Microsoft Word - BN-OA-0201-03 Karalija, Paric 29 Original Article ! The effect of BA and IBA on the secondary metabolite production by shoot culture of Thymus vulgaris L. Erna Karalija, Adisa Parić* Faculty of Science, University of Sarajevo, Zmaja od Bosne 33-35, 71 000 Sarajevo, Bosnia and Herzegovina * E-mail: adisacausevic@hotmail.com Abstract: Karalija, E., Parić, A.: The effect of BA and IBA on the secondary metabolite production by shoot culture of Thymus vulgaris L.. Biologica Nyssana, 2 (1), September 2011: 29-35. In vitro shoots of the common thyme (Thymus vulgaris L.) were established, and the effects of different concentrations of the cytokinin BA and the auxin IBA on secondary metabolite production were investigated. The highest number of shoots was obtained through cultivation on the MS medium containing 2 mg/L BA and 0.1 mg/L IBA and 4 mg/L BA and 0.1 mg/L IBA (14.3 and 13.3 respectively). Quantitative changes in chlorophyll a, chlorophyll b and carotenoid content were recorded in response to the effect of varying concentrations of growth regulators in the medium. Furthermore, after addition of plant growth regulators (0.5 mg/L BA + 0.1 mg/L IBA) to the medium, elicitation of phenolic compounds was recorded in plantlets. The concentration range of BA from 2 to 4 mg/L improved the production of flavonoids (0.61 and 0.64 mg/g FW). In contrast, plantlets cultivated on the same treatment, showed a decrease in monomeric anthocyanins . Key words: common thyme, in vitro, micropropagation, secondary metabolite production Introduction ! The common thyme or Thymus vulgaris L., a member of the Lamiaceae family, is an aromatic herb growing up to 30 cm in height and flowering from July to September (G r u e n w a l d et al., 2004). It is native to southern Europe, where it is often cultivated as a culinary herb. Thyme is used as spice and medicinal herb almost everywhere in the world (M o r a l e s , 2002). Many pharmacological in vitro experiments carried out during the last decade revealed well defined pharmacological properties of both, the thyme essential oil and the plant extracts. The non-medicinal use of thyme is worthy of attention, because thyme is used in the food and aroma industries and it is widely used as a culinary ingredient and it serves as a preservative for foods especially due to its antioxidant effect (Z a r z u e l o & C r e s p o , 2002). The active substances in the thyme essential oil are thymol, carvacrol, p-cymene, β - pinene, γ- terpinene, β-caryophyllene, 1-borneol, 1, 8-cineole, etc (J u v e n et al., 1994). Recent studies showed that thyme essential oils have strong antibacterial, antifungal, and antioxidative properties (C r u z et al., 1989; P i c c a g l i a et al., 1993; C o n s e n t i n o et al., 1999; K a r a m a n et al., 2001; V a r d a r et al., 2003, P r a b u s e e n i v a s a n et al., 2006; B u d k a & K h a n , 2010). Essential oils of different species of genus Thymus inhibit a broad spectrum of bacteria, with Gram-positive bacteria generally being more susceptible than Gram-negative bacteria (B l a k e w a y , 1986; F a r a g et al., 1986; D e a n s & R i t c h i e , 1987; K n o b l o c h et al., 1988). Recently, the antibacterial property of thyme oil against some important food-borne pathogens, like Salmonella enteritidis, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, and 2 (1) • September 2011: 29-35 BIOLOGICA NYSSANA 2 (1) September 2011: 29-35 Karalija E., Parić A. The effect of BA and IBA on the secondary metabolite... 30 Campylobacter jejuni, has been tested (S m i t h - P a l m e r et al., 1998). Several in vitro and in vivo screenings have shown that volatile oils may be used against fungal diseases, especially those from plants of the genus Thymus (B l a k e w a y , 1986; F a r a g et al., 1986; D e a n s & R i t c h i e , 1987). A few studies demonstrate the antiviral effects of thyme extracts (H e r r m a n n & K u c e r a , 1967). Micropropagation techniques play an important role in conservation and genetic improvement of pharmaceutical and medicinal plants. Today, there is a tendency to develop in vitro systems of production as an