High level resistance to Turnip mosaic virus in Chinese cabbage (Brassica campestris ssp. pekinensis (Lour) Olsson) transformed with the antisense NIb gene using marker-free Agrobacterium tumefaciens infiltration

A TuMV-NIb gene fragment was amplified from the total RNA of Chinese cabbage leaves infected with TuMV-C4 by RT-PCR. This fragment was cloned into the marker-free plant binary vector pROK (a derivative of pROK2) in the antisense orientation and transformed into Chinese cabbage [Brassica campestris ssp. pekinensis (Lour) Olsson] by the Agrobacterium tumefaciens strain LBA4404 using infiltration gene-transformation system. Forty-three transgenic T0 plants were obtained. PCR, ELISA and Southern blot analysis showed that the TuMV-NIb gene had integrated into the genome of Chinese cabbage and the heredity of this gene in some lines followed the 3:1 ratio. Both T0 and T1 transgenic plants showed high levels of resistance to TuMV when artificially inoculated with TuMV. RT-PCR analysis of T0 transgenic Chinese cabbage revealed the integrated TuMV-NIb gene can be expressed normally. The infiltration gene-transformation system had proved effective in transformation of Chinese cabbage.