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 μEms, 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 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. The best rooting of shoots was obtained on MS medium supplemented with 2 mgdm 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%).


Introduction
Thymus vulgaris L. (garden thyme) from the Lamiaceae family is one of the most widely used aromatic plants.The dried leaves and flowering tops are employed as flavouring agents for food and beverages, and as sources of essential oil for the pharmaceutical and cosmetic industries.Thyme is an important medicinal plant, and essential oils extracted from the plant, commonly known as thyme oils, have a wide range of therapeutic applications and properties including antirheumatic, antiseptic, antibacterial, carminative, diuretic and expectorant (Deans and Ritchie, 1987;Tabak et al., 1996;Cosentino et al., 1999;Grigore et al., 2010;Mirzaei-Aghsaghali et al., 2012;Nikolič et al., 2014).Essential oils stimulate the immune system to fight colds and flu and aid in tackling infectious diseases (Nikolič et al., 2014).Moreover, essential oils are also used in food as well as the textile industry (Hazzit et al., 2009).The oils are also applied in a wide range of oral hygiene products, and as fragrance components in soaps, cleansers and other toiletries (Leung and Foster, 1996).Thyme essential oil is well recognised for its medicinal properties.The principal constituents of thyme oil are: thymol (20-80%), p-cymene (9-43%), limonene (0.2-24%), γ-terpinene (0-13%) and carvacrol (0.5-6%) (Blum et al., 1997;Lee et al., 2005;Nickavar et al., 2005).
Plant tissue culture, known as micropropagation, offers independence from climatic factors, elimination of geographical and political boundaries, shorter production cycles and novel products not found in nature.In recent years, there have been numerous publications on micropropagation of various species of the thyme genus: T. bleicherianus (Nordine and El Meskaoui, 2014), T. caespititius (Mendes et al., 2013), T. daenensis (Bernard et al., 2015;Hassannejad et al., 2012), T. hyemalis (Nordine et were rinsed three times with sterile distilled water and blotted dry on sterile absorbent paper, and then each seed was put into a 20 ml test tube filled with 5 ml of MS medium of mineral concentration as specified by Murashige and Skoog (1962).At this stage of the experiment, as well as at subsequent stages, the medium was supplemented with 30 gdm −3 sucrose, 100 mgdm −3 inositol, and agar at a concentration of 7 gdm −3 (Plant Propagation Agar Biocorp); the pH of the medium was adjusted to 5.7 with 0.1 M HCl and NaOH solutions.Depending on the stage of the experiment, the test tubes or jars were autoclaved at a temperature of 121 °C for 20min following the potential addition of plant growth regulators to the medium.The established cultures were transferred to a phytotron in which air humidity was adjusted to 70-80% and temperature to 24 ± 1 °C.Lighting time was 16 h a day, and light intensity was maintained at 40µEM −2 s −1 .At the initiation stage, half of the cultures were subjected to the aforementioned conditions, and the other half was cultured in the dark.Each combination comprised 100 seeds.After 4 weeks, the percentage of germinating seeds was determined.

Multiplication stage
Sterile shoots were initiated to grow, and multiplied twice on a medium of mineral composition as specified by Murashige and Skoog (1962).Single-node shoot fragments of 1-1.5 cm in length were collected from multiplied plants and cultured on media containing macro-and microelements according to Murashige and Skoog (1962) and supplemented with BAP, 2iP or KIN at concentrations from 0.5 to 5 mgdm −3 .For each combination of media, 7 jars were prepared (each jar constituted one replication) and 5 explants were transferred to each medium.The controls were explants cultured on MS medium without growth regulators.After 6 weeks, the following morphological characteristics of the analysed plants were determined: mass, number of shoots and plant height.