alternative to field production of plants used as a source of active compounds. Such system is not limited by sources of nutrients or climate changes and can thus be better controlled. For effective induction of secondary metabolite production, plant growth regulators (PGRs) are required (Z h o n g et al., 1996; A l -S a n e ' et al., 2005; Shilpashree & Ravishankar, 2009), since they have significant effects on the metabolism of secondary metabolites. The quality, as well as the quantity, of plant growth regulators, plays a major role in the production capability of a given in vitro culture. PGRs affect cell multiplication and division, which increases production of secondary metabolites (K u r z & C o n s t a b e l , 1979; S t a b a , 1980). The effects of different PGRs on secondary metabolite production have previously been studied and confirmed in vrious plants (F u r u y a et al., 1971; I k u t a et al., 1975; I k e d a et al., 1976; I k u t a & I t o k a w a , 1982; N a i r et al., 1992; W e a t h e r s et al., 2005; K h a n et al., 2008). The aim of this study was to develop an efficient protocol for mass multiplication of T. vulgaris, through manipulation of different concentrations of plant growth regulators, and to examine the relationship between PGRs and the production and accumulation of antioxidants, polyphenols and chlorophyll. Materials and methods Plant Material and Treatments - Commercially purchased seeds of T. vulgaris L. (Sjemenarna Ljubljana d.d., Slovenija) were surface-sterilised with 20% commercial bleach for 15 min followed by 3x5 min rinsing in sterile distilled water and were then germinated on a MS basal medium (M u r a s h i g e & S k o o g , 1962). Shoot multiplication was investigated on the MS basal medium (BH; control) or on media supplemented with 0.5, 2, 4 mg/L BA and 0.1 mg/L IBA (BI1; BI2 and BI4). The pH of all media was adjusted to 5.8 with 1 M KOH or 1 M HCl before autoclaving at 121°C for 20 minutes. All cultures were incubated at 24±1°C under cool white fluorescent lights (3000 lux) and with the 16 h light photoperiod. The aerial parts of T. vulgaris were collected for analysis. Photosynthetic pigments – Extraction was undertaken in accordance to Porra et al. (1989). Quantification was done by spectrophotometric determination of absorbance on 663 nm (chlorophyll a), 646 nm (chlorophyll b) and 440 nm (carotenoids). The concentrations of photosynthetic pigments were expressed as mg of pigments per g of fresh weight (mg/g FW). Secondary Metabolite Production – Samples used for the analysis of concentrations of phenols, flavonoids and anthocyanins were frozen in liquid nitrogen and stored at -20°C. Extraction of phenols and flavonoids was performed in 100% methanol. Total phenol content was determined according to W o l f e et al. (2003), with Folin-Ciocalteu reagent through spectrophotometric readings of absorbance at 765 nm. Total phenol content was expressed as mg of catechin per g of FW. Total flavonoid content was analysed according to O r d o n et al. (2006) by absorbance readings at 415 nm. Total flavonoid content was expressed as mg of ruthin per g of FW. Monomeric anthocyanins were determined according to M a n c i n e l l i (1984). Extraction was performed in acidified methanol (1% HCL in methanol), and the concentration was expressed as mg of cyanidin 3-glucoside per L of extract. Observations and Statistical Analysis - Each treatment had 5 replicates containing 10 explants and all experiments were repeated three times. All analyses were done after 30 days of cultivation with no subcultivation. The data was analysed using SPSS 15.0 by employing parametric (Pearson correlation, one tailed) and non-parametric (Kruskal-Walis analysis) tests. Results and discussion Shoot Multiplication – The induction of shoot formation started after a week of culture cultivation with initiation of shoot meristemswhich was followed by growth of shoot buds. No multiplication was recorded on the MS medium without plant growth regulators (control) (Fig. 1a), while a high rate of root induction was observed on the same medium. Shoot apices grown in the presence of 0.5 mg/L BA and 0.1 mg/L IBA, produced on average 8.2 axillary buds at the base of the first pair of leaves (Tab. 1, Fig. 1b). Treatments with higher concentrations of BA (2 mg/L) increased the number of shoots, but shoots were much shorter and BIOLOGICA NYSSANA 2 (1) September 2011: 29-35 Karalija E., Parić A. The effect of BA and IBA on the secondary metabolite... 