Cryoprervation techniques.............pptx

Cryoprervation techniques.pptx

1. CRYOPRESERVATION TECHNIQUES

2. Why preservation is important? Until two decades ago the genetic resources were getting depleted owing to the continous depredation by man. It was imperative therefore that many of the elite, economically important and endangered species are preserved to make them available when needed. The conventional methods of storage failed to prevent losses caused due to various reasons. A new methodology had to be devised for long term preservation of material.

3. There are various methods of storage: 1. Cryopreservation - generally involves storage in liquid nitrogen. 2. Cold storage - it involves storage in low and non freezing temperature.19733. 3.Low pressure - it involves partially reducing the atmospheric pressure of surrounding. 4. Low oxygen storage - it involves reducing the oxygen level but maintaining the pressure.

4. Cryopreservation Cryo is Greek word. ( krayos -frost) It literally means preservation in “frozen state”. The principle to bring plant cells or tissue to a zero metabolism and non dividing state by reducing the temperature in the presence of cryoprotectant. It can be done : Over solid carbon dioxide (at-79 degree) Low temperature deep freezer (at -80 degree) In vapor phase nitrogen (at -150 degree) In liquid nitrogen (-196 degree).

5. Cryopreservation refers to the storage of cells, tissues & organs at the ultra-low temperature of liquid nitrogen. At such low temperatures, the stored material enters in a state of "absolute quiescence" as all the physical & the biochemical reactions are practically halted. Thus, cryopreservation is a long term storage techniques with very low temperatures to preserve the structurally intact living cells & tissues for extended period of time at a relatively low cost. The science pertaining this activity is known as "CRYOBIOLOGY

6. PRECULTURE Culture of cells / tissues/ organs in the presence of amino acid like proline, sugars or at low temperatures prior to freezing initiates in them physiological changes. These changes facilitate dehydration & accumulation of substances like proline, which protect the cells from toxicity of high solute concentration. Plantlets hardened by growing them at near freezing temperature(for 1 week at 22°C). Cold hardened tissues show water efflux, permits dehydration of cells. A 3-4th day preculture on 2-4% proline either alone or in combination with cryoprotectants like DMSO improves the survival of suspension cultures. Sugars acting as osmotically effective agents, although they don't penetrate inside the cells.

7. Mechanism of cryopreservation The cryopreservation technique followed by the regeneration of plants involves following steps: 1. Selection of material. 2. Addition of cryoprotectant. 3. Freezing. 4. Storage in liquid nitrogen. 5. Thawing. 6. Washing and reculturing . 7. Measurement of viability.

8. SELECTION OF A PLANT MATERIAL Material chosen for cryopreservation should be far as possible in meristematic state. For selecting a material, a number of facts are taken into account: 1. The nature of cells. 2. The density of cells in the vials to be preserved. Cell cultures are generally preserved in lag or early exponential phase of growth. Young, highly cytoplasmic & small cells which are non- vacuolated & in small aggregates are good materials to be selected for cryopreservation. In some species, it may be important to use highly embryonic cell cultures since non- embryonic or poorly embryogenic cultures show poor or no regrowth after thawing.

9. ADDITION OF CRYOPROTECTANTS They are chemical which prevent cryodestruction . CRYODESTRUCTION" refers to the formation of large ice crystals inside the cells that rupture cells itself & cell organelles. These are sucrose, alcohols, glycols, some amino acid (proline), DMSO (dimethyl sulfoxide). Generally two cryoprotectant should be used together instead of single one as they are more effective.

10. FREEZING After addition of cryoprotectants, freezing is done in such a a way that it does not cause intracellular freezing & crystal formation. • The following types of freezing is done in the process of cryopreservation: This method is simple & easy. RAPID FREEZING :- Freezing is done quickly so that there should be least change or development of intracellular crystals. SLOW FREEZING :- In this method the rate of freezing is slow (0.1-10°C/ min). It is commonly used for animal germplasm.In this due to cooling below freezing point, extra cellular crystals are formed not intracellular. STEPWISE FREEZING :- In this temperature gets lowered by -20°C to 40°C & allows protective freezing of the cells.Freezing stopped for 30 mins & then rapidly freezed in liq.N ; this results in decline in temperature & good results are obtained.

11. STORAGE IN LIQUID NITROGEN If the cells are not tored at sufficiently low temperature, an additional injury to the material be caused. The storage temperature be such that it stops all the metabolic activities & prevents biochemical activity; this can be achieved with the help of liq. N₂ at 196°C.

