Morocco is a huge provider of the world market in aromatic and medicinal plants, and thanks to its geographical position, it offers a varied range of Mediterranean bioclimate allowing a rich flora made up of more than 4200 species and varied vegetation. Aromatic and /or medicinal species are estimated at 500 to 600 species, many of which are endemic and among them, Origanum compactum.

Oreganos are aromatic and medicinal plants, dedicated to various purposes such as alimentation, drinks, and traditional medicine, used in a number of different ways due to their essential oils characterized by antioxidant, antimicrobial, cytotoxic, antitumoral, anti-corrosion activities, etc. [1]. Oreganos are native to Asia and Europe, currently distributed worldwide [2] but widespread in the Mediterranean region [3]. 80% of species are located in the western Mediterranean [4], some species being specific to a well-defined territory, such as O. saccatum, O. boissieri, O. hypericifolium, O. sipyleum, O. acutidens, O. haussknechtii, O. srevidens in Turkey [5], O. syriacum in the Middle East or O. dictamnus in Greece where it is very popularly known in the field of medicine.

In Morocco, there are few taxa belonging to the genus Origanum (Zaatar in Arabic): O. elongatum(Bonnet) Emb. & Maire (included O. grosii Pau & Font Quer), a Morrocan endemic species, O. vulgare ssp. virens (Hoffmanns. & Link) Ietsw. (= Origanum virescens Poir.) and O. compactum Benth. [6]. Origanum x font-queri Pau, considered a hybrid between O. grosii and O. compactum, and possibly included in O. elongatum, is close to O. grosii with larger bracts and calyx and more or less glabrous stems and leaves [7].

The geographical distribution of oreganos includes the western Rif area and the middle Atlas regions. Due to drought and mainly overexploitation, the second region is shrinking more and more, as nowadays only the Rif region offers the current wide range of exploitation of oregano [6]. Origanum compactum is widely exploited for marketing purposes and O. elongatum on a smaller scale. Morocco exported 23 Million tons of dried oregano in 2021 [8]. Origanum vulgare ssp. vulgare could also be cultivated [9].

Origanum compactum is a native herb in the Mediterranean region belonging to the stages of thermo-Mediterranean and meso-Mediterranean vegetation with semi-arid and sub-humid bioclimate. It is the main source of production of oregano OHC in Morocco [10]. Origanum compactum is rich mainly in phenolic compounds and widely used in traditional Moroccan medicine to cure certain diseases, such as diarrhea, respiratory diseases, cutaneous and urinary infections [11]. Several studies have shown the biological activities of Origanum compactum, antioxidant and antimalarial activities [12], antibacterial activity [13] [14], antifungal activity [15], cytotoxic activity and antitumoral activity [16].

In the Rif region of Morocco, Origanum compactum is overexploited because of its commercial value: the species is threatened with extinction because of abusive exploitation mode (uprooting of the whole plant with its roots). Therefore, operational rationalization measures for regeneration and conservation of the species are needed. Biotechnological tools can provide solutions for the protection and domestication of the species, vegetative propagation or in vitro culture. Tissue culture and micropropagation have been completed in some Oregano species. Several studies use nodes from seedlings as a type of explant for micropropagation [9] [17] - [26]. In the present study, nodes with two axillary buds of Origanumcompactum originating from Ouazzane region were used as explants with the objective to establish a protocol for vegetative multiplication in vitro by proceeding different tests in order to assure the farmer's needs and introducing an alternative culture of Origanumcompactum.

