plant
(McInroy and Kloepper, 1994; Sturz et growth promoting Rhizobacteria (PGPR),
al., 1997a; Rosenblueth and Martnez-
Romero, 2004; Gagn et al. (1987).
isolated from different parts of chilli pro- Pseudomonas spp. Isolated from Serbia
duced 23g/ml, 21g/ml, 111.5g/ml able to solubilize TCP (Stajkovi et al.,
and 125g/ml of IAA, respectively. It 2011; Djuric et al., 2011; Josic et al.,
was much higher than that of found in 2012b). Amrutha et al. (2012) reported
other reports (Long et al. 2008). that Pseudomonas aureginosa CEFR3
Harish et al., (2008) assessed the (198ppm/ml), Bacillus sp CEFR19
plant growth promotion efficacy of 45 (1354ppm/ml) isolated from ripened fruit
endophytic bacteria isolated from corm and isolated from green fruit and Bacil-
and root of banana. 12 strains isolated lus cereus AVSCE 5(137ppm/ml) isolat-
from gingseng plant, endophytic P. fluo- ed from leaf of chilli are able to solubil-
rescens WCS365 as biocontrol s bacteria ize inorganic phosphate efficiently. In-
isolated from Lycopersicon esculentum crease in the yield of canola by endophyt-
produced significant amounts of IAA ic Bacillus sp. was reported by de Freitas
(Thamizh Vendan et al., 2010, Patel et et al. (1997). Sundara et al. (2002) report-
al., 2012). Three strains isolated from ed that enhancement in available phos-
sugar beet roots produced indole-3-acetic phorus and yield of sugarcane by applica-
acid (IAA) promote plant growth signifi- tion endophytic bacteria. Pseudomonas
cantly increased plant height, fresh and spp. are able to increase the growth and
dry weights and number of leaves per phosphorus content of maize by endo-
plant (Long et al., 2008; Yingwu Shi et phytes (Vyas and Gulatti, 2009).
al., 2009). Vetrivelkalai et al., 2010 also
reported significant enhancement in the 4.3. Siderophore
germination percentage, shoot and root Researchers reported that endophytic
length and vigour index of bhendi seed- fungal siderophore have lower affinity
lings by seed bacterization. than bacteria to sequester iron and deprive
pathogenic fungi (Whipps, 2001; Loper
4.2. Phosphate solubilization and Henkels 1999). Endophytic bacterial
Earlier reports revealed endophyt- isolates associated with hyacinth and
ic bacteria also solubilize phosphate from Genseng plants produce siderophore
organic or inorganic bound phosphates (Jafra et al., 2009; Thamizh Vendan et
and type of organic acid produced during al., 2010). Rajkumar et al., (2010) report-
phosphate solubilizaton depends on the ed that the siderophore play a pivotal role
carbon source utilized as substrate. High- in nitrogen fixation under iron deficiency.
est P solubilization has been observed
when glucose, sucrose or galactose has 4.4. Nitrogen fixation
been used as sole carbon source in the According to earlier studies en-
medium (Khan et al., 2009; Park et al., dophytic bacteria better express their ni-
2010). Endophytic bacteria able to solu- trogen fixation potential inside plant tis-
bilize inorganic phosphate and extracel- sues due to the lower competition for nu-
lular tricalcium phosphate effectively in trients and protection against high levels
presence of glucose (Kuklinsky - Sobral of O2 present on the root surface. Many
et al., 2004; Long et al., 2008; Thamizh diazotrophic bacteria are able to establish
Vendan et al., 2010; Patel et al., 2012). a symbiotic relationship with plants for
Endophytic Bacillus, Pseudomonas, biological nitrogen fixation (Robson et
Klebsiella and Acinetobacter are also re- al., 1986; Chisnell et al., 1988; Dekas et
ported as potential phosphate solubilizers al., 2009). Earlier studies reveled that en-
(Huang et al., 2010). Endophytic Bacillus dophytic diazotrophs constitute only a
cereus and B. megaterium isolated of small proportion of total endophytic bac-
Ginseng plant also showed significantly teria include Azospirillum lipoferum,
high P solubilization (ThamizhVendan et Klebsiella pnemoniae and Azorhizobium
al., 2010). Endophytic and rhizosphere caulinadans (Schloter et al., 1994; Bar-
raquio et al,. 1997; Martnez et al., 2003). and studied growth response of A. thali-
Unlike symbiotic diazotrophes, endophyt- ana. Each strain showed significant vola-
ic bacteria are unable to form nodules. tile-mediated plant growth modulation
Gluconacetobacter diazotrophicus was and also reported that Burkholderia pyr-
identified as first N2-fixing endophytic rocinia as significant plant growth-
bacteria associated with sugarcane stem promoter. The volatiles indole, 1-hexanol
(Cavalcante and Dobereiner, 1988) and and pentadecane promotes growth only
confirmed by other scientists in USA, under stress conditions
UK, and Germany and two more N2-
fixing endophytes Herbaspirillum sero- 5. Endophytes as biological control
pedicae and H. rubrisubalbicans were agents (BCA)
reported by Boddey et al., (1995). James
(2000) reported Herbaspirillum sp. as en- Endophytes are potential biocon-
dophytic diazotroph in sugarcane and trol agents like other biocontrol agents
rice. Azoarcus sp. in rice and Kallar grass such as associative nitrogen fixing PGPB
and endophytic Klebsiella sp. Kp342 on sugarcane (Boddy, 2003) or Burkhold-
strain of wheat identified as nitrogen fix- eria phytofirmans PsJN, non-symbiotic
ers (Iniguez et al., 2004). Silva-Froufe endophytic bacteria (Sharma and Nowak,
(2009) reported Glucanoacetobacter dia- 1998) endophyte holds potential of BCA
zotrophicus as endophytic diazotrophic may be due to self-perpetuating nature of
bacteria in sugarcane, sweet potato, and endophytes inside the host by coloniza-
pineapple. Endophytic Bacillus species of tion and being transfer to progeny (Bod-
soybean nodule showed potential N fixing dy, 2003). According to Backman et al.
and reported to improve root growth and (1997), the effectiveness of endophytes as
function, (Bai et al., 2002; Asis et al., biological control agents (BCA) is de-
2004; Matiru and Dakora, 2004). 23 en- pendent on many factors. These factors
dophytic bacteria are identified as poten- include: host specificity, the population
tial ammonia producers in chilli (Amrutha dynamics, pattern of host colonization,
et al., 2012) and reported that Pseudomo- ability to move within host tissues and the
nas, Curtobacterium and Bacillus sp iso- ability to induce systemic resistance. Cer-
lated from chilli were found to be maxi- tain endophytic bacteria trigger induced
mum producers of Ammonia. systemic resistance (ISR) which is pheno-
typically similar to systemic-acquired re-
4.5. Volatile compounds sistance (SAR). SAR develops when
Earlier literature reveled that en- plants successfully activate their defense
dophytic bacteria can produce a wide mechanism in response to primary infec-
range of volatiles. Biological function of tion by a pathogen and induces a hyper-
most of these volatiles is not yet under- sensitive reaction in the form of a local
stood. It is assumed that volatile com- necrotic lesion of brown desiccated tissue
pounds involved in a number of processes (van Loon et al., 1998). ISR is effective
including cell-cell signaling, inter-species against different types of pathogens bu
signaling, promote plant growth and act differs from SAR because in ISR the in-
as microbial inhibiting agents (Wheatley, ducing bacterium does not cause visible
2002; Vesperman et al., 2007; Kai et al., symptoms on the host plant (van Loon et
2009). Ryu et al., (2003) reported that al., 1998).
Bacillus sp. produce 2, 3 butanediol and The first record of an endophyte
acetoin and promote plant growth in Ara- affecting a plant disease was reported by
bidopsis thaliana. 38 volatile compounds Shimanuki (1987) who showed that Phle-
were reported from rhizobacteria (Farag um pratense plants infected with the
et al., 2006). Blom et al. (2011) screened Epichloe typhina were resistant to the
42 strains in four different growth media fungus Cladosporium phlei. In some cas-
es, endophytes also accelerate seedling Serratia marcescens 90-166 reduced Cu-
emergence and promote plant establish- cumber Mosaic Virus (CaMV) in toma-
ment under adverse conditions and en- toes and cucumbers (Raupach et al.,
hance plant growth and development (Pil- 1996), anthracnose and Fusarium wilt in
lay and Nowak, 1997). Several endophyt- cucumber (Liu et al., 1995).
ic bacterial species including Achromo- Sixty one (61) endophytic bacteria
bacter sp., Acinetobacter baumannii, A. isolated from potato stem tissues were
lwoffii, Alcaligenes, Moraxella sp., Alcal- identified as effective biocontrol agents
igenes sp., Arthrobacter sp., Bacillus sp., against Clavibacter michiganensis subsp.
Burkholderia cepacia, Citrobacter freun- Sepedonicus (Sturz et al., 1999). Bacillus
dii, Corynebacterium sp., Curtobacterium mycoides BacJ and Bacillus pumilis 203-7
flaccumfaciens, Enterobacter cloacae, E. isolates from different host plants sup-
aerogenes, Methylobacterium extorquens, pressed Cercospora leaf spot in sugar
Pantoea agglomerans, Pseudomonas ae- beet (Bargabus et al., 2002: 2004). Araujo
ruginosa, and Pseudomonas sp. isolated et al. (2002) reported Curtobacterium
from the xylem of lemon roots (Citrus flaccumfaciens, citrus endophyte help
jambhiri) have been reported as antago- citrus plants to better resist against the
nistic bacteria against root phytopatho- pathogenic infection of Xylella fastidi-
gens (Arajo et al., 2001; Lima et al., osa. Endophytes isolated from potato
1994). plants produce antibiotics and siderophore
Cabbage treated with endophytes and showed antagonismic against fungal
did not reach the economic threshold for pathogens (Berg et al., 2005a; Sessitsch
the disease approximately 50 days after et al., 2004) and bacterial pathogens Er-
inoculation with Xanthomonas campestris winia and Xanthomonas (Sessitsch et al.,
pv. Campestris due to the induction of 2004). Of 2,648 bacterial isolates from
defense mechanisms (Jetiyanon, 1994). the rhizosphere, phyllosphere, en-
Several bacterial endophytes have been dosphere, and endorhiza, only Serratia
reported to support growth and improve plymuthica root endophyte was a highly
the health of plants (Hallmann et al., effective against Xanthomonas sp. in
1997). Erwinia caratovora, for example, Brassica seeds (Berg et al., 2005b).
is inhibited by numerous endophytic bac- Endophytic actinobacteria also
teria, including several strains of Pseu- show effective antagonism against the
domonas sp., Curtobacterium luteum, and pathogenic fungus Gaeumannomyces
Pantoea agglomerans (Sturz et al., 1999). graminis of wheat (Coombs et al. 2004)..
Wilhelm et al. (1997) demonstrat- A number of endophytic actinobacteria
ed that Bacillus subtilis strains isolated isolated by culture dependent methods
chestnut trees exhibit antifungal effects belong to the genera of Streptomyces, Mi-
against Cryphonectria parasitica causing crobispora, Micromonospora, and No-
chestnut blight. The ability of endophytic cardioides (Coombs and Franco, 2003a)
bacteria to inhibit pathogen has been de- were capable of suppressing Rizoctonia
creased in potato tubers due to deep colo- solani, Pythium spp., and Gaeumannomy-
nization interior to the plant host (Struz et ces graminis var tritici, fungal pathogens
al., 1999). Barka et al. (2002) demon- of wheat in vitro and in planta, (Coombs
strated the ability endophytes to colonize et al., 2004).
in divergent hosts. Pseudomonas sp. an Aravind et al. (2009) reported against.
onion endophyte colonized in grape vine Native endophytes IISRBP 35, IISRBP 25
inhibited Botrytis cinerea Pers. and pro- and IISRBP 17 isolated from black pep-
moted growth in grapevines. Coloniza- per exhibited 70% disease suppression of
tion of multiple hosts has also been ob- Phytophthora capsici in black pepper in
served with other species of endophytes greenhouse trials. The intimate relation-
such as Pseudomonas putida 89B-27 and ship between endophytic bacteria and
their hosts make the endophytes as natural ammonia lyase, , oxidase and/or chalcone
candidates for selection as biocontrol synthase) and phytoalexins to protect the
agents with high level of competition host plant from future infections (Nowak
(Chen et al., 1995; Van Buren et al., and Shulaev, 2003). Elicitors include lip-
1993). opolysaccharides, flagella, siderophore,
Twenty two (22) endophytic bac- antibiotics, VOCs or quorum-sensing sig-
teria isolated from different parts of chilli nals produced by endophytic bacteria
plant showed antagonism against Colle- elicit ISR in plants (Van Loon, 2007).
totrichum capsici, C. gloeosporioides and ISR activated by PGPB is mediated by
C. acutatum (Amrutha et al., 2014). An- jasmonate or ethylene in majority of
tagonism of endophytic bacteria against plants. Thickening of the outer tangential
Clavibacter michiganensis subsp. and outermost part of the radial side of
sepedonicum cause rot on tomato(Van the first layer of cortical cell walls was
Buren et al., 1993), Pseudomonas chlo- also noticed in tomato roots with the
roraphis, P. fluorescens, P. graminis, P. treatment of endophytic P. fluoresces
putida, P. tolaasii and P. veronii agaist WCS417 (Duijff et al.,1997).
bacterial pathogens (Chen et al., 1995; Application of endophytic bacteria
Adhikari et al., 2001) Invitro inhibition of by stem injection in cotton plants reduced
various fungal pathogens by Bacillus sub- the root rot caused by Rhizoctonia solani
tilis ME488nd (Chung et al., 2008) hace and vascular wilt caused by F.oxysporum
also been reported. f. sp. vasinfectum (Chen et al., 1995).
Pleban et al. (1995) reported that endo-
6. Induced systemic resistance (ISR) phytic bacteria move upward and down-
ward from the point of application before
Endophytes have a natural and in- colonizing the internal tissues and check
timate association with plants because the entry of F. solani in cotton and Scle-
the internal tissues of plant provide rela- rotium rolfsii in beans. Application of
tively uniform and protected environment Pseudomonas florescence prevented the
and favors endophytic bacteria to be a entry Pythium ultimum in the roots of pea,
potential agent of ISR (Chen et al. 1995). P. florescence restrict the growth of
Viswanathan (1999) reported that applica- Fusarium oxysporum f. sp. pisi and
tion of endophytic strain is more benefi- P.ultimum in pea plant (Benhamou et al.,
cial in vegetatively propagated crops like 1996b, 1998 ). Pseudomonas strain 63-28
banana, sugarcane and tapioca for induc- also induced resistant in tomato against F.
ing systemic resistance and also reported oxysporum f. sp. radicislycopersici
that endophytic P.fluorescens strain EP1 (MPiga et al., 1997). Inoculation of en-
isolated from stalk tissues of sugarcane dophytic Pseudomonas denitrificans
induced systemic resistance against red strain 1-15 and P.putida strain 5-48 pro-
rot caused by Colletotrichum falcatum tected the oak tree against Ceratocystis
(Viswananthan and Samiyappan ,1999a). fagacearum (Brooks et al., 1994) .Recent
van Loon (2007) reported that the phe- reports have been reviewed mechanisms
nomenon of ISR has been noted to be ex- of ISR induced by endophytic Psuedomo-
hibited both associative and endophytic nas (Jankiewicz and Koltonowicz, 2012).
bacteria. ISR triggered by endophytes for- Bacillus cereus AR156 in A.thaliana
tifies plant cell wall strength, alters host (Niu et al., 2011) and different endophyt-
physiology and metabolic responses and ic bacillus sp. from vegetable crops in-
enhance synthesis of plant defense chem- duced systemic resistance . Combination
icals such as phenolic compounds, patho- of three endophytic isolates resulted in
genicity related protein (PR-1, PR-2, PR- significant growth promotion and ISR in
5), ROS enzymes (chitinases, peroxidas- tomato, bell pepper, cucumber and tobac-
es, polyphenyoxidase, phenyl alanine co (Kloepper et al., 2004). Mixed formu-
lation of B. subtilis GB03, B. amylolique- principle behind the plant and endophytic
faciens IN937a and B. subtilis IN937b interaction to elicit ISR or plant growth
together with chitosan in tomato and dif- promotion and development of endophyt-
ferent endophytic bacillus sp. isolated ic bioformulations will be an index of
from vegetable crops induced systemic growing scientific knowledge in agricul-
resistance in Theobroma cacao seedlings. ture and horticulture.
Harish et al. (2008) reported mixed for-
mulation of rhizobacterial and endophytic 8. Concluding remarks
bacterial (EPB5 + EPB22 + Pf1 + CHA0)
was significantly effective in suppressing According to the research con-
Banana Bunchy top virus under field con- ducted so far, the use of chemical fertili-
ditions. zation is necessary because biological fer-
tilization has not yet proven to be good
7. Perspectives enough in providing complete plant nutri-
ent requirements. It has been indicated
Microorganisms with phytopatho- that about less than 50% of chemical fer-
genic antagonism act as Biocontrol agents tilizers is absorbed by plant and the rest
(BCA). Most of the biocontrol agents would not be accessible by plant as it is
have not fulfilled their initial promise be- subjected to leaching, runoff, and emis-
cause of poor rhizosphere competence. sion from the soil surface. Hence, the use
The failure of BCA being attributed the of biological fertilizers as supplementary
difficulties in long-term culture. It would fertilization to chemical fertilization is
obviate the need for selecting bacterial necessary with the above mentioned ad-
types with high levels of rhizosphere vantages. Accordingly, the right and
competence and successful seed or root proper application of chemical and bio-
bacterization treatments before or at logical fertilization is very much depend-
planting (Schroth et al., 1984; Weller, ent on realizing the interactions between
1988). The intimate relationship between soil, plant and microorganisms. Endo-
endophytic bacteria and their hosts make phytic bacteria a big help to plant and the
the endophytes as natural candidates as environment as they own great abilities
biocontrol agents with high level of com- that collectively enhance plant growth
petence (Chen et al., 1995; Van Buren et and ISR. Among such abilities, enhanced
al., 1993). nutrient uptake by plant is also of great
Selection of endophytic bacteria that importance; in the presence of soil mi-
can elicit plant growth promotion and ISR crobes, plant absorbs higher amounts of
in plants and research on such endophytes nutrients and less risk of environmental
in understanding plant responses that oc- pollution is likely. Some of the most im-
cur during the signal transduction path- portant functions of endophytic bacteria
ways that culminate in disease protection were reviewed in this article. However,
is essential to delineate the pathosystems. the particular emphasis has been on the
In many cases, elicitation of ISR by en- use of endophytic bacteria for biological
dophytic bacilli is associated with in- fertilization and biological control of phy-
creased plant growth, and the relationship topathogens. PGPR bio-fertilization is a
between ISR and growth promotion very common method because of its rapid
should be further investigated. Elucida- effects on plant growth and yield produc-
tion of specific bacterial determinants that tion. But there are issues regarding the
account for elicitation of ISR is just be- use of PGPR fertilizers, as their compe-
ginning, and further work is needed to tence in soil and interaction with plant
understand why one strain of a given bac- vary with the rhizosphere environment.
terial species can elicit ISR while another Since the endophyte is metabolically ac-
strain of the same species cannot. The climatized to the host plant with mutual-
trophicus Pal5 BMC Genomics, 10, Brunel, B., Prissol, C., Fernandez,
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Abstract: Global climate change and green house effects are very serious and contro-
versial problems that have severe negative impacts on environment, society, energy in-
dustry and government policies and sustainability. Global environment and climate
challenges are directly connected to the accumulation of green house gases which has
caused concerns related to the usage of traditional and fossil fuels as the key energy re-
source. To mitigate climate and environment changes, one solution is to utilize the po-
tential of metabolic engineering of microbes for biofuels production from renewable
sources. For long-term economic sustainability of the energy, industries and transporta-
tion sectors should adopt renewable and sustainable fuels produced by metabolically
engineered microorganisms. The biofuels produced from renewable sources by meta-
bolically engineered microbes carry good energy contents with minimal emission of
greenhouse gases and causes minimal impact on the environment, food chain, water
supply and land use. Toxic organic and inorganic chemicals are also one of the main
reasons for environment contamination and also present major risk for climate change.
Avoiding of upcoming contamination from these chemicals poses a huge technical
challenge. Currently, metabolically engineered microbes have been explored only for
selective and high capacity bioremediation of heavy toxic metals and chemicals. This
chapter will shed light on current trend and developments in metabolic engineering of
microbes for biofuel and bio-based chemicals production from renewable resources.
This chapter also highlights the potential of metabolically engineered microbes for bio-
remediation, a possible futuristic solution for sustainable development for energy and
reduction of global climate change and green house effects.
ide concentration will reach to 500 ppm in combination with rising global crises
by doubling of the carbon dioxide emis- provides the platform for microbial meta-
sion rate without remediation. The carbon bolic engineering applications and inno-
dioxide concentration of 500 ppm will vation on a large scale. Scientists from
lead to a global warming of around 2C industries and academics strongly believe
above the level in year 1900 (Pacala and that microbial metabolic engineering in
Socolow, 2004). This level of increase in combination with other technologies such
temperature would raise the threat of dis- as synthetic biology and systems biology
integration of the West Antarctic Ice can help to solve the growing concerns of
Sheet (WAIS) along with other negative climate and environment (Zhang et al.,
effects. It is estimated that increase in 2011).
temperature by 2C would lead to disrup- From the beginning microbial
tive rise of sea level by 46 meters metabolic engineering had aimed the pro-
(Stocker, 2013). duction of fuels and chemicals as chief
The atmospheric CO2 concentra- goals of the rising field. The metabolic
tion, CO2 emission and global tempera- engineering of yeast Saccharomyces
ture are having severe negative effects on cerevisiae and E. coli are the classic ex-
the environment, climate, economy and amples of metabolic application in the
society and we need to deal with it. To field of biofuel production (Kuyper et al.,
avoid the rise in the temperature, it is 2003, Bro et al., 2006, Ingram et al.,
necessary that we should reduce the car- 1987). Microbial metabolic engineering
bon emission from the fossil fuel usage applications have expanded in the past
and increase the sources of renewable few years because of the growing atten-
energy and remove the toxic chemicals tion on biofuels and chemical production
and metals present in the environment. through biomass conversion. There are
When compared with different renewable many reports stating that the combination
energy sources, biofuels are well-suited of microbial metabolic engineering along
with present infrastructure and have an with combinatorial approaches supported
advanced energy density. Thus there has by high throughput systems had given
been much curiosity in establishing biore- good results in biofuel production. The
fineries for the production of fuels and key advantage of utilizing microorganism
chemicals from renewable resources. for the production of biofuels from re-
We are in the modern age of newable sources is the metabolic diversity
microbial metabolic engineering which of fungi, algae and bacteria facilitate us
comprises of progressively more efforts at the use of diverse substrates as the start-
cell, and pathway design. One of the rea- ing point for biofuel generation.
sons for the more success of microbial Bioremediation is a very cost
metabolic engineering is development of effective and eco-friendly method and is
additional omics tools which provide steadily making inroads for environmen-
both temporally and spatially analyzing tal clean-up applications. Bioremediation
opportunity for cellular systems at the depends on enhanced detoxification and
level of protein, metabolite, RNA and degradation of toxic metals and degrada-
DNA (Peralta-Yahya et al., 2012). Mi- tion of toxic pollutants either through en-
crobial metabolic engineering have been zymatic transformation or intracellular
evolved to solve crucial international accumulation to less or non-toxic com-
problems such as global warming, biore- pounds. There are several physio-
mediation, food and human health. If chemical processes for treating toxic pol-
properly utilized microbial metabolic en- lutants in environment, but these process-
gineering can play an important role in es are non specific, very costly and some-
facing the global challenges. By devel- times they may introduce secondary con-
opment of novel technological innovation tamination. Microbes naturally have the
ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 249
Biotech Sustainability (2017)
Microbial Metabolic Engineering for Sustainability Meerza et al.
sugars, glucose and xylose (Sanny et al., oped an E. coli strain which can fermenta-
2010, Wang et al., 2008). tively produce 1- butanol by transferring a
group of six genes from C. acetobutyli-
2.3. Isopropanol production cum 1- butanol pathway and removal of
E. coli do not have some of the the competing pathway. This metabolical-
necessary pathways which are very much ly engineered E. coli strain produced 552
necessary pathways for the production of mg/L of 1- butanol under semi-anaerobic
advanced fuels, metabolic engineering conditions from a rich medium (Atsumi
has provided the chance to produce non- and Liao, 2008a). 1- Propanol is univer-
traditional biofuels by the construction of sal solvent with several industrial applica-
non native biosynthesis pathways (Atsumi tions which can be converted to propylene
et al., 2008b). The microorganisms such and diesel and it is a promising gasoline
as Clostridium can naturally produce Iso- substitute. The wild strain organisms can-
propanol which is one of the secondary not produce the 1-propanol is considera-
alcohols. Isopropanol has several diverse ble amounts (Shen and Liao, 2008). 2-
applications. In Clostridium several at- ketobutyrate is a precursor of 1-propanol
tempts have been made to enhance the and isoleucine. It is also can be converted
production ability, but product inhibition to 2-methy 1-butanol and 1- butanol
and low titer. As a result, metabolic engi- through several multi steps enzymes reac-
neering of E. coli for Isopropanol produc- tions. E. coli was metabolically engi-
tion become a promising substitute for neered to produce 1-propanol and 1-
industrial production of Isopropanol. butanol through 2-ketobutyrate (Shen and
Metabolically engineered strain of E. coli Liao, 2008).
was constructed that produced a 13.6 g/L Metabolically engineered
isopropanol from glucose under vigorous strain was developed by overexpres-
aerobic culture conditions (Jojima et al., sion of the genes such as thrAfbBC,
2008). Isobutanol has same physical leuABCD, ilvA from E. coli, kivd
properties like isopropanol, except it has from L. lactis and adh2 from S.
higher ocatane number. Metabolic engi- cereviasiae followed by deletion of
neering of E. coli resulted in the produc- the competing genes such as adhE,
tion of isobutanol using non-fermentative ilvB, metA and tdh. This engineered
pathways through 2-ketoisovalerate as strain produced 2 g/L propanol and
precursor. butanol in 1: 1 ratio. The drawback
this developed strain is it production
2.4. 1- butanol and 1- Propanol pro- of unwanted fermentative by prod-
duction ucts. In another study, a more direct
1-Butanol is very attractive and pathway to produce 2-ketobutyrate
alternative biofuel with high energy den- through citramalte pathway was engi-
sity and good compatibility with the abil- neered in Methanococcus jannaschii
ity to completely substitute gasoline. by directed evolution to produce 1-
Generally, C. acetobutylicum produces 1- butanol and 1- propanol. CimA spe-
butanol along with butyrate, ethanol and cific activity was enhanced by the
acetone. But due to absence of genetic DNA shuffling and error prone PCR
information, complex physiology and un- in E. coli strain. This E. coli strain
availability of genetic tools the C. aceto- with enhanced CimA activity pro-
butylicum had not been improved to the duced more than 524 mg/L and
level of usage in large scale industrial 3.5g/L propanol. The production of
production. The complete information of other side products is drawback of
the 1-butanol metabolic pathway, it be- this strain.
come possible to construction of butanol
pathway in E. coli. Atsumi et al. devel-
ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 251
Biotech Sustainability (2017)
Microbial Metabolic Engineering for Sustainability Meerza et al.
2.5. 2-Methyl-1-butanol and 3-methyl- the less toxic dialkyl phosphates and p-
1-butanol production nitrophenol is by metabolic engineering
The five carbon alcohols such as of microbes with the required degradation
3-methyl-1-butanol and 2-Methyl-1- pathways (Cook et al., 1980; de la Pena
butanol have characteristics like high en- Mattozzi et al., 2006).
ergy density and lower vapor pressure. The p-nitrophenol can be degrad-
When compared with ethanol, 3-methyl- ed by metabolically engineered P.putida
1-butanol and 2-Methyl-1-butanol are and Moraxella species. In Moraxella spe-
more suitable to replace gasoline and cies organophosphate hydrolase was ex-
more well-suited for the present fuel in- pressed on the cell surfaces and this strain
frastructure. 3-methyl-1-butanol and 2- showed to degrade paraoxon, parathion
Methyl-1-butanol can be naturally pro- and methyl parathion and their hydrolysis
duced by as by- products and some meta- product p-nitrophenol. In P. putida
bolic approaches have been tried to strain, natural p-nitrophenol degrading
enhance the production of 2-Methyl-1- operon was introduced. A synthetic oper-
butanol in S. cerevisiae (Abe and on for expression of alkaline phosphatase
Horikoshi, 2005). Table 1 illustrates the (PhoA), phosphodiesterase (Pde) and or-
biofuels production by metabolically en- ganophosphate hydrolase was introduced
gineered E. coli. to improve the diethyl phosphate (DEP)
mineralization and hydrolysis of oragan-
3. Bioremediation by metabolical- ophosphates. The resulting metabolically
ly engineered microorganisms engineered P.putida strain was able to
totally degrade p-nitrophenol in 78 hours,
3.1. Bioremediation of organic pollu- paraoxon in 24 hours and diethyl phos-
tants phate within 142 hours (de la Pena Mat-
The extensive use of extremely tozzi, et al., 2006). This research study
toxic compound oraganophosphates presents an effective novel approach to
(OPs) in agriculture as pesticide had led create an artificial metabolite pathway for
to a serious environmental pollution. Ora- total mineralization.
ganophosphates are used in the insecti- The present hydrodesulfurization
cides; generally oraganophosphates are in method for decreasing the level of organic
the form of phosphoric acid. The bacteria sulfur compounds is not capable to please
growing in the soil had naturally acquired the strict government regulations. The
the ability to degrade oraganophosphates present advances in desulfurization have
with the help of enzyme called organo- therefore been focused on biological pro-
phosphate hydrolase (McDaniel et al., cesses using microbes. Dibenzothiophene
1988; Kulkarni and Chaudhari 2006). P-O which is a popular organosulfur com-
linkage is hydrolysed by the organophos- pound is not removed by the hydrodesul-
phate hydrolase, releasing p-nitrophenol furization from fossil fuels. The desulfu-
as leaving group. As the toxicity of ora- rization of dibenzothiophene requires a
ganophosphates is extensively diminished group of enzymes (Kilbane, 2006).
by hydrolysis of phoshoester bonds, sev- Dibenzothiophene monooxygenase
eral scientists have concentrated on the (DszC) is the first enzyme that converts
primary hydrolysis by organophosphate dibenzothiophene to dibenzothiophene
hydrolase. Even though oraganophos- sulfone. The dibenzothiophene sulfone is
phates are transformed into dialkyl phos- converted into 2-hydroxybiphenyl-2-
phates and p-nitrophenol (PNP) by initial sulfinate by the catalysis of dibenzothio-
hydrolysis. Still these degraded products phene-5,5-dioxide monooxygenase. 2-
are resistant to biodegradation and they hydroxybiphenyl-2-sulfinate is converted
are toxic. One solution to remove even
nuclear plant leakage is one of the major metabolically engineered bacteria Dein-
environmental problems. Naturally occur- ococcus geothemalis is able to decrease
ring bacteria which are more resistant to mercury at higher temperature and ioniz-
radiation are ideal metabolic engineering ing radiation, it is also able to reduce
candidates for enhanced radionuclide re- Cr(VI), U(VI) and Fe(III). This study re-
moval. Metabolic engineering of thermo- ported the possibility of using metaboli-
philic bacterium Deinococcus geothe- cally engineered microorganisms for re-
malis was done by over expressing of mer moval of versatile radioactive wastes at
operon from E. coli coding for Hg2+ re- high temperatures. In P. aeruginosa, radi-
duction, which resulted in radiation re- onuclide precipitation can be achieved as
sistant bacterium (Brim et al., 2003). The metal phosphate by over expressing of
ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 255
Biotech Sustainability (2017)
Microbial Metabolic Engineering for Sustainability Meerza et al.
1 2
Wild type strain Wild type strain
A B C A B C
Metabolically Metabolically
engineered strain engineered strain
A B C A B C D
Wild type strain 3
C
A B
Metabolically
D Figure 1: Strategies for metabolic engineer-
engineered strain ing for the production of a desired chemical;
SYSTEMS BIOLOGY
SYNTHETIC BIOLOGY Metabolic models
Heterologous expression of - Omics analysis
natural pathways
- de novo pathway design
METABOLIC DIRECTED
ENGINEERING EVOLUTION
PROTEIN
GENETIC ENGINEERING ENGINEERING
Deletion or silencing
- Transcriptional tuning Random mutagenesis
- Translational tuning - Rational modification
Energy Materials Lab, #120, School of Physics, Alagappa University, Karaikudi-630 004,
Tamil Nadu, India; *Correspondence: susiva73@yahoo.co.in; Tel: +91 4565 223304
Abstract: Diminishing petroleum reserves and increasing environmental regulations are the
main driving forces to search for renewable fuel. Biodiesel is a renewable substitute fuel for
petroleum diesel fuel and produced by transesterification in which oil or fat is allowed to
react with a monohydric alcohol in the presence of a catalyst. This paper gives an overview
of the work carried out by researchers in the field of biodiesel production from different
types of oil. The different methods of biodiesel production techniques and factors affecting
biodiesel production are also highlighted.
Table 2: Projections of biodiesel demand and corresponding Jatropha area required for
meeting the blending targets in India (Area in Mha, Demand in Mt)
For 5% blending For 10 % blending For 20 % blending
Diesel
Year Biodiesel Jatropha Biodiesel Jatropha Biodiesel Jatropha
demand
demand area demand area demand area
2011- 64.19 3.21 3.42 6.42 6.85 12.84 13.69
12
2016- 92.15 4.61 4.91 9.21 9.83 18..43 19.66
17
2020- 123.06 6.15 6.56 12.31 13.13 24.61 26.25
21
-ion hectare is classified as waste and curcas is the most known variety; it
degraded land (Dwivedi et al., 2014). requires little water or additional care;
therefore, it is adequate for warm regions
4.1. Typical oil crops useful for biodiesel with little fertility. Productivity may be
production reduced by irregular rainfall or strong
The main characteristics of typical winds during the flowering season. Yield
oil crops that have been found useful for depends on climate, soil, rainfall and
biodiesel production are summarized in treatment during sowing and harvesting.
the following paragraphs. Jatropha plants become productive after 3
or 4 years, and their lifespan is about 50
4.1.1. Castor seed years. Oil yield depends on the method of
The castor oil plant grows in extraction; it is 2832% using presses and
tropical climates, with temperatures in the up to 52% by solvent extraction. Since the
range 2030C; it cannot endure frost. It seeds are toxic, jatropha oil is nonedible.
is important to note that once the seeds The toxicity is due to the presence of
start germinating, the temperature must curcasin (a globulin) and jatrophic acid
not fall below 12 C. The plant needs a (as toxic as ricin).
warm and humid period in its vegetative
phase and a dry season for ripening and 4.1.4. Microalgae
harvesting. It requires plenty of sunlight Microalgae have great potential for
and adapts well to several varieties of biodiesel production, since the oil yield
soils. The total rainfall during the growth (in liters per hectare) could be one to two
cycle must be in the range 7001,400 orders of magnitude higher than that of
mm; although it is resistant to drought, other raw materials. Oil content is usually
the castor oil plant needs at least 5 months from 20 to 50%, although in some species
of rain during the year. Castor oil is a it can be higher than 70%. However, it is
triglyceride, ricinolenic acid being the important to note that not all microalgae
main constituent (about 90%). The oil is are adequate for biodiesel production.
non-edible and toxic owing to the High levels of CO2, water, light, nutrients
presence of 15% of ricin, a toxic protein and mineral salts are necessary for the
that can be removed by cold pressing and growth of microalgae. Production
filtering. The presence of hydroxyl groups processes take place in raceway ponds
in its molecules makes it unusually polar and photobiological reactors.
as compared to other vegetable oils.
5. Biodiesel production techniques
4.1.2. Jojoba
Although jojoba can survive There are different processes which
extreme drought, it requires irrigation to can be applied to synthesize biodiesel
achieve an economically viable yield. such as direct use and blending, micro
Jojoba needs a warm climate, but a cold emulsion process, thermal cracking
spell is necessary for the flowers to process and the most conventional way is
mature. Rainfall must be very low during transesterification process (Gashaw et al.,
the harvest season (summer). The plant 2015).
reaches its full productivity 10 years after
planting. The oil from jojoba is mainly 5.1. Direct use and blending
used in the cosmetics industry; therefore, The direct use of vegetable oils in
its market is quickly saturated. diesel engine is not favorable and
problematic because it has many inherent
4.1.3. Jatropha failings. Even though the vegetable oils
Jatropha is a shrub that adapts have familiar properties as biodiesel fuel,
well to arid environments. Jatropha it required some chemical modification
before can be used into the engine. It has proportions. The equipment for thermal
only been researched extensively for the cracking and pyrolysis is expensive for
past couple of decades, but has been modest biodiesel production particularly
experimented with for almost hundred in developing countries. Furthermore, the
years. Although some diesel engine can removal of oxygen during the thermal
run pure vegetable oils, turbocharged processing also removes any
direct injection engine such as trucks are environmental benefits of using an
prone to many problems. oxygenated fuel. Another disadvantage of
pyrolysis is the need for separate
5.2. Microemulsion process distillation equipment for separation of
A micro emulsion is defined as the the various fractions. Also the product
colloidal equilibrium dispersion of obtained is similar to gasoline containing
optically isotropic fluid microstructures Sulphur which makes it less ecofriendly.
with dimensions generally in the range of The pyrolyzed material can be vegetable
1150 nm formed spontaneously from oils, animal fats, natural fatty acids and
two normally immiscible liquids and one methyl esters of fatty acids.
or more ionic or non-ionic. The problem
of the high viscosity of vegetable oils was 5.4. Transesterification
solved by micro-emulsions with solvents Generally, biodiesel is produced
such as methanol, ethanol, and 1-butanol. by means of transesterification.
The components of a biodiesel micro- Transesterification is the reaction of a
emulsion include diesel fuel, vegetable lipid with an alcohol to form esters and a
oil, alcohol, surfactant and cetane byproduct, glycerol. It is, in principle, the
improver in suitable proportions. action of one alcohol displacing another
Alcohols such as methanol and ethanol from an ester, referred to as alcoholysis
are used as viscosity lowering additives, (cleavage by an alcohol). In
higher alcohols are used as surfactants Transesterification mechanism, the
and alkyl nitrates are used as cetane carbonyl carbon of the starting ester
improvers. Microemulsions can improve (RCOOR1) undergoes nucleophilic attack
spray properties by explosive by the incoming alkoxide (R2O) to give
vaporization of the low boiling a tetrahedral intermediate, which either
constituents in the micelles. Micro- reverts to the starting material, or
emulsion results in reduction in viscosity proceeds to the transesterified product
increase in cetane number and good spray (RCOOR2). Transesterification consists of
characters in the biodiesel. However, a sequence of three consecutive reversible
continuous use of microemulsified diesel reactions. The first step is the conversion
in engines causes problems like injector of triglycerides to diglycerides, followed
needle sticking, carbon deposit formation by the conversion of diglycerides to
and incomplete combustion. monoglycerides, and finally
monoglycerides into glycerol, yielding
5.3. Thermal cracking (pyrolysis) one ester molecule from each glyceride at
Pyrolysis is defined as the each step. The reaction is represented in
conversion of one substance into another equation 1.
by means of heat or heating with the aid
of a catalyst. Pyrolysis involves heating in
absence of air or oxygen and cleavage of
chemical bonds to yield small molecules.
The pyrolysis of vegetable oil to produce
biofuels has been studied and found to
produce alkanes, alkenes, alkadienes,
aromatics and carboxylic acids in various
as the molar ratio of alcohol to vegetable found that reaction mixture containing
oil and reaction temperature were 65ml of methanol along with 2.4 g of
investigated during the transesterification catalyst (KOH) took a good start in half
within this supercritical media. Increasing an hour at30C. In this reaction, amount
the reaction temperature within the of glycerine removed as well as ester
supercritical regime resulted in increased content produced was considerably
ester conversion. increased with rise in temperature of
mixture up to 70C by extending time
6. Previous work done on production of period (180-360 minutes). The removal of
biodiesel from edible oil glycerine increased by two and half times
and ester content by four times,
Leung and Guo (2006) compared respectively. When castor oil was
the transesterification reaction conditions subjected to acid esterification, prior to
for fresh canola oil and used frying oil. transesterification (a separate
Higher molar ratio (7:1, methanol/used investigation), it was found that ester
frying oil), higher temperature (60 C) contents up to 95% could be obtained.
and higher amount of catalyst (1.1 wt% Hasan et al. (2013) produced biodiesel
NaOH) was maintained in used frying oil from neem seeds, its properties were
when compared to fresh canola oil where close to diesel. The methodology of
optimal conditions maintained were 315- esterification process was selected and
318 K, 1.0 wt% NaOH and 6:1 carried out by 1000 ml raw neem oil,
methanol/oil molar ratio. However, less 300ml methanol and sodium hydroxide
reaction time (20 min) was observed for on mass basis as a catalyst usually kept in
used frying oil when compared to fresh oven to form methyl ester, and initially to
canola oil reaction time (60 min). Ying et reach equilibrium condition at
al. (2011), developed a new method temperature 55-66C. The ester and
catalyst, benzyl bromide-modified glycerine were separated by stimulating
calcium oxide (CaO) for production of continuously and allow settling under
biodiesel from rapeseed. The improved gravity for 24 h. Thus the separated ester
catalytic activity was obtained by better contains 3% to 6% methanol and soap
fat diffusion to the surface of the benzyl agents. The methanol was removed by
bromide-modified CaO. Further, a 99.2% vaporization. The biodiesel had some
yield of fatty acid methyl esters in 3h was catalyst; it was removed by warm water
obtained in comparison to by better fat mix with ester. Kinematic viscosity lay
diffusion to the surface of the benzyl between 1.9 and 6.0 according to the
bromide-modified CaO. Wakil et al. ASTM D6751 specification. It was
(2012), chosen Cottonseed oil, Mosna oil reported that, 0.95 L biodiesel was
and Sesame oil for producing biodiesel. produced from 1 L neem oil.
Abstract: Most fast developing countries in Asia face severe pollution problems due to in-
dustrialization and modernization. Analytical chemical measurements are common in pollu-
tion monitoring. However, the ecological (biological) effects of those chemicals are diffi-
cult to understand. The latest generation of bioanalytical tools, such as in vitro transactiva-
tion bioassays, show great promise as water and sediment quality monitoring tools. A bat-
tery of in vitro cell bioassays has been developed in recent years that are effective, specific,
fast, less expensive and easy to handle. One example is, Aryl hydrocarbon Receptor (AhR)
mediated CYP1A1 protein expression that has been utilized effectively as a biomarker of
pollution stress to marine biota. Hence, AhR binding potential, subsequent induction, geno-
toxic expression and cytotoxic potential of pollutants are reviewed. The list of in vitro cell
bioassays reviewed will hopefully be utilized in ecotoxicological studies in developing
countries in Asia.
recently been shown to effectively screen tants that exist in environmental extracts
environmental contaminants based on is made possible. Moreover, in compari-
their biological mode of action. In partic- son to the limited identification of pollu-
ular, a number of in vitro cell bioassays tants provided through instrumental anal-
have been adopted to measure the inte- ysis, in vitro techniques provide an as-
grated response of bioactive contami- sessment of the total biological impact
nants, such as estrogens, in recycled and exerted by complex mixtures of environ-
surface waters as well as in wastewater mental contaminants that are mediated
effluents (van der Linden et al. 2008; through a common mechanism of action.
Leusch et al. 2010; Escher et al. 2014; Various bioassays have been developed
Mehinto et al. 2015). These emerging bi- over the years to evaluate the toxic poten-
oanalytical tools can indicate which clas- cy of environmental extracts with refer-
ses of chemicals are of concern, thus nar- ence to a specific target receptor. Bioas-
rowing the field of targeted chemical says are reliable tools to measure the re-
analysis needed and toxicity endpoints to sponse of a cell in terms of protein ex-
evaluate (Brack et al. 2015; Maruya et al. pression and enzyme activity when bio-
2016). In general, in vitro bioassays are logical organisms are exposed to envi-
good markers of biological toxicity as ronmental pollutants. These responses are
they are sensitive, quick, less expensive reportedly triggered by specific genes that
(Mehinto et al. 1993; Qiao et al., 2006). mediate the transcription of the particular
The advantage of effect-based bioassay target protein. Cell lines developed from
measurements is that they determine the mammals and sh utilizing CYP1A1 in-
integrated toxic potency of the complex duction as a biomarker of exposure to
mixture of micro contaminants in the en- Poly aromatic hydrocarbons (PAHs), Pol-
vironment. In addition, bioassays may ychlorinated biphenyls (PCBs) and other
detect mixture effects of compounds, Halogenated aromatic hydrocarbons
even when the individual constituents of (HAHs) are effective in vitro tools for
the mixture are present at concentrations cumulative impact of sediment extracts
too low to cause an effect or to be detect- (Jung et al., 2012; Schnell et al. 2013).
ed by chemical analysis (Hamers et al., Thus, various cell lines developed from
2010). The impact of unmonitored con- mammals (He et al., 2011; Willet et al.,
taminants (contaminants of emerging 1997 a, b) and fish (Fernandes et al.,
concern) remains largely uncharacterized. 2014; Schnell et al. 2013; Fent, 2001) in-
Considering these points, one can safely ducing CYP1A monooxygenases enzyme
conclude that in-vitro bioassays are com- belonging to the cytochrome P450 family
prehensive and holistic and can act as ear- have been utilized.
ly warning signals of environmental deg-
radation. 3. Aryl hydrocarbon Receptor (AhR)
mediated toxic response
2. Significance of cell in vitro bioassays
The most potent inducer of Aryl hydro-
In vitro cell bioassays utilizing either carbon Receptor (AhR) is 2,3,7,8-
wild type cells or genetically engineered tetrachlorodibenzo-p-dioxin (TCDD).
eukaryotic cells providing an assessment Thus chemicals that elicit response simi-
on the potency of contaminants extracted lar to TCDD are generally clustered as
from environmental matrices. Applica- dioxin-like chemicals. The ability of these
tions of in vitro cell bioassays are highly chemicals to cause hepatotoxicity, em-
effective in terms of cost and time. A no- bryotoxicity, teratogenicity, immunotoxi-
table advantage is that through in vitro city, dermal toxicity, lethality, carcino-
bioassays the detection and assessment of genesis, and tumor promotion in many
toxicity of a complex mixture of pollu- dierent species at low concentrations
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Biotech Sustainability (2017)
In vitro Cell Bioassays in Pollution Assessment Kannan et al.
have generated much concern (Giesy et AhR signaling pathway through the ex-
al; 2002; Ahlborg et al., 1992; Peterson et pression of the protein CYP1A and its
al., 1993). These chemicals are largely enzymatic activity has been developed
mediated through AhR dependent mecha- and constantly improved with modifica-
nism of action in biological system (Po- tions to evaluate and applied in environ-
land and Knutson., 1982; Willet et al., mental assessment.
