Double haploid (DH) genotypes of canola (Brassica napus
L.) have a high level of genetic uniformity but have not been previously
tested for genetic transformation. Transgenic plants from three of four DH
genotypes derived from cv. Westar were obtained by inoculation of either
hypocotyl segments or root explants with
Agrobacterium tumefaciens. For hypocotyl transformation,
A. tumefaciens strain LBA4404 containing a binary
plasmid with the neomycin phosphotransferase gene
(nptII) and a CaMV 35S-peroxidase gene cassette was
co-cultivated with hypocotyl segments taken from the 5–6-day-old
seedlings. Transformation frequencies for hypocotyl explants of two DH
genotypes were 0.3–3%. Direct evidence for genetic transformation
of hypocotyl explants was obtained through molecular hybridisation analysis.
Using this protocol, mature transformed plants were obtained within 4–6
months of co-cultivation. A method of root transformation was successfully
modified for one DH genotype of canola and transgenic plants were obtained at
a frequency of 2%. Using this protocol, a peroxidase gene
promoter–GUS fusion construct was introduced into a DH genotype. Tissue
specific GUS expression driven 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 agronomic and physiological traits.
Genetic analysis of nonpathogenic Agrobacterium tumefaciens mutants arising in crown gall tumors.