Cent. Eur. J. Biol. • 9(9) • 2014 • 853-863 DOI: 10.2478/s11535-014-0320-3

Central European Journal of Biology

Enhanced production of valerenic acid in hairy root culture of Valeriana officinalis by elicitation

Research Article Mohammad Reza Dini Torkamani1, Morad Jafari2,3*, Nasser Abbaspour1, Reza Heidary1, Naser Safaie4

1Department of Biology, Faculty of Science, University of Urmia, P.O. Box 165, Urmia, Iran 2 Department of Plant Breeding and Biotechnology, Faculty of Agriculture, University of Urmia, P.O. Box 165, Urmia, Iran 3Department of Agricultural Biotechnology, Institute of Biotechnology, University of Urmia, P.O. Box 165, Urmia, Iran 4Department of Plant Pathology, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran Received 29 November 2013; Accepted 03 April 2014

Abstract: Valerenic acid (VA) is a pharmacologically-active found in (Valeriana officinalis L., Valerianaceae) roots and rhizomes. The plant produces only small amounts of this metabolite naturally. So, induction of hairy roots as well as elicitation can be useful to increase its commercial production. In this study, Wild-type strain ‘A13’ of Agrobacterium rhizogenes was used to induce hairy roots in valerian. The influence of three different elicitors including Fusarium graminearum extract (FE), methyl jasmonate (MJ) and salicylic acid (SA) on VA production in the selected hairy root line ‘LeVa-C4’ was also investigated. The 23-day-old cultures were treated with different concentrations of the elicitors at exposure time of 3 and 7 days. FE (1%) and MJ (100 µM L-1) highly promoted VA production at 7 days after elicitation, to a level of 12.31- and 6-fold higher than that of non-elicited controls, respectively, and FE did not exert any negative effects on biomass yield of hairy root. SA did not significantly increase the production of VA. This is the first time study to assess the elicitation of hairy root cultures to promote VA biosynthesis in valerian and the resulting experiments demonstrated that F. graminearum extract and MJ were indeed a potent inducer of VA biosynthesis.

Keywords: Elicitation • Hairy root culture • Valerenic acid production • Valeriana officinalis © Versita Sp. z o.o.

1. Introduction as well as a number of other constituents [4]. Although the identity of the active components responsible for Valerian (Valeriana officinalis L.), a perennial herb the beneficial health effects in valerian is still uncertain, from the Valerianaceae family, is native to Europe and in recent findings the main pharmacological effects have Asia. It has been used as a medicinal herb worldwide been attributed to valerenic acid and its derivatives [1,2]. Pharmaceutical application of valerian is due including acetoxyvalerenic acid and hydroxyvalerenic to its , anticonvulsant, effects, and acid [5,6]. Also, VA along with its derivatives is often anxiolytic activity. Lately, the potential cytoprotective used to indicate medicinal quality [4,7]. effect of an aqueous extract of valerian on human The production of medicinal compounds from neuroblastoma cells has also been demonstrated [3]. extraction of wild or cultivated plants can be limited The compounds accumulated mainly in the roots and by various problems: plants difficult to cultivate, risk rhizomes of valerian include valepotriates (iridoids), of extinction for overexploited plants, and geopolitical the components of the volatile oil, as problems, among other causes [8]. In vitro culture valerenic acid (VA) and its derivatives, monoterpenes of plants can overcome these problems, since the