1997, Yoo et al, 2006). Several reviews
have been published on the potency of 4. AhR active compounds
dioxin-like environmental xenobiotics
(Gillesby and Zacharewski, 1998; Ankley AhR ligands are made by hydropho-
et al., 1998; van den Berg et al., 1998; bic compounds such as polychlorinated
Giesy et al, 2002). dibenzo-p-dioxins and dibenzofurans
AhR is described as a ligand dependent (PCDDs and PCDFs), chlorinated azo-
transcription factor located in the cytosol, benzenes and azoxybenzenes, polychlo-
chaperoned with heat shock proteins rinated biphenyls (PCBs), several polycy-
(Giesy et al., 2002). Increased toxicity, clic aromatic hydrocarbons (PAHs), poly-
enhanced gene transcription and enzyme chlorinated naphthalenes (Giesy et al.,
activity are indicators of the binding 2002; Blankenship et al., 2000; Jung et
strength of congeners to the AhR (Giesy al., 2012) and various halogenated aro-
et al., 2002; Safe, 1995). The AhR medi- matic hydrocarbon (HAHs) (Willet et al.,
ated mechanism is initiated once the lig- 1997). Relatively weak AhR ligand has
ands bind to cytosolic AhR. The receptor been identified for natural and synthetic
ligand complex is then translocated into compounds (Giesy et al., 1998; Denison
the nucleus leading to the dissociation of and Heath-Pagliuso, 1998).
heat shock proteins followed by for-
mation of a dimer by binding to Ah Re- 5. Dioxin responsive (DR) CALUX bio-
ceptor Nuclear Traslocator (ARNT) pro- assay
tein. The heteromeric ligand AhR:ARNT
complex then binds to dioxin-responsive The CALUX (chemically activated
element (DRE) a specic DNA sequenc- luciferase expression) is a reporter-gene
es. This binding in turn will stimulate based cell bioassay that is increasingly
transcriptional activation of adjacent re- applied in screening of dioxin and dioxin
sponsive genes leading to production of like chemicals in environmental matrices
specific protein such as CYP1A1 (Giesy and in food materials (Tsutsumi et al.,
et al., 2002; Denison and Heath-Pagliuso, 2003; Cederberg et al., 2002). To perform
1998; Hankinson, 1995; Celander et al., this assay, recombinant mammalian cell
1996). CYP1A1, belonging to the super- lines that have been stably transfected
family of cytochrome P450 plays a signif- with one of two different AhR-responsive
icant role in the biotransformation of xe- luciferase reporter gene plasmids that
nobiotic such as PAHs and PCBs in or- responds to dioxin and related chemicals
ganisms. Induction of CYP1A has been are utilized (Denison et al., 2004;
the most useful biomarker for environ- Garrison et al., 1996). Briefly, these cells
mental contamination and had been ap- contains AhR, that when bound to
plied in various pollution monitoring pro- activating ligands will innitiate the
grams (Bucheli and Fent, 1995; Celander transcription of the luciferase genes and
et al., 1996). the induction of the luciferase activity that
Biotransformation is a complex process will be determined by measuring
of excretion of hydrophobic substrate by luminescence. Induction of luciferase
converting them to hydrophilic metabolite activity is directly proportional to the
through monooxygenation (Czeka, 2000). amount and potency of inducing chemical
Bioassays that measure the activation of to which the cells are exposed (Han et al.,
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Biotech Sustainability (2017)
In vitro Cell Bioassays in Pollution Assessment Kannan et al.
2004; Denison et al., 2004). However, the provides a highly responsive and sensitive
results obtained will vary significantly bioassay system for the detection and
depending on the type of cell lines used. relative quantitation of very low levels of
This is due to the species and tissues dioxin-like chemicals in sample extracts
specific differences and functionality of (He et al., 2011). This bioassay is an
the AhR as well as the antagonistic and essential assay to characterize AhR active
synergistic effects of some compounds compounds in environmental matrices as
that are cell line dependent. it accounts for the total AhR mediated
The analysis of the luminescence activities in the test samples including
measured through this assay is converted non-dioxin-like chemicals such as PAHs
to CALUX TEQ toxic equivalents. The and HAHs. Several advantages of
luminescence value given by a sample is CALUX assay are discussed by Windal
compared to the dose response curve of a et al. (2005) such as the reliability of
reference standard such as 2,3,7,8- CALUX assay to analyzes the overall
tetrachlorodibenzo-p-dioxin. Briefly, the biological activity of all AhR ligands
sample concentration that induce (agonists and antagonists) present in an
luciferase to 25% of the TCDD-induced extract, in comparison to chemical
maximum luciferase activity is designated analyses that only focuses on a selected
as the EC25 TCDD for that sample. The number of compounds. Besides, for
TEQ concentrations were calculated as; enviromental screening CALUX and
chemical analysis are complimentary
reflecting the mangitude of dioxin like
activity induced by other compounds that
were undetected in chemical analaysis;
particularly, when large numbers of envi-
ronmental samples are to be screened.
H4IIE-luc cells are among the most CALUX ensures a rapid and cost effec-
common cells utilised in luciferse based tive analysis for dioxin-like chemicals.
assay to characterize the induction
potency of AhR active compounds such 6. EROD Assay
as halogenated aromatic hydrocarbon
(HAH) mixtures containing Induction of 7-ethoxyresorun-O-
polychlorinated biphenyls (PCBs), deethylase (EROD) activity has been ap-
dibenzo-p-dioxins (PCDDs) in plied in numerous environmental toxicol-
environmental matrices (Yoo et al., 2006; ogy studies as to indicate pollution stress
Giesy et al., 2002; Whyte et al., 2004; (Fernandes et al., 2014; Kim et al., 2013;
Willet et al., 1997 a,b; Schmitz et al. Schnell et al., 2013; Huuskonen et al.,
1995). H4IIE-luc cells have been studied 2000). EROD activity is based on the
and suggested as an alternative deethylation of the synthetic model sub-
bioanalytical tool to the wild-type cells strate 7-ethoxyresorun by CYP1A1, an
for the detection of AhR agonists in enzyme that belong to the phase-1 group
environmental samples (Sanderson et al., of biotransformation enzymes. This reac-
1996). tion will produce a uorescent product
Progressive studies in this particular resorun that is quantied uorometrical-
assay lead to the development of a third ly and normalized to the total protein con-
generation (G3) CALUX cell bioassays. tent. In biological system, the induction of
In such studies mouse hepatoma CYP1A1 is triggered when dioxin-like
(hepa1c1c7) cells transfected with G3 chemicals such as PAHs and HAHs bind
CALUX plasmids with increased to the AhR. As an immediate response, a
numbers of dioxin response elements binding enhanced gene expression of
(DREs) are utilized. This development CYP1A1 protein occurs to detoxify the
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Biotech Sustainability (2017)
In vitro Cell Bioassays in Pollution Assessment Kannan et al.
tion of CYP1A and mechanism of toxic ronmental sample. Comet assay (single
action. Enzyme-linked immunosorbent cell gel electrophoresis) is a technique of
assay (ELISA) is a useful technique in measuring DNA damage in eukaryotic
immunologically detecting CYP1A pro- cells or disaggregated tissues when ex-
tein. ELISA provides complementary in- posed to hazardous agents (Azqueta and
formation on the induction of CYP1A in Collins, 2013). In this assay the DNA is
the presence of inducing substances. Pre- drawn out towards the anode through
viously quantification of CYP1A protein electrophoresis, forming a comet-like im-
content was made possible through the age that is observed with fluorescence
preparation of subcellular fractions in- microscopy (Azqueta and Collins, 2013).
volving scrapping of cells from culture In general, this assay refers to the relaxa-
flask, followed by sonication and centrif- tion of supercoiled DNA in agarose-
ugation and eventually quantification embedded nucleoids (the residual bodies
through western blotting (Hahn et al., remaining after lysis of cells with deter-
1993). This technique is rather time con- gent and high salt), which then allows the
suming. Hence, a much rapid and sensi- DNA to be drawn out towards the anode
tive detection of CYP1A protein was de- under electrophoresis, forming comet-like
veloped in which the detection of the images as seen under fluorescence mi-
amount of CYP1A protein extracted was croscopy. DNA break frequency is indi-
measured directly in the microwells (Bru- cated through the relative amount of DNA
schweiler et al., 1995). This technique in the comet tail (Azqueta and Collins,
allows a semiquantitave determination of 2013). The extent of DNA migration are
the amount of immunoreactive CYP1A determined as a percentage of DNA in the
protein directly in the microwells in tail (% tDNA) using an image analysis
which the cells are cultured. The data of system. For statistical analysis, the induc-
absolute absorption values are presented tion factor (IF) was calculated using the
as the percent of maximal induction of following equation:
CYP1A and EC50 is determined. Studies
have utilized ELISA to quantify CYP1A
protein to provide a complementary quan-
tification on CYP1A based EROD activi-
ty (Herrero and Castel, 1994; Jung et al.,
2001). (6) (rut et al., 2011)
midine DNA glycosylase (FPG), provides tact metabolism ensures the existence of
a greater yield for strand breaks and in- full biotransformation capacity within the
creases the sensitivity of the assay in gen- cells that is being exposed to the samples.
eral (Azqueta and Collins, 2013). In the Recent research suggests that the results
case of low level of strand breaks at non- of in vitro EROD assay to be presented by
cytotoxic concentrations of genotoxic reference to a truly physiological cytotox-
chemical, the yield of breaks was greatly icity assay, the 3-(4,5-dimethylthiazol-2-
enhanced after incubation with (FPG) yl) 2,5-diphenyltetrazo-lium bromide
(Azqueta et al., 2013). (MTT) test replacing protein normaliza-
This is a sensitive, simple and versatile tion which will enable an optimized in
assay performed to visualize the single vitro EROD protocol to a reference com-
strand break in nuclear DNA of single pound (Heinrich et al., 2014).
cells (Wilkening et al., 2003). Besides
providing the overall level of damage in 9. Conclusion and recommendations
the cells being analysed, comet assay also
provides the data on how the individual Pollution is a major setback in devel-
cells respond to the xenobiotic. Comet oping countries with heavy emphasis on
assay has been applied in studies on industrialization and modernization. Con-
PLHC-1 fish cell line (rut et al., 2011) stant and consistent pollution monitoring
and characterization of the genotoxicity is crucial in order to maintain pollution
of sediment extracts from the Baltic and level in control. In pursuit of the effort,
North Sea on EPC (epithelioma papu- extensive environmental toxicology stud-
losum cyprini) sh cell line (Kammann et ies are essential because they provide the
al., 2001, 2004). actual impact situation before the expen-
sive instrumental analysis is conducted.
8.2. Cytotoxicity assay Ecotoxicological studies in Malaysia are
To assess the acute toxicity of pollu- extremely rare. Hence, there is a need for
tants on biological organisms, various cy- immediate exploration and application of
totoxicity tests are usually performed. these techniques and this will aid the rel-
Among them, tetrazolium salt reduction evant authorities to improve the environ-
(MTT) assay is widely used as an end- mental status monitoring.
point for the cytotoxicity measurement of
chemicals/pollutants in monolayer cell Acknowledgement
cultures (Vakharia et al., 2001; Bru-
schweiler et al., 1995; Heinrich et al., This work was supported by grants-in-
2014). The MTT assay detects the reduc- aid from Research University Grant
tion of soluble MTT tetrazolium salt to a Scheme by University Putra Malaysia
blue insoluble MTT formazan product by (9331400) to Prof. Ahmad Zaharin Aris.
mitochondrial succinate-dependent dehy-
drogenase (Bruschweiler et al., 1995). References
The percentage of viability was calculated
relative to DMSO treatment control wells Ahlborg, U. G., Brouwer, A., Fin-
from triplicate observations that are des- gerhut, M. A., Jacobson, J. L.,
ignated as 100% viable cells. Only cells Jacobson, S. W., et al. (1992). Im-
with active mitochondria are able to cata- pact of polychlorinated dibenzo-p-
lyze this reaction. Cellular stress determi- dioxins, dibenzofurans, and bi-
nation is crucial when analyzing complex phenyls on human and environ-
samples such as environmental samples mental health, with special empha-
because enzymatic assays such EROD sis on application of the toxic
assay requires an intact metabolism for equivalency factor concept. Euro-
the in-vitro live cell approaches. This in-
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Biotech Sustainability (2017)
In vitro Cell Bioassays in Pollution Assessment Kannan et al.
Keiter, S., Grund, S., Bavel, B.V., Hag- emerging concern in aquatic eco-
berg, J., Engwall, M., et al. systems. Integrated Environmental
(2008). Activities and identifica- Assessment Management 12:540-
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agonists in sediments from the Mehinto, A. C, Jia, A., Snyder, S. A.,
Danube River. Anals of Bioanal Jayasinghe, B. S., Denslow, N. D.,
Chem 390: 20092019 et al. (1993). Developmental and
Kim, H. N., Park, C. I., Chae, Y. S., reproductive toxicity of dioxins
Shim, W. J., Addison, R. F., et al. and related compounds: Cross spe-
(2013). Acute toxic responses of cies comparisons. Critical Review
the rocksh (Sebastes schlegeli) to in Toxicology 23, 283335.
Iranian heavy crude oil: Feeding Mehinto, A. C., Jia, A., Snyder, S. A.,
disrupts the biotransformation and Jayasinghe, B. S., Denslow N. D.,
innate immune systems. Fish and et al. (2015). Interlaboratory com-
Shellsh Immunology 35, 357-365. parison of in vitro bioassays for
Krishnan, P., Kannan, N., Aris, A. Z., screening of endocrine active
Arulselvan, P., Fakurazi S. chemicals in recycled water. Water
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bio-analytical assays in the biolog- Poland, A., Knutson, J. C. (1982).
ical impact assessment of Persis- 2,3,7,8-Tetrachlorodibenzo-pdioxin
tent Organic Pollutants (POPs) in and related halogenated aromatic
mangrove sediments in South East hydrocarbons: examination of the
Asia with particular reference to mechanism of toxicity. Annual Re-
Malaysia. In: Persistent Organic view of Pharmacology 22, 517
Chemicals in the Environment: 554.
Status and Trends in the Pacific Qiao, M., Chen, Y., Zhang, Q., Huang,
Basin Countries II. ACS Symposi- S., Ma, M., et al. (2006). Identifi-
um Series; American Chemical So- cation of Ah receptor agonists in
ciety. Loganathan, B. G., Khim, J. sediment of Meiliang Bay, Taihu
S., Prasada Rao S. Kodavanti, P. R. Lake, China. Environmental Sci-
S., Masunaga, S. (eds.) Publication ence and Technology 40,1415
Date (Web): December 7, 2016 | 1419.
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Lapa, N., Barbosa, R., Morais, J., and molecular biology of aryl hy-
Mendes, B., Mhu, J., et al. drocarbon (AH) receptor mediated
(2002). Ecotoxicological assess- gene expression. Archives of. Toxi-
ment of leachates from MSWI bot- cology. 7, 99115.
tom ashes. Waste Management 22, Sanderson J. T., Aarts, M. M. J. G.,
583593 Brouwer, A., Froese, K. I., Den-
Leusch, F. D, de Jager, C., Levi, Y., ison, M. S., et al. (1996). Compari-
Lim, R., Puijker, L., et al. (2010). son of Ah Receptor-Mediated Lu-
Comparison of five in vitro bioas- ciferase and EthoxyresorunO-
says to measure estrogenic activity deethylase Induction in H4IIE
in environmental waters. Environ- Cells: Implications for Their Use as
mental Science and Technology Bioanalytical Tools for the Detec-
44:3853-3860. tion of Polyhalogenated Aromatic
Maruya KA, Dodder NG, Mehinto AC, Hydrocarbons. Toxicology and ap-
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In vitro Cell Bioassays in Pollution Assessment Kannan et al.
Department of Plant Pathology, Centre for Plant Protection Studies, Tamil Nadu Agricul-
tural University, Coimbatore 641-003, Tamil Nadu, India; *Correspondence:
meepath@rediffmail.com; Tel: +91 422 6611226
Abstract: Biosurfactants are surface active biological compounds that are produced by
broad range of microorganisms. They are ecofriendly with low toxicity, no residual effects
with high biodegradable properties and known to suppress the growth of pathogenic fungi.
Bacillus genus is considered microbial factories for the production of a huge number of bio-
logically active molecules that are inhibitory for plant pathogen growth. In rhizobacteria
Bacillus subtilis, an average of 4 5% of its genome is dedicated to antibiotic synthesis and
has the possibility to produce more than two dozen of structurally diverse antimicrobial
compounds. Because of the surfactant properties, the antimicrobial peptide compounds or
cyclic lipopeptide antibiotics of the surfactin, iturin and fengycin families are well-
recognized for their potential applications in biotechnology. Different groups of lipopep-
tides can give an advantage to the Bacillus strains in specific environmental niches. The
ability to induce systemic resistance in plants and their use in the spreading of the bacterial
cells that leads to rhizosphere colonization could open new fields of applications for their
use in phytopharmaceutical products. In this review article, we are highlighting the role and
functions of some major biocontrol lipopeptide biosurfactants present in the Bacillus spe-
cies.
Sankari Meena et al., 2016) and anti- amount, to the production of iturin A
microbial activity have been reported (Tsuge et al., 2001). Gene clusters in-
from other Bacillus strains. volved in iturin A production have been
intensively investigated (Hiraoka et al.,
2. Bacillus lipopeptides 1992, Huang et al., 1993, Kunst et al.,
1997, Yao et al., 2003). The iturin A op-
The lipopeptides in Bacillus are eron spans a region more than 38 kb long
classified into three families namely Itu- and it contains four open reading frames
rin, Surfactin and fengycin family. There viz., ituD, ituA, ituB and ituC (Tsuge et
are several Bacillus strains isolated and al., 2001). The ituD gene encodes a puta-
reported to produce all the three tive malonyl coenzyme A transacylase,
families of lipopeptide biosurfactants whose distraction results in deficiency of
simultaneously (Ramyabharathi and Ra- iturin A production (Kunst et al., 1997).
guchander, 2014). Ramyabharathi and Raguchander (2014)
reported that the B. subtilis strain EPCO
2.1. Iturin lipopeptide 16 contains ItuC, ItuD, BmyA, BacD,
Iturins are a member of an anti- BacAB and FenD genes involved in the
fungal lipopeptide group comprises iturin, biosynthesis of Iturin, Bacillomycin,
bacillomycin and mycosubtilin which are Bacilysin and Fengycin, respectively. Itu-
cyclic lipoheptapeptides and are linked by rin and surfactin were detected in culture
beta amino acid residue. Iturin family filtrates from isolate EPCO16 by thin lay-
contains iturins A-E, bacillomycins D, F, er chromatography that showed tremen-
and L, and mycosubtilin. Members of this dous control over Fusarium oxysporum
family have powerful antibiotic activity pathogen infecting tomato.
but moderate surfactant activity and en- Bacillomycin D which is a mem-
hanced swarming motility (Leclere et al., ber of the Iturin family along with myco-
2006). Hence the presence of iturin in subtilin and iturin A, is made of one -
Bacillus is responsible for disease sup- amino fatty acid and seven -amino acids
pression and growth inhibition of wide exhibits a strong antifungal activity
number of phytopathogens. Bacillus sub- against a broad range of plant pathogenic
tilis produces a diversity of antibiotics fungi. Biosynthesis of bacillomycin D is
that are effective against phytopathogenic independent of the ribosomal process and
fungi and bacteria (Phae et al., 1990). Itu- the enzymes responsible for
rin production seems to be constrained to bacillomycin D production are complex
B. subtilis and B. amyloliquefaciens peptide synthetases. Bacillomycin D and
(Bonmatin et al., 2003). In control of fengycin jointly contributed to the inhibi-
phyllosphere diseases, Podosphaera fusca tion of conidial germination of Monilinia
infecting melon leaves the iturins and fructicola and fengycin played a major
fengycins lipopeptides produced by role in suppressing mycelial growth of the
B. subtilis contributed more in disease fungal pathogen. Luo et al., 2011 per-
suppression. Vater (1986) reported that formed bioassay of antifungal compound
the iturin is produced, during slow, sta- bacillomycin against Magnaporthe ory-
tionary growth phase. Sandrin et al. zae, Rhizoctonia solani and Botrytis ci-
(1990) reported that iturin A, one member nerea. The hyphae of the pathogenic fun-
of the iturin group, shows a strong antibi- gi treated with bacillomycin L showed
otic activity with a broad antifungal activ- abnormal growth, and conidia produced
ity, making it an ideal potential biological enlarged and constricted germ tube. When
control agent with the aim of reducing the bacillomycin L used in high concentration
use of chemical pesticides in agriculture. there is a possibility for cellular leakage.
The antimicrobial action of Bacillomycin D was detected in
B. subtilis can be attributed, to a certain B. subtilis (Moyne et al., 2001; Ramara-
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Biotech Sustainability (2017)
Lipopeptide Biosurfactants as a Weapon for Plant Disease Management Ramyabharathi et al.
Abstract: Andaman and Nicobar Islands are known to harbor large diversity of plant and
seaweed species, a number of them being endemic. Micropropagation could be used as an
effective tool for large scale multiplication of economically important plants and seaweeds.
The technique could also help in multiplying threatened species to conserve them. In vitro
production of pharmaceutical macromolecules could be a viable option for avoiding de-
structive harvesting of plant species. Somaclonal variation, in vitro mutagenesis and trans-
genic could be useful in some cases. Molecular markers could help in assessment of genetic
diversity, DNA barcoding, marker assisted selection etc. The article highlights the im-
portance and relevance of various biotechnological tools in the management of biodiversity
of the fragile ecosystem of Andaman and Nicobar Islands. Various research activities un-
dertaken to conserve species of these islands are also highlighted.
Keywords: Andaman and Nicobar Islands; Bay of Bengal; biodiversity; endemism; sustain-
able development
are strategically placed between two such actives which are of great interest in the
biodiversity hotspots viz. Arakan Yoma medical field. Andaman and Nicobar Is-
ranges of Myanmar and the Sumatra. This lands, with 1/3rd of Indias coastal line,
has resulted in unique confluence of flora supports good diversity of seaweeds and
of both these regions in ANI (Pandey and so far about 206 species including com-
Diwakar, 2008). The islands are charac- mercially important agarophytes and al-
terized by lush green forests occupying ginophytes have been reported from here.
about 81.8% of the total geographical ar- Interestingly, different parts of
ea. There are more than 2,314 species of these islands are inhabited by six native
flowering plants reported from these is- tribes since centuries. Two Mongoloid
lands so far (Murugan et al., 2016) and tribes, Shompen and Nicobarese, reside in
the number may still increase considering the Nicobar groups of islands, while the
the larger unexplored areas. Furthermore, tribes of Negrito origin i.e. Jarawa, Great
these islands are known to harbor a large Andamanese, Onge and Sentinelese, are
number of endemic species in a relatively residing in the Andaman islands. These
smaller geographical area of about 8,249 tribes differ in most of their cultures and
km2. So far, about 300 species of endemic habits. Some of the local species are pres-
plants have been reported from ANI ently being used by the native tribes for
(Murugan et al., 2016). Majority of the food, medicine, fodder, fuel and other
diversity is still unexplored and consider- purposes. Similarly, the settler population
able scope exists for utilizing these spe- migrated from different parts of mainland
cies for the betterment of humankind. India are utilizing these plants for variety
Since, horticulture has been the major of purposes. Underutilized fruits, indige-
source of livelihood and nutritional secu- nous leafy vegetables and tuber crops
rity for the island dwellers (Singh et al., have immensely contributed in the liveli-
2016), the present article focuses on the hood and nutritional security of the island
management of genetic resources of hor- dwellers. Further, a large number of wild
ticultural crops including their wild rela- relatives of cultivated crop plants have
tives. been reported to occur in these islands.
Similarly, seaweeds are important This diversity needs to be assessed and
component of the diversity of these is- utilized sustainably to strengthen our re-
lands. The macro algae mainly belonging source base, while striking the fine bal-
to Chlorophtya, Phaeophtya and Rhodo- ance between development and ecological
phyta are found attached to the substra- soundness (Waman and Bohra, 2016).
tum in benthic zone. They are non- Biotechnology could be an effec-
flowering plants with true roots, stem and tive tool for achieving this target through
leaves, and are known to contribute sub- the application of techniques namely mi-
stantially to the primary production in the cropropagation, in vitro production of
marine environment. Seaweeds have been secondary metabolites, in vitro mutation,
used for centuries as food either in raw or in vitro conservation, marker assisted se-
processed form in many of the South East lection, genetic diversity assessment, de-
Asian countries and the trend is picking velopment of trait specific markers etc.
up in the western countries as well. Sea- (Waman et al., 2015; Waman and Bohra,
weeds are the only known natural sources 2016). Present chapter concerned explor-
of hydrocolloids viz. agar, algin and car- ing the possibility of utilizing various bio-
rageenan. These multipurpose products technological tools for management of
find application in industrial, pharmaceu- biodiversity of the tropical Bay Islands of
tical and medicinal fields. Besides, sea- India.
weeds are used as animal feed and biofer-
tilizers in crop production. The current 2. Relevance of biotechnological ap-
research on seaweeds is centering on bio- proaches and tools for island ecosystem
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Biotech Sustainability (2017)
Genetic Resources Sustainability Bohra et al.
2.1. Micropropagation and in vitro con- hardening has been emphasized in low
servation price-high value crops, especially medic-
Andaman and Nicobar Islands are inal plants (Waman and Bohra, 2016).
home to many endemic species belonging In vitro conservation is a tech-
to different botanical families, which are nique, wherein plant tissues are cultured
of potential economic/ ecological signifi- in vitro under sub-optimal growth condi-
cance and require timely attention for tions in order to reduce the frequency of
their conservation. A number of species sub-culturing. The technique has proven
belonging to rare, endangered and threat- to be very efficient for short to medium
ened (RET) category are also found dis- term storage of a number of species. Con-
tributed in these islands. Some of these sidering vulnerability of the islands to
species have problems in natural regener- natural disasters, the endemic species
ation owing to the damage caused by could be conserved under in vitro condi-
birds/animals, poor seed viability, anthro- tions and copies of the same could be
pogenic pressure etc. For example, Myris- maintained at other laboratories in main-
tica andamanica is a vulnerable wild land India. Some horticulturally important
nutmeg species endemic to the islands endemic species of islands have been
and micropropagation could help to mul- listed in Table 1.
tiply it in large number. Similarly, natu-
ral populations of an underutilized fruit 2.2. In vitro production of secondary me-
species blood fruit (Haematocarpus tabolites
validus) are dwindling (Bohra et al., A large number of species are val-
2016a) and micropropagation could help ued for their medicinal properties. How-
in saving the species from extinction from ever, the yield of bioactive molecules is
the region. Experiments are in progress to very low in most of the cases. At times,
standardize micropropagation protocol for complete plants are destroyed for obtain-
this species. ing the desired active ingredients. Such
Secondly, banana is a major crop practice of destructive harvesting has
of the islands covering more than half of been a cause of concern as it tends to
the area under fruit crops cultivation. threaten the natural populations to a great
However, the islands are largely depend- extent (Waman and Bohra, 2013). Bio-
ent on the planting material supplies from technological tools could help in large
mainland India. This has probably result- scale quality production of secondary me-
ed in inadvertent introduction of dreadful tabolites under in vitro conditions. Induc-
banana bunchy top virus in the pristine tion of callus and extraction of active in-
islands. Developing protocols for in vitro gredients from them has been suggested
multiplication of locally suitable varieties as an important alternative for obtaining
would help in production of their quality the desired molecules without disturbing
planting material. The importance of tis- the wild populations (Waman et al.,
sue culture technology for the island 2015).
farmers has been emphasized earlier
(Bohra et al., 2016b). Considering this, 2.3. Creation of variability
experiments have been initiated at au- There are a few species e.g. man-
thors institute for optimization of proto- gosteen (Garcinia mangostana), which
cols for locally popular banana varieties are economically important for the islands
of the islands. Other commercializable but have narrow genetic base. For im-
crops of the islands include variety of or- provement of such crops, creation of vari-
chids and ornamental plants, which need ability is possible through the induction of
further attention. Use of low cost options somaclonal variations in tissue culture or
including concurrent ex vitro rooting cum using the technique of in vitro mutation
Table 1: List of selected endemic species of horticultural importance reported from ANI
Family Species
Anacardiaceae Mangifera andamanica, M. nicobarica, Semecarpus kurzii
Apocyanaceae Carissa andamanensis
Arecaceae Phoenix andamanensis
Clusiaceae Garcinia andamanica, G. cadelliana, G. calycina, G. dhanikhariensis,
G. kingii, G. kurzii, G. microstigma
Dilleniaceae Dillenia andamanica
Dioscoreaceae Dioscorea vexans
Musaceae Musa bulbisiana var. andamanica, M. indandamanensis, M. sabuana,
M. paramjitiana
Myristicaceae Myristica andamanica, Knema andamanica
Myrtaceae Syzygium andamanicum, S. manii
Pandanaceae Pandanus lerum var. lerum
Tiliaceae Grewia indandamanica
Orchidaceae Vanilla andamanica, Eulophia nicobarica
Zingiberaceae Kaempfaria siphonantha
breeding. Being cornerstone of breeding species, which play pivotal role in the
activity, variability created will also be lives of island dwellers. The generated
useful in development of island suitable information from such studies could be
varieties. useful for selection of parents for carrying
out conventional crop improvement pro-
2.4. Estimation of genetic diversity grammes.
The ANI is considered as a centre
of origin or diversity for a number of spe- 2.5. Evolutionary studies
cies e.g. wild populations of Piper betle From time to time, a number of
are present in these islands. Both inter and new species have been reported by vari-
intra specific diversity occurs for a num- ous research workers in the ANI, howev-
ber of species of ecological and economic er, absence of requisite scientific infor-
importance (Singh et al., 2016). This di- mation about their genetic relationship
versity needs to be tapped in such a way with their existing commercial counter-
that commercially viable types are identi- parts could delay the process of their
fied for the benefit of island farmers. Se- commercial utilization. Molecular phylo-
lection of such elite types needs systemat- genetic studies could help in this regard.
ic characterization. Molecular characteri- For example the presence of Indo-
zation is one of the most reliable methods Myanmarese as well as Indonesian forms
of estimating such diversity. Further, of Erianthus arundinaceus (Retz.)
available diversity could also be com- Jeswiet, a wild relative of sugarcane
pared with their counterparts occurring in (Saccharum officinarum) was confirmed
mainland India or other parts of the through the use of molecular markers
world. Through this information, the stud- (Nair and Mary, 2006). They concluded
ied population could be categorized in a that collections from North Andaman
way to pave the way for future breeding were more similar to Indo-Myanmarese
programmes. A few attempts were initiat- form, while that from Nicobar were of
ed at the authors Institute or other organ- Indonesian form. This report supports the
izations in country in this direction, which fact that ANI harbor confluence of flora
have been summarized in Table 2. The of two different regions. Similarly, genet-
similar technique could also be employed ic diversity between the collections of
for diversity estimation and further stud- Musa balbisiana from mainland India and
ies in non-traditional and underutilized the islands suggested that ANI is one of
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Biotech Sustainability (2017)
Genetic Resources Sustainability Bohra et al.
the centres of diversity of the species fied during early stages of development.
(Uma et al., 2005). This new piece of in- In case of medicinal plants, identification
formation would go a long way in devis- of markers linked to the presence of their
ing conservation strategy of Musa spp. active ingredients would be very useful.
from ANI. Flowering behavior related markers in
mango have been identified, which could
2.6. Development of trait linked markers be of practical utility (Damodaran et al.,
As previously mentioned, a num- 2006).
ber of perennial dioecious species are
known to occur in the islands. Important 3. Applications of biotechnological in-
being Myristica andamanica, Knema an- terventions in seaweeds
damanica, Horsfieldia glabra, Piper
betle, Carica papaya, Garcinia spp., In the wake of increasing demand
Momordica spp. etc. Development of sex for seaweeds for various applications, col-
linked markers would be a boon for the lections from the natural habitats are not
conservation and utilization of these spe- sufficient to meet the requirement. Artifi-
cies as the desired plants could be identi- cial culture of commercially exploited
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Biotech Sustainability (2017)
Genetic Resources Sustainability Bohra et al.
seaweeds still remains the major means of be still in nascent stage. In vitro derived
raw material supply to the industries. calli of economically important seaweeds
However, on farm culture of seaweeds have been commonly used for mainte-
using vegetative fragments generally re- nance and clonal propagation of seed
sults in reduced growth rate and produc- stock for mariculture (Dawes and Koch,
tivity over time. In order to overcome the- 1991; Reddy et al., 2003; Rajakrishna et
se problems and to develop strains with al., 2004; Reddy et al, 2008). Studies
improved growth and yield parameters, suggested that growth and quality of car-
biotechnological techniques such as mi- rageenan obtained from tissue cultured
cropropagation, transgenics and molecu- Kappaphycus alvarezii were superior
lar markers have been tried in seaweed when compared with conventional vege-
breeding and genetic studies. Microprop- tative fragments (Rajakrishna et al.,
agation is a tool to produce large number 2007). Non-availability of standardized
of seeding material from explants of sea- protocols for obtaining viable axenic cul-
weed possessing desirable traits. Among tures from wild, lack of knowledge about
the three cellular organization types in the role of culture incubation conditions,
seaweeds, most of the work on micro- media supplements, explanting season
propagation has been reported on paren- etc. on callus induction are the major lim-
chymatous and pseudo-parenchymatous iting factors in the development of sea-
types (Aguirre-Lipperheide et al., 1995). weed micropropagation. Table 3 repre-
Although Gibor attempted to cultivate sents list of selected seaweed species re-
seaweeds axenically in as early as 1950, ported from ANI in which micropropaga-
the first successful attempt is considered tion has been attempted elsewhere.
to be made during 1978 by Chen and Tay- Molecular marker assisted breed-
lor in Chondrus crispus (Yokoya and ing based on quantitative trait loci (QTLs)
Valentin, 2011). Seaweed micropropaga- offers several advantages over traditional
tion protocols have been developed in phenotypic based breeding. Development
similar lines with that of higher plants and of markers linked with genes of desirable
so far micropropagation protocols have traits could increase accuracy and effi-
been developed in about 85 species (Red- ciency of the breeding process. Several
dy et al, 2008). Considering the diversity molecular markers such as Random Am-
present and diversified applications of plified Polymorphic DNA (RAPD), Inter
seaweeds, the technique is considered to Simple Sequence Repeat (ISSR), Se-
Table 3: List of seaweed species reported from ANI in which cell and tissue culture has
been accomplished elsewhere
Seaweed Reference
Chlorophyta Boergesenia forbessi Enomoto and Hirose, 1972
Bryopsis plumosa Tatewaki and Nagata, 1970
E. intestinalis Polne-Fuller and Gibor, 1987; Russig and Cosson,
2001
Rhodophyta Gracilaria corticata Subbaraju et al., 1981; Rajakrishna et al., 2007
G. verrucosa Gusevet al., 1987; Kaczyna and Megnet, 1993
Gelidiella acerosa Rajakrishna et al., 2004
Phaeophyta Grateloupia filipina Huang and Fujita, 1997; Baweja and Sahoo, 2009
Hypnea musciformis Rajakrishna et al., 2007
Sargassum tenerri- Rajakrishna et al., 2007
mum
Turbinaria conoides Rajakrishna et al., 2007
Abstract: Antibodies are the important part of adaptive immune system. Plants do not natu-
rally make the antibodies; but, they can be produced in plants by introducing antibody-
coding genes from humans and animals. Plant derived antibodies are called as plantibodies
and known to work in the same way as mammalian antibodies. The plantibodies bioproduc-
tion offers several advantages over the production of antibodies using mammals. Plants
are more economic than all other forms of creating antibodies and the technology for ob-
taining and maintaining them is already present. Plantibodies are safer in use because,
plants reduce the chance of coming in contact with pathogens. Plantibodies can be made at
an affordable cost using plants as the genetic engineering methods are well established for
agricultural crops such as tobacco, tomato, potato, soyabean, alfalfa, rice, and wheat. Plan-
tibodies production is cost effective and safe. This review highlights the methods of produc-
tion and purification of plantibodies as well as the various types of pharmaceutical antibod-
ies produced in transgenic plants.
ability of plant cells to correctly fold and cells with the cost of plantibody produc-
assemble antibody fragments, single chain tion in plants are substantially lesser
peptides and also full-length multimeric (Oluwayelu et al., 2016). Different types
proteins and heterologous proteins such as of plantibodies are produced from plants
antibodies accumulate in large amount to (Figure 1).
plant cells. Protein synthesis, secretion
and folding as well as posttranslational
modifications like signal peptide cleav-
age, di-sulphide bond formation and the
initial stages of glycosylation are very
similar in plants and animals. There is
very low risk of product contamination by
mammalian viruses, bacterial toxins,
blood-borne pathogens and oncogenes Figure 1: Different types of plantibodies
(Jain et al., 2011). Use of these plantibod- are produced in plants.
ies avoids the ethical issue of producing
transgenic animals, elimination of purifi- 2. Plants used as expression host for
cation requirement when the plant con- plantibody production
taining recombinant protein is edible and
production of disease resistant plants by A range of different plant systems
raising antibodies in them. The plantibod- have been developed for antibody produc-
ies bioproduction process offers several tion (Table 1). The choice of expression
advantages over the conventional method
of antibody production in mammalian
Table 1: Pharmaceutical antibodies produced in transgenic plants
Antigen Plant Antibody form Application Reference
Human chorion- Tobacco scFv, diabody, Diagnos- Kathuria et al.,
ic Gonadotro- chimeric, IgG1 tic/contraceptive 2002
phin
Glycophorin Barley, po- ScFv-fusion Diagnostic (HIV) Schunmann et
tato, tobac- al., 2002
co
Streptococcus Tobacco SigA/G Therapeutic (topical Ma et al., 2003
surface antigen (CaroRx)
SAI/II
Sperm Corn IgG Contraceptive (topi- Cone and
cal) Whaley, 2002
Rhesus D Arabidop- IgG Diagnostic Bouquin et al.,
sis 2002
Human IgG Alfalfa IgG Diagnostic Khoudi et al.,
1999
Rabies Tobacco IgG Therapeutic Ko et al., 2003
Herpes simples Soyabean, IgG Therapeutic (topi- Zeitlin, 1998
virus rice cal)
CD40 Tobacco ScFv- Therapeutic Francisco et al.,
cell culture immunotoxin 1997
fusion
Herpes simplex Algae One-chain anti- Therapeutic Mayfield et al.,
virus chlamydo- body 2003
monas
chloroplast
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Biotech Sustainability (2017)
Plant Tissue Culture for Sustainability Minakshi et al.
Table 1: Continued
Antigen Plant Antibody form Application Reference
Colon cancer Tobacco IgG Therapeutic Verch et al.,
antigen /Diagnostic 1998
Carcinoembry- Tobacco, scFv, diabody, Therapeu- Stoger et al.,
onic antigen rice, wheat, chimeric, IgG1 tic/Diagnostic 2002
(CEA) pea, tomato
Herpes simplex Corn sIgA Therapeutic Hood et al.,
virus 2002
Non-Hodgkins Tobacco scFv Personalized vac- McCormick et
lymphoma idi- Virus vec- cines al., 2003
otypes tor
Clostridium dif- Corn IgG Therapeutic (oral) www.epicyte.co
ficile m
Hepatitis B vi- Lettuce IgG Vaccine Kapusta et al.,
rus 1999
New castle dis- Corn Surface glyco- Vaccine Guerrero-
ease virus protein F Andrade et al,
2006
Cholera Tomato Cholera toxin B Oral vaccine Jiang et al.,
subunit (ctb 2007
gene)
Enterovirus Tomato Serum IgG VP1 Oral vaccine Chen et al.,
2006
Porcine repro- Banana IgG and IgA Oral immunization Chan et al.,
ductive and res- 2013
piratory syn-
drome virus
system depends upon many factors such crop several times in a year (Fischer et
as suitability for scale-up, storage and al., 2003). Tobacco grows quickly and
downstream processes. Other considera- has been shown to produce comparatively
tions attributed to host choice are antici- large quantity of antibodies. Additionally,
pated production scale, the value and use tobacco is a non-food/non-feed crop if
of the product, geographical area of pro- grown separately; there is less chance of
duction, proximity of processing facility, cross-contamination food chain by phar-
biosafety, intellectual property right and maceuticals (Schillberg et al., 2002).
economical aspects (Twyman et al., Chloroplast engineering in tobacco, an
2003). Several works have shown that alternative to nuclear transgenics, trans-
tobacco, potatoes, soya beans, corn, alfal- plastomic plants are produced by intro-
fa and similar kind of crops are the alter- ducing DNA into the chloroplast genome
native ways for production of recombi- instead of nuclear genome, and this will
nant proteins (Hiatt et al., 1989; Mason be achieved by particle bombardment
and Amtzen, 1995). Figure 2 showed that (Daniell et al., 2002). Chloroplast trans-
many benefits of plants used for planti- formation is more advantageous, includes
body production. high transgene copy number, absence of
position effects and transgene silencing.
2.1. Tobacco Combination of these properties leads to
Among leafy crops tobacco have extraordinary levels of expression, ex-
the greatest biomass yield per hectare and ceeding 25 percent of the total soluble
allow rapid scale up because they can protein (Tregoning et al., 2003). Other
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Biotech Sustainability (2017)
Plant Tissue Culture for Sustainability Minakshi et al.
benefit of chloroplast engineering in- which was immunized with leaf extracts
cludes the ability to express several genes conferred specific antibodies (Shao et al.,
as operons and the accumulation of re- 2008).
combinant protein in the chloroplast,
which reduces the toxicity of host plant. 2.2. Alfalfa and other legumes
Biologically active and structurally accu- Another leafy crop used to pro-
rate Human growth hormone and serum duce recombinant antibodies are alfalfa
albumin produced at high levels in tobac- and soya bean. Alfalfa is a perennial crop
co chloroplasts (Staub et al., 2000; Fer- can propagated easily and also have good
nandez et al., 2003). Recently tetanus tox- biomass yield. The Biotechnology Com-
in fragment (Tregoning et al., 2003) and pany Medicago selected it as a platform
cholera toxin B subunit (Daniell et al., technology. The strong advantage of this
2001) has been expressed in tobacco chlo- crop is its tendency to synthesize homog-
roplast and was shown that tetanus toxin enous N-glycans, which improves the
fragment induce protective levels of anti- consistency of recombinant proteins batch
tetanus antibodies and cholera B subunit to batch (Bardor et al., 2003). One of the
shows that plastids can fold and assemble potential advantages of alfalfa is that re-
oligomeric proteins perfectly. Fischer et combinant antibodies produced as a single
al. (1999) reported that recombinant pro- glycoform rather than heterogeneous col-
teins can also be produced from tobacco lection of different glycoforms that is
cell culture and several recombinant pro- found in other plant systems. Pea, a grain
teins including antibody derivatives were legume also a useful production crop, rea-
derived from a suspension cell line of to- son that of its high protein content of
bacco strain BY-2. The expression of seed. Although at present only low yield
classical swine fever virus E2 protein was is possible with this species (Perrin et al.,
expressed in tobacco chloroplast elicited 2000).
protective immune response in mice
dental carries (Larrick et al., 2001). Treatment or cure for rabies through plan-
CaroRx is intended for regular topical tibodies has been investigated by Ko et al.
preventive administration by both dental 2003. A plantibody based rabies vaccine
hygienists and patients allowing a thor- produced in tobacco was experimentally
ough cleaning and intervention for any administered in hamsters to check wheth-
existing decay. Plantibodies have been er it could effectively target rabies. The
investigated for inflammatory disease and plantibody proved to be safe and econom-
to induce tolerance (Jain et al., 2011). A ically feasible alternative method com-
humanised antibody, another plantibody pared to the current antibody production
with human medical application was ex- in animal systems. Another study, tobac-
pressed in soya bean against herpes sim- co-derived plantibodies were experimen-
plex virus (Zietlin et al., 1998). Anti- tally administered in mice against the
tumour antibodies against Burkitt`s lym- Lewis Y antigen found on tumour cells in
phoma was expressed in rice and wheat mice and also in lung, breast, ovarian and
(Ghasempour et al., 2014). Antibodies colorectal cancer. According to Brodzick
engineered to bind to Bacillus anthracis et al (2006), the plantibodies showed a
was extracted from transgenic strains of definite positive effect on the cancer-
tobacco and tested in mice in a study con- striken mice by preventing tumour for-
ducted by Hull et al in 2005. The result of mation in them (Figure 3).
this study showed that the antibodies were
effective in fighting B. anthracis strain 5.2.1. Immunization
and bodes well for the future in any an- One of the most interesting appli-
thrax epidemic will be a cheap and effec- cations of this technology is production of
tive prevention against the disease. edible vaccines or oral vaccines. Produc-
Potential proteins such as cytokines, hor- progress has been made towards engineer-
mones, enzymes, epidermal growth fac- ing residence against insects (Schillberg
tors, interferons, human protein C, and et al., 2001; Jaeger et al., 2000). Im-
pharmaceutical foodstuff are produced, munomodulation is a dynamic tool for
which are considered for oral immuniza- altering the function of an antigen in vivo.
tion (Mason and Amtzen, 1995). Trans- When an artificial abscisic acid sink was
genic plants that express antigens might created by the production of an anti- ab-
be used as an inexpensive oral vaccine scisic acid specific scFv in the endoplas-
production and delivery system, so im- mic reticulum of potato and tobacco
munization might be possible through plants, both physiological and morpholog-
consumption of an edible vaccine to pro- ical changes were noticed (Conrad and
vide immunization. Due to all these rea- Manteuffel, 2001). Moreover, agro-
sons transgenic plants are considered as filtration of tobaco was used to produce a
better alternative for oral vaccines. This diabody against carcinoembryonic antigen
offers convenient immunization strategies (Vaquero et al., 2002). In addition, plan-
for implementing universal vaccination tibodies may also prove useful as feed
programmes throughout the world (Tack- additives or for phytoremediation in hu-
et et al., 1998). In human host the patho- man health care (Mason and Arntzen,
gens usually attack mucosal sites in the 1995).
respiratory tract, gastro-intestinal tract or
genital tract. Stimulation of immune re- 6. Pathogen resistance in plants
sponses in these sites through mucosal
vaccines to protect against illness is desir- Antibody mediated pathogen re-
able and this can be achieved by applying sistance in plants is a novel strategy for
vaccine to the mucosal surface directly, generating transgenic plants resistant to
inducing systemic and cellular immune pathogens have been developed in many
responses as well as local immune re- cases for therapeutic applications, for
sponses at the initial site of interaction Immunomodulation. Peschen et al.,
between the pathogen and host directly (2004) demonstrated antibody-mediated
(Kusnadi et al., 1997). Oral vaccines resistance against fungal pathogens and
must be protected during passage through protecting plants against fungal diseases.
the hostile environment of the stomach Schillberg et al (2000), targeted anti-
and intestine to the sites of immune TMV antibodies to the plasma membrane
stimulation. in vivo (in planta) results in evolvement
of transgenic plants resistant to TMV in-
5.2.2. Immunomodulation fection. A study conducted by Boonrod et
Immunomodulation is a technique al (2004), paved the way for engineering
that allows interference with cellular me- broad-range virus resistance by expres-
tabolism, signal transduction or pathogen sion of scFv antibodies in vivo that are
infectivity by the ectopic expression of specific and highly conserved motifs in
gene encoding antibodies or antibody viral replicase or polymerases. Boonrad
fragments (Jaeger et al., 2000). Applica- and his colleagues demonstrated that
tions that are relaying on modulating an- transgenic tobacco plants expressing scFv
tigen levels in vivo are dependent on ex- antibodies against a conserved domain in
pression and accumulation in specific sub plant viral RNA dependent RNA poly-
cellular compartments and specific tis- merase either in the cytosol or endoplas-
sues. Development of crop resistance and mic reticulum, showed high levels of re-
passive immunization of plants by expres- sistance to four plant viruses from differ-
sion of pathogen-specific antibodies re- ent genera. Malembic-Maher et al. (2005)
duces infection and symptoms caused by studied the scFv 2A10 protein expression
viruses and mollicutes, and significant in engineered tobacco plants and for their
ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 313
Biotech Sustainability (2017)
Plant Tissue Culture for Sustainability Minakshi et al.
resistance to stobular disease. It was ob- na benthamiana, was used for the produc-
served that tobacco plants producing se- tion of mAb (6D8) that protected animals
creted scFvs shown a short delay in symp- from Ebola virus infection (Chen et al.,
tom appearance, reduction to pathogen 2002). An Ebola immune complex (EIC)
susceptibility and phyloplastoma multi- was produced by fusing Ebola glycopro-
plication. Isolated antipectinase scFv an- tein GP1 to the C-terminus of the heavy
tibodies directed against extracellular pro- chain of humanized 6D8 mAb that binds
teins from Rhizoctonia solani secluded specifically to a linear epitope on GP1
from a page display library. Soluble scFv using the geminivirus-based expression
antibodies shown to inhibit polygalac- system and Nicotiana benthamiana (Bhoo
turonase in the culture supernatants of a et al., 2011). The recombinant immuno-
range of fungal pathogens such as asco- globulins were produced in leaves of Ni-
mycetes, basidiomycetes and oomycetes. cotiana benthamiana, which was purified
This soluble antibody also inhibited mac- by protein G affinity chromatography.
eration in potatoes (Manatunga et al., The resultant recombinant antibody
2005). First time antibody mediated fun- bound the the complement facto C1q, in-
gal resistance was demonstrated by Wu et dicating immune complex formation.
al in wheat and cereal grains. This fungal Therefore subcutaneous immunization of
resistance in transgenic plants is mediated mice with purified EIC showed high level
by generating specific antibodies against of production of anti-Ebola virus antibod-
Fusarium graminearum, a predominant ies that provides protection against Ebola
fungal species infecting wheat and small virus (Figure 4). This was the first pub-
cereal grains in china (Wu et al., 2005). lished report of an Ebola virus candidate
vaccine to be produced in plants.
