Conservation of Embryo and

Ovules, cell, Ovule culture,

Suspension culture, Protoplast and
Callus culture, Micro propagation
method, its Advantage and
Disadvantage
 What is Germplasm?

• Germplasm broadly refer to the hereditary

material (total content of gene) transmitted to the
offspring through germ cell.
• It is also used to describe a collection of genetic
resources for an organism. For plants, the
germplasm may be stored as a seed collection or
,for trees, in a nursery.
 What is germplasm conservation?
Plant germplasm is the genetic source material used by the
plant breeders to develop new cultivars.
They may include :-
• Seeds
Other plant propagules such as
• Leaf
• Stem
• Pollen
• Cultured cells
Which can be grown into mature plant.
Germplasm provide the raw material (genes) which the
breeder used to develop commercial crop varieties.
Need for Conservation of plant Germplasm
 Loss of genetic diversity among crop plant species.
Human dependence on plant species for food and many
different uses. e.g ; Basic food crops, building materials, oils,
lubricants, rubber and other latexes, resins, waxes,
perfumes, dyes fibres and medicines.
Species extinction and many others are threatened and
endangered – deforestation.
Great diversity of plants is needed to keep the various
natural ecosystems functioning stably – interactions
between species.
Aesthetic value of natural ecosystems and the diversity of
plant species.
 Mode of conservation
It is of two methods:-
 In situ Conservation
• In situ conservation is on-site conservation or conservation of
genetic resources in a natural population of plants, such as
forests genetic resources in natural population of tree species.
• It is the process of protecting an endangered plant in its natural
habitat either by protecting or cleaning up the habitat itself, or by
defending the species from predators.
• It is applied to conservation of agriculture biodiversity in agro
ecosystem by farmers, especially those using unconventional
farming practice.
 Ex situ Conservation
Ex-situ conservation means literally, "off-site conservation". It is
the process of protecting an endangered species of plant or
animal outside of its natural habitat; for example, by removing
part of the population from a threatened habitat and placing it
in a new location, which may be a wild area or within the care of
humans.
Ex situ conservation can be carried out by
using several methods
 Seed gene bank
 In vitro storage
 DNA storage
 Pollen storage
 Field gene bank
 Botanical gardens
 CRYOPRESERVATION
Cryopreservation (Greek, krayos-frost) literally mean in the
frozen state. The principal involved in cryopreservation to bring
the plant cells and tissue cultures to a zero metabolism or non-
dividing state by reducing the temperature in the presence of
caryoprotectants.
Cryopreservation broadly means the storage of
germplam at very low temperature.
• Over solid carbon dioxide (at 79⁰C)
• Low temperature deep freezers (at -80⁰C)
• In liquid nitrogen (at -196⁰C)
•Among these the most commonly used cryopreservation is by
employing liquid nitrogen. At the temperature of liquid nitrogen
(-196⁰C), the cell stay in a completely inactive state and thus can
be conserved for longer period. Infact cryopreservation has
been successfully applied for germplasm conservation . Plant
species e.g. rice, wheat, peanut, sugarcane ,coconut.
 Cryopreservation
-
 Cryo is Greek word. (krayos – frost)

 It literally means preservation in “frozen state.”

It is a process where tissues, organelles, cells, extracellular

matrix, organs or any other biological constructs susceptible to
damage caused by unregulated chemical kinetics are preserved by
cooling to very low temp(typically-80degree Celsius using solid
carbon dioxide or -196 degree Celsius using liquid nitrogen.
The principle - to bring plant cells or tissue to a zero
metabolism and non dividing state by reducing the
temperature in the presence of cryoprotectant(anti freeze).

It can be done :

Over solid carbon dioxide (at -79

degree)

Low temperature deep freezer (at -80

degree )

 In vapour phase nitrogen (at -150

degree)

 In liquid nitrogen (at -196 degree

Until two decades ago the genetic resources were getting depleted
owing
to the continous depredation by man.

