Molecular Techniques and Methods

Transformation of Protoplast-Derived Cells
with Agrobacterium

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

This transformation procedure is less labor intensive than methods involving (1) uptake of isolated plasmids, (2) fusion of plant protoplasts with bacterial spheroplasts, or (3) liposome delivery.




MATERIALS AND SOLUTIONS

Protoplast Preparation Solution (1 liter)
KH2PO4 ------------------------------------------- 27.2 mg
KNO3 --------------------------------------------- 101 mg
CaCl2.2H2O --------------------------------------- 1.48 g
KI -------------------------------------------------- 0.16 mg
MgSO4.7H20 --------------------------------------- 0.246 g
CuS04.5H20 ---------------------------------------- 0.025 mg
13% Mannitol -------------------------------------- 130 g
Add deionized H2O to make a final volume of ----- 1 liter
  • Adjust pH to 5.8
  • 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. Purify the enzymatically released protoplasts as follows.
  • Overlay the protoplasts on 0.6 M Sucrose solution in screw-capped centrifuge bottles.
  • Centrifuge at 100g for 5 minutes.
  • Remove the supernatant.
  • Resuspend the pellet with Protoplast Preparation Solution.


  • 2. Repeat step 1.

    3. Resuspend the protoplast pellet in MS-H Liquid Medium-9.

    4. Count protoplasts.

    5. Culture protoplasts (2.0 X 105/ml) using 9-cm petri dishes in 3.0 ml of MS-H Liquid Medium-9 over 12 ml of MS-H Solid Medium at 25oC (700 lux, daylight fluorescent illumination) for 7 days.

    6. Transfer cells regenerated from protoplasts to MS-H Liquid Medium-7.

    7. Culture for 3 days at 25oC.

    8. Inoculate each dish with 0.1 ml of a overnight-grown culture of Agrobacterium tumefaciens, giving 5.0 x 107 bacteria/ ml and 0.8 x 102 bacteria/ protoplast-derived cell.

    9. Incubate cultures for 36 hours at 25oC in the dark.
    Include control plates which are uninoculated or inoculated with heat-treated (110oC, 15 min) bacteria.

    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 MS-H Liquid Medium-3.5.

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

    19. At day 8, transfer cells to MS-H Liquid Medium-0.

    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

  • Cells derived from mesophyll protoplasts of Nicotiana tabacum cv. Xanthi are best inoculated 7-14 days after protoplast isolation.

  • Liquid cultures of mesophyll protoplasts of Petunia 'Mitchell' can be inoculated 48 hours after protoplast isolation.

  • Protoplast-derived cells of Nicotiana tabacum v. Petit Havana SR1 (streptomycin resistant) can be inoculated with Agrobacterium after 72 hours of protoplast culture.



  • KIT INFORMATION


    REFERENCES

  • Aerts, M. Jacobs, M., Hemalsteens, J.-P., van Montagu, M., and Schell, J., 1979, Plant Sci. Lett. 17, 43.

  • Chilton, M-D., Currier, T.C., Fan, S.K., and, Bendich, A.J., Gordon, M.P. and Nester, E.W., 1974, PNAS 71, 3672.

  • Davey, M.R., Cocking, E.C., Freeman, J., Pearce, N., and Tudor, I., 1980, Plant Sci. Lett. 18, 307.

  • DeBlock, M., Herrera-Estrella, L., van Montagu, M., Schell, J., and Zambryski, P., 1984, EMBO J. 3, 1681.

  • Draper, J., Davey, M.R., Freeman, I.P., Cocking, E.C., and Cox, B.J., 1982, Plant Cell Physiol. 23, 451.

  • Hain, R., Steinbiss, H-H, and Schell, J., 1984, Plant Cell Rep. 3, 60.

  • Hasezawa, S., Nagata, T., and Syono, K., 1981, Mol. Gen. Genet. 182, 206.

  • Herrera-Estrella, L., De Block, M., Messens, E., Hemalsteens, J.P., van Montagu, and Schell, J., 1983, EMBO J. 2, 987.

  • Horsch, R.B., Fraley, R.T., Rogers, S.G., Sanders, P.R., Lloyd, A., and Hoffmann, N., 1984, Science 223, 496.

  • Horsch, R.B., and Jones, G.E., 1980, In Vitro 16, 103.

  • Krens, F.A., and Schilperoort, R.A., 1984, Cell. Cult. Somatic Cell Genet. Plants 1, 522.

  • Krens, F.A., Molendijk, L., Wullems, G.J., and Schilperoort, R.A., 1982, Nature (London) 296, 72.

  • Murashige, T., and Skoog, F., 1962, Physiol. Plant. 15, 473.

  • Paszkowski, J., Shillito, R.D., Saul, M., Mandak, V., Kohn, T., Hohn, B., and Pokus, I., 1984, EMBO J. 3, 2717.

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

  • Salomon, F., Deblaere, R., Leenians, J., Hemalsteens, J.-P., van Montagu, M., and Schell, J., 1984, EMBO J. 3, 141.

  • Senda, M., Takeda, J., Abe, S., and Nakamura, T., 1979, Plant Cell Physiol. 20, 1441.

  • Zimmermann, J., and Scheurich, P., 1981, Planta 151, 26.



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