Hardening and Germplasm preservation.pptx

CLass- M.Sc-II SEM-IV
MSBOSC401T- Plant Tissue Culture-II
Unit-II: Hardening And Germplasm
Preservation
Presented By- MS. SHEETAL SHARMA

TO Study-
● Hardening stages
● Role of Polyhouse, Net House, Compost, Chemical
fertilizer, Cocopeat, Soil in hardening.
● Germplasm preservation- Definition, Importance and
Methods.
● In-situ and Ex-situ conservation, Centers of
germplasm preservation in India.

General stage of Micropropagation

Stage 0: (Pre-propagation Stage):
● Requires proper maintenance of the mother plants in the greenhouse under disease
and insect free conditions with minimal dust.
● Clean enclosed areas, glasshouses, plastic tunnels and net covered tunnels, provide
high quality explant source plants with minimal infection.
Stage 1: Initiation of Aseptic Culture:
● In this stage sterilization of explants and establishment of explants is done.
● The plant organ used to initiate a culture is called explant.
● The choice of explant depends on the method of shoot multiplication to be followed.

Stage 2: Multiplication of Culture:
● This is the most important stage.
● The rate of multiplication determines the large success of micropropagation system.
● This can be achieved by-Enhanced axillary branching by Adventitious bud formation or Through
callusing
Stage 3: In Vitro Rooting of Shoots:
● In-vitro grown shoots lack root system.
● For induction of roots explants are transferred to rooting medium.
● For rooting half strength MS medium supplemented should be supplemented with 1.0mg/l auxin.

Stage 4: Hardening and Acclimatization of Tissue Culture Plantlets
● This is the final stage and requires careful handling of plants.
● The plants grown in tissue culture vessels in laboratories can not be
directly taken to the field.
● In laboratory condition, the plants grow in high humidity, low light
intensity, high nutrition supply and in a narrow temperature range.
● The transplantation from completely controlled conditions should be
gradual.
● This process of gradually preparing the plants to survive in the field
conditions is called acclimatization.
● This gradual acclimatization process is known as Hardening.

● Tissue culture plantlets do not rely on photosynthesis; instead, they use sucrose
as a source of energy.
● Therefore, an early hardening-off process could start while the plants are still in
vitro.
● This process could gradually adjust the plantlets to rely on photosynthesis, and
activate the stomata so that loss of water through the cuticle is minimized.
● Also inside the culture vessels humidity is very high and thus the natural
protective covering of cuticle is not fully developed.
● Therefore immediately after transfer plants should be maintained under high
humidity.
● Optimum conditions should be provided to plants in the Greenhouse.

In the laboratory, plantlets can be hardened through the following measures either singly or in
combination:
1. Lowering the mineral salts (e.g. half strength MS media);
2. Increasing the concentration of the gelling agent. This has the effect of hardening the root
structure;
3. Changing the concentration of sucrose (higher or lower) in the growth media. By increasing the
sucrose content the plantlet stabilizes, does not grow so fast and becomes more robust. By
reducing the sucrose content the plantlet struggles to locate nutrients, with the effect of hardening
the root structure;
4. Lowering the relative humidity in the vessel to stimulate the formation of the wax layer on the
leaves limits the loss of water through evaporation.
The hardening process is generally done in two stages. In the first primary hardening stage, they are
prepared for photo-autotrophic growth and then in the secondary hardening stage they are gradually
exposed to harsher environment for acclimatization to field conditions.

Primary hardening :
● After adequate development of shoot and roots, the plants are taken out from the nutrient medium
and they are washed under running water to remove all traces of solid media from their roots.
● Then they are transplanted in small plastic or ceramic pots containing sterilized inert porous
substrate like vermiculite, pumice chips, ash free charcoal chips or dried sphagnum moss.
● The inert medium in the pots are then soaked with basal MS medium. The pots are incubated under
defused light for 2 – 3 weeks in a specific primary hardening area in the laboratory.
● As the inert substrate dry out with time, it is re-soaked with MS medium but the concentration of
carbon source is gradually reduced.
● Relatively high humidity is maintained during this stage to prevent drying out of the tender plants
and enhance survival.

Secondary hardening :
● In this stage, the primary hardened plants (in the pots used for primary hardening) are
transferred to green house and they are incubated under shade with regular misting for
maintenance of high humidity.
● After one week, the plants are transferred to poly tubes or seed trays filled with appropriate
potting mix.
● Different combinations of compost, peat moss, coco-peat, perlite, vermiculite or loamy soil
are used as potting mix.
● The plants are raised in the potting mix in green house conditions under 70% shade for 3 – 4
weeks.
● The optimal moisture condition of the potting mix is to be maintained throughout the growing
phase (preferably 90% of the field capacity).
● Then the plants are to be transferred to the hardening yard under full sunlight and to be kept
there for another two weeks.
● After this stage, the plants are ready to transplant in the field.

