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.