Micropropagation from shoot tips and nodal segments was carried out for the conservation and domestication of spontaneous Moroccan thyme, Thymus broussonetii Boiss. subsp. broussonetii (endemic threatened). The mineral composition of the culture medium, as well as the succession of different growth regulators, influenced the in vitro growth of this species. Sterilized achenes of T. broussonetii were able to germinate on an agar medium containing Gautheret macronutrients with a rate of 25% and a degree of contamination of less than 4%. Shoot apices of 15-day seedlings (two cotyledon leaves) were cultivated on SD + 0.46 μM Kin medium and the explants obtained were transplanted every month. Six macronutrients (MS, B 5 , SH, SD, MS m and N30K) were tested and N30K was chosen for the following experiments. Seven cytokinins (Kin, BAP, 2iP, DPU, adenine, Zeat and TDZ) at 0.46, 0.93 and 2.32 μM/l were evaluated and the addition of 0.93 μM adenine to N30K medium favored significantly the induction of buds and the elongation of explants. Three polyamines (putrescine, spermidine and spermine) at 2, 5, 10 and 20 μM/l were tested. A better multiplication of buds, shoots and roots was noted for N30K + 10 μM spermine. Cytokinin-auxin combinations led to better root multiplication and an increase in the number of buds a nd the length of explants, particularly for 0.46 μM Kin + 2.85 μM IAA. Acclimatization was successfully carried out using vitroplants developing a good root system. One month after the start of acclimatization, 97% of T. broussonetii plantlets were healthy. Three months later, they were transplanted into larger pots. 100% of the acclimatized plants developed flowers in the 2 nd year between June and August. Re-initiation of the in vitro culture was carried out from sterilized twig segments collected from the acclimatized plants of T. broussonetii with 1 - 2 nodes on the medium N 30 K + 0.46 μM Kin, and 52.1% of the explants healthily proliferated. Finally, two micropropagation prototypes were developed: shoot tip culture from seedlings obtained after germination of achenes and node culture from acclimatized plants.
Thymus broussonetii Boiss. is a very rare medicinal and aromatic plant which exists in the form of two subspecies: subsp. hannonis (Maire) R. Morales endemic to Morocco and subsp. broussonetii endemic to Morocco, Algeria and Tunisia [
Thymus broussonetii is a small shrub 12 to 40 cm height with flat leaves ciliated at the base with whole edges; flowers are gathered towards the top of shoots in dense oval-cylindrical inflorescences, with floral leaves wider than the leaves, often colored with purple, attenuated and sharp at the end, with cilia on the edges; the calyx, 6 - 7.5 mm long, has 2 lips, the upper not very toothed; the corolla is pink to purple, 2 to 3 times longer than the calyx, with a distinctly prominent narrow tube. The subsp. hannonis differs from the type by its smaller size (12 vs. 15 - 40 cm), its shorter leaves (5 - 10 vs. 10 - 18 mm) not subpetiolate (2.5 vs. 1 mm), and floral bracts densely covered by longer hairs and not only on abaxial side [
Moreover, the essential oil of Thymus broussonetii is mainly rich in thymol, carvacrol, borneol, ρ-cymene, α-pinene and γ-terpinene [
Actually, the excessive pressure exerted on wild plant populations, by harvesting from their natural habitat, combined with land conversion, has resulted in a reduced and scattered distribution of this species. Its harvest takes place mainly during the flowering period before the formation of seeds. This causes a decrease in the rate of regeneration and a progressive degradation of wild populations [
The present study completes the already established micropropagation protocol of this species, through the evaluation of the effect of several macronutrients, cytokinins, polyamines and auxins, in addition to the re-initiation of in vitro culture from acclimatized plants.
Thymus broussonetii Boiss. subsp. broussonetii achenes were provided by the National Institute of Agronomic Research (INRA) of Rabat and were used as a source of plant material.
Achene 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 minutes;
- Rinsing with sterile distilled water for 5 minutes;
- Immersion in mercury chloride (HgCl2) solution 0.1% (w/v) for 2 minutes;
- Three successive rinses with sterile distilled water (5, 10 and 15 minutes).
Achenes were then soaked for 48 h in sterile distilled water in 250 ml conical flasks installed on a rotary shaker (12 rpm, Fisher Scientific).
After imbibition, seeds were germinated in vitro into glass test tubes (18 × 180 mm), one seed per tube, this latter containing 15 ml of a culture medium composed of Gautheret macronutrients [
The 2-week-old seedlings resulting from in vitro germination were used in the following experiments, since their organs (hypocotyls, cotyledons and shoot tip) have developed and their roots were short.
Thus, cultures were induced from nodal segments (5 - 6 mm) with axillary buds obtained from 4-week-old aseptic seedlings, on a medium solidified with 0.7% bacteriological agar, containing Shah and Dalal (SD, 1980) [
Six solutions of macronutrients differing in nitrogen content (NO− 3 and NH+ 4,) and in potassium, all added with MS micronutrients and vitamins, were tested: MS [
Seven cytokinins (Sigma-Aldrich): Kin (kinetin), BAP (6-benzylaminopurine), 2iP (2-isopentenyladenine), DPU (1,3-diphenylurea), adenine, Zeat (zeatin) and TDZ (thidiazuron) were evaluated on Thymus broussonetii plantlets growth. Three concentrations were tested: 0.46, 0.93 and 2.32 μM/l, plus a control medium containing no growth regulator.
Three polyamines (putrescine, spermidine and spermine, Sigma-Aldrich) were tested at four concentrations: 2, 5, 10 and 20 µM/l.
