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Conservation of Thymus pallidus Cosson ex Batt. by shoot tip and axillary bud in vitro culture
J Plant Biotechnol 2020;47:53-65
Published online March 31, 2020
© 2020 The Korean Society for Plant Biotechnology.

Zineb Nejjar El Ansari · Ibtissam Boussaoudi · Rajae Benkaddour · Ouafaa Hamdoun · Mounya Lemrini · Patrick Martin · Alain Badoc · Ahmed Lamarti

Laboratory of Plant Biotechnology, Biology Department, Faculty of Sciences, Abdelmalek Essaadi University, Tetouan, Morocco
Université d’Artois, UniLaSalle, ULR7519 - Unité Transformations & Agroressources, F-62408 Béthune, France
Axe MIB (Molécules d’Intérêt Biologique), Unité de Recherche (Enologie EA 4577, USC 1366 INRA), UFR des Sciences Pharmaceutiques, Université de Bordeaux, ISVV (Institut des Sciences de la Vigne et du Vin), Bordeaux, France
Correspondence to: e-mail: zainabnejjar@gmail.com
Received February 2, 2020; Revised March 22, 2020; Accepted March 24, 2020.
Abstract
Here, we describe an efficient and rapid protocol for the micropropagation of Thymus pallidus Cosson ex Batt., 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 acid (GA3) or auxins, on T. pallidus plantlet growth. We found that Margara macronutrients (N30K) had the best effect on the in vitro development of the plantlets. The addition of 0.93 μM/L 1,3-diphenylurea (DPU), 0.46 μM/L adenine (Ad), and 0.46 and 0.93 μM/L kinetin (Kin) resulted in the best shoot multiplication and elongation. In addition, the combination of 0.46 μM/L Kin, DPU, or Ad with gibberellic acid, in particular, 0.46 μM/L Ad + 0.58 μM/L GA3 and 0.46 μM/L Kin + 1.15 μM/L GA3, led to better bud and shoot multiplication. Moreover, the integration of the combinations of 0.46 μM/L Kin and auxins, namely 0.46 μM/L Kin + 2.85 μM/L indole-3-acetic acid (IAA), 0.46 μM/L Kin + 2.85 or 5.71 μM/L indole-3-butyric acid (IBA), and 0.46 μM/L Kin + 0.3 or 0.57 μM/L 1-naphthaleneacetic acid (NAA), in the culture medium led to better root development and optimized aerial growth. Finally, the in vitro plants from the medium containing N30K + 0.46 μM/L Kin + 2.85 μM/L IAA were successfully acclimatized; these plants served as a source for repeating in vitro culture.
Keywords : Auxins, Cytokinins, Gibberellic acid, Macronutrients, Micropropagation, Thymus pallidus
Introduction

Thymus pallidus Cosson ex. Batt is a medicinal and aromatic plant found exclusively in Morocco, Algeria and Spain (The Euro + Med Plant Base 2019). In Morocco, it is encountered in Tagmoute, north of Taliouine, Siroua mountains, Ourika and the central Anti Atlas (Bellakhdar 1997; Fennane and IbnTattou 1998). The leaves of this species are greenish and petiolate, with a full-margin branch (Bennouna et al. 2012). The subspecies encountered are: Thymus pallidus Batt. subsp. pallidus (synonym of Thymus pallidus var. vulcanicus Maire & Weiller), characterized by an inflorescence densely covered with fine glandular hairs, but not very hairy and, Thymus pallidus subsp. eriodontus (synonym of Thymus pallidus var. eriodontus Maire), characterized by a villous inflorescence. Both subspecies are heterotypic synonyms of Thymus willdenowii Boiss. (The Euro + Med Plant Bas 2019; World Checklist of Selected Plant Families; Morales 1994).

The essential oil of T. pallidus is characterized by its diversity and its composition varies depending on the geographic location of the studied plant (Bennouna et al. 2012; Figueiredo et al. 2010). Therefore, the most encountered compounds are α-Terpinene, thymol, carvacrol, β-ocimene, menthone, borneol, ρ-cymene, β-linalol and caryophyllene. These bioactive molecules are responsible for its antioxidant, antifungal, antibacterial and anti-tumor properties (Fadli et al. 2011; Jaafari et al. 2007; Jamali et al. 2012; Sqalli et al. 2009).

Actually, T. pallidus is a very rare species in Morocco, represented by small dispersed populations (Fennane and IbnTattou 1998). Therefore, it is necessary to apply tools and techniques for the propagation and conservation of this species. Indeed, micropropagation has been considered as a good tool for ex situ conservation programs, applied for species with a very small populations or low seed production. This technique facilitates the rapid establishment of a large number of mother plants with minimal impact on endangered wild plants (Abdallah et al. 2017). Subsequently, several studies have focused on the in vitro propagation of different species of the genus Thymus (Bernard et al. 2015; Macro-Medina and Casas 2015; Mirshekar et al. 2014; Nordine and El Meskaoui 2014) and, the present study is the first to establish a micropropagation protocol for T. pallidus Coss., through the evaluation of the effect of different compositions of culture media, to determine the best ones for good growth of vitro-plants.

Material and Methods

Plant material

T. pallidus Coss. seeds were provided by the National Institute of Agronomic Research of Marrakech and were used as a source of plant material.

