Acta Physiol Plant (2013) 35:5563 DOI 10.1007/s11738-012-1047-2

ORIGINAL PAPER

Callus-mediated organogenesis and effect of growth regulators on production of different valepotriates in Indian valerian (Valeriana jatamansi Jones.)Jayashankar Das Ashiho A. Mao Pratap J. Handique

Received: 11 February 2012 / Revised: 29 May 2012 / Accepted: 19 June 2012 / Published online: 3 July 2012 rski Institute of Plant Physiology, Polish Academy of Sciences, Krako w 2012 Franciszek Go

Abstract A reproducible and efcient callus-mediated shoot regeneration system was developed for the large-scale production of Valeriana jatamansi Jones., a highly medicinal plant species of global pharmaceutical importance. Effect of Murashige and Skoog (MS) medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), a-naphthaleneacetic acid (NAA) and indole-3-butyric acid (IBA) on callus induction and production of valepotriates accumulation was studied by using different explants. In V. jatamansi, the degree of callus induction varied signicantly depending on explants type and the growth regulators used. Among different explants used, rhizomes have the highest callus induction potential followed by leaf. The callus induction frequency was found to be optimum in rhizome explants on media supplemented with 0.5 mg/l 2,4-D. The regenerative ability of proliferated compact calli was studied by the application of cytokinins alone and in combination with auxin. MS medium fortied with 0.75 mg/l thidiazuron in combination with 0.5 mg/l NAA showed the highest regeneration

Communicated by J. Van Huylenbroeck. J. Das A. A. Mao Plant Tissue Culture Laboratory, Botanical Survey of India, ERC, Shillong 793 003, India J. Das P. J. Handique Department of Biotechnology, Gauhati University, Guwahati 781084, India Present Address: J. Das (&) Plant Bioresources Division, Regional Centre of IBSD, Tadong, Gangtok 737102, India e-mail: biotechjay@yahoo.co.in

frequency (88.6 %) and produced the maximum number of shoot buds (15.20 0.20) capable of growing into single plants. Vigorous callus obtained from MS medium supplemented with different concentrations of 2,4-D, NAA and IBA were used for industrially important valepotriates [acevaltrate (ACE), valtrate (VAL) and didrovaltrate (DID)] analysis. High performance liquid chromatography analysis of callus revealed that medium with 2,4-D (1 mg/l) was found responsible for increasing ACE and DID yield, whereas VAL production was higher in case of medium supplemented with NAA (1 mg/l). However, the accumulation of valepotriates in callus decreased in logarithmic phase after 8 weeks. IBA was not benecial for the valepotriate synthesis, as it helped to accumulate signicantly lower concentration of ACE, VAL and DID. Micropropagated plantlets with well-developed root system were successfully acclimatized in greenhouse condition, in root trainers containing garden soil with a survival frequency of 100 %. As Indian valerian is a highly traded medicinal plant due to extensive use of its industrially important secondary metabolites, the present system can be utilized to obtain mass multiplication of the species as well as for the stable biomass and continuous valepotriate production for the pharmaceutical industries. Keywords Indian valerian Callus Regeneration Valepotriates

Introduction Valeriana jatamansi Jones. syn. V. wallichi (Fam: Valerianaceae) commonly known as Indian valerian is a perennial herb, distributed in Himalayas from Kashmir to Bhutan and Khasi hills of Northeast India at an altitude