Testing the effects of macronutrients on the micropropagation of Origanum compactum allows us to choose N₃₀K macronutrients for the experiments that follow. This possibility is made by the complete absence of hyperhydric plants as well as the successful development of the culinary and rooting part. However, in many cases with other species of oregano and plants belonging to Lamiaceae family, MS medium has been the basal one in most researches. Studies that reflect this include the micropropagation in Origanum minutiflorum [33] , O. vulgare [34], O. majorana [35], O. sipyleum [36] , O. acutidens [26], O. syriacum and O. ehrenbergii [37], Lavandula angustifolia [38], Salvia rosmarinus [39], Thymus vulgaris [40], Salvia officinalis, Melissa officinalis and Mentha longifolia [41]. Furthermore, Morone-Fortunato and Avato (2008) [42] opted for the micropropagation of Origanum vulgare in the (BM) Buffered Minimum medium

containing the macronutrients of MS and the micronutrients of Nitsh and Nitsh [43]. On the other hand, few studies used modified MS as a less concentrated media as in the case of Thymus mastichina [44]. Basal medium N₃₀K is demonstrated to be chosen by less authors, such as Nobre (1996) [45], for the micropropagation of Lavandula stoechas from single nodes.

Moreover, the explant response varies according to the concentration and the type of cytokinins, as, indeed, a higher concentration of BAP, Zeatin and 2ip promoted the best multiplication of buds and nodes. However, less concentration of Kin (1.33 µM) remained the best, not only for the bud and node proliferation, but also for the elongation of the culinary part. This result is similar to BAP and Zeatin, as well as in the case of 2ip. Additionally, elevated concentrations of Kin, DPU and TDZ improved the multiplication of shoots, unlike Adenine and Zeatin, BAP and 2IP where lower concentrations remained efficient. Furthermore, the highest concentration of Kin and the lowest concentration of Zeatin, 2ip, and BAP provided best root development and the optimal rate of rhizogenesis is mentioned in case of 3.11 µM Kin. Moreover, Zeatin and adenine was more effective at concentration of 3.11 µM in terms of all the parameters evaluated.

The study of both the concentration and type of cytokinin effects on the organogenesis is reported in several species belonging to Origanum genus. The findings reveal the importance of these chemicals in the culture’s micropropagation and reproduction. Thus, the investigation of Özkum (2007) [33] showed that high concentration of BAP (2 µM) was efficient for the shoot regeneration; in addition, Cristea et al. (2008) [34] revealed that BAP at 2.97-5.94 µM gave the best shoot proliferation and regeneration of Origanum majorana and its replacement with Kin did not promote explants regeneration, unlike Abdallah et al. (2017) [22] who demonstrated that 2 µM Kin gave the highest number of shoots, shoot length, number of leaves and number of leaves per shoot. Zayova et al. (2016) [46] proved that 2.28 and 4.56 µM Zeatin remained the best for multiple shoot formation of Greek oregano (Origanum heracleoticum) with 70% shoot formation. On the other hand, Korkor et al. (2017) [35] demonstrated that 5.94 µM BAP had negative effect on shoot elongation of Origanum majorana explants but favorise their multiplication. Moreover, the study of Baricevic and Padulosi [47] indicated that the micropropagation of Origanum vulgare is independent of cytokinins and the integration of Kin in the culture medium had no effect on proliferation unlike Borovec (1988) [48] who revealed that a very high concentration of BAP (23.76 µM) exhibited good development of cultures. In addition, Morone-Fortunato and Avato (2008) [42] reported that 5.94 µM BAP was suitable for the production of multiple shoots of Origanum vulgare ssp. hirtum. Furthermore, Socorro et al. (1998) [49] noted a maximum elongation of main and axillary stems, an increasement of leaves number and axillary stems also a good development of root system of Origanum bastetanum (=O. vulgare ssp. virens) vitroplants when the MS media is added with 2.22 µM BAP. The effect of cytokinins is evaluated in other species belonging to Lamiaceae family as Lavandula angustifolia where lower concentration growth regulators was efficient for induction of buds, elongation and multiplication of shoots [38]; for the case of Salvia fructicosa, the multiplication of shoots, buds and nodes, and the elongation of stems were optimum in medium containing 0.75 µM BAP [50]. The integration of cytokinins on media culture had a positive effect on development of aerial parts more specifically. 2.22 µM BAP that gave the best results in terms of budding, growth, regeneration rate and absence of hyperhydric vitroplants but on the other hand the growth of the roots was not good enough to succeed the acclimatization.