Seeds germination

Seeds sterilization

Seeds surface was sterilized according to the following protocol:

- Immersion in a filtered solution of calcium hypochlorite (Ca(ClO)2) 7% (w/v), containing a few drops of Tween-80 for 15 min;

- Rinsing with sterile distilled water for 5 min;

- Immersion in mercuric chloride solution (HgCl2) 0.1% for 2 min;

- Three successive rinses with sterile distilled water (5- 10-15 min).

The seeds are soaked for 48 hr in sterile distilled water before germination.

In vitro germination

After imbibition, seeds were germinated in vitro into glass test tubes (18×180 mm), one seed per tube, this latter containing 15 mL of the culture medium composed of Gautheret macronutrients (Gautheret and Longchamp 1959) and Murashige and Skoog (MS 1962) micronutrients, solidified with 0.7% (w/v) bacteriological agar, previously sterilized at 121°C. The tubes were placed in a culture room, with a temperature of 24±1°C and 60% of relative humidity. The lighting was supplied 18 hr a day by fluorescent tubes (4,000 lux). After a few days, the seeds germinate and give a root tip.

Germinated seeds were counted 24 hr after the beginning of the experiment. A seed was considered germinated when the radicle pierced the seminal envelopes.

The 4-week-old seedlings resulting from the in vitro germination of T. pallidus seeds were used in the following experiments, since their organs (hypocotyls, cotyledons and apex) have developed and their roots were short.

Thus, cultures were induced from nodal segments (5~6 mm) obtained from 4-week-old aseptic seedlings, on a medium solidified with 0.7% bacteriological agar, containing Shah 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 medium until enough plantlets were available to establish experiments.

Effect of macronutrients

Three solutions of macronutrients differing in nitrogen content (NO3- and NH4+) and in potassium, all added with MS micronutrients and vitamins, were tested: MS, B5 (Gamborg et al. 1968) and N30K (Margara 1978). The medium composed of N30K macronutrients was selected and used for all the following experiments.

Multiplication and elongation phase

Effect of cytokinin type

Three cytokinins: Kinetin (Kin), 1,3-diphenylurea (DPU) and Adenine (Ad) were evaluated on T. pallidus plantlets growth. Three concentrations were tested: 0.46, 0.93 and 2.32 µM/L, plus a control medium containing no growth regulator.

Effect of cytokinins and gibberellic acid combinations

Three cytokinins (Kin, DPU and Ad) at a concentration of 0.46 µM/L were tested alone or combined with two concentrations of gibberellic acid (GA3): 0.58 and 1.15 µM/L.

Rooting phase: effect of Kinetin and auxins combinations

The Kinetin at 0.46 µM/L was tested alone or combined to three auxins: Indole-3-acetic acid (IAA), Indole-3-butyric acid (IBA) and 1-Naphthaleneacetic acid (NAA) at: 0.057, 0.3, 0.57, 2.85 or 5.71 µM/L.

Acclimatization phase

After removal from the culture medium (N30K + 0.46 µM/L Kin + 2.85 µM/L IAA), 30 rooted plantlets were gently washed to remove the rest of the agar medium from roots and then acclimatized in 250 mL plastic pots, containing a mixture of sterilized peat and vermiculite (2:1, v/v). Each pot was covered by a transparent plastic cup, incubated under specific conditions (photoperiod: 18/6 hr, humidity: 90~100%, temperature: 24±1°C) and watered, if necessary, with distilled water. After three weeks, the humidity was gradually reduced until the cups were completely eliminated at the end of the fourth week. Regular irrigation was performed during the first two weeks, at intervals of two days from the fifteenth to the twentieth day and as needed until transplantation into larger pots.

Re-initiation of in vitro culture of T. pallidus from acclimatized plants

Twigs were cut from the acclimatized plants of T. pallidus, thoroughly washed with tap water, then surface sterilized under a laminar flow hood according to five methods:

Method 1

- Rinsing with 10% Ca(ClO)2 with 4 to 5 drops of Tween-80 for 20 min;

- Rinsing with 10% Mercryl with 4 to 5 drops of Tween-80 for 10 min;

- Rinsing three times with sterile distilled water for 5 min.

Method 2

-Rinsing with ethanol 70° for 30 s;

-Rinsing with 10% Ca(ClO)2 with 4 to 5 drops of Tween-80 for 30 min;

-Rinsing with 0.1% HgCl2 with 4 to 5 drops of Tween-80 for 5 min;

-Rinsing three times with sterile distilled water for 5 min.

Method 3

-Rinsing with ethanol 70° for 30 s;

-Rinsing with 10% Ca(ClO)2 with 4 to 5 drops of Tween-80 for 30 min;

-Rinsing with 10% Mercryl with 4 to 5 drops of Tween-80 for 5 min;

-Rinsing three times with sterile distilled water for 5 min.

Method 4

-Rinsing with 10% Ca(ClO)2 with 4 to 5 drops of Tween-80 for 30 min;

-Rinsing with 0.1% HgCl2 with 4 to 5 drops of Tween-80 for 5 min;

-Rinsing three times with sterile distilled water for 5 min.

Method 5

-Rinsing with 10% Ca(ClO)2 with 4 to 5 drops of Tween-80 for 30 min;

-Rinsing with 10% Mercryl with 4 to 5 drops of Tween-80 for10 min;

-Rinsing three times with sterile distilled water for 5 min.

