Explain the enzymatic method of isolation of protoplast.

Protoplasts are cells without a cell wall. It is bounded by a cell membrane or plasma membrane. The protoplasts are used for the regeneration of the whole plant and also for the production of somatic hybrids. The first step of this process is the isolation of the protoplast. The protoplast is isolated from the cell mainly by using enzymes. Small bits of plant tissue like leaf tissue is used for the isolation of protoplast. The leaf tissue is immersed in 0.5% macrozyme and 2% cellulase enzyme dissolved in 13% sorbitol or mannitol at pH 5.4. It is then incubated overnight at 25 0 C. After a gentle teasing of cells, protoplasts are obtained and these are then transferred to 20% sucrose solution to retain their viability. They are then centrifuged to get pure protoplasts. The isolated protoplasts are either transferred to the culture medium for protoplast culture or use in somatic hybridization. In somatic hybridization, the protoplasts fused with the help of PEG (polyethylene glycol).