Cryo-preservation of seeds

CRYO STORAGE - CONCEPTS,
MERITS , DEMERITS AND ITS
APPLICATION IN SEED INDUSTRY

1. WEL COME

2. Seminar Topic CRYO STORAGE - CONCEPTS, MERITS , DEMERITS AND ITS APPLICATION IN SEED INDUSTRY NAME: MALLIKARJUN SHERAKHANE I.D.No : PALB 8333 Sr.M.Sc . (Agri.) DSST, CoA, UAS, GKVK Bengaluru

3. What is Cryopreservation ? Viable freezing of biological material and their subsequent storage at ultra low temperatures (-150 to -196° C) in liquid nitrogen R epresents safe and cost-effective option for long-term conservation of germplasm

4. Science of cryobiology initiated since mid 20 th century Cryopreservation - Utilized earlier for creating gene banks of animal sperms, embryos, cells and micro organisms Sakai (1960)- Successful cryopreservation of woody plant i.e. winter-hardy mulberry (  M orus spp .) Quatrano (1968)- Cell suspensions Nag and Street (1973) -Somatic embryos

5. Biological , biochemical and physiological activities stopped due to less temperature then, plant materials could be stored for unlimited years Principle- removal of all freezable water from tissues by physical or osmotic dehydration, followed by ultra-rapid freezing Goal of cryopreservation - Replace some of the water with other compounds ( Cryoprotectants ) that will not form intracellular ice crystals when frozen i.e., protects cellular integrity of cell Zero-metabolism rate - Ultra lower temperature (-196 ° C )

6. Types of tissue preserved under cryopreservation Seeds and pollen Zygotic embryos / embryonic axes Embryonic cell suspensions Somatic embryos Meristem / shoot tip cultures etc.

7. Materials used Liquid nitrogen is most widely used material for cryopreservation Dry ice ( Solid carbon dioxide) can also be used Why liquid nitrogen ? Chemically inert Relatively having low cost Non-toxic Nonflammable Readily available

8. Methods of cryopreservation Conventional method Addition of an appropriate cryo protectant Subjection of culture to super low temperatures Storage of frozen culture in liquid nitrogen Thawing Removal of cryoprotectant by washing Viability Determination Reculture Induction of growth and plant regeneration

9. 2. Recently Developed or Modified Methods Vitrification P hysical process of transition of an aqueous solution into an amorphous and glassy state or Process in which ice formation cannot take place because aqueous solution is too concentrated to permit ice crystals nucleation. Instead, water solidifies into an amorphous ‘glassy’ state

10. Avoids most damaging event i.e. formation of intercellular ice crystals during cryopreservation Cells are dehydrated by treatment in a highly concentrated solution such as PVS2 (Plant Vitrification Solution) solution (Sakai et al ., 1990 )

11. Protocol of Vitrification Method Sakai et al., 1990

12. B. Encapsulation-dehydration M ethod first reported by Fabre and Dereuddre (1990 ) using shoot apices of potato This involves encapsulation of tissues in Calcium / Sodium alginate beads which are pre-grown in liquid culture media containing high concentration of sucrose After these treatments tissues are able to withstand exposure to liquid nitrogen without application of chemical cryoprotectants

13. Encapsulation-dehydration procedure Adriana et al. , 2004

14. C. Droplet vitrification Technique- modification of basic vitrification protocol Involves placing the sample within a droplet of 1-10 µl cryoprotective solution on a piece of aluminum foil before immersion in liquid nitrogen A pproach achieves higher cooling and re-warming rates , as small volume of liquid allows higher rate of heat transfer to and from sample (Sakai & Engelmann, 2007)

15. D roplet vitrification procedure Anja et al., 2012

16. Cryopreservation steps P lant material selection Pre-growth Freezing Addition of cryoprotectants Storage Thawing Viability determination

