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 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 L. (; 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 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 (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 the mother clump was divided hot air oven at 60 °C for 48 h. The fine powder of mycelial into four pieces and sub-cultured on same fresh medium. On mass and commercially available chitin [poly(N-acetyl-1,4- subculture to fresh medium at 4-week intervals, multiple β-D-glucopyranosamine)] and yeast extract (Hi Media, shoot cultures were maintained over a period of 18 months. Mumbai, India) were added at varying concentrations (0, 100, 200, 300, 400, and 500 mg/l) separately in MS medium Root initiation and acclimatization with 7.5 μM BA. Before autoclaving, different concentra- tions of salicylic acid (0, 50, 100, 150, 200, and 250 μM), Regenerated shoots that were approximately 5 cm in height mannitol and sorbitol (0, 100, 200, 300, 400, and 500 mM), were separated from shoot clusters and transferred for root- PEG-6000 (0, 1, 2, 3, 4, and 5 mM), NaCl (0, 20, 40, 60, 80, ing on MS basal medium incorporated with NAA or IAA and 100 mM), and KCl (0, 40, 80, 120, 160, and 200 mM) (0.0–20.0 μM). Data were recorded on percent rooted were incorporated in liquid MS medium fortified with shoots and mean number of roots per shoot. After 28 days 7.5 μM BA. Progesterone, cholesterol (0, 100, 200, 300, of incubation, culture tubes with well-rooted shoots were 400, and 500 mg/l), and squalene (0, 1, 2, 3, 4, and 5 mM), dipped in water for 10 min to lose the agar-solidified medi- the precursors in biosynthetic pathway of cardiac glycosides um and the culture tubes were slowly inverted in water to (digitoxin and digoxin), were incorporated before autoclav- separate the plants from medium. Individual plants were ing in liquid MS medium supplemented with 7.5 μM BA. transferred to pots (23 cm diameter×12 cm height) contain- ing sand: soil mixture (1:1). Initially for 1 week, pots con- Growth analysis taining plants were covered with polythene bags. Plantlets were maintained in a glass house (temperature 25±5 °C; Shoots grown in control and under various treatments were humidity 80±10 %; maximum light 200 μmolm−2s−1) and harvested after 28 days and fresh weight was recorded. were watered (20 mL) three times a day (8 am, 1 pm, and Shoot biomass was dried at 60 °C until constant dry weight 6 pm) initially. was recorded.