Role of Polyhouse, Net House, Compost, Chemical
fertilizer, Cocopeat, Soil in hardening

Summary of Hardening stages
1. Initiation: This is the first stage of plant tissue culture. In this stage, small pieces of plant tissue are
explanted from the donor plants and cultured in sterile media.
2. Multiplication and Rooting: In this stage, the explanted tissue is further cultured and multiplied
to form a cluster of explants. The explants are then subcultured and transferred to different media to
get more plants with desired characteristics.
3. Selection: In this stage, the explants are carefully selected based on the desired characteristics. The
selected explants are then transferred to the next stage.
4. Hardening: In this stage, the explants are hardened by gradually exposing them to the
environment. This helps the explants to adapt to the environment and become more resilient to
external conditions.
5. Acclimatization: In this stage, the hardened explants are transferred to the soil where they can
grow and develop into plants. The explants are monitored closely to ensure their successful

Role of Polyhouse in Hardening
● Polyhouse is a type of greenhouse, typically made of
plastic sheeting, that is used to create a controlled
environment for growing plants.
● It helps to maintain a higher temperature and humidity
level than the outside air, as well as protect plants from
pests, diseases and extreme weather.
● This helps to increase the growth rate of plants and also
helps to harden them off before they are transplanted into
the garden.
● The controlled environment also allows growers to be
able to control the amount of light, water and fertilizer
that the plants receive, which helps to produce a higher
quality crop.

Role of Net house in Hardening
● Net house is a type of greenhouse with a roof and
walls made of a very fine netting material.
● The netting helps to regulate the amount of light
and air that reaches the plants, and it also helps to
protect them from pests, wind, and other
environmental factors.
● The netting also helps to create a microclimate
that is ideal for the plants, allowing them to
harden off and become acclimatized to their new
environment.
● The net house can also be used to protect plants
from extreme temperatures, wind, and pests.

Role of Compost in Hardening
● Compost is organic matter that has been decomposed and
recycled as a fertilizer and soil amendment.
● Compost is also used to harden plants, as it can help to
improve soil structure, aeration, and nutrient holding capacity.
● Compost is an important source of nutrients for plants, and it
can also help in the hardening process.
● Compost increases the soil’s ability to retain water, which can
help reduce stress during droughts or extreme weather.
● Compost also helps reduce soil compaction, allowing for
better root growth and development.
● Compost adds organic matter to the soil, which improves soil
structure and drainage.
● It can also help to buffer soil pH and improve nutrient
availability for plants.
● Finally, compost increases the microbial activity in the soil,
which in turn helps to break down organic matter, improving
soil aeration and drainage.

Role of Chemical Fertilizer in Hardening
● Chemical fertilizers are substances containing essential plant
nutrients that are applied to soil or plant tissues to increase crop
yields.
● They are typically composed of nitrogen, phosphorus, and
potassium, as well as other micronutrients.
● Chemical fertilizers can be inorganic, such as ammonium nitrate or
sulfate, or organic, such as manure or compost.
● Chemical fertilizers can help to harden plants by providing essential
nutrients for plant growth.
● Plants need a variety of macronutrients and micronutrients for
proper growth and development, and chemical fertilizers can
provide these nutrients in the right amounts and ratios.
● By providing the right combination of nutrients, chemical fertilizers
can help to strengthen a plant’s roots and stems, leading to a hardier
overall structure.
● In addition, chemical fertilizers can help to improve the soil
structure, making it easier for plants to absorb the nutrients
necessary for healthy growth.

Role of Cocopeat in Hardening
● Coco peat, also known as coir pith, is a by-product of the
coconut industry and is made up of the coir fiber pith or
coir dust which is obtained by processing coconut husks.
● It is a multi-purpose growing medium used mainly for
plants, vegetables, and flowers.
● Coco peat helps in hardening of plants by providing them
with a good soil structure, adequate aeration and optimum
water-holding capacity.
● It is also used for seed germination and rooting, as it
provides better aeration for root growth and helps to retain
moisture.
● It helps to keep the soil loose and crumbly and prevents
compaction, thus allowing the roots to easily spread out
and grow.
● Coco peat also facilitates faster root development and
encourages healthy root growth.

