Application of Tissue Culture With Relation to Valuable Germplasm

8/8/2019 Application of Tissue Culture With Relation to Valuable Germplasm

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Application of Tissue culture with relation to Valuable Germplasm

Jnanendra Narayan sengupta

Germplasm : Germplasm refers to the sum total of all the genes present in a crop and its related species.

The conservation of germplasm involves the preservation of the genetic diversity of a particular plant or

genetic stock for its use at any time in future. It is important to conserve the endangered plants or else

some of the valuable genetic traits present in the existing and primitive plants will be lost.

Genetically improved cultivated plants and domesticated animals provide an assured food supply. To

meet the current demands of an increasing world population there is always a need to increase the

production potential.

Germplasm is the source of the genetic potential of living organisms. Diversified germplasm

allows organisms to adapt to changing environmental conditions. No single individual of any species

contains all the genetic diversity of that species. Hence the total genetic potential is represented only in

populations made up of many individuals. Such genetic potential is referred to as the genepool. The

potential represented in a genepool is the foundation for our crop plants.

Germplasm is only maintained in living tissue, most often the embryo of seeds. When the seed

dies the germplasm is also lost. The limited number of plants that has historically fed the human

population is approximately 1 percent of the flora of the world, and the number that have entered

agriculture is a small fraction of that percent. Today we are depending upon about 150 plant species.

Extinction of a species or a genetic line represents the loss of a unique resource. This type of

genetic and environmental impoverishment is irreversible. Any reduction in the diversity of resources

narrows societys scope to respond to new problems and opportunities. To the extent that we cannot be

certain what needs may arise in the future : new plant diseases or pests, climatic change due to the

greenhouse effect, etc.

Germplasm or plant genetic resources:

The sum total of genes in a crop species is referred to as genetic resources or gene pool or genetic stockor germplasm. In other words, gene pool refers to a whole library of different alleles of a species.

Germplasm or gene pool is the basic material with which a plant breeder has to initiate his breeding

progrmme. Some important features of plant genetic resources or gene pool are given below:

1. Germplasm represents the entire genetic variability or diversity available in a crop species.



2. Germplasm is collected from the centres of diversity, gene banks, gene sanctuaries, farmers

fields, markets and seed companies.

3. Germplasm includes both cultivated and wild species or relatives of crop plants

4. Germplasm consists of land races, modern cultivars, obsolete cultivars, breeding stocks and

wild forms and species of cultivated crops.

5. Germplasm is the basic material for launching a crop improvement programme

Classification of Genepool

Various criteria are used for the classification of genetic resources or gene pool or germplasm.

Gene pool can be classified on the basis of: (1) area of collection, (2) domestication, (3) duration of

conservation and (4) cross ability in breeding programmes. A brief classification of germplasm on the

basis of these criteria is presented below.

Area of collection

Based on area of collection germplasm is divided into two groups: (1) Indigenous and (2) exotic.

The germplasm which is collected with in the country is referred to as indigenous germplasm. On the

other hand, the germplasm which is received or collected from other countries is known exotic

germplasm.

Domestication

On the basis of domestication again germplasm can be divided into two groups, viz,

(1) Cultivated and

(2) Wild.

The germplasm of domesticated spices is known as cultivated germplasm, whereas that of

uncultivated species is referred to as wild germplasm. Cultivated germplasm offers greater

opportunities for use in the breeding programmes than wild germplasm.

Duration of conservation

Depending upon the duration of conservation, germplasm is of three types:

(a) Base collection,

(b) Active collection, and



(c) Working collections.

(a) Base collections: Plant materials which are meant for long term storage or conservation are referred

to as base collections. Such collections are disturbed only for the purpose of regeneration. The

regeneration is carried out after a long time depending on the viability of seeds. The seed viability

should not drop to less than 95% before regeneration. Base collections contain seeds with 5+1 %

moisture content. Seeds are stored in sealed containers at 18 to 20oC.

(b) Active collections: The germplasm which is meant for medium term storage (10 to 15 years) is

referred to as active collection. Such collections are subjected to regeneration, multiplication,

evaluation, distribution and documentation after every 10-15 years. Such collections are stored at

around zero degree Celsius temperature and seed moisture should be around 8%. In these collections,

routine germination tests are carried out after every 5 to 10 years to assess the reduction in germinationpercentage. Large samples (50 to 100) have to be grown for regeneration to prevent genetic drift.

(c) Working collections. The genetic resources which are stored for short term (3 to 5 years) are known

as working collections. Such materials are regularly used in crop improvement programmes. There is no

need to grow such material every year. The seed is stored at 5oC to 10

oC with seed moisture content of

8 to 10%.

