The genus Thymus is very common in the Mediterranean region, where certain species form a particular type of dense vegetation not exceeding 50 cm high, well adapted to the hot summers [1]. Generally, this plant presents gynodioecy, which means that it produces two types of individuals, some with female flowers without stamens and others with hermaphrodite flowers [2].

Plants of the genus Thymus have been the subject of numerous chemical and pharmacological studies and their main characteristic is the production of essential oil, accumulated at the level of pelted glandular trichomes [3]. In particular, Thymus vulgaris, is a perennial shrub present in the Mediterranean region with at least six different chemotypes [4] ; the major compounds reported by the studies carried out on the essential oil of this species are phenolic monoterpenes, namely thymol and carvacrol, in addition to ρ-cymene, γ-terpinene, oxygenated monoterpenes such as borneol and linalool, and sesquiterpenes represented mainly by β-caryophyllene [5] [6] [7] [8] [9]. Also, Thymus vulgaris contains tannins, saponins, flavonoids, phenolic acids, steroids and tripterpenes [10] [11] [12].

Actually, the great morphological and chemical variability of Thymus vulgaris makes it possible to select plants with the most interesting characteristics for producers. The development of tissue culture methods can contribute to the production of secondary metabolites in several ways. One of them is the rapid multiplication of selected plants by a process to obtain plants genetically identical to the donor plant. Another method could be the production of secondary metabolites by culturing cells and tissues from selected plant explants. With regard to the great variability of thyme, micropropagation could contribute to the production of useful plants for the selection of new varieties, thus providing large quantities of valuable plant material [13] [14] [15]. Thereby, different micropropagation methods of Thymus vulgaris were presented, most commonly by axillary buds and using shoot tips or nodal segments to establish cultures [16] [17] [18] [19].

In this way, the present study describes detailed research on the micropropagation of Thymus vulgaris, extinct species in Morocco, by axillary buds, shoot tips and nodal segments, through the evaluation and comparison of the effect of different compositions of the culture media.

Results obtained concerning the effect of macronutrients on the micropropagation of Thymus vulgaris allowed us to choose MS macronutrients for the rest of our experiments, since they ensure a good regeneration of the aerial part, especially in terms of elongation, with complete absence of hyperhydricity.

Indeed, MS macronutrients have been the most used in the micropropagation protocols established for Thymus vulgaris [16] [17] [18] [19] [26] and for species of the genus Thymus in general [27] - [36]. However, less concentrated media were opted in some studies, namely 1/2 MS for Thymus satureioides Coss. [37] and Thymus longicaulis C. Presl [17], DKW medium (1984) [38] for Thymus membranaceus Boiss. [39], modified MS according to Gavazzi et al. [40] for Thymus mastichina (L.) L. [41] and CMS [42] for Thymus piperella L. [43]. Furmanowa and Olszowska [15], the first to establish micropropagation of Thymus vulgaris, used Nitsch and Nitsch (1969) [44] medium.

Moreover, our results are in concordance with Abdallah et al. [45], who obtained the best number of shoots, leaves and leaves/shoots for Origanum syriacum on MS medium, compared to NN and B₅, as well as with Ӧzkum [46], who found that MS generates the highest number of parameters, such as the number of shoots and the number of leaves/shoots for Origanum minutiflorum, compared to B₅. Also, Nordine et al. [47] found that MS medium is the best for survival, multiplication and elongation of Thymus hyemalis Lange shoots, compared to 1/2 MS, B₅ and White (1963) [48]. In addition, Arikat et al. [49] found that B₅ medium produced shortest shoots compared to MS for Salvia fruticosa. However, Yavuz [50] found that culturing Sideritis stricta on B₅ results in longer shoots than those grown on MS.

Concerning the effect of cytokinins on the multiplication and elongation of cultures, Kin, BAP, DPU, Zeat. and TDZ at 0.46 µM have the best lengths on the one hand and on the other hand, 2iP, Ad. and Zeat. offer the best multiplication of buds. Also, 2iP, Ad., Zeat. and TDZ in the same concentration show an optimal multiplication of shoots. In addition, Kin, 2iP, Ad. and DPU ensure better root regeneration. Again, root development is absent in the case of BAP and weak in the case of Zeat. and TDZ.

