Interaction Effect of Plant Growth Regulators on Shoot Micropropagation of Aromatic Plant Origanum elongatum (Bonnet) Emberger & Maire

Origanum elongatum (Bonnet) Emb. & Maire, is a medicinal, aromatic and endemic plant of Morocco, characterized by its pharmacological effects, and is commonly used for the production of essential oils and aromas, resulting in high harvest and overexploitation pressure. This is why the present study aims to im-plement the in vitro micropropagation of Origanum elongatum for optimal vi-troplant production. Six macroelements were tested (SH, SD, N30K, MS, MSm and B5) and the SD medium was selected for vegetative propagation of the explants. Seven cytokinins: adenine (Ad), N6-(2-Isopentenyl) adenine, zeatin (Zeat), kinetin (Kin), benzyladenine (BAP), 1,3-diphenylurea (DPU) and thidiazuron (TDZ) were then evaluated at five concentrations (0.44, 1.33, 2.22, 3.11 and 4.44 µM/L) on growth, development, budding, rooting and hyperhydricity. 0.44 µM Kin was selected and combined with three auxins: indole-3-acetic acid (IAA), indole-3-butyric acid (AIB), and 1-naphthaleneacetic acid (NAA) at four concentrations (1.14, 2.85, 4.56 and 6.27 µM/L) to improve rooting and association with 1.14 µM IAA was shown to be efficient for roots development. Different concentrations of gibberellic acid (0.29, 1.5, 2.60 and 2.89 µM/L), combined with 0.44 µM/L Kin and 1.14 µM/L IAA, were tested and 2.60 µM/L GA 3 gave maximum buds and shoots. Then, the combination of three polyamines at five concentrations (1.134, 3.402, 5.67, 7.938 and 11.34 µM/L) with 0.44 µM Kin and 1.14 µM/L IAA showed an increase in the number of buds and shoots for 7.938 µM/L putrescine and 3.402 µM/L spermine. Finally, seedlings with good foliar and root development were acclima-tized.

It is an aromatic species of limited geographical distribution in northeastern Morocco and extends from the Middle Atlas to the Rif mountain ranges, mainly at high altitude on the mountains) [2]. It grows in forests, rocks and matorals (thicker than scrub), more on siliceous substrates and deep, well-drained soils [3]. It is characterized by a fairly high bioclimatic plasticity ranging from semiarid to wet per, with the thermo-Mediterranean and meso-Mediterranean vegetation stages being the most favorable [4]. In Morocco, its common Arabic name is Zaatar [3].
It is a 90 cm tall subshrub with light or dark upright stems, has glabrescent leaves, more or less glaucous, and a simple panicle of verticillasters about 40 mm long and 3 mm wide) [5]. It flowers from June to October [3]. White inflorescences are attached to vertical stems [6]. The abundance of inflorescences gives the species an ornamental interest [3]. Harvesting is possible in the first year but with a low yield of dry matter [7]. Germination is conditioned by several abiotic factors: a temperature of 20˚C, pH 6 and 1 g/L salinity are optimal for its germination [8]. Volatile compounds and extracts of Origanum elongatum exhibit antibacterial, antifungal, antiviral, antioxidant, vasodilator, anticorrosive and hepatoprotective effects [9]. O. elongatum contains several classes of bioactive compounds, including terpenoids, hydrocarbons, flavonoids, and phenolic compounds [10] [11].
Origanum elongatum is used in Morocco as an antibacterial, antifungal, antiviral, antioxidant, condiment and for the preservation of local food products such as melted butter and olives ([12] [13] [14]). It is a plant commonly used in Morocco for the production of essential oil and flavorings [15], which causes its overexploitation, especially since Morocco is ranked twelfth world exporter of medicinal and aromatic plants and the species is endemic to Morocco. High harvest pressure makes the species vulnerable and leads to its inclusion in Morocco's national red list ( [16] [17]). A rapid assessment of its vulnerability by [18] classified it as a species in urgent need of conservation, restoration and sustainable management. Plant tissue culture can provide agriculture and industry with the plants needed to meet growing global demand, and is a powerful means of conserving, protecting, and domesticating vulnerable species. In this context, the aim of the present study is to establish an efficient protocol for the in vitro culture of O. elongatum originating in the Taza region, by the axillary bud technique.

