Sclerotinia Transformation Procedure

(Based on Rollins. 2003. Mol. Plant-Microbe Interact 16:785-795)

  1. 5X106 protoplasts (50µl) in STC are added to each chilled DNA (1-5 µg) suspension and incubated on ice for 30 min.
  2. One ml of PEG solution is added to the protoplast-DNA suspension with gentle mixing by pipetting or just twirling the tube. Tubes are incubated at room temperature for 20 min.
  3. Each suspension is evenly spread on the surface of a RM bottom agar plate by pipetting the solution to the center of the plate and tilting the plate to let the thick solution cover the entire surface. Bottom agar plate does not contain the selective agent. Plates are incubated overnight (15 hr) at room temperature. Pour plates, incubate PEG soln and add top agar, all on leveling table.
  4. First thing in the morning, plates are overlayed with 5 ml of RM top agar containing the selective agent. To achieve a final hygromycin concentration of 100 µg/ml, 5 ml of RM top agar containing 3000 µg of hygromycin is used. The final concentration of hygromycin in 30 ml (5 ml top agar + 25 ml bottom agar) will be 100 µg/ml after the hygromycin has diffused throughout the medium. To achieve a final bialaphos concentration of 10 µg/ml, 5 ml of RM top agar containing 300 µg of bialaphos is used. The final concentration of bialaphos in 30 ml (5 ml top agar + 25 ml bottom agar) will be 10 µg/ml after the bialaphos has diffused throughout the medium.
  5. Allow the overlay to solidify and incubate the plates at room temperature. Regenerating colonies emerging through the top agar on selection plates should be visible in 2-3 days with the aid of a dissecting scope and in 5-7 days regenerating colonies should be visible by eye. Protoplasts which went through transformation but did not receive DNA will regenerate on the bottom agar but will rarely emerge through the top agar.
  6. Pick vigorously growing colonies to grid plates (PDA + 100µg/ml hygromycin or modified Fries with 10µg/ml bialaphos). After transferring, cut rest of the colony out of the primary plate so that it does not grow over slower ones. Fast growing colonies are then transferred to small (50 mm Ø) PDA plates that have 100µg/ml hygromycin or 10-50 µg/ml bialaphos. Tip each transformant at least three times. Maintenance concentration for bialaphos is 1µg/ml.


  • STC: 1M Sorbitol, 50 mM Tris pH8, 50 mM CaCl2
  • PEG: Sol’n: Prepare a solution of 60% w/v PEG 4000 in water and autoclave for 30 min. Mix 2 parts of 60% PEG with 1 part of sterile KTC (1.8 M KCl, 150 mM Tris pH 8, 150 mM CaCl2).
  • RM for hygromycin: 0.7 M sucrose, 0.5 g yeast extract

RM for bialaphos: MF NO3 = Modified Fries
Carbon source: 0.7 M Sucrose (239.6g/L), 10g/L for maintenance plates
NaNO3 5g
KH2PO4 1g
MgSO4 7H2O 0.51g
NaCl 0.5 g
CaCl2 2H2O 0.065g
Make 10xstock of the salts that can be autoclaved together.
Agar: 15g/L for bottom, 8g/L for top agar. For instance: 420 ml bottom and 2×40 ml top agar

Updated 28 May 2007 by Jeffrey Rollins.

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