The sterilized twigs were divided into 2~3 cm segments with at least two axillary buds, and these segments were used as explants. For re-initiation of the in vitro culture, the explants were placed in glass test tubes (18×180 mm), one per tube, containing 15 mL of N30K culture medium supplemented with 0.46 µM/L Kin. After multiplication, plantlets were transferred to bigger flasks.

Culture conditions

The culture media were supplemented with 3% sucrose and 0.7% bacteriological agar. The pH of the media was adjusted to 5.6~5.8 using sodium hydroxide (NaOH). Sterilization of the culture media was carried out at 121°C for 20 min. The in vitro culture was performed under aseptic conditions in a horizontal laminar flow hood. The vitro-plants were incubated in a culture room (photoperiod: 18/6 hr with 4,000 lux light density, temperature: 24±1°C).

Evaluation of plantlets growth

After one month of growth, the following parameters were evaluated:

-Regeneration rate (%) (Plantlets that have generated new buds and shoots);

-Mean plantlets length (cm);

-Mean number of buds per plantlet;

-Mean number of shoots per plantlet;

-Rooting rate (%);

-Mean number of roots per plantlet;

-Hyperhydricity rate (%).

Statistical analysis

All measurements were run in triplicates (n = 3); 24 samples were used for each replicate: 24 seeds and 24 plantlets per each of three replicates. The values were averaged and given along with standard error (± SE). Analyses were performed with Statistica 6, averages were compared by Duncan test and values beyond p ≤ 0.05 were considered significant.

Results

Seeds germination

The germination of T. pallidus seeds begins after four days of culturing, the final rate is about 25%, and the degree of contamination does not exceed 4% (Fig. 1).

Fig. 1. In vitro germination of Thymus pallidus Coss. Ex Batt. Seeds

Effect of medium type

The results mentioned inTable 1 show that N30K macronutrients ensure total survival of T. pallidus plantlets (100%), followed by MS (97.2), while just 68.1% regenerated on B5 medium. On the other hand, this latter guaranteed the best rooting rate (84.1), but the analysis of the variance showed that there is not a significant difference between the three media. In addition, some plantlets have developed a translucent appearance, especially in the case of MS medium (15.5).

Effect of three macronutrients on the micropropagation of Thymus pallidus Coss. ex Batt

Medium Regeneration (%) Shoot length (cm) Number of buds Number of shoots Rooting (%) Number of roots Hyperhydricity (%)
B5 68.1 ± 10.0b 2.35 ± 0.16b 28.20 ± 1.59a 3.00 ± 0.22a 84.1 ± 1.7a 4.44 ± 0.45b 5.1 ± 1.1b
MS 97.2 ± 2.8a 3.64 ± 0.13a 29.14 ± 1.03a 3.27 ± 0.21a 73.5 ± 15.8a 5.96 ± 0.49a 15.5 ± 6.8a
N30K 100.0a 3.53 ± 0.18a 23.22 ± 0.91b 2.39 ± 0.16b 77.8 ± 9.1a 6.32 ± 0.48a 4.2 ± 1.2c

The data represent the mean ± SE of replicates (n = 3). Values in the same rows carrying different letters are significantly different between treatments and compared by Duncan's multiple range tests at p ≤ 0.05.


Furthermore, we note better shoots elongation in the case of MS and N30K (3.64 and 3.53 cm, respectively), while better multiplication of buds (28.20 and 29.14) and shoots (3 and 3.27) is observed in B5 and MS, respectively. Also, the maximum number of roots is noticed in the case of MS and N30K (5.96 and 6.32, respectively) (Fig. 2).

Fig. 2. Effect of three macronutrients on the micropropagation of Thymus pallidus Coss. Ex Batt. (A) MS, (B) B5, and (C) N30K Bars = 1 cm

Although B5 and MS macronutrients provided better bud and shoot multiplication, they showed a lower regeneration rates and more important hyperhydricity compared to N30K. For this reason, we chose N30K medium for the following experiments.

Multiplication and elongation phase

Effect of cytokinins type

The integration of cytokinins into the culture media caused several changes, both in the aerial and in the root part (Table 2 andFig. 3).

