PROJECT REPORT

PLANT CELL TISSUE AND ORGAN

CULTURE

Submitted in partial fulfillment of

B.Tech.Biotechnology

Semester VIII

Institute of Biomedical Education & Research

Mangalayatan University
Aligarh
2012

Submitted to: Submitted By:

Dr. Nishi Sharma Geetanjali Verma
Lecturer
Mangalayatan University, Aligarh

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 Acknowledgement

I would like to express my special thanks of gratitude to my guide Mrs.Seema Garg

for his direction, assistance, and guidance, who gave me the golden chance to create
this wonderful paper on the topic PLANT CELL TISSUE AND ORGAN CULTURE
and also helped me in doing a lot research and I came to know about so many new
things.

Without her encouragement and constant guidance, I could not have finished this
dissertation. She was always there to meet and talk about my ideas, to proofread and
mark up my paper.

I have left no stone unturned to make this paper informative, useful and at the same
time interesting by using pictures. I hope this paper of mine prove beneficial to all its
readers.

I would like to thanks my teachers for their valuable suggestions in finishing this
paper within the limited time.

I would also like to thanks my colleagues who have taught me techniques of writing
and presentation.

Very special thanks should be given to my parents and god who helped me in many
ways.

Geetanjali Verma

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 List of Abbreviations

A fatua L. Avena fatua L.

MS media Murashige and Skoog media
IAA 1H indole-3-acetic acid
NAA Naphthaleneactetic acid
IBA- 1H- indole-3-butyric acid
2,4-D 2,4-dichlorohenoxyacetic acid
NOA Naphthoxyacetic acid
BAP 6-benzylpurine
ABA Abscisic acid
MgSO4.7H2O Magnesium sulfate heptahydrate.
KH2PO4 Monopotassium phosphate

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 LIST OF FIGURES AND TABLES

S.No. Table/Figure Name Page No.

1 Different media used for plant tissue culture 6-7

2 Selection of explants 7

3 Sterilization of explants 7

4 Inoculation of explants in the Laminar Air Flow 8

5 Callus Culture 11

6 Figure to show types of the culture 11

7 Figures to show differentiation of callus into short 12

plantlets

8 Figure to show Rooting of Shoots 12

9 Figure to show Stages in somatic embryogenesis 14

10 Figure to show Direct somatic embryogenesis 15

11 Figure to show cycle of regeneration of plant 16

12 Figure to show Embryo Maturation and Plantlet 17

Development

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Content

I. Introduction 01

Principle of Tissue culture .1-2

Review of Literature...............................3-4

II. Materials and Method ......05

Nutrient Media ....5-10
Selection of Explants ...10
Sterilization of Explants ..11
Inoculation of Explants.11-12
Incubation of Cultures ..13

III.Regeneration of plants from cultured plant tissues ..13

IV.Plantlet formation through organogenesis 14

Micropopagation.15
Multiplication by Auxiliary and Apical Shoots .15
Multiplication by Adventitious Shoots.. ....15
Multiplication through Callus Culture16-17
Differentiation of callus into short plantlets ..17
Rooting of Shoots ..18
Greenhouse acclimatization ...18-19

V.Plantlet formation through somatic embryogenesis ..19

Types of somatic embryogenesis .....................21
Direct somatic embryogenesis...21
Advantages of somatic embryogenesis . ...24-25

VI. Clonal propagation ..25

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VII. Artificial Seeds .25-26

What are Artificial Seeds? .24-25

Advantages of Synthetic Seeds.. 25-26

VIII. Genetic Transformation .....28

IX. Somatic Hybridization ............28

Isolation of Protoplasts ..........29

Sources of Protoplasts ... ............29
Culture of Protoplast...............30

X. Somaclonal variation .31

Advantages of Somaclonal Variations.31-32

XI. Applications of tissue culture ...........32-36

XII. Reference ..36-39

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 1

INTRODUCTION
Development of the Science of tissue culture is historically linked to the discovery of the cell and
subsequent propounding of the cell theory. Henri-Louis Duhamel du Manceau (1756) first
demonstrated callus formation by his experiments on wound healing. Further, contributions to
plant tissue culture could be attributed to the cell doctrine, which implicitly admitted that a cell is
capable of autonomy and even demonstrated the potential for totipotency. Plant tissue culture is
a collection of techniques used to maintain or grow plant cells, tissues or organs under sterile
conditions on a nutrient culture medium of known composition. Plant tissue culture is widely used
to produce clones of a plant in a method.
Advantages over traditional methods of propagation, including:
a) The production of exact copies of plants that produce particularly good flowers, fruits, or
have other desirable traits.
b) To quickly produce mature plants.
c) The production of multiples of plants in the absence of seeds or necessary pollinators to
produce seeds.
d) The regeneration of whole plants from plant cells that have been genetically modified.
e) The production of plants in sterile containers that allows them to be moved with greatly
reduced chances of transmitting diseases, pests, and pathogens.
f) The production of plants from seeds that otherwise have very low chances of germinating
and growing, i.e.: orchids and nepenthes.
g) To clean particular plants of viral and other infections and to quickly multiply these plants
as 'cleaned stock' for horticultur and agriculture.

Plant tissue culture relies on the fact that many plant cells have the ability to regenerate a
whole plant (totipotency). Single cells, plant cells without cell walls (protoplasts), pieces of
leaves, or (less commonly) roots can often be used to generate a new plant on culture media given
the required nutrients and plant hormones.

Gottlieb Haberlandt made the first serious attempt to culture monocot cells in 1902 on an
artificial nutrient medium. The idea presented was called totipotentiality,
Means that plant cells are able to give rise to a complete plant.
With the help of plant tissue culture advanced knowledge can be gained in different fields like
fundamental Agriculture, Horticulture, Plant Breeding, Forestry, Somatic Cell Hybridization,
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Physiopathology and production of plant metabolites on commercial scale. In plant tissue culture
technique cells, tissues or organs of a plant are separated. These separated cells are grown
aseptically in containers with a nutrient media under controlled conditions of temperature and
light. The cultured plant material requires a source of energy from sugar, salts, a few vitamins,
amino acids, etc. that are provided in the nutrient media. Generally, these give rise to an
unorganized mass of cells called callus. From callus, an embryo or a shoot may develop, which
then grows into a whole new plantlet.

Principle
Plant tissue culture relies on the fact that many plant cells have the ability to regenerate a whole
plant, known as Totipotency. Usually all cells, pieces of leaves, stem or root possess this ability
and used for the in vitro germination of the plant. Plant tissue culture simply directs and assists
the natural potential within the plant to put forth new growth and to multiply in a highly efficient
and predictable way. Thus plant tissue culture works on two concepts of plasticity and
totipotency.

