Plant biotechnology

Chemodemes:
Chemically distinct population within a species and have similar phenotypes, but different genotypes
and as such are identical in external appearance but differ in chemical constituents.

Before the existence of chemical race certain fundamental observance are necessary.

The observation of chemodemes can be confirmed by growing different plant of species in identical
conditions preferably from seeds and for many generations.by this way it shows variations either in
type or content of certain constituents, like the secondary metabolitesof medicinal importance.

The chemical characteristics of chemodemes are hereditary.

The discovery of chemodemes has enabled to select high yielding chemical strains or to eliminate
chemical strains that contain toxic metabolites. The chemodemes are induced by breeding in some
species so as to manipulate active constituent to enhance therapeutic efficacy.

For eg –

Normal rape seed oil contains acyl-glycerol (20-40%eracic oil) which makes the oil unsuitable for
edible purpose but varieties are now grown presently that contain no eracic oil. The oil is very useful
for manufacture of lubricants and artificial fibres.

Sunflower oil constitutes an important oil seed crop and its genetic variability which facilitates the
breeding of variety with widely differing oil constituent proportion. High oleic varieties are used for
human consumption and high linoleic comp varieties are used for industrial protein and lubricants.

The above examples examples improve plants with a short life span breeding by classical method is
relatively rapid procedure however this is not so with plants such as coconut ,palm , olive .in this
cases modern techniques involving gene transfer is advancement for introduction of new or
modified oil characteristics.

Polyploidy – deviation from normal genome.

Hyperploidy increase in number of chromosomes.vigorous growth.

Hypoploidy decrease in number of chromosomes.stunted growth.

Mutation – sudden changes in heritable characters.

Chromosomal abbreviations – shape ,size of chromosomes

Plants are grown in concentric circles and divided into different selections in each selection different
groups of plants can be grown. Once radiation is started amount is monitored by radiation monitor
and one of the monitor is kept at the centre and portable monitor near plants. We can select the
radiation that are more beneficial.after irradiation these plants are planted in specific fields
surrounded by control material to avoid inter crossing with non irradiated plants.

The plants obtained are called R1 generation. Seeds are irradiated sown similarly.R1 plants are
selected with desired characteristics and these seeds are grown further till R5, R6 generation.

By that time one can know whether the characters are heritable and constituents.

Adv of ¥ radiations - -

Large number of plants can be employed and amount of radiation received is different and therefore
we get different mutations from which desired character can be selected.

Using chemicals :

The most powerful mutagenic chemicals discovered so far are the mustard gas and it’s related
compounds like ethylene oxide , 8-ethoxy caffeine. The seeds can be sowed in these chemical or
seedling itself can be treated in young condition . with solutions they bring about changes in
chromosomes.

They induce gene and chromosomal mutation.

Different groups of compounds are

1) DNA PRECURSORS – adenine , deoxy adenosine and ethoxy caffeine. They generally bring
about mutational changes by interfering with dna synthesis and also sometimes by
degradation & denaturation of dna.
2) DNA analogues – it includes thymidine , uridine . generally these produce abnormal DNA.
3) Certain antibiotics like streptomycin and actinomycin bring out degradation and
denaturation of DNA.
4) Certain alkylation agents like nitrogen mustard produce abnormal dna. Some nitroso
compounds inorganic cyanides , bring about mutation.
Nitroso compounds combine with heavy metals ,fe,cu, which inturn combine with DNA
forming abnormal dna. They are supported to interfere with enzyme synthesis, amt of
changes produced is less and also individual plants must be handled.

LIMITATIONS:---
Unpredictable
May not be beneficial
Once canges takes place ,must be grown for 5-6 generations.
Can be carried out at those places only where facilities are available.
 Person working with radiation has to be careful.

ADVANTAGES:__
We can get different varities of inexhaustible variations & mutation breeding can make plant
breeders free from complete dependence , on nature for raw material .

Time, labour, land and expenses needed in this are considerably less.
Widely used in plant improvement both for higher yield and to get resistant plants.

Work on medicinal plants

Poppy seeds treated with cobalt-60has produced different varieties some with very high amount of
morphine.

