Research article Comparative study of withanolide production and the related transcriptional responses of biosynthetic genes in fungi elicited cell suspension culture of Withania somnifera in shake flask and bioreactor Seema Ahlawat, Parul Saxena, Athar Ali, Shazia Khan, Malik Z. Abdin * Department of Biotechnology, Faculty of Science, Centre for Transgenic Plant Development, Jamia Hamdard, New Delhi 110062, India article info Article history: Received 3 September 2016 Received in revised form 16 December 2016 Accepted 13 February 2017 Available online 17 February 2017 Keywords: W. somnifera Withanolides Bioreactor P. indica Cell suspension cultures abstract Ashwagandha (Withania somnifera) is one of the most reputed medicinal plants in the traditional me- dicinal system. In this study, cell suspension culture of W. somnifera was elicited with cell homogenates of fungi (A. alternata, F. solani, V. dahliae and P. indica) in shake flask and the major withanolides like withanolide A, withaferin A and withanone were analysed. Simultaneously expression levels of key pathway genes from withanolides biosynthetic pathways were also checked via quantitative PCR in shake flask as well as in bioreactor. The results show that highest gene expression of 10.8, 5.8, 4.9, and 3.3 folds were observed with HMGR among all the expressed genes in cell suspension cultures with cell homogenates of 3% P. indica, 5% V. dahliae, 3% A. alternata and 3% F. solani, respectively, in comparison to the control in shake flask. Optimized concentration of cell homogenate of P. indica (3% v/v) was added to the growing culture in 5.0-l bioreactor under optimized up-scaling conditions and harvested after 22 days. The genes of MVA, MEP and withanolides biosynthetic pathways like HMGR, SS, SE, CAS, FPPS, DXR and DXS were up-regulated by 12.5, 4.9, 2.18, 4.65, 2.34,1.89 and 1.4 folds, respectively in bioreactor. The enhancement of biomass (1.13 fold) and withanolides [withanolide A (1.7), withaferin A (1.5), and withanone (1.5) folds] in bioreactor in comparison to shake flask was also found to be in line with the up- regulation of genes of withanolide biosynthetic pathways. © 2017 Elsevier Masson SAS. All rights reserved. 1. Introduction Withania somnifera (Ashwagandha), also recognised as winter cherry, Indian ginseng or rasayana, is a member of “Generally Regarded As Safe” plants and a popular home medication in the Indian pharmacopoeia (Mishra et al., 2000; Padmavathi et al., 2005). The herb is termed a rasayana in Ayurvedic practice, which means it acts as a tonic for vitality and longevity. Withania is used as herbal medicine in various forms (infusions, ointments, powder and syrup) in different parts of the world (Davis and Kuttan, 2001; Kumar et al., 2007), for all age groups of patients without any side effects even for pregnant women (Gupta and Rana, 2007). The extracts as well as different isolated bioactive constituents of W. somnifera have been shown to possess adapto- genic, anticancer, anti-convulsant, antioxidative, immunomodula- tory and neurological effects. The plant is also considered efficacious in the treatment of arthritis, geriatric, behavioural and stress related problems (Dhuley, 2001; Gupta and Rana, 2007; Kaur et al., 2001; Mishra et al., 2000; Newman et al., 2003). Several bioactive alkaloids and sterollactone based phytochemicals, e.g. ashwagandhine, cuscohygrine, anaferine, anhygrine, iso- pelletierine, tropine, sitoindosides (saponins), the diversely func- tionalized withanolides, withanamides, and glycowithanolides have been extracted from different parts of this plant (Matsuda et al., 2001; Mishra et al., 2005, 2008; Rahman et al., 2003). Its increasing therapeutic benefits continuously attract the attention of pharmacologists for biomedical analysis on plant extracts and isolated phytochemicals (Kaieh et al., 2007; Mulabagal et al., 2009; Nair and Jayaprakasam, 2007a,b; Pan et al., 2009). The demand of Withania drugs in India has been estimated about 12,120 tonnes per Abbreviations: CAS, Cycloaretenol synthase; DW, Dry weight; DXS, 1-deoxy-d- xylulose 5-phosphate synthase; DXR, 1-deoxy-d-xylulose 5-phosphate reduc- toisomerase; FPPS, Farnesyl pyrophosphate synthase; HMGR, 3-Hydroxy-3- methylglutaryl coenzyme A reductase; HPLC, High performance liquid chroma- tography; MEP, Methyl erythritol pathway; MS, Murashige and Skoog; MVA, Mevalonate pathway; qPCR, quantitative polymerase chain reaction; SE, Squalene epoxidase/cytochrome P450 reductase; SS/SQ, Squalene synthase. * Corresponding author. E-mail address: mzabdin@rediffmail.com (M.Z. Abdin). Contents lists available at ScienceDirect Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy http://dx.doi.org/10.1016/j.plaphy.2017.02.013 0981-9428/© 2017 Elsevier Masson SAS. All rights reserved. Plant Physiology and Biochemistry 114 (2017) 19e28