17. 1. Plant material selection Explant’s morphological and physiological condition influences ability to survive during cryopreservation Considerable f actors Tissue selection- from healthy plants Small , young , rich in cytoplasm and meristematic cells - Can survive better than larger and highly vacuolated cells Callus- freezing damage resistant Cell or tissue should contain low water content for cryopreservation then only tissues withstand extreme low temperatures

18. 2. Pre-growth Protect plant tissues against exposure to liquid nitrogen Involves application of additives ( Abscisic acid , Proline , Trehalose etc. ) to enhance plant stress tolerance Partial tissue dehydration achieved by application of osmotically active compounds

19. 3. Freezing Three types 1. Rapid / Fast freezing E mployed for shoot tip cryopreservation- Potato , Strawberry , Brassica species etc. Material placed in vials/tubes and plunged into liquid nitrogen Temperature reduction from -300 to - 1000°C/min or more occurs P revents growing of big ice crystals

20. 2 . Slow freezing Successfully employed for meristem cryopreservation- Peas, Potato, Cassava, Strawberry etc. Tissue slowly frozen with decrease in temperature from - 0.1 to - 10° C/ min Permits water flow from cells to outside- thereby promotes extracellular ice formation instead of lethal intracellular freezing

21. Difference between slow freezing and fast freezing

22. 3. Step-wise freezing Give excellent results with suspension cultures Slow freezing down to -20 to 40 O C A stop for period of approximately 30 minutes then Additional rapid freezing to - 196 O C done by plunging in liquid nitrogen Slow freezing permits protective dehydration of the cells

23. 4. Addition of cryoprotectants C ryoprotectant - Substance used to protect  biological tissue from freezing   damage (i.e. damage due to  ice crystal  formation ) A cts like antifreeze L owers freezing temperature Increases viscosity P revents cell damage

24. P otential sources of cell damage during cryopreservation Large ice crystal f ormation inside the cell Intracellular concentration of solutes increase to toxic levels before or during freezing as a result of dehydration Various cryoprotectants used are :- Glycerol Dimethyl Sulphoxide (DMSO) Sugars Mannitol Sorbitol Propylene Glycol (PEG) etc .

25. Dimethyl Sulphoxide (DMSO) An organosulfur compound with formula (CH3 ) 2 SO An excellent cryo-protectant Features Non-toxic Easily permeable Low molecular weight Easily washable from the cells Freezes within 18.5°C ( typical property) Below room temperature transformed into solids Usage concentration- 5 to 10 %

26. 5. Thawing D one by putting ampoule/tube containing frozen tips of sample in warm water bath (35 to 40°c ) with vigorous swirling action T ubes should not be left in warm water bath after ice melts for survival of tissue Tissues frozen by encapsulation/dehydration is frequently thawed at ambient temperature At point of thawing- quick transfer of tubes to water bath maintained at room temperature, continue swirling action for 15 sec to cool the warm walls of tube

27. 6. Storage Storage of frozen material at correct temperature is as important as freezing Frozen cells/tissues kept for storage at temperature ranging from - 70 to - 196°c Low temperature for longer period- To stop all metabolic activities and prevent biochemical injury Best done at - 196°C

28. 7. Survival / Viability determination Regrowth of plants from stored tissues or cells is only test of plant material survival Viability tests Fluorescein diacetate (FDA) staining Growth measurement by cell number Dry and fresh weight Staining of immature pollen at the “late” stage with fluorescein diacetate (FDA) for cell viability (A and B)

29. Staining methods Triphenyl tetrazolium chloride (TTC) Cell survival measured by amount of red formazan product formed due to reduction of TTC assay which is measured spectrometrically Only viable cells which contain enzyme dehydrogenase reduces TTC to r ed f ormazan will be stained Dead cells will not take up the dye/stain 2. Evan’s blue staining One drop of 0.1% solution Evan’s blue added to cell suspension on a microscope slide and observed under light microscope Only non viable cells (dead cells) stain with Evan’s blue