Menthol – cobalt -60 M.piperita species produces the infection resistant varieties.

Rose produced different sized flowers and oil contents.

Plant tissue culture:

Plant tissue culture is a collective term for the technique of growing plant cells , tissues, organs or
even plantelets on a nutrient medium in a suitable container under sterile and controlled conditions.

The technique of plant tissue culture has developed around the concept of totipotency.

When an explants from differentiated cells / tisssues is used for culture on a nutrient medium, the
non-dividing and inactive cells undergo changes to achieve meristematic state. The phenomenon of
reversion of mature cells to meristemetic state leading to the formation of callus is called

de-differentiation.

The component of cells of callus have ability to form a whole plant and this phenomenon is called re-
differentiation.

These 2 phenomenon of re and de- differentiation are basic components of capacity plant cells
described as totipotency , a property .

When an explants from differentiated cells/tissues is used for culture on a nutrient medium, the non
dividing and inactive cells undergo changes to achieve meristematic state. The phenomenon of
reversion of mature cells to meristematic state leading to the formation of callus is called de-
differentation. The component of cells of callus have the ability to form a whole plant and this
phenomenon is called re-differentiation.

These two phenomenon of re- & de differentiation are basic component of capacity of plant cells
described as totipotency, a property found only in plant cells and not in animals.in other words a
differentiated plant cells retains its capacity to give rise to a whole plant but an animal cell loses this
capacity of regeneration after de-deffferentiation.

Tissue culture is a clean and rapid way for genetic engineers to grow material for
identify,manipulate,or transfer genes from one plant to another.it plays a vital role in field of
botany,molecular biology,hybrid development and food science.

Types
1) Callus culture
2) Meristem culture
3) Organ culture
Vegetative organ culture ,Root culture, Shoot culture, Leaf culture

Reproductive organ culture

Flower organ culture
Isolated ovary culture
Seed culture
Fruit culture- 1)whole fruit 2) isolated tissue
Pollen mother culture
Isolated ovule & embryo culture
4)protoplast culture
5) haploid culture

Essentially all organs can be used as explants .however degree of success with different tissue can
vary and calluses with different morphologies are frequently obtained. Callus culture require sub –
culturing at about 1 month intervals by maintaining them at 25°c +-2°. And provision of light at
prescribed intensityand photo period as per requirement of plant species.

Nutrient medium is supplied with auxins to induce cell division. The unique feature of callus is its
ability to develop normal root and shoot , forming a plant .
Callus is formed through 3 stages of development
1) Induction
2) Cell division
3) Cell differentiation

During induction stage metabolic rate of the cell will be increased due to this the cell
accumulates organic contents and finally divides into a no of cells. After cell diff will occur in
this stage many metabolic pathways start leading to formation of secondary metabolites.
Callus culture in some medicinal plant produces the selected compounds with a yield equal to
or higher than that of parent plant. However the capacity of callus culture often differ
substantially from that of fully differentiation tissue both qualitatively & quantatively.
 As a result tissue specific patterns of differentiate and biosynthetic do not persist when
tissues are culyured . however these are exceptions persist when tissues are cultured.
However there are exception for persistant tissue specific production of secondary
metabolites. CALLUS CULTURE INITIATED FROM ROOTS of tuta graveolens produce root
specific essential components whereas callus culture from stem produce stem specific
components.
The potential usefulness of callus in propagation lies in the possibility of greatly extending the base
for adventitious shoots or embryo production.
This can be achieved by lowering the concentration of auxins & increasing the conc of cytokines
than those required for callus induction and maintainance.
Many of the industrial medicinal plants are being micro propagated through callus culture.
Plant regeneration using callus is of great significance in species like bergenia crassifolia,hypericum
perforatum. When natural supply is limited due to low yield adn slow growth rates invitro cultures
provides an alternative method. Most plants can be cultivated by di- or re – differentiation.

