PROTOPLAST ISOLATION BY MECHANICAL METHOD

Objective:- To isolate protoplast by mechanical method.

Protoplast:- Protoplast is the living material of the cell where as an isolated protoplast is the cell from which the cell wall is removed. In plant breeding programme many desirable combination of characters could not be transmitted through the conventional method of genetic manipulation.

Principle:- Protoplast can be isolated from almost all plant parts: roots, leaves, fruits, tuber, root nodules, pollen mother cell etc. Protoplast isolated by mechanical is a crude and tedious procedure. Cells are plasmolysed causing the protoplast to shrink from the cell wall. The protoplast obtained from this method is then cultured on suitable culture medium. The principle deficiency of this approach is that the protoplast released is few in number. Mechanical isolation was that of only historical event now.

Materials Required:-

i. Plant leaves

ii. Mortar and pestle

iii. Phosphate buffer pH-7.0

iv. 0.3 M sorbitol

v. 0.3 M mannitol

vi. Glass slides

vii. Microscope.

Procedure:-

1. Young leaves were obtained from plants growing outdoors and initially washed with tap water to remove any dust particles.

2. The leaves were washed with phosphate buffer and homogenized gently with the mortar and pestle.

3. The crude protoplast suspension was centrifuged at very low 50-100 rpm for 10 minutes.

4. The supernatant containing intact protoplast was carefully pipetted out and the pellet containing cell debris and other cell organelles were discarded.

5. Small volume of supernatant was placed in the slides and covered with coverslip.

6. The slide was observed in light microscope to find out viable protoplast.

Result:- The spherical shaped protoplasts were observed using the microscope.