Effect of plant growth regulators on production of multiple Quantitative studies of digitoxin and digoxin shoot biomass and glycosides One gram of biomass powder was soaked in 10 ml of Multiple shoots were raised from nodal explants on MS distilled water for 2 h, and the mixture was squeezed. medium containing 7.5 μM BA. After separation of indi- Plant residue was extracted thrice in conical flask containing vidual shoots, the mother explant clumps was divided into 20 ml of 95 % (v/v) ethanol for 24 h on a rotary shaker at four pieces. These mother explant pieces were inoculated on 80 rpm at 25 °C. The pooled filtrate was evaporated to dry MS medium containing different concentrations (0 to under vacuum. The dried residue was dissolved in methanol 15 μM) and combinations of cytokinins (BA, Kin, and at a concentration of 1 mg/ml and used for high performance TDZ) and auxins (IAA, NAA, and 2,4-D). The production thin layer chromatography (HPTLC) analysis (CAMAG, of biomass and glycosides was analyzed per culture at Switzerland). The standard digitoxin and digoxin were pro- weekly intervals for 5 weeks. It is noted that 4 weeks of cured from Sigma-Aldrich, Bangalore, India and dissolved growth was the best for biomass production as well as for in HPLC grade methanol at the concentration of 1 mg/ml. glycoside accumulation. Therefore, in subsequent experi- HPTLC was performed on 20×10 cm aluminum foil plates ments, shoots were harvested after 4 weeks of culture. coated with 200 μm layer of silica gel 60F254 (E. Merck, Germany). Standard and samples were loaded with Linomat Effect of elicitors and precursors on production of biomass 5 semiautomatic applicator (CAMAG, Switzerland) fitted and glycosides with 100 μl syringe as bands of 8 mm width. The delivery speed of the syringe was 90 nl/s. HPTLC plates were de- Fungal strains viz., Aspergillus niger (NCIM 545), veloped in twin-trough chambers (20×10×4 cm; CAMAG, Helminthosporium sp. (NCIM 1280), and Alternaria sp. Switzerland). Chamber saturation was carried using 20× (NCIM 1079) were procured from the National Collection 10 cm Whatman filter paper for 20 min. The plates were of Industrial Microorganisms and National Chemical developed in ethyl acetate–methanol–water (75:10:7.5 v/v), Laboratory, Pune, India. The strains were grown on liquid until 80 mm from the lower edge of the plate. After Appl Microbiol Biotechnol development, plates were dried using hair dryer. Then, the MS+7.5 μM BA was found superior. After separation of plates were immersed in anisaldehyde reagent for 2 s fol- individual shoots, the mother explant clumps from liquid lowed by drying in an oven for 5 min at 100 °C. The plates medium was divided into four pieces. These mother explant were then documented using CAMAG TLC visualizer at pieces were inoculated on fresh MS liquid medium containing 254 nm and white light. Evaluation of the plates was per- 7.5 μM BA. It was observed that the shoot regeneration formed at 366 nm using CAMAG TLC scanner III system frequency was slightly higher (18 per explant) in mother with winCATS software 1.4.3. The content in the samples explant as compared to the fresh nodal explants (12.7±0.6). was quantified by comparing the peak area with that of To further maintain the multiple shoot induction, every time standard digitoxin and digoxin. The digitoxin and digoxin mother tissue was cut into four equal parts and repeatedly sub- contents were calculated and expressed in terms of micro- cultured on fresh shoot induction liquid medium. On adapta- grams per gram of dry weight (DW). tion of this strategy, shoot cultures were maintained over a period of 18 months by sub-culturing at 28-day intervals. Data analysis Rooting of shoots and acclimatization A completely randomized design was used in all experi- ments. All experiments were repeated thrice with at least The micro shoots when cultured on MS medium without seven replicates per treatment. Data were subjected to anal- supplementation of plant growth regulators produced an av- ysis of variance (ANOVA) followed by Duncan’s multiple erage of 14.0±1.2 roots per shoot in 14.4 % shoots with an range test (DMRT) at P≤0.05. average length of 1.9±0.06 cm (Table 2). Inclusion of differ- ent concentrations of IAA and NAA (2.5 to 15 μM) in MS medium induced roots in 23.4 to 85.6 % of the shoots Results (Table 2). Among different concentrations of IAA and NAA (2.5 to 20 μM), 15 μM IAA was found as the best for Shoot regeneration and maintenance of shoot culture induction of rooting in the in vitro raised shoots. Mean num- ber of roots per shoot and mean root length increased with Shoot regeneration response of node, internode, and leaf increasing concentration of IAA. Mean number of roots per explants cultured on MS basal media supplemented with shoot was found higher (35.4±0.6) in MS medium supple- various concentrations of BA, Kin, or TDZ is shown in mented with 15 μM IAA (Table 2;Fig.1b). The well rooted Table 1. The first visible sign of shoot initiation with swell- plantlets were taken out of the medium, washed with water ing at the cut ends of explants was observed 10–12 days thoroughly, and transferred to pots containing sand–soil mix- after inoculation. Node, internode, and leaf explants, when ture (1:1). To maintain humidity for initial 1 week, the pots inoculated on MS basal medium without growth regulators, containing plantlets were covered with polythene bags. These showed shoot proliferation in 28.6, 14.3, and 12.8 % of bags were perforated at the top for ventilation. On acclimati- cultures, respectively (Table 1). Incorporation of cytokinins zation in glass house for 1 month, a survival percentage of promoted the induction of shoot proliferation from node as about 80 % was achieved and then transferred to field con- well as internode and leaf explants. Among the different ditions (Fig. 1c). The in vitro propagated plantlets grew well explants, nodal explants were found more responsive for and did not show morphological abnormalities. shoot regeneration than internode and leaf explants. Maximum shoot regeneration frequency (85.7 %) and aver- Effect of plant growth regulators on production of shoot age number of shoots per explants (12.7±0.6) were ob- biomass and glycosides served in nodal explants on media supplemented with 7.5 μM BA (Fig. 1a). Comparatively low response of shoot Shoot cultures were incubated for different time periods (7, regeneration was observed in internode (74.3 %; 9.4±0.1 14, 21, 28, and 35 days); the optimum growth of shoots was shoots per explant) and leaf (57.1 %; 7.2±0.1 shoots per recorded at 28 days of incubation, and it was slightly de- explant) explants. Incorporation of Kin at 7.5 μM and TDZ creased with further increase in incubation period up to at 2.5 μM was effective for induction of shoot regeneration, 35 days (Fig. 2). HPTLC analysis of the shoot biomass but the regeneration frequency and average number of showed maximum accumulation of digitoxin (53.6± shoots in node, internode, and leaf explants were compara- 1.1 μg/g DW) and digoxin (28.8±1.3 μg/g DW) 28 days tively poor (Table 1). Inclusion of auxins IAA, NAA, and after inoculation (Fig. 2). Therefore, shoot biomass was 2,4-D together with BA, Kin, and TDZ inhibited the re- harvested after 28 days and used for quantification of digi- sponse of shoot regeneration in these explants. toxin and digoxin. Among the cytokinins used, vigorous For maintenance of shoots, solid and liquid MS media growth of shoots and shoot dry weight was observed on supplemented with 7.5 μM BAwere evaluated of which liquid MS medium fortified with BA followed by Kin and TDZ. plMcoilBiotechnol Microbiol Appl

Table 1 Effect of different concentrations of cytokinins (BA, Kin, and TDZ) on in vitro shoot multiplication in D. purpurea L.

