Protoplasting Sclerotinia Hyphae

(Based on procedure from Kohn et al. 1991. Phytopathol. 81:480-485)

  1. Grow a starter culture of Sclerotinia in a PDA plate.
  2. Inoculate 25 ml of PDB in a glass petri dish with actively growing hyphae, several 2-3 mm pieces; four plates for each strain.
  3. Harvest the hyphae in 3-5 days, when it covers the surface of the liquid medium, but before sclerotial initials emerge.
  4. Pick up the confluent mass with forceps and wash 1x with sterile water over powder funnel with 4 layers of sterile cheesecloth, then 1x with protoplast buffer.
  5. Coarsely chop the washed mycelium (usually from one plate) on the sterile cover of the petri dish and transfer to a sterile 125 ml Erlenmeyer.
  6. Dissolve 200 mg Glucanex in 3 ml Novozyme buffer and filter sterilize with 0.45 µm filter. Add that to 17 ml protoplast buffer and pour it to the flask with the chopped mycelium.
  7. Incubate at 28°C, slowly (100 rpm) shaking for 1-3 hours. Start checking protoplast formation after one hour. When most hyphae have been digested, proceed.
  8. Separate protoplasts from the residual hyphae by filtration through sterile glass wool over a sterile 125 ml flask. Pour 30 ml of 0.6 M KCl over.
  9. Pellet protoplasts in the 50 ml disposable conical tubes, 3’000xg (AB50.10 5000rpm) at 4°C, 10 min.
  10. Wash and pellet protoplasts with 10 ml STC 2x and centrifuge as before.
  11. Count the protoplasts in STC, record the volume and spin them down one more time and suspend them in STC to make a concentration 1× 108/ml. Keep on ice.
  12. Store protoplasts in –80°C: to 1 ml of protoplasts add 12.5µl DMSO, 62.5 µl Heparin (5mg/ ml in STC) and 250 µl 40% PEG in water and 1 part KTC”>PEG.


  • Protoplast buffer: 0.8 M MgS04 •7H2O (FW 246.48), 0.2 M Sodium citrate• 2H2O (FW 294.1), pH 5.5
  • Novozyme buffer: 1 M Sorbitol (FW 182.2), 50 mM Sodium citrate, pH 5.8
  • STC: 1M Sorbitol, 50 mM Tris, pH 8, 50 mM CaCl2 •2H2O (FW 147.02)
  • KTC: 1.8M KCl (FW 74.56), 150 mM Tris, pH 8, 150 mM CaCl2
  • PEG: Prepare a 60% w/v PEG 4000 in water and autoclave.
  • 0.6 M KCl

Updated 28 May 2007 by Jeffrey Rollins.

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