Molecular Techniques and Methods

Transformation of Protoplast by
Chemically Stimulated Uptake of Isolated DNA

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

The methodology for transformation of protoplasts by isolated Ti plasmid is based upon the use of chemical agent used to induce protoplast fusion, e.g., polyethylene glycol (PEG). The technique described here should also be applicable to the transformation of protoplasts by recombinant plasmids, provided the selectable markers capable of expression in plant cells.




MATERIALS AND SOLTIONS

Protoplast Suspension Solution (100 ml)
50 mM Glycine-NaOH buffer (pH 10.1) ------------ 5 ml of 1 M Glycine-NaOH Buffer
20 mM CaCl2 --------------------------------------- 2 ml of 1 M CaCl2
9% Mannitol ---------------------------------------- 9 g
Add deionized H2O to make a final volume of ----- 100 ml
  • Autoclave.


    PEG-Transformation Solution (100 ml)
    47% PEG 6000 ------------------------------------- 47 g
    50 mM glycine-NaOH Buffer (pH 10.1) ------------- 5 ml of 1 M Glycine-NaOH Buffer
    20 mM CaCl2 --------------------------------------- 2 ml of 1 M CaCl2
    3% Mannitol ---------------------------------------- 3 g
    Add deionized H2O to make a final volume of ----- 100 ml
  • Autoclave.


    MS-H Liquid Medium-9 (1 liter)
    MS salts -------------------------------------------- 4.3 g
    NAA ----------------------------------------------- 2 mg
    6-BAP (benzylaminopurine) ------------------------- 0.5 mg
    9% Mannitol ---------------------------------------- 90 g
    Add deionized H2O to make a final volume of ----- 1 liter
  • Adjust pH to 5.8
  • Autoclave.


    MS-H Solid Medium-9 (1 liter)
    MS salts -------------------------------------------- 4.3 g
    NAA ----------------------------------------------- 2 mg
    6-BAP (benzylaminopurine) ------------------------- 0.5 mg
    9% Mannitol ---------------------------------------- 90 g
    0.6% Phytagar -------------------------------------- 6 g
    Add deionized H2O to make a final volume of ----- 1 liter
  • Adjust pH to 5.8
  • Autoclave.


    MS-H Liquid Medium-7 (1 liter)
    MS salts -------------------------------------------- 4.3 g
    NAA ----------------------------------------------- 2 mg
    6-BAP (benzylaminopurine) ------------------------- 0.5 mg
    7% Mannitol ---------------------------------------- 70 g
    Add deionized H2O to make a final volume of ----- 1 liter
  • Adjust pH to 5.8
  • Autoclave.


    MS-H Liquid Medium-3.5 (1 liter)
    MS salts -------------------------------------------- 4.3 g
    NAA ----------------------------------------------- 2 mg
    6-BAP (benzylaminopurine) ------------------------- 0.5 mg
    3.5% Mannitol -------------------------------------- 35 g
    Add deionized H2O to make a final volume of ----- 1 liter
  • Adjust pH to 5.8
  • Autoclave.


    MS-H Liquid Medium-0 (1 liter)
    MS salts -------------------------------------------- 4.3 g
    NAA ----------------------------------------------- 2 mg
    6-BAP (benzylaminopurine) ------------------------- 0.5 mg
    Add deionized H2O to make a final volume of ----- 1 liter
  • Adjust pH to 5.8
  • Autoclave.


    MS-H Solid Medium-0 (1 liter)
    MS salts -------------------------------------------- 4.3 g
    NAA ----------------------------------------------- 2 mg
    6-BAP (benzylaminopurine) ------------------------- 0.5 mg
    0.6% Phytagar -------------------------------------- 6 g
    Add deionized H2O to make a final volume of ----- 1 liter
  • Adjust pH to 5.8
  • Autoclave.


    MS Solid Medium-0 (1 liter)
    MS salts -------------------------------------------- 4.3 g
    0.6% Phytagar -------------------------------------- 6 g
    Add deionized H2O to make a final volume of ----- 1 liter
  • Adjust pH to 5.8
  • Autoclave.




    PROCEDURES

    1. Resuspend protoplasts (3.0 X 106) in 0.5 ml of Protoplast Suspension Solution containing an appropriate amount (up to 5.0 ug/106 protoplasts) of plasmid DNA.

    2. Incubate at 25oC for 15 seconds in a screw-capped tube.

    3. Mix the suspension with 1.5 ml of PEG-Transformation Solution.

    4. Incubate at 34oC for 2 min.

    5. Dilute the protoplasts with 10 ml Protoplast Suspension Solution.

    6. Incubate for 20 minutes at 34oC.

    7. Pellet the protoplasts by centrifugation at 90g for 2 minutes.

    8. Wash thoroughly with three changes of MS-H Liquid Medium-9.

    9. Culture in 3.0 ml of MS-H Liquid Medium-9 over 12 ml of MS-H Solid Medium containing 1.0 mg/ml carbenicillin for 3 days at 25oC in the dark.

    10. Harvest cells from the incubation dishes by centrifugation at 90g for 2 minutes in screw-capped tubes.

    11. Remove the supernatant.

    12. Add MS-H Liquid Medium-7 containing 1.0 mg/ml carbenicllin.

    13. Centrifuge at 90g for 2 minutes in screw-capped tubes.

    14. Repeat steps 11 to 13.

    15. Replate the cells in 3.0 ml of MS-H Liquid Medium-7 over 12 ml of MS-H Solid Medium containing 1.0 mg/ml carbenicillin.

    16. Culture for 3 days at 25oC in the dark.

    17. At day 4, transfer cells to 3.0 ml of MS-H Liquid Medium-3.5 over 12 ml of MS-H Solid Medium containing 1.0 mg/ml carbenicillin.

    18. Culture for 7 days at 25oC in the dark.

    19. At day 8, transfer cells to 3.0 ml MS-H Liquid Medium-0 over 12 ml of MS-H Solid Medium containing 1.0 mg/ml carbenicillin.

    20. Culture for 7 days at 25oC in the dark.

    21. Harvesat cells from the liquid layer of each dish, and divided into four aliquots.

    22. Mix each aliquot with 3.0 ml of MS-H Solid Medium-0 containing carbenicillin (1 mg/ml).

    23. Cells spread in MS-H Solid Medium-0 containing carbenicillin (1 mg/ml) and antibiotics (kanamycin, 50 ug/ml) should develop into colonies which appear above the surface of the agar medium within 4-6 weeks of plating.

    24. Transfer individual colonies using the tip of a scalpel, to MS Solid Medium containing carbenicillin (1 mg/ml) and kanamycin (50 ug/ml).

    25. Transfer colonies to fresh MS Solid Medium containing carbenicillin (1 mg/ml) and kanamycin (50 ug/ml) at 4-week intervals.

    26. Those colonies which continue to proliferate after 4 to 5 subcultures are putative transformants.

    27. Regenerate plants as described in Protoplast Preparation and Regeneration.


    NOTES



    KIT INFORMATION



    REFERENCES

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  • Power, J.B., Chapman, J.V., and Wilson, D., 1984, "Laboratory Manual: Plant Tissue Culture." University of Nottingham.

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