PGRs (μM) Explants

BA Node Internode Leaf

Shoot regeneration No. of shoots Length of Shoot regeneration No. of shoots Length of Shoot regeneration No. of shoots Length of frequency (%) per explant shoots (cm) frequency (%) per explant shoots (cm) frequency (%) per explant shoots (cm)

0.0 28.6 4.8±0.2e 4.8±0.3bcd 14.3 3.0±0.3g 4.7±0.1bc 12.8 2.1±0.1hi 4.5±0.1abcd 2.5 57.1 6.2±0.3de 5.6±0.2bc 32.3 4.8±0.2cdefg 5.1±0.1abc 44.2 6.1±0.4abc 5.8±0.1ab 5.0 68.4 9.2±0.2b 6.0±0.6bc 58.2 6.2±0.1bcd 5.3±0.1abc 38.6 5.5±0.5abcd 5.1±0.1abc 7.5 85.7 12.7±0.6a 7.9±0.1a 74.3 9.4±0.1a 6.8±0.1a 57.1 7.2±0.1a 6.3±0.1a 10.0 37.1 8.0±0.6bcd 5.8±0.1bc 42.8 5.0±0.1cdef 4.6±0.2bc 28.1 4.0±0.2defgh 4.3±0.1bcd 15.0 24.2 5.3±0.3e 5.1±0.3bcd 23.4 4.6±0.1defg 3.6±0.1bc 21.1 3.0±0.1fghi 3.9±0.2bcd Kin 2.5 27.4 5.3±0.2e 5.7±0.6bc 25.1 5.7±0.3bcde 4.5±0.3bc 21.4 5.4±0.2bcde 4.1±0.3bcd 5.0 32.5 7.2±0.2cd 5.3±0.2bcd 27.2 6.0±0.1bcde 5.2±0.1abc 21.6 5.2±0.1bcde 4.5±0.1abcd 7.5 42.8 8.6±0.1bc 6.7±0.3ab 38.6 7.0±0.1b 5.8±0.1ab 34.1 6.4±0.1ab 5.3±0.1abc 10.0 24.6 6.4±0.3de 4.2±0.6cde 18.6 5.6±0.2bcde 4.0±0.1bc 16.7 4.7±0.1bcdefg 3.7±0.2cde 15.0 17.1 5.1±0.2e 3.9±0.2de 16.4 4.2±0.1efg 3.5±0.2cd 15.8 4.8±0.1bcdefg 2.6±0.1def TDZ 2.5 35.4 6.3±0.6de 2.9±0.3ef 28.6 6.6±0.1bc 2.3±0.1de 27.4 4.9±0.1bcdef 2.7±0.1def 5.0 28.6 5.3±0.1e 2.6±0.1ef 24.3 5.6±0.1bcde 2.2±0.1de 18.9 4.3±0.2cdefg 1.9±0.1efg 7.5 14.3 4.7±0.2e 2.4±0.1ef 12.6 5.0±0.2cdef 1.3±0.2ef 11.8 3.3±0.1efgh 1.0±0.1fg 10.0 11.6 3.0±0.5f 1.7±0.2f 10.8 4.3±0.2defg 1.1±0.1ef 09.1 2.9±0.1ghi 1.9±0.1fg 15.0 8.6 2.0±0.3f 1.5±0.6f 08.3 3.5±0.2fg 0.7±0.1f 07.1 1.2±0.1i 0.7±0.5g

Values represent mean±SE calculated on three independent experiments, each based on minimum seven replicates. Means followed by same letters within columns are not significantly different at 5 % level (DMRT) Appl Microbiol Biotechnol

Table 2 Effect of auxins (NAA and IAA) on rooting of in vitro regenerated shoots of D. purpurea L.

Auxins Rooted Mean no. of Mean root Survival (%) (μM) shoots (%) roots/shoot length (cm)

0.0 14.4 14.0±1.2f 1.9±0.06c 20 IAA 2.5 28.6 16.4±0.6f 3.2±0.1bc 35 5.0 44.7 21.3±1.7c 3.3±0.06bc 42 7.5 57.1 24.5±1.2b 3.5±0.1bc 58 10.0 71.4 25.2±0.6b 4.4±0.2ab 65 15.0 85.6 35.4±0.6a 5.4±0.1a 80 20.0 68.4 18.4±0.5de 4.2±0.2ab 63 NAA 2.5 23.4 15.2±1.7f 2.8±0.1bc 30 5.0 42.8 17.4±0.6e 2.9±0.1bc 39 7.5 52.1 19.3±0.1d 3.7±0.1abc 46 10.0 58.6 18.5±1.0de 4.2±0.3ab 58 15.0 67.1 24.3±0.6b 4.5±0.1ab 72 20.0 56.7 17.5±0.8e 4.1±0.2ab 54