Plant Material
The explants used in this study were obtained from the apex of 3 to 4 centimeters of young plantlets of Origanum elongatum (Bonnet) Emberger & Maire.
aged four weeks preserved in the Laboratory of Plant Biotechnology.

Effect of Mineral Nutrients
The mediums on which we performed the test are MS [19], SD [20], modified MS (MSm) [21], N30K [22], B5 [23] and SH [24], all of them were added with MS micronutrients and vitamins and 3% sucrose. The macronutrients gave the best results in terms of being served for all the following tests.

Effect of Cytokinins Combined with Auxins
Three auxins: IAA (indole-3-acetic acid), NAA (1-naphthalene acetic acid) and IBA (indole-3-butyric acid) at four concentrations (1.14, 2.85, 4.56 and 6.27 µM/L) were tested with the most appropriate cytokinin determined in the preceding test. The medium containing only cytokinin served as a double control.

Effect of Cytokinins and Auxins Combined with Gibberellic Acid
Four concentrations of gibberellic acid (0.29, 1.5, 2.60 and 2.89 µM/L) were tested with the best combination of cytokinin and auxin. The medium containing only cytokinin was considered the control medium number 1 and the medium supplemented with the best combination of cytokinin and auxin served as double control.

Effect of Cytokinins and Auxins Combined with Polyamines
Three polyamines (putrescine, spermidine and spermine) at four concentrations each (1.134, 3.402, 5.670, 7.938 and 11.340 µM), were tested with the best combination between cytokinin and auxin. The medium containing only cytokinin served as the control medium number 1 and the medium supplemented with the best combination of cytokinin and auxin served as double control.

Culture Conditions
The tubes were hermetically wrapped with aluminum foil and autoclaved 21 mn at 121 • C and 1 bar pressure. The cultures were incubated under specific con-

Acclimatization of Plantlets
The rooted explants, one month old and about 15 cm were removed from the tubes and their roots were freed of the agar. They were transferred to plastic pots filled with autoclaved peat. The plantlets were covered with plastic transparent plastic to prevent the loss of moisture and placed in a culture room (photoperiod: 18/6 h, humidity: 90% -100%, temperature: 24˚C ± 1˚C). The leaves were sprayed with water twice a week. After four weeks, the transparent plastics were removed, and after three weeks, the surviving ones were transferred to large pots. Afterwards, they were placed under natural conditions of illumination and temperature. After ten days, the number of acclimated plants and the percentage of survival were determined.

Statistical Analysis
36 explants were used for each experiment and data were processed by analysis of variance (ANOVA) to detect significant differences between means using the IBM SPSS 20 and Statistica 18 PSW software. Significant differences were compared using Tukey's HSD. Values above p ≤ 0.05 are considered significant.

Effect of Macronutrients
The maximum number of buds is marked in the case of the N 30 Figure 1).
Furthermore, root multiplication was at its maximum for SD medium (7.81), followed by MSm medium (5.64) and N 30 K medium (4.91) and at its minimum for MS and B5 medium (3.00). For the elongation of the stem, SD medium gave American Journal of Plant Sciences better results (2.13 cm), followed by Mm (2.08) and N 30 K (2.00) and the shortest explants were generated in the B5 medium (Table 1, Figure 1).  In the other hand, the SD medium allowed total regeneration of the explants, followed by N 30 K (88.90%) and SH (80.55), while B5 gave a minimum percentage of regeneration (44.44). The maximum rate of rhizogenesis is marked in MSm medium (88.90%), followed by MS (94.44) and SD (93.75) and the minimum rate is mentioned for B5 (75.00) ( Table 1). The absence of hyperhydric explants is marked in the six culture media (Table 1).
In conclusion, the SD medium is the best for the development and growth of vitroplants of Origanum elongatum and allows a total regeneration of explants.
It has been selected for the following experiments.

Effect of Cytokinins
The addition of cytokinins to the SD medium gave better results. In terms of bud  Figure 2).  To sum up, the integration of cytokinins into the culture medium proves advantageous for the development of explants and more particularly the aerial part. 0.44 µM Kin was chosen for the following experiments because it gave good results in terms of elongation of the vitro plants and multiplication of shoots and buds. In addition, it gave a maximum rate of regeneration with no hyperhydric plants. The root part will be improved by combining 0.44 µM Kin with three auxins at increasing concentrations.

