Research Article Received: 10 January 2012 Revised: 12 June 2012 Accepted: 26 June 2012 Published online in Wiley Online Library: (wileyonlinelibrary.com) DOI 10.1002/jsfa.5825 Sulfate determines the glucosinolate concentration of horseradish in vitro plants (Armoracia rusticana Gaertn., Mey. & Scherb.) Mohammad Alnsour, Maik Kleinw ¨ achter, Julia B ¨ ohme and Dirk Selmar * Abstract BACKGROUND: Horseradish plants (Armoracia rusticana) contain high concentrations of glucosinolates. Former studies have revealed that Armoracia plants cultivated in vitro have markedly lower glucosinolate concentrations than those grown in soils. Yet, these studies neglected that the sulfate concentration in the growth medium may have had a strong impact on glucosinolate metabolism. Accordingly, in this study horseradish in vitro plants were cultivated with differing sulfate concentrations and the glucosinolate concentrations were quantified by ion pair HPLC. RESULTS: Cultivation in 1.7 mmol L -1 sulfate (as used in the prior studies) resulted in the accumulation of 16.2 μmol g -1 DW glucosinolates, while the glucosinolate concentration increased to more than 23 μmol g -1 DW when 23.5 mmol L -1 sulfate was used in the medium. Correspondingly, the glucosinolate concentration decreased to 1.6 μmol g -1 DW when sulfate concentration was lowered to 0.2 mmol L -1 . CONCLUSION: Since the glucosinolate accumulation in relation to the sulfate concentration follows a typical saturation curve, we deduce that the availability of sulfate determines the glucosinolate concentration in horseradish in vitro plants. c  2012 Society of Chemical Industry Keywords: Armoracia rusticana; Brassicaceae; glucosinolates; sinigrin; gluconasturtiin; sulfate; in vitro plants INTRODUCTION Horseradish plants (Armoracia rusticana Gaertn., Mey. & Scherb.) contain high concentrations of glucosinolates. These sulfur- containing secondary metabolites are derived from amino acids and consist of a sulfonated oxime moiety, a β -thioglucose moi- ety and a variable side chain. Usually, horseradish contains eight different glucosinolates and when considering the entire plant (Fig. 1), sinigrin (2-propenyl glucosinolate), gluconasturtiin (2-phenylethyl glucosinolate), glucobrassicin (3-indolylmethyl glu- cosinolate) and neoglucobrassicin (N-methoxy-3-indolylmethyl glucosinolate) represent the major glucosinolate components. 1 In fully developed horseradish roots, sinigrin (∼83%) and gluconas- turtiin (∼11%) account for more than 90% of the total concen- tration of glucosinolates. 1 Glucosinolates are part of a preformed defence system against herbivores and pathogens. 2,3 Upon tis- sue disruption, glucosinolates are hydrolysed by myrosinases (EC 3.2.1.147) to yield glucose and unstable thiohydroximate- O-sulfonates. The latter compounds react to a wide array of further products (e.g. isothiocyanates, nitriles or thiocyanates), the so- called mustard oils, which have strong biological activity. 4 More than 20 different mustard oil components have been detected in grated horseradish root tissue. 5 Apart from their pungent flavour, the mustard oils of horseradish also show strong antibiotic activity and preparations of horseradish roots in combination with nas- turtium leaves (Tropaeolum majus L.) are successfully used for the treatment of urinary tract infections. 6 In the last three decades, a number of papers have been published dealing with the in vitro cultivation of horseradish. 7–9 Most of these studies have focused on the bulk propagation and on the generation of virus-free plant materials. Only few further studies have dealt with the glucosinolate content and composition of horseradish in vitro cultures. These works primarily addressed the impact of the cellular differentiation and were aimed to apply in vitro systems for the biotechnological production of these pharmaceutical interesting compounds. Radojˇ ci´ c Redovnikovi´ c et al. reported that fully differentiated in vitro plants contain nearly the same mix of glucosinolate compounds as soil grown plants, i.e. sinigrin represents the principal component and only minor amounts of gluconasturtiin and three indole glucosinolates (glucobrassicin, neoglucobrassicin and 4-OH-glucobrassicin) are present. 10 In contrast, in less differentiated tissues (e.g. callus, cell suspension and tumour and teratoma tissue), only indole glucosinolates are accumulated considerably, whereas sinigrin and gluconasturtiin were completely lacking. 10,11 Mevy et al. explained the unusual occurrence of indole glucosinolates in cell suspension and callus cultures on the basis of the biochemical differentiations related to their developmental status. 11 Radojˇ ci´ c Redovnikovi´ c et al. assumed that the interaction between the biosynthesis of ∗ Correspondence to: Professor Dr Dirk Selmar, Institute for Plant Biology, Department of Applied Plant Biology, Technische Universit¨ at Braunschweig, Mendelssohnstraße 4, 38106 Braunschweig, Germany. E-mail: d.selmar@tu-bs.de Institute for Plant Biology, Technische Universit¨ at Braunschweig, 38106 Braunschweig, Germany J Sci Food Agric (2012) www.soci.org c  2012 Society of Chemical Industry