Appl Microbiol Biotechnol DOI 10.1007/s00253-012-4489-y BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING In vitro propagation and production of cardiotonic glycosides in shoot cultures of Digitalis purpurea L. by elicitation and precursor feeding Jitendra Gopichand Patil & Mahendra Laxman Ahire & Kirti Manik Nitnaware & Sayantan Panda & Vijay P. Bhatt & Polavarapu B. Kavi Kishor & Tukaram Dayaram Nikam Received: 18 July 2012 /Revised: 16 September 2012 /Accepted: 1 October 2012 # Springer-Verlag Berlin Heidelberg 2012 Abstract Digitalis purpurea L. (Scrophulariaceae; Fox To further maintain the multiple shoot induction, mother glove) is a source of cardiotonic glycosides such as digitox- tissue was cut into four equal parts and repeatedly sub- in and digoxin which are commercially applied in the treat- cultured on fresh shoot induction liquid medium after each ment to strengthen cardiac diffusion and to regulate heart harvest. On adaptation of this strategy, an average of 18 shoots rhythm. This investigation deals with in vitro propagation per explant could be produced. This strategy was applied for and elicited production of cardiotonic glycosides digitoxin the production of biomass and glycosides digitoxin and di- and digoxin in shoot cultures of D. purpurea L. In vitro goxin in shoot cultures on MS medium supplemented with germinated seedlings were used as a primary source of 7.5 μM BA and several treatments with plant growth regu- explants. Multiple shoot formation was achieved for three lators, incubation period, abiotic (salicylic acid, mannitol, explant types (nodal, internodal, and leaf) cultured on sorbitol, PEG-6000, NaCl, and KCl), biotic (Aspergillus Murashige and Skoog (MS) medium with several treatments niger, Helminthosporium sp., Alternaria sp., chitin, and of cytokinins (6-benzyladenine—BA; kinetin—Kin; and yeast extract) elicitors, and precursors (progesterone, cho- thidiazuron—TDZ) and auxins (indole-3-acetic acid— lesterol, and squalene). The treatment of KCl, mycelial IAA; α-naphthaleneacetic acid—NAA; and 2,4-dichloro- mass of Helminthosporium sp., and progesterone were phenoxy acetic acid—2,4-D). Maximum multiple shoots highly effective for the production of cardenolides. In the (12.7±0.6) were produced from nodal explants on MS+ presence of progesterone (200 to 300 mg/l), digitoxin and 7.5 μM BA. Shoots were rooted in vitro on MS containing digoxin accumulation was enhanced by 9.1- and 11.9-folds 15 μM IAA. Rooted plantlets were successfully acclimatized. respectively. : : : : Keyword Cardiotonic glycosides . Digitalis purpurea . J. G. Patil M. L. Ahire K. M. Nitnaware S. Panda Digitoxin . Digoxin . Shoot cultures T. D. Nikam (*) Department of Botany, University of Pune, Pune 411 007, Maharashtra, India e-mail: [email protected] Introduction V. P. Bhatt Herbal Research and Development Institute, Digitalis purpurea L. belongs to the family Scrophula Mandal, Gopeshwar, riaceae and is a biennial or perennial herb widely distributed 246 401, Chamoli, Uttarakhand, India in the Western Europe, Asia, Northwest Africa, South America, New Zealand, and Canada (Hultén 1968). The P. B. K. Kishor Department of Genetics, Osmania University, entire plant including the roots and seeds is poisonous. It Hyderabad 500 007, India contains several deadly physiological and chemically related Appl Microbiol Biotechnol cardiac and steroidal glycosides (Budavari et al. 1989). 1992), and shoot cultures (Hagimori et al. 1983; Seitz and However, it is commercially cultivated to obtain cardiotonic Gärtner 1994; Gurel et al. 2011). Further, it is shown that the glycosides namely digitoxin and digoxin (Mastenbroek cardenolide biosynthesis is basically dependent on morpho- 1985). Both glycosides are the best known products to logical differentiation (Eisenbeiβ et al. 1999; Pérez-Alonso strengthen cardiac diffusion and to regulate heart rhythm et al. 2009). To date, to the best of our knowledge, no (Navarro et al. 2000; Pérez-Bermúdez et al. 2010; Sharma information is available on in vitro propagation protocol and Purkait 2012). Actually, they function by inhibiting the and on the influence of elicitors and precursors on the sodium–potassium ATPase activity which results in in- production of Digitalis cardenolides on the in vitro grown creased intracellular concentration of sodium. This helps in shoot cultures. the elevation of intracellular calcium and creation of posi- The aim of the present investigation was to develop in tive inotropic effect (Rahimtoola and Tak 1996; Xie and vitro propagation protocol using different explants of in Askari 2002; Mohammadi et al. 2003; López-Lázaro 2007; vitro germinated seedlings. Attempts were made to test the Kuate et al. 2008; Wu et al. 2012). The Digitalis glycoside is