Secondary metabolites which are large varied & mysterious group of molecules while some are
mostly extraneous by- products of metabolic pathways.due to enzymatic activity. Many do serve
important function in defence or protection in plants and thus considered biologically active.
Most secondary met. Are often present in extremely low amounts in the plants.the scarcity can
make natural harvestation impractical for bulk production, especially in the case of slow growing
species. The significance engineering challanges is to find a means by which we can produce
desired natural products ,in a way.i.e both sustainable and financially feasible.

Production of these products in microbial / fungal host by transferring biosynthetic

pathway is possible but is limited due to the complexity and laws of complete
knowledge regarding many of these pathways.
Un-differentiated cultures often accumulate sec metabolites to a lesser extent and sometimes not at
all. The anticancer compounds camptothecin from camptotheca accuminta. Nothapodytes foetida &
ophiorrhiza pumila among other species has been shown to accumulate in undifferentiated culture
in very low or even in undectable amounts compared to root cultures in which production levels
were comparable to intact plants.

Applications of plant tissue cultures

1) In agriculture production of high yielding , drought resistant , salt resistant and insect
resistant plants.
2) Horticulture- micro propagation of aromatic and medical plants, production of seeds, prod
of phytopharmaceuticals, food colours and flavours etc from plant cell culture. The yield of
sec metabolites are very poor in some of the species.it can be improved by strain
improvement i.e seletion, screening & med variation. Culture conditions like inoculums size,
pH, temp, light, agitation, special techniques such as immobilisation, permeabilisation etc,
biotransformation , genetic trans. The valuable preditions from transgenic ..e.g – antigens,
antibiotics, edible vaccines, monoclonal antibodies.
Plant tissue culture media :]
Inorganic nutrients (macro & micro )
Carbon sources, antibiotic, growth regulators, buffers, vehicles, jellying and solidifying
agents.
Names of media:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::
Gautheratemedia(1942)
Nitsch media (1951)
Whites media
Gaunbergs media
N6 meddia
E1 media (gamberg)

Macro nutrients – ca+2, cl-1,mg, k+, nh4+, po32-, no3-, etc

Micronutrients – fe2+, na+, zn+2, computer, ni, kio3, b,vitamins,biotin, glycine, inositol,
pyridoxine,thiamine, folic acid, EDTA.

Cell suspension culture:-

The plant cells in agitated liquid meddis has been reported by caplin and stward in 1949,
nickel (1956). These workers showed the feasibility by growing plant cells in a similar way as
that of m.o.
Nickel in 1959 studied the production of mass amounts of cell suspension by using
submerged and also described it’s potential use for the study of biosynthesis of sec met.
Plants cell cultures are becoming of increased importance as an exceptance material for
various biochemical and physiological studies. Its also useful for various biochemical and
physiological studies.its useful in enzyme purification. Till recent time , cell susp of higher
plants were grown in media containing coconut milk and water, yeast and malt extract. But
nowdays they have been successful grown on purely synthetic media.

Characteristic of suspension culture:

All cell suspension cultures consists of single cells , small cell group and large cell aggregates
dispersed in a liquid medium. Cell suspension culture actively grows in a agitated and
aeration. The cells divides in an incubation medium leading to an increase in material.
Suspension culture can be subcultured by pippeting out and by adding to fresh medium.
These cultures are propagated at regular intervals showing similar patterns of growth and
yield . it is different to obtain a suspension culture consisting only free cells as the daughter
cells adhere together soon after division to form aggregate.
For the suspension culture cell homogenate are made by subjecting sterile seedlings or
young embryos to a hand operated glass homoginiser consists of intact living cells debris ,
and dead cells. The homogenate is added to liquid medium on shakers and supernatant
layers containing cell and aggregates is pipettted out into the medium and & incubated in
diffused light.
Agitation is necessary for gaseous exchange & to maintain cells in dispersed condition.
Techniques
The selection of culture media for cell suspension culture is ddone on basis of
optimal growth for recording growth rate.
The culture med for callus culture may not be suitable for suspension cultures as the
nutritional requirements may vary.
Sucrose is most common sugar used for carbon source
Auxin/cytokinin are suitably modified and also vitamins.
Cells suspension cultures are maintained by sequential sub culturing during early
starting phase and at the time when cell aggregation reaches maximum level.