7. Treatment of Ebola patients The Ebola virus disease outbreak
in West Africa has provided a great op-
Recently, antibodies against Ebola portunity for the use of plantibodies in
virus have been explored in plants. A resolving global human health challenges
high yielding geminivirus-based expres- as
sion system in the tobacco plant, Nicotia-
cines, which are cheap and can be easily and Vezina, L.P. (2003). Monoclo-
administered. Plants can be easily ex- nal C5-1 antibody produced in trans-
plored by pharmaceutical industries and genic alfalfa plants exhibits a N-
Indian climate helps for the production of glycosylation that is homogenous
diversified crops. Engineering of in- and suitable for glyco-engineering
creased pathogen resistance and alteration into human compatible structures.
of phenotypes by immunomodulation Plant Biotechnology Journal 1, 451-
provide a great platform to develop the 462.
prevention strategies. Various other strat- Begley, S. (2014). Tobacco-derived 'plan-
egies have been developed to exploit tibodies' enter the fight against Ebo-
plants as bioreactors for the production of la.
pharmaceutical antibodies and many plant http://www.reuters.com/article/2014/
produced antibodies are proved increased 08/06/healthebolatobaccoidUSL2N0
disease resistance. Recent developments QB24N20140806.
focus on the detailed characterisation of Bhoo, S.H., Lai, H., Ma, J., Arntzen,
recombinant products. Recent indications C.J., Chen, Q. and Mason, H.S.
that tissue specific and physiological fac- (2011). Expression of an immuno-
tors may have an impact on the quality genic Ebola immune complex in Ni-
and glycosylation pattern of a plantibod- cotiana benthamiana. Plant Biotech-
ies will perhaps lead to new insights and nology Journal 9, 807 -816.
production strategies. Although some Boonrod, K., Galetzka, D., Nagy, P.D.,
plant-derived antibody products have suc- Conrad, U. and Andkrczal, G.
cessfully completed early phase clinical (2004). Single-chain antibodies
trials, several issues including regulatory against a plant viral RNA-dependent
guidelines and public acceptance must RNA polymerase confers virus re-
still be resolved. Currently, more than 200 sistance. Nature Biotechnology 22,
novel antibody-based potential products 856-862.
are in clinical trials worldwide, and mar- Bouquin, T., Thomsen, M., Nielsen,
ket demand will certainly strain the capa- L.K., Green, T.H., Mundy, J. and
bilities of existing production systems. Hanefeld, D. (2002). Human anti-
Moreover, adoption of plants as bioreac- rhesus D IgG1 antibody produced in
tors on a larger scale would reduce the transgenic plants. Transgenic Re-
cost of antibody therapy and simultane- search 11, 115-122.
ously increase the number of patients who Brodzick, R., Glogowska, M., Ban-
access to these treatments. durska, K., Okulicz, M., Deka, D.
and Ko, K. (2006). Plant-derived
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Malembic-Maher, S., Le Gall, F., (2004). Fusion proteins comprising a
Danet, J.L., Borne, F.D., Bove, Fusty-nun-specific antibody linked to
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ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 319
Biotech Sustainability (2017)
Plant Tissue Culture for Sustainability Minakshi et al.
Abstract: The continued use of fossil-derived fuels has resulted in progressive depletion of
non-renewable energy resources and environmental deterioration that led to the develop-
ment of an alternative renewable source of energy for sustainability. Agricultural and horti-
cultural crops waste processing offer an option among alternatives for generating renewable
energy (bio-refinery) due to its potential sustainable supply and abundance. The conversion
of agro-industrial processing wastes typically contains mixed hexoses and pentoses with
significant proportions of polymeric sugars (lingo-celluloses and cellulose) which are diffi-
cult to degrade and require advances in the technology. This article is highlighting the ma-
jor developments in various biomass-based agro-industrial processing waste-based fuel-
generating processes and suggests a possible major solution to provide energy (bio-energy)
in an eco-friendly way for the reduction of greenhouse gases and pollution.
es (rice husk, coffee wastes, sugar cane losses occur at retail and consumer levels.
biogases, maize harvesting wastes, and Among them, lignocellulosic wastes de-
bamboo cellulose pulp, etc.,) at the retail rived from cereals, oil seeds, pulses and
and consumer levels, of which the post- plantation crops, account for 222 million
harvest and processing level wastages ac- tons in industrialized countries. Fruits,
count for the major share (Singh et al., vegetables and root crop processing
2012).The different residues/biomass re- wastes alone are available to the extent of
sulting from the production of agricultural 12 million tons (Auer et al., 2017). How-
crops might contribute to the achieve- ever, over 86 x 106 t of fruits and 162 x
ments of the renewable energy target as 106 t vegetables are produced annually in
proposed for further uses (Scarlat et al., India, contributing 12.6% and 14.0% of
2010). The use of biomass for transport the total world production of fruits and
fuel, heat and electricity production will vegetables respectively (Source FAO
have to increase substantially to meet the website- February 2014-15; Horticulture
proposed binding target of renewable en- Database, India-2015). Out of the total
ergy in the EU energy mix of 20% by production, nearly 76% is consumed in
2020 (Scarlat et al., 2010). The chapter fresh form, while wastage, and loss ac-
discusses the agricultural/horticultural count for 20-22 percent. Of which only 2-
resources and food processing wastes for 4% is processed in the fruits and vegeta-
potential production of bioenergy. bles processing industries. This is in sharp
contrast to the extent of processing of
2. Agricultural and food processing fruits and vegetables in several other de-
wastes veloping countries such as Brazil (70%),
Malaysia (83%), Philippines (78%), and
There are various forms of agricultural Thailand (30%) (Horticulture Database,
and horticultural (agro-industrial) re- India- 2015). Disposal of these putresci-
sources in the world. Among the agro- ble fruits and vegetables processing
industrial wastes, horticulture processing wastes (organic refuse) leads to environ-
industries form a major share throughout mental and economic problems (Viswa-
the world. Horticultural wastes mainly nath et al., 1992; Scano et al., 2014). The
generate from fruits and vegetable pro- nutrients of food waste may be re-used in
cessing, starchy roots and tubers, coconut, agriculture by composting or by biotrans-
olive and palm oil mills and fruit-based formation of food waste into animal feed
fermentation industries (Panda and Ray, and/or converted to biofuels (Table 1).
2015; Panda et al., 2017). The fermenta- These wastes contain a high amount cel-
tion industries of solid waste include lulose, hemi-cellulose, and pectin, which
items removed from fruits and vegetables provide a suitable substrate for fermenta-
during cleaning, processing, cooking, tion process (Khalid et al., 2011). These
and/or packaging. These items may in- polymers can be hydrolyzed enzymatical-
clude leaves, peels, pomace, skins, rinds, ly by cellulose, -glucosidase and pa-
cores, pits, pulp, stems, seeds, twigs, and tience to their corresponding soluble car-
spoiled fruits and vegetables (Bouallagui bohydrates and subsequently to biofuels
et al., 2005; Panda et al., 2016). Globally, (Ariunbaatar et al., 2014; Behera and Ray
more than billion tons of agro- industrial 2016).
and food processing solid wastes are
available. The losses in industrial coun- 2.1.Bio-ethanol production
tries are as high as in developing coun- Bio-ethanol has been proved to be
tries, but in developing countries more a most promising alternate energy source
than 40% of losses occur at post-harvest with various added advantages (Manzano-
and processing levels, while in industrial- Agugliaro et al., 2013). The worldwide
ized countries, more than 40% of the
Table 1: Current status of biofuel technologies utilizing agricultural and horticultural sub-
strates and wastes. (Lynd et al., 2015, modified)
No Crop Industry status
Example
category
1. Starch-rich Maize, wheat, About 50 billion L ethanol in the US based on
sorghum maize
2. Sugar -rich Sugar cane, sug- About 23 billion L ethanol in Brazil based on sug-
ar beet ar cane
3. Oil-rich Rapeseed, soy, About 23 billion L produced worldwide, mostly in
sunflower, palm the EU, US and Brazil
oil
4. Cellulosic Grass trees, vari- Liquid fuel capacity about 175 million L world-
ous horticultural wide
wastes
ids (Table 2). Microbes are well-suited beet, molasses and fruits) converted into
natural agents for recycling of organic ethanol directly. The starches (corn, cas-
wastes including VFWs (Zupancic and sava, potatoes, and root crops) are initial-
Grilc, 2012; Panda et al., 2016). The mi- ly hydrolyzed to fermentable sugars by
croorganisms such as Bacillus, Pseudo- the action of enzymes (Ray and Naskar,
monas, Rhizopus and Trichoderma, well 2007; Ray et al., 2008). Celluloses (agri-
known for production of hydrolytic en- cultural residues, wood, waste sulfite liq-
zymes, are employed for biodegradation uor from pulp and paper mills) are con-
of VFWs (organic matter) to reduce the verted into sugars by the action of mineral
biological oxygen demand and chemical acids. The simple sugar so formed, can
oxygen demand of the liquid wastes readily fermented to ethanol by the action
(Panda and Ray, 2015). More specifically, of microbial enzymes (Lin and Tanaka,
microorganisms, such as fungi (Mucor 2006; Monlauet al., 2013).
indicus), bacteria (Zymomonas mobilis),
and yeasts (Candida utilis, Kluyueromy- 2.2.1. Anaerobic digestion and methano-
ces marxianus) have been used for etha- genesis
nol production from VFWs. However, Anaerobic digestion is a biochem-
yeast is most common microorganisms ical degradation process that is widely
grown on VFWs as substrates (Stabniko- used for the treatment and energy recov-
va et al., 2005).Among the yeast, Can- ery from many kinds of biomass, espe-
dida utilis is selected for cultivation of cially agricultural products and agro-
concentrated effluents of the food indus- industrial wastes (Scano et al., 2014).
try after its anaerobic acidogenic treat- Hydrolysis and acidification are the ad-
ment (Stabnikova et al., 2005). In con- vantages in two-phase in anaerobic diges-
trast, Saccharomyces cerevisiae is the tion processes. FVWs are characterized
most commonly used yeast in industrial by a high percentage of moisture (>80%),
ethanol production from red beet (juice high organic content (volatile solids>95%
and bagasse) (Jimenez-Islas et al., 2014). of total solids), are readily biodegraded
Moreover, biotransformation of cellulose- and are therefore suited to energy recov-
to-ethanol can be conducted by various ery through anaerobic digestion (Jiang et
anaerobic thermophilic bacteria, such as al., 2012). Scano et al. (2014) studied the
Clostridium thermocellum, and some fil- biogas production through an anaerobic
amentous fungi, including Monilia sp., digestion pilot plant by using VFWs as
Neurosprora sp., Zygosaccharomyces single substrate. The experimental study
rouxii, Aspergillus sp., Paecilomyces sp., was carried out using most suitable oper-
and Trichoderma viride (Lin and Tanaka, ating parameters and optimum organic
2006). Others efficient microbes and ge- loading rate was reported at 2.5-3.0 kg
netically modified microbes may also en- volatile solids/m3d with maximum biogas
hance the enzymatic hydrolysis. The ap- production of 0.78 Nm3/kg volatile solids
proach of using depolymerizing enzymes (CH4 content of 55%).
producing co-culture or construction of
engineered microorganisms are attractive 2.3. Factors affecting biogas production
for low capital cost and enhanced yield of Various aspects/factors influence
fermentable sugars (Arora et al., 2015). the biogas production from the feed
stocks. The C: N ratio, pH, temperature,
2.2. Fermentation process total solid contents, organic loading rates,
The varied raw materials used in hydraulic retention time, design of digest-
the fermentation are classified into three er, inoculum quality and volatile fatty ac-
categories: sugars, starches, and cellulose id content also regulate and impact biogas
materials (Mohapatra et al. 2017). The production.
main sources of sugars (sugar cane, sugar
Table 2: Continued..
(LNH-ST)
2.3.1. Carbon to nitrogen (C: N) ratio ture has significant effect on the microbial
A C/N ration between 22 and 25 community, process kinetics and stability
seemed to be better for anaerobic co- and methane yield (Patil and Deshmukh,
digestion of FVWs with its co-substrates. 2015). The high temperature cooking
The most significant factor for enhanced (140-180oC) is very effective for fermen-
FVWs digestion performance is the im- tation of ethanol from starchy materialin
proved organic nitrogen content provided industrial-scale. The high temperature
by the additional wastes (Bouallagui et enhances the efficiency of starch sacchar-
al., 2009). ification and achieves high levels of etha-
nol under complete sterilization of harm-
2.3.2. PH ful microorganisms (Krishania et al.,
The optimum pH and pH range 2013). However, to resolve the difficul-
differs with substrate and bio methanation ties of high production costs and require-
technique. The optimum pH values for ment of additional amounts of enzymes
the acidogenesis and methanogenesis (amylase), non-cooking and low-
stages are different. During acidogenesis temperature cooking fermentation system
stage, lactic, propanoic, and acetic acids have been recently developed (Ghimire et
are formed and thus, the pH falls. The low al., 2015).
pH (about 6.4) can be toxic for methane-
forming bacteria (optimum between 6.6- 2.3.4. Total solid content
7.0). Most of digested carbohydrate sub- Total solid content influences bio-
strates are acidic and developing pH of gas production from fruits and vegetable
6.2 or less and thus becoming toxic wastes. Abbassi-Guendouz et al. (2012)
(Chanakya and Malayil, 2012). A suitable investigated the role of total solid content
amount of lime is added to neutralize the in anaerobic digestion in batch reactors.
acid accumulated in the carbohydrate res-
idues of processing digesters. 2.3.5.Organic loading rate /Volatile sol-
ids
2.3.3. Temperature The organic loading rate deter-
The temperature is an important mines the input of organic matter per unit
parameter for bio methanation. Tempera- volume of digester capacity per day
with the methane yield of 240 mL-CH4/g genes eutrophus and Bacillus licheniform-
volatile solids-added. is when held under anoxic conditions, can
produce hydrogen from organic sub-
3.2. Acetone-butanol-ethanol production strates/wastes (Sivagurunathan et al.,
There is also renewed interest in 2016).
reviving the acetone-butanol-
ethanolprocess through application of the 3.4. Biodiesel
recombinant strains (Ltke-Eversloh and The technology implemented for
Bahl, 2011) and process development and production of liquid biofuels is based on
using cheaper agricultural transformation of food-grade biomass
wastes/substrates (Green, 2011). The bio- (carbohydrates) into bioethanol and vege-
conversion of lignocellulosic sub- table oils into biodiesel fuel. The main
strate/wastes to monomeric sugars and its sources of juices of sugar cane, sugar
consequent fermentation has been sug- beet, and sweet sorghum, as well as
gested for economic production of ace- starches of corn, wheat, potato, and some
tone-butanol-ethanol (Amiriet al., 2014). other agricultural plants (Ioelovich,
A variety of bacterial strains, such as 2015). Oil-seed crops are the largest
Clostridium aurantibutyricum, C. bei- sources of exploitable biomass to produce
jerinckii and C. butyricumparticipatein liquid fuel, bio-diesel (i.e., fatty esters).
acetone-butanol-ethanol production and Bio-diesel offers enhanced safety charac-
utilize a variety of substrates including teristics as compared to diesel fuel, hav-
pentose, hexose, starch, and xylan but not ing no emission of explosive air/fuel va-
cellulose (Bellido et al., 2014). Further, pors (Bhuiya et al.,2014; Kumar and
development can be directed by manipu- Sharma, 2015). Considerable research has
lating and controlling the fermentation been progressed on the use of vegetable
conditions by reducing the toxic effect of oils as diesel fuel. Vegetable oils such as
products (repression) on cell physiology soybean oil, sunflower oil, coconut oil,
and promoting one dominant solvent rapeseed oil, Tung oil, and palm oil are
product during production of acetone- the best choice (Carlsson, 2009). The
butanol-ethanol. most common way to produce bio-diesel
is by transesterification, which refers to a
3.3. Microbial hydrogen production catalyzed chemical reaction of vegetable
Hydrogen is produced by several oil and an alcohol to yield fatty acid alkyl
processes, such as electrolysis of water, esters (i.e., biodiesel) and glycerol (Sha-
thermocatalytic reformation of hydrogen- hid and Jamal, 2011). Indigenous to cen-
rich organic compounds, and biological tral-south America, Jatropha was intro-
processes. Currently, biological produc- duced to Africa a few centuries ago. It is
tion of hydrogen (bio-hydrogen) from currently widely distributed throughout
horticultural residues, using microorgan- these areas where rural inhabitants gener-
isms, is an exciting new area of technolo- ally make extensive use of it. Oil from the
gy development (Levin et al., 2004). seeds of jatropa is used as a bio-diesel
Asian countries possess significant poten- substitute (Osseweijeret al., 2015).
tial for producing bio-hydrogen from crop
residues. Bio-hydrogen production by 4. Challenges and further prospective
culture of bacteria is highly attractive for
larger-scale applications (Kumar et al., The production of bioenergy and
2015). Microbes, including strict anaer- food production is interrelated and is af-
obes (clostridia, ruminococci and ar- fected by global change of atmospheric
chaea) and facultative anaerobes, includ- (rising CO2 and tropospheric
ing Escherichia coli and Enterobacter ozone),climate (temperature and soil
aerogenes and aerobes, including Alcali- moisture), and land degradation (saliniza-
Abstract: Agricultural inputs and farming systems are playing vital role in the consumption
of fossil fuels and climate change. Organic and non-organic production in terms of energy
use is essential for understanding the energy inefficiencies of different agro systems and
their potential for minimizing energy consumption and mitigating environmental impacts
particularly climate change. Organic agriculture can provide a more energy efficient ap-
proach which leads to sustainable agricultural productions. Organic agro productions is
contributing less greenhouse gas emissions and have a superior potential to sequester car-
bon available in biomass than conventional agro production. The energy efficiency of or-
ganic agriculture in terms of bioenergy production and thereby renewable fuel source is to
reduce dependency of fossil fuel energy and mitigate environmental pollution caused by
emissions. The industrialized production of agro products is responsible for a heavy impact
on the environment and playing a major role in increasing global Green House Gas (GHG)
emissions in respect to the usage of synthetic fertilizers and pesticides. More than 480 mil-
lion tons of GHG is released to the atmosphere each year by the synthetic fertilizers and
pesticides factories. Climate change is a serious environmental issue and has broad impacts
on sustainable development and the future of our economy, health, and agricultural produc-
tion sector. Integrated farm management system is reducing and sequestering GHG emis-
sions. Organic farming is essential to ensure the future of our environment and food pro-
duction in a sustainable manner. This article highlights the importance of organic agricul-
ture and its role in mitigating climatic change.
Keywords: Carbon sequestration; climatic change; greenhouse gases; organic farming; sus-
tainable production
the atmosphere over time. This increased the fermentation that takes place in the
concentration has been caused by the digestive tract of ruminants observed in
emission of GHGs as a result of economic livestock resulted the emission of 40% of
activities, including energy, industry, CO2eq (Figure 1). Photosynthesis is the
transport, and land use, many of which natural process in which the CO2 is con-
rely upon fossil fuels (Banuri and Op- verted to organic carbon and it is convert-
schoor, 2007). Climatic change is influ- ed to CO2 by respiration and decomposi-
encing the food caching behavior of a tion. Carbon restoration to the soil is hap-
wide range of animals to store food for pened by many agricultural practices in-
future use. (Sutton et al, 2016). Migrat- clude, conservation tillage, recycling ag-
ing animals are vulnerable to climate ricultural residues, cover cropping and
change and it affects the migratory bird crop rotations.Biological carbon seques-
movement. (Seebacher and Post, 2015). tration is a process by which the green-
Mollusks, and coral reefs are observed house gases generated during agricultural
negative impacts of climatic change activities have been removed from the
(Tschakert 2015). Prolonged exposures to atmosphere. Agriculture recorded from
elevated CO2 have affected structural pro- 10 to 12 percent of total global human
teins like actin (Ertl et al., 2016). The caused emissions of greenhouse gases
core objective of the this chapter is to elu- during 2005, according to the report by
cidate the importance of organic farming Intergovernmental Panel on Climate
as the alternative to modern farming sys- Change (IPCC, 2007). India is recorded
tem and leads to the sustainable agricul- the second largest emitter of CO2 from
tural system to overcome the recent total agricultural practices and by the ap-
changes in the environment and energy plication of the synthetic fertilizers fol-
crisis faced by the present generation. lowed by China (Figure 2 and 3).
Figure 1: Average CO2 emission by sector observed during the year 2000 and 2014
(Source: FAOSTAT 2014).
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Mitigation of Climatic Change by Organic Agriculture Mani et al.
Figure 2: Top 10 CO2 emitters during agricultural production (Source: FAOSTAT 2014).
Figure 3: Top 10 CO2 emitters by the application of synthetic fertilizers (Source: FAO-
STAT 2014).
farmers who are doing organic agriculture farming practices. The demand for food
were efficiently utilized the land resource and renewable energy sources are in-
and appropriately utilizing the SOIL creased with the increase of population.
(Soul Of Infinite Life). The impacts of climate change by the us-
Organic agriculture is a holistic pro- age of fossil fuel supported chemical fer-
duction management system that avoids tilizers, herbicides and pesticides will cre-
use of synthetic fertilizers, pesticides and ate deleterious issue on agricultural pro-
genetically modified organisms, minimiz- duction.
es the pollution of air, soil and water and Organic agriculture is proved to be a
optimizes the health and productivity of remedy for climate change and the global
interdependent communities of plants, food insecurity and potentially solve the
animals and people (Codex Alimentarius issues related to vulnerability, unsustain-
Commission, 2001). ability and social inequity of agricultural
Organic agriculture is playing a signif- production. Climate change has its most
icant role in climate change and food se- significant impacts on agriculture because
curity to the worlds population.Climate of its broad geographic dispersion and
change mitigation and food security are highly dependent on climate and envi-
vital management mediated by organic ronmental factors. Developing countries
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Mitigation of Climatic Change by Organic Agriculture Mani et al.
are the least responsible for climate work Convention on Climate Change
change, yet the most at risk from its ef- (UNFCCC) held in Paris in December
fects (UNESCO, 2010).There is a need of 2015 focused to lower the greenhouse gas
a policies and practices to protect the ever (GHG) by launching a voluntary action
changing environmental conditions and plan named 4 per 1000 initiative - Soils
ecosystems that ensure the sustainable for food security and climate (4o/oo Initi-
development. Organic agriculture is ef- ative). It initiated to increase soil carbon
fectively retaining soil organic matter, stock annually by 0.04 % through carbon
soil carbon and there by balancing of eco- sequestration. The annual growth rate 4
system. Organic agriculture is the tradi- parts per thousand of the soil carbon stock
tional practice that mitigates climate would make it possible to stop the present
change, generate evergreen farming sys- increase in atmospheric CO2. Food securi-
tems, eradicate poverty and augment the ty is confirmed by providing proven tech-
level of food security. Organic agriculture nologies to the farmers in developing
discharges very minimum levels of countries to repair and sustain the soils.
greenhouse gases (GHG) and efficiently To feed 9.5 billion in 2050, it is essen-
sequesters carbon in the soil. Organic ag- tial to keep our soils alive. Figure 4 shows
riculture makes faming system and people various steps to ensure soil organic matter
dependent on this sector more resilient to by providing various ecosystem services
climate change due to its water use effi- like capable of resisting the soil from ero-
ciency, tolerance to extreme weather con- sion, retaining water, increasing soil fer-
ditions and lower or no risk of total crop tility and proliferating soil biodiversity as
failure. Organic agriculture is extensively per the initiation suggested by 4 per 1000.
practiced agro-ecological farming system It can meet three fold challenges includ-
that accomplishes the main objectives of ing food security, adaptation to food sys-
enabling people to thrive while improving tem and people to climate change and
our eco-systems and their natural cycling mitigation of anthropogenic emission.
process. Soils under organic management will of-
ten increase the level of soil organic car-
4. Soils for food security and climate - bon more than conventional management
4 per 1000 initiative (FAO 2011 and 2011a).
Figure 4: Ecosystem services by soil organic matter and its effects and challenges.
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Mitigation of Climatic Change by Organic Agriculture Mani et al.
Abstract: Farming of various marine shrimp species has been developed and commercial-
ized in the last three decades. In the beginning, marine shrimp were cultivated in South-east
Asia by farmers who raised them as incidental crops in tidal fish ponds. Over the years,
enormous progress in developing shrimp culture techniques has been made. Shrimp culture
evolved from an extensive farm using tidal zones to a super-intensive one in the 2000s us-
ing ponds more inland. Outbreak of disease such as Vibriosis is considered as one of the
important constraint and challenge in aquaculture industry of the world.Conventional ap-
proaches such as the use of antibiotics, disinfectants and other antimicrobial drugs have
shown limited success in the disease prevention. In this context, there are sound reasons for
studying the beneficial eco-friendly actions of probiotics and phyto-medicine in boosting the
shrimp aquaculture production without or minimal adverse impacts on environment. This
chapter highlights the anti-vibrio and anti-QS nature of probionts and plants against the Vib-
rio pathogen of luminous Vibriosis, a major bacterial disease in shrimp aquaculture.
combination of both disease and pollution. The disease produced by Vibrio in shrimp
While disease played the prominent role culture is commonly called as vibriosis.
in declining aquaculture stocks, both fac- The other names of bacterial vibriosis are
tors caused massive mortality in the lead- luminescent vibriosis, penaeid bacterial
ing aquaculture countries. Among the septicaemia and red-leg disease (Aguirre-
groups of microorganisms that cause seri- Guzmn et al., 2004). With the rapid de-
ous losses, the best known are bacteria velopments in aquaculture particularly in
because of the devastating economic ef- Asia and South America, V. harveyi and
fects they have on affected farms. Bacteri- related bacteria have become recognized
al diseases mainly due to Vibriosis have as a serious cause of disease (Austin and
been reported in Penaeid shrimp culture Zhang, 2006). In many cases, Vibrios are
systems implicating several species of opportunists causing disease when the
Vibrios which includes Vibrio harveyi, host organism is immune-suppressed or
V.splendidus, V. parahaemolyticus, V. al- otherwise physiologically stressed (Peddie
ginolyticus, V. anguillarum, V. vulnificus, and Wardle, 2005). Even though all crus-
V. campbelli, V. fischeri, V. damsella, V. taceans are suffered by this bacterium,
pelagicus, V. orientalis, V. ordalii, V. most serious struggle was reported in Pe-
mediterrani, V. logei etc. From the all naeid shrimp culture (Austin and Zhang,
above species V. harveyiis the main causa- 2006).
tive agent causing luminous vibrosis to The ill effects of adult animal due to
larva in hatchery and pond cultivation vibriosis includes reddening body with red
(Won and Park, 2008). Being an important to brown gills, swimming lethargy and
etiological agent, it causes mass mortali- reduced feeding behaviours (Nash et al.,
ties in penaeid shrimp culture and leads to 1992). Similarly infected PL showed emp-
huge economic losses.They continue to ty gut and reduced motility and phototax-
cause chronic mortalities of up to 30% is. Based on the syndrome,they are ex-
among P. monodon larvae, post larvae and pressed as localised and systemic vibri-
adult under stressful conditions (Le osis, oral and enteric vibriosis, septic
Groumellec et al., 1996). In production hepatopancreatitis and appendage and cu-
unit it causes 100% losses at a time due to ticular vibriosis (Lightner, 1996). Lumi-
various virulence factors (Chythanya et nescent V. harveyi appears to release exo-
al., 2002). toxins and may cause 80-100% mortality
The genus Vibrio is a gram negative in P. monodon hatcheries (Harris, 1995).
motile rod shaped gamma proteobacter. Vibrio species also cause red-leg disease
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Sensing Technology for Shrimp Aquaculture Sustainability Kandasamy et al.
characterised by red colouration of the Bassler, 2001). These molecules are con-
pleopods, periopods and gills in juvenile stantly produced and received at a basal
to adult shrimps. Shrimps suffering vibri- level by bacterial cells. With high popula-
osis may display localised lesions of the tion density, there is a surplus of signal-
cuticle typical of bacterial shell disease, ling molecules in the environment. These
localised infections from puncture signals diffuse back into the cell where
wounds, loss of limbs, cloudy muscula- they facilitate the regulation of gene ex-
ture, localised infection of the gut or pression (Hastings and Greenberg, 1999).
hepatopancreas and general septicemia The quorum sensing signal molecules are
(Lightner, 1993). Virulence factors asso- found to be involved in the regulation of
ciated with V. harveyi pathogenicity is due various physiological processes such as
to the production of bacteriocin-like sub- the bioluminescence, biofilm formation,
stance, phage induced haemolytic activity, pigment production, toxin production, ex-
extracellular products such as chitinases, opolysaccharide production, motility and
cysteine protease, haemolysin, luciferase, virulence factor production in many
metalloprotease, proteases, phospholipas- Gram-negative bacteria including fish and
es, siderophores and proteinaceous exo- shrimp pathogens like V. harveyi (Bruhn
toxins. et al., 2005; Kennedy et al., 2006).
High loads of either V. parahae- In each QS system, the autoin-
molyticus or V. harveyi induced the round- ducer attaches to a gene that is known as
ing up and detachment of epithelial cells the transcriptional activator and this at-
from the basal lamina of the mid gut trunk tachment can lead to alterations in DNA
(MGT) and can cause high mortality in and activation of virulence factors (Waters
shrimp by eliminating two layers that pro- and Bassler, 2005). Gram-negative bacte-
tect the shrimp from infections: the epithe- ria use N-acyl homoserine lactones
lium and the peritrophic membrane. In (AHLs) as autoinducers, while Gram-
addition, loss of the epithelium may affect positive bacteria use oligopeptides to
the regulation of water and ion uptake into communicate (Miller and Bassler, 2001).
the body. It is well-known that there are The most extensively investigated inter-
significant differences between different cellular signaling molecules are the AHLs.
V. harveyi isolates in terms of pathogenic- Quorum sensing in V. harveyi is regulated
ity with some strains being highly virulent via a multichannel phosphorylation /
and others being non-pathogenic (Austin dephosphorylation cascade. This bacte-
and Zhang, 2006). Even if many virulence rium produces and responds to two auto-
factors have been already reported in V. inducers namely (i) harveyi autoinducer 1
hrarveyi, complete pathogenic mecha- (HAI-1) and (ii) autoinducer 2 (AI-2),
nisms should be elucidated. which regulate the expression of biolumi-
nescence and other virulence fac-
5. Quorum sensing in V. harveyi tors.Miller and Bassler (2001) have thor-
oughly studied the mechanism of QS in V.
The term Quorum Sensing re- harveyi. HAI-1 is an AHL and its biosyn-
fers to the process of bacterial cell-to-cell thesis is catalysed by the luxM enzyme
communication. It is a population depend- (Figure 2). AI-2 is a furanosyl borate
ent phenomenon first characterized in the diester and its biosynthesis is mediated by
1970s in luminescent marine species of the luxS enzyme. Both HAI-1 and AI-2
Vibrio (Nealson et al., 1970). Through are detected at the cell surface by the
this mechanism bacteria coordinate gene LuxN and LuxP-LuxQ receptor proteins
expression in a density-dependent manner. respectively. In the absence of the signals,
It is solely depends on the production, re- LuxN and LuxQ autophosphorylate and
lease and detection of chemical signal transfer phosphate to LuxO via LuxU. The
molecules called autoinducers (Miller and phosphorylated luxO is an active repressor
for the target genes. In the presence of the occur between resistant bacteria and viru-
signal molecules, LuxN and LuxQ interact lent pathogens that re-enter the culture
with their autoinducers and change from facilities after the treatment. Thus, antibi-
kinases to phosphatases that drain phos- otic-resistant strains of pathogenic V. har-
phate away from LuxO via LuxU. The veyi can evolve rapidly. In view of the in-
dephosphorylated LuxO is inactive. Sub- discriminate use of antibiotics in aquacul-
sequently, transcription of the target genes ture, it cannot be surprising that many re-
is activated by LuxR. Recently, a third QS ports have mentioned multiple resistance
component, a Vibrio cholerae-like autoin- of V. harveyi strains to several antibiotics
ducer CAI-1 was discovered and identi- (Table 1).From all this, it might be clear
fied as (S)-3-hydroxytridecan-4-one in V. that the efficacy of antibiotics to treat lu-
Harveyi (Henke and Bassler, 2004). Sev- minescent vibriosis is very poor. The
eral investigations have been made to find presence of residual antibiotics in com-
the effect of V. harveyi quorum sensing on mercialized aquaculture products consti-
the production of the virulence factors like tutes another problem with respect to hu-
caseinase, gelatinase, lipase, hemolysin man health as this can lead to an alteration
and phospholipase by determining their of the normal human gut microbiota and
expression levels both in vitro and invivo can also generate problems of allergy and
during infection of gnotobiotic brine toxicity (Cabello, 2006). Anbiotics resi-
shrimp (Natrah et al., 2011; Darshanee et dues in animals are also regarded as haz-
al., 2011). ardous for exporting quality. In order to
limit the use of antibiotics, many workers
6. Antibiotics vs anti-vibrio probiotics have been exploring the use of new bioac-
tive compounds for controlling bacterial
Traditionally antibiotics have diseases of shrimp particularly that of
been used in attempts to control bacterial caused by V. harveyi. Various solutions
disease in aquaculture. When antibiotics have been proposed such as the use of
are used, the resistant strains can multiply probiotics, immunostimulation, vaccina-
rapidly because their (sensitive) competi- tion, specific pathogen-free (SPF) and
tors get removed. Moreover, horizontal specific pathogen-resistant (SPR) shrimp.
exchange of resistant determinants can
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Sensing Technology for Shrimp Aquaculture Sustainability Kandasamy et al.
and seaweeds (Kim et al., 2007; Skinder- C. violaceum CV017. Various fruits and
soe et al., 2008; Daglia et al., 2010; herbs were shown to possess anti-QS ac-
Musthafa et al., 2010). tivity in a C. violaceum biomonitor strain
The QS interference from novel and on the swarming motility of E. coli
sources may also be an important as the and P. aeruginosa (Vattem et al., 2007).
antibacterial effects. Anti-QS agents were The subsequent discovery of com-
first characterized in the red marine alga pounds that inhibit cell-to-cell communi-
D. pulchura (Manefield et al., 1999). cation, dubbed anti-quorum sensing (anti-
This alga was investigated for its anti- QS) agents could provide a novel method
fouling properties and was found to con- of combating infection. It is possible that
tain halogenated furanones which block several terrestrial plants also produce
AHLs via competitive inhibition and de- quorum signal mimics capable of control-
stabilization of LuxR (Manefield et al., ling bacterial quorum sensing (Gao et al.,
2002). Delisea furanones have been 2003). Even bacteria themselves produce
shown to reduce light emission in Vibrio QSI substances (Nithya et al., 2010).
species (Givskov et al., 1996), inhibit Spices such as garlic, ginger and turmeric
pigment production in C. violaceum have been reported for their QSI potential
(Martinelli et al., 2004) and attenuate exo- (Vattem et al., 2007). Similarly, the essen-
enzyme production and swarming motility tial oils of cinnamon (Niu et al., 2006) and
in Serratia liquefaciens (Rasmussen et al., clove (Khan et al., 2009) are also known
2000). The quorum sensing-disrupting to possess QSI potentials. Acyl-homo ser-
natural furanone, (5Z)-4-bromo-5- ine lactone analogs and other quorum
(bromomethylene)-3-butyl-2(5H)- sensing inhibitors (QSI) have been inves-
furanone was found to block autoinducer tigated to determine their ability to pre-
2 quorum-sensing in V. harveyi in a con- vent expression of quorum sensing con-
centration-dependent way (Defoirdt et al., trolled genes. The complex of signalling
2006). Persson et al., (2005) reported that molecules (AHLs) and receptor proteins
toluene extracts of garlic contained several trigger the expression of specific genes
compounds with varying levels of QSI responsible for bioluminescence in V.
against Gram-negative transcriptional reg- harveyi (LuxM/N). Hence the disintegra-
ulators Lux R or Lux R. Sergey et al., tion of signals with receptor by plant de-
(2011) have evaluated 78 natural products rived QSI prevents the bioluminescence
from chemical libraries containing com- and other virulence factors in V. harveyi.
pounds from marine organisms (Sponges, Several compounds have been identified
algae, fungi and cyanobacteria) and terres- that have the ability to interfere with QS-
trial plants were screened for the inhibi- mediated gene expression through com-
tion of bacterial QS using a reporter strain
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Biotech Sustainability (2017)
Sensing Technology for Shrimp Aquaculture Sustainability Kandasamy et al.
petitive inhibition thus reducing biofilm Balczar, J. L., de Blas, I., Ruiz-Zarzuela,
thickness (Hentzer et al., 2002). I., Cunningham, D., Vendrell, D. and
Muzquiz, J.L. (2006). The role of pro-
9. Perspectives biotics in Aquaculture. Veterinary Mi-
crobiology 114, 173-186.
The oceans cover more than 70% of Browdy, C. L. (1998). Recent developments
the earths surface and they are a promis- in penaeid broodstock and seed produc-
ing source of novel pharmacologically tion technologies: improving the outlook
active compounds. Although macroorgan- for superior captive stocks. Aquaculture
isms of the ocean have proved to be good 164, 3-21.
sources of novel bioactive metabolites, Bruhn, J. B., Dalsgaard, I., Nielsen, K. F.,
large-scale productions of these bioactive Buchholtz, C., Larsen, J. L. and
metabolites have been difficult. Microor- Gram, L. (2005). Quorum sensing sig-
ganisms isolated from marine sources nal molecules (acylated homoserine lac-
have been reported to produce anti- tones) in Gram-negative fish pathogenic
bacterial, anti-fungal, anti-viral and anti- bacteria. Disease of Aquatic Organism
tumor substances. Several studies have 65, 43-52.
suggested that such marine bacteria can be Cabello, F. C. (2006). Heavy use of prophy-
used as bio-control to combat epizootics lactic antibiotics in aquaculture: a grow-
in aquaculture systems.During the past ing problem for human and animal
two decades, the use of probiotics as an health and for the environment. Envi-
alternative to antibiotics has shown to be ronmental Microbiology 8, 1137-1144.
promising in aquaculture. Data about the Chythanya, R., Karunasagar, I. and
impact of quorum sensing on virulence of Karunasagar, I. (2002). Inhibition of
aquatic pathogens are still lacking. Few shrimp pathogenic vibrios by a marine
reports that deal with disruption of quor- Pseudomonas I-2 strain. Aquacul-
um sensing of aquatic pathogens indicate ture208, 1-10.
that this new approach has potential in Czajkowski, R. and Jafra, S. (2008).
fighting infections in aquaculture. The Quenching of acyl-homoserine lactone-
furanones reported earlier as quorum dependent quorum sensing by enzymatic
quenchers (QS inhibitors) are toxic and disruption of signal molecules. Journal
chemically synthetic (non-degradable). of Acta Biochimica Polonica 56, 1-16.
Consequently the invention of non-toxic, Daglia, M., Stauder, M., Papetti, A., Si-
broad spectrum QS inhibitors is needed gnoretto, C., Giusto, G.,Canepari, P.,
for its successful exploitation against bac- Pruzzo, C. and Gazzani, G. (2010).
terial infections due to their drug re- Isolation of red wine components with
sistance. anti-adhesion and anti-biolm activity
against Streptococcus mutans. Food
References Chemistry 119, 1182-1188.
Darshanee, R. H. A., Bhowmick, P. P.,
Aguirre-Guzmn, G., Meija Ruz, H. and Karunasagar, I., Bossier, P. and
Ascencio, F. (2004). A review of extra- Defoirdt, T. (2011). Quorum sensing
cellular virulence product of Vibrio sp. regulation of virulence gene expression
important in disease of cultivated in Vibrio harveyiin vitro and in vivo dur-
shrimp. Aquaculture Research 35, 1395- ing infection of gonotobiotic brine
1404. shrimp larvae. Environmental Microbi-
Austin, B. and Zhang, X. H. (2006). Vibrio ology Report 3, 597-602.
harveyi: a significant pathogen of ma- Decamp, O., Moriarty, D. J. W. and Lav-
rine vertebrates and invertebrates. Letter ens, P. (2008). Probiotics for shrimp
of Applied Microbiology 43, 119-124. larviculture: review of field data from
plants and elicit the nodule formation ers. The Bio-fertilizers are bacteria, algae
where they colonize and differentiate into and fungi and may broadly be classified
non-dividing endocellular symbionts. into two categories viz. Nitrogen fixing
These symbionts convert atmospheric di- like Rhizobium, Azotobactor, Azospiri-
nitrogen into NH3 through the induction lum, Acetobacter, Blue Green Algae and
of the nitrogenase complex (Patriarca et Azola and Phosphorous solubilis-
al., 2002). Rhizobium species have been ers/mobilisers like PSM and Mycorrizae
successfully used worldwide as a bio- (Figure 3). Rhizobia and legumes estab-
inoculant leading to effective establish- lish a mutualistic symbiosis. Host speci-
ment of nitrogen fixing symbiosis with ficity is an important characteristic of
leguminous crop plants (Miller et al., symbiosis, where specific species of rhi-
2007). Nitrogen applied as fertilizers usu- zobia forms nodules on defined legumes
ally provides high yields to plants. There- (Ampomah et al., 2008). Rhizobia cur-
fore efficient monitoring of biological ni- rently consist of 61 species belonging to
trogen fixation and status of chemical fer- 13 different genera, namely Rhizobium,
tilizers are essential to balance the yield Bradyrhizobium, Mesorhizobium, Azorhi-
of crops and need to minimize environ- zobium, Allorhizobium, Sinorhizobium,
mental pollution, especially water and soil Methylobacterium, Cupriavidus,
quality (Jaynes et al., 2001). The role of Burkholdera, Devosia, Ochrobactrum,
BNF, especially in legumes, is well estab- Herbaspirullum and Phyllobacterium.
lished and documented but Legume- Some Rhizobia have a narrow host range
Rhizobium symbiosis is not so extensively and form nodules with specific legume.
studied the system of nitrogen-fixation. For example Azorhizobium caulinodans,
Soil containing adequate and diverse Sinorhizobium saheli and sesbaniae
communities (Figure 1) of rhizobia and biovar of Sinorhizobium terange nodulate
become less effective at nitrogen fixing. only Sesbania rostrata (Boivin et al.,
The application of sufficiently high 1997) and Rhizobium galegae is the only
numbers of improved inoculant strains symbiont of Galega offcinalis and Galega
can successfully compete with established orientalis (Lindstrom, 1989). In contrast
soil rhizobia and replace them (Figure 2). some rhizobia are capable to infect a
The aim is to increase, the percentage of spectrum of legumes as they have broad
crops that are inoculated in terms of bio- host range (various degree of promiscui-
mass yield and extra nitrogen in the soil. ty). For example, Sinorhizobium sp.
Improved Rhizobium strains for greater NGR234 and closely related Sinorhizobi-
symbiotic association and ability to infect um fredii USDA257 nodulate at least 112
across strict host specificity would be of and 77 legumes from two different tribes,
greater importance for the farming com- respectively (Pueppke and Broughton.
munity. Farmers can enhance BNF by in- 1999).
oculating such correct rhizobia to their
legume crops. Such promiscuous Rhizo- 3. Symbiotic infection is a regulated
bium strains with improved efficiency to pathway
fix nitrogen would acts as a single inocu-
lum for all the legumes and may add Symbiosis is a developmental process
higher amount of nitrogen per unit area. driven by bacteria but ultimately under
the control of host plant (Murray, 2011).
2. Classification of biofertilizers as per The successful establishment of infection
host specificity requires several factors such as nod fac-
tors and plant exudates (flavonoids). The-
Biofertiliser are the low cost source of se flavonoids activate different kinds of
plant nutrients, eco-friendly and have nitrogen fixing genes and the interaction
supplementary role with chemical fertiliz- takes place between bacteria and
introduction of strain NGR234 nodD1 hybrid genes it was concluded that a cen-
into R. Meliloti increases Nod factor pro- tral region determine different host rang-
duction by about two-fold and permits the es. Louise et al. (2002) have mutagenised
nodulation of V. Unguiculata, a non-host Rhizobium strains with transposon Tn5 to
(Relic et al., 1994). In S. Meliloti these determine if additional negatively-acting
NodD proteins respond to different traits exist that can alleviate cultivar-
groups of flavonoids, suggesting that specific nodulation failure. They reported
NodD redundancy allows the bacterium to two new mutants, proficiently nodulate
infect multiple hosts secreting a wide cv. Woogenellup. They suggested that
range of flavonoids (Maillet et al., 1990). simple gene to gene interaction is not suf-
To demonstrate that nod is a key determi- ficient for symbiosis but there are at least
nant of host specificity, Melicent et al. two independent mechanisms which me-
(2006) expressed nod genes from differ- diated the cultivar-specificity. Although
ent species of rhizobia in a strain of S. much information is available on the in-
Meliloti which was lacking endogenous fluence of Nod factors on the host range
nodD activity. They observed that nod yet no strict correlation can be drawn the
gene expression was initiated in response types of LCOs produced by rhizobia and
to distinct set of flavonoid inducers. Fur- host plants.
thermore, data from several researches
suggest that nodD controls the response 5. Molecular factors control the sym-
of rhizobia to flavonoids in species- biosis
specific manner (Hovath et al., 1987)
Herman et al. (1989) found that node The nitrogen fixation and nodulation
product is the main factor that distinguish by Rhizobium strains is controlled at vari-
the host range for symbiosis. Hybrid nodE ous levels by certain factors (Hooykaas et
genes, which consist of a 5 part Rhizobi- al., 1981). The importance of each indi-
um leguminosarum nodE genes and a 3 vidual step would depend on the specific
part of the Rhizobium trifolii gene, were legumeRhizobium combination and Nod
constructed. From the properties of these factors (NF) in the rhizosphere (Figure 5).
