The basal production of secondary metabolites in medicinal plants is limited. One of the effective approaches that encourages plants to produce a remarkable amount of precious compounds is an application of elicitors. Our work was focused on the elicitation of Eschscholzia californica Cham. suspension cultures using various concentrations of MnCl2 (5; 10; 15 mg/L) with the aim of evaluating its effect on sanguinarine, chelerythrine, and macarpine production and gene expression of enzymes involved in the biosynthesis of mentioned secondary metabolites (BBE, 4′-OMT, CYP80B1) or in defense processes (LOX). Suspension cultures were exposed to elicitor for 24, 48, and 72 h. The content of alkaloids in phytomass was determined on the basis of their fluorescence properties. The relative mRNA expression of selected genes was analyzed using the ΔΔCt value method. PCR products were evaluated by melting curve analysis to confirm the specific amplification. Our results demonstrated that Eschscholzia californica Cham. cell suspension cultures evince sensitivity to the presence of MnCl2 in growth media resulting in the increased production of benzophenanthridine alkaloids and gene expression of selected enzymes. Manganese chloride seems to be a potential elicitor supporting natural biosynthetic properties in plant cell cultures and can be applied for the sustained production of valuable secondary metabolites.

Macarpine is a minor benzophenanthridine alkaloid with interesting biological activities, which is produced in only a few species of the Papaveraceae family, including Eschscholzia californica. Our present study was focused on the enhancement of macarpine production in E. californica suspension cultures using three elicitation models: salicylic acid (SA) (4; 6; 8 mg/L) elicitation, and simultaneous or sequential combinations of SA and L-tyrosine (1 mmol/L). Sanguinarine production was assessed along with macarpine formation in elicited suspension cultures. Alkaloid production was evaluated after 24, 48 and 72 h of elicitation. Among the tested elicitation models, the SA (4 mg/L), supported by L-tyrosine, stimulated sanguinarine and macarpine production the most efficiently. While sequential treatment led to a peak accumulation of sanguinarine at 24 h and macarpine at 48 h, simultaneous treatment resulted in maximum sanguinarine accumulation at 48 h and macarpine at 72 h. The effect of SA elicitation and precursor supplementation was evaluated also based on the gene expression of 4′-OMT, CYP719A2, and CYP719A3. The gene expression of investigated enzymes was increased at all used elicitation models and their changes correlated with sanguinarine but not macarpine accumulation.

Our recent study is focussed on the investigation of the influence of biotic (Botrytis cinerea, methyl jasmonate) and abiotic (cadmium and cobalt chloride) elicitors on Melissa officinalis L. (lemon balm) suspension cultures production properties. The plant material was treated with different concentrations of methyl jasmonate (10 and 100 μmol.l−1), with a phytopathogenic fungus hydrolyzate (Botrytis cinerea, 1 and 2 ml with glucose equivalent 24 μg.ml−1), cadmium and cobalt (II) chloride (both at concentrations 100 μmol.l−1 and 1 μmol.l−1). Elicited suspension cultures were harvested after 24, 48 and 72 h of elicitor treatment. The elicitation effect was evaluated based on hydroxycinnamic acid derivatives content expressed as rosmarinic acid in dose- and time-dependent manners. The Botrytis cinerea hydrolyzate exhibited the best effect on Melissa officinalis L. suspension cultures production properties among biotic elicitors (glucose equivalent 24 mg.ml−1, 1 ml, after 72 h of treatment). The hydroxycinnamic acid derivatives content increased in triplicate compared to non-elicited samples. Methyl jasmonate affected the suspension cultures production properties moderately at concentration 10 μmol.l−1. Chlorides salts of cobalt and cadmium stimulated phenolic compounds production effectively at concentrations 100 μmol.l−1. The presence of cobalt and cadmium ions in suspension cultures growth media increased the hydroxycinnamic acid derivatives content twofold and quadruple, respectively.