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.
Agrobacterium Plasmid DNA minipreps
To facilitate plasmid DNA minipreps a single Agrobacterium colony should be inoculated in 5ml YT containing appropriate antibiotics, and grown at 30°C for 24-36 hours. 3ml of cells are then pelleted by centrifugation, and resuspended in 100 ml GTE buffer (25mM Tris.Cl, 10mM EDTA, 50mM Glucose, pH 8.0, 4mg/ml lysozyme). 200 m l of freshly prepared solution II (0.2M NaOH, 1% SDS) is added, and mixed by slowly inverting the tube five times.
Following a 5 minute incubation at room temperature 30 ml of alkaline phenol (6 ml 10N NaOH in 1ml phenol:chloroform:isoamyl alcohol (25:24:1; v:v:v)) is added and mixed by vortex . 150 ml of ice-cold solution III (3M/5M KOAc, pH 4.8) is added, then shook by hand. Samples are incubated at –20°C for 20 minutes, and then centrifuged at 14000 rpm (Eppendorf microcentrifuge) to remove cell debris. The supernatant is transferred to an Eppendorf tube, and DNA ethanol precipitated. The DNA pellet is dissolved in 10 ml TE pH8.0 or dH2O. To check the plasmid, 1 ml of DNA solution should be transformed into E.coli, from which DNA is extracted, and analysed by restriction enzyme digest or by sequencing.
Agrobacterium tumefaciens Mediated Plant Transformation
Plants are transformed with Agrobacterium using a protocol based upon Clough and Bent (1998). To facilitate plant transformation 1ml of Agrobacterium glycerol stock solutions should be used to inoculate 50ml YT solution containing appropriate antibiotics. Following incubation at 30°C for 24Hrs with constant shaking, the culture is added to a 400ml YT solution and incubated for a further 24-48 hours.
Agrobacterium cells are collected by centrifugation for 10 min at 4°C. The cells re-suspended in 250ml of infiltration media (Murashige and Skoog salts, 5% (w/v) sucrose, 0.5 g/l MES pH 5.7 with NaOH) containing 50m l Silwett L-77. Plants are submerged in this solution for 3-4 minutes. Excess liquid is removed from the plants by allowing them to dry on their sides. A plastic sheet is used to cover the plants for two days to aid infiltration. Plants should then be transferred to a greenhouse.