Influence of growth regulators and sucrose concentrations on growth and rosmarinic acid production in calli and suspension cultures of Coleus blumei

Abstract

Rosmarinic acid, which is reported to have adstringent, antibacterial, antiviral and antioxidant activities, is one of the most prominent secondary compounds in Coleus blumei (Lamiaceae). Rosmarinic acid (RA) production in different hybrids of C. blumei was estimated by HPLC. Conditions for HPLC were as follows: column, 150 × 4.6 mm; solvent system, methanol −0.1% phosphate (45 : 55); flow rate, 0.9 mL/min; detection: 325 nm. Two out of four hybrids of C. blumei (hy1; hy2) contain better rosmarinic acid production (0.9 and 1.0% dry weight, respectively) and the leaves have the highest rosmarinic acid production, followed by stems and roots. The hydroxyphenylpyruvate reductase (HPPR) gene expression levels were analysed 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 was probably a result of the fact that the RA biosynthetic pathway was regulated by interactions of several enzymes necessary for biosynthesis. The explants from the hy1 leaves were used in subsequent studies on the effect of different growth regulators (2.0 mg L⁻¹ 6-benzyl-aminopurine (6-BA), different 2,4-dichlorophenxyaretic acid (2,4-D) and α-naphthaleneacetic (NAA) concentrations) and sucrose contents (1, 2, 3, 4, 5 or 6%) on culture growth and rosmarinic acid accumulation. On the effect of different growth regulators, the best result is obtained when the B5-medium supplemented is with 2.0 mg L⁻¹ 6-BA, 0.5 mg L⁻¹ NAA, 0.8 mg L⁻¹ 2,4-D and 2% sugar, and solidified with 0.8% agar. In this case, both growth index and rosmarinic acid accumulation reach a maximum, which is 49.7 and 25.3% (dry weight), respectively. The optimal medium for suspension culture growth contains 2.0 mg L⁻¹ 6-BA, 0.5 mg L⁻¹ NAA, 0.8 mg L⁻¹ 2,4-D, 600 mg L⁻¹ inositel and 2% sugar, and the rosmarinic acid production is 1.7% (dry weight). The rosmarinic acid accumulation is enhanced by the sucrose concentrations. When the sucrose content is 6%, the calli and cell culture accumulate 33.7% (dry weight) and 10.1% (dry weight) rosmarinic acid, respectively. Higher sucrose content (≥5%) has a negative influence on culture growth. B5-medium supplemented with 2.0 mg L⁻¹ 6-BA, 0.5 mg L⁻¹ NAA, 0.8 mg L⁻¹ 2,4-D, 4% sugar and 0.8% agar is best for both culture growth and rosmarinic acid accumulation. On the basis of the above results, we can establish a two-phase culture system by calli of C. blumei for the biosynthesis of RA. In the growth phase, the calli grow well, but with lower RA production in a modified B5-1A medium with 2% sucrose. The calli are then subcultured to B5-1A medium with 6% sucrose where the calli start to accumulate RA, but grow slowly.

Keywords:

• calli
• cell cultures
• HPLC
• HPPR
• semi-quantitative RT-PCR

Acknowledgements

We would like to thank the Laboratory of Biochemistry and Molecular Biology, College of Life Sciences, Shandong Agriculture University, for funding this study, and the College of Chemistry and Materials for their excellent technical assistance.