Totipotency
When plant cells and tissues are cultured in vitro they generally exhibit a very high degree of
plasticity, which allows one type of tissue or organ to be initiated from another type. In this way,
whole plants can be subsequently regenerated. This regeneration of whole organisms depends
upon the concept that all plant cells can, given the correct stimuli, express the total genetic
potential of the parent plant. This maintenance of genetic potential is called totipotency. Plant
cell culture and regeneration do, in fact, provide the most compelling evidence for totipotency. In
practical terms though, identifying the culture conditions and stimuli required to manifest this
totipotency can be extremely difficult and it is still a largely empirical process. Totipotent cells
serve the same role in plants that stem cells do in animals. They are found in shoot and root
growing tips as meristems, and in the cambium layer (the layer of cells between the bark and the
wood) of woody plants and trees. The activity of these meristematic regions gives rise to new
roots, stems, leaves, and flowers or fruiting structures in plants, as well as increasing the diameter
of woody plant trunks and branches. All of the structures found in a mature or growing plant are
the result of cellular material produced by meristematic tissue. Knowing this helps in
understanding how a cutting can root or how tissue culture labs can produce hundreds or
thousands of young plants using just the meristematic cells.

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Review of Literature

German botanist Gottlieb Haberlandt (1902) developed the concept of in vitro cell culture. He
cultured isolated, fully differentiated cells in a nutrient medium containing glucose, peptone and
Knops salt solution. Haberlandt is regarded as the father of tissue culture. Few years later, Hannig
(1904) initiated a new line of investigation involving the culture of embryogenic tissue, which
later became an important applied area of investigation, using in vitro techniques. He excised
nearly mature embryos of some crucifers and successfully grew them to maturity on mineral salts
and sugar solution.
H.W. Rines and T.J. McCoy (1981), worked on tissue culture and regenerated plants
from three hexaploid oat species- the cultivated oat, Avena sativa L., and two wild oats A. sterilis
L. and A. fatua L. They concluded that since genotype influences culture initiation frequency and
culture type, screening of genotypes and selection among segregating populations should be an
approach in the improvement of cell culture capabilities in oats. Thirty nine genotypes of winter
wheat (Triticum aestivum L.) were examined for potential use in tissue culture studies (R.G. Sears
and E. L. Deckard, 1982). Five essential oil components (Anethole, Carvacrol, Cinnamaldehyde,
Eugenol & Safrole) were tested for fungitoxic activity (D.P. Thompson, 1989) against different
species of Rhizopus like R. arrhizus, R. chinensis, R. circinans, R. japonicus,. Virendra K Gautam,
Kanan Nanda & Shrish C. Gupta (1993) worked on Neem and found that callus originated in
microsporangial wall layers and connective tissues of anthers containing uninucleate microspores
on Nitschs or Murashige & Skoogs medium supplemented with growth regulators. Elizabeth E.
Hansen, John F. Hubstenberger and Gregory C.
Phillips (1995) initiated cell suspension from callus obtained from pre-selected
regenerating lines of Allium cepa and A. fistulosum. A modified culture protocol has been
developed by Wei Wen Su, Wen-Ing Hwang, Se Young Kim & Yoneo Sagawa (1997) for the
induction of somatic embryogenesis in Azadirachta indica (Neem). C. C. Eady, R. C. Butler & Y.
Suo (1998) obtained somatic embryos and regenerated plants from immature embryos of onion
following culture on embryogenic induction media. In vitro micropropagation of holy basil
(Ocimum sanctum L.), an Indian medicinal herb, has been accomplished on Murashige and Skoog
(MS) medium utilizing young inflorescence explants. Rakhi Chaturvedi , M. K. Razdan and S. S.
Bhojwani (2004) achieved in vitro clonal propagation of a 50-year-old neem tree through axillary
shoot proliferation. Mutasim M. Khalafalla, sheikh M. Seiki Sato, Norio Katoh, Sumio Iwai and
Manabu Hagimori (2005), investigated the proportion of haploids, diploids and polyploids in B.

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rapa and B. oleracea plants regenerated in anther culture and microspore culture..P Dharmani &
Gautam Patil (2006) reported some important plants of antiulcer and ulcer healing properties.
Neelofar Jabeen, A.S. Shawl et al (2007), developed a rapid in vitro regeneration protocol
induction of multiple shoots from nodal segments of Inula racemosa Hook.f. was developed. Leaf
and nodal segments were inoculated on MS medium containing different concentrations of
Benzylaminopurine (BAP) either alone or in combination with Naphthalene Acetic Acid
M.Viuda-Martos et al, 2007, worked on the antifungal activities of thyme, clove and oregano
essential oils. The essential oils of oregano (Origanum vulgare) , thyme (Thymus vulgaris) and
clove (Syzyjium aromaticum) presented inhibitory effect on A. niger and A. flavus. Ali R. Alan,
Wansang Lim, Martha A. Mutschler, Elizabeth D. Earle (2007) investigated three complementary
strategies for increasing the frequency of DH onion plants obtained via gynogenesis.

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Materials and Method

Whole plant can be regenerated virtually from any plant part or cells. Plant tissue culture
techniques involve following steps:
Preparation and selection of suitable nutrient media.
Selection of explants such as root tip, auxiliary buds, anthers etc.
Surface sterilization of the explants by disinfectants followed by washing with sterile
distilled water.
Inoculation of explants onto the suitable sterile nutrient medium under aseptic conditions.
Incubation or growing the cultures in the growth chamber or plant tissue culture room at
optimum physical conditions of light, diurenal illumination, temperature and relative
humidity.
Regeneration of plants from cultured plant tissues.
Hardening: It is the process of gradual exposure of plantlets for acclimatization to
environment conditions.
Transfer of plants to the field conditions.

Nutrient Media
Growth and morphogenesis of plant tissues in vitro are largely governed by the composition of
the culture media. Various media compositions are used for tissue culture. No single medium can
support the growth of all tissues.
Functions of medium-
Provide water
Provide mineral nutritional needs
Provide vitamins
Provide growth regulators
Access to atmosphere for gas exchange
Removal of plant metabolite waste
Factors that affect the media-
Temperature -Practically it is impossible to optimize temperature for each specific plant and
each specific stage. For most plants the set point for room temperature during the day is 22 2C.
1. Night temperature is usually a few degrees lower.
2. Temperature in the culture container is higher (up to 5C) than room temperature due to the
greenhouse effect.

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3. One of the major problems is to have a uniform temperature in the culture room. There are
gradients.
4. Safety device: a maximum thermostat needs to be installed, which switches off the lights in
case of too high temperature.

