CRYOPRESERVATION

1. CRYOPRESERVATION
PRESENTED BY:-
DR AKANKSHA JAIN
BIOTECHNOLOGY

2. SYNOPSIS
1.INTRODUCTION
2.CRYOPRESERVATION OF PLANT STOCK CELL
Cryopreservation of plant protoplast
Cryopreservation of shoot tips & meristems
Cryopreservation of seeds
3.GENERAL MATERIALS USED IN CRYOPRESERVATION
4.GENERAL METHODS OF CRYOPRESERVATION
5.DETAIL DISCUSION OF:-
Cryopreservation of plant protoplast
Cryopreservation of seeds
6.PLANT CELL BANK/GERMBANK/CELL CRYOBANK
7.POLLEN BANK
8.ACHIEVEMENT MADE THROUGH CRYOPRESERVATION
9.APPLICATION OF CRYOPRESERVATION
10.CENTER OF CRYOPRESERVATION
11.DIFFICULTIES IN CRYOPRESERVATION
12.CONCLUSION
13.REFERENCES

3. INTRODUCTION:-
Cryopreservation (Gr.kryos means frost) refers to
“preservation in the frozen state”. It means storage at very low
temperature such as over solid CO2 (-79° c) in deep freezers
(- 80 c ) in vapour phase nitrogen (- 150° c or in liquid nitrogen
- 196° c) .The plant material is generally preserved and
maintained in liquid nitrogen.
Cryobiology deals with the study of metabolic activities and
their response in plant materials stored at low temperature
(- 196° c) by using liquid nitrogen in the presence of
cryoprotectants.

4. HISTORY
First cryopreservation of sperm in 1953 and of
embryo thirty years later,these techniques have
become routine.
Dr.Christopher chen of Australia reported the
World’s first pregnancy in 1986 using previously
frozen oocytes

5. FREEZABLE TISSUE
•Blood: Special cells for transfer
•Stem cell
•Umbilical cord blood
•Tissue sample: Tumors & histological cross
section
•Egg(oocyte)
•Embryo that are 2,4 or 8 cells
•Plant seeds & shoots
•Semen:21year
•Semen

6. GENERAL MATERIALS USED IN
CRYOPRESERVATION:-
Materials which are preserved
Culture medium
Cryoprotectants- DMSO, glycerols, sorbitol,
mannitol, glucose, sucrose.
Polypropylene ampules.
Pasture or volumetric pipettes
Freezing and storage apparatus
Thermostatted water bath

7. GENERAL METHOD OF CRYOPRESERVATION:-
•Selection of materials
•Addition of cryoprotectors
•Freezing
RAPID FREEZING:-
This method is simple and easy to handle .After placing the plant
material the cryovials are put into liquid Nitrogen which cause a
decrease in temp.
Rapid freezing of several plant materials has been done somatic
embryos & shoot tips of Brassica napus, strawberry, potato etc.

8. SLOW FREEZING:-
In this method the rate of freezing is slow 0.1-10 °c
per minute. Meristem of potato, cassava , strawberry have
successfully been cryopreserved.
STEPWISE FREEZING:-
In this method temp gets lowered by -20° to --40° c
further freezing is rapidly freezed in liq. N2 to get --196 °c.

9. Storage in liquid nitrogen:-
Prolonged storage of frozen materials is possible only
when the temp is lower than -130° c . This is achieved by
liq nitrogen . Popove stored the culture of carrot cells for
about 5 years by doing so.
• Storage of frozen material at the correct temp. is
as imp. as freezing.
• The frozen cells are immediately kept for storage
at temp. ranging from -70°C to -196°C.

10. Thawing
Thawing is usually carried out by putting the vial or ampoule
containing the sample in a warm water bath for thawing.
Thawing is the process of releasing the vials containing
culture from the frozen state to elevate the temp
between 35-45° c.

11. VITRIFICATION:-
At a sufficiently low temp , highly concentrated
aqueous solution of cryoprotective agents become so viscous
that they solidify into a metastable glass state , without ice
crystal formation at practical cooling rates, this phenomenon is
call as vitrification.
Vitrification solution (PVS) themselves may cause to
toxicity which depends mainly on their osmotic potential.
Vitrification has been used to the greatest effect for
cryopreservation of germplasm of such plant species that are
recalcitrant to the traditional cryopreservation methods based
on controlled freezing.

12. ENCAPSULATION DEHYDRATION:-
In other approach, explants are first encapsulated in a
suitable matrix like alginate and then subjected to
dehydration.
SLOW GROWTH CULTURE:-
Slow growth of plantlets in vitro provides an
attractive alternation to freeze preservation of germplasm as
it is simpler, cheaper & very effective. Slow growth may be
achieved by maintaining the plantlets either at a low
temperature (4-9°c or ca, 15ºc) or on a medium having high
osmotic concentration.

16. CRYOPRESERVATION OF PLANT PROTOPLAST:-
INTRODUCTION:-
Isolated plant protoplasts are most commonly liberated from
tissue or cultured cells, by enzymatic digestion of the retaining
cell wall material.
The ideal candidate as a conservation technique is
cryopreservation using liquid nitrogen (-196ºc) as the storage
medium, which will provide an effective cessation of all
aspects of growth and development & provide the maximum
available genetic stability.
MATERIALS USED IN CRYOPRESERVATION:-
Slow cooling:-
Protoplast culture
Culture medium
Cryoprotectants
Ampules
Pipets

17. •Freezing & storage apparatus
•Thermostatted water bath at an appropriate
temperature.
•A freshly made stock solution of fluoroscein
diacetate in acetone the immediate survival of
the protoplast is determined by microscopical
examination of a sample using UV light.

