Cryopreservation of germplasm

BY HEENA BEPARI
FIRST YEAR M. PHARM PHARMACOGNOSY
bepari.heena@gmail.com
10/14/2014 CRYOPRESERVATION OF GERMPLASM 1

Cryopreservation
 Cryopreservation is a non-lethal storage of biological
material at ultra-low temperature. At the temperature
of liquid nitrogen (-196 degree) almost all metabolic
activities of cells are ceased and the sample can then
be preserved in such state for extended peroids.
 However, only few biological materials can be frozen to
(-196 degree) without affecting the cell viability.

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.

HISTORY
 Theoreticians of cryopreservation was James Lovelock
(born 1919).
 Osmotic stress
 Salt concentration
 Christopher Polge, carried out cryopreservation of
first fowl sperm.
 Later, in 1950s they tried it on humans where
pregnancy was obtained after insemination of frozen
sperm.

 However, the rapid immersion of the samples in liquid
nitrogen did not produce the necessary viability to
make them usable after thawing.
 Importance of controlled or slow cooling to obtain
maximum survival on thawing of the living cells.
 Cryoprotectants were introduced.

INTRODUCTION
 In recent years with tremendous increase in
population:
 Forest
 Land resources
 Population of medicinal plants
 Aromatic plants species

The conventional methods of germplasm
preservation are prone to possible catastrophic
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 be done
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 costly process.

Advantages
 Small areas can store large amount of material.
 Protection from environmental methods.

 Liquid nitrogen is most widely used material for
cryopresevation.
 Dry ice can also be used.
 Why Liquid nitrogen ?
Chemically inert
 Relatively low cost
 Non toxic
Non flammable
Readily available

STEPS INVOLVED IN CRYOPRESERVATION
SELECTION OF PLANT MATERIAL
PREGROWTH
ADDITION OF
CRYOPROTECTANTS
VITRIFICATION
CRYOPROTTECTIVE
DEHYDRATION

ENCAPSULATION AND
DEHYDRATION
FREEZING
RAPID FREEZING
SLOW FREEZING
STEPWISE FREEZING

STORAGE
THAWING
DETERMINATION OF
SURVIVAL OR VIABILITY

SELECTION OF PLANT MATERIAL
 Morphological and physiological conditions of plant
material influence the ability of explants to survive
during cryopreservation.
 Different types of tissues can be used for
cryopreservation such as:
 Ovules
 Anther/pollen
 Embryos
 Endosperm
 Protoplast, etc.

 FACTORS:
o 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.

o Callus derived from tropical plant is more resistant to
freezing damage.
o A rapidly growing stage of callus shortly after 1 or 2
weeks of subculture is best for cryopreservation.
o Old cells at the top of callus and blackened area should
be avoided.
o cultured cells are not ideal for freezing. Instead,
organized structures such as shoots apices, embryos or
young plantlets are preferred.

o Water content of cell or tissue used for
cryopreservation should be low freezable water,
tissues can withstand extremely low temperatures.

PREGROWTH
 Pregrowth treatment protect the plant tissues against
exposure to liquid nitrogen.
 Pregrowth involves the application of additives known
to enhance plant stress tolerance.
E.g.
 abscisic acid
 praline
 trehalose
(OTHER EXAMPLES??)

 Partial tissue dehydration can be achieved by the
application of osmotically active compounds.
 The addition of low concentration of DMSO (1-5%)
during pre-growth often improves shoot tip recovery,
 E.g.
 C. roseus cells are precultured in medium containing
1M sorbitol before freezing. (Chen et al., 1984)
 Digitalis cells were precultured on 6% Mannitol
medium for 3 days before freezing. (Seitz et al., 1983)
 Nicotiana sylvestris with 6% sorbitol for 2-5 days
before freezing. (Maddox et al., 1983)
 (GET MORE EXAMPLES HERE)

ADDITION OF A CRYOPROTECTANT
 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.

