Journal of Biotechnology 143 (2009) 157–168 Contents lists available at ScienceDirect Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec Elicitation studies in cell suspension cultures of Cannabis sativa L. Isvett Josefina Flores-Sanchez a , Jaroslav Peˇ c b , Junni Fei a , Young Hae Choi a , Jaroslav Duˇ sek b , Robert Verpoorte a,∗ a Pharmacognosy Department/Metabolomics, Institute of Biology, Gorlaeus Laboratories, P.O. Box 9502, Leiden University, 2300 RA Leiden, The Netherlands b Pharmacognosy Department, Faculty of Pharmacy, Heyrovského 1203, Charles University in Prague, CZ-500 05 Hradec Králové, Czech Republic article info Article history: Received 30 July 2008 Received in revised form 8 May 2009 Accepted 12 May 2009 Keywords: Cannabis sativa Elicitation NMR spectroscopy THCA synthase gene Metabolomics Glutamyl-tyramine abstract Cannabis sativa L. plants produce a diverse array of secondary metabolites. Cannabis cell cultures were treated with biotic and abiotic elicitors to evaluate their effect on secondary metabolism. Metabolic profiles analysed by 1 H NMR spectroscopy and principal component analysis (PCA) showed variations in some of the metabolite pools. However, no cannabinoids were found in either control or elicited cannabis cell cultures. Tetrahydrocannabinolic acid (THCA) synthase gene expression was monitored dur- ing a time course. Results suggest that other components in the signaling pathway can be controlling the cannabinoid pathway. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Cannabis sativa L. is an annual dioecious plant from Central Asia. Two hundred and forty-seven secondary metabolites have been identified in this plant. Cannabinoids are a well-known group of natural products and more than 70 different cannabinoids have been found so far (ElSohly and Slade, 2005; Radwan et al., 2008). Several therapeutic effects of cannabinoids have been described (Williamson and Evans, 2000) and the discovery of an endo- cannabinoid system in mammals marks a renewed interest in these compounds (Di Marzo et al., 2007; Mackie, 2008; Moreira and Lutz, 2008). C. sativa cell cultures have been used for breeding (Jekkel et al., 1989; Mandolino and Ranalli, 1999), for studying sec- ondary metabolite biosynthesis (Itokawa et al., 1977; Loh et al., 1983; Hartsel et al., 1983) and for secondary metabolite produc- tion (Veliky and Genest, 1972; Heitrich and Binder, 1982). However, cannabinoids have not been detected in cell suspension or callus cultures so far. Some of the strategies used to stimulate cannabi- noid production from cell cultures involved media modifications and a variety of explants. Although, elicitation has been employed for inducing and/or improving secondary metabolite production in the cell cultures (Bourgaud et al., 2001) it would be interesting to observe elicitation effect on secondary metabolite production in C. sativa cell cultures. ∗ Corresponding author. Tel.: +31 71 52 74 528; fax: +31 71 52 74 511. E-mail address: verpoort@chem.leidenuniv.nl (R. Verpoorte). Metabolomics has facilitated an improved understanding of cellular responses to environmental changes and analytical plat- forms have been proposed and applied (Sanchez-Sampedro et al., 2007; Hagel and Facchini, 2008; Zulak et al., 2008). 1 H NMR spec- troscopy is one of these platforms which is currently being explored together with principal component analysis (PCA), the most com- mon method to analyse the variability in a group of samples. In this study biotic and abiotic elicitors were employed to eval- uate their effect on secondary metabolism in C. sativa cell cultures. Metabolic profiles were analysed by 1 H NMR spectroscopy. Expres- sion of the THCA synthase gene was also monitored by reverse transcription-polymerase chain reaction (RT-PCR). 2. Materials and methods 2.1. Chemicals CDCl 3 (99.80%) and CD 3 OD (99.80%) were obtained from Euriso- top (Paris, France). D 2 O (99%) was acquired from Spectra Stable Isotopes (Columbia, MD, USA). NaOD was purchased from Cortec (Paris, France). The cannabinoids  9 -THCA, CBGA,  9 -THC, CBG and CBN were isolated from plant material previously in our laboratory (Hazekamp et al., 2004). All chemical products and mineral salts were of analytical grade. 2.2. Plant material and cell culture methods Seeds of C. sativa, drug type variety Skunk (The Sensi Seed Bank, Amsterdam, The Netherlands) were germinated and maintained 0168-1656/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2009.05.006