Effect of cytokinins on the micropropagation of Thymus pallidus Coss. ex Batt

Cytokinins (µM/L) Regeneration (%) Shoot length (cm) Number of buds Number of shoots Rooting (%) Number of roots Hyperhydricity (%)
Control 89.6 ± 2.1c 2.16 ± 0.10f 20.48 ± 1.07bc 2.00 ± 0.14bc 93.1 ± 2.2ab 5.80 ± 0.82d 4.6 ± 0.1b
Kin 0.46 98.6 ± 1.4ab 3.85 ± 0.22de 24.74 ± 1.26a 2.69 ± 0.19a 100.0 ± 0.0a 8.66 ± 0.85abc 10.0 ± 1.4a
0.93 98.6 ± 1.4ab 4.67 ± 0.43cd 25.61 ± 1.18a 2.14 ± 0.16bc 100.0 ± 0.0a 10.69 ± 0.77ab 0.0c
2.32 76.9 ± 1.9d 2.33 ± 0.16f 20.54 ± 1.06bc 1.61 ± 0.14c 100.0 ± 0.0a 8.23 ± 0.85bc 0.0c
DPU 0.46 100.0a 4.96 ± 0.26bc 24.11 ± 1.30ab 2.36 ± 0.17ab 100.0 ± 0.0a 10.14 ± 0.71ab 9.7 ± 1.4a
0.93 100.0a 7.47 ± 0.46a 23.08 ± 1.49abc 1.81 ± 0.16bc 100.0 ± 0.0a 9.31 ± 0.61abc 0.0c
2.32 95.8 ± 1.4b 3.11 ± 0.21ef 21.89 ± 1.00abc 1.83 ± 0.14bc 91.4 ± 5.7b 7.03 ± 0.74cd 0.0c
Ad 0.46 100.0a 4.50 ± 0.31cd 19.33 ± 1.02c 2.12 ± 0.15bc 91.7 ± 4.2b 11.19 ± 0.92a 12.5 ± 4.2a
0.93 100.0a 5.82 ± 0.51b 22.71 ± 1.51abc 2.33 ± 0.21ab 100.0 ± 0.0a 9.33 ± 0.89abc 0.0c
2.32 100.0a 4.85 ± 0.37bcd 24.17 ± 1.08ab 1.87 ± 0.16bc 100.0 ± 0.0a 10.75 ± 0.87ab 0.0c

The data represent the mean ± SE of replicates (n = 3). Values in the same rows carrying different letters are significantly different between treatments and compared by Duncan's multiple range tests at p ≤ 0.05.



Fig. 3. Effect of cytokinins on the micropropagation of Thymus pallidus Coss. Ex Batt. (A) Control, (B) 0.46 µM DPU, (C) 0.46 µM Kin, (D) 0.93 µM Ad, (E) 0.93 µM DPU, (F) 0.93 µM Kin, and (G) 2.32 µM Ad Bars = 1 cm

Thus, addition of cytokinins to N30K medium had a significant impact on the regeneration ability of the plantlets and the highest value (100%) was noted with Ad at all concentrations and DPU at 0.46 and 0.93 µM/L compared to 76.9~98.6% in the media added with 2.32 DPU and Kin at all concentrations. Moreover, a total rooting ability was observed for almost all cytokinins at different concentrations compared to lower values (91.4 and 91.7%) on media added with 2.32 DPU and 0.46 Ad. In addition, absence of hyperhydricity (0%) was noticed for all cytokinins at 0.93 and 2.32 µM/L compared to values ranging from 9.7 to 12.5% with the concentration 0.46 µM/L.

In general, the addition of cytokinins contributed to an increase in shoots length compared to the control and the highest values was observed with medium added with 0.93 DPU (7.47 cm) and 0.93 Ad (5.82) compared to the rest of cytokinins and concentrations (2.33~4.96 cm). Furthermore, Kin at 0.46 and 0.93 µM/L provided the higher number of buds (24.74 and 25.61, respectively) in comparison with the rest of cytokinins and concentrations (19-24). Also, there is no significant difference between cytokinins at different concentrations in regenerating shoots (2~3).

Besides, the addition of cytokinins to N30K medium had a significant impact in rooting ability and the highest number of roots was observed with Ad (9~11) and Kin (8~11) compared to DPU (7~10).

Effect of cytokinins and gibberellic acid combinations

Addition of GA3 had no significant impact on regeneration ability of the plantlets, but a decrease in shoot length was observed in media added with combinations between GA3 and cytokinins (2.57~4.19 cm) compared to Kin (3.85), DPU (4.96) and Ad (4.50), alone in the culture media. Besides, higher number of buds and shoots was noticed on media with 0.46 Kin + 1.15 GA3 (31 buds and 4 shoots) and 0.46 DPU + 1.15 GA3 (26 buds and 3 shoots) compared to 21~24 buds and 2~3 shoots for the rest of combinations.

Furthermore, addition of GA3 in the presence of cytokinins had no significant impact on rooting ability of the plantlets, but the highest number of roots (10~13) was observed on medium with combination of GA3 and DPU or Ad compared to 8~9 roots developed on medium with GA3 and Kin.

Moreover, an absence of hyperhydricity was noted on media with 0.46 Kin and 0.46 Ad combined to 0.58 GA3 compared to the rest of combinations (12.5~25%) (Table 3 andFig. 4).

Effect of cytokinins combined with gibberellic acid on the micropropagation of Thymus pallidus Coss. ex. Batt

