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Protoplast culture, Production of haploid(in vitro), Monoploid, Somatic…

- - - - Mechanical cutting(remove lower epidermis by forceps)
        
        Enzyme digestion(cellulase, Pectinase)
        
        Combination of both
        
        Plasmolysed the cell
    - - Obtain protoplast suspense
        
        Remove enzyme
        
        Wash the suspense
        
        Protoplast suspense in washing medium(sucrose)
        
        Filtered with nylon mesh
        
        Centrifugation 75xg
        
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        Resuspend in culture medium
        
        remove small sample for haemocytometer count
        
        Resuspend in washing medium to give correct density
        
        18 hrs Incubation in enzyme solution with osmotic stabilizer and wait for protoplast sink from the plant(eg.leaf)
      - Plating
        
        Place 1 volume Protoplast(known density) in culture medium
        
        Place 1 volume of culture medium with 1-2% agar (40 C)
        
        Close the petridish and invert it and incubate at 25C with illumination
        
        Multiplication and rooting
        
        Subculturing
- - - - Surface sterilize (5% NaOCl for 10 mins)
        
        Aseptically excise Anther/squeeze out the pollen by pestle
        
        Pretreatment of anther
        
        Heat(30C for 24hrs) to stimulates embryogenesis
        
        Chemical (ethrel) induce parthenogenesis
        
        Cold (3-6 C for 3-15 days) to kill non viable anther & change from gametophyte --> sporophyte
        
        Multiplication (solid media- gently press; liquid- not sink)
        
        Direct Embryogenesis/ Indirect Organogenesis/ Indirect Embryogenesis
- - - - Surface sterilization of ovary
        
        Solid medium is used to culture
        
        Multiplication and rooting
        
        Doubling of chromosome number can be done by Colchicine
- - - - Cells aggregate
        
        Globular stage
        
        Heart stage
        
        Torpedo Stage
        
        Cotyledonary stage
        
        Mature embryo(germination can occur)
      - Auxin is applied for initiation for Embryogenesis(2,4-D followed by NAA)
        
        Reduced Nitriogen
        
        Active charcoal to absorb phenylacetic acid
    - - High Auxin level can inhibit the shoot development
      - Addition little amount of cytokinin + Abscisic acid
      - Physical factor: light, humidity, temperature
      - Somatic embryo can germinate on agar medium without PGR
- - - - Hybrid selection
        
        Induction of cell division in hybrid cells
        
        Regeneration of plant from calli
        
        Characterization of regenerated cells to confirm hybrid nature
      - Fusion of cells(Heterokaryon)
        
        Fusion of nucleus(Hybrid)
        
        Spontaneous fusion
        
        protoplast fused through plasmodesmata during protoplast isolation
        
        Induced fusion
        
        Electrical depolarization/Electporation
        
        Dielectric dipole--> pearl chain arrangement--> surface charge of membrane altered(short pulse)
        
        Chemical Fusing Agent (Polyethylene glycol with high Ca+ and pH) to cause agglutination occurs
        
        add PGE into pallet of protoplast
        
        Incubate in room temperature with occasional agitation
        
        After fusion, add culture medium and centriduge
        
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