BOTANY Higher Secondary Second Year - Textbooks Online

4.4. Protoplast fusion
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.
Isolation of protoplast
Protoplast can be isolated from a variety of plant tissues using either
mechanical (or) enzymatic methods.
Mechanical method
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.
Enzymatic method
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.
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