Elementary plant Biochemistry and Biotechnology (2+1)

Protoplast Isolation

• In eukaryotes the transfer of genetic material form one individual to another is conventionally achieved through sexual breeding. In plants, where fairly distant species could be crossed, it has not always been possible to obtain full hybrids between desired individuals because of sexual incompatibility barriers. In this respect cell fusion offers a novel approach to distant hybridization through somatic hybridization. Fusion of cells must occur through the plasma membrane. Unlike animals, in plants the plasma membrane is bound by a rigid cellulosic wall and the adjacent cells are cemented together by a pectin rich matrix. That’s why somatic cell genetics is more advanced in animals than plants. In 1960, E. C. Cocking demonstrated the feasibility of enzymatic degradation of plant cell walls to obtain large quantities of viable cells – called as protoplasts.


• Besides being able to fuse with each other, higher plant protoplasts can also take up foreign DNA, through their naked plasma membrane under specific chemical and physical treatments. Protoplasts also provide an experimental system for a wide range of biochemical and molecular studies ranging from investigations into the growth properties of individual cells to membrane transport.

Isolation of protoplast
1.Mechanical method:

• Klecker in 1892, has first initiated the protoplast isolation by mechanical means- the cells were kept in a suitable medium plasmolyticum and cut with a fine knife. In this process some of the plasmolyzed cells were cut only through the cell wall, releasing intact protoplasts.

Limitation:

1. applicable only to vacuolated cells
2. yields are extremely low.

2.Enzymatic method

• In 1960, Cocking used a concentrated solution of cellulase enzyme, prepared from cultures of the fungus, Myrothecium verrucaria, to degrade the cell walls. However, real progress in this area was made after 1968 when cellulase and macerozyme enzymes became commercially available. The commercial preparations of the enzymes for protoplast isolation were first employed by Takebe et al., (1968). The tobacco leaf species were first exposed to macerozyme to liberate single cells which were then treated with cellulase to digest the cell walls and release the protoplasts. Later, these two enzymes were used together and this is found as faster method and also reduces the chances of microbial contamination by cutting down a few steps.


• A range of enzyme preparations are now available commercially (Table) and depending on the nature of the tissue these are used in different combinations. The use of commercially available enzymes has enabled the isolation of protoplasts from virtually every plant tissue as long as cells have not acquired lignification. Protoplast isolation has been reported from mesophyll cells of in vivo and in vitro growing plantlets (Figure), aseptic seedlings, microspore mother cells, young microspores, pollen grain calli and embryogenic and non-embryogenic suspension cultures. More recently, viable protoplasts have been obtained from male and female gametes.