Get Complete Information on Protoplast Preparation and Fusion

1. Objective :

Preparation of protoplasts from plant tissue and demonstration of somatic fusion.

2. Introduction :

ADVERTISEMENTS:

Protoplast is a plant cell devoid of cell wall. These are useful for the study of physiological problems of the cells and isolation of organelles and study of the exogenously applied material oh cell activity. Protoplasts are prepared from the plant tissues, callus and cultures of suspensions.

When the plant tissue is cut into small sized pieces and treated with pectinase enzyme, the cells are separated from the connective tissue by the enzymes. Cellulase and hemicellulase enzymes breakdown the cell wall leaving only the cell membrane.

Consequently, protoplasts are liberated where cells lose their original shape and turns into round shape. The osmotic potential of the cells is maintained by using mannitol or sorbitol in the digesting medium. This prevents the bursting and shriveling of the protoplasts.

When the protoplasts from the same species are kept in close contact in proper buffer solution, they may fuse spontaneously. The frequency of fusion may be increased by using specific chemicals called fusogens (e.g. sodium nitrate, polyethylene glycol, Ca2+, high pH).

ADVERTISEMENTS:

The fusogens lower the surface charge and thus permit the protoplasts to come into the close proximity for fusion. Protoplast fusion is a useful tool for plant agriculturists to use it in making crosses between sexually incompatible species.

Through protoplast fusion one can transfer cytoplasmic traits between the species (interspecific), within the species (intraspecific) and withing the genera (intergeneric).

3. Principle :

When plant tissues are treated with cell wall lysing enzymes and incubated properly, the plant tissues are digested by the enzymes and protoplasts are released. In the presence of fusogen these protoplasts can fuse with each other. When the protoplasts are obtained from coloured plant tissue, the protoplasts also appear coloured and can easily be seen under the microscope.

ADVERTISEMENTS:

When protoplasts are obtained from two differently coloured tissues, the fusion products can easily be seen and identified as homokaryo (fusion of protoplasts from same tissue) or heterokaryon fusion of protoplasts obtained from different plant tissue.

4. Requirements :

i. Plant tissues such as petals of flower, in vitro grown green tissue, young leaves, a microscope

ii. Pasteur pipettes, rubber bulbs, mannitol

ADVERTISEMENTS:

iii. Macerozyme (a mixture of pectinase, cellulase and hemicellulase) (2% in 0.6 M mannitol, pVL 5.8)

iv. Polyethylene glycol (PEG) (5 g/10 ml)

5. Procedure :

A. Isolation of Protoplasts :

ADVERTISEMENTS:

(i) Collect healthy leaves/petals of coloured flowers/m vitro grown plant tissues.

(ii) Peel off epidermis and shred the leaf tissue (grown preferably under aseptic conditions) into small pieces. The coloured petals of flowers (that have anthocyanin) can also be used.

(iii) Put 200 mg of tissue in 2 ml of enzyme solution taken in a Petri dish.

(iv) Cover the Petri dish and incubate for 4-6 hours at 30°C at 50 rpm. Observe the isolated protoplasts under a simple microscope.

ADVERTISEMENTS:

B. Fusion of Protoplasts :

(i) Put one drop of suspension containing protoplasts and one drop of petal protoplasts on a slide.

(ii) Add 1 drop of PEG in the center of slide to allow the drops of protoplasts to come closer. The process of protoplast fusion is given in Fig. 12.5. Observe the slides under the microscope after 10 minutes.

6. Results :

The isolated protoplasts appear as rounded structures of various sizes. The heterokaryon fusion products appear of different colour.