Conservation of Embryo and Ovules, cell, Ovule culture, Suspension culture, Protoplast and Callus

culture, Micro propagation method, its Advantage and Disadvantage

An Assignment

on

Present By : Sohil K PrajapatiReg No. : 04-2652-2015

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 :-• SeedsOther plant propagules such as• Leaf • Stem• Pollen • Cultured cellsWhich 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 Conservation• In situ conservation is on-site conservation or conservation of

genetic resources in a natural population of plants, such as forests genetic resources in natural population of tree species.• It is the process of protecting an endangered plant in its natural

habitat either by protecting or cleaning up the habitat itself, or by defending the species from predators.• It is applied to conservation of agriculture biodiversity in agro

ecosystem by farmers, especially those using unconventional farming practice.

Ex situ Conservation

Ex-situ conservation means literally, "off-site conservation". It is the process of protecting an endangered species of plant or animal outside of its natural habitat; for example, by removing part of the population from a threatened habitat and placing it in a new location, which may be a wild area or within the care of humans.

Ex situ conservation can be carried out by using several methods

Seed gene bank In vitro storage DNA storage Pollen storage Field gene bank Botanical gardens

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 conservationLarge 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 hazardsFrom the germplasm stock large number of plants can be

obtained whenever needed.

Various Methods Have Been Shown To Reduce The Growth Rate Of anther, embryo, cell and suspension Cultures And Thus To Delay Or Reduce The Frequency Of Subculture.

• 1. Minimal Media and Growth Retardants: The use of minimal media and growth retardants, such as abscisic acid (Henshaw et al. 1978) or the growth of cultures in a sucrose-free medium (Jones 1974) has helped to delay the subculture/transfer period.

• 2. Mineral Oil Overlay: Caplin reported in 1959 that the mineral oil overlay of carrot callus tissue considerably reduced the rate of growth and subsequently delayed the transfer frequency. Later, by using mineral oil overlay, tissue cultures of various medicinal plants were stored for 4-6 months without subculture (Augereau et al. 1986).

• 3. Desiccation: Nitzsche (1980) reported the growth of dried callus after 1 year of storage. Recently (see Gray et al. 1995), extensive studies have been conducted on the desiccation of somatic embryos of carrot, grape, alfalfa etc.

• 4. Low Pressure/Low Oxygen: Bridgen and Staby (1981) stored tissue cultures under low atmospheric pressure and low oxygen and achieved four fold reductions in the growth rate of callus cultures of tobacco, tomato, and chrysanthemum.

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

1. Cryopreservation 2. Cold storage3. Low – pressure and low oxygen - storage

CRYOPRESERVATION Cryopreservation (Greek, krayos-frost) literally mean in the frozen state. The principal involved in cryopreservation to bring the plant cells and tissue cultures to a zero metabolism or non-dividing state by reducing the temperature in the presence of caryoprotectants.Cryopreservation broadly means the storage of germplam at very low temperature.• Over solid carbon dioxide (at 79⁰C)• Low temperature deep freezers (at -80⁰C)• In liquid nitrogen (at -196⁰C)•Among these the most commonly used cryopreservation is by employing liquid nitrogen. At the temperature of liquid nitrogen (-196⁰C), the cell stay in a completely inactive state and thus can be conserved for longer period. Infact cryopreservation has been successfully applied for germplasm conservation . Plant species e.g. rice, wheat, peanut, sugarcane ,coconut.

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.

2.Long term conservation of meristem or shoot tip

Technique of cryopreservationThe cryopreservation of plant cell culture followed the regeneration of plants broadly involves the following stages1. Development of sterile tissue culture.2. Addition of cryoprotectant and pretreatment3. Freezing 4. Storage5. Thawing6. Reculture7. Measurement of survival/viability8. Plant regeneration

1:- Development of sterile tissue cultureThe selection of plant species and the tissue with particular reference to the morphological and physiological characters largely influence the ability of the explants to survive in cryopreservation . Any tissue from a plant can be used for cryopreservation e.g. meristems, embryos, endosperm, ovules, seeds, culture plants cells, protoplast, callus.

2:- Addition of cryoprotectant Cryoprotectant are the compound that can be prevent the damage caused to cells by freezing or thawing. There are several cryoprotectant which include (DMSO), glycerol, ethylene, propylene , sucrose, mannose, glucose , proline and acetamide. Among these DMSO, sucrose and glycerol are most widely used.

3:- Freezing The sensitivity of the cell to low temperature is variable and largely depends on the plant species. Four different types of freezing method are used:Slow freezing method : The tissue is slowly frozen at 0.5-5°C/min from 0°C to -100°C,and

then transferred to liquid nitrogen.Rapid freezing method : Decrease in temperature up to -300 to -1000°C.Stepwise freezing method: Intermediate temperature for 30 min. and rapidly cool.Dry freezing method :Reported that non- germinated dry seeds can survive freezing at low

temperature in contrast to water imbibing seeds which are susceptible to cryogenic injuries.