Cytokinins (µM/L) GA3 (µM/L) Regeneration (%) Shoot length (cm) Number of buds Number of shoots Rooting (%) Number of roots Hyperhydricity (%)
0.46 Kin 0 98.6 ± 1.4a 3.85 ± 0.22bc 24.74 ± 1.26ab 2.69 ± 0.19a 100.0 ± 0.0a 8.66 ± 0.85bcde 10.0 ± 1.4bcd
0.58 100.0a 2.72 ± 0.17d 20.58 ± 2.21bc 1.42 ± 0.19c 100.0 ± 0.0a 8.17 ± 0.85cde 0.0d
1.15 100.0a 2.57 ± 0.19d 30.50 ± 3.11a 3.50 ± 0.50a 87.5 ± 4.2b 7.70 ± 1.40de 25.0 ± 8.3a
0.46 DPU 0 100.0a 4.96 ± 0.26a 24.11 ± 1.30ab 2.36 ± 0.17abcd 100.0 ± 0.0a 10.14 ± 0.71abcd 9.7 ± 1.4bcd
0.58 100.0a 3.43 ± 0.30cd 22.83 ± 2.08bc 2.17 ± 0.34bc 100.0 ± 0.0a 11.00 ± 1.12abcd 12.5 ± 4.2bc
1.15 95.8 ± 4.2a 3.33 ± 0.39cd 26.00 ± 2.85ab 2.73 ± 0.70ab 86.7 ± 4.9b 11.89 ± 0.98ab 17.4 ± 0.8ab
0.46 Ad 0 100.0a 4.50 ± 0.31ab 19.33 ± 1.02cde 2.12 ± 0.15abcde 91.7 ± 4.2ab 11.19 ± 0.92abc 12.5 ± 4.2bc
0.58 100.0a 4.19 ± 0.34abc 22.67 ± 2.06bc 2.08 ± 0.26bc 100.0 ± 0.0a 12.67 ± 1.16a 0.0d
1.15 100.0a 4.02 ± 0.37abc 23.50 ± 3.37bc 2.67 ± 0.63ab 100.0 ± 0.0a 12.17 ± 1.11a 12.5 ± 4.2bc

The data represent the mean ± SE of replicates (n = 3). Values in the same rows carrying different letters are significantly different between treatments and compared by Duncan's multiple range tests at p ≤ 0.05.



Fig. 4. Effect of gibberellic acid combined with cytokinins on the micropropagation of Thymus pallidus Coss. Ex Batt. (A) 0.46 µM/L Ad + 0.58 µM/L GA3, (B) 0.46 µM/L Ad + 1.15 µM/L GA3, (C) 0.46 µM/L DPU + 0.58 µM/L GA3, (D) 0.46 µM/L DPU + 1.15 µM/L GA3, and (E) 0.46 µM/L Kin + 1.15 µM/L GA3 Bars = 1 cm

Rooting phase: effect of Kinetin and auxins combinations

The combination of the three auxins (IAA, IBA and NAA) with 0.46 µM Kin resulted in a number of changes in the in vitro growth of T. pallidus plantlets (Table 4 andFig. 5).

Effect of 0.46 µM/L Kin and auxin combinations on the micropropagation of Thymus pallidus Coss. ex. Batt

Auxins (µM/L) Regeneration (%) Shoot length (cm) Number of buds Number of shoots Rooting (%) Number of roots Hyperhydricity (%)
Control 98.6 ± 1.4a 3.85 ± 0.22bcde 24.74 ± 1.26cd 2.69 ± 0.19a 100.0a 8.66 ± 0.85e 10.0 ± 1.4ab
IAA 0.057 100.0a 3.17 ± 0.22ef 16.00 ± 1.50e 1.67 ± 0.14b 100.0a 9.00 ± 0.70e 0.0c
0.3 100.0a 3.90 ± 0.32bcde 27.67 ± 2.16bc 2.33 ± 0.28ab 100.0a 10.00 ± 0.95bcde 0.0c
0.57 100.0a 2.91 ± 0.18f 22.92 ± 1.81cd 2.42 ± 0.34ab 95.8 ± 4.2ab 9.36 ± 1.11de 20.8 ± 4.2a
2.85 100.0a 3.27 ± 0.28def 21.00 ± 1.68cde 1.58 ± 0.15b 100.0a 14.67 ± 1.09a 0.0c
5.71 100.0a 5.09 ± 0.39ab 25.50 ± 2.06cd 2.00 ± 0.21ab 100.0a 10.17 ± 0.76bcde 0.0c
IBA 0.057 100.0a 3.51 ± 0.38cdef 24.00 ± 1.59cd 2.42 ± 0.19ab 100.0a 9.67 ± 1.07cde 12.5 ± 4.2ab
0.3 100.0a 4.46 ± 0.59bcd 21.75 ± 1.76cde 2.08 ± 0.31ab 95.8 ± 4.2ab 9.00 ± 1.29e 0.0c
0.57 100.0a 4.70 ± 0.34abc 27.33 ± 2.35bcd 2.67 ± 0.35a 100.0a 9.92 ± 0.85bcde 8.3 ± 0.0b
2.85 100.0a 3.65 ± 0.41cdef 22.17 ± 1.44cde 1.83 ± 0.11ab 100.0a 13.42 ± 1.41abc 0.0c
5.71 100.0a 2.93 ± 0.33f 23.17 ± 1.38cd 2.25 ± 0.22ab 100.0a 12.50 ± 1.26abc 0.0c
NAA 0.057 100.0a 4.14 ± 0.39bcde 26.17 ± 2.78bcd 1.92 ± 0.29ab 100.0a 10.08 ± 1.09bcde 0.0c
0.3 95.8 ± 4.2ab 5.65 ± 0.47a 36.09 ± 3.77a 2.45 ± 0.37ab 100.0a 13.73 ± 1.39ab 0.0c
0.57 100.0a 5.73 ± 0.57a 32.33 ± 3.13ab 2.17 ± 0.32ab 95.8 ± 4.2ab 14.00 ± 0.60a 0.0c
2.85 100.0a 4.27 ± 0.46bcde 26.67 ± 1.75bcd 2.25 ± 0.22ab 100.0a 9.58 ± 1.08cde 0.0c
5.71 100.0a 4.58 ± 0.43abc 20.50 ± 1.96de 1.58 ± 0.29b 100.0a 11.33 ± 1.74bcd 0.0c

The data represent the mean ± SE of replicates (n = 3). Values in the same rows carrying different letters are significantly different between treatments and compared by Duncan's multiple range tests at p ≤ 0.05.