Kin or adenine at 0.46 µM/l were tested, alone or combined to three auxins (Sigma-Aldrich): IAA (Indole-3-acetic acid), IBA (Indole-3-butyric acid) and NAA (1-Naphthaleneacetic acid) at five concentrations: 0.057, 0.3, 0.57, 2.85 or 5.71 µM/l.
After removal from the culture media, 30 rooted plantlets were gently washed to remove the rest of the agar medium from roots and then acclimatized in plastic pots (9 × 6 × 8 cm), 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 h, humidity: 90% - 100%, temperature: 24˚C ± 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. After complete elimination of the cups, 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 (17 × 13 × 15 cm).
Twigs were cut from the acclimatized plants of Thymus broussonetii, thoroughly washed with tap water, then surface sterilized under a laminar flow hood. Several sterilization methods, differing by the products used for this purpose, were tested. The chosen method is based on soaking twigs first in 10% Ca(ClO)2 (w/v) with 4 to 5 drops of Tween 80 for 30 min, then in 0.1% HgCl2 (w/v) with 4 to 5 drops of Tween 80 for 5 min and finally dipping three times in 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 a medium composed of N30K [
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 1 N). 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 vitroplants were incubated in a culture room (photoperiod: 18/6 h with 4000 lux light intensity, temperature: 24˚C ± 1˚C).
After one month of growth, the following parameters were evaluated:
- Regeneration rate (%);
- 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 (%).
All measurements were run in triplicates (n = 3); 24 samples were used for each replicate and 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 to be significant.
Germination of Thymus broussonetii achenes does not begin until 48 hours. The final germination rate is 25% and the degree of contamination does not exceed 4% (
The differences between the six macronutrients in terms of regeneration and rooting are not significant contrarily to hyperhydricity. N30K macronutrients ensure total regeneration of the vitroplants, followed by MS and MSm (93.1). SD macronutrients show the best rooting rate (71.2%), followed by SH (67.7) and N30K (62.5). Hyperhydricity appears for MSm (31.2%) and MS (41.6) and is absent for B5 and SD.
In addition, a better shoots elongation is noticed in the case of MS (3.04 cm) and SD (2.91). The minimum length is observed for SH (2.08). The multiplication of shoots and buds is approximately similar for the six macronutrients, with a maximum number of shoots in the case of N30K (3.6), followed by MS (3.1). The best number of buds is noted in the case of N30K (28.6), followed by B5 (27.5). Root multiplication is optimal in the case of SD and MS (4.58 and 4.44, respectively), followed by SH and N30K (4.02 and 3.80, respectively).
In short, B5 and SD macronutrients have lower regeneration rates and MS and MSm show very high hyperhydricity rates (41.6% and 31.2%, respectively). Between SH and N30K macronutrients, the second ones were selected, since they ensure total regeneration and better multiplication of buds and shoots, with a low hyperhydricity rate (
The integration of cytokinins into the culture media has produced several changes in the aerial as in the root parts of the vitroplants (
Thus, a total regeneration of the explants is noted for cytokinins 2iP and adenine at all concentrations, at 0.46 µM for DPU, Zeat and TDZ, as well as 0.93 µM DPU and 2.32 µM Zeat. The lowest rates are observed in the case of 2.32 µM Kin (80.6%), as well as BAP (83.3) and TDZ (87.5) at 0.93 µM.
Among the regenerated explants, 100% develop roots in the case of DPU and Kin at 2.32 µM, as well as BAP and adenine at 0.93 µM; 98.6% for 2.32 µM adenine; 97.2% for 0.46 µM adenine and 0.93 µM DPU. Very low rooting rates were noted in the case of 0.93 and 2.32 µM TDZ (11.7 and 4.5, respectively), besides 0.46 µM Zeat (37.5).
Macronutrients | Regeneration rate (%) | Shoots length (cm) | Number of shoots | Number of buds | Rooting rate (%) | Number of roots | Hyperhydricity rate (%) |
---|---|---|---|---|---|---|---|
MS | 93.1 ± 6.9 a | 3.04 ± 0.15 a | 3.15 ± 0.22 ab | 27.08 ± 1.01 ab | 45.8 ± 23.0 a | 4.44 ± 0.54 a | 41.6 ± 4.1 a |
B5 | 82.0 ± 9.7 a | 2.14 ± 0.12 b | 2.62 ± 0.18 b | 27.50 ± 1.19 a | 46.0 ± 7.4 a | 3.14 ± 0.37 ab | 0.0 ± 0.0 c |
SH | 93.0 ± 2.8 a | 2.08 ± 0.08 b | 2.75 ± 0.17 b | 25.82 ± 0.89 abc | 67.7 ± 11.0 a | 4.02 ± 0.59 ab | 2.1 ± 0.0 c |
SD | 87.5 ± 8.7 a | 2.91 ± 0.16 a | 2.64 ± 0.15 b | 23.37 ± 0.68 c | 71.2 ± 7.6 a | 4.58 ± 0.50 a | 0.0 ± 0.0 c |
MSm | 93.1 ± 3.7 a | 2.14 ± 0.12 b | 3.06 ± 0.21 ab | 24.96 ± 1.10 bc | 56.4 ± 6.2 a | 2.70 ± 0.35 b | 31.2 ± 2.0 b |
N30K | 100.0 ± 0.0 a | 2.36 ± 0.11 b | 3.64 ± 0.25 a | 28.64 ± 0.95 a | 62.5 ± 4.2 a | 3.80 ± 0.36 ab | 2.1 ± 0.0 c |
The data represent mean ± SE of replicates (n = 3). Values in a same row carrying different letters are significantly different by Duncan’s multiple range test at p ≤ 0.05.
