A modified Agrobacterium-mediated transformation method for Indonesian soybean varieties with Cry1Ab-1Ac fusion genes

Abstract

Grobogan and Wilis are major Indonesian soybean varieties, but their productivity is constrained by pod borer (Etiella zinckenella) susceptibility. While genetic transformation can aid in developing insect-resistant cultivars, the protocols for these local genotypes are limited by low regeneration efficiency. This study aims to develop an Agrobacterium-mediated transformation protocol using plumular meristem explants by optimizing infection duration and acetosyringone concentration, along with the evaluation of an ex vitro approach for post-transformation recovery. To establish a baseline for transformation, regeneration was optimized; shoot induction on MS medium with 1 mg/L BAP reached 85%, averaging 3.6 shoots per explant. Although this optimal BAP concentration was subsequently applied to the in vitro transformation system, it yielded no surviving putative plants after infection. In contrast, the modified ex vitro approach revealed a clear trade-off: a short 15-min infection with 100 µM acetosyringone maximized shoot elongation (67.5%), whereas a longer 30-min infection with 200 µM acetosyringone achieved transformation efficiency up to 17.5%. PCR analysis confirmed transgenes in 10–13 T0 plants, producing 37 T1 seeds, predominantly from Wilis, while Grobogan exhibited lower performance. The study demonstrates that the ex vitro approach is a practical strategy for soybean transformation.