Rooting stage
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 control was MS medium without plant growth regulators.For each concentration, 7 jars were prepared and 5 thyme explants were placed in each culture vessel.After 4 weeks of culturing, the following features of the analysed plants were determined: mass, height, number of roots and their length.
Even though T. vulgaris is the most common species of this genus and is grown in large areas, scientific literature on the possibilities of propagation of this species in in vitro cultures is not extensive and the results presented therein are often divergent.Research on propagation of in vitro thyme cultures was initiated by Olszowska and Furmanowa (Furmanowa and Olszowska, 1980;Olszowska, 1982;Olszowska and Furmanowa, 1987).The aforementioned authors multiplied the plants on modified NN medium with addition of 10 mgdm −3 adenine sulphate (AS) and 0.5 mg•dm −3 IBA.Their research also allowed for determination of the concentration of essential oils in plant tissues regenerated in in vitro cultures.Ozudogru et al. (2011) investigated the effect of plant growth regulators on the multiplication of selected thyme species including common thyme (T.vulgaris) in in vitro cultures.The experiments conducted by the researchers allowed for determination of the effect of Murashige and Skoog (MS) medium with cytokinins (BAP, KIN and TDZ), used separately and in combination with auxins and gibberellins, on the development of the plant.In order to optimise the process of shoot proliferation, silver nitrate was added to the culture medium.The highest growth rate was obtained using semi-solid MS medium supplemented with 1 mgdm −3 GA3, and MS medium supplemented with 0.05 mgdm −3 2,4-D showed greatest efficiency at the rooting stage.
Selection of the appropriate medium is crucial for obtaining desired results in in vitro cultures.Therefore, this study attempts to identify the most efficient method of micropropagation of thyme (T.vulgaris) in in vitro plant cultures.On the other hand, the quantity of essential oil and its composition may be dependent on the regeneration conditions (Andrys et al., 2017;Andrys and Kulpa, 2017).Moreover, biologically active compounds may be accumulated in in vitro cultures athigher concentrations than in intact plants (Gounaris, 2010, Matkowski, 2008).The aim of the conducted study was to determine the method of thyme multiplication in in vitro cultures at the initiation, multiplication and rooting stages, and to determine the chemical composition of essential oils isolated by hydrodistillation in Deryng and Clevenger apparatus from in vitro shoot cultures of Thymus vulgaris L.

Culture initiation
Seeds of the 'Słoneczko' cultivar of thyme (T.vulgaris) were used to set up the experiment.The seeds were washed for 15 min under running tap water and soaked for 10 s in 70% ethyl alcohol and then for 10 min in 10% sodium hypochlorite solution (NaOCl).Decontaminated seeds anhydrous sodium sulphate, filtered and stored in dark sealed vials at 4 °C until gas chromatography-mass spectrometry (GC-MS) analysis.Two replicates were carried out.Essential oil percentage was calculated based on the dry weight of plant material.

GC-MS analysis of essential oils
The qualitative GC-MS analysis of the essential oils was carried out using an HP 6890 gas chromatograph equipped with an HP-5MS capillary column (30 m × 0.25 mm, film thickness 0.25 μm) and directly coupled to an MSD 5973 mass selective detector.Helium (1 mlmin −1 ) was used as a carrier gas.Samples of 2 μl (40 mg of oil dissolved in 1.5 ml of dichloromethane) were injected in the split mode at a ratio of 5:1.The injector and transfer line were kept at 280 °C.The ion source temperature was 230 °C.
The initial temperature of the column was kept at 40 °C for 5min, then increased to 60 °C at a rate of 30 °C min −1 , next to 230 °C at a rate of 6 °C min −1 (kept constant for 10 min), and then increased to a final temperature of 280 °C at a rate of 30 °C min −1 .The oven was held at this temperature for 5 min.Mass spectra were taken at 70 eV.The mass range was from 40 to 550 m/z.Solvent delay time was 4 min.The total running time for a sample was about 51 min.The relative percentage of the essential oil constituents was evaluated from the total peak area (TIC) by the apparatus software.

