Eryngium maritimum L. is a valuable medicinal species, but since it is protected plant, collection from natural populations is forbidden. Therefore, establishing an efficient system for micropropagation of this species is desirable. To determine the optimal nutritional factors needed for shoot multiplication, root development and secondary metabolites accumulation, different media and plant growth regulators were tested. The highest plant regeneration efficiency (over 96 %), with 4.4 shoots per explant was induced on Murashige and Skoog (MS) medium supplemented with 1.0 mg L -1 benzyladenine (BA) and 0.1 mg L -1 indole-3-acetic acid (IAA). The in vitroregenerated shoots were rooted (83.3-100 %) and transferred to an experimental plot with 62 % efficiency. Flow cytometric analysis revealed no variation in nuclear DNA content in field-and in vitro-delivered plant material. Ultra high performance liquid chromatography (UHPLC) indicated that multiple shoots and roots from in vitroregenerated plantlets and adventitious root cultures maintained the production of rosmarinic (RA) and chlorogenic (CGA) acids and triterpenoid saponins found in the rosette leaves and roots of E. maritimum intact plants. UHPLC revealed a 12-fold increase of RA and CGA and 3.2-fold higher accumulation of triterpenoid saponins in roots of in vitro-derived plantlets in comparison to roots from fieldgrown plants. Adventitious root cultures allowed continuous growth of excised root in liquid media with or without exogenous auxins. The roots grown in liquid medium supplemented with 0.1 mg L -1 IAA showed higher (227-fold) phenolic acids accumulation than those without auxin. Obtained results confirmed that micropropagation is a useful strategy in the protection of endangered species and a renewable source of a high quality plant material for secondary metabolites production.

Phenolic acids and flavonoids, important bioactive compounds of polyphenols, play a significant role in plants; their impact, mainly as antioxidants, on human health have been of great interest in recent years. The genetically uniform shoots of Eryngium alpinum L. cultured in vitro, developed via axillary buds and regenerated from callus tissue, maintained on the media supplemented with various plant growth regulators, were subjected to the phenolic acids and flavonoids quantitative analysis applying HPLC-DAD technique. In vitro cultures give the opportunity to perform the phytochemical studies on the protected species without harvesting the plant material from natural environment. Depending on the hormonal supplementation, the biomass from the shoot cultures accumulated from 11.41 to 25 times more phenolic acids [the total content ranged from 158.66 to 1817.96 mg/100 g of dry weight (DW)] and from 6.8 to 11.8 times more flavonoids (the total content ranged from 29.30 to 51.30 mg/100 g DW) than the shoots from the soil-grown plant. The polyphenols present in the shoot cultures include two phenolic acids: 3,4-dihydroxyphenylacetic and caffeic, four depsides: caftaric, neochlorogenic, chlorogenic, isochlorogenic, and rosmarinic acids, and flavonoids: aglycone-isoquercetin and glucoside-quercitrin. Most of them (apart from chlorogenic and rosmarinic acids) were detected for the first time in this species cultured in vitro. To our best knowledge, the present report is the first one that discusses establishment of Eryngium alpinum L. in vitro cultures and the shoot and callus biomass capacity to produce two subgroups of polyphenols i.e. phenolic acids and flavonoids. Key message This is the first report on establishment of Eryngium alpinum in vitro cultures and the shoot and callus biomass capacity to produce polyphenols-phenolic acids and flavonoids.

An efficient micropropagation protocol for production of genetically uniform clones of Eryngium campestre L. was developed. To determine the effect of nutritional and hormonal factors on shoot and root development and bioactive compounds production, three variants of media differing in the content of macro-and micronutrients, as well as plant growth regulators of various types and concentrations were tested. The highest regeneration (100%), with over 13 shoots per explant, was induced on Murashige and Skoog (MS) medium with 1.0 mg l -1 benzyladenine (BA) and 0.1 mg l -1 indole-3-acetic acid (IAA). The in vitro derived shoots multiplied through axillary bud formation were rooted and transferred to an experimental plot with 78% frequency of survival. Flow cytometry showed no variation in nuclear DNA between the seedlings and micropropagated plants. Preliminary thin layer chromatography (TLC) analysis indicated that phenolic acids, saponins, flavonoids and acetylenes were present in plant biomass. Ultra high performance liquid chromatography (UHPLC) analysis revealed that shoots and roots from in vitro derived plants and root cultures maintained the ability to produce rosmarinic acid (RA), rosmarinic acid hexoside (RA-HEX) and chlorogenic acid (CGA). The highest phenolic acid content was detected in roots of in vitro regenerated plants. The extract from those roots expressed the highest inhibitory effect against bacteria Staphylococcus aureus, as well as dermatophytes Trichophyton mentagrophytes and T. rubrum.

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