Figure 5: Molecular factors which are involved in symbiosis (Okazaki et al., 2013).
Bacterial Nod factors functions as a key a particular host and are involved in vari-
to opens the door of its host (Perret et al., ous modification of the chitin backbone
2000), and there is a high degree of strin- (Gibson et al., 2008). The nodulation
gency for chemical structure of Nod fac- gene expression is activated when bacte-
tor that determine whether the host allows ria perceive flavonoids that are secreted
bacterial invasion to proceed. Nod factor by plant roots (Perret et al., 2000). nodD,
elicits significant changes in the expres- gene is central to the regulation of nod
sion of host gene (DHaeze and Holster, box which activates other nod gene ex-
2002; Oldroyd and Dowine, 2008). Nod pression (Loh and Stacey, 2003). It in-
factors are complex signaling molecules duces the transcription of nodulation
secreted from bacteria as a cocktail of genes involved in the synthesis of nod
1-4-linked N acetyle D-glucosamine factors (Capela et al., 2005 and Peck et
(GlcNAc) trimers, tetramers or pentamers al., 2006). In response of isoflavone sig-
(DHaeze and Holster, 2002). The host- nals which are produced by plants
rhizobia co-evolution involved modifica- NodVW, positively regulate nod genes,
tions of Nod factor structure such as re- thus these are thought to activate tran-
placement of fucosyl, which made the in- scription via a series of phosphorylation
teraction more specific and increased af- steps. Mutation in either of these two
finity between partners (Mario et al., genes results in the complete loss of
2006). nodulation activity in certain plant hosts.
All the rhizobial species have The proposed role of nodV as a sensor of
common nod genes (nod A, B, and C), plants signals adds another point of com-
which are capable of cross species com- plexity of nod gene regulation. In re-
plementation and responsible for the syn- sponse of plant signal, phosphorylation of
thesis of nod factor backbone. These NodV takes place which subsequently
genes confer specificity for nodulation of activate NodW via the transfer of phos-
phoryl group to an aspartate residue in its portant for induction of different compo-
receiver domain. The nodW play a key nents of the nodulation pathway but also
role in the regulation of nod gene expres- some bacterial cell surface components
sion and in the ability of B. Japonicum to such as LPS, cyclic--glucans, EPS, cap-
infect the host plant (Sanjuan et al., sular proteins and K antigens recognised
1994). The phosphorylation of NodV and by plants help to determine host specifici-
NodW is essential not only for nod gene ty (Mathis et al., 2005). These additional
expression but also for the nodulation. factors must have role in symbiotic de-
Nod factors permit rhizobia to enter their velopment between rhizobia and plant.
hosts and certain additional factors proba-
bly act within plant (DHaeze et al., 6. Broadening of host range (promis-
1998). Thus a number of physiological cuity) of rhizobia
responses to nod factor are observed
when rhizobium applied to the plant roots The host specificity concept has
(Gibson et al., 2008). now almost defunct, because many over-
Said et al., (1998) have found that no- lapping host ranges have observed so the
lO factor is the principal host range de- concept of host specificity has been chal-
terminant. They have mobilized large lenged. A single legume e.g. Acacia, Gly-
fragments of the symbiotic plasmid of cine max or Leucaena can be associated
Rhizobium spp. NGR234 into heterolo- with genetically dissimilar symbionts.
gous rhizobia. The trans-conjugants nodu- Closely related rhizobia infect legumes
late V. unguiculata at low frequency and from different tribes and distantly related
extended the host range. They have con- rhizobia infect closely related legumes
firmed that the conjugation of nolO into (Quesada et al., 1997). The Rhizobium sp.
Rhizobium fredii extends the host range strain NGR234 is good examples of this
of the recipient to the non-hosts. Nod fac- phenomenon. It has broad host range and
tors are essential to the nodulation and nodulate legume species from 112 genera
their modification contributes to host and the non-legume Parasponia (Pueppke
specificity, thus these signaling molecules and Broughton, 1999). Zhu et al., (2002)
probably one of the several elements characterized the rhizobia that nodulate
specifying host range. It should also be legume species of the genus Lespedeza by
noted that although nodFE mutants of analysing whole cell proteins, and cross-
Rhizobium melilotii secrete nod factors in nodulation with selected legume species.
which C16 unsaturated fatty acids are re- They have observed that the strains iso-
placed by vaccenic acid, the mutant still lated from Sesbania spp. and Lespedeza
form nodules on various Medicago culti- spp. represent a cross-nodulating group of
vars (Ardourel et al., 1994). Several other bacteria. Hernandez-Lucas et al., (1995)
examples contradict the dogma that Nod also found two strains of R. etli and three
factors determine host specificity. Despite stains of R. tropici and tested on 43 leg-
the fact that predominant nod factors se- ume species. Out of these 22 of the tested
creted by R. Leguminosarum bv. trifolii legume species were nodulated by three
and R. Leguminosarum bv. viciae are or more of these strains. These strains
identical yet these two bacteria have dis- have broad host range and nodulate
tinct host ranges (Orgambide et al., 1995). woody species also such as Albizia
In contrast, two rhizobia that secrete dif- lebbeck.
ferent nod factor may nodulate the same Setiyo Hadiwaluyo (2011) charac-
plant: R. Tropici and R. etli produce dif- terized forty one cross inoculating rhizo-
ferent nod factors but both effectively bial isolates from Java and Sumatra and
nodulate Phaseolus vulgaris (Poupot et these isolates were used to inoculate soy-
al., 1995). It would thus seem that nod bean and mungbean plants. He found 19
factors in absolute level are not only im- isolates from Java and 15 isolates from
leguminosarum bv. trifolii attached to the hair wall of the host and making a local-
root surface of white clover and produce ized hole of sufficient size to allow rhizo-
pit erosions in epidermal wall that follow bial cell penetration. This leads to develop
the penetration of the bacterium, suggest- more nodules for successful nitrogen-
ing that wall-degrading enzymes are in- fixation.
volved in symbiosis. Vashishat et al., Hussain et al. (1995) studied the in-
(1985b) observed that R. trifolii strains volvement of hydrolytic enzymes in the
were capable of producing hydrolytic en- nodulation of berseem (Trifolium alexan-
zymes like pectinase, hemicellulase and drinum). They selected a single mutant
cellulose and these enzymes play im- hrt20m7 in wild type strain hrt20 of R.
portant role in symbiosis. Considering trifolii by screening for reduction in activ-
these evidence Angle in 1986 tried to de- ity of degradative enzymes, the relative
termine whether differences exists be- activities shown by the mutant for pecti-
tween fast and slow growing soyabean nases and cellulase were 33 and 4 per-
rhizobia to produce pectinase and proteo- cent, respectively of wild type strain. It
lytic enzymes. It was proposed that wide was observed that mutants unlike its par-
spread production of proteolytic enzymes ent failed to nodulate clover seedlings.
indicates indirect evidences for their in- Aggarwal et al. (2000) found rhizobia be-
volvement in the invasion of host. Al- having as super nodulating rhizobia. They
Mallah et al. (1990) pre-treated clover suggest cellulases over pectinases in the
roots with an enzyme mixture of 1% process of symbiotic infection of berseem
(w/v) cellulase and 0.1% pectolase before by R. leguminosarum bv. trifolii. They
inoculating clover with R. trifolii. In- derived rhizobia mutants showing better
crease in nodulation indicated the role of growth on CMC and /or pectin i.e. behav-
cellulase and pectinase in nodulation pro- ing as super-nodulating rhizobia. Emtiazi
cess of clover. et al. (2007) also studied the cellulase ac-
In 1992, Pedro et al., verify the pro- tivities in nitrogen fixing Paenibacillus
duction of R. leguminosarum bv. trifolii isolated from nitrogen free media. The
enzymes that deteriorate polygalac- cellulase positive Paenibacillus were se-
turonate and carboxymethyl cellulose lected by reduction of congored color on
(CMC) as model substrates of plant cell CMC medium. They have observed that
wall polymers. Similarly Mateos et al. nitrogen fixing strains with cellulase ac-
(1992) reported the production of en- tivities grow well on nitrogen free media
zymes from R. leguminosarum bv. trifolii with sucrose or manitol as the only
that degrade carboxymethyl cellulose and sources of carbon. They have concluded
polypectate substrates. Their studies that most plant associated microorganism
shows that R. leguminosarum bv. trifolii might have cellulase activities for adop-
produces multiple enzymes that cleave tion or establishment of a plant microbe
glycosidic bonds in the plant cell wall. interaction. Egamberdieva et al. (2010)
Mateos et al., (2001) also found that cel- determined the bacterial cellulase activity
lulase (Cel2) enzyme is important for on media containing the substrate car-
symbiotic development because rhizobial boxy-methylcellulose. They have found
symbionts require its activity to breach that cellulase producing bacteria were
the host barrier to establish nitrogen fix- significantly increase nodule numbers and
ing association with legumes. Robledo et nitrogen content of the plants. Fouts et al.
al. (2008) have purified cell bound cellu- (2008) studied the complete genome se-
lase (Cel2) isozymes and analysed its quence of the nitrogen fixing broad host
symbiotic function by reverse genetics range endophyte Klebsiella pneumonia
and plant microscopy approaches. These 342. They have found the gene related to
results provide compelling evidence that carbohydrates, including pectins and cel-
this enzyme could erode the tip of root lulosic compound degradation are essen-
tial for Kp342 to form endophytic associ- Better nitrogen fixation may be
ations. So, it was concluded that hydrolyt- brought by manipulating both rhizobia
ic enzyme activity in the form of cellulase and plant host by eventually creating an
and pectinase is an essential property of artificial rhizosphere. Schlaman et al.,
Rhizobium for infection of white clover (1998) observed nodulation and the levels
during symbiosis. of nitrogen fixation can be significantly
higher when plants are infected with rhi-
8. Improvement in symbiotic efficien- zobia containing the hybrid gene
cy of rhizobia nodD604, which activates the transcrip-
tion of nod genes independent from fla-
The rhizobium-legume symbiosis vonoids. For introduction of foreign DNA
accounts for a significant proportion of into bacterial species electroporation is a
nitrogen available to leguminous plants. novel approach (Chassy et al., 1988).
Thus there is a need to improve rhizobia Garg et al. (1999) successfully carried out
to increase their symbiotic efficiency and electro-transformation of R. legumi-
host range. The traditional method for ob- nosarum with 15.1kb plasmid, pMP154
taining Rhizobium strains with improved (Cmr), containing a nodABC-lacZ fusion
properties has been the selection of natu- by electroporation. Chitchanok et al.
rally occurring field isolates that best ex- (2011) derived mutants from wild type
hibit the trait desired (Figure 6). An alter- Rhizobium sp. 6-1C1 through 0.8 and 1.0
native approach is to construct improved kGy gamma radiation. They have ob-
Rhizobium strains by genetic transfer of served that Rhizobium meliloti nodH gene
symbiotically favorable determinants. mutants result in a change of host range.
Genomic rearrangements have been re- They infect vetch with this mutated strain
ported to occur frequently in R. legumi- but fail to nodulate their normal host, al-
nosarum phaseoli (Flores et al., 1988, falfa.
Garg et al., 1999).
The nodQ on the other hand, are pMH682 for increasing NF production),
able to infect both alfalfa and vetch. and the R. tropici (pGMI149)
Mostly mutations results in the alteration (pGMI1962) hybrid strain and then tested
or extension of the host range (Faucher et the ability of these NFs to form nodules
al., 1989 and Horvath et al., 1986). In R. on alfalfa. NFs produced by the R. tropici
leguminosarum bv. viciae and bv. trifolii, hybrid strain were able to induce nodule
the nod product is the main factor that formation. The presence of R. meliloti
distinguishes the host range for nodula- nodulation genes therefore enables R.
tion. In contrast to wild type, R. legumi- tropici to produce new NFs that can in-
nosarum bv. Trifolii nodEF mutants nodu- duce nodule formation. Their results show
late white and red clover poorly but have that allelic variation of the common nod-
acquired the ability to infect peas. When ABC genes is a genetic mechanism that
these nodEF mutants harbour the node plays an important role in signaling varia-
gene of R. leguminosarum bv. Viciae, tion and in the control of host range. They
they have an extended host range to vicia have also found that mutations in the reg-
and lathyrus species (Spaink et al., 1989). ulatory genes nodD1 and nodD3 did not
Transfer of nodD1 gene of strain result in a detectable decrease in nodula-
NGR234 to restricted host range rhizobia tion.
extend the nodulation capacity of the re- Falguni et al., (2009) amplified 2.4 kb
cipients to new hosts, including the non- fegA gene (encoding ferrichrome recep-
legume Parasponia andersonii (Horvath tor) along with its native promoter from
et al., 1987). Nonetheless, nodD gene rep- Bradyrhizobium japonicum 61A152 and
resents a molecular interface between the cloned in a broad host range plasmid vec-
bacterium and the plant. Plasmid transfer tor pUCPM18. The plasmid construct pFJ
may increase nodulation or nitrogen fixa- was transferred by conjugation into Rhi-
tion in R. Leguminosarum bv. viciae zobium sp. ST1 to give trans-conjugant
strains (DeJonj et al., 1982), and there is ST1pFJ12. Inoculation of pigeon pea
one report of a plasmid loss that improves seedlings with trans-conjugant ST1pFJ12
symbiotic properties in Rhizobium loti led to a marked increase in plant growth
(Pankhurst et al., 1986). In R. meliloti, a parameters as compared to plants inocu-
non-symbiotic plasmid enhances nodula- lated with the parent strain ST1, Nodule
tion of the strains harboring it (Urban, J. occupancy on pigeon pea plant when in-
1988). Esperanza and Monica (1990) ge- oculated with the trans-conjugant was in-
netically modified R. leguminosarum bv. creased. Gene fegA not only supports the
phaseoli CFN42, through transfer of a growth of the trans-formants rhizobia un-
225kb plasmid from typeII strain der iron limited laboratory conditions, but
CFN299. They have observed that more also increases its survivability under natu-
nodules were obtained with the transcon- ral soil conditions, which led to higher
jugants on P. vulgaris. These strains also nodulation on peanut plant. Yoshitake et
have a diminished competitive ability. al., (2010) introduced vktA into R. legu-
Philippe et al., (1996) introduced an IncP minosarum cells and the strain with a re-
plasmid, pGMI149, carrying the main R. markably high catalase activity was con-
meliloti nodulation region into R. tropici. structed. The vktA trans-formant was in-
The R. tropici (pGMI149) transconjugants oculated to the host plant P. vulgaris and
poorly nodulate M. sativa. When a second the nodulation efficiency was evaluated.
plasmid, of the IncQ group (compatible The nitrogen-fixing activity of nodules
with pGMI149), carrying the nodL gene was increased 1.7 to 2.3 times as com-
(pGMI1962) was introduced into R. pared to the parent. Results show that the
tropici (pGMI149), a better nodulation increase of catalase activity in rhizobial
was observed. They have also prepared cells could be a valuable way to improve
NFs from R. tropici, R. meliloti (with the nodulation and nitrogen-fixing ability
tracellular signals. Molecular Plant Heidstra, R., Geurts, R., Franssen, H.,
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potential changes in alfalfa root va. Plant Physiology 105, 787797
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Yoshitake, O.,Yoshinobu, N., Takuji, 22192230.
O., Hidetoshi, O., Nobutoshi, I.,
Abstract: Everyone wants to consume safe and healthy food. Also, the producers want to
position their products according to the customers demands. In the context of safe and
healthy foods, among others, there are two different terminologies, Organic and Halalan
Toyyiban. However, our understandings on these two terminologies are not clear enough
especially when it comes to relate them. Therefore, this research work was undertaken to
better understand the terminologies - organic and halalan toyyiban, and find out the rela-
tionship between them, if any. The research methodology involves both primary data by
visiting an organic farm and face-to-face interviewing farmers, and secondary data. The
findings might help the consumers in selecting the produce/product and business people in
promoting their products. Research informants were farmer, volunteer, and intern at the Sri
Lovely Farm, a government-certified organic farm at Sik, Kedah, Malaysia. The research
reveals new insights on the relationship of characteristics of organic farming with halalan
toyyiban. The three commonly found characteristics are: 1) quality; 2) healthy; 3) environ-
mental friendly. Based on the findings, we are proposing a model on the relationship of or-
ganic farming with halalan toyyiban. This study is the first of its kind and undertaken as an
exploratory research; therefore, further study should be conducted to obtain more under-
standing and knowledge on this subject.
Keywords: Environment; halal food; halalan toyyiban; organic farming; organic food
Table 1: Key differences between conventional and organic farming-Sri Lovely Farm fo-
cusing on the seed used
Key practices Conventional Sri Lovely Farm
1. Seed preparation Seed not selected Seed selected: Seeds soaked for 24
hours prior to sowing to eliminate
non-viable ones
2. Quality of seed- All kinds of seedlings Only healthy seedlings transplanted
ling at transplant
iii) It does not cause any negative effect hereditary which, when we planted the
upon intake seeds can later use to return to replant-
iv) The food contents are nutritious; ing- (Farmer 4, personal communication,
therefore, beneficial to human. October 24, 2016)
Relationship between
organic farming and
Characteristics of halalan toyyiban foods:
organic farming:
Characteristics of -Quality
halalan toyibban: -Healthy seed
-Healthy
-Quality -Natural fertilizer
-Environmental
-Healthy -Quality soil
friendly/Natural
-Clean (safe) -Natural insect control
-Environmental friendly
Figure 1: A proposed model to show relationship between organic farming and halalan
toyyiban foods
ICAR-Indian Institute of Sugarcane Research, Lucknow- 226 002, Uttar Pradesh, India;
*Correspondence: Ashutosh.Mall@icar.gov.in / misra.varucha@gmail.com; Tel: +91 522-
2480726
helps in reducing the cost of production Another most important constrain is the
as well as in overcoming the environmen- time required for a new variety to develop
tal pollution (Baker and Pretson, 2003). In and commercialize that generally takes a
case of sugarcane crop, on worldwide ba- long time of 12-15 years. As mentioned
sis there is high pressure to augment cane before that biotechnology helps in trans-
productivity for sustaining the profits of ferring a desired trait of gene from one
sugar mills (Halon et al., 2000). In this plant to other so in case of sugarcane
regards, various sugarcane researchers crop, there is certain desired traits which
have been showing effort in developing would not be able to introduced into it
new hybrid cane varieties that posses high through the normal plant breeding meth-
yield and high sugar contents under con- ods. The victory of improving the crop
ventional breeding programmes of sugar- production by biotechnological tools lies
cane at different institutes. With the use in the high levels of the trans-gene ex-
of these approaches, new cultivars are pression. In this aspect, promoters have
being able to develop which possess high been identified in driving the high levels
sugar content, better ability of ratooning of gene expression in transgenic sugar-
as well as resistance towards various dis- cane, particularly in stem and leaves. The
eases. These newer techniques and meth- first identified promoter was obtained
odology have paved new way in the field from Cestrum yellow leaf curling virus
of breeding for improving varieties and which impels the elevated level of consti-
also helped in rapid multiplication of the- tutive trans-gene expression significantly
se varieties. A common breeding con- higher than the ones obtained by the
strain in developing new varieties is its maize polyubiquitin-1 (Zm-Ubi1) promot-
slow multiplication rate as well as its rap- er (a well known benchmark). Another
id spread. This creates a problem in not identified promoter was the
fulfilling the seed requirement of the new- maize phosphonenolpyruvate carbox-
ly developed varieties, biotechnology in ylate promoter which facilitates the ex-
this aspect, had helped in faster multipli- pression levels, particularly in the leaf
cation of new varieties (Source access: region of sugarcane, compared to Zm-
http://shodhganga.inflibnet.ac.in/bitstrea Ubi1. By the process of gene modifica-
m/10603/42274/7/07_chapter%202.pdf, tion, the transgenic expression was en-
3.05.2017). hanced by approximately 50-fold for bet-
ter cane production (Kinkema et al.,
2. Biotechnological achievements 2014). Bower and Birch (1992) had
achieved success in the sugarcane trans-
2.1. In improving cane production formation trailing with the development
Being a major food and fuel of micro-projectile system. Some studies
source all over the world, biotechnology had showed improved resistance in de-
in this regard has the power for improving veloping a transgenic sugarcane crop to-
the economically important traits in this wards micro-organisms acting as patho-
crop. The key approach in improving gens (Joyce et al., 1998a, b; Ingelbrecht et
sugarcane production lies in the classical al., 1999; Zhang et al., 1999; Gilbert et
plant breeding method but plant breeders al. 2005;), towards pests like stem borer
always encounter difficulty in this regard (Arencibia et al., 1999; Braga et al.,
as cane genome is highly complex and 2003) and towards herbicide (Gallo-
possess narrow genetic base (Roach, Meagher and Irvine, 1996; Enriquez-
1989; Lima et al., 2002). The biotechno- Obregon et al., 1998).
logical approaches had successfully im- The use of techniques of genetic
proved cane production especially the in- engineering in the past two decades by
ter-specific Saccharum officinarium and plant breeders had caused transmission of
S. spontaneous hybrids (Usman, 2015). noble gene into the plant for developing
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better characteristics in them. The tech- raise and secondly cost of production will
nique involves insertion of foreign genes be reduced (Shanthi, 2016).
into the parent plant, use of protoplasmic
cells or tissues for the development of 2.3. Towards abiotic stress
transgenic plant having normal physiolog- For tolerance to a particular stress
ical and biological functions. Several ad- or multiple stresses, biotechnology had
vances have been seen from past several showed a new way for developing trans-
years in field of molecular biology as well genic plants. The best short-term ap-
as genetic engineering pertaining to crops proach for development of stress tolerant
(Jenes et al., 1993). With the use of a cane variety lies on the base of selection
technology based on recombinant DNA it and breeding wherein wide crosses are
is now even achievable to clone a gene, being made. For the development of the
modify or mobilize it and even integrate it stress tolerant variety certain steps have
in any other without any discrimination been outlined (Figure 1) (Epstein and
from where the gene has been taken Rains, 1987). Researchers had identified
(Chakrabarty et al., 2002). About 50 % candidate genes in sugarcane for impart-
losses are being occurring by different ing tolerance to various abiotic and biotic
types of borers but the use of Bacillus stresses. In this view, some of the exam-
thurigenesis by these technologies have ples of candidate genes which have been
shown harmful effects towards these bor- identified in case of drought/water deficit
ers. In the present scenario, by the use of condition are DREB (dehydration respon-
cry genes, greater than 30 species of dif- sive transcription factor), HSP (heat
ferent plants have been transformed shock proteins), LEA (late embryo-
(Schuler et al., 1998). genesis), RAB (responsive to abscisic ac-
id), osmotin, choline oxidase and annexin
2.2. In developing high yielding sucrose (Nair, 2011), stress-related clusters show-
cane varieties ing differential expression (>2-fold) dur-
Biotechnological approaches had ing biotic and abiotic stress conditions
made possible in increasing the frequency (Gupta et al., 2010), sugarcane ethylene-
of capability of parental clones possess responsive factor, SodERF3 (Trujillo et
with very high sucrose content which in al., 2009), up-regulation of genes regulat-
prevailing breeding programmes is at a ing intracellular redox status (Prabu et al.,
very low level. In case of sugarcane crop, 2011) and presence of LEA (late embryo-
Sugarcane Breeding Institute had initiated genesis abundance)-related proteins and
programme in this aspect. The target of dehydrin (Iskandar, et al., 2011), accumu-
this programme is to develop genetic lation of trehalose and proline (Molinari
stocks that consist of high sucrose by the et al., 2007; Guimares et al., 2008), oth-
use of recurring cycles of intensive cross- er stress-inducible proteins (Jangpromma
ing and selection. In cycle I, about 5420 et al., 2010), early response to dehydra-
seedlings obtained from 30 bi-parental tion protein 4 (ERD4) (McQualter et al.,
crosses were considered. Out of all the 2007). Wahid and Close (2007) had iden-
crosses performed, crosses between CoC tified various expressions of genes or pro-
671 x CoT 8201, Co 86002 x Co 62198, teins in sugarcane grown under tempera-
Co 85002 x CoT 8201, CoC 671 x Co ture and salinity induced stress. Some of
94019, PR 1080 x Co 94008 and PR 1080 the instances of such gene expression un-
x CoT 8201 had shown high levels of su- der stress are heat stress-induced DHNs
crose contents. The approach of biotech- (Wahid and Close, 2007), genes encoding
nology in this aspect of developing high for O-/OH- radicals and reduction of H2O2
sugarcane varieties have opened up a new by peroxidase/ catalase under heat stress
option which will reward to sugar millers (McQualter et al., 2007; Chagas et al.,
in two ways, firstly productivity will
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Biotech Approaches for Sustainable Sugarcane Productivity Mall and Misra
Figure 1: A diagrammatic sketch depicting the steps involved in using molecular ap-
proaches for the development of stress tolerant sugarcane variety (Source: Data from
Shrivastava et al., 2016).
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in identifying the enzymes that gives a these small products which in turn con-
start to these processes, however, through verts the complete cane plant into a bio-
the modern technique of genetic engineer- factory. A number of high value products
ing these enzymes cane be hasten or are being produced like therapeutic pro-
slowed down to attain more efficient stor- teins (Wang et al., 2005) and biopolymers
age and accumulation of sucrose in cane (Petrasovits et al., 2007; McQualter et al.,
stalks. The application of genetic manipu- 2005). Another important production of
lation in cane stalks are being conducted small product is production of isomaltose
one step at a time. Towards first step for which was possible by insertion of a bac-
success South African scientists had terial gene into the cane plant for produc-
knocked down a certain enzyme by genet- tion of an enzyme responsible for conver-
ic means which enhanced the sucrose sion of sucrose into iso-maltose, an alter-
content in young stalks of sugarcane native sweetener (Wu and Birch, 2007).
(Groenewald and Botha, 2008). Another Biotechnological efforts gave positive
perspective is making cellulosic bio-fuel results in stream of characterization of
production easier. It is well known that genome structure, specific traits mapping,
the sucrose is an essential component for marker assisted selection in resistance
production of bio-ethanol, an alternative towards insect/disease, variability of
for fossil fuels, through the process of pathogens on molecular basis, transfor-
fermentation. Breeders are focusing now mation, pathogen detection in precise
on enhancing the sucrose yield for in- manner in plants and many more. The
creasing production of ethanol without sharpness in sensitivity of the assays
compromising the sucrose content as food made them more rapid for routine analy-
commodity. By the modern use of bio- sis of plant pathogen detection and identi-
technology tools the cellulose content in fication. Besides, the assay has become
cane leaves as well as bagasse are being even more economical. The better ability
used for the production of ethanol thereby to detect the infection at early or latent
not utilizing the main cane product, sugar. stages help in improving the management
The intricate structure of cellulose can be of disease as well as restricting the
broken down into simpler molecules of movement of the various diseases and
carbohydrates by number of enzymes even assist in solving the phylogenetic
which later can be used for production of relationship amongst the various patho-
ethanol by fermentation nevertheless cel- gens. This also assists in developing new-
lulosic structure is guarded by lignin. This er strategies for enhancing the resistance
hard guarding material requires a costly activity of the host by genetic transfor-
procedure for its removal. Brazilian scien- mation methods. There is a need to im-
tists are taking initiatives through genetic prove the technologies for development
engineering in modifying the cellulosic of transgenic before making it a part of
structure so that it could be separated the varietal developmental programmes of
from bagasse without any difficulty sugarcane. There is still a challenge for
(http://agencia.fapesp.br/en/16756, ac- the researchers in developing a pathogen-
cessed on 03.04.2017). Adding to it, Aus- ic free transgenic as this requires com-
tralian researchers developed transgenic plete understanding of interaction of plant
canes by inserting genes capable of pro- and pathogen, however, gene cloning, to
ducing the cellulose degrading enzymes some extent, is making a new revolution
in leaves of mature plants (Harrison et al., in this aspect. There is still a need to iden-
2011). Besides production of small prod- tify and characterize the genes responsi-
ucts through sugarcane bio-factory which ble for the antifungal activity in case of
involves the tweaking of genetic mecha- disease resistance. In grassy shoot disease
nism occurring in cells of sugarcane plant of sugarcane and Sugarcane yellow leaf
that instructs them for the production of syndrome, a common occurring disease
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Biotech Sustainability (2017)
Biotech Approaches for Sustainable Sugarcane Productivity Mall and Misra
development of PCR diagnostic kits are short time, however, a major problem in
needed for Phytoplasma. this technique is its cost of production
For improving the crop quality as (Usman, 2015) .
well as productivity and even providing One of the most talked topics in
resistivity to pathogen, tissue culture is biotechnology achievements is the devel-
playing an effective role. The use of cryo- opment of transgenic varieties and the
therapy has enhanced the likelihood of area under the transgenic obtained plants
attaining healthy plants. In case of attain- has increased to approximately greater
ing virus resistant plants, micropropaga- than 81 million hectares but there are cer-
tion is the best tool which requires the use tain limitations too in developing it. Singh
of shoot apical meristem (acting as ex- et al. (2013) showed that the drawbacks
plants). In minimizing the somaclonal in developing cultivars are not even re-
variation in times to come, application of moved by the process of transformation;
clonal propagation as well as research in however, other methods for precise inte-
transgenic are needed. There is a need to gration and control trans-gene expression
work on the structure of genomes so that are still to be performed in sugarcane
identification of markers associated with crop. Furthermore, in studies related to
important agronomic characters may be sugarcane association, there is still need
performed. Transgene constructs that help for developing high throughput markers
in lessening the hazardous effect of envi- as well as producing more markers and
ronment and even bio-safety jeopardize of even ensure the proper availability of the-
these plants are need of the upcoming se markers. The newly developed markers
times. The way researchers are moving will enhance the knowledge and mystery
towards the biotechnological techniques of complex structure of sugarcane ge-
many mysteries will unravel (Tiwari et nome. DNA-based molecular markers of
al., 2010). progenitor plants have the potential to
show the prevailing genetic polymor-
4. Biotechnological challenges in en- phism that may be helpful in case of this
hancing the cane production and crop as parental genome is much less
productivity complex in comparison to the hybrids
ones (Henry et al., 2012).
The use of biotechnology in sug-
arcane crop has drawn researchers as well 5. Constrains in improving cane pro-
as entrepreneurs towards itself but its ap- duction using biotechnological tools
plication over this crop on commercial
basis had always be a regulatory chal- Biotechnology is generating
lenge particularly in case of field cultiva- enormous information which had played
tion. There is higher probability of trans- an important role in transforming the
ferring of genes along with unwanted world of science. Biotechnological inter-
genes from the source plant to other ference in sugarcane crop provides a
plants used as a food commodity. There- chance for sugar producers and cane
fore, the practicability of these techniques farmers to enhance the production and
on commercial cane bio-factory will be sustainability. These approaches act as a
dependent on how much amount of risk proactive approach in alleviation of the
containment as compared to the non-food troubles occurring in cane production
product plants, for example tobacco (Usman, 2015). Though researchers are
(http://isaaa.org/resources/publications/po attaining much success in improving the
cketk/45/default.asp, 27.3.2017). A com- cane production and yield yet there are
monly used technique now-a-days is tis- certain constrain in this regard using bio-
sue culture technique that helps in devel- technological tools. Some of these con-
oping uniform disease free plantlets in strains are: High-throughput sugarcane
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Abstract: The term bioremediation refers to the use of natural biological agents such as
microbes (bacteria, fungi, and yeast), plants or the enzymes released by them to return the
polluted natural environment to its original uncontaminated state. Bioremediation is a
novel, safe, cost effective, and ecologically feasible technology to detoxify accumulated
pesticides, toxic chemicals, fertilizers, aromatic compounds, xenobiotics, hydrocarbons (oil
spills), heavy metals in soil and water. Both in situ and ex situ bioremediation are being
used at a large number of sites all around the world with varying level of success. Although
bioremediation cannot degrade all the toxicity, particularly all the inorganic contaminants,
but it is still more eco-friendly and less expensive as compared to other remediation
methods like incineration, chemical treatment and thermal recovery of pollutants.
Furthermore, advances in bioremediation are being achieved by coupling this biological
method with molecular, genetic engineering, microbiology, pathway engineering, and
enzyme design and immobilization tools. In this chapter we have discuss the general
process for bioremediation, in situ and ex situ classes of bioremediation followed by the
types of bioremediation techniques being used till date. The role of bioremediation to deal
with different types of pollutions and comprehend the advantages, disadvantages and
sustainable use of its approaches is also highlighted.
function of bioremediation are known as bacteria. Also oil spills can be treated by
Bioremediators (Kumar et al., 2011). bacteria (Agarwal and Liu, 2015).
Bioremediation is a natural, effective and Recently it has been noticed that
environment friendly alternative to the awareness of the dangers of many
previously used methods for degradation chemicals used in our society has led to
of harmful contaminants, such as physical research on formulation of products that
removal, absorbents, catalytic destruction, are more easily degraded in the
incineration etc. which are high cost and environment. The process of
nonspecific methods (Head and Swannell, bioremediation involves the degradation
1999; Gillespie and Philp, 2013) of contaminants using microorganisms
Although use of microbial that have adverse impact on environment
consortia have proved their capability for and humans. Bioremediation includes the
remediation, application of biotechnology actions of several microorganisms that are
and genetic engineering tools is further acting in parallel or sequence, in order to
improving the efficiency and decreasing complete the process of degradation. In
the cost involved in treating toxic this process, both in situ as well as ex situ
substances . Bacteria, fungi, yeast and remediation are used. Hence,
algae along with several plants are being bioremediation is a technology applied in
used for this purpose. case of different environmental conditions
We will discuss the general where the numerous and versatile
process for bioremediation, in situ and ex microbes degrades a vast array of
situ classes of bioremediation followed by pollutants (Majone et al., 2015).
the types of bioremediation techniques On the basis of ecological point of
being used till date. The role of view, the term bioremediation involves
microorganisms in bioremediation as well the interactions between three factors that
as their genetic modification by the use of is substrate (pollutant), organisms, and
recombinant DNA technology will be environment, as shown in Figure 1. The
understood. Further we will discuss the interactions between these three factors
role of bioremediation to deal with primarily affect the biodegradability and
different types of pollutions and bioavailability of pollutants, and
comprehend the advantages, physiological requirements of microbes,
disadvantages and sustainable use of which plays a vital role in the assessment
bioremediation approaches. of the feasibility of bioremediation.
Biodegradability defines whether any
2. Bioremediation chemical can be degraded by microbes or
not, whereas bioavailability refers to the
Bioremediation is the branch of availability of a pollutant to organisms
biotechnology that deals with the that are capable of degrading it. For
solutions of problems related to the instance, the substrate has low
environment. Bioremediation also bioavailability if it is tightly bound to soil
involves the process of cleaning the organic matter or it is trapped inside its
environment from different types of aggregates. The conditions that are
pollutants as well as contaminants by required by microorganisms to carry out
using bacteria, fungi etc. Bacteria play a the process of bioremediation include
vital role in the process of bioremediation different factors like nutrient availability,
since it break down the dead materials optimal pH, and availability of electron
into organic matter and nutrients. Several acceptors, such as oxygen and nitrate etc.
types of contaminants such as chlorinated are referred to as physiological
pesticides etc. can be easily digested by requirements.
Plants tolerant to PAN: Acer platanoides, his research in this field in the early
Chrysanthemum spp. 1950s.
In another breakthrough, Dr.
4.2. Rhizofiltration Ananda Mohan Chakrabarty of the
In this form of phytoremediation General Electric Company (USA)
roots of plants act as biofilters i.e. these developed a genetically engineered oil
absorb toxic metals from the eating bacterium (Pseudomonas putida).
contaminated water and accumulate them. The patent was registered to him in
Therefore, the water which passes 1980s. It was welcomed by the scientists
through root zone becomes free from community as an answer to pollution
pollutants (Macek et al., 2004). Later, the problem. The Environment Protection
contaminants are removed from the Agency (EPA) reported that
system by harvesting root biomass. bioremediation eliminated both soil and
Members of family Brassicaceae (the water borne oil contamination at about
family of scavengers) have shown 1/5th cost of previous method. Since then,
tremendous potential for the cleanup of bioremediation has been increasingly
polluting metals. The plants roots used to clean up oil pollution in United
accumulate metals from the surrounding States and in other countries. In 1989, oil
soil giving a metal content as high as 30% spilled from the Exxon Valdez tanker off
of the dry weight of the plant roots. the coast of Alaska where these oil eaters
Alan Baker, a geobotanist from helped in clean-up of oil. They degraded
University of Sheffield, England, the oil efficiently trapped between rocks
discovered a tree Sebertia acuminate (the and under gravel beaches where all other
nickel lover) which hyper-accumulates Ni means had failed.
so much that when slashed, it bleeds a
jade green liquid. The tree is a native of 4.4. Zooremediation
New Caledonia and accumulates Ni as It is the process of
high as 20% of its dry body weight. decontamination of polluted environment
Besides, Acedium plant absorbs by using animals as bioagents. Animals so
Vanadium and some plants of family far used for bioremediation purposes are
papilionaceae (e.g. Pea) are known to fish, different arthropods and other filter
absorb higher amounts of Molybdenum feeders in aquatic systems and
from soil. earthworms in solid organic waste
management systems. Use of animals in
4.3. Microbial bioremediation bioremediation is not very encouraging
Microorganisms like bacteria are except earthworms because there are so
proving very important tools for the many limitations with them e.g. fish and
removal of pollutants from the arthropods may bioaccumulate the toxic
environment and are thus continuously compounds and metals which may be
doing their job of detoxification, biomagnified in the food chain creating
tirelessly, with or without coming into the many problems to the environment
attention of man, who is the only culprit (Gifford et al., 2007).
of dumping more and more pollutants into Solid organic waste generation is a
the environment. In the recent past, major problem of cities which are facing
microbes were first used to treat industrial the threat of being overrun by garbage
wastewater as early as 1930s. There was a and the piled up waste and is adversely
significant movement in the field of threatening our health, environment and
microbial bioremediation when Dr. wellbeing. In this context,
Howard Worne (USA) first discovered vermicomposting - waste degradation
phenol degrading microbes, when began through earthworms has proved to be very
promising. The principles behind this are grinding gizzard, an organ that all
relatively simple and related to those earthworms possess. The earthworms
involved in traditional composting derive their nourishment from the
(Figure 2 summarizes composting as a microorganisms that grow upon the
basic Ex-situ bioremediation process). In organic materials. At the same time they
general, vermicomposting consists of 4 promote further microbial activity in the
major phases: Phase I: Collection of the residues so that the faecal matter or casts
waste, separation of metal, glass, that they produce are much more
ceramics etc. from the organic waste, and fragmented. During this process, the
storage of the organic waste. important plant nutrients in the organic
Phase II: Earthworm beds are material particularly nitrogen,
maintained and the earthworms are fed phosphorus, potassium and calcium are
with the organic waste. released and converted into forms that are
Phase III: After the organic waste much more soluble and available to plants
has been worked over by the earthworms, than those in the parent compounds.
the vermicompost, cocoons, earthworms Worms can digest waste several times
and the undigested material are separated. their own weight each day.
Phase IV: Packaging of the
vermicompost and reintroduction of 4.5. Bioventing
undigested material into the vermipits. Bioventing is a process of
stimulating the natural in situ
Certain species of earthworms biodegradation of contaminants in soil by
(Eisenia fetida, E. Andrei, Lumbricus providing air or oxygen to existing soil
rubellus, L. hortensis, L. terristris etc.) microorganisms. Bioventing uses low air
can consume organic residues very flow rates to provide only enough oxygen
rapidly and fragment them into much to sustain microbial activity in the vadose
finer particles by passing them through a zone (Hinchee, 1994). This is an on-site
all conditions that may be controlled. metals, and stones. The stones will then
Land Farming differs from composting be crushed into smaller pieces and then
because it actually incorporates depending on the degree of contamination
contaminated soil into soil that is will either be added to a pile or sent out
uncontaminated. Landfarming is most for reuse. The soil is then homogenized,
successful in removing polycyclic meaning that the pollution concentration
aromatic hydrocarbons (PAH) and is averaged out across the entire soil
pentchlorphenol (PCP). sample. Homogenization allows for
biopiling to be more effective (Schulz
4.8. Bioreactor Berendt, 2000). Once the soil is piled,
Bioreactors treat contaminated nutrients, microbes, oxygen, and substrate
soils in both solid and liquid (slurry) are added to start the biological
phases. The solid phase treatment process degradation of the contaminants. The
mechanically decomposes the soil by results of the initial laboratory tests
attrition and mixing in a closed container. indicate to the operators which substrates
The objective of the mixing is to such as bark, lime, or composts needs to
guarantee that the pollutants, water, air, be added to the soil. Nutrients such as
nutrients, and microorganisms are in mineral fertilizers may also be added.
permanent contact. An acid or alkalinity Additionally, microorganisms such as
may also be added to control the pH (van fungi, bacteria, or enzymes could be
Deuren and Lloyd, 2002). Infixed bed added (Schulz-Berendt, 2000).
reactors, compost are added and
significantly increase the degradation rate. 4.10. Bio-stimulation
In rotating drum reactors, the drum has a Bio stimulation could be
screw like mechanism in the middle of it perceived as including the introduction of
that rotates to mix and transport the soil. adequate amounts of water, nutrients, and
The liquid phase treatment process uses oxygen into the soil, in order to enhance
suspension bioreactors and treats as the activity of indigenous microbial
slurry. The slurry feed enters the system degraders. The concept of bio stimulation
and is rinsed through a vibrating screen to is to boost the intrinsic degradation
remove debris. Sand is then removed potential of a polluted matrix through the
using a sieve or hydrocyclone. If a accumulation of amendments, nutrients,
hydrocyclone is used to remove the sand, or other limiting factors and has been
the sand falls to the bottom of the cyclone used for a wide variety of xenobiotics
and the fines remain on top. The fines are (Kadian et al., 2008). Microorganisms do
then treated in a bioreactor. After the extremely well in thriving on herbicide
treatment, the slurry must be dewatered compounds in the soil by utilizing them as
and the water is then treated with standard a supply of nutrients and energy. Many
wastewater techniques (Kleijntjens and herbicides serve as good carbon and/or
Luyben, 2000). nitrogen sources for soil microorganism
(Qiu et al., 2009). Evidence for their
4.9. Biopiling remarkable range of degradative abilities
It is an in situ process that is also can be seen in the recycling rather than
known as the heap technique. The first accumulation of vast quantities of
step in the biopiling process is to perform biological materials that have been
laboratory tests that will determine the produced throughout the history of life on
biological degradation capabilities of the earth (Dua et al., 2002).
soil sample. The next step involves the
mechanical separation of the soil, which 4.11. Bio-augmentation
will homogenize the sample and remove Bio augmentation is the
any disruptive material such as plastics, enhancement of biodegradation of waste
and contaminants in the media by the following key site characteristics are
introduction of adapted competent required to evaluate the likely success of
microbes and nutrients. Microorganisms intrinsic remediation; the bioavailability
from Geobacteraceae family due to their of contaminants, levels of nutrients, the
physiological characteristics can play an presence of minerals to buffer the pH of
important role in the bioremediation of the matrix, adequate levels of electron
subsurface environments contaminated acceptors (either oxygen, nitrate, ferric
with organic or metal contaminants iron, or sulphate), and site specific
(Lovley et al., 2004). In some instances, contamination migration rates. This
the rate of biological degradation can be approach deals with stimulation of
increased through the addition of indigenous or naturally occurring
microorganisms that have been shown to microbial populations by feeding them
degrade the contaminants of concern at nutrients and oxygen to increase their
high rates or are particularly well suited to metabolic activity.
remain active under prevailing site
conditions. This process is referred to as 5. Microbes involved in bioremediation
bio augmentation. This can be useful if
the contaminants are particularly Microorganisms are responsible
recalcitrant to degradation or if site for biodegradation in various diverse
conditions are extreme (for example: high environmental conditions. These
concentrations or toxicity of microorganisms include: Acinethobacter,
contaminants). To be effective, the Actinobacter, Acaligenes, Arthrobacter,
introduced organism(s) must become Bacillins, Berijerinckia, Flavobacterium,
distributed throughout the contaminated Methylosinus, Mycrobacterium,
matrix and compete with the indigenous Mycococcus, Nitrosomonas, Nocardia,
microorganisms for available nutrients. If Penicillium, Phanerochaete,
they are not distributed throughout the Pseudomonas, Rhizoctomia, Serratio,
matrix the positive effect will be Trametes and Xanthofacter. Individual
localized. On the other hand if the microorganisms are not efficient in
introduced organisms compete poorly, mineralization of harmful substances.
they will not persist and the treatment Thorough mineralization results in a
effect will be short lived. The problems progressive degradation by a group of
encountered using this approach include microorganisms (or microbial
biofouling of equipment, injection wells consortiums) and involves coaction and
and seepage beds. Adjustments to the co-metabolism actions. Microorganisms
system, such as the use of new discharge in various habitats have remarkable
areas, may be required to prevent this physiological flexibility, so they are able
from occurring. This approach to to make use of and often mineralize an
bioremediation must be evaluated on a enormous number of organic molecules.
site specific basis. Several other requirements for microbial
growth in biodegradation process are
4.12. Intrinsic bioremediation listed in Table 2. Some microbes with
Often bioremediation can be specific biodegradation capabilities are
accomplished without human intervention discussed below.
by microorganisms that are naturally Pseudomonas putida: In context
found in the contaminated matrix. For of bioremediation, it is a microorganism
this approach to be used, it is usually found in farmland soil involving high
necessary for the rate of contaminant impact xenobiotics including
degradation to exceed the rate of organophosphate insecticides, petroleum
contaminant migration. Knowledge of the hydrocarbons, and both monocyclic and
polycyclic aromatics (Iyer and Damania, organisms like bacteria, plant, virus or
2016). animal and thus these are also referred to
Dechloromonas aromatic: This as transgenic organisms (Ozcan et al.,
bacterium is involved in degradation of 2012).
aromatic compounds like benzene in
nitrate reducing conditions as well as 6.1. Role of GMOs in environmental
physiological and molecular management
characterization in anaerobic mixed Genetically modified organisms
cultures (Ulrich and Edwards, 2003). can be used to clean up the environment
Deinococcus radiodurans In by bioremediation. Effects of some
field of development of bioremediation genetically modified microorganisms are
strategies, this bacterium plays a role as a unstable and vary according to species,
radiation resistant organism. It is used for changes in population structure and loss
the treatment of mixed radioactive wastes of some functions, to the formation of
containing ionic mercury (Brim et al., toxic metabolites. Presence of high and
2000). The radioactive waste sites can be active microorganisms makes the process
treatedby this strategy of bioremediation. of bioremediation more operative and
Methylibium petroleiphilum they must adapt with the changing
Also known as PM1 strain that is environmental conditions. Deinococcus
involved in methyl tert butyl ether radiodurans that exhibit toluene
(MTBE) bioremediation. MTBE is dioxygenase to clear-out toxic elements
degraded by this strain using the that are found in radioactive waste sites
contaminants as source of carbon and was used by Lange (1998) as a
energy (Hanson et al., 1999). recombinant. Deinococcus radiodurans is
Alcanivorax borkumensis is a rod- known to have two properties, first it is
shaped bacterium having capability of resistant to radiation and secondly it can
consuming hydrocarbons and produces degrade chlorobenzene in radioactive
carbon dioxide. Hence it can be used environments (Lange et al., 1998). Then
readily in oil damaged environment again, it can only be produced in an
(Santisi et al., 2015). environment at temperatures less than
Phanerochaete chrysosporium It 39C and as radioactive sites generally
is the first fungi involved in degradation have high temperatures, so a bacterium is
of organic pollutants (Kadri et al., 2017). required that can function at higher
temperatures. Another well-known
6. Genetically modified organisms example for the application of GMOs in
the management of environmental issues
Bioremediation by means of can be cited through certain bacteria that
microorganisms is not significant for can yield biodegradable plastics and this
treatment of all types of pollutants. For quality of bacteria were transferred to
example, heavy metals such as cadmium microbes which were cultured in the
and lead are not freely absorbed or taken laboratory and now a days they have
by organisms. The role of genetically enabled the wide scale greening of plastic
modified organisms in the process of industry.
bioremediation has emerged as a new tool In the early 1990s, Zeneca, a
(Jafari et al., 2013). A genetically British company, established a
modified organism, or GMO, is an microbially manufactured biodegradable
organism that has an altered DNA plastic called Biopol
configuration made through genetic (polyhydroxyalkanoate, or PHA). The
engineering. Most of the genetically plastic was made using a GM bacterium,
modified organisms have been Ralstonia eutropha, to transform glucose
transformed with DNA from other and a variety of organic acids into a
Abstract: Sea urchin gonads usually called as Sea urchin Roe or Uni, are important
food delicacies in different parts of the world. In Asian, Mediterranean and Western Hemi-
sphere countries, the roe of sea urchins is considered as a highly prized delicacy sea food
because of its tastes and also have long been utilized as extravagance foods in Japan. Peo-
ples of the Asian Pacific region have long been utilizing it for improving general body tone
and also treatment for a number of diseases. It has been reported that, sea urchin gonads are
found to be rich with high-quantities of bioactive compounds, such as polyunsaturated fatty
acids (PUFAs) and -carotenes. The PUFAs, particularly eicosapentaenoic acid (EPA,
C20:5) (n-3)) and docosahexanoic acid (DHA C22:6 (n-3)), have profound significant ef-
fects on arrhythmia, cardiovascular diseases and cancer. -carotene and some xanthophylls
have strong pro-vitamin activity and can be used to prevent tumor development and light
sensibility. The sea urchin fisheries in recent years have extended so impressively that the
natural populations of them have been overexploited to meet-up the increasing demand. Not
surprisingly, the continued high demand and the decrease in supply have headed towards a
pronounced interest for the commercial aquaculture of sea urchins. Global sea urchin har-
vesting, having peaks at 120,000 metric tons in 1995, are presently in the scale of around
82,000 metric tons. However, these declining arrays evidently mirror the overfishing of ma-
jor fishery grounds and focus the necessity for conservation measures, aquaculture devel-
opment and sustainable fisheries management. Once the natural stocks decrease, the higher
market demand for foodstuff, nutraceuticals, pharmaceuticals and cosmeceuticals, increases
the value of the manufactured goods and therefore, culturing seems to become economical-
ly feasible. As per this assessment exhibits, there have been intense progresses in the aqua-
culture protocols of sea urchins during the past 15-20 years, we can come to the end that
presently the main impediments to successful farming are actually managerial, cultural,
conservational and economical rather than biological and ecological. Expected that demand
is implausible to decline, the commercial value of future product will be increased. Hence,
the fortune of sea urchin is strictly connected to those fisheries, whose prospect would
eventually depend on the stock improvement, aquaculture production, fishery management,
roe enhancement and market forces that will play a significant role to give a structure of
this expanding industry.
