Describe the method of Isolation of protoplast.

Explanation

Protoplasts are the plant cells without cell wall, encircled by a cell membrane.
The small plant bits or tissue like leaf tissue are used to prepare protoplasts. The leaf bits are immersed in 0.5% macrozyne or onozuka cellulase enzymes dissolved in 13% sorbitol or mannitol at pH 5.4 . It is then kept for overnight incubation at `25^@C`. The protoplasts are obtained by gentle teasing of cell. The protoplasts are transferred to 20% sucrose solution in order to retain their viability. The pure protoplasts are obtained by the centrifugation , which removes all debris of cell walls.