Q.Explain the enzymatic method of isolation of protoplast.

The enzymatic method isolates wall-less protoplasts by digesting plant tissue with cellulase and pectinase in an osmotic solution, then filtering and purifying them.

Protoplasts (plant cells stripped of their cell wall) can be isolated either mechanically (an old, low-yield method) or, much more efficiently, enzymatically -- a technique pioneered by E.C. Cocking in 1960 using fungal cell-wall-degrading enzymes.

Procedure:

1. Choice and preparation of tissue -- healthy plant tissue (most commonly leaf mesophyll, but also callus, cell suspension cultures, etc.) is surface-sterilized, and the lower epidermis is peeled off or the tissue is cut into thin strips to expose the maximum surface area for enzyme penetration.
2. Enzyme treatment -- the tissue is incubated in an enzyme mixture containing:
   • Cellulase -- breaks down cellulose of the primary cell wall.
   • Pectinase (macerozyme) -- dissolves the pectin of the middle lamella, separating individual cells (maceration).
   • Sometimes hemicellulase is also included. Two approaches are used: the sequential (two-step) method (macerozyme first to separate cells, then cellulase to remove the wall) or the mixed enzyme (one-step) method, where both enzymes act together.
3. Osmoticum -- the enzyme solution is prepared in an osmotically stabilizing medium (commonly mannitol or sorbitol at an appropriate molarity) to prevent the naked, wall-less protoplasts from bursting due to osmotic uptake of water.
4. Incubation conditions -- the tissue is incubated for a few hours (often overnight for gentler digestion) at a suitable temperature (around 25 degrees C) and pH, usually with gentle or no agitation to avoid damaging fragile protoplasts. …