Cryopreservation is a method for long-term conservation of plant genetic resources by storing plant materials like seeds, tissues, cells, pollen, etc. at ultra-low temperatures, usually in liquid nitrogen at -196°C. This preserves the viability and genetic integrity of the materials. There are several advantages like maintaining a large number of accessions in a small space and providing pathogen-free plant materials. Successful cryopreservation involves pretreatment with cryoprotectants, controlled freezing and thawing, then regeneration of plants from the stored materials. It allows preservation of plant genetic diversity for future use in breeding programs.

1. PRESENTED BY :-
Anita Devi
pre-PhD
13098015

2. 1. Abstract
2. History
3. Definition
4. Way of conservation
5. Cryopreservation
6. Advantages and Disadvantages
7. References

3. ABSTRACT
The ever growing human population demands increased
agricultural productivity if possible through methods that
don’t harm the natural environment. The genetic
improvement of crop species is one answer to this
problem.
However, to successfully do so, a diverse array of
genotypes would be needed. Since continued overuse and
misuse of our natural resources together with intensive
agriculture are causing tremendous amount of genetic
erosion.
It is imperative that crop genetic resources be conserved

4. Ever since primitive man learned the art of learning and realised the economic
utility of plants, he started saving selected seeds or vegetative propagules from
one season to next. Conservation of forest resources was taught and decreed in
part of India and China as far back as (700 B.C).
The concept of physical basis of heredity expressed by the 19th-
century Biologist August Weismann. According to his theory, germplasm, which
is independent from all other cells of the body (somatoplasm), is the essential
element of germ cells (eggs and sperm) and is the hereditary material that is
passed from generation to generation. Weismann first proposed this theory in
1883, it was later published in (1892; The Germplasm: A Theory of Heredity ).
Bajaj 1995 and Staristky 1997 reported that some of the valuable gene pools
might be lost unless co-ordinated efforts are made towards the conservation of
genetic stock all over the world.
Realising the danger of genetic resources the U.N Conference on
Human Environment held in Stockholm in 1972, recommended conservation of
the habitat that are rich in genetic diversity.
Two year later in 1974 (CGIAR) established the (IBPGR), subsequently
(IPGRI) with the objective of providing necessary support to the collection,
conservation and utilisation of the plant genetic resources anywhere in the
world.

5. Germplasm broadly refer to the hereditary material (total
content of gene)transmitted to the offspring through germ
cell.
It is also used to describe a collection of
genetic resources for an organism. For plants, the
germplasm may be stored as a seed collection or ,for
trees, in a nursery.

6. Plant germplasm is the genetic source material used
by the plant breeders to develop new cultivars.
They may include :-
• Seeds
Other plant propagules such as
• Leaf
• Stem
• Pollen
• Cultured cells
Which can be grown into mature plant.
Germplasm provide the raw material (genes) which
the breeder used to develop commercial crop
varieties.

7. • Loss of genetic diversity among crop plant species.
• Human dependence on plant species for food and
many different uses. E.g. : Basic food crops, building
materials, oils, lubricants, rubber and other latexes,
resins, waxes, perfumes, dyes fibres and medicines.
• Species extinction and many others are threatened and
endangered – deforestation.
• Great diversity of plants is needed to keep the various
natural ecosystems functioning stably – interactions
between species.
• Aesthetic value of natural ecosystems and the diversity
of plant species.

8. It is of two methods:-

9. In situ conservation is on-site conservation or
conservation of genetic resources in a natural population
of plants, such as forests genetic resources in natural
population of tree species.
It is the process of protecting an endangered plant in its
natural habitat either by protecting or cleaning up the
habitat itself, or by defending the species from predators.
It is applied to conservation of agriculture biodiversity in
agro ecosystem by farmers, especially those using
unconventional farming practice.

11. Ex-situ conservation means literally, "off-site conservation". It is the process of
protecting an endangered species of plant or animal outside of its natural
habitat; for example, by removing part of the population from a
threatened habitat and placing it in a new location, which may be a wild area or
within the care of humans.

12.  Seed gene bank
 In vitro storage
 DNA storage
 Pollen storage
 Field gene bank
 Botanical gardens

13. In vitro method employing shoots, meristems and embryos are ideally suited for
the conservation of germplasm. The plant with recalcitrant seeds and
genetically engineered can also be preserved by this in vitro approach.
There are several advantages associated with in vitro germplasm conservation
Large quantities of material can be preserved in small space
The germplasm preserved can be maintained in an environment free from
pathogens.
It can be protected against the nature’s hazards
From the germplasm stock large number of plants can be obtained whenever
needed.

14. 1. Cryopreservation
2. Cold storage
3. Low – pressure and low oxygen - storage

15. Cryopreservation (Greek, krayos-frost) literally mean in the frozen state. The
principal involved in cryopreservation to bring the plant cells and tissue cultures
to a zero metabolism or non-dividing state by reducing the temperature in the
presence of caryoprotectants.
Cryopreservation broadly means the storage of germplam at very low
temperature.
• Over solid carbon dioxide (at 79⁰C)
• Low temperature deep freezers (at -80⁰C)
• In liquid nitrogen (at -196⁰C)
•Among these the most commonly used cryopreservation is by employing liquid
nitrogen. At the temperature of liquid nitrogen (-196⁰C), the cell stay in a
completely inactive state and thus can be conserved for longer period. Infact
cryopreservation has been successfully applied for germplasm conservation .
Plant species e.g. rice, wheat, peanut, sugarcane ,coconut.

