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

2. 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
 Represents safe and cost-effective option for long-term
conservation of germplasm

4.  Science of cryobiology initiated since mid 20th 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 ( Morus 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
1. 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
A. Vitrification
Physical 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
• Method 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
 Approach 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. Droplet vitrification procedure Anja et al., 2012

16. Cryopreservation steps
Plant 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 factors
 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
 Employed 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
• Prevents 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 40OC
• A stop for period of approximately 30 minutes then
• Additional rapid freezing to -196OC done by
plunging in liquid nitrogen
• Slow freezing permits protective dehydration of the
cells

23. 4. Addition of cryoprotectants
Cryoprotectant- Substance used to protect biological tissue from
freezing
damage (i.e. damage due to ice crystal formation)
 Acts like antifreeze
 Lowers freezing temperature
 Increases viscosity
 Prevents cell damage

24. Potential sources of cell damage during cryopreservation
1. Large ice crystal formation inside the cell
2. 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
 Done by putting ampoule/tube containing
frozen tips of sample in warm water bath (35
to 40°c) with vigorous swirling action
 Tubes 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
1. 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 red formazan 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
Type 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
 Seeds 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
• Slow 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
• Endangered 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
 Elimination of viruses from infected plants through apex
cryopreservation
7. Genetic stability maintenance
 Ultra low temperature stops metabolic deterioration
during storage of tissues and seeds.
 Extends longevity by which genetic stability can be
maintained
8. Require less space and energy inputs
 Method 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 𝒐C), refrigeration (-20o C) and
cryopreservation (-196o 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
• Used 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
oMany plant species successfully cryopreserved through
development of various cryopreservation methods
oCryopreserved plants found to be genetically stable in most of the
cases
oPollen stored under ultra low temperatures used for pollination
without affecting pollen germination
oHelps in reducing the cost of seed production and ensure
germplasm security
oSuccessfully used in artificial insemination, in-vitro fertilization,
in molecular biology study and most recently in identifying
unknown transmissible disease or pathogen