31 Table 1. The induction of adventive shoots formation in Thymus vulgaris L. Treatment Number of shoots per explant Rate of regeneration (%) Induction of root formation (%) BH 2.9 (±0,71) 100 81.25 BI1 8.2 (±0,56)* 100 0 BI2 14.3 (±0,82)* 100 0 BI4 13.3 (±0,84)* 100 0 The results represent mean values of three replications (±standard deviation) after 30 days on the MS basal medium with no plant growth regulators and with addition of different concentrations of BA and IBA. *statistically important differences at 0.01 level with respect to control. BH 0 m g/L BA + 0 mg/L IBA; BI1 0.5 mg/L BA + 0.1 mg/L IBA; BI2 2 mg/L BA + 0.1 mg/L IBA; BI4 4 mg/L BA + 0.1 mg/L IBA; Table 2. The effect of BA and IBA on photosynthetic pigments in shoot cultures of T. vulgaris Treatment Chlorophyll a (mg/g) Chlorophyll b (mg/g) Total Chlorophylls (mg/g) Carotenoids (mg/g) BH 0.29 (±0.02) 0.16 (±0.01) 0.45 (±0.02) 0.51 (±0.03) BI1 0.27*(±0.01) 0.40* (±0.02) 1.25* (±0.03) 0.20* (±0.03) BI2 0.24*(±0.24) 0.39* (±0.01) 1.14* (±0.01) 0.22* (±0.01) BI4 0.10*(±0.10) 0.17* (±0.02) 0.48* (±0.01) 0.65* (±0.02) The results represent mean values of three replications (±standard deviation) after 30 days on the MS basal medium with no plant growth regulators and with addition of different concentrations of BA and IBA. *statistically important differences at 0.01 level with respect to control. BH 0 m g/L BA + 0 mg/L IBA; BI1 0.5 mg/L BA + 0.1 mg/L IBA; BI2 2 mg/L BA + 0.1 mg/L IBA; BI4 4 mg/L BA + 0.1 mg/L IBA; Table 3. The Effect of BA and IBA on secondary metabolite production in shoot cultures of T. vulgaris Treatment Phenols (mg/g) Flavonoidis (mg/g) Monomeric anthocynins (mg/L) BH 2.09 (±0.14) 0.50 (±0.01) 6.62 (±0.32) BI1 3.05* (±0.11) 0.55 (±0.02) 3.38*(±0.05) BI2 1.70* (±0.01) 0.61*(±0.01) 2.95*(±0.01) BI4 2.32 (±0.04) 0.64*(±0.01) 2.13* (±0.04) The results represent mean values of three replications (±standard deviation) after 30 days on the MS basal medium with no plant growth regulators and with addition of different concentrations of BA and IBA. *statistically important differences at 0.01 level with respect to control. BH 0 m g/L BA + 0 mg/L IBA; BI1 0.5 mg/L BA + 0.1 mg/L IBA; BI2 2 mg/L BA + 0.1 mg/L IBA; BI4 4 mg/L BA + 0.1 mg/L IBA; had developed necrosis (Fig. 1c). On the medium with 4 mg/L BA and 0.1 mg/L IBA, an average of 13.3 shoots was obtained directly from the apical or the axillary buds (Tab. 1, Fig. 1d). Similar results were observed in previous studies on the common thyme (B e h e s h t i & M o r t e z a , 2005). A high capability of explant multiplication was observed on the medium with the highest cytokinin/auxin ratio (2 and 4 / 0.1 mg/L) and leads us to consider these combinations as efficient promoters of T. vulgaris multiplication. Photosynthetic pigments – The content of the total chlorophylls ranged from 0.45 mg/g FW for control to 1.25 mg/g FW for BI1 treatment (Tab. 2). The increase in the total chlorophyll content was a result of a significant increase in chlorophyll b content, while chlorophyll a content decreased significantly when compared to the control. According to our results an increase in the cytokinin concentration in the medium (BI1 and BI2) significantly increases the amount of chlorophyll b produced, except for BI4 medium. On the other hand it shows reverse correlation with the chlorophyll a content. It was not possible to establish a direct link between concentrations of PGRs used and the content of photosynthetic pigments in all cases (S t e t l e r & L a e t s c h , 1965; E d e l m a n & H a n s o n , 1971). Cytokinins as well as auxins can improve concentration of photosynthetic pigments (V e r m a & S e n , 2008; P a r s a e i m e h r et al., 2010; V a m i l et al., 2010). Furthermore, it was shown that the BIOLOGICA NYSSANA 2 (1) September 2011: 29-35 Karalija E., Parić A. The effect of BA and IBA on the secondary metabolite... 