* E-mail: [email protected] 853 Valerenic acid production in hairy roots of valerian by elicitation

environmental conditions that affect plant metabolism germination rate, seeds were pre-treated with can then be strictly controlled. Working with plant cells commercial grade sulfuric acid (10 %) for 15 min. After drastically reduces preparation time, handling and complete washing with sterile distilled water, the seeds storage costs compared with traditional whole plant were surface-sterilized in 70% (v/v) ethanol for 1 min, approaches [9]. Although the plant cell is considered rinsed with sterile distilled water for 3 min and immersed to be totipotent, the biosynthesis of many secondary in 2% (v/v) solution of sodium hypochlorite (commercial metabolites requires a certain level of differentiation bleach) for 10 min, followed by three rinses with sterile of the tissues. Hairy roots produced by Agrobacterium distilled water. The seeds were blotted dry with sterilized rhizogenes infection are characterized by fast growth, filter paper and cultured on MS[29] basal medium biosynthetic stability and ability to grow in hormone–free containing 3% sucrose and 0.7% plant agar (Duchefa, medium, all advantages that make transformed roots Netherlands). The cultures were maintained in a growth efficient sources for secondary metabolite production chamber at 22±2°C and 70% humidity and a 16/8-h [10]. The greatest advantage of hairy roots is the ability light/dark photoperiod with a photon flux density (PFD) to produce higher levels of secondary metabolites as of 60 μmol/m2s. After seed germination, seedlings were compared to intact plants [11]. Medicinal plants have transferred to fresh medium for further growth. The pH been widely explored for potential hairy root culture and of the medium was adjusted to 5.9 with KOH (0.1 N) or production of their bioactive compounds [12]. HCl (1 N) prior to autoclaving at 121°C temperature for In vitro plant secondary metabolite production is 20 min. enhanced through elicitation in plant cell or tissue culture systems [13]. Elicitors as compounds or 2.1.2. Infection of leaf explants and hairy root induction treatments have been proved to be an effective method Among the different explants tested (data not shown), to increase secondary metabolite production. A number 5-week-old leaf explant was used for hairy root induction of elicitors such as methyl jasmonate (MJ) [14-17] and because of its capacity for higher transformation salicylic acid (SA) [14,18] have been used successfully efficiency. So, leaf explants obtained from 5-week-old for enhanced production of secondary metabolites in in vitro grown seedlings were cut into small segments hairy root cultures of many plant species. In addition, (5–10 mm) and were dipped in the suspension of fungal elicitors are significant in triggering phytoalexin Agrobacterium rhizogenes A13 strain for 30 min and production in cultured cells and have been used for the explants were then blotted with sterile filter paper to enhanced production of secondary metabolites in some remove excess liquid. Then explants were transferred medicinal plants [19-22]. High biosynthetic capability onto 100×15-mm sterile petri dishes (15 explants per of valepotriate production has been reported in hairy dish) containing agar-solidified MS co–cultivation root cultures of Valeriana wallichii DC [23], Valerianella medium and maintained in the dark at 24±2°C for 2 days. discoidea L. Loisel [24], Valeriana officinalis L. var. As a control, a few explants were immersed in sterile sambucifolia Mikan [25] and Valerianella locusta [26] and distilled water and were cultured the same way. After in adventitious root cultures of Valeriana glechomifolia 2 days, the explants were rinsed with sterile distilled [27] and V. officinalis L. [28]. water and placed on solid hormone-free MS medium To our knowledge, there is no published study on containing 500 mg/l cefotaxime. After the appearance of elicitor-induced production of secondary metabolites hairy roots at the wound sites of the explants, they were in hairy root cultures of Valerian. In the present study, subcultured at intervals of 2 weeks on a fresh medium for the first time, the effects of three different elicitors and the concentration of cefotaxime was gradually i.e. MJ, SA and Fusarium graminearum extract with reduced and then omitted after the third subculture. different time exposure were investigated on growth rate and VA content of valerian hairy root cultures. This paper 2.1.3. Establishment of hairy roots suggests the optimal concentration of elicitors for high- Hairy roots with more than four lateral branches were cut level VA production in hairy root cultures of valerian. and transferred to 100 ml Erlenmeyer flaks containing 25 ml of hormone–free MS liquid medium supplemented with 3% (w/v) sucrose. The root cultures were incubated 2. Experimental procedures on an orbital shaker (100 rpm) in darkness at 22°C and subcultured on fresh liquid medium at 2 week- 2.1 Hairy root production intervals. To measure hairy root growth rate in terms of 2.1.1. Plant materials fresh weight (FW, mg/25 ml culture medium), they were Seeds of valerian were provided by Pakan Bazr harvested after 4 subcultures and blotted dry on sterile Co. (Isfahan, Iran) at late August 2011. To increase filter paper to remove excess moisture.