CRYOPROTECTANTS
PENETRATING
EXERT THEIR
PROTECTIVE
COLLIGATIVE
ACTION
NON-PENETRATING
EFFECT THROUGH
OSMOTIC
DEHYDRATION

VITRIFICATION
 The term “vitrification” refers to any process
resulting in “glass formation”, the transformation from
a liquid to a solid in the absence of crystallization.
 According to this definition, cells that are properly
slow frozen become “vitrified”.
 A process where ice formation cannot take place
because the aqueous solution is too concentrated to
permit ice crystal nucleation. Instead, water solidifies
into an amorphous ‘glassy’ state.

CRYOPROTECTIVE DEHYDRATION
 Dehydration can be achieved by growth in presence
of high concentration of osmotically active compounds
like
 sugars
 polyols and/or
 In a sterile flow cabinet
 over silica gel.

 Dehydration reduces the amount of water
Depresses
its freezing
temperature and
Promotes
vitrification
Increases the Ice formation
osmotic pressure
 If cells are sufficiently dehydrated they may be able to
withstand immersion in liquid hydrogen.

ENCAPSULATION AND DEHYDRATION
 This involves the encapsulation of tissues in calcium
alginate beads.
 Which are pre-grown 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.


Cryoprotectants used in cryopreservation

FREEZING: RAPID FREEZING
 The plant material is placed in vials and plunged into
liquid nitrogen and decrease of -300 to -10000c or
more occurs.
 The quicker the freezing is done , the smaller the
intracellular ice crystals are.
 Dry ice can also be used in a similar manner.

 This method is technically simple and easy to handle.
 Rapid freezing has been employed for
cryopreservation of shoot tips of potato , strawberry ,
brassica species

SLOW FREEZING
 Tissue is slowly frozen with decrease in temperature
from -0.1 to 10°c/min.
 Slow cooling permits the flow of water from the cells
to the outside , thereby promoting extracellular ice
formation instead of lethal intracellular freezing.
 This method has been successfully employed for
cryopreservation of meristems of peas , potato ,
cassava , strawberry etc.

 In a normal ice making process, the surface of the cube
freezes up much faster than the interior.
 Which “cramps” the interior, clouding it.
 By using very hot (and pure) water inside an insulated
environment, you are assuring yourself a very slow
freezing that allows the interior to cool down at a rate
far closer to that of the exterior, and that lack of
“cramping” is what produces such clear ice.


STEPWISE FREEZING
 In this method slow freezing down to -20 to 40c.
 A stop for a period of approximately 30 min and then
additional rapid freezing to -196c is done by plunging
in liquid nitrogen.
 Slow freezing permits protective dehydration of the
cells and rapid freezing prevents the growing of big ice
crystals.
 The Stepwise freezing gives excellent results in
strawberry and with suspension cultures.

STORAGE
 Storage of frozen material at correct temperature is as
important as freezing.
 The frozen cells/tissues are kept for storage at
temperature ranging from -70 to -196°c.
 Temperature should be sufficiently low for long term
storage of cells to stop all the metabolic activities and
prevent biochemical injury.
 Long term storage is best done at -196°c. …


THAWING
 It is done by putting ampoule containing the sample in
a warm water bath (35 to 40°c).
 Frozen tips of the sample in tubes or ampoules are
plunged into the warm water with a vigorous swirling
action just to the point of ice disappearance.
 It is important for the survival of the tissue that the
tubes should not be left in the warm water bath after
ice melts .


 just a point of thawing quickly transfer the tubes to a
water bath maintained at room temperature and
continue the swirling action for 15 sec to cool the warm
walls of the tube.
 Tissue which has been frozen by
encapsulation/dehydration is frequently thawed at
ambient temperature.

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 Fluorescien diacetate
(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.


MEASUREMENT OF GROWTH OF CELL
CULTURES
 Fresh and dry weight measurements
 Increase in cell number
 Packed cell volume (PCV)
 Molecular protien and DNA
 Mitotic index
 Medium component calibration
 Conductivity of medium
 Cellular protien

FRESH AND DRY WEIGHT
MEASUREMENT
For callus
 Transfer the entire callus to pre-weighed weighing
boat.
 Determine the fresh weight.
For cell suspension culture
 Collect the cells on a pre weighed nylon membrane
 Determine the fresh weight

ALTERNATIVE METHOD
 Weigh an empty centrifuged tube.
 Add cell suspension culture to the tube.
 Centrifuged the tube for 5-10 minutes.
 Check the supernatant fluid, it should not show any
cells.
 Later without disturbing the pellet separate the
supernatant solution with the help of pipette.
 Weigh the tube along with the cells to determine the
fresh weight.

