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

作者：Verma Amit Kumar; Patil Virupaksha U; Bhat Ramesh S^*

来源：Plant Cell, Tissue and Organ Culture, 2011, 107(1): 181-185.

DOI：10.1007/s11240-011-9967-x

• Sorghum
• Transposon-mediated gene tagging
• Transient expression of transposase
• Ds-tagged mutants
• Functional genomics

Six T-DNA/Ds launch pad lines (T(0)) previously generated by Agrobacterium-mediated transformation of M 35-1 genotype of sorghum were confirmed by PCR. T(1) 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-T(0)(1) primary transformant was co-infected with Agrobacterium-carrying iAc construct for transient expression of transposase to generate stable Ds-tagged mutants in the T(0) 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-T(0)(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-T(0)(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.

• 出版日期2011-10