30. Individual cell viability assayed with Evan's blue dye

31. Merits Organ/cell Preservation In molecular biology Cryosurgery Blood transfusion Artificial insemination In-vitro fertilization Recently in identifying unknown transmissible disease or pathogen . Bone marrow transplantation Pollen preservation- Quality seed production

32. Gene bank T ype of  biorepository which preserve genetic material Used to store and conserve plant genetic resources of major crop plants and their  wild relatives This could be by freezing plant cuts or seed stocking Svalbard Global Seed Vault (Norway)- famous gene banks of world

33. India's doomsday vault in frozen Himalayas Located at Chang-La of Ladhak in western Himalayas Located at New Delhi

34. Seed bank It stores  seeds  as a source for planting in case seed reserves destroyed A type of gene bank S eeds stored may be food crops , or rare species to protect  biodiversity Seeds are dried to a moisture content of less than 5 % and stored in freezers at -18°C or below Because seed (DNA) degrades with time - Seeds need to be periodically replanted ; fresh seeds collected for long-term storage

35. Demerits At −196°C in liquid nitrogen, cell stops metabolizing leads to unavoidable side effects S low genetic changes within biological cells associated with lipids and proteins, could disfigure integrity of cells Cryoprotective agents could damage chromosome stability of cells Cryoprotectant makes cell susceptible towards infections Cost action like “ CRYOPLANT” could make difference

36. Applications 1. Genetic material conservation E ndangered plant species could be conserved Used to store wide range of tissues- meristems, anthers/pollens and embryos 2. Freeze storage of cell cultures and sub-culturing Cryopreservation- an ideal approach to suppress cell division which avoids periodical sub culturing

37. 3. Maintenance of disease free stock Pathogen free stocks of rare plant material could be frozen and propagated when needed 4 . Cold acclimatization and frost resistance Cryopreserved tissue culture provide suitable material for selection of cold resistant mutant cell lines This could later differentiate into frost resistance plants 5. Cryo selection Selection through freezing of samples with special properties

38. 6 . Cryotherapy E limination of viruses from infected plants through apex cryopreservation 7 . Genetic stability maintenance U ltra low temperature stops metabolic deterioration during storage of tissues and seeds. E xtends longevity by which genetic stability can be maintained 8 . Require less space and energy inputs M ethod relies on liquid nitrogen in self contained tanks Independent from refrigeration or constant electricity supply

39. CASE STUDIES

40. Rakesh et al., 2015

41. Made an effort to preserve pollen of hot pepper under normal room temperature (25 C), refrigeration (-20 o C) and cryopreservation (-196 o C), which can be used to direct supply of pollen, instead of bud to longer distances They selected flower buds when they were minimum 4-6.5 mm in length ( bud stage-III ) as described by Erickson and Markhart They concentrated mainly on study of pollen viability and its germination  

42. Fig. 1 Effect of storage time on pollen germination Fig. 2 Effect of storage time on fruit set Fig. 3 Effect of storage time on seed set Fig. 4 Effect of storage time on seed germination

43. Fig. 4 Effect of storage time on seed germination

44. Inference Pollen stored under ultra low temperatures can be used for pollination without affecting seed germination Helps in reducing the cost of seed production and ensure germplasm security Pollen storage facilitates crop breeding , genetic conservation and artificial pollination

45. Dutta et al ., 2013

46. Investigated pollen viability of three polleniser mango cultivars, viz. ‘Sensation’, ‘Tommy Atkins’ and ‘Janardan Pasand’ Stored up to 24 weeks under four storage conditions (room temperature, −4 ◦ C, −20 ◦ C and −196 ◦ C ) Pollen viability confirmed by , in-vitro germination , fluorescein diacetate (FDA) and acteocarmine tests Room temperature storage of pollen showed very low pollen viability Cryo-stored (−196 ◦ C) pollens showed significantly higher viability compared to other storage conditions

47. Pollen viability of three polliniser mango cultivars stored at room temperature ,− 4 ◦ C, −20 ◦ C and −196 ◦ C, by in-vitro germination test

