Callitriche cophocarpa as a biotechnological resource: optimizing in vitro cultures and elicitation to boost biologically active phenolic acids and phenylethanoid and iridoid glycosides

Callitriche cophocarpa Sendtn. (water starwort; Plantaginaceae) is an aquatic higher plant common in natural environments worldwide. The plant is used in folk medicine due to its ability to synthesize large amounts of secondary metabolites with a broad range of biological activity. This study used various types of tissue culture to produce safe plant material for medical purposes. The plants were subjected to elicitation through nitrogen deficiency, and their extracts were examined for biological activity. Qualitative phytochemical analysis of shoots in vitro showed the presence of phenolic acids and phenylethanoid and iridoid glycosides. Based on the results, cultures (stationary and agitated) grown in liquid medium were selected due to their greater effectiveness, i.e., plant growth and higher production of secondary metabolites, compared to a temporary immersion bioreactor. In the second part of the research, tissue cultures (stationary and agitated) were subjected to elicitation, where the stressor was a decreased level (¼N) or lack (0 N) of available nitrogen in the form of nitrate and ammonium ions. Compared to unelicited tissues, a greater accumulation of phenolic acids and phenylethanoid and iridoid glycosides was observed. However, when individual secondary metabolites within these classes were considered, some compounds showed reduced accumulation under elicitation with a medium containing a reduced concentration of N ions, which also resulted in decreased fresh weight growth. The antioxidant and antibacterial properties of extracts from both control and elicited plants were evaluated. The induction of synthesis of the phenolic acids and phenylethanoid and iridoid glycosides resulted in an increased reducing capacity and antibacterial properties against reference, antibiotic-resistant, and antibiotic-sensitive strains of Staphylococcus aureus. Callitriche cophocarpa grown in vitro is a rich source of valuable secondary metabolites. Elicitation by nitrogen deficiency activates biochemical pathways connected with phenolic compounds and phenylethanoid glycosides. The strong bactericidal properties of C. cophocarpa relate to a high level of accumulation of verbascoside and phenolic acids