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Protoplast Fusion

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Protoplast Fusion

  1. 1. Presented by Melika M.Razzaz
  2. 2. PROTOPLAST  Protoplasts are the 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. 3. PROTOPLAST FUSION  By protoplast 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. 4. INTRODUCTION  Two genetically different 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. 5. ENZYMES  various enzymes used 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. 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. 7. MECHANICAL FUSION  In this process the isolated protoplast are brought into intimate physical contact mechanically.  under microscope and using micromanipulator or perfusion micropipette.
  8. 8. CHEMOFUSION  Several chemicals has 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.
  9. 9. ZETA POTENTIAL  Zeta potential (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.
  10. 10. FUSION  The negative charge 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.
  11. 11. ELECTROFUSION  mild electric stimulation 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.
  12. 12. 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.
  13. 13. PROTOPLAST FUSION IN BACTERIA  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.

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