Molecular Techniques and Methods

Preparation of Leaf Mesophyll Protoplasts
from Greenhouse Grown Plants

Copy Right © 2001/ Institute of Molecular Development LLC



Osmoticum-A (100 ml)
0.3 M Mannitol ---------------------------------------- 5.5 g
0.04 M CaCl2 ----------------------------------------- 4 ml of 1 M CaCl2
Add deionized H2O to make a final volume of -------- 100 ml
  • Adjust pH to 5.8 with 1 M MES [2-(N-morpholino)ethanesulfonic acid].

    Osmoticum-B (100 ml)
    0.375 M Mannitol ------------------------------------- 6.8 g
    0.05 M CaCl2 ----------------------------------------- 5 ml of 1 M CaCl2
    Add deionized H2O to make a final volume of -------- 100 ml
  • Adjust pH to 5.8 with 1 M MES [2-(N-morpholino)ethanesulfonic acid].

    Enzyme Solution (100 ml)
    2% Cellulase ------------------------------------------- 2 g
    1% Hemicellulase -------------------------------------- 1 g
    1% Pectinol -------------------------------------------- 1 g
    Osmoticum-A ------------------------------------------ 96 ml

    Protoplast Culture Medium (1 liter)

    MS (Murashige and Skoog) Salts
    4.32 g
    2% Coconut milk
    20 g
    68.4 g
    0.25 g
    0.25 g
    0.25 g
    Fructose 0.25 g
    Mannose 0.25 g
    Rhamnose 0.25 g
    Ribose 0.25 g
    Xylose 0.25 g
    Vitamin Solution 1 ml
    2,4-D (2,4-dichlorophenoxyacetic acid) 0.2 mg
    BAP (6-benzylaminopurine) 0.05 mg
  • Add deionized H2O to make a final volume of 1 liter
  • Adjust pH to 6.0.
  • Autoclave.

    Regeneration Medium (1 liter)

    MS (Murashige and Skoog) Salts 4.32 g
    2% Sucrose 20 g
    Vitamin Solution 1 ml
    NAA (naphthaleneacetic acid) 1 mg
    BAP (6-benzylaminopurine) 1 mg
    0.8% Agarose 8 g
    Deionized H2O 990 ml
  • Adjust pH to 5.7-5.8.
  • Autoclave.

    Vitamin Solution (100 ml)

    Biotin 1 mg
    Pyridoxine-HCl 100 mg
    Thyamine-HCl 1 g
    Nicotinamide 100 mg
    Folic acid 40 mg
    D-Ca-pantothenate 100 mg
    p-Aminobenzoic acid 2 mg
    Choline chloride 100 mg
    Riboflavin 20 mg
    Ascorbic acid 200 mg
    Vitamin D3 1 mg
    Vitamin B12 2 mg
  • Add deionized H2O to make a final volume of 100 ml.


    1. Collect young leaves at approximately two-thirds of their final size.

    2. Sterilize leaves as follow.
  • Wash leaves with tap water.
  • Sterilize leaves by immersion for 10 seconds in 70% ethanol.
  • Sterilize leaves by immersion for10 minutes in 0.01% HgCl2 solution containing 0.05% Tween 80.
  • Wash leaves with 5 changes of sterile distilled water (each change 5 minutes).

  • 3. Cut leaves in half by removing midribs.

    4. Wet leaf halves with Osmoticum-A and arrange in a stack of 6 on the lid of a petri dish ready for cutting.

    5. Cut leaves into clean sections 0.5 mm wide.

    6. Transfer sectioned leaves into a small screw top flask containing 10 ml of Enzyme Solution.

    7. Vacuum infiltrate at 13-15 in3Hg until the leaf tissue is translucent. (for 5-15 minuets)

    8. Place the leaf slices in fresh Enzyme Solution in a sterile petri dish.

    9. Seal the petri dish with parafilm and incubate at 28oC for 3 hours.

    10. Check the incubation mixture periodically under the inverted microscope for the release of protoplasts.
  • The time required may vary, especially with greenhouse grown material.

  • 11. Gently agitate the digest, filter through a 100 um mesh stainless-steel sieve, and transfer in 5-ml aliquots into 15 ml sterile centrifuge tubes.

    12. Add 5 ml Osmoticum-B to each tube and mix gently.

    13. Centrifuge for 5 minutes at 60g to sediment the protoplasts.

    14. Pipette off the supernatant.

    15. Shake the sediment gently to free the protoplasts before resuspension in 10 ml of Osmoticum-B.

    16. Repeat steps 13-15 two times.
  • If necessary, the suspension is overlaid on 0.6 M Sucrose to remove debris and the protoplasts collecting at the interface recovered and resuspended in Osmoticum-B.

  • 17. Count the protoplasts.

    18. Sediment the protoplasts by centrifugation at 60g for 5 minuets.

    19. Resuspend the protoplasts in medium at 1-2 X 105/ml.

    20. Transfer 3 ml protoplast suspension into 9-cm-diameter petri dish.

    21. Add 3 ml of liquefied Protoplast Culture Medium with 1.2% Agarose at 40oC.

    22. Distribute the protoplasts evenly by gentle swirling and allow the medium to set.

    23. Incubate the cultures 6 days in the dark at 26oC.

    24. Transfer half the protoplast-containing agarose gel to each of two 10-cm-diameter containers each containing 40 ml of Protoplast Culture Medium.

    25. Incubate at 26oC on a gyratory shaker (80 rpm, 0.6 cm throw) in the light (1,000 lux).

    26. Replace the Protoplast Culture Medium weekly, each time reducing the glucose concentration in the original medium by one-quarter so as to reduce the osmotic pressure.

    27. After 6-8 weeks the colonies are 2-3 mm in size and can be cultured further.

    28. Transfer colonies directly to Regeneration Medium.

    29. Where shoot regeneration occurs, this is manifested as a mossy type growth of many shoot primordia.
  • On transfer to the same medium with the addition of 0.1 mg/L GA3, these normalize, eventually to be rooted, potted on, and moved to the greenhouse.




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