Role of soil in Hardening
● Soil is the uppermost layer of the Earth’s surface, consisting of
rock and mineral particles mixed with organic matter.
● Soil is the mixture of organic matter, minerals, gases, liquids,
and organisms that together support life on Earth.
● Soil provides plants with essential nutrients, water, and air to
grow and thrive.
● Soil also serves as a foundation for the construction of
buildings and roads.
● The role of soil in hardening of plants is to provide the
necessary support, nutrients, and water to help plants grow and
develop.
● Soil provides a medium for the roots to grow and absorb water
and nutrients from the soil.
● Additionally, soil can help protect plants from extreme
temperatures and can serve as a buffer against wind and other
environmental factors that can damage the plants.
● Finally, soil can provide insulation for plants and help them
survive in cold weather.

Germplasm preservation- Definition, Importance
and Methods

Plant Genetic Resources-
● The sum totals of hereditary material i.e. all the alleles of various genes, present in a crop species
and its wild relatives is referred to as germplasm. This is also known as genetic resources or gene
pool or genetic stock.
● Important features of plant genetic resources are given below-
1. Genetic pool represents the entire genetic variability or diversity available in a crop species.
2. Germplasm consists of land races, modern cultivars, obsolete cultivars, breeding stocks, wild forms
and wild species of cultivated crops.
3. Germplasm includes both cultivated and wild species and relatives of crop plants.
4. Germplasm is collected from centres of diversity, gene banks, gene sanctuaries, farmer’s fields,
markers and seed companies.
5. Germplasm is the basic material for launching a crop improvement programme. Germplasm may
be indigenous (collected within country) or exotic (collected from foreign countries)

What is Germplasm Conservation?
● Germplasm broadly refers to the hereditary material (total content of genes) transmitted to the offspring
through germ cells.
● Germplasm provides the raw material for the breeder to develop various crops.
● As the primitive man learnt about the utility of plants for food and shelter, he cultivated the habit of
saving selected seeds or vegetative propagules from one season to the next one. In other words, this
may be regarded as primitive but conventional germplasm preservation and management, which is
highly valuable in breeding programmes.
● The very objective of germplasm conservation (or storage) is to preserve the genetic diversity of a
particular plant or genetic stock for its use at any time in future and to protect them from genetic
erosion.
● In recent years, many new plant species with desired and improved characteristics have started
replacing the primitive and conventionally used agricultural plants.
● It is important to conserve the endangered plants or else some of the valuable genetic traits present in
the primitive plants may be lost.

Importance of Germplasm Conservation
● In recent years, the primitive and conventionally used agricultural plants are being replaced by many new
plant species with desired and improved characteristics.
● It is very crucial to conserve the endangered plants otherwise some of the important genetic traits possessed
by the primitive plants may be lost.
● It has been estimated that up to 100,000 plants, depicting more than one third of all the world’s plant species,
are currently threatened or face extinction in the wild.
● Biodiversity is seriously threatened, particularly, in Europe.
● Biotechnological approaches provide several conservation possibilities which have the potential to support in
situ protection strategies and provide complementary conservation options.
● International Board of Plant Genetic Resources (IBPGR), a global body has been established for germplasm
conservation.
● Its main aim is to provide essential support for collection, conservation and utilization of plant genetic
resources all over the world.

In-situ and Ex-situ conservation

Types of Germplasm Conservation
● There are two approaches for germplasm conservation of plant genetic materials: 1. In-situ
conservation 2. Ex-situ conservation
1. In-situ Conservation
● Conservation of germplasm under natural conditions is referred to as in situ conservation.
● The in-situ conservation is considered as a high priority germplasm preservation programme.
● This is achieved by protecting the area from – human interference, such an area is often called natural park,
biosphere reserve or gene sanctuary etc.
● Thus, The conservation of germplasm in their natural environment by establishing protective areas is
regarded as in-situ conservation.
● This approach is particularly useful for preservation of land plants in a near natural habitat along with
several wild relatives with genetic diversity.
● NBPGR, New Delhi, established gene sanctuaries in Meghalaya for citrus, north Eastern regions for musa,
citrus, oryza and saccharum.

Merits of In-situ conservation
● It helps to protect the genetic integrity of the species. This is because the variety of a species
is preserved in its natural environment, where it has adapted to the conditions of that
particular habitat.
● It preserves the biodiversity at the species level.
● It helps to protect the habitats of the species, as well as other species associated with that
habitat.
● It helps to maintain the balance of the ecosystems.
● It allows for the species to evolve naturally and adapt to changing environmental conditions.
● It helps to reduce the risk of extinction of the species.
● Itallows for local communities to benefit from the species and its associated products.
● In this method of conservation, the wild species and the complete natural or semi-natural
ecosystems are preserved together.

