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Turkish Journal of Biology Turk J Biol (2015) 39: 469-478 © TÜBİTAK doi:10.3906/biy-1412-37 http://journals.tubitak.gov.tr/biology/ Research Article Influence of carbon sources on growth and GC-MS based metabolite profiling of Arnica montana L. hairy roots 1, 1 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 2 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: flavones, phenolic acids, organic acids, fatty acids, amino acids, sugars, sugar alcohols, 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 The perennial herb Arnica montana L. (Asteraceae) is one of the most important medicinal species worldwide. The plant is used for treatment of hematomas, contusions, sprains, rheumatic disease, and superficial inflammations of the skin (Willuhn, 1998). Its pharmacological value is due to the presence of sesquiterpene lactones, flavonoids, essential oils, and other active compounds in various parts of the plant. The roots contain essential oils, phenolic acids, oligosaccharides, lignans, etc (Willuhn, 1972a, 1972b; Rossetti et al., 1984; Schmidt et al., 2006; Pljevljaušić et al., 2012). The chemical composition of hairy roots of A. montana obtained by genetic transformation of plant tissue with Agrobacterium rhizogenes is less studied. There are few publications related to transformed roots essential oil constituents (Weremczuk-Jezyna et al., 2006, 2011). The greatest advantages of hairy roots are their genetic and biochemical stability, fast auxinindependent growth, and the ability to synthesize natural compounds at levels comparable to intact plants. These are the reasons that in recent years hairy roots of various medicinal and aromatic plants are being cultured for the production of secondary compounds (Murthy et al., 2008; Danphitsanuparn et al., 2012; Nagella et al., 2013; * Correspondence: marry_petrova@yahoo.com Thiruvengadam et al., 2014; Shakeran et al., 2015). It is known that the addition of a carbon source in the nutrient medium is necessary for the growth of tissue cultures, including hairy roots, because plants autotrophic ability at in vitro conditions is limited. It was recently found that sugars (monosaccharide and disaccharide) induce signals that affect metabolism, development, growth, and gene expression of plants (Praveen and Murthy, 2012). The production of numerous biologically active compounds through hairy roots (such as hyoscyamine, isoflavones, sennosides A and B, pyranocoumarins, gymnemic acid, ginsenoside) was found to be affected by initial concentrations of carbon sources in the nutrient media (Liang et al., 2004; He et al., 2005; Pavlov et al., 2009; Xu et al., 2009; Romero et al., 2009; Nagella et al., 2013; Kochan et al., 2014). To date, there have been no studies regarding the effects of type and concentration of sugars on growth and accumulation of metabolites of hairy root cultures of A. montana and this motivated our study. By manipulating the type and concentration of the carbon sources, our goal was to determine the optimal conditions for promoting growth and biomass accumulation of hairy roots and improving the synthesis of important secondary metabolites in them. 469 PETROVA et al. / Turk J Biol 2. Materials and methods 2.1. Plant material The induction of hairy roots culture was described by Petrova et al. (2013). The transgenic clone (T4) that showed the best growth characteristics was used to study the effects of sucrose, maltose, and glucose (added to the agar solidified Murashige and Skoog (1962) nutrient medium (MS) at concentrations of 1%, 3%, 5%, 7%, and 9%) on the accumulation of biomass and metabolite production of A. montana hairy roots. The root segments (20 pieces) with a length of approximately 2 cm and a fresh weight of 0.2 g were cultivated on all tested media in petri dishes (9 cm in diameter) containing 20 mL of medium. The treatments were replicated three times. To characterize hairy root growth, root length, number of lateral branches per 1 cm root, length of branches, and fresh weight were recorded after 40 days of cultivation. The hairy roots were grown in a culture room at a temperature of 25 ± 1 °С, relative humidity of 65%–70%, and a photoperiod of 16 h/8 h under diffused light (20 μmol m–2 s–1). 