Advantages of Enzymatic Method of Isolation of Protoplast,Isolation of Protoplasts,Cell Suspension Cultures,Enzymatic Treatment

	Advantages of Enzymatic Method of Isolation of Protoplast - The many advantages of the enzymatic method of isolation of protoplasts include: 1) large scale reproducible isolation of protoplasts from various tissues and more or less universal application; 2) osmotic shrinkage is minimum and the deleterious effects of excessive plasmolysis are minimized; 3) cells remain intact and are not injured as is the case of mechanical methods of isolation; and 4) protoplasts are readily obtained.
Commercially available enzymes have enabled isolation of protoplasts from practically every plant tissue in which cells have not acquired lignification. However, the reproducible potential for regeneration has been observed mostly in protoplasts isolated from leaf mesophyll tissues or cell suspension cultures. The isolation and viability of protoplasts depend on a number of factors: age and physiological state of the plant, concentration and purity of the enzyme, pH, period of incubation in enzyme mixture, and the plasmolyticum.
Since the protoplasts isolated for various critical experiments are naked and more highly susceptible to osmotic damage and shock, exposure of the tissue to enzymatic treatment should be , as brief as necessary and the plasmolyticum used should ensure osmotic stability of the protoplasts. Generally, sorbitol and mannitol are used as osmotic stabilizers at an osmolality of 0.5-0.6 osm.