Demerits of In-situ conservation
● Each protected area will cover only very small portion of total diversity of a crop species, hence several
areas will have to be conserved for a single species.
● The risk of losing germplasm due to environmental hazards
● The cost of maintenance of a large number of genotypes is very high.
● It is often difficult to manage in areas that are intensively used by humans, as it requires large areas of land.
● Thus it is is expensive and labour-intensive.
● In situ conservation is vulnerable to environmental changes, such as climate change, natural disasters, and
deforestation.
● In situ conservation is often difficult to monitor and control, as wild populations may be spread out over a
large area.
● In situ conservation may not be able to protect the full genetic diversity of the species, as some genes may
be lost due to natural selection.

2. Ex-situ Conservation
● Ex-situ conservation is the chief method for the preservation of germplasm obtained
from cultivated and wild plant materials.
● The genetic materials in the form of seeds or in form of in- vitro cultures (plant cells,
tissues or organs) can be preserved as gene banks for long term storage under suitable
conditions.
● For successful establishment of gene banks, adequate knowledge of genetic structure of
plant populations, and the techniques involved in sampling, regeneration, maintenance
of gene pools etc. are essential.
● This is the most practical method of germplasm conservation.

Merits of Ex-situ Conservation
● It is a more reliable way to protect plant species than in situ conservation, as it is less vulnerable to man-made
and natural risks.
● It enables researchers to study and understand the genetic diversity of a species more effectively.
● It can help to prevent or slow the process of extinction, which is particularly important in cases where the
species is threatened by habitat destruction.
● It can also help to introduce new varieties of plants into the environment, giving farmers more options when it
comes to crop selection and production.
● It can provide a source of seeds for replanting after natural disasters, or for restocking depleted species.
● It can also serve as a backup system in case of major environmental changes, such as climate change or the
introduction of invasive species.
● Finally, it can help to improve the quality of the gene pool and increase the biodiversity of an area.
● It is possible to preserve entire genetic diversity of a crop species at one place.
● Handling of germplasm is also easy. This is a cheap method of germplasm conservation.

Demerits of Ex-situ Conservation
● It is a costly process as it requires extensive resources for collection, storage, transportation and maintenance of the
germplasm.
● It requires large areas of land for storing the germplasm, which is not always available.
● There is a risk of contamination of germplasm during transportation or storage due to diseases or pests.
● Risk of genetic erosion: loss of genetic diversity between and within populations of the same species over time; or
reduction of the genetic basis of a species due to human intervention, environmental changes, etc. There is a risk of
genetic erosion due to the limited number of individuals stored in ex situ conservation.
● It may not be possible to recreate the exact conditions of the natural environment in ex situ conservation, which can
lead to the deterioration of the germplasm.
● Viability of seeds is reduced or lost with passage of time.
● This approach is exclusively confined to seed propagating plants, and therefore it is of no use for vegetatively
propagated plants e.g. potato, Ipomoea, Dioscorea. Thus, It is difficult to maintain clones through seed conservation.

Forms/ classification of Ex-situ Conservation
1. Seed banks
● Germplasm is stored as seeds of various genotypes.
● Seed conservation is quite easy, relatively safe and needs minimum space.
● Seeds are classified, on the basis of their storability into two major groups- i. Orthodox and ii. Recalcitrant
i. Orthodox seeds: Seeds which can be dried to low moisture content and stored at low temperature without
losing their viability for long periods of time is known as orthodox seeds. (eg.) Seeds of corn, wheat, rice, carrot,
papaya, pepper, chickpea, cotton, sunflower.
ii. Recalcitrant: Seeds which show very drastic loss in viability with a decrease in moisture content below 12 to
13% are known as recalcitrant seeds. (e.g) citrus, cocoa, coffee, rubber, oil-palm, mango, jack fruit etc.

Seed storage:
Based on duration of storage, seed bank collects are classified into three groups-(i) Base
collections, (ii) Active collections and, (iii) Working collection.
i) Base collections: Seeds can be conserved under long term (50 to 100 years), at about -20°C
with 5% moisture content. They are disturbed only for regeneration.
ii) Active collection: Seeds are stored at 0°C temperature and the seed moisture is between 5
and 8%. The storage is for medium duration, i.e., 10-15 years. These collections are used for
evaluation, multiplication, and distribution of the accessions.
iii) Working collections: Seeds are stored for 3-5 years at 5-10°C and usually contain about
10% moisture. Such materials are regularly used in crop improvement programmes.

