Research article Calcium and magnesium elimination enhances accumulation of cardenolides in callus cultures of endemic Digitalis species of Turkey G. Sahin * , S.K. Verma, E. Gurel Abant Izzet Baysal University, Department of Biology, 14280 Bolu, Turkey article info Article history: Received 2 July 2013 Accepted 10 September 2013 Available online 24 September 2013 Keywords: Calcium Magnesium Cardenolides accumulation Digitalis cariensis Boiss. ex Jaub. et Spach Digitalis davisiana Heywood Digitalis lamarckii Ivanina Digitalis trojana Ivanina abstract Elimination of calcium (Ca), magnesium (Mg) or both from the medium of callus cultures of Digitalis davisiana Heywood, Digitalis lamarckii Ivanina, Digitalis trojana Ivanina and Digitalis cariensis Boiss. ex Jaub. et Spach increased cardenolides production. Callus was induced from hypocotyl segments from one-month old seedlings were cultured on MS medium containing 0.5 mg ml 1 thidiazuron (TDZ) and 0.25 mg ml 1 indole acetic acid (IAA). After 30 days of culture, callus was transferred in hormone-free MS medium (MSO) as well as Ca or Mg or both were completely eliminated from same medium. The amount of five cardenolides from D. davisiana Heywood, D. lamarckii Ivanina, D. trojana Ivanina and D. cariensis Boiss. ex Jaub. et Spach were compared. Higher amounts of five cardenolides and total cardenolides were obtained when callus of four Digitalis species were incubated on MS medium lacking both Ca and Mg. The mean contents of total cardenolides obtained were in the order of D. lamarckii (2017.97 mgg 1 ) > D. trojana (1385.75 mgg 1 ) > D. cariensis (1038.65 mgg 1 ) > D. davisiana (899.86 mgg 1 ) when both Ca and Mg were eliminated from the medium, respectively. This protocol is useful for development of new strategies for the large-scale production of cardenolides. Ó 2013 Elsevier Masson SAS. All rights reserved. 1. Introduction Digitalis, commonly known as foxgloves, belongs to the family Plantaginaceae. The genus consists of about 20 species of herba- ceous perennials, shrubs and biennials. Members of the genus Digitalis L. are medicinally and economically important plants as they contain cardiac glycosides that are used as heart medicines [1] and effective agents in cancer chemotherapy [2e4]. Because of their importance, studies have focused on in vitro culture of several Digitalis species including Digitalis davisiana [5], Digitalis lanata [6], Digitalis obscura [7], Digitalis purpurea [8,9] and Digitalis thapsi [10e13]. Digitalis species is distributed in Europe, Western Asia and the Mediterranean region. D. davisiana Heywood, Digitalis lamarckii Ivanina, Digitalis trojana Ivanina and Digitalis cariensis Boiss. ex Jaub. et Spach, which are the most widespread members of the nine Digitalis species growing in Turkey and are endemic to Turkey [14,15]. D. trojana contains the highest amount of cardiac glycoside in leaves among the endemic Digitalis species in Turkey [16]. Large- scale plant cell and tissue culture has been considered as an alternative technique for the production of bioactive secondary metabolites since their production by traditional agriculture is inefficient [17]. The production of cardenolides using tissue culture techniques for Digitalis species has been studied for several years, however, the productivity has not yet reached economically desired levels. Most scientists have reported that tissue cultures estab- lished from Digitalis or other cardenolide containing plants either did not produce cardenolides or contained only at trace amounts. On the other hand, to improve the production of such secondary metabolites, some strategies have been developed [18]. One of them is the employment of various kinds of abiotic stress factors such as changing nutrient conditions, temperature range, UV and chemical application [19]. The aim of this work was to achieve the enhancement of car- denolide accumulation in D. davisiana Heywood, D. lamarckii Iva- nina, D. trojana Ivanina and D. cariensis Boiss. ex Jaub. et Spach, using calcium and magnesium deprived cultures and to determine the content of digitoxin, digoxin, lanatoside C, gitoxigenin and digoxigenin as secondary metabolites of commercial interest for the pharmaceutical industry. 2. Results Callus was readily initiated within one week when hypocotyl segments excised from one-month old seedlings were cultured on Abbreviations: IAA, indole-3-acetic acid; TDZ, thidiazuron; MS, Murashige and Skoog; Lan C, lanatoside C. * Corresponding author. Tel.: þ90 5544509688; fax: þ90 3742534642. E-mail address: guncesahin@gmail.com (G. Sahin). Contents lists available at ScienceDirect Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy 0981-9428/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.plaphy.2013.09.007 Plant Physiology and Biochemistry 73 (2013) 139e143