Protoplast isolation and immobiliz by Dr.U.Srinivasa


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introduction on protoplast

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Protoplast isolation and immobiliz by Dr.U.Srinivasa

  2. 2.  Definition : Protoplast is a cell without a cell wall are called protoplast. They contain all the normal cell organelles plus the nucleus. The cell wall of a plant cell can be decomposed and removed by the treatment of the lytic enzymes like cellulose and pectinase
  3. 3. Isolation of protoplast Protoplasts can be isolated from all types of actively growing young and healthy tissues. METHODS OF ISOLATION : 1.Mechanical method 2. Enzymatic method 3. Combination of above methods
  4. 4.  During the process of isolation, the cells are first separated by mechanical method and subsequently protoplasts are isolated by enzymatic method
  5. 5. Mechanical method The cells are first placed in a suitable plasmolyticum .This treatment makes the protoplasms of these plasmolysed cells shrink away from their cell walls( this makes the removal of cell wall easy) Further ,they are cut with a knife.
  6. 6.  Then the protoplasts are released from the cells through the cell wall , and then the tissue is again deplasmolysed.
  7. 7.  Advantages : It is suitable method for the isolation of protoplasts from vacuolated cells. Eg onion bulbs, scales, radish roots Dis advantages : Poor yield Unsuitable for the isolation of protoplasts from meristematic cells Unsuitable for the isolation of protoplasts from less vacuolated cells
  8. 8. Enzymatic method Protoplasts can be isolated from aIt is widely used method.The isolation of protoplasts requires digestion of cell wall and middle lamellae. This is affected by the use of enzymes variety of tissues including leaves, roots, in vitro shoot culture and cell suspension culture
  9. 9. Steps 1.Sterilization of leaves 2. Peeling of the epidermis 3. Enzymatic treatment 4. Isolation and cleaning of the protoplasts
  10. 10.  Sterilization of leaves – Fully expanded leaves are sterilized by the following procedure A. dipping in 70% ethyl alcohol for about a minute and then with 2% solution of sodium hypochlorite for 20-30 minutes B. rinsing with sterile distilled water three times.
  11. 11.  Peeling of the epidermal layer : The lower epidermis is carefully peeled off and the stripped leaves are cut into small pieces. This operation must be carried out under aseptic conditions Mesophyll protoplasts can be isolated from the peeled leaf segments while epidermis yields epidermal protoplasts
  12. 12.  Enzymatic treatment : There are two methods 1.Direct method ( 1 step) 2.Sequential method (2 steps)
  13. 13.  Direct method : Here simultaneous treatment with macerase ( pectinase ) and cellulose enzymes is carried out. 0.5% macerase and 2% cellulose enzyme in 13% sorbitol or mannitol at pH 5.4
  14. 14. • Sequential method :• 1 step – Sample is treated with macerase ( pectinase ) enzyme for isolation of cells 2 step – Isolated cells are treated with cellulose enzyme for protoplast isolation In both cases , peeled leaf segments are placed with the lower surface downwards in a petridish containing enzyme mixture
  15. 15. Purification The isolated protoplasts are usually associated with a range of cell debris and broken cell organelles . Methods used for purification : 1. Sedimentation 2. Flotation
  16. 16. Applications Suitable for the isolation of cell organelles and chromosomes Suitable for the isolation of mutants To affect genetic transformations through DNA or organelle uptake Study of cell wall formation, membrane transport , ultra structures
  17. 17. Immobilization of EnzymeDefinition“Enzymes physically confined or localized in a certain defined region of space with retention of their catalytic activities , and which can be used repeatedly and continuously". Immobilization is a process of aggregate formation and adhesion on a matrix under controlled conditions.
  18. 18. Advantages 1) No purification of enzyme after production 2) High enzyme activity (high reactor activity) 3) Enhanced operational stability 4) Low enzyme cost 5) Application of multienzyme reaction is possible
  19. 19. How to stabilize enzyme?• Addition of substrate analogues• Addition of sugar alcohols (e.g. sorbitol)• Addition of cofactors (e.g. calcium ion)• Immobilization: reuse & long-term stability !
  20. 20. Methods of immobilization1.Direct intercellular binding due to natural affinity . Eg: Adhesion, adsorption,agglutination2.Covalent bonding on inert matrices3.Embedding4.Cross linking with biopolyfunctional reagents5. Purely physical retensions in diverse pore size eg: entrapment, microencapsulation
  21. 21. Agents used Agarose gel Alginate gel Chitosan Polyacrylamide Polyurethane foam Polyethylene oxide
  22. 22. Enzyme Immobilization
  23. 23. Immobilization by Binding• Adsorption:• Electrostatic interactions (van der Waals forces, ionic and hydrogen bonds betweeen the cell surface and the support materials• cell wall composition: determined by distribution of carboxyl and amino groups of the peptide amino acids of cell wall surface (ex) yeast cells are negative charge, thus choose a positively charged support• Advantages: ability to regenerate the support• Disadvantsges: low stability (desorption of cells due to changes of pH and/or ionic strength)
  24. 24. • Covalent-binding methods• Advantages:• Free of diffusional limitations• High operational stability• Uniform binding• Disadvantages:• Toxicity of the coupling agents(loss of activity and cell viability, not acceptable in food and pharmaceutical fields)• Hard to regenerate the supports