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1,8003,500 m (Bos et al. 1997). V. jatamansi has been used in Ayurvedic, Unani and modern system of medicine due to its high medicinal and aromatic values. The essential oil components of Indian valerian make it one of the most demanded plant in the drug industry. Major constituents of its volatile oil are patchouli alcohol and bornyl isovaltrate (Bos et al. 1997). Essential oil from rhizomes exhibited antifungal and antibacterial activities (Girgune et al. 1980). 6-Methylapigenin and hesperidin isolated from rhizome of Indian valerian showed anxiolytic and sedative activities (Wasowski et al. 2002). The sedative and tranquilizing properties of the plant are also due to the presence of nonglycosidic iridoid esters known as valepotriates. The major valepotriates are valtrate (VAL), acevaltrate (ACE) and didrovaltrate (DID) due to which V. jatamansi possesses antispasmodic, anticonvulsive and antidepressant properties (Gupta et al. 1986). DID, VAL and their degraded product baldrinal were found to be cytotoxic in rat hepatoma cells (Bounthanh et al. 1981). In addition, the antitumor activity of DID was demonstrated in vivo on female mice KREBS II ascetic tumors (Marder et al. 2003). Due to extensive use in the modern pharma and perfumery industries, V. jatamansi is listed as one of the most exploited plant of Himalayan range. However, it is not yet cultivated anywhere in India for the large-scale production and all demands for its domestic and foreign trade are met from its wild population (Gupta et al. 2006). Over the years its indiscriminate collection has led to its large-scale depletion in the wild and has necessitated its replenishment and cultivation. During the last several years, there is an increase demand of in vitro culture techniques which offer a feasible tool for rapid clonal propagation and germplasm conservation of rare, endangered and threatened medicinal and aromatic plants (Abraham et al. 2010). Cultured plant cells synthesize, accumulate and sometimes exude many classes of secondary metabolites. Numerous alkaloids, saponins, cardenolides, anthraquinones, polyphenols and terpenes have been reported from in vitro cultures (Verpoorte et al. 2002; Vanisree and Tsay 2004). In dedifferentiated cells, some biosynthetic potential typical for the developed organs from which they were initiated can be conserved. In Pueraria lobata callus cultures, the bioactive isoavonoid content depended on the source organ, reecting relations in the mature plant (Matkowski 2004). A range of environmental and nutritional factors are known to inuence the biosynthetic pathways of secondary metabolites (Stintzing and Carle 2004). Plant growth regulators have been widely used in promoting the biosynthesis of both inducible and constitutive secondary metabolites, including medicinal compounds such as anticancer alkaloids (Vanisree and Tsay 2004; Verpoorte et al. 2002). The continuous monitoring of a chosen metabolite is a

prerequisite for the successful development of production technology. Also, the super-efcient clones of cultured cell or tissues can be selected by monitoring the level of the compound of interest, or can be complemented by a selecting agent facilitating the process. Though an earlier fragmentary report was published on micropropagation of V. jatamansi (Kaur et al. 1999), the result was scanty towards standardization of the protocol for the commercial scale. The present research work is based on twofold objectives, viz., to develop an efcient and rapid propagation protocol of V. jatamansi for the fulllment of market demand and to quantify the industrially important valepotriates (VAL, ACE and DID) accumulated in the calli of different ages. This is a rst attempt to study the effect of different growth regulators on in vitro accumulation of valepotriates in the callus of V. jatamansi.

Materials and methods Callus induction Leaf (ca. 1 9 1 cm), petiole (ca. 0.50.7 cm) and rhizomes (ca. 1 9 1 cm) explants were taken from a 12-month-old single genotype of V. jatamansi maintained in the greenhouse, BSI, ERC, Shillong, for callus induction studies. The explant cuttings of 2.02.5 cm long were rinsed in running tap water three times and washed in a 2 % (v/v) Tween 20 detergent solution for 15 min. Then the plant materials were surface-sterilized in a solution of 10 % (v/v) sodium hypochlorite for 5 min followed by 0.1 % (w/v) mercuric chloride for 1 min. Finally, the explants were rinsed 3 times with sterilized distilled water. The explants were established in Murashige and Skoogs (1962) basal medium supplemented with 3 % (w/v) sucrose and 0.8 % (w/v) agar-agar (Hi-media, Mumbai, India). The pH of media was adjusted to 5.8 before autoclaving at 15 psi and 121 C for 20 min. All the explants were cultured on MS medium supplemented with different concentrations of 2,4-D, NAA and IBA (0.25, 0.5, 1.0, 2.0, 3.0 mg/l). Callus cultures were subcultured at 4-week intervals on respective media. Regeneration of multiple shoot bud from callus Randomly selected compact calli were transformed to growth regulator-free MS basal medium to overcome the carryover effect of auxins. To evaluate the effect of growth regulators on the callus potential for shoot regeneration, calli were excised, divided into small pieces (0.5 9 0.5 cm) transferred to the regeneration medium for shoot induction. MS medium supplemented with different concentrations of TDZ (0.5, 0.75 mg/l) and kinetin (Kn; 2.0,