influence of plant growth regulators, different biotic and still the only safe inotropic drug treatment for oral use, abiotic elicitors, and precursors on the accumulation of which improves hemodynamics in patients with a compro- medicinally important cardiotonic glycosides, digitoxin mised cardiac function (Schwinger et al. 2003; Pérez- and digoxin in shoot cultures of D. purpurea L. Alonso et al. 2009). Recently, it is also noted that both the glycosides were effective against several types of cancer (Haux 1999; Stenkvist 2001;Hauxetal.2001; López- Materials and methods Lázaro et al. 2003; López-Lázaro 2007; Sharma and Purkait 2012; Wu et al. 2012). The chemical synthesis of Plant material and culture conditions these glycosides is so far not cost effective due to complex chemical nature (Pérez-Bermúdez et al. 2010). The pharma- Seeds of D. purpurea were obtained from Herbal Research ceutical industries are still relying on natural sources of the and Development Institute, Mandal-Gopeswar, Chamoli, plant material which contains very low amounts of these Uttarakhand. Seeds were washed in running tap water for glycosides. In such a situation, it necessitates the application 5 min followed by five rinses in sterilized distilled water. of biotechnological approaches to enhance the yield of Then, seeds were surface-sterilized with 0.1 % (w/v)mercuric digitoxin and digoxin. chloride (HgCl2) solution for 5 min and washed five to six Plant cell and tissue culture can be used as an alternative times with sterilized distilled water to remove the traces of source for the production of medicinally important metabo- HgCl2. Surface-sterilized seeds were inoculated onto the MS lites (Verpoorte and Memelink 2002; Vanisree and Tsay (Murashige and Skoog 1962) medium for germination. Node, 2004). In vitro grown cell and organ cultures furnish a ready internode, and leaf explants were excised from 21-day-old source of uniform, sterile, and compatible material for bio- aseptic seedlings and used as explants. The explants were chemical characterization and identification of active con- placed on MS medium supplemented with different concen- stituents (Sajc et al. 2000). This process may condense trations (0.0–15.0 μM) of 6-benzyladenine (BA), kinetin biosynthetic cycle and facilitates the higher rate of metabo- (Kin), or thidiazuron (TDZ) individually or in combination lism than field grown plants (Rao and Ravishankar 2002; with auxins indole-3-acetic acid (IAA), α-naphthalene acetic Naik et al. 2011). Production of digitoxin and digoxin has acid (NAA), or 2,4-dichlorophenoxyacetic acid (2,4-D) and been investigated from cultured cells from Digitalis,but 30 gl−1 sucrose. pH of the medium was adjusted to 5.8 and most of the workers reported that undifferentiated cells solidified with 0.8 % (w/v)agar–agar (Hi Media, Mumbai, either did not produce cardenolides (Graves and Smith India) prior to autoclaving at 121 °C for 15 min. Cultures were 1967; Elze et al. 1974; Hirotani and Furuya 1977) or pro- incubated under controlled conditions such as 25±2 °C tem- duce only in trace amounts (Rücker et al. 1976; Lui and perature, 60±10 % relative humidity, and 8-h photoperiod Staba 1979). Attempts were made to use differentiated tis- (PFD 40 μmolm−2s−1) provided by white fluorescent tubes sues or organ cultures and showed the production of medic- (Philips, Kolkata, India). inally important compounds (Wilken et al. 2005;Pérez- Alonso et al. 2009). Redifferentiating organ cultures Maintenance of multiple shoot culture reported to accumulate considerable amount of cardinolides in Digitalis (Hirotani and Furuya 1977; Lui and Staba 1979; In preliminary experiments, shoot cultures showed signifi- Grave et al. 1980; Hagimori et al. 1982). Efforts were made cantgrowthin50mlliquidMSmediumfortifiedwith for in vitro production of cardiotonic glycosides from the 7.5 μM BA in culture glass bottles (400 ml capacity). genus Digitalis using leaf cell (Kreis et al. 1986), embryonic Therefore, multiple shoot cultures were maintained by sub- cell (Lindemann and Luckner 1997), root (Shimomura et al. culturing onto the same fresh medium after 28 days of Appl Microbiol Biotechnol interval. After separation of individual shoots, the basal potato-dextrose medium under controlled conditions as de- clump of mother explants was divided into four pieces and scribed earlier. After 28 days of growth, cultures were were inoculated separately in a bottle containing 50 ml MS autoclaved at 121 °C for 12 min. Cultures were allowed to medium fortified with 7.5 μM BA. The control was main- cool to room temperature, mycelial mats were harvested, tained on medium lacking BA. After 4 weeks of culture, washed five times with sterile distilled water, and dried in shoots were harvested and