Cryopreservation

Introduction:
Cryopreservation (Greek, Kryos means frost) refers to preservation in the
frozen state. It means storage at very low temperature such as over solid carbon
dioxide (-790C), in deep freezers (-800C), in vapor phase nitrogen (-1500C) or in liquid
nitrogen (-1960C). The plant material is generally preserved and maintained in liquid
nitrogen. Cryopreservation of animal stock cells and cell lines is preferred to protect
them from genetic drift, microbial contamination, cross contamination by other cell
lines, incubator failure, senescence, etc.
Cryopreservation of plant stock cells:
Due to gradual disappearance of economic and rare plant species, the necessity
for storage of genetic resources of plant is general and agricultural plants in particular
was realized.
Cryobiology deals with the study of metabolic activities and their responses in
plant materials and animal cells stored at low temperature (-1960C) by using liquid
nitrogen in the presence of cryoprotectants. Storage at reduced temperature has been
very effective for tissue culture of most of the plant species such as potato, cassava,
pea, rice, wheat, coconut, strawberry and sugarcane.
The difficulties in cryopreservation are as follows:
High specific feature of plant cells such as their large size, strong
vacuolization and abundance of water.
Cell damage during freezing and subsequence thawing caused by ice
crystals formed inside the cells.
Gradual formation of large crystals of more than 0.1 m which rupture
many cell membranes.
These difficulties could be overcome in the presence of cryoprotectants (the
chemicals decreasing cryodestruction).
Methods of cryopreservation:
The freezing-storage-thawing cycle is an external procedure which includes
the following stages:

a) Selection of materials:
For selecting the plant materials a number of factors are taken into
account. Young, meristematic, highly cytoplasmic and small cells which are
non-vacoulated and thin walled and in small aggregates are good materials to
be selected for this purpose. Cell density in vials should be high as it shows
prolonged survival at high cell density. The cultured cells are not the ideal
system that can be cryopreserved. Organized structures like shoot pieces,
young plantlets, embryos, ovules, anther, pollen, protoplasts, etc, are preferred
for cryopreservation.
b) Addition of cryoprotectants:
Cryoprotectants are the chemicals which decrease cryodestruction.
Example sugars, glycols, sugar alcohols, alcohols, polyvinylpyrrollidone,
polyethylene glycol (PEG), polyethylene oxide (PEO), dextrans, hydoxystarch,
glycerine, sucrose and some amino acid like proline. It is advised to use a
mixture of two or three cryoprotectants at low concentrations rather than a
single cryoprotectant at a high concentration as it may be toxic. Dimethyl
sulfoxide (DMSO) is an excellent cryoprotectant because:
It has low molecular weight.
It is easily miscible with solvent.
It is toxic at low concentration.
It is easily permeable into cells and easily washable.
c) Freezing:
Freezing should be done in such a way that it does not cause intracellular
freezing and crystal formation. The following types of freezing can be done:
i)

Rapid freezing: this method is simple and easy to handle. After

placing the plant material, the cryovials are put into liquid nitrogen
which causes a decrease in temperature. Freezing is done quickly so
that there should be least change or development of intracellular
crystals. Ultra cooling prevents ice crystals.

ii)

Slow freezing: in this method, the rate of freezing is slow i.e. 0.1
100C per minute. This facilitates flow of water from inside to outside.
Thus extra cellular ice crystals are formed but not intracellular crystals.

Thus cell cytoplasm gets concentrated to cause cellular dehydration

and these dehydrated cells survive for a longer duration.
iii)

Step wise freezing: in this method, temperature gets lowered by -20 to

-400C which allows protective freezing of cells. Further, freezing is
stopped for 30 minutes and then it is rapidly freezed in liquid nitrogen
to get -1960C. By doing so, formation of big crystals is increased and
good results are obtained.

d) Storage in liquid nitrogen:

The storage temperature should be such that it stops all metabolic
activity and prevents biochemical injury.

Prolonged storage of frozen

materials is possible only when the temperature is lower than -1300C. This
can be achieved with the help of liquid nitrogen, which keeps the temperature
-1960C.
e) Thawing:
Thawing is the process of releasing the vials containing cultures from
the frozen state to raise the temperature between 35 and 450C. It should be
done quickly but without overheating.

As soon as the last ice crystals

disappear, the vials are transferred into a water bath maintained at 20 25 0C.
During freezing and thawing, major biophysical changes occur in the cell and
the thawed cells need suitable nourishment because they are prone to further
damage.
f)

Washing and reculturing:

Washing of plant materials is done to remove the toxic cryoprotectants.
When low toxic or non-toxic cryoprotectants are used, the cultures should not
be washed before reculturing.

Washing becomes necessary only when

cryoprotectants have toxic effects on cells.