Values represent mean±SE calculated on three independent experiments, each based on minimum seven replicates. Means followed by the same letters within columns are not significantly different at 5 % level (DMRT)

medium containing 5 μM NAA. On 2,4-D supplemented medium, accumulation of glycoside was low compared to that of shoots growing on IAA and NAA (Fig. 4b, c). Incorporation of auxins (IAA, NAA, or 2,4-D) together with optimum concentration of cytokinins (BA, Kin, and TDZ) Fig. 1 In vitro propagation of D. purpurea L. a multiple shoot regen- did not promote the growth of shoots and glycoside accu- eration from nodal explants on MS medium supplemented with 7.5 μM mulation (Table 3). BA (bar01.0 cm), b rooting of in vitro regenerated shoots on MS medium containing 15 μM IAA (bar01.0 cm), c acclimatized plantlets Influence of elicitors on growth and accumulation in earthen pot (bar010.0 cm) of cardiotonic glycosides

Supplementation of 7.5 μM BA was found suitable for Increasing concentrations of salicylic acid, mannitol, sorbitol, obtaining maximum shoot biomass (1.69±0.1 g DW) after PEG-6000, NaCl, and KCl reduced the growth but resulted in 28 days of incubation (Fig. 3a). These cultures also showed higher accumulation of digitoxin (53.6±3.1 μgg−1 DW; Fig. 3b) and digoxin (28±1 μgg−1 DW; Fig. 3c). Among different types (IAA, NAA, and 2,4-D) and concentrations (0.0–15 μM) of auxins, MS medium supplemented with 5 μM IAA exhibited higher shoot dry weight (0.89±0.4 g; Fig. 4a). But the dry weight was less compared to the cytokinin containing media. Shoots grown on medium lack- ing growth regulators displayed 21.6±2.3 μgg−1 DW digi- toxin and 15.9±1 μgg−1 DW digoxin after 28 days of culture. Incorporation of auxins in the medium promotes the cardiac glycoside accumulation. About two-fold in- crease in digitoxin (Fig. 4b) and 1.8-fold increase in digoxin (Fig. 4c) content was observed in the medium fortified with μ Fig. 2 Effect of incubation period on growth and accumulation of 5 M IAA. While the increase in digitoxin was 1.7-folds cardiotonic glycosides in shoot cultures of D. purpurea. DW dry (Fig. 4b), digoxin was augmented by 1.55-folds (Fig. 4c)on weight Appl Microbiol Biotechnol

Fig. 3 Effect of cytokinins on growth and production of digitoxin and digoxin in shoot cultures of Digitalis purpurea. a Dry weight of shoots, b digitoxin content, and c digoxin content. The error bars indicate the standard error (SE). Bars with different letters revealed significantly different values at p≤0.05, according to DMRT. DMRT was applied to each cy- tokinin separately

enhanced accumulation of cardiac glycosides (Table 4). mannitol in the medium resulted in 4.8-fold and 2.57-fold Addition of 50 μM salicylic acid promoted 2.17-fold higher higher accumulation of digoxin and digitoxin in the shoot digitoxin (Table 4). But increasing concentrations of salicylic biomass, respectively (Table 4). Similarly, supplementation of acid drastically reduced the digitoxin content. On the contrary, 100 and 200 mM sorbitol was found optimum for 7.48- and digoxin accumulation was increased in the shoots (134.3± 3.45-fold higher accumulation of digoxin and digitoxin, respec- 1.6 μgg−1 DW) with an increase in the salicylic acid concen- tively (Table 4). Compared to the control, inclusion of PEG- tration up to 200 μM(Table4). Presence of 200 and 300 mM 6000 in the medium resulted in decreased accumulation of Appl Microbiol Biotechnol

Fig. 4 Effect of auxins on growth and production of digitoxin and digoxin in shoot cultures of D. purpurea. a Dry weight of shoots, b digitoxin content, and c digoxin content. Bars indicate standard error (SE). Bars with different letters revealed significantly different values at p≤0.05, according to DMRT. DMRT was applied to each auxin separately

digitoxin. At the same time, digoxin content increased with of KCl (up to 200 mM) was found beneficial for accumulation increasing concentration of PEG-6000 up to 5 mM level of digoxin in the shoot cultures. About 8.7-folds higher digoxin (Table 4). NaCl was found ineffective in stimulating the syn- accumulation was noticed in the shoot cultures grown on thesis of cardiotonic glycosides (Table 4). In contrast to it, 200 mM KCl (Table 4). incorporation of 80 mM KCl in the medium influenced the The results recorded in Fig. 5a revealed that fungal my- accumulation of digitoxin by 7.75-folds over control. Higher celia, commercially available yeast extract, and chitin pow- concentrations of KCl, however, were inhibitory for growth and der were inhibitory for the growth of shoots. But, inclusion digitoxin accumulation. Interestingly, increasing concentration of elicitor derived from Helminthosporium sp. (500 mg/l) Appl Microbiol Biotechnol

Table 3 Effect of cytokinins in combination with auxins on growth and Table 4 Effect of abiotic elicitors on growth and accumulation of accumulation of digitoxin and digoxin in shoot cultures of D. purpurea L. digitoxin and digoxin in shoot cultures of D. purpurea L.