Figure 1: Global production of sea urchin fisheries from 1950 to 2008 (FAO, 2010).
ible (Hagen, 2000; Kelly, 2000, 2005; inhabiting in temperate zones mostly have
Sivertsen, 2004; Rahman et al., 2014b). moderate longevity between 10 and 20
Nonetheless, the continuous declining years, even though highest longevity of
patterns noticeably reveal the overexploi- 100 years has been reported. Growth and
tation of most of the fishery grounds and production performances are flexible and
focus the necessity for proper conserva- greatly dependent on quality food and nu-
tion policies, sustainable management trition. In conditions with lower density,
strategies and appropriate aquaculture growth rates are usually high, while at
practices. high densities, growth rates are low.
However, the density of sea urchins is not
2. Biology and ecology of major spe- the single factor that starts the construc-
cies tion of barrens or flats. Inter-annual
events that trigger warming (e.g., El
The most parts of the world involved Nino), are known to cause kelp dieback
in sea urchin production are linked with which then leads to overgrazing and the
main sites of primary productivity. In the maintenance of barrens in the extended
subtropical and tropical regions, these are period. The exclusion of predators that
usually associated with seagrass beds, would then cause moderate urchin densi-
while in the temperate regions, with kelp ties may also endorse conditions leading
forests. Urchins commonly occur at lower to overgrazing. Usually, densities of sea
densities within the kelp communities, urchins are the highest in barrens, but in-
overgrazing on kelps and then lead to the dividual growth performance and overall
establishment of flats dominated by en- productivity is low owing to competition
crusting macroalgae (called as coralline for food and perhaps due to the deficient
flats and barrens). Both of the communi- nourishment.
ties (barrens and kelp forests) are often The most efficient predator of sea ur-
found near to each other, making either a chins, particularly the sea otter and its re-
mosaic of stable patches or strata. establishment around the North is having
The biological features of sea urchins main effects on sea urchin fisheries. In
vary largely among species. The species areas where populations have been re-
established, predation can far exceed fish- 2.2. Red sea urchin (Strongylocentrotus
ing pressures and urchin densities have franciscanus)
been reduced to levels below that which Strongylocentrotus franciscanus usu-
is required for fishing to be profitable ally referred to as red sea urchin, is the
(Andrew et al., 2002). Recruitment mor- biggest echinoid in the world and com-
tality is also affected by different types of monly occurs along the West Coast of
habitats. It has been reported that mortali- North America, extending from Baja Cal-
ty of juveniles among a number of species ifornia to the Aleutian Archipelago and
is higher in kelp forests because of the the coast of Siberia and northern. The
occurrences of micro-predators in their ranges of their habitats encompass north-
habitats (Tegner, 2001). ern-wards up the west coast to Sitka and
Given the similar habitat types, a Kodiak AK at 58oN (Tegner, 2001). This
number of edible sea urchin species share species is also occurs in the subtidal zone
some similarities in their distribution, to a depth of 50 m seawards and is in-
abundance and reproduction. Yet, they tensely associated with kelp forests.
are differed in their fundamental biologi- Growth performance in the earlier stage
cal characteristics such as growth, surviv- of urchin is comparatively fast and the
al, production, maturity and longevity. species shows the highest longevity. TD
at recruitment is about 90 mm, which is
2.1. Green sea urchin (Strongylocentrotus usually attains in around 6-8 years of the
droebachiensis) age. The maximum size is around 200
Green sea urchin has a circumpolar mm and the individuals over 150 mm TD
distribution and occurs through the North are older than 100 years (Tegner, 2001).
Atlantic to the North Pacific. The fisher- Spawning commonly occurs over the
ies of S. droebachiencis have been fo- spring and summer months when the ur-
cused in Maine and the Canadian Mari- chins attain sexual maturity at around 50
times but smaller fisheries are concentrat- mm TD and the spawning usually over
ed in Alaska, British Colombia, Washing- summer and spring months.
ton and Iceland. The species is mostly
common in the intertidal zone to a depth 2.3. Japanese green sea urchin (Strongy-
of 50 m, where it is closely related with locentrotus intermedius)
kelp beds (Scheibling and Hatcher, 2001). Strongylocentrotus intermedius com-
Two distinct growth rates have been iden- monly known as Japanese green sea ur-
tified, but the overall growth rates are chin is the 2nd most economically im-
found to be moderate. The fast growing portant regular echinoid in Japan. This
form attains the minimum legal size be- species is distributed along the Asian and
tween 4 and 6 years and can live for 16- Siberian coast of the Pacific. This species
20 years. On the other hand, the slow is common in intertidal shallow waters
growing one inhabits for 8-12 years and around Hokkaido and is exploited com-
never attains the optimum legal size (An- mercially from Aomori, Irate and Hok-
drew et al., 2002). In British Colombia, kaido (Agatsuma, 2001a). It generally
the minimum legal size is not more than occurs in shallower stony substratum and
55 mm TD (test diameter) and in most is usually associated with kelp forests
cases, the time needed to grow to this size (Agatsuma, 2001a). The matured adult of
is supposed to range from 4 to 8 years S. intermedius contains small reddish-
(Taylor 2004). This sea urchin attains yellow gonads, which do have a good
sexual maturity within 1-2 years and the taste and thus listed on Tsukiji as Japa-
first spawning occurs between mid- nese. It is well-adapted to cold water and
winters to early spring when it reaches to the growth restriction does not appear to
45-50 mm in TD. be related to the temperature limits
(Agatsuma, 2001a). Density and nutrition
are the two major factors influencing closely associated with kelp beds and its
growth of urchins. In appropriate culture growth is extremely flexible and reliant
conditions, the urchin attains 40 mm TD on the availability of algae. The maxi-
within 2-4 years and the maximum size of mum size has been recorded to be 100
55 mm TD at ages between 6 and 10 mm TD. S. purpuratus usually attains
years. It obtains sexual maturity at the age sexual maturity around 2 years of age,
of 2 years with the size of 30-35 mm TD and spawns during their natural breeding
and the spawning usually occurs in au- season, extending from January to March.
tumn and spring.
2.6. Purple crowned sea urchin (Cen-
2.4. Strongylocentrotus nudus trostephanus rodgersii)
This species has been recorded on The purple crowned urchin experienc-
Tsukiji as Japanese and also considered as es a subtropical distribution throughout
the most commonly harvested edible sea the water areas of Australia and New Zea-
urchin in Japan and accounts for ~ 44% of land, but most abundantly occurs in East-
the total commercial catch (Agatsuma ern Australia. This species has also been
2001b). It is found on inter- and sub-tidal reported to be extended into Bass Strait
rocky bottoms extending from Dalian, and the East Coast of Tasmania (Rdger,
China northwards to Primorskyi Kray, 1999) and is possibly related with the
Russia and in Japan where it is found in warming of coastal waters around the re-
the Pacific from Sagami Bay to Cape Er- gion (Andrew and Byrne, 2001). It is one
imo on Hokkaido and in the Sea of Japan of the large urchins with long dark purple,
from Omi Island in Yamaguchi to Soya black to red spines that have iridescent
Cape northern Hokkaido. The urchins blue/green sheen. Centrostephanus rodg-
generally reach the legal size (40 mm) in ersii is usually seen in large numbers and
2 to 4 years when feeding on perennial plays an important ecological role in in-
Laminarians whereas they take 7 to 8 tertidal near shore rocky reefs by cleaning
years on coralline flats. It occurs in the the areas of kelp. Growth performances
intertidal to subtidal rocky reefs and is follow the medium trends and the indi-
strongly associated with kelp communi- viduals attain 70-90 mm TD within the
ties. Juveniles recruit to coralline flats and age between 4 and 10 years. The longest
move to adjacent kelp forests. In kelp for- size (120 mm TD) achieved when the ur-
ests individuals reach 50 mm TD in 2 to 4 chin become 20 years of age. The adult
years, whereas 7 to 8 years reported in urchin attains sexual maturity at the size
coralline flats (Agatsuma, 2001b). Maxi- between 40 and 60 mm TD and spawning
mum longevity is reported as 14 to 15 occurs during the winter months.
years. Sexual maturity is attained at 40 to
45 mm TD, and spawning takes place in 2.7. Kina (Evechinus chloroticus)
autumn. Evechinus chloroticus well-known as
kina is a sea urchin endemic to New Zea-
2.5. Purple sea urchin (Strongylocentro- land waters. The distribution of kina is
tus purpuratus) intensely linked within kelp beds or ag-
The sea urchin S. purpuratus, usually gregating in nearby barrens. It is typically
recognized as purple sea urchin, inhabits occurs from the intertidal area to a depth
along the eastern edge of the Pacific coast of 14 m; even some are found in 60 m.
of North America, extending from British Growth rate is moderate and the individu-
Columbia, Canada to Ensenada, Mexico. als reach to a size of 50 mm within the
It occurs abundantly in lower intertidal age of 4 years. However, E. chloroticus
and nearshore subtidal communities but attains the maximum size of 80-100 mm
has been found to a maximum depth of in 8-9 years of age. Depending on the site,
150 m (Tegner, 2001). This species is the longevity of this urchin varies be-
tween 10 and 20 years (Barker, 2001). It age of 3 years, this species can reach to a
is predominantly herbivorous, feeding maximum size of 92 mm TD. Average
mainly on brown algae, red algae and en- longevity ranged from 1 to 2 years. When
crusting substrate (Barker, 2007). Kina the urchin attains a size of 40 mm TD, it
usually reaches sexual maturity within 3- gets sexual maturity within a year after
4 years of age having a size range be- metamorphosis. No reasonability of
tween 40 and 50 mm TD and spawns in spawning was observed in this species.
spring, extending from November to Feb-
ruary (Barker, 2007). 2.10. Rock sea urchin (Paracentrotus
lividus)
2.8. Chilean red sea urchin (Loxechinus Paracentrotus lividus is a species of
albus) sea urchin belongs to the family Parechin-
The Chilean red sea urchin, Loxechi- idae and commonly known as rock or
nus albus, is one of the reasonably slow- purple sea urchin. It occurs in the Medi-
growing urchins, commonly found around terranean Sea and eastern Atlantic Ocean,
the Pacific coasts of South America from extending from western Scotland and Ire-
Isla Labos de Afuera in Peru to the land to the Azores, Canary Islands and
Southern tip of South and usually occurs Morocco, and most common in the west-
within the depths ranged from the inter- ern Mediterranean, the coasts of Portugal
tidal zone to a maximum of depth of 340 and the Bay of Biscay, where the water
m (Vasquez, 2001). This urchin attains temperature in winter months varies with-
sizes up to 130 mm TD and can live until in 10 to 15oC. This species usually inhab-
20 years of age (Andrew et al., 2002). It is its in the shallow sub-littoral area to a
considered as one of the important com- maximum depth of 20 m. Paracentrotus
mercial species along the south-west lividus is intensely related to the seagrass
coast of South America due to high taste meadows and mainly existed on encrusted
qualities. The distribution of L. albus is rocky substratum where it makes perma-
mostly on rocky substrates and closely nent burrows to live in. It experiences
related with the kelp beds. It is an herbi- with moderate growth rates and the indi-
vore and seems most likely to feed on viduals having 40 mm TD are usually 4-5
whatever species of alga grow nearby. years old, and the adults with a size of
The urchin is comparatively slow- 70+ mm TD are older than 12 years. The
growing, attaining a maximum size of largest size of 15 mm TD is reported by
130 mm TD. Spawning period differs de- Boudouresque and Verlaque (2001). The
pending upon its destitution patterns; species gets sexual maturity at the size
happening in spring to summer in the range between 13 and 20 mm TD and the
north, summer in the south and spring in spawning usually occurs during spring to
the extreme south. the early summer months.
2.9. Variegated sea urchin (Lytechinus 2.11. Collector sea urchin (Tripneustes
variegatus) gratilla)
The variegated sea urchin occurs in Tripneustes gratilla is commonly rec-
the shallow waters and widely distributed ognized as collector, cake or Parsons hat
throughout the tropics and subtropics of sea urchin and has a circumtropical distri-
the western Atlantic, from Florida, bution, encompassing to the subtropics.
through the Caribbean to Brazil and Pan- This species is usually occurs in the Indo-
ama (Watts et al., 2001). Lytechinus var- Pacific, Hawaii, the Red Sea and Baha-
iegatus is usually inhabits on seagrass mas, and is widely distributed from Red
beds and hard bottoms covered with sea westward to Hawaii and Clarion Is-
macroalgae. It is a fast-growing urchin land, eastward to Paumotu, as far south as
but the longevity is limited. Within the Port Jackson, and at Sharks Bay on the
west coast of Australia. It is regarded as a can live for more than 10 years (Sander-
shallow-water sea urchin and usually in- son, 1995). It attains sexual maturity at
habits on a diversity of substrates and oc- TD sizes of 4050 mm within 56 years
curs in the depth range between 2 and 30 of age (Sanderson et al., 1996). The best
meters (Lawrence, 2007). Tripneustes roe production was found to be 10-14%
gratilla grazes continuously during day during AugustDecember and spawning
and night and its diet comprises of algae, usually occurs between summer and au-
periphyton and seagrass. It has higher tumn (Sanderson, 1994).
growth rate with lower longevity. Within
the age of 4 to 5 years, the urchin attains a 2.13. Shore sea urchin (Psammechinus
maximum size of 160 mm TD. However, miliaris)
it can grow to 75 mm TD in the first year Psammechinus miliaris is a species of
of age. The collector sea urchin has an sea urchin under the family Parechinidae
annual reproductive cycle mediated by and sometimes known as shore of green
seawater temperature, length of day and sea urchin. It shows restricted distribu-
feeding activity. Spawning mainly occurs tions in the southern and eastern waters of
during mid to later winter months, when the North Sea, and the eastern Atlantic
water temperatures and day lengths be- Ocean from Scandinavia south to Moroc-
come the lowest and each clutch contains co, where it occurs from the low tide
approximately 2 million eggs. Similar to mark down to a maximum depth of 100 m.
the other regular sea urchins, the fertilized Its abundance is strongly associated with
eggs develop into pluteus larvae, which the presence of Laminaria kelp. This sea
then stay in the water column for almost urchin is often found on or under Saccha-
30 days. They then settle on the sea floor, rina lastissima, a large brown seaweed
undergo metamorphic induction and then with which it shares its range of distribu-
become the tiny young juveniles. This tions and also occurs in a variety of other
species attains the sexual maturity at habitats including under boulders and
around 2-5 years of age to become a rocks, among seaweeds, on rough ground
complete reproducing adult. and on the rhizomes of Zostera marina in
seagrass meadows. It is an omnivore and
2.12. Purple sea urchin (Heliocidaris mainly feeds of macroalgae, diatoms, hy-
erythrogramma) droids, worms, small crustaceans, mol-
Heliocidaris erythrogramma or lusks and detritus. Longevity is relatively
the purple sea urchin is usually distributed short and the growth rates are observed to
in the shallower coastal communities, ex- be moderate. It has been reported that in-
tending from intertidal to a maximum dividuals can reach to a maximum size
depth of 35 m in the southern Australia. (45 mm TD) with an age between 3 and 4
In the coastal waters of Tasmania, this years. Psammechinus miliaris gets sexual
species is commonly occurs in kelp com- maturity in the first year at 6-7 mm TD
munities and barrens, where it feeds by and usually spawns in the months of
grazing and capturing drift weeds. It can spring and early summer.
also be occurred in high densities in asso-
ciation with sea grass beds (Keesing, 2.14. White sea urchin (Salmacis
2001). Growth rates of this sea urchin sphaeroides)
usually vary depending on food availabil- The short-spined white sea urchin (S.
ity and nutrition, but are mostly moderate. sphaeroides) belonging to the family
Individuals of H. erythrogramma attain a Temnopleuridae, is considered as one of
size of 40 mm TD within one year as well the rare species under the group of regular
as a harvestable size (60 mm TD) at 3 to 5 Echinoids. It usually occurs in the tropical
years. It has been reported that individu- Indo-West Pacific Ocean, extending from
als having maximum size of 122 mm TD China to Solomon Islands and Australia
including Singapore and Malaysia (Tan small-bodied Echinoids, having the max-
and Ng, 1988; Schoppe, 2000; Miskelly, imum size of 85 mm TD and can live for
2002; Rahman et al., 2012b, 2013b). This 8 to 10 years. They inhabit burrows and
species has almost white cloudy test of crevices and thereby defend themselves
55-80 mm TD with plentiful small white from strong wave action and predators.
spines, the size of which are between 10 Echinometra spp. are active herbivorous
and 15 mm. Some individuals have white in nature and without the presence of
spines with marron bands, some with all predators, they can occur in densities that
maroon species, while the others with ma- exceed the primary production potential
roon and green bands. Salmacis (McClanahan and Muthiga, 2001). Breed-
sphaeroides can be found in the depth, ing takes place in any time throughout the
ranging from 0 to 90 m seawards, and al- year but they usually spawn during sum-
so be occurred in shallow intertidal zone, mer and autumn in warmer waters. Simi-
particularly among seagrass meadows, lar to the other regular Echinoids, Echi-
coral reef substrates and in muddy sublit- nometra spp. release their eggs and
toral areas or washed ashore Schoppe, sperms in the water column, where fertili-
2000). This species feeds different types zation occurs externally and the plankton-
of seaweeds, bryzoans and detritus. ic echinopluteus larvae are produced
Spawning is found to be around the year. through the embryonic and early larval
Following fertilization in the water col- stages. The time when the competent lar-
umn, the embryo develops into a blastula vae gets suitable substratum, they first
in about 9 hours. A series of larval stages settle on the seabed then undergo meta-
follows, in which the larvae acquire more morphosis to produce juvenile urchins.
and more arms, and develops tube feet
and spines within the larval body. Up to 2.16. Long-spined black sea urchin (Di-
this point, the process takes about 35 days. adema setosum)
Competent larvae swim near the surface The tropical sea urchin, Diadema se-
of the substrate to determine a suitable tosum commonly referred to as long-
site for settlement. After attachment, lar- spined black sea urchin, is a member of
val structures are either discarded or re- regular Echinoids under the family Di-
sorbed, and adult features continue to de- adimatidae. It is broadly distributed
velop in the juvenile (Rahman et al., throughout the Indo-Pacific region, from
2012b). Australia and Africa to Japan and Red
Sea, extending to the Gulf of Aqaba, Gulf
2.15. Rock boring sea urchin (Echi- of Suez and Arabian/Persian Gulf (Lessi-
nometra spp.) os et al., 2001). This species has charac-
A number of recently diverged spe- teristics long black spines land five white
cies of rock boring sea urchins belonging spots on the aboral side. The distinctive
to the genus Echinometra, are widely dis- orange ring around its anal cone com-
tributed throughout the Worlds marine pletes the special visual features of this
ecosystems. They occur commonly within species. It is usually an omnivorous scav-
and around coral reefs from central Japan enger and detritus eater and scraps films
in the north to southwest Australia in the of hard substrates. They are generally
south, from Clarion Island off Mexico in found in coral reefs and shallow rocky
the east, and to the Gulf of Suez in the habitats at depths from 1 to 6 m. This
west (Rahman et al., 2000; 2005). Vari- species has a wide range of diets, which
ous species of Echinometra exhibits cir- includes microalgae, seaweeds, coral
cumtropical distribution and usually occur polyps and encrusting animals (Grignard
in shallow intertidal habitats, however a et al., 1996). Gametogenesis begins in
few has been recorded at a maximum AprilMay, when the seawater tempera-
depth of 20. These species are usually ture rises above 25oC in the Gulf of Suez
and spawning take place between June Sea urchin aquaculture has successfully
and September (Pearse, 1970). It has also been accomplished on a large scale in Ja-
been known to spawn both seasonally and pan for many decades. In order to en-
thought out the year depending upon the hance the natural stocks, millions of juve-
locations and sites of the spawning adults. nile urchins are being produced in hatch-
Temperature levels higher than 25oC have eries, for releasing to the managed areas
been observed to be a potential spawning of seafloor on the intertidal seashore are-
cue (Pease, 1974). Throughout the year, as. The nationally co-ordinated reseeding
the equatorial populations are found to be program has been developed to the extent
spawn without following any particular that over 66 million juveniles were re-
times. This is eventually true for the Ma- leased on the reefs within which, over
laysian and Philippine populations of D. 80% were S. intermedius (Agatsuma et
setosum (Tuason and Gomez, 1979). In al., 2004). The contribution of released
the Persian Gulf, spawning usually occurs sea urchin juveniles to the overall catch
between the months of April and May has been estimated to be between 62 and
(Alsaffar and Khalid, 2000). Some other 80%. There have also been much small-
cues, such as the moon phases have been scale reseeding programs functioning in
found to influence the spawning of D. se- South Korea and Luzon Islands in the
tosum. This urchin has also been observed Philippines (Andrew et al., 2002). The
to trigger spawning activities in concord- farm entrepreneurs and researchers
ance with the entrance of a full moon around the southern Ireland have been
(Lessios, 1981). developing techniques for commercial sea
urchin (P. lividus) cultivation for more
3. Culture, management and stock than 20 years (Leighton, 1995), and com-
enhancement paratively newly in France (Grosjean et
al., 1998). Culture of 3 commercially im-
3.1. Aquaculture portant sea urchins (P. miliaris, E. escu-
Mostly, the edible sea urchins are lentus and P. lividus) has been conducted
regular Echinoids (Lawrence, 2007), ex- in Scotland since 1995 and there are also
periencing separate male and female sex- well-established research teams in the
es and are generally broadcast spawners. east coast of North America including
At the onset of the breeding season, the Florida, Alabama, Maine, New Hen-
sexually matured adults release their isphere, New Brunswick and Newfound-
gametes in the water column where ferti- land working on S. droebechiensis and
lization takes place. The pluteus larvae L. variegatus; On the west coast of North
form through the embryonic and early America, including California and British
larval development of the fertilized eggs, Columbia (S. droebechiensis, S. francis-
which after a period of planktonic devel- canus, S. purpuratus); in Chile (L. albus);
opment, feed on unicellular diatom, settle in New Zealand (Evechinus chloroticus),
to a suitable substratum and undergo met- Norway (S. droebachiensis) and in Israel
amorphosis to produce small juvenile ur- (P. lividus) (Kelly, 2005).
chins. At 26-28oC, almost 1 month is re- Sea urchin brood stocks are regu-
quired to complete the larval life cycle, larly collected from the wild stocks when
comprising the feeding or 4-armed stage, they reach proper sexual maturity. Ma-
the 6 to 8-armed stages and finally com- tured gametes are obtained by injecting
petent stage for settlement (Figure 2). The 0.5 M KCl into the coelomic cavity of
newly born metamorphosed juveniles both female and male urchins. Sperms in
grow on macroalgae until attain the mar- its most concentrated from are pipetted
ketable size (4050 mm) within the age off the genital pores, while eggs are col-
ranged from 1 to 3 years, depending upon lected by inverting the female urchins
the species (Kelly, 2005). over a glass beaker filled with filtered sea
Figure 2: Breeding, development and complete life-cycle of ball-like white sea urchin
(Salmacis sphaeroides) (Rahman et al., 2012b).
water (FSW) (Rahman et al., 2000, 2001, they are reared in glass bottles on a roll-
2004, 2005, 2012b). At limited sperm ing roller keeping a larval density of 1-2
concentrations, fertilization is usually individual/ml of medium. The unicellular
done by mixing a few drops of diluted cultured diatoms (Chaetoceros calcitrans,
sperm (10-4 dry sperm dilutions) with egg Isochrysis galbana) are commonly used
suspensions in a petri dish and the result- as supplementary larval food at the con-
ing embryos are reared. Hatching of ferti- centrations of 5,000, 10,000 and 15,000
lized eggs usually takes 10-15 hours after cells per ml of medium daily at 4-, 6- and
insemination, to develop into a ciliated 8-armed pluteus stages, respectively until
blastula. When the swimming larvae attaining metamorphic competence within
achieve feeding stage (4-armed pluteus), around 30-35 days post-fertilization
(Rahman et al., 2016) (Figure 3). In Japan, diatom (50:50) in petri dishes containing
partial water exchange system (Sakai et FSW (Rahman et al., 2012b). Within 1
al., 2004) and continuous flow-through day after the settlement induction, majori-
system (Hagen, 1996) are used for the ty of the competent larvae are found to
large scale cultivation. The most costly metamorphose into young juvenile (Fig.
aspect of captive larval culture is the need 4A). They are then reared on the encrust-
for the concurring production of the ing coralline algal rocks in the aerated
above microalgae as live food for larvae. glass/plastic aquaria for at least three
Nevertheless, larvae of the variegated sea months by which they attains appropriate
urchin (L. variegatus) have just been con- juvenile (hereafter referred to as sea ur-
firmed to be appropriate for culturing chin seed) sizes (Fig. 4D) for stocking in
with artificial diets (Gorge at al., 2004). grow-out aquaculture system. In the coun-
Settlement and metamorphic in- tries like Japan, South Korea, Ireland,
duction are considered as the most crucial Norway, Scotland, and in British Colom-
stages for the development and culture of bia, Canada, sea urchin juveniles have
sea urchin larvae. The higher survival rate been produced on a commercial or semi-
is always dependent on the larvae to be- commercial scale by some well-
come competent to metamorphose and developed hatcheries and nurseries. Al-
then responding to the exact settlement most all the culturists use natural biofilm
cues. In small-scale culture, induction for or a specially seeded diatom substratum
metamorphosis of competent larvae has made from species locally isolated and
recently been performed on coralline red then grown on a PVC wave plate. How-
algal extracts + Chaetoceros calcitran ever, one of the most challenging areas of
Figure 3: Developmental stages for larvae of short-spined white sea urchin, Salmacis
sphaeroides: A) 4-arm pluteus, B) 6-arm pluteus, C) 8-arm pluteus, D) Pre-competent larva,
E) Competent larva with complete rudiment growth, and F) Just newly metamorphosed ju-
venile with adult spines and tubefeets (Rahman et al., 2012b).
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Biotech Sustainability (2017)
Sea Urchin - A New Potential Bio-resource for Human Health Rahman et al.
Figure 4: Juveniles of the white sea urchin, Salmacis sphaeroides: A) 1-day-old juvenile,
B) 1-month-old juvenile, C) 2-minth-old juvenile, and D) 3-month-old juvenile for grow-
out culture (Rahman et al., 2012b).
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Biotech Sustainability (2017)
Sea Urchin - A New Potential Bio-resource for Human Health Rahman et al.
Table 1: Comparison of growth and production of S. sphaeroides fed with different types
of algae. Mean SE, n = 30
Parameters Treatments
T1 (Red alga) T2 (Brown alga) T3 (Sea grass)
a a
Initial length (cm) 10.04 0.70 10.04 0.70 10.04 0.70a
Final length (cm) 46.49 1.01a 43.56 1.04b 38.67 0.35c
Initial weight (g) 0.49 0.11a 0.49 0.11a 0.49 0.11a
Final weight (g) 51.17 1.17 a
31.91 1.42 b
20.80 0.65c
Weight gain (g) 50.67 1.93a 31.39 1.44b 20.31 0.43c
Length gain (cm) 36.46 1.01a 33.85 0.66b 28.63 0.35c
SGR (%/day) 0.73 0.01 a
0.65 0.01 b
0.58 0.01c
DGR (%/day) 7.92 0.30a 4.91 0.22b 3.17 0.10c
Wet gonad weight (g) 6.01 0.37a 3.56 0.26b 2.32 0.10c
Gonad index (%) 18.26 0.51 a
16.44 0.19 b
14.84 0.25c
Survival (%) 88.89 1.93a 73.33 3.34b 56.67 5.77c
Means sharing the same superscripts within the same row are not significantly different
from each other (P > 0.05).
completely changed at every 23 months. from the relocation from poor to good
After two years of culture, the urchins feeding grounds (Moylan, 1997) to the
attain sexual maturity (Figure 5B) and ranching of urchins caged on the seafloor
those fed red alga, performed the best (Cuthbert et al., 1995). Juveniles reared in
over the brown- and sea grass-fed urchins hatcheries have been grown under sus-
with regard to body growth and edible pended culture condition (Kelly, 2002,
gonad production (Table 1; Rahman un- 2005) in closed recirculation systems
published data). On the contrary to the (Grosjean et al., 1998) and in demand
Japanese culture system, where hatchery- rock pools in southern Ireland. The sea-
reared juveniles are mainly released to cage culture system of stacking baskets
managed seafloor (Hagen, 1996; Sakai et suspended from a ladder-like structure
al., 2004; Kelly, 2005), scientists of other over which a work barge or raft can oper-
countries have conducted research with a ate has been developed by Norwegian re-
wide range of grow-out culture systems searchers (Aas, 2004). The time taken for
for the juvenile and adult urchins, ranging juveniles of most species to reach market-
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Biotech Sustainability (2017)
Sea Urchin - A New Potential Bio-resource for Human Health Rahman et al.
able size is in the range from 1 to 3 years limited entry (moratoriums) followed
(Kelly, 2005, Rahman et al., 2014b). by active programs to reduce latent ef-
fort,
3.2. Management resource surveys at various levels of
The sea urchin fisheries around the world complexity,
have consistently shown their susceptibil- the use of annual Total Allowable
ity to overfishing and to the problems as- Catches based on resource assessment,
sociated with readjusting effort following zoning and area management, which
a fishing-down phase. Through the late may be developed to the point of rota-
1980s and 1990s, a number of fisheries tional harvest,
have developed to cater for the expanding the use of minimum legal sizes.
demand of the lucrative Japanese market.
This has driven the development of new Classical fisheries science was de-
fisheries off Chile, both coasts of North veloped in consideration of offshore, open
America and Australia. These fisheries access and industrial fishing situations
A
commenced B
on virgin stocks and as a re- and the resulting management systems are
sult have needed to deal with the issues of not well adapted, or particularly robust,
rising expectations during fishing-down when applied to more complex spatial
phase and adjustment of the fishery for structure of small scale, inshore fishery
long-term sustainability. resources (Orensanz and Jamieson, 1998).
The current position of the various The social significance of small scale in-
American fisheries provides the full range shore fisheries is much greater, given the
of outcomes in dealing with this problem. numbers of fishermen and other players
For instance, the fisheries of Canada, involved, than the sometimes more pro-
Alaska and Washington have had active ductive offshore fisheries, which general-
programs to readjust effort levels and are ly involve larger enterprises, higher capi-
now managed on the basis of catch limits tal investment and limited numbers of
based on sustainable harvest strategies fishermen. As a result, much of the chal-
using regular population surveys. The lenge in ensuring the sustainability of
Californian fishery has yet to adequately shellfish fisheries lies in developing and
adjust levels of effort and there is evi- applying appropriate utilization, assess-
dence that the fishery is now being over- ment and management models. According
fished. The Chilean fishery has little ca- to Orensanz and Jamieson (1998), the
pacity to readjust effort levels and it management measures that explicitly
would appear that once the fish-down acknowledge spatial structure of fishery
phase is completed there is the potential resources, and are therefore the most suit-
for significant overfishing. Of the fisher- able for these sorts of fisheries, include
ies reviewed here, there are numerous ex- (but not limited to):
amples of fisheries that have collapsed to i) territorial property and use rights in-
levels of one or two magnitudes below cluding lease, traditional tenure sys-
their peak production. These include the tems etc.;
fisheries of France (Mediterranean and ii) harvest rotation coupled with pulse
Atlantic), Iceland, Ireland, South Korea fishing and/or thinning;
and Philippines. Significant parts of the iii) reproductive refugia and Marine Pro-
Chinese fishery have also collapsed. tected Areas;
Those fisheries that are currently being iv) experimental management with spa-
managed for long term sustainability tial control, contrasting treatments and
share a number of characteristics. They replication;
are: v) localized enhancement including habi-
tat manipulation, seeding and predator
control.
Systems that accelerate growth to seeding did not have any benefit on the
market size sea urchins while producing a subsequent urchin production and crabs
uniform size class would give an econom- and sea stars are removed from the
ic advantage One probable way to obtain grounds using baited traps prior to the
sustainable and environmentally friendly release of the urchins to control mortality
systems for urchin culture is to further in the immediate period after release.
examine their potential in integrated sys- Government has taken considerable in-
tems. They have already been shown to volvement in the management of coastal
thrive in polyculture with Atlantic salmon fisheries, particularly in the provision of
(Kelly et al., 1998) and to have a role in subsidies for enhancement and infrastruc-
land-based integrated systems (Shpigel et ture development as well as management
al., 2004). Nevertheless, many species are coordination. A couple of studies have
true omnivores, so the potential for their been looked at the contribution of re-
integration into systems where natural seeding or habitat enhancement to the ac-
prey items, for instance, mussels and tual abundance of urchins in harvest areas
clams, are already produced should be in a sort of round-about way at localized
explored. sites around Hokkaido and estimated that
re-seeded urchins comprised 62%, 66%
3.3. Stock enhancement and 80% of the total catch in 1994, 1995
Decline in world production and over- and 1996, respectively (Agatsuma, 2004;
fishing have prompted increasing in en- Rahman et al., 2014b).
hancement as a means of maintaining Translocation of the urchins is
production. It is most developed in Japan used for a number of related reasons in
where the 1974 Coastal Fishing Ground Japan. In areas where kelp forest devel-
Improvement and Development Law pro- opment is held back by excessive urchin
vide the basis for stock enhancement densities, urchins are sometimes removed
(Agatsuma et al., 2004). The goal of this and replaced with adult kelps to permit
program is to develop and improve rapid development of complex kelp for-
coastal fishing grounds systematically by ests (Agatsuma, 2004). The urchins may
the construction of artificial reefs and the then be placed into intensive sea ranching
release of seedlings. Enhancement can pens where they are fed ad libitum and
comprise a number of different activities prepared for harvest some months down
including direct stock enhancement the road. Experiments have shown that
through seeding of hatchery-raised juve- Green Sea Urchins at densities up to 35
niles, habitat improvement or restoration, kg/m have recorded recovery increases
creation of artificial reefs, predator con- from 6% to over 18% in 11 weeks on an
trol, thinning and/or roe enhancement artificial diet (Aas, 2004), although fur-
through supplemental feeding to increase ther finishing for about 6 weeks on a nat-
the product recoveries etc. Re-seeding has ural kelp diet is still required to get an
been especially applied in Japan since late acceptable taste profile at this point. The
1980's. The numbers have been fairly sta- sea urchin Evechinus chloroticus is wide-
ble since 1997 with about 70-85 million ly distributed around New Zealand but
juveniles reared to about 5-10 mm TD attempts to establish a commercial fishery
and released each year primarily in the have, like Norway, not succeeded because
areas with the largest historical harvest. of the poor product quality and low re-
Strongylocentrotus intermedius accounts coveries. Experiments with ponding over
for about 85% of the urchins released by 2 month periods have seen recoveries to
the Japanese in Hokkaido (Andrew et al., increase near 20% and produced other
2002). Predator removal is required as quality improvements that promise well
excess predation by sea stars etc. has been for the future but further research is still
implicated in the few cases where the re- needed to reach an economic breakeven.
There are a number of aquaculture sites the fore in a number of countries (Robin-
around New Zealand which are currently son, 2004).
considered marginal for mussel farming,
which would be suitable for urchin cul- 4. Bioactive compounds and human
ture (Barker and Fell, 2004). Multi- health benefits
disciplinary approaches are therefore
needed for stock enhancement and both Alike many other marine inverte-
scientific and user group advisors should brates, sea urchins have been considered
be involved (Masuda and Tsukamoto, as a source of biologically active com-
1998). One method of enhancement that pounds with biomedical applications
apparently works very well with Green (Kelly, 2005, Rahman et al., 2014b).
Sea Urchins simply requires the presence However, the full potential of echinoids
of a salmon net pen. The urchins can ap- as a source of biologically active products
parently settle out quite abundantly on is largely unexplored (Bragadeeswaran et
such structures and grow quite nicely by al., 2013). The marine environment is an
feeding on the fouling organisms on the exceptional reservoir of natural bioactive
mesh. This has provided some opportuni- compounds, many of which exhibit struc-
ties for Canadian fishermen in British Co- tural and chemical features not detected in
lombia (BC) for some easy harvests. terrestrial derived natural products. The
The current market system used richness of diversity offers a great oppor-
for the urchin trade developed in tandem tunity for the discovery of new bioactive
with the wild fishery but this will no compounds. Modern technologies have
doubt change dramatically once cultured opened huge extents of research for the
product is available in substantial quanti- extraction of bioactive compounds from
ties. Cultured production is more tightly seas and oceans to treat the fatal diseases.
controlled than from the wild fishery so The number of natural products isolated
that, as with the cultured salmon, the con- and purified from marine organisms in-
sistent availability of an invariably high creases rapidly and currently surpass with
quality product throughout the year will hundreds of new compounds being dis-
have a tremendous impact on the urchin covered every year (Proksch and Muller,
markets throughout the world. Traditional 2006). The isolated secondary metabolites
harvesters of sea urchins do not generally have numerous functions; it is likely that
know much about the potential of aqua- some of them may be pharmacologically
culture (Robinson, 2004) and will likely active compounds for humans and useful
tend towards obstructing its development as medicines (Briskin, 2000). A number
as opposed to recognizing the available of such compounds have been isolated
advantages and applying them to their from echinoderms (Carballeria et al.,
own benefit. This will be unfortunate be- 1996). There has also been much valuable
cause if the wild and cultured urchin fish- information available for new antibiotics
eries could be more closely integrated, and give new insights into bioactive com-
both would stand to benefit. For example, pounds in sea urchins. Sea urchins shells
the gonad size and quality are quite easy are containing various polyhydroxylated
to manipulate and the economic yield of naphtoquinone pigments, spinochromes
the roe can be dramatically and fairly eas- (Anderson et al., 1969) as well as their
ily increased. This knowledge is probably analogous compound echinochrome A, of
directly applicable to the wild fishery and which was showed bactericidal effect as
could contribute to an increase in quality, reported by Service et al. (1984). The
value and profitability. Already, fisheries phenolic hydroxyl groups in these mole-
and aquaculture are blurring together with cules also suggested that they could par-
respect to product (gonad) enhancement ticipate in particular antioxidant activity
and re-seeding of juveniles is coming to as was observed in other well-known an-
pending on their type and location. Ta- This is mainly the case for the worlds
yum eggs are usually eaten raw and the largest sea urchins fishery in Chile, where
selling price is from RM 2 to RM 5 per the risks of collapse cannot be discounted.
pack. Meanwhile, tehe-tehe is sold at Given that this fishery alone contributes
RM 1 to RM 2 per plate i.e., containing upwards of 55% of the global harvest, a
three urchins with their skin intact to be significant decline in Chiles fishery
cooked into oku-oku, a traditional Bajau would likely lead to structural realign-
delicacy. In comparison, price of sea ur- ment in the market and higher prices for
chin eggs is RM 36 to RM 60 for every mid-range products until aquaculture pro-
80 g in America, while in Japan, an ur- duction ramped up. There is also general
chin can cost as much as RM 18 (Parvez agreement that some form of exclusive
et al., 2016b). Aesthetically, the diverse access as a prerequisite condition to pro-
forms of the sea urchins, and their beauti- mote meaningful enhancement and intel-
ful coloring, are often providing not only ligent harvesting to maximize roe value
a source of joy and recreation but also will provide the best hedge against uncer-
increase the additional revenue to humans tainties in fisheries productivity and mar-
observing them. Thus, sea urchins play a ket stability. In the short term it is likely
significant role in livelihood development that global production of sea urchin roe
and income generation to the local coastal from wild fisheries will decline, with the
communities. major production being provided by those
fisheries that have supported active man-
7. Concluding remarks agement strategies to readjust the effort
and contain catches to levels that provide
This paper has been presented as a back- long-term sustainability. Given that de-
ground document and review of the mand is unlikely to decline; future pro-
Worlds sea urchin fisheries. To summa- duction will be increasingly valuable. In
rize the views, reports and publications of order to make sea urchins fisheries viable
other scientists/researchers, it is apparent and profitable, the following actions are
that sea urchin fisheries have a poor rec- suggested:
ord of sustainability, as evidenced by the Refinement of artificial diet formula-
declines recorded in Japan, Maine, Cali- tions for juveniles and adults to max-
fornia and South Korea among others, as imize the growth rates and survivor-
well as by the ad hoc and/or ineffective ship and produce gonads of the de-
management in many sea urchin fisheries. sired taste, texture, flavor and color.
Very few stocks have been formally as- Optimization of grow-out facilities for
sessed, meaning it is near impossible to juveniles and adults either at sea (in
qualify declines as the fish-down of ac- containers and ranched) or land-
cumulated biomass, which does not arrest based.
the productivity of the stock, or as a case Regulations regarding fishing meth-
of over-fishing in which case its produc- ods, fishing areas and protection of
tivity may be forced into permanent de- company investments need to be de-
cline. Small-scale management is men- veloped.
tioned time and again as offering the most Better surveillance of sea urchin den-
promise for ensuring long term sustaina- sity to guarantee a steady flow of raw
bility. The strong and consistent spatial materials.
structure inherent in sea urchin stocks Areas need to be thinned out to get the
combined with excessive effort from mo- best possible product for the market,
bile fleets and inappropriately large scale, this is also necessary for the kelp for-
and therefore ineffective management all est to grow back.
contribute to declining production in More capital needs to be directed to-
many of the worlds sea urchin fisheries. wards investing in technology for
ly related tropical sea urchins (ge- from the coastal waters of Johor,
nus Echinometra): evidence Malaysia. Asian Journal of Ani-
against postzygotic isolating mal and Veterinary Advances 8(2),
mechanisms. Biological Bulletin 301308.
200, 97106. Rahman, M. A., Yusoff, F. M. and Ar-
Rahman, M. A., Uehara, T. and Pearse, shad, A. (2014a). Potential and
J. S. (2004). Experimental hybrid- prospect for sea urchin resource
ization between two recently di- development in Malaysia. Fish-
verged species of tropical sea ur- mail 21, 1618.
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Echinometra oblonga. Inverte- F. M. (2014b). Sea urchins (Echi-
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Rahman, M. A., Uehara, T. and Law- pounds. In: Kao, J. C. M. and
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fecundity estimates of long-spined val and juvenile development of
sea urchin, Diadema setosum, tropical sea urchin, Diadema se-
from the Pulau Pangkor, Peninsu- tosum. Iranian Journal of Fisher-
lar Malaysia. Aquatic Ecosystem ies Sciences 14(2), 409424.