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2.2 Polymerase chain reaction (PCR) analysis At the end of the exposure times, the root cultures were for the rolB and virD2 genes harvested and subjected to VA content analysis. Growth The extraction of DNA from both hairy roots and rate of elicitor-treated hairy root cultures was also untransformed roots (control) was carried out using determined in term of dry weight (DW, mg/25 ml culture the CTAB method [30]. For molecular confirmation medium) and for DW measurement, the samples were of the transgenic nature of hairy roots, the presence lyophilized. of the rol genes located on the T-DNA as the main determinants of hairy root development were examined 2.4 VA quantification by PCR analysis using corresponding gene-specific For VA extraction, hairy roots and untransformed roots primer pairs. The Ri plasmid of A. rhizogenes strains were lyophilized and ground to a fine powder. The root ‘A13’ was used as a positive control. The primer powder (approx. 200 mg) was extracted three times with sequences to amplify a 780-bp portion of the rolB gene 5 ml of 70% MeOH, sonicated for 10 min and then diluted were 5ʹ-TTAGGCTTCTTTCTTCAGGTTTACTGCAGC- to a final volume of 30 ml with MeOH. All samples were 3ʹ (forward primer) and filtered through a 0.45 µm micro-filter (Merck, Germany) 5ʹ-TGGATCCCAAATTGCTATTCCTTCCACGA- and this solution was used for chemical analysis. 3ʹ (reverse primer). The primers High-performance liquid chromatography (HPLC) (5ʹ-ATGCCCGATCGAGCTCAAGT-3ʹ and analysis was carried out using a Knauer HPLC 5ʹ-CCTGACCCAAACATCTCGGCTGCCCA-3ʹ as system (Germany) equipped with a UV detector forward and reverse primers, respectively) specific to k-2501. Separation was achieved by reverse-phase the virD2 gene present in the non-transferred virulence isocratic C18 column (250×3 mm), with a pre-column region of the A. rhizogenes Ri plasmid were also used (25×4.6 mm, particle size 5 µm). The mobile phase A in PCR analysis to ensure transformed root tissue was was 0.5% (v/v) phosphoric acid and the mobile phase B free of bacterial cells. PCR reaction profiles involved: was a 0.5% phosphoric acid: mixture (27:73). initial denaturation at 94°C for 5 min, followed by 35 The flow rate was 1.5 ml min−1 and the injection volume cycles of 94°C for 1 min, 58°C for 1 min, 72°C for 1 min, was 10 µl. Detection was by UV at wavelength 225 nm. and a final extension at 72°C for 10 min. The amplified VA was obtained from Sigma Aldrich Co. (USA). Standard were analyzed on 0.8% agarose gel at 90 V for solution was prepared by dissolving VA in methanol to 1 h and stained with ethidium . obtain solutions with 7 different concentrations in the range of 0.01562-1 µg L-1. The calibration curves were 2.3 Elicitor preparation and treatment established based on measuring their respective peak A stock solution of MJ was obtained by dissolving it areas. All analyses were repeated three times employing in 96% ethanol after filter sterilization using 0.22 µm three replicates each. The results were expressed Sartorius filters. A stock solution of SA was prepared as mean values ± SE. The metabolite contents were in methanol, and then filter-sterilized. F. graminearum calculated as mg of the compound per gram of root DW. [Teleomorph: Gibberella zeae (Schwenitz) Petch] was also used as fungal elicitor. Fungal extract (FE) 2.5 Statistical analysis was prepared as described by Esmaeilzadeh et al. All experiments were repeated three times and for each [19]. Briefly, one-week-old mycelia of F. graminearum set of experiments, four replicates (each replication were harvested and rinsed with sterile distilled water. consisted of one Erlenmeyer flask) were included. The collected mycelia were ground under liquid The data are expressed as the average of results of and suspended with water to a final concentration of triplicate experiments and obtained data were subjected 250 mg L-1. The suspension was centrifuged at 10,000 g to analysis of variance (ANOVA) using the general linear for 10 min and then the supernatant was autoclaved for models (PROC GLM) procedure in the Statistical Analysis 10 min. System (SAS 9.1) programme package. The means For the elicitation treatment, 23-day-old hairy root were compared using Fisher’s least significant difference cultures of the LeVa-C4 line were inoculated into 100-ml (FLSD) test at the 5% probability level. Erlenmeyer flasks containing 25 ml of liquid MS medium supplemented with various concentrations of MJ (50, 100, 200 µM L-1), SA (50, 100, 150 µM L-1) or FE 3. Results and Discussion (0.5, 1, 2% v/v) for different exposure times (3 and 7 days). The control cultures received the same amount 3.1 Establishment of hairy root cultures of 96% ethanol or water. All cultures were incubated Leaf explants were infected by strain A13 of on an orbital shaker at 100 rpm in darkness at 25°C. A. rhizogenes. Hairy root formation was observed