Washing includes dilution,

resuspension, centrifugation and removal of cells. Some cells may die due to
storage stress and only the most stable ones survive. Therefore, determination
of cell viability by culturing them on growth medium. The parameters of
growth measurements are counting cell number, dry and fresh weight, mitotic
index etc. Mitotic index (MI) is counted using the following formula:
MI = No. of cells destined to cell divisions (P+M+A+T)/ total no. of cells both dividing and undividing.
Where P = cells in prophase, M metaphase, A Anaphase and T Telophase.

g) Regeneration of plantlets:

The viable cells are cultured on non-specific growth media to regenerate into
plantlets.
PLANT CELL BANK OR GERMPLASM BANK/ CELL CRYOBANK
Introduction:
Cryopreservation of genetic stock i.e. germplasm (or vegetatively propagated crops,
recalcitrant producing plants, rare plant species, medicinal, horticultural and forest
plants and VAM fungi) is a novel approach for their conservation in liquid nitrogen on
a long term basis. To achieve this goal, a plant cell bank or germ plasm bank and cell
cryobank have been established and these are attached to some of the International
Research Institutes that would hold responsibility for the storage, maintenance,
distribution (at national and international level) and exchange of these disease free
germplasm of the important plants. The flow chart given below shows the potential
and prospects of cryopreservation of plant cell, tissue and organ and establishment of
germplasm bank.
Anther and pollen

Tissue and organ

Pollen embryo

Callus

Cell
suspension

Store disease free stocks

International exchange of
materials
Prolonged pollen longevity
Cold acclimatization

Meristem tip

Shoot apex

Somatic
embryo

long term storage

Germplasm bank
- 1960C

Preservation of haploids

save manpower
save space
save medium
No need to subculture
retain morphogenetic
Potential

Conservation of important
and rare germplasm

prevent aging
Maintaining genetic stability

Facilities for storage of genetic stock of plants can be developed in large sized
cylinders (30- 50 liters capacity) where liquid nitrogen does not require refilling for 6

 8 months.

Thus, germplasm bank is such a device where facilites of

cryopreservation of genetic resources of a variety of plants are available and on

demand, the germplasm can be supplied nationally and internationally.
POLLEN BANK
Besides germplasm bank, the storage of pollen grains in liquid nitrogen and
establishment of pollen bank have been suggested to retain their viability for various
lengths of time. The freeze storage of pollen would enable the following:

Hybridization between plants with flowers at different times.

Growth at different places.

Reducing the dissemination of diseases by pollination agents.

Maintenance of germplasm and enhancement of longevity.

ACHIEVEMENT MADE THROUGH CRYOPRESERVATION

Various forms of plant materials such as cell suspensions, clones, callus, tissues,
somatic embryos, root/ short tips, propagules (tubers), pollen grains etc, have been
preserved in liquid nitrogen for prolonged time and tested for their survival and
regeneration potential. Some of the plant materials that have been cryopreserved are
as follows:

Cryopreservation of cell lines- example cell suspensions (soybean, tobacco,

carrot) and somatic hybrid protoplasts (Rice x Pea; Wheat x Pea).

Cryopreseration of pollen and pollen embryos example, fruit crops, trees,

mustard, carrot, pea nut etc.

Cryopreservation of excised meristems example potato, sugarcane, chickpea,

pea nut etc.

Cryopreservation of germplasm of vegetatively propagated crops example

potato, sugarcane etc.

Cryopreservation of recalcitrant seeds and embryos example, large sized

seeds that are short lived and abortive, such as oil palm, coconut, walnut,
mango and cocoa.

Applications of germplasm conservation or storage

The success of germplasm conservation depends on mainly three factors

1) Innate capability of the species for storage i.e. inherent longevity and
physiological storage behavior.
2) The initial quality like moisture content and
3) Method of storage being employed.
The in vitro methods of germplasm storage are useful in the following:
1) Many asexually propagated crop species do not produce seeds example,
banana, potato, sweet potato, yam, cassava etc.
2) Many crops example mango, rubber, cocoa, coffee etc. produce recalcitrant
seeds which lose viability when they are desiccated.
3) The asexually propagated crops are highly heterozygous and thus seeds
produced by them are genetically highly variable. Therefore, the genetic
worth of such seeds and their value as germplasm are entirely unknown.
4) The materials developed by genetic transformation may sometimes show
instability; seed storage in such cases would not be desirable.
During the last two decades, many regional and international GRCs (Genetic
Resource Centers) for gene or germplasm bank has been set up in different countries.
International Rice Research Institute, Manila (Philippines) has rice germplasm bank,
where 25,000 varieties of rice germplasm has been collected throughout the world.
Maize germplasm have been stored at Maize and Wheat Improvement Centre,
Mexico. Potato germplasm is stored at International Potato Centre, Lima (Peru). A
seed bank has been set up at the National Bureau of Plant Genetic Resources
(NBPGR), New Delhi which is associated with a world network of gene resource
centers coordinated by the International Bureau of Plant Genetic Resources of FAO.