Generation of selectable marker-free sheath blight resistant transgenic rice plants by efficient co-transformation of a cointegrate vector T-DNA and a binary vector T-DNA in one Agrobacterium tumefaciens strain

Abstract

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.

Co-expression of VpROMT gene from Chinese wild Vitis pseudoreticulata with VpSTS in tobacco plants and its effects on the accumulation of pterostilbene

“…To investigate the synthesis of pterostilbene, the two genes were over-expressed in tobacco by co-transformation. Numerous researches about co-transformation were successfully reported (Daley et al 1998;Sripriya et al 2008;RamanaRao and Veluthambi 2010). The highest co-transformation efficiency was achieved with an equal mixture of the two Agrobacterium strains (Depicker et al 1985;Komari et al 1996).…”

Plant Genetic Engineering: Problems and Applications

“…Selection of transformed cells from a number of non-transformed ones is done by the marker genes and for visual selection of transformed tissues a reporter gene like the green fl uorescent protein gene is required (Miki and McHugh 2004 ;Sripriya et al 2008 ) . Sometimes selectable marker gene affects the plant growth but an optimistic level of antibiotic is essential to reduce the false positives (Zapata et al 1999 ;Zhang et al 2001 ;Jin et al 2005 ;Wu et al 2006 ) .…”

Establishment of homozygous transgenic rice lines after elimination of unlinked albino traits from transgenic events through segregation

“…Transposable elements, explant type, genotype, culture medium, multiplicity of callus cycles, and extended periods in the callus phase are the important factors that cause somaclonal variations (Jain 2001). Since embryogenic callus is frequently used in rice transformation experiments (Hiei et al 1997;Nishimura et al 2006;Sripriya et al 2008), protocols emphasize a short callus phase in order to avoid somaclonal variations in transgenic plants (Toki 1997). Albinism is of common occurrence in the tissue culture of Poaceae members (Caredda and Clément 1999).…”