There are however, certain limitations in the conservation of seeds:
i. Viability of seeds is reduced or lost with passage of time.
ii. Seeds are susceptible to insect or pathogen attack, often leading to their destruction.
iii. This approach is exclusively confined to seed propagating plants, and therefore it is
of no use for vegetatively propagated plants e.g. potato, Ipomoea, Dioscorea.
iv. It is difficult to maintain clones through seed conservation.
Certain seeds are heterogeneous and therefore, are not suitable for true genotype
maintenance.

2. Field/Plant bank
It is an orchard or a field in which accessions of fruit trees or vegetatively propagated crops are grown and
maintained.
Limitations:
1. Require large areas
2. Expensive to establish and maintain
3. Prone to damage from disease and insect attacks
4. Man – made
5. Natural disasters
6. Human errors in handling
3. Cell and Organs banks
A germplasm collection based on cryopreserved (at – 196 °C in liquid nitrogen) embryogenic cell cultures, somatic/
zygotic embryos are called cell and organ bank.

4. Shoot tip banks
Germplasm is conserved because of slow growth cultures of shoot-tips and nodal segments.
Conservation of genetic stocks by meristem cultures has several advantages as given below-
● Each genotype can be conserved indefinitely free from virus or other pathogens.
● It is advantageous for vegetatively propagated crops like potato, sweet potato, cassava etc., because seed
production in these crops are poor.
● Vegetatively propagated material can be saved from natural disasters or pathogen attack.
● Long regeneration cycle can be envisaged from meristem cultures.
● Regeneration of meristems is extremely easy. Plant species having recalcitrant seeds can be easily
conserved by meristem cultures.
5. DNA banks
In these banks, DNA segments from the genomes of germplasm accessions are maintained and conserved.

In-vitro methods of Ex-situ Conservation
● In vitro methods employing shoots, meristems and embryos are ideally suited for the conservation of germplasm of
vegetatively propagated plants.
● The plants with recalcitrant seeds and genetically engineered materials can also be preserved by this in vitro approach.
There are several advantages associated with in vitro germplasm conservation:
i. Large quantities of materials can be preserved in small space.
ii. The germplasm preserved can be maintained in an environment, free from pathogens.
iii. It can be protected against the nature’s hazards.
iv. From the germplasm stock, large number of plants can be obtained whenever needed.
v. Obstacles for their transport through national and international borders are minimal (since the germplasm is maintained under
aseptic conditions).
There are mainly three approaches for the in vitro conservation of germplasm:
I. Cryopreservation (freeze-preservation)
II. Cold storage
III. Low-pressure and low-oxygen storage

I. Cryobank & Cryopreservation (freeze-preservation)
Cryobank
A cryobank is a facility that is capable of storing germplasm ( normally in cell/ tissue form) under a such low
temperatures that the cells are prevented from damaging themselves or evolving from their initial state.
Cryopreservation:
● Cryopreservation (Greek word- krayos-frost) literally means preservation in the frozen state.
● The principle involved in cryopreservation is to bring the plant cell and tissue cultures to a zero metabolism
or non-dividing state by reducing the temperature in the presence of cryoprotectants.
● Cryopreservation techniques are probably the most promising approach for preserving ex situ plant genetic
resources and are of extreme importance when applied to plant species in danger of extinction.
● The ultra low temperatures of liquid nitrogen (-196ºC) allow the conservation of germplasm for a long period
of time without deterioration, because at these temperatures all metabolic processes are drastically reduced.
● Different plant materials, such as seeds, shoot tips, nodal explants, cell suspensions, dormant buds and others,
can be cryopreserved.

● Plant germplasm stored in liquid nitrogen does not undergo cellular divisions. In addition, metabolic and most
physical processes are stopped.
● Therefore, plant germplasm preserved under cryogenic storage can be maintained for very long periods of
time and problems that are typical for storage in the active growth state, like genetic instability and the loss of
accessions due to contamination, loss of vigour and totipotency and human error during continual sub-
culturing, are overcome.
Forms of Cryopreservation :
★ Over solid carbon dioxide (at -79°C)
★ Low temperature deep freezers (at -80°C)
★ In vapour phase nitrogen (at -150°C)
★ In liquid nitrogen (at -196°C)
Among these, the most commonly used cryopreservation is by employing liquid nitrogen. At the temperature of
liquid nitrogen (-196°C), the cells stay in a completely inactive state and thus can be conserved for long periods.

