Cryopreservation

ppt on Cryopreservation

1. Seminar Topic Cryopreservation Pt. Ravishankar Shukla University

2. Outline…. 2 What is Germplasm Germplasm Conservation method In-vitro method of germplasm Conservation Cryopreservation Step involve in Cryopreservation Selection of plant material Pre- freezing treatment Freezing Storage Thawing Viability Assays Other techniques for pre-freezing Advantages & Disadvantages New research References

3. What is Germplasm Germplasm is the total content of the genes transmitted to offspring by germ cell. Or The collection of genetic resources for an organism. 3

4. Germplasm Conservation method Insitu conservation:- The conservation of germplasm in their natural habitats by establishing biosphere reserves. Exsitu conservation:- The conservation of germplasm in outside of its natural habitats like in the form of seed or invitro culture. 4

5. Invitro method for germplasm conservation 5 Germplasm Conservation Cryopreservation Cold storage Low oxygen storage

6. Cryopreservation Cryopreservation is defined as the viable freezing of biological material and their subsequent storage at ultra low temperatures, preferably in liquid nitrogen. Plant cryopreservation involves the storage of plant tissues (usually seed or shoot tips) in liquid nitrogen (LN) at -196°C . Principle :- Around 90% of water is free while the remaining 10% bound to other molecules component of cell. This water does not freeze & called hydrated water. To bring plant cell or tissue to a zero metabolic & non dividing state by reducing the temperature in the presence of cryoprotectant . 6

7. Why only liquid nitrogen It is chemically inert Relatively low cost Non toxic Non flammable Readily available 7

8. Step involve in Cryopreservation 8 Selection of plant material Pre-freezing treatment Freezing Storage Thawing Reculture Viability Assays Plant regeneration

9. Selection of plant material Material chosen for cryopreservation should be as far as possible in meristematic state. Cell cultures are generally preserved in lag or early exponential state of growth. Cells in the early lag or stationary phase may be susceptible to cryoinjury because of their arrest in G1 phase. Cell suspension, protoplasts, pollens, shoot tips, & embryos have been successfully preserved. 9

10. Pre-Freezing Treatment Pre-Culture Desiccation Addition of Cryoprotectants 10

11. 1) Pre-Culture Before freezing culture are exposed to low temp. because freshly harvested cells or tissue may not survive in super cooling and require to be conditioned by their brief culture.Prefreezing treatment of this type beneficial for potato shoot apices This process enhance the survival frequency of the excised tissue. Ex- white clover cultures at 4°C for 2 days. 11

12. 2) Desiccation Exclusion of freezable water from the cells before freezing. Dehydration increase the intracellular conc. of solutes and thus makes them to toxic to cells, to protect cells from this toxic solution effect the amorphous cryoprotectants are added to the freezing mixture. Ex. Carrot culture – somatic embryo - 3%water is removed. - In melon somatic embryogenesis- 11.8% Dehydration Reduce the amount of water Increase the osmotic pressure Reduce ice formation Depresses its freezing temperature & promotes vitrification 12

13. 3) Addition of Cryoprotectant Cryoprotectant are the compounds that can prevent the damage caused to cells by freezing or thawing. To check the intracellular conc. of solutes during dehydration, some Cryoprotectants are added in culture medium. They protect cells against osmotic shock. They also prevent ice crystal formation in cells. DMSO – dimethyl sulphoxide (5-8%) alone or with glycerol. Proline is also used (~10%) 13

14. Characteristics of Cryoprotectant Lower the freezing point. Protect cell membrane from freeze-related injury. High solubility. Low toxicity or high concentration Low molecular weight. Ability to interact with water via hydrogen bonding. 14

15. Freezing Slow Cooling Method – 0.5 - 4°C per min to reach from -40°C to -100°C in LN. Generally used for cell suspension culture. Slow freezing allows for cytoplasmic dehydration. Rapid Cooling Method – Successful for the plantlet, shoot tip. 10°C per min to -196°C with the combination of desiccation, in this cryoprotectants are not required. Pre-Freezing Method – When cultures are gradually cool to -30°C to -50°C at the rate of 1-5°C/min. Then held for 30 min. and rapidly cooled into liquid nitrogen. It is also used for shoot apices & bud. Especially the germplasm of strawberry is freeze by this method. 15