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1. Marine ecosystem and its resources goons, salty marshes, mangroves, deep
sea and sea floor ecosystems which are
The ocean occupy 71 percentages important for marine (and terrestrial) liv-
of earths surface; they are interconnected ing organism (Barange et al., 2010). They
and traditionally divided into four large provide goods and various services to the
basins including North and South Atlan- human society such as good climate, vital
tic, North and South Pacific, Arctic and foods, medicines, bio processing and em-
Indian oceans. The average depths are ployments including fishing, process in-
13,216, 10,932, 12,786 and 3,665 feet for dustries, aquaculture and coastal tourism
Pacific, Atlantic, Indian and Arctic etc.
oceans, respectively. Marine environment
is most important for life on earth, the 2. Marine pollution
living organisms originated in marine and
they emigrated to terrestrial and freshwa- The oceans are susceptible for
ter bodies. Oceans are the main regulators pollution ever by human activities by pol-
of climate and temperature to the terres- luting with different agents and physical
trial ecosystem. Phytoplanktons are im- destruction ocean environments. The def-
portant for oxygen production; they yield inition of Marine Pollution as Introduc-
around eighty percentages of oxygen tion by man, directly, or indirectly, of
which is used by the animals and plants substances or energy to the marine envi-
for breathing in terrestrial and aquatic ronment resulting in deleterious effects
ecosystems (Bigg et al., 2003). Marine such as: hazards to human health, hin-
ecosystem is the largest ecosystem with drance to marine activities, impairment of
intertidal zones, coral reefs, estuaries, la- the quality of seawater for various uses
ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 444
Biotech Sustainability (2017)
Marine Pollution and Its Impacts on Living Organisms Citarasu and Babu
and reduction of amenities. Marine pol- their feathers (Kachel, 2008). The crude
lution is mainly classified to coastal oil is highly toxic to the marine organisms
sources by riverine inputs, deposition because of it contains toluene, xylene,
from atmosphere and offshore inputs etc. benzene and polycyclic aromatic hydro-
Coastal diffuse sources are contaminants carbons and these chemicals bioaccumu-
via coastal industry, sewages and devel- lated to planktons, fishes, shellfishes, sed-
opment sites (Clark, 2001). Alternative iments constitute a long lasting threaten-
inputs are site-specific discharges, agri- ing to the benthic animals. Polycyclic Ar-
culture lands and forests, mainly for nu- omatic Hydrocarbons (PAHs) is having
trients leakage to groundwater bodies, many pathetic effects to the living organ-
further they transported into the marine isms including mutagenic, carcinogenic
ecosystem by backwater bodied and final- and act as a teratogens (Kachel, 2008).
ly huge quantities of contaminants depos-
ited marine environments (GESAMP, 3.2. Persistent toxic substances (PTS)
1993). Municipal wastes and sewage wa- Persistent Toxic Substances
ter effluents are not properly treated and (PTS) also called Persistent Organic Pol-
discharged in to sea, these problems lutants (POPs) are noxious, long-lived
mainly happened in developing countries. and less persistent. The prolonged usage
Emissions through air crafts are main and dispersion of PTS may cause serious
sources of atmospheric pollution to sea problems and responsible for chemical
and the pollutants are dispersed a vast ar- and biological degradation. Their physical
eas by the wind flow and weather chang- characteristics are chlorinated or halogen-
es. Marine pollution in offshore caused by ated affected to water solubility levels and
discharges of pollutants from vessel based high lipid solubility, no degradability
at least 10 % of the total marine pollution leading to fatty tissues bioaccumulationsn
and they contribute by different ways (El-Shahawi et al., 2010). POPs constitute
(Anon, 2005). The other sources are in- remarkable societal advantages, or not
cluding crude oil extraction and of miner- planned by-products of burning process-
al extraction etc. es, such as dioxin. The halogenated hy-
drocarbons derivatives of tributyl tin
3. Pollutant Sources (TBT), dibutyl tin and monobutyltin that
are the disruptors of endocrine organs
3.1. Oil pollutants (Kachel, 2008).
Hydrocarbons are classified into
alkanes, naphthenes and aromatics. The 3.3. Heavy metal pollution
crude oil also contains nitrogen, oxygen Through riverine input, heavy
and vanadium compounds. The spillage metals are entering the sea and accumu-
of oils from the ships/ cargo, platforms of late in marine sediments as well as flora
offshore oil and on-shore refineries, it and fauna. The important heavy metals
may be produced serious effects to the are mercury (Hg), cadmium (Cd) and lead
marine environments in multiple ways. (Pb), accumulations are important for tox-
The causes of oil pollution producealtera- icity (Burger and Gochfeld, 2002). They
tions in physic chemical levels, also in- generally share the features of PTS, be-
volved toxication of marine habitats and cause they are bioaccumulate, non-
the flora and fauna seriously affected af- degradable and generate stringent or long
termath of large spills. The turbidity of oil standing toxic effects. The toxicity effect
prevents the light penetrations and lead to are vary based on the heavy metal types,
photosynthesis processes for phytoplank- for example if absorption of mercury as in
tons. Lose of waterproofing qualities very little doses, cause severe harmto
faced by the larger animals, aquatic birds brain and the central nervous system. The
getting lost of waterproofing by losing main sources of oceanic and atmospheric
contamination heavy metal contamina- ing the toxin secretion from the microbes
tions provided by electric utilities, fuel and harmful algal blooms in the marine
combustion, iron manufacturing, incinera- water create other environmental prob-
tion of urban refuse and fuel additives. lems. Environmental changes causespar-
Marine anti-fouling paints are very dan- ticular animals can change their living
gerous to marine organisms because it place to alternate place due to warmer
contains. Lead is another big poison cre- waters and reproduction also affected by
ates the problems like neurotoxicity, men- increasing temperature. Marine organisms
tal health problems, lead is mainly re- lay of undeveloped eggs or the tempera-
leased from batteries, sewage and fuel ture prevents the normal development of
combustions. Electroplating factories, particular eggs due to rise of temperature
batteries and sewages are also responsible level in marine waters. Enzymatic activity
for cadmium toxicity and it affects bones and metabolic rate also raised and in-
by deformities and kidney dysfunction. creased food consumption due to increase
Nickel poisoning is also a potent carcino- temperature. This is seriously affecting
gen that is toxic at relatively low concen- the stability of food chain and modifies
tration. Crude oil contains selenite with the balance of species composition (Brett,
sulfur, reported as a toxic metal found out 1970).
in marine environment at a huge level.
The other dangerous heavy metals con- 3.5. Nuclear radiation
taminations released through various In marine environments, radia-
routes including rain of pollutants from tions may classified in to natural and hu-
the atmospheric air, fallout from ship de- man activities, natural radiations are
struction and contaminated land runoff emission of cosmic rays, potassium-40
creating dangerous environmental prob- by earth's crust and decay products of
lems (Kennish, 1998). uranium etc. Human activities including
nuclear power plants and reprocessing,
3.4. Thermal pollution nuclear weapons testing and accidents,
Nuclear reactors release huge fallouts of nuclear wastes, radiation using
amount of heat to the marine environ- food conservation, medical diagnosis
ments leading to decreased level of oxy- combustion, land-based mining, phos-
gen can disastrous effects on ecosystems phate production, and oil exploration etc
and its communities.The decreased level (Shinde and Gawande, 2015). High level
of oxygen in marine water, degrade the radioactive waste dumping is not permit-
quality of wildlife animals that lives un- ted in the ocean, even low level wastes is
derwater. The increased temperature still permitted to the marine trenches, be-
holds less oxygen and creates suffocation cause low level wastes containing low
to marine fauna including copepods and radioactivity than high level waste. High
amphibians (Gautam et al., 2016). Indus- level nuclear waste had half-life for
tries are responsible for decrease the qual- 24,100 years whereas the short-lived ra-
ity marine life and destroy habitats by al- dioactive elements had the half life of 30
tering the natural environments. Natural years. The radioactivity may absorbed by
destruction including geothermal activity micro algae, zooplankton, and other small
and volcanoes under the marine can in- marine organisms and then this will be
duce warm lava to increase the tempera- transmitted or biomagnified to the food
ture of marine waters. Thunders and chain, to fish, marine mammals, and hu-
lightening is also responsible for tremen- mans. In human concerns, the radiations
douslevel of heat into the marine that cause cancer, changes in DNA that en-
leads to raise the temperature of marine sures cell repair (Kachel, 2008).
water. The high temperature discharge
from industries is responsible for induc- 3.6. Excess nutrients
Emissions and discharges of agri- get the diseases such cholera, typhoid etc
culture sources are a significant pollu- (Yazhini et al., 2015).
tions to the coastal
zone particularly affects biogeochemical 3.8. Noise pollution
cycles and primary producers. Eutrophi- Life in marine can be susceptible
cation is created by the pollutants includ- for noises from different noise pollutants
ing discharge of ammonia and methane, including oil exploration, seismic surveys,
use of insecticides and other pesticides passing ships, and naval low-frequency
affecting marine flora and fauna. Excess sonar waves. Sound is very fast in water
nutrients discharges including phosphorus than in the atmosphere and the noise pol-
and compounds are responsible to eu- lution in marine are increased at ten folds
trophication. High concentrations of nu- during the period of 1950 to 1975. Marine
trients, based on the physico-chemical mammals including dolphins, porpoises
properties of the affected marine envi- and whales are using sound as communi-
ronments, may lead to increased growth cation and sensation because sound is not
of phytoplanktons (Sarkar and Malchow, or less effective for their communications.
2005). Hydrogen sulphides also induce Also the particulates scattering the light
the negative impacts, if the oxygen level and it interferes the communications in
decreases, hydrogen sulphides levels also marine mammals. Also it is impossible to
increased. Hydrogen sulphides induced using smell as communications, because
low resistance to the aquatic living organ- the chemical molecules in water diffuse
isms leading to die off. The dead micro more slowly than in air are not possible
algae due to hydrogen sulphide toxicity (Payne, 1983). Sound speed is four times
floats on the sea surface leading to inter- higher in water than atmosphere so it is
fere the penetration of sunlight. Fertilizer opt to communicate the marine mammals.
runoffduring heavy rainthe organic ferti- Sonar waves is very dangerous to marine
lizers run off from the agricultural field mammals it interfere the sound wave
and it affects the marine environment and communications and causes severe inju-
back water bodies. ries including lose of sense organs leading
to death. Also the sounds emanated from
3.7. Microbial contamination the passing ships other instruments for
Generally microorganisms are re- marine mining activities also disturbed
leased from waste water, waste products the dolphin and whale populations.
and sometimes human and animal wastes
into the environment seriously spoiled the 3.9. Ship based threatening
marine ecosystem. The viruses do not Ship based threatening mainly
replicate without the help of host animals. caused by operationally or accidentally
First it may infect some other host cells and releases the pollutants to the marine
and multiply. Seafood contaminations are ecosystem and these pollutants mainly
the important pollution. The effluents damage the flora and fauna. The pollu-
from fish processing units, shrimp farms, tants also somewhat released more in ship
hatcheries are creating big problems. The are on voyage than accidental ships. The
effluents contain serious bacterial patho- pollutants released including tank resi-
gens such as salmonella, pathogenic Vib- dues, bunker oils, chronic discharge of
rio, WSSV and the fungal pathogen sewage, garbage, exchange of ballast wa-
Fusarium. Due to the broader host range ter, other emissions from vessel and anti-
of these pathogens it may infect very easi- fouling paints etc. The sewage discharges
ly in the marine animals and affect the causes severe problems including micro-
food chain. This also affects the consumer bial pollution especially bacterial infec-
levels very easily and there is an easy to tions it will be seriously damage the bio-
diversity, fisheries and food chains etc.
The excess nutrient discharges also affect and deposited the sediments may add
the primary production etc (Katchel, dead organic matter load. Further dead
2008). Pollutants are released by acci- organic matter may cause bad effects to
dental pollution including contacts with the normal microbial flora and depleted
external objects, due to collisions, cargo- the oxygen and this will affect the de-
transfer failures, sinking or loss of cargo, composition process. Shrimp farm efflu-
explosions and groundings etc. It is also ents including algal bloom die off, large
very hazardous, the toxic materials spill- amount of dead organic load accumulate,
ing from cargoes which carry large quan- affect filter feeding animals, shrimps and
tities. The oil tankers happened in acci- lobsters.
dent may spill larger quantities of crude
oil to the marine environments. The oil 3.11. Ocean mining
may drift to the sea shore and seriously Deep sea mining is the mineral re-
affected the ecosystem particularly the trieval process from the ocean floor by
rocky animals including oyster and mus- disturbing the marine organism from ben-
sel beds, backwater etc. The places which thic regions. Ocean mining takes place
affected by oil spills may suffer a long about from 1,400 to 3,700 meters below
times, until the oil pollution completely the oceans surface for extinct or active
degraded or solved (Katchel, 2008). The hydrothermal vents (Ahnert and Bor-
hydrocarbons form the oil spills pene- owski, 2000). The mine deposits are drill
trates the marine and related ecosystems out from the ocean deeps by hydraulic
sediments alter the community structure pumps or buckets that take ore to the sur-
especially the phytoplanktons and worms. face. Removal of marine floors disturbs
Ship can also harm to the marine habitats the benthic habitats. Removing parts of
and wildlife by damaging the marine an- the seafloor disturbs the habitat of benthic
imals by physical destruction by anchor- organisms depending upon the places and
ing. Anchoring may seriously affected by type of mining; sometimes it may disturb-
the coral and sponge beds. Another phys- ances to the marine benthos permanently.
ical hindrance including collisions by Leakage, corrosion and spilling might be
ships, ships propellers and ship strikes altering the area of mining bycontamina-
are cause the damages and death to the tionof chemicals. Surface plumes may
marine mammals including whales etc. cause more serious effects and based on
the particles size and water currents by
3.10. Harmful algal blooms (HABs) the plumes could spread over vast areas
Harmful algal bloom (HABs) of the floor.Sand mining is a prac-
causes pathetic effects to other marine tice mined from beaches, inland dunes
animals by mechanical damage through and dredged from ocean beds and river
the secretion of toxins.HABs are involved beds. Sand mining also emits radiation
in large-scale marine mortality of marine problems and pathetic effects to the living
animals and they secreted with different organisms in sea shore peoples (Pitchaiah,
toxins responsible for shellfish poisonings 2017).
(Nancy, 2012). Red tides caused by
HABs that can affect estuarine and salt 3.12. Plastics and polythenes
lakhs areas by depletion of oxygen, in- Plastic pollution is one of the seri-
creasing the carbon dioxide and secrete ous concerns in recent years because the
toxins. Thousands of harmful algal spe- plastic pollutions in ocean become signif-
cies were identified among that one hun- icant environmental issues related to gov-
dred species ware produce toxins that will ernmental and nongovernmental organi-
seriously affect the shellfish species by zations, scientists and members of the
ingested the HABs by filter feeding. The public worldwide. The main risk to ma-
excess amount algal bloom may dies off rine biota posed by different activities in-
cluding marine vessels, commercial fish- head sea turtles, Caretta caretta from
ing, marine-industries and recreational Central Mediterranean sea (Gramentz,
coastal tourism. These are the major 1988). Polythenes is the another danger-
sources for generating the plastic and pol- ous pollutants Generated from household,
ythene wastes hat can directly enter the industrial wastes and Recreational beach-
marine environment. During World War es. Sea turtles mistakenly easting the pol-
II end, approximately eighty percent of ythene bags. During degradation of poly-
the marine debris is considered as plastic thenes, it releases phenol, bisphenol,
wastes. phathalate and gases it causing cancer,
Marine vessels have been reported as a heart failures in humans.
major contributor of marine litter and it is
estimated that the commercial fishing 3.13. Microplastics
fleet dumped more than 23,000 tons of Microplastic contamination in
plastic packaging materials during 1970s ocean has been a serious issue nowadays
(Pruter, 1987). The waste materials in- with harmful effects to marine biota.
cluding nylon netting, plastic monofila- They are very tiny plastic granules de-
ment line and discarded or waste fishing rived from the breakdown of macroplas-
gears are neutrally buoyant can therefore tics and the plastic particles used in cos-
drift at variable depths within the oceans. metics and detergents. Primary microplas-
The waste plastic materials are problem- tics in personal care and cosmetic prod-
atic to marine animals because they cause ucts are a minor source of releases of mi-
entanglement of marine biota, known as croplastics to the environment. Micro-
ghost fishing (Lozano and Mouat, plastics which used in detergents, cosmet-
2009). ics and also in air-blasting media are en-
Waste six pack rings, plastic bags tering the freshwater bodies and reached
and other forms of plastic waste cause to domestic or industrial drainage systems
dangerous effects to wildlife and fisher- (Derraik, 2002). Most of the microplastic
ies. Plastic fishing nets can be lost or left granules are trapped the waste-water
by fishermen to the ocean are dangerous treatment plants unfortunately the small
and create so many problems including microplastic granules from cosmetics and
entangle to fishes, sea turtles, sharks, dol- detergents are passing through such filtra-
phins, crocodiles, crabs, seabirds, and tion systems and finally reached the ma-
other creatures, restricting movement, rine environments (Browne et al., 2007).
causing starvation and infection etc. Plas- The small size nature, microplastics are
tic waste debris during bulky or tangled, it considered bio-available to marine organ-
is very difficult to move, and may become isms throughout the food-web. Microplas-
permanently trapped in the digestive tics in intertidal sediments have been
tracts of marine animals finally leading to shown to reduce the thermal conductive
blocking the food passage and causing properties and alter drainage. Due to the
death through infection or starvation. tinny size and availability of microplas-
Moser and Lee (1992) conducted a study tics in pelagic and benthic ecosystems,
for plastic pollution in sea birds, they col- they have easily ingested by the marine
lected 1033 birds and among the birds, biota including microalgae, crustaceans,
fifty five percentages of the birds guts fishes and sea birds etc (Blight and Burg-
containing plastic particles. Carpenter et er, 1997). Devriese et al. (2015) studied
al. (1972) observed different species of the microplastic contamination in the
fishes had plastic wastes in their guts es- brown shrimp, Crangon crangon sampled
pecially white plastic spherules had been Europe and the study revealed that, the
ingested, indicating that they feed selec- shrimps are heavily contaminated with
tively. The same pattern of white plastic microplastics. Ingestion of microplastics
debris ingestion also observed in logger- may release toxins to the food chain and
pacts to various ways. Huge quantities of living organisms and passes through the
waters are loaded by cruise ships, cargos food chain.
and large tankers from one place for bal-
ancing and discharge to some other plac- 3.18. Marine littering
es. Ballast water have various biological Littering to marine environment is
materials such as seaweeds, sea grass, a global concern, they affect ecosystem
phytoplanktons, zooplanktons, small fish- very seriously. Several million tons of
es, invertebrates, microbes including bac- litters are discharged to the worldwide
teria, fungi and virus etc. The biological every year creating economic, environ-
materials are nuisance, non-native and mental, health and aesthetic issues
exotic species can create extensive eco- (Strieker, 1998).The important land based
logical and economic problems to the disposals including industrial outfalls,
aquatic ecosystem with human health is- rivers and floodwaters, discharge from
sues etc. The important preventive storm water drains, untreated municipal
measures for treating the ballast water sewerage, land-fills and littering from
bytreating with ultraviolet irradiation, fil- coastal tourism. The marine based litters
tration, heat, gas super saturation and including shipping transport, offshore
electrical fields etc (Silva et al., 2004), mining and extraction, fishing industry,
chemical treatments byiodides, borates, illegal dumping at sea and discarded fish-
chlorates, ozone, ionization, copper ions, ing gear etc. The vast majority of marine
hydrogen peroxide, adjustment of litter is plastic, which never truly breaks
pH,changes in salinity for eradication of down. Another serious problem is nuclear
organisms, ozonization and deoxigeniza- waste disposal to the marine trenches,
tion etc (Wright et al., 2003). Mariana trench is the main site
for nuclear waste disposal because of its
3.17. Natural calamities huge depth 36, 201 feet which is situated
Hurricanes and floods can induce in the western Pacific Ocean, to the east
waste transportation from land to the ma- of the Mariana Islands (Sheavly and Reg-
rine environment. Earthquakes and tsu- ister, 2007). Ocean is used as a dumping
nami can cause ground, air, and water ground for disposing the nuclear materials
pollution (Shaw, 2006). The pollutants for many decades after Second World
including detergents, chemicals from pro- War II, and then it was banned interna-
cessing plants, farm wastes and fertilizers tionally. Thirteen nuclear capable coun-
from crops are swept downstream and tries are dumped their nuclear/radioactive
deposited to marine through large river waste including started from 1946 to
floods. Volcanic eruptions have also been 1993. The important nuclear waste mate-
creating fluorine-containing compounds rials are medical products, industrial
deposition to sea and also harm to the ma- products, weapons, house hold containers,
rine flora and fauna. High amount of min- reactor vessels, with and without spent or
erals are accumulated to the marine envi- damaged nuclear fuel etc. The discarded
ronments from demolishing of the build- wastes emit nuclear radiation leading to
ings, bridges during natural disasters and health issues to the marine living organ-
small amount of hazardous materials isms.
reached to marine environments that
threatening to marine living organisms. 4. Impact of pollutants to marine life
Heavy floods in rivers also washed out
the sewages to the accumulated in the ma- The pollutants which are affect a
rine environments that contains pathogen- broad range to the environments as well
ic bacteria fungi and virus which causes as the living organism in the entire world
serious pathological effects to the marine directly or indirectly. Climate changes
are spoiled by extreme weather conditions
as well as rising of sea level, sea surface pesticides may change hormonal system,
warming by temperatures and ocean acid- reduce fertility, weaken immune system
ification. Corals reefs are important for and create cancer. The degraded deriva-
preventing acidification etc, loss by an- tives like phenol, bisphenols and phath-
thropogenic stress activities, collection alates also creating heart diseases and
and recreational activities and coral beds cancers to humans by consuming sea
may be affected. Mangrove forest is im- foods.
portant for preventing barrier for the natu-
ral calamities like tsunami. Degradation 5. Remedies to solve the pollutants by
of mangroves by over exploitation may biotechnological approach
affect the marine environments. Illegal
and over exploitation of capture fisheries Bioremediation is defined asThe
by trawlers may spoil the fishery re- act of adding materials to contaminated
sources leading to decline the particular environments such as oil spill sites, to
fish species. The pollution in beaches by cause an acceleration of the natural bio-
coastal tourism causes several environ- degradation process. The microbes uti-
mental impacts and the oil pollution caus- lize the nutrients from the polluted water
es reduction in benthic organisms. Biodi- bodies and reduce or neutralize the pollu-
versity is important for produce organic tants. The extremophilic microbes includ-
material, decompose organic material, ing halophilic, thermophilic, acidophilic
capture and store energy, cycling water and alkaliphilic are useful to bioremedia-
and nutrients and helps to regulate climate tion purposes for to neutralize the pollu-
and atmospheric gases. The pollutants tants, because they withstand high tem-
seriously interrupt the food chains leading perature, low and high pH and higher sa-
to several problems. In concern with the linity. The polluted effluents/ water bod-
human health point of view, the chemical ies, fish farm effluents, effluents from any
poisoning in food chain creates the bioac- waste sources may remediate with mi-
cumulation and biomagnifications prob- crobes before reaching the water bodies
lems (Table 1). The harmful chemical like river, back water may help to reduce
contamination in seafood crate several or remove the pollutants and solve the
problems to humans. The pathogenic bac- pollution problems. Genetically modified
teria including pathogenic vibrios, salmo- microbes like super bug also helps de-
nella contaminations from waste water grade the oil from oil contaminated water
disposal from fish processing industry bodies.
may cause the disease problems in the The water probiotic microbes like
fishes/ shrimps from the sea. The con- Lactobacillus sp and Bifidobacterium sp
sumption of infected fish/ shrimp may also clean the aquaculture ponds by re-
cause diseases to humans. The residue of ducing or removal pathogenic microbes
educated publics to restrict / stop the pol- Caldeira, K. and Wickett, M. E. (2003).
lution will definitely help in minimizing Anthropogenic carbon and ocean
the pollution. pH". Nature 425 (6956), 365365.
Carpenter, E.J., Anderson, S.J., Har-
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International Max Plank research
Abstract: The present study was carried out to determine the influence of ecological char-
acteristics and bioluminescent bacterial distribution in the seawater and sediment of Palk
Strait, Southeast coast of India during July 2010 - June 2012. The physico-chemical param-
eters such as., atmospheric temperature range was varied from 24.3 - 35.3C, surface wa-
ter temperature 23.2 - 33.5C, pH (6.2 - 8.91), dissolved oxygen concentration ranged from
3.84 - 7.73ml l-1, salinity fluctuated between 23.12 and 39 ppt, POC concentration ranged
from 0.78 - 2.56mg dry wt.l-1. The seawater was analysed and results suggest that it con-
tains, inorganic phosphate (4.12 - 21.6M), reactive silicate (4.3 - 19.26M), inorganic ni-
trate (1.95 - 12.25M), inorganic nitrite (1.1 - 5.5M), total nitrogen (1.5 - 10.39M), cal-
cium (120 - 990 mg l-1) and magnesium (730 - 2460mg l-1). Bottom water temperature
ranged from 20.4 to 27.1C, sediment temperature (17 to 25C), sediment pH (6.2 to 9.1),
concentration of sediment nutrients and heavy metals phosphorus concentration ranged
from 0.065 to 0.315%, potassium (3.18 to 8.57%), calcium (4.08 to 25.44%), magnesium
(0.29 to 1.7%), silicon (33.2 to 56.53%), sodium (1.56 to 3.35%), sulphur (0.32 to 2.06%),
chloride (1.3 to 5.71%), aluminium (4.63 to 11.87%), titanium (1.55 to 9.15%), manganese
(0.08 to 0.26% ), iron (3.2 to 11.13%), cobalt (3 to 11ppm), copper (4 to 29ppm), chromium
(26 - 85ppm), nickel (5 to 18ppm) and lead (11 to 27ppm). Seawater colony forming unit
(CFU) for the bioluminescent bacterial population density was varied from 1.06 x 104 to
9.44 x 104 CFU/ml and sediment (2.6 x104 to 23.2 x104 CFU/g). Statistical analysis sea-
water colony forming unit (CFU) of bioluminescent bacteria showed a positive correlation
with salinity and water pH. Sediment colony forming unit of bioluminescent bacteria
showed a positive correlation sediment temperature, sediment pH, sediment silicon, sodium
and chloride. It can be concluded that the ecological parameters were observed in water and
sediment which highly influence the bioluminescent bacterial populations and their diversi-
ty.
versity, India. The soil particles were dis- 5g, Agar 15g, Seawater 1000ml, pH
aggregated, crushed and sieved through 7.201). The plates were incubated at
10mm nylon sieve and then stored in pol- 32C for 24 hrs. After incubation, the
ythene bags and labelled at 4C until the colonies were counted in the dark room
analysis. All the elemental present in the and expressed in CFU ml/g and identified
sediments were analyzed by using by the standard procedure (Nealson,
Bruker S4-Pioneer model wavelength 1978). The isolated bioluminescent colo-
dispersive X-ray fluorescence spectropho- nies were purified and stored at 4C with
tometer (WD-XRF). The samples were 40% glycerol until analysis (Kita-
ground to particle size well below 100m Tsukamoto et al., 2006). All the data were
using ball mill in order to minimize the computed using SPSS v 16.0 statistical
grain size interference on XRF- software to obtain Pearsons correlation
measurement. The major elements viz., co-efficient (r) for the statistical interpre-
Phosphorus (P), Potassium (K), Calcium tation. The correlation coefficient and
(Ca), Magnesium (Mg), Silicon (Si), So- standard deviation (SD ) were calculated
dium (Na), Sulphur (S), Chloride (Cl), between the physico-chemical variables
Aluminium (Al), Titanium (Ti), Manga- and CFU of bioluminescent bacterial
nese (Mn), Iron (Fe), Cobalt (Co), Copper population in all samples collected from
(Cu), Chromium (Cr), Nickel (Ni) and all the stations from June 2010 to May
Lead (Pb) were analyzed. 2012.
Then, 1ml seawater/ 1g sediments
of samples were mixed with 99/100ml of 3. Results
50% aged sterile seawater mixed vigor-
ously and used for serial dilution in test Atmospheric temperature was var-
tubes upto 10-2 dilutions up to 10-7. About ied from 24.3 to 35.3C (Figure 1), the
0.1ml from the each dilution was spread surface water temperature was varied
in luminescent agar (LA) medium (Dehy- from 23.2 to 33.5C (Figure 2). The pH
drated Nutrient broth 8g, Sodium Chlo- range was from 6.2 to 8.91 (Figure 3), the
ride 30g, Glycerol 3g, Calcium Carbonate
Figure 4: Monthly variations of dissolved oxygen (ml l-1) recorded at stations I, II and III
from July2010 to June 2012.
Figure 5: Monthly variations of salinity (S%o) in seawater recorded at stations I, II and III
from July 2010 to June 2012.
Figure 6: Monthly variations of particulate organic carbon (mg dry wt.l-1) recorded at sta-
tions I, II and III from July 2010 to June 2012.
Figure 10: Monthly variations of inorganic nitrite (M) concentration in seawater recorded
at stations I, II and III from July 2010 to June 2012.
Figure 11: Monthly variations of total nitrogen (M) concentration in seawater recorded at
stations I, II and III from July 2010 to June 2012.
ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 460
Biotech Sustainability (2017)
Ecology, Distribution and Diversity of Bioluminescent Bacteria Srinivasan et al.
calcium (120 to 990 mg l-1; Figure 12) 25.44%; Figure 20), magnesium (0.29 to
and magnesium (730 to 2460mg l-1; Fig- 1.7%; Figure21), silicon (33.2 to 56.53%;
ure13). The bacterial diversity revealed Figure 22), sodium (1.56 to 3.35%; Fig-
that, the bioluminescent bacterial popula- ure 23), sulphur (0.32 to 2.06%; Figure
tion density was varied from 1.06 x 104 to 24), chloride (1.3 to 5.71%; Figure 25),
9.44 x 104 CFU/ml in seawater (Figure aluminium (4.63% to 11.87%; Figure 26),
14). titanium (1.55 to 9.15%; Figure 27), man-
Bottom water temperature was in ganese (0.08 to 0.26%; Figure28), iron
the range of 20.4 to 27.1C during the (3.2 to 11.13%; Figure 29), cobalt (3 to
study period at all three stations (Figure 11ppm; Figure 30), copper (4 to 29ppm
15). Sediment temperature varied from 17 Figure 31), chromium (26 to 85ppm; Fig-
to 25C (Figure 16). Sediment pH varied ure 32), nickel (5 to 18ppm; Figure 33)
from 6.2 and 9.1 at all stations (Figure and lead (11 to 27ppm; Figure 34). In sed-
17). The results for sediment nutrients iment, the bioluminescent bacterial popu-
showed that phosphorus (0.065 to lation density varied from 2.6 x104 to 23.2
0.315%; Figure 18), potassium (3.18 to x104 CFU/g (Figure 35).
8.57%; Figure 19), calcium (4.08 to
Figure 12: Monthly variations of calcium (mg l-1) concentration in seawater recorded at
stations I, II and III from July 2010 to June 2012.
Figure 13: Monthly variations of magnesium (mg l-1) concentration in seawater recorded at
stations I, II and III from July 2010 to June 2012.
Figure 15: Monthly variations in bottom water temperature (C) recorded at stations I, II
and III from July 2010 to June 2012.
Figure 16: Monthly variations in sediment temperature (C) recorded at stations I, II and III
from July 2010 to June 2012.
Figure 17: Monthly variations of hydrogen ion concentration (pH) in sediment recorded at
stations I, II and III from July 2010 to June 2012.
Figure 19: Monthly percentage variations of potassium (%) concentration in sediment rec-
orded at stations I, II and III from July 2010 to June 2012.
Figure 20: Monthly percentage variations of calcium (%) concentration in sediment rec-
orded at stations I, II and III from July 2010 to June 2012.
Figure 22: Monthly percentage variations of silicon (%) concentration in sediment record-
ed at stations I, II and III from July 2010 to June 2012.
Figure 23: Monthly percentage variations of sodium (%) concentration in sediment record-
ed at stations I, II and III from July 2010 to June 2012.
Figure 24: Monthly percentage variations of sulphur (%) concentration in sediment record-
ed at stations I, II and III from July 2010 to June 2012.
Figure 25: Monthly percentage variations of chloride (%) concentration in sediment rec-
orded at stations I, II and III from July 2010 to June 2012.
Figure 26: Monthly percentage variations of aluminum (%) concentration in sediment rec-
orded at stations I, II and III from July 2010 to June 2012.
Figure 27: Monthly percentage variations of titanium (%) concentration in sediment rec-
orded at stations I, II and III from July 2010 to June 2012.
Figure 29: Monthly percentage variations of iron (%) concentration in sediment recorded at
stations I, II and III from July 2010 to June 2012.
Figure 30: Monthly variations of cobalt (ppm) concentration in sediment recorded at sta-
tions I, II and III from July 2010 to June 2012.
Figure 31: Monthly variations of copper (ppm) concentration in sediment recorded at sta-
tions I, II and III from July 2010 to June 2012.
Figure 33: Monthly variations of nickel (ppm) concentration in sediment recorded at sta-
tions I, II and III from July 2010 to June 2012.
Figure 34: Monthly variations of lead (ppm) concentration in sediment recorded at stations
I, II and III from July 2010 to June 2012.
temperature to minimum (Kannan and fall and the resultant freshwater mixing.
Kannan, 1996). In addition, the surface The minimum dissolved oxygen was
water temperature showed positive corre- found during summer months, which
lation with colony forming unit (CFU) at could be mainly due to reduced agitation
station - II (r= 0.635; P< 0.02) and at sta- in the coastal and estuarine water (Ruby
tion - III (r= 0.627; P< 0.02). Further, the and Nealson, 1978; Nealson and Hasting,
statistical analysis revealed that the sedi- 1979). Further, this is evidenced by the
ment colony forming unit of biolumines- negative correlation between the dis-
cent bacteria showed a positive correla- solved oxygen and seawater CFU at sta-
tion with sediments temperature at station tion-III (r= -0.823; P< 0.001).
- I (r= 0.806; P< 0.001), station-II (r= Maximum particular organic car-
0.842; P< 0.001) and station-III (r= 0.784; bon (POC) was recorded during the
P< 0.001). month of November (2010) at station-II
Surface water salinity was reduced and minimum POC was observed during
greatly during the monsoon and it was the month of May (2012) at station-I. The
gradually increased from postmonsoon to maximum POC content in water was
summer at all stations. The maximum sa- mainly due to the organic matter brought
linity could be due to low amount of rain- in from the land through run-off. Further,
fall and higher rate of evaporation in the it could be also due to the presence of
shallow coastal area owing to high at- plant (seagrass and seaweeds) and animal
mospheric temperature (Govindasamy organic matter within the seagrass ecosys-
and Kannan, 1991). Significantly positive tem and exported from the adjacent eco-
correlation was observed between sea- system by wind and wave action. Further,
water salinity and CFU of bioluminescent the seasonal variation in POC content in
bacteria at station-I (r= 0.773; P< 0.001), the water could be related to the plankton
station-II (r= 0.903; P< 0.001) and sta- productivity (Kannapiran et al., 2008).
tion-III (r= 0.837; P< 0.001). The present This is further evidenced by the negative
study results are in line with the research correlation between POC and seawater
findings of Abraham et al. (2003). CFU at station-I (r= -0.786; P< 0.001),
The high pH values recorded dur- station-II (r= -0.841; P< 0.001) and sta-
ing summer and this might be due to the tion-III (r= -0.832; P< 0.001).
influence of seawater penetration and Maximum inorganic phosphate
high biological activity. These findings was observed during the monsoon season
are in accordance with the previous report (December 2010) at station-III and mini-
(Smith and Key, 1975). The statistical mum was recorded during the summer
analysis shows the positive correlation season (May 2011) at station-I. Possibly,
between pH and CFU station - I (r=0.726; the maximum concentration of phosphate
P< 0.01) and station - III (r= 0.874; P< was due to invasion of upwelling of wa-
0.001) to seawater colony forming unit. ter, which increased the level of phos-
The statistical analysis shows the positive phate. Low values of phosphate observed
correlation with sediments pH station-I to utilization by phytoplankton, seagrass-
(r= 0.692; P< 0.001), station-II (r= 0.641; es and other primary producers (Rajaseg-
P< 0.001) and station-III (r= 0.813; P< ar, 2003). The statistical analysis shows
0.001) to sediment colony forming unit. the negative correlation to seawater colo-
Dissolved oxygen is one of the most im- ny forming unit with inorganic phosphate
portant abiotic parameters influencing the at station - I (r= - 0.734; P< 0.01), station-
life in the coastal environment. In the pre- II (r= -0.832; P< 0.001) and station-III (r=
sent study, the maximum dissolved oxy- -0.828; P< 0.001).
gen was recorded during monsoon and Minimum concentration of silicate
this might be due to the cumulative effect was observed during the summer season
of higher wind velocity coupled with rain- (May 2012) at station II and maximum
during the monsoon season (November was recorded during the summer (May
2010) at station III. It might be due to the 2011) at station I. It might be due to
heavy rain, land runoff water mixing was freshwater inflow was high in the mon-
high level. It has been reported that the soon season so high level of nitrogen was
silicate from the bottom sediment might recorded. The statistical analysis showed
have been exchanged with overlaying wa- total nitrogen a negative correlation with
ter in this mangrove environment (Go- seawater CFU of bioluminescent bacteria
vindasamy and Kannan, 1996). The low at station-I (r= -0.829; P< 0.001), station-
value observed in summer and post- II (r= -0.929; P< 0.001) and station-III (r=
monsoon season could be attributed to -0 .896; P< 0.001).
uptake of silicates (Saravanakumar et al., Maximum and minimum level of
2008). The statistical analysis of silicate seawater calcium was observed during the
showed negative correlation with sea- monsoon (December 2010) and summer
water colony forming unit at station-II (r= seasons (May 2011) at station-III. Maxi-
-0.786; P< 0.001) and station-III (r= - mum level of sediment calcium was ob-
0.841; P< 0.001). served during the monsoon season (De-
Nitrate concentration was found cember 2010) at station-II and minimum
lower than that of nitrate and same trend level of calcium was observed during the
of fluctuation was reported in Muthpettai summer (May 2011) at station-II. Maxi-
mangrove (Ashokkumar et al., 2011). mum level of seawater magnesium was
The statistical analysis showed nitrate a recorded during the monsoon season De-
negative correlation with seawater colony cember (2010) at station I and minimum
forming unit at station-I (r= -0.868; P< was recorded during the summer season
0.001), station-II (r= -0.888; P< 0.001) May (2011). Maximum level of sediment
and station-III (r= -0.926; P< 0.001). magnesium was observed during the post
Maximum level of nitrite was rec- monsoon season (January 2011) at sta-
orded during December 2011 at station-II tion-I and minimum level of magnesium
and minimum was observed during the was recorded during the summer season
May 2011 at station-I. It might be due to (May 2011) at station-II. The statistical
the minimum nitrite were recorded during analysis showed seawater calcium a nega-
the summer may be due to high salinity. tive correlation to seawater colony form-
The higher nitrate value in monsoon sea- ing unit at station-II (r= -0.816; P< 0.001)
son could be due to the increased phyto- and station-III (r= -0.762; P< 0.001). In
plankton excretion, oxidation of ammo- addition, to that the seawater magnesium
nia, reduction of nitrate, the recycling of a negative correlation with seawater colo-
nitrogen and bacterial decomposition of ny forming unit at station-II (r= -0.829;
planktonic detritus (Govindasamy et al., P< 0.001) and station-III (r= -0.840; P<
2000; Asha and Diwakar, 2007) and also 0.001). Sediment calcium a negative cor-
due to denitrification and air-sea interac- relation with sediment colony forming
tion exchange of chemicals were also re- unit at station-I (r= -0.843; P< 0.001), sta-
sponsiple for this increased values (Raja- tion-II (r= -0.832; P< 0.001) and station-
segar, 2003; Ashok Prabu et al., 2008). III (r= -0 .909; P< 0.001); Sediment mag-
The statistical analysis which showed ni- nesium showed a negative correlation
trite a negative correlation with seawater with sediment colony forming unit at sta-
colony forming unit at station-I (r= - tion-I (r= -0.710; P< 0.001), station-II (r=
0.767; P< 0.001), station-II (r= -0.834; P< -0.675; P< 0.001) and station-III (r= -
0.001) and station-III (r= -0.882; P< 0.721; P< 0.001). It might be due to the
0.001). calcium and magnesium higher level was
Maximum level of total nitrogen recorded in monsoon and post monsoon
was recorded during the monsoon (De- seasons. Calcium and magnesium concen-
cember 2010) at station III and minimum trations could be increased due to the in-
put of freshwater (Sundararajan and Nate- ment colony forming unit at station-I (r= -
san, 2010). 0.661; P< 0.001), station-II (r= -0.856; P<
Maximum sediment total phos- 0.001) and station-III (r= -0.882; P<
phorus was observed during the month of 0.001).
December-2010 at station-I and minimum Maximum concentration of sedi-
was recorded during the month of May- ment sodium was observed during the
2011 at station-III. and this might be due month of 2011 at station-III and minimum
to the domestic, municipal and agricultur- was recorded at station-II and this might
al waste (non-pointed sources). The re- be due to the high counts of microbial
generation and releases of total phospho- populations depending on the sodium
rus from bottom mud into the water col- concentration and it was varied spatially.
umn by turbulence and mixing was also The statistical analysis which showed sed-
attributed to recorded higher values iment sodium a positive correlation with
(Chandran and Ramamoorthy, 1984). The sediment colony forming unit at station-I
statistical analysis showed sediment total (r= 0.834; P< 0.001), station-II (r= 0.824;
phosphorus a negative correlation with P< 0.001) and station-III (r= 0.845; P<
sediments colony forming unit at station-I 0.001). Maximum and minimum level of
(r= -0.848; P< 0.001), station-II (r= - chloride was recorded during the summer
0.843; P< 0.001) and station-III (r= - and post monsoon seasons at station III
0.861; P< 0.001). Maximum concentra- and station I respectively. The statistical
tion of sediment potassium was observed analysis of sediments chloride shows a
during the monsoon season (December positive correlation with sediment colony
2010) at station-I and minimum concen- forming unit station-I (r= 0.665; P<
tration of potassium was recorded during 0.001), station-II (r= 0.840; P< 0.001) and
the summer (June 2010) at station-II. It station-III (r= 0.811; P< 0.001).
might be due to high concentration of po- The maximum sediment alumini-
tassium are inflected by heavy rainfall um was observed recorded during the post
inflow on in peak values was recorded monsoon month of January 2012 at sta-
during the monsoon seasons. Further, evi- tion-I and minimum was observed during
denced by the statistical analysis showed the month of April 2011 at station-III. It
potassium a significant negative correla- might be due to contribution from detrital
tion with sediments colony forming unit mineral grains supplied from through the
at station-II (r= -0.656; P< 0.001) and sta- rivers in addition to the precipitation of
tion-III (r= -0.751; P< 0.001). their dissolved species under prevailing
Maximum and minimum sediment estuarine condition. The statistical analy-
silicon was observed during the May - sis which showed a negative correlation
2012 at station II and November -2011 at to sediment colony forming unit with al-
station-III. This might be due to its bio- uminium at station-II (r= -0.706; P<
logical significance; the flux of particulate 0.001). Removal of dissolved river-borne
silica from surface waters plays an im- aluminum by coagulation in the coastal
portant role in the cycling of other ele- areas is common (Holliday and Liss,
ments in the marine environment such as 1976).
radium, barium and germanium (Li et al., In sediment, the maximum and
1973; Froelich and Andreae, 1980). The minimum titanium level was recorded
statistical analysis showed silicon a sig- during the monsoon 2010 and summer
nificant positive correlation with sediment April 2011 at stations III and I respective-
colony forming unit at station-I (r= 0.745; ly. Moreover, the statistical analysis re-
P< 0.001), station-II (r= 0.824; P< 0.001) vealed that the sediment titanium shows a
and station-III (r= 0.836; P< 0.001). The negative correlation to sediment colony
statistical analysis showes sulphur forming unit at station-I (r= -0.732; P<
showed a negative correlation with sedi- 0.001), station-II (r= -0.792; P< 0.001)
and station-III (r= -0.888; P< 0.01). Max- al. (2004) has reported that effluent of
imum and minimum level of sediment metal finishing industry and corrosion of
manganese was recorded during the mon- building materials play the main role to
soon (2010) at station III and summer increase chromium level in marine envi-
(2012) seasons at station I. The statistical ronment. The statistical analysis showed
analysis showed sediment manganese a sediment chromium a negative correlation
negative correlation with sediment colony with sediment microbial count at station-I
forming unit at station-I (r= -0.732; P< (r= -0.760; P< 0.001), station-II (r= -
0.001), station-II (r= -0.819; P< 0.001) 0.768; P< 0.001) and station-III (r= -
and station-III (r= -0.800; P< 0.001). 0.896; P< 0.001).
Maximum and minimum concen- Maximum sediment nickel con-
tration of sediment iron was recorded dur- centration was recorded during the mon-
ing the post monsoon (2011) and summer soon (2010) at station I and minimum was
(2012) seasons at station I and III. In gen- recorded during the summer seasons
eral, marine environment and discharge of (2012) at station II. The statistical analy-
aquatic ponds, domestic wastes, land and sis sediment nickel showed a negative
agricultural drainage, boating activities correlation with sediment colony forming
such as loading and unloading of materi- unit at station-I (r= -0.801; P< 0.001), sta-
als, antifouling paints from boating activi- tion-II (r= -0.778; P< 0.001) and station-
ties contribute to enhance the metal level III (r= -0.684; P< 0.001). Maximum and
in marine environment (Govindasamy and minimum concentration of sediment lead
Azariah, 1999; Ashokkumar et al., 2009). was recorded during the monsoon (2010)
The statistical analysis showed sediment and summer (June 2010) seasons at sta-
iron a negative correlation with sediment tion-III. It might be due to the heavy rain
colony forming unit at station-I (r= - river runoff and sewage discharges in the
0.643; P< 0.01), station-II (r= -0.695; P< coastal region. Chatterjee et al. (2006)
0.001) and station-III (r= -0.758; P< reported that 25.3 to 33.4mg/kg were ob-
0.001). served in Hugi (Ganges) estuary, north-
Maximum and minimum level of east coast of Bay of Bengal. The statisti-
copper was observed during the month of cal analysis sediment lead showed a nega-
January 2012 at station III and summer tive correlation with sediment colony
month of May (2012) at station I. It might forming unit station-I (r= -0.778; P<
be due to the discharge of maximum level 0.001), station-II (r= -0.835; P< 0.001)
of freshwater in the central west coast of and station-III (r= -0.801; P< 0.001).
India (Sankaranarayanan and Reddy,
1973). The statistical analysis showed 5. Conclusion
sediment copper a negative correlation
with sediment colony forming unit sta- The marine environment supports
tion-I (r= -0.675; P< 0.001), station-II (r= the survival, reproduction and the meta-
-0.699; P< 0.001) and station-III (r= - bolic reactions of the flora and fauna.
0.770; P<; 0.001). Among them, the microbial community
Maximum sediment chromium plays a vital role in the food webs, bioge-
was recorded monsoon season (2010) at ochemical cycles and the energy flow
station III and minimum was recorded mechanisms in the marine ecosystem. But
during summer season (2011) at station- the microbial diversity in seagrass ecosys-
II. It might be due to huge amount of tem is structured and determined by the
freshwater river flow carry agricultural prevailing temporal and spatial variability
water, domestic sewage, animal and of ecological parameters in the native en-
chromium in river-borne solids transport- vironment. The physico-chemical pa-
ed in estuaries is relatively higher than rameters of seawater were highly influ-
that of near shore sediments. Mannan et encing the bioluminescent bacterial popu-
lation in the Palk Strait region. Most of behavior of zinc and copper. Ma-
the parameters such as the atmospheric hasagar Bulltion National Institute
temperature, salinity, pH were significant- of Ocanography 22, 185-191.
ly correlated with the bioluminescent bac- Chandran, R and K. Ramamoorthi.
terial population in all the three stations (1984). Hydrobiological studies in
studied. the gradient zone of the Vellar es-
tuary. I. Physicochemical parame-
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Abstract: The synthesis of nanoparticles has become a matter of great interest in recent
times due to their various advantageous properties and applications in a variety of fields.
We have reported biological synthesis of nano-sized silver particles. The nanoparticles of
silver were formed by the reduction of silver nitrate to aqueous silver metal ions while ef-
fect of marine seaweed U. reticulata extract was investigated; silver nanoparticles were
characterized using UV-visible absorption and room temperature photoluminescence. The
X-ray diffraction results revealed that the synthesized silver nanoparticles were in the cubic
phase. The existence of functional groups was identified using Fourier transform infrared
spectroscopy. The morphology and size of the synthesized particles were studied with
atomic force microscope. Further, the photocatalytic degradation of methyl orange was
measured spectrophotometrically by using silver as nanocatalyst under visible light illumi-
nation. Silver nanoparticles synthesized from U. reticulata by facile method from can able
to degrade dyes in the presence of visible light and paves way for ecological health and en-
vironmental bioremediation. Despite numerous studies conducted over the last decade,
there are still considerable gaps in our knowledge about the biotechnological potential of
green-synthesized nanoparticles. Furthermore, the precise basis of their antibiotic activity
has yet to be defined. The biological methods are generally cost effective, nontoxic, and
ecofriendly. This chapter focuses on the methods involved in algal-synthesized nanoparti-
cles and its applications.