Mechanism of Cryopreservation:
The technique of freeze preservation is based on the transfer of water present in the cells from a liquid to a 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 a
standstill.
Precautions for successful cryopreservation :
i. Formation ice crystals inside the cells should be prevented as they cause injury to the organelles and the cell.
ii. High intracellular concentration of solutes may also damage cells.
iii. Sometimes, certain solutes from the cell may leak out during freezing.
iv. Cryoprotectants also affect the viability of cells.
v. The physiological status of the plant material is also important.

Technique of Cryopreservation:
The cryopreservation of plant cell culture followed by the regeneration of plants broadly involves the following
stages :
1. Development of sterile tissue cultures
2. Addition of cryoprotectants and pretreatment
3. Freezing
4. Storage
5. Thawing
6. Re-culture
7. Measurement of survival/viability
8. Plant regeneration.

1. Development of sterile tissue cultures
● The selection of plant species and the tissues with particular reference to the morphological and physiological
characters largely influence the ability of the explant to survive in cryopreservation.
● Any tissue from a plant can be used for cryopreservation e.g. meristems, embryos, endosperms, ovules, seeds, cultured
plant cells, protoplasts, calluses.
● Among these, meristematic cells and suspension cell cultures, in the late lag phase or log phase are most suitable.
1. Addition of cryoprotectants and pretreatment
● Cryo-protectants are the compounds that can prevent the damage caused to cells by freezing or thawing.
● The freezing point and super-cooling point of water are reduced by the presence of cryoprotectants. As a result, the ice
crystal formation is retarded during the process of cryopreservation.
● There are several cryoprotectants which include dimethyl sulfoxide (DMSO), glycerol, ethylene, propylene, sucrose,
mannose, glucose, proline and acetamide.
● Among these, DMSO, sucrose and glycerol are most widely used.
● Generally, a mixture of cryoprotectants instead of a single one is used for more effective cryopreservation without
damage to cells/tissues.

3. Freezing methods
3.1. Slow-freezing method:
The tissue or the requisite plant material is slowly frozen at a slow cooling rates of 0.5-5°C/min from
0°C to -100°C, and then transferred to liquid nitrogen. The advantage of slow-freezing method is that some amount
of water flows from the cells to the outside. This promotes extracellular ice formation rather than intracellular
freezing. As a result of this, the plant cells are partially dehydrated and survive better. The slow-freezing procedure
is successfully used for the cryopreservation of suspension cultures.
3.2. Rapid freezing method:
This technique is quite simple and involves plunging of the vial containing plant material into liquid
nitrogen. During rapid freezing, a decrease in temperature -300° to -1000°C/min occurs. The freezing process is
carried out so quickly that small ice crystals are formed within the cells. Further, the growth of intracellular ice
crystals is also minimal. Rapid freezing technique is used for the cryopreservation of shoot tips and somatic
embryos.

3. Freezing methods
3.4. Stepwise freezing method:
This is a combination of slow and rapid freezing procedures (with the advantages of both), and is
carried out in a stepwise manner. The plant material is first cooled to an intermediate temperature and maintained
there for about 30 minutes and then rapidly cooled by plunging it into liquid nitrogen. Stepwise freezing method has
been successfully used for cryopreservation of suspension cultures, shoot apices and buds.
3.6. Dry freezing method:
Some researchers have reported that the non-germinated dry seeds can survive freezing at very low
temperature in contrast to water-imbibing seeds which are susceptible to cryogenic injuries. In a similar fashion,
dehydrated cells are found to have a better survival rate after cryopreservation.

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 -70 to -196°C.
● However, with temperatures above -130°C, ice crystal growth may occur inside the cells which reduces
viability of cells.
● Storage is ideally done in liquid nitrogen refrigerator — at 1 50°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.
● A regular and constant supply of liquid nitrogen to the liquid nitrogen refrigerator is essential.
● It is necessary to check the viability of the germplasm periodically in some samples.
● Proper documentation of the germplasm storage has to be done.

5. Thawing:
● Thawing is usually carried out by plunging the frozen samples in ampoules into a warm water (temperature 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.
● For the cryopreserved material (cells/tissues) where the water content has been reduced to an optimal level before
freezing, the process of thowing becomes less critical.
6. Re-culture:
● In general, thawed germplasm is washed several times to remove cryoprotectants.
● This material is then re-cultured in a fresh medium following standard procedures.
● Some workers prefer to directly culture the thawed material without washing.
● This is because certain vital substances, released from the cells during freezing, are believed to promote in vitro cultures.

