Turkish Journal of Biology Turk J Biol (2015) 39: 469-478 http://journals.tubitak.gov.tr/biology/ © TÜBİTAK Research Article doi:10.3906/biy-1412-37

Influence of carbon sources on growth and GC-MS based metabolite profiling of Arnica montana L. hairy roots

1, 1 2 2 2 Maria PETROVA *, Ely ZAYOVA , Ivayla DINCHEVA , Ilian BADJAKOV , Mariana VLAHOVA 1 Institute of Plant Physiology and Genetics, Bulgarian Academy of Sciences, Sofia, Bulgaria 2 AgroBioInstitute, Sofia, Bulgaria

Received: 08.12.2014 Accepted/Published Online: 17.02.2015 Printed: 15.06.2015

Abstract: Arnica montana L. (Asteraceae) is an economically important herb that contains numerous valuable biologically active compounds accumulated in various parts of the plant. The effects of carbon sources (sucrose, maltose, and glucose) at different concentrations (1%, 3%, 5%, 7%, and 9%) on growth were studied and GC-MS based metabolite profiling of A. montana hairy roots was conducted. The optimal growth and biomass accumulation of transformed roots were observed on an MS nutrient medium containing 3% or 5% sucrose. GC-MS analysis of hairy roots of A. montana showed the presence of 48 compounds in polar fractions and 22 compounds in apolar fractions belonging to different classes of metabolites: , phenolic acids, organic acids, fatty acids, amino acids, sugars, sugar , hydrocarbons etc. Among the various metabolites identified, only the sugars and sugar alcohols were influenced by the concentration of the respective carbon sources in the nutrient medium.

Key words: Arnica, transformed roots, carbon sources, GC-MS analysis

1. Introduction Thiruvengadam et al., 2014; Shakeran et al., 2015). It is The perennial herb Arnica montana L. (Asteraceae) is one known that the addition of a carbon source in the nutrient of the most important medicinal species worldwide. The medium is necessary for the growth of tissue cultures, plant is used for treatment of hematomas, contusions, including hairy roots, because plants autotrophic ability sprains, rheumatic disease, and superficial inflammations at in vitro conditions is limited. It was recently found of the skin (Willuhn, 1998). Its pharmacological value is that sugars (monosaccharide and disaccharide) induce due to the presence of sesquiterpene lactones, , signals that affect metabolism, development, growth, essential oils, and other active compounds in various parts and gene expression of plants (Praveen and Murthy, of the plant. The roots contain essential oils, phenolic acids, 2012). The production of numerous biologically active oligosaccharides, , etc (Willuhn, 1972a, 1972b; compounds through hairy roots (such as hyoscyamine, Rossetti et al., 1984; Schmidt et al., 2006; Pljevljaušić et , sennosides A and B, pyranocoumarins, al., 2012). The chemical composition of hairy roots of gymnemic acid, ginsenoside) was found to be affected by A. montana obtained by genetic transformation of plant initial concentrations of carbon sources in the nutrient tissue with Agrobacterium rhizogenes is less studied. media (Liang et al., 2004; He et al., 2005; Pavlov et al., There are few publications related to transformed 2009; Xu et al., 2009; Romero et al., 2009; Nagella et al., roots essential oil constituents (Weremczuk-Jezyna et 2013; Kochan et al., 2014). To date, there have been no al., 2006, 2011). The greatest advantages of hairy roots studies regarding the effects of type and concentration are their genetic and biochemical stability, fast auxin- of sugars on growth and accumulation of metabolites independent growth, and the ability to synthesize natural of hairy root cultures of A. montana and this motivated compounds at levels comparable to intact plants. These our study. By manipulating the type and concentration are the reasons that in recent years hairy roots of various of the carbon sources, our goal was to determine the medicinal and aromatic plants are being cultured for optimal conditions for promoting growth and biomass the production of secondary compounds (Murthy et al., accumulation of hairy roots and improving the synthesis 2008; Danphitsanuparn et al., 2012; Nagella et al., 2013; of important secondary metabolites in them.