Compound identification
The components of the isolated essential oils were identified by comparison of their mass spectra with those stored in NIST 2002 and Wiley NBS75K mass spectral libraries and confirmed by comparison of their calculated retention indices (RI) with data available online in the NIST Chemistry WebBook (http://webbook.nist.gov/chemistry/), the literature (Adams 2007) and our own data for standards (p-cymene, carvacrol and thymol purchased from Fluka and Sigma-Aldrich).
RI were calculated for all volatile constituents using a homologous series of n-alkanes (C7-C40) under the same chromatographic conditions used for analysis of the essential oils.

Statistical analysis
The biometric measurement results obtained were developed on the basis of analysis of variance and assessment of the significance of difference at the levels p=0.05 and p=0.01.The confidence half-intervals calculated using Tukey's test allowed identification of homogenous groups with significant differences between them.Statistical analysis of the GC-MS analysis (results given in Table 3) was conducted for 20 selected constituents of content greater than 1% of the essential oil.

Culture initiation
The culture initiation stage usually requires the primary explants, in this case seeds, to be decontaminated.The decontamination method applied in this experiment (soaking in 70% ethanol solution, then for 10min in 10% NaOCl) proved to be efficient (Fig. 1a, Fig. 2).The seeds started to germinate as soon as 3 days after transferring onto a medium, and after the first week of the experiment, the percentage of germinated seeds cultured in the 16h photoperiod was 60% against 45% for seeds cultured in the dark.After 4 weeks, in the lighted culture the percentage of seeds which germinated was 80% in comparison to 50% in the dark-grown cultures.For most species of the Thymus genus, decontamination of seeds is carried out by soaking in NaOCl solution ata concentration of 1% as for T. hyemalis (Nordine et al., 2013a) to 5% as for T. persicus (Bakhtiar et al., 2014) and T. lotocephalus (Coelho et al., 2012), or in 0.1% HgCl2 solution as for T. mastichina (Fraternale et al. 2003).Other agents used for decontamination of T. vulgaris seeds were 5% calcium hypochlorite (Furmanowa and Olszowska, 2009), 20% NaOCl solution (Karalija and Parić, 2011), or 10% H2O2 solution (Ozudogru et al., 2011).

Effect of cytokinins on multiplication of thyme in in vitro cultures
Our studies show that all applied concentrations of cytokinins had a significant effect on growth reduction.The use of control medium without growth regulators resulted in development of the tallest plants.The shortest plants were cultured on media supplemented with BAP, and the Fig. 1.Micropropagation and callus culture initiation of thyme: a).Germination of seeds on MS medium in cultures lighted with the intensity of 40 µE .m -2.s -1 ; b).Hyperhydric shoots (arrow) forming on node explant on MS medium with 1 mg•dm -1 TDZ; c) Thyme shoots rooted on MS medium with 2 mg•dm -1 IBA; d).Well developed shoots proliferated on medium with 5 mg•dm -3 2iP; e).Thyme plant rooted on MS medium supplemented with 1 mg•dm -1 IAA with visible roots developed on shoots height of the plant decreased with the increase in BAP concentration in the medium (2.6 cm for the plants on medium with 5 mgdm −3 ).According to many authors, multiplication of thyme on media supplemented with cytokinins results in a decrease in shoot height (Nordine and El Meskaoui, 2014).However, the use of this medium also resulted in the development of shoots showing symptoms of hyperhydricity (Table 1, Fig. 1b).
Growth regulators added to media at the multiplication stage are mostly cytokinins added separately or, for the purpose of shoot elongation, in combination with GA3 or auxins.In their extensive research on the effect of cytokinins (6-benzyladenine, kinetin, and thidiazuron) on the multiplication of T. vulgaris and T. longicaulis, Ozudogru et al. (2011Ozudogru et al. ( , 2012) ) found semi-solid MS medium supplemented with 1 mgdm −3 kinetin and 0.3 mgdm −3 GA3 to be the most efficient medium at the multiplication stage.Also, Nordine et al. (2013a) claimed that the best medium to be used at this stage of the experiment was the medium supplemented with 0.5 mgdm −3 KIN.The results of our own research show that KIN at a concentration of 0.5 mgdm −3 resulted in development of plants of the lowest mass and smallest number of side shoots.The results obtained indicate that the cytokinin which had the most positive impact on plant development at the multiplication stage was 2iP (medium supplemented with 5 mgdm −3 , Fig. 1d).The plants cultured on medium supplemented with 528 this growth regulator at the aforementioned concentration were marked by the greatest mass and number of shoots, which allowed for excision of a higher number of explants at the subsequent stage of the experiment.

