Plant Cell Tiss Organ Cult DOI 10.

1007/s11240-010-9840-3

RESEARCH NOTE

Adventitious shoot regeneration and in vitro biosynthesis of steroidal lactones in Withania coagulans (Stocks) Dunal
Rohit Jain Arunima Sinha Devendra Jain Sumita Kachhwaha S. L. Kothari

Received: 5 June 2010 / Accepted: 30 August 2010 Springer Science+Business Media B.V. 2010

Abstract A micropropagation system through leaf explant culture has been developed for Withania coagulans. Shoot bud proliferation occurred through both adventitious and de novo routes depending on the hormonal regime of the culture medium. Green compact nodular organogenic callus developed on Murashige and Skoog (MS) medium supplemented with 2.3 lM kinetin (Kn) and lower levels of 6benzyladenine (BA) (13.3 lM) while multiple adventitious shoot bud differentiation occurred on medium fortied with 2.3 lM kinetin (Kn) and higher levels of BA (22.2 lM). Shoot buds were transferred to proliferation medium containing 2.2 lM BA, 2.3 lM Kn, and 3.9 lM phloroglucinol (PG) for further growth and development of shoot system. Elongated shoots were rooted using a two-step procedure involving pulse treatment of 7 days in a medium containing 71.6 lM choline chloride (CC) and 3.9 lM PG and then transferred to rooting medium containing MS, 1.2 lM IBA, 3.6 lM PAA, and 14.3 lM CC for 3 weeks. Well-rooted plants were transferred to a greenhouse for hardening and further growth. Random amplication of polymorphic DNA (RAPD) showed monomorphic bands in all the plants thereby conrming clonality of the regenerants. Thin layer chromatography (TLC) showed the presence of withanolides in the regenerated plants. Quantication through reverse-phase HPLC revealed increased concentration of withanolides in the regenerated plants compared to the eld-grown mother plant. Accumulation of withaferin A and withanolide A
R. Jain A. Sinha D. Jain S. Kachhwaha S. L. Kothari Department of Botany, University of Rajasthan, Jaipur 302004, India S. Kachhwaha S. L. Kothari (&) Centre for Converging Technologies (CCT), University of Rajasthan, Jaipur 302004, India e-mail: slkothari@lycos.com

increased up to twofold and that of withanone up to tenfold. Direct regeneration via leaf explants will be useful for Agrobacterium-mediated genetic transformation, and will facilitate pathway manipulation using metabolic engineering for bioactive withanolides. Keywords Micropropagation HPLC TLC RAPD Withania coagulans Withanolides Abbreviations BA 6benzyladenine CC Choline chloride DAD Diode array detector IAA Indole3acetic acid IBA Indole3butyric acid Kn Kinetin MS Murashige and Skoog NAA anaphthaleneacetic acid PAA Phenylacetic acid PG Phloroglucinol RAPD Random amplication of polymorphic DNA TLC Thin layer chromatography

Introduction Withania coagulans (fam. Solanaceae) is commercially important for its ability to coagulate milk, in the treatment of ulcers, rheumatism, dropsy, consumption and sensile debility (Bhandari 1995). Antimicrobial, anti-inammatory, antitumor, hepatoprotective, antihyperglycemic, cardiovascular, immunosuppressive, free radical scavenging and central nervous system depressant activities of the

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plant have also been demonstrated (Maurya and Akanksha 2010). Pharmacological investigations have elucidated association of these activities with the specic steroidal lactones known as withanolides present in Withania (Attaur-Rahman et al. 1998). Withaferin A, withanolide A and withanone are the major withanolides present in W. somnifera and W. coagulans. Overexploitation and the reproductive failures forced the species W. coagulans towards the verge of extinction (Jain et al. 2009b). The in vitro shoot cultures could provide an alternative to eld plant harvesting for the production of therapeutically valuable compounds (Sangwan et al. 2007). There are no reports of in vitro plant regeneration in W. coagulans except our earlier report using nodal and shoot tip explant cultures (Jain et al. 2009b). Here, we report regeneration from leaf explants and production of withanolides from the regenerated plants for the rst time.

