A great mullein ( Verbascum thapsus L.) was a famous species in Scrophulariaceae family. It was generally used as herbal medicine. Explants of V. thapsus (leaves and petioles) were cultured in vitro on Murashige and Skoog (MS) medium for shoot proliferation.Plantlet explants were cultured on MS medium supplemented with combination of Benzyl adenine (BA) and Naphthalene acetic acid (NAA) for callus induction. The best fresh and dry weight of callus formation was achieved using 0.5 mg/l BA. Quantitative analyses with High-performance liquid chromatography (HPLC) showed the content of phenols like Coumarin, Eugenol and Thymol were relatively low in leaves of mother plant, (10, 41, 310 ppm) respectively. The addition of different concentrations of amino acids as a precursor adding separately to the tissue culture medium led to raise the accumulation levels of phenolic compounds in callus tissue. Generally, the enhancement of accumulation depended on the type of amino acids and their concentration. The results showed 150 mg/l of Proline encouraged production of Comarin to 2752%, while 50 mg/l of Proline promoted accumulation of Eugenol to 290%. Whilst 150 mg/l of Tryptophan increased production of Thymol to 390%, in comparison with mother plant.
Verbascum is a genus of about 360 species of flowering plants in the Scrophulariaceae family. They are native to Europe and Asia, with the highest species diversity in the Mediterranean and Iranian Altoranih [
V. thapsus is rich plant in Phenolsso, it may be used wisely as an alternative medicine, it has been used as antiseptic, demulcent, narcotic, diuretic and has anti-microbial, anti-malarial, anti-oxidant and anti-inflammatory activities [
Plant phenols are secondary metabolites with diverse chemical nature and potential including: phenolic acids, flavonoids, tannins, coumarins, lignans and xanthones. Phenols are providing essential functions in the reproduction and the growth of the plants acting as defense mechanisms against pathogens, parasites, and predators [
Several endeavors have been recorded for enhanced synthesis of secondary metabolites in vitro cultures of different plant species, e.g. flavonoid productionin cultured tissue of Hydrocotyle bonariensis [
Many researchers refer that metabolic engineering seems a promising approach to improve the cells production. So this study is conducted to experience the effect of amino acids as precursor feeding on the enhancement of phenolic compound accumulation in vitro.
Common mullein plants, Verbascum thapsus L. were collected at flowering stage from Al-Sulaimania mountains, Iraq on April 2013. In vitro plantlets were established according to Turker et al. [
Leaves of mother plant and callus tissue were dried at 45˚C in oven for 48 hrs, and then kept at 4˚C. A 200 mg of dried plant material was taken for phenols extraction; the aliquots were shaken in 5 ml of methanol and incubated at room temperature for overnight. Plant material was centrifuged and filtered out using Whatman No.1, and 3 ml aliquots from each filtrate were filtered again using 0.22 µm syringe filters [
The RP-HPLC (Sykum-German) system with C18 reversed-phase column (250 × 4.6 mm) was used to detect of phenolic compound, Coumarin, Eugenol and Thymol. Acetonitrile and water in 75:25 (v/v) ratios was chosen as the mobile phase under a column temperature of 30˚C. The detection wavelength was set at 210 nm with a flow rate of 1.4 ml/min; the auto sampler injection volume was 20 µl. Quantitative method was analyzed by external standard. All standards were obtained from Sigma-Aldrich (USA).
All experiments were carried out in 15 replicates. The experiment results were statistically analyzed by ANOVA with Two-way Analysis of Variance test using MINITAB11 statistical program. Experiments were carried out with completely randomized block design and the differences between groups were compared using LSD at P ≤ 0.05.