Cytokinins (µM) | Regeneration rate (%) | Shoots length (cm) | Number of shoots | Number of buds | Rooting rate (%) | Number of roots | Hyperhydricity rate (%) | |
---|---|---|---|---|---|---|---|---|
Control | 100.0 ± 0.0 a | 5.58 ± 0.49 b | 1.96 ± 0.20 abc | 20.67 ± 1.45 bc | 91.7 ± 2.4 bcd | 8.81 ± 1.10 bc | 16.7 ± 2.4 b | |
Kin | 0.46 | 94.4 ± 1.6 cd | 2.94 ± 0.25 hij | 1.88 ± 0.14 bc | 19.97 ± 1.01 bc | 94.1 ± 1.7 abcd | 5.61 ± 0.57 cde | 4.9 ± 0.9 cdef |
0.93 | 96.3 ± 1.8 bc | 3.29 ± 0.35 fghij | 1.59 ± 0.13 cd | 18.88 ± 0.90 c | 82.3 ± 1.7 ef | 6 .74 ± 0.87 bcd | 5.9 ± 1.7 cdef | |
2.32 | 80.6 ± 1.6 g | 3.87 ± 0.36 efgh | 2.07 ± 0.14 ab | 21.14 ± 1.34 abc | 100.0 ± 0.0 a | 7.18 ± 0.71 bcd | 10.7 ± 2.1 c | |
BAP | 0.46 | 91.9 ± 1.6 de | 2.57 ± 0.13 ij | 2.36 ± 0.19 ab | 21.27 ± 1.17 ab | 66.7 ± 1.7 g | 5.36 ± 0.84 de | 5.0 ± 1.0 cdef |
0.93 | 83.3 ± 1.6 g | 2.37 ± 0.26 j | 1.66 ± 0.12 cd | 17.38 ± 1.02 c | 100.0 ± 0.0 a | 4.93 ± 0.57 def | 0.0 ± 0.0 f | |
2.32 | 89.5 ± 1.5 ef | 2.35 ± 0.11 j | 1.94 ± 0.16 abc | 18.82 ± 0.93 c | 61.8 ± 1.7 g | 4.05 ± 0.42 def | 3.9 ± 0.9 def | |
2iP | 0.46 | 100.0 ± 0.0 a | 3.58 ± 0.25 efghi | 1.97 ± 0.15 ab | 19.44 ± 0.94 bc | 94.4 ± 1.6 abc | 7.58 ± 0.69 bcd | 8.3 ± 1.6 cd |
0.93 | 100.0 ± 0.0 a | 4.98 ± 0.39 bcd | 1.64 ± 0.20 cd | 21.53 ± 1.00 ab | 91.7 ± 1.6 bcd | 9.39 ± 0.81 bc | 6.5 ± 2.4 cde | |
2.32 | 100.0 ± 0.0 a | 5.24 ± 0.37 bc | 1.75 ± 0.12 c | 19.83 ± 0.70 bc | 88.9 ± 3.2 cd | 8.03 ± 0.62 bcd | 18.5 ± 2.4 b | |
DPU | 0.46 | 100.0 ± 0.0 a | 4.04 ± 0.24 defg | 1.92 ± 0.15 abc | 23.11 ± 1.06 ab | 94.4 ± 1.6 abc | 7.67 ± 0.99 bcd | 1.8 ± 0.9 ef |
0.93 | 100.0 ± 0.0 a | 5.65 ± 0.46 b | 1.75 ± 0.14 c | 24.83 ± 1.25 a | 97.2 ± 1.6 ab | 10.03 ± 0.83 ab | 0.0 ± 0.0 f | |
2.32 | 94.4 ± 1.1 cd | 5.59 ± 0.37 b | 2.07 ± 0.21 ab | 24.50 ± 1.59 a | 100.0 ± 0.0 a | 9.75 ± 0.55 abc | 3.6 ± 2.1 def | |
Adenine | 0.46 | 100.0 ± 0.0 a | 4.20 ± 0.24 cdef | 1.96 ± 0.18 abc | 21.96 ± 1.51 ab | 97.2 ± 1.4 ab | 6.04 ± 0.86 bcde | 0.0 ± 0.0 f |
0.93 | 100.0 ± 0.0 a | 6.65 ± 0.54 a | 1.71 ± 0.20 c | 22.08 ± 1.20 ab | 100.0 ± 0.0 a | 8.38 ± 0.98 bc | 4.2 ± 2.4 def | |
2.32 | 100.0 ± 0.0 a | 5.07 ± 0.42 bcd | 2.46 ± 0.23 a | 23.42 ± 1.42 a | 98.6 ± 1.4 a | 13.26 ± 1.13 a | 37.5 ± 2.4 a | |
Zeat | 0.46 | 100.0 ± 0.0 a | 3.32 ± 0.16 fghij | 2.00 ± 0.19 ab | 19.63 ± 1.19 bc | 37.5 ± 2.4 h | 2.11 ± 0.39 ef | 0.0 ± 0.0 f |
0.93 | 95.8 ± 2.4 c | 4.60 ± 0.31 bcde | 1.39 ± 0.12 cd | 17.91 ± 0.93 c | 91.3 ± 2.5 bcd | 5.00 ± 0.78 def | 0.0 ± 0.0 f | |
2.32 | 100.0 ± 0.0 a | 4.30 ± 0.36 cdef | 2.25 ± 0.19 ab | 21.92 ± 1.34 ab | 87.5 ± 2.4 de | 7.95 ± 0.89 bcd | 33.3 ± 2.4 a | |
TDZ | 0.46 | 100.0 ± 0.0 a | 3.40 ± 0.29 fghij | 2.21 ± 0.22 ab | 18.75 ± 1.18 c | 79.2 ± 2.4 f | 4.50 ± 0.74 def | 33.3 ± 2.4 a |
0.93 | 87.5 ± 2.4 f | 2.99 ± 0.28 ghij | 1.15 ± 0.08 d | 15.20 ± 0.69 d | 11.7 ± 4.4 i | 2.25 ± 0.63 ef | 0.0 ± 0.0 f | |
2.32 | 94.2 ± 1.4 cd | 3.66 ± 0.26 efghi | 1.50 ± 0.11 cd | 13.64 ± 0.58 d | 4.5 ± 2.6 j | 1.00 ± 0.00 f | 9.1 ± 2.6 cd |
The data represent mean ± SE of replicates (n = 3). Values in a same row carrying different letters are significantly different by Duncan’s multiple range test at p ≤ 0.05.