32 biosynthesis pathway of chlorophyll is highly insensitive to light and cytokinins (Y a r o n s k a y a et al., 2006). Changes in the content of chlorophylls, caused by PGRs, could, most likely, be related to growth rate and primary and secondary metabolic activities (L i c h t e n t h a l e r , 1987). Secondary Metabolite Production – The total phenol content was significantly higher on BI1 treatment, while total flavonoid content was higher on all treatments when compared with the control (Tab. 3). It was previously reported that the maturation of cells always corresponds to an increase in secondary metabolites (S h e t t y & L a b b e , 1998; B i o n d i et al., 2002; P a r s a e i m e h r et al., 2010). On the other hand, the quality and the quantity of applied plant growth regulators have a significant effect on the metabolism of secondary metabolites (K h a n et al., 2008). In plant cell cultures the production of secondary metabolites supports cell multiplication and division, hence plant growth regulators are bound to have a major role in determining the production of potentiality of a given culture. During our research presence of higher concentrations of BA promoted shoot proliferation and flavonoid production. Accumulation of flavonoids is usually correlated to morphogenesis during plant development (P a s q u a et al., 2003). However, no such positive influence of higher BA concentrations was observed for phenol content. Only the BA concentration that tested the lowest (0.5 mg/L) induced phenol accumulation. The concentration of monomeric anthocyanins was significantly lower on all treatments in comparison to the control (Tab. 3). Anthocyanins exhibited positive correlation for chlorophyll a and carotenoids, but a negative correlation for flavonoids, with the significance at the 0.01 level. The reverse relation between the in vitro shoot proliferation and the production of anthocyanins production, which was found in this study, is a general complication found in in vitro secondary metabolites production. Several studies dealing with this problem observed that 2.4-D or NAA increase anthocyanin production and decrease growth rate. On the other hand some of the studies observed that BA or KIN enhance production of anthocyanins, while inhibiting cell growth (M e y e r & V a n S t a d e n , 1995). The difficulties expressed through the reverse relationship between the production of anthocyanins and cell growth can cause problems in all secondary products induction media and additional research is needed to establish specific requirements of each plant species. a b c d Figure 1. Induction of adventive shoots formation on MS medium with BA and IBA a – BH 0 m g/L BA + 0 mg/L IBA; b – BI1 0.5 mg/L BA + 0.1 mg/L IBA; c – BI2 2 mg/L BA + 0.1 mg/L IBA; d – BI4 4 mg/L BA + 0.1 mg/L IBA; Conclusion Our research shows that the shoot multiplication of common thyme is depended upon the treatment that is used. The highest number of adventive shoots was obtained on the MS media supplemented with 2 or 4 mg/L BA and 0.1 mg/L IBA, which employed the highest concentration of cytokinins. An increase in the concentration of cytokinins above 2 mg/L was followed by a decrease in photosynthetic pigments content, and total monomeric anthocyanins content. Cytokinins (0.5 mg/L) in combination with low auxin levels (0.1 mg/L) can enhance production of phenolic compounds, and higher concentrations of cytokinins can induce an increase in total BIOLOGICA NYSSANA 2 (1) September 2011: 29-35 Karalija E., Parić A. The effect of BA and IBA on the secondary metabolite... 