Effect of Cytokinins Combined with Auxins
The combination of 0.44 µM Kin and the three auxins shows a favorable effect on the growth of Origanum elongatum explants.
Thus, the medium added with 1.14 µM of IAA generates the maximum number of buds (28.26), followed by 6.27 µM of IAA (26.52) and 4.56 µM of NAA (25.4). In contrast, the medium supplemented with 1.14 µM NAA provides a minimum number of buds (17.42). For shoot proliferation, it is highest in the presence of 1.14 µM IAA (1.97), followed by 6.27 µM IAA (1.88) and 4.56 µM NAA (1.86). The IBA at 1.14 µM records the lowest value (1.40) (Table 3, Figure  3). In addition, the combination of 1.14 µM IBA and 1.14 µM IAA respectively with 0.44 µM Kin provides best results in terms of explant elongation (4.34 and 4.06 cm), followed by 4.56 µM NAA (3.95 cm), while the shortest explants is obtained in the presence of 4.56 NAA (2.34 cm) (Table 3, Figure 3).  On the other hand, the addition of auxins to the culture medium improves the propagation of the roots. In fact, the maximum number of roots is recorded in the presence of 1.14 µM IAA (8.67), followed by 1.14 µM IBA (7.66) and 4.56 µM IAA (6.90) while the minimum number of roots is noted in the presence of 6.27 µM IBA (3.90) (Table 3, Figure 3).
All in all, the combination of 0.44 µM Kin and 1.14 µM IAA is the most advantageous for the development of both parts of the plant. Also, it allows a relatively high rate of regeneration and rhizogenesis.

Effect of Cytokinins and Auxins Combined with Gibberellic Acid
Among the four concentrations of GA 3 combined at 0.44 µM Kin + 1.14 µM IAA, we find that the culture medium supplemented with 2.60 µM of GA 3 is the best in terms of bud multiplication (20.  (Table 4).  (2.30) ( Table 4, Figure 4).   Figure 5).

Discussion
The oregano Origanum x majoricum Cambess [42]. In addition, Kizil [49] and the elongation and multiplication of the roots of Tectona grandis L.f. vitroplants was better in the medium supplemented with 18.08 mM putrescine and 2.46 µM IBA, in addition, the presence of putrescine accelerated shoot growth and development [50]. Application of polyamines alone, including spermine and spermidine at concentrations greater than 10 µM, inhibits the root formation of Lavandula × intermedia "Grosso" vitroplants (Erland and Mahmoud 2014) [51]. El Ansari et al. (2019) [36] found that the best propagation of buds, shoots and roots of Thymus vulgaris L. was marked in the medium supplemented with 10 µM spermine and demonstrated that 50 µM spermidine was effective for better propagation of roots whereas 10 µM of the same polyamine was favorable for root propagation.

Conclusions
This study is the first experiment of micropropagation of Origanum elongatum, endemic to Morocco.
The SD medium ensured good results in terms of bud and root multiplication and elongation of the stem and root parts while allowing total regeneration of the vitroplants and the absence of hyperhydricity. Kin at 0.44 µM has provided optimal results in term of bud proliferation and shoots elongation and multiplication; in addition, it afforded a total regeneration of vitroplants. Thus, the optimization of growth and development of the root part is ensured by the combination 0.44 µM Kin and 1.14 µM IAA.
Moreover, the combination of polyamines with 0.44 µM Kin and 1.14 µM IAA did not significantly improve the micropropagation of Origanum elongatum, particularly the proliferation of the culinary part. Certainly, a small increase in the number of buds and shoots is observed with 7.938 µM of putrescine and spermine. However, high concentrations of putrescine and spermidine favored better root elongation, and the combination of 2.60 µM GA 3 with 0.44 µM Kin and 1.14 µM IAA provided a maximum number of buds and shoots, and the medium supplemented with 1.14 µM GA 3 allowed a better root multiplication and elongation.
Finally, vitroplants from SD + 0.44 µM/L Kin/L + 1.14 µM/L IAA showing good foliar development and characterized by a persistent root system were selected for acclimatization and it was successfully carried out, in addition, a high survival rate after acclimatization was marked. As a result, the protocol followed in this study is efficient for the conservation, protection and domestication of this vulnerable species, in order to meet increasing market demand in a short time, however, the main disadvantage plant tissue culture through micropropagation methods is the relatively higher costs involved as compared to other methods, therefore recent findings on low-cost methods used in plant tissue culture for the in vitro propagation of plant are investigated.