Actually, knowing the effect of types and concentrations of cytokinins is indispensable for cultures multiplication, as reported for several species of the genus Thymus. Thus, Nordine and El Meskaoui [30] mentioned that regeneration rate of vitroplants of Thymus bleicherianus Pomel was not affected by the addition of low concentrations of cytokinins, whereas the addition of higher concentrations of BAP (8.88 μM) or Kin (9.6 μM) significantly reduced regeneration rate. Also, shoot multiplication was favored by the addition of 4.44 μM BAP, unlike Kin which has no effect. In addition, stressed appearance of plantlets was reported for Thymus piperella L. and T. vulgaris, characterized by smaller leaves and shorter internodes than in vivo, on CMS basal medium and after addition of increasing concentrations of BAP (6.6 to 8.9 μM). In this way, plantlets were subcultured onto the basal medium supplemented with only 2.2 μM BAP [43] [51]. Furthermore, Fraternale et al. [41] noted a maximum elongation of Thymus mastichina shoots after addition of 0.1 mg/l BAP to MS medium. Moreover, high concentration of Kin (9.3 μM) resulted in minimal survival of Thymus satureioides Coss. explants with callus formation at their base and the maximum number of shoots was obtained after addition of 2.22 μM BAP, while their number decreases with the increase of this concentration to 8.88 μM. Also, Kin proved to be less effective than BAP for shoot multiplication [37]. Besides, the addition of Kin at 4.6 or 6.9 μM ensured complete regeneration of Thymus hyemalis plantlets, whereas this rate decreased with higher concentrations (9.3 μM) for explants regenerated from apex. Similarly, the addition of BAP at concentrations higher than 2.2 μM resulted in a decrease in the number of shoots [47]. As well, Bakhtiar et al. [29] reported that Kin and TDZ are less effective for the micropropagation of Thymus persicus (Ronniger ex Rech.f.) Jalas. Cytokinins types and concentrations effect were established during the micropropagation of several other Lamiaceae too [45] [52] [53] [54].

The combination of the four cytokinins (Kin, BAP, DPU and Ad.) at 0.46 µM and the three auxins (IAA, IBA and NAA) at 0.57 µM did not always contribute to the optimization of in vitro growth of Thymus vulgaris plantlets, though, we obtained interesting results for the majority of evaluated parameters. Thus, Kin, in combination with auxins, did not always ensure better elongation of the number of buds and the number of shoots indeed increased after integration of 0.57 IAA and 0.57 NAA in MS + 0.46 Kin medium. Also, the number of roots and their length generally improved after addition of auxins. In addition, the combination between BAP and auxins is marked mainly by the development of roots and by a better multiplication of buds. As well, apart from shoots length, the combination of 0.46 DPU + 0.57 IBA has the best values for all morphological evaluated criteria. Generally, the combination of Ad. with auxins resulted in better development of rooting part and the best buds and shoots multiplication is observed after addition of 0.57 IBA to MS + 0.46 Ad.

Actually, the choice of the best cytokinin/auxin balance ensures not only a better development of roots, but a better proliferation of all parts of the plant. Thus, a better multiplication of Thymus caespititius Brot. was obtained by combining 1 mg/l BAP with 0.25 mg/l IBA [33], as well as 8.9 μM BAP with 2.7 μM NAA for Thymus persicus [29], as for Sáez et al. [43], the best multiplication of Thymus piperella vitroplants was carried out on the CMS medium supplemented with 6.66 µM BAP and 2.85 µM IAA. For Sideritis stricta, B₅ medium supplemented with 4.44 µM BAP + 0.54 µM NAA or 8.88 µM BAP + 2.68 µM NAA, was determined to be the most effective medium for shoot formation, while the best rooting was obtained on B₅ medium supplemented with 22.15 µM IBA [50]. Nordine and El Meskaoui [30] reported that the combination of cytokinins and auxins did not significantly improve the micropropagation of Thymus bleicherianus and carried out the rooting phase on growth regulators-free MS medium, as did Karalija and Parić [16] for Thymus vulgaris. Also, rooting of several Lamiaceae was completed on MS or 1/2 MS medium free of growth regulators [54] [55] [56] [57]. In other studies of Thymus and other Lamiaceae species, it was noted that the combination of IBA with BAP or TDZ inhibits rooting [28] [43] [58] [51]. Macro-Medina and Casas [28] obtained the best rooting of Thymus moroderi Pau ex Martínez on a double-phase medium consisting of a solid MS medium without growth regulators and a solution composed of 1/2 Knop macronutrients, 5% sucrose and 0.3% or 0.6% activated charcoal. In addition, rooting of other species of Thymus was carried out on media supplemented only with auxins at different concentrations [17] [29] [31] as well as other Lamiaceae [49] [59] [60] [61].

The micropropagation protocol established in this study showed that biomass production of Thymus vulgaris plantlets differs depending on the medium and growth regulators used. Thus, we found that shoots growing on MS, B5 and SH media hold higher dry weights. Also, the integration of certain cytokinins at specific concentrations resulted in an increase of the dry weight, both for shoots (0.93 TDZ and 0.93 2iP) and roots (0.46 2iP). In addition, combinations between the four cytokinins and the three auxins resulted in an increase in biomass, except for 0.46 Kin + 0.57 IBA, 0.46 DPU + 0.57 IBA and 0.46 Ad + 0.57 IAA.