Very high hyperhydricity rates are observed in the case of 2.32 µM adenine (37.5%), 2.32 µM Zeat and 0.46 µM TDZ (33.3). Values ranging from 1.8% to 18.5% are noted in the rest of cases, except for BAP, DPU, Zeat and TDZ at 0.93 µM, in addition to adenine and Zeat at 0.46 µM, where an absence of hyperhydricity is noticed.
The maximum shoots length is observed for 0.93 µM adenine (6.65 cm), followed by 0.93 and 2.32 µM DPU (5.65 and 5.59, respectively) and 2.32 µM 2iP (5.24). A low elongation is recorded in the case of 0.93 µM TDZ (2.99 cm), 0.46 µM Kin (2.94), as well as 0.46, 0.93 and 2.32 µM BAP (2.57, 2.37 and 2.35, respectively).
A maximum number of shoots is regenerated in the case of 2.32 µM adenine (2.46) and 0.46 µM BAP (2.36), followed by 2.32 µM Zeat (2.25), 0.46 µM TDZ (2.21), plus Kin and DPU at 2.32 µM (2.07). The minimum of shoots is observed in the case of 0.93 µM TDZ (1.15).
The multiplication of buds is maximum for 0.93 and 2.32 µM DPU (24.83 and 24.50, respectively), 2.32 µM adenine (23.42) and 0.46 µM DPU (23.11). The minimum number of buds is noted for 2.32 µM TDZ (13.64).
Also, the greatest number of roots grow for explants regenerated on N30K medium supplemented with 2.32 µM adenine (13.26), followed by 0.93, 2.32 µM DPU (10.03 and 9.75, respectively) and 0.93 µM 2iP (9.39). The minimum number of roots is noted in the case of 2.32 µM TDZ (1.00).
In conclusion, 0.93 µM adenine and 0.93 µM DPU alone in N30K medium are the best for the micropropagation of T. broussonetii: they ensure total regeneration of the vitroplants, optimal elongation and maximum multiplication of buds. In addition, a good development of the root part is noted, with high rooting rates and high multiplication of roots.
The addition of polyamines to the culture media resulted in some modifications in the in vitro growth of T. broussonetii vitroplants (
Polyamines (µM) | Regeneration rate (%) | Shoots length (cm) | Number of shoots | Number of buds | Rooting rate (%) | Number of roots | Hyperhydricity rate (%) | |
---|---|---|---|---|---|---|---|---|
Control | 91.7 ± 0.0 b | 4.69 ± 0.42 b | 1.73 ± 0.24 ab | 21.00 ± 1.07 cd | 100.0 ± 0.0 a | 6.45 ± 0.65 ef | 9.1 ± 0.0 a | |
Putrescine | 2 | 100.0 ± 0.0 a | 5.82 ± 0.41 a | 1.92 ± 0.23 ab | 23.83 ± 1.58 abc | 91.7 ± 0.0 bc | 7.96 ± 0.74 def | 0.0 ± 0.0 c |
5 | 95.8 ± 4.2 ab | 4.05 ± 0.35 bc | 1.96 ± 0.17 ab | 25.78 ± 1.31 ab | 100.0 ± 0.0 a | 8.52 ± 0.80 def | 0.0 ± 0.0 c | |
10 | 91.7 ± 0.0 b | 4.30 ± 0.29 bc | 1.59 ± 0.16 b | 25.18 ± 1.90 abc | 100.0 ± 0.0 a | 13.86 ± 0.87 a | 0.0 ± 0.0 c | |
20 | 95.8 ± 4.2 ab | 2.74 ± 0.14 d | 1.68 ± 0.26 ab | 18.48 ± 1.28 d | 100.0 ± 0.0 a | 10.56 ± 0.70 bc | 0.0 ± 0.0 c | |
Spermidine | 2 | 91.7 ± 0.0 b | 6.24 ± 0.53 a | 2.27 ± 0.30 a | 25.50 ± 1.31 abc | 100.0 ± 0.0 a | 8.55 ± 0.78 cde | 4.5 ± 4.5 b |
5 | 91.7 ± 0.0 b | 3.82 ± 0.32 bc | 1.46 ± 0.14 b | 19.41 ± 1.43 bcd | 86.4 ± 4.5 d | 7.47 ± 0.94 bcd | 0.0 ± 0.0 c | |
10 | 100.0 ± 0.0 a | 4.76 ± 0.31 b | 1.38 ± 0.12 b | 23.83 ± 1.62 abc | 100.0 ± 0.0 a | 12.21 ± 0.84 ab | 0.0 ± 0.0 c | |
20 | 95.8 ± 4.2 ab | 3.92 ± 0.21 bc | 2.35 ± 0.27 a | 21.65 ± 1.45 bcd | 100.0 ± 0.0 a | 11.09 ± 0.87 b | 0.0 ± 0.0 c | |
Spermine | 2 | 100.0 ± 0.0 a | 5.99 ± 0.42 a | 1.83 ± 0.12 ab | 21.58 ± 1.09 bcd | 95.8 ± 4.2 ab | 5.96 ± 0.59 f | 0.0 ± 0.0 c |
5 | 91.7 ± 0.0 b | 3.63 ± 0.33 cd | 2.00 ± 0.22 ab | 22.18 ± 1.26 bcd | 90.9 ± 0.0 cd | 7.35 ± 0.81 ef | 0.0 ± 0.0 c | |
10 | 100.0 ± 0.0 a | 4.18 ± 0.28 bc | 2.29 ± 0.30 a | 27.42 ± 1.48 a | 100.0 ± 0.0 a | 10.50 ± 0.84 bc | 8.3 ± 0.0 a | |
20 | 95.8 ± 4.2 ab | 2.82 ± 0.14 d | 2.35 ± 0.22 a | 23.00 ± 1.36 abc | 100.0 ± 0.0 a | 10.65 ± 0.77 bc | 0.0 ± 0.0 c |
The data represent mean ± SE of replicates (n = 3). Values in a same row carrying different letters are significantly different by Duncan’s multiple range test at p ≤ 0.05.