and shoot buds developed per explant were recorded and shoot buds were subcultured on rst stage proliferation medium (MS, 2.2 lM BA, and 2.3 lM Kn) containing 3.9 lM phloroglucinol (PG) to further enhance growth and development of shoot buds. Regenerated shoots of appropriate length ([3 cm) were subjected to a two-step rooting procedure involving pulse treatment of 7 days on MS, 71.6 lM choline chloride (CC) and 3.9 lM PG and then transferred to rooting medium containing MS, 1.2 lM IBA, 3.6 lM PAA, and 14.3 lM CC prior to hardening as described previously (Jain et al. 2009b). The data on shoot bud formation and rooting were collected after 4 weeks. Three explants per ask and single explant per test tube was cultured. All experiments were repeated twice. RAPD analysis DNA was extracted from the leaves of 17 randomly selected regenerated plants and from the leaves of mother plant (WM). The leaf samples were powdered in liquid nitrogen and stored at -20C until used for DNA extraction by CTAB method (Doyle and Doyle 1990). The PCR amplication conditions were: an initial denaturation at 94C for 4 min followed by 40 cycles of 94C for 45 s, 37C for 45 s and 72C for 2 min, and a nal extension at 72C for 10 min. The amplicons were separated through 1.2% agarose (Himedia, India) gel electrophoresis and photographed using Gel Documentation System (Bio-Rad, Germany). Extraction of withanolides All the analytical and HPLC grade solvents, reagents and precoated silica gel TLC plates were purchased from Merck. Isolation of withanolides from various tissues was performed using the method described by Sangwan et al. (2007). Qualitative and quantitative analysis of withanolides Qualitative withanolide proling was done through TLC while quantication was carried out through HPLC as described by Sangwan et al. (2007). For TLC, 10 ll sample was loaded on precoated silica gel G-60 plates, performed in a solvent system consisting of chloroform:ethyl acetate:methanol:toluene (74:4:8:30, v/v), and development was done with anisaldehyde reagent (250 ll anisaldehyde in a mixture of 20 ml acetone, 80 ml water and 10 ml 60% perchloric acid) followed by heating at 110C. HPLC analysis was performed on Agilent (Germany) model 1200 and separation was achieved by a reverse-phase column (Eclipse XDB c-18, 4.5 mm 9 150 mm, particle size 1.8 lm; Agilent) using water (A) and methanol (B), each containing 0.1% acetic acid, as solvent and online UV-Diode Array

Materials and methods Plant material and establishment of in vitro cultures from leaf explants Leaf explants (0.82 cm) were collected from the eldgrown plants spotted in Ajmer (Rajasthan) in 2007. The species was identied by the Herbarium, Dept. of Botany, University of Rajasthan, Jaipur. Explants were thoroughly washed under running tap water for 15 min followed by treatment with 20% Extran (liquid detergent; Merck, India) for 5 min. Eventually, the explants were aseptically surface sterilized with 0.1% (w/v) HgCl2 (Merck, India) solution for 3 min. Explants were rinsed 45 times with sterile distilled water and cultured on full- and half-strength MS (Murashige and Skoog 1962) medium supplemented with 3% sucrose (Merck, India) and 0.9% agar (bacteriological grade; Merck, India). Various concentrations and combinations of different plant growth regulators (Sigma, India) including 6benzyladenine (BA; 2.2, 4.4, 8.8, 13.2 and 22.2 lM), kinetin (Kn; 2.3, 4.6, 9.2, 13.9 and 23.2 lM), indole-3-acetic acid (IAA; 1.1, 1.7 and 2.8 lM), indole-3butyric acid (IBA; 0.9, 1.4 and 2.4 lM), phenylacetic acid (PAA; 1.4, 2.2 and 3.6 lM) and anaphthaleneacetic acid (NAA; 1.0, 1.6 and 2.6 lM) were added in the medium to optimize growth and differentiation. The pH of the medium was adjusted to 5.8 followed by sterilization at 1.2 kg/cm2 pressure and 121C temperature for 20 min. Leaf explants with or without petiolar parts were placed abaxially on the medium. Cultures were maintained at 26 1C under 16/ 8 h photoperiod with 25 lmol m-2 s-1 photosynthetic photon ux density provided by white uorescent tubes (40 W; Philips, India). Twenty replicates were maintained for each treatment. The numbers of responding explants

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Detector (UV-DAD) at 227 nm. The solvent gradient was set as A:B, 60:4025:75, 045 min; 10:90, 4560 min at a ow rate of 0.6 ml min-1. Sample volume of 10 ll was injected and the column temperature maintained at 27C during the run. Authentic withanolides including withaferin A, withanone and withanolide A (Chromadex, CA, USA) were used as markers to ascertain their discrete resolution from each other under these conditions for both TLC and HPLC. Computation of withanolide concentration in the samples was done through a calibration curve of concentration versus detector response (peak area) using different concentrations of standard solutions of withaferin A, withanolide A and withanone in methanol. The data was analyzed statistically using one-way analysis of variance (ANOVA) by Fischers least signicant difference (P = 0.05) (Gomez and Gomez 1984). HPLC data was analyzed with the Chemstation LC 3D software (Agilent).

