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

 

 

WARNING EMS IS A POTENT MUTAGEN

Use proper clothing, gloves, containment tray, etc.
Sodium thiosulfate 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 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
  • Material for spore sterilization and culture inoculation

 

Method

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

2. 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.

3. 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. Remove EMS Solution and wash spores three times with 2 ml distilled H2O using the same technique as described for spore sterilization.

4. Sterilize and sow spores using standard procedures.