Cryopreservation | BiotechStudies

CRYOPRESERVATION

• Cryo is Greek word. { krayos= Frost }.
• It literally means preservation in “ frozen state ".
• Principle –  To bring plant cells or tissue to a zero metabolism and non dividing state by reducing the temperature in the presence of cryoprotectant.

 It can be done :

       Over solid carbon dioxide (at-79 degree)

       Low temperature deep freezer (at-80 degree)

       In vapour  phase nitrogen (at-150 degree)

       In liquid nitrogen (at-196 degree)

• There are various method of storage :

1. Cryopreservation – generally involves storage in liquid nitrogen.
2. Cold storage – it involves 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.

Mechansim of cryopreservation:

The cryopreservation technique  followed by regeneration of plants 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 factor depend on it such as:
•     Nature and Density
•     Any tissue can be selected for this purpose. e.g. Meristem ,embryo, ovules, seeds etc.
•     The density should be high.

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.

3. Feezing:

The sensitivity of cells to low temperature depends on the plant species.

There are four different types of methods.

1. Slow Freezing method – the tissue or plant material is slowly frozen at slow cooling rate. The advantage is the plant cells are partially dehydrated and survive better.
2. Rapid freezing method -  it involves plunging the vials in liquid nitrogen. The temperature decreases from -300 to-1000 degree rapidly.
3. 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.
• 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 the 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.
• Its 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.

Major advantages are:

•  Once the material is successfully conserved  to particular temperature it can be preserved indefinitely.
• Once in storage no chance of new contamination of fungus or bacteria.
• Minimal space required.
• Minimal labour required.