 It was imperative therefore that many of the elite, economically

important
And endangered species are preserved to make them available when
needed.

 A new methodology had to be devised for long term preservation of

material.
 Mechanism of cryopreservation
The technique of freeze preservation is based on the transfer of
water present in the cells from a liquid to solid state. Due to the
presence of salts and organic molecules in the cells, the cell
water requires much more lower temperature to freeze (even
up to -68°C) compared to the freezing point of pure water
(around 0°C) . When stored at low temperature , the metabolic
processes and biological deteriorations in the cells/tissues
almost come to standstill.
Mechanism of cryopreservation
The cryopreservation technique followed by the regeneration of
plants involves following steps :

Selection of material

Addition of cryoprotectant

Freezing

Storage in liquid

nitrogen Thawing

Washing and reculturing

Measurement of

viability
1.Selection of plant material :

Selection of proper plant material is important.

Two important factors depend on it such as

(a) nature
and
(b)density.

Any tissue can be selected for this purpose. e.g. meristem,

embryo, ovules, seeds etc..
2. Addition of cryoprotectant

 They are chemical which prevent

cryodestruction.

These are sucrose, alcohols, glycols, some amino

acid (proline), DMSO (dimethyl sulfoxide).

Generally two cryoprotectant should be used

together instead of single one as they are more
effective.
3. Freezing
The sensitivity of cells to low temperature depends on the plant
species.

There are four different types of methods :

Slow freezing method - the tissue or plant material is slowly

frozen at slow cooling rate. The advantage is the plant cells are
partially dehydrated and survive better.

Rapid freezing method - it involves plunging the vials in liquid

nitrogen. The temperature decreases from -300 to -1000 degree
rapidly.

Combine freezing method - this is combination of both slow and

rapid freezing method. The process is carried out in step wise like
manner.

Dry freezing method - in this method dehydrated cells and seeds

are stored.
4. Storage

The maintenance of the frozen cells or material at

specific temperature is very important.

In general the temperature is kept -70 to -196 degree.

Prolong storage is done at temperature of -196 degree in

liquid nitrogen.

To prevent damage, continous supply of nitrogen is done.

5. Thawing
Usually carried out by plunging the vials into warm
water bath with vigorous swirling.

As thawing occurs the vials are transferred to another

bath at 0 degree.

6. Washing and reculturing

The preserved material is washed few times to remove the
cryoprotectant.

This material is then recultured in a fresh medium

There are three main approaches for the In
vitro conservation of germplasm
1. Cryopreservation
2. Cold storage
3. Low – pressure and low oxygen - storage
7. Measurement of viability
There is possibility of death of cells due to storage stress.

Thus viability can be found at any stage.

It is calculated by formula :
No of cells growing / no of cells thawed
* 100

8. Plant regeneration
The viable seeds are cultured on non specific growth medium.

Suitable environmental conditions are maintained.

 Major advantages are :
1. Once the material is sucessfully conserved to particular
temperature it can be preserved indefinately.

2. Once in storage no chance of new contamination of

fungus or
bacteria.

3. Minimal space required.

4. Minimal labour required.

It is ideal method for long term conservation of material.

Disease free plants can be conserved and propagated.

Recalcitrant seeds can be maintained for long time.

Endangered species can be maintained.

Pollens can be maintained to increase longitivity.

Rare germplasm and other genetic manipulations can

be stored.
 In vitro method for germplasm
conservation
In vitro method employing shoots, meristems and embryos are
ideally suited for the conservation of germplasm. The plant with
recalcitrant seeds and genetically engineered can also be
preserved by this in vitro approach.
There are several advantages associated with in vitro germplasm
conservation
 Large quantities of material can be preserved in small space.
The germplasm preserved can be maintained in
an environment free from pathogens.
 It can be protected against the nature’s hazards
From the germplasm stock large number of plants can
be obtained whenever needed.
Various Methods Have Been Shown To Reduce The Growth Rate Of anther, embryo, cell
and suspension Cultures And Thus To Delay Or Reduce The Frequency Of Subculture.