855 Valerenic acid production in hairy roots of valerian by elicitation

directly 1 to 2 weeks after inoculation on the wounded well growth and biomass increment (Figure 1D) and the sites of the leaf explants of valerian without callus hairy roots showed a progressive increase in biomass formation (Figure 1A). The leaf explants of the 5 during the culture period (Figure 1E-H). The presence weeks age seedlings were shown to be susceptible of the 780 bp rolB amplified product in the hairy root to inoculation with A. rhizogenes, as a transformation samples was showed by PCR analysis confirming frequency of 86.5% was achieved. Hairy roots of the transgenic nature of the roots (Figure 2A). valerian showing typical transformed root properties had The rolB gene is absolutely essential for the induction extensive lateral branching and fast and plagiotropic of hairy roots [31]. To diagnosing the presence of any growth (Figure 1B). Hairy roots of valerian were easily remaining agrobacteria in the roots; the virD2 gene, grown in a hormone-free MS medium (Figure 1C) in located outside the T-DNA, is amplified by PCR. Non compared to the untransformed root (as control) with no PCR amplification for the gene demonstrated that Ê

Ê

FigureÊ 1. Hairy root induction in valerian through inoculation with Agrobacterium rhizogenes strain A13. A) profuse hairy roots induced from leaf segments on solidified MS medium, 3 weeks after inoculation. B) a typical hairy root phenotype characterized by fast growth, high lateral branching and lack of geotropism at 1 month of culture of the root segment in the dark after elimination of bacteria. C) fast Ê and mat-like growth of a hairy root line in a hormone-free liquid MS medium compared to unsatisfactory growth of the untransformed normal root (D). E-H) progressive increment in biomass of hairy root line LeVa-C4 cultured in hormone-free liquid MS medium during the culture period of 35 days. Ê 856 Ê

Ê M.R.D. Torkamani et al.

