The development of clonal propagation and determination of phenolic profiles of in vitro-raised and field-raised leaves of Astragalus brachypterus Fischer (milkvetch) by LC-ESI-MS/MS analysis

Abstract

Astragalus brachypterus Fischer (milkvetch) is a perennial medicinal plant in the family Fabaceae. Astragalus species have been utilized as antiviral, antibacterial, antifungal, antioxidant, anticancer, hepatoprotective, wound healing, anti-diabetic, and diuretic for centuries in folk medicine. Objectives of this study were to determine an in vitro regeneration system for A. brachypterus and then to compare phenolic constituents of in vitro-raised and naturally raised leaves. Various plant explants (leaf, petiole, stem, and node) were taken from sterile seedlings that were obtained from germinated seeds. The explants were transferred to Murashige and Skoog (MS) medium supplemented with different cytokinins (benzyladenine (BA), kinetin (KIN), and thidiazuron (TDZ)), auxins (indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), naphthaleneacetic acid (NAA), and 2,4-dichlorophenoxy acetic acid (2,4-D)), and gibberellic acid (GA3) at various concentrations. Regeneration was obtained only with explants containing preexisting meristem (node). The highest mean number (29.16 ± 4.91) and frequency of shoot proliferation (100%) were obtained with nodal explants on MS medium containing 3.0 mg L−1 BA, 1.0 mg L−1 IAA, and 0.5 mg L−1 GA3 (P ≤ 0.05). Regenerated shoots were transferred to rooting medium and root formation was only observed with 1.0 mg L−1 IAA after 6 wk. At the second stage of the study, methanolic extracts of in vitro-raised and field-raised leaves of A. brachypterus were analyzed by using liquid chromatography-electrospray ionization multistage tandem mass spectrometry (LC-ESI-MS/MS). Although naturally raised leaves contained higher amount of caffeic acid, chlorogenic acid, gallic acid, isorhamnetin, myricetin, quercetin, rutin hydrate, and vanillic acid, in vitro-raised leaves had higher amount of luteolin and apigenin. It was observed that A. brachypterus leaves were remarkable source of rutin hydrate. This study provides highly efficient method for the conservation of germplasm and mass multiplication of A. brachypterus, and helps to reveal the quality of in vitro-grown plants in terms of phenolic content production for biotechnological and commercial purposes.