Conservation of Thymus Pallidus Cosson Ex Batt. by Shoot Tip and Axillary Bud in Vitro Culture

J Plant Biotechnol (2020) 47:53–65 ISSN 1229-2818 (Print) DOI:https://doi.org/10.5010/JPB.2020.47.1.053 ISSN 2384-1397 (Online) Research Article Conservation of Thymus pallidus Cosson ex Batt. by shoot tip and axillary bud in vitro culture Zineb Nejjar El Ansari ･ Ibtissam Boussaoudi ･ Rajae Benkaddour ･ Ouafaa Hamdoun ･ Mounya Lemrini ･ Patrick Martin ･ Alain Badoc ･ Ahmed Lamarti Received: 2 February 2020 / Revised: 22 March 2020 / Accepted: 24 March 2020 ⓒ Korean Society for Plant Biotechnology Abstract Here, we describe an efficient and rapid protocol Keywords Auxins, Cytokinins, Gibberellic acid, Macro- for the micropropagation of Thymus pallidus Cosson ex Batt., nutrients, Micropropagation, Thymus pallidus a very rare medicinal and aromatic plant in Morocco. After seed germination, we tested the effect of different macronutrients, cytokinins alone or in combination with gibberellic Introduction acid (GA3) or auxins, on T. pallidus plantlet growth. We found Thymus pallidus Cosson ex. Batt is a medicinal and aromatic that Margara macronutrients (N30K) had the best effect on the in vitro development of the plantlets. The addition of 0.93 plant found exclusively in Morocco, Algeria and Spain (The Euro + Med Plant Base 2019). In Morocco, it is encountered µM/L 1,3-diphenylurea (DPU), 0.46 µM/L adenine (Ad), and in Tagmoute, north of Taliouine, Siroua mountains, Ourika and 0.46 and 0.93 μM/L kinetin (Kin) resulted in the best shoot the central Anti Atlas (Bellakhdar 1997; Fennane and Ibn- multiplication and elongation. In addition, the combination of Tattou 1998). The leaves of this species are greenish and 0.46 µM/L Kin, DPU, or Ad with gibberellic acid, in particular, petiolate, with a full-margin branch (Bennouna et al. 2012). 0.46 µM/L Ad + 0.58 µM/L GA3 and 0.46 µM/L Kin + 1.15 µM/L The subspecies encountered are: Thymus pallidus Batt. subsp. GA3, led to better bud and shoot multiplication. Moreover, the pallidus (synonym of Thymus pallidus var. vulcanicus Maire μ integration of the combinations of 0.46 M/L Kin and auxins, & Weiller), characterized by an inflorescence densely covered namely 0.46 µM/L Kin + 2.85 µM/L indole-3-acetic acid (IAA), with fine glandular hairs, but not very hairy and, Thymus 0.46 µM/L Kin + 2.85 or 5.71 µM/L indole-3-butyric acid pallidus subsp. eriodontus (synonym of Thymus pallidus var. (IBA), and 0.46 µM/L Kin + 0.3 or 0.57 µM/L 1-naphthaleneacetic eriodontus Maire), characterized by a villous inflorescence. acid (NAA), in the culture medium led to better root devel- Both subspecies are heterotypic synonyms of Thymus will- opment and optimized aerial growth. Finally, the in vitro denowii Boiss. (The Euro + Med Plant Bas 2019; World plants from the medium containing N30K + 0.46 µM/L Kin + Checklist of Selected Plant Families; Morales 1994). 2.85 µM/L IAA were successfully acclimatized; these plants The essential oil of T. pallidus is characterized by its served as a source for repeating in vitro culture. diversity and its composition varies depending on the geo- graphic location of the studied plant (Bennouna et al. 2012; Figueiredo et al. 2010). Therefore, the most encountered com- Z. Nejjar El Ansari () ･ I. Boussaoudi ･ R. Benkaddour ･ O. Hamdoun ･ M. Lemrini ･ A. Lamarti pounds are α-Terpinene, thymol, carvacrol, β-ocimene, men- Laboratory of Plant Biotechnology, Biology Department, Faculty thone, borneol, ρ-cymene, β-linalol and caryophyllene. These of Sciences, Abdelmalek Essaadi University, Tetouan, Morocco e-mail: [email protected] bioactive molecules are responsible for its antioxidant, antifungal, antibacterial and anti-tumor properties (Fadli et P. Martin Université d’Artois, UniLaSalle, ULR7519 - Unité al. 2011; Jaafari et al. 2007; Jamali et al. 2012; Sqalli et Transformations & Agroressources, F-62408 Béthune, France al. 2009). Actually, T. pallidus is a very rare species in Morocco, A. Badoc Axe MIB (Molécules d’Intérêt Biologique), Unité de Recherche represented by small dispersed populations (Fennane and (Enologie EA 4577, USC 1366 INRA), UFR des Sciences IbnTattou 1998). Therefore, it is necessary to apply tools and Pharmaceutiques, Université de Bordeaux, ISVV (Institut des Sciences de la Vigne et du Vin), Bordeaux, France techniques for the propagation and conservation of this This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 54 J Plant Biotechnol (2020) 47:53–65 species. Indeed, micropropagation has been considered as of 24±1°C and 60% of relative humidity. The lighting was a good tool for ex situ conservation programs, applied for supplied 18 hr a day by fluorescent tubes (4,000 lux). After species with a very small populations or low seed production. a few days, the seeds germinate and give a root tip. This technique facilitates the rapid