2.4 Generation of transgenic rice plants overexpressingbiuret hydrolase
Transgenic Nipponbare plants that over-expressed bacterial biuret hydrolase under the control of the cauliflower mosaic virus 35s promoter were generated. A biuret decomposing soil bacterium,Rhizobium sp. KaB01, which was isolated in this study, was used as a biuret hydrolase donor (see Supporting methods). Insert DNA was amplified from the genomic DNA of KaB01 via PCR, using Prime Star polymerase (Takara Bio, Shiga, Japan) and primers 5´-CACCATGAAGACACTTTCCAGCGC-3´ and 5´-TGGCAAATGCCTCTCAAGG-3´. Subcloned PCR products in the vector pENTR/D-TOPO (Life Technologies, Carlsbad, CA) were then transferred to a binary vector pGWB502omega (Nakagawa et al., 2007), via the LR reaction. The transformation of rice was mediated by an agrobacterium, as described by Toki et al. (2006), using theAgrobacterium tumefaciens strain EHA105. The presence of the transgene in regenerated T0 plants was confirmed by PCR, using the Blend Taq polymerase (Toyobo, Osaka, Japan) and primers 5´-ATGAAGACACTTTCCAGCGC-3´ and 5´-TGGCAAATGCCTCTCAAGG-3´.