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The first transcriptomes, expressed sequence tags (ESTs) in a leaf and root from Withania somnifera plant referenced in this report are the first of its kind. A cDNA library was constructed from samples of the 2-months-old, in vitro cultured leaves and roots, which generated 1,047 leaf cDNA and 1,034 root cDNA clones representing 48.5% and 61.5% unique sequences. The ESTs from leaf and root grouped into 239 and 230 clusters representing 22.8% and 22.2% of total sequences. Of these, about 70% encoded proteins found similar (E-value > or =10(-14)) to characterized or annotated proteins from the NCBI non-redundant database and diverse molecular functions and biological processes based on gene ontology (GO) classification. We identified genes with potential role in photosynthesis (cytochrome p-450), pathogenesis (arginine decarboxylase, chitinase) and withanolide biosynthesis (squalene epoxidase, CDP-ME kinase). Highly expressed transcripts, with a particularly high abundance of cytochrome p-450 (0.85% in leaf) were noticed. Pfam analysis revealed the presence of functional domains in selected sequences. W. somnifera is a source of multifarious and beneficial alkaloids referred as withanolides. High levels of withanolides accumulate in mature leaves and roots. Since, the knowledge for synthesis and presence of some of these important biochemical constituent is limited, identification of the genes involved in two different pathways of secondary metabolite synthesis (MVA and MEP), in different tissue will be requisite for articulation of withanolide biosynthesis. This investigation aimed at elucidating the differential gene expression in two vital sites where withanolides essentially found and leaf and root transcriptomes were comparatively analyzed. The comparative analysis of the sequences provides a framework for future research in proteomics and evolutionary genomics in the withanolide biosynthesis.

Withania somnifera is one of the most important medicinal plants of Ayurveda and finds extensive uses in Indian traditional herbal preparations. In this investigation, selected accessions of the plant were examined for diversity through RAPDs, isoenzymes, polypeptide polymorphism and withanolide profiles. The accessions clustered together with respect to their characteristic profile of major withanolides and represented withaferin A, withanone, withanolide D or withanolide A rich groups. This level of phytochemical diversity as discrete chemotypes is widest and is being first ever documented to occur in Indian population of the plant.

Phytochemical investigations of multiple shoot cultures of selected accessions AGB002 and AGB025 of Withania somnifera established in vitro utilizing shoot tip apices cultured on Murashige and Skoog's medium supplemented with BAP (1 mg/L) have been carried out. This has lead to isolation of four glycowithanolides viz. Withanoside IV (WSG-3), Withanoside VI (WSG-3A), Physagulin D (WSG-P) and Withastraronolide (WSC-O). The structures of these have been confirmed on the basis of spectroscopic data. Multiple shoot cultures could be an alternative renewable resource for production of these biologically active molecules.