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Trasncscriptome sequencing of leaves (WcL) and roots (WcR) of micropropagated W. coagulans plantlets was done to identify the putative gens involved in the withanolide biosynthesis under in vitro growth conditions, which produced 8.08 and 6.35 GB of raw reads, assembled into 292,074 and 16,474 high quality (HQ) reads, out of which 267,119 and 15,758 unigenes were identified in WcL and WcR, respectively. Further, 40.6% WcL and 55.05% WcR unigenes were annotated using more than one database. Metabolic process and cellular components were identified as dominant categories in gene ontology. 20,927 WcL and 2,474 WcR unigenes were mapped to different biological pathways. KEGG classification aided in identification of genes involved in biosynthesis of withanolide precursor, 24-methylenecholesterol. All the genes related to withanolide precursor biosynthesis were present only in WcL, indicating de novo biosynthesis of withanolides, while absence of some rate limiting enzymes in WcR suggests biosynthesis of withanolides through salvage pathways. GTs, MTs and CYP450s were identified as putative genes involved in conversion of 24-methylenecholesterol to different withanolides. Differential expression of these genes further revealed details of enzymes involved in biosynthesis of tissue-specific withanolides. Further, withanolide profiling through HPLC analysis ascertained the differential biosynthesis and accumulation of withanolides in both the tissues and confirmed salvage biosynthesis of withanolides due to their lesser/negligible quantities in roots. Therefore, the present study can be fruitful for future research and …