| Coleus blumei Benth. (Lamiaceae) is an ornamental plant, growing all over the world in an enormous number of different cultivars that vary in colour and shape of the leaves. It is used in India, Indonesia and Mexico as a medical plant. Rosmarinic acid, which was reported to have adstringent, antibacterial, antiviral and antioxidant activities, is one of the most prominent secondary compounds in Coleus blumei. The rosmarinic acid production in different hybrids(hy1; hy2; hy3; hy4)and tissues of Coleus blumei was estimated by HPLC (High Performance Liquid ChromatoGraphy) to analysis the rosmarinic acid distribution in plants. In order to obtain the best medium for growth index and rosmarinic acid accumulation, the explants from leaves were used in subsequent studies on the effect of different growth regulators (2.0mg/L 6-BA, different 2, 4-D and NAA concentrations) and sucrose contents (1, 2, 3, 4, 5 or 6%) on cultures growth and rosmarinic acid accumulation. For the establishment of suspension cultivation, the calli were then subcultured to above medium with different inositel concentrations. At the same time, we studied the effect of sucrose concentrations (1, 2, 3, 4, 5 or 6%) on cultures growth and rosmarinic acid accumulation. Finally, the hydroxyphenylpyruvate reducatase (HPPR) gene expression levels were analyzed by semi-quantitative RT-PCR. The results are as follows:1. The rosmarinic acid production in different hybrids and tissues of Coleus blumei was estimated by HPLC to analysis the rosmarinic acid distribution in plants. Conditions for HPLC were as follows: column, 150×4.6mm; solvent system, methanol-0.1% phosphate (45:55); flow rate, 0.9mL/min; detection: 325nm. The results vary significantly with different hybrids and tissues. Two out of four hybrids of Coleus blumei (hy1, mauve leaves with green borders; hy2, the dark red leaves with green borders) contain more rosmarinic acid production (0.946% and 1.010% dry weight, respectively) and the leaves of all the hybrids show higher rosmarinic acid production than roots and stems.2. The explants from the hy1 leaves were used in subsequent studies on the effect of different growth regulators (2.0mg/L 6-BA, different 2, 4-D and NAA concentrations) on cultures growth and rosmarinic acid accumulation. On the effect of different growth regulators, the best result is obtained when the B5-medium supplemented with 2.0mg/L6-BA, 0.5mg/LNAA, 0.8mg/L2, 4-D and 2% sugar, solidified with 0.8% agar. In this case, both growth index and rosmarinic acid accumulation reach to the maximum, which is 49.7 and 25.325 % (dry weight), respectively.3. The optimal medium for suspension cultures growth contains 2.0mg/L 6-BA, 0.5mg/L NAA, 0.8mg/L 2, 4-D, 600mg/L inositel and 2% sugar, and the rosmarinic acid production is 1.738% (dry weight).4. The rosmarinic acid accumulation is enhanced by the sucrose concentrations. When the sucrose content is 6%, the calli and cell cultures accumulate 33.732% (dry weight) and 10.145 % (dry weight) rosmarinic acid, respectively. Higher sucrose contents (≧5%) have a negatively influence on cultures growth. B5-medium supplemented with 4% sugar is best for both cultures growth and rosmarinic acid accumulation. In the forward experiments, we can establish a two-phase culture system by calli of Coleus blumei for the biosynthesis of rosmarinic acid. In the growth phase, the calli grow well but with lower rosmarinic acid production in a modified medium with 2% sucrose. The calli are then subcultured to medium with 6% sucrose where the calli start to accumulated rosmarinic acid.5. HPPR gene expression levels were analyzed by semi-quantitative RT-PCR. Hy3 shows highest level of HPPR gene expression out of four hybrids on genotype-specific patterns and stems represent the highest level of HPPR gene expression among leaves, roots and stems. This is probably that the rosmarinic acid biosynthetic pathway is regulated by interactions of several enzymes necessary for biosynthesis. |
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