Genetic transformation of the bast fiber plant ramie (Boehmeria nivea Gaud.) via Agrobacterium tumefaciens

Hypocotyls and cotyledons of three ramie (Boehmeria nivea Gaud.) cultivars (Zhongzhu No.1, Zhongsizhu No.1 and NC01), important plants for textile fiber, were pre-cultured on callus-inducing medium for 1 day before co-cultivation for 2 days with Agrobacterium tumefaciens strain LBA4404 harboring the plasmid pGBI4ABC carrying two insect resistance (CryIA and CpTI), gus, and neomycin phosphotransferase (npt II) genes. Calli were induced from both hypocotyl and cotyledon explants grown on a kanamycin selection medium. Regenerated shoots were obtained after two cycles of culture and transferred to rooting medium. Kanamycin-resistant plantlets were rooted in 2 weeks, and then transplanted to soil. Transgenic plants were subsequently confirmed by polymerase chain reaction, Southern blot hybridization, and GUS assays. More than 100 transgenic plants carrying insect-resistance genes were produced. A transformation frequency of 8.8 to 10.3% was obtained using hypocotyls as explants, which was higher than all previously reported transformation frequencies. The whole protocol, from transformation recovery of plants grown in soil, was completed within 2-4 months. Therefore, a simple, efficient, and robust Agrobacterium tumefaciens -mediated transformation system for ramie has been developed.