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The present study investigated the biosynthesis of major and minor withanolides of Withania somnifera in cell suspension culture using shake-flask culture and bioreactor by exploiting elicitation and precursor feeding strategies. Elicitors like cadmium chloride, aluminium chloride and chitosan, precursors such as cholesterol, mevalonic acid and squalene were examined. Maximum total withanolides detected [withanolide A (7606.75 mg), withanolide B (4826.05 mg), withaferin A (3732.81 mg), withanone (6538.65 mg), 12 deoxy withanstramonolide (3176.63 mg), withanoside IV (2623.21 mg) and withanoside V (2861.18 mg)] were achieved in the combined treatment of chitosan (100 mg/l) and squalene (6 mM) along with 1 mg/l picloram, 0.5 mg/l KN, 200 mg/l L-glutamine and 5% sucrose in culture at 4 h and 48 h exposure times respectively on 28th day of culture in bioreactor. We obtained higher concentrations of total withanolides in shake-flask culture (2.13-fold) as well as bioreactor (1.66-fold) when compared to control treatments. This optimized protocol can be utilized for commercial level production of withanolides from suspension culture using industrial bioreactors in a short culture period. [ABSTRACT FROM AUTHOR]

Withanolide is one of the most extensively exploited steroidal lactones, which are biosynthesized in Withania somnifera. Its production from cell suspension culture was analyzed to defeat limitations coupled with its regular supply from the plant organs. In order to optimize the different factors for sustainable production of withanolides and biomass accumulations, different concentrations of auxins or cytokinins and their combinations, carbon sources, agitation speed, organic additives and seaweed extracts was studied in cell suspension culture. Maximum biomass accumulation (16.72 g fresh weight [FW] and 4.18 g dry weight [DW]) and withanolides production (withanolide A 7.21 mg/g DW, withanolide B 4.23 mg/g DW, withaferin A 3.88 mg/g DW and withanone 6.72 mg/g DW) were achieved in the treatment of Gracilaria edulis extract at 40 % level. Organic additive l-glutamine at 200 mg/l in combination with picloram (1 mg/l) and KN (0.5 mg/l) promoted growth characteristics (11.87 g FW and 2.96 g DW) and withanolides synthesis (withanolide A 5.04 mg/g DW, withanolide B 2.59 mg/g DW, withaferin A 2.36 mg/g DW and withanone 4.32 mg/g DW). Sucrose at 5 % level revolved out to be a superior carbon source yielded highest withanolides production (withanolide A 2.88 mg/g DW, withanolide B 1.48 mg/g DW, withaferin A 1.35 mg/g DW and withanone 2.47 mg/g DW), whereas biomass (7.28 g FW and 1.82 g DW) was gratefully increased at 2 % level of sucrose in cell suspension culture. This optimized protocol can be utilized for large scale cultivation of W. somnifera cells in industrial bioreactors for mass synthesis of major withanolides. [ABSTRACT FROM AUTHOR]

Chitosan is a polycationic polysaccharide derived from chitin, and β-cyclodextrin is a type of macrocyclic oligosaccharide linked by α-1,4 glycosidic bonds. These compounds are recognized as effective elicitors in the biosynthesis of secondary metabolites in plants. These elicitors were studied to assess the growth of shoots and the synthesis of glucosinolates (GSLs) from elicited shoots in Chinese cabbage under controlled in vitro conditions for the first time. Chitosan at 150 mg L−1 supplemented in the optimized shoot induction recovered maximum quantities of total GSLs (7.344 μmol g−1 DW) at the end of 5th week of culture duration, followed by β-cyclodextrin (15 mg L−1) with the synthesis of GSLs (6.379 μmol g−1 DW) at the end of 4th week of culture. The application of chitosan completely deteriorated the growth of shoots, whereas β-cyclodextrin did not affect and even increased the growth of shoots (4.66 g DW). Upon elicitation, the individual got GSLs contents exhibited various fold changes (1.78-to-23.5-fold). Real-time PCR analysis of essential GSLs biosynthesis genes like MAM1, ST5b, AOP2, FMOGS-OX1, CYP83B1, CYP81F2, ST5a , and CYP81F4 revealed substantial higher expression upon elicitation. This present study would provide a steady production of GSLs in Chinese cabbage shoots with the influence of carbohydrate-based elicitors for pharmaceutical or food companies in the future. • An effective elicitor for enhancing glucosinolates content was optimized under in vitro condition. • Chitosan treatment increased glucosinolates in Chinese cabbage shoot culture. • Chitosan treatment triggered expression of glucosinolates biosynthesis genes. • Concentration and incubation time with shoot culture played an important role. [ABSTRACT FROM AUTHOR]

The investigation of seaweeds, Gracilaria edulis and Sargassum wightii extracts was carried out for the estimation of growth characteristics and major withanolides production in hairy root culture of Withania somnifera . The extract of G. edulis (50%) in MS liquid basal medium enabled maximum production of dry biomass (5.46 g DW) and withanolides contents (withanolide A 5.23 mg/g DW; withaferin A 2.24 mg/g DW and withanone 4.83 mg/g DW) in hairy roots after 40 days of culture with 48 h contact time. The obtained withanolides contents were significantly higher (2.32-fold–2.66-fold) in hairy root culture when compared to the control. RT PCR analysis of important pathway genes such as SE, SS, HMGR and FPPS exhibited substantial higher expression upon the seaweed extracts treatment in hairy root culture. This experiment would paw a platform for withanolides production in hairy root culture with the influence of sea weed extracts for pharmaceutical companies in the future. [ABSTRACT FROM AUTHOR]