CRYOPRESERVATION AND GERMPLASM STORAGE

CRYOPRESERVATION AND GERMPLASM STORAGE

In situ hybridization is defined as conservation of genetic resources with their eco system and natural habitat. Ex situ conservation means conservation of components of genetic material of biological diversity outside their naturall habitat. This refers to man made gene bank conservation that includes ex situ seed conservation in seed gene bank, ex situ plant conservation in field gene bank and ex situ in vitro conservation of explants/organs in in vitro bank and cryo bank, DNA library and DNA bank.

In vitro conservation: Conservation of genetic diversity under aseptic condition using culture methods is called in vitro conservation

Strategies

1.Normal growth: short to medium storage

2. Slow growth: Using different enclosures for culture vessels, reduction in temperature, inclusion of osmoticum, growth retardants, modification of gaseous environment, induction of storage organs, minimal growth media

3.Suspended growth: Cryo stroage./ Cryopreservation is defined as the viable freezing of biological material and their subsequent storage at ultra low temperature preferably at that of liquid nitrogen..

Cryo preservation-Steps

1. Rising sterile tissue cultures and cell suspensions.

2. Addition of cryoprotective agent.

3. Subjecting cell cultures to super low temperature by regulated slow rate of cooling or after pre freezing.

4. Storage of frozen cells in liquid nitrogen.

5. Thawing or rapid re warming of cells.

6. Removal of cryoprotectant by repeated washing.

7. Determination of viability.

8. Re culture of the retrieved cells.

9. Induction of growth and regeneration of plants.

Cryo protectants

Most of the experimental systems (meristem, shoot tips, cultured cells etc) contain high amount of cellular water and hence are extremely sensitive to freezing, injury protection from freezing and thawing injury has to be imposed artificially. A number of compounds such as glycerol, DMSO, ethylene glycol, polyethylene glycol, sugars and sugar alcohols either alone or in combination protect cells against damage during freezing and thawing.

Freezing Methods

1. Slow freezing: This is the most common method of freezing meristems, somatic embryos and cell cultures which is by regulated slow cooling at a rate of 0.5-1.0° C/min to either -30, -35 (or) -40°C followed by storage in liquid nitrogen.

2. Rapid Freezing: Meristems and somatic embryos for few plant species have been successfully cryo preserved by rapid freezing. Here, the temperature is rapidly lowered.

3. Droplet freezing: Mostly suitable for meristems E.g. Cassava. Here, the cryo protectant solution, (15% DMSO and 3% sucrose) is dispensed into droplets of 2-3µl in an aluminum foil contained in a petri dish. The advantage of droplet freezing on aluminum foil is homogeneous cooling due to efficient thermal conductivity.

4. Vitrification: This is based on the ability of highly concentrated solutions of cryoprotectants to super cool to very low temperature upon imposition of rapid cooling rates to become viscous at sufficiently low temperatures and solidity without the formation of ice. The advantage is being simple and doesn’t require regulated cooling. The disadvantage is toxicity owing to high concentration of cryoprotectants.

e.g. Cell cultures of Brassica, naval orange, Somatic embryos of Asparagus and Daucus, mesophyll protoplasts of Secale and shoot tips of Mint, Potato, Papaya and Carnation.

Cryostorage and Thawing Long term preservation is accomplished at ultra low temperatures such as that of liquid nitrogen (-196°C). Thawing is generally carried out by immersing specimens rapidly for 1-2 minutes in 35-40°C water bath.

Viability assays: Viability can be assayed by fluorescent di acetate staining (FDA), Triphenyl tetrazolium chloride(TTC) reduction assay, and using other parameters like mitotic index, Cell number Cell volume, dry and fresh weight and plating deficiency.

Applications

1. In vegetatively propagated crops, to avoid high level of heterozygosity, they are clonally propagated through tubers or cuttings which have a limited life span, labor intensive and expensive and risks are associated with field maintenance. In seed crops problems on non-viability recalcitrance to storage, deterioration and heterozygosity exists. To circumvent the problems, Cryo preservation technology can be used.

2. Preservation of rare genomes.

3. Freeze storage of cell cultures.

4. Conservation of genetic uniformity.

5. Maintenance of Disease Free Material which is ideal for the international exchange.

6. Cold Acclimation and Frost Resistance.

7. Retention of Morphogenetic Potential.

8. Slow metabolism, which would prevent or virtually 'stop' the ageing process.