Fig. 5. Effect of three auxins combined with 0.46 µM Kin on the micropropagation of Thymus pallidus Coss. Ex Batt. (A) 0.46 µM/L Kin + 0.3 µM/L NAA, (B) 0.46 µM/L Kin + 0.57 µM/L NAA, (C) 0.46 µM/L Kin + 5.71 µM/L NAA, (D) 0.46 µM/L Kin + 2.85 µM/L IAA, (E) 0.46 µM/L Kin + 2.85 µM/L IBA, and (F) 0.46 µM/L Kin + 5.71 µM/L IBA Bars = 1 cm

Thus, a total regeneration (100%) was observed for media added with combinations between 0.46 Kin and auxins, but a lower value was recorded for medium added with 0.46 Kin + 0.3 NAA (95.8). Also, the addition of auxins had a significant impact on shoot elongation and higher lengths were observed on medium added with combination of Kin and NAA (4.14~5.73 cm) compared to Kin combined to IAA (2.91~5.09 cm) or IBA (2.93~4.70 cm). Similarly, higher number of buds was noted for medium with combination of Kin and NAA (26~36) compared to Kin combined to IAA (16~28) or IBA (22~27). However, no significant difference was observed in the number of shoots between the media added with combinations between Kin and auxins (2~3).

Furthermore, addition of auxins to N30K+0.46 Kin medium had no significant impact on rooting ability of the plantlets, but the highest number of roots (9~15) was observed on medium with combination of Kin and IAA or NAA, compared to 9~13 roots on medium with Kin and IBA.

In addition, an absence of hyperhydricity (0%) was noted for almost all the combinations between Kin and auxins, but higher rates were observed for media added with Kin combined to 0.57 IAA (20.8), 0.057 and 0.57 IBA (12.5 and 8.3, respectively).

Acclimatization phase

The thirty explants developing roots were successfully acclimatized to ex-vitro conditions. Actually, one month after the start of acclimatization, 93.33% of acclimatized plantlets appeared to be in good condition. Three months later, we transplanted them into larger pots. After one year, the acclimatized plantlets were indistinguishable from the wild plants of T. pallidus and 96% developed flowers during the 2nd year, between June and September (Fig. 6).

Fig. 6. Acclimatization phase (A and B) Acclimatization after 4 weeks; (C and D) acclimatization after 8 weeks; (E and F) acclimatization after one year; (G) acclimatization after two years; and (H and I) Thymus pallidus Coss. Ex Batt. inflorescences Bars = 1 cm

Re-initiation of in vitro culture of Thymus pallidus from acclimatized plants

Surface sterilization of twigs from T. pallidus acclimatized plants proved to be very difficult (Table 5).

Comparison of sterilization methods of shoot segments from the acclimatized Thymus pallidus Coss. ex. Batt. Plants

Bacterial contamination (%) Fungal contamination (%) Death rate (%) Survival rate (%)
Method 1 39.8 ± 1.9b 62.9 ± 2.9a 100.0a 0.0d
Method 2 0.0c 6.2 ± 2.1b 60.4 ± 2.1c 35.4 ± 2.1b
Method 3 91.7 ± 0.0a 10.4 ± 2.1b 100.0a 0.0d
Method 4 0.0c 10.4 ± 2.1b 27.1 ± 2.1d 56.2 ± 2.1a
Method 5 81.2 ± 2.1a 10.4 ± 2.1b 93.7 ± 2.1b 6.2 ± 2.1c

The data represent the mean ± SE of replicates (n = 3). Values in the same rows carrying different letters are significantly different between treatments and compared by Duncan's multiple range tests at p ≤ 0.05.


In this way, three out of five sterilization methods tested caused 94~100% mortality of plant material, while the rates of bacterial and fungal contaminations were still high. Method 4 provided the highest number of surviving plants (56%), and bacterial and fungal contamination rates were reduced to 0 and 10%, respectively.

The healthy and alive explants were multiplied by subculturing them on N30K + 0.46 Kin medium. The vitroplants obtained (Fig. 7) present the morphological criteria mentioned inTable 6.

Fig. 7. In vitro plants obtained after sterilization of shoot segments from the acclimatized Thymus pallidus Coss. Ex Batt. plants and their multiplication on N30K + 0.46 µM/L Kin medium Bar = 1 cm

Morphological characteristics of in vitro plants obtained after sterilization of shoot segments from the acclimatized Thymus pallidus Coss. ex. Batt. plants and their multiplication on N30K + 0.46 µM/L Kin medium

Mean plantlet length (cm) 5.62 ± 0.12
Mean number of buds 20.64 ± 0.40
Mean number of shoots 2.04 ± 0.06
Mean number of roots 6.99 ± 0.22

Discussion

The protocol used for seed decontamination was efficient, with a fungal and bacterial contamination rate not exceeding 4% and a final germination rate of approximately 25%. Seed sterilization was the starting point in other in vitro culture studies of Thymus species, like T. hyemalis Lange (Nordine et al. 2013a), T. satureioides Coss. (Nordine et al. 2013b) and T. lotocephalus (Coelho et al. 2012), but with different stages.

