Genetic Transformation and Withanolides Analysis of Withania Coagulans Dunal .

Withania coagulans is a valuable medicinal plant that holds a key place in folk medicine due to its extensive use in the treatment of many ailments. Biological activities like antimicrobial, anti-inflammatory, antitumor, hepatoprotective, antihyperglycemic, immunosuppressive, and antitumor, free radical scavenging and anti-depressant have been attributed to the plant due to the presence of secondary metabolites. Withanolides are the major class of secondary metabolites found in W. coagulans. In the present study, bioactive withanolides of Withania coagulans collected from 11 different sites in Pakistan were analyzed. Total withanolide concentration differed by ~17-fold between sites, ranging from 1.01 ± 0.01 mg/g dry weight (mean ± SE) at Jand to 16.83 ± 0.02 mg/g at Mohmand Agency. Different tissues varied in their total withanolide content and there were strong inverse correlations between site annual precipitation versus withanolide amounts in fruits (r = -0.84, P = 0.001), leaves (r = -0.88, P< 0.001), roots (r = -0.91, P< 0.001), and total (r = -0.89, P< 0.001), but not stems (r = -0.20, P = 0.556). Leaf extracts of plants collected from Mianwali and Mohmand Agency possessed high anti-cancer activity in terms of increased induction of apoptosis and decreased cell viability, cell proliferation, cell invasion, and cell migration of different prostate cancer cell lines. These results are useful for the selection of withanolide rich germplasm with potent anti-cancer properties. Owing to the therapeutic importance of these secondary metabolites in W. coagulans, it would be useful to enhance its withanolide content. Therefore, W. coagulans was transformed with the rol A gene which is known to enhance secondary metabolites. Prior to producing transgenic plants, an effective protocol for in vitro regeneration was developed. Maximum embryogenic callus induction response was observed at MS(Murashige and Skooj) enriched with 4.0 μM naphthalene acetic acid and 6.0 μM benzyl adenine, whereas maximum shoot formation response (100%) was observed at 8 μM benzyl adenine. Elongated shoots demonstrated good in vitro rooting (80%) with 2.5 μM indole butyric acid. To produce transgenic plants, pre-culturing of explants was done for 3 days. The GV3101 strain of A. tumefaciens, containing pCV002-A vector harboring the rolA gene of Agrobacterium rhizogenes, was used for infection for 48 hours. Three independent transgenic lines were generated that showed stable integration of the transgene confirmed by PCR analysis. These transgenic lines were selected and screened for eight biologically active withanolides using ultra-highpressure liquid chromatography (UHPLC) with mass spectrometry (MS). Results revealed that transformed lines possessed higher withanolide content as compared to untransformed ones. Extracts of these transgenic lines, which possessed higher levels of total withanolide content, were tested for bioactivity against different prostate cancer cell lines. The results showed that transformed plants extracts had higher anticancer activity in terms of decreased cell viability of prostate cancer cell lines. The data presented could be useful for the selection of potent and withanolide-rich germplasm from natural populations or transformed cell culture.