Enzymes for plant culture
Protoplasts are spherical naked plant cells produced by the removal of the cell wall with digestive enzymes. Plant protoplasts can be cultured in defined media and will form a new cell wall, divide, and in many cases regenerate complete plants. Protoplasts provide a unique experimental system for studies of the structure and function of plant cells.
Enzymatic method is a very widely used technique for the isolation of protoplasts. The advantages of enzymatic method include good yield of viable cells, and minimal or no damage to the protoplasts. Protoplasts can be isolated from a wide variety of tissues and organs that include leaves, roots, shoot apices, fruits, embryos and microspores.
The enzymatic isolation of protoplasts can be carried out by two approaches:
Two step or sequential method:
The tissue is first treated with pectinase (macerozyme) to separate cells by degrading middle lamella. These free cells are then exposed to cellulose to release protoplasts. Pectinase breaks up the cell aggregates into individual cells while cellulose removes the cell wall proper.
One step or simultaneous method:
This is the preferred method for protoplast isolation. It involves the simultaneous use of both the enzymes — macerozyme and cellulose.