Utilisation in breeding (crossability)

Depending upon the crossability, gene pool is of three types: viz,

1. Primary gene pool,

2. Secondary gene pool and

3. Tertiary gene pool.

Primary gene pool (GP1)

The gene pool in which intermating (crossing) is easy and leads to production of fertile hybrids is

known as primary gene pool. It includes plants of the same species or of closely related species which

produce completely fertile off spring on intermating. In such gene pool, genes can be exchanged

between lines simply by making normal crosses. This is also known as gene pool one (GP1). This is the

material of prime breeding importance.



Secondary gene pool (GP2)

The genetic material that leads to partial fertility on crossing with GP1 is referred to as

secondary gene pool. It includes plants that belong to related species. Such material can be crossed

with primary gene pool, but usually the hybrids are sterile and some of the progeny to some extent are

fertile. Transfer of gene from such material to primary gene pool is possible but difficult. This type of

gene pool is also known as gene pool two (GP2).

Tertiary gene pool (GP3)

The genetic material which leads to production of sterile hybrids on crossing with primary gene

pool is termed as tertiary gene pool or gene pool three (GP3). It includes material which can be crossed

with GP1, but the hybrids are sterile. Transfer of genes from such material to primary gene pool is

possible with the help of special techniques.

Components of Genetic Resources

Various plant materials which constitute genepool or germplasm are known as components of

genetic resources.

There are seven major components of genepool, viz,

(1) Land races,

(2) Obsolete cultivars,

(3) Modern cultivars,

(4) Advanced breeding materials,

(5) Wild forms of cultivated species,

(6) Wild relatives and

(7) Mutants.

Land races

Land races are nothing but primitive cultivars which were selected and cultivated by the farmers

from many generations. Main features of land races are given below.



1. Land races were not deliberately bred like modern cultivars. They evolved under

subsistence agriculture.

2. Land races have high level of genetic diversity which provides them high degree of

resistance to biotic and abiotic stresses. Biotic stress refers to hazards of diseases and

insects, whereas abiotic stress means, drought, salinity, cold, frost, etc.

3. Land races have broad genetic base which again provides them wider adaptability and

protection from epidemic of diseases and insects.

Land Races according to J.R. Harlan (1975) have certain gene integrity. There are recognizable

morphologically; farmers have names for them and different land races are understood to differ in

adaptation to type, time of seeding, date of maturity, height, nutritive value and other properties. Most

important, they are genetically diverse. They are balanced populations variable in equilibrium with both

environment and pathogen and genetically dynamic.

Land races even respond to selection for high yield, but to certain extent. Since land races

possess valuable alleles, their conservation is essential. The main drawbacks of land races are that they

are less uniform and low yielders. Land races were first collected and studied by N.I. Vavilov in rice.

Obsolete cultivars

Improved varieties of recent past are known as obsolete cultivars. They are the varieties which

were popular earlier and now have been replaced by new varieties. These varieties have several

desirable characters and constitute an important part of genepool. For example, wheat varieties K68,

K65 and Pb 591 were most popular traditional tall varieties before introduction of high yielding Mexican

wheat varieties. These varieties are well known for their attractive grain colour and chapatti making

quality. Now these varieties are no more cultivated. They are good genetic resources and have been

widely used in wheat breeding programmes especially in India for improvement of grain quality. Now

such old varieties are found in the gene pool only.

Modern cultivars

The currently cultivated high yielding varieties are referred to as modern cultivars. Modern

cultivars are also known as improved cultivars or advance cultivars. These varieties are high yield

potential and uniformity as compared to obsolete varieties and land races. Modern cultivars constitutea major part of working collections and are extensively used as parents in the breeding programmes for

further genetic improvement in various characters. Hence these cultivars are in great demand. These

varieties are the outcome of scientific plant breeding and have been developed for modern intensive

agriculture. However, modern cultivars have narrow genetic base and low adaptability compared to

land races.



Advanced breeding lines

Pre-released plants which have been developed by plant breeders for use in modern scientific

plant breeding are known as advanced lines, cultures and stocks. They include advanced cultures which

are not yet ready for release to farmers. Sometimes advanced breeding lines and stocks are not very

much productive, but constitute valuable part of gene pool for various economic characters.

Wild forms of cultivated species

Wild forms of cultivated species are available in many crop plants. Such plants have generally

high degree of resistance to biotic and abiotic stresses and are utilized in breeding programmes for

genetic improvement of resistance to biotic and abiotic stresses. They can easily cross with cultivated

species. However, wild forms of many crop species are extinct. Moreover, entire range of diversity of

available wild forms is rarely lapped. They constitute small part of gene pool.

Wild relatives

Those naturally occurring plant species which have common ancestry with crops and can cross

with crop species are referred to as wild relatives or wild species. Wild relatives are important sources

of resistance to biotic diseases and insects and abiotic (drought, cold, frost, salinity, etc) stresses.