7. Measurement of survival/viability:
● The viability/survival of the frozen cells can be measured at any stage of cryopreservation or after thawing or re-
culture.
● The techniques employed to determine viability of cryopreserved cells are the same as used for cell cultures.
● Staining techniques using triphenyl tetrazolium chloride (TTC), Evan’s blue and fluorescein diacetate (FDA) are
commonly used.
● The best indicator to measure the viability of cryopreserved cells is their entry into cell division and regrowth in
culture. This can be evaluated by the following expression-
8. Plant regeneration:
● The ultimate purpose of cryopreservation of germplasm is to regenerate the desired plant.
● For appropriate plant growth and regeneration, the cryopreserved cells/tissues have to be carefully nursed, and grown.
● Addition of certain growth promoting substances, besides maintenance of appropriate environmental conditions is
often necessary for successful plant regeneration.

II. Cold Storage
● Cold storage basically involves germplasm conservation at a low and non-freezing temperatures (1-9°C) The
growth of the plant material is slowed down in cold storage in contrast to complete stoppage in
cryopreservation. Hence, cold storage is regarded as a slow growth germplasm conservation method. The
major advantage of this approach is that the plant material (cells/tissues) is not subjected to cryogenic injuries.
● Long-term cold storage is simple, cost-effective and yields germplasm with good survival rate. Many in vitro
developed shoots/plants of fruit tree species have been successfully stored by this approach e.g. grape plants,
strawberry plants.
● Virus- free strawberry plants could be preserved at 10°C for about 6 years, with the addition of a few drops of
medium periodically (once in 2-3 months). Several grape plants have been stored for over 15 years by cold
storage (at around 9°C) by transferring them yearly to a fresh medium.

● As alternatives to cryopreservation and cold storage, low-pressure storage (LPS) and low-oxygen storage
(LOS) have been developed for germplasm conservation.
● A graphic representation of tissue culture storage under normal atmospheric pressure, low-pressure and low-
oxygen is depicted in figure Below-
III. Low-pressure and low-oxygen storage

A. Low-Pressure Storage (LPS): In low-pressure storage, the atmospheric pressure surrounding the plant
material is reduced. This results in a partial decrease of the pressure exerted by the gases around the
germplasm. The lowered partial pressure reduces the in vitro growth of plants (of organized or
unorganized tissues). Low-pressure storage systems are useful for short-term and long-term storage of plant
materials. The short-term storage is particularly useful to increase the shelf life of many plant materials e.g. fruits,
vegetables, cut flowers, plant cuttings. The germplasm grown in cultures can be stored for long term under low
pressure. Besides germplasm preservation, LPS reduces the activity of pathogenic organisms and inhibits spore
germination in the plant culture systems.
A. Low-Oxygen Storage (LOS):
In the low-oxygen storage, the oxygen concentration is reduced, but the atmospheric pressure (260 mm Hg) is
maintained by the addition of inert gases (particularly nitrogen). The partial pressure of oxygen below 50 mm Hg
reduces plant tissue growth (organized or unorganized tissue). This is due to the fact that with reduced availability of
O2, the production of CO2 is low. As a consequence, the photosynthetic activity is reduced, thereby inhibiting the
plant tissue growth and dimension.

Centers of germplasm preservation in India

● Germplasm refers to the genetic material of plants, animals, and microorganisms that can be used for breeding and developing new
varieties with desirable traits.
● India is a mega-diverse country with a rich genetic diversity of plant and animal species.
● To conserve this diversity, India has established several centers for germplasm preservation that are responsible for collecting,
conserving, and distributing the germplasm of different crops and their wild relatives.
● These centers are located across India and are responsible for the conservation and sustainable utilization of genetic resources of
different crops, animals, and microorganisms.
● The main objective of germplasm conservation is to preserve genetic diversity and prevent the loss of valuable genetic resources due
to natural calamities, human activities, or genetic erosion.
● Germplasm conservation centers use various methods to conserve genetic resources, including seed storage, in vitro conservation,
cryopreservation, and field gene banks.
● Germplasm conservation centers play a crucial role in the conservation and sustainable utilization of plant, animal, and microbial
genetic resources.
● They help in the development of new varieties with desirable traits, the improvement of agricultural productivity, and the promotion
of food security.
● Germplasm centres aim to preserve and protect the unique genetic makeup of various species to prevent their extinction, enhance
agricultural productivity, and provide a source of genetic material for future breeding programs.

