Micropropagation and HPLC-DAD, UPLC MS/MS analysis of oenothein B and phenolic acids in shoot cultures and in regenerated plants of fireweed (Chamerion angustifolium (L.) Holub)

Abstract

In this study, a micropropagation protocol using nodal explants from in vitro grown plants of Chamerion angustifolium (L.) Holub was developed and analysis of oenothein B and selected phenolic acids in shoot cultures was performed for the first time. For shoot induction and multiplication Murashige and Skoog’s (MS) basal medium supplemented with 2-isopentenyladenine (2iP), zeatin (Z) and 6-benzyloaminopurine (BAP) was used. 2iP was the most responsive in terms of promoting shoots per explant with the maximum (6.57 ± 1.14) recorded at a concentration of 2.0 mg L−1 after 6 weeks of culture. After two subcultures the multiplication rate was increased up to 19 shoots per explant on medium with 2iP (1.0 mg L−1). To prevent tissue browning, ascorbic acid and casein hydrolysate were added to the induction medium, resulting in a reduction of browning by 30%. The rooted plantlets were successfully transferred to soil and acclimatized with 97% frequency. Quantitative and qualitative assessments of oenothein B and phenolic acid contents in in vitro regenerated shoots as well as in ex vitro plants were performed using high-performance liquid chromatography with a diode-array detector (HPLC-DAD) and ultra-performance liquid chromatography–tandem mass spectrometry (UPLC MS/MS) methods. Oenothein B (1.62‒4.55 g 100 g−1 DW), ellagic acid, gallic and caffeic acids were identified in in vitro regenerated plants. The results of this study confirm that the oenothein B-producing plantlets can be obtained using the micropropagation method with axillary shoots being a valuable source of oenothein B and phenolic acids.