Question

Select the correct sequence of steps involved in the isolation of protoplasts from the mesophyll cells of a plant leaf:
A. Culture of protoplast suspension
B. Infiltration of the enzyme into the leaf tissue
C. Incubate leaf pieces in filter-sterilized enzyme solution
D. Release of mesophyll protoplasts in solution
Choose the correct answer from the options given below:

• A. B, C, D, A
• B. A, D, C, B
• C. C, B, D, A
• D. C, D, B, A

Hint:

Think of the process as: Inject the enzymes $\rightarrow$ Wait (Incubate) $\rightarrow$ Collect (Release) $\rightarrow$ Grow (Culture).

Answer 1

The Correct Option is A

Concept: Protoplasts are "naked" plant cells where the cell wall has been enzymatically removed. To isolate them from leaves, enzymes must penetrate the tissue to reach the mesophyll.

Step 1: Enzyme Penetration.
The first critical step is Infiltration (B). Because leaves have a protective cuticle, the enzyme solution must be forced into the intercellular spaces (often via vacuum infiltration) to ensure contact with the cell walls.

Step 2: Digestion.
The leaf pieces are then Incubated (C) in a cocktail of Cellulase and Pectinase. This provides the time necessary for the enzymes to degrade the cellulose and pectin of the cell walls.

Step 3: Release and Culture.
As the walls dissolve, the Release of protoplasts (D) occurs into the osmoticum solution. Once isolated and cleaned, the final step is the Culture (A) of these cells to regenerate the cell wall or for fusion experiments.