Thus, a total regeneration is noticed in the case of 2 µM putrescine, 10 µM spermidine, 2 and 10 µM spermine. In addition, the rooting rate is 100%, except for the control (91.7%), 2 µM putrescine (91.7), 5 µM spermidine (86.4), 2 and 5 µM spermine (95.8 and 90.9, respectively). Also, a certain number of plants show a translucent appearance in the case of 2 µM putrescine (4.5%) and 10 µM spermine (8.3), but lower than that of the control (9.1).
Compared with shoot length on the control medium (4.69 cm), an improvement is recorded after adding 2 µM putrescine, 2 µM spermine and 10 µM spermidine (5.82; 5.99 and 6.24, respectively). On the other hand, it decreases for the rest, especially after the addition of 20 µM putrescine (2.74) and 20 µM spermine (2.82).
An increase in the number of shoots is noted for 2 and 20 µM spermidine (2.27 and 2.35, respectively), as well as 10 and 20 µM spermine (2.29 and 2.35, respectively), while this number decreases in the case of 10 µM spermidine (1.37).
The number of buds increases for most polyamines, particularly for 5 µM putrescine (25.78), 2 µM spermidine (25.50) and 10 µM spermine (27.42). The lower number of buds is observed in the case of 20 µM putrescine and 5 µM spermidine (18.48 and 19.41, respectively).
Furthermore, vitroplants growing on N30K medium supplemented with polyamines develop in most cases greater number of roots, essentially for 10 µM putrescine and spermidine (13.86 and 12.21, respectively). The lower number of roots is noticed in the case of 2 µM spermidine (5.96).
In conclusion, the addition of 10 µM putrescine influences positively the multiplication of buds and roots, while 2 µM spermidine optimizes elongation as well as buds and shoots multiplication. Besides, 10 and 20 µM spermine contribute to increase the number of buds, shoots and roots.
The combination of the three auxins (IAA, IBA and NAA) with 0.46 Kin resulted in some changes in the in vitro growth of T. broussonetii vitroplants (
Auxins concentration (µM) | Regeneration rate (%) | Shoots length (cm) | Number of shoots | Number of buds | Rooting rate (%) | Number of roots | Hyperhydricity rate (%) | |
---|---|---|---|---|---|---|---|---|
Control | 0 | 94.4 ± 1.6 ab | 2.94 ± 0.25 b | 1.88 ± 0.14 abc | 19.97 ± 1.01 bc | 94.1 ± 1.7 b | 5.61 ± 0.57 e | 4.9 ± 0.9 b |
IAA | 0.057 | 100.0 ± 0.0 a | 3.02 ± 0.24 b | 1.67 ± 0.26 bc | 19.00 ± 2.05 c | 100.0 ± 0.0 a | 8.67 ± 1.24 de | 0.0 ± 0.0 c |
0.3 | 100.0 ± 0.0 a | 2.92 ± 0.26 b | 1.83 ± 0.21 abc | 20.50 ± 2.41 bc | 100.0 ± 0.0 a | 8.50 ± 0.92 de | 0.0 ± 0.0 c | |
0.57 | 100.0 ± 0.0 a | 2.96 ± 0.27 b | 2.50 ± 0.42 ab | 21.17 ± 1.66 bc | 86.1 ± 2.8 c | 7.70 ± 1.32 de | 13.9 ± 2.8 a | |
2.85 | 94.4 ± 2.8 ab | 3.03 ± 0.27 b | 1.91 ± 0.21 abc | 21.64 ± 2.36 abc | 100.0 ± 0.0 a | 14.54 ± 1.31 a | 0.0 ± 0.0 c | |
5.71 | 100.0 ± 0.0 a | 5.37 ± 0.51 a | 2.00 ± 0.17 abc | 26.17 ± 2.25 ab | 100.0 ± 0.0 a | 10.58 ± 0.66 bcd | 0.0 ± 0.0 c | |
IBA | 0.057 | 100.0 ± 0.0 a | 3.56 ± 0.34 b | 2.42 ± 0.26 abc | 25.33 ± 1.91 abc | 100.0 ± 0.0 a | 13.08 ± 1.41 abc | 0.0 ± 0.0 c |
0.3 | 100.0 ± 0.0 a | 4.99 ± 0.41 a | 2.58 ± 0.40 a | 28.50 ± 2.72 a | 100.0 ± 0.0 a | 12.92 ± 1.20 abc | 0.0 ± 0.0 c | |
0.57 | 100.0 ± 0.0 a | 3.40 ± 0.42 b | 1.67 ± 0.19 bc | 20.67 ± 0.90 bc | 100.0 ± 0.0 a | 11.42 ± 0.83 abcd | 0.0 ± 0.0 c | |
2.85 | 100.0 ± 0.0 a | 3.18 ± 0.32 b | 2.00 ± 0.27 abc | 23.67 ± 2.89 abc | 100.0 ± 0.0 a | 13.33 ± 1.72 abc | 0.0 ± 0.0 c | |
5.71 | 100.0 ± 0.0 a | 3.62 ± 0.37 b | 2.17 ± 0.37 abc | 24.83 ± 2.12 abc | 100.0 ± 0.0 a | 14.00 ± 0.71 ab | 0.0 ± 0.0 c | |
NAA | 0.057 | 100.0 ± 0.0 a | 4.81 ± 0.52 a | 1.58 ± 0.26 c | 23.83 ± 2.34 abc | 100.0 ± 0.0 a | 9.92 ± 1.18 cd | 0.0 ± 0.0 c |