Humidity
1. Relative humidity (RH) is usually not controlled in the culture room
2. In a standard container the headspace is saturated with water vapour
3. RH in the container can be controlled by different devices: ventilation, bottom cooling, agar
concentration.

Media Composition
The principal components of most plant tissue culture media are inorganic nutrients
(macronutrients and micronutrients), carbon source, organic supplements, growth regulators
and a gelling agent. Some tissues can grow on simple media containing only inorganic salts and
utilizable carbon (sugar) source, but for most of the other plants supplementation of medium with
vitamins, amino acids and growth substances is necessary.
a) Vitamins: Plants synthesize vitamins endogenously and these are used as catalysts in various
metabolic processes. While in vitro growth also plants produce some vitamins but only in
suboptimal quantities. Hence the supplementation of the medium with required vitamins and
amino acids is necessary. Therefore, to achieve the best growth of the tissues; use of required
vitamins and amino acids is essential. Thiamine (B 1), Nicotinic Acid (B3), Pyridoxine (B6),
Calcium pentothenate (B5) and Myoinositol are the most commonly used vitamins. Thiamine is
the basic vitamin which is required by all the cells and tissues.

b) Other Organic Supplements: Other organic extracts are also added in the medium. These
include Casein Hydrolysates, Coconut milk, yeast and Malt Extracts, ground banana, orange juice
and tomato juice etc. Casein hydrolysates and coconut milk are most commonly used and gave
given significant results.

c) Activated Charcoal: Activated charcoal shows inhibitory as well as stimulating effect on the
cells and tissues. It also helps to reduce toxicity by removing toxic compounds (Phenols),
produced during the culture and permits unhindered cell growth.

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d) Antibiotics: The various fungicides and bactericides are added in case plant explants on
cultures are excessively contaminated. The antibiotics are soluble in water. Streptomycin or
Kanamycin at low concentration effectively controls systemic infection and media supplemented
with these antibiotics do not adversely inhibit the growth of cell cultures.

1. Growth regulators: The four classes of growth regulators are commonly used in tissue
culture media i. e. Auxins, cytokinins, gibberellins and abscisic acid. The type of growth
regulators and concentration used will vary according to the cell culture purpose. The
growth, differentiation and organogenesis of tissues become feasible only on the addition of
one or more of these classes of hormones to a medium.

a)Auxins: Various auxins are added in the media like 1H indole-3-acetic acid (IAA), 1-
naphthaleneactetic acid (NAA), 1H- indole-3-butyric acid (IBA), 2,4-dichlorohenoxyacetic acid
(2,4-D) and naphthoxyacetic acid (NOA). IAA occurs naturally in the plant tissues. Auxins induce
cell division and root initiation in cultured tissues.
b) Cytokinins: Cytokinins are adenine derivatives which are mainly concerned with cell division,
modification of apical dominance and shoot differentiation in tissue culture. The most frequently
used cytokinins are 6-benzylpurine (BAP) or 6-benzyladenine (BA), 6---dimethylaminopurine
(2ip)2, N- (2-furfurylamino)-1-H-purine-6-amine (kinetin) and 6-(4-hydroxy-3-methyltrans-2-
butanylamino) purine (zeatin). Zeatin and 2-iP are naturally occurring cytokinins while BA and
kinetin are synthetically derived cytokinins.
c) Gibberellins: The gibberellins are infrequently used in plant tissue cultures as it can inhibit
callus growth. GA3 is most commonly used.
Functions of Gibberellins-
1.Stimulate stem elongation by stimulating cell division and elongation.
Breaks seed dormancy in some plants which require stratification or light to induce
germination.
Stimulates enzyme production (a-amylase) in germinating cereal grains for
mobilization of seed reserves.
Induces maleness in dioecious flowers (sex expression).
Can cause parthenocarpic (seedless) fruit development.
Can delay senescence in leaves and citrus fruits.

d) Abscisic acid (ABA) in the culture medium either stimulates or inhibits the callus

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growth depending on the species. The ABA is useful in embryo culture and somatic
embryogenesis. ABA is heat stable but light sensitive.
Functions of Abscisic acid
Stimulates the closure of stomata (water stress brings about an increase in ABA synthesis).
Inhibits shoot growth but will not have as much affect on roots or may even promote growth
of roots.
Induces seeds to synthesize storage proteins.
Inhibits the affect of gibberellins on stimulating de novo synthesis of a-amylase.
Has some effect on induction and maintanance of dormancy.
Induces gene transcription especially for proteinase inhibitors in response to wounding which
may explain an apparent role in pathogen defense.

v) Solidifying agents: Gelling agents or solidifying agents are used while preparing semi-solid or
solid tissue culture media. Normally 0.5 to 1% agar is used in the medium to form a firm gel at
the pH typical of plant cell culture media.Gelatin is used at high concentrations (10%) but its use
is limited as it melts at lower temperature (25C). Other compounds successfully tested include
metacel, alginate, phytagel and gelrite.

vi) pH: Optimum pH is required for the growth of plant cells and tissues in cultures. The pH
affects the uptake of ions and for most of the culture media pH 5.0 to 6.0 before sterilization is
considered optimal. Higher pH is likely to give a hard medium while a low pH results in
unsatisfactory solidification of the agar.

Media Preparation
Nowadays the plant tissue culture media which are most commonly used are available in the market
as dry powders. These powders are dissolved in certain quantity in the distilled water. After the
contents have been thoroughly mixed in water, sugar, agar (melted) and other organic supplements
are added. Finally the volume is made up to 1 litre. The pH is adjusted and the medium is autoclaved.
The experiments in which changes in the quantity and quality of media constituents become
necessary, it is desirable to weigh and dissolve each ingredient separately before mixing them
together. Another convenient method is to prepare stock solutions which when mixed together in
appropriate quantities, constitute a basal medium. Four stock solutions are prepared i) Major Salts
(20X concentrated), ii) Minor Salts (1000X concentrated), iii) Iron (100X concentrated) and iv)
Organic Nutrients except Sucrose (100X concentrated). All the stock solutions are stored in proper

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plastic or glass containers at low temperatures. Iron stock should be stored in coloured (amber) bottles.
Containers should be shaked before us and any contaminant or suspension in the form of precipitate
must be discarded. Extra care is required in storing coconut milk. The liquid extract (endosperm) from
the fruit is boiled to deproteinise it, filtered and stored in plastic bottles in a deep freeze at -20C.

