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Home » Biology Homework Help » Biotechnology » Cryopreservation
Cryopreservation
It has been suggested that tissue cultures may be frozen and stored in liquid nitrogen at -196˚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 tuber crops, or where it may not be desirable to store seeds. The preservation of cells, tissues and organs in liquid nitrogen is called cryopreservation, and the science pertaining to this activity is known as 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 or freeze preservation involves four steps. (i) freezing, (ii) storage, (iii) thawing and (iv) reculture.

Freezing and storage

It may be either slow (cooling rate 0.5-4˚C min-1 from 0˚C to -100˚C and then transfer to liquid nitrogen, LN) or rapid (> 1000˚C min-1), or it may be initially slow, e.g. 5˚C min-1 upto -30 to -50 ˚C, held at this temperature for about 30 min and then cooled rapidly by plunging the vials into LN. A partial dehydration of the material (in vacuum or by plunging 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 tissues may also be encapsulated in matrices the 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. Cryoprotectants prevent the formation of large ice crystals in cells and protect them from toxic solution effect due to water loss from the cytoplasm.

Thawing

Thawing of the frozen materials is achieved by plunging the vials into water at 37-40˚C (thawing rate of 500-750˚C min-1) for 90 seconds. The material is then transferred into an ice bath till it is recultured. The thawing has to be rapid in order to avoid ice crystal formation. Generally, the thawed material is washed several times to remove the cryoprotectant before it is recultured. But the recent trend is to reculture the material in the presence of the cryoprotectant either in diluted (more common) or undiluted state. The cells/tissues may be recultured on filter paper discs kept on a suitable culture medium. The disc alongwith cells is frequently transferred to fresh medium; this dilutes the cryoprotectants out. Finally, cells are scrapped off the filtered disc and cultured directly on the medium. At least in some species, this approach is dramatically more successful than others.

Reculture

Materials subjected to cryopreservation may show some special requirements during reculture. For example, shoot-tips from freeze-reserved seedlings of tomato required Gas for developing into shoots (they formed callus in absence of GA3), while normal shoot tips of tomato do not require GA3. Similarly, survival of carrot plantlets 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.

Cell cultures, shoot tips, somatic/zygotic embryos and even plantlets of a number of species have been successfully frozen and stored for variable periods (from a few minutes to several months). In general meristematic cells survive better during freeze preservation than mature differentiated cells. The techniques for freeze preservation of shoot-tips are being refined for germplasm storage over very long periods.

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