1Department of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Madurai 625 021, Tamil Nadu, India
2Department of Plant Pathology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India
3Department of Biochemistry, Kansas State University, Manhattan, KS 66506–3702, USA
*Corresponding author. E-mail: veluthambi@mrna.tn.nic.in
Abbreviations: AS, acetosyringone; BCIP, 5-bromo-4-chloro-3-indolyl phosphate; chi11, rice chitinase gene; Cl, callus-induction; 2,4-D, 2,4-dichlorophenoxyaceticacid; gus, β-glucuronidase; hyg, hygromycin; hph, hygromycin phosphotransferase; int-gus, intron-containing gus gene; LB, left T-DNA border; MS, Murashige and Skoog; NAA, α-naphthaleneacetic acid; NBT, nitroblue tetrazolium, PB1, Pusa Basmati 1; PR, pathogenesis-related; RM, regeneration medium; RB, right T-DNA border; TTBS, Tween-Tris buffered saline; Ubi1, maize ubiquitin promoter-intron.
Agrobacterium-medialed transformation of an elite indica rice variety, Pusa Basmati 1, was performed using LBA4404 (pSB1, pMKU-RF2) that harbours a rice chitinase gene (chi11) under the control of the maize ubiquitin (Ubi1) promoter-intron. Right border (gus) and left border (hph) flanking sequences and the transgene (chi11) in the middle of the T-DNA were used as probes in Southern analysis. Out of eleven independent T0 plants regenerated, three had single copy T-DNA insertions and eight had multiple T-DNA insertions. Nine T0 plants carried the complete T-DNA with the chitinase transgene. Two T0 plants did not carry chi11, though they had other T-DNA portions. Three plants harbouring single copy insertions and one plant harbouring two inserted copies were analyzed in detail. A segregation ratio of 3:1, reflecting T-DNA insertion at a single locus, was observed in the progeny of all the four T0 plants. Northern and western blot analyses of T, plants revealed constitutive expression of chitinase at high levels. Bioassays of T, plants indicated enhanced resistance to the sheath blight pathogen, Rhizoctonia solani, in comparison to control plants. A homozygous transgenic line was established from one T0 line, which exhibited the maximum resistance to R. solani.
sheath blight, Rhizoctonia solani, chitinase, transgenic rice, Agrobacterium