Table 1 Shoot bud formation from leaf explants of W. coagulans cultured on MS medium supplemented with BA and Kn BA (lM) 2.2 4.4 8.9 13.3 22.2 SE Standard error Means in a column followed by different letters are signicantly different from each other at P = 0.05 Kn (lM) 2.3 2.3 2.3 2.3 2.3 % response 80 86 73 93 80 Shoot buds (Mean SE) 4.6 0.5 e 7.7 0.6 d 9.3 0.6 c 12.1 0.2 b 17.6 0.5 a

Results and discussion Leaf explants cultured in the absence of growth regulators senesced without producing callus or adventitious buds, whereas they responded with enlargement and swelling at the cut petiolar end followed by callus formation on MS medium supplemented with Kn (2.3 lM) or BA (2.213.3 lM). Kn alone (Murch et al. 2004) or in combination with auxins (Kachhwaha and Kothari 1996; Reddy et al. 2004) and BA alone (Kulkarni et al. 2000; Sharma et al. 2003; Tilkat et al. 2009) or in combination with auxins (Koroch et al. 2002; Jain et al. 2009a; Kothari et al. 2010; Sinha et al. 2010) have most frequently been reported to induce in vitro plant regeneration in a wide range of monocotyledonous and dicotyledonous plants. Therefore, we also examined the effect of IAA, NAA or PAA in combination with BA or Kn on organogenesis. The combination of BA or Kn with auxins was not conducive to organogenesis. Brown, compact, nodular callus was observed on medium supplemented with BA (13.322.2 lM) and IAA (1.1 lM) or IBA (0.9 lM) or PAA (1.4 lM), but it could not induce any shoot buds. The amount of callus increased with increasing concentration of auxins. Rhizogenesis was observed all along the lamina cultured on medium with BA (2.222.2 lM) with NAA (1.0 2.6 lM). Kn in combination with auxins initiated formation of pale and nonmorphogenic callus. The use of 2.3 lM Kn in combination with BA (2.213.3 lM) promoted the initiation and development of shoot buds along with callus (Fig. 1a). Clusters of adventitious shoots (17.6 0.5) regenerated mostly from petiolar base of leaf explants or at leaf midrib region on medium supplemented with 22.2 lM BA and 2.3 lM Kn (Table 1, Fig. 1b). This clearly demonstrated that the combination of

BA and Kn was the most important factor for shoot regeneration from leaf explants of W. coagulans. Combination of BA with Kn for inducing shoot bud differentiation from the explants has also been reported in several other plants (Dayal et al. 2003; Baskaran and Jayabalan 2005; Sreedhar et al. 2008). Presence of petiolar part along with lamina was essential for morphogenesis as no response was observed when lamina without petiolar part was cultured. Previous reports have shown the same impact including petioles for enhancing shoot regeneration in several other plant species such as Paulownia tomentosa (Corredoira et al. 2008), Prunus persica (Gentile et al. 2002; Zhou et al. 2010), and P. serotina (Liu and Pijut 2008). Shoot buds induced on explants in the primary cultures were transferred to the proliferation medium containing 2.2 lM BA and 2.3 lM Kn for further differentiation of new shoot buds, but the elongation of the shoot buds did not occur (Fig. 1c). A combination of 2.2 lM BA, 2.3 lM Kn and 3.9 lM PG was required in the proliferation medium for the elongation of shoot buds up to 23 cm, a length which was required for rooting (Fig. 1d). PG has similarly been used by other workers (Sarkar and Naik 2000; Feeney et al. 2007). Elongated shoots ([3 cm) were transferred to MS medium containing 1.2 lM IBA, 3.6 lM PAA, and 14.3 lM CC after 7 days of pulse treatment with 71.6 lM CC and 3.9 lM PG for rooting. The incorporation of CC and PG enhanced rooting signicantly. These compounds have been reported to act as auxin protectors and increase the endogenous IAA levels during the inductive phase of rooting (Faivre-Rampant et al. 2004). Use of CC and PG in enhancing rooting has also been reported in Dendrocalamus hamiltonii (Sood et al. 2002) and Bambusa tulda (Mishra et al. 2008). The rooted plantlets (Fig. 1e) were successfully transferred to the greenhouse for hardening. The regenerated plants were subjected to RAPD analysis to check their clonality. Twenty random primers (OPF 110 and OPT 110) were used, of which 15 produced distinct and reproducible bands. A total of 1,197 amplicons were obtained and primer OPF-3 generated a highly

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Fig. 2 Agarose gel electrophoresis of RAPD fragments showing banding pattern amplied by OPF3 primer. M Molecular marker, C control