Although N30K macronutrients didn’t provide the best shoots elongation, nor the best buds and shoots multiplication, we recorded a total regeneration of the plantlets and a minimal hyperhydricity rate. For this reason, we chose N30K macronutrients for the rest of our experiments.

Actually, MS basal medium was the most used for Thymus in vitro culture (Bakhtiar et al. 2014; Bernard et al. 2015; Coelho et al. 2012; Costa et al. 2012; Hassannejad et al. 2012; Macro-Medina and Casas 2015; Mendes et al. 2013; Mirshekar et al. 2014; Nordine and El Meskaoui 2014; Nordine et al. 2014), but less concentrated media were used in other studies (Fraternale et al. 2003; Furmanowa and Olszowska 1992; Kirillov et al. 2014; Pérez-Tortosa et al. 2012; Sáez et al. 1994) or MS with reduced levels of macronutrients (Nordine et al. 2013b; Ozudogru et al. 2011). Also, N30K macronutrients were used for the micropropagation of Lavandula stoechas (Nobre 1996).

The addition of DPU at 0.46 and 0.93 µM, as well as 0.93 Ad contributed to a better shoots elongation of T. pallidus plantlets. In addition, an improved buds multiplication was observed, more specifically after the addition of 0.46 and 0.93 Kin; 0.46 DPU and 2.32 Ad. In addition, 0.46 Kin provided the higher number of shoots. For the root part, we recorded an improvement in the rooting rate for most cytokinins at different concentrations and in the number of roots for all cytokinins and concentrations. Nevertheless, high levels of hyperhydricity were observed in the case of 0.46 Kin and 0.46 Ad.

In fact, cytokinins are necessary for the multiplication of cultures. Some authors have reported the sensitivity of Thymus plantlets to high concentrations of cytokinins, namely Thymus blecherianus Pomel for [6-Benzylaminopurine (BAP)]> 8.88 µM and [Kin]> 9.6 µM. The best growth of shoots was noted on MS + 4.44 µM BAP medium (Nordine and El Meskaoui 2014). In addition, Thymus piperella L. and Thymus vulgaris L. plantlets acquired a stressed morphology, characterized by small leaves and short internodes than in vivo, on CMS medium (Collet 1985) supplemented with 6.6 to 8.9 µM BAP. For this reason, plantlets were multiplied on CMS + 2.2 µM BAP (Lê, 1989; Sáez et al. 1994). Moreover, Macro-Medina and Casas (2015) concluded that Thymus moroderi is a sensitive species to cytokinins, since low concentrations produced a negative effect on their morphology. Similarly, Kin and Thidiazuron (TDZ) were less effective for the regeneration, multiplication and elongation of Thymus persicus shoots (Bakhtiar et al. 2014). Also, a total regeneration of the plantlets obtained from shoot tips and nodal segments of Thymus hyemalis Lange was observed after the addition of 4.6 or 6.9 µM Kin to MS medium, but a lower rate was noted in the case of 4.4 or 8.8 µM BAP. As well, BAP concentrations higher than 2.2 µM, caused a reduction in the number of shoots for the two types of explants (Nordine et al. 2013a). Moreover, Mendes and Romano (1999) reported that the increase in BAP concentration didn’t improve shoot proliferation of Thymus mastichina L. On the other hand, higher concentrations of BAP (> 2.22 µM) were found to be the most favorable for the multiplication of Thymus lotocephalus shoots (Coelho et al. 2012).

The use of gibberellic acid and cytokinins combinations did not contribute to the improvement of T. pallidus shoots length. However, we noted an increase in the number of buds, especially for 0.46 Kin + 1.15 GA3 and 0.46 DPU+1.15 GA3. In addition, the number of shoots increased for 0.46 Kin + 1.15 GA3. Besides, the addition of these combinations didn’t influence the development of the root part. Also, an increase in the hyperhydricity rate was noted for 0.46 Kin + 1.15 GA3 and 0.46 DPU + 1.15 GA3.

Indeed, the use of 0.58 µM GA3 combined with 0.44 µM BAP + 0.98 µM IBA in MS medium didn’t bring a significant improvement during the in vitro culture of Mentha pulegium (Aid et al. 2003). Contrariwise, the incorporation of 1 µM of GA3 in ½ MS medium supplemented with 2.22 µM BAP contributed to the elongation of Thymus satureioides Coss. shoots (Nordine et al. 2013b). Also, maximum regeneration of Thymus vulgaris L. and T. longicaulis C. Presl subsp. longicaulis var. subisophyllus (Borbás) Jalas plantlets was obtained on ½ MS medium supplemented with 4.65 µM Kin + 0.87 µM GA3 combination (Ozudogru et al. 2011). El-Banna (2017) obtained the best elongation of Thymus vulgaris L. shoots on MS medium supplemented with 8.88 µM/L BAP + 1.44 µM/L GA3. Furthermore, the imbibition of Tectona grandis nodal segments in a liquid solution of gibberellic acid (100 mg/L), before their culture on a modified MS medium ([NH4NO3] halved), added with 6.66 µM BAP; 0.049 IBA and 0.29 µM GA3, resulted in high quality shoots (De Gyves et al. 2007).