0.3 | 100.0 ± 0.0 a | 3.54 ± 0.36 b | 1.75 ± 0.22 abc | 26.17 ± 2.56 ab | 100.0 ± 0.0 a | 10.50 ± 1.20 bcd | 0.0 ± 0.0 c | |
0.57 | 88.9 ± 2.8 bc | 2.52 ± 0.27 b | 1.91 ± 0.21 abc | 22.18 ± 1.84 abc | 100.0 ± 0.0 a | 10.45 ± 1.53 bcd | 0.0 ± 0.0 c | |
2.85 | 83.3 ± 4.8 c | 3.45 ± 0.49 b | 1.90 ± 0.23 abc | 22.80 ± 2.00 abc | 100.0 ± 0.0 a | 9.10 ± 1.11 de | 0.0 ± 0.0 c | |
5.71 | 91.7 ± 4.8 b | 2.39 ± 0.26 b | 1.90 ± 0.31 abc | 20.00 ± 2.15 bc | 100.0 ± 0.0 a | 7.70 ± 1.48 de | 0.0 ± 0.0 c |
The data represent mean ± SE of replicates (n = 3). Values in a same row carrying different letters are significantly different by Duncan’s multiple range test at p ≤ 0.05.
Thus, a total regeneration is observed for all phytohormonal combinations, except 2.85 µM IAA (94.4), as well as 0.57, 2.85 and 5.71 µM NAA (88.9, 83.3 and 91.7, respectively). In addition, the rooting rate increases for all combinations between 0.46 µM Kin and auxins (100%), but decreases for 0.57 µM IAA (86.1%). Besides, a total absence of hyperhydricity is noted, except for 0.57 µM IAA (13.9%).
Shoots length increases after integration of auxins in the medium N30K + 0.46 µM Kin, especially in the case of 5.71 µM IAA (5.37 cm); 0.3 µM IBA (4.99) and 0.057 µM NAA (4.81). However, it decreases insignificantly for 0.3 µM IAA (2.92), 0.57 and 5.71 µM NAA (2.52 and 2.39, respectively).
Moreover, the number of shoots increases for certain phytohormonal combinations, namely 0.57 µM IAA (2.50), 0.057 and 0.3 µM IBA (2.42 and 2.58, respectively), but decreases in the case of 0.057 µM NAA (1.58), 0.57 µM IBA (1.67) and 0.057 µM IAA (1.67).
In addition, an increase in the number of buds is noted for almost all the combinations between 0.46 µM Kin and auxins, in particular for 0.3 µM IBA (28.50), 0.3 µM NAA (26.17) and 5.71 µM IAA (26.17). However, this number decreases in the case of 0.057 µM IAA (19.00).
Besides, the number of roots increases significantly after adding the auxins to the medium N30K + 0.46 µM Kin, in particular in the case of 2.85 µM IAA (14.54), 5.71 µM IBA (14.00) and 0.3 µM NAA (10.50).
After adding IAA to N30K + 0.46 µM adenine medium, the regeneration rate decreases from 100% to 86.1% for 0.3 µM IAA and IBA, to 88.9 for 2.85 µM IBA and NAA, while it is maintained at 100% for all the other auxins and concentrations. All of the regenerated explants develop roots after integration of auxins into the culture media, except for 0.3 and 0.57 µM IBA (93.3% and 91.7%, respectively) and 0.57 µM NAA (83.3). In addition, a total absence of hyperhydricity is noted.
Shoots length decreases compared to the control medium (4.20 cm) after the addition of IAA. However, a small increase is noted in 5.71 µM IAA (4.32). Also with IBA, the highest value is mentioned in the case of 0.057 (4.63) and 0.57 µM (5.24). For NAA, shoots length increases only for 0.57 µM (4.90). On the other hand, the lowest values are recorded at 5.71 µM NAA (2.27) and 0.057 µM IAA (2.22).
In comparison with the number of shoots on the control medium (1.96), an important increase is noted for 0.3 µM IBA (2.80), as well as 0.57 µM NAA (2.75). On the other hand, their number decrease with 5.71 µM IBA (1.60) and 0.3 µM NAA (1.18).
The number of buds increases after the addition of IBA and NAA, with maximum values at 0.57 µM IBA (27.33) and 2.85 µM NAA (28.00). However, this number decreases with 5.71 µM IBA and NAA (21.00 and 20.00, respectively) compared to the control (21.96). Even for IAA, a larger number of buds are observed with 2.85 and 5.71 µM (24.00 and 24.83, respectively). However, their number decreases in the case of 0.057 and 0.3 µM (17.33 and 19.80, respectively).
