Efficient Plant Regeneration from Explants of Compact Oregano (Origanum compactum Bentham)

Origanum compactum Benth., a species endemic to Morocco characterized by its biological activities, is overexploited because of its commercial value and threatened with extinction. Accordingly, measures for its conservation are needed. Micropropagation serves as a solution for the protection and the domestication of this species. In this investigation, we established a protocol for vegetative multiplication in vitro of Origanum compactum by the axillary bud technique. Six culture media (SH, SD, N30K, MS, MSm, B5) were tested to determine the most suitable mineral medium for growth and development of explants. Four cytokinins: Kinetin, Zeatin, BAP (6-Benzylaminopurine), 2ip (2-Isopentenyladenine) and three compounds with cytokinin activity: Adénine, 1,3-Diphenylurea (DPU) and Thidiazuron (TDZ) at five concentrations (0.44, 1.33, 2.22, 3.11, 4.44 μM) were tested on budding, growth, hyperhydria and rooting. Then three auxins, Indole-3-acetic acid (IAA), Naphthalene acetic acid (NAA) and Indole butyric acid (IBA) at four concentrations (1.14, 2.85, 4.56, 6.27 μM) in presence of 2.22 μM BAP were evaluated. The combination of Gibberellic acid (0.29, 1.5, 2.60, 2.89 μM GA3) and three polyamines (Putrescine, Spermidine, Spermine) at five concentrations (1.134, 3.402, 5.67, 7.938, 11.34 μM) with cytokinins and auxins were considered. Our results show that Margara medium is the most efficient and BAP at 2.22 μM is the best cytokinin for development of the aerial parts, with a regeneration rate of 88, 90%;rhizogenesis is successful with the combination of 6.27 μM IAA and 2.22 μM BAP. Moreover, the integration of 2.89 μM GA3 with 2.22 μM BAP and 6.27 μM IAA promotes vitroplant growth, bud and shoot multiplication and elongation of the aerial part. The addition of polyamines with 2.22 μM BAP and 6.27 μM IAA does not improve the root part, but Spermine at 5.67 μM promotes bud and shoot multiplication with a high percentage of regeneration, and spermidine at the same concentration gives long explants. Finally, plantlets with good root development were successfully acclimatized to natural conditions and served as a source to establish in vitro culture again.

Micropropagation and Acclimatization of Common Oregano (Origanum vulgare L. Subsp. vulgare) by Shoot Tip Culture

Origanum vulgare L. is a commercially valued species with remarkable biological properties. It is subject to over-exploitation practices that seriously threaten its sustainability for future generations. Thus, micropropagation serves as a tool for the protection and domestication of this species. In this study, we established an in vitro vegetative propagation protocol for Origanum vulgare. This is done through the axillary bud technique by carrying out various tests. Six culture media (MS, MSm, N30K, SD, SH and B5) were tested. Therefore, SD was chosen for the following experiments. Seven cytokinins (adenine (Ad), N6-(2-isopentenyl) (2ip), zeatin (Zeat), kinetin (Kin), benzyladenine (BAP), 1,3-diphenylurea (DPU) and thidiazuron (TDZ) at 5 concentrations (0.44, 1.33, 2.22, 3.11 and 4.44 μM/L) were evaluated. Thus, Kin at 3.11 μM allowed high regeneration of vitroplants, optimal elongation, total rooting of explants, maximum bud multiplication, and absence of hyperhydric explants. In fact, the integration of auxins (indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), and 1-naphthaleneacetic acid (NAA)) into the culture medium and their combinations with 3.11 μM Kinetin contributed to the optimization of the root part. Thus, it was improved in particular in the case of 3.11 μM Kin and 6.27 μM IBA. Three polyamines (Putrescine, Spermidine and Spermine) at different concentrations (1.134, 3.402, 5.67, 7.938 and 11.34 μM/L) combined at 3.11 μM Kin and 6.27 μM IBA were tested. In fact, 1.304 μM putrescine was considered to be the most suitable for in vitro culture of explants, since it allowed optimal propagation of buds and roots, also a high rate of regeneration and rhizogenesis. GA3 at 1.15 μM combined with 3.11 μM Kin and 6.27 μM IBA permitted maximum bud multiplication. The acclimatization was carried out successfully using vitroplants showing good foliar and root development. Thus, three months after acclimatization, the seedlings were transferred into large pots under natural light and temperature conditions. Almost all acclimatized plants developed flowers in the first year between May and July.

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 implement the in vitro micropropagation of Origanum elongatum for optimal vitroplant 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 GA3 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 acclimatized.

Influence of Amino Acids on the Secondary Somatic Embryogenesis Proliferation Process of Moroccan Cork Oak (<i>Quercus suber</i>L.)

The present study aims to explore the regeneration potential of Moroccan cork oak through the secondary somatic embryogenesis process. Particularly, we focus on the analysis of amino acids influence on the quantity and quality of the regenerated secondary embryos. The amino acids tested are: Glutamine, asparagine, arginine, tryptophane, methionine, casein hydrolysate and urea. Each amino acid is added in the Margara (N30K) medium at different concentrations ranged between 10 and 500 mg/l. The results are collected after 2 months of culture. First analysis shows that the glutamine and the casein hydrolysate gives a maximum number of somatic embryos, clusters and pre-embryos newly formed on the clusters. By comparison to the control medium, the increase of the secondary embryos number directly formed exceed 36% in the case of casein hydrolysate and 35% of in the case of glutamine both at the concentration of 30 mg/l. However, the test of the combination of these amino acids did not have any significant results. In terms of quality, the influence of amino acids on the morphology of secondary embryos was analyzed.

Macronutrients Effect on Secondary Somatic Embryogenesis of Moroccan Cork Oak (<i>Quercus suber</i>L.)

To define the preliminary embryogenesis culture conditions of Moroccan Cork Oak (Quercus suber L.) in secondary propagation systems, secondary embryos formation from primary embryos were analyzed using seven macronutrient medias: (Chalupa) (BTM), Murashige and Skoog (MS), Schenk and Hildebrant (SH), Schenk and Hildebrant with half content macronutrients (SH ?), full Gamborg (G), Margara (N30K) and Woody Plant Media(WPM). Mature primary embryos at cotyledonal stage of 8 - 10 mm, were placed in each culture medium, and supplemented with 30 g/l of glucose and 7 g/l of agar without PGR. The experimental design consisted of a Petri dish containing three embryos explants. Each one of the seven treatments was composed of ten Petri dishes. Mean number of secondary somatic embryos, clusters and new embryogenic formation on clusters were recorded after 8 weeks, and evaluated by statistical analysis. There were no significant differences (p ≤ 0.05) in clusters and new embryos on clusters formation among evaluated media;but mean number of secondary embryos was significantly higher in N30K (4.37 ± 0.48) compared with control media (1.37 ± 0.15). The morphology of secondary embryos grown in the N30K medium exclusively showed the presence of three embryogenic stages: early cotyledonal with translucide aspect, white opaque, or green, and mature embryos. These results indicate that the medium do influence the morphogenic characteristics of produced embryos. Our finding revealed that secondary somatic embryos produced in N30K medium presented better morphogenic potential, with different stages of embryogenic formation.