Fig. 1 Shoot bud induction from leaf explants of W. coagulans. a Indirect induction on MS, 13.3 lM BA and 2.3 lM Kn. b Direct induction from petiolar end on MS, 22.2 lM BA and 2.3 lM Kn. c Shoot buds developed on the rst stage proliferation medium. d Proliferation and elongation of shoots on MS, 2.2 lM BA, 2.3 lM Kn and 3.9 lM PG. e Rooting on MS, 1.2 lM IBA, 3.6 lM PAA and 14.3 lM CC

reproducible banding pattern (Fig. 2). DNA ngerprinting proles of regenerants revealed that there was no variation amongst mother and tissue culture-raised plants. There are many reports demonstrating the suitability of enhanced axillary branching for raising true-to-type plants (Rani and Raina 2000). Analysis of withanolide content in in vitro shoot cultures of W. somnifera has been reported by several workers (Ray and Jha 2001; Sangwan et al. 2004, 2007), but there are no such reports for W. coagulans. The study used an analytical reverse phase HPLC system providing symmetrical and high resolution peaks of three important withanolides in the plant. TLC of different extracts revealed that withaferin A, withanolide A and withanone were biosynthesized in regenerated plants of W. coagulans (Fig. 3). Withanolide content was analyzed by HPLC, and standard samples of withaferin A, withanolide A and withanone were used to construct a calibrated graph by plotting peak areas versus

Fig. 3 TLC prole of W. coagulans. Lanes 1 standard withaferin A, 2 standard withanolide A, 3 standard withanone, 4 sample extracted from in vitro shoots, 5 samples extracted from eld leaves, 6 samples extracted from callus, 7 samples extracted from eld roots

the amount of respective withanolide over a range of 501,000 ng ll-1. The response was linear over the tested concentration range. The identication of withanolides was conrmed on the basis of retention time and absorption spectra on UV-DAD (32.46 min, 215 nm; 38.38 min,

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Fig. 4 DADHPLC chromatogram of standards. a Withaferin A, b withanolide A, c withanone. Samples from d in vitro developed shoots, e eld leaves, and f eld roots (insets are UV-DAD spectra of the specied withanolide)

230 nm; and 40.90 min, 230 nm for withaferin A (Fig. 4a), withanolide A (Fig. 4b) and withanone (Fig. 4c), respectively). The accumulation of all the three withanolides was higher in regenerated plants than in the samples taken from eld-grown plants (Fig. 4d, e). A shift towards organ differentiation resulted in improved potential of the cultures to synthesize withanolides. The quantities of withaferin A and withanolide A increased up to two-fold while the withanone content increased up to ten-fold in the regenerated plantlets as compared to eld-grown plants (Table 2). Withanolide A accumulates in small amounts in shoots (Fig. 4e) and more in roots (Fig. 4f) in eld-grown plants, but in the present study the amount of withanolide A was as good in regenerated shoots as in the roots of eld plants (Table 2, Fig. 4d). Several factors, e.g., the difference in chemotype utilized as source for initiation of multiple shoot buds, and culture conditions such as basal media composition and growth regulator types utilized to establish cultures might have contributed to withanolide production. The positive correlation between withanolide synthesis and morphological differentiation suggests that

Table 2 Withanolide content in different tissues of W. coagulans Sample Withanolide Content (mg gfw-1) Mean SE Withaferin A Field leaves In vitro leaves Field roots 0.084 0.004 0.192 0.005 Nil Withanolide A 0.059 0.014 0.123 0.009 0.113 0.009 Withanone 0.031 0.001 0.282 0.006 Nil

synthesis is regulated in a tissue-specic way and organogenesis is the key regulatory factor which stimulates production of withanolides in vitro. The detection of higher content in differentiated cultures also points out that the enzymes responsible for biogenesis of withanolides in vitro might be optimally active in the culture conditions as has been shown earlier in W. somnifera (Sharada et al. 2007). Taken as a whole, our results demonstrate that leaves of W. coagulans have a great organogenic potential for shoot bud formation; however, the response is highly sensitive and directly related to the combinations of exogenous growth regulators in the culture medium. The results also

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conrm the potential of this plant to biosynthesize the active principle (withanolides) under in vitro culture conditions. In vitro regeneration of adventitious shoots is an essential component for most of the genetic transformation protocols. The system described here will be useful in this respect and for conservation of elite germplasm of this important medicinal plant species.
Acknowledgments Financial support from Council of Scientic and Industrial Research (CSIR) in the form of R&D project: CSIR 38(1178) EMRII/2007 is gratefully acknowledged. Rohit Jain, Arunima Sinha and Devendra Jain thank CSIR for the award of Senior Research Fellowships.

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