PROTOPLAST ISOLATION BY CHEMICAL METHOD

Objective:- To isolate protoplasts by enzymatic 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:- Protoplasts are isolated by treating tissues with a mixture of cell wall degrading enzyme in solution, which contain osmotic stabilizer. A most suitable source of protoplasts is mesophyll tissue from fully expanded leaves of young plants or new shoots. The release of protoplast is very much dependent on the nature and composition of enzymes used to digest the cell wall. There are three primary components of the cell wall which have been identified as cellulose, hemicellulose and pectin substance. Pectinase (macrozyme) mainly degrades the middle lamella while cellulose and hemicellulase degrades the cellulose and hemicellulosic components of the cell wall. During this enzymatic treatment, the protoplast obtained should be stabilized because the mechanical barrier of the cell wall which offered support has been broken. For this reason an osmoticm is added which prevents the protoplast from bursting.

Materials Required:-

i. Young leaves

ii. 70% ethanol

iii. 2% cellulase

iv. 13% mannitol

v. 0.5% macrozyme

vi. CPW salt solution:

KH2PO4 - 27.2mg/l

KNO3 - 101mg/l

CaCl2 - 1480mg/l

MgSo4 - 246mg/l

KI - 0.16mg/l

CaSo4 - 0,026mg/l

pH - 5.8.

Procedure:-

1. The young leaves were collected and washed in sterile distilled water thrice.

2. The leaves were cut into small bits.

3. Then the leaves were kept immersed in 13% mannitol for 1 h for pre-plasmolysis.

4. Mannitol was removed after incubation ant sterilized enzyme mixture (Cellulase + macerozyme) was added and incubated at 25ºC in a shaker for 12 h.

5. The filtrate was centrifuged at 100g for 5 min to sediment the protoplast.

6. The supernatant was removed and the protoplast pellet was suspended in 10ml of CPW + 21% sucrose solution.

7. The mixture was centrifuged at 100g for 5 min. The viable protoplast will float to the surface of the sucrose solution.

8. The supernatant was collected and viewed under microscope.

9. The protoplasts were visualized in microscope.

Result:- Protoplasts were isolated by enzymatic method and viewed under the microscope.