Protoplasts are cells with no a cell wall but bound through a plasma membrane. The isolated protoplasts are totipotent. Due to this unique asset, plant protoplasts play an important role in the field of genetic engineering. Protoplast technology involves the culture, isolation, and fusion of higher plant protoplasts leading to the making of complete plants.
A hybrid generated through the fusion of somatic cells of two varieties (or) species is termed as somatic hybrid. This procedure of generating somatic hybrids is termed as somatic hybridization. The first step in somatic hybridization is the isolation of protoplast.
Isolation of protoplast:
Protoplast can be isolated from a range of plant tissues by using either mechanical or enzymatic methods.
In this technique, cells are kept in an appropriate plasmolyticum (protoplast shrink away from cell wall in a plasmolysed cell) and cut with a fine knife, that is why protoplasts are released from cells by the opening of the cell wall. This method provides poor yield of protoplast and it is being used seldom.
Leaves from a 10 week old plant are sterilized with 70 % alcohol and after that treating them with 2 % solution of sodium hypochlorite for 20 to 30 minutes. The leaves after that are washed with sterile water and consequent processes are completed within aseptic conditions (by using laminar air flow chamber). The lower epidermis of the leaf is peeled off and the leaf is cut into small fragments. The protoplasts are isolated from the peeled leaf segments. For isolation of protoplast, peeled leaf segments are positioned with their lower surface down in a petridishes consisting of the enzyme mixture that contains 0.5 % macerozyme, 2 % cellulase in 13% sorbitol or mannitol at pH 5.4. At last the protoplasts are released and are maintained in the isotonic solution.
Diagram: Protoplasmic fusion
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