1 ABSTRACT Charles University in Prague Faculty of pharmacy in Hradec Králové Department of pharmacognosy Student: Kateřina Šandová Supervisor: PharmDr. Marie Kašparová, PhD. Title of diploma thesis: Plant tissue cultures as a potential source of phenylpropanoids II. This diploma thesis deals with the monitoring of jasmonic acid biotic elicitation impact on the production of podophyllotoxin in Juniperus virginiana suspension culture. The monitoring was carried out in two varieties of J. virginiana: var. 'Glauca' (7th and 19th passage) and var. 'Hetzii' (7th passage). The elicitation of the suspension culture was performed in 4 jasmonic acid concentrations: 0.005 mmol/l, 0.05 mmol/l, 0.5 mmol/l, 5 mmol/l. Samples were taken after 6, 24, 48 and 168 hours of elicitation. The best podophyllotoxin production increase was observed in the suspension culture of Juniperus virginiana var. 'Glauca' (7th passage). To achieve this result, the concentration of elicitor was 5 mmol/l and elicitor was applied for 168 hours. The podophyllotoxin production percentage was 0.067% and production increased by 179% over the control. Keywords: Juniperus virginiana, suspension cultures, podophyllotoxin, elicitation

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacognosy Student: Jiří Olšar Supervisor: doc. PharmDr. Tomáš Siatka, CSc. Title of diploma thesis: Podophyllotoxin in eastern red cedar (Juniperus virginiana) This thesis describes the optimisation of the extraction process for quantity determination of podophyllotoxin in the leaves of Juniperus virginiana L. This determination was conducted through three different methods: the extraction in ultrasound bath, the extraction in the orbital shaker and the maceration in cold environment of 4 řC. Extractions were affected by various factors which were monitored, for instance time of extraction, temperature, and extraction solvents. The most efficient method appears to be the extraction in ultrasound bath with the use of methanol or a combination of phosphate buffer with ethyl-acetate.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacognosy Student: Lenka Srbová Supervisor: PharmDr. Marie Kašparová, Ph.D. Title of diploma thesis: Tissue culture of Juniperus virginiana L. as a promising source of podophyllotoxin. The diploma thesis deals with the cultivation of Juniperus virginiana tissue cultures. A growth was observed at Juniperus virginiana two years old culture (variety 'Glauca' and 'Grey Owl') after adding various concentrations of phenylalanine (1 mmol.l-1, 10 mmol.l-1 and 100 mmol.l-1 ) at selected time intervals. The results show that the highest increase in callus fresh weight was detected at Juniperus virginiana variety 'Grey Owl', particularly on the 14th day after adding 10 mmol.l-1 phenylalanine. Suspension culture was successfully derived from the Juniperus virginiana two years old callus culture (variety 'Glauca').

Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmacognosy Candidate: Gabriela Dohnalová Supervisor: PharmDr. Marie Kašparová, Ph.D. Title of diploma thesis: Plant tissue cultures as a potential source of phenylpropanoids I Explant cultures are the source of plant secondary metabolites. However, the production of secondary metabolites is usually low in explant cultures. Production can be increased by a method called elicitation. The basic prerequisite for successful elicitation is, among other things, finding a suitable elicitor, its concentration and optimal time of elicitor action on plant culture in vitro. The aim of this study was to observe the influence of lead chloride and ferrous sulfate (in four concentrations) on the production of podophyllotoxin in the suspension cultures of Juniperus virginiana L. (variety Hetzii and Glauca). The culture was cultured on Schenk and Hildebrandt nutrient medium with addition of 3.0 mg.l-1 α-naphthylacetic acid, 0.2 mg.l-1 kinetin and 15 mg.l-1 ascorbic acid. Cultivation proceed in 25 řC temperature and 16 hours light/8 hours dark period. Subsequently, the determination of the content of podophyllotoxin by HPLC was performed. Juniperus virginiana L. variety Hetzii produces a higher amount of podophyllotoxin than the Glauca...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacognosy Student: Nikola Urbanová Supervisor: PharmDr. Marie Kašparová, Ph.D. Title of diploma thesis: The study of secondary metabolites in plant tissue cultures II The aim of this work was to determine the effect of the potential elicitor chitosan on production of podophyllotoxin in callus and suspension cultures of Juniperus virginiana Glauca variety and suspension cultures of Juniperus virginiana Hetzii variety. Schenk and Hildebrandt medium supplemented with α-naphtylacetic acid (3.0 mg/l), kinetin (0.2 mg/l) and ascorbic acid (15.0 mg/l) was used for the cultivation. Chitosan solutions of various concentrations (0.001; 0.01; 0.1; 1 g/100 ml) were affecting the cultures for 6, 24, 48 and 168 hours. The content of chitosan was determined by HPLC. Higher values of podophyllotoxin content were measured in cultures derived from the Glauca variety. The best chitosan effect on podophyllotoxin production was manifested in callus cultures after 24 hours application of 0.001 g/100ml concentration. A podophyllotoxin content of 0.210 % was determined, which was about 320 % higher in comparison with the control. The maximum content (0.140 %) in suspension culture was induced by 24-hours application of a 0.1 g/100 ml concentration;...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacognosy Student: Veronika Vargovčíková Supervisor: PharmDr. Marie Kašparová, Ph.D. Title of diploma thesis: Production of podophyllotoxin by plant tissue cultures of Juniperus virginiana Key words: Juniperus virginiana, callus cultures, suspension cultures, podophyllotoxin Elicitor and precursor adding method can be used to enhance the production of secondary metabolites in explantate cultures. This master's thesis is focused on the evaluation of the effects of biotic elicitor (salicylic acid) and phenylpropanoid metabolism precursor (cinnamic acid) on podophylotoxin production in Juniperus virginiana (var. 'Glauca' and 'Hetzii') suspension cell culture. Suspension cultures were cultivated on Schenk and Hildebrandt medium at 25řC and circadial period of 16 hours light and 8 hours dark. Medium contained 15 mg.l-1 ascorbic acid and growth regulators 3.0 mg.l-1 , α-naphthaleneacetic acid, 0.2 mg.l-1 kinetin. The effects of salicylic acid aqueous solution 0.01 mmol.l-1 , 0.10 mmol.l-1 , 1.00 mmol.l-1 , 10.0 mmol.l-1 and cinnamic acid 60% alcohol solution 0.10 mmol.l-1 , 1.00 mmol.l-1 , 10.0 mmol.l-1 , 100 mmol.l-1. were examined after 6, 24, 48 and 168 hours. Podophylotoxin content was evaluated using HPLC method. In 'Glauca'...