Agrobacterium-mediated transformation of Brassica napus and Brassica oleracea.
Agrobacterium-mediated transformation is widely used for gene delivery in plants. However, commercial cultivars of crop plants are often recalcitrant to transformation because the protocols established for model varieties are not directly applicable to them. The genus Brassica includes the oil seed crop, canola (B. napus), and vegetable crop varieties of Brassica oleracea, including cauliflower, broccoli and cabbage. Here, we describe an efficient protocol for Agrobacterium-mediated transformation using seedling explants that is applicable to various Brassica varieties; this protocol has been used to genetically engineer commercial cultivars of canola and cauliflower in our laboratory. Young seedling explants are inoculated with Agrobacterium on the day of explant preparation. Explants are grown for 1 week in the absence of a selective agent before being transferred to a selective medium to recover transgenic shoots. Transgenic shoots are subjected to an additional round of selection on medium containing higher levels of the selective agent and a low-carbohydrate source; this helps to eliminate false-positive plants. Use of seedling explants offers flexible experiment planning and a convenient explant source. Using this protocol, transgenic plants can be obtained in 2.5 to 3.5 months.
Bhalla
Prem L
PL
Australian Research Council Centre of Excellence for Integrative Legume Research, Faculty of Land and Food Resources, The University of Melbourne, Parkville, Victoria 3010, Australia. premlb@unimelb.edu.au
Singh
Mohan B
MB
D016960:Agrobacterium tumefaciens / Q000235:genetics*; D001937:Brassica / Q000235:genetics* / Q000382:microbiology; D018014:Gene Transfer Techniques; D030821:Plants, Genetically Modified; D014170:Transformation, Genetic