An efficient in vitro regeneration system for Bellis perennis L. and comparison of phenolic contents of field-grown and in vitro-grown leaves by LC-MS/MS.

摘要

Bellis perennis L. (common daisy) is a medicinal plant in the family Asteraceae. It has been used in the treatment of common cold, wounds, rheumatism and inflammation in traditional medicine. A highly efficient and rapid regeneration system was developed for common daisy for the first time. Field-grown plants and in vitro-grown plantlets were compared in terms of their regeneration capacity. The field-grown plants were found to be much more responsive than the in vitro-grown plantlets when used as explant source. Leaf, pedicel and root explants obtained from field-grown plants, and leaf, petiole and root explants obtained from in vitro-grown plantlets were cultured on Murashige and Skoog's minimal organics medium (MSMO) with various plant growth regulator combinations. The best shoot proliferation was obtained with pedicel explants on media containing 0.5 mg l-1 thidiazuron (TDZ) and 0.5 mg l-1 indole-3-acetic acid (IAA). Plant regeneration was observed trough indirect organogenesis. Regenerated shoots were rooted on MSMO medium containing different concentrations of IAA, indole-3-butyric acid (IBA), naphthalene acetic acid (NAA), and 2,4-dichlorophenoxyacetic acid (2,4-D). IAA (1.0 mg l-1) was determined as the most effective auxin for rooting. Rooted explants were transferred to vermiculite in Magenta containers for acclimatization period. After 2 weeks, they were planted in to plastic pots containing potting soil and maintained in the plant growth room. Approximately 3 months after the transfer to room conditions, the flowering of the regenerated plants could be observed. Quantification of the chosen phenolics in dichloromethane (DCM) and methanol (MeOH) extracts of field-grown leaves and in vitro-grown leaves was performed using high-performance liquid chromatography coupled with tandem mass spectrometry (LC-ESI-MS/MS) method.