Q.Explain the enzymatic method of isolation of protoplast.

Wall-degrading enzymes strip plant cells of their cell wall to release living, membrane-bound protoplasts.

1. Selection of explant: a suitable, actively growing tissue -- most commonly young leaf mesophyll -- is surface-sterilised and cut into small pieces to maximise the surface area exposed to the enzyme solution.
2. Plasmolysis/osmotic stabilisation: the tissue is placed in an osmoticum (an osmotically balanced solution, typically containing mannitol or sorbitol) to prevent the naked protoplasts from bursting once the rigid cell wall is removed.
3. Enzymatic digestion: the tissue is incubated in a mixture of cell-wall-degrading enzymes dissolved in the osmoticum:
   • Cellulase -- degrades cellulose microfibrils of the primary wall.
   • Hemicellulase -- degrades hemicellulose components.
   • Pectinase (macerozyme) -- degrades the pectin-rich middle lamella, helping separate individual cells (this step alone, without cellulase, is used for cell separation/maceration).
4. The mixture is incubated for a few hours with gentle shaking, at a suitable temperature and pH, allowing the enzymes to completely digest the wall and release spherical, membrane-bound protoplasts into suspension. …