AGROBACTERIUM-TUMEFACIENS-MEDIATED TRANSFORMATION OF DOUBLE HAPLOID CANOLA (BRASSICA-NAPUS) LINES

Double haploid (DH) genotypes of canola (Brassica napus L.) have a hig h level of genetic uniformity but have not been previously tested for genetic transformation. Transgenic plants from three of four DH genoty pes derived from cv. Westar were obtained by inoculation of either hyp ocotyl segments or root explants with Agrobacterium tumefaciens. For h ypocotyl transformation, A. tumefaciens strain LBA4404 containing a bi nary plasmid with the neomycin phosphotransferase gene (nptII) and a C aMV 35S-peroxidase gene cassette was co-cultivated with hypocotyl segm ents taken from the 5-6-day-old seedlings. Transformation frequencies for hypocotyl explants of two DH genotypes were 0.3-3%. Direct evidenc e for genetic transformation of hypocotyl explants was obtained throug h molecular hybridisation analysis. Using this protocol, mature transf ormed plants were obtained within 4-6 months of co-cultivation. A meth od of root transformation was successfully modified for one DH genotyp e of canola and transgenic plants were obtained at a frequency of 2%. Using this protocol, a peroxidase gene promoter-GUS fusion construct w as introduced into a DH genotype. Tissue specific GUS expression drive n by the peroxidase gene promoter in transgenic plants was analysed by GUS staining. Transformation systems for double haploid canola lines will permit the assessment of introduced genes for their effect on agr onomic and physiological traits.