Transformation of Brassica napus L. (oilseed rape) using Agrobacterium tumefaciens and Agrobacterium rhizogenes — A comparison

Abstract

We have produced transgenic oilseed rape plants by two different transformation strategies. The first method utilised an A. rhizogenes binary system comprising of p Ri1855 and p Bin19 plasmids, to induce hairy root proliferation at the cotyledonary nodes. Clonal hairy root explants selected on kanamycin were then used to regenerate whole transgenic rape plants which though exhibiting the hairy root phenotype to varying degrees, were fertile. These plants were outcrossed with “wild type” rape lines and the F 1 progeny analysed to show segregation of hairy root and antibiotic resistance genes. In the second method, an A. tumefaciens binary system was used to transform inflorescence stalks of different varieties of rape, using modifications of existing protocols which resulted in more efficient transformation. These modifications included the use of acetosyringone, the use of N. plumbaginifolia feeder cells, the inclusion of Seaplaque agarose in the media, and the postponement of kanamycin selection in the shooting medium for 2–3 weeks. Regenerated shoots were rooted and whole rape plants produced. Putative transgenic plants were subjected to phenotypic and Southern analyses to confirm the presence or absence of the introduced genes. The methods are effective with both Spring and Winter varieties of rape. Details of the two methods are presented and the advantages and disadvantages of each method are discussed.