33 flavonoid content. This study successfully established the optimal conditions for the in vitro growth of common thyme, with a potential for secondary metabolite production. Further investigations are necessary in order to increase the production of secondary metabolites by in vitro cultures. References Al-Sane', K.O., Shibli, R.A., Freihat, N.M., Hammouri, M.K. 2005: Cell Suspension Culture and Secondary Metabolites Production in African Violet (Saintpaulia ionantha Wendl.). Jordan Journal of Agricultural Sciences, 1 (1): 84-92. Beheshti, H.B., Morteza, K. 2005: Effects of media and growth regulators on garden thyme (Thymus vulgaris L.) Micropropagation. Iranian Journal of Horticultural Science and Technology, 6 (2): 61-68. Biondi, S., Scaramagli, S., Oksman-Caldentey, K., Poli, F. 2002: Secondary metabolism in root and callus cultures of Hyoscyamus muticus L.: the relationship between morphological organization and response to methyl jasmonate. Plant Science, 163 (2): 563−569. Blakeway, J. 1986: The antimicrobial properties of essential oils. Soap, Perfumery & Cosmetics, 59 (4): 201-203. Budka, D., Khan, N.A. 2010: The Effect of Ocimum basilicum, Thymus vulgaris, Origanum vulgare Essential Oils on Bacillus cereus in Rice-Based Foods. European Journal of Biological Sciences, 2 (1): 17-20. Consentino, S., Tuberoso, C.I.G., Pisano, B., Satta, M., Arzedi, E., Palmas, F. 1999: In vitro antimicrobial activity and chemical composition of Sardinian Thymus essential oils. Letters in Applied Microbiology, 29 (2): 130–135. Cruz, T., Cabo, M.P., Cabo, M.M., Jiménez, J., Cabo J., Ruiz, C. 1989: In vitro antibacterial effect of the essential oil of Thymus longiflorus Boiss. Microbios, 60 (242): 59–61. Deans, G.G., Ritchie, G. 1987: Antibacterial properties of plant essential oils. International Journal of Food Microbiology, 5 (2): 165-180. Edelman, J., Hanson, A.D. 1971: Sucrose suppression of chlorophyll synthesis in carrot callus culture. Planta, 98: 150-156. Farag, R.S., Salem, H., Badei, A.Z.M.A., Hassanein, D.E. 1986: Biochemical studies on the essential oils of some medicinal plants. Fette, Seifen, Anstrichmittel, 88 (2): 69-72. Furuya, I., Kojima, H., Syono, K. 1971: Regulation of nicotine biosynthesis by auxins in tobacco callus tissue. Phytochemistry, 10: 1529-1532. Gruenwald, J., Bendler, T., Jaenicke, C. 2004: PDR for Herbal Medicines. Medical Economics Company. Montvale (NJ). Herrmann, E.C., Kucera, L.S. 1967: Antiviral substances in plants of the Mint Family (Labiatae) 111. Peppermint (Mentha piperita) and other Mint plants. Proceedings of the Society for Experimental Biology and Medicine, 124: 874-878. Ikeda, T., Matsumoto, T., Noguchi, M. 1976: Formation of ubiquinone by tobacco plant cells in suspension culture. Phytochemistry, 15: 954- 959. Ikuta, A., Itokawa, H. 1982: Berberine and other protoberberine alkaloids callus tissue of Thalictrum minus. Phytochemistry, 21: 1419- 1421. Ikuta, A., Syono, K., Furuya, T. 1975: Alkaloids in plants regenerated from Coptis callus cultures. Phytochemistry, 14: 1209-1210. Juven, B.J., Kanner, J., Schveol, F., Weisslowicz, H. 1994: Factors that interact with the antibacterial action of thyme essential oils and its active constituents. The Journal of Applied Bacteriology, 76 (6): 626-631. Karaman, S., Digrak, M., Ravid, V., Iclim, A. 2001: Antibacterial and antifungal activity of the essential oils of Thymus revolutus Celak from Turkey. Journal of Ethnopharmacology, 76 (2): 183–186. Khan, T., Krupadanam, D., Anwar, S.Y. 2008: The role of phytohormone on the production of berberine in the calli cultures of an endangered medicinal plant, turmeric (Coscinium fenestratum l.). African Journal of Biotechnology, 7 (18): 244-3246. Knobloch, K., Pauli, A., Iberl, B., Weis, N. Weigand, H. 1988: Mode of action of essential oil components on whole cell of bacteria and fungi in plate tests. In: Schreier, P. (ed.), Bioflavour 87: 287-299, De Gruyter, Berlin, New York. Kurz, W.E.W., Constabel, F. 1979: Plant cell cultures, a potential source of pharmaceuticals. Advances in Applied Microbiology, 25: 209-240. Lichtenthaler, H. K., 1987: Chlorophylls and carotenoids, the pigments of photosynthetic biomembranes. In: Douce, R., Packer, L. (ed.), Methods in Enzymology 350–382, Academic Press Inc., New York. Mancinelli, A.L. 1984: Photoregulation of anthocyanin synthesis. Plant Physiology, 75: 447-453. BIOLOGICA NYSSANA 2 (1) September 2011: 29-35 Karalija E., Parić A. The effect of BA and IBA on the secondary metabolite... 