However, wild relatives are used as the last resort in crop improvement programmes, because their use

in crossing leads to: (1) hybrid sterility (2) hybrid viability and (3) transfer of several undesirable genes to

the cultivated species along with desirable alleles. This group constitutes a minor part of gene pool.

Interspecific derivatives are added to the gene pool.

Mutants

Mutation breeding is used when the desired character is not found in the genetic stocks of

cultivated species and their wild relatives. Mutations do occur in nature as well as can be induced

through the use of physical and chemical mutagens. The extra variability which is created through

induced mutations constitutes important component of gene pool. Mutants for various characters

sometimes may not be released as a variety, but they are added in the gene pool. For example, mutant

genepool Dee-Geo-Woo-Gen in rice and Norin 10 in wheat proved to be valuable genetic resources inthe development of high yielding and semi dwarf varieties in the respective crop species. In seed

propagated crops, 410 varieties have been released through the use of mutants in the crosses (IAEA,

1991).



The strategies for germplasm conservation is based on

1) Genetic erosion , 2) genetic wipeout, 3) genetic vulnerability, 4) gene banks and 5) training for

plant breeding.

Genetic erosion: Current elite varieties yield better than older varieties. If the older varieties is no longer

planted its genes are lost to future generations unless it is conserved. This genetic erosion leaves a

diminished genepool. Eg. Bt cotton

y Replacement of land races with improved cultivars. The main features of modern cultivars are

high yield, uniformity, narrow genetic base and narrow adaptability. On the other hand land

races and primitive cultivars have more genetic diversity, broad genetic base, under adaptability

and low yield potential. Thus replacement of land races with modern cultivars has resulted in

reduction of genetic diversity because land races are disappearing.

y Modernization of agriculture. Clean and modern agriculture has resulted in the elimination of

wild and weedy forms of many crops. These weedy forms enhance the genetic diversity through

introgression of genes from crop to weedy forms and weedy forms to crop plants.y Extension of farming into wild habitats. It has resulted in destruction of wild relatives of various

crops resulting in reduction of their genetic diversity.

y Grazing into wild habitats. Grazing of animals in the wild habitats also reduces genetic diversity

by destroying the wild and weedy forms of crop plants.

y Growth of towns, cities, roads, air ports and industrial areas as a part of development activities

also lead to genetic erosion of crop plants. Because vast areas are cleaned for such activities.

Genetic wipeout: Genetic erosion is a slow gradual process while genetic wipeout is the rapid and

complete destruction of genetic resources. Social disruptions such as political instability or crop failure

and famine due to natural disasters can eliminate genetic resources rapidly.

Genetic vulnerability: It means the extent to which crops are unprotected from potential damage from

unsuspected pathogens and pests. This is especially most important when the crop is highly and

uniformly susceptible. Eg corn blight, green ear in pearl millet.

Gene bank or germplasm bank:

Gene banks are an institutional solution to genetic erosion and genetic wipeout.

There are five important activities related to plant genetic resources were

(1) Exploration and collection

(2) Conservation,



(3) Evaluation

(4) Documentation

(5) Distribution and

(6) Utilization.

1. EXPLORATION AND COLLECTION

(a) Exploration refers to collection trips and collection refers to tapping of genetic diversity from

various sources and assembling the same at one place. Collection is essential to reduce the loss of

genetic diversity which is taking place due to genetic erosion and extinction.

The exploration and collection is a highly scientific process. This process takes into account six

important items, viz.,

(1) Sources of collection,

(2) Priority of collection

(3) Agencies of collection

(4) Methods of collection

(5) Methods of sampling and

(6) Sample size.

1. Sources of collection.

There are five important sources of germplasm collection viz (1) centres of diversity (2) gene

banks (3) gene sanctuaries (4) seed companies and (5) farmers fields. Moreover, collections can be

made by local exploration trips to the regions of crop diversity. Gene banks are organizations where

genetic diversity is maintained in living state. Gene banks are also known as germplasm banks or gene

pools. In India, gene banks are generally maintained by respective crop research institute of ICAR (Table

.)

Table . Gene banks of various crops in India

Crop species Location of gene bank centre

Wheat DWR, Karnal (Haryana)



Rice CRRI, Cuttack (Orissa)

Potato CPRI, Shimla (Himachal Pradesh)

Cotton CICR, Nagpur (Maharashtra)

Sugarcane SBI, Coimbatore (Tamil Nadu)

Tobacco CTRI, Rajahmundry (Andhra Pradesh)

Pulses IIPR, Kanpur, (Uttar Pradesh)

Forage Crops IGFRI, JhansiPlantation Crops CPCRI,Kasargod, (Kerala)

Tuber Crops (except Potato) CTCRI, Trivandrum (Kerala)

Oilseed Crops DOR, Hyderabad (Andhra Pradesh)

Horticultural Crops IIHR, Bangalore (Karnataka)

Sorghum NRC Sorghum, Hyderabad (Andhra Pradesh)

Groundnut NRC Groundnut, Junagadh (Gujarat)

Soybean NRC Soybean, Indore (Madhya Pradesh)

Maize IARI, New Delhi

Besides Crop Research Institutes, State Agricultural Universities at private seed companies also

maintain huge collection of different crops. On global basis, the gene banks are maintained by

International Crop Research Institutes. International Research Institute maintains the gene bank of

concerned crops (Table .).