establishment of a large Germinated seeds were counted 24 hr after the beginning number of mother plants with minimal impact on endangered of the experiment. A seed was considered germinated when wild plants (Abdallah et al. 2017). Subsequently, several the radicle pierced the seminal envelopes. studies have focused on the in vitro propagation of different The 4-week-old seedlings resulting from the in vitro species of the genus Thymus (Bernard et al. 2015; Macro- germination of T. pallidus seeds were used in the following Medina and Casas 2015; Mirshekar et al. 2014; Nordine experiments, since their organs (hypocotyls, cotyledons and and El Meskaoui 2014) and, the present study is the first apex) have developed and their roots were short. to establish a micropropagation protocol for T. pallidus Coss., Thus, cultures were induced from nodal segments (5 ~ 6 through the evaluation of the effect of different compositions mm) obtained from 4-week-old aseptic seedlings, on a medium of culture media, to determine the best ones for good growth solidified with 0.7% bacteriological agar, containing Shah of vitro-plants. and Dalal (SD 1980) macronutrients, MS micronutrients and vitamins, 100 mg/L myo-inositol, 3% (w/v) sucrose and 0.46 µM/L Kinetin. Seedlings were transplanted in the same Material and Methods medium until enough plantlets were available to establish experiments. Plant material Effect of macronutrients T. pallidus Coss. seeds were provided by the National Institute of Agronomic Research of Marrakech and were used as a Three solutions of macronutrients differing in nitrogen con- - + source of plant material. tent (NO3 and NH4 ) and in potassium, all added with MS micronutrients and vitamins, were tested: MS, B5 (Gamborg Seeds germination et al. 1968) and N30K (Margara 1978). The medium composed of N30K macronutrients was selected and used for all the Seeds sterilization following experiments. Seeds surface was sterilized according to the following protocol: Multiplication and elongation phase - Immersion in a filtered solution of calcium hypochlorite (Ca(ClO)2) 7% (w/v), containing a few drops of Effect of cytokinin type Tween-80 for 15 min; Three cytokinins: Kinetin (Kin), 1,3-diphenylurea (DPU) and - Rinsing with sterile distilled water for 5 min; Adenine (Ad) were evaluated on T. pallidus plantlets growth. - Immersion in mercuric chloride solution (HgCl2) 0.1% Three concentrations were tested: 0.46, 0.93 and 2.32 µM/L, for 2 min; plus a control medium containing no growth regulator. - Three successive rinses with sterile distilled water (5- 10-15 min). Effect of cytokinins and gibberellic acid combinations The seeds are soaked for 48 hr in sterile distilled water before germination. Three cytokinins (Kin, DPU and Ad) at a concentration of 0.46 µM/L were tested alone or combined with two concen- In vitro germination trations of gibberellic acid (GA3): 0.58 and 1.15 µM/L. After imbibition, seeds were germinated in vitro into glass Rooting phase: effect of Kinetin and auxins combinations test tubes (18×180 mm), one seed per tube, this latter containing 15 mL of the culture medium composed of Gautheret The Kinetin at 0.46 µM/L was tested alone or combined macronutrients (Gautheret and Longchamp 1959) and Mura- to three auxins: Indole-3-acetic acid (IAA), Indole-3-butyric shige and Skoog (MS 1962) micronutrients, solidified with acid (IBA) and 1-Naphthaleneacetic acid (NAA) at: 0.057, 0.7% (w/v) bacteriological agar, previously sterilized at 121°C. 0.3, 0.57, 2.85 or 5.71 µM/L. The tubes were placed in a culture room, with a temperature J Plant Biotechnol (2020) 47:53–65 55 Acclimatization phase Method 4 - Rinsing with 10% Ca(ClO)2 with 4 to 5 drops of Tween-80 After removal from the culture medium (N30K + 0.46 µM/L for 30 min; Kin + 2.85 µM/L IAA), 30 rooted plantlets were gently - Rinsing with 0.1% HgCl2 with 4 to 5 drops of Tween-80 washed to remove the rest of the agar medium from roots for 5 min; and then acclimatized in 250 mL plastic pots, containing - Rinsing three times with sterile distilled water for 5 min. a mixture of sterilized peat and vermiculite (2:1, v/v). Each pot was covered by a transparent plastic cup, incubated Method 5 under specific conditions (photoperiod: 18/6 hr, humidity: 90 ~ 100%, temperature: 24±1°C) and watered, if necessary, - Rinsing with 10% Ca(ClO)2 with 4 to 5 drops of with distilled water. After three weeks, the humidity was grad- Tween-80 for 30 min; ually reduced until the cups were completely eliminated at - Rinsing with 10% Mercryl with 4 to 5 drops of the end of the fourth week. Regular irrigation was per- Tween-80 for10 min; formed during the first two weeks, at intervals of two days - Rinsing three times with sterile distilled water for 5 from the fifteenth to the twentieth day and as needed until min.

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