Protoplast culture

Protoplast culture

1. Protoplast and its isolation

2. Introduction Is a naked cell surrounded by a plasma membrane. It can regenerate cell wall, grow and divide. Is fragile but can be cultured and grow into a whole plant. Is used by biotechnologists, microbiologists, pathologists. In 1960, E.C Cocking contributed to the enzymatic isolation and culture of protoplast

3. A typical Plant cell wall The Plant cell wall is multilayered structure The middle lamella - Pectin compounds and proteins. The primary wall - Cellulose and hemicelluloses. The secondary wall - Cellulose, hemicelluloses and lignin.

4. Definition Protoplast : Unit of biology which is composed of a cell's nucleus and the surrounding protoplasmic materials. Hanstein (1880). OR It is a plant cell that has its cell wall completely removed using either mechanical or enzymatic means.

5. Why do we need to remove cell wall ? Large molecules can not pass through the cell wall. Somatic hybridization and genetic Manipulation can not be done . So we have to remove the cell wall. After removing it, we will get protoplasts

6. ISOLATION OF PROTOPLAST Important to isolate as gently and as quickly as possible viable and uninjured protoplast. There are two methods for the isolation of protoplast from plant tissues. Mechanical method Enzymatic method

7. 1. Mechanical method

8. Mechanical method In this method, the cells are kept in suitable plasmolyticum (for example CPW salts containing 13% w/v mannitol or immersed in 1.0 M sucrose). Once the plasmolysis is complete , while remaining in the osmoticum , the leaf lamina would be cut with a sharp-edged knife Now, cells are cut only through the cell wall, releasing intact protoplasts. The released protoplasts then have to be separated from damaged ones and cell debris.

9. Drawbacks Used for vacuolated cells like onion bulb scale, radish and beet root tissues only. Tissue damage is caused by mechanical method maximum osmotic shrinkage Low yield. Laborious and tedious process. Low viability.

10. 2. Enzymatic method: One step method (direct method) Refers to the use of enzymes to dissolve the cell wall for releasing protoplasts. Cocking 1960. Advantages: Used for variety of tissues and organs such as fruits, roots, petioles, leaves, shoots, etc. Osmotic shrinkage is minimum Cells remain intact and not injured Protoplast readily obtained

11. Protocol for direct method Incubation of leaf segments over night in enzyme solution ( pectinase and cellulase ) Treated with enzymes to liberate protoplasts , Mixture is filtered and Centrifugation at 600rpm for 5mins. Protoplast forms pellet These are washed with sorbitol 3X , recentrifuge Cleaned protoplast float Pipettes out.

12. Protocal : Sequential method (indirect method) Two enzyme mixtures ( mixture A and 'mixture B ) are used one after the other. Leaf segments with mixture A ( macerozyme in manifold at pH 5.8 ) are vacuumed infiltrated for 5 min. (WASHING) After 15 min. the enzyme mixture is replaced by fresh 'enzyme mixture A ' and leaf segments are incubated for another hour.

13. The mixture is filtered using nylon mesh , centrifuged at 600rpm for 1 min. Washed three times with 13% mannitol to get a pure sample of isolated cells. Cells are then, incubated with 'enzyme mixture B ' ( cellulase in a solution of mannitol at pH 5.4) for above 90 min, at 30° C. After incubation, the mixture is centrifuged for 1 min , so that protoplasts form a pellet, which are cleaned three times as in 'one step method.

14. Purification of protoplasts Protoplasts are purified by removing: Undigested material (debris) Bursts protoplasts(damaged protoplasts) Enzymes( pectinase+cellulase ) ● Debris are removed by filtering the preparation through a nylon mesh of 100µ pore size. ● Enzymes are removed by centrifugation at 600rpm for 5 mins whereby the protoplasts settle to the bottom of the tube and the supernatant removed with the help of a pipette ● Intact protoplasts are separated from broken protoplasts through centrifugation and removed by a pipette as they are collected at the top of tube

15. Protoplast viability and density To check the viability of isolated protoplasts, 1 . Fluorescein diacetate test : It accumulates only inside the plasma lemma of viable protoplasts , can be detected with fluorescence/UV microscopy. 2 . Evans blue test : Intact viable protoplasts, exclude the Evans blue stain. Impermeability of the cell to Evans blue indicates a living cell. 3. Cyclosis test : P rotoplasmic streaming can be a measure of viability. 4. Density test: The density of the protoplasts in the suspension ( number/unit volume ) is determined by counting with a modified haemocytometer such as Neubauer with a field depth of 0.2mm.

16. Application of protoplast Protoplasts can be used to study; Metabolic studies including photosynthesis. For DNA transformation. For somatic hybridization. Ingesting "foreign" material into the cytoplasm. Single cell systems. Cell Wall synthesis.

17. Application

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