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3.0 mg/l) alone and in combination with NAA (0.5 mg/l) was used for shoot proliferations from callus in 150-ml culture asks (Borosil, India). Callus along with the initiated multiple shoot buds were subcultured on respective regeneration medium after 4 weeks interval to obtain healthy shoots. Culture conditions

explants each. All the valepotriates were analyzed with 3 replicates. To analyze the effect of various treatments, the data were subjected to analysis of variance (ANOVA) and statistical signicance between mean values was assessed by Duncans multiple range test at P \ 0.05 by using statistical software SPSS ver. 15 (SPSS Inc., Chicago, USA).

Results and discussion The cultures were maintained at 24 2 C and relative humidity (RH) of 50 5 % under 16 h photoperiod with 30 lmol m-2s-1 photosynthetic photon ux density (PPFD) provided by cool-white uorescent light tubes (Philips, India). Extraction and HPLC analysis Vigorous callus obtained from rhizome explants supplemented with 2,4-D (0.5, 1.0 mg/l), NAA (0.5, 1.0 mg/l) and IBA (1 mg/l) were used for valepotriates (ACE, VAL and DID) analysis. The calli were dried and subjected to dichloromethane extraction 3 times at room temperature (25 2 C). The extract was dissolved in methanol to obtain a concentration of 1 mg/ml, ltered through a membrane lter (0.22 lm pore size, Merck), analyzed by HPLC and compared with the reference compounds (Sigma Aldrich, USA). HPLC analysis of valepotriates was performed in a Shimadzu LC-10A gradient HPLC coupled with 2 LC10AD pumps, 10A UV detector and manual injector with a 20 ll sample loop. The separation conditions were: Waters Nova pack C18 column (25 9 4.6 mm i.d.); mobile phase acetonitrile: water; 80:20 (v/v); ow rate 1 ml/min; detector wavelength 254 nm and sensitivity 0.04 Aufs. Rooting and acclimatization Healthy shoots of 45 cm were excised and cultured for rooting on MS medium supplemented with different concentrations of auxins viz. indole-3-acetic acid (IAA), NAA and IBA (0.05, 0.10, 0.20 mg/l) in 150-ml culture asks (Borosil, India). MS medium without growth regulators was used as control. Plantlets with well-developed roots were removed from the culture medium, washed gently under running tap water, and transferred to root trainers containing garden soil and acclimatized under greenhouse condition (24 2 C temp. and 80 5 % RH) without use of any organic fertilizers. Data analysis The callus induction, shoot regeneration and rooting experiments were repeated three times with minimum 24 Callus induction The disinfection treatment used was efcient for in vitro establishment with approximately 94 % of the explants remaining aseptic. In V. jatamansi callus induction varied signicantly depending on the explant type. Among different explants used, rhizomes have the highest callus induction potential followed by leaf. Rhizome explants started swelling within 68 days, whereas leaf and petiole materials started within 1012 days of inoculation. MS basal medium without growth regulators exhibited no callus proliferation. All the calli in the growth regulatorssupplemented media were observed to be initiated from the cutting edge of the explants. The induced calli were fast growing, yellowish green and compact (Fig. 1a, b). The callus induction frequency was found optimum in rhizome explants (Table 1) on media supplemented with 0.5 mg/l 2,4-D (86 %) followed by 0.5 mg/l NAA (75.8 %). In our study, 0.5 mg/l 2,4-D was the best auxin responsible for obtaining the vigorous and compact callus from rhizome (0.23 0.06 g), leaf (0.17 0.06 g) and petiole (0.13 0.03 g) explants. Effect of different explants and growth regulators on callus induction of Allium chinense was observed by Yan et al. (2009). Different tissues may have different levels of endogenous hormones, and therefore, the type of explants source would have a critical impact on the callus induction and its regeneration success. The selection of proper donor plants and organs should already be considered when starting the culture, unless it can be overcome by a suitable treatment. However, in most circumstances the dedifferentiation apparently also involves some of the biochemical properties of the cells. The modication of relative biosynthesis to degradation ratios of a desired product can also inuence the nal levels of a desired compound in the culture (Stintzing and Carle 2004). The degree of callus formation was varied within all the explants as well as PGRs used. Within different concentrations of NAA and IBA, 0.5 mg/l NAA produced the richest callus from rhizomes (0.21 0.07 g) with callus induction frequency 75.8 % followed by 1.0 mg/l NAA (0.16 0.01 g) with 68.2 %. No somatic embryos or adventitious roots formed from any callus. 2,4-D was