PGR (μM) DW (g) Digitoxin Digoxin Abiotic elicitors DW (g) Digitoxin Digoxin (μgg−1 DW) (μgg−1 DW) (μgg−1 DW) (μgg−1 DW) Cytokinins Auxins Salicylic acid (μM) BA IAA Control 1.69±0.10a 53.6±3.1c 18.0±1.0f 7.5 1.25 0.49±0.06c 16.9±1.1r 17.1±1.1qr 50 0.67±0.11b 117.6±2.7a 23.0±0.9e 2.5 0.38±0.08f 28.6±0.6k 24.0±2.2no 100 0.66±0.10b 63.6±2.0b 34.9±1.3d 5.0 0.38±0.14f 24.9±1.2n 21.0±2.3p 150 0.60±0.15c 33.3±1.9d 58.9±1.9c NAA 200 0.49±0.19d 28.4±1.2e 134.3±1.6a 1.25 0.41±0.22e 31.9±0.7j 23.2±1.7a 250 0.34±0.10e 23.0±0.3f 117.9±0.6b 2.5 0.26±0.08j 27.9±0.7kl 12.0±1.3c Mannitol (mM) 5.0 0.19±0.01n 25.7±0.7m 08.1±1.6f Control 1.69±0.10da 53.6±3.1f 18.0±1.0f 2,4-D 100 0.57±0.02b 135.9±1.4b 26.2±1.2e 1.25 0.33±0.20h 47.3±1.1b 25.2±1.2n 200 0.49±0.20c 98.9±0.9e 87.0±1.1a 2.5 0.21±0.03qr 26.5±1.0lm 23.8±0.9i 300 0.42±0.10d 140.1±2.2a 62.5±1.1b 5.0 0.18±0.03no 23.6±1.1kl 20.1±2.2h 400 0.24±0.06e 122.4±1.7c 51.2±1.4c Kin IAA 500 0.11±0.01f 101.0±1.7d 46.1±1.3d 7.5 1.25 0.31±0.08i 20.4±2.1op 19.5±2.2p Sorbitol (mM) 2.5 0.27±0.09f 19.0±1.2q 12.4±1.8no Control 1.69±0.10a 53.6±3.1e 18.0±1.0f 5.0 0.25±0.07g 15.1±1.1h 10.2±1.4l 100 0.71±0.16b 157.5±1.2b 134.3±1.7a NAA 200 0.37±0.12c 186.1±1.7a 114.1±1.2b 1.25 0.25±0.07k 37.4±1.5d 22.7±1.2o 300 0.21±0.02d 155.9±1.4b 106.3±1.0c 2.5 0.24±0.14gh 21.4±1.3o 17.4±1.8j 400 0.17±0.05e 120.1±1.6c 92.1±1.7d 5.0 0.20±0.11i 29.3±1.0k 13.0±2.2k 500 0.13±0.01f 100.3±1.3d 88.0±1.2e 2,4-D PEG – 6000 (mM) 1.25 0.23±0.06l 41.5±1.0g 15.8±1.7j Control 1.69±0.10a 53.6±3.1a 18.0±1.0f 2.5 0.19±0.06rs 40.3±0.8g 12.3±1.5im 1 1.19±0.05b 23.8±1.3e 19.2±1.1e 5.0 0.12±0.08pq 47.7±1.2f 11.8±0.6m 2 1.09±0.05c 25.2±1.6e 26.8±1.5d TDZ IAA 3 0.85±0.10d 47.9±2.3b 31.1±1.6c 2.5 1.25 0.49±0.03c 34.4±1.4i 15.9±1.2b 4 0.73±0.20e 28.5±1.8d 44.8±1.4b 2.5 0.41±0.07a 24.4±1.8e 17.8±1.8e 5 0.64±0.05f 38.2±2.4c 54.5±1.7a 5.0 0.35±0.22d 27.4±1.7b 11.8±1.5d NaCl (mM) NAA Control 1.69±0.10a 53.6±3.1a 18.0±1.0d 1.25 0.42±0.09b 36.0±1.9a 06.8±1.5g 20 0.23±0.10b 34.7±0.8c 23.3±1.3c 2.5 0.37±0.02o 27.8±1.2b 04.7±1.3i 40 0.18±0.03c 36.6±1.5b 23.7±2.1c 5.0 0.21±0.00m 24.7±2.2c 02.7±2.3l 60 0.16±0.03d 26.8±1.8d 28.7±0.9b 2,4-D 80 0.11±0.04e 22.3±1.3e 31.8±1.9a 1.25 0.18±0.03no 41.3±1.8g 15.6±1.2r 100 0.10±0.03f 22.1±1.2e 32.7±1.3a 2.5 0.13±0.03p 37.1±1.2h 17.7±1.5q KCl (mM) 5.0 0.09±0.04s 27.8±1.2f 19.9±1.2p Control 1.69±0.10a 53.6±3.1f 18.0±1.0f 40 0.75±0.23b 383.0±2.9b 87.0±1.2e Values represent mean±SE calculated on three independent experi- ments, each based on minimum 3 replicates. Means followed by the 80 0.66±0.10c 413.6±2.1a 102.2±1.3d same letters within columns are not significantly different at 5 % level 120 0.50±0.21d 212.1±1.2c 135.0±1.4c (DMRT) 160 0.48±0.11e 179.7±0.9d 146.0±0.8b 200 0.24±0.12f 106.3±1.1e 156.4±1.5a was highly effective in stimulating both digitoxin and di- Values represent mean±SE calculated on three independent experi- goxin as compared to the control and other elicitors (Fig. 5b, ments, each based on minimum three replicates. Means followed by c). Less, but considerable accumulation of digitoxin was the same letters within columns are not significantly different at 5 % level (DMRT) observed on inclusion of 300 mg/l elicitor derived from Alternaria sp. However, supplementation of elicitor derived Appl Microbiol Biotechnol