Nutritional components of some plant tissue culture media are as follows:

Table: 2 Different media used for plant tissue culture
Amounts (mg/l)
Components
Whites MS B5 Nitschs N6 E1
Macronutrients
MgSO4.7H2O 750 370 250 185 185 400
KH2PO4 - 170 - 68 400 250
NaH2PO4.2H2O 19 - 150 - - -
KNO3 80 1900 2500 950 2830 2100
NH4NO3 - 1650 - 720 - 600
CaCl2.2H2O - 440 150 - 166 450
(NH4)2.SO4 - - 134 - 463 -
Micronutrients
H3BO3 1.5 6.2 3 - 1.6 3
MnSO4.4H2O 5 22.3 - 25 4.4 -
MnSO4.H2O - - 10 - 3.3 10
ZnSO4.7H2O 3 8.6 2 10 1.5 2
Na2MoO4 - 0.25 0.25 0.25 - 0.25
CuSO4.5H2O 0.01 .025 .025 .025 - .025
CoCl2.6H2O - .025 .025 .025 - .025
KI 0.75 0.83 0.75 - 0.8 0.8
FeSO4.7H2O - 27.8 - 27.8 27.8 -
Na2EDTA.2H2O - 37.3 - 37.3 37.3 -
EDTA Na Ferric
- - 43 - - 43
salt
Sucrose (g) 20 30 20 20 50 25
Organic
supplements
Vitamins
Thiamine HCl 0.01 0.5 10 0.5 1 10
Pyridoxine HCl 0.01 0.5 1 0.5 0.5 1
Nicotinic Acid 0.05 0.5 1 5 0.5 1
Myoinositol - 100 100 100 - 250
Others
Glycine 3 2 - 2 - -
Folic Acid - - - 0.5 - -
Biotin - - - 0.05 - -
Ph 5.8 5.8 5.5 5.8 5.8 5.5

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Selection of Explant
The tissue or plant part removed from the plan t body for the experimental purpose is called
explant and the plant from which it removed is known as Stock Plant. The age of stock plant and
location on the stem from which the explants are removed can greatly effect the establishment of
callus tissue in vitro. It is assumed that whole plant can be developed from any cell of a plant.
But, in many species explants of various organs vary in their rates of growth and regeneration,
while some do not grow at all. The most commonly used tissue explants are the meristematic ends
of the plants like the stem tip, auxiliary bud tip and root tip. These tissues have high rates of cell
division and either concentrate or produce required growth regulating substances including auxins
and cytokinins. The choice of explant material also determines if the plantlets developed via
tissue culture are haploid or diploid.
An alternative for obtaining uncontaminated explants is to take explants from seedlings which are
aseptically grown from surface-sterilized seeds.

Selection of explant Sterilization of explant

Sterilization of Explant
Al the cultures will become contaminated if explants used for the culture are not obtained from
properly disinfected plant material. Its not an easy process because during sterilization living
materials should not lose their biological activity; only Bacterial and fungal contaminants should
be eliminated. Plant organs or tissues are therefore, only surface-sterilized by treatment with a
disinfectant solution at suitable concentrations for a specific period. Hard expalnts are directly
treated with disinfectants. Various sterilizing agents are used for this purpose like Mercuric

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Chloride, Chlorine water, Sodium Hypochlorite, Ethanol etc. Ethyl alcohol is used to surface
sterilize delicate tissues such as shoot apices, pollen grains and shoot or flower buds. Such
explants are given a rinse in 70% ethanol for a few seconds and then washed 3-4 times in sterile
distilled water under aseptic conditions. Mercuric chloride is used for the sterilization of hard
materials like seed.

Inoculation of Explant
Successful control of contamination largely depends upon the technique used for the transfer of
sterile explants to the sterile culture media under aseptic conditions. Dust, hair, hands and clothes
are the potential source of contamination during this process. Therefore, to control the
contamination hands are washed with 95% ethanol before inoculation of the expalnt. Talking or
sneezing while the culture material is being inoculated or transferred from one container to the
other should be avoided. Cultured tissue should never touch inside the edges of culture vessels.
Precautions which should be taken to check contamination while inoculation of explants are as
follows:
UV light should be switched on 15 min before making transfers.
Working surface of the Laminar should be cleaned with 95% ethanol.
Only sterile instruments, appliances and culture tubes and vessels should be placed inside
the laminar.
After work, the surface of the laminar should be rewiped with ethanol.
Pieces of plant material should be collected in a suitable container and immersed in a
solution of the disinfectant at suitable concentrations.
This should be followed by sterilization with appropriate sterilizing agent and sterile
distilled water to remove it.

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Inoculation of explants in the Laminar Air Flow

After washing, the surface sterilized plant material is transferred to presterilized petri-
dishes for the cutting of the explants.
The cap of the Culture tubes should be removed and inoculums should be transferred into
the tube with the help of forceps.
All operations should be carried out in laminar air flow.

Incubation of Cultures
Proper incubation of culture tubes is necessary for the in vitro growth of explants. Appropriate
temperature, light, relative humidity etc. are the important factors that affect the growth of the
plant cells and tissues. During culture initiation and shoot multiplication phases, the cultures are
generally kept at a constant temperature of 25C and are illuminated with about 1000 lux white
light from fluorescent tubes. In some cases, a high light intensity, e.g., 3000 lux may have
detrimental effects, but during rooting, higher light intensities, e.g., 3,000-10,000 lux are
commonly used since it has a beneficial effect on rooting and plant survival on transfer to soil.

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In vitro culture of plants in a controlled, sterile environment

Regeneration of plants from cultured plant tissues

Under favourable conditions plants are regenerated by using different technique of plant tissue
culture. These techniques include somatic embryogenesis, single cell culture, protoplast culture,
root culture, shoot culture, organogenesis etc. This can be carried out by three ways. These are as
follows:
1. Plantlet formation through organogenesis
2. Plantlet formation through somatic embryogenesis
3. Somatic hybridization

Plantlet formation through Organogenesis or Micropropagation

In nature plants may reproduce sexually or asexually. Sexually propagated plants show a high
degree of heterogeneity since their seed progeny are not true-to-type unless they have been
derived from inbred lines. Tissue culture methods also offer an alternative means of plant
vegetative propagation. Through tissue culture or micropropagation clones can be achieved in a
shorter time and minimal space. Thus, it is possible to produce a large number of plants starting
from a single individual and maintain exactly same genetic characteristics of the plant.
Techniques

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In vitro clonal propagation is a complicated process which is carried out in various steps.
Murashige (1978) proposed four distinct stages for micropropagation of plants. Stages I-III are
carried out under in vitro conditions while stage IV is accomplished in greenhouse environment.
These are as follows-

Stage 0Selection and maintenance of stock plants for culture initiation

Stage I Initiation and establishment of aseptic culture (Main steps: Explant isolation,
surface sterilization, washing and establishment on appropriate culture medium)

Stage II Multiplication of shoots or rapid somatic embryo formation using a defined culture
medium

Stage III Germination of somatic embryos and/or rooting of regenerated shoots in vitro

Stage IV Transfer of plantlets to sterilized soil for hardening under greenhouse environment
(in a few cases this stage may also include the in vivo rooting of regenerated shoots by skipping
Stage III)

Micropropagation

1.Multiplication by Axillary and Apical Shoots

Axillary and apical shoots contain quiescent or active meristem depending on the physiological
state of the plant. Apical shoots are cultured on media supplemented with the mixture of auxin

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(0.01-0.1 mg/l) and cytokinin (0.05-0.5 mg/l). The level of cytokinin is raised subsequently to
induce an acceptable rate or proliferation without yellowing or distortion of shoots. If the
presence of cytokinin in the media inhibits root development, cultured material is often
transferred in Stage III to a rooting medium which contains either no or reduced levels of
cytokinin.