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Fig. 1 Callus induction and regeneration of plantlets from callus in V. jatamansi Jones. a 8-week-old vigorous callus (bar 10 mm) induction from leaf explant on MS medium with 0.5 mg/l 2,4-D. b 8week-old vigorous callus induction (bar 10 mm) from rhizome explant on MS medium with 0.5 mg/l 2,4-D. c, d Regenerated

plantlets (bar 15 mm) from callus on MS medium with 0.75 mg/l TDZ and 0.5 mg/l NAA. e 8-week-old in vitro-raised plantlet with a healthy root system. f Acclimatized plants in the greenhouse condition and g 1-year-old established plants

considered as one of the most effective auxins for the callus induction and found effective in case of Pennisetum glaucum (Jha et al. 2009), Pinus caribaea (Akaneme and Ene-Obong 2005), Tylophora indica (Thomas 2009) and Juncus effusus (Xu et al. 2009). Medium fortied with

different concentrations of IBA produced signicantly less biomass than the other auxins used. However, the rate of callus proliferation was inversely proportional to the concentrations of auxins in V. jatamansi (Table 1). Compact calli are important in the in vitro cultures as they have

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Acta Physiol Plant (2013) 35:5563 Table 1 Effect of different concentrations of auxins on callus induction from different explants of V. jatamansi Plant growth regulators (mg/l) 2,4-D 0.25 0.5 1.0 2.0 3.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 NAA 0.0 0.0 0.0 0.0 0.0 0.25 0.5 1.0 2.0 3.0 0.0 0.0 0.0 0.0 0.0 IBA 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.25 0.5 1.0 2.0 3.0 Callus induction frequency (%) Rhizome 54.6 86.0 72.5 62.6 55.2 70.5 75.8 68.2 54.0 50.6 44.7 64.8 70.8 42.8 36.5 Petiole 48.2 64.1 62.7 48.2 42.8 52.6 59.0 51.6 46.2 40.5 38.9 50.5 56.2 40.1 30.5 Leaf 51.0 74.2 66.0 50.4 44.4 61.4 68.9 58.5 48.7 42.8 42.4 56.9 62.0 42.2 32.6 Mean weight of the callus (gm) Rhizome 0.19 0.09c 0.23 0.06a 0.21 0.04b 0.17 0.08d 0.14 0.01f 0.17 0.01d 0.21 0.07b 0.16 0.01e 0.12 0.01h 0.10 0.01j 0.11 0.01i 0.12 0.01h 0.13 0.07g 0.10 0.05j 0.09 0.04jk Petiole 0.12 0.02b 0.13 0.03a 0.12 0.03b 0.12 0.04b 0.11 0.05c 0.12 0.02b 0.11 0.06c 0.11 0.06c 0.11 0.06c 0.08 0.09ef 0.09 0.04de 0.10 0.04d 0.09 0.04de 0.07 0.04efg 0.07 0.04efg Leaf

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0.14 0.07b 0.17 0.06a 0.14 0.06b 0.13 0.06c 0.11 0.06e 0.13 0.03c 0.14 0.09b 0.13 0.08c 0.11 0.07e 0.09 0.03fg 0.10 0.05f 0.11 0.05e 0.12 0.07d 0.08 0.08gh 0.07 0.04ghi

