No abstract

The 42-kDa endochitinase (cht42) gene from the mycoparasitic fungus, Trichoderma virens, driven by CaMV 35S promoter, was introduced into rice by Agrobacterium-mediated transformation. Eight transgenic plants harboring single copies of complete T-DNA were identified by Southern blot analysis. Homozygous transgenic plants were identified for five lines in the T(1) generation by Southern blot analysis. Homozygous T(2) plants constitutively accumulated high levels of the cht42 transcript, showed 2.4- to 4.6-fold higher chitinase activity in total leaf extract and 1.6- to 1.8-fold higher chitinase activity in the extracellular fluid. Infection assays performed on the homozygous T(2) plants with Rhizoctonia solani showed up to 62% reduction in the sheath blight disease index.

Co-transformation of Oryza sativa L. var. Pusa Basmati1 was done using an Agrobacterium tumefaciens strain harbouring a single-copy cointegrate vector and a multi-copy binary vector in the same cell. The T-DNA of the cointegrate vector pGV2260::pSSJ1 carried the hygromycin phosphotransferase (hph) and beta-glucuronidase (gus) genes. The binary vector pCam-chi11, without a plant selectable marker gene, harboured the rice chitinase (chi11) gene under maize ubiquitin promoter. Co-transformation of the gene of interest (chi11) with the selectable marker gene (hph) occurred in 4 out of 20 T(0) plants (20%). Segregation of hph from chi11 was accomplished in two (CoT6 and CoT23) of the four co-transformed plants in the T(1) generation. The selectable marker-free (SMF) lines CoT6 and CoT23 harboured single copies of chi11. Homozygous SMF T(2) plants were established in the lines CoT6 and CoT23. Northern and Western blot analysis of the homozygous SMF lines showed high level of transgene expression. In comparison to untransformed controls, chitinase specific activity was 66- and 22-fold higher in the homozygous SMF T(2) plants of lines CoT6 and CoT23, respectively. The lines CoT6 and CoT23 exhibited 38 and 40% reduction in sheath blight disease, respectively.