A non-antibiotic selection system uses the phosphomannose-isomerase (PMI) gene for Agrobacterium-mediated transformation of Chinese cabbage

To establish a non-antibiotic selection system that utilizes the phosphomannose-isomerase (PMI) gene for Chinese cabbage transformation, we first determined the optimum mannose concentration for selecting transformed cells. Hypocotyl and cotyledon explants that were grown on media containing more than 5 g L-1 mannose did not induce green calli but, rather became chlorotic and withered before dying. In contrast, media containing 20 g L-1 sucrose plus 5 g L-1 mannose proved suitable for selection. We then used this particular level of mannose to transform hypocotyl tissues. Within 6 weeks, shoots were regenerated from some of the calli; subsequently, these plants were transplanted to pots and grown in the greenhouse. A 514-bp PCR fragment was obtained from most transformants but not from the non-transformed plants. Southern blot analysis also revealed the expected PMI gene in those PCR-confirmed transgenic plants. RT-PCR of total RNA was performed to confirm PMI expression. We have now demonstrated that this gene does not inhibit the growth of transgenic plants, and that this selection system can be applied to Chinese cabbage transformation.