48. Pollen viability of three polliniser mango cultivars stored at room temperature ,−4 ◦ C, −20 ◦ C and −196 ◦ C, by fluorescein diacetate (FDA) and acteocarmine tests

49. Inference Study revealed −196 ◦ C cryo-storage of mango pollen could be best Efficient conservation of genetic resources could be achieved Pollens might be used for commercial fruit production and breeding

50. Nipawan et al., 2012

51. Aim - To study the effect of V. tricolor seed stored in liquid nitrogen on germination V. tricolor is an outstanding vandaceous orchid found on rocks or trees, native to East Java U sed solid New Dogashima (ND) medium supplemented with Benzyladenine, Naphthaleneacetic acid (NAA) and 2% sucrose as growing medium Mature seeds- harvested 7 months after self-pollination, were directly plunged into liquid nitrogen

52. Immature seeds- harvested 6 months after self-pollination Treated with or without loading solution (LS) i.e . glycerol and sucrose Dehydrated with PVS2 (Plant Vitrification Solution) Lastly cryo -preserved by vitrification

53. Effect of seed cryopreservation by vitrification on the germination of non-cryopreserved seeds (- LN) and cryopreserved seeds (+LN) of V. tricolor after 90 days of sowing

54. Germination of cryopreserved mature seeds of V. tricolor after cryopreservation by directly plunging into liquid nitrogen. (a) 15 days, (b) 20 days, (c) and (d) 28 days of sowing

55. Germination of non-cryopreserved and cryopreserved seeds (6 months old) and development of protocorms of V . tricolor after cryopreservation by vitrification Germination of ( a) non-cryopreserved seeds:- Its protocorms development ( b ) and (c) after 150 days Germination of (d ) cryopreserved seeds after 120 days of sowing :- Its protocorms development (e ) and (f ) after 180 days

56. Inference Study showed liquid nitrogen induced germination of mature seeds of Vanda tricolor The LS treatment was very efficient in inducing dehydration and freezing tolerance in tissues Liquid nitrogen did not affect growth and development of protocorms from cryopreserved seeds when compared with non-cryopreserved seeds

57. Usman and Abdulmalik, 2010

58. Studied response of isolated embryonic axes of five maize genotypes using plant vitrification solution (PVS2) at different concentrations ( 50 %, 100 % and 150 % ) Embryonic axes were aseptically excised from surface sterilized seeds Embryo axes of elite maize genotypes were used as explants material The embryonic axes were pre-cultured for three (3) days on Murashige and Skoog ( MS) with 0.7 M sucrose They were then transferred to the different levels of plant vitrification solution (PVS2) for 30min

59. Effect of plant vitrification solution (PVS2) treatment on takeoff, survival and recovery of maize embryo

60. Response of maize genotypes to vitrification treatments

61. Inference Results-cryopreservation protocol by vitrification has potential for improving conservation of maize germplasm Using either 50% or 100% PVS2 is ideal for cryopreservation of maize germplasm

62. Rekha et al., 2010

63. They used organic solvent Cyclohexane for pollen collection Successfully adopted cryopreservation method for storing of pollens of mango and litchi crops up to four years There by, sufficient quantity of pollen could be used for large scale pollination in these two crops Transport of pollen in viable conditions over long distances was successfully devised Stored pollens showed high percentage viability

64. In vitro germinated litchi pollen after 4 years cryostorage of different cultivars: A- CHES-6; B- Chaina, C- Kasba

65. Inference Use of organic solvent (Cyclohexane) for pollen collection proved improved Large amounts of pollen are needed for pollination, viability testing , storage and future distributions

66. Conclusion Many plant species successfully cryopreserved through development of various cryopreservation methods Cryopreserved plants found to be genetically stable in most of the cases Pollen stored under ultra low temperatures used for pollination without affecting pollen germination H elps in reducing the cost of seed production and ensure germplasm security Successfully used in artificial insemination , in-vitro fertilization , in molecular biology study and most recently in identifying unknown transmissible disease or pathogen

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