1. Cryopreservation
Plant Tissue Culture
By
KAUSHAL KUMAR SAHU
Assistant Professor (Ad Hoc)
Department of Biotechnology
Govt. Digvijay Autonomous P. G. College
Raj-Nandgaon ( C. G. )

2. Index
2
 Introduction
 History
 Applications
 Cryopreservation Components
 Pre-treatment
 Freezing Methods
 Cryostorage
Thawing
 Viability Assays
 Recent Developments in Cryopreservation
 Advantages and Disadvantages
 Conclusion
 References

3. Introduction
3
• 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 or
in the vapour phase of LN at -135°C in such a way that the
viability of stored tissues is retained following re-warming.
• It is estimated that 6 million samples of plant genetic
resources are held in national, regional, international and

4. History
• A major breakthrough was the finding that glycerol was
capable of protecting Avian Spermatozoa from freezing injury
by Polge et al., in 1949.
• In early 1950’s a number of low molecular weight neutral
solutes have been identified as potential cryoprotectants,
most common one being Dimethyl Sulfoxide (DMSO) and
Glycerol.
• DMSO used by Lovelock and Bishop in 1959 to prevent
freezing damage to human blood cells and bull spermatozoa,
has become a universal cryoprotectant.
4

5. Applications
• Cryopreservation is usually applied to species with recalcitrant (i.e.
Dehydration sensitive) seeds that are not storable by any other
means, or preservation of specific cultivars of vegetatively
propagated crop plants like banana or potato, or for unique
ornamental genotypes.
• Another reason is to conserve endangered plant species,
particularly where seeds may be extremely scarce or of doubtful
quality and/or the species is threatened with imminent extinction.
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6. Cryopreservation Components
• Candidates for Cryopreservation
• Prefreezing treatment
• Freezing
– Slow freezing
– Rapid freezing
• Storage
• Thawing
• Recovery/Reculture
• Viability Assays
6

7. Candidates for
Cryopreservation
• 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 succeptible to
cryoinjury because of their arrest in G1 phase.
• Calli, cell suspension, protoplasts, pollens, shoot tips, & embryos
have been successfully preserved.
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8. Pre-Freezing Treatment
• Pre-Culture - Before freezing, cultures are exposed to low temp.
– Ex- white clover cultures at 4°C for 2 days. It is called pre culture.
• Dessication – Exclusion of freezable water from the cells.
– Ex. Carot culture – somatic embryo – 3%water is removed.
– In melon somatic embryogenesis- 11.8%
– Kiwi fruit shoot apices – 29%
• Use of Cryoprotectants - To check the intracellular conc. of solutes
during dehydration, some Cryoprotectants are added in culture
medium. They also prevent ice crystal formation in cells.
– DMSO – dimethyl sulphoxide(5-8%) alone or with glycerol.
– Proline is also used (~10%)
8

9. Freezing
• Slow Cooling Method – 0.5-4C 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 dessication, in this even
cryoprotectants are not required. Quick freezing results in fast
intercellular freezing with little dehydration.
• Pre-Freezing Method – When cultures are gradually cool to -30°C to
-50C at the rate of 1-5C/min. Then held for 30 mins and rapidly
cooled into liquid nitrogen. 9

10. 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. Ease of availability of
liquid nitrogen makes this
strategy feasible in most
countries.
• 4000 ampoules of 2ml each, we 10

11. 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
11

12. Recovery/Reculture
• Generally thawed materials are
washed to remove cryoprotectant &
then they are cltured 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 , 12

13. Viability Assays
• A number of viability tests are available for the estimation of viability
of cryopreserved cells.
• These include FDA test, TTC test, etc.
• FDA Test – 0.01% FDA solutn in acetone is added to the cell. After 5
mins of incubation it is enterd into the living cell and it is cleaved by
the esterase activity, releasing flourescent flourescien. This
flourescien is not permeable across the memb., 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 the13

14. Recent Developments
• In recent certain new developments have occured in
cryopreservation . These include :
• Vitrification
• Encapsulation-Dehydration
• Dessication
• Droplet Freezing Method
14

15. 15Fig. Vitrification Fig. Dessication

16. 16Fig. Droplet Method Fig. Encapsulation Dehydration

17. 17

18. Advantages
(a) Relatively little space is needed for the preservation of large numbers of clonally multiplied
plants (as 'vegetative seeds').
(b) The plants are maintained free from pests, pathogens, virus and other natural hazards.
(c) The plants are not exposed to the threat of changing government policies and urban
development.
(d) Under special storage conditions the plants do not require frequent splitting and pruning.
(e) The material could serve as an excellent form of nucleus stock to propagate large numbers of
plants rapidly, when required.
(f) Being free from known viruses and pathogens the clonal plant material could be sent from
country to country. In cultures, plants can be maintained by serial.
18

19. Disadvantages
(a) Some crops such as cacao, 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) Discrete clones cannot be maintained in the form of seeds
except for apomictic species.
(d) It is not applicable to vegetatively propagated crops such as
Dioscorea, Ipomoea and potato.
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20. 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 and droplet vitrification are widely applied.
• Shoot tips are the preferred material for cryostorage as they contain the
meristem and an organised structure.
• Success in cryopreservation cannot be guaranteed for all plants, as some
species are recalcitrant to tissue culture or the cryopreservation process.
• Storage of desiccated seeds at low temperature, the most convenient
method to preserve plant germplasm, is not applicable to crops that do not20

21. References
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Books –
S. S Purohit – Plant Tissue Culture
Plant Cell and Tissue Culture – Indra K. Vasil and Trevor A. Thorpe, Springer International
Edition, 2010 2nd Edition.
Pdf’s –
Current Frontiers in Cryobiology
Edited by Prof. Igor Katkov
Publisher - InTech
Published in print edition March, 2012
Websites –
http://www.biotecharticles.com/Agriculture-Article/Cryopreservation-and-Conservation-
of-Plant-Genetic-Material-225.html
http://www.fao.org/biotech/docs/panis.pdf