Protoplasts are cells without a cell wall but bound by a plasma membrane. The isolated protoplasts are totipotent. Because of this unique property, plant protoplasts play a vital role in the field of genetic engineering. Protoplast technology includes the isolation, culture and fusion of higher plant protoplasts leading to the production of entire plants. You will be studying the method of isolation and fusion of protoplast in this chapter.
A hybrid produced by fusion of somatic cells of two varieties (or) species is called somatic hybrid. This process of producing somatic hybrids is known as somatic hybridization. The first step in somatic hybridization is the isolation of protoplast.
Protoplast can be isolated from a variety of plant tissues using either mechanical (or) enzymatic methods.
In this method, cells are kept in a suitable plasmolyticum (protoplast shrink away from cell wall in a plasmolysed cell) and cut with a fine knife, so that protoplasts are released from cells through the opening of the cell wall. This method gives poor yield of protoplast and it is being rarely used.
Leaves from a 10 week old plant are sterilized with 70 per cent alcohol and then treating them with 2 per cent solution of sodium hypochlorite for 20 to 30 minutes. The leaves are then washed with sterile water and subsequent procedures are done under aseptic conditions (using laminar air flow chamber). The lower epidermis of the leaf is peeled off and the leaf is cut into small fragments. From the peeled leaf segments, the protoplasts are isolated. For isolation of protoplast, peeled leaf segments are placed with their lower surface downwards in a petridishes containing the enzyme mixture, which consists of 0.5 per cent macerozyme, 2 per cent cellulase in 13 per cent sorbitol or mannitol at pH 5.4. Finally the protoplasts are released and are kept in the isotonic solution.
Protoplast fusion facilitates mixing of two genomes and could be exploited in crosses which are not possible by conventional techniques due to incompatibility. Even though transfer of a single gene from one plant to another is desirable and protoplast fusion facilitates easy monitoring of cell genetic changes. Protoplast fusion could be spontaneous during isolation of protoplast or it can be induced by mechanical, chemical and physical means.
The isolated protoplasts are kept in isotonic solution (mannitol and enzyme mixture) to prevent damage. The isolated parent protoplasts are fused with a fusogenic agent like Polyethylene glycol (PEG). It is followed by nuclear fusion and results in a somatic hybrid. The somatic hybrids are allowed to grow in the same culture medium. The fused protoplast are then induced to regenerate the cell wall by transferring it into a suitable medium. This is followed by callus formatiom which leads to regeneration and organization of tissues.
Due to the existence of incompatibility prevailing between different species, protoplasmic fusion greatly compensates for interspecific hybridization. Somatic hybrids between rice and carrot were produced only through the process of protoplasmic fusion. Somatic hybrids may be used for gene transfer, transfer of cytoplasm and production of useful allopolyploids.