Cryopreservation Cryo is Greek word (krayos means “frost”). It means preservation in “frozen state”. Principle – to bring plant cells or tissue to zero metabolism and non dividing state by reducing the temperature in the presence of cryoprotectant. Materials : Over solid carbon dioxide (at -79 degree). Low temperature deep freezer (at -80 degree). In vapour phase Nitrogen (at -150 degree). In liquid nitrogen (at -196 degree).

1. FSC 622 BIOTECHNOLOGY OF
FRUIT CROPS (1+1)
Cryopreservation techniques in fruit
crops

2. Cryopreservation
• Cryo is Greek word (krayos means “frost”).
• It means preservation in “frozen state”.
• Principle – to bring plant cells or tissue to
zero metabolism and non dividing state by
reducing the temperature in the presence of
cryoprotectant.

3. Materials
• Over solid carbon dioxide (at -79 degree).
• Low temperature deep freezer (at -80 degree).
• In vapour phase Nitrogen (at -150 degree).
• In liquid nitrogen (at -196 degree).
Liquid nitrogen storage tank

4. Advantages
• Once the material is successfully conserved to
particular temperature it can be preserved
indefinitely.
• Once in storage no chance of new
contamination of fungus or bacteria.
• Minimal space is required.
• Minimal labour is required.

5. Mechanization
• Selection of material.
• Addition of cryoproctectents.
• Freezing.
• Storage.
• Thawing.
• Washing and Reculturing.
• Measurement of viability.
• Regeneration of plants

6. Selection of materials
• Selection of proper plant material is important.
• Two important factors depend on it such as
(a) nature and
(b) density.
• Eg: Meristem, Embryo, Ovules, Seeds, etc.,
• Density of plant material should be high.
Addition of cryoprotectants
• Chemicals - it prevents cryodestruction.
• Eg: Sucrose, Alcohols, Glycols, Amino acids like
proline, DMSO (dimethyl sulfoxide).
• Generally two cryoprotectants should be used
together.

7. Freezing
• Sensitivity of cells to low temperature depend
on the plant species.
Freezing
Rapid
freezing
Stepwise
freezing
Slow
freezing
Dry
freezing

8. Contd....
• Rapid freezing:- plunging the vials in liquid
nitrogen. The temperature decreases from -300
to -1000 degree rapidly.
• Slow freezing :- the tissue or plant material is
slowly frozen at slow cooling rate. The advantage
is the plant cells are partially dehydrated and
survive better.
• Stepwise freezing :- combination of both slow
and rapid freezing method.
• Dry freezing :- dehydrated cells and seeds are
stored.

9. Storage
• Storage of frozen material at correct temperature
is as important as freezing.
• The frozen cells/tissues are kept for storage at
temperature ranging from -70 to -196 0C.
• Low temperature is sufficient for long term
storage of cells to stop all the metabolic activities
and prevent biochemical injury.
• Long term storage is best done at -196 0C.
• To prevent damage, continuous supply of
nitrogen is done.

10. • Storage container will vary with the size of the
seeds or tissues. Store seeds and tissues in 1.8
and 2.0 ml cryogenic vials for cryopreservation:
1) meristem and shoot tips - 10-25 tissues/vial.
2) small seeds-1500 to 3000 / vial.
3) Larger vials - larger seeded orthodox seeds.
Frozen banana meristems
in liquid nitrogen

11. Thawing
• It is done by putting ampoule containing the sample
in a warm water bath (35 to 40°c).
• Frozen tips of the sample in tubes or ampoules are
plunged into the warm water with a vigorous
swirling action just to the point of ice
disappearance.
• It is important for the survival of the tissue that the
tubes should not be left in the warm water bath
after ice melts.
• just a point of thawing quickly transfer the tubes to
a water bath maintained at room temperature and
continue the swirling action for 15 sec to cool the
warm walls of the tube.

12. Washing and Reculturing
• The preserved material is washed few times to
remove the cryoprotectant.
• This material is then recultured in a fresh
medium.

13. Measurement of viability
• There is possibility of death of cells due to
storage stress.
• Thus viability can be found at any stage.
• It is calculated by formula :
(No of cells growing / no of cells thawed) X 100.
• Various viability tests include Triphenyl
Tetrazolium Chloride assay, Evan’s Blue
Staining.

14. Triphenyl Tetrazolium Chloride assay
• Cell survival is measured by amount of red
formazan product formed due to reduction of
TTC assay which is measured
spectrometrically.
• Only the viable cells which contain the
enzyme mitochondrial dehydrogenase which
reduces TTC to red formazan will be stained
and dead cells will not take up the dye.

15. Evan’s Blue Staining
• One drop of 0.1% solution of Evan’s blue is
added to cell suspension on a microscope slide
and observed under light microscope.
• Only non viable cells (dead cells) stain with
Evan’s blue. % of viable cells = Number of
fluorescent cells × 100 total no of cells(viable +
non-viable).
• Individual cell viability assayed with Evan's blue
dye and fluorescein diacetate.

16. Plant regeneration
• The viable seeds are cultured on non specific
growth medium.
• Suitable environmental conditions are
maintained.

17. Applications
• It is ideal method for long term conservation of
material.
• Disease free plants can be conserved and
propagated.
• Recalcitrant seeds can be maintained for long
time.
• Endangered species can be maintained.
• Pollens can be maintained to increase longitivity.
• Rare germplasm and other genetic manipulations
can be stored.

19. Thank you......