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Root Culture of Valeriana Officinalis by Elicitation

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Root Culture of Valeriana Officinalis by Elicitation 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 sesquiterpene found in valerian (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 sedative, anticonvulsant, hypnotic 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, sesquiterpenes 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. 854 M.R.D. Torkamani et al. 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.
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