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A transiently expressed transposase system to generate Ds-tagged mutants for functional genomics in sorghum

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Abstract

Six T-DNA/Ds launch pad lines (T0) previously generated by Agrobacterium-mediated transformation of M 35-1 genotype of sorghum were confirmed by PCR. T1 plants of all six lines showed 3:1 segregation when sprayed with 12 ppm Basta herbicide, indicating single copy insertion, which was also confirmed by left border flanking sequence tag. Calli derived from pNU435-T0(1) primary transformant was co-infected with Agrobacterium-carrying iAc construct for transient expression of transposase to generate stable Ds-tagged mutants in the T0 generation. All nine regenerants were PCR-positive for Ds. However, four contained intact T-DNA/Ds launch pad, while five plants carried empty launch pad, indicating transposition of the Ds. One of these plants, IDs-T0(8), was negative for iAc PCR, indicating that it was a stable Ds-tagged mutant. Of the four plants with intact T-DNA/Ds, IDs-T0(5) carrying iAc was a double transformant and mutagenic, which can generate mutants in the subsequent generation. Hence, the transient expression of transposase system in sorghum reported here can be employed for high throughput mutagenesis.

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Acknowledgments

We thank Dr. Narayana M. Upadhyaya of CSIRO Plant Industry, Canberra, Australia for sending the Ds and iAc constructs. We also thank Head, Sorghum scheme, University of Agricultural Sciences, Dharwad, India, for providing the seeds of M 35-1. We are grateful to the Department of Biotechnology, New Delhi, India for the research grant.

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Authors and Affiliations

  1. Department of Biotechnology, University of Agricultural Sciences, Dharwad, 580 005, India

    Amit Kumar Verma, Virupaksha U. Patil & Ramesh S. Bhat

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  1. Amit Kumar Verma
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  2. Virupaksha U. Patil
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  3. Ramesh S. Bhat
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Correspondence to Ramesh S. Bhat.

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Verma, A.K., Patil, V.U. & Bhat, R.S. A transiently expressed transposase system to generate Ds-tagged mutants for functional genomics in sorghum. Plant Cell Tiss Organ Cult 107, 181–185 (2011). https://doi.org/10.1007/s11240-011-9967-x

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  • DOI: https://doi.org/10.1007/s11240-011-9967-x

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