Here is a list of all the germplasm preservation centers in India:
● National Bureau of Plant Genetic Resources (NBPGR)
● Central Potato Research Institute (CPRI)
● Central Tuber Crops Research Institute (CTCRI)
● National Bureau of Fish Genetic Resources (NBFGR)
● Central Institute of Freshwater Aquaculture (CIFA)
● Central Marine Fisheries Research Institute (CMFRI)
● Central Plantation Crops Research Institute (CPCRI)
● Indian Institute of Horticultural Research (IIHR)
● Central Citrus Research Institute (CCRI)
● National Research Centre on Seed Spices (NRCSS)
● National Bureau of Soil Survey and Land Use
Planning (NBSS&LUP)
● Central Arid Zone Research Institute (CAZRI)
● National Research Centre on Camel (NRCC)
● Central Institute for Research on Goats (CIRG)
● National Research Centre on Equines (NRCE)
● National Research Centre on Pig (NRCP)
● National Bureau of Agriculturally Important
Microorganisms (NBAIM)
● National Bureau of Animal Genetic Resources
(NBAGR)
Insects (NBAII)
Fungi (NBAIF)
● National Bureau of Plant Protection (NBPP)

Here are some of the Germplasm Conservation Centres in India in detail:
1. National Bureau of Plant Genetic Resources (NBPGR): The NBPGR is the premier institute in India
responsible for the collection, conservation, and documentation of plant genetic resources. It is located in New
Delhi it also has regional stations in different parts of India to collect, conserve, and distribute germplasm of
crops adapted to specific agro-climatic conditions.The NBPGR's gene bank is the largest gene bank in India,
with over 450,000 accessions of plant genetic resources.
2. National Agri-Food Biotechnology Institute (NABI): It is located in Mohali, Punjab, and is a research
institute that focuses on agri-food biotechnology. The center has a gene bank that stores plant and microbial
genetic resources for research and conservation.
3. Central Potato Research Institute (CPRI): It is located in Shimla, Himachal Pradesh, and is responsible for
the development of potato varieties and their conservation. The institute maintains a potato gene bank that
preserves more than 10,000 accessions of potato germplasm.
4. National Rice Research Institute (NRRI): It is located in Cuttack, Odisha, and is responsible for the
development of improved rice varieties and their conservation. The institute has a rice gene bank that
preserves over 40,000 accessions of rice germplasm.

5. Indian Council of Agricultural Research (ICAR): It is an apex body for coordinating, guiding, and managing
research and education in agriculture in India. The ICAR has several institutes and centers across the country that
focus on different aspects of agriculture, including the conservation and preservation of plant genetic resources.
6. National Centre for Cell Science (NCCS): Located in Pune, NCCS maintains a repository of microbial strains,
including bacteria, fungi, and viruses. It also conducts research on the diversity and ecology of microorganisms.
7. Indian Agricultural Research Institute (IARI): Located in New Delhi, IARI maintains a germplasm bank of
various crop plants and conducts research on crop improvement, including the development of high-yielding and
disease-resistant varieties.
8. Central Institute of Medicinal and Aromatic Plants (CIMAP): It is located in Lucknow and is responsible for
the conservation of medicinal and aromatic plants. It has a germplasm bank that houses around 30,000 accessions of
290 species.
9. National Research Centre on Plant Biotechnology (NRCPB): It is located in New Delhi and is responsible for
the conservation of genetically modified plants. It has a plant tissue culture laboratory and a germplasm repository.

11. National Gene Bank: The National Gene Bank is the main centre for germplasm conservation and is located in New
Delhi. It is responsible for collecting, conserving, characterizing, evaluating and documenting plant genetic resources.
12. National Bureau of Fish Genetic Resources (NBFGR): NBFGR is located in Lucknow, Uttar Pradesh, and is
responsible for the conservation and management of fish genetic resources. It maintains a collection of over 20,000
accessions of fish germplasm, including both freshwater and marine species.
13. National Bureau of Animal Genetic Resources (NBAGR): NBAGR is located in Karnal, Haryana, and is responsible
for the conservation and management of animal genetic resources. It maintains a collection of over 4,000 accessions of
livestock germplasm, including cattle, buffalo, sheep, goat, and pig.
14. National Bureau of Soil Survey and Land Use Planning (NBSS&LUP): NBSS&LUP is located in Nagpur,
Maharashtra, and is responsible for the conservation and management of soil genetic resources. It maintains a collection of
more than 70,000 soil samples representing different soil types from all over India.
15. Institute of Forest Genetics and Tree Breeding (IFGTB): IFGTB is located in Coimbatore, Tamil Nadu, and is
responsible for the conservation and management of forest genetic resources. It maintains a collection of over 2,000
accessions of tree species, including both indigenous and exotic species.

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