2.2. Extraction procedure Prior to the GC-MS analysis an extraction was performed according to Nikiforova et al. (2005) with some modification: lyophilized drugs (50 mg from each samples) were put in 2 mL Eppendorf tubes and mixed with 500 μL of methanol. The samples were vortexed for 2 min and incubated for 30 min in a water bath at 70 °C. The solution was cooled to room temperature (22 °C). Chloroform (500 µL) was added and the mixture was vortexed for 2 min. Then 300 μL of H2O (ultrapure 18 MΩ cm) was added and the mixture was again vortexed for 2 min. The samples were centrifuged at 12,000 rpm for 5 min at room temperature. The obtained two fractions (polar and apolar), each 300 µL, were placed in glass vials for analysis. They were dried under vacuum and subjected to derivatization by silylation (20 μL of Pyridine + 20 μL of N,O-Bis(trimethylsilyl) trifluoroacetamide (BSTFA, Sigma)/70 °C/30 min). 2.3. Gas chromatography–mass spectrometry (GC-MS) analysis The GC-MS analysis was performed on a Hewlett Packard 7890 instrument coupled with MSD 5975 equipment (Hewlett Packard, Palo Alto, CA, USA) operating in EI mode at 70 eV. An HP-5 MS column (30 m × 0.25 mm × 0.25 µm) was used. All samples were analyzed in three replicates. A split ratio of 1:20 was used for the injection of 1 μL of the solutions. Helium was used as the carrier gas at a flow rate of 1.2 mL/min. The analysis was performed using the following temperature program: 2 min at 100 °C followed by a ramp of 15 °C/min up to 180 °C, which was sustained for 1 min. Finally, a second ramp of 5 °C/min up to 300 °C was applied and then sustained for 10 min. Mass 470 spectra were recorded at 2 scans/s with an m/z of 50–550 scanning range. The temperatures of the ion source and the interface were adjusted to 230 °C and 280 °C, respectively. The components in the samples were identified by comparison of their mass spectra with mass spectra from the NIST 08 database (NIST Mass Spectral Database, PCVersion 65.0, 2008) of the National Institute of Standards and Technology (Gaithersburg, MD, USA) and the specific database: the Golm Metabolome Database (Kopka et al., 2005). The amounts of the components are shown as a percentage of the Total Ion Current (TIC). 2.4. Statistical analysis The data were subjected to one-way ANOVA for comparison of means, and significant differences were calculated according to the Fisher LSD test at the 5% significance level, using a statistical software package (Statgraphics Plus, version 5.1 for Windows). Data were reported as means ± standard error. 3. Results 3.1. Effects of different carbon sources on growth and development of hairy roots The influence of different carbon sources on the growth, development, and accumulation of biomass in clone T4 was studied. Data on the effects of sucrose, maltose, and glucose in different concentrations in the MS medium on the growth parameters of hairy roots are presented in Table 1. The comparative analysis showed that the best growth and development of roots took place in media containing sucrose (Figure 1). It was observed that the transgenic roots started their growth on the 3rd day of their cultivation on MS nutrient media with this carbon source. Of all tested concentrations, the best results were obtained by using concentrations of 3% and 5% sucrose, where parameters such as average length of the root (4.52 and 4.22 cm, respectively) and number of branches per cm root (8.35 and 7.40, respectively) were the highest, which resulted in greater fresh