Genetic engineering of secondary metabolic pathways is an emerging area of research for production and improvement of natural products in plant biotechnology. Here, we describe a systematic approach to manipulate a key regulatory step of isoprenoid biosynthetic pathway in Withania somnifera to study its effect on withanolide production. We generated T 0 W. somnifera plants overexpressing squalene synthase (WsSQS) by Agrobacterium tumefaciens mediated transformation, which were analyzed by Gus biochemical assay and PCR of hygromycin phosphotransferase (hptII) and WsSQS. qRT-PCR analyses of various transformed tissues indicated 2-5 fold increase in WsSQS transcripts in both T 0 and T 1 generations. The tissue specific protein expression studies revealed 2-3 fold increase in WsSQS, which was further confirmed by enzyme activity. These observations were corroborated with the 1.5-2 fold increase in total withanolide content of the transformed tissues. However, in leaf tissue, the levels of Withaferin A and Withanolide A increased significantly up to 4-4.5 fold. These findings demonstrate genetic engineering of isoprenoid pathway in W. somnifera resulting in enhanced production of withanolides, and also provide insights into such metabolic pathways for their manipulation to improve the pharmacological content of different medicinally important plants.

Complete plants were grown from zygotic embryos cultured on Y3 basal liquid medium supplemented with coconut milk, BA and NAA. Explants from stem, leaf and rachilla of mature coconut trees turned green and swelled on Y3 semi-solid basal media supplemented with 2,4-D, K, NAA, BA and activated charcoal. Callus was initiated in explants from the subapical regions of the stem on Y3 basal medium supplemented with 2,4-D (4.52×10(2)μM). Globular embryo-like structures were obtained when this callus was subcultured to auxinless medium. Root formation was obtained from leaf explants on Y3 basal medium containing citric acid, ascorbic acid and 2,4-D (4.52×10(2) μM). Globular embryo-like structures were also obtained directly from leaf explants on a Y3 basal medium supplemented with 2,4-D (2.26×10(2) μM). Callus isolated from rachilla explants on Y3 basal medium containing 2,4-D(4.52×10(2) μM), formed nodular structures when transferred to medium with 2,4-D (2.3×10(1) μM). These nodules developed roots from the base of the nodular growth whereas from the upper portion shoots were observed on Y3 basal liquid medium.

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