4 : Storage

Maintenance of the frozen cultures at the specific temperature is as important as freezing . In general the frozen cells/tissues are kept for storage at temperatures in the range of -72 to -196°C. Storage is ideally done in liquid nitrogen refrigerator – at 150°C in the vapour phase, or at -196°C in the liquid phase. The ultimate objective of storage is to stop all the cellular metabolic activities and maintain their viability. For long term storage temperature at -196°C in liquid nitrogen is ideal.

5 : Thawing Thawing is usually carried out by plunging the frozen samples in ampoules into a warm

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

the rate of 500-750°C min¯¹) occurs, and this protects the cells from the damaging effects

ice crystal formation.

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

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

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

6 : Reculture

In general thawned germplasm is washed several times to remove

cryoprotectant. The material is then recultured in a fresh media.

7 : Plant regeneration•The ultimate purpose of cryopreservation of germplasm is to regenerate the desired plant . For appropriate plant growth and regeneration , the cryopreserved cell/tissues/culture have to be carefully nursed, grown. •Addition of certain growth promoting substances, besides maintenance of appropriate environmental conditions is often necessary for successful plant regeneration

Applications of germplasm conservation

Plant materials (cell/tissue) of several species can be cryopreserved and maintained for

several years, and used as and when needed.

Cryopreservation is an ideal method for long term conservation of cell culture which

produce secondary metabolites e.g. medicines

Disease (pathogen) free plant material can be frozen and propagated whenever

required.

Recalcitrant seeds can be maintained for long .

Conservation of somaclonal and gametoclonal variation in culture.

Plant material from endangered species can be conserved.

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

which could develop into frost resistant plant .

Limitations of Germplasm Conservation

The expensive equipment needed to provide controlled and variable rates

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

application of in vitro technology for large scale germplasm conservation.

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

injury to the cell.

Sometimes certain solutes from the cell leak out during freezing .

Cryoprotectant also effect the viability of cells.

MICROPROPAGATION• Micropropagation is the practice of rapidly multiplying stock plant

material to produce a large number of progeny plants, using modern plant tissue culture methods.

• Micropropagation is used to multiply noble plants such as those that have been genetically modified or bred through conventional plant breeding methods. It is also used to provide a sufficient number of plantlets for planting from a stock plant which does not produce seeds, or does not respond well to vegetative reproduction.

• Multiplication is the taking of tissue samples produced during the first stage and increasing their number. Following the successful introduction and growth of plant tissue, the establishment stage is followed by multiplication. Through repeated cycles of this process, a single explant sample may be increased from one to hundreds and thousands of plants. Depending on the type of tissue grown, multiplication can involve different methods and media.

Micropropagation has a number of advantages over traditional plant propagation techniques:

• The main advantage of micropropagation is the production of many plants that are clones of each other.

• Micropropagation can be used to produce disease-free plants.• It can have an extraordinarily high fecundity rate, producing thousands

of propagules while conventional techniques might only produce a fraction of this number.

• It is the only viable method of regenerating genetically modified cells or cells after protoplast fusion.

• It is useful in multiplying plants which produce seeds in uneconomical amounts, or when plants are sterile and do not produce viable seeds or when seed cannot be stored

• Micropropagation often produces more robust plants, leading to accelerated growth compared to similar plants produced by conventional methods - like seeds or cuttings.

• Some plants with very small seeds, including most orchids, are most reliably grown from seed in sterile culture.

• A greater number of plants can be produced per square meter and the propagules can be stored longer and in a smaller area.

Disadvantages• It is very expensive, and can have a labour cost of more than 70%.• A monoculture is produced after micropropagation, leading to a lack of overall

disease resilience, as all progeny plants may be vulnerable to the same infections.

• An infected plant sample can produce infected progeny. This is uncommon as the stock plants are carefully screened and vetted to prevent culturing plants infected with virus or fungus.

• Not all plants can be successfully tissue cultured, often because the proper medium for growth is not known or the plants produce secondary metabolic chemicals that stunt or kill the explant.

• Some plants are very difficult to disinfect of fungal organisms.• The major limitation in the use of micropropagation for many plants is the cost

of production; for many plants the use of seeds, which are normally disease free and produced in good numbers, readily produce plants (see orthodox seed) in good numbers at a lower cost. For this reason, many plant breeders do not utilize micropropagation because the cost is prohibitive. Other breeders use it to produce stock plants that are then used for seed multiplication.

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