there was no bacterial contamination of the hairy root to 1.5% decreased growth (Figure 3A). Decrease (50%) tissues (Figure 2B). in growth in suspension cultures of Linum album using Measuring the fresh biomass of transformed (hairy) F. graminearum extract has been reported [19]. roots and the normal root under the same condition MJ treatment caused a slight decline in biomass after 4 subcultures showed that the transformed throughout the exposure period and maximum inhibition samples produced more biomass (maximum biomass of of growth (decrease by 12.7%) was found after 7th days 4133 mg FW/25 ml culture medium) in comparison with of exposure time at 200 µM MJ (Figure 3B). The addition the untransformed samples (132 mg FW/25 ml culture of MJ to the cultures also changed the color of the roots medium). The strain of Agrobacterium influences the from white to dark brown. This negative effect may be development, growth rate and secondary metabolite due to a direct toxic effect of MJ and loss of viability of the production in transformed root cultures [32]. The A13 culture [38]. These results are in agreement with earlier strain of A. rhizogenes is one of the aggressiveness reports [19,32,35]. SA decreased biomass 3 days after strains whose superiority for hairy root induction and treatment but did not affect the growth rate at exposure bioactive compound production have been reported in time of 7 days, and DW of elicited roots at the end of several other medicinal plants [33,34]. the culture period was similar to the control (Figure 3C). This is in accordance with previous studies reporting 3.2 Effects of elicitors on biomass production that SA treatment has no significant negative effects on of hairy roots growth of Fagopyrum esculentum suspension cultures As a result of the transformation with A. rhizogenes and [35] and root cultures of Angelica gigas [13]. However, hairy root production, the LeVa-C4 hairy root line was a negative effect of SA on growth of plant cell culture selected for elicitation studies, because roots of the line has also been reported by several studies [36,37]. were a light yellow, giving fast and stable growth without In the present study, to enhance overall productivity a tendency to develop callus. 23-day-old hairy root and limit negative effects of elicitors (especially MJ) on cultures of the LeVa-C4 line was treated with various growth of roots, elicitation in valerian hairy roots were elicitors and all the elicitors were shown to influence tested when the roots were in the late linear or early the growth of hairy roots in a dose-dependent and stationary growth phase (23rd day of the culture period). point time manner (Figure 3). FE did not inhibit growth at a time exposure of 3 days; but 7 days after treatment 3.3 Effects of elicitors on VA production the fungal extract at a concentration of 1% significantly All concentrations of F. graminearum extract led increased biomass by 13% compared to the non- to a significant increase in VA level at both exposure times elicited control, whereas increasing of FE concentration compared to the control, however the highest content of

Figure 2. PCR analysis for hairy roots of valerian using the rolB gene (A) and virD2 gene (B) specific primers. M– 1 Kb DNA Ladder (Fermantas), Ri plasmid– the plasmid from A. rhizogenes strain A13 as a positive control, LeVa-A6, LeVa-C4 and LeVa-G7– Transgenic hairy root lines raised from leaf explants, UTR– Untransformed normal root as first negetive control, NDT– Non-DNA template PCR reaction as second negative control.

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Valerenic acid production in hairy roots of valerian by elicitation

26Ê

Figure 3. Effect of Fusarium graminearum extract (FE) (A), methyl jasmonate (MJ) (B) and salicylic acid (SA) (C) on growth of hairy root line LeVa-C4 of valerian. 23-day-old hairy root cultures were exposed to various concentrations of FE, SA and MJ for 3 and 7 days. Error bars represent the standard error of the mean of three repeated experiments. Means with the same letter are not significantly different according FLSD test (P < 0.05).

VA (3.02 mg/g DW) was observed after treatment with as intermediate signals in elicitor-induced secondary 1% FE for 7 days (Figures 4, 5A). The levels of VA were metabolite accumulation in plant cells [39]. Few studies 11.06- and 12.31 times higher than that of non-elicited refer to the plant secondary metabolism mediated by the control (0.24 mg g-1 DW) at exposure times of 3 and 7 days, fungal elicitor. It has been speculated that recognition respectively. This substantial effect of FE has also been of the fungi elicitor by host plants triggers a cascade of reported for podophyllotoxin production in suspension signal transduction, which gives rise to a series of plant culture of Linum album [19]. Accumulation of JA and its defense responses similar to plant pathogen interaction, derivatives, such as methyl jasmonate (MJ) is one of the thus leading to a noticeable change in plant metabolic common responses of plant cells to fungal elicitors and state [40].

