presentation on

on
• Cryopreservation and it’s Application in
Seed Science and Technology

By:
P.Anitha
1st Ph.D , Dept. of Horticulture
I. D. No. PALB 8081
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WHY PRESERVATION IS IMPORTANT
WHAT IS CRYOPRESERVATION
MATERIALS USED
STEPS INVOLVED IN CRYOPRESERVATION
APPLICATIONS
SEED BANK
GENE BANK
CASE STUDIES
CONCLUSION
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 Why preservation is important ?
Until two decades ago the genetic resources were getting
depleted owing to the continuous degradation by man.

It was imperative therefore that many of the elite, economically

important and endangered species are preserved to make them
available when needed.

The conventional methods of storage failed to prevent losses

caused due to various reasons.

A new methodology had to be devised for long term

preservation of material.
 Cryopreservation is defined as the viable freezing of
biological material and their subsequent storage at
ultra low temperatures (-150 to -196°c) in liquid
nitrogen.
 Long – term storage of plant germplasm

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 Principle of cryopreservation
Cryopreservation, developed during the last 25 years, is an important
and the most valuable method for long-term conservation of
biological materials.
cryopreservation are simplicity and the applicability to a wide
range of genotypes

Cryopreservation technique is based on the removal of all

freezable water from tissues by physical or osmotic dehydration,
followed by ultra-rapid freezing. Cryopreservation includes
classical and new techniques.
 Liquid nitrogen is most widely used material for cryopreservation.
 Dry ice can also be used.

Why Liquid nitrogen ?

Chemically inert
Relatively low cost
Non toxic
Non flammable
Readily available

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 Type of Tissue preserved under

• Seeds and pollen.

• Zygotic embryos/embryonic axes.
• Embryonic cell suspensions.
• Somatic embryos.
• Meristem/shoot tip cultures

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 NBPGR, New Delhi
PGR Conservation : Cryo Gene Bank
Category Accessions
Recalcitrant\in 5,969
termediate
seed
Orthodox 3,182
seeds
Dormant buds 373
Pollen grains 345

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Cryotank
There are various methods of storage :
1. Cryopreservation - generally involves storage in liquid
nitrogen.
2. Cold storage - it involves storage in low and non freezing
temperature.
3. Low pressure – it involves partially reducing the atmospheric
pressure of surrounding.
4. Low oxygen storage - it involves reducing the oxygen level
but maintaining the pressure.
Germplasm
A germplasm is a collection of genetic resources for
an organism.
For plants, the germplasm may be stored as a seed
collection (even a large seed bank).
For trees, in a nursery.
Animal as well as plant genetics may be stored in a
gene bank or cryobank.
The conventional methods of germplasm preservation are
prone to possiblecatastrophic losses because of:

1. Attack by pest and pathogens

2. Climate disorder
3. Natural disasters
4. Political and economic causes
 The conservation of germplasm can bedone by two
methods.

1. In-situ preservation: Preservation of the

germplasm in their natural environment by
establishing biospheres, national parks etc.
2. Ex-situ preservation: in the form of seed or in vitro
cultures.
Ex-situ has following disadvantages
Some plants do not produce fertile seeds.
Loss of seed viability
Seed destruction by pests, etc.
Poor germination rate.
This is only useful for seed propagating plants.
It’s a costlyprocess.
Advantages:

Small areas can store large amount of material.

Protection from environmental methods.
 Mechanism of cryopreservation
The cryopreservation technique followed by the regeneration of plant
involves following steps :
1. Selection of material.
2. Addition of cryoprotectant.
3. Freezing.
4. Storage in liquid nitrogen.
5. Thawing.
6. Washing and reculturing.
7. Measurement of viability.
8. Regeneration of plants.
1.Selection of plant material :
Selection of proper plant material is important.
Two important factors depend on it such as
(a) nature and
(b) density.
Different types of tissues can be used for cryopreservation
such as:
Ovules
Anther/pollen
Embryos
Endosperm
Protoplast, etc.
Tissue must be selected from healthy plants.
o Small
o Young
o Rich in cytoplasm
o Meristematic cells can survive better than the
larger
o Highly vacuolated cells
2. Addition of cryoprotectant
They are chemical which prevent cryodestruction.
These are sucrose, alcohols, glycols, some amino
acid
(proline), DMSO (dimethyl sulfoxide).
Generally two cryoprotectant should be used together
instead of single one as they are more effective.
A cryoprotectant is a substance that is used to protect
biological tissue from freezing damage(damage due to ice
formation).
They acts like antifreeze. They lower freezing temperature
Increase viscosity and Prevents damage to the cells.

