Phosphomannose-isomerase (pmi) gene as a selectable marker for Agrobacterium-mediated transformation of rapeseed

A transformation method using the phosphomannose-isomerase (pmi) gene as a selectable marker was developed for Brassica napus. The pmi-gene, which converts mannose-6-phosphate to fructose-6-phosphate allowing for selection of transgenic plants on mannose-selective medium, was transferred to B. napus hypocotyl explants by Agrobacterium-mediated transformation. More than 350 transgenic plants from three rapeseed varieties were obtained with transformation frequencies up to 24.2% when a combination of 4.5 g l(-1) mannose and 10 g l(-1) sucrose was used in the selection medium. For early identification of transgenic plants, histochemical staining with 5-bromo-4-chloro-3-indolyl-beta-d-glucuronide (X-Gluc) was used. Stable integration of the transgene was confirmed by PCR and Southern blot analysis. Mannose can be used as selective agent to identify transgenic plants in progeny i.e. segregation analysis. These results indicate that the mannose-selection system can be successfully used for Agrobacterium-mediated transformation of rapeseed.