Agrobacterium-Mediated Genetic Transformation of Withania coagulans (Dunal) with rol A Genes and Its Antioxidant Potential

Abstract

In ancient times, Withania coagulans Dunal was used as a therapeutic plant for the treatment of several diseases. This report aims to examine the effect of Agrobacterium tumefactions-mediated transformation of W. coagulans with the rolA gene to enhance secondary metabolite production, antioxidant activity, and anticancer activity of transformed tissues. Before transgenic plant production, the authors designed an efficient methodology for in vitro transformation. In this study, leaf explants were cultured on Murashage and Skoog (MS) media containing different amounts of naphthalene acetic acid (NAA) and benzyl adenine (BA). The best performance for inducing embryogenic callus was in MS medium containing 4 μM NAA and 6.0 μM BA, while the best results for shooting (100%) were obtained at 8 μM benzyl adenine. On the other hand, direct shooting was attained by subculturing leaves on MS medium supplemented with 8 μM benzyl adenine. Prolonged shoots showed excellent in vitro rooting results (80%) with 12 μM indole-3-butyric acid (IBA). The samples were precultivated for 3 days and were followed by 48 h infection with A. tumefaciens strain GV3101 having pCV002. Then, a vector expressed the rol A gene of strain Agrobacterium rhizogenes. Furthermore, three independent transgenic shoot lines and one callus line (T2) were produced and exhibited stable integration of transgene rol A genes, as revealed by PCR analysis. Transgenic strains showed a significant increase in antioxidant potential as compared to untransformed plants. Additionally, LC-MS analysis showed that the transformed strains have a higher withanolide content as compared to untransformed ones. Moreover, the reduced proliferation of prostate cancer cells was observed after treatment with extracts of transgenic plants. Furthermore, these transformed plants exhibited superior antioxidant capability and higher withanolide content than untransformed ones. In conclusion, the reported data can be used to select withanolide-rich germplasm from transformed cell cultures.