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Transcript of Protoplast Fusion
-Have their cell wall entirely removed
-More generally protoplast refers to that unit of biology which is composed of a cell's nucleus and the surrounding protoplasmic materials.
Protoplasts are formed from bacteria, fungi, yeasts and actinomycetes when dividing cells are caused to lose their cell walls.
Fusion from mixed populations of protoplasts is greatly enhanced by the use of polyethylene glycol
Protoplasts are isolated from cells by two methods-
ISOLATION OF PROTOPLAST
Various enzymes used for this process
Bacterial cell wall are degraded by lysozyme.
Fungal wall degraded by Novozyme -234 ( glucanase and chitinase)
Streptomyces cell wall degraded by lysozyme and achromopeptidase
Enzymes used for breaking of cell walls:
to study the control of cell division and gene expression,
to investigate malignant transformations,
to obtain viral replication
for gene or chromosome mapping
production of monoclonal antibodies by producing hybridoma (hybrid cells between an immortalised cell and an antibody producing lymphocyte), etc.
Application of protoplast fusion
Isolated protoplast are brought into intimate physical contact mechanically
under microscope, using micromanipulator or perfusion micropipette.
Isolation of protoplast:
In bacteria protoplast can be obtained.
Fusion can be carried out with low frequency in some gram positive organisms.
For gram negative bacteria it is possible to obtain protoplast but regeneration is difficult
The procedure is highly efficient and yields upto 80% transformants.
Protoplast fusion in bacteria
Studies on the fate of plasma genes
Production of unique hybrids of nucleus and cytoplasm
Transfer gene for disease resistance, abiotic stress resistance, herbicide resistance and many other quality characters
The cells are kept in a suitable plasmolyticum & cut with a fine knife, so that protoplast are released from cells cut through the cell wall, when the tissue is again deplasmolysed.