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17. Storage Storage temp. Should permit total immobilisation of metabolic activities of cells arrest them in a particular stage. This is accomplished at ultra-low temps such as that of liquid Nitrogen. 4000 ampoules of 2ml each, we need 20-25L of LN/week. 17

18. Thawing Thawing means bringing back of cryopreserved materials back to the normal state in such a way that damaging ice crystal formation does not take place. Rapid Thawing – From -196°C the stored vials are plunged into the water of 37-40°C for 1-2 mins . Slow Thawing – This may be fatal for the tissue because of ice formation. 18

19. Recovery/ Reculture Generally thawed materials are washed to remove cryoprotectant & then they are cultured in their respective medium & cultured in normal tissue culture medium. Under 25°C ±2, 16hr light and 8hr dark, 1000 lux pressure. Sometimes GA (Tomato apices) or activated charcoal (carrot) is added , to improve internodal growth and to absorb phenolic compound respectively. 19

20. Viability Assays FDA Test – 0.01% FDA solution in acetone is added to the cell. After 5 mins of incubation it is entered into the living cell and it is cleaved by the esterase activity, releasing flourescent flourescien . This flourescien is not permeable across the membrane, so it accumulates in the cytoplasm of living cells. When illuminated in UV it gives green flourescence . TTC Test – It oxidizes the cells & forms red formazan . It checks the respiratory efficiency of the cells. If cells are alive and respiring then this 2,3,5-triphenyl tetrazolium chloride converts to red colour. No. of cells / organs growing No. of cells / organ thawed 20 x 100

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23. Other techniques for pre-freezing Vitrification Desiccation Encapsulation-Dehydration Droplet Freezing Method 23

24. Vitrification Vitrification is a physical process by which a concentrated aqueous solution cooled to low or ultralow temperature directly solidifies into an amorphous ‘glassy’ state, without crystallization. The significance of this phenomena in cryopreservation of plant materials is that the cell applied with a highly concentrated solution of osmotically active compound are protected from internal damage from ice crystal formation during freezing. 24

25. 25 Fig. Vitrification Fig. Desiccation

26. 26 Fig. Droplet Method Fig. Encapsulation Dehydration

27. Advantages Relatively little space is needed for the preservation of large numbers of clonally multiplied plants (as 'vegetative seeds'). The plants are maintained free from pests, pathogens, virus and other natural hazards. Once the material is successfully conserved to particular temperature it can be preserved indefinitely. The material could serve as an excellent form of stock to propagate large numbers of plants rapidly, when required. 27

28. Disadvantages Some crops such as coconut, and mango , produce large recalcitrant (short-lived) seeds which lack a dormancy mechanism and cannot bear subjection to desiccation or exposure to low temperature. The seeds may be destroyed by internal pathogen and pest attacks. It is not applicable to vegetatively propagated crops such as Dioscorea , and potato. 28

29. New researches New cryogenic technique could keep donor organs on ice for years. A new nanopartical ( nanowarming ) technology warmed cryopreserved heart valves & blood vessels without damaging the tissue A nanowarming technology could someday extend life of human organ through cryopreservation. Currently, donor organ such as heart & kidney must be transplanted within hours because the cell begins to die when organ are cut from blood supply. The minimum tolerant organ preservation for transplantation by hypothermic storage is around 4 hours for heart & 8 hours for lungs, 12 for liver. 29

30. Conclusion Hence these Techniques may have few disadvantages but these are the only developed techniques by which the cultures can be stored. Many plant species have been successfully cryopreserved through the development of various cryopreservation methods. As a standard protocol, & vitrification are widely applied. Shoot tips are the preferred material for cryostorage as they contain the meristem and an organised structure. 30

31. References 31 Books – S. S Purohit – Plant Tissue Culture Plant Cell and Tissue Culture – Indra K. Vasil and Trevor A. Thorpe, 2 nd Edition. Plant Tissue Culture, a Revised Edition – S. S. Bhojwani and M. K. Razdan Websites – http://www.biotecharticles.com/Agriculture-Article/Cryopreservation-and-Conservation-of-Plant-Genetic-Material http://www.fao.org/biotech/docs/panis.pdf

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