Multigene Engineering in Rice Using High-Capacity Agrobacterium tumefaciens BIBAC Vectors
The high-capacity binary bacterial artificial chromosome (BIBAC) vector system permits the insertion of large fragments of DNA, up to 150 kb, into plants via Agrobacterium-mediated transformation. Here, we describe an optimized protocol for transformation of japonica rice (Oryza sativa L.) using this system. Calli derived from mature embryos are transformed using Agrobacterium strain LBA4404 that carries the BIBAC vector and the super-virulent helper plasmid pCH32. Transformed calli are then regenerated using optimized media and tested for transgene integration by PCR, GUS assay, and Southern blot analyses.
KeywordsBinary bacterial artificial chromosome (BIBAC) Transformation of large DNA fragment Agrobacterium tumefaciens Rice (Oryza sativa L.)
We thank the Plant Science Center of Cornell University for providing the BIBAC2 vector and pCH32 plasmid. This work was supported by the National Natural Science Foundation of China (No. 30470922), the Natural Science Foundation of Hubei Province (No. 2004ABA117), and the National Program of High Technology Development of China (No. 2004AA227120).