* Correspondence: [email protected] 469 PETROVA et al. / Turk J Biol

2. Materials and methods spectra were recorded at 2 scans/s with an m/z of 50–550 2.1. Plant material scanning range. The temperatures of the ion source and the The induction of hairy roots culture was described by interface were adjusted to 230 °C and 280 °C, respectively. Petrova et al. (2013). The transgenic clone (T4) that showed The components in the samples were identified by the best growth characteristics was used to study the comparison of their mass spectra with mass spectra from effects of sucrose, maltose, and glucose (added to the agar the NIST 08 database (NIST Mass Spectral Database, PC- solidified Murashige and Skoog (1962) nutrient medium Version 65.0, 2008) of the National Institute of Standards (MS) at concentrations of 1%, 3%, 5%, 7%, and 9%) on the and Technology (Gaithersburg, MD, USA) and the specific accumulation of biomass and metabolite production of A. database: the Golm Metabolome Database (Kopka et al., montana hairy roots. The root segments (20 pieces) with 2005). The amounts of the components are shown as a a length of approximately 2 cm and a fresh weight of 0.2 percentage of the Total Ion Current (TIC). g were cultivated on all tested media in petri dishes (9 cm 2.4. Statistical analysis in diameter) containing 20 mL of medium. The treatments The data were subjected to one-way ANOVA for were replicated three times. To characterize hairy root comparison of means, and significant differences were growth, root length, number of lateral branches per 1 cm calculated according to the Fisher LSD test at the 5% root, length of branches, and fresh weight were recorded significance level, using a statistical software package after 40 days of cultivation. The hairy roots were grown (Statgraphics Plus, version 5.1 for Windows). Data were in a culture room at a temperature of 25 ± 1 °С, relative reported as means ± standard error. humidity of 65%–70%, and a photoperiod of 16 h/8 h under diffused light (20 μmol –2m s–1). 3. Results 2.2. Extraction procedure 3.1. Effects of different carbon sources on growth and Prior to the GC-MS analysis an extraction was development of hairy roots performed according to Nikiforova et al. (2005) with The influence of different carbon sources on the growth, some modification: lyophilized drugs (50 mg from each development, and accumulation of biomass in clone T4 samples) were put in 2 mL Eppendorf tubes and mixed was studied. Data on the effects of sucrose, maltose, and with 500 μL of methanol. The samples were vortexed for glucose in different concentrations in the MS medium 2 min and incubated for 30 min in a water bath at 70 °C. on the growth parameters of hairy roots are presented in The solution was cooled to room temperature (22 °C). Table 1. The comparative analysis showed that the best (500 µL) was added and the mixture was growth and development of roots took place in media containing sucrose (Figure 1). It was observed that the vortexed for 2 min. Then 300 μL of 2H O (ultrapure 18 MΩ cm) was added and the mixture was again vortexed transgenic roots started their growth on the 3rd day of for 2 min. The samples were centrifuged at 12,000 rpm for their cultivation on MS nutrient media with this carbon 5 min at room temperature. The obtained two fractions source. Of all tested concentrations, the best results were (polar and apolar), each 300 µL, were placed in glass vials obtained by using concentrations of 3% and 5% sucrose, where parameters such as average length of the root (4.52 for analysis. They were dried under vacuum and subjected and 4.22 cm, respectively) and number of branches per cm to derivatization by silylation (20 μL of Pyridine + 20 μL root (8.35 and 7.40, respectively) were the highest, which of N,O-Bis(trimethylsilyl) trifluoroacetamide (BSTFA, resulted in greater fresh weight and accumulated biomass Sigma)/70 °C/30 min). (3.47 and 2.79 g, respectively) (Figures 1 and 2). Maltose 2.3. Gas chromatography–mass spectrometry (GC-MS) proved to be the second most efficient carbon source for analysis growth and development of roots. The highest values of The GC-MS analysis was performed on a Hewlett Packard all investigated parameters and maximum growth were 7890 instrument coupled with MSD 5975 equipment observed when the hairy roots were cultivated on nutrient (Hewlett Packard, Palo Alto, CA, USA) operating in EI medium supplemented with 5% maltose (Table 1; Figure mode at 70 eV. An HP-5 MS column (30 m × 0.25 mm 1). The average fresh weight at this concentration was × 0.25 µm) was used. All samples were analyzed in three 3.14 g. Glucose had a smaller effect on the rate of growth replicates. A split ratio of 1:20 was used for the injection of and accumulation of biomass. Its optimal concentration 1 μL of the solutions. Helium was used as the carrier gas was 3%, at which the measured parameters were higher at a flow rate of 1.2 mL/min. The analysis was performed in comparison with the other tested concentrations. All using the following temperature program: 2 min at 100 °C investigated carbon sources at a concentration of 1% followed by a ramp of 15 °C/min up to 180 °C, which was showed slower growth of roots, resulting in lower fresh sustained for 1 min. Finally, a second ramp of 5 °C/min up weight (Figure 1). High concentrations of sugars (9%) to 300 °C was applied and then sustained for 10 min. Mass led to a change in the color of roots (accumulation of

