The principle underlying this technique involves bringing the plant cells and tissue cultures to a non-dividing or zero metabolism sate by subjecting them to superflow temperature in the presence of cryoprotectants. In this technique the plant material is frozen and maintained at the temperature of liquid nitrogen ( LN) which is around -196 0C. It is essential that cells during freeze –preservation be protected against cryogenic injuries. Formation of ice-crystals inside the cell can cause the rupture of organelles and cell itself. Increase in concentration of intracellular soultes to toxic levels is another potential source of cell damage. The general practise is to suspend material in the culture medium and after treating with a suitable cryopreservation transfer it to sterile polypropylene ampoules with screw- caps and freeze by one of the following methods.
A) Slow-freezing Method:
In this method the material is frozen at slow cooling rates of 0.5-4 0C per minute, starting from 0 0C until the temperature reaches – 100 0C and finally transferred to liquid nitrogen. It is useful for suspenion culture.
B) Rapid- freezing Method:
This method is simple as the vials containing the plant material are directly lowered into a tank filled with liquid nitrogen. The temperature decreases rapidly at the rate of 300 0C to 11000 0C per minute. Dry ice (CO2), used instead of LN, exerts a similar effect.
C) Step-wise –freezing Method:
The plant material is cooled step-wise to an intermediate temperature, maintained at that temperature for 30 minutes and then rapidly cooled by ploughing it into LN. This method can be used for shoot apices, buds and suspension cultures.
D) Dry-freezing Methods:
Materials dehydrated by drying in oven or under vacuum demonstrate remarkable resistance to cryogenic damage. Dry seeds are able to survive freezing at super low temperature in contrast to water imbibing seeds which show susceptibility to cryogenic injuries. Similarly, dehydration of cells under vacuum also leads to a better survival of plant organs after freezing at 196 0C.
Various studies have shown that freshly harvested cells or tissues may not survive super cooling and required to be conditioned by their brief cultures before freezing. Prefreezing treatment of this type proved beneficial for potato shoot apices only when they were cultured for 48 hours in the presence of 5% dimethylsulphoxide( DMSO). The process of hardening is also important as a prefreezing treatment in tissues culture. Plants grown at low temperature ( 4 0C) for 3 days to a week before taking shoot apices for freeze preservation are reported to considerably enhance the survival frequency of the excised tissues. Along with DMSO3 glycerol and proline are also used as cryoprotectants.
Normally a dilute solution of cryoprotectants ( 5-10 % DMSO) is prepared and added gradually at interval of 5 minutes to prevent plasmolysis of the cells. Use of an ice bath while adding a cryopreservation is beneficial since room temperature may affect the viability of cells and tissues. After the last addition of cryoprotectants these should be an interval of 20-30 minutes prior to freezing.
The storage of culture material at low and non-freezing temperature ( 1-9 0C) has been practised for some plants. Virus free strawberry plants could be maintained for 6 years at 4 0C provided a few drops of liquid medium were added to the cultures after every three months. About 800 cultivars of grape plants have been stored for 15 years at 9 0C by yearly transferred to fresh medium. the use of ABA and high levels of sucrose may help to prolong the interval between two subcultures.
Low Pressure and Low Oxygen Storage:
Attempts have been made to develop low pressure storage (LPS) and low oxygene storage ( LOS) techniques. In LPS, the atmospheric pressure surrounding the tissue culture is reduced, resulting in partial decrease of pressure created by gases in contact with the plant material. On the other hand, in LOP the atmospheric pressure (760 mm hg) is not reduced but the inert gases are combined with oxygene to create low oxygene pressure.
The principle objectives of germplasm conservation using cell and tissue cultures have been:
1. Conservation of Somaclonal and gametoclonal variation in cultures.
2. Maintain of recalcitrant seeds.
3. Conservation of cell lines producing medicines.
4. Storage of pollens for enhancing longevity.
5. Conservation of rare germplasm arsing through somatic hybridization or other methods of genetic manipulations.
6. Delaying the process of ageing.
7. Storage of meristem culture for micropropagation, micro grafting and production of disease free plants.
8. Conservation of plant material from enhanced species.
9. Establishment of germplasm bank.
10. Exchange of information as well germplasm at the international level.