There are two potential sources of cell damage

during cryopreservation.
1. Formation of large ice crystals inside the cell.
2. Intracellular concentration of solutes increase to toxic levels
before or during freezing as a result of dehydration.
3. Freezing
The sensitivity of cells to low temperature depends on the plant
species.
There are four different types of methods :
Slow freezing method - the tissue or plant material is slowly
frozen at slowcooling rate. The advantage is the plant cells are
partially dehydrated and survive better.
Rapid freezing method - it involves plunging the vials in liquid
nitrogen. The temperature decreases from -300 to -1000 degree
rapidly.
stepwise freezing method - this is combination of both slow and
rapid freezing method. A stop for a period of approximately 30
min and then additional rapid freezing to -196c is done by
plunging. The process is carried out in step wise like manner.
in liquid nitrogen
Dry freezing method - in this method dehydrated cells and seeds
are stored.
4. Storage
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.
Temperature should be sufficiently low for long term
storage of cells to stop all the metabolic activities and
prevent biochemical injury
To prevent damage, continous supply of nitrogen is
done
5. Thawing:
Usually carried out by plunging the vials into warm water bath
with vigorous swirling.
As thawing occurs the vials are transferred to another bath at 0
degree.
6. Washing and reculturing
The preserved material is washed few times to remove the
cryoprotectant.
This material is then recultured in a fresh medium.
7. 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 * 100
8. Plant regeneration
The viable seeds are cultured on non specific growth medium.
Suitable environmental conditions are maintained.
 Application

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.
Cryopreservation has been used successfully to store a
range of tissue types , including meristems ,anthers/pollens
and embryos.
2. Recently Developed or Modified Methods

a) Vitrification-

b) Encapsulation\ Alginate Coating-

c) Dehydration\ Silica Gel-

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Methods help in preventing damage to the cells:

It is a process in which ice formation cannot take place because the

aqueous solution is too concentrated to permit ice crystals nucleation.
Instead, water solidifies into an amorphous ‘glassy’ state.

..

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 Dehydration can be achieved by growth in presence of high concentration of
osmotically active compounds like sugars , salt, and in a sterile flow cabinet or
over silica gel.

 Dehydration reduces the amount of water available for the ice formation and
increases the osmotic pressure of the intracellular solution (the cytoplasm)
which depresses its freezing temperature and promotes vitrification.

 If cells are sufficiently dehydrated they may be able to withstand immersion in

liquid nitrogen.

..

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This involves the encapsulation of tissues in calcium alginate beads
which are pregrown in liquid culture media containing high
concentration of sucrose.
After these treatments the tissues are able to withstand exposure to
liquid nitrogen without application of chemical cryoprotectants.

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DETERMINATION OF SURVIVAL/VIABILITY

Regrowth of the plants from stored tissues or cells is

the only test of survival of plant materials.
Various viability tests include (FDA) staining ,
growth measurement by cell number ,dry and fresh
weight.
Important staining methods are:
Triphenyl Tetrazolium Chloride (TTC)
Evan’s blue staining
TRIPHENYL TETRAZOLIUM CHLORIDE (TTC) 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.
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 ×
1oo total no of cells(viable + non-viable).
Individual cell viability assayed with Evan's blue dye
and fluorescein diacetate.
MAINTAINENCE OF DISEASE FREE STOCK

