Effect of Agrobacterium infection duration on transformation efficiency in tuberose Agave amica [(Medik.) Thiede & Govaerts] cv. Phule Rajani

Abstract

Agrobacterium tumefaciens mediated genetic transformation of tuberose Agave amica [(Medik.) Thiede & Govaerts] cv. Phule Rajani was optimized by evaluating the influence of infection duration on transformation efficiency. Callus explants derived from tepal segments were pre-cultured for one week, then infected with Agrobacterium strain LBA4404 carrying the pCAMBIA-1305.1 vector. Infection durations of 5, 10, and 15 minutes were tested at 200 µM Acetosyringone, followed by co-cultivation and selection on medium containing 10 mg/L Hygromycin and Cefotaxime. Transformation efficiency, assessed by explant survival and PCR confirmation, peaked at 18% with a 10-minute infection, where 13 of 50 calli survived and 9 were PCR positive. Shorter infection (5 minutes) yielded only 6% efficiency, while extended exposure (15 minutes) reduced efficiency to 10%, likely due to bacterial overgrowth and tissue necrosis. These results highlight that a 10-minute infection period achieves optimal T-DNA delivery without compromising explant viability, providing a reliable protocol for genetic transformation in tuberose.