The combination of 0.46 µM Kin and auxins ensured in the majority of cases a better development of the root part of T. pallidus plantlets. Thus, we noted an increase in the number of roots, especially for 0.46 Kin + 2.85 IAA; 0.46 Kin + 2.85 or 5.71 IBA; 0.46 Kin + 0.3 or 0.57 NAA. In addition, better growth of the aerial part is observed, especially shoots elongation and buds multiplication.

Actually, cytokinins, occasionally combined with low concentrations of auxins, have been used during micropropagation, to multiply cultures of several species of the genus Thymus and Lamiaceae. Auxins alone in culture media were used to optimize rooting. Thus, Furmanowa and Olszowska (1992) obtained the best multiplication of Thymus vulgaris L. shoots on NN medium (Nitsch and Nitsch 1969), added with 0.46 µM Kin + 0.54 µM NAA and 0.46 µM Kin + 1.48 or 2.46 µM IBA, while the best rooting was obtained on NN medium supplemented with only 2.46 µM IBA. In addition, the most suitable growth regulators for elongation and multiplication of Thymus piperella shoots were found to be 4.44 or 6.66 µM BAP without auxins and, 2.85 µM IAA without cytokinins to promote rooting (Sáez et al. 1994). Furthermore, the inclusion of 0.54 µM NAA resulted in a decrease in the regeneration rate of Thymus vulgaris L. (cultured on MS medium) and T. longicaulis C. Presl subsp. longicaulis var. subisophyllus (Borbás) Jalas (cultured on ½ MS medium) compared to 4.65 µM Kin alone. However, this formulation produced the best condition for shoots proliferation. The best root multiplication was obtained after adding, alone in the culture medium, 0.23 µM 2,4-Dichlorophenoxyacetic acid (2,4-D) (Ozudogru et al. 2011). Also, ½ MS medium supplemented with 14.76 µM/L IBA produced the highest rooting percentage of Thymus capitatus L. shoots, and the highest number of roots was recorded on ½ MS containing 9.84 µM/L IBA (El-Makawy et al. 2008). Additionally, the use of adenine associated with low levels of NAA in a medium containing N30K macronutrients (Margara 1978) improved the multiplication of Lavandula stoechas shoots (Nobre 1996). On the other hand, the addition of NAA to MS medium supplemented with BAP reduced considerably the number of shoots of Lavandula vera (Andrade et al. 1999) and Lavandula dentata (Echeverrigaray et al. 2005) plantlets. Moreover, for other species such as Thymus lotocephalus (Coelho et al. 2012), Thymus hyemalis Lange (Nordine et al. 2013a), Lavandula vera (Andrade et al. 1999), Lavandula viridis (Dias et al. 2002) and Teucrium stocksianum (Bouhouche and Ksiksi 2007), it was observed that a reduction in the concentration of macronutrients improved the root formation.

Re-initiation of the in vitro culture of T. pallidus from acclimatized plants has shown that it is as difficult to ensure a high survival rate of healthy plantlets, as to have the minimum of contamination. This remark was reported for the in vitro culture of plants of the genus Thymus from nodal segments, namely T. moroderi (Macro-Medina and Casas 2015), T. blecherianus Pomel (Nordine and El Meskaoui 2014), T. caespititius (Mendes et al. 2013) and T. longicaulis (Ozudogru et al. 2011), as well as other Lamiaceae such as Lavandula viridis (Dias et al. 2002), Salvia pratensis and Salvia nemorosa (Ruffoni et al. 2009), with survival rates ranging from 8 to 36%.

Conclusions

The present study is the first about the micropropagation of Thymus pallidus Coss., through shoot tips and nodal segments culture. Thereby, several compositions of culture media were evaluated in order to determine the best ones for the multiplication, elongation and rooting of T. pallidus plantlets.

In vitro germination resulted in about 25% of germinated seeds and the obtained plantlets were multiplied on SD + 0.46 Kin medium. N30K macronutrients were the most effective, since they ensured a total regeneration and a minimum hyperhydricity rate. Also, the addition to N30K medium of 0.93 DPU; 0.46 Ad; 0.46 and 0.93 Kin resulted in better multiplication and elongation of shoots. Moreover, 0.46 Ad + 0.58 GA3 and 0.46 Kin + 1.15 GA3 led to an optimization of buds and shoots multiplication. In addition, 0.46 Kin combined to 2.85 IAA, 2.85 or 5.71 IBA; 0.3 or 0.57 NAA resulted in better development of the root part and to an optimization of the growth of the aerial part.

Finally, acclimatization was successfully carried out for vitro-plants from N30K + 0.46 µM/L Kin + 2.85 µM/L IAA medium, and the in vitro culture was re-established, once again, after sterilization of nodal segments from acclimatized plants.

Actually, this micropropagation protocol could be established for the multiplication of selected genotypes and chemotypes of medicinal and aromatic plants. Moreover, plants cultured in vitro can then be used for various studies, avoiding their collection from their natural habitat and, in addition to their importance for facilitating the propagation of plants, in vitro culture techniques provide models of systems allowing studying the production, accumulation and metabolism of important bioactive metabolites.

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