Keywords: AFM; Ag nanoparticles; antibacterial activity; cubic phase; methyl orange; pho-
tocatalytic degradation; Ulva reticulata
interest of researchers on microbial re- pieces and boiled in 100 ml of sterile dis-
sistance. Among the developed nanopar- tilled water for 5 min. The crude extract
ticles, silver (Ag) nanoparticles are per- was passed through Whatman No.1 filter
taining to have a wide range of applica- paper and the filtrate was stored at 4C
tion in the fields of physical, chemical for further use the methods suggested by
and biological sciences. In the past dec- Jha et al. (2009).
ade, several kinds of the biological organ-
isms (microbes, plants and seaweeds) 2.3. Synthesis of silver nanoparticles
have been employed and well-studied for Analytical grade (AR) Silver ni-
the ability of silver nanoparticles (Ag- trate (AgNO3) was purchased from E.
NPs) synthesis (Ramanathan et al., 2011; Merck (India). In the typical synthesis of
Ahmad et al., 2003; Shankar et al., 2003; silver nanoparticles, 10 ml of the aqueous
Mohanpuria et al., 2008; Kumar et al., extract of Ulva reticulata was added to 90
2012a). Application of green chemistry in ml of 1 mM aqueous AgNO3 solution in
synthesizing nanomaterials has vital role 250 ml conical flask and incubated at
in medicinal and all technological aspects room temperature for 72 h by agitating at
(Mondal et al., 2011, Begum et al., 2009). 120 rpm. Suitable controls were main-
Biologically synthesized Ag-NPs have tained throughout the experiments (Para-
wide range of applications because of shar et al., 2009). The bio-reduction of
their remarkable physical and chemical AgNO3 into AgNPs can be confirmed
properties. The literature on the extra cel- visually by the change in colour from
lular biosynthesis of Ag-NPs using plants light yellow to brown indicating the for-
and pure compounds from plants are in- mation of silver nanoparticles (Figure 1).
significant (Kattumuri et al., 2007; Song
and Kim 2008; Gilaki 2010). 2.4. Characterization techniques
In this article, we describe a sim- The colour change in reaction
ple one step method for the synthesis of mixture (metal ion solution + seaweed
Ag-NPs by the reduction of aqueous Ag- extract) was recorded through visual ob-
ions using extracts of green seaweed, at servation. The bio reduction of silver ions
direct sunlight conditions. in aqueous solution was monitored by pe-
riodic sampling of aliquots (0.5 ml) and
2. Materials and methods subsequently measuring UV-Vis spectra
of the solution. UV-vis spectra of these
2.1. Screening and selection of sample aliquots were monitored as a function of
Fresh sample of Ulva reticulata time of reaction on UV-Vis spectropho-
green seaweed was collected in the month tometer UV-2450 (Shimadzu).
of January 2013 from Pudumadam coastal The Ag-NPs solution thus ob-
region (78.99E, 9.27N), in Gulf of tained was purified by repeated centrifu-
Mannar, Tamil Nadu, India. Sample was gation at 5000 rpm for 20 min followed
immediately brought to the laboratory in by resuspention of the pellet of Ag-NPs in
polythene bags and cleaned thoroughly 10 ml of deionized water. After freeze
with fresh water to remove adhering de- drying of the purified Ag-NPs, its struc-
bris and associated biota. The alga sample ture and composition was analyzed by
was cleaned with distilled water using XRD. The dried mixture of Ag-NPs was
brush for the removal of the epiphytes. collected for the determination of the
formation of Ag-NPs by an XPert Pro x-
2.2. Preparation of aqueous extract ray diffractometer (PAN analytical BV,
The whole U. reticulata samples The Netherlands) operated at a voltage of
were initially rinsed thrice in distilled wa- 40 kV and a current of 30 mA with Cu K
ter and dried on paper toweling. Twenty radiation in - 2 configurations. The
five (25) gram sample was cut into fine crystallite domain size was calculated
Figure 1: Silver nitrate (AgNO3) solution and others colour changes during the reduction of
AgNO3 into AgNPs by the extract of U. reticulata after 20 min of incubation.
from the width of the XRD peaks, assum- thesized Ag-NPs (Rashed and El-Amin
ing that they are free from non-uniform 2007). All the experiments were per-
strains, using the Scherrers formula. formed outdoor with sun as the main
D= 0.94 / Cos source of light (Wang et al., 2000). Prior
where D is the average crystallite domain to the experiment, a suspension was pre-
size perpendicular to the reflecting planes, pared by adding 20 mg of Ag-NPs to 50
is the X-ray wavelength, is the full ml of methyl orange solution (Fisher Sci-
width at half maximum (FWHM), and entific). Later, the mixture was allowed to
is the diffraction angle. To eliminate addi- stir constantly for about 30 min in dark-
tional instrumental broadening the ness to ensure constant equilibrium of Ag-
FWHM was corrected, using the FWHM NPs in the organic solution. During the
from a large grained Si sample. reaction, the mixture was kept under sun-
corrected = (FWHM2 sample- light within a Pyrex glass beaker and
FWHM2si) 1/2 stirred constantly. The mean temperature
This modified formula is valid only when was found to be 29C with 10 h mean
the crystallite size is smaller than 100 nm. shine duration. The absorption spectrum
of the suspension mixture was measured
The silver nanoparticles were ob- periodically using a UVvisible spectro-
served using SEM. Sample was prepared photometer (Shimadzu, UV-2450, Japan)
by placing a drop of AgNPs on carbon after centrifugation to ensure the degrada-
coated copper stuff and subsequently dry- tion of methyl orange solution.
ing air, before transferring it to the micro-
scope operated at an accelerated and volt- 2.6. Antibacterial activities
age of 120KV (JOEL Model JSM-5010 Experimental pathogens namely,
LV with INSA EDS) and followed for Gram positive bacterium Staphylococcus
Energy Dispersive Spectrophotometer aureus, Gram negative bacteria Pseudo-
analysis. monas aeruginosa, Escherchia coli, Pro-
The morphology of the product teus mirabilis and Proteus vulgaris were
was observed by Nano Surf Easy Scan 2 obtained from Raja Sir Muthaiya Medical
Atomic Force Microscope (AFM) meas- College, Annamalai University. Patho-
urement to study the morphology and size gens were loaded in inoculated in medium
of the Ag-NPs (Al-Warthan et al., 2010). (Muller Hinton Agar medium) [100
g/ml, 50 g/ml, 25 g/ml (Ag-NPs)] and
2.5. Photocatalytic degradation AgNO3 was used as negative control.
The photocatalytic degradation of Each culture was spread on to Muller
methyl orange was evaluated by biosyn- Hinton Agar plates. Sterile paper discs of
synthesized nano-Ag was capped with tern revealed that the sample contains a
bimolecular compounds which are re- mixed phase (cubic and hexagonal) struc-
sponsible for reduction of silver ions tures of silver nanoparticles. The average
(Jegadeeswaran et al., 2012). The above- estimated particle size of this sample was
mentioned shift was also observed in 10 nm derived from the FWHM of peak
Codium capitatum (Kannan et al., 2013). corresponding to 90 plane (Figure 4). X-
ray diffraction showed the average parti-
3.3. Crystal structures analysis and de- cle size of 15 nm as well as revealed their
termination of crystallite size cubic structure (Geethalakshmi and Sara-
The XRD pattern showed three da 2010).
intense peaks in the whole spectrum of 2
value ranging from 10 to 80. Average size 3.4. Particles morphology (SEM and
of the synthesized particles was 10 nm AFM measurements)
with size range 10 - 50nm with cubic and The SEM image (Figure 5 a and b)
hexagonal shape. The typical XRD pat- depicts the high density Ag-NPs synt -
Figure 5: (a) SEM micrograph of Silver nanoparticles synthesized from the extracts of U.
reticulata; (b) energy dispersive spectrometer analysis
-hesized by the U. reticulata and confirms are spherical shaped and well distributed
the development of silver nanostructures without aggregation in solution. Ag-NPs
with energy dispersive spectrometer. The predominantly spherical well distributed
SEM micrographs of nanoparticle ob- with an average size 15 nm (Saraniya De-
tained in the filtrate showed that Ag-NPs vi et al., 2013). It is known that the shape
Figure 6: (a). AFM images of synthesized silver nanoparticles using extract of U. reticula-
ta; (b) corresponding 3D view.
ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 481
Biotech Sustainability (2017)
Synthesis of Biocompatible Silver Nanoparticles Ganapathy Selvam et al.
Figure 8: Antibac-
terial activity of sil-
ver nanoparticles
synthesized from U.
reticulata against
human pathogens.
morphology of silver nanoparticles was Begum, N.A., Mondal, S., Basu,S., Las-
characterized by SEM and AM. The na- kar, R.A. and Mandal, D. (2009).
noparticles were found to be active in de- Biogenic synthesis of Au and Ag
grading methyl orange solution with visi- nanoparticles using aqueous solu-
ble light illumination. The antimicrobial tions of Black Tea leaf extracts.
activity of synthesized Ag-NPs is promis- Colloids Surf B Biointerfaces. 71,
ing. In a nutshell, synthesis and character- 113118.
ization of Ag-NPs with regard to novel Binupriya AR, Sathishkumar M, Vi-
morphology are of great interest in the jayaraghavan K, Yun SI. (2010).
fabrication of antibacterial materials. Bioreduction of trivalent aurum to
nano-crystalline gold particles by
Acknowledgments active and inactive cells and cell-
free extract of Aspergillus oryzae
The authors wish to thank Profes- var. viridis. Journal of Hazardous
sor and Head, Department of Botany, An- Materials. 177, 539-545.
namalai University. We also thank Dr. S. Chandran SP, Chaudhary M, Rasricha
Barathan, Professor and Head, Depart- R, Ahmad M, and Sastry. (2006).
ment of Physics. Authors cordially thank Synthesis of gold nanotriangales
Dr. B.Shanthi and Dr. G. Sivakumar, and silver nanoparticles using Al-
CISL Lab, Department of Physics, An- oevera plant extract. Biotechnology
namalai University, for their help in Progress. 22, 577.
providing access to SEM and AFM and Elechiguerra JL, Burt JL, Morones JR,
for his suggestions while performing the Bragado AC, Gao X, Lara HH,
research work. Yocaman M. (2005). Interaction of
silver nanoparticles with HIV- 1.
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Venckatesha R. (2012). Green syn- particles by filamentous cynaobacte-
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Kannan RRR, Stirk WA, Van Staden lic Au/Ag alloy nanoparticles using
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anthophora spicifera to encumber Wokaun A. (2005). Nanoparticles
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Ganeshkumar RS, Govindaraju RY, Smooker PM, Bhargava SK,
M. (2012b). Synthesis of silver na- Bansal V. (2011). Bacterial kinet-
noparticles from Sargassum ten- ics-controlled shape-directed bio-
errimum and screening phytochemi- synthesis of silver nanoplates using
man, through his trial and error, has tries, but also in developed countries, where
selected many wild fruits which are modern medicines are predominantly used.
edible and subsequently domesticated While the traditional medicines are derived
them which played a very vital part from medicinal plants, minerals, and organic
in supplementary diet knowingly or matter, the herbal drugs are prepared from
medicinal plants only. Use of plants as a
unknowingly. Although due to the igno- source of medicines has been inherited and is
rance of modern generation the im- an important component of the health care
portance of wild plants were recently system in India. There are about 45,000 plant
have been decreasing yet many people species in India, with high concentration in
especially in rural areas still use them the region of Eastern Himalayas, Western
extensively as a supplementary to their Ghats and Andman and Nicobar Island. The
basic food requirement. A scientific study officially documented plants with medicinal
of wild fruits is important for the po- potential are 3,000 but traditional practioners
tential sources which are protective use more than 6,000. India is the largest pro-
foods. The nutrients/pigments present ducer of medicinal herbs and is appropriately
in the fruits prevent different degrada- called the botanical garden of the world. In
rural India, 70 percent of the population is
tive/ageing process in our body and thus dependent on the traditional system of medi-
via restoring health offer longevity cine, the Ayurveda, which is the ancient Indi-
(Singh, 2011). These wild fruits would be an therapeutic measure renowned as one of
utilized at the time of scarcity or culti- the major systems of the alternative and com-
vated as a source of food material for plementary medicine (Bhatia, et al., 2013).
ever increasing population (Rashid et In Katarniaghat Wildlife Sanctuary
al., 2008). there are fifty five tree plant species repre-
India has been considered as one of senting forty five genera belonging to thirty
the 17 mega-diversity centers of the word one families. Moraceae was found to be the
with a wide range of phyto-geographical var- largest family with seven plant species
iations. It consists of about 64 million hec- whereas Euphorbiaceae and Mimosaceae
tares forest covers out of which 86% is tropi- with five; Anacardiaceae, Myrtacea and Ru-
cal forest comprising 54% dry deciduous, biaceae with three; Caesalpiniaceae, Eh-
37% moist deciduous and 9% wet evergreen retiaceae, Papilionaceae and Louraceae with
& semi-evergreen (Kaul and Sharma, 1971). two and rest twenty one families, viz., Ru-
As a characteristic feature, the tropical forest taceae, Apocyanaceae, Baringtoniaceae,
shows a huge variation in tree species diversi- Bombocaceae, Dilleniaceae, Ebenaceae, Til-
ty place to place (Pitman et al., 2002). iaceae, Ulmaceae, Malvaceae, Lythraceae,
Among the different phyto diverse regions Sapotaceae, Annonaceae, Rutaceae, Sapin-
found in the country, the Terai region is one daceae, Dipterocarpaceae, Sterculeaceae,
of them existing from Uttarakhand to West Bignoniaceae, Verbinaceae, Combretaceae,
Bengal. It is the transition zone between two Meliaceae and Rhamnaceae with single plant
eco-climatic zones, the Gangetic plain to- species only. The available literature reveals
wards south and Bhabhar towards north, that most of the tree plants found in Katar-
along with the sub- Himalayan tracts (Tripa- niaghat Wildlife Sanctuary (KWS) are multi-
thi and Singh, 2009). The region has lost ma- purpose, ethno-botanical, nutrimental, ethno-
jority of its natural forest due to deforestation medicinal, ethno-veterinary and of environ-
chiefly for agriculture and lack of sustainable mental use in agro-forestry which provide
forest management in last many centuries shade, habitat for organisms, soil improve-
(Bajpai et al., 2012a, b). Now the natural for- ment, etc., many useful products are also ob-
ests of the region have been restricted to the tained such as fruits, timber, fire wood and
wildlife protected areas only. Katerniaghat variety of metabolic chemicals which may be
Wildlife Sanctuary (KWS) is also one of used in the form of home remedies and for
them. traditional medicine. Considering the multi-
Traditional medicines are used by purpose importance of these trees of KWS,
about 60 percent of the worlds population. the present overview is an attempt to summa-
These are not only used for primary health rize the informations available on these
care just in rural areas, in developing coun- plants which are yet not popular due to one
reason or the other despite providing an array while description of the plant. Since the
of benefits. list of the trees is big, the description of
all is beyond the scope of this manuscript
2. Study area so we have taken thirteen plants, viz.,
Acacia catechu, Acacia concinna, Aegle
The study area Katerniaghat Wild- marmelos, Albizia lebbeck, Albizia
life Sanctuary (KWS) is situated in Bah- procera, Alstonia scholaris, Bombax cei-
raich district of Uttar Pradesh in India. It ba, Diospyros cordifolia, Ficus racemosa,
lies along Indo-Nepal international board- Madhuca latifolia, Shleichera oleosa,
er and is situated between 27 41 27 Syzygium cumuni and Ziziphus mauritia-
56 N and 81 48 81 56 E covering na for detail description.
an area of 440 km2 with 116 to 165 m el-
evation. The sanctuary comes under the 4.1. Acacia catechu Willd.Khair, Catechu
tropical moist deciduous forest of the (Mimosaceae)
Himalayan Terai-Bhabar region (Cham- It is a moderate size tree. Leaves
pion and Seth, 1968; Rodgers and are pinnate. Flowers yellow in globose,
Panwar, 1988). The forest of the sanctu- peduncled axillary heads. Pods strap
ary area has been classified into two ma- shaped, dark brown. Phenology - August
jor forest types (i) The Sal forest and (ii) to February. In Katarniaghat Wildlife
The miscellaneous forest (Champion and Sanctuary it is found only in miscellane-
Seth, 1968). Pedagogically the study area ous forest with IVI value 18.3.
is made up of the alluvial soil of the
Kaudiyala and Saryu rivers and its tribu- Ethnobotanical potential
taries flowing adjoining to it. Geological- Catechu, a multipurpose tree species
ly the sanctuary area has been divided is widely used by the inhabitants for
into high and low land areas. fodder, fuel, building material and in
health care.
3. Climate The heartwood of the tree is mainly
used for extracting Katha and Cutch
A typical tropical monsoonal cli- (decoction obtained after filtration)
mate with three distinct seasons, i.e., which are sold in the market.
summer (April to June), winter (Novem- Katha is commonly used in ayurve-
ber to February) and warm-rainy (July to dic preparations.
September) prevails in the study area. Katha serves as one of the major
March and October are considered as components in masticatory, i.e.,
transition months between the seasons. chewing of betel leaf (pan) in India.
The mean maximum temperature ranges
catechu is a valuable bio-resources
from 22C in January to 40C in May and
and has been exploited commercially
the mean minimum temperature ranges
in tannin and Katha industry for dec-
from 8C in January to 27C in June. The
ades.
annual rainfall ranges from 36 to 142 cm
Besides its commercial importance,
in winter, 34 to 662 cm in summer and
it is equally significant for the people
1294 to 1689 cm in warm-rainy seasons
particularly rural communities living
(Bajpai et al., 2012).
in the vicinity of catechu forests as it
is a subsidiary source of income to
4. Observations
them. To a certain extent, these peo-
ple are dependent on this plant to ful-
At present the KWS has been di-
fill their day to day need of fuel, fod-
vided in to three types of forests- miscel-
der, building material, etc.
laneous forest, sal forest and teak planta-
tion forest. The IVI of the plants in all the
Ethno-medicinal potential
three forests are presented respectively
ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 488
Biotech Sustainability (2017)
Diversity and Ethno-Botanical Potential of Tree Plants Mall
The decoction of bark mixed with handles of axe, saw, sickle, hammer,
milk is taken to cure cold and cough. spade and combs.
The bark decoction is either alone or
used in combination with opium to Socio-religious beliefs
cure severe diarrhea. Khair is considered one of the sa-
Katha after drying is applied on lem- cred trees by the natives and wood
on slice and taken regularly with is used in the religious ceremonies
empty stomach to cure piles. at the time of havans (yagya).
Heartwood of khair is boiled with Wood is considered sacred and used
other ingredients to prepare the de- as one of the religious plants along
coction. It is taken as tea by the with bhoj patra (Betula utilis) at the
pregnant ladies to keep warm their funeral ceremony. It is believed to
body. It is also given to cure fever provide mukti or moksha (peace to
due to cold during the pregnancy. the heavenly soul).
A decoction is served to women after
2-3 days of child delivery, prepared Fencing
by boiling Katha along with carda- Cut branches are extensively used
mom. It is believed that it provides for fencing purpose by the farmers
strength to the body and also helps in to protect agricultural fields and lo-
secretion of milk. cal grasslands from domestic live-
The water boiled with the heartwood stock and wild animals.
chips of Khair, is used to take bath
by women after delivery. It is con- Tanning
sidered beneficial to cure the body The cutch is used locally for tanning
pains. leather and as dye to a great extent.
Katha or decoction of heartwood is
applied in mouth and on tongue to Economic Importance
cure mouth ulcer. It is also applied Besides traditional utility, A. cate-
externally on ulcers, boils, skin erup- chu is widely utilized commercially
tions and on gums as disinfectant. for extracting Katha from the heart
wood which costs around US $ 4-6
Fuel per kilogram in Indian markets.
The dried logs, twigs and branches Cutch is used as adhesive in ply-
are largely used as fuel. wood industry and it is also used in
preparing polishes and paints (Singh
Fodder and Lal, 2006).
The trees are lopped heavily for
their leaves used as fodder particu- From the present study, it is en-
larly for sheep and goats. visaged that A. catechu has a great socio-
economic importance as it is widely used
Building and furniture material for different purposes by the natives. Be-
The wood is considered durable and sides, traditional and commercial im-
widely used by the inhabitants for portance, it has tremendous ecological
house building material as pole and significance. Because of its leguminous
to prepare furniture like bed poles, nature and soil binding abilities, it is a
tables etc. suitable species for wasteland develop-
ment.
House hold articles
Wood of khair is preferred to pre- 4.2. Acacia concinna (Willd.) DC
pare various parts of local plough,
dian gooseberry (Amia), two hun- & heart tonic, ulcer, antiviral, intesti-
dred g Acacia concinna (Shika- nal parasites, gonorrhoea, epilepsy.
kai), twenty g Bacopa monnieri Root: Dog bite, gastric troubles, heart
(Bhrahmi), one g Asphaltum disorders, intermittent fevers, ant-
(Shilajit), forty g dried pomegran- amoebic, hypoglycemic, rheumatism.
ate peel (Anar ka chilka), twenty Bark: Stomach disorders, intermittent
g Eclipta alba (Bhringraaj), Soak fevers, heart disorders.
them overnight in any iron bowl. Seed: Febrifuge.
Grind to make paste. Apply it on Flower: Expectorant, epilepsy.
your hair and Scalp. Wash after 3 Whole Plant: Abdominal pain, ab-
hours with lukewarm water. Do scess, astringent back ache, dog bite,
not use shampoo. Shampoo may breast pain, cholera constipation, con-
be used on next day. vulsions, cramp, diabetes, diarrhoea,
dysentery, fevers, eye complaints,
4.3. Aegle marmelos (L.) Corr gastric trouble, abdominal disorders,
It is a medium-sized, deciduous, jaundice, laxative, nausea night fever,
armed tree. Leaves are tri- heart disorders, snakebite, stomach
foliolate. Flowers are yellowish. disorder, vomiting tonic, cut &
Fruits are large, globose. Phenolo- wounds.
gy is April-May & March-July. In Root bark: Fish poison.
Katerniaghat Wildlife Sanctuary
Seed mucilage: Plaster for walls.
Aeglemarmelos is found only in
Seed oil: Laxative.
teak plantation forest.
Wood: Beads worn by low caste, spe-
cial couches for rheumatic patients.
Ethnobotanical potentiality
Bael is one of the most important Gum around seed: To improves adhe-
tree species used in various indige- sive strength of water paints.
nous system of medicine in India, Unripe fruit rind, Bark: Yellow dye.
China, Burma, and Sri Lanka. Bael is Stem: Pestles of oil and sugar mills.
used in all tridosa- vista (air), Pitta The medicine is prepared in the form
(phlegm) and kapha (cough). Out of of pills, powder and paste. Ayurvedic
more than 66 ethno-botanical uses of practitioners commonly use the roots
bael, 48 are exclusively for medicinal of bael as an ingredient of dasmula
purposes. Almost all parts of bael are (ten roots), which is useful in recover-
used in preparing medicine (Kala, ing the loss of appetite and use fruits
2006). in the preparation of chawanprash.
Leaf: Abscess, backache, eye com- Bael fruits regarded as an astringent
plaints, abdominal disorders, vomit- are frequently used by various ethnic
ing, cut & wounds, ulcer, destroy, ber- communties for the treatment of diar-
iberi, weakness of heart, cholera, diar- rhoea, dysentery, constipation, stom-
rhoea, cardio tonic, blood sugar, inju- ach ache, intestinal ulcer, diabetes,
ries caused by animals, nervous disor- dyspepsia, heart diseases and cholera
ders, hair tonic, acute bronchitis, child due to its digestive and carminative
birth, veterinary medicine for wounds, properties.
killing worms, fodder for sheep, goat Bael is highly valued in Ayurvedic
and cattle, stimulation of respiration medicine for the treatment of chronic
and contraction of de-nerved nictitat- diarrhoea and dysentery and as brain
ing membrane in anaesthetized cats. tonic. Bael possesses antiviral, anti-
Fruit: Astringent, diarrhoea, gastric helminthic anti-inflammatory, anti-
troubles, constipation, laxative, tonic, bilious, anti-parasitical, anti-pyretic,
digestive, stomachic, dysentery, brain anti-scorbutic, aromatic, astringent, di-
gestive, febrifuge, haemostatic, anti-
ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 493
Biotech Sustainability (2017)
Diversity and Ethno-Botanical Potential of Tree Plants Mall
diarrheal, laxative and nutritive proper- For Hindus, the Bael is sacred tree,
ties. which they dedicate to the lord Shiva
Ripe bael fruit is sweat, aromatic by offering of Bael leaves. Its three
and nutritive, whereas fresh fruit is leaflets are assumed by the symbols of
stringent and has laxative properties. three gunas or attributes (e.g., satva,
Bael fruit powder exhibits anti- rajas and tamas, literally meaning mo-
cancerous and anti-proliferative activi- rality, superiority and immorality, re-
ties. The combinations of five parts of spectively); three Gods (Brahma,
bael, such as, fruit, leaf, bark, root and Vishnu and Mahesh); and three lives
flower is assumed to be effective for (past, present and future). Bael is con-
certain mental disorders. sidered to be extremely auspicious and
Unripe fruits pulp mixed with boiled cultivated around most of the Hindu
rice water is taken twice a day to cure temples.
vomiting in pregnancy. Unripe fruits
pulp mixed with sugar is taken with Phytochemicals of Aegle marmelos
milk twice daily for curing urinogenital A. marmelos has been reported to con-
disorders. tain several phyto-constituents mainly
Half roasted unripe fruit pulp mixed marmenol, marmin, marmelosin, mar-
with equal quantity of sugar is taken melide, psoralen, alloimperatorin, ruta-
twice a day to cure dysentery. Unripe retin, scopoletin, aegelin, marmelin, fa-
fruit pulp powder is taken twice daily garine, anhydromarmelin, limonene, a-
to cure abscess. phellandrene, betulinic acid, marmesin,
Bael leaf extract is taken twice a day to impertorin, marmelosin, luvangentin and
remove the intestinal worms. Leaf auropetene Rahman and Parvin .
poultice is used as remedy in ophthal- Due to the presence of various
mic problems and ulcer. phyto-constituents the plant has an-
Leaf juice is reported to have multiple ti-diarrhoeal, anti-microbial, anti-
medicinal uses, including controls of cancerous, anti-pyretic, anti-
diabetes. Cooling delicious drink pre- genotoxic, anti-fertility, anti-
pared from fruit pulp along with sugar inflammatory anti-diabetic and diuret-
and tamarind diluted with water is use- ic activities.
ful for health. The essential oil isolated from the
Baelroot decoction is given twice daily leaves of A. marmelos tree has proved
to cure fever and cold. Extract of bael to have antifungal activity against an-
root, pyaz (Allium cepa Linn.), and imal and human fungi like Tri-
haldi (Curcuma domestica Valeton) chophyton mentagrophytes, Tri-
mixed in equal proportion is put in the chophyton rubrum, Microsporum
ears to relive earache and secretion gypseum, Microsporum audounii,
from ears. Root decoction is used in Microsporumcookie, Pidermophyton
the treatment of intermittent fevers and floccosum, Aspergillus niger, Asper-
heart palpitation. gillus flavus and Histoplasma capsu-
Root and stem bark decoction is used latum.
in the treatment for fever and various The leaf extracts and fractions have
types of heart disorders. Bael root is fungicidal activity against various
used in the treatment of abdominal clinical isolates of dermatophytic fun-
pain, heart palpitation and urinary gi.
troubles. Various extracts of A. marmelos
Bael tea is good for health and is used leaves, roots ad fruits have been re-
for flatulence, gastrointestinal prob- ported to be active against many bac-
lems, cough and chronic intestinal dis- terial strains.
eases in children.
Research in plants with medicinal gland near the base of the petiole, leaflets
properties and identification of the rigidly sub coriaceous, grey beneth, gla-
chemical components responsible for brous obliquely truncate at the base.
their activities have corroborated the Flowers whitish in copiously panicled
traditional uses of ancient healing heads. Pods thin, brown, glabrous. White
wisdom and lore and have proven the siris is a large, fast growing tree with an
enduring healing potential of many open canopy that is almost evergreen but
plant medicines even in todays hi- becomes leafless for a short time in the
tech community. dry season. It grows up to 30 meters tall.
It is previously reported that the al- The bole can be straight or cooked; it can
coholic extract of Albizia lebback be branchless for up to nine meters and up
protects the guinea pig against the an- to sixty cm in diameter. An ornamental
tigen induced challenge (Tripathi et tree, it is often planted along avenues and
al., 1977; Barua et al., 1997). in gardens to beautify them. Phenology is
Further there it also reduced the level in May-June and September-March. In
of histamine and raised the plasma Katarniaghat Wildlife Sanctuary it is
cortisol in antigen challenged guinea found both in miscellaneous forest and
pigs (Tripahti and Shukla, 1979) as Sal forest with IVI of 2.8 and 0.5, respec-
well as in bronchial asthma patients tively.
(Tripathi et al., 1978). Das et al.,
(2003) and Pramanik et al., (2005) Ethno-botanical potential
previously reported the anti- The tree is extensively harvested
inflammatory activity of the metha- from the wild for its timber, many
nol extract of Albizia Lebback bark. natural forests being managed on a
Many saponins, such as lebbekanin forty year rotation.
A-H (Varshney and Khan, 1961; The tree is also grown as a plantation
Varshney and Sharma, 1969; Varsh- crop in Asia, Africa and the Ameri-
ney et al., 1973, 1976) and Albizzi- cans.
asaponin A-C (Pal et al., 1995), Fuel wood plantations are managed
which contain oleanolic acid, echino- on a 20-year rotation.
cystic acid or acacic acid as sapogen- The cooked leaves are eaten as a
ins were reported from various parts vegetable. In times of scarcity the
of this plant. Further, melanoxetin bark can be ground into a powder,
okenin-3-one, (+) pinitol, (-) leuco- mixed with flour and eaten.
pelargonidin (Gupta et al., 1966). The tree is widely planted for its
Alternative medicine for the treat- good soil binding capacity.
ment of various diseases is getting It is occasionally cultivated as shade
more popular. Many medicinal plants tree for tea and coffee plantations,
provide relief of symptoms compara- where it also acts as a wind and fire-
ble to that of conventional medicinal break.
agents (Verpoorte, 1999). It is popular for the rehabilitation for
seasonally dry, eroded and degraded
4.5. Albizia procera (Roxb.) Benth. soils. Its ability to grow on dry,
Synonym: Mimosa procera Benth sandy, stony and shallow soils makes
The habitat ranges from monsoon it a useful species for reforestation
forest, mixed deciduous forest, savannah for difficult sites.
woodlands, pyrogenic grasslands, road- Good survival and rapid early growth
sides and dry gullies, to stunted, seasonal have been reported in reforestation
swamp forest. It is commonly found in trials on both saline and alkaline
open secondary forest. It is a large decid- soils, which are widely cultivated in
uous tree. Leaves bi-pinnate with a large agro-forestry system.
The bark can provide tanning materi- bre length is 0.9 mm, mean fibre di-
al. It is used in India for tanning and ameter is 0.021mm).
dying. However, its low tanning con- The calorific value of dried sapwood
tent (12-17%), considerable weight is 4870 kcl/kg, and that of heartwood
loss in dying and difficult harvesting 4865 kcl/kg. An excellent charcoal
has limited its importance. (39.6%) can be prepared from the
When injured, the stem exudes large wood, and it is widely used as a fuel.
amounts of a reddish-brown gum that
is chemically similar to, and used as a Ethno-medicinal potential
substitute for, gum arabic (obtained White siris is commonly used in tra-
from Acacia senegal and other spe- ditional medicines. Some research
cies). has been carried out into the medici-
The leaves are known to have insec- nal activities of the plant and a num-
ticidal and pesticidal properties. ber of active compounds have been
The branches (twigs) are used by tea recorded.
planters as stakes for lying out tea All parts of the plant are anti-
gardens. These are found to split cancerous.
well. The species is popular along The roots contain alpha-spinasterol
field borders. and a saponin that possess spermi-
Pods and fallen leaves should be con- cidal activity at a dilution of 0.008%.
sidered not as undesirable litter but as A decoction of the bark is given for
potential energy sources. It seems the treatment of rheumatism and
probable that if the pods of the relat- haemorrhage.
ed species A. lebbeck can yield ten It is also considered useful in treating
barrels of ethanol per hectare, then problems of pregnancy and for stom-
this species could as well. ach-ache. The leaves are poultice on
The timber has large amount of non- to ulcers.
durable, yellowish-white sapwood.
The heartwood large and heavy, light 4.6. Alstonia scholaris (Linn.) R.Br.
or dark brown with light and dark Synonym: Echites scholaris Linn.
bands. Due to the broadly interlocked Alstonia scholaris found in India,
nature of the grain, it is more suitable Sri Lanka, Pakistan, Nepal, Thailand,
for use in large section where a bold- Burma, South East Asia, Africa, Northern
er effect is desired, such as in large- Australia, Solomon Islands, and Southern
sized panels and tabletops. China. Alstonia scholaris is an evergreen
It seasons and polishes well. The tropical tree up to 80 ft in height, having
wood is used chiefly for construction, greyish rough bark with lenticels, secret-
furniture, veneer, cabinet work, floor- ing white milky latex-rich in poisonous
ing, agricultural implements, mould- alkaloid, lateciferous which is bitter in
ing, carts, carriages, cane crushers, taste. Leaves grow in clusters of seven,
carvings, boats, oars, oil presses and coriaceous, elliptic-oblong, 10 to 20 cm
rice pounders. It is resistant to several long, 3 to 4.5 cm wide, pointed at the
species for termites. base, rounded at the apex, glossy green on
The chemical analysis of the wood the upper surface, white or greyish on the
indicates that it is a suitable material underside. The tip of the leaf is rounded
for paper pulp. Bleached pulp in sat- or shortly pointed, tapering towards the
isfactory yields (50.3%) can be pre- base. The blokes of larger trees are
pared from A. procera wood by the strongly fluted to 10 m. the outer blaze is
sulphate process. It is suitable for cream to yellowish in colour with abun-
writing and printing paper (mean fi- dant, milky latex that flows rapidly when
cut. The inflorescence is a much-branched
terminal panicle, up to 120 cm long; The bark and roots are boiled with
flowers 7-10 mm long white, cream or rice and eaten by girls daily for sever-
green, the tube hairy, lobes sparsely or al weeks to treat excessive vaginal
densely pubescent, 1.5-4 mm long, the discharge.
left margins overlapping, strongly per- The roots and bark are used in tradi-
fumed. The fruits are thin pods that can tional medicine as an anthelmintic, as-
grow up to 20 inches long. Fruit is made tringent tonic, alternative antidiar-
up two slender follicles which are pendu- rhoeaticum, antiperiodicum, etc.
lous and cylindrical follicles, 20 to 40 cm The latex is used to clean wounds and
long, 4-5 mm in diameter. Seeds are 3 to can we used for chewing gum.
4 mm long, with brown ciliate hairs on
the ends. Phenology- December-June. In 4.7. Bombax ceiba DC.
Katarnia Ghat Wildlife Sanctuary it is It is a large deciduous tree. Leaves are
found only in miscellaneous forest with digitate, leaflets 5-7, flowers red or yel-
IVI 0.4. lowish, capsules ovoid. Phenology is
March-April. In Katarniaghat Wildlife
Ethno-botanical potential Sanctuary is being found only in miscel-
The wood is too soft for making laneous forest with IVI 11.00.
anything- so it is usually used in making
packing boxes, blackboards, etc. Alstonia Ethno-botanical potential
scholaris tree has been used to make pa- The silk cotton tree is often re-
per. ferred to as the silent doctor for the
host of medicinal benefits that is of-
Ethno-madicinal potential fers almost each part of the tree, in-
Alstonia scholaris has many medici- cluding the bark, flowers, fruits, seed
nal properties like antimicrobial, anti- and leaves, gums, thorns have
amoebic, anti-diarrheal, antihyperten- therapeutic potential.
sive, anti-malarial, febrifuge, stimu- A herbal composition made from the
lant, hepoprotective, immune- bark of the tree, for example is admin-
modulatory, anti-cancer, anti- istered for the treatment of male sexu-
asthmatic, antioxidant, analgesic, anti- al and gastro-intestinal disorders like
inflammatory, anti-fertility, anti- dysentery and diarrhoea. The pharma-
diabetic, etc. cological benefits are basically due to
Alstonia scholaris used in the treat- the presence of Glycosides and tan-
ment of fevers, chronic diarrhea, dys- nins in the root and stem.
entery, ulcers, rheumatic pains, can- It has haemostatic properties and is
cer, malarial fever etc. administered during menorrhagia.
The ripe fruit of the plant are used in Silk cotton extracts are used in eve
syphilis and epilepsy. care, tentax forte, acne pimple
The milky juice of Alstonia Scholaris cream. The plant is also being used
has been applied to treat ulcers. for general debility, diabetes, impo-
The bark of the Alstonia scholaris is tence, spermatorrhoea, urinary stones
used in Ayurvedic medicine to treat and liver disorders.
fever, malaria, troubles in digestion, Some of the diseases for example di-
tumors, ulcers, asthma and so forth. arrhoea, dysentery, asthma, rheuma-
The leaves and the latex are applied tism, leprosy, leucorrhoea, body
externally to treat tumors. pain, wounds are included in anti-
The dried leaves of the Alstonia inflammatory, analgesic, anti-
scholaris are used as an expectorant. microbial and oxytocic activities
The leaves can be used to treat skin of plant as indirect evidence of
diseases.
scientific validation (Jain and Ver- tings of stem and root shakers or by seeds
ma, 2014). also. The flowers are pollinated by very
small wasps. Phenology: April July. In
4.8. Diospyros cordifolia Linn. Katarniaghat Wildlife Sanctuary is being
It is a large shrub or small tree. found both in miscellaneous forest and
Leaves are ovate-oblong, ovate lanceo- Teak Plantation forest with IVI of 9.7 and
late, acute, base cordate or rounded and 1.9, respectively.
hirsute on both surface. Flowers pale
white in axillary cymes. Fruits are glo- Ethnobotanical potentiality
bose yellow at maturity. Phenology- Traditionally it is used in Indian
March-June and June-September, In medicinal practice as astringent,
Katarniaghat Wildlife Sanctuary is being carminative, stomachic, vermicide
found both in Sal forest and Teak Planta- etc (Mall and Tripathi, 2017).
tion forest with IVI of 3.2 and 3.0 respec- The extract of fruit is used in leprosy,
tively. diarrhoea, menorrhagia. It is useful in
the treatment of leucorrhoea, blood
Ethnobotanical potentiality disorder, burning sensation, fatigue,
It is of great use for human being. urinary discharge, intestinal worms
It has commercial value being used in and as carminative.
Bidi industry as a raw material. Leaves are astringent to bowels and
Leaves are being used in stupefying good in case of bronchitis; leaves are
fishes. used in dysentery young tender leaves
It is being used in several ailments are used for fair complexion. The de-
either as a cure or for the well-being, coction of leaves is used to wash the
viz., used for lever disorders, whoop- wounds and ulcers.
ing cough, leprosy, ulcers, gonor- Bark is useful in asthma and piles.
rhoea, fever as emetic and anti- The latex or milky juice is adminis-
helminthic. Alcoholic extract are an- tered in chronic infected wounds,
ti-inflammatory, antipyretic and an- haemorrhoids, boils, traumatic swell-
algesic. It is depressant, spasmolytic ing, toothache, vaginal disorder,
producing bradycardia and hypoten- wounds it promote healing very soon.
sion. Aqueous extract is being used The root sap is used for treating dia-
in critical jaundicised condition. betes.
The fruits are consumed because of its Phytochemical properties: The leaf of
juicy and sweet nature by local inhab- this plant contains sterols, triterpe-
itants (Mall, 2016). noides (lanosterol) and alkaloids, tan-
nins and flavonoids. Stem bark gives
4.9. Ficus racemosa Linn gluanol acetate, -sterol, lupenol,
stigmasterol. Fruit contains gluanol
Synonym: Ficus glomerata Roxb. acetate, glucose, tiglic acid, esters of
A large deciduous tree, buttressed taraxasterol, lupeol acetate and other
at the base. Bark smooth reddish brown; phytosterols.
blaze pink, fibrous with white latex turn-
ing yellow on exposing. Leaves are 5-15 4.10. Madhuca latifolia Roxb.
x 2.5-6.5 cm, alternate, ovate or elliptic- Madhuca is a large deciduous tree
lanceolate, entire, sub-acute, base round- reaching a height up to 20m. Leaves are
ed or acute, glabrous above, minutely dot- large and broadly elliptic 12-20cm long.
ted beneath. Receptacles 2-3.2 x 2-3.5 The bark is 1.2 cm thick. Flowers white to
cm, clustered on leafless branches, cream colour with tubular, fleshy and
smooth or pubescent, red or pink at ma- juicy corolla. Fruit berry, ovoid, green
turity. Plant is propagated by using cut-
at maturity and turn pinkish yellow osa is derived from the Latin word ole-
when ripe. osa meaning oil, as the seed kernels are
rich in oil. Synonymously the tree is also
Ethnobotanical potentiality reffered as Schleichera trijugaWilld.,the
The madhuca contains protein, carbo- word trijuga stands for three pairs,based
hydrate, fat, minerals, calcium, phos- on thepresence of three pair of leaflets in
phorus, iron, carotenes, sugar. Vita- a leaf. Kusum is a large forest tree with
mins and many other nutrimental dense green foliage. Leaves pinnate with
chemical constituents. three pairs of leaf lets .Inflorescence ra-
The bark of Madhuca is used to ceme. Flowers are white and fruits small.
cure leprosy and to heal wound. The fruits are berry shaped, globose or
The flower decoction is used for ovoid with a hard skin. The seeds are
headache due to cough and cold. brown, irregular elliptic, slightly com-
Paste of fresh bark is useful on joint pressed oily and enclosed in a succulent
and muscles pain. aril. The oil content of the seed is around
Whole plant decoction is taken orally 59-72% with yellowish green color. Phe-
which is useful in joint and muscles nology is in October-November.
pain (Mall and Tripathi, 2017a). It is locally known as kusum. The
other common names are kusum, kusumb,
4.11. Syzygium cumini Skeels. kosumb, koshamara, Celon oak kosamara,
It is a large evergreen tree with whit- lac tree, honey tree, gum lac tree, macas-
ish brown bark. Every year the bark is sar oil tree, sukoshka, skrataka, jatud-
shed off. Its leaves are simple pointed at ruma,koshamra, jantu vriksha and kshu-
the tip, somewhat leathery, oval to rec- dra maukkuli, etc. It occurs in the Indian
tangular and somewhat shiny. Flowers are sub-continent and south East Asia. There
mostly white and appear in cluster from are many trees that are grown for multiple
axil to leaves.The fruit is berry. products. They are known as multipur-
pose trees (MPTS), a term widely used in
Ethnobotanical potentiality: agro-forestry. Kusum is also one among
The fruit contains 88% moisture, the multipurpose trees which has been
0.7% protein, 0.1% fat, 19.7% car- proved to be useful in numerous ways
bohydrate and 0.4% minerals. Fresh from times immemorial.
fruit had the antioxidants 708 In Katarniaghat Wildlife Sanctu-
mg/100g AEAC units. The ripe fruit ary (KWS) it occurs in all the three types
contains anthocyanin pigment (Rao et of forests with different IVI values. In
al., 2006). Syzygium cumini is a well- miscllaceous forest, Sal forest and Teak
known anti-diabetic herb. plantation the IVI value of the kusum
plant is 1.5, 4.3 and 4.4, respectively.
It is a good immune modulator. It is
The available literature reveals
also used in blood pressure, dysen-
that this multipurpose ethno-botanical,
tery, diarrhoea and gingivitis.
nutrimental, ethno-medicinal, ethno-
veterinary and plant of environmental use
4.12. Schleichera oleosa (Lour.) Oken.
in agro-forestry which provide shade,
Synonims: Pistacia oleosa Lour.
habitat for organisms, soil improvement,
Schleichera trijuga Willd., Cussambium
etc., many useful products are also ob-
oleosum Kuntze., Melicocca trijuga Juss.
tained such as fruits, timber, fire wood
It is a monotypic genus belonging
and variety of metabolic chemicals which
to the same family to which the popular
may be used in the form of home reme-
fruit Litchi belongs. The generic name
dies and for traditional medicine. Con-
of kusum, Schleichera is derived after the
sidering the multipurpose importance of
Swiss botanist J. C. Schleicher who first
the tree, which is yet not popularise due
descrived the tree. The species name ole-
to one reason or the other despite provid- duced in winter and summer season.
ing an array of benefits (Mall and Tripa- About 34-38% of the total lac pro-
thi, 2017b). duction of India is shared by the
Kusum tree as lac host. There are al-
Ethno-botanical potential so other lac hosts but the quality of
The leaves, twigs and the seed- cake kusum lac is far superior. The dence
are used as fodder to feed cattle. foliage of the mature kusum tree pro-
The wood is suitable as firewood and vides an additional advantage of sup-
makes excellent charcoal. porting brood lac (inoculums stick
Pressed oil cakes from kusum tree are lac with emerging larvae from the
rich source of crude protein, carbo- female resin cells) viability even dur-
hydrate, fibre and other minerals ing the very hot summer season, oth-
and serves as nutritive cattle feed. erwise summer mortality of lac in-
The oil extracted from the seed, sects is a common problem with oth-
called as kusum oil is used for culi- er host plants like Butea monosperma
nary and lighting purposes. (palas) and Ziziphus mauritiana
The kusum oil is being used to cure (ber).
itching, acne, burn and other skin The seeds of kusum are a very rich
problems. source of oil (60-72%) for industrial
The oil is used in rheumatism by ex- implications. The seed oil called
ternal massage. kusum oil is an important component
Kusum oil is used in hair dressing as of the Makassar oil used for hair
well as for promoting hair growth. dressing and cooling bath oil.
The pinkish-brown heart wood is Kusum oil is used in textile industry
very hard, durable and excellent to for batik applications and also for
make pestles, cartwheels, axles, making soap.
plows, tool handles, and rollers of The bark of kusum tree produces tan-
sugar mills and oil presses. nins and dyes that are occasionally
Kusum plant is known for lac cultiva- used in small-scale industries like
tion. It is one of the major host plant tanning in leather industry.
commercially exploited for cultiva- Young leaves and shoots-raw cooked
tion of the Indian lac insect (Kerria in soups or steamed and served with
lacca). It supports the kusmi strain of rice.
lac insect, which produces good qual- The ripe fruit is eaten raw which has
ity, natural, biodegradable and com- a pleasant acid flavor.
mercially important, light colored lac The unripe fruits are pickeled.
resin of demand by lac industry, thus Oil obtained from the seed called
fetching high remunerative prices to macassar oil, is sometimes used for
lac growers. The lac resins serves as culinary purposes. It contains cyano-
a livelihood support to millions of genic compounds, which may cause
poor farmers in states like Jharkhand, giddiness and should be removed if
Chattisgarh, Orissa, Andhra Pradesh the oil is used for human consump-
and West Bengal. Immature lac in- tion.
sects prefer semi-tender twig of The kusum tree is also grown as an
kusum tree for sap sucking and start avenue tree or wayside tree.
secreting resins surrounding their The tree is utilized for multifarious
body. The resinous coatings of close- purposes and is a boon for a subsist-
ly settled sessile insects eventually ence farmer.
coalesce together to form an encrus- The extended foliage and canopy of
tation in five to six months. On the kusum tree provides good shade
kusum tree, two lac crops are pro- and is therefore, suitable for mixed
ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 501
Biotech Sustainability (2017)
Diversity and Ethno-Botanical Potential of Tree Plants Mall
farming with other heat susceptible and Gandhi et al., 2011). Therefore, it is
economic plants. used in production of biodiesel. In a re-
port by Gandhi et al.,2011 methyl ester
Importance of S. oleosa as a biodiesel was produced using S. oleosa seeds.
fuel
The depletion of the conventional Phytoremediation properties
petroleum resources has become a prob- Callophyllum inophyllum L. and Bixa
lem of major concern in recent years. Ex- orellana L. (Chaturvedi et al., 2012).
tensive research is going on to find an al- Mining, smelting of metalliferrous,
ternative fuel. Since vegetable oils have dumping of waste, chemicals used in
properties similar with that of diesel, they agriculture etc. Are the different
are replacing diesel in the field of com- source of soil pollution, but the waste
mercial transportation and agricultural rocks generated by mining is the main
machinery. But the direct use of vegetable source of the metal pollution of soil.
oil is having adverse effects on the com- The direct consequences of the depo-
bustion engine. Therefore, these vegetable sition of waste rocks on the surface
oils are converted to biodiesel. Blending, are the loss of cultivatable lands, for-
emulsification, thermal cracking, and est and grazing land (Clemente et al.,
trans-esterification are the few techniques 2007, Rio et al., 2006 and Freitas et
used for the conversion of crude vegeta- al., 2004). Activity such as grinding,
ble oil into biodiesel. At present, biodiesel crushing, washing and smelting, used
is produced by sunflower oil, palm oil and to extract and concentrate metals,
soybean oil by trans-estrification process. generate waste rocks and tailings.