18. CRYOPRESERVATION OF SEEDS:-
MATERIALS USED :-
Freezing of dry seeds
Seeds
Stainless steel or aluminum weighing dishes with lids
Fan oven
A dry room or relative humidity chamber.
Crypstorage containers
A programmable freezer
Storage system
A water bath 40-45°c
1% triphrnyltetrazolium chloride (TTC) in phosphate
buffer is required for the vital, histochemical staining test.
Dissecting instruments
Incubator with temp & lighting control
Protective equipment
Freezing of cryoprotected seeds
Seed rehydration
Cryoprotectants

19. METHOD:-
FREEZING OF CRYOPROTECTED SEED:-
Rehydrate seed on moist filter paper or agar . or above water to
desired moisture content at room temp.
Surface sterilize the seed in a dilute solution of commercial sodium
hypochlorite for 30 min . Higher conc. And longer times may be
necessary depending on the level of coat borne infection and surface
topography.
Following rehydration , soak the seed in cryoprotectant for 1 h at 25
or 4°c .
Remove seed from cryoprotectant surface dry on filter/ tissue paper
transfer to appropriate container and place directly in liq N2 then
transfer to cryostorage vessel.
Thaw in water bath at 40-45°c for 30S 2min or in air at RT for 20
min.
Sow seeds for germination without removal of cryoprotectant ,
although when high conc. have been employed remove by washing
alternatively asses seed quality by tetrazolium staining.

20. CELL CRYOBANK:-
Cryopreservation of genetic stock germplasm is a novel
approach for their conservation in liq. Nitrogen on a long term basis
. To achieve this goal, a plant cell bank has been suggested by Bajaj
& reinert and popov.
Suggestions have also been made that germplasm bank
should be attached to some of the international research institutes
that would hold responsibility for the storage, maintenance,
distribution and exchange of these disease free germplasm of the
important plants.
Thus, germplasm bank is such a device where facilities of
cryopreservation of genetic resources of a variety of plants are
available and on demand the germplasm can be supplied nationally
& internationally.
POLLEN BANK:-
Besides germplasm bank the storage of pollen grains in liq.
Nitrogen and establishment of pollen bank have also been
suggested to retain their viability for various length of time.

21. ACHIEVEMENT MADE THROUGH CRYOPRESERVATION:-
CRYOPRESERVATION OF CELL LINES:- For ex. Cell suspentions (soyabean,
tobacco, dhatura, carrot) and somatic hybrid protoplasts (rice x pea, wheat x pea.)
CRYOPRESERVATION OF POLLEN & POLLEN EMBRYOS:- fruit crops ,
trees, mustard.
CRYOPRESERVATION OF EXCISED MERISTEMS:- potato,
sugarcane,chickpea, peanut.
CRYOPRESERVATION OF RECALCITRANT SEEDS AND EMBRYOS:- large
sized seeds that are short lived and abortive, such as oil palm, coconut, walnut,
mango,cocao.

22. DIFFICULTIES OF CRYOPRESERVATION:-
High specific features of plant cells, such as their large
size, strong vacuolization and abundance of water.
Cell damage during freezing and subsequent thawing
caused by ice crystals formed inside the cells & by cell
dehydration.
Gradual formation of large crystals of more than 0.1 um
whose facets rupture many cell membrane.

23. CRYOPRESERVATION CENTRE:-
NCGRP cryopreservation lab (National center for
genetic resources preservation).
US department of agriculture, research service
Research center of ROME
Hosts in field fruit germplasm collection
Advance in potato cryopreservation at the international
potato centre, PERU.
USDA-ARS national center for genetic resources
preservation USA>
ISHS plant cryopreservation :- germplasm in INDIA
Cryopreservation of crop species in EUROP
NBPGR biodiversity (National bureau of plant genetic
resources)
Germplasm center lucknow.

24. APPLICATION:-
Conservation of genetic materials:- like cultured
embryos, tissue, cells.
Maintenance of disease free stocks:-Pathogen free
stocks of rare plant materials could be frozen,
revived & propagate.
3.Cold acclimation & frost resistance:-Tissue
cultures would provide a suitable material for
selection of cold resistant mutant cell lines, which
could later differentiates in to frost resistance
plants.

25. CONCLUSION:-
•Hence we conclude that cryopreservation is
the successful technique for the preservation of
cells tissues, seeds, embryos etc.
•Human oocyte cryopreservation (egg freezing)
is a novel technology in which a woman’s eggs
are extracted, frozen & stored. Later when is
ready to become pregnant the eggs can be
fertilized & transferred to the uterus as embryo.

26. BOOKS AUTHOR EDITION
PLANT BIOCHEMISTRY P.M.DEY & J.B
HARBORNE
(2005)
CRYOPRESERVATION
AND FREEZE DRYING
PROTOCOLS
JOHN G DEY AND
MARK R MCLELLAN
(2003)
A TEXT BOOK OF
BIOTECHNOLOGY
R.C. DUBEY (2007)
EXPANDING HORIZON B.D. SINGH (2007)
REFERENCES