Table . Gene banks maintained by various International Crop Research Institutions

Location of gene bank Crops maintainedIRRI, Philippines Rice

CIMMYT, Mexico Maize, Wheat, Triticale and Barley

CIAT, Colombia Cassava, beans, Rice and Maize

IITA, Nigeria Cowpea, Soybean, Lima bean, Cassava, Sweet Potato, yam Rice and

Maize

CIP, Peru Potato

ICRISAT, India Sorghum, Pearl Millet, Pigeon pea, Chick pea, Groundnut

ICARDA, Syria Durum wheat, Barley and Beans

Florida, USDA Sugarcane

Gene Sanctuaries

Gene sanctuaries refer to protected areas of great genetic diversity under conditions. Various

plant species are protected in such areas, hence gene sanctuaries are important sources of germplasm

collection. Farmers fields especially in the tribal areas are also important sources of germplasm

collection, because tribals still grow old cultivars which have great genetic diversity.



2. Priority of collection

The next important step in the germplasm collection is to fix priority of collection. Some areas

of diversity have been threatened more than others by the danger of extinction. Similarly, some crop

species have more risk of extinction than others. Hence, endangered areas and endangered species

should be given priority for germplasm collection.

3. Agencies of collection

The task of germplasm collection is undertaken by crop Research institutes and State

Agricultural Universities in collaboration with National Bureau of Plant Genetic Resources, New Delhi for

Indigenous collections. For global collection, the task is undertaken in collaboration with International

Plant Genetic Resources Institute (IPGRI), Rome Italy.

4. Method of collection.

Germplasm collections are made in four principal ways viz., (1) through expeditions to the areas

or regions of genetic diversity (2) by personal visit to gene bank centre (3) through correspondence (4)

through exchange of material.

5. Method of sampling

There are two sampling methods for collection of germplasm from the regions of diversity viz.,(1) random sampling, and (2) biased sampling. Random sampling is effective in capturing of alleles for

biotic and abiotic stresses, whereas non random or biased sampling is useful in collection of

morphologically distinct genotypes. Hence, it is advised that both random as well as biased sampling

procedures should be adopted to tap the maximum genetic diversity of a crop species.

6. Sample size.

The sample size should be such that 95% of the total genetic diversity can be captured from the

area of collection. To achieve this goal, 50 to 100 individuals should be collected per site with 50 seeds

per plant.

Merits and Demerits

There are several merits and demerits of exploration and collection of germplasm, some of

which are as discussed below:



Merits

1. Collection helps in tapping crop genetic diversity and assembling the same at one place. It

reduces the loss of genetic diversity due to genetic erosion.

2. Sometimes, we get material of special interest during exploration trips

3. Sometimes, we come across a new plant species during the process of collection

4. Collection also helps in saving certain genotypes from extinction. Once the material is

collected, it can be maintained further in a germplasm

Demerits

1. Collection of germplasm especially from other countries, sometime leads to entry of new

diseases, new insects and new weeds.

2. Collection is a tedious job. The collection has to be made generally from uncultivated

areas like hills, mountains, river valleys and forests, where the collector faces problems of

boarding, lodging and transportation.

3. In the remote areas, the collector, sometimes has to encounter with wild animals likeelephants, rhinos, tigers, lions and snakes which involves risk of life.

4. Transportation of huge collections also poses difficulties in the exploration and collection.

2. CONSERVATION

Conservation refers to protection of genetic diversity of crop plants from genetic erosion. There

are two important methods of germplasm conservation or preservation, viz (1) in situ conservation, and

(2) ex situ conservation. These are described below

In situ Conservation

Conservation of germplasm under natural conditions is referred to as in situ conservation. It

requires establishment of natural or biosphere reservoir, national parks or protection of endangered

areas or species. In this method of conservation, the wild species and the complete natural or semi

natural ecosystems are preserved together. This method of preservation has following main

disadvantages.

1. 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.

2. The management of such areas also poses several problems

3. This is a costly method of germplasm conservation.

Ex situ Conservation

It refers to preservation of germplasm in gene banks. This is the practical method of germplasm

conservation. This method has following main advantages.



