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

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.