Data recorded in
Callus inductions depended on kind and concentration of plant growth regulators, type of explant also played a considerable role in callus induction [
All treatments of amino acids (Proline, Glutamine, Tryptophan and Phenylalanine), gave different mass value of callus tissue. Generally, 150 mg/L of Glutamine added to MS medium done high response of fresh and dry weight reached to (3065 and 160 mg) respectively, (
BA (mg/l) | 2,4-D (mg/l) | Mean of BA | |||
---|---|---|---|---|---|
0.0 | 0.5 | 1 | 1.5 | ||
Callus fresh weight (mg) | |||||
0.0 | 688 | 654 | 379 | 217 | 484 |
0.5 | 3672 | 2409 | 2669 | 2664 | 2854 |
1 | 3128 | 2514 | 2517 | 2250 | 2603 |
1.5 | 3020 | 2773 | 2855 | 3611 | 3065 |
Mean of 2,4-D | 2624 | 2088 | 2105 | 2186 | |
LSD at P ≤ 0.05 | BA = 126 2,4-D = 126 Interaction = 252 | ||||
Callus dry weight (mg) | |||||
0.0 | 49 | 54 | 32 | 30 | 41 |
0.5 | 218 | 153 | 159 | 162 | 173 |
1 | 150 | 144 | 143 | 136 | 143 |
1.5 | 164 | 149 | 150 | 167 | 157 |
Mean of 2,4-D | 145 | 126 | 122 | 123 | |
LSD at P ≤ 0.05 | BA = 6.5 2,4-D = 6.5 Interaction = 13.1 |
Concentration of amino acid (mg/l) | Amino acid | |||
---|---|---|---|---|
Proline | Glutamine | Tryptophan | Phenyl alanine | |
Callus fresh weight (mg) | ||||
0.0 | 2548 | 2548 | 2548 | 2548 |
50 | 2758 | 2887 | 2857 | 2244 |
100 | 2193 | 2923 | 2951 | 1923 |
150 | 2112 | 3065 | 2709 | 2045 |
Mean | 2402 | 2855 | 2766 | 2190 |
No statistically significant differences at P ≤ 0.05 | ||||
Callus dry weight (mg) | ||||
0.0 | 141 | 141 | 141 | 141 |
50 | 159 | 153 | 157 | 139 |
100 | 138 | 156 | 165 | 138 |
150 | 137 | 160 | 158 | 141 |
Mean | 143 | 152 | 155 | 139 |
No statistically significant differences at P ≤ 0.05 |
The use of amino acids as an organic source of nitrogen was not usually desired in modern media, where a proper balance between
The results in
Although Eugenol was scarcely detected (15.0 ppm) in callus culture of control treatment, (
The results illustrated in
Attempts to induce the yield of the secondary product by supplying precursors or abiotic elicitors are found to be effective in many cases. Amino acids have been used as organic nitrogen source in in vitro cultures of several
Concentration of amino acid (mg/l) | Coumarin (ppm) | Percentage (%) of Increase compared with mother plants (+) | Percentage (%) of decrease compared with mother plants (−) | |
---|---|---|---|---|
Mother plants (leaves) | 10 | |||
Control (callus without amino acid) | 0.0 | 90.2 | +802 | |
Proline | 50 | 178.9 | +1689 | |
100 | 121.8 | +1118 | ||
150 | 285.2 | +2752 | ||
Glutamine | 50 | 113.2 | +1032 | |
100 | 70.0 | +600 | ||
150 | 41.1 | +311 | ||
Tryptophan | 50 | 70.7 | +607 | |
100 | 70.4 | +604 | ||
150 | 88.2 | +782 | ||
Phenyl alanine | 50 | 45 | +350 | |
100 | 122.3 | +1123 | ||
150 | 146.8 | +1368 |
Concentration of amino acid (mg/l) | Eugenol (ppm) | Percentage (%) of Increase compared with mother plants (+) | Percentage (%) of decrease compared with mother plants (−) | |
---|---|---|---|---|
Mother plants (leaves) | 41.0 | |||
Control (callus without amino acid) | 0.0 | 15.0 | −63 | |
Proline | 50 | 160.3 | +290 | |
100 | 57.8 | +40 | ||
150 | 0.0 | −100 | ||
Glutamine | 50 | 71.8 | +75 | |
100 | 68.0 | +65 | ||
150 | 74.9 | +82 | ||
Tryptophan | 50 | 56.0 | +36 | |
100 | 51.0 | +24 | ||
150 | 0.0 | −100 | ||
Phenyl alanine | 50 | 68.2 | +66 | |
100 | 57.6 | +40 | ||
150 | 0.0 | −100 |
Concentration of amino acid (mg/l) | Thymol (ppm) | Percentage (%) of Increase compared with mother plants (+) | Percentage (%) of decrease compared with mother plants (−) | |
---|---|---|---|---|
Mother plants (leaves) | 310.0 | |||
Control (callus without amino acid) | 0.0 | 519.4 | +67 | |
Proline | 50 | 255.8 | −17 | |
100 | 290.0 | −6 | ||
150 | 224.4 | −27 | ||
Glutamine | 50 | 272.8 | −12 | |
100 | 250.0 | −19 | ||
150 | 286.0 | −7 | ||
Tryptophan | 50 | 185.5 | −40 | |
100 | 699.7 | +125 | ||
150 | 1518.8 | +390 | ||
Phenyl alanine | 50 | 153.6 | −50 | |
100 | 296.2 | −4 | ||
150 | 763.7 | +146 |
species like sorghum [
This research was carried out in biotechnology research center/Al Nahrain University and funded by grants from Ministry of Higher Education and Scientific Research.
Abedaljasim M.Jasim Al-Jibouri,AshwaqS. Abed,Abdal-JabbarAbass Ali,Duha M.Majeed, (2016) Improvement of Phenols Production by Amino Acids in Callus Cultures of Verbascum thapsus L.. American Journal of Plant Sciences,07,84-91. doi: 10.4236/ajps.2016.71010