Fig. 5 Effect of biotic elicitors on growth and production of digitoxin and digoxin in shoot cultures of D. purpurea L. a Dry weight of shoots, b digitoxin content, and c digoxin content. Error bars indicates standard error (SE). Bars with different letters revealed signif- icantly different values at p≤ 0.05, according to DMRT. DMRT was applied to each elicitor separately

from Aspergillus niger, commercial chitin powder, and yeast digoxin varied significantly with the and concentration extract could not show any effect on digitoxin accumulation of precursors. The best response for the content of digitoxin (Fig. 5b). (492.6±1.8 μgg−1 DW) and digoxin (214.2±1.2 μgg−1 DW) was on medium fortified with 200 and 300 mg/l of progester- Influence of precursors on growth and accumulation one, respectively. On this medium, the increase in digitoxin of glycosides was about nine-folds and digoxin was around 12-folds over the control (Table 5). However, when compared to progester- Growth of shoots in terms of dry weight on MS+7.5 μMBA one, cholesterol and squalene were less effective. devoid of any precursor was 1.69±0.1 g DW per culture. Compared to control, incorporation of precursors associated with the biosynthesis of cardiotonic glycosides such as Discussion progesterone, cholesterol, and squalene in the medium resulted in significant decline in growth (Table 5). Shoot dry Incorporation of cytokinins promoted the induction of shoot weight per culture remained in the range of 0.3 to 0.48 g in the proliferation in all the three explants of D. purpurea. Among presence of precursors. But, the content of digitoxin and different explants, nodal explants were found more Appl Microbiol Biotechnol

