Requirements-materials and reagents
- Plant materials- leaf of Nicotiana tabacum
- Cell and protoplast washing (CPW) solution
- 10% sodium hypochlorite
- Tween 20
- 13% mannitol-inorganic salts
- Enzyme mixture: macerozyme (0.1–0.5%) and cellulase (0.5–1%)
- Protoplast culture media: Kao and Michayluk media
- Pasteur pipette
- petri dish
Procedure: Isolation of protoplast from Nicotiana tabacum
- Isolate fully expanded leaves including petiole.
- Use 10% sodium hypochlorite solution containing two drops of Tween 20 for 10 min to sterilize it.
- Wash 3–4 times with sterile de-ionized water and blot dry on sterile tissue paper.
- With fine forceps, peel the lower epidermis from sterilized leaves and cut into pieces by using fine scalpel.
- Transfer the leaf pieces (peeled areas only) with lower surface down into 30 ml of 13% mannitol-inorganic salts of cell and protoplast washing media (CPW) solution contained in a petri dish for 30 min–1 hr for plasmolysis.
- Composition of Cell and protoplast washing (CPW) solution:
- Ingredients mg/litre
- KH2PO4—————————–27.2
- KNO3——————————–101.0
- CaCl2·2H2O———————- 1480.0
- MgSO4·7H2O———————-246.0
- KI ————————————–0.16
- CuSO4·5H2O———————–0.025
- *pH maintained at 5.8
- Take out the mannitol-CPW salt solution with a Pasteur pipette and replace it with filter sterilized enzyme mixture( macerozyme and cellulase)in 13% mannitol solution (~ 20 ml).
- Gently stir the leaf pieces with sterile Pasteur pipette to enables the separation of protoplasts, pushing the larger pieces of leaf material to one side and keeping the petri dish at an angle of 15°C. Or, remove the larger debris by filtering through a 60–80 μm mesh.
- Transfer the filtrate to a screw cap centrifuge tube.
- Centrifuge at 100x g for 5–10 min to sediment the protoplasts.
- Remove the supernatant and resuspend the protoplast pellet in CPW + 21% sucrose (prepared in CPW) and gently spread the protoplasts in the solution.
- Centrifuge again at 100x g for 10 min. The viable protoplasts will float to the surface of the sucrose solution while the remaining cells and debris will sink to the bottom of tube.
- Remove the band of protoplasts from the top with a Pasteur pipette and transfer into another centrifuge tube.
- Resuspend in CPW + 10% mannitol. Centrifuge again at 100x g for 10 min to separate the contaminating debris.
- Repeat the washing procedure at least 3 times.
- After the final washing, add enough MS protoplast culture medium with 9% mannitol to achieve a protoplast density of ~ 5 × 104/ ml.
- Kao and Michayluk media has been widely used for protoplast culture. Plate the protoplasts as small droplets (100–150 μl) or a thin layer in small petri dishes for 24h at 25°C.
- Keep the cultures at low light intensity of 500 lux for the next 2 days and then for rest of the experiment at 2000 lux.
- Protoplasts form cell walls and begin to divide after 3–5 days.
- Protoplasts may also be cultured in an agarose medium by mixing 1.5 ml of protoplast suspension with an equal volume of 1.2% agarose medium at 45°C in 35 mm petri dish.
- Cultures continue to grow in the media and the first colonies become visible after 3–4 weeks.
- Transfer the colonies to new medium with a reduced mannitol level.
- Transfer the colonies to a suitable medium for embryo differentiation and plantlet regeneration.
