How to perform Ethyl Methane-Sulphonate (EMS) Mutagenesis
To facilitate EMS mutagenesis approximately 6500 seeds were wrapped in miracloth package. They were then pre-imbibed in 0.1% KCL overnight at 4°C. The miracloth package was transferred to a Duran bottle containing 100ml of dH2O. 300 ml of Ethyl Methane-Sulphonate (EMS) was added to the flask in a fume hood. The bottle was sealed and placed in a sealed plastic bag. The bottle was placed on a shaking table for a period of 9 hours. The EMS containing solution was then neutralised by the addition of 100ml 0.1M Na2SO3. The miracloth package was transferred to a bottle containing 100ml 0.1M Na2SO3 for 15 minutes. It was then placed in a series of wash bottles (3 X 30 min) containing dH2O.
Following EMS mutagenesis
The seeds were unwrapped from the miracloth and added to a dilute agar solution (0.7%) at a density of ~650 seeds per litre. The seeds were then sowed onto 9cm 2 pots (260 pots of ~25 seeds/pot; 38ml agar/seed solution) and grown under long-day conditions in the greenhouse.
The M2 seed was harvested into 260 separate pools (Families) each containing ~25 M1 plants. 313 individual seeds from each pool were sown under long-day conditions in the greenhouse. Wild-type seeds are sown were sown as controls. Plants can then be screened for the required phenotype.