<i>WsCAS</i> transcript expression analysis and withanolide accumulation in Withania silencing and overexpressing lines.

<p>(A) RealTime PCR analysis of <i>WsCAS</i> silenced lines. (B) Withanolide content in silenced lines. (C) Real-Time PCR analysis of <i>WsCAS</i> overexpressing lines. (D) Withanolide content in <i>WsCAS</i> overexpressing lines.</p

Withanolide biosynthetic pathway.

<p>IPP, isopentenyl pyrophosphate; MVA, mevalonate; MEP, 2-C-methyl-D-erythritol 4-phosphate GPPS, geranyl pyrophosphate synthase; FPPS, farnasyl pyrophosphate synthase; SS, squalene synthase; SE squalene epoxidase; CAS, cycloartenol synthase; LAS, lanosterol synthase; LS, lupeol synthase; BAS, beta amyrin synthase; AAS, alpha amyrin synthase.</p

Full length <i>WsCAS</i> gene with three selected silencing sites for silencing constructs preparation in <i>Withania somnifera</i>.

<p>19 bp silencing sites were enclosed in red colored boxes. Arrows indicate the region of start of amplification of particular fragment incorporating silencing sites.</p

Profiling of <i>WsCAS</i> expression and withanolide in Withania seedlings under different elicitor conditions.

<p>(A) Seedling before and after treatment of cold, wounding, SA 2mM, SA 5mM, MeJ 200μM, MeJ 500μM and heating (a-i) respectively. (B) Real time-PCR analysis of <i>WsCAS</i>. (C) Quantitative withanolide profile.</p

Molecular characterization of Withania silenced lines and overexpressing lines of <i>WsCAS gene</i>.

<p>(A) PCR amplification with <i>bar</i> primers showing amplicon of ≈400 bp. Lane M 100 bp ladder; lane–C untransformed shoot; lane +C plasmid DNA; lane 1–9, <i>in vitro</i> transformed shoots; (B) Southern blot analysis of transgenic silenced lines of <i>Withania</i> with <i>bar</i> PCR probe. (C) PCR amplification of pIGCAS transformants with <i>npt</i>II primers showing amplicon of 500bp. Lane- 1 to 6 transformed lines. (D) Southern blot analysis of overexpresing lines.</p