Asymmetric somatic hybridization between Arabidopsis thaliana and Brassica nigra

The regeneration and transformation protocol for protoplasts of A. thaliana ecotype C24 was modified for asymmetric protoplast fusion. This modified protocol was used to fuse protoplasts of A. thaliana with X-ray irradiated protoplasts of B. nigra. The marker gene hygromycin resistance was introduced to the latter species by transformation and was used for selection of asymmetric somatic hybrids. The B. nigra lines used for fusion were resistant to two important Brassica pathogens (Leptosphaeria maculans, Plasmodiophora Brassicae). The maximal relative fusion rate using this protocol was estimated at 28 % by a fusion between two A. thaliana lines. Sixty six hygromycin resistant, putative hybrid-calli were obtained, which corresponds to a relative fusion rate of 0.003 % in regard to the total number of treated A. thaliana protoplasts. Shoots could be permanently regenerated from four calli, but the hybrid character was proven for only three of this lines by RAPD-analysis and Southern-blot hybridizations with repetitive sequences of A. thaliana and B. nigra. Two lines showed morphological characters of both fusion partners and revealed a nearly symmetric hybrid character, whereas one line showed a Brassica-morphology and a highly asymmetric hybrid character. Several subclones were obtained by regeneration of shoots from protoplast-derived calli of the hybrids. Resistance tests against P. Brassicae with the hybrid lines revealed that these plants were not suitable for analysis of resistance genes, although none of the tested hybrid plants showed any clubroot disease symptoms.