ppt on Cryopreservation

1. Seminar Topic

2. Outline….
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 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.
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4. Germplasm Conservation method
1) Insitu conservation:- The conservation of germplasm in
their natural habitats by establishing biosphere reserves.
2) Exsitu conservation:- The conservation of germplasm
in outside of its natural habitats like in the form of seed or
invitro culture.
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5. Invitro method for germplasm
conservation
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Germplasm
Conservation
Cryopreservation Cold storage Low oxygen
storage

6. • 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.
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7. Why only liquid nitrogen
• It is chemically inert
• Relatively low cost
• Non toxic
• Non flammable
• Readily available
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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.
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10. Pre-Freezing Treatment
• Pre-Culture
• Desiccation
• Addition of Cryoprotectants
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11.  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.
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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
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13. • 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%)
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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.
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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.
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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.
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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.
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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
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x 100

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23. Other techniques for pre-freezing
• Vitrification
• Desiccation
• Encapsulation-Dehydration
• Droplet Freezing Method
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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.
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25. 25Fig. Vitrification Fig. Desiccation

26. 26Fig. 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.
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28. Disadvantages
(a) 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.
(b) The seeds may be destroyed by internal pathogen and pest
attacks.
(c) It is not applicable to vegetatively propagated crops such as
Dioscorea, and potato.
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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.
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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.
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 Books –
S. S Purohit – Plant Tissue Culture
Plant Cell and Tissue Culture – Indra K. Vasil and Trevor A. Thorpe,
2nd 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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