• 1. Minimal Media and Growth Retardants: The use of minimal media

and growth retardants, such as abscisic acid (Henshaw et al. 1978) or the
growth of cultures in a sucrose-free medium (Jones 1974) has helped to
delay the subculture/transfer period.
• 2. Mineral Oil Overlay: Caplin reported in 1959 that the mineral oil
overlay of carrot callus tissue considerably reduced the rate of growth
and subsequently delayed the transfer frequency. Later, by using
mineral oil overlay, tissue cultures of various medicinal plants were
stored for 4-6 months without subculture (Augereau et al. 1986).
• 3. Desiccation: Nitzsche (1980) reported the growth of dried callus after 1
year of storage. Recently (see Gray et al. 1995), extensive studies have
been conducted on the desiccation of somatic embryos of carrot, grape,
alfalfa etc.
• 4. Low Pressure/Low Oxygen: Bridgen and Staby (1981) stored tissue
cultures under low atmospheric pressure and low oxygen and achieved four
fold reductions in the growth rate of callus cultures of tobacco, tomato, and
chrysanthemum.
2.Long term conservation of meristem or shoot tip
 Technique of cryopreservation
The cryopreservation of plant cell culture followed the
regeneration of plants broadly involves the following
stages
1. Development of sterile tissue culture.
2. Addition of cryoprotectant and pretreatment
3. Freezing
4. Storage
5. Thawing
6. Reculture
7. Measurement of survival/viability
8. Plant regeneration
 1:- Development of sterile tissue culture
The selection of plant species and the tissue with particular
reference to the morphological and physiological characters
largely influence the ability of the explants to survive in
cryopreservation . Any tissue from a plant can be used for
cryopreservation e.g. meristems, embryos, endosperm, ovules,
seeds, culture plants cells, protoplast, callus.
 2:- Addition of cryoprotectant
Cryoprotectant are the compound that can be prevent the
damage caused to cells by freezing or thawing. There are several
cryoprotectant which include (DMSO), glycerol, ethylene,
propylene , sucrose, mannose, glucose , proline and acetamide.
Among these DMSO, sucrose and glycerol are most widely used.
 3:- Freezing
The sensitivity of the cell to low temperature is variable and
largely depends on the plant species. Four different types of
freezing method are used:
Slow freezing method :
The tissue is slowly frozen at 0.5-5°C/min from 0°C to -
100°C,and then transferred to liquid nitrogen.
Rapid freezing method :
Decrease in temperature up to -300 to -1000°C.
Stepwise freezing method:
Intermediate temperature for 30 min. and rapidly
cool. Dry freezing method :
Reported that non- germinated dry seeds can survive freezing at
low temperature in contrast to water imbibing seeds
which are susceptible to cryogenic injuries.
 4 : Storage
Maintenance of the frozen cultures at the specific temperature
is as important as freezing . In general the frozen cells/tissues
are kept for storage at temperatures in the range of -72 to -
196°C. Storage is ideally done in liquid nitrogen refrigerator – at
150°C in the vapour phase, or at -196°C in the liquid phase.
The ultimate objective of storage is to stop all the cellular
metabolic activities and maintain their viability. For long term
storage temperature at -196°C in liquid nitrogen is ideal.
 5 : Thawing
 Thawing is usually carried out by plunging the frozen samples in ampoules into a

warm water (temp 37 – 45°C) bath with vigorous swirling. By this approach, rapid

thawing (at the rate of 500-750°C min¯¹) occurs, and this protects the cells from the

damaging effects ice crystal formation.

 As the thawing occurs (ice completely melts ) the ampoules are quickly transferred

to a water bath at temperature 20-25°C. This transfer is necessary since the cells

get damaged if left for long in warm (37-45°C) water bath.

 6 : Reculture
In general thawned germplasm is washed several times to remove
cryoprotectant. The material is then recultured in a fresh media.