34 Meyer, H.J., Van Staden, J. 1995: The in vitro production of anthocyanin from callus cultures of Oxalis linearis. Plant Cell, Tissue and Organ Culture 40: 55-58. Morales, R. 2002: The history, botany and taxonomy of the genus. In: Stahl-Biskup, E., Sáez, F. (ed.), Medicinal and Aromatic Plants – industrial profiles 17: 1-43, Taylor & Francis, London. Murashige, T., Skoog, F. 1962: A revised medium for rapid growth and bioassays with tobacco tissue culture, Physiologia Plantarum, 15: 473- 497. Nair, A.J., Sudhakaran, P.R., Madhusudana, J.R., Ramakrishna, S.V. 1992: Berberine synthesis by callus and cell suspension cultures of Coscinium fenestratum. Plant Cell, Tissue and Organ Culture, 29: 7-10. Ordon, A.A.L., Gomez, J.D., Vattuone, M.A., Isla, M.I. 2006: Antioxidant activities of Sechium edule (Jacq.) Swart extracts. Food Chemistry, 97: 452–458. Parsaeimehr, A., Sargsyan, E., Javidnia, K. 2010: A comparative study of the antibacterial, antifungal and antioxidant activity and total content of phenolic compounds of cell cultures and wild plants of three endemic species of ephedra. Molecules, 15 (3): 1668-1678. Pasqua, G., Avato, P., Monacelli, B., Santamaria, A.R., Argentieri, M.P. 2003: Metabolites in cell suspension cultures, calli, and in vitro regenerated organs of Hypericum perforatum cv. Topas. Plant Science, 165: 977–982. Piccaglia, R., Marotti, M., Giovanelli, E., Deans, S.G., Eaglesham, E. 1993: Antibacterial and antioxidant properties of Mediterranean aromatic plants. Industrial Crops Production, 2 (1): 7–50. Porra, R.J., Thompson, W.A., Kriedemann, P.E. 1989: Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectrometry. Biochimica et Biophysica Acta, 975: 384–394. Prabuseenivasan, S., Jayakumar, M., Ignacimuthu, S. 2006: In vitro antibacterial activity of some plant essential oils. BMC Complementary and Alternative Medicine, 6 (1): 39. Shetty, K., Labbe, R.G. 1998: Food-borne pathogens, health and role of dietary phytochemicals. Asia Pacific Journal of Clinical Nutrition, 7 (3/4): 270-276. Shilpashree, H.P., Ravishankar, R. 2009: In vitro plant regeneration and accumulation of flavonoids in Hypericum mysorense. International Journal of Integrative Biology, 8: 43-49. Smith-Palmer, A., Stewart, J., Fyfe, L. 1998: Antimicrobial properties of plant essential oils and essences against five important food-borne pathogens. Letters in Applied Microbiology, 26 (2): 118-122. Staba, E.J. 1980: Plant Tissue Culture as a source of biochemicals.CRC Press, Boca Raton, Florida. Stetler, D.A., Laetsch, W.M. 1965: Kinetin-induced chloroplast maturation in cultures of tobacco tissue. Science, 149: 1387-1388. Vamil, R., Aniat-ul-Haq, Agnihotri, R.K. 2010: Plant growth regulators as effective tool for germination and seedling growth for Bambusa arundinaceae. Research Journal of Agricultural Sciences, 1 (3): 233-236. Vardar, U.G., Candan, F., Sokmen, A., Daferera, D., Polissiou, M., Sokmen, M., Donmez, E., Tepe, B. 2003: Antibacterial and antioxidant activity of the essential oil and methanol extracts of Thymus pectinatus Fisch. et Mey var. pectinatus (Lamiaceae). Journal of Agricultural and Food Chemistry, 51 (14): 63–67. Verma, P., Sen, N.L. 2008: The impact of plant growth regulators on growth and biochemical constituents of coriander (Coriandrum sativum L.). Journal of Herbs, Spices & Medicinal Plants, 14 (3/4): 144 – 153. Weathers, P.J., Bunk, G., McCoy, M.C. 2005: The effect of phytohormones on growth and artemisinin production in Artemisia annua hairy roots. In Vitro Cellular & Developmental Biology, Plant, 41 (1): 47-53. Wolfe, K., Wu, X., Liu, R.H. 2003: Antioxidant activity of apple peels. Journal of Agricultural and Food Chemistry, 51 (22): 609–614. Yaronskaya, E., Vershilovskaya I., Poers, Y., Alawady, A.E., Averina, N., Grimm, B. 2006: Cytokinin effects on tetrapyrrole biosynthesis and photosynthetic activity in barley seedlings. Planta, 224 (3): 700–70. Zarzuelo, A., Crespo, E. 2002: Medicinal and aromatic plants – industrial profiles. In: Stahl- Biskup, E., Sáez, F. (ed.), Thyme 24: 278, Taylor & Francis, United Kingdom. Zhong, J.J., Bai, Y., Wang, S.J. 1996: Effects of plant growth regulators on cell growth and ginsenoside saponin production by suspension cultures of Panax quinquefolium. Journal of Biotechnology, 45 (3): 227-234.