 Dry the samples in an oven for 60 degree untill no
change in the dry weight is measured.
 Growth index (GI) = Final weight of callus or cells
 Initial weight of the callus or cells

INCREASE IN CELL NUMBER
 Haemocytometer can be used
 Callus or cells suspension is needed to be macerated
 Maceration fluid is an equal volume of 10% chromic
acid and 10% nitric acid.
 Treat this mix foe 5 to 10 minutes at 60 degree.
 After cooling, shake the container vigorously to loosen
the cell clumps.
 1% macerozyme incubate flask overnight under
growth condition.

PACKED CELL VOLUME
 Suitable for suspension culture only.
 Simple and can be determined at any stage of growth.
 A small volume(10ml) is aseptically sampled and
placed in graduated conical centrifuged tube.
 Total volume of a cell pellet is determined after
centrifuging at a constant speed of (500g) for specific
time (5 mins).
 PCV is typically expressed as a percentage of the total
volume in tube.

MOLECULAR PROTIEN AND DNA
 The components like DNA and protien are usually
determined by using well established techniquese.g.
Barford’s reagent for protien.

MITOTIC INDEX
 Mitotic index is an estimate of the number of cells of
population in the stages like prophase., metaphase,
anaphase and telophase.
 It is simple but time cnsuming.
 Mitotic index:
 (Number of nuclei in mitosis/ total number of nuclei
scored) × 100

CRYPPRESERVATION EQUIPMENT
 A reliable source of liquid nitrogen
 Safety equipment ( gloves, apron, face shield, pumps
for dispensing liquid nitrogen from a large storage
dewars, trolleys for the transport of dewars).
 Small (1-2 litre) liquid nitrogen resistant dewar(s)
 Dewar(s) for the routine storage of liquid nitrogen.
 Dewar(s) for the long term storage of spicemens
 Cryovilas, straws, boxes, canes, racks.
 A refrigerent (-20 degree)
 A programmable feezer wit dewar and pump
 A water bath for thawing at 40 to 50 degree.

APPLICATIONS OF CRYOPRESERVATION:
CONSERVATION OF GENETIC MATERIAL
 Cryopreservation provides an opportunity for
conservation of endangered medicinal plants.
 Cryopreservation has been used successfully to store a
range of tissue types , including meristems ,
anthers/pollens and embryos.

FREEZE STORAGE OF CELL CULTURES
 A cell line to be maintained has to be subcultured and
transferred periodically and repeatedly over an
extended period of time.
 cryopreservation is an ideal approach to suppress cell
division to avoid the need for periodical subculturing

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 seeds
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 wild
relatives.
 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.

QUESTIONS
 Callus derived from tropical plant is more resistant to freezing damage??
 What is low freezable water??
 Rapid freezing has been employed for cryopreservation of shoot tips of potato ,
strawberry , brassica species. Why??
 Macerozyme : Description of Macerozyme R-10
Macerating enzyme from Rhizopus sp. suitable for the isolation of plant cells
and can be used in combination with cellulase.
Macerozyme R-10 is used in plant protoplast preparation to digest cell wall
prior to oranelle isolation.
Macerozyme R-10 is a multi-component enzyme mixture containing
approximately: Cellulase: 0.1 U/mg; Hemicellulase: 0.25 U/mg; Pectinase: 0.5
U/mg
Macerozyme R-10 is suitable for the isolation of plant cells, and can be used in
combination with cellulase. Used in plant protoplast preparation to digest cell
wall prior to organelle isolation.

BY HEENA BEPARI
FIRST YEAR M. PHARM PHARMACOGNOSY
bepari.heena@gmail.com

Cryopreservation of germplasm

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