Indeed, indicators of biomass growth have been used in some micropropagation studies on Thymus and other Lamiaceae species. Thus, the fresh weight of Thymus caespititius vitroplants increased by 7.7% - 8.2% on MS medium supplemented with 1.77 µM BAP in combination with 0.49 µM IBA [33]. In addition, an increase in water content and a loss of dry weight were observed on Thymus daenensis Celak. plantlets treated with 4.4 μM BAP on MS medium; these cultures developed morphological characteristics of hyperhydricity, with translucency, very short shoots, rigid and abnormal leaf appearance [34]. Also, Fraternale et al. [41] reported a maximum biomass increase of Thymus mastichina plantlets, estimated through their fresh and dry weight, on an MS medium supplemented with 0.44 µM BAP, alone or in combination with 10 or 20 μg/l of triacontanol. Likewise, it was found that the fresh and dry mass production of the Thymus vulgaris vitroplants is maximum on MS medium supplemented with 5 μM BAP, whereas the increase of the concentration of the medium in IAA, Kin or Zeat. causes a decrease in biomass production [18]. Pourebad et al. [62] found that increasing MS medium content in TDZ resulted in a decrease in the fresh weight of Lallemantia iberica shoots. Besides, the maximum increase in the fresh weight of Lavandula dentata vitroplants was noted on MS medium supplemented with 0.44 µM BAP [63]. Moreover, dry weight of Ocimum basilicum shoots grown in vitro on MS medium is higher than that of those grown on B₅ and WPM, too, this parameter increased after addition of growth regulators, especially in the case of 2.22 µM BAP combined to 11.42 µM IAA, 9.84 µM IBA or 10.70 µM NAA [52].

Moreover, the difficulty of establishing in vitro culture from acclimatized plants did not show up only in the high levels of contamination obtained, but also in ensuring a high rate of survival uncontaminated plantlets. This remark was also reported in the case of other Thymus species, namely Thymus moroderi [28] with a survival rate not exceeding 28.6%, Thymus bleicherianus with 36% [30], Thymus caespititius with 8% [33] and Thymus longicaulis with 29.4% [17], as well as other Lamiaceae such as Lavandula viridis [64], Salvia pratensis and S. nemorosa [65] with about 30%. Actually, explants from field grown plants contain many contaminants that affect the survival and growth of vitroplants, bacteria in most cases. These contaminants are usually introduced with initial explants and are resistant to surface sterilization protocols. Furthermore, contamination may reappear after a long culture period if it is endogenous [66].

Despite these difficulties, we noticed that plantlets that survived following established sterilization protocols have a high growth capacity and allow us to obtain a large number of vitroplants after several subcultures.

The present study is a complete and effective protocol for micropropagation of Thymus vulgaris, extinct species in Morocco. First, the study of the effect of macronutrients allowed us to conclude that, as with most Lamiaceae, MS macronutrients are the most suitable for in vitro vegetative propagation of this species. Then, we found that the addition of certain cytokinins, even at low concentrations, to the culture medium, ensures better multiplication and growth of vitroplants, namely 0.46 Kin, 0.46 and 0.93 BAP, 0.46 2iP, 0.46 DPU, 0.46 Ad. and 0.46 Zeat. Then, we optimized the multiplication and rooting of cultures by establishing combinations between Kin, BAP, DPU and Ad. at 0.46 μM with auxins IAA, IBA and NAA at 0.57 μM. Thus, we obtained interesting results for shoot multiplication and rooting for the combinations 0.46 Kin + 0.57 IAA or NAA, 0.46 DPU + 0.57 IBA and 0.46 Ad. + 0.57 IBA.

Finally, the successful acclimatization and obtaining of plants similar to wild plants, plus the possibility to initiate again in vitro culture from acclimatized plants, ensure that the present protocol is an interesting tool for large-scale multiplication of Thymus vulgaris plantlets and for the selection and preservation of interesting genotypes.

This work is carried out as part of the project in the priority areas of scientific research and technological development (PPR2/2016/35), financed by the Ministry of National Education, Vocational Training, Higher Education and Scientific Research, as well as the National Center for Scientific and Technical Research (CNRST, Morocco).

The authors declare no conflicts of interest regarding the publication of this paper.

El Ansari, Z.N., El Mihyaoui, A., Boussaoudi, I., Benkaddour, R., Hamdoun, O., Tahiri, H., Badoc, A., El Oualkadi, A. and Lamarti, A. (2019) Effect of Macronutrients, Cytokinins and Auxins, on in Vitro Organogenesis of Thymus vulgaris L. American Journal of Plant Sciences, 10, 1482-1502. https://doi.org/10.4236/ajps.2019.109105