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Table 1. Effect of carbon sources on the growth parameters of A. montana hairy roots.

Type of carbon source, % Length of root, cm Number of branches, cm Length of branches, cm Sucrose 1 2.38 ± 0.06 a 3.50 ± 0.32 a 0.44 ± 0.03 a 3 4.52 ± 0.14 d 8.35 ± 0.52 d 0.86 ± 0.05 d 5 4.22 ± 0.12 d 7.40 ± 0.41 c 1.05 ± 0.06 e 7 3.21 ± 0.12 c 4.65 ± 0.31 b 0.67 ± 0.03 c 9 2.83 ± 0.36 b 3.65 ± 0.37 a 0.56 ± 0.04 b Maltose 1 2.20 ± 0.03 a 2.85 ± 0.24 a 0.29 ± 0.02 a 3 3.04 ± 0.11 b 3.80 ± 0.31 b 0.75 ± 0.05 c 5 3.82 ± 0.14 d 6.55 ± 0.43 e 1.06 ± 0.05 d 7 3.56 ± 0.12 c 5.40 ± 0.40 d 0.80 ± 0.04 c 9 2.95 ± 0.09 b 4.25 ± 0.36 c 0.52 ± 0.04 b Glucose 1 2.46 ± 0.06 a 2.70 ± 0.28 a 0.77 ± 0.04 b 3 3.42 ± 0.34 bc 6.15 ± 0.44 c 1.10 ± 0.05 d 5 3.11 ± 0.13 b 5.05 ± 0.38 b 0.96 ± 0.06 c 7 3.73 ± 0.15 c 4.75 ± 0.39 b 0.42 ± 0.04 a 9 2.75 ± 0.04 a 3.35 ± 0.34 a 0.34 ± 0.03 a

4 MS nutrient medium containing 3% sucrose yielded the 1% highest biomass of 3.47 g fresh weight, while those cultured 3.5 3% on 1% maltose or 1% glucose accumulated only 0.45 and 5% 0.48 g fresh weight, respectively. 3 7% 3.2. GC-MS based metabolite profiling of Arnica 2.5 9% montana L. hairy roots t, g GC-MS analysis of polar fractions of hairy roots of 2 A. montana showed the presence of 48 compounds

esh we gh 1.5 belonging to different classes of metabolites: one Fr flavone (), five phenolic acids (caffeic acid, 1 4-hydroxyphenyllactic acid, protocatechuic acid, 2-phenyl lactic acid, and m-hydroxybenzoic acid), ten organic 0.5 acids (γ-hydroxybutyric acid, malonic acid, fumaric 0 acid, malic acid, succinic acid, α-hydroxyglutaric acid, sucrosemaltose glucose L-(+)-tartaric acid, 2-keto-l-gluconic acid, isocitric Nutr ent med um acid, and galactaric acid), six fatty acids (palmitic Figure 1. Growth of A. montana hairy roots (expressed by fresh weight) depending on the type and concentration of the carbon acid, myristic acid, linoleic acid, stearic acid, trans-13- source in the nutrient medium after 40 days of culture. octadecenoic acid, and arachidic acid) and two ethers of fatty acids (2-monostearin trimethylsilyl ether and anthocyanin) and their growth was inhibited. The highest 1-monolinoleoylglycerol trimethylsilyl ether), one sterol accumulation of biomass, expressed by fresh weight, (β-sitosterol trimethylsilyl ether), four amino acids was observed at 3% and 5% concentrations, irrespective (L-asparagine, L-proline, L-glutamine, and cystathionine), of carbon type (Figure 1). The hairy roots grown on an five sugars (D-xylofuranose, D-mannopyranose, β-DL-

