A transiently expressed transposase system to generate <Emphasis Type="Italic">Ds</Emphasis>-tagged mutants for functional genomics in sorghum |
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Authors: | Amit Kumar Verma Virupaksha U Patil Ramesh S Bhat |
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Institution: | (1) Department of Biotechnology, University of Agricultural Sciences, Dharwad, 580 005, India; |
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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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