The objective of this study was to analyze the chemical profile of extracts from different fruit parts of the perennial arborescent cactus Opuntia leucotricha by applying ultra-high-performance liquid chromatography–high-resolution mass spectrometry (UHPLC-HRMS) and to evaluate their biological properties (antioxidant and enzyme inhibitory capacities). Extracts were obtained from the fruit pulp (without or with seeds), seeds, and peel by ultrasound-assisted extraction (UAE) using the conventional solvent methanol 50% and two Natural Deep Eutectic Solvents (NADESs) (glycerol:urea, 1:1 and citric acid:sorbitol, 1:2). A total of 33 compounds were identified, including phenolics, fatty acids, and others. Phenolic acids were the most abundant class of phenolics identified in all fruit parts, with the highest concentration observed in the methanol extracts from peel (593.02 µg/gDW), followed by seed-containing pulp (69.03 µg/gDW), pulp (57.83 µg/gDW), and seeds (39.97 µg/gDW). The second most effective extractant was NADES 1 (glycerol:urea, 1:1), which was also successful in extracting compounds with antioxidant capacity. Overall, the extracts demonstrated considerable enzyme inhibitory activities, with the greatest effects observed against α-amylase and α-glucosidase. The results indicate that O. leucotricha fruits could be a promising source of bioactive compounds, and NADES a viable alternative to organic solvents for their industrial exploitation.

Quercus suber L. is the main woody tree species in the Mediterranean basin. The in vitro regeneration from adult material, through primary somatic embryogenesis, is a well-known process, but the use of secondary somatic embryos for plant regeneration remains a very sparsely studied process. The main objective of this work is to explore the cork oak regeneration potential by using the secondary somatic embryogenesis process. Mainly, in this work, we report the polyamine effect. Explants used consisted on primary mature embryos, derived from leaves rejuvenated by epicormic shoot of the Moroccan Quercus suber. Three different polyamines were added to the basal medium, which was composed by macronutrients of N30K, 30 g/l glucose, and 7 g/l agar. Three polyamines, Putrescine, Spermine, and Spermidine, were added to the basal medium at 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mg/l. Explants were tested after 8 weeks. Morphological analysis showed that the medium with 0.4 mg/l Spermidine provided the best result for secondary embryos, which corresponds to a very significant (p < 0.05) increase of 375%. The number of secondary embryos directly formed was 2.70 ± 0.51. Similarly, the optimum concentrations for high number of clusters (0.50 ± 0.11) and embryo clusters (1.43 ± 0.35) were increased by 145% and 158%. The addition of the polyamine also acted on the quality of embryos formed. A very significant (p < 0.05) increase in the size of secondary embryos was observed compared to the medium without polyamines. Spermidine showed the greatest increase (about 38%).

Cork oak (Quercus suber L.) is one of the most important Mediterranean forest tree species, and the Maamora forest-Morocco is considered to be the world largest cork oak stand. Clonal propagation of Quercus suber via somatic embryogenesis is an alternative to conventional tree propagation methods. However, complete maturation of somatic embryos is considered the major bottleneck for mass propagation of Quercus suber during somatic embryogenesis. This study focuses on influences of cytokinins on secondary somatic embryogenesis. Cytokinins showed a negative effect on the induction of secondary somatic embryogenesis. For the various concentrations tested, the number of secondary somatic embryos was significantly reduced except in the case with low dose of 2iP, which showed a slight increase. Abscisic acid (ABA) may be beneficial at low concentrations between 0.3 and 0.5 mg/l. The best number of secondary embryos was given at 0.5 mg/l ABA with an average number of 5 embryos from each primary embryo, which represents a slight increase below 10% compared to control medium. Also, we found that the number of responsive embryos presenting secondary somatic embryos based on their cotyledons decreased once adding ABA. Phenylurea derivatives TDZ and DPU do not promoted the process of secondary somatic embryogenesis. DPU showed no significant effect with a similar response to that of the control medium and TDZ showed an inhibitory effect. Gibberellic acid (GA(3)) partially blocked the process of somatic embryos multiplication, but promoted remarkably the germination of cotyledonary embryos without requesting a particular protocol. Our results provide a new insight into embryo development, establishing the basis for further research toward improvement of secondary somatic embryogenesis in Moroccan cork oak.

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.

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.

Zineb Nejjar El Ansari · Ibtissam Boussaoudi · Rajae Benkaddour · Ouafaa Hamdoun · Mounya Lemrini · Patrick Martin · Alain Badoc · Ahmed Lamarti. J Plant Biotechnol 2020;47:53-65. https://doi.org/10.5010/JPB.2020.47.1.053

The present study reports an efficient protocol for in vitro propagation of Thymus vulgaris L., an aromatic and medicinal plant in Morocco.Initially, we performed in vitro multiplication of Thymus vulgaris explants existing in the laboratory and obtained from micropropagation by shoot tip culture.Afterwards, we have evaluated the effect of six macronutrients.After that, seven cytokinins (Kin, BAP, 2iP, DPU, Adenine, Zeatine and TDZ) in three different concentrations (0.46, 0.93, 2.32 µM) have been evaluated to optimize cultures multiplication and elongation.Moreover, the effect of three auxins (IAA, IBA and NAA) at 0.57 µM, combined to 4 cytokinins (Kin, BAP, DPU and Ad.) at 0.46 μM, on shoot rooting has been studied.Thereby, MS medium has been proved the most favorable for plantlets growing.Also, we found that the addition of certain cytokinins, specifically 0.46 Kin, 0.46 and 0.93 BAP, 0.46 2iP, 0.46 DPU, 0.46 Ad. and 0.46 Zeat., ensures better multiplication and growth of vitroplants.In addition, multiplication and rooting of cultures were well optimized after addition 0.46 Kin + 0.57 IAA or NAA, 0.46 DPU + 0.57 IBA and 0.46 Ad. + 0.57 IBA combinations to the culture medium.Lastly, plantlets with roots were successfully acclimatized to ex-vitro conditions and these latter served as a source to establish in vitro culture again.