7 : Plant regeneration
•The ultimate purpose of cryopreservation of germplasm is to regenerate
the desired plant . For appropriate plant growth and regeneration , the
cryopreserved cell/tissues/culture have to be carefully nursed, grown.
•Addition of certain growth promoting substances, besides maintenance
of appropriate environmental conditions is often necessary for successful
plant regeneration
 Applications of germplasm conservation
 Plant materials (cell/tissue) of several species can be cryopreserved
and maintained for several years, and used as and when needed.
 Cryopreservation is an ideal method for long term conservation of cell culture
which produce secondary metabolites e.g. medicines
 Disease (pathogen) free plant material can be frozen and propagated
whenever required.
 Recalcitrant seeds can be maintained for long .

 Conservation of somaclonal and gametoclonal variation in culture.

 Plant material from endangered species can be conserved.

 Cryopreservation is a good method for the selection of cold resistant mutant

cell lines which could develop into frost resistant plant .
 Limitations of Germplasm Conservation
 The expensive equipment needed to provide controlled and variable rates of

cooling/warming temperatures can however be a limitation in the application

of in vitro technology for large scale germplasm conservation.

 Formation of ice crystal inside the cell should be prevented as they cause

injury to the cell.

 Sometimes certain solutes from the cell leak out during freezing .

 Cryoprotectant also effect the viability of cells.

 MICROPROPAGATION
• Micropropagation is the practice of rapidly multiplying stock plant
material to produce a large number of progeny plants, using
modern plant tissue culture methods.
• Micropropagation is used to multiply noble plants such as those that
have been genetically modified or bred through conventional plant
breeding methods. It is also used to provide a sufficient number
of plantlets for planting from a stock plant which does not produce
seeds, or does not respond well to vegetative reproduction.
• Multiplication is the taking of tissue samples produced during the
first stage and increasing their number. Following the successful
introduction and growth of plant tissue, the establishment stage is
followed by multiplication. Through repeated cycles of this process,
a single explant sample may be increased from one to hundreds and
thousands of plants. Depending on the type of tissue grown,
multiplication can involve different methods and media.
 Micropropagation has a number of advantages over traditional plant propagation
techniques:
• The main advantage of micropropagation is the production of many plants that are
clones of each other.
• Micropropagation can be used to produce disease-free plants.
• It can have an extraordinarily high fecundity rate, producing thousands
of propagules while conventional techniques might only produce a fraction of this
number.
• It is the only viable method of regenerating genetically modified cells or cells
after protoplast fusion.
• It is useful in multiplying plants which produce seeds in uneconomical amounts, or
when plants are sterile and do not produce viable seeds or when seed cannot be
stored
• Micropropagation often produces more robust plants, leading to accelerated growth
compared to similar plants produced by conventional methods - like seeds or
cuttings.
• Some plants with very small seeds, including most orchids, are most reliably grown
from seed in sterile culture.
• A greater number of plants can be produced per square meter and the propagules
can be stored longer and in a smaller area.
 Disadvantages
• It is very expensive, and can have a labour cost of more than 70%.
• A monoculture is produced after micropropagation, leading to a lack of overall
disease resilience, as all progeny plants may be vulnerable to the same
infections.
• An infected plant sample can produce infected progeny. This is uncommon as
the stock plants are carefully screened and vetted to prevent culturing plants
infected with virus or fungus.
• Not all plants can be successfully tissue cultured, often because the proper
medium for growth is not known or the plants produce secondary metabolic
chemicals that stunt or kill the explant.
• Some plants are very difficult to disinfect of fungal organisms.
• The major limitation in the use of micropropagation for many plants is the cost
of production; for many plants the use of seeds, which are normally disease free
and produced in good numbers, readily produce plants (see orthodox seed) in
good numbers at a lower cost. For this reason, many plant breeders do
not utilize micropropagation because the cost is prohibitive. Other breeders use
it to produce stock plants that are then used for seed multiplication.