Moreover, a significant increase is remarked in the number of roots after the addition of auxins, in particular for 2.85 and 5.71 µM IAA (13.92 and 11.50, respectively), 0.057 and 2.85 µM IBA (12.08 and 12.00, respectively), as well as 2.85 µM NAA (12.80) (
Auxins concentration (µM) | Regeneration rate (%) | Shoots length (cm) | Number of shoots | Number of buds | Rooting rate (%) | Number of roots | Hyperhydricity rate (%) | |
---|---|---|---|---|---|---|---|---|
Control | 0 | 100.0 ± 0.0 a | 4.20 ± 0.24 abcd | 1.96 ± 0.18 abc | 21.96 ± 1.51 abcd | 97.2 ± 1.4 ab | 6.04 ± 0.86 e | 0.0 ± 0.0 |
IAA | 0.057 | 100.0 ± 0.0 a | 2.23 ± 0.20 g | 1.92 ± 0.26 abc | 17.33 ± 1.29 d | 100.0 ± 0.0 a | 8.17 ± 0.85 cde | 0.0 ± 0.0 |
0.3 | 86.1 ± 2.8 bc | 2.89 ± 0.35 efg | 2.00 ± 0.39 abc | 19.80 ± 1.72 cd | 100.0 ± 0.0 a | 6.20 ± 1.08 e | 0.0 ± 0.0 | |
0.57 | 100.0 ± 0.0 a | 2.55 ± 0.20 fg | 2.17 ± 0.27 ab | 21.83 ± 1.47 abcd | 100.0 ± 0.0 a | 9.58 ± 1.14 bcd | 0.0 ± 0.0 | |
2.85 | 100.0 ± 0.0 a | 2.47 ± 0.18 g | 2.08 ± 0.19 abc | 24.00 ± 1.92 abcd | 100.0 ± 0.0 a | 13.92 ± 0.98 a | 0.0 ± 0.0 | |
5.71 | 100.0 ± 0.0 a | 4.32 ± 0.38 abcd | 1.83 ± 0.11 abc | 24.83 ± 1.24 abc | 100.0 ± 0.0 a | 11.50 ± 1.51 abc | 0.0 ± 0.0 | |
IBA | 0.057 | 100.0 ± 0.0 a | 4.63 ± 0.68 abc | 2.33 ± 0.23 ab | 24.17 ± 2.12 abcd | 100.0 ± 0.0 a | 12.08 ± 0.97 ab | 0.0 ± 0.0 |
0.3 | 86.1 ± 2.8 bc | 3.34 ± 0.67 defg | 2.80 ± 0.47 a | 25.00 ± 1.69 abc | 93.3 ± 3.3 b | 10.00 ± 0.97 bcd | 0.0 ± 0.0 | |
0.57 | 100.0 ± 0.0 a | 5.24 ± 0.58 a | 2.08 ± 0.36 abc | 27.33 ± 3.52 ab | 91.7 ± 4.8 b | 11.36 ± 0.93 abc | 0.0 ± 0.0 | |
2.85 | 88.9 ± 2.8 bc | 3.84 ± 0.43 bcde | 2.18 ± 0.44 ab | 25.46 ± 3.70 abc | 100.0 ± 0.0 a | 12.00 ± 1.04 ab | 0.0 ± 0.0 | |
5.71 | 91.7 ± 4.8 b | 3.74 ± 0.57 bcdef | 1.60 ± 0.31 bc | 21.00 ± 3.20 abcd | 100.0 ± 0.0 a | 10.00 ± 0.93 bcd | 0.0 ± 0.0 | |
NAA | 0.057 | 100.0 ± 0.0 a | 3.84 ± 0.27 bcde | 1.75 ± 0.31 bc | 25.83 ± 1.77 abc | 100.0 ± 0.0 a | 9.42 ± 1.04 bcd | 0.0 ± 0.0 |
0.3 | 83.3 ± 4.8 c | 3.40 ± 0.26 cdefg | 1.18 ± 0.12 c | 24.18 ± 1.13 abcd | 100.0 ± 0.0 a | 10.64 ± 1.11 abcd | 0.0 ± 0.0 | |
0.57 | 100.0 ± 0.0 a | 4.90 ± 0.65 ab | 2.75 ± 0.35 a | 26.17 ± 3.02 abc | 83.3 ± 4.8 c | 9.73 ± 0.73 bcd | 0.0 ± 0.0 | |
2.85 | 88.9 ± 5.6 bc | 3.47 ± 0.33 cdefg | 2.20 ± 0.39 ab | 28.00 ± 2.39 a | 100.0 ± 0.0 a | 12.80 ± 1.01 ab | 0.0 ± 0.0 | |
5.71 | 100.0 ± 0.0 a | 2.28 ± 0.19 g | 1.92 ± 0.23 abc | 20.00 ± 1.67 bcd | 100.0 ± 0.0 a | 7.42 ± 0.73 de | 0.0 ± 0.0 |
The data represent mean ± SE of replicates (n = 3). Values in a same row carrying different letters are significantly different by Duncan’s multiple range test at p ≤ 0.05.
The thirty explants developing roots respond well to the applied acclimatization protocol. One month after the start of acclimatization, 97% of the plantlets appear healthy. Three months later, they are transplanted into larger pots and 100% develop flowers during the 2nd year, between June and August (
Surface sterilization of twigs from T. broussonetii acclimatized plants proved to be very difficult. Several sterilization methods using different products were tested. The method that consists of soaking first in Ca(ClO)2 for 30 minutes, then in 0.1% HgCl2 for 5 minutes and finally dipping three times in sterile distilled water for 5 minutes, proves to be the most adequate, with a total absence of bacterial contamination, 6.2% of fungal contamination, 37.5% of mortality and 52.1% of the explants tested have proliferated healthily.
