Micropropagation and Composition of Essentials Oils in Garden Thyme (Thymus vulgaris L.)
Thymus vulgaris L. is an important aromatic plant, because of the synthesis and production of its essential oils for the pharmaceutical and cosmetic industries. In this study, we developed a micropropagation protocol for T. vulgaris ‘Słoneczko’ and evaluated the potential of micropropagated plants for essential oil production with industrial application. The seeds were soaked for 10 min in 10% sodium hypochlorite (NaOCl) solution. Then, each seed was put into a 20 ml test tube filled with 5ml of Murashige and Skoog (MS) medium. Half of the cultures were subjected to light intensity which was maintained at 40 µEm−2s−1, and the other half was cultured in the dark. Shoot explants were multiplied in vitro using MS medium supplemented with BAP, 2iP or KIN. The results obtained indicate that the cytokinin which had the most positive impact on plant development at the multiplication stage was 5 mg dm−3 2iP. Single-node fragments of shoots cultured on MS medium without plant growth regulators were transferred to MS medium supplemented with IAA, IBA and NAA at concentrations of 0.2, 0.5, 1 and 2 mgdm−3. The best rooting of shoots was obtained on MS medium supplemented with 2 mgdm−3 IBA. The essential oils obtained by hydrodistillation in Deryng and Clevenger apparatus from in vitro shoot cultures of T. vulgaris L. were analysed using gas chromatography-mass spectrometry (GC-MS). Analysis revealed the presence of 54 components represented mainly by oxygenated monoterpenes (56.81-57.28%) and monoterpene hydrocarbons (31.90-33.72%). Among identified constituents, the most abundant were thymol (33.37-34.05%), γ-terpinene (11.62-11.91%), p-cymene (9.81-10.07%), carvacrol (5.63-5.96%), carvacrol methyl ether (3.86-3.87%) and linalool (3.16-3.36%).
Adams RP (2007). Identification of essential oil components by gas chromatography/mass spectrometry. Allured Publishing Corporation, 4th ed. USA pp 54-401.
Andrys D, Kulpa D (2017). In vitro propagation affects the composition of narrow-leaved lavender essential oils. Acta Chromatographica 1-6.
Andrys D, Kulpa D, Grzeszczuk M, Bihun M, Dobrowolska A (2017). Antioxidant and antimicrobial activities of Lavandula angustifolia Mill. field-grown and propagated in vitro. Folia Horticulturae 29(2):161-180.
Bakhtiar Z, Mirjalili MH, Sonboli A, Moridi Farimani M, Ayyari M (2014). In vitro propagation, genetic and phytochemical assessment of Thymus persicus – a medicinally important source of pentacyclictriterpenoids. Biologia 69(5):594-603.
Bernard F, Moghadam NN, Mirzajani F (2015). The effect of colloidal silver nanoparticles on the level of lignification and hyperhydricity syndrome in Thymus daenensis in vitro shoots: a possible involvement of bonded polyamines. In Vitro Cellular and Developmental Biology – Plant 51:546-553.
Blum C, Kubeczka KH, Becker K (1997). Supercritical fluid chromatography-mass spectrometry of thyme (Thymus vulgaris L.). Journal of Chromatography A 773:377-380.
Coelho N, Goncalves S, Gonzalez-Benito ME, Romano A (2012). Establishment of an in vitro propagation protocol for Thymus lotocephalus, a rare aromatic species of the Algarve (Portugal). Plant Growth Regulation 66:69-74.
Cosentino S, Tubeerso CIG, Pisano B, Satta M, Mascia V, Arzedi E, Palmas F (1999). In vitro antimicrobial activity and chemical composition of Sardinian Thymus essential oils. Letters in Applied Microbiology 29:130-135.
Deans SG, Ritchie G (1987). Antibacterial properties of plant essential oils. International Journal of Food Microbiology 5:165-180.
European Pharmacopoeia (2010). European Pharmacopoeia, 7th edition. Strasbourg, Council of Europe pp 1252-1254.
Fraternale D, Giamperi L, Ricci D, Rocchi MBL, Guidi L, Epifano F, Marcotullio MC (2003). The effect of triacontanol on micropropagation and on secretory system of Thymus mastichina. Plant Cell Tissue Organ Cultures 74:87-97.
Furmanowa M, Olszowska O (1980). Thymus vulgaris L. propagation through tissue culture. Acta Poloniae Pharmaceutica 37(2): 242-247.
Furmanowa M, Olszowska O (2009). Micropropagation of thyme (Thymus vulgaris L.) III. Biotechnology in agriculture and forestry. High-Tech and Micropropagation 19:230-243.
Gounaris Y (2010). Biotechnology for the production of essential oils, flavours and volatile isolates. A review. Flavour and Fragrance Journal 25:367-386.
Grigore A, Parasachiv I, Colceru S, Babuneau C, Draghici E, Ichim M (2010). Chemical composition and antioxidant activity of Thymus vulgaris L. volatile oil obtained by two different methods. Romanian Biotechnology Letters 15:5436-5443.
Hassannejad S, Bernard F, Mirzajani F, Gholami M (2012). SA improvement of hyperhydricity reversion in Thymus daenensis shoots culture may be associated with polyamines changes. Plant Physiology and Biochemistry 51:40-46.
Hazzit M, Balliouamer A, Verissimo AR, Falerio ML, Miguel MG (2009). Chemical composition and biological activities of Algerian Thymus oils. Food Chemistry 116:714-721.
Hemada M, El-Darier S (2011). Comparative study on composition and biological activity of essential oils of two Thymus species grown in Egypt. American-Eurasian Journal of Agricultural & Environmental Sciences 11:647-654.
