1. Protoplast Fusion Technology Presented by MONA ALBUREIKAN
2. What is protoplast fusion?• cell walls are removed.• They only have a cell membrane.• can be obtained by specific lytic enzymes.• It is a physical phenomenon.• it is possible to transfer some useful genes.• The hybrid protoplast = cytoplasm + two fused parent nuclei.• important tools in strain improvement.• Used for prokaryotic and eukaryotic cells.
3. Why use protoplast fusion?• create strains with desired properties.• industrial properties.• one of the most frequently used research tools.• a great potential for genetic analysis.• for strain improvement.
4. Enzymes used for breaking of cell walls:• Various enzymes used for this process.• Cellulase and pectinase or macerozyme for plant cell wall.• Bacterial cell wall are degraded by lysozyme.• Fungal wall degraded by Novozyme -234 ( glucanase and chitinase).• Streptomyces cell wall degraded by lysozyme and achromopeptidase
5. Methods of protoplast fusion:Protoplast fusion classified into two categories:• Spontaneous fusion: - often fuse spontaneously. - fuse through their plasmodesmata.• Induced fusion: - Fusion with the help of fusion inducing chemicals agent. - Normally , do not fuse with each other.
6. How the isolated protoplast can be induced to fuseby three ways;1- Mechanical fusion:• Isolated protoplast are brought into intimate physical contact mechanically• under microscope• using micromanipulator or perfusion micropipette.
7. How the isolated protoplast can be induced to fuse2- Chemofusion:• Several chemicals has been used to induce protoplast fusion.• such as sodium nitrate ,polyethylene glycol, Calcium ions(Ca++ ).• cause the isolated protoplast to adhere each other.• It is a non specific.• It is inexpensive.• It can cause massive fusion product.• It can be cytotoxic and non selecetive and having less fusion frequency.
8. How the isolated protoplast can be induced to fuse3- Electrofusion;• Used electric stimulation.• Used An electric field of low strength (10Kvm-1).• Electrofusion is easy to control.• It is having fusion frequency up to 100%.• It is gives reproducibility.• It is less cytotoxic.• The equipment is sophisticated and expensive.
9. Mechanism of protoplast fusion:
10. Protoplast fusion in bacteria:• 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.