It has been suggested that tissue cultures may be frozen and stored in liquid nitrogen at -1960 C for long term storage of germplasm. This would be of great value in the conservation of germplasm of those crops which normally do not produce seeds, e.g. root and tube crops, or where it may not be desirable to store seeds.
The preservation of cells, tissue and organs in liquid nitrogen is called cryobiology. Many studies have been carried out on cryopreservation of plant cells and organs and the approach appears to have considerable promise in germplasm conservation.
The technique of cryopreservation of freeze preservation involves four steps. (i) Freezing, (ii) storage, (iii) thawing and (iv) re-culture.
Freezing and storage:
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It may be either slow (cooling rate 0.5-4oC min-1 from 0o C to -100o C and then transfer to liquid nitrogen LN), or rapid (>1000o C min-1) or it may be initially slow, e.g. 50 C min-1 up to -30 to 500 C, held at this temperature for about 3- min and then cooled rapidly by plunging the vials into LN. A partial dehydration of the material (in vacuum or by treating them a specially formulated concentrated solution called plant vitrification solution, PVS) before freezing has been found to increase the survival of cells tissues.
The cells and tissue may also be encapsulated in matrices like alginate before freezing. A cryoprotectant, e.g. DMSO (5-8%), glycerol or proline (10%), must be added to the culture medium to protect the cells from injury.
Thawing: Thawing of the frozen materials is achieved by plunging into the water at 37-40o C (thawing rate of 500-7500 C min-1) for 90 seconds. The material is then transferred into an ice bath trill it is re-cultured. The thawing has to be rapid order to avoid ice crystal formation.
Generally, the thawed material is washed several times to remove the cryoprotectant before it is re-cultured. But the recent trend is to re-culture the material in the presence of the cryoprotectant either in diluted (more common) or undiluted state. The cells tissues may be re-cultured on filter paper discs kept on a suitable culture medium. The disc along with cells is frequently transferred to fresh medium this dilutes the cryoprotectants out. Finally cells are scraped off the filtered disc and cultured directly on the medium. At least in some species, this approach is dramatically more successful than others.
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Re-culture:
Materials subjected to cryopreservation may show some special requirements during reculture. For example, shoot-tips from freeze preserved seeding of tomato required GA for developing into shoots (they formed callus in the absence of GA3), while normal shoots tips of tomato do not require GA3) while normal shoot tips of tomato do not require GA3.
Similarly, survival of carrot plantlet was greatly improved by activated charcoal. It is therefore necessary to determine the optimum conditions for reculture of different plant species, particularly when commonly used regimes fail.