weight and accumulated biomass (3.47 and 2.79 g, respectively) (Figures 1 and 2). Maltose proved to be the second most efficient carbon source for growth and development of roots. The highest values of all investigated parameters and maximum growth were observed when the hairy roots were cultivated on nutrient medium supplemented with 5% maltose (Table 1; Figure 1). The average fresh weight at this concentration was 3.14 g. Glucose had a smaller effect on the rate of growth and accumulation of biomass. Its optimal concentration was 3%, at which the measured parameters were higher in comparison with the other tested concentrations. All investigated carbon sources at a concentration of 1% showed slower growth of roots, resulting in lower fresh weight (Figure 1). High concentrations of sugars (9%) led to a change in the color of roots (accumulation of PETROVA et al. / Turk J Biol 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 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 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 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 Sucrose Maltose Glucose 4 1% 3.5 3% 5% Fresh weight, g 3 7% 2.5 9% 2 1.5 1 0.5 0 sucrose maltose Nutrient medium glucose Figure 1. Growth of A. montana hairy roots (expressed by fresh weight) depending on the type and concentration of the carbon source in the nutrient medium after 40 days of culture. anthocyanin) and their growth was inhibited. The highest accumulation of biomass, expressed by fresh weight, was observed at 3% and 5% concentrations, irrespective of carbon type (Figure 1). The hairy roots grown on an MS nutrient medium containing 3% sucrose yielded the highest biomass of 3.47 g fresh weight, while those cultured on 1% maltose or 1% glucose accumulated only 0.45 and 0.48 g fresh weight, respectively. 3.2. GC-MS based metabolite profiling of Arnica montana L. hairy roots GC-MS analysis of polar fractions of hairy roots of A. montana showed the presence of 48 compounds belonging to different classes of metabolites: one flavone (chrysin), five phenolic acids (caffeic acid, 4-hydroxyphenyllactic acid, protocatechuic acid, 2-phenyl lactic acid, and m-hydroxybenzoic acid), ten organic acids (γ-hydroxybutyric acid, malonic acid, fumaric acid, malic acid, succinic acid, α-hydroxyglutaric acid, L-(+)-tartaric acid, 2-keto-l-gluconic acid, isocitric acid, and galactaric acid), six fatty acids (palmitic acid, myristic acid, linoleic acid, stearic acid, trans-13octadecenoic acid, and arachidic acid) and two ethers of fatty acids (2-monostearin trimethylsilyl ether and 1-monolinoleoylglycerol trimethylsilyl ether), one sterol (β-sitosterol trimethylsilyl ether), four amino acids (L-asparagine, L-proline, L-glutamine, and cystathionine), five sugars (D-xylofuranose, D-mannopyranose, β-DL- 471 PETROVA et al. / Turk J Biol a b c d e 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 sugar alcohols (glycerol, pentitol, l-(-)-arabitol, d-(+)arabitol, d-glucitol, dulcitol, scyllo-inositol, and myoinositol), two sugar acids (L-threonic acid and D-gluconic acid), one nonorganic acid (phosphoric acid), and three hydrocarbons ((9Z)-9-octadecenyl trimethylsilyl ether, octadecyl trimethylsilyl ether, and trans-squalene) (Tables 2 and 3). The identification of compounds was carried out based on a comparison of their mass spectra and retention index relative to those published in the available databases. Table 2 shows the compounds in polar fractions with a constant amount that did not vary depending on the type and concentration of the sugar source used in the nutrient medium. Secondary metabolites presented from the flavone chrysin and the five phenolic acids were not affected by the type of culture medium. The predominant phenolic acid was m-hydroxybenzoic acid (0.8% of TIC). From the detected six fatty acids, saturated palmitic acid (5.7% of TIC) and stearic acid (5.1% of TIC) were the most abundant. The dominant amino acid in the studied samples of hairy roots was L-proline (1.3% of TIC), while the other three amino acids were 0.2% of TIC. The identified organic acids showed similar amount (from 0.2% to 0.4% of TIC). The compounds with the highest abundance were the sugar alcohols pentitol and d-glucitol, at 22.7% and 17.5% respectively. Of the various classes of compounds, only the sugars and sugar alcohols were influenced by the concentration of the respective carbon sources in the culture media (Table 3). With increasing content of sucrose, maltose, or glucose in the culture medium, the amounts of sugars and sugar alcohols increased. For 472 example, by increasing the glucose concentration from 1% to 9% in the culture medium, the glycerol content was increased from 0.6% to 1%. The relative content of D-glucitol (sorbitol) and dulcitol (galactitol) increased 1.06 and 1.44 times by increasing the glucose concentration in the nutrient medium. The same trend was observed when increasing the content of sucrose and maltose in the medium. The levels of D-mannopyranose, d-(-)-fructose, and D-turanose were influenced by the concentration of the carbon sources in the nutrient media, while that of D-xylofuranose and β-DL-lyxopyranose remained constant irrespective of carbon source concentration. The dominant sugar was d-(-)-fructose (1.6% to 2% of TIC). GC-MS analysis of apolar fractions of hairy roots showed the presence of 22 compounds (one sugar alcohol, eight hydrocarbons, one fatty alcohol, eight fatty acids, and four ethers of fatty acids) (Table 4). The compounds bis (trimethylsilyl) monostearin and 1-monopalmitin trimethylsilyl ether were present in the highest amounts at 23.0% and 23.8% of TIC, respectively (Table 4). Among the fatty acids, saturated stearic acid (octadecanoic acid) and palmatic acid (hexadecanoic acid) were present in the greatest quantity at 4.9% and 4.4% of TIC, respectively. 4. Discussion The appropriate type of carbon source and its optimal concentration at which hairy roots of A. montana accumulate the most biomass were established. Although carbohydrates are essential for in vitro culture, their metabolism in in vitro conditions is not sufficiently studied (Kozai, 1991). It was found that the requirement PETROVA et al. / Turk J Biol 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 Amino acids Organic acids Flavons and phenolic acids Fatty acids and esters of fatty acids Sugar acids Others Compounds RT (min) Total ion current, % L-Asparagine 5.1 0.2 Cystathionine, di-TMS 5.0 0.2 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 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 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 trans-13-Octadecenoic acid 17.6 0.9 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 D-Gluconic acid 14.7 0.2 Phosphoric acid 5.7 1.4 Octadecyl trimethylsilyl ether 16.6 1.6 (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 474 22.4 24.4 5.6 8.2 10.3 10.4 13.6 13.8 14.0 14.4 d-(-)-Fructose D-Turanose Glycerol Pentitol l-(-)-Arabitol d-(+)-Arabitol d-Glucitol Dulcitol Scyllo-Inositol Myo-Inositol 14.3 D-Mannopyranose 15.2 14.1 D-Xylofuranose β-DL-Lyxopyranose RT (min) Compounds 0.4 a 0.3 a 0.7 a 15.9 a 2.1 a 0.9 a 21.9 a 0.6 a 0.9 a 1.6 a 0.2 a 0.5 a 0.2 a Suc 1% 0.4 a 0.3 a 0.7 a 16.2 ab 2.1 a 1.1 b 0.4 a 0.3 a 0.8 a 16.4 bc 2.2 a 1.2 bc 22.2 a 0.7 ab 0.6 a 22.1 a 1.0 ab 1.8 bc 0.2 a 0.6 ab 0.2 a Suc 5% 1.0 ab 1.7 ab 0.2 a 0.5 a 0.2 a Suc 3% Total ion current, % 0.4 a 0.3 a 0.8 a 16.5 bc 2.3 a 1.2 bc 22.4 a 0.7 ab 1.1 b 1.9 cd 0.2 a 0.7 ab 0.2 a Suc 7% 0.4 a 0.3 a 0.8 a 16.8 c 2.4 a 1.3 c 22.5 a 0.8 b 1.0 ab 2.0 d 0.2 a 0.8 b 0.2 a Suc 9% 0.4 a 0.3 a 0.8 a 16.1 a 2.0 a 1.0 a 22.0 a 0.6 a 1.0 a 1.6 a 0.2 a 0.9 a 0.2 a Mal 1% 0.4 a 0.3 a 0.8 a 16.3 a 2.1 ab 1.1 ab 22.1 a 0.6 a 1.1 ab 1.6 a 0.2 a 0.9 a 0.2 a Mal 3% 0.4 a 0.3 a 0.9 a 16.7 