It is a process of preservation of cells or whole tissues , which are preserved by cooling the temperature to low- subzero .(-196c ) the boiling point of liquid nitrogen . in this all biological process are suspended and material does not decompose .Cryopreserved cells are fragile it should be handle carefully .
Advantage of freezing cells-
1. Reduced risk of Genetic change .
2.Reduced risk of Senescence leading to extinction of cell line.
3.Reduced risk of Transformation to tumor related properties.
4.Reduced risk of Contamination
5.Saving reagents time
6.Prevent Cross contamination by other cell line.
Process of cryopreservation -
1.The selection of plant species or tissues with particular morphological and physiological characters . ( 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.
2.Cryoprotectants are the compounds that can prevent the damage caused to cells by freezing .The freezing point of water are reduced by the presence of cryoprotectant. As a result, the ice crystal formation is retarded during the process of cryopreservation.
Cryoprotectants are dimethyl sulfoxide (DMSO), glycerol, ethylene, propylene, sucrose, mannose, glucose, proline and acetamide. Among these, DMSO, sucrose and glycerol are most widely used.
Advantage of freezing cells-
1. Reduced risk of Genetic change .
2.Reduced risk of Senescence leading to extinction of cell line.
3.Reduced risk of Transformation to tumor related properties.
4.Reduced risk of Contamination
5.Saving reagents time
6.Prevent Cross contamination by other cell line.
Process of cryopreservation -
1.The selection of plant species or tissues with particular morphological and physiological characters . ( 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.
2.Cryoprotectants are the compounds that can prevent the damage caused to cells by freezing .The freezing point of water are reduced by the presence of cryoprotectant. As a result, the ice crystal formation is retarded during the process of cryopreservation.
Cryoprotectants are dimethyl sulfoxide (DMSO), glycerol, ethylene, propylene, sucrose, mannose, glucose, proline and acetamide. Among these, DMSO, sucrose and glycerol are most widely used.
3.The sensitivity of the cells to low temperature is variable and largely depends on the plant species.
4.The frozen cells/tissues are kept for storage at temperatures in the range of -70 to -196°C. Storage is ideally done in liquid nitrogen refrigerator — at 1 50°C in the vapour phase, or at -196°C in the liquid phase to maintain viability .For long term storage, temperature at -196°C in liquid nitrogen is ideal. A regular and constant supply of liquid nitrogen to the liquid nitrogen refrigerator is essential.
5.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.
6.Thawed germplasm is washed several times to remove cryoprotectants and then re-cultured in a fresh medium following standard procedures.
7.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 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.
8.Regeneration - For appropriate plant growth and regeneration thr cryopreserved cells should be carefully nursed, and grown. Addition of certain growth regulators also help in regeneration.
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