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ab c

de Figure 2. Hairy roots of A. montana cultivated on an MS nutrient medium supplemented with: a) 1% sucrose; b) 3% sucrose; c) 5% sucrose; d) 7% sucrose; e) 9% sucrose. lyxopyranose, d-(-)-fructose, and D-turanose), eight example, by increasing the glucose concentration from sugar alcohols (glycerol, pentitol, l-(-)-arabitol, d-(+)- 1% to 9% in the culture medium, the glycerol content arabitol, d-glucitol, dulcitol, scyllo-inositol, and myo- was increased from 0.6% to 1%. The relative content of inositol), two sugar acids (L-threonic acid and D-gluconic D-glucitol (sorbitol) and dulcitol (galactitol) increased 1.06 acid), one nonorganic acid (phosphoric acid), and three and 1.44 times by increasing the glucose concentration hydrocarbons ((9Z)-9-octadecenyl trimethylsilyl ether, in the nutrient medium. The same trend was observed octadecyl trimethylsilyl ether, and trans-squalene) (Tables when increasing the content of sucrose and maltose in the 2 and 3). The identification of compounds was carried medium. The levels of D-mannopyranose, d-(-)-fructose, out based on a comparison of their mass spectra and and D-turanose were influenced by the concentration retention index relative to those published in the available of the carbon sources in the nutrient media, while that databases. Table 2 shows the compounds in polar fractions of D-xylofuranose and β-DL-lyxopyranose remained with a constant amount that did not vary depending on constant irrespective of carbon source concentration. The the type and concentration of the sugar source used in the dominant sugar was d-(-)-fructose (1.6% to 2% of TIC). nutrient medium. Secondary metabolites presented from GC-MS analysis of apolar fractions of hairy roots the flavone chrysin and the five phenolic acids were not showed the presence of 22 compounds (one sugar , affected by the type of culture medium. The predominant eight hydrocarbons, one fatty alcohol, eight fatty acids, phenolic acid was m-hydroxybenzoic acid (0.8% of TIC). and four ethers of fatty acids) (Table 4). The compounds From the detected six fatty acids, saturated palmitic acid bis (trimethylsilyl) monostearin and 1-monopalmitin (5.7% of TIC) and stearic acid (5.1% of TIC) were the most trimethylsilyl ether were present in the highest amounts abundant. The dominant amino acid in the studied samples at 23.0% and 23.8% of TIC, respectively (Table 4). Among of hairy roots was L-proline (1.3% of TIC), while the the fatty acids, saturated stearic acid (octadecanoic acid) other three amino acids were 0.2% of TIC. The identified and palmatic acid (hexadecanoic acid) were present in the organic acids showed similar amount (from 0.2% to 0.4% greatest quantity at 4.9% and 4.4% of TIC, respectively. of TIC). The compounds with the highest abundance were the sugar alcohols pentitol and d-glucitol, at 22.7% and 4. Discussion 17.5% respectively. Of the various classes of compounds, The appropriate type of carbon source and its optimal only the sugars and sugar alcohols were influenced by concentration at which hairy roots of A. montana the concentration of the respective carbon sources in accumulate the most biomass were established. Although the culture media (Table 3). With increasing content carbohydrates are essential for in vitro culture, their of sucrose, maltose, or glucose in the culture medium, metabolism in in vitro conditions is not sufficiently the amounts of sugars and sugar alcohols increased. For studied (Kozai, 1991). It was found that the requirement

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Table 2. Metabolites identified in polar fractions of A. montana hairy roots by GC-MS that were not influenced by the type and concentration of the carbon source in the nutrient medium.

Classes of metabolites Compounds RT (min) Total ion current, % L-Asparagine 5.1 0.2 Cystathionine, di-TMS 5.0 0.2 Amino acids L-Proline 8.0 1.3 L-Glutamine 11.0 0.2 Malonic acid 4.9 0.3 γ-Hydroxybutyric acid 4.5 0.2 Succinic acid 6.0 0.3 Fumaric acid 6.3 0.3 Malic acid 7.6 0.4 Organic acids α-Hydroxyglutaric acid 8.5 0.2 L-(+)-Tartaric acid 8.6 0.4 2-Keto-l-gluconic acid 11.3 0.3 Isocitric acid 11.7 0.4 Galactaric acid 15.4 0.2 Chrysin 13.4 0.2 2-Phenyl lactic acid 8.6 0.3 m-Hydroxybenzoic acid 9.0 0.8 Flavons and phenolic acids Protocatechuic acid 11.6 0.2 4-Hydroxyphenyllactic acid 12.8 0.5 Caffeic acid 16.5 0.2 Myristic acid 11.8 0.6 Palmitic acid 14.9 5.7 Linoleic acid 17.5 0.4