b 2.2 abc 1.2 bc 22.3 a 0.6 a 1.2 b 1.7a b 0.2 a 1.1 ab 0.2 a Mal 5% 0.4 a 0.3 a 0.9 a 16.8 b 2.3 cd 1.3 c 22.3 a 0.6 a 1.2 b 1.7 ab 0.2 a 1.2 b 0.2 a Mal 7% 0.4 a 0.3 a 0.9 a 17.2 c 2.4 d 1.3 c 22.5 a 0.6 a 1.2 b 1.8 b 0.2 a 1.3 b 0.2 a Mal 9% 0.4 a 0.3 a 0.9 a 16.4 a 2.3 a 0.8 a 22.0 a 0.6 a 1.0 a 1.7 a 0.2 a 0.4 a 0.2 a Glu 1% 0.4 a 0.3 a 1.1 b 16.7 b 2.3 a 0.9 ab 22.2 a 0.7 ab 1.1 ab 1.7 a 0.2 a 0.4 a 0.2 a Glu 3% 0.4 a 0.3 a 1.1 b 16.8 b 2.4 ab 0.9 ab 22.3 a 0.8 bc 1.1 ab 1.8 ab 0.2 a 0.5 ab 0.2 a Glu 5% 0.4 a 0.3 a 1.2 bc 17.3 c 2.5 bc 1.0 ab 22.5 a 0.9 cd 1.2 b 1.8 ab 0.2 a 0.6 ab 0.2 a Glu 7% 0.4 a 0.3 a 1.3 c 17.5 c 2.6 c 1.1 b 22.7 a 1.0 d 1.2 b 1.9 b 0.2 a 0.7 b 0.2 a Glu 9% RT- retention time. Values are expressed as percentages of the total ion current (TIC). Data are presented as means of three independent analyses. Different letters indicate significant differences assessed by the Fisher LSD test (P ≤ 0.05) after performing ANOVA multifactor analysis. Sugar alcohols Sugars Classes of metabolites Table 3. Metabolites identified in polar fractions of A. montana hairy roots by GC-MS that were influenced by the type and concentration of the carbon source in the nutrient medium. PETROVA et al. / Turk J Biol PETROVA et al. / Turk J Biol 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 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 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 Hydrocarbons Fatty acids and ethers of fatty acids RT- retention time. Values are expressed as percentages of the total ion current (TIC). for carbohydrates depends on the stage of development of the culture, and there may be differences depending on the type of carbohydrate (Thompson and Thorpe, 1987). In our studies, the best growth of hairy roots is achieved on an MS nutrient medium containing 3% or 5% sucrose. Sucrose is the most widely applied carbon source in tissue cultures. This is due to its effective absorption through the cell membrane (Borkowska and Szezebra, 1991). Sucrose is hydrolyzed to glucose and fructose by the plant cells during assimilation and the rate of uptake varies in different plant cells (Srinivasan et al., 1995). Sugars act as carbon sources and as osmotic regulators (Neto and Otoni, 2003). There is an inverse relationship between carbon source osmotic contribution and carbon source concentration. The increase of carbon concentration leads to an initial increase, followed by a reduction in the values of the examined parameters. This reduction can be caused by an excessive osmotic contribution or by toxicity of the carbohydrate (Slesak et al., 2004). The maximum biomass accumulation of A. montana hairy roots was recorded in the medium containing 3% sucrose (3.47 g fresh weight). The fresh weight of transgenic roots decreased at higher sucrose contents (7% and 9%). Similar observations were made by Udomsuk et al. (2009) and Praveen and Murthy (2012) in studies of the growth of Pueraria candollei Wall. ex Benth. and Withania somnifera (L.) Dunal hairy roots. Hairy roots of bitter melon (Momordica charantia L.) cultured in MS liquid medium supplemented with 3% sucrose showed the highest accumulation of biomass and phenolic compound 475 PETROVA et al. / Turk J Biol production (Thiruvengadam et al., 2014). The authors reported that hairy root growth dramatically decreased in media containing concentrations of sucrose either above or below the 3% level. Sivanesan and Jeong (2009) found that when using fructose or glucose (3%, w/v) (instead of sucrose) in the culture medium, the growth rate of hairy roots of Plumbago zeylanica L. is significantly slower. Weremczuk-Jezyna and Wysokinska (2006) reported that the growth of hairy roots of A. montana improved significantly when the medium B5, containing 50 g L–1 sucrose, was used. The sucrose concentration of 40 g L–1 was the best for fresh weight accumulation and hairy root growth