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Figure 4. Chromatograms of standard valerenic aicd (A), non-elicited control (B) and hairy root cultures exposed with 1% (v/v) Fusarium graminearum extract (for a 7 days) which was the best treatment of elicitation (C).

Jasmonic acid and its analogues are known to induce of valerian is shown in Figure 5B. MJ had positive the of genes involved in plant defense effect on VA accumulation at both timepoints examined. metabolism [41]. The effects of MJ concentrations at two However, increasing MJ concentration from 100 to exposure times on VA production in hairy root cultures 150 µM L-1 significantly decreased VA content

859 Valerenic acid production in hairy roots of valerian by elicitation

28Ê

Figure 5. EffectÊ of Fusarium graminearum extract (FE) (A), methyl jasmonate (MJ) (B) and salicylic acid (SA) (C) on valerenic acid (VA) production in hairy root line LeVa-C4 of valerian. 23-day-old hairy root cultures were exposed to various concentrations of FE, SA and MJ for 3 and 7 days. Error bars represent the standard error of the mean of three repeated experiments. Means with the same letter are not significantly different according FLSD test (P < 0.05).

even below (0.16 mg g-1 DW) that of the control roots (0.246 mg g-1 DW). MJ has also been reported cultures (0.246 mg g-1 DW) after 7 days of exposure. to promote production of valtrate in the Valeriana The highest increase in VA accumulation amurensis adventitious root cultures [42]. (2.296 mg g-1 DW) was obtained with the addition of It is known that secondary metabolites increase 100 µM L-1 MJ for a time period of 7 days, which was through MJ elicitation, but to reach the highest level about 6-fold higher than that of non-elicited control hairy the time required is different for each biosynthetic

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pathway and even each compound type. For example metabolite production when SA accumulation is the diterpenoid content in Salvia sclarea hairy root impaired and it has been suggested that an unusual cultures reached a maximum after 7 days of exposure to complementary relationship exists between JA and SA 125 µM MJ [38]. But a longer period (10 days) was in mediating plant secondary metabolite accumulation, required for reaching the highest level of valepotriate however SA does not have similar effects on JA production in hairy roots of Valerianella locusta [26]. In signaling in mediating the fungal elicitor-induced the present study, exposure to MJ for 7 days enhanced secondary metabolite biosynthesis [46]. VA accumulation two-fold higher than 3 days of exposure In conclusion, the results obtained in this study time (1.266 mg g-1 DW). demonstrated that F. graminearum extract at 1% SA slightly induced VA accumulation in the hairy root (v/v) and MJ at 100 µM L-1 significantly enhanced the cultures in both exposure times compared to controls biosynthesis of valerenic acid from hairy root cultures. (Figure 5C). VA content at the highest concentration Furthermore, SA does not appear to be an effective of SA used (150 mg L-1) was significantly higher (by elicitor to increase VA content in this study. This work 50%) than other concentrations at both exposure may help further investigate the optimal concentrations times, however SA did not improve VA contents of and exposure times of fungal elicitor and MJ for the hairy roots compared with FE and MJ (Figures 5A, B). possible large-scale production of valerenic acid from The inefficiency of SA in promoting valtrate production hairy root cultures of valerian in a bioreactor system. in adventitious roots of Valeriana amurensis has also been reported [42]. However, several studies have shown the effectiveness of SA in improving target Conflict of interests metabolites in the plant cells [35,43]. SA and Jasmonic acid (JA)/MJ are messengers in the signal transduction The authors have no conflict of interest to disclose. chains involved in different pathways of biosynthesis and in activation of secondary metabolites and enzymes that participate in the plant stress response Acknowledgements [44]; and exogenous application of MJ and SA may also induce the expression of many defense genes in plants The authors would like to acknowledge the Biotechnology [45]. JA signaling might be reinforced as a substitute Research Center of Urmia University for providing the for SA signaling to mediate elicitor-induced secondary necessary laboratory facilities.

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