Values represent mean SE of three replicates with 24 explants each. Means within a column followed by different letters differ signicantly at P B 0.05 as compared by Duncans multiple range test

ability for organogenesis. They are more efcient to develop chlorophyll than friable calli from the same explants; this might be due to the chloroplast development and integrity favored by cell aggregation (George and Sherrington 1984). Shoot regeneration from callus The regenerative ability of proliferated compact calli was studied by the application of cytokinins alone and in combination with auxin. After 2 weeks of culture, most of

the calli turned green in order to give response towards regeneration of plantlets. Shoot primordia appeared after 2 weeks in the regeneration medium. MS medium supplemented with 0.75 mg/l TDZ in combination with 0.5 mg/l NAA showed the highest regeneration frequency (88.6 %) and produced the highest number of shoot buds (15.20 0.20) capable of growing into single plants (Table 2). Medium fortied with equal concentration (0.5 mg/l) of TDZ and NAA produced 12.30 0.21 shoots having 80.4 % regeneration potential. Addition of NAA at low concentration with TDZ-supplemented medium

Table 2 Inuence of different plant growth regulators on regeneration of shoot buds and elongation of proliferated shoots from callus of V. jatamansi Plant growth regulators (mg/l) TDZ 0.5 0.75 0.5 0.75 0.0 0.0 0.0 0.0 0.0 0.0 Kn 0.0 0.0 0.0 0.0 1.0 2.0 3.0 1.0 2.0 3.0 NAA 0.0 0.0 0.5 0.5 0.0 0.0 0.0 0.5 0.5 0.5 56.3 64.2 80.4 88.6 31.0 34.8 52.1 53.0 66.2 48.2 7.20 0.24d 11.90 0.31c 12.30 0.21b 15.20 0.20a 3.50 0.09g 3.70 0.15g 5.10 0.17e 5.30 0.14e 5.40 0.30e 4.70 0.21ef 2.20 0.17de 2.50 0.08c 2.80 0.07b 3.60 0.07a 2.10 0.11cd 2.30 0.12cd 2.40 0.11cd 2.10 0.10cd 2.40 0.12cd 2.30 0.06cd Regeneration frequency (%) Mean number of shoots/callus Mean shoot length (cm)

Values represent mean SE of three replicates with 24 explants each. Means within a column followed by different letters differ signicantly at P B 0.05 as compared by Duncans multiple range test

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60Acevaltrate contents (g% dry weight)

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1.2 1

a4 wk

0.8 0.6 0.4 0.2 0

8wk 12wk

0.5 mg/l 2,4-D 1.0 mg/l 2,4-D 0.5 mg/l NAA 1.0 mg/l NAA 1.0 mg/l IBA

Auxin type and concentration Valtrate contents (g% dry weight)0.6 0.5 0.4 0.3 0.2 0.1 0 0.5 mg/l 2,4-D 1.0 mg/l 2,4-D 0.5 mg/l NAA 1.0 mg/l NAA 1.0 mg/l IBA

