Molecular Techniques and Methods

Transformation of Protoplasts
by Fusion of Bacterial Spheroplasts

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

Treatment of Agrobacterium spheroplast-plant protoplast mixtures with a polyvinyl alcohol resulted in the uptake of Agrobacterium spheroplasts into cell suspension protoplasts, and expression of T-DNA in 0.1-0.2% of protoplast-derived cell colonies. Since it is most convenient to perform genetic manipulations in E. coli, it is useful to be able to transfer genes directly from E. coli to higher plant protoplasts. This has been achieved by fusing E. coli spheroplasts with tobacco mesophyll protoplasts, giving a transformation frequency of 2.0 in 105.




MATERIALS AND SOLUTIONS

AB Minimal Medium (1 liter)
K2HPO4 -------------------------------------------- 3 g
NaH2PO4 ------------------------------------------- 1 g
NH4Cl ---------------------------------------------- 1 g
MgSO4.7H2O -------------------------------------- 0.3 g
KCl ------------------------------------------------- 0.15 g
CaCl2 ----------------------------------------------- 0.01 g
FeSO4.7H20 ---------------------------------------- 2.5 mg
Glucose --------------------------------------------- 5 g
Add deionized H2O to make a final volume of ------ 1 liter


Lysozyme Solution for Agrobacterium tumefaciens (1 liter)
Lysozyme ------------------------------------------- 0.4 g
EDTA-Na2 ------------------------------------------ 3.72 g
2.5 mM Tris-HCl (pH 8.2) --------------------------- 2.5 ml of 1 M Tris-HCl
0.4 M Sorbitol --------------------------------------- 73 g
Add deionized H2O to make a final volume of ------ 1 liter


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.


    Tris-Sucrose Buffer (1 liter)
    20% Sucrose --------------------------------------- 200 g
    0.05 M Tris-HCl (pH 8.0) -------------------------- 50 ml of 1 M Tris-HCl
    Add deionized H2O to make a final volume of ----- 1 liter
  • Autoclave.
  • Store at 4oC.


    Lysozyme Solution for E. coli (1 liter)
    Lysozyme -------------------------------------------- 5 mg
    0.25 M Tris-HCl (pH 8.0) --------------------------- 250 ml of 1 M Tris-HCl
    Add deionized H2O to make a final volume of ------ 1 liter


    Magnesium-Sucrose Solution (1 liter)
    10% Sucrose --------------------------------------- 100 g
    10 mM MgCl2 -------------------------------------- 10 ml of 1 M MgCl2
    Add deionized H2O to make a final volume of ----- 1 liter
  • Autoclave.


    PEG (Polyethylene Glycol) Fusion Solution (100 ml)
    40% PEG (MW 4000) -------------------------------------- 40 g
    Protoplast Suspension Solution to make a final volume of --- 100 ml


    PVA (Polyvinyl Alcohol) Fusion Solution (100 ml)
    20% PVA (degree of polymerization 300, 88% hydrolyzed) -- 20 g
    Protoplast Suspension Solution to make a final volume of --- 100 ml


    Glycine-NaOH Buffer (1 liter)
    0.05 M Glycine-NaOH (pH 10.0) ------------------ 50 ml of 1 M Glycine-NaOH
    0.05 M CaCl2 -------------------------------------- 50 ml of 1 M CaCl2
    0.4 M Mannitol ------------------------------------- 72.9 g
    Add deionized H2O to make a final volume of ----- 1 liter
  • 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

    Preparation of Agrobacterium tumefaciens Spheroplasts
    1. Grow Agrobacterium overnight in AB Minimal Medium at 30oC.

    2. Add carbenicillin (500 ug/ml) during the logarithmic growth phase, 1.5 hour before harvesting the cells.

    3. Centrifuge 50 ml of overnight culture for 10 minutes at 3,000g and 4oC.

    4. Resuspend the cells in 2.5 ml of Tris-Sucrose Buffer.

    5. Add 0.5 ml of Lysozyme Solution and incubate for 2 hours at 30oC.

    6. Monitor the conversion of bacterial rods to spheres by phase contrast microscopy.

    7. Dilute spheroplasts in Protoplast Suspension Solution at a density of 4.0 x 109 cells/ml.

    8. Go to step 9.



    Preparation of E. coli Spheroplasts
    1. Grow E.coli in LB with a suitable antibiotic for plasmid selection and chloramphenicol or spectinomycin (250 ug/ml) for plasmid amplification.

    2. Centrifuge 50 ml of overnight culture for 10 minutes at 3,000g and 4oC.

    3. Resuspend the cells in 2.5 ml of prechilled Tris-Sucrose Buffer.

    4. Add 0.5 ml of Lysozyme Solution and incubate at 4oC for 5 minutes.

    5. Add 1.0 ml of 0.25 M EDTA (pH 8.0) slowly with continuous stirring on ice.

    6. Add 1.0 ml of 0.05 M Tris-HCl (pH 8.0) slowly on ice.

    7. Monitor the conversion of bacterial rods to spheres by phase contrast microscopy after 10-15 minutes at 37oC.

    8. Resuspend spheroplasts in Protoplast Suspension Solution at 8.0 x 109/ml.




    Treatment of Plant Protoplasts and Bacterial Spheroplasts with Fusogens
    9. Resuspend protoplasts (1.0 x 106) in 0.25 ml of Protoplast Suspension Solution.

    10. Resuspend Agrobacterium (1.0 x 109) or E. coli (2.0 x 109) spheroplasts in 0.25 ml of the Protoplast Suspension Solution.

    11. Mix bacterial spheroplasts from step 10 with the protoplasts from step 9.

    12. Add 0.5 ml of PEG or polyvinyl alcohol (PVA) Fusion Solution dropwise to the mixture with gentle swirling.

    13. After 15 min at 22oC, add 5.0 ml of Glycine-NaOH buffer to dilute the viscous mixture.

    14. Incubate for 20 min at 30oC.

    15. Wash the protoplasts three times with MS-H Liquid Medium-7 to remove free spheroplasts.

    16. Plate the cells in 3.0 ml of MS-H Liquid Medium-7 over 12 ml of MS-H Solid Medium-9 containing 1.0 mg/ml carbenicillin.

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

    18. At day 4, transfer cells to 3.0 ml of MS-H Liquid Medium-3.5 over 12 ml of MS-H Solid Medium-9 containing 1.0 mg/ml carbenicillin.

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

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

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

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

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

    24. 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.

    25. Transfer individual colonies using the tip of a scalpel, to MS Solid Medium-0 containing carbenicillin (1 mg/ml) and kanamycin (50 ug/ml).

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

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

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


    NOTES



    KIT INFORMATION



    REFERENCES

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  • 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.

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



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