Cryopreservation is an important method of long-term preservation that involves storing plant materials like pollen, meristems, embryos, and seeds at ultra-low temperatures, typically in liquid nitrogen at -196°C. The process includes selecting healthy plant materials, treating them with cryoprotectants to prevent ice crystal formation, freezing the samples, storing them long-term in liquid nitrogen, then thawing, washing, testing viability, and regenerating plants. Cryopreservation allows for the long-term conservation of endangered species and economically important plants while preventing disease and genetic contamination.

1. “CRYOPRESERVATION”
BY
TAWSEEF AHMAD MIR
MSC BOTANY IV SEM.
UCBMS&H D.DUN

2. WHY IS PRESERVATION IMPORTANT
• Before the cryopreservation the important
resources were getting depleted and it was
not possible to use them in future.
• Many of the elite and economically
important endangered species are to be
preserved for their availability in the future.
• Due to various reasons the conventional
methods failed to prevent losses to the
important materials.

3. VARIOUS METHODS OF PRESERVATION
• Cryopreservation:- Generally involves
storage in liquid nitrogen.
• Cold storage:- It involves storage in low
and freezing temperature.
• Low pressure:- It involves partially
reducing the atmospheric pressure.

4. CRYOPRESERVATION
Cryo is a Greek word (krayos-frost)
It literally means preservation in “frozen state.”
It involves the principle of bringing the plant cells to
low temperature to stop the metabolic activities in
presence of the cryoprotectants.
It can be done:-
• Over solid carbon dioxide ( -79 degree).
• Deep freezer ( -80 degree).
• Vapour phase nitrogen ( -150 degree).
• Liquid nitrogen ( -196 degree ).

5. STEPS INVOLVED
1) Selection of material.
2) Addition of cryoprotectant.
3) Freezing.
4) Storage in liquid nitrogen.
5) Thawing.
6) Washing.
7) Viability test.
8) Regeneration.

6. 1) Selection Of Plant Material
Selection of the materials is important.
• The materials which are to be selected
should have high meristimatic activity as far
as possible.
• They should not be diseased and should not
have any problem.
• The materials which are mainly selected are
the pollens, meristem, embryo, ovules,
seeds etc…

7. 2) Addition Of Cryoprotectants
• These are the chemicals which prevent the
cryodestruction.
• Cryoprotectants are added to reduce the
formation of the crystals in the cell and to
reduce the dislocation of the cell organelles.
• These are sucrose, alcohols, glycols, amino
acids (proline), DMSO (dimethylsulfoxide).
• Generally two cryoprotectants should be
used together instead of the single one as
they are more effective.

8. 3) Freezing
Cells are exposed to the freezing temperature in
the way that the damaging ice crystals are not
formed.
There are few methods of freezing process.
• Slow freezing:- The tissue or plant material is
slowly frozen at slow cooling rate. Temperature
decreases from -40 to -100 degree.
• Rapid freezing:- it involves plunging the vials in
liquid nitrogen. The temperature decreases
from -300 to -1000 degree rapidly.
• Combine freezing:- combination of both slow
and rapid freezing method. The process is
carried out in step wise manner.

9. 4) Storage In Liquid Nitrogen
• The maintenance of the frozen cells or
material at specific temperature is very
important.
• In general the temperature is kept -70 to
-196 degree.
• Prolong storage is done at temperature of
-196 degree in liquid nitrogen.
• To prevent damage, continuous supply of
nitrogen is given.

10. 5) Thawing
• Thawing is the process of bringing back the
cryopreserved cells to normal state.
• It is usually carried out by plunging the vials
into warm water bath.
• This is to be done on 37-40 degree.

11. 6) Washing
• The preserved material is to be washed few a
times to remove the cryoprotectants.
• Its important to wash the preserved materials
because some cryoprotectants are toxic in
nature.

12. 7) Measurement Of Viability
• There is the possibility of death of cells due to
storage stress.
• Thus viability can be found at any stage.
• This can be calculated by the formula
• No. of cells growing /No. of cells thawed * 100
• This is also done by chemical tests like staining
with various dyes such as FDA(fluorescein
diacetate).

13. 8) PLANT REGENERATION
• After the viability test plant material is
cultured on the growth media .
• Before transferring the regenerated plant to
the natural environment hardening is done
so as to make the plant suitable for growth
in the natural conditions.
• By doing this the a desired variety is
obtained and is used again.

14. APPLICATIONS
• It is ideal method for long term conservation.
• Disease free plants can be preserved and
propagated.
• Endangered species can be maintained.
• Desired characters of plants can be conserved
for future.
• Once in preservation no chance of new
contamination of fungus and bacteria is
experienced, hence disease to plant material
is prevented.

15. Selection of
material
Addition of
cryoprotectant
Freezing
Storage
Thawing
Washing.Viability testRegeneration
FLOW CHART OF CRYOPRESERVATION