Agrobacterium tumefaciens-mediated Transformation of Double Haploid Canola (Brassica napus) Lines

Abstract

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.

A seed coat-specific promoter for canola

“…They showed transformation efficiencies varied from 0.3 to 2%. Transformation efficiencies of 0.3-3% from Agrobacterium-mediated transformation of double haploid canola lines have been previously reported (Kazan et al 1997). Cardoza and Stewart (2003) …”

Agrobacterium -mediated transformation of a doubled haploid line of cabbage

“…DH lines have been used for plant breeding, mutagenesis, molecular mapping, and plant transformation (Kasha et al 1995), and they are believed to be superior material for physiological studies and plant-microbe interactions at different stages or environments (Kazan et al 1997). For this purpose, Kazan et al (1997) established Agrobacterium-mediated transformation of DH lines of Brassica napus.…”

The Use of Herbicides in Biotech Oilseed Rape Cultivation and in Generation of Transgenic Homozygous Plants of Winter Oilseed Rape (Brassica napus L.)

“…Doubled haploids, obtained by tissue cultures of microspores and microspore-derived embryos (MDEs), show uniformity and complete homozygosity, thus they are particularly useful for breeding programmes and have been extensively used to produce homozygous breeding lines and cultivars of oilseed rape (Cegielska et al, 2002;Friedt and Zarhloul, 2005;Takahata et al, 2005). DH plants can also be efficiently used in basic studies, for example in genetic analysis and genetic transformation (Cegielska-Taras et al, 2008;Fukuoka et al, 1998;Huang, 1992;Jardinaud et al, 1993;Kazan et al, 1997;Nehlin et al, 2000;Oelck et al, 1991;Swanson & Erickson, 1989;Takahata et al, 2005;Troczyńska et al, 2003). It is postulated that in the process of genetic transformation, the gene introduced into the haploid genome, in this case MDE and subsequent chromosome doubling (diploidization), it will give rise to homozygous transgenic oilseed rape (Dorman et al, 1998;Huang, 1992).…”