Fatty acids and trans-13-Octadecenoic acid 17.6 0.9 esters of fatty acids Stearic acid 18.0 5.1 Arachidic acid 21.1 0.2 2-Monostearin 25.8 0.3 1-Monolinoleoylglycerol trimethylsilyl ether 28.8 0.2 L-Threonic acid 8.4 0.2 Sugar acids D-Gluconic acid 14.7 0.2 Phosphoric acid 5.7 1.4 Octadecyl trimethylsilyl ether 16.6 1.6 Others (9Z)-9-Octadecenyl trimethylsilyl ether 16.1 0.3 trans-Squalene 26.7 4.6 β-Sitosterol 33.6 0.4 RT- retention time. Values are expressed as percentages of the total ion current (TIC).

473 PETROVA et al. / Turk J Biol a c a b a b b d b c c a a 0.2 0.7 0.2 1.9 1.2 1.0 22.7 1.1 2.6 17.5 1.3 0.3 0.4 Glu 9% a c a ab a ab b cd ab bc bc a a 0.2 0.6 0.2 1.8 1.2 0.9 22.5 1.0 2.5 17.3 1.2 0.3 0.4 Glu 7% a b a ab a ab ab bc ab ab b a a 0.2 0.5 0.2 1.8 1.1 0.8 22.3 0.9 2.4 16.8 1.1 0.3 0.4 Glu 5% a b a a a a ab ab ab a b a a 0.2 0.4 0.2 1.7 1.1 0.7 22.2 0.9 2.3 16.7 1.1 0.3 0.4 Glu 3% a a a a a a a a a a a a a 0.2 0.4 0.2 1.7 1.0 0.6 22.0 0.8 2.3 16.4 0.9 0.3 0.4 Glu 1% a c a b a b b a c d a a a 0.2 1.3 0.2 1.8 1.2 0.6 22.5 1.3 2.4 17.2 0.9 0.3 0.4 Mal 9% a b a b a ab b a c cd a a a 0.2 1.2 0.2 1.7 1.2 0.6 22.3 1.3 2.3 16.8 0.9 0.3 0.4 Mal 7% a b a ab a b a bc abc a a a a b 0.2 1.1 0.2 1.7 1.2 0.6 22.3 1.2 2.2 16.7 0.9 0.3 0.4 Mal 5% a a a a a a ab a ab ab a a a 0.2 0.9 0.2 1.6 1.1 0.6 22.1 1.1 2.1 16.3 0.8 0.3 0.4 Mal 3% a a a a a a a a a a a a a 0.2 0.9 0.2 1.6 1.0 0.6 22.0 1.0 2.0 16.1 0.8 0.3 0.4 Mal 1% a c a b a d ab b c a a a a 0.2 0.8 0.2 2.0 1.0 0.8 22.5 1.3 2.4 16.8 0.8 0.3 0.4 Suc 9% a bc a ab a cd b ab bc a a a a 0.2 0.7 0.2 1.9 1.1 0.7 22.4 1.2 2.3 16.5 0.8 0.3 0.4 Suc 7% a bc a ab a bc ab ab bc a a a a 0.2 0.6 0.2 1.8 1.0 0.7 22.2 1.2 2.2 16.4 0.8 0.3 0.4 Suc 5% hairy roots by GC-MS that were influenced by the type and concentration of the carbon source in the nutrient nutrient in the of source carbon the by concentration typeand the influenced were GC-MS that hairy by roots a ab a a a ab ab a b a a a a 0.2 0.5 0.2 1.7 1.0 0.6 22.1 1.1 2.1 16.2 0.7 0.3 0.4 Suc 3% a a a a a a a a a a a a a 0.5 0.2 1.6 0.9 21.9 0.9 2.1 15.9 0.7 0.3 0.4 Total ion current, % current, ion Total Suc 1% 0.6 0.2 14.3 15.2 22.4 24.4 8.2 10.3 10.4 13.6 13.8 14.0 14.4 RT (min) 5.6 14.1 D-Mannopyranose β-DL-Lyxopyranose d-(-)-Fructose D-Turanose Pentitol l-(-)-Arabitol d-(+)-Arabitol d-Glucitol Dulcitol Scyllo-Inositol Myo-Inositol Compounds Glycerol D-Xylofuranose Metabolites identified in polar fractions of A. in montana fractions polar identified 3. Metabolites Table Classes metabolites of alcohols Sugar assessed differences significant indicate letters Different analyses. independent three of means as presented are Data (TIC). current ion the total of percentages as expressed are Values time. retention RT- analysis. multifactor ANOVA performing (P ≤ 0.05) after test LSD the Fisher by medium. Sugars Sugars