1. It is possible to preserve entire genetic diversity of a crop species at one place

2. Handling of germplasm is also easy

3. This is a cheap method of germplasm conservation

The germplasm is conserved either (1) in the form of seed, or (2) in the form of meristem

cultures. Preservation in the form of seed is the most common and easy method. Seed conservation is

relatively safe, requires minimum space (except coconut, etc) and easy to maintain. Glass, tin or plastic

containers are used for preservation and storage of seeds. The seeds can be conserved under long term

(50 to 100 years), medium term (10 to 15 years) and short term (3-5 years) storage conditions.

Roberts (1973) has classified seeds into two groups for storage purpose: viz

(1) Orthodox and

(2) Recalcitrant.

Orthodox

The seeds which can be dried to low moisture content and stored at low temperature without

loosing their viability are known as orthodox seeds. This group includes seeds of corn, wheat, rice,

carrot, beets, papaya, pepper, chcickpea, lentil, soybean, cotton, sunflower, various beans, egg plant

and all the brassicas.

Recalcitrant

The seeds which show very drastic loss in viability with a decrease in moisture content below 12

to 13% are known as recalcitrant seeds. This group includes cocoa, coconut, mango, tea, coffee, rubber,

jackfruit and oil plam seeds. Such seeds cannot be conserved in seed banks and, therefore, require in

situ conservation. Crop species with recalcitrant seeds are conserved in field gene banks. These are

simply areas of land in which collection of growing plants are assembled.

For conservation of meristem cultures, meristem or shoot tip bank are established.

Conservation of genetic stocks by meristem cultures has several advantages as given below:



1. Exact genotypes can be conserved indefinitely free from virus or other pathogens and

without loss of genetic integrity

2. It is advantageous for vegetatively propagated crops like potato, sweet potato, cassava, etc.,

because seed production in these crops is poor.

3. Vegetatively propagated material can be saved from natural disasters or pathogen attack.

4. Long regeneration cycle can be envisaged from meristem cultures.

5. Perennial plants which take 10 to 20 years to produce seeds can be preserved any time by

meristem cultures.

6. Regeneration of meristems is extremely easy

7. Plant species having recalcitrant seeds can be easily conserved by meristem cultures.

3. EVALUATION

Evaluation refers to screening of germplasm in respect of morphological genetical, economic,

biochemical, physiological, pathological and entomological attributes. Evaluation of germplasm isessential from following angles.

1. To identify gene sources for resistance to biotic and abiotic stresses viz, earliness,

dwarfness, productivity and quality characters.

2. To classify the germplasm into various groups

3. To get a clear picture about the significance of individual germplasm line

Evaluation requires a team of specialists from the disciplines of plant breeding, physiology,

biochemistry, pathology and entomology. First of all a list of descriptors (characters) for which

evaluation has to be done is prepared. This task is completed by a team of experts from IPGRI, Rome,

Italy. The descriptors are ready for various crops. The material is evaluated at several locations to get

meaningful results. Moreover, evaluation is done in a phased manner. The variation for polygenic

characters is assessed by three different methods as given below:

1. By simple measures of dispersion (range, standard, deviation standard error and coefficient

of variation)

2. By metroglyph analysis of Anderson (1957) and

3. By D2 statistics of P.C. Mahalanobis (1936)

The evaluation of germplasm is done in three different places viz, (1) in the filed, (2

) in greenhouse, and (3) in the laboratory. Observations on morphological characters, productivity attributes,

resistance to biotic and abiotic stress, and some physiological parameters like photosynthetic efficiency

and transpiration rate can be recorded under field conditions using portable instruments. The

resistance to biotic and abiotic stresses can also be screened under green house conditions. Evaluation

for biochemical characters like, protein, oil and amino acid contents, and technological characters is



completed under laboratory conditions. Both visual observations and metric measurements are used

for evaluation.

4. DOCUMENTATION

Documentation refers to completion, analysis, classification, storage and dissemination of

information. In plant genetic resources, documentation means dissemination of information about

various activities such as collection, evaluation, conservation, storage and retrieval of data.

Now the term documentation is more appropriately known as information system.

Documentation is one of the important activities of genetic resources. Information system is useful in

many ways as given below:

1. It provides information about various activities of plant genetic resources

2. It provides latest information about characterization, conservation, distribution and

utilization of genetic resources

3. It helps explorers, evaluators and curators in the conservation of genetic resources

4. It helps in making genetic resources accessible to plant breeders and other users

Large number of accessions is available in maize, rice, wheat, sorghum, potato and other major

crops. About 7.3 million germplasm accessions are available in 200 crops species. Handling of such

huge germplams information is only possible through electronic computers. For uniformity of information IPGRI has designed descriptors (characters) and descriptor state for majority of crops. The

entire data is put in the computer memory and the desired information can be obtained any time from

the computer.

5. DISTRIBUTION

The distribution of germplasm is one of the important activities of genetic resources centres.The specific germplasm lines are supplied to the users on demand for utilization in the crop

improvement programmes.

1. Distribution of germplasm is the responsibility of the gene bank centre where the

germplasm is maintained and conserved.