b4 wk 8wk 12wk

potential use of TDZ in the regulation of adventitious shoot proliferation and hypothesized on the synergism existing between TDZ and other endogenous and exogenous auxins. However, signicant decreased shoot regeneration found in case of medium supplemented with cytokinin alone than in combination with auxin. The efciency of TDZ on shoot regeneration in many medicinal plants was reported (Thomas 2003, 2007; Mithila et al. 2003; Sanikhani et al. 2006). According to George and Sherrington (1984) cell differentiation and morphogenesis may be promoted by insufcient oxygen environment such as in compact calli. On the other hand, they reported that due to this factor friability of callus is mostly associated with somatic embryogenesis while compact callus can be readily used for organogenesis. Also, in our case no somatic embryogenesis occurred during plant regeneration. Effect of PGRs on valepotriates accumulation in callus Callus derived from medium supplemented with different concentrations of 2,4-D (0.5, 1.0 mg/l), NAA (0.5, 1.0 mg/l) and IBA (1 mg/l) were subjected to HPLC analysis for quantifying ACE, VAL and DID and the result revealed that all treatments showed the presence of valepotriates. Medium with 2,4-D (1 mg/l) was found to be responsible for increasing ACE (Fig. 2a) and DID (Fig. 2c) yield, whereas VAL (Fig. 2b) production was higher in case of medium supplemented with NAA (1.0 mg/l). The increased concentration of auxins like 2,4-D and NAA could have caused a mild but chronic oxidative stress response in undifferentiated mass of cells potentially capable of inducing valepotriate accumulation. Reactive oxygen species have been shown to trigger the production of various secondary metabolites, including terpenes (Zhao et al. 2005). 8-week-old cultures produced optimum biomass and valepotriates yield (Fig. 2). The accumulation of valepotriates in callus decreased in logarithmic phase after 8 weeks. Previously, the presence of valepotriates was characterized in callus of Valeriana glechomifolia (Maurmann et al. 2009). In our study, ACE and DID were the major valepotriates found in V. jatamansi callus. IBA was not benecial for the valepotriate production, as it helped to accumulate signicantly lower concentration of ACE, VAL and DID than 2,4-D and NAA. The observed impact of auxins on valepotriate metabolism was not necessarily dependent on phytohormone-induced developmental changes on callus (De Klerk et al. 1999). The benets of auxin exposure were apparently correlated to auxin stability, since metabolically stable type of auxins, such as IBA was not benecial for valepotriate yield. Differences in auxin stability and auxin-induced secondary effects could also explain the concentration and auxin-type dependence of valepotriate content responses in calli (Bello

Auxin type and concentration Didrovaltriate content (g% dry weight)

1.4 1.2 1 0.8 0.6 0.4 0.2 0

c4 wk 8wk 12wk

0.5 mg/l 2,4-D 1.0 mg/l 2,4-D 0.5 mg/l NAA 1.0 mg/l NAA

1.0 mg/l IBA

Auxin type and concentration

Fig. 2 Time-course effect of different plant growth regulators on valepotriate accumulation in callus of V. jatamansi from rhizome explants: a inuence of different efcient auxins on acevaltrate accumulation, b inuence of different efcient auxins on valtrate accumulation, c effect of different efcient auxins on didrovaltrate accumulation. Bar indicates mean of 3 replicates SE (n = 10)

enhanced the callus regeneration frequency with more number of shoot response (Fig. 1c, d). The same effect was observed in case of Kn supplemented media in combination with NAA. However, 2 mg/l Kn along with 0.5 mg/l NAA showed 66.2 % regeneration frequency proliferating 5.40 0.30 shoots (Table 2). Synergistic effect of auxin and cytokinin on shoot regeneration from calli were observed in Solanum tuberosum (Shirin et al. 2007), Cynondon dactylon (Zhang et al. 2007), Ipomoea obscura (Mungole et al. 2009), Juniperus excels (Shanjani 2003), Aframomum corrorima (Tefera and Wannakrairoj 2006) and Pennisetum glaucum (Jha et al. 2009). Huetteman and Preece (1993) and Gyves et al. (2001) emphasized the

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de Carvalho et al. 2004). However, the comparative analysis of individual valepotriate accumulation within every 4-week interval showed signicant variation among all the treatments analyzed. Active secondary metabolite production through callus culture is known to be an effective process in pharmaceutical industries. Several industrially important secondary metabolites, viz., anthocyanin from Panax sikkimenesis (Mathur et al. 2010), cerpegin from Ceropegia juncea (Nikam and Savant 2009), cynarin from Cynara cardunculus (Silvia et al. 2006), rosmarinic acid from Lavandula ofcinalis (Georgiev et al. 2006), rutin from Hemidesmus indicus (Misra et al. 2005), crocin from Crocus sativus (Chen et al. 2003), lithospermic acid from Salvia miltiorrhiza (Morimoto et al. 1994) and piceatannol from Arachis hypogaea (Ku et al. 2005) were produced by in vitro callus culture. Rooting and acclimatization In vitro shoots, regenerated from shoot clusters proliferated on multiple shoot induced medium and callus regeneration medium were separated and used for the rooting experiments. Rooting initiated after 2 weeks in all cultures including control. But, the response of rooting (%), number and length of roots were achieved signicantly higher when cultured with auxin-supplemented media. Among different auxins used, NAA (0.05, 0.1 mg/l) and IAA (0.1 mg/l) were effective in producing longer and healthy roots with 100 % response (Table 3). IBA showed significantly lower root-inducing potential both in response to root numbers and root length. MS medium fortied with 0.1 mg/l NAA produced the highest number of roots (17.30 2.01) followed by 0.05 mg/l NAA (12.40 1.20). NAA when used as low concentrations was considered as an effective rooting hormone in many plant systems