The healthy and alive explants are multiplied by subculturing them on N30K + 0.46 µM Kin medium. The vitroplants obtained (
Plantlets mean length (cm) | 4.26 ± 0.19 |
---|---|
Mean number of buds | 19.30 ± 0.75 |
Mean number of shoots | 1.92 ± 0.11 |
Mean number of roots | 3.68 ± 0.34 |
The results obtained, following the germination of Thymus broussonetii achenes, show that the protocol used for decontamination was efficient, since the rate of fungal and bacterial contamination did not exceed 4%. The use of achenes for initiation of in vitro culture can avoid most of the decontamination problems, often associated with the nature of the starting plant part used [
Concerning the effect of macronutrients, we opted for N30K, since they ensured total regeneration of the explants, as well as the best multiplication of buds and shoots, with a low hyperhydricity rate. Also, Nobre (1996) [
Furthermore, the addition of some cytokinins at determined concentrations to the culture medium contributed to the improvement of the morphology of T. broussonetii vitroplants. Thus, we noted an improvement in shoots length in the case of 0.93 µM adenine and 0.93 µM DPU. Also, the number of buds and rooting rate increased after the addition of 0.93 and 2.32 µM adenine, 0.93 µM DPU, as well as 2.32 µM Kin. Besides, the addition of 2.32 µM adenine and 0.93 µM DPU to the culture medium contributed to better root multiplication.
The effect of cytokinins has been tested in other studies on the genus Thymus, especially in the multiplication phase. In this way, Bakhtiar et al. (2014) [
Moreover, the use of polyamines contributed, in most cases, to the improvement of the morphology of T. broussonetii vitroplants. Thus, a better shoot elongation occurred in the case of 2 µM putrescine, 2 µM spermidine and 2 µM spermine. Also, a better multiplication of buds was noted after adding 5 and 10 µM putrescine, 2 µM spermidine and 10 µM spermine. As well, we noticed a maximum shoot multiplication after adding 2 and 20 µM spermidine, as well as 10 and 20 µM spermine. In addition, we found better root multiplication, especially for 10 and 20 µM putrescine, 10 and 20 µM spermidine, as well as 10 and 20 µM spermine.
The first application of exogenous polyamines aimed to increase rooting and to improve the quality of roots in olives. When polyamines were combined with auxins, this promoted early rooting and increased the percentage of final rooting and the number of roots per explant [
For most combinations of 0.46 µM Kin and 0.46 µM adenine with auxins, better development of the root part was observed. Thus, the maximum of roots grew in the media added with the combinations 0.46 µM Kin + 2.85 µM IAA and 0.46 µM adenine + 2.85 µM IAA. In addition, the integration of auxins in the culture media optimized the development of the aerial part, especially in terms of bud multiplication. The most notable combinations are 0.46 µM Kin + 0.3 µM IBA and 0.46 µM adenine + 2.85 µM NAA.
Alone or combined with cytokinins, auxins were integrated into the culture media for the induction of roots and to optimize multiplication of Thymus vitroplants. Subsequently, Sáez et al. (1994) [
During the establishment of the in vitro culture from the acclimatized plants, two difficulties were encountered: high contamination rate and low survival of the non-contaminated explants. It was the same case for other species of the genus Thymus, for which the culture was carried out in vitro after sterilization of shoot tips and nodes, namely T. moroderi [
The present study represents a complete description, from in vitro achenes germination until acclimatization to ex-vitro conditions, for the micropropagation of T. broussonetii, very rare and endemic species in Morocco.
First, the protocol used to decontaminate achenes before in vitro germination was effective, since the rate of fungal and bacterial contamination did not exceed 4%. Then, the study of the effect of macronutrients resulted in the choice of N30K, since they ensure total regeneration of the vitroplants, as well as best multiplication of buds and shoots, with a low hyperhydricity rate.
After multiplication of cultures, the evaluation of the effect of seven cytokinins at three concentrations showed that 0.93 µM adenine, added to N30K medium, favored significantly the induction of buds and the elongation of explants of T. broussonetii.
In addition, the use of polyamines contributed, in most cases, to the improvement of the morphology of T. broussonetii vitroplants, namely a better multiplication of buds, shoots and roots in N30K + 10 µM spermine.
Then, after integration into the culture media of 0.46 µM Kin or 0.46 µM adenine, combined with auxins, better roots multiplication and an increase in the number of buds and the length of T. broussonetii explants were noted, particularly for 0.46 µM Kin + 2.85 µM IAA and 0.46 µM adenine + 2.85 µM IAA.
Finally, acclimatization was successfully carried out using vitroplants developing a good root system. One month after the start of acclimatization, 97% of T. broussonetii plantlets were healthy and 100% of the acclimatized plants developed flowers in the 2nd year between June and August.
Re-initiation of the in vitro culture was carried out from sterilized segments of twigs collected from the acclimatized plants of T. broussonetii, with 1 - 2 nodes on the medium N30K + 0.46 µM Kin and 52.1% of the explants healthily proliferated.
In practice, this protocol can ensure a sustainable supply of this important rare species in a limited time and space, regardless of seasonal variations and thus, meet the growing demand for its essential oil. Also, regenerated plants could serve as potential sources for the extraction of bioactive compounds.
At the end of this work, two prototypes were established. The first (
The authors declare no conflicts of interest regarding the publication of this paper.
El Ansari, Z.N., Boussaoudi, I., Benkaddour, R., Tahiri, H., El Oualkadi, A., Badoc, A., Martin, P. and Lamarti, A. (2020) Micropropagation of the Moroccan Endemic Plant Thymus broussonetii Boiss. with Aromatic-Medicinal Value and Conservation Concern. American Journal of Plant Sciences, 11, 913-938. https://doi.org/10.4236/ajps.2020.116067