2.Multiplication by Adventitious Shoots

Adventitious shoots are stem and leaf structures that arise naturally on plant tissues located in
sites other than at the normal leaf axil regions. These structures include stems, bulbs, corms,
tubers and rhizomes etc. These can be used for the micropropagation. Bulbs and corms grow from
meristem at the base of leaves and scales

3.Multiplication through Callus Culture

Explants, when cultured on the appropriate medium, usually with both an auxin and a cytokinin,
can give rise to an unorganised, growing and dividing mass of cells which is known as Callus.

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Callus cultures are extremely important in plant biotechnology. Callus cultures can also be used to
initiate cell suspensions, which are used in a variety of ways in plant transformation studies.
Manipulation of the auxin to cytokinin ratio in the medium can lead to the development of shoots,
roots or somatic embryos from which whole plants can subsequently be produced. This is known
as Organogenesis

Differentiation of callus into short plantlets

The callus is a mass of highly vacuolated, unorganized cells resulting as a consequence of
wounding in plants, and in tissue culture with the use of sophisticated techniques.

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Figures to show differentiation of callus into short plantlets

When an explant from differentiated tissue is used for culture on a nutrient medium, the non-
dividing, quiescent cells first undergo certain changes to achieve a meristematic state. The
Phenomenon of the reversion of mature cells to the meristematic state leading to the formation of
callus is called dedifferentiation. The component cells of callus have the ability to form a whole
plant, a phenomenon described as redifferentiation. These two phenomenons of dedifferentiation
and redifferentiation are inherent in the capacity described as cellular totipotency, a property
found only in plant cells.Callus cultures need to be subcultured every 3-5 weeks due to cell
growth, nutrient depletion and medium drying. Calluses are easy to maintain and most widely
used for in vitro micropropagation. Higher concentrations (>2mg/l BAP) of cytokinin induce
adventetitious buds and retards shoot growth.

Rooting of Shoots

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Figure to show Rooting of Shoots

Usually, the rooting medium has low salt, e.g., 1/2 or even 1/4 salts of the MS medium, and
reduced sugar levels (usually 1 g/l). Reduced salts being essential for rooting in some species like
Narcissus. In some species, e.g., Narcissus, strawberry, etc., rooting occurs on basalmedium. But
in most species 0.1 -1 mg/I NAA or IBA is required for rooting. In plants like Citrus, however, a
pulse treatment with an auxin (10 min with 100 mg/I NAA or IBA) gives optimum rooting. Shoots
are usually rooted in an agar medium, but the recent trend is to root them directly in vermiculite or
potting mix. The cut ends of shoots are treated with a suitable auxin solution or powder mix,
transplanted in pots and kept under high relative humidity and low light intensity. This saves cost
as rooting and soil transfer stages are combined and the rooting medium is eliminated. Rooting
takes about 10-15 days, depending mainly on species. Plantlets with 0.5 to 1 cm roots are usually
transplanted into pots since longer roots tend to get damaged
Greenhouse acclimatization
Microcuttings without roots or in vitro rooted plantlets are both planted in any of several well-
drained soilless potting mixes in the greenhouse under high humidity. Various types of fogs, tents,
domes and mist systems are used to wean the tender shoots into the real by gradually reducing the
humidity. The time requirement for weaning process is about 3 days to 6 weeks. It depends on the
type of plants. Fertilization is not recommended until the plantlets become established and begin
to grow.

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Plantlet formation through somatic embryogenesis

Somatic embryogenesis is the process of a single cell or a group of cells initiating the
developmental pathway that leads to reproducible regeneration of non-zygotic embryos capable of
germinating to form complete plants. Under natural conditions this pathway is not normally
followed, but from tissue cultures somatic embryogenesis occur most frequently and as an
alternative to organogenesis for regeneration of whole plants. Adherence to this pattern of
morphogenesis depends on co-ordinated behaviour of a cell or cells t establish polarity as a unit
and thereby initiate gene action sequentially specific to emerging tissue regions.
Somatic Embryogenesis was first demonstrated by Steward et al. in 1958 and Reinert in 1959 in
carrot suspension and agar cultures. Sharp in 1980 showed that it may be initiated directly from
explant or indirectly from callus.
Somatic embryos are also called embryoids or nonzygotic embryos.
Auxins are known to induce somatic embryo formation.
Somatic embryos may develop from single cells or from a small group of cells.
Repeated cell divisions lead to the production of group of cells that develop into an
organized structure called globular-stage embryo(formed after 16-cell stage).
Further development results in heart- and torpedo-stage embryos.
Polarity is established in early embryo development.
Signs of tissue differentiation become apparent at globular stage and apical meristems are
apparent in heart-stage embryos.
According to Kohlenbach (1978) embryos are of following types:

Non-zygotic embryos
These are formed by the cells other than the zygote. These are of following types:

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i)Somatic embryos:
These are formed by sporophytic cells (except zygote) either in vitro or in vivo. Somatic embryos
which arise directly from other embryos (stem embryos in carrot and buttercup) are known as
Adventive Embryos.

Figure to show Torpedo stage somatic embryo

ii)Parthanogenetic embryos: These are formed by unfertilized eggs.

iii)Androgenic embryos: These embryos are formed by the male gametophyte (microspore
pollen grains).
Types of somatic embryogenesis:
It is of two types which are as follows-

A. Direct Somatic Embryogenesis

B. Indirect Somatic Embryogenesis

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Figure to show Stages in somatic embryogenesis

A.Direct Somatic Embryogenesis :
In direct somatic embryogenesis, the embryo is formed directly from a cell or small group of cells
without the production of an intervening callus. Embryo is formed directly from a cell or small
group of cells without production of an intervening callus.
e.g. young trifoliate leaves of alfalfa (Medicago falcata) are used as explant and then are
removed from plant and chopped into pieces.
The pieces are washed in plant growth regulator-free medium The pieces are placed in
liquid medium (B5) supplemented with 2,4-D, kinetin, adenine and glutathione.
Cultures are maintained in agitated liquid medium for about 10-15 days.
Washing the explants and replacing the old medium with B5 medium supplemented with
maltose and polyethylene glycol results in development of somatic embryos.
These somatic embryos can be matured on solid medium containing abscisic acid.
Usually rare than the other

Figure to show Direct somatic embryogenesis

B. Indirect Somatic Embryogenesis

In indirect somatic embryogenesis, callus is first produced from the explant. Embryos can then be
produced from the callus tissue or from a cell suspension produced from that callus. Somatic
embryogenesis from carrot is the classical example of indirect somatic embryogenesis
a) This callus can be used to produce a cell suspension by placing it in agitated liquid MS
medium containing 2,4-D.

