Effectively mutagenize C-Fern spores for use in selection protocols.

WARNING EMS IS A POTENT MUTAGEN

• Use proper clothing, gloves, containment trays, supplies, and equipment.
• Sodium thiosulfate solution should be used to neutralize any EMS spills and to neutralize treatment solutions after mutagenesis.
• All mutagenesis procedures should be carried out in an appropriate exhaust/fume hood.

Materials

• 0.1 M Phosphate Buffer, pH 7.0 (Prepare buffer using 0.2 M solutions of monobasic and dibasic sodium phosphate, as follows, then diluting to 0.1 M with distilled H₂O)
  • 39 mL 0.2 M NaH₂PO₄ (FW 137.99; 2.76 g / 100 ml)
  • 61 ml 0.2 M Na₂HPO₄ (FW 141.96; 2.84 g / 100 ml)
  • 100 ml distilled H₂O
• 200 mM EMS (ethyl methane sulfonate) Solution
  • 43.3 ml EMS (Sigma) MW = 124.2, d = 1.1452, 1 M = 108.5 ml/l
  • 1957 ml 0.1 M Phosphate Buffer, pH 7.0
• 0.4 M Sodium thiosulfate (Na₂S₂O₃)
• C-Fern spores
• Materials and supplies for C-Fern spore sterilization and culture inoculation

Method

PREPARE SPORES Weigh out spores to be mutagenized and transfer to a 15 ml conical centrifuge tube.

PREPARE EMS SOLUTION Mix appropriate amounts of previously prepared 0.1 M Phosphate Buffer (pH 7.0) and EMS to prepare the 200 mm EMS Solution, and vortex very thoroughly and carefully.

NOTE: It is very important to thoroughly vortex the mixture because EMS is difficult to dissolve in buffer. If EMS solution is properly mixed, spores should mostly sink to the bottom after wetting and settling. If spores are mostly floating, mixing was not adequate.

TREAT SPORES Add at least 1 ml of EMS Solution or approximately twice the volume of the spores being treated. Treat spores for 36 hours at room temperature.

WASH, STERILIZE AND INOCULATE SPORES Remove EMS Solution and wash spores three times with 2 ml distilled H₂O using the same technique as described for spore sterilization. Sterilize and sow spores using standard procedures.