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A Cointegrate Ti Plasmid Vector for Agrobacterium tumefaciens - mediated Transformation of indica Rice cv Pusa Basmati 1

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Abstract

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 C58C1RifR. The resulting A. tumefaciens strain C58C1RifR (pGV2260::pSSJ1) stably transformed Oryza sativa L. cv Pusa Basmati 1 scutellum-derived calli at 26% frequency. Introduction of the plasmid pSSJ3 (3′virB, virG and virC of pTiB0542) into A. tumefaciens C58C1RifR (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 pSSJ3 and from 26% to 35% in the presence of pToK47 (complete virB, virG and virC). T-DNA integration in To 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.

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Abbreviations

AS:

acetosyringone

CaMV:

cauliflower mosaic virus

CIM:

callus-induction medium

2,4-0:

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

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Authors and Affiliations

  1. Department of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Madurai, 625 021, India

    Subha S. Jacob & K. Veluthambi

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  1. Subha S. Jacob
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  2. K. Veluthambi
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Correspondence to K. Veluthambi.

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Jacob, S.S., Veluthambi, K. A Cointegrate Ti Plasmid Vector for Agrobacterium tumefaciens - mediated Transformation of indica Rice cv Pusa Basmati 1. J. Plant Biochem. Biotechnol. 12, 1–9 (2003). https://doi.org/10.1007/BF03263152

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  • DOI: https://doi.org/10.1007/BF03263152

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