2. The germplasm is usually supplied to the workers who are engaged in the research work of a

particular crop species

3. Germplasm samples are generally supplied free of cost to avoid cumbersome work of book

keeping

4. The quantity of seed samples to be sent is usually small, depends on the availability of seed

material and demands received for the same and several other factors

5. Proper records are maintained about the distribution of material. After evaluation users

should send a report about important characters of the accessions to the distributor who

will record the information in the germplasm register for documentation purpose.

6. The germplasm is usually distributed after evaluation by collection centre for one or two

crop seasons. It helps in acclimatization and purification of the material.

7. Without distribution to the actual users, there is no point in collection the germplasm.

6. UTILIZATION

Utilization refers to use of germplasm in crop improvement programme. The germplasm can be

utilized in various ways. The uses of cultivated and wild species of germplasm are briefly discussed

below:

Cultivated germplasm

The cultivated germplasm can be used in three main ways (1) as a variety, (2) as a parent in the

hybridization, and (3) as a variant in the gene pool. Some germplasm lines can be released directly as

varieties after testing. If the performance of an exotic line is better than a local variety, it can be

released for commercial cultivation. In some cases, new variety is developed through selection from the

collection.

Some germplasm lines are not used as such, but have some special characters, such as disease

resitance, quality of economic produce, or wider adaptability. These characters can be transferred to

commercial cultivars by incorporating such germplasm lines in the hybridization programme. Transfer

of desirable characters from cultivated germplasm to the commercial cultivars is very easy because of

cross compatibility.



Wild germplasm

The wild germplam is used to transfer resistance to biotic and abiotic stresses, wider

adaptability and sometimes quality such as fibre strength in cotton. However, utilization of wild

germplasm poses three main problem viz (1) hybrid inviability - inability of a hybrid to survive, (2) hybrid

sterility inability of a hybrid to produce off spring, and (3) linkage of undesirable characters with

desirable ones. Thus utilization of wild germplasm for crop improvement is a difficult task.

.

Basic Pathways of germplasm preservation : The germplasm is preserved by the following two

ways:(a) In-situ conservation- The germplasm is conserved in natural environment by establishing

biosphere reserves such as national parks, sanctuaries. This is used in the preservation of land plants in a near natural habitat along with several wild types.

(b) Ex-situ conservation- This method is used for the preservation of germplasm obtained fromcultivated and wild plant materials. The genetic material in the form of seeds or in vitro cultures

are preserved and stored as gene banks for long term use. In vivo gene banks have been made to preserve the genetic resources by conventional methods

e.g. seeds, vegetative propagules, etc. In vitro gene banks have been made to preserve the

genetic resources by non - conventional methods such as cell and tissue culture methods. Thiswill ensure the availability of valuable germplasm to breeder to develop new and improvedvarieties.

The methods involved in the in vitro conservation of germplasm are:

(a) Cryopreservation- In cryopreservation (Greek-krayos-frost), the cells are preserved in the

frozen state. The germplasm is stored at a very low temperature using solid carbon dioxide (at -



790C), using low temperature deep freezers (at -800C), using vapour nitrogen (at- 1500C) andliquid nitrogen (at-1960C). The cells stay in completely inactive state and thus can be conserved

for long periods. Any tissue from a plant can be used for cryopreservation e.g. meristems,embryos, endosperms, ovules, seeds, cultured plant cells, protoplasts, calluses. Certain

compounds like- DMSO (dimethyl sulfoxide), glycerol, ethylene, propylene, sucrose, mannose,

glucose, praline, acetamide etc are added during the cryopreservation. These are calledcryoprotectants and prevent the damage caused to cells (by freezing or thawing) by reducing thefreezing point and super cooling point of water.

(b) Cold Storage- Cold storage is a slow growth germplasm conservation method and conserves

the germplasm at a low and non-freezing temperature (1-90C). The growth of the plant materialis slowed down in cold storage in contrast to complete stoppage in cryopreservation and thus

prevents cryogenic injuries. Long term cold storage is simple, cost effective and yieldsgermplasm with good survival rate. Virus free strawberry plants could be preserved at 100C for

about 6 years. Several grape plants have been stored for over 15 years by using a cold storage attemperature around 90C and transferring them in the fresh medium every year.

(c) Low pressure and low oxygen storage- In low- pressure storage, the atmospheric pressure

surrounding the plant material is reduced and in the low oxygen storage, the oxygenconcentration is reduced. The lowered partial pressure reduces the in vitro growth of plants. In

the low-oxygen storage, the oxygen concentration is reduced and the partial pressure of oxygen below 50 mmHg reduces plant tissue growth. Due to the reduced availability of O2, and reduced

production of CO2, the photosynthetic activity is reduced which inhibits the plant tissue growthand dimension. This method has also helped in increasing the shelf life of many fruits,

vegetables and flowers.

The germplasm conservation through the conventional methods has several limitations such asshort-lived seeds, seed dormancy, seed-borne diseases, and high inputs of cost and labour. The

techniques of cryo-preservation (freezing cells and tissues at -1960c) and using cold storageshelp us to overcome these problems.

17. E x situ and in situ conservation of PGR

Based on the place, size of sample, nature and breeding behaviors of species and the objectives

of conservation, the conservation strategies can be broadly classified into two categories - in situ

conservation and ex situ conservation.

I n situ: In situ conservation is defined as conservation of genetic resources within their ecosystem and

natural habitat. It means conservation of biodiversity in nature where species are allowed to grow in

their habitat. This method of conservation is good for

y Species about to extinct



y Wild relatives of crop plants, tree crops, forest crops, etc. where ex situ conservation is not

effective and

y Those species that cannot be grown outside their habitat : (i) members of complex ecosystem-

moist tropical forest trees, which are interdependent for survival, (ii) species with high

dormancy that cannot be broken by available methods and (iii) species with highly specialized

breeding system (dependent on specific insect).

E x situ: Ex situ conservation is defined as conservation of genetic material outside their natural habitat.

The e x situ conservation refers to man-made gene bank conservation that includes

y e x situ seed conservation in seed gene bank;

y e x situ plant conservation in field gene bank; and

y e x situ in vitro conservation of ex plants or organs in in vitro bank and cryo bank, and DNA

bank.

In vitro conservation:

Conservation of genetic diversity under ascetic condition using culture methods is called in vitro

conservation. It is especially suited for vegetatively propagated and recalcitrant species and whose ex

situ conservation involves high cost, vulnerability to environment and space requirement beside quick

loss of viability. The advantages of in vitro conservation are that large number of material can be stored

in a small space, under pathogen free condition and for exchange of germplasm.

There are certain limitations of in vitro conservation:

y Genetic instability

y Culture induced variability

y Lack of availability of regeneration of protocols.

Methods of in vitro conservation:

For the purpose of conservation of PGR, the growth of cultures should be kept to the minimum, if not

completely arrested. This is essential to avoid frequent transfer of cultures to fresh media.

Technical approaches to in vitr o storage

A. Germplasm in growing state

a. Maintenance of in vitro cultures under growth limitation

b. Maintenance of in vitro cultures under normal growing conditions



B. Germplasm under suspended growth (Cryopreservation)

Maintenance of in vitro cultures under growth limitation

The aim of maintenance of cultures under growth limitation is to extend the subculture interval thereby

to reduce the requirement of frequent sub culturing. The various approaches to achieve this include

the following:

i) maintenance under reduced temperature and /or reduced light intensity

ii) Use of growth retardants

iii) Use of minimal growth media and restrictive growth conditions

iv) Other approaches

Maintenance under reduced temperature and/ or reduced light intensity

The basic principle of this method is that if the in vitro plants are maintained at a temperaturesignificantly below the temperature required for optimum growth, the metabolic activities are affected

and thereby the growth of plants becomes restricted. In most cases the storage temperature is species

specific. Usually, temperate crops are stored at 1-10 C where as tropical crops are stored with in the

range of 15-22 C.

A reduction of light intensity or a complete suppression is often used in combination with temperature

reduction. Efficient in vitro storage of banana has involved incubation at reduced temperature (15 C)

and low light intensity (1000 lux).

Use of growth retardants:

The basic principle of using growth retardants such as maleic hydrazide (MH), abscisic acid (ABA), n-

dimethyl succinamic acid (DSA), cycocel (CCC), and phosphone-D to reduce the overall growth rate of in

vitro plantlets and enhance the sub culture interval. In potato shoot cultures, ABA, DSA, MH were found

effective in reducing growth. This method is very simple and cheap. Using these compounds, cultures

could be stored at normal culture room temperature (25 C) for longer period. However usage of growth

retardants is likely to induce the genetic variation due to its mutagenic effects or changes in physiology

of the cultures.

Use of minimal growth media

Lowering mineral content and /or sucrose in the medium have proved to be a successful and simple

method to restrict the growth of cultures. Plantlets of Coffea arabica can be stored for two years in a



medium devoid of sugar and having only half of the mineral solution of the standard medium. The

major difficulty with the application of minimal growth medium is that different genotypes may react

differently under these conditions.

Reduction in oxygen concentration:

Lowering the available oxygen level to tissues can reduce growth of cultures. The simple method is to

cover the tissues with mineral oil layer. Several ginger species could be conserved upto two years under

paraffin overlay.

Other approach:

Judicious use of a combination of two or more methods may be more useful for in vitro conservation.

Maintenance of in vitro cultures under normal growing conditions

Using normal growing conditions:

In this technique, cultures are maintained normal growing conditions. It involves frequent sub culturing

which is labour intensive, costly and poses risks of losses due to contamination and error. Cultures of

Musa, yams, piper and Alocasia could be maintained on their respective simple shoot culture media for

10-12 months at 25 C without subjected to growth inhibitory treatments.

Using induced storage organs:

Alteration of culture conditions and/or culture media helped in the induction of storage organs in

several species, which in turn increased the shelf life of cultures at 25 C. In ginger, shoot cultures

produced rhizomes on a high sucrose medium (9-12 per cent) and survived upto 24 months.

Storage using Cryopreservation

Cryopreservation, the non-lethal storage of biological material at ultra-low temperature, usually that of

liquid nitrogen (-196 C) is the only method currently available for the long term conservation of

germplasm of problem species. At this temperature, all cellular divisions and metabolic processes are



arrested. The plant material can thus be stored without alteration or modification for a theoretically

unlimited period of time. Moreover, cultures are stored in a small volume, protected from

contamination and they require a very limited maintenance.

Problems in cryopreservation:Solution effects

Solution effects caused by concentration of solutes in non-frozen solution during freezing assolutes are excluded from the crystal structure of the ice. High concentrations can be very

damaging.Extracellular ice formation

When tissues are cooled slowly, water migrates out of cells and ice forms in the extra cellular space. Too much extra cellular ice can cause mechanical damage due to crushing

DehydrationThe migration of water causing extra cellular ice formation can also cause cellular dehydration.

The associated stresses on the cell can cause damage directly.Intracellular ice formation

While some organisms and tissues can tolerate some extra cellular ice, any appreciableintracellular ice is almost always fatal to cells.

Cryopreservation techniques:

The experimental systems usually employed in cryopreservation (cell suspensions, calli, shoot tips,

embryos) contain high amount of cellular water and are thus extremely sensitive to freezing injury since

most of them are not inherently freezing tolerant. Cells have thus to be dehydrated artificially to

protect them from the damages caused by the crystallization of intracellular water.

There are various techniques available to achieve dehydration of cellular water to the required extent

for successful cryopreservation without loss of viability. The techniques employed and the physical

mechanisms upon which they are based are different in classical and new cryopreservation techniques.

Classical techniques involve freeze-induced dehydration, whereas new techniques are based on

vitrification (the transition of water in a concentrated solution directly from the liquid phase into an

amorphous phase) by avoiding the formation of crystalline ice.

Classical techniques:

Classical cryopreservation techniques involve slow cooling down to a defined prefreezing temperature,

followed by rapid immersion in liquid nitrogen. They are operationally complex in nature since they

require the use of sophisticated and expensive programmable freezers. In some cases the slow cooling



step with a domestic or laboratory freezer can be used. Recently use of alcohol bath in deep freezer has

become a cost effective method for classical cryopreservation procedure.

New cryo preservation techniques:

In the new vitrification based procedures, cell dehydration is performed prior to freezing by exposure of

samples to concentrated cryoprotective media and/or air desiccation followed by rapid freezing.

Intracellular ice formation is thus avoided. Vitrification procedures offer practical advantages in

comparison to classical freezing techniques.

Various types of vitrification :

y Encapsulation dehydration

y Vitrificationy Encapsulation-vitrification

y Desiccation

Desiccation : Zygotic embryos are extracted from the seeds, dehydrated in laminar flow or anair-tight container with silica gel. Freezing is rapidly done by placing the embryos in liquid

nitrogen and re-growth of embryos after freezing on a suitable medium.Encapsulation and Dehydration : This technique is based on the development of synthetic seeds,

ie., somatic embryos, shoot buds, and shoot spices are encapsulated in a gel matrix (e.g., sodiumalginate). For cryopreservation, explants are dissected under microscope, cultured for a definite

period on a defined medium, subsequently encapsulated in alginate beads and allowed to grow inliquid medium with high concentration of sucrose (0.5-125 M). Encapsulated explants are

dehydrated using slow or rapid desiccation techniques under a laminar flow to decrease themoisture of beads to 20 %. Freezing is done by placing the beads in liquid nitrogen. Recovery

can be made after slow thawing and using a standard protocol for a particular species.Vitrification : Vitrification refers to the physical process of transition of an aqueous solution into

an amorphous and glassy. The plant material is treated with extremely concentrated solutions of cryoprotectant (DMSO) and rapid freezing is done.

Cryopreservation for vegetatively propagated species :

Generally techniques based on vitrification are used for meristems and shoot tips.

Cryopreservation for recalcitrant seed species:

Cryopreservation protocols mainly based on desiccation have been developed for embroyos or

embryonic axes of a limited number of recalcitrant species and a large number of intermediate seed



species. However some times there is a problem in recovery growth of these explants after

cryopreservation.

Documents

Application of Tissue Culture With Relation to Valuable Germplasm