16. The technique of freeze preservation is based on the
transfer of water present in the cells from a liquid to solid
state. Due to the presence of salts and organic molecules
in the cells, the cell water requires much more lower
temperature to freeze (even up to -68°C) compared to the
freezing point of pure water (around 0°C) . When stored at
low temperature , the metabolic processes and biological
deteriorations in the cells/tissues almost come to standstill.

18. The cryopreservation of plant cell culture followed the regeneration
of plants broadly involves the following stages
1. Development of sterile tissue culture.
2. Addition of cryoprotectant and pretreatment
3. Freezing
4. Storage
5. Thawing
6. Reculture
7. Measurement of survival/viability
8. Plant regeneration

19. The selection of plant species and the tissue with particular
reference to the morphological and physiological
characters largely influence the ability of the explants to
survive in cryopreservation . Any tissue from a plant can be
used for cryopreservation e.g. meristems, embryos,
endosperm, ovules, seeds, culture plants cells, protoplast,
callus.

20. Cryoprotectant are the compound that can be prevent the
damage caused to cells by freezing or thawing. There are
several cryoprotectant which include (DMSO), glycerol,
ethylene, propylene , sucrose, mannose, glucose , proline
and acetamide. Among these DMSO, sucrose and glycerol
are most widely used.

21. The sensitivity of the cell to low temperature is variable and largely depends on
the plant species. Four different types of freezing method are used:
Slow freezing method :
The tissue is slowly frozen at 0.5-5°C/min from 0°C to -100°C,and then
transferred to liquid nitrogen.
Rapid freezing method :
Decrease in temperature up to -300 to -1000°C.
Stepwise freezing method:
Intermediate temperature for 30 min. and rapidly cool.
Dry freezing method :
Reported that non- germinated dry seeds can survive freezing at low
temperature in contrast to water imbibing seeds which are susceptible to
cryogenic injuries.

22. Maintenance of the frozen cultures at the specific
temperature is as important as freezing . In general the
frozen cells/tissues are kept for storage at temperatures in
the range of -72 to -196°C. Storage is ideally done in liquid
nitrogen refrigerator – at 150°C in the vapour phase, or at
-196°C in the liquid phase.
The ultimate objective of storage is to stop all the
cellular metabolic activities and maintain their viability. For
long term storage temperature at -196°C in liquid nitrogen
is ideal.

23. Thawing is usually carried out by plunging the frozen samples in ampoules into
a warm water (temp 37 – 45°C) bath with vigorous swirling. By this approach,
rapid thawing (at the rate of 500-750°C min¯¹) occurs, and this protects the cells
from the damaging effects ice crystal formation.
As the thawing occurs (ice completely melts ) the ampoules are quickly
transferred to a water bath at temperature 20-25°C. This transfer is necessary
since the cells get damaged if left for long in warm (37-45°C) water bath.

24. In general thawned germplasm is washed several times to
remove cryoprotectant. The material is then recultured in a
fresh media.
7 : Plant regeneration
The ultimate purpose of cryopreservation of germplasm is
to regenerate the desired plant . For appropriate plant
growth and regeneration , the cryopreserved cell/tissues
have to be carefully nursed, grown. Addition of certain
growth promoting substances, besides maintenance of
appropriate environmental conditions is often necessary for
successful plant regeneration

25. Plant material
Cell suspensions
Callus
Protoplast
Meristems
Zygotic embryo
Somatic embryo
Pollen
Plant species
Oryza sativa
Glycine max
Zea mays
Nicotiana tabacum
Saccharum sp.
Oryza sativa
Zea mays
Nicotiana tabacum
Solanum tuberosum
Cicer arietnum
Zea mays
Citrus sinensis
Daucus carota
Citrus sp.
Nicotiana tabacum

27.  Plant materials (cell/tissue) of several species can be cryopreserved and
maintained for several years, and used as and when needed.
 Cryopreservation is an ideal method for long term conservation of cell culture
which produce secondary metabolites e.g. medicines
 Disease (pathogen) free plant material can be frozen and propagated
whenever required.
 Recalcitrant seeds can be maintained for long .
 Conservation of somaclonal and gametoclonal variation in culture.
 Plant material from endangered species can be conserved.
 Cryopreservation is a good method for the selection of cold resistant mutant
cell lines which could develop into frost resistant plant .

28. The expensive equipment needed to provide controlled
and variable rates of cooling/warming temperatures can
however be a limitation in the application of in vitro
technology for large scale germplasm conservation.
Formation of ice crystal inside the cell should be
prevented as they cause injury to the cell.
Sometimes certain solutes from the cell leak out during
freezing .
Cryoprotectant also effect the viability of cells.

29. M.K Razdan (1993) “An plant Introduction to
plant tissue culture” published by Oxford and IBH
publishing Co.Pvt.Ltd New Delhi - 110049
S.S Purohit (1997) “Plant tissue culture”
published by Axis Books Chopasani Road,
Jodhpur -342003
 Kalyan Kumar De (1992) “Plant Tissue culture”
published by New central book agency Pvt. Ltd
Chintamoni Das Lane , Kolkata- 700009
http://en.wikipedia.org/wiki/Germplasm

30. THANK YOU