Table 5 Effect of precursors on growth and digitoxin and digoxin of auxins (IAA, NAA, and 2,4-D) alone or in combination with accumulation in shoot cultures of D. purpurea L. cytokinins (BA, Kin, and TDZ). In contrast, earlier Hagimori Precursors DW (g) Digitoxin (μgg-1 Digoxin (μgg-1 et al. (1982; 1984) established shoot forming cultures of D. DW) DW) purpurea on MS medium containing 1 mg/l BA+1 mg/l IAA. Similarly, Pérez-Alonso et al. (2009) also established shoot Control 1.69±0.10a 53.6±3.1k 18.0±1.0k forming cultures on MS medium supplemented with Progesterone 1 mg/l BA in conjunction with 0.1 mg/l IAA in D. (mg/l) 100 0.48±0.11b 378.3±1.1b 149.1±1.4d purpurea. Sales et al. (2002) reported adventitious bud 200 0.38±0.06c 492.6±1.8a 184.4±1.5b differentiation with BAP, but addition of auxins increased 300 0.31±0.04d 216.4±1.5c 214.2±1.2a the frequency from leaf explants in D. minor. Recently, a 400 0.30±0.02d 145.1±1.6d 162.2±1.3c combination of 1 mg/l BA and 0.1 mg/l IAA was also used 500 0.30±0.03d 91.4±1.1g 138.1±1.2e for establishment of shoot cultures in Digitalis lanata by Cholesterol (mg/ Pérez-Alonso et al. (2012). For maintenance of shoots, solid l) and liquid MS media supplemented with 7.5 μMBAwere 100 0.30±0.01d 77.2±1.4i 32.7±1.2i evaluated of which liquid MS+7.5 μM BA was found supe- 200 0.30±0.01d 136.9±1.6e 54.2±1.2g rior. On this medium, shoot cultures were maintained over a 300 0.30±0.03d 112.4±1.3f 78.2±1.9f period of 18 months by sub-culturing at 28-day intervals. 400 0.30±0.01d 56.7±1.5j 49.1±1.1h Shoots sub-cultured on rooting media containing either 500 0.30±0.02d 19.7±1.3o 32.4±1.3i IAA or NAA produced a mass of numerous fibrous, thin Squalene (mM) roots at the base of the shoots within 4 weeks of culture. The 1 0.36±0.07c 26.9±1.7n 14.4±1.1l frequency of rhizogenesis on auxin-containing media was 2 0.30±0.03d 34.1±1.7m 18.3±1.3k more as compared to medium devoid of it. Gurel et al. 3 0.30±0.01d 78.7±1.2i 27.1±1.7j (2011) reported rooting of shoots on LS medium supple- 4 0.30±0.01d 81.4±1.3h 31.9±1.4i mented with IAA or IBA, and IAA was found more effective 5 0.30±0.01d 45.2±1.8l 26.5±1.0j than IBA in D. davisiana. IAA at 0.5 mg/l concentration was also reported as optimum for rooting of in vitro regen- Values represent the mean±SE calculated on three independent experi- erated shoots of Digitalis lamarckii (Verma et al. 2011). ments, each based on minimum three replicates. Means followed by the same letters within columns are not significantly different at 5 % level Plants subjected to hardening under ex vitro conditions (DMRT) revealed the highest percent of survival (80 %) with elon- gation of shoots. After 1 month of glass house acclimatiza- responsive for shoot regeneration than internode and leaf tion, plants transferred to field conditions survived and grew explants. Nodal explants are generally preferred for micro- well (Fig. 1c). Similar results were reported in D. lamarckii propagation of most of the plant species due to the presence (Verma et al. 2011) and in D. davisiana (Gurel et al. 2011). of pre-existing meristems, which can be easily developed In vitro raised plants did not show any morphological var- into shoots. Such shoots also maintain clonal fidelity iation but did not show flowering. (Frabetti et al. 2009). Of the cytokinins tested, BA was Cultures treated with MS medium fortified with 7.5 μM found superior to Kin and TDZ for shoot formation from BA were found better for shoot growth and accumulation of nodal explants. Earlier reports showed that BA was the most cardiotonic glycosides (Fig. 3). Several strategies like media efficient cytokinin for shoot regeneration in many plant composition, light, temperature, incubation period, different species (Stefaan et al. 1994). Kin was also found suitable types, and concentrations of growth regulators have been for shoot regeneration frequency (42.8 %) and number of used previously to improve growth and secondary metabo- shoots (8.6±0.1) from nodal as well as in internodal and leaf lite accumulation in different plants (Hagimori et al. 1983; explants (Table 1). Shoots produced from TDZ containing Gavidia and Pe′rez-Bermu′dez 1997; Debjani and Brattati medium remained, however, stunted. TDZ was effective for 2005; Pérez-Alonso et al. 2009;Chavanetal.2011). Earlier, organogenesis in woody plant species (Fiola et al. 1990; Hagimori et al. (1982) obtained maximum biomass from Malik and Saxena 1992). In the present investigation, TDZ shoot forming cultures after 20 days of incubation. Identical showed poorer response for shoot organogenesis. Similar results were observed in the present study. Hagimori et al. results were reported by Ahire et al. (2011)inUraria picta. (1982, 1984) and Pérez-Alonso et al. (2009)alsousedBAin Whereas, Gurel et al. (2011) reported the regeneration of combination with IAA for establishment of shoot cultures of Digitalis davisiana on LS medium supplemented with D. purpurea. 0.5 mg/l TDZ in combination with 0.25 mg/l IAA. In vitro cultures like callus provide a promising alterna- Poor response for shoot formation was observed in node, tive for the production of metabolites besides direct extrac- internode, and leaf explants cultured on various concentrations tion from shoot cultures (Zhao et al. 2005). One of the major Appl Microbiol Biotechnol constraints is the lower yield of these metabolites in in vitro associated with the production of these glycosides. To in- grown callus or suspensions or shoot cultures. Elicitors, vestigate the effect of salinity stress on glycoside accumu- signal compounds, and abiotic stresses have been employed lation, different concentrations of NaCl (0–200 mM) were earlier for enhancing the metabolites (Yukimune et al. 1996; added to the medium. Salt stress suppressed the shoot Zhao et al. 2000, 2005; Zhang et al. 2004). Elicitors at growth while no difference in glycoside production was appropriate concentrations might act as signaling molecules noticed. Both Na+ and