Investigation on primary and secondary metabolism of Datura innoxia Mill. tissue cultures.

摘要

Datura innoxia Mill, contains several secondary metabolites with pharmacological and toxicological importance, including more than 30 tropane- and pirrolidin-base alkaloids. Total amount of these alkaloids varies between 0.05%-0.5% in different organs of the plant. Main alkaloids are scopolamine and hyoscyamine which have parasympatholytic effect.; The aim of our experiments was to study different---genetically modified and non-transformed---in vitro cultures of D. innoxia . The growth (fresh and dry weight, dry matter content, growth value and pH of liquid culture media) and tropane alkaloid content (scopolamine, hyoscyamine and apoatropine, using HPLC technique) of in vitro plants, callus tissues and genetically modified, so called hairy root cultures were investigated. The transferred character of hairy root tissues obtained by Agrobacterium rhizogenes A4 microinjection was demonstrated by using polimerase chain reaction (PCR) and opine detection (paper electrophoresis). We have established that the total alkaloid content of hairy root clones (#410, #411 and #415, 0.23% hyoscyamine and 0.21% scopolamine) reached that of the root of the in vivo plants.; Growth and alkaloid content of in vitro cultures has been effected by several circumstances. We studied the effects of culturing in light, the sucrose and Mg2+ content of liquid MS basal medium on the growth, biomass production and tropane alkaloid content of the tissues. The presence of apoptotic processes in cells of different tissues (callus and root) accompanied with the shrinkage of the nuclei was investigated by TUNEL reaction.; To study the relationship among abiotic stress, growth, alkaloid metabolism and measurable endogenous HCHO concentration different molecules affecting transmethylation reactions have been administered to the culture media (dimedone, semicarbazide, aminoguanidine and hidralazine, 1ppm-1000ppm) of tissues.; The endogenous HCHO concentration of callus (4.6mug-27.0mug) and hairy root cultures (345.0mug) was determined by OPLC and HPLC techniques as formaldemethone. Following administration of dimedone and semicarbazide the apoptotic processes were investigated by TUNEL reaction.; The results obtained might help deepening our knowledge about the growth of D. innoxia in vitro cultures, tropane alkaloid metabolism, apoptotic processes in plant cells and the effect of abiotic stress (special chemical stress).