Genetic transformation of Brassica napus via Agrobacterium spp. and the development of herbicide resistance

This thesis describes the genetic transformation of Brassica napus using Agrobacterium spp. and the application of this technology to achieve sulfonylurea and imidazolinone herbicide resistance. In planta tumourigenesis induced by Agrobacterium was studied to identify strains that were efficient in the transformation of B. napus. The efficiency of A. tumefaciens nopaline Ti plasmids in contrast with the relative inefficiency of octopine Ti plasmids was demonstrated. The disarmed Ti plasmid pGV3850 derived from the nopaline pTiC58 was used to recover transgenic B. napus plants from stem explants at a frequency of c.a. 2%. Intermediate vectors containing chimeric plant genes coding for kanamycin and chloramphenicol resistances as selectable markers were employed. The nopaline synthase gene was also used as a screenable marker for genetic transformation. The chloramphenicol acetyltransferase gene was not a practical marker for Brassica genetic transformation. The transgenic B. napus plants were morphologically normal and fertile. The introduced genes were transmitted to the progeny. The mutant gene csr1-1 from A. thaliana was used to recover transgenic N. tabacum plants which were resistant to chlorsulfuron and also to other short residual sulfonylurea herbicides. When used with chlorsulfuron as a selective agent, this gene was not as efficient as the neomycin phoshotransferase gene in both N. tabacum and B. napus. The overexpression of the ALS gene fused to a 35S promoter yielded tolerance to both sulfonylurea and imidazolinone herbicides in transgenic N. tabacum. In B. napus, analysis of transgenic callus and plant obtained with the mutant csr1-1 gene indicated the presence of ALS enzyme resistant to chlorsulfuron.