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b) This cell suspension can be maintained by repeated subculturing into 2,4-D containing
medium.
c) Removal of the old 2,4-D containing medium and replacement with fresh medium
containing abscisic acid results in production of embryos.
d) Usually common than others.

Rapid and high efficiency regeneration system (organogenesis and somatic embryogenesis) from
isolated shoot apices in vitro. (A) One day old isolated shoot apices with primordial leaves LP =
leaf primordia; SM = shoot meristem. (B) One- week old explant showing bulged meristem
portion and expanded primordial leaves. (C) Three- week old meristematic mass showing
multiple buds and leaf initials. (D) Individual buds producing 2-8 translucent tissue strata. (E)
Each of the tissue stratum giving rise to many somatic embryos. (F) Meristematic clumps
showing differentiating buds. (G) Germinating somatic embryos showing shoot apex (SA)
surrounded by a pair of primary leaves (PL). (H) Differentiation of somatic embryos into
platelets. (I) Plantlets with well formed roots in magenta box. (J) Acclimatization of plantlets in
the growth chamber. (K) Regenerated plants in greenhouse.

Embryo Maturation and Plantlet Development

Somatic embryos germinate only on maturation; when its root and shoot apices become capable
of meristematic growth. High auxin levels can inhibit development and growth of the shoot
meristem and often embryos mature when the embryogenic cultures are transferred to the medium

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lacking auxin. Progressive increase in sucrose level is also used to achieve maturation. The
maturation of somatic embryos proceeds more normally in complete darkness although light
seems essential for somatic embryogenesis in some cultures like tomato, egg plant. Somatic
embryos germinate on agar medium in the absence of any growth regulator.

Figure to show Embryo Maturation and Plantlet Developmen

.
Somatic embryogenesis and plant regeneration of cotton (Gossypium hirsutum L.) variety Simian-
3. A, nonembryogenic callus; B, embryogenic callus; C, somatic embryos at
variousdevelopmental stages; D, germination of somatic embryos and plant regeneration; E,
regenerative plant; F, regenerated plant after transferred to soil .

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Advantages of Somatic embryogenesis:

a) Somatic embryogenesis is an ideal technique for clonal propagation of woody and fruit
plants and genetic gain can now be captured through it.
b) ability to produce large numbers of plants
c) potential for automation
d) the opportunities for synthetic seed
e) long-term storage (cryopreservation)
f) packaging
g) direct delivery systems
h) genetic manipulations.
i) Somatic embryogenesis is highly genotypic dependent

Clonal propagation
Somatic embryogenesis can be used for clonal propagation. As somatic embryogenesis is usually
related to suspension cultures, it is possible to combine to combine somatic embryogenesis with
engineering technology to create large-scale mechanized or automated culture systems. Such
systems can produce propagules i.e. somatic embryos, repetitively with low labour inputs. The
process of repetitive somatic embryogenesis which is also known as Accessory, Adventive or
Secondary Embryogenesis; initiates a cycle in which somatic embryos proliferate from the
previously existing somatic embryo in order to produce clones.

Figure to show Clonal propogation

Artificial Seed

What are Artificial Seeds?

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A seed is a living embryo with a hard coat, which on germination gives rise to a plant utilizing
stored nutrients from its endosperm.
Artificial seeds are synthetic seeds which are developed from callus tissues to form somatic
embryos.
They may be derived from single cells from any part of plant that later develop to form cell mass
containing dense cytoplasm, large nucleus, starch grains, proteins, oils etc.
They are encapsulated by a cuticle or induced to gain a protective covering of chemicals and lose
connection with the surrounding cells.
They are, in fact, somatic cells having a full potential to develop into embryoids and then into
complete plantlets.
Viable embryos are called somatic or synthetic seeds.
Are tissue culture derived somatic embryos encased in a protective coating.Somatic embryos are
bipolar structures with both apical and basal meristematic regions capable of forming shoot and
root respectively. Somatic embryos are structurally similar to zygotic embryos but they are
derived from somatic cells and thus they can be used to produce duplicate copies (clones) of a
single genotype. High quality somatic embryos are produced and encapsulated in a sodium
alginate hydrogel with essential nutrients and hormones.Alginate is a stright chain, hydrophilic,
colloidal polyuronic acid composed of hydro-beta-D-mannuronic acid residues with 1-4 linkage.
The sodium alginate droplets containing the somatic embryos and nutrients when dropped into
CaCl2.2H2O solution form round and firm beads due to ion exchange between Na and Ca.

These artificial seeds can be utilized for the rapid and mass propagation of desired plant species as
well as hybrid varieties. The major benefits of synthetic seeds are:
a) They can be stored up to a year with out loss of viability
b) Easy to handle and useful as units of delivery

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c) Can be directly sown in the soil like natural seeds and do not need
acclimatization in green house.
A second coat covering the artificial endosperm is required to stimulate the seed coat.
The encapsulation is necessary to protect artificial seeds from microbial contamination and
desiccation.
In vitro embryoids develop from callus tissue in a suitable medium which is supplied with
growth promoting substances to initiate somatic embryogenesis.
Somatic embryoids develop and isolated for transfer to alginate solution for encapsulation
to take place.
Most effective encapsulation medium consists of sodium alginate (2%) and gelatin (5%),
complexed with calcium chloride or ammonium chloride (100um).
The encapsulated embryoids are then dropped into calcium nitrate solution for about 20
minutes, rinsed with distilled water, and stored in air tight containers.
The capsules or artificial seeds are collected by decanting off the complexation solution and
rinsed in water. The artificial seeds should be pliable enough to cushion and protect the embryo,
yet allow germination and growth of the shoot bud or somatic embryo. It should be rigid enough
to withstand rough handling during manufacture, transportation and planting. For the artificial
seeds to remain dormant until planting, a thin layer of water-soluble resin is used to coat the
encapsulation matrix.

Advantages of Synthetic seeds over true seeds

Production of artificial seeds is especially useful for plants which do not produce viable
seeds.