Cl− ions are toxic and hence could which could be perceived by a receptor present on plasma affect the shoot growth. Potassium is beneficial to plant membrane and thus initiate the signal transduction network growth but at high concentrations, it can elevate the osmotic involving regulation of gene expression responsible for pressure on the cultured cells, thus acts as a stress agent or biosynthesis of target compounds (Zhao et al. 2005; abiotic elicitor. In the present study, addition of 80 mM KCl Savitha et al. 2006; Chavan et al. 2011). Elicitors have been improved the accumulation of digitoxin by 7.75-folds. effective in inducing the synthesis of secondary metabolites Higher concentration of KCl (200 mM) improved the con- in cell and organ cultures of different species, e.g., berberine tent of digoxin by 8.7-folds. It has previously been shown in Thalictrum rugosum (Brodelius et al. 1989), thiophenes that osmotic or salt stresses could efficiently improve sec- in Tagetes patula (Buitelaar et al. 1992), taxol in Taxus ondary metabolite production in in vitro as well as in vivo sp. (Ciddi et al. 1995), tanshinone in Salvia miltiorrhiza (Zhao et al. 2001). Compact callus clusters of Catharanthus (Zhao et al. 2010), and hypericin and pseudohypericin in roseus treated with 3 and 4 gl−1 KCl also promoted cathar- Hypericum hirsutum and Hypericum maculatum (Coste et anthine and serpentine production by three- to four-fold over al. 2011). Salicylic acid is one of the most widely studied the control (Zhao et al. 2001). signaling molecules mediating plant resistance to pathogens Fungal elicitation altered the expression of over 40 and other stress factors (Draper 1997). It is also well known transcripts, including representatives from the phenylpro- for its role as an elicitor for the enhanced production of panoid, pentose phosphate, glycolytic, and fatty acid many secondary metabolites (Kang et al. 2006; Dong et al. metabolic pathways (Batz et al. 1998), suggesting that 2010). Salicylic acid enhanced the accumulation of digitox- the response to elicitation is much more than the simple in in the present study (2.2-fold) at lower (50 μM), but at induction of biosynthetic enzymes of secondary metabo- higher concentrations (100–250 μM) its accumulation de- lism (Broeckling et al. 2005). In the present study, addi- clined. On the contrary, salicylic acid at 200 μMimproved tion of different biotic elicitors significantly affected the the accumulation of digoxin by 7.5-folds. Similarly, Ghanem biomass production of the shoot cultures at the concentrations et al. (2010) reported decreased total cardiac glycosides in tested. Supplementation of Helminthosporium sp. (500 mg/l) plantlets of D. lanata with the addition of salicylic acid. improved the digitoxin content by 4.5- and digoxin by 3-folds. To investigate the influence of non-metabolic sugars, The kinetics of induction or accumulation levels of the metab- different concentrations of mannitol and sorbitol (0– olites depends on the plant species and elicitors used 500 mM) were added to the culture medium. Shoots grown (Vasconsuelo and Boland 2007). Such a difference in the under osmotic stress displayed delayed growth and reduced kinetics of induction and levels of release of hyoscyamine shoot biomass. But, digitoxin and digoxin production in and scopolamine were recorded in hairy root cultures of sorbitol treated shoots was enhanced by 3.47- and 7.5-folds, Brugmansia candida (Pitta-Alvarez et al. 2000). respectively, compared to the corresponding control. Similar Addition of progesterone, cholesterol, and squalene results were also reported by Kim et al. (2001) on paclitaxel resulted in significant decrease in biomass production. But, production in cell cultures of Taxus chinensis. Contrarily, accumulation of digitoxin and digoxin were improved by Gertlowski et al. (1993) reported no accumulation of ros- 9.1- and 11.9-folds in the presence of 200 and 300 mg/ marinic acid in a medium incorporated with 2 % sucrose and l progesterone, respectively. Hagimori et al. (1983)also 2 % mannitol. To confirm the effect of osmotic pressure and reported 183 % improvement in digitoxin content over that to avoid the possible side effect of non-metabolic sugars on of control by incorporation of 0.lmg/l of progesterone in cardiotonic glycoside production in the present study, we shoot forming cultures of D. purpurea. Earlier, Lui and analyzed the effect of osmotic pressure generated by PEG Staba (1979) reported increased digoxin content in leaf 6000 (0–5 mM). In the presence of this non-permeable and root cultures by the addition of progesterone and cho- osmotic agent, both the dry weight of shoots as well as lesteryl acetate in D. lanata. Progesterones are the group of accumulation of digitoxin was reduced. Surprisingly, en- steroids with 21 carbon atoms and include pregnenolone, hanced digoxin accumulation was noticed with increasing progesterone, and deoxycorticosterone. They are the inter- concentrations of PEG-6000. About three-fold higher di- mediates in the biosynthesis of other steroids like cardeno- goxin accumulation was recorded at 5 mM PEG-6000 over lides, oestrogens, and androgens. Cholesterol as a precursor that of control, indicating that these osmotic agents influ- in the formation of steroids has been demonstrated previ- ence differentially the biosynthetic pathway enzymes ously by Heftmann (1975). In the present study, addition of Appl Microbiol Biotechnol cholesterol in the medium improved the accumulation of Coste A, Vlase L, Halmagyi A, Deliu C, Coldea G (2011) Effects of both digitoxin and digoxin. Transformation of exogenously plant growth regulators and elicitors on production of secondary metabolites in shoot cultures of Hypericum hirsutum and supplied cholesterol into cardenolides was first demonstrat- Hypericum maculatum. Plant Cell Tiss Org Cult 106:279–282 ed by Wickramasinghe et al. (1968). Hagimori et al. 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