These oil due to their non-toxic, biode- Most of the tailings exhibit acidic pH
gradable and renewable nature, have due to which the microbial activity
gained a lot of alteration by the research- decreases which in turn leads to the
ers. Cetane number for biodiesel is higher death of plants. Tailings fo not con-
than that of petroleum. Moreover, bio- tain organic matter and are character-
diesel does not contain aromatic compo- ized by the high concentration of ar-
nents. The emission of carbon monoxide, senic, cadmium, copper, manganese,
hydrocarbon and particulate matter is also lead, zinc and other heavy metals
less as compared to that of diesel fuel. (Mukhopadhyay and Maiti, 2010).
High cast of the above mentioned oil is However some plants can exist in the
the basic disadvantage associated with region of high concentration of metals
them. Hence, the non-edible type of oils (Das and Maiti, 2008). Such plants
yielded from tress such a mahua, sal, in- can be used to restore the contaminat-
seed, caster, karanji neem, rubber, ed sites by the process of phytoreme-
jatropha, kusum, cashew, restaurants diation. Phytoremediation is an envi-
waste oils and greases slong with animal ronmental friendly and cost efficient
fats are best suited for the production of technique used to treat the contami-
biodieses, for instance, S. oleosa seed oil, nated soil, air or water through the use
one of the many non-edible seed oil is of plant without employing any soil
found to have many cyanogenitic materi- excavation or mechanical clean up
als and free fatty acides (FFA) such as method. Although many physic-
myristic acid, palmitic acid, palmitoleic chemical techniques are also available
acid, cis oleic acid, trans linolelaidic acid, to extract metals such as acid leaching
cis linoleic acid, alpha linolenic acid, and electro-osmosis, but these tech-
eicosadienoic, heneicosanoic, behenic niques are quite costly and can decon-
acid, erucic acid, lingoceric acid, do- taminate only small portions of land
cosahexaenoic acid (Mikolajczek and (Ramadhas and Murleedharan, 2005).
Smith, 1971, Canacki and Gerpen, 2001 Moreover, these techniques also dete-
riorate biological activity of the soil The bark is known to contain medical-
and adversely affect its physical struc- ly important compounds like lupeol
ture. Therefore, the phyto-remediation used in preparing analgesic and anti-
is the preferred technique to decon- tumerous agents like betulin and betu-
taminate the soil. This approach to lic acids.
remove the metal is called green min- It balances kaph, useful in productive
ing because further extraction of met- cough and asthema.
als can be done from the plant tissue It cleanses intestine.
(Clemente et al., 2007). It is used in bleeding disorders like
nasal bleeding and heavy periods.
Use as livestock feed The unripe fruits are absortant, useful
A few non-conventional agro- in diarrhea, neuralgia, paralysis, con-
industrial by-products including S. stipation and bloating.
oleosa cake were checked for their ef- The fruit pulp improves hair strength
fectiveness a livestock feed (Punj, and promotes hair growth.
1988). The presence of tannins ad- The ripe fruit improves digestion
versely effects the utilization of vari- strength, improve taste and relieves
ous nutrients (Mc Leod, 1974). In ad- anorexia.
dition, tannins are believed to create
The leaf, seed, oil, and bark are used
toxic effects by breaking down the al- for treating rheumatoid arthritis,
imentary canal tissues and the hydro- headache, myalgia,,skin disease, ma-
lysable tannins make pathological larial fever and prophylactic against
changes in liver, kidney, heart, etc.
cholera.
When their concentration in blood in-
The bark is astringent and is used in
creases further than the competence of
fever as antipyratic, useful in pruritus.
the liver to deify them. The levels of
The kusum oil is bitter, sour, sweet
tannins were determined using various
which improves strength and immuni-
chemical and biological methods. It
ty and can be taken regularly. It im-
was observed that in S. Oleosa, tannin
proves taste and relieves anorexia.
levels in terms of total phenols (TP)
and condensed phenols (CP) were The oil is digestive, induce mobility,
low, and protein-precipitation capaci- causes diarrhea, purgative and re-
ty (PPC) could not be detected be- lieves constipation.
cause of its very low level. Hence, it The fine paste of the bark which is
can be considered safe for incorpora- astringent is mixed with oil is applied
tion in livestock feed since the harm- to cure itch and acne and other skin
ful factors are absent (Makkar, 1990). eruptions.
The oil is useful in worm infection,
Ethno-medicinal potential skin diseases, in toxic conditions, poi-
Different plant parts (stem bark, seed, soning, ulcer and wounds.
fruit and seed oil) of kusum are used The seed oil is also used for the cure
in traditional medicines. of itch and acne.
The seed oil is used by the local vaids The seed oil is stimulating and has
for curing skin diseases like scabies, cleansing applications.
itching, and acne. The ripe fruit is often served with salt
The bark decoction is also used which improves digestion, useful in
against skin inflammation and ulcers. anorexia and nourishing.
The bark decoction is also infused for
curing malaria. Ethno-veterinary potential
The fine paste of the bark of Kusum is The seed is grinded so as to make
often used to control tissue swelling. fine powder. It is mixed with wa-
ter and given to cattle for remov- tion of an extract prepared from the bark
ing worms from the stomach. and stem of Sri lankan tree S. oleosa re-
The fine powder of the seeds is sults in the isolation of seven sterols,
applied to wounds and ulcers of Scheicherastins (1-7) and two related
cattles to remove maggots. sterols 8 and 9 designed as Schleicheols 1
and 2. The isolated Scheicherastins exhib-
Phyto-chemical constituents ited cancer cell growth inhibitory proper-
Phytochemical studies have ties (Pettit et al., 2000).
shown that its bark contains lupeol, lupeol
acetate, betulin, betulinic acid, beta- Antioxidant activity
sitosterol, and scopoletin (Dan and Dan, Oxygen is used for generating
1986). A very recent report have also metabolic energy in our body but it also
shown the existence of taraxerone and produces reactive oxygen as by products
tricadenic acid A in the outer bark of the during its various reactions in the body.
above plant (Ghosh et al., 2011). The Reactive oxygen species are usually at-
bark also contains about 10% tannin and oms or a group of atoms having odd (un-
antitumor agents such as betulin and betu- paired) electrons, in aerobic cells these
linic acid have also been isolated from it. are produced during mitochondrial elec-
tron transport and several oxidation reac-
Anticancer activity tions (Forman and Torres, 2002). These
Cancer is a term used for a disease reactive species acan, react with DNA
in which abnormal cells trend to prolifer- and several other bio-molecules causing
ate in an uncontrolled way and, in some what is called oxidative damage to DNA
cases metastasize. Extensive research has this damage causes changes in DNA such
been done in order to find therapeutic as stand breaks; changes at cross links
drug for the treatment of cancer. Plant between DNA and protein; changes as
based products have been frequently ex- base tree sites among other changes (Diz-
amined as potential anticancer agents. daroglu et al., 2002). Several medicinal
The screening of various medicinal plants plants, fruits, vegetable can decrease the
results in the isolation of bioactive com- risk of oxidative damage as they comprise
pounds which have been reported as ef- of vitamins, carotenes, phenolic com-
fective chemopreventive as well as chemo pounds, flavanoids, alkanoids, tannins,
therapeutic agents (Kawamori et al., etc. which act as chemo-preventive agents
1999, Choi et al., 2001, Kirana et al., (Dhir et al., 1993, Cozzi et al., 1997 and
2003 and Sandhya et al., 2006). The phy- Thind et al., 2012). These phyto-
tochemical screening of S. oleosa re- chemicals can prevent damage by their
vealed the presence of lupeol and butilinic radical scavenging ability. Thind et al.
acid type triterpene which have antineo- evaluated the hydroxyl radical scavenging
plastic activity (Bhatia et al., 2013). This potential of S. oleosa. Extracts of roots of
study provides a step toward the explora- S. oleosa with different solvents were
tion of S. oleosa as a chemo preventive tested for their anti-proliferative activity.
agent against cancer. A bulk of research Antioxidants are molecules which
revealed that the phyto-chemicals exhibit can safely interact with free radicals and
their anticancer properties either by sup- terminate the chain reaction before vital
pressing the proliferation of tumor cells molecules get damaged. The free radical
via suppression of various cell signalling damage can be prevented by several en-
pathways or by induction of apoptotic zymes and the principle antioxidants such
death in tumor cells by generation of free as vitamin E, beta-carotene, and vitamin
radical, such as reactive oxygen/nitrogen C, present in the defence system of our
species (Bharti et al., 2003 and Pettit et body. Several studies have shown that
al., 2000). A report involving the separa- plant phenolics also have antioxidant
tide alkaloids, found to have sedative, Bajpai, O., Kumar, A., Mishra, A. K.,
antimicrobial, hypoglycaemic, anti- Sahu, N., Pandey, J., Behera, S.
plasmodial, anti-infectious, anti-diabetic, K. and Chaudhary, L. B. (2012b).
diuretic, analgesic, anticonvulsant and Recongregation of tree species of
anti-inflammatory activities (Goyal et Katarniaghat Wildlife Sanctuary,
al.,2012). Uttar Pradesh, India. Journal of Bi-
In spite of the fact that Ziziphus mau- odiversity and Environmental Sci-
ritiana having medicinal properties it is ences 2, 24-40.
neither considered as important medicinal Babu, N. P., Pandikumar, P. and Igna-
plant nor utilized for medicinal use in cimuthu, S. W. (2009). Anti-
main stream therapeutic. inflammatory activity of Albizia
From the present study, it is envisaged lebbeck Benth., an ethnomedicinal
that the trees of Katarniaghat Wildlife plant, in acute and chronicanimal
Sanctuary has great socio-economic im- models of inflammation. Journal of
portance as they are being widely used for Ethnopharmacology. 125, 356-360.
different purposes by the natives. Nature Balasubramaniam, P. (1992). Observa-
has provided a complete store house of tion on the utilization of forest
remedies to cure ailments of mankind. plants by the tribals of point Cali-
Besides, traditional and commercial im- mere wild life sanctuary, Tamil Na-
portance, they have tremendous ecologi- du. Bulletin of Botanical survey of
cal significance. Trees which are of le- India 34, 100-111.
guminous nature and soil binding abili- Barua, C. C., Gupta, P. P., Patnaik, G.
ties, they all are suitable species for K., Kulsherestha, D. K. and Dha-
wasteland development. However, other wan, B. N. (1997). Studies on the
uses of many trees are currently underex- antianaphylactic activity of
ploited but hold promise to benefit human franctions of Albizzia lebbeck. Cur-
life in many spheres. The plantation of rent science 25, 397-399.
plants in suitable areas needs to be pro- Bharti, A. C., Donato, N., Singh, S. and
moted in view of their advantages as Aggrawal, B. B. (2003). Curucu-
malty purpose trees (MPT). There is a min down regulates the constitutive
tendency to prefer quick growing trees in activation of nuclear factor-kappa B
the forestation programmes, which may and Ikappa B alpha kinase in human
not always be advantageous or even eco- multiple myeloma cells, leading to
friendly in long run. The trees must be suppression of proliferation and in-
conserved and more plantations should be duction of apotosis. Blood 101,
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Abstract: The present research work was aimed to evaluate the free radical scavenging po-
tential and anticancer property of Primula denticulata ethanol extract (PDEE) against dif-
ferent human cancer cell lines. The antioxidant potential was determined by total phenolic
and flavonoid content, DPPH free radical scavenging assay, hydroxyl radical scavenging
assay and DNA damage assay. The anticancer activity was determined by SRB assay.
Apoptotic induction in MiaPaca-2 cells was analysed by propidium iodide staining cell-cycle
and DNA content analysis, and mitochondrial membrane potential loss was measured using
rhodomine-123 as fluorescent dye through flow cytometry. The total phenolic and flavo-
noid content in PDEE was found to be 12.242.11 (mg GAE/g dry extract) and 7.06 1.31
(mg catechin/g dry extract) respectively. The extract at 600 g/ml concentration induces
69.35% DPPH free radical inhibition. In hydroxyl radical scavenging, the extract showed
54.51% inhibition at 120 g/ml concentration. PDEE prevents DNA damage against oxida-
tive stress in concentration dependent manner. Anticancer activity was evaluated against six
human cancer cell lines (MiaPaca-2, A-549, PC-3, THP-1, HCT-116 and HOP-620). The
extract showed significant anticancer activity in concentration dependent pattern. The high-
est activity was shown against MiaPaca-2 cell line. PDEE induces significant apoptotic in-
duction in cells. Exposure of MiaPaca-2 cells to PDEE (0-100 g/ml) caused dose dependent
cell cycle arrest at G0/G1 phase and induced apoptosis by increasing accumulation of cells at
G0/G1 phase. PDEE increased the apoptotic cell population from 11.4% in case of control to
49.6% at 100 g/ml. Further, PDEE induces loss of mitochondrial membrane potential (m)
to 99.5% at 100 g/ml from 24.4% in control cells. These primary results depict the free radical
scavenging potential and anticancer activity of P. denticulata extracts. These findings may
serve as foundation to develop an anticancer drug from medically important P. denticulata.
gen peroxide (H2O2), ferric chloride, di- tude of 2650 m. The healthy plant species
methyl sulfoxide (DMSO), potassium fer- were randomly collected by hand-picking
ricyanide were purchased from Merck. and later identified by Dr. Anzar A.
The other reagents used were all of ana- Khuroo at department of Botany, Univer-
lytical grade. sity of Kashmir. A specimen under
voucher number KASH-1743 was pre-
2.2. Collection and identification of plant served for future reference.
Primula denticulata Sm. at flow-
ering stage was collected from Gulmarg 2.3. Extract preparation
region of Kashmir Himalaya (latitude Fresh and healthy leaves of P.
343'27" N; longitude 7423'9" E) at alti- denticulata (1Kg) were cleaned with dou-
ble distilled water, dried under shade (25 pable of donating hydrogen or electron.
2C) for 5-6 days. The dried plant materi- 100 l of different concentrations (100-
al was ground to powder form. The plant 600 g/ml) of plant extract or standard
powder was extracted in soxhelt apparatus antioxidant was added to 1 ml DPPH so-
using ethanol as solvent at desirable tem- lution (0.5 mM). The solution was slight-
perature. The filtered extract was concen- ly shaken and kept stand for 30 min at
trated using Buchi rotavapour and stored room temperature under dark conditions.
in glass vials at 4oC until used. The yellow colour solution was read at
517 nm against ethanol (Brand-Williams
2.4. Estimation of total phenolics et al., 1995). The free radical inhibition
The total phenolic content in leaf was calculated as:
extract of P. denticulata was determined Percentage inhibition = [(Ac-As)/Ac] x
by FolinCiocalteu method as adopted by 100
Slinkard and Singleton (1977) with slight Where, Ac and As are the absorbance of
modifications. To 0.2 ml of plant extract control and sample respectively
(1mg/ml) was added to 2.5 ml of 10% di- Butylated hydroxytoluene and - tocoph-
luted FolinCiocalteu reagent and 2 ml of erol were used as positive control.
2.5% aqueous Na2CO3. The reaction mix-
ture was incubated at room temperature 2.7. Hydroxyl radical (HO) scavenging
with intermittent shaking. The blue colour assay
solution was read at 765 nm on UV Deoxyribose assay was used to
visible spectrophotometer. The absorb- evaluate the hydroxyl radical scavenging
ance of solution was compared against potential of P. denticulata leaf extract
standard Gallic acid (50 mg %) calibra- (Halliwell et al., 1987). The HO generat-
tion curve. ed in Fenton reaction attack deoxyribose
to form products that upon heating with
2.5. Estimation of total flavonoids thiobarbituric acid at low pH yield a pink
The aluminium chloride colori- chromogen (TBARS). A reaction mixture
metric method as described by Mcdonald containing deoxyribose (25 mM), FeCl3
et al., (2001) was used to determine the (10 mM), ascorbic acid (100 mM), H2O2
total flavonoid content of leaf extract. The (2.8 mM) in 10 mM KH2PO4 (pH 7.4)
principle of this method is based on fla- with or without plant extract at various
vonoidaluminium complex formation, concentrations (20-120 g/ml) and incu-
which shows absorbance maximum at 430 bated at 37 0C for 1h. Then 1 ml of TBA
nm. Briefly 0.5 ml (1mg/ml) of extract (1% w/v) and 1 ml of TCA (3% w/v)
was mixed with 1.5 ml of ethanol, 0.1 ml were added and heated at 100 0C for 20
of 10% AlCl3, 0.1 ml of 1M potassium min. Absorbance of TBARS was read at
acetate and 2.8 ml of distilled water. After 532 nm. Deoxyribose oxidation inhibition
5 min of incubation, the absorbance was was calculated as:
read at 430 nm. Flavonoid concentration Percentage inhibition = [(A-B)/A] 100
was expressed as milligrams of catechin Where, A is malonaldehyde produced
equivalents per gram dry weight. when treated with extract and B is malo-
naldehyde produced without extract. Bu-
2.6. DPPH assay tylated hydroxytoluene and - tocopherol
DPPH assay is one of the most ex- were taken as the positive control.
tensively used method for determining the
antioxidant potential of any biological 2.8. DNA damage assay
sample. DPPH is a purple stable free radi- The Prevention of oxidative DNA
cal which is reduced to yellow colour damage by PDEE was determined by
complex 1,1-diphenyl-2-picrylhydrazine method as previously described by Ghan-
(DPPH-H) by compounds which are ca- ta et al., (2007). Calf thymus DNA
(0.37g) with and without plant extract at room temperature. The optical density
(10, 30, 50, 80 and 100 g) was incubated was read at 570 nm using ELISA reader.
with 20 mM ferric nitrate 30 mM H2O2 in The experiments were done in triplicates.
20.0 mM phosphate buffer (pH 7.4) in a Percentage cell growth was calculated as:
final reaction mixture volume of 20 l for Percentage cell viability = [At/Ac] 100
1h at 37 oC. Oxidative DNA damage was Percentage cell growth inhibition = (100-
induced by hydroxyl radicals generated in percentage cell viability)
Fenton reaction (Ani et al., 2006). Bro- Where At and Ac are absorbance of treat-
mophenol blue (0.25%) and glycerol ed and control cells, respectively.
(30%) were added to terminate reaction
mixture, followed by gel electrophoresis 2.11. DNA content and cell cycle phase
in 0.7% agarose. The gel was then visual- distribution
ized and photographed on gel doc. Human pancreatic (MiaPaca-2)
cells were seeded in 6-well culture plates
5
2.9. Cells culture with cell density 2x10 cells/ml/well and
Pancreatic cell line (MiaPaca-2), incubated for 24 hrs. After incubation, the
Lung cell line (A-549), Prostate cell line cells were treated with PDEE (0, 30, 50
(PC-3), Leukaemia cell line (THP-1), Co- and 100 mg/ml) and again incubated for
lon cell line (HCT-116) and Lung cell line 48 hrs. After 48 hrs treatment cells were
(HOP-62) were purchased from National collected by 5 min centrifugation at 1000
Cancer Institute, U.S.A and European rpm. The harvested cells were washed
collection of cell culture, UK. Cells were twice with phosphate buffer solution and
cultured in RPMI-1640 and MEM medi- fixed with 70 % ethanol at -20 C for 1h.
um supplemented with nutrients and anti- The cells were then stained with DNA
biotics. Cells were grown at 37 oC and 5% staining solution containing propidium
CO2 level with relative humidity of 98%. iodide (20 mg/ml) and triton X-100 (1%)
in PBS for 30 min in dark. FACScan was
2.10. Anticancer activity used to measure DNA content. For each
The anticancer activity of PDEE data file, data was collected from 10,000
against different human cancer cell lines cells. Cell Quest (Becton, USA) was used
was evaluated by SRB assay as described for analysis of histograms.
by Monks et al., 1991. Briefly 100l of
cell suspension (1x105 cells/well) were 2.12. Loss of Mitochondrial Membrane
cultured in 96-well plates and incubated Potential (m)
overnight at 37 0C and 5% CO2 level. 20 Flow cytometry was used to meas-
l test material at various final concentra- ure the mitochondrial membrane potential
tions (10-100 g/ml) was added. Paclitax- loss (m). Human pancreatic (MiaPaca-
el (1 M) and 5-fluorouracil (20 M) 2) cells were plated in 6-well cultural
6
were used as standard anticancer drugs. plates with cell density of 1x10
Cell growth was stopped after 48 hrs of cells/ml/well and incubated for 24 hrs at 5
incubation by adding 50 l of 50% TCA % CO2 level. The cells were then treated
in each well and incubated further for 1h with PDEE (0, 30, 50 and 100 mg/ml) and
at 4 0C. The plates were then washed, air again incubated for 48 hrs. Rhodamine-
dried and stained with 50 l of 0.4% SRB 123, a cell permeable cationic dye was
dye in 1% acetic acid, followed by incu- added one hour before termination of ex-
bation for 30 min at room temperature. periment and again incubated for 30 min.
The unbound dye was then removed by The cells were washed with PBS and pel-
washing with 1% acetic acid and kept lets were collected by centrifugation. The
overnight for drying. In each well, 100 l collected pellets were re-suspended in 300
of 10 mM tris-base was added to solubil- ml of PBS. Florescence of Rh-123 in cells
ise the dye followed by stirring for 5 min
were analysed at 485 nm by flow cytome- them to act as strong reducing agents, hy-
ter (Jung et al., 2006). drogen donors metal chelaters and singlet
oxygen quenchers (Miguel, 2010). The
2.13. Statistical analysis total flavonoid content in DPEE was
All of the experiments were done in found to be 7.06 1.31 (mg catechin/g
triplicate. The data were recorded as dry extract). Flavonoids are known exhib-
means standard deviations and were it many biological activities like antioxi-
analysed with SPSS software. dant, anticancer, antimicrobial and anti-
inflammatory properties (Hodek et al.,
3. Results and discussion 2002).
3.1. Total phenolic and flavonoid content 3.2. DPPH free radical scavenging activi-
Polyphenolic compounds are very ty
important plant bioactive constituents be- DPPH is a purple stable free radi-
cause of their scavenging potential due to cal at room temperature with characteris-
the presence of hydroxyl groups (Hatano tic absorbance at 517 nm. The nitrogen
et al., 1989). Folin-Ciocalteu method is free radical of DPPH is easily quenched
most widely used to measure the poly- by an antioxidant to yellow coloured
phenol contents, with the basic mecha- complex (1,1-diphenyl-2-picrylhydra-
nism of electron transfer and reducing zine). The decolourization of purple col-
ability (Prior and Schaich, 2005). Using our is stoichiometric depending on the
this quantitative assay, we found that the number of electrons gained (Soares et al.,
total phenolic content (TPC) of ethanolic 1997; Mokbel et al., 2006; Singh et al.,
leaf extract of P. denticulate was found 2002). DPPH radical scavenging potential
to be 12.24 2.11 (mg GAE/g dry ex- of PDEE at different concentrations in-
tract) as depicted in Figure 1. Considera- vestigated in the present study was deter-
ble attention has been received by poly- mined together with standard antioxidants
phenols for their physiological role as an- (BHT and -tocopherol) at the same con-
tioxidant and anticancer agents (Othman centrations (Figure 2). PDEE showed sig-
et al., 2007). Polyphenolic compounds h- nificant scavenging effect on DPPH free
Figure 3: Effect of PDEE and known antioxidant BHT and -tocopherol on hydroxyl radi-
cal scavenging potential. Values are means of triplicate experiments (n = 3) standard de-
viation.
Figure 4: PDEE Protects calf thymus DNA f oxidative damage. Lane 1: Native calf thymus
DNA; Lane 2: DNA + 20mM Ferric Nitrate + 100mM Ascorbic Acid + 30mM H2O2; Lane
3: DNA + 20mM Ferric Nitrate + 100mM Ascorbic Acid + 30mM H2O2 + 10g of extract;
Lane 4: DNA + 20mM Ferric Nitrate + 100mM Ascorbic Acid + 30mM H2O2 + 30g of
extract; Lane 5: DNA + 20mM Ferric Nitrate + 100mM Ascorbic Acid + 30mM H2O2 +
50g of extract; Lane 6: DNA + 20mM Ferric Nitrate + 100mM Ascorbic Acid + 30mM
H2O2 + 80g of extract; Lane 7: DNA + 20mM Ferric Nitrate + 100mM Ascorbic Acid +
30mM H2O2 + 100g of extract; Lane 8: DNA + 20mM Ferric Nitrate + 100mM Ascorbic
Acid + 30mM H2O2 + 10g of catechin.
ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 519
Biotech Sustainability (2017)
Free Radical Scavenging Potential and Anticancer Activity of Primula sp. Wani et al.
Figure 5: PDEE induces apoptosis of human pancreatic cancer (MiaPaca-2) cells via cell
cycle arrest. Flow cytometric analysis of MiaPaca-2 cells after propidium iodide staining.
Cells were incubated for 48 h in presence of PDEE (0, 30, 50 and 100 g/ml). Figures show
the representative staining profile of one of two similar experiments. P1 is the population of
apoptotic cells, which increases from 11.4% in case of control to 57.00% in case of 100
g/ml of PDEE.
Figure 6: PDEE induced loss of mitochondrial membrane potential (m) in human pan-
creatic cancer cell line (MiaPaca-2) incubated with extract at different concentrations (0, 30
50 and 100 g/ml) in 6 well plate for 48 h treatment.
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Othman, A., Ismail, A., Ghani, N.A., and Traditional Medicine in Afri-
Adenan, I. (2007). Antioxidant ca. 2nd Edn. Spectrum Books Lim-
capacity and phenolic content of ited, Ibadan, Nigeria 1-153.
cocoa beans. Food Chemistry. Sreejayan, N., Rao, M.N.A. (1996). Free
100,15231530. radical scavenging activity of cur-
Prior, R.L., Wu, X., Schaich, K. (2005). cuminoids. Arzneimittel forschung
Standardized methods for the de- 46, 169-171.
termination of antioxidant capaci- Vandana, S., Arvind, S.N., Kumar,
ty and phenolics in foods and die- J.K., Gupta, M.M., Suman,
tary supplements. Journal of Agri- P.S.K. (2005). Plant-based anti-
cultural and Food Chemistry. 53, cancer molecules: A chemical and
42904303. biological profile of some im-
portant leads. Bioorganic and Me- Yang, J., Liu, R.H., Halim, L. (2009).
dicinal Chemistry 13, 58925908. Antioxidant and antiproliferative
Vanicha, V., Kanyawim, K. (2006). Sul- activities of common edible nut
forhodamine B colorimetric assay seeds. LWT Food Science and
for cytotoxicity screening. Nature Technology 42, 18.
protocols 3, 1112-1116.
Figure 1: Global map of phosphorus fertilizer application rates (kg per ha of grid cell area)
[source: https://ourworldindata.org/fertilizer-and-pesticides/#note-6].
Figure 2: Global map of nitrogen fertilizer application rates (kg per ha of grid cell area)
[Source: https://ourworldindata.org/fertilizer-and-pesticides/#note-7].
These are mixed in proper ratio and then tender coconut, is believed to be a po-
allowed to ferment. Panchamrita is a tent organic pesticide and growth promot-
similar mixture that replaces dung and er - this is considered to
urine with honey and sugar. The mixture be pseudoscience.
which is made using yeast as a fermenter, The Sanskrit word Panchagavya m
bananas, groundnut cake, and the water of eans "mixture of five cow products". It is
ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 527
Biotech Sustainability (2017)
Use Organic Fertilizers for Sustainability Ramya and Karpagam
minated seeds were counted, the radical melongena was studied in sterile Petri
and plumule length were measured (cm) plates. Seeds of 40 numbers were soaked
and tabulated. in 2ml of water (Control), in 2ml of
Chemical control, (Cartap hydrochloride
2.5. Seedling length 50% radon sp + tata tafgor dimethoate
Two days after seed germination, 30% ec = 2:1 ratio and dissolved in 10
ten normal seedlings were taken out care- ml of water), and 2 ml of panchakavya;
fully at random from each treatment and 2ml of Panchakavya + neem cake (2
seedling length was measured from the tip gm), 2ml of vermicompost as treatment,
of primary root to the tip of apical shoot. separately and was observed for 5 days.
The average length (cm) of ten seedlings The germinated seeds were counted and
was calculated and expressed as mean the germination percentage was calculat-
seedling length. ed in each of the treatment (Table 1 and
2). The germination frequency was 100%
2.6. Seedling vigour index in panchakavya and the adjuvant treat-
The seedling vigour index was ment in A. esculentus while it was only
calculated adopting the method suggested 90% in S. melangena. The germination
by Abdul-Baki and Anderson (1973) and frequency was lesser in all the other
expressed whole number treatment wise treatments and it was only 50% in control
for S. melangena. Among the pan-
Vigour index = Germination percentage chakavya treated seeds, maximum germi-
Seedling length nation was found in both A. esculentus
and S. melongena.
2.7. Effect of panchakavya on cell divi- After germination the seedling
sion and cell growth vigour index was calculated by measur-
Cytological studies of root tip of ing the length of shoot and root. The data
Allium cepa was studied for 7 days. On- on shoot and root length of different
ion bulbs were treated with diluted pan- treatment were measured by cm scale.
chakavya (50%, 25%, 10% and 1%).and The plants treated with 2ml pan-
tap water as control. Allium cepa din chakavya produced the longest shoot
(2n=16) (Rank and Nielsen 1994) were legnth (2.3) and root length (2) in Abel-
used as test system. The root tip from mochus esculentus. While in Solanum
control and experimental setup were thor- melongena longest shoot (1.2), root
oughly washed in distilled water and length (1.5) was in panchakavya treat-
fixed in Carnoys fixative (ethanol, chlo- ment which was comparatively higher
roform glacial acetic acid 6:3:1 v/v/v) and than control. The seedling vigour index
chromosome studies were done by aceto- was higher in panchakavya treated seed-
carmine staining technique. The fixed lings in both the plants which was 430 for
root tip were washed in distilled water A. esculentus and 243 for S. melangena
and hydrolyzed in IN HCL for 10 min which was higher than control and all
then treated in 45% acetic acid for 5 min other treatments (Table 1 and 2).
and stained in acetocarmarine stain for Among the panchakavya and pan-
10-15 minutes. After staining the root tips chakavya + neem cake treated seeds,
were squashed and observed under phase maximum germination frequency was
contrast microscope. observed in Abelmoschus esculents and
Solanum melongena. The shoot length
3. Results and discussion was 2.5 and 1.2 in A. esculentus and S.
melangena and root length was 2 and
The effect of panchakavya on 1.1in A. esculentus and S. melangena.
seed germination of two plants namely There was a slight difference in 2ml of
Abelmoschus esculentus and Solanum panchakavya + neem cake. The pan-
ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 529
Biotech Sustainability (2017)
Use Organic Fertilizers for Sustainability Ramya and Karpagam
chakavya and panchakavya + neem cake, was measured in (cm) scale (Table 3 and
is more effective, when compared to con- 4).
trol as well as other treatments. The 100 The onion bulb developed more
% germination was observed in number of roots in 1% panchakavya
A.esculentus in panchakavya and pan- which was 48, and the root length was
chakavya + neem cake and while only 90 7.5, and the shoot length was 7 which
% germination was seen in Solanum was higher when compared to other con-
melongena (Table 1 and 2). centrations. At 1% concentration of pan-
The growth of onion roots was ob- chakavya the number of cells in the mi-
served on panchakavya at the concentra- croscopical field was 250 cells, of which
tions of, (50%, 25%, 10%, and 1%). The 48 of them was in metaphase stage.
highest number of onion roots, shoots Whereas, in control it was lesser, 22 cells
length and root length was observed in 1 in metaphase stage out of the 250 cells in
% concentration of panchakavya, and it the microscopical field. The 1% concen-
tration of panchakavya increased the
Table 1: Pesticide consumption (in lakh tones) during, 1994-1997 (Heisey and Norton,
2007)
Country/Region Herbicides Insecticides Fungicides
China NA NA NA
India 6.8 37.2 9.4
Other Asia 24.4 41 19.2
Middle East/North Africa 9.7 19.5 14.1
Sub-Saharan Africa 11.7 9.7 9
Latin America/Caribbean 85.8 39.8 31.8
All developing 138.4 147.2 83.6
Transitional 35.6 7.9 23.2
Industrialized 337.8 163.4 190.4
World 511.8 318.4 297.2
number of onion roots, when compared panchakavya, adjuvated with neem cake,
with control as well as other different and vermicompost.
concentration of panchakavya. The effect of adjuvants namely
The greatest challenge of the na- vermicompost and neem cake showed a
tion in the coming years is to provide safe slight difference, whereas the pan-
food for the growing population in the chakavya alone was sufficient to enhance
country without degrading the environ- the seed germination. The panchakavaya
ment. In this regard organic farming favors cell growth, cell division (Table 5)
which is a holistic production manage- which is known from the mitotic index.
ment system for promoting and enhancing The onion bulbs produced longer roots
health of agro ecosystem, has gained wide and shoots when compare to control. The
recognition as a valid alternative to con- panchakavya at 1% concentration fa-
ventional food production and ensure voured the production of larger number of
safer food supply for human consump- roots. The presence of growth promoting
tion. This farming system avoid large use substance present in the panchakavya fa-
of synthetic fertilizer, pesticides, growth vored rooting. The present study reveals
regulators and livestock feed additives that panchakavya preparation is easy and
and relies on green manures, crop rota- it is cost effective and could be prepared
tion, crop residues, animal manures, bio easily by a farmer with his household ex-
fertilizers, bio pesticides, different kinds penses and availability. Rahul kumar et
of cow based liquid organic manures such al. (2014) studied the effect of pan-
as panchakavya, sanjibani, kunapajala, chakavya fortified with Bauhinia plant
amrit pani etc. extract which showed a positive response
Many advanced countries mainly as an anthelminthic preparation.
depend upon the dairy products because
of their commercial, agricultural and nu- Table 5: Effect of Panchakavya on cell
tritive properties. The dairy industries division [onion root tips]
play a vital role in the development of the No. of cells No.
Concentration
country. When a new house or building or No in of
[%]
even a temple constructed in India, the Metaphase cells
first to enter the premises would be the 1 Control 22 250
cow because this is considered to be aus- 2 50 46 250
picious. Cows urine (Comiyam) is used 3 25 44 250
in almost all the Hindu rituals. The poten- 4 10 45 250
tial of using panchakavya as growth pro- 5 1 48 250
motor and biofertilizer is revealed in this
work. The present study revealed the Sangeetha and Thevanathan
germination frequency of Abelmoschus (2010) studied the potential of panchaga-
esculentes, and Solanum melongena vya as biofertilizer against certain pulses
grown under the different treatments by growing in soil amended with seaweed
namely, Control, Chemical + fertilizer, extract and panchagavya. Experimental
ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 531
Biotech Sustainability (2017)
Use Organic Fertilizers for Sustainability Ramya and Karpagam
seedling recorded higher rates of linear only enhances the plant yield and growth
growth of both shoots and roots as com- rate, it also reduces the insect invasion
pared to controls. These seedlings pro- and fungal attack. The results of the pre-
duced 264 to 390% more lateral roots sent study clearly show that panchakavya
than the control and maximum lateral root is an organic fertilizer and growth pro-
production was always observed in seed- moter. It also increases the soil fertility.
lings grown in soil amended with sea- It is very much essential to develop a
weed based panchagavya at low concen- strong workable and compatible package
trations of nutrient management through organic
In recent years the people have resources for various crops, based on sci-
recognized a number of commercial, me- entific facts, local conditions, and eco-
dicinal and agricultural values from the nomic viability for the sustainability.
various products of dairy forms. Tharun
et al., (1983) carried out extensive works Acknowledgement
in this aspects and the environmental
management in developing countries. The authors would like to thank
The number of new methods of Dr. S.Karpagam, Head of the department
recycling and controlling measures of or- of Botany, Queen Marys College for
ganic waste in urban and rural habits was providing the laboratory facilities.
proposed by (Furedy et al., 1989). The
current global scenario firmly emphasizes References
the need to adopt eco-friendly agricultural
practices for sustainable agriculture. Abdul-Barki, A.A, and Anderson, J.D,
Chemical input had an adverse impact on (1973). Vigour determination in soy-
the health-care of not only soil but also bean seed by multiple criteria. Crop
the beneficial soil microbial communities Science 13, 630 63.
and the crops. This eventually led to a Furedy,C, Bluemental,u.J, Strauss.M,
high demand for organic products. Farm- Carnicross, L. (1980). Model for the
ers all over the world realize the need to effect of different control measures
detoxify the land by switching over to in reducing health risks from waste
organic farming dispensing with chemical water. Sci.Tec., 21, 567 577.
fertilizers, pesticides, fungicides and Gomathi, R., Isaivani Indrakumar and
herbicides. In India, organic farming was S. Karpagam (2014). Larvicidal ac-
a well developed and systematized agri- tivity of Monstera adansonii plant
cultural practice during the past and this extracts against Culex quinequefacia-
ancient wisdom obtained through Indian tus. Journal of Pharmacognosy and
knowledge is the use of panchagavya in Phytochemistry, 3(3), 160-162.
agriculture for the health of soil, plants Gosavi D.D. and Jhon, P.S. (2012). Ef-
and humans. fect of panchakavya Ghritra on some
neurological parameters in albino
4. Conclusion rats. Asian journal of pharma ceutical
science clinical Research, 5, 154
Panchakavya is used in different 156.
forms. Such as foliar spray, soil applica- Gupta and Gopal (2001).
tion along with irrigation, seed treatment http:/www.epa.gov/oecaagct/ag101/c
or seedling treatment etc. For foliar spray roppesticidesuse
3% concentration is being used by organ- Natarajan, K. (2002). Panchakavya a
ic farmers. Panchakavya was an important manual. Other India press, Mapusa,
one that enhanced the biological efficien- Goa, India, pp, 33.
cy of crop and the quality of fruits and Nene,Y.L. (1999). Seed health ancient
vegetable production. Panchakavya not and medicinal history and its rele-
ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 532
Biotech Sustainability (2017)
Use Organic Fertilizers for Sustainability Ramya and Karpagam
Short Communication
Increasing Human Interference in Katarniaghat
Wildlife Sanctuary
Shiv Pratap Singh*
Department of Geography, Kisan P.G. College Bahraich, Affiliated to Dr. R.M.L. Awadh
University, Faizabad, Uttar Pradesh, India;*Correspondence: principalkpgc@yahoo.co.in;
Tel.: +919984146483
portant part of Himalayan Tarai - Bhabhar Tiger, Cheetal, Stages, Rhinocerous, Ele-
region. This wildlife sanctuary is included phant, Leopard, Boar, Padha etc. Under
in the Project Tiger in 2003 for the con- the endangered aquatic birds we find Lal-
servation of tigers, Saryu (Kaudiyala and sar, Surkhab, Nilsar, Gugral, Kaj kurchhia
Girwa) river Dalphins, massive Croco- and little Mew. Some other birds like
diles, Alligator and Turtles biodiversity. Peacock, Moorcock, Pheasant, Dhanesh,
The forest of the sanctuary area has Log log, Hairns, Submarine bird, Crane,
been classified into two major types: (1) Wilture, Hawak, Kite, Owl, Caprimulgid,
The Sal forest and (2) the miscellaneous Magpia, Woodpecker, Khanjan, Mynah
forest. This area is rich in biodiversity by bird, Crow, Nightangle, Satbahin are also
including vast grasslands, dense forests of observed.
Sal, Sakhu and Teak trees and Aquatic Endangered reptiles namely, Iguanas,
areas. Pythans, Black Cobras and Cobras are
Pedagogically, the study area is made found here. For conserving crocodiles and
up of the alluvial soil of the Saryu River alligator a Crocodile Project has been es-
and its tributaries flowing adjoining to it. tablished in the Katarniyaghat. Currently,
The study area has a tropical mon- this wild life division is being developed
soon type climate with three distinct sea- as a tourist place of Bahraich district.
sons i.e. summer (April to June) winter Tourists come here to see and watch wild
(November to February) and warm-rainy animals and birds natural beauty as well
(July to September). March and October from for-off places. The main tribe living
are considered as transition months be- here is Tharu which lives in the buffer
tween the seasons. The mean maximum zone of the forest area. They are able to
temperature ranges from 22 degree Celsi- earn their livelihood by working in agri-
us in January to 40 degree Celsius in May cultural or non-agricultural sectors.
and the mean minimum temperature
ranges from 8 degree Celsius in January 3. Katarniaghat wildlife sanctuary and
to 27 degree Celsius in June. The annual the concept of biosphere reserve
rainfall ranges from 36 to 142 cm in win-
ter, 34 to 662 cm in summer and 1294 to Under the concept of bio-sphere re-
1689 cm in warm-rainy seasons. serve the conservation and enhancement
It is a natural aesthesis to see roaring of endangered environmental condition is
Tigers, Leopard resting on tree, leaping implied. The entire zone is divided into
Cheetal, Padha, Stage, Kakad and Boars three zones. First zone is the specific zone
digging the forest land with their long related to the internal part; where human
snout. entries are restricted. Second zone is
To conserve the abundance of wild lives, known as transitional zone or mid-zone
U.P. government has divided it in 6 sub surrounded to the core zone; this zone is
divisions. Four sub divisions (Katarnia, related to research centers, tourist places
Nishangara, Murtiha, Bharthapur) are de- and utilization of fuels and non-living re-
clared as a core zone and remaining two sources. Third zone which is situated
(Motipur, Kakraha) are declared as a round the mid zone is related to the habi-
buffer zone. North-east railway line pass- tats, agriculture, tourist places and utiliza-
es through this division connecting the tion of non-living resources. But in the
Bichhia-Katarniya tourist place. This line Bio-sphere reserve (Katarniyaghat Wild-
is parallel to the roadway that divides the life Sanctuary), the core zone (Katarnia,
zone into two parts (Figure 1). Nishangara, Murtiha, Bharthapur ) is be-
There is greatest biodiversity under ing developed as Guest House (GH) and
the Katarniya zone. If we have a glimpse Tourist Centre (TC). Meanwhile, in the
on biodiversity of biosphere reserve then buffer zone, the activities of forestry and
we find endangered Ganga Dolphin, agriculture are growing for the earning of
Figure 1: A diagrammatic sketch (map) showing the Katarniaghat Wildlife Sanctuary loca-
tion in India. Katarniaghat Wildlife Sanctuary map shows the roads, railway lines and
guest houses located within the sanctuary.
lation on the Nepal side. There are some responsible for the fragmentation are; as
forest villages (erstwhile Taungya villag- follows:
es) and a Central State Seed Research Burgeoning human population
Farm near Girijapuri, situated inside the (and therefore the escalating pres-
wildlife sanctuary covering 38.42 sq Km sure for resources)
area that causes considerable disturbance Changed socio-economic scenario
to the wildlife, creating a break in the for- (e.g. changed lifestyle of the Guj-
est connectivity. jars and Khatta holders, therefore
Generally, we know that the har- overgrazing over cutting of fire-
nessing of timber and fuel is right and to wood timber and fodder species)
cut the forest for trade is illegal. It cannot Encroachment of forest lands ( by
be restricted without giving the employ- agriculture monoculture plantation
ment to the effected people and maintain- and other land use)
ing facultative measures. In the studied Infrastructure development (like
area, the most of the human activities are rail, road, hydroelectric and irriga-
done by rural people; because, the activi- tion projects)
ties of cattle rearing and forestry are done Various illegal/ legal (timber har-
directly. Along with these activities, ille- vesting, boulder mining in river
gal wood cutting and hunting is found in beds etc.)
abundant measure. In the buffer zone of approach of various departments
this sanctuary, the rural population and
Little/ no community participation
unplanned habitats are growing continu-
Obsolete policies less amenable to
ously. So the local people are overleaping
adapt to changes, and
the forest land and converting it into agri-
Lack of adequate scientific
cultural land by cutting the forest. In fact
knowledge and proper monitoring
is a challenge of present time for the sanc-
plants.
tuary. In addition to this, the fast running
The magnitude of the above stated prob-
vehicles and north-east railway line cause
lems leading to habitat fragmentation and
the accident of wild lives of the forest ar-
biological invading varies. There are dif-
ea. The ecological system is widely dis-
ferent responses from various stakehold-
turbed by the noise pollution and savage
ers. The government forest department,
pollution caused by the tourists. As the
local communities, and the main stake-
Sanctuary is situated at the border of Ne-
holders that largely manage and use the
pal; Nepalies from the border region are
natural resources of the landscape. There
involved in lot of illegal activities related
are some broad responses, which could be
to forestry; which is also disturbing to the
generalized across the sites for the bet-
wildlife and forest of the Sanctuary.
terment of the sanctuary.
5. Response to cope with the changing
6. Perspectives
theme (human activity)
This paper highlighted the human ac-
Though the management plant of the
tivities in which are disturbing wildlife
Protect Area Sanctuary (PAS) and work-
sanctuary. To protect the biosphere re-
ing plant of Reserve Forest Sanctuary
serve people should be educated to incul-
(RFS) have not been specifically oriented
cate the importance of conserving wildlife
to cope with the identified Global Change
for the sustainability. The human interfer-
Factors per se yet, Many initiatives are
ence in the buffer and core zone generat-
taken up to minimize the factors that con-
ed the danger to the bio-diversity. Forest
tribute to habitat fragmentation and infes-
department has recently started the plans
tation of invasive species. Major factors
to supply income to the local people but it
is well know that only forest department
cannot do this. Other institution will have McComb, C. Benda (2007). Book: Wild-
to take responsibility to make more life Habitat Management Concepts
chances to generate employments for the and Application in Forestry, Secand
people in the vicinity of the sanctuary. Edition, pp-162-168.
Jawahar Rojgar Yojna, Samekit Gramin Verma, S. (2010). The Eco-Friendly
Vikas Karyakram and MGNAREGA will Tharu Tribe: A Study in Socio- Cul-
be helpful in providing proper jobs to lo- tural Dynamics; Joural Of Asia Pa-
cals to establish the right situation to con- cific Studies 1, 177-187.
serve the wild lives. Task is challenging; Pandy,R.K. (IFS 2005-2008). Tiger Pro-
but, it is essential for the sustainable tection and Conservation in Indian
growth and development of the people Tarai: Project Work: New Delhi.
and the region. Praveer, R. (2001). Book: Tharu Tribe;
Bihar Hindi Granth Akadme, Patna.
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