Isolation of protoplasts
Protoplasts (cell minus cell wall) is the biologically active and most significant material of cells. When cell wall is mechanically or enzymatically removed the isolated protoplast is known as "naked plant cell" on which most of recent researches are based.
Plant cell wall acts as physical barrier and protects cytoplasm from microbial invasion and environmental stress. It consists of a complex mixture of cellulose, hemicellulose, pectin, lignin, lipids, protein, etc. For dissolution of different components of the cell wall it is essential to have the respective enzymes.
Until suitable methods were developed, protoplasts were isolated by cutting the plasmolysed plant tissues and releasing protoplast through deplasmolysis of cells. Cooking (1960) for the first time isolated the protoplasts of plant tissues by using cell wall degrading enzymes viz., cellulase, hemicellulase, pectinase, and protease extracted from a saprophytic fungus Trichoderma viride. Later on protoplasts were cultured in vitro.
Microorganisms are well equipped with a system to produce substrate specific extracellular enzymes, the extent of which depends on the genetic variability of the specific species and strains. A detailed account of enzyme preparation and their uses in isolation of protoplast has been given by Cooking (1972 ); Bajaj (1977b) and Patnaik et al (1981). However, the basic techniques of isolation and culture of protoplast are given in Figs. 8.5. and 8.6 with a brief description.
Surface Sterilization of Leaf Samples : Mature leaves are collected from healthy plants which are washed in tap water to remove adhering soil particles and sterilized with sodium hypochlorite solution.
Rinsing in Suitable Osmoticum : After 10 min, sample is properly washed with sterile distilled water or MS medium adjusted to a suitable pH and buffer to maintain osmotic pressure. Washing should be done for about 6 times to remove the traces of sodium hypochlorite.
Plasmolysis of Cells : The lower epidermis covered by thin wax cuticle is removed with a forcep. Stripping should be done from midrib to margin of lamina. The stripped surface of leaf is kept in mannitol solution (13% W/V) for 3 hours to allow plasmolysis of cells.
Peeling of Lower Epidermis : Thereafter, about 1 gm leaves are peeled off and transferred into enzyme mixture already sterilized through a Seitz filter (0.45 mm). This facilitates the penetration of enzyme into tissue within 12-18 hours at 25Â°C.
Isolation and Purification of Portoplasts : Leaf debris are removed with forcep, and enzyme solution containing protoplasts are filtered with a nylon mesh (45mm). Filtrate is centrifuged at 75 X g for 5 min and supernatant is decanted. Again a fresh MS medium plus 13% mannitol is added to centrifuge. Repeated washing with nutrient medium, centrifugation and decantation are done for about three time. Finally specific concentration of protoplast suspension is prepared.Protoplast culture and regeneration
From the protoplast solution of known density (about 105 protoplast/ml) about 1 ml suspension is poured on sterile and cooled down nutrient medium in Petri dishes. The plates are incubated at 25Â°C in a dim white light.
The protoplasts regenerate a cell wall, undergo cell division and form callus. The callus can also be subcultured. Embryogenesis begins from callus when it is placed on nutrient medium lacking mannitol and auxin. The embryo develops into seedlings and finally mature plants.