The cryopreservation of plant cell culture followed by the regeneration of plants broadly involves the following stages
1. Development of sterile tissue cultures
2. Addition of cryoprotectants and pretreatment
7. Measurement of survival/viability
8. Plant regeneration.
Development of sterile tissue culture:
The selection of plant species and the tissues with particular reference to the morphological and physiological characters largely influence the ability of the explant to survive in cryopreservation. Any tissue from a plant can be used for cryopreservation e.g. meristems, embryos, endosperms, ovules, seeds, cultured plant cells, protoplasts, calluses. Among these, meristematic cells and suspension cell cultures, in the late lag phase or log phase are most suitable.
Addition of cryoprotectants and pretreatment:
Cryoprotectants are the compounds that can prevent the damage caused to cells by freezing or thawing. The freezing point and super-cooling point of water are reduced by the presence of cryoprotectants. As a result, the ice crystal formation is retarded during the process of cryopreservation.
There are several cryoprotectants which include dimethyl sulfoxide (DMSO), glycerol, ethylene, propylene, sucrose, mannose, glucose, proline and acetamide. Among these, DMSO, sucrose, and glycerol are most widely used. Generally, a mixture of cryoprotectants instead of a single one is used for more effective cryopreservation without damage to cells/tissues.
The sensitivity of the cells to low temperature is variable and largely depends on the plant species.
Four different types of freezing methods are used:
1. Slow-freezing method:
2. Rapid freezing method:
3. Stepwise freezing method:
4. Dry freezing method:
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 temperatures in the range of -70 to -196°C. However, with temperatures above -130°C, ice crystal growth may occur inside the cells which reduce the viability of cells. 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, the temperature at -196°C in liquid nitrogen is ideal. A regular and constant supply of liquid nitrogen to the liquid nitrogen refrigerator is essential. It is necessary to check the viability of the germplasm periodically in some samples. Proper documentation of the germplasm storage has to be done.
Thawing is usually carried out by plunging the frozen samples in ampoules into a warm water (temperature 37-45°C) bath with vigorous swirling. By this approach, rapid thawing (at the rate of 500- 750°C min-1) occurs, and this protects the cells 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 (37-45°C) water bath. For the cryopreserved material (cells/tissues) where the water content has been reduced to an optimal level before freezing, the process of thawing becomes less critical.
In general, thawed germplasm is washed several times to remove cryoprotectants. This material is then re-cultured in a fresh medium following standard procedures. Some workers prefer to directly culture the thawed material without washing. This is because certain vital substances, released from the cells during freezing, are believed to promote in vitro cultures.
Measurement of survival/viability:
The viability/survival of the frozen cells can be measured at any stage of cryopreservation or after thawing or re-culture.The techniques employed to determine the viability of cryopreserved cells are the same as used for cell cultures.Staining techniques using triphenyl tetrazolium chloride (TTC), Evan’s blue and fluorescein diacetate (FDA) are commonly used. The best indicator to measure the viability of cryopreserved cells is their entry into cell division and regrowth in culture. This can be evaluated by the following expression.
The ultimate purpose of cryopreservation of germplasm is to regenerate the desired plant. For appropriate plant growth and regeneration, the cryopreserved cells/tissues have to be carefully nursed and grown. Addition of certain growth promoting substances, besides maintenance of appropriate environmental conditions is often necessary for successful plant regeneration.