The combination of different auxins at different concentrations with 2.22 µM BAP ensured a better development of roots similarly for the culinary part, except NAA that had a negative response on root development, unlike the investigation of Cristea et al. (2008) [34] who showed that the rooting medium containing lower concentration of NAA gave a good development of foliar and root system of Origanum vulgare explants, and those who are exposed to highest concentration of NAA 10.74 µM or more have become necrotic and have lost their leaves with shoot fragments dying gradually. Therefore, 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 and the response to growth regulators could change for species to an another. Indeed, in case of Origanum acutidens [26], the regeneration of roots was successful in case of medium containing a high concentration of BAP (10.69 µM) and a low concentration of NAA (1.07 µM) and shoots regenerated in the medium containing different concentrations of BAP and 1.07 µM NAA easily developed roots, in agreement with Socorro et al. (1998) [49] and Goleniowski et al. (2003) [26]. On the other side, for the micropropagation of Lippia graveolens performed on media containing a combination of BAP and IAA, no significant differences in shoot numbers were found, and addition of IAA allowed only the stimulation of the roots [51]. Contrariwise, in our study, the combination between BAP and IAA stimulates higher rate of root formation with satisfactory shoot formation. Besides, the protocol established for the micropropagation of O. sipyleum showed that the best rooting medium contained higher concentrations of IBA (7.38 or 12 µM) (Sevindik et al., 2017) [23], whereas in our case lower concentration of IBA (1.14 µM) was sufficient, result similar to that reported in the study of Oluk and Çakýr (2009) [36] which demonstrated that 96% of Origanum sipyleum explants developed roots in the medium supplemented with 246 µM IBA with a very important root length. It has also been shown that the rooting of O. minutiflorum was successful in the MS medium supplemented with 14.76 µM IBA [33]. Contrariwise, in our case, higher concentration of IBA had a negative influence on root development. Arafeh et al. (2003) [52] also demonstrate that lower concentration of IBA (1.96 µM) gave acceptable length of roots and shoots of Origanum syriacum L. but higher percentage of rhizogenesis (90%) was mentioned in MS medium supplemented with 4.56 µM IAA in agreement with our study where the addition of high IAA concentration generate an important percentage of rooted explants.

The combination of GA₃ with 2.22 µM BAP and 6.27 µM IAA results in some changes in the growth of vitroplants of Origanum compactum, particularly in the aerial part. Control medium 2, compared with the different combinations, is the best for root growth and development, and generates a high percentage of rooted vitroplants. Few studies rely on the effect of GA₃ alone or in combination with cytokinins and auxins. Goleniowski et al. (2003) [26] showed that combining 0,25 µM GA₃ with BAP had a positive effect on the elongation of O. vulgare x applici shoots but had a negative effect on the multiplication of shoots of Origanum sipyleum. In addition, Harfi et al. (2019) [53] reported that the addition of 2.22 µM BAP to the medium supplemented with 0.58 µM GA₃ allowed maximum elongation of the vitroplants and that the combination of 1,44 µM GA₃ with 2.22 µM BAP and 1.425 µM IAA induced callus formation. Moreover, they showed that the absence of cytokinins in the medium supplemented with GA₃ and IAA promoted root development and regeneration of a significant percentage of rooted vitroplants of Origanum compactum. In addition, El-Antably et al. (1975) [54] demonstrated that the addition of 100 µM GA₃ to the culture medium containing or not 116 µM Kin allowed the increase of the length of Origanum majorana vitroplants.

The effect of GA₃ alone or in combination with cytokinins or auxins have been explored in other Lamiaceae species, such as the study by Mandal (2013) [55] which showed that the addition of 4.44 µM BAP and 1.44 µM GA₃ to the culture medium is favorable for the multiplication of Hyptis suaveolens and allows a total regeneration of the explants. In addition, Samantaray et al. (2013) [56] obtained a maximum regeneration rate of Vitex trifolia explants in the medium containing 1.14 µM GA₃, 0.54 µM NAA, and 271.5 µM Adenine. On the other hand, Aicha et al. (2013) [57] reported that the presence of 1 µM GA₃ alone in MS 1/2 medium improves the elongation of Thymus saturejoides vitroplants. Similarly, Paul et al. (2010) [58] demonstrated that the incorporation of 1 µM GA₃ into MS medium has a positive effect on the elongation of Pogostemon cablin.

