cryopreservation

 Cryopreservation

Why Preservation is important?

• Until tow decades ago the genetic resources were getting depleted owing to the continuous depredation 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 of storage failed to prevent losses caused to various reason.
• A new methodology had to be devised for long term preservation of material.

There are various methods of storage

1. Cryopreservation:- Generally involves storage in liquid Nitrogen.
2. Cold storage:- It involve 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.

Cryopreservation;-

• Cryo is greek word, (Kroyes -frost)
• It literally means preservation in "frozen state"

The 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°C)
• Low temperature deep freezer (at-80°C)
• In vapor phase nitrogen (at-150°C)
• In liquid nitrogen (at-196°C)

Major advantages are:-

• Once the material is successfully conserved. Particular temperature it can be preserved indefinitely.
• Once in storage no chance of new contamination of fungus or Bacteria.
•  Minimal space required.
• Minimal labor required.

Mechanism of Cryopreservation:-

 

                             There cryopreservation technique followed by the regeneration of plants involves following steps:-

1. Selection of 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:-

             Two important factors depends 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, alcohol's, glycols, Some amino acid (proline), DMSO (dimethyl sulfoxide)
• Generally tow 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 Freezing:-

• 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.
• Rapid freezing method:- It involves pluming the vials in liquid nitrogen. The temperature decreases from -300°C to -1000°C  rapidly.
• Rapid freezing method:- This is combination of both slow and rapid freezing method. The process is carried out in step wise like manner.
• 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 kept -70°C to -196°C
• Prolong storage is done at temperature of -196°C 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°C 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 Environment Conditions are maintained. 

                                                        Application

• It is ideal method for long term conservation of material.
• DISEASE FREE PLANT CAN BE CONSERVED AND PROPAGATE.
• Peculcitrant 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.