Cryopreservation Short Notes | Biotechnology

In this article, we will discuss about:- 1. What is Cryopreservation? 2. Mechanism of cryopreservation and 3. Application of cryopreservation

1. What is Cryopreservation?

Cryopreservation (Greek, krayos-frost) literally means preservation in the frozen state. The principle involved in cryopreservation is to bring the plant cell and tissue cultures to a zero metabolism or non-dividing state by reducing the temperature in the presence of cryoprotectants.

Cryopreservation broadly means the storage of germplasm at very low temperatures:

i. Over solid carbon dioxide (at -79°C)

ii. Low temperature deep freezers (at -80°C)

iii. In vapour phase nitrogen (at -150°C)

iv. In liquid nitrogen (at -196°C)

Among these, the most commonly used cryopreservation is by employing liquid nitrogen. At the temperature of liquid nitrogen (-196°C), the cells stay in a completely inactive state and thus can be conserved for long periods.

In fact, cryopreservation has been successfully applied for germplasm conservation of a wide range of plant species e.g. rice, wheat, peanut, cassava, sugarcane, strawberry, coconut. Several plants can be regenerated from cells, meristems and embryos stored in cryopreservation.

2. Mechanism of cryopreservation

The cryopreservation technique followed by the 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 factors depend on it such as (a) nature and (b) 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. Freezing:

The sensitivity of cells to low temperature depends on the plant species. There are four different types of methods :
i) 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.
ii) Rapid freezing method - it involves plunging the vials in liquid nitrogen. The temperature decreases from -300 to -1000 degree rapidly. 
iii) Stepwise freezing method - this is combination of both slow and rapid freezing method. The process is carried out in step wise like manner.
iv) 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, continuous 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.

3. Application of cryopreservation:

• 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.