Furthermore, the in vitro growth of Origanum compactum explants is optimized in the case of a combination of 2.22 µM BAP and 6.27 µM IAA and the integration of three polyamines at different concentrations in the N₃₀K medium supplemented with 2.22 µM BAP and 6.27 µM IAA leads to some modifications in the micropropagation of Origanum compactum, especially the aerial part. In fact, compared with the two controls media and other combinations, 5.67 µM Spermine is the most suitable for bud and shoot propagation and provides a high percentage of regeneration. On the other hand, Spermidine at the same concentration produced the longest explants. However, the addition of polyamines has no improving effect on root multiplication, and Spermidine at four concentrations has an inhibitory effect on root development. As well for 11.34 µM Spermine. In contrast, root multiplication is optimal for control medium 2.

Our study (9) is the one which has examined the effect of polyamines on the in vitro culture of Origanum vulgare L. ssp. vulgare; we have found that 1.304 µM Putrescine provides better multiplication of buds and roots and gives a high percentage of regeneration and rhizogenesis. However, investigations into the influence of these growth regulators have been undertaken on other Lamiaceae. Thus, Bajaj (2013) [59] showed that the presence of Spermidine alone in the culture medium did not induce the morphogenesis of Sideritis angustifolia but had an effect on growth when combined with NAA, IAA or BAP. In addition, the combination of 0.01 mM Spermidine with 27 µM NAA had a remarkable effect on rhizogenesis and reversed the 27 µM NAA inhibitory response on root formation.

The protocol established in this study for the micropropagation of Origanum compactum Benth. by the technique of axillary bud is reliable and original. N₃₀K medium was chosen because it ensures good development of the stem part, provides a relatively high rate of regeneration and rhizogenesis, and guarantees a total absence of hyperhydric explants.

Subsequently, the evaluation of the effect of seven cytokinins at five concentrations showed that 2.22 µM BAP gave the best results in terms of growth, regeneration and absence of hyperhydric plants.

Root-part development was optimized only after the 2.22 µM BAP was combined with auxins, more specifically 6.27 µM IAA, which ensured not only root-part development but also whole plant growth.

Subsequently, the incorporation of GA₃ into the culture medium in the presence of 2.22 µM BAP and 6.27 µM IAA resulted in some changes in the growth of vitroplants, particularly in the aerial part. Bud and shoot multiplication and aerial elongation were at their maximum for a combination with 2.89 µM GA₃. However, control medium 2 (2.22 µM BAP + 6.27 µM IAA) remained the best for root growth and development, generating a high percentage of rooted vitroplants.

The integration of polyamines at 2.22 µM BAP and 6.27 µM IAA showed that Spermine at 5.67 µM was the most efficient for bud and shoot propagation and provided a high percentage of regeneration. On the other hand, Spermidine at the same concentration produced the longest explants. However, the addition of polyamines did not improve the root part.

Finally, acclimatization of rooted seedlings from 12 to 16 weeks 3 was successfully established. These seedlings allow a re-initiation of micropropagation after sterilization and the proliferation of a large number of the vitroplants. The protocol followed in this study thus makes it possible to ensure the conservation of this species, to avoid its disappearance and to introduce plants on the market with a high added value.

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

Benkaddour, R., Ali, N.B., Badoc, A., Hamdoun, O., Boussaoudi, I., Azaroual, L., Martin, P. and Lamarti, A. (2022) Efficient Plant Regeneration from Explants of Compact Oregano (Origanumcompactum Bentham). American Journal of Plant Sciences, 13, 1311-1334. https://doi.org/10.4236/ajps.2022.1310089