of Angelica gigas Nakai (Xu et al., 2009), while 50 g L–1 sucrose mostly stimulated the growth of hairy roots of Senna alata (L.) Roxb. (Putalun et al., 2006) and Datura quercifolia Kunth (Dupraz et al., 1994). Among the various sources of carbons, maltose at a concentration of 3% induces the highest percentage of hairy root cultures in leaves of Camellia sinensis (L.) Kuntze tea, followed by dextrose, sucrose, and fructose (John et al., 2009). Although essential oils are the most intensively studied biologically active substances in roots of A. montana (Willuhn, 1972a, 1972b; Weremczuk-Jezyna et al., 2006, 2011; Pljevljaušić et al., 2012), they were not a subject in the current study. In a previous analysis of normal dried roots of the plant, certain classes of metabolites were established and some of them were present in our study. They include phenolic acids (caffeic acid, cynarin, chlorogenic acid), some free amino acids, and soluble carbohydrates (glucose, fructose, sucrose) (Rossetti et al., 1984). The qualitative and quantitative compositions of the samples were determined applying different methods (thin layer chromatography and spectrophotometry) as variations in the contents of the respective components are dependent on the developmental stage of the plant. In our study GC-MS analysis of hairy roots of A. montana showed the presence of 48 compounds in polar fractions and 22 compounds in apolar fractions. Among the various metabolites identified, only the sugars and sugar alcohols were influenced by the concentration of the respective carbohydrate in the culture medium. Similar results were reported by Sivakumar et al. (2005), who observed an increase in the content of reducing sugars, total sugars, and starch in the hairy roots of Panax ginseng (ginseng) with increases in the concentration of sucrose in the culture medium. Many primary and secondary metabolites have been recognized to be sugar responsive (Arsenault et al., 2010). It was found that sucrose and glucose may influence directly (such as signaling molecules) or indirectly a variety of metabolic pathways (Krapp et al., 476 1993). Sugars in the culture media are the substrate for the synthesis of various sugar alcohols, and maybe this is the reason that increases in their concentration lead to increased sugar alcohol contents. By using fructose instead of glucose, the production of catharanthine was doubled in Catharanthus roseus (Jung et al., 1992), but growth was reduced by about 40%. Therefore, a two-stage culture should be used: the roots must first be grown on MS medium containing sucrose, followed by a transfer to a medium containing fructose for secondary metabolism optimization. High concentrations of sucrose in the culture medium inhibit the growth of root cultures of Hypericum perforatum L., but stimulate the production of total phenols, flavonoids, chlorogenic acid, and hypericin by osmotic stress (Cui et al., 2010). In a study on the influence of different contents of sucrose in the nutrient medium, 3% sucrose in the medium increased growth and stimulated the accumulation of isoflavones and puerarin in Pueraria phaseoloides (Roxb.) Benth hairy roots (He et al., 2005). Liu et al. (2013) showed that nutritive factors, like the carbon source, the nitrogen source, and the pH of the culture medium, are important parameters influencing the alkaloid production of Anisodus acutangulus hairy roots. Thiruvengadam et al. (2014) reported that MS basal liquid medium supplemented with 3% sucrose was superior for phenolic compound production in Momordica charantia hairy roots. Our GC-MS analysis detected a flavone (chrysin) in the hairy roots. It is known that flavonoids are not common for roots of A. montana and are accumulated only in the aerial parts of the plant (leaves and flower heads) (Merfort and Wendisch, 1985, 1992). 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