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Millions of artificial seeds can be produced at any time at low cost.

They provide an easy method to produce genetically engineered plants with desirable traits.
It is easy to test the genotypes of plants.
Artificial seeds produce identical plants.
The period of dormancy of artificial seeds is greatly reduced, hence growth is faster with a
shortened life cycle.

Genetic Transformation
In seed embryogenesis, zygotic embryos are seated deep inside the nucellar tissue. They live in a
protected environment besides being genetically heterogenous. On the other hand, somatic
embryos remain virtually unprotected and more or less give rise to genetically uniform plants.
Leaf-disc transformation systems have made it possible to successfully engineering of species in
which tissues are capable of regeneration via somatic embryogenesis. Repetitive embryos are
formed by single epidermal cells which can be readily exposed to Agrobacterium. Thus, the
transformation technique applied to a primary somatic embryo, instead of a zygotic embryo,
should give rise to totally transgenosic somatic embryos.
Repetitive embryogenesis is also ideally suited to practicle gun-mediated genetic transformation.
In this technique, the particle gun literally shoots DNA that has been precipitated onto particles of
a heavy metal, into the plant cells. The transformed cell lines can be induced to form an unlimited
number of transformed somatic embryos.

Somatic Hybridization
Many desirable combinations of characteristics can not be transmitted through the conventional
process of genetic manipulation. Therefore, another process, other than the sexual cycle, is used
that can lead to genetic recombination. This non-conventional genetic procedure involves fusion
of isolated somatic protoplasts (wall less naked cells) under in vitro conditions and subsequent
development of their product which is known as Heterokaryon, to a hybrid plant. This process is
known as somatic hybridization. Plastids and mitochondrial genomes are inherited maternally in
sexual crossings. Through the fusion process the nucleus and cytoplasm of both the parents are
mixed in the hybrid cell. This results in various nucleo-cytoplasmic combinations. Sometimes
interactions in the plastome and genome contribute to the formation of cybrids which are
cytoplasmic hybrids. Cybrids unlike the hybrids possesses nuclear genome from only from one

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parent but cytoplasmic genes from both parents. The process of protoplast fusion resulting in the
development of cybrids is known as cybridisation.

Isolation of Protoplasts
Protoplasts can be isolated by using three methods which are as follows-
Mechanical (Non-enzymatic)
Sequential Enzymatic (Two Step)
Mixed Enzymatic (Simultaneous)
Mechanical isolation involves the cutting of plasmolysed tissue with a sharpedged knife and
releasing the protoplasts by deplasmolysis. The main drawback of this system is that very few
protoplasts are released. Therefore it is not used so frequently for protoplast isolation. The
sequential approach involves initial incubation of macerated plant tissues with pectinase which, in
turn, are then converted into protoplasts by a cellulose treatment. However, Power and Cocking
(1968) mixed two enzymes together (simultaneous procedure) and isolated protoplasts in one-
step. In this mixed enzymatic approach, plant tissues are plasmolysed in the presence of a mixture
of pectinases and celulases, thus inducing concomitant separation of cells and degradation of their
walls to release the protoplasts directly.
Sources of Protoplasts: These are as follows-
Leaves: The leaves are the most convenient and popular source of plant protoplasts because it
allows isolation of a large number of relatively uniform cells. It involves following steps:
Callus Cultures: Young callus cultures are an ideal material for obtaining a large quantity of
protoplasts. Older callus cultures tend to form giant cells with thick cell walls which are usually
difficult to digest; therefore, young actively growing callus is subcultured and used after two
weeks for protoplast isolation.
Cell Suspension Cultures: Cell suspension cultures can also be used for protoplast isolation. A
high density cell suspension is centrifuged. After removing the supernatant, cells are incubated in
an enzyme mixture (Cellulase + Pectinase) in a culture flask placed on a platform shaker for 6 hr
to overnight depending on the concentration of enzymes.
Viability of Protoplast: Isolated protoplasts should be healthy and viable in order to undergo
sustained divisions and regenerations. Methods which are used for checking the viability of
protoplasts are as follows-
Observation of cyclosis or cytoplasmic streaming
Oxygen uptake by protoplasts measured by an oxygen electrode
Photosynthetic activity

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These tests indicate the active metabolism in the cells.

Culture of Protoplasts:
First step includes the formation of cell wall around the membrane of isolated protoplasts. This is
followed by induction of divisions in the protoplasts-derived new cell giving rise to a small cell
colony. By manipulation of nutritional and physiological conditions in the nutrient media, cell
colonies may be induced to grow a callus continuously or to regenerate whole plants. Isolated
protoplasts or their hybrids are cultuted either in a liquid or agar medium. The common practice
of using a liquid culture medium includes either incubating protoplasts/ heterokaryons in a thin
layer or as small drops of nutrient medium inside a petridish which in turn is covered by another
petriplate and finally sealed with parafilm. The culture dish is incubated at 25-28C.
For culturing the protoplasts in the nutrient medium containing agar 2 ml aliquots of isolated
protoplasts of suitable density are mixed with an equal volume of agar nutrient medium, the
temperature of which should not exceed 45C. On solidification of agar, the culture plates are
sealed and maintained in an inverted positon at 25-28C. With this method, individual protoplasts
or heterokaryons can be conveniently observed under a microscope and plating efficiency readily
determined.

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SOMACLONAL VARIATION

Genetic variations in plants that have been produced by plant tissue culture and can be detected
as genetic or phenotypic traits.
The genetic variations found in the in vitro cultured cells are collectively referred to as
somaclonal variation and the plants derived from such cells are called as somaclones. It has
been observed that the long-term callus and cell suspension culture and plants regenerated from
such cultures are often associated with chromosomal variations. It is this property of cultured cells
that finds potential application in the crop improvement and in the production of mutants and
variants (e.g. disease resistance in potato). Larkin and Scowcroft (1981) working at the division of
Plant Industry, C.S.I.R.O., Australia gave the term 'somaclones' for plant variants obtained from
tissue cultures of somatic tissues. Similarly, if the tissue from which the variants have been
obtained is having gametophytic origin such as pollen or egg cell, it is known as 'gametoclonal'
variation. They explained that it may be due to:
(a) reflection of heterogeneity between the cells and explant tissue,
(b) a simple representation of spontaneous mutation rate, and
(c)activation by culture environment of transposition of genetic materials.

