Question

Give the method for isolation of protoplast.....

Answer 1

isolation of protoplasts can be done by two ways :

1. Mechanical method

2. Enzymatic method

Mechanical Method:

Protoplas isolation by mechanical method is a crude and tedious procedure. This results in the isolation of a very small number of protoplasts.

The technique involves the following stages (Fig. 44.1):



1. A small piece of epidermis from a plant is selected.

2. The cells are subjected to plasmolysis. This causes protoplasts to shrink away from the cell walls.

3. The tissue is dissected to release the protoplasts.

Mechanical method for protoplast isolation is no more in use because of the following limitations:

i. Yield of protoplasts and their viability is low.

ii. It is restricted to certain tissues with vacuolated cells.

iii. The method is laborious and tedious.

However, some workers prefer mechanical methods if the cell wall degrading enzymes (of enzymatic method) cause deleterious effects to protoplasts.

Enzymatic Method:

Enzymatic method is a very widely used technique for the isolation of protoplasts. The advantages of enzymatic method include good yield of viable cells, and minimal or no damage to the protoplasts.

Sources of protoplasts:

Protoplasts can be isolated from a wide variety of tissues and organs that include leaves, roots, shoot apices, fruits, embryos and microspores. Among these, the mesophyll tissue of fully expanded leaves of young plants or new shoots are most frequently used. In addition, callus and suspension cultures also serve as good sources for protoplast isolation.

Enzymes for protoplast isolation:

The enzymes that can digest the cell walls are required for protoplast isolation. Chemically, the plant cell wall is mainly composed of cellulose, hemicellulose and pectin which can be respectively degraded by the enzymes cellulose, hemicellulose and pectinase. The different enzymes for protoplast isolation and the corresponding sources are given in Table 44.1.

In fact, the various enzymes for protoplast isolation are commercially available. The enzymes are usually used at a pH 4.5 to 6.0, temperature 25-30°C with a wide variation in incubation period that may range from half an hour to 20 hours.

The enzymatic isolation of protoplasts can be carried out by two approaches:

1. Two step or sequential method:

The tissue is first treated with pectinase (macerozyme) to separate cells by degrading middle lamella. These free cells are then exposed to cellulose to release protoplasts. Pectinase breaks up the cell aggregates into individual cells while cellulose removes the cell wall proper.

2. One step or simultaneous method:

This is the preferred method for protoplast isolation. It involves the simultaneous use of both the enzymes — macerozyme and cellulose.

Isolation of protoplasts from leaves:

Leaves are most commonly used, for protoplast isolation, since it is possible to isolate uniform cells in large numbers.

The procedure broadly involves the following steps (Fig. 44.2):



1. Sterilization of leaves.

2. Removal of epidermal cell layer.

3. Treatment with enzymes.

4. Isolation of protoplasts.

Besides leaves, callus cultures and cell suspension cultures can also be used for the isolation of protoplasts. For this purpose, young and actively growing cells are preferred.

hope it will help you!!!!

Answer 2

Hey mate ☺️

$\huge \red {Ur Answer}$

Isolation of protoplast can be done by three methods:

(i) Mechanical (non-enzymatic)

(ii) Sequential enzymatic (two-step)

(iii) Mixed enzymatic (simultaneous) GOVT.

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