(Mao et al. 1995; Sanches-Gras and Calvo 1996; Rout et al. 2000). Also, IAA was reported to enhance the root formation in case of Hedeoma multiorum (Koroch et al. 1997) and Woodfordia frusticosa (Krishnan and Seeni 1994). However, MS basal medium without auxins produced signicantly lower number of roots and were not found healthy for hardening in greenhouse. There are many reports on the microshoots of various medicinal plants rooted on only MS medium without the growth regulators (Christine and Chan 2007; Mao et al. 1995). Explants having a functional rooting system are more likely to survive transition to greenhouse. Roots were washed thoroughly before being transferred to root trainers. Micropropagated plantlets with well-developed root system were successfully acclimatized in greenhouse condition, in root trainers containing garden soil with a survival frequency of 100 % (Fig. 1f). The in vitro-derived plants were phenotypically similar to the parental stock and no morphological abnormalities have been observed in the micropropagated plants.

Conclusion A highly efcient and reproducible protocol was developed for the large-scale production of V. jatamansi. The indirect organogenesis system of Indian valerian can provide a mass production of disease-free and genetically uniform plant materials throughout the year for the fulllment of market demand globally and conservation of the species. The standardized protocol can offer stable biomass and continuous valepotriate production for the pharmaceutical industries. Furthermore, using bioreactor, callus induction protocol can be useful for continuous callus culture and the extraction of valepotriate on a commercial scale.

Table 3 Effect of different concentrations of auxins on rooting of in vitro-raised elongated shoots in V. jatamansi Auxins (mg/l) NAA 0.0 0.05 0.10 0.20 0.0 0.0 0.0 0.0 0.0 0.0 IBA 0.0 0.0 0.0 0.0 0.05 0.10 0.20 0.0 0.0 0.0 IAA 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.05 0.10 0.20 34.2 100 100 64.6 38.5 60.8 42.0 75.2 100 52.5 3.30 0.61e 12.40 1.20b 17.30 2.01a 7.20 0.82d 6.15 0.98d 10.80 1.85bc 5.20 0.61d 7.80 0.38d 12.10 1.29b 5.80 0.61d 4.10 0.21e 8.30 0.24b 9.30 0.39a 7.90 0.29bc 7.70 0.21bc 8.10 0.21bc 6.60 0.20d 7.90 0.32bc 9.20 0.39a 7.60 0.20bc Response of rooting (%) Mean number of roots/explant Mean root length (cm)

Values represent mean SE of three replicates with 24 explants each. Means within a column followed by different letters differ signicantly at P B 0.05 as compared by Duncans multiple range test

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However, the valepotriate accumulation mainly depends on the growth regulators used and age of the calli. As V. jatamansi is a highly traded medicinal plant due to extensive use of its industrially important secondary metabolites, the present micropropagation system can be utilized for further research on metabolic pathways and transgenic approach to build up the value added products and production of quality material. Author contribution J. Das performed the experiment, analyzed the data and wrote the manuscript. A.A. Mao and P.J. Handique designed the work, edited the manuscript and supervised the entire research.Acknowledgments The authors are thankful to the Joint Director, BSI, Shillong, for providing the facilities and to the Department of Biotechnology (DBT), Government of India, New Delhi, India, for the award of research fellowship to J.D.

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