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Table 4. Metabolites identified in apolar fractions of A. montana hairy roots by GC-MS analysis.

Classes of metabolites Compounds RT (min) Total ion current, % Heptadecane 7.6 0.3 Hexadecane 8.1 0.6 Butane 8.2 1.5 Crocetane 13.0 0.3 Hydrocarbons Octadecane 13.6 0.6 n-Pentacosane 16.9 0.5 Heptacosane 19.6 0.2 Hentriacontane 20.3 0.3 Sugar alcohol Trimethylsilyl ether of glycerol 5.6 2.4 to 2.6 Fatty alcohol 1-Hexadecanol 5.9 0.4 n-Tetradecanoic acid, 11.8 0.3 n-Pentadecanoic acid 13.3 0.2 Hexadecanoic acid 14.8 4.4 Lauric acid 17.5 0.6 Octadecanoic acid 18.0 4.9 Fatty acids and ethers of fatty acids Myristic acid 20.6 1.1 Eicosanoic acid, trimethylsilyl ester 21.1 0.3 Eicosanoic acid, 2,3-bis[(trimethylsilyl)oxy]propyl ester 28.9 0.3 2-Monopalmitin trimethylsilyl ether 23.0 4.5 1-Monopalmitin trimethylsilyl ether 23.6 23.8 2-Monostearin trimethylsilyl ether 25.8 4.7 Bis(trimethylsilyl)monostearin 26.3 23.0

RT- retention time. Values are expressed as percentages of the total ion current (TIC). for carbohydrates depends on the stage of development leads to an initial increase, followed by a reduction in of the culture, and there may be differences depending on the values of the examined parameters. This reduction the type of carbohydrate (Thompson and Thorpe, 1987). can be caused by an excessive osmotic contribution or In our studies, the best growth of hairy roots is achieved by toxicity of the carbohydrate (Slesak et al., 2004). The on an MS nutrient medium containing 3% or 5% sucrose. maximum biomass accumulation of A. montana hairy Sucrose is the most widely applied carbon source in tissue roots was recorded in the medium containing 3% sucrose cultures. This is due to its effective absorption through (3.47 g fresh weight). The fresh weight of transgenic the cell membrane (Borkowska and Szezebra, 1991). roots decreased at higher sucrose contents (7% and 9%). Sucrose is hydrolyzed to glucose and fructose by the plant Similar observations were made by Udomsuk et al. (2009) cells during assimilation and the rate of uptake varies in and Praveen and Murthy (2012) in studies of the growth different plant cells (Srinivasan et al., 1995). Sugars act of Pueraria candollei Wall. ex Benth. and Withania as carbon sources and as osmotic regulators (Neto and somnifera (L.) Dunal hairy roots. Hairy roots of bitter Otoni, 2003). There is an inverse relationship between melon (Momordica charantia L.) cultured in MS liquid carbon source osmotic contribution and carbon source medium supplemented with 3% sucrose showed the concentration. The increase of carbon concentration highest accumulation of biomass and phenolic compound