Pathogen free stocks of rare plant material could be

frozen and propagated when needed.
Cold acclimatization and frost resistance
A cryopreserved tissue culture would provide a suitable material for
selection of cold resistant mutant cell lines , which could later
differentiate into frost resistance plants.
SEED BANK

A seed bank stores seeds as a source for planting in

case seed reserves elsewhere are destroyed.
It is a type of gene bank.
The seeds stored may be food crops, or those of rare
species to protect biodiversity.
The reasons for storing seeds may be varied
Seeds are dried to a moisture content of less than 5%.
The seeds are then stored in freezers at -18°C or below.
Because seed (DNA) degrades with time, the seed need to be
periodically replanted and fresh seeds collected for another round
of long-term storage.

GENE BANK
Gene banks are a type of biorepository which preserve genetic
material.
In plants, this could be by freezing cuts from the plant, or
stocking the seeds.
In animals, this is the freezing of sperm and eggs
in zoological freezers until further need..
In an effort to conserve agricultural biodiversity,
gene banks are used to store and conserve the plant
genetic resources of major crop plants and their crop
wildrelatives.
There are many gene banks all over the world,
with the
Svalbard Global Seed Vault being probably the most
famous one.
National Seed Storage Laboratory (NSSL) (Fort Collins,
Colorado, USA): 2,100 accessions of apple (dormant buds) .
National Clonal Germplasm Repository (NCGR) of Corvallis
(USA): 104 accessions of pear (shoot tips).

International Potato Centre (CIP) (Lima, Peru) : 345 potato

accessions.

Tissue Culture BC Research Inc .(Vancouver, BC, Canada) :

5000 accessions representing 14 conifer species.
AFOCEL (Association Foret Cellulose) of France, with over
100 accessions of elm (dormant buds).

National Institute of Agro biological Resources (NIAR) of

Japan, with about 50 accessions of mulberry.

IRD (Montpellier, France) : 80 accessions of oil palm .

German Collection of Micro-organisms and Cell

Cultures (DSMZ) (Braunschweig, Germany) : 519 old

potato varieties.
INIBAP, Laboratory of Tropical Crop Improvement,
K.U.Leuven (Heverlee, Belgium) : 440 banana
accessions.
CASE STUDIES

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Seed

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Table 1. viability and germination of ‘Bosc’ pear control seed and seed subjected to
four liquid nitrogen immersion and removal treatments including 30 min exposure to
liquid nitrogen (LN) as determined by triphenyl tetrazolium chloride (TZ) and
germination tests.
LN immersion and removal treatments
Viability test Dz in, D out D in, VP out VP in, D out VP in, VP out Control not
frozen

TZ 87 a 93 a 90a 97 a 87 a

Germination 100 a 93 b 87 b 83 b 77 c

D = direct immersion or removal from liquid nitrogen.

VP = held in the vapor phase of liquid nitrogen for 2 min prior to immersion or 1 min
after removal from liquid nitrogen

Barbara et al., 2001

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Fig.6. Triphenyl tetrazolium chloride staining patterns for Pyrus communis seed.
Black sections indicate pink or red staining of living cell and white areas indicate
dead cells. Drawings 1 to 5 illustrate germinable seeds and 6 to 14 nongerminable
seeds
Barbara et al., 2001
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 By using cryoprotectants
• Cryopreservation of germplasm at liquid nitrogen (-196°C).

• Lily Seeds were subjected to a rapid freezing protocol in liquid

nitrogen following dehydration and treatment with 0.75 M
sucrose for 1 h.

• Survival after freezing was nil for control seeds and 75% for
seeds treated with sucrose and dehydration.

Behzad et al.,2009

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Fig. 7. Effect of precooling temperature on the germination rate and normal
seedlings(%) of C. arabica precooling control seeds (O) and cryopreserved seeds
(O).

Dussert et al.,1997
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Fig. 8. Response of maize genotypes to vitrification treatments.

Usman et al., 2010

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Effect of seed moisture content and cryopreservation on germination of papaya seeds.

Azimi et al.,2005
Thank you