Improvement of shoot proliferation and comparison of secondary metabolites in shoot and callus cultures of Phlomis armeniaca by LC-ESI-MS/MS analysis

Abstract

Phlomis armeniaca Willd. is a medicinal plant in the Lamiaceae family endemic to Turkey. The present study describes efficient plant regeneration and callus induction protocols for P. armeniaca and compares phenolic profiles, total phenol and flavonoid contents, and free radical scavenging activity of in vitro-derived tissues. Stem node explants from germinated seedlings were cultured on Murashige and Skoog medium (MS) supplemented with 75 plant growth regulator (PGR) combinations. The highest shoot number per explant, frequency of shoot proliferation, and frequency of highly proliferated, green, compact callus were obtained on MS medium containing 0.25 mg L−1 thidiazuron (TDZ) and 0.25 mg L−1 indole-3-acetic acid (IAA). The best root formation was on MS basal medium (control). Methanol extract of leaves obtained from regenerants contained higher total phenol and flavonoid contents than the callus extract. The callus extract showed stronger free radical scavenging activity than leaves with IC50 [concentration inhibiting 50% of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical] values of 4.30 ± 0.08 and 2.21 ± 0.04 mg g−1 dry weight in leaves and callus, respectively. Apigenin, caffeic acid, p-coumaric acid, luteolin, rutin hydrate, vanillic acid, ferulic acid, salicylic acid, sinapic acid, and chlorogenic acid were detected by liquid chromatography–electrospray ionization multistage tandem mass spectrometry (LC-ESI-MS/MS) analysis in in vitro-grown leaves and callus tissue. Rutin hydrate, p-coumaric acid, and vanillic acid were found at approximately tenfold higher levels in callus than in leaves. This new micropropagation protocol, the first for P. armeniaca, could be used in industrial production for new herbal tea and germplasm conservation.