Department of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Madurai 625 021, India
*Corresponding author. E-mail: veluthambi@mrna.tn.nic.in
Abbreviations: AS, acetosyringone; CaMV, cauliflower mosaic virus; CIM, callus-induction medium; 2,4-D, 2,4-dichlorophenoxyacetic acid; GUS, (β-glucuronidase; Hyg, hygromycin; LB, left T-DNA border; MS, Murashige and Skoog; NAA, α-naphthaleneacetic acid; RB, right T-DNA border; RIM, root-induction medium; SIM, shoot-induction medium.
An intermediate vector pSSJ1 was constructed by cloning a hph gene and a gus gene with catalase intron in pGV1500. pSSJ1 was cointegrated into a disarmed receptor Ti plasmid pGV2260 harboured in Agrobacterium tumefaciens strain C58C1 RifR. The resulting A. tumefaciens strain C58C1 RifR (pGV2260::pSSJ1) stably transformed Oryza sativa L. cy Pusa Basmati 1 scutellum-derived calli at 26% frequency. Introduction of the plasmid pSSJS (3′virB, virG and virC of pTiBo542) into A. tumefaciens C58C1 RifR (pGV2260::pSSJ1) resulted in the elevation of acetosyringone-induced T-strand accumulation. Rice transformation efficiency of the cointegrate plasmid pGV2260::pSSJ1 increased from 26% to 33% in the presence of pSSJS and from 26% to 35% in the presence of pToK47 (complete virB, virG and virC). T-DNA integration in T0 plants was confirmed by Southern hybridization analysis. Inheritance analysis of the T0 plants with single-copy T-DNA insertions revealed segregation of hygromycin resistance in 3:1 ratio. The feasibility of rice transformation with a cointegrate Ti plasmid vector is clearly established.
Agrobacterium, cointegrate vector, pTiBo542, T-strands, transgenic rice