12. STORAGE The maintenance of the frozen cells or material at specific temperature is very important. In general the temperature is kept -70 to 196 degree. Prolong storage is done at temperature of -196 degree in liquid nitrogen. To prevent damage, continous supply of nitrogen is done.

13. THAWING It is the process of releasing the vials containing the material from the frozen state at elevate temperature. As soon as the last crystals disappear, the vials are transferred into a water bath maintained at 20°C- 25°C. Thawing has to be rapid to avoid ice- crystal formation.

14. WASHING AND RECULTURING Washing of materials is done to remove the toxic cryoprotectants. When low toxic or non-toxic cryoprotectants are used, the cultures should not be washed but simply recultured .

15. Measurement of viability There is possibility of death of cells due to storage stress. Thus viability can be found at any stage. It is calculated by formula:No of cells growing/no of cells thawed *100 Plant regeneration The viable seeds are cultured on non specific growth medium.Suitable environmental conditions are maintained

16. VITRIFICATION Researchers Greg Fahy & William F.Rall helped to introduce vitrification to reproductive cryopreservation in mid 1980s. It means the addition cryoprotectants prior to cooling. They act as anti-freeze i.e. they decrease the freezing temperature & the viscosity . Researchers claim that vitrification provides the benefits of cryopreservation without damage due to ice crystal formation. Rather than a phase change from liquid to solid by the crystallization the amorphous state is like a 'solid- liquid' & the transformation is over a small temperature range described as “glass transition” temperature.

17. CRYOPRESERVATION OF ANIMAL STOCK CELL Development of animal cell line is expensive, time consuming & labour intensive. It is essential to protect this considerable investment by preserving the cell line so that it can be further used when required. Selection of cell line & standardization of culture: A cell line have different properties; continuous cell line are clone; suitable clone is selected & grown to get suitable bulk of cell required for freezing. Continuous cell line have several advantages over fertile cell line such as: 1. They survive indefinitely. 2. They grow more rapidly. 3. They can clone more easily, but they may be less stable genetically. 4. Usually the finite cell line are diploid & stable but harder to clone. They grow more slowly & eventually die or transfer.

18. CRYOPRESERVATION OF PLANT STOCK CELL Due to gradual disappearance of economic & rare species the necessity for storage of genetic resources increase. The conventional methods of storage fail to prevent from losses caused by: 1. Attack of pathogen & pest. 2. Climatic disorders. 3. Natural disorders. 4. Political & economic causes.

19. CRYOPRESERVATION OF PLANT STOCK CELL The materials to be preserved are stored at low temperatures due to which growth rate of cells retards; consequently biological activities are conserved for long time. 3 principal methods are: 1. Alteration of physiological condition of culture i.e. temperature of gas composition in the vessels. 2. Changing the composition of basal medium. EX: Using sub or supra optimal concentration of nutrients. 3. Supplementing the nutrients with growth retarders or osmoregulatory compounds.

20. SIGNIFICANCE OF CRYOPRESERVATION Cryopreservation of gametes, embryos etc. prevents genetic drift. It safeguards genetic integrity of valuable stains. It offers generation time & allows further contribution of genetics. It eases transportation of genetic stock. It causes decrease of disease transmission

21. ADVANTAGES Once the material is successfully conserved to particular temperature it can be preserved indefinitely. Once in storage no chance of contamination of fungus or bacteria. Minimal space required Minimal labour required. DISADVANTAGES High cost Social issues

22. APPLICATION It is ideal method for long term conservation of material. Disease free plants can be conserved and propagated. Recalcitrant seeds can be maintained for long time. Endangered species can be maintained. Pollens can be maintained to increase longitivity . Rare germplasm and other genetic manipulations can be stored

23. REFERENCE 1. Bhojwani, S. S., & Razdan, M. K. (1986), Plant tissue culture: theory and practice Elsevier. 2. Razdan, M.K. (2000). An introduction to plant tissue culture, Oxford &IBH publishing Co. Pvt. Ltd. New Delhi, Calcutta, 3. Kumar, D. (2010). Plant breeding Biometry Biotechnology, New Central Book Agency Pvt. Ltd. Calcutta. 4. Misra, S. P. (2009), Plant tissue culture. Arne Books Pvt. Ltd. New Delhi.

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