The effect of substituted amides of pyrazine-2-carboxylic acids on flavonolignan production in Silybum marianum culture in vitro Lenka Tumova 1 , Katerina Gallova 1 , Jana Rimakova 1 , Martin Dolezal 2 , Jiri Tuma 3 1 Department of Pharmacognosy, School of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic, e-mail: tumova@faf.cuni.cz 2 Department of Pharmaceutical Chemistry and Drug Control, School of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic 3 Department of Biology, Faculty of Education, University of Hradec Kralove, Vita Nejedleho 573, 500 03 Hradec Kralove, Czech Republic Key words: abiotic elicitors, amides of carboxylic acids, culture in vitro, flavonolignan, milk thistle Abstract In vitro plant tissue and cell cultures were used to study herbi- cide effects on growth, selected metabolic activities and other phenomena. The effect of abiotic elicitors, two newly synthe- sized substituted amides of pyrazine-2-carboxylic acids (syn- thesized at the Department of Pharmaceutical Chemistry and Drug Control, School of Pharmacy in Hradec Kralove), on the flavonolignan accumulation in callus and suspension culture of Silybum marianum (L.) Gaertn. was investigated. The com- pounds markedly influenced production of flavonolignans in an in vitro culture. Particularly after the elicitation with a solu- tion of compound 3-methylamide 5-tert-butylpyrazine- -2-carboxylic acid at a concentration of 3.71x10 -7 mol·l -1 and within 72 h of elicitation, an increase in flavonolignan produc- tion by 893 % in suspension culture versus control took place. The flavonolignan accumulation in callus culture after the elic- itation with a solution of 5-brom-2-hydroxyphenylamide of 5-tert-butyl-6-chloropyrazine-2-carboxylic acid was also in- creased by about 1039 % (24 hour elicitation and concentration of 2.59x10 -4 mol·l -1 ). List of abbreviations: DNOC 2-methyl-4,6-dinitrophenol 2,4-D - 2,4-dichlorophenoxyacetic acid HPLC - high-performance liquid chromatography NAA - α-naphthaleneacetic acid Introduction Plant tissue and cell cultures provide model sys- tems for the study of various molecular, physiologi- cal, systemic and genetic problems. These systems were used in studies with herbicides and other xenobiotics (Dwight Camber and McDonald 1989). Herbicides are generally considered to be inhibi- tors, thus different inhibitory responses have been studied in various culture systems. Callus tissues, particularly of tobacco, have been studied to test various compounds for cytokine ac- tivity (Linsmaier and Skoog 1965, Schmitz et al. 1972). It presents a convenient bioassay for sub- stances that inhibit cell division because differenti- ation is minimized and cell division is the primary morphological event. In a study of the response of tobacco callus to 11 different dinitroanilines, 357 ACTA PHYSIOLOGIAE PLANTARUM Vol. 27. No. 3B. 2005: 357-362