475 PETROVA et al. / Turk J Biol production (Thiruvengadam et al., 2014). The authors 1993). Sugars in the culture media are the substrate for reported that hairy root growth dramatically decreased in the synthesis of various sugar alcohols, and maybe this media containing concentrations of sucrose either above is the reason that increases in their concentration lead to or below the 3% level. Sivanesan and Jeong (2009) found increased sugar alcohol contents. that when using fructose or glucose (3%, w/v) (instead of By using fructose instead of glucose, the production of sucrose) in the culture medium, the growth rate of hairy catharanthine was doubled in Catharanthus roseus (Jung et roots of Plumbago zeylanica L. is significantly slower. al., 1992), but growth was reduced by about 40%. Therefore, Weremczuk-Jezyna and Wysokinska (2006) reported a two-stage culture should be used: the roots must first be that the growth of hairy roots of A. montana improved grown on MS medium containing sucrose, followed by a significantly when the medium B5, containing 50 g –1L transfer to a medium containing fructose for secondary sucrose, was used. The sucrose concentration of 40 g –1L metabolism optimization. High concentrations of sucrose was the best for fresh weight accumulation and hairy root in the culture medium inhibit the growth of root cultures of growth of Angelica gigas Nakai (Xu et al., 2009), while 50 Hypericum perforatum L., but stimulate the production of g L–1 sucrose mostly stimulated the growth of hairy roots total , flavonoids, chlorogenic acid, and hypericin of Senna alata (L.) Roxb. (Putalun et al., 2006) and Datura by osmotic stress (Cui et al., 2010). In a study on the quercifolia Kunth (Dupraz et al., 1994). Among the influence of different contents of sucrose in the nutrient various sources of carbons, maltose at a concentration of medium, 3% sucrose in the medium increased growth and 3% induces the highest percentage of hairy root cultures stimulated the accumulation of isoflavones and in in leaves of Camellia sinensis (L.) Kuntze tea, followed by Pueraria phaseoloides (Roxb.) Benth hairy roots (He et al., dextrose, sucrose, and fructose (John et al., 2009). 2005). Liu et al. (2013) showed that nutritive factors, like Although essential oils are the most intensively studied the carbon source, the nitrogen source, and the pH of the biologically active substances in roots of A. montana culture medium, are important parameters influencing the (Willuhn, 1972a, 1972b; Weremczuk-Jezyna et al., 2006, alkaloid production of Anisodus acutangulus hairy roots. 2011; Pljevljaušić et al., 2012), they were not a subject Thiruvengadam et al. (2014) reported that MS basal liquid in the current study. In a previous analysis of normal medium supplemented with 3% sucrose was superior for dried roots of the plant, certain classes of metabolites phenolic compound production in Momordica charantia were established and some of them were present in our hairy roots. study. They include phenolic acids (caffeic acid, cynarin, Our GC-MS analysis detected a flavone (chrysin) in the chlorogenic acid), some free amino acids, and soluble hairy roots. It is known that flavonoids are not common carbohydrates (glucose, fructose, sucrose) (Rossetti et al., for roots of A. montana and are accumulated only in the 1984). The qualitative and quantitative compositions of aerial parts of the plant (leaves and flower heads) (Merfort the samples were determined applying different methods and Wendisch, 1985, 1992). Lawsone and artemisinin, (thin layer chromatography and spectrophotometry) as which are typical for the aerial parts of the plants, were also variations in the contents of the respective components are reported to accumulate in hairy root cultures of Lawsonia dependent on the developmental stage of the plant. inermis L. and Artemisia annua L., respectively (Bakkali et In our study GC-MS analysis of hairy roots of A. al., 1997; Liu et al., 1999). montana showed the presence of 48 compounds in polar In conclusion, the influence of carbon sources on fractions and 22 compounds in apolar fractions. Among the growth and production of primary and secondary the various metabolites identified, only the sugars and metabolites of Arnica montana L. hairy roots was reported sugar alcohols were influenced by the concentration of the for the first time. This information may be useful for more respective carbohydrate in the culture medium. Similar extensive studies of the influences of nutritive factors and results were reported by Sivakumar et al. (2005), who culture conditions on the metabolite profile of hairy root observed an increase in the content of reducing sugars, cultures of the species. total sugars, and starch in the hairy roots of Panax ginseng (ginseng) with increases in the concentration of sucrose Acknowledgments in the culture medium. Many primary and secondary This work was supported by grant no. metabolites have been recognized to be sugar responsive BG051PO001-3.3.06-0025, financed by the European (Arsenault et al., 2010). It was found that sucrose and Social Fund and Operational Programme Human glucose may influence directly (such as signaling molecules) Resources Development (2007–2013) and co-financed by or indirectly a variety of metabolic pathways (Krapp et al., the Bulgarian Ministry of Education and Science.

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