Culture apparatus:
A culture apparatus by steward consists of flask of 250-1000ml capacity fitted with 6-
8 small tubes at the outer end and open at the base.
These are also called as the nipple flasks or tumble tube culture vessels. The tubes
have side necks to facilitates introduction of inocolum. The tubes are mounted at
mid point held by clips along the periphery of the circular disk fine to horizontal
central drive shaft. It is rotated at low speed of 1-2 rpm. The ex – planted tissues are
introduced into these tubes on slow rotation and alternatively immersed and
exposed to air. The apparatus is placed in a temp. Controlled room, shakers have
been imparted to impart circular motion.each platform bears a dozenor more
cylindrical culture bottles of varying capacities which can be held in position by clips
or rubber pads. For the preparation of cell suspension. The shakers are fitted with
variable aped is adjusted to optimum growth rate,.
Total cell yield at highest cell separation. Various mods of shaker culture and
continuous culture synthesis for large batches have been used in practice.
Recent methods of these culture apparatus for continuous culture system which
makes use of inverted cylindrical culture bottles for intiment renewal of medium ,
harvesting of cells, refilling of fresh batches.
Single cell clones :- single cells could be grown in the medium for establishing single
cell clone . in these cases 3 methods are generally used.
1)paper raftners technique
2)plating method
3) growth of isolated cells in microchambers.
In all these methods the essential nutrient in the medium diffuses through the
barrier and bring about the division and growth of single cells on the uppersurface.
The medium is conditioned dddue to release of conditioned metabolites.
In the microchambers method division of single cell is initiated as a consequence of
enrichment of micromedium. Enhanced planting efficiency has been observed from
from the use of minimal media supplements by selected metabolites. Single cell
clone can be subcultured but it has been observed that single cell loses its
totipotency when it is continuously subcultured.
Single cell clone differ from another in texture , friability and growth rate on different
media. Primarily single cell clones have also shown difference with their secondary
derivatives with different conc of sugars or ar a carbon source . it has shown the
variation in organo genetic potential.

Growth of cells in microchambers :

Single cells are grown efficiently by hanging drops in microchambers. The cells in
isolation grown in microchambers undergo cell division under appropriate nutritional
and hormonal conditions and the calli that develops could regenerate new plant s.
Maximors double coverslip method and the modified micro chambers devised are
used to observe the growth of cell .
Conditioning of the nuclei is needed in order to trigger initial cell division . certain
essential acids like glutamine and serine , gases like co2, & growth regulators like
cytokinines are essential factors concerned with conditioning of the media.

Large scale culture of cells:---------------

Several studies have been carried out in order to obtain accumulated products of
metabolism using large scale cultures of plants cells for industrial scale,
Plant cells are grown as suspension culture in a wide range and bioreactors of 2-
2000litres.plant cells are quiet large in then microbial cells . during low
growth in batch cultures cells excretes polysaccharides ,so media becomes highly
viscous. Cells forming aggregates increases the biomass .plant cells being aerobic
need oxygen supply at low level. Cell culture system form foams and surface
adhesions but it doesn’t affect cultures . most large scale cultures are run as batch
cultures but conventional stirred tank reactors, air liff system and bubble column are
also in use but not a single is applicable for plant cell culture.
Cell suspension cultures can be maintained at steady growth rates as homogenous
system using the parameters like cell weight, no, total cell proteins, metabolic
stability etc. A liquid culture sys under agitation permits the metabolic pathways to
be altered to suit particulars objectives.
Auspension culture offer advantages over stn culture as the cells are uniformly
placed in the medium containing nutrients, plant growth regulators etc. Precise
manipulation of ingredients is possible and handling off cells , cells aggregates
embrioids becomes easy.

