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 uded to cover the plants for two days to aid infiltration. Plants should then be transferred to a greenhouse.