Karalija E, Paric A (2011). The effect of BA and IBA on the secondary metabolite production by shoot culture of Thymus vulgaris L. Biology Nyssana 2(1):29-35.
Lee SJ, Umano K, Shibamoto T, Lee KG (2005). Identification of volatile components in basil (Ocimum basilicum L.) and thyme leaves (Thymus vulgaris L.) and their antioxidant properties. Food Chemistry 91:131-137.
Leung AY, Foster S (1996). Encyclopedia of common natural ingredients used in food, drugs and cosmetics. John Wiley & Sons, New York.
Marco-Medina A, Casas JL (2015). In vitro multiplication and essential oil composition of Thymus moroderi Pau ex Martinez, an endemic Spanish plant. Plant Cell Tissue Organ Cultures 120:99-108.
Matkowski A (2008). Plant in vitro culture for the production of antioxidants – a review. Biotechnology Advances 26:548-560.
Mendes MD, Figueiredo AC, Oliveira MM, Trindade H (2013). Essential oil production in shoot cultures versus field-grown plants of Thymus caespititius. Plant Cell Tissue Organ Cultures 113:341-351.
Mendes ML, Romano A (2009). In vitro cloning of Thymus mastichana L. field-grown plants. International Society Horticultural Sciences 505: 303-306.
Mirzaei-Aghsaghali A, Alireza Syadati S, Fathi H (2012). Some of thyme (Thymus vulgaris) properties in ruminant’s nutrition. Annals of Biology Researches 3(2):1191-1195.
Muraskige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum 15(3):473-497.
Nickavar B, Mojab F, Dolat-Abadi R (2005). Analysis of the essential oils of two Thymus species from Iran. Food Chemistry 90:609-611.
Nicolic M, Glamoclia J, Ferreria ICFR, Calhelha CR, Fernandes A, Markovic D, Markovic T, Giweli A, Sokovic M (2014). Chemical composition, antimicrobial, antioxidant and antitumor activity of Thymus serpyllum L. Thymus algeriensis Boiss. and Reut and Thymus vulgaris L. essential oils. Industrial Crop Products 52:183-190.
Nordine A, Bousta D, El Khanchoufi A, El Meskaoui A (2013a). An efficient and rapid in vitro propagation system of Thymus hyemalis lange, a wild medicinal and aromatic plant of Mediterranean region. International Journal of Pharma Bioscience and Technology 1(3):118-129.
Nordine A, Rachida TCh, El Meskaoui A (2013b). Micropropagation of Thymus satureioides Coss. an endangered medicinal plant of Morocco. Journal of Agricultural Science and Technology 9(2):487-501.
Nordine A, El Meskaoui A (2014). Rapid in vitro regeneration and clonal multiplication of Thymus bleicherianus Pomel, a rare and threatened medicinal and aromatic plant in Morocco. Medicinal and Aromatic Plants 3:145.
Olszowska O, Furmanowa M (1987). Micropropagation of thyme (Thymus vulgaris L.) from nodal segments. Herba Polonica 33:137-144.
Olszowska O (1982). Vegetative propagation of Thymus vulgaris L. in tissue culture and estimation of selected strains. PhD Thesis. Medical Academy Warsaw.
Ozcan M, Chalchat JC (2004). Aroma profile of Thymus vulgaris L. growing wild in Turkey. Bulgarian Journal of Plant Physiology 30:68-73.
Ozudogru EA, Ergun K, Ermah K, Issever-Oztruk S (2011). In vitro propagation from young and mature explants of thyme (Thymus vulgaris and T. longicaulis) resulting in genetically stable shoots. In Vitro Cellular and Developmental Biology – Plant 37:309-320.
Pérez-Tortosa V, López-Orenes A, Martínez-Pérez A, Ferrer MA, Calderón AA (2012). Antioxidant activity and rosmarinic acid changes in salicylic acid-treated Thymus membranaceus shoots. Food Chemistry 130:362-369.
Polish Pharmacopoeia (2001). Polish Pharmacopoeia VI. Warszawa pp 897-900.
Porte A, Ronoel L, Godoy O (2008). Chemical composition of Thymus vulgaris L. (thyme) essential oil from Rio de Janeiro State (Brazil). Journal of the Serbian Chemical Society 73:307-310.
Sáez F, Sánchez P, Piqueras A (1994). Micropropagation of Thymus piperella. Plant Cell, Tissue and Organ Culture 39:269-272.
Sargsyan E, Vardanyan A, Ghalachyan L, Bulgadaryan S (2011). Cultivation of thymus by in vitro and hydroponics combined method. World Academy of Science, Engineering and Technology 80:129-132.
Syamasundar KV, Srinivasulu B, Stephen A, Ramesh S. Rao RR (2008). Chemical composition of volatile oil of Thymus vulgaris L. from Western Ghats of India. Journal of Herbs, Spices and Medicinal Plants 17:255-258.
Tabak M, Armon R, Potasman I, Neeman I (1996). In vitro inhibition of Helicobacter pyroli by extracts of thyme. Journal of Applied Microbiology 80:667-672.
Tavan M, Mirjalili MH, Karimzadeh G (2015). In vitro polyploidy induction: changes in morphological, anatomical and phytochemical characteristics of Thymus persicus (Lamiaceae). Plant Cell, Tissue and Organ Culture 122:573-583.
Zawislak G (2007). Analysis of chemical composition of essential oil in the herb of thyme (Thymus vulgaris L.) grown in south-eastern Poland. Herba Polonica 53(3):241-245.
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