FACULTY OF ENGINEERING &
TECHNOLOGY
Course: B. Tech Biotechnology Semester: 5th
Sub Code: BBT-515 Sub Name: Plant Biotechnology
LECTURE 6
Dr. NIHARIKA SINGH
Assistant Professor
Dept. of Biotechnology
Cryopreservation
Cryopreservation (Greek,krayos-frost)literally means in the frozen state. The
principle involved in cryopreservation to bring the plnt cells and tissue cultures
to a zero metabolism or non-dividing state by reducing the temperature in the
presences of cryoprotectants (DMSO (dimethyl sulfoxide), glycerol, ethylene,
propylene, sucrose, mannose, glucose, praline, acetamide etc ).
CRYOPRESERVATION broadly means the storage of germplasm at very low
temperature using :-
Over solid carbon dioxide(at 79°C)
Low temperature deep freezer(at -80°C)
Using vapour nitrogen (at- 150°C)
In liquid nitrogen( at -196°C)
Among these, the most commonly used cryopreservation is by employing liquid
nitrogen. At the temperature of liquid nitrogen(at -196°C), 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 of some plant species e.g
rice, wheat, peanut, sugarcane, coconut.
MECHANISM OF CRYOPRESERVATION
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.
TECHNIQUE OF CRYOPRESERVATION
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
1.DEVELOPMENT OF STERILE TISSUE CULTURE
The selection of plant species and the tissue with particular references to the
morphological and physiological characters largely influences 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.
2.ADDITION OF CRYOPROTECTANT
Cryoprotectant are the compound that can prevent the damage caused to cells
by freezing or thawing. There are several cryoprotectant which include:
(DMSO, GLYCEROL, ETHYLENE, PROPYLENE, SUCROSE, MANNOSE,
GLUCOSE…..)
3.FREEZING
The sensitivity of the cells to low temperature is visible and largely depends on
the plant species.Four different types of freezing are used. Slow freezing
method Rapid freezing method Stepwise freezing method Dry freezing
method
4.STORAGE
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
temperature 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.
5.THAWING
Thawing is usually carried out by plunging the frozen sample in ampoules into the
warm water (temp 35- 45°C) bath with vigorous swirling. By this approach,rapid
thawing(at the rate of 500-750°Cmin-1)occurs, and this protects the cell 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(35-45°C) water bath.
6.RECULTURE
In general thawed germplasm is washed several times to remove cryoprotectant.
The material is then cultured 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/tissue have to be carefully nursed and grown. Addition of certain growth
promoting substances ,besides maintenance of appropriate environmental
condition is often necessary for successful plant regeneration.
QUIZ
