J Plant Biotechnol 2020; 47(1): 53-65
Published online March 31, 2020
https://doi.org/10.5010/JPB.2020.47.1.053
© The Korean Society of Plant Biotechnology
Correspondence to : e-mail: zainabnejjar@gmail.com
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
The essential oil of
Actually,
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.
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
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.
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.
Three cytokinins: Kinetin (Kin), 1,3-diphenylurea (DPU) and Adenine (Ad) were evaluated on
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.
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.
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.
Twigs were cut from the acclimatized plants of
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.
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).
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 (%).
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.
The germination of
The results mentioned inTable 1 show that N30K macronutrients ensure total survival of
Table 1 Effect of three macronutrients on the micropropagation of
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).
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.
The integration of cytokinins into the culture media caused several changes, both in the aerial and in the root part (Table 2 andFig. 3).
Table 2 Effect of cytokinins on the micropropagation of
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.
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).
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).
Table 3 Effect of cytokinins combined with gibberellic acid on the micropropagation of
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.
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
Table 4 Effect of 0.46 µM/L Kin and auxin combinations on the micropropagation of
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.
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).
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
Surface sterilization of twigs from
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.
Table 6 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 |
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
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
The addition of DPU at 0.46 and 0.93 µM, as well as 0.93 Ad contributed to a better shoots elongation of
In fact, cytokinins are necessary for the multiplication of cultures. Some authors have reported the sensitivity of
The use of gibberellic acid and cytokinins combinations did not contribute to the improvement of
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
The combination of 0.46 µM Kin and auxins ensured in the majority of cases a better development of the root part of
Actually, cytokinins, occasionally combined with low concentrations of auxins, have been used during micropropagation, to multiply cultures of several species of the genus
Re-initiation of the in vitro culture of
The present study is the first about the micropropagation of
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.
J Plant Biotechnol 2020; 47(1): 53-65
Published online March 31, 2020 https://doi.org/10.5010/JPB.2020.47.1.053
Copyright © The Korean Society of 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
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
The essential oil of
Actually,
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.
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
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.
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.
Three cytokinins: Kinetin (Kin), 1,3-diphenylurea (DPU) and Adenine (Ad) were evaluated on
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.
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.
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.
Twigs were cut from the acclimatized plants of
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.
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).
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 (%).
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.
The germination of
The results mentioned inTable 1 show that N30K macronutrients ensure total survival of
Table 1 . Effect of three macronutrients on the micropropagation of
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).
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.
The integration of cytokinins into the culture media caused several changes, both in the aerial and in the root part (Table 2 andFig. 3).
Table 2 . Effect of cytokinins on the micropropagation of
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..
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).
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).
Table 3 . Effect of cytokinins combined with gibberellic acid on the micropropagation of
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..
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
Table 4 . Effect of 0.46 µM/L Kin and auxin combinations on the micropropagation of
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..
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).
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
Surface sterilization of twigs from
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.
Table 6 . 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 |
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
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
The addition of DPU at 0.46 and 0.93 µM, as well as 0.93 Ad contributed to a better shoots elongation of
In fact, cytokinins are necessary for the multiplication of cultures. Some authors have reported the sensitivity of
The use of gibberellic acid and cytokinins combinations did not contribute to the improvement of
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
The combination of 0.46 µM Kin and auxins ensured in the majority of cases a better development of the root part of
Actually, cytokinins, occasionally combined with low concentrations of auxins, have been used during micropropagation, to multiply cultures of several species of the genus
Re-initiation of the in vitro culture of
The present study is the first about the micropropagation of
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.
Table 1 . Effect of three macronutrients on the micropropagation of
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..
Table 2 . Effect of cytokinins on the micropropagation of
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..
Table 3 . Effect of cytokinins combined with gibberellic acid on the micropropagation of
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..
Table 4 . Effect of 0.46 µM/L Kin and auxin combinations on the micropropagation of
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..
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..
Table 6 . 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 |
D. Ramakrishna, G. Chaitanya, V. Suvarchala, and T. Shasthree
J Plant Biotechnol 2018; 45(1): 55-62Linh Minh Hong Tran
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