Effect of auxins on rooting of thyme in in vitro cultures
The results of our research showed that shoots rooted on MS medium without auxins produced the shortest plants with the smallest number of roots (Table 2).In plant in vitro cultures, auxins are responsible for stimulation of root development and cell elongation.However, excessive auxin concentrations added to media result in inhibition of root system development.In the course of our own research it was found that the tallest plants with the greatest mass and the longest and most developed root systems were cultured on the medium supplemented with IBA at a concentration of 1 mgdm −3 (Fig. 1e).According to Nordine and El Meskaoui (2014), Nordine et al. (2013a) and Coelho et al. (2012), the most suitable medium to be used for rooting Thymus sp. is MS medium without plant growth regulators or medium supplemented with IBA at a concentration of 0.5 or 1.5 mgdm −3 .
The auxin often added to medium at the multiplication stage is NAA.Ozudogru et al. (2011) claimed that MS medium supplemented with 0.01 mgdm −3 NAA is the most efficient for rooting thyme shoots.Hosseini Behesthi and Khoushkhoui ( 2005) indicated that at the rooting stage of thyme in in vitro culture, the best results are obtained when the MS medium is supplemented with 0.2 mgdm −3 NAA.However, results of the research conducted by Coehlo et al. (2012) proved that the best medium used at the rooting stage is a combination of MS medium with 1 mgdm −3 NAA or 0.5 mgdm −3 IAA.It was found in the course of our own research that NAA added to the medium at a concentration from 0.2 to 2 mgdm −3 results in development of less tall plants with roots shorter than those found in plants multiplied on the control medium.

Composition of essential oils
The essential oils obtained by hydrodistillation from in vitro shoot cultures of T. vulgaris were found to be yellow liquids and were obtained in a yield of 1.77% (v/w) and 1.75% (v/w) for Deryng and Clevenger apparatus,  respectively.Obtained results are in conformity with the European Pharmacopoeia standard for Thymi herba (a yield of at least 1.2%) (European Pharmacopoeia 2010).The relative amounts of the volatile components identified in the extracted oils are presented in Table 3, in the order of their elution from the HP-5MS column.
In total, 54 different compounds were identified in the essential oils obtained by hydrodistillation in Deryng and Clevenger apparatus from in vitro shoot cultures of thyme, constituting 99.56-99.57% of the oils.
The amounts of p-cymene and thymol recorded in the investigated oils were slightly lower than the European Pharmacopoeia limit; however, the percentage of linalool was found to be within the desired range.The percentage content of γ-terpinene and carvacrol was found to be much higher than that specified by the European Pharmacopoeia.

Fig. 2 .
Fig. 2. The percentage of seeds germinated in vitro in the dark and illuminated with light of intensity 40 µM•m 2 •s -1

Table 1 .
Effect of cytokinin type and concentration on proliferation of Thymus vulgaris aValues followed by the same letter are not significantly different at P ≤ 0.05 according to the LSD Duncan test.

Table 2 .
Effect of auxin type and concentration on shoot rooting of Thymus vulgaris Values followed by the same letter are not significantly different at P ≤ 0.05 according to the LSD Duncan test.

Table 4 .
Statistical analysis of the content of main constituents of essential oil of thyme in dependence on distillation apparatus

Table 3 .
Composition of Thymus vulgaris L. oil in dependence on distillation apparatus RI: retention indices relative to n-alkanes (C7-C40) on HP-5MS column