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Protoplast Fusion
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- 2. PROTOPLAST Protoplasts arethe cells of which cell walls are removed and cytoplasmic membrane is the outermost layer in such cells. during fusion two or more protoplasts come in contact and adhere with one another either spontaneously or in presence of fusion inducing agents.
- 3. PROTOPLAST FUSION Byprotoplast fusion it is possible to transfer some useful genes from one species to another. Protoplast fusion is an important tools in strain improvement. These are the powerful techniques for engineering of microbial strains for desirable industrial properties.
- 4. INTRODUCTION Two geneticallydifferent protoplast isolated from the somatic cells and are fused to obtain hybrid protoplasts. The hybrid protoplast contained heteroplasoic cytoplasm and two fused parent nuclei. Protoplast fusion technique has a great potential for strain improvement (Specially for industrially microorganisms)
- 5. ENZYMES various enzymesused for breaking of cell walls: Type of cell Enzyme Plant cells Cellulase , pectinase , macerozyme , Xylanase Gram-positive bacteia Lysozyme Fungal cells Novozyme -234 ( includes glucanase and chitinase)
- 6. METHODS OF PROTOPLAST FUSION Protoplast fusion can be broadly classified into two categories: 1. Spontaneous fusion (fuse through their plasmodesmata) 2. Induced fusion (needs a fusion inducing chemicals): a) b) c) Mechanical fusion Chemofusion Electrofusion
- 7. MECHANICAL FUSION Inthis process the isolated protoplast are brought into intimate physical contact mechanically. under microscope and using micromanipulator or perfusion micropipette.
- 9. CHEMOFUSION Several chemicalshas been used to induce protoplast fusion such as sodium nitrate ,polyethylene glycol, Calcium ions. Chemofusion is a non specific. It is inexpensive. can cause massive fusion product. Disadvantages: It can be cytotoxic and non selecetive and having less fusion frequency.
- 10. ZETA POTENTIAL Zetapotential (ZP) is the electrical potential that exists at the around of a particle and cell. There is an electrical double layer in the region of the particle-liquid interface: 1. Each particle dispersed in a solution is surrounded by oppositely charged ions called fixed layer. 2. Outside the fixed layer, there are varying compositions of ions of opposite polarities, forming a cloud-like area.
- 12. FUSION The negativecharge carried by protoplast is mainly due to intramembranous phosphate groups. Cacium ions causes reduction in the zeta potential of plasma membrane and under this situation protoplasts are fused. On elution of the PEG ,the surface potential are disturbed, leading to intramembrane contact and subsequent fusion.
- 13. ELECTROFUSION mild electricstimulation is being used to fuse protoplast. An electric field of low strength (10Kv/m) gives rise to dielectrophoretic dipole generation within the protoplast suspension. high strength of electric fields (100 kvm-1) for some microseconds are applied. Electrofusion is easy to control and less cytotoxic. It is having fusion frequency upto 100%. It is gives reproducibility. Disadvantages: the equipment is sophisticated and expensive.
- 16. MECHANISM OF PROTOPLAST FUSION The mechanism of protoplast fusion is not fully known. when the protoplasts are closely adhered, the external fusogens cause disturbance in the intramembranous proteins and glycoproteines. This increases membrane fluidity and creates a region where lipid molecule intermix,allowing coalescence of adjacent membranes.
- 17. PROTOPLAST FUSION INBACTERIA 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.