Advantages of Somaclonal Variations

1. Methodology of introducing somaclonal variations is simpler and easier as compared to
recombinant DNA technology.
2. Help in crop improvement

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3. Creation of additional genetic varitions

4. Selection of plants resistant to various toxins, herbicides, high salt concentration
5. and mineral toxicity Development and production of plants with disease resistance e.g.
rice, wheat, apple, tomato etc.
6. Develop biochemical mutants with abiotic stress resistance e.g. aluminium tolerance in
carrot, salt tolerance in tobacco and maize.
7. Development of somaclonal variants with herbicide resistance e.g. tobacco resistant to
sulfonylurea
8. Development of seeds with improved quality e.g. a new variety of Lathyrus sativa seeds
(Lathyrus Bio L 212) with low content of neurotoxin.
9. Bio-13 A somaclonal variant of Citronella java (with 37% more oil and 39% more
citronellon), a medicinal plant has been released as Bio-13 for commercial cultivation by
Central Institute for Medicinal and Aromatic Plants (CIMAP), Lucknow, India.
10. Supertomatoes- Heinz Co. and DNA plant Technology Laboratories (USA) developed
Supertomatoes with high solid component by screening somaclones which helped in
reducing the shipping and processing costs.
11. Increased and improved production of secondary metabolites
12. Suitable for breeding of tree species

Applications of tissue culture:

There are certain advantages of plant tissue culture over traditional method of propagation like:
The production of exact copies of plants that produce particularly good flowers, fruits or
possess other desirable traits.
To quickly produce mature plants.
For the production of multiple copies of plants in the absence of seeds or necessary
pollinators to produce seeds.
For the regeneration of whole plant from the genetically modified cells.
The production of plants under sterile conditions which lowers the chances of transmitting
diseases, pests & pathogens.
The plants which have very low chances of germination & growth by seeds for e.g.
Orchids & Nepenthes are propagated by using plant tissue culture techniques.
To clean particular plant of viral and other infections and to quickly multiply these plants
as Cleaned Stock for Horticulture and Agriculture.
Plants whose particular sex is commercially important are produced by using techniques
of plant tissue culture. Example- female plants of Papaya and male plants of Asparagus.

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Some species of plants vary markedly in their traits for e.g. Eucalyptus. The selected trees
are marked from the variant population for the desirable trait such as disease resistance,
higher productivity etc.
Tissue culture or mass cloning methods of elite tree species is done for increasing land
productivity. They are being modified or adapted for large-scale modification.

1.Production of disease free plant:

Most of the horticultural and forest crops are infected by systemic diseases caused by fungi,
viruses, bacteria, mycoplasma and nematodes. Pathogen attack does not always lead to the death
of the plant but very often the infection caused by pathogens considerably reduces the yield and
quality of crops. While pathogens are nearly always transferred in plants through vegetative
propagation, viral disease occur in virtually all seed-propagated as well as vegetatively
propagated crop species. Eradication of pathogens is highly desirable to optimize the yields and
also to facilitate the movement of materials across the international boundaries. While plants
infected with bacteria and fungi may respond to treatments with bactericidal and fungicidal
compounds. To produce disease-free plants a healthy nucleus stock could be prepared by selecting
out one or more healthy plants then multiplying them vegetatively, but where the entire
population of a clone is infected the only way to obtain a pathogen-free plant is through tissue
culture. Apical meristems in the infected plants are generally either free or carry a very low
concentrations of viruses. The titre of the viruses increases in the older tissues corresponding to
the increase in the distance from the meristem tips. Various reasons attributed to the escape of the
meristems by virus invasion are-
Viruses move readily in a plant body through the vascular system which in meristems is
absent.
A high metabolite activity in the actively dividing meristematic cells does not
allow virus replication.
A high endogenous auxin level in shoot pices may inhibit virus multiplication.
Method of Virus Elimination:
Earlier heat treatment was used to given the whole plants for the elimination of viruses. At higher
temperature most of the viruses became partially or fully inactivated without little or no injury to
the host tissues. Heat treatment was given through hot water or hot air. But it was observed the
percentage of survival of heat treated plants was very low. Therefore, techniques of tissue culture
were applied for the complete eradication of viruses. It involves following steps-
i) Meristem tip culture: The size of the explants is critical for virus elimination since in
most of the systemic viral diseases the pathogen establishes a gradient in plant tissues. It has been

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observed that virus-free plants regenerated in vitro are inversely proportional to the size of the
meristem culture.

1. Production of Haploid Plants: An important aspect of plant breeding is the induction of

maximum genetic variability of germplasm sources to secure a wider scope for selection and
introduction of better trait qualities in existing crop species. Therefore, efforts have been done for
the production of haploid plants. The in vivo techniques used for the production of haploid plants
are-

i) Gynogenesis: Production of a haploid individual by the development of an unfertilized

egg-cell as a result of delayed pollination (through use of abortive pollen pre-exposed to ionising
irradiation or by using an alien pollen).
ii)Androgenesis: A haploid individual is produced by the development of an egg-cell containing
the male nucleus. In this case elimination or inactivation of the egg nucleus occurs before
fertilization.

2. Triploid Production: Endosperm is produced as a result of double fertilization or triple

fusion in angiosperm. The chromosome number of triploid plants are 3n. Triploid plants are more
vigorous than the diploid counter parts. Conventionally triploid plants are produced by crossing
tetraploid with diploid plant. This method is cumbersome and may not be successful always.
However, endosperm culture will produce triploid and consequently hexaploid plantlet by giving
colchicines treatment
3.Micropropagation is a method by which millions of clones can be produced from small
amount of plant tissue within a short span of time.
4.Ornamental plants are now in high demand world-wide, so micropropagation has caught up on
a commercial scale.
5.Not only new varieties can be developed by this method, their propagation can be carried out
within a short space and time to meet the global demand of horticulture species such as
eucalyptus, gerbera, ferns, orchids and rhododendron.
6.Ornamental flowering plants and vegetables are being propagated.
7.Micropropagation is also effective in germplasm storage to save endangered species.

Germplasm conservation
It is important to save endangered species as conservation programme is necessary to save
biodiversity.

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The term GERMPLASM is defined as the total genome (all the genes) present in a crop plant or its
related species.
Steps for germplasm conservation
In vivo gene banks have been established to preserve germplasm of endangered species in the
form of seeds and vegetative parts
In vitro gene banks are being used to preserve genetic resources by utilizing cell and plant tissue
culture methods. This is to ensure their availability to agriculturists and plant breeders to further
improve the species for higher yield.

Species are selected on the basis of following categories for the in vitro studies :
Rare and endangered species.
Species with traditional use in medicinal, religious and cultural practices.
Species with specific propagation difficulties.
Species of economic interest to the host country.
Species with unusual growth characteristics that are interesting model systems for basic
research into plant growth and development.

Figure to show Clonal germplasm conservation using tissue culture technique

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A standard procedure for the micropropagation of the neem tree (Azadirachta indica A.
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