Agrobacterium-Mediated Transformation of Protoplasts from Oilseed Rape (Brassica napus L.)

Although a number of protocols are available for the regeneration of oilseed rape protoplasts (1 –4 ), there are almost no reports of successful Agrobacterium tumefaciens -mediated transformation of protoplasts resulting in transgenic plants. Plant regeneration from protoplast-derived calli of Brassica napus generally occurs at a low frequency and depends on the genotype used (4 –5 ). In addition, shoot regeneration is significantly reduced in protoplast-derived calli after cocultivation and kanamycin selection (6 ). The methods for the transformation and regeneration of B. napus hypocotyl protoplasts described in this chapter are suitable for obtaining transgenic plants by an A. tumefaciens -mediated gene transfer. However, a prerequisite is the use of oilseed rape genotypes possessing a sufficient shoot regeneration capability, more than 20%, and the application of a three-step kanamycin selection. In this protocol a marker gene coding for kanamycin resistance is introduced into hypocotyl protoplasts of winter-type oilseed rape. Genes, possessing relevant qualities for improving the agronomic character of oilseed rape varieties, such as stilbene synthase and T4 phage lysozyme, have also been cointroduced together with a chimeric kanamycin resistance gene using this protocol (6 ).