Protocol for Protoplast fusion

June 24, 2020 Gaurab Karki Biotechnology practical 0




Protocol for Protoplast fusion
Protocol for Protoplast fusion

Protoplast fusion by Polyethylene glycol (PEG) method

Material:

  • Green leaf (mesophyll) and cultured cells (albino) of N. tabacum
  • PEG fusion solution contains:
    • 22.5% (w/v) PEG molecular weight 6000
    • 1.8% (w/v) sucrose
    • 154 mg/l CaCl2·2H2O
    • 9.52 mg/l KH2PO4
    • pH 5.8 (adjust with 1M KOH or IN HCl, autoclaved and stored in dark at 4°C)

Protocol:

  • In continuation with protoplast isolation experiment, perform the fusion experiment.
  • Take 4–6 ml of freshly isolated protoplast suspension from two sources in protoplast washing media CPW salt medium –13 % mannitol (CPW13M) with a density of 5 × 105/ml.
  • Use a Pasteur pipette to place a small drop (~50l) of sterile silicone on to the center of a plastic petri dish (35 mm) and gently lower a cover glass on to the drop of silicone fluid.
  • Pipette ~150l of protoplast suspension directly on to the middle of cover glass and allow it to settle for 10–15 min. (Protoplasts suspended in CPW13M for convenience are mixed in screw capped centrifuge tube in a 1:1 ratio).
  • Add 450l of PEG fusion solution drop-wise to the edge of the protoplast culture, placing the last drop in the centre by using a Pasteur pipette.
  • Leave the protoplasts undisturbed for 20–40 min at room temperature. Then add one drop of washing solution very gradually whilst withdrawing some of the fusion solution from the coalesced drops.
  • Repeat this procedure at 5 min intervals for the next 20 min. During this procedure it is important that the protoplasts are subjected to minimal disturbance. Washing solution: CPW13M medium to which 0.74g/l CaCl2·2H2O is added (sterile).
  • Five min after the last wash, carefully suck off the solution with a Pasteur pipette leaving the protoplasts with a thin film of medium and replace it with culture medium (MS protoplast medium with 9% mannitol). Wash at least 3 times with culture medium.
  •  For the culture of protoplasts flood the cover glass with culture medium and scatter several drops of medium on the base of petri dish prior to sealing with parafilm.
  • Incubate the cultures initially in the dark for 24 h at 25°C and then transfer to white light (1000 lux).
  •  The fusion products can be easily observed using an inverted microscope.
  • Subsequently transfer the colonies for regeneration.
  • Test the hybridity of fusion product and somatic hybrid as per equipment available in the lab, which has been explained in the text.