Applications:
An appreciation of basic technique of cell suspension culture is essential to any
assement of their realised and potential application. Following a critical evaluation of
present state of tech in the field where the suspension cultures have contributed the
basic of physiology of plant cells in cultures.
 It opens up new approaches to the study of secondary products biosynthesis in plant
cells culture.
Esters adn amides of cinnamic acid form a major widespread group of phenyl
propanoid metabolites in plants which accumulates spontaneously often at high
level in cell suspension cultures.
Eg:- rosmarinic acid from coleus blumei. These cultures were maintained in
suspension for several years without loosing their capacity for syn and accumulating
rosmarinic acid. The syn of rosmarinic acid can be stimulated by increasing sucrose
level to 5-8% in which content of r.acid increased upto 15%. Recently it has been
shown that r.acid and enzyme in biosy. Can be induced in withocapermucan
erythrorirhizon and orthosiphon aristatus by elecitors such as yeast and methyl
jasmonate from nearly 0-1.5% of dry mass.
Napthoquinones ans anthraquinones sometimes accumulate in culture cells at levels
for exceeding the amount found in intact plant.

Different approaches are used to produce secondary metabolites from plants tissues
1)cell cloning
2)mass cultivation
3)immobilised plant cell culture
4)fungal elicitus induced production
5)biotransformation using plants cells.
Selection of high yielding cells , growing them on suitable medium , screening cells to
produce sec. Metabolites is the technique to develop high yielding cell lines.
Successful selection of clones i.e increased producers of sec metabolites are
achieved by visual, chemical, radio immunoassay tech. These selected cell lines are
mass cultivated to achieve incred level prodn.

There are two basic problem in such cultivation :

Biological barriers that includes low growth rate & genetic instability.

Technical barriers which includes shear sensitivity of cell oxygen transfer,cell

aggregation,cell wall growth by addition of cells,.

Drought tube , air liff reactor, ait lift loop reactor, stirred tank reactor, rotating drum reactor
are used for for large scale production of secondary metabolites from plant cell culture.

Problems of cell culture are resolved by newly developed tech surface immobilisation of
plant cells which protects cells from high shear stresses optimising production.

Immobilisation:
It is the newest culture technology in plant cells and it has been defined as a technique
which confines to catalytically active enzyme or to a cell within a reactor system and
prevents its entry into the mobile phase which carries the product and the substrate.

The first successful immobilisation of plant cell was reported in 1979 by brodelius. And the
entrap catharanthus roseus and Decius carota cells in alginate weeds.

Immobilisation has been subjected as strategy to enhance the over all productivity of
secondary metabolites in plant cell culture. Immobilisation of plant cells has been used for
wide range of reactors which can be divided in three groups.

1)biotransformation

2)synthesis from precursors

Denovo synthesis

Advantages

---Retention of bio mass enables its continuous reatialisation as a product system , a definite
advantage with slow blowing plant cells.

Eg.- papavera somnifera have remained stable and active form upto 6 months.

---the immobilisation cells allow the use of higher bio mass level compareddd to cell
suspension culture because of the limit of mass transfer and setteling

Eg-bead densities of 110gm dry weight per litre have been obtained with calcium alginate
entrapped cells well as 30 gmdry wt per litre in suspension culture the high density allows a
reduction in contact time in black bed catalyst leading to an increased volumetric
productivity.

--- separation f cells from medium the immmobilasition seperates cells from medium and
desired product is extracellular which will simplify downstream processing compared to
extraction from tissues.

----immobilisation allows a continuous process which increases volumetric productivity and

allows down stream processing compared to extraction from tissue.

---decoupling of growth and product formation immobilisation is compatible with non

growth associated product formation reduces problems such as aggregate growth and
foaming.

The immobilisation reduces some of the physical problems associated with cultivation of
plant cells such as formation of aggregrates and susceptibility to mechanical cell damage or
shear stress are problems which do not affect immobilised system as compared to cell
culture.

---Bette stability granules can be used upto 60 days-

Disadvantages:

1)the catalysing capacity of immobilised cells is just half of suspended cells

2)secretion of secondary metabolites require cellular transport or artificially altered

membrane

3)the efficiency of production process depends on the rate of release of products rather
than actual rate of biosynthesis.

4)may reduce biosynthetic capacity

5)product must be released from cells into the media

6) release of single cells from cell aggregate may make processing of product difficult.

7) the micro environment favouring optimal production can be unfavourable for secondary
met and can cause its degradation or metabolisation.

NEED FOR IMMOBILISATION: