SEMINAR ON GERMPLASM CONSERVATION

PRESENTED BY :- MOHD ABDULLAH HAIDERY m.SC-II (SEM-III)

DEPARTMENT OF BOTANY SANT GADGE BABA AMRAVATI

UNIVERSITY AMRAVATI

CONTENT :-

1. Abstract

2. History

3. Definition

4. Way of conservation

5. Cryopreservation

6. Advantages and Disadvantages

7. References

ABSTRACT

The ever growing human population demands increased agricultural productivity if possible through methods that don’t harm the natural environment. The genetic improvement of crop species is one answer to this problem.

However, to successfully do so, a diverse array of genotypes would be needed. Since continued overuse and misuse of our natural resources together with intensive agriculture are causing tremendous amount of genetic erosion.

It is imperative that crop genetic resources be conserved

History about germplasmEver since primitive man learned the art of learning and realised the economic utility of plants, he started saving selected seeds or vegetative propagules from one season to next. Conservation of forest resources was taught and decreed in part of India and China as far back as (700 B.C).

The concept of physical basis of heredity expressed by the 19th-century Biologist August Weismann. According to his theory, germplasm, which is independent from all other cells of the body (somatoplasm), is the essential element of germ cells (eggs and sperm) and is the hereditary material that is passed from generation to generation. Weismann first proposed this theory in 1883, it was later published in (1892; The Germplasm: A Theory of Heredity ). Bajaj 1995 and Staristky 1997 reported that some of the valuable gene pools might be lost unless co-ordinated efforts are made towards the conservation of genetic stock all over the world.

Realising the danger of genetic resources the U.N Conference on Human Environment held in Stockholm in 1972, recommended conservation of the habitat that are rich in genetic diversity.

Two year later in 1974 (CGIAR) established the (IBPGR), subsequently (IPGRI) with the objective of providing necessary support to the collection, conservation and utilisation of the plant genetic resources anywhere in the world.

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 conservationIt is of two methods:-

In situ ConservationIn 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

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.

There are three main approaches for the In vitro conservation of germplasm

1. Cryopreservation

2. Cold storage

3. Low – pressure and low oxygen - storage

CRYOPRESERVATIONCryopreservation (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 79:C)• Low temperature deep freezers (at -80: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 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.

Mechanism of cryopreservationThe 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.

Technique of cryopreservationThe 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 regenerationThe ultimate purpose of cryopreservation of germplasm is to regenerate the desired plant . For appropriate plant growth and regeneration , the cryopreserved cell/tissues 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

A selected list of the plants in various forms that are successfully cryopreserved

Plant material

Cell suspensions

Callus

Protoplast

Meristems

Zygotic embryo

Somatic embryo

Pollen

Plant species Oryza sativaGlycine maxZea maysNicotiana tabacum

Saccharum sp.Oryza sativa

Zea maysNicotiana tabacum

Solanum tuberosumCicer arietnum

Zea mays

Citrus sinensisDaucus carota

Citrus sp.Nicotiana tabacum

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

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.

References

M.K Razdan (1993) “An plant Introduction to plant tissue culture” published by Oxford and IBH publishing Co.Pvt.Ltd New Delhi - 110049

S.S Purohit (1997) “Plant tissue culture” published by Axis Books Chopasani Road, Jodhpur -342003

Kalyan Kumar De (1992) “Plant Tissue culture” published by New central book agency Pvt. Ltd Chintamoni Das Lane , Kolkata- 700009

http://en.wikipedia.org/wiki/Germplasm