1 ABSTRACT Charles University in Prague Faculty of pharmacy in Hradec Králové Department of pharmacognosy Student: Václav Předota Supervisor: PharmDr. Marie Kašparová, PhD. Title of diploma thesis: Plant tissue culture of Juniperus virginiana The derivation of callus cultures from leaves of Juniperus virginiana (varieties Hetzii, Glauca and Grey Owl) and determination of their growth curves were studied in this work. The cultures were cultivated at the temperature of 25 řC and light period of 16 hours light/8 hours dark on the Schenk and Hildebrandt medium with the addition of 3.0 mg.l-1 α-NAA and 0.2 mg. l-1 kinetin. It is clear from the growth curves, that all three varieties reached their maximum in growth on 35th day of the cultivation. The best results were achieved by variety Glauca.

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').

Master of Science / Department of Horticulture, Forestry, and Recreation Resources / Cheryl R. Boyer / Pine bark (PB) has been the principal component of nursery crop substrates in the United States for more than 60 years. Substrate material used for the purpose of growing ornamental plants in the Great Plains is generally shipped a great distance, primarily from the Southeastern U.S., due to a lack of pine plantations in this region. Eastern redcedar (Juniperus virginiana L.; ERC), an aggressively weedy tree species, has been identified as a possible alternative to PB for nursery substrates. The objective of this thesis was to evaluate the establishment of Miscanthus sinensis N.J. ‘Little Kitten’ (dwarf maiden grass), Rosa (L.) ‘Radtkopink’ (Knockout® rose), Ilex glabra (L.) A. Gray ‘Compacta’ (holly), Ulmus parvifolia Jacq. ‘Emer II’ (Allee® lacebark elm), Sedum telphium L. ‘Autumn Joy’ (sedum), Hosta Tratt. ‘Sum and Substance’ (hosta), and Hemerocallis L. ‘Charles Johnston’ (daylily) in three substrate mixes. These substrate mixes consisted of 80% PB: 20% sand, 80% ERC: 20% sand, and 40% PB: 40% ERC: 20% sand. At the end of the production phase differences in growth were observed in maiden grass, holly, lacebark elm, and sedum. At the end of the landscape establishment phase, no differences in growth were observed in any species except holly and hosta. To attempt to overcome the sub-optimal physical properties of ERC (high air space and low container capacity), cyclic irrigation was used to evaluate growth of Sedum spectabile Boreau ‘Autumn Fire’ (sedum), Rudbeckia fulgida Aiton ‘Goldstrum’ (black-eyed susan), and Hibiscus moscheutos L. ‘Luna White’ (hibiscus) in the same 3 substrates. The objective of this study was to evaluate the affect of irrigation frequency (1x, 2x, 3x, or 6x per day) and substrate ERC content on plant growth. Sedum had the greatest growth index (GI) shoot dry weight in PB and when irrigated 1x and 2x per day had the greatest root dry weight regardless of substrate. Irrigation frequency had no significant difference for GI and shoot dry weight Black-eyed susan had the greatest GI and shoot dry weight when grown in PB. Irrigation frequency only had an impact on shoot dry weight of black-eyed susan; plants irrigated 1x, 2x, and 3x per day had the greatest growth. Hibiscus had greatest growth in PB: ERC mix. Irrigation frequency had no impact on growth of hibiscus. Cyclic irrigation does not overcome low water holding capacities in these three species. Overall, ERC can be used as a partial replacement to PB (up to 50%). However, further research evaluating effects of different fertility regimes on ERC substrate is warranted.

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

Anthropogenic change in the Great Plains of North America within the past two centuries has facilitated extensive woody encroachment by eastern redcedar (Juniperus virginiana, hereafter redcedar). Conversion of grassland to redcedar woodland occurs rapidly as a result of the interaction between changing disturbance and land use patterns. In addition, redcedar encroachment causes compositional shifts in grassland floral and faunal assemblages, and may alter abiotic factors such that grassland restoration efforts are limited. The objectives of this study were to: 1) track vegetation and faunal assemblages in grasslands along a gradient of redcedar encroachment, 2) assess the capacity for experimental tree removal to promote re-colonization of grasslands by associated flora and fauna, and 3) examine the influence of scale on spatial relationships between small-mammal abundance and redcedar cover. During the first phase of my study, I examined herbaceous vegetation, woody vegetation, and small-mammal assemblages at grassland sites along a redcedar encroachment gradient in north-central Oklahoma. I noted hump-shaped trends in the capture rate, species diversity, and species evenness of small mammals along the redcedar encroachment gradient. In addition, higher levels of encroachment were associated with compositional shifts from grassland- to woodland-associated small mammals. Characteristics of the small-mammal assemblage along the gradient corresponded to increases in redcedar cover and the frequency of episodic management events within the past two decades. Experimental redcedar removal during the second phase of my study generally increased vegetation and faunal diversity in 2 years following treatment, and treatment sites having the highest pre-treatment levels of redcedar cover exhibited the greatest responses to tree removal. The results of my study suggested that within the time frame I examined, redcedar encroachment had altered the biotic characteristics of this system, but did not facilitate abiotic shifts capable of constraining rapid grassland recovery. Tree removal also appeared to modulate the effects of consecutive drought and flood years on small-mammal diversity during post-treatment. During the third phase of my study, I examined the strength of association between differences in percent redcedar cover and spatial abundance patterns of 4 common small-mammal species. I examined small-mammal abundance at three relatively small spatial scales (38, 154, and 616 m2) to determine how redcedar-mammal associations were stronger with increases in spatial scale. In addition, I determined whether the influence of spatial scale on redcedar-mammal associations varied along a gradient of redcedar encroachment. The strength of redcedar-mammal associations increased with spatial scale, but was strongest at sites having the lowest levels of encroachment. These results corresponded to variation in the spatial distribution of redcedar cover and compositional differences in the small-mammal species assemblage along the encroachment gradient. Studies examining the effects of woody encroachment will extend our understanding of successional processes and ensure that appropriate management is implemented in the conservation of these imperiled grassland ecosystems.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacognosy Student: Vendula Damborská Supervisor: PharmDr. Marie Kašparová, Ph.D. Title of diploma thesis: The Optimization of the growth of the plant tissue culture of Juniperus virginiana The main goal of this diploma thesis lies in the optimization of cultivation medium composition for the tissue culture of Juniperus virginiana (varieties 'Hetzii', 'Glauca', 'Grey Owl'). The best results of all tested media were achieved by using Schenk and Hildebrandt medium with an addition of α-NAA (3.0 mg.l-1 ), kinetin (0.2 mg.l-1 ) and ascorbic acid (15.0 mg.l-1 ). The cultures were cultivated at the temperature of 25 řC and light period of 16 hours light/8 hours dark. The growth curve for the tissue culture of Juniperus virginiana 'Glauca' was set for 2 different cases: with and without the addition of phenylalanin (biogenetic precursor of phenylpropans biosynthesis). The maximal growth was reached on 35th day of the cultivation in both cases, phenylalanin increases the growth rate by 29.65 %.

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'...

The growth rings of eastern redcedar (Juniperus virginiana L.) often contain a high frequency of false intra-annual growth bands, which complicates the dendrochronology of this species. However, exactly dated false rings replicated among many trees can reflect major weather changes during the growing season. Sixty-one trees from two sites (Oklahoma and Kansas) were dated and used to compile replicated chronologies of false rings at both locations extending from AD 1700–2000. False-ring events during the modern instrumental era were compared with the daily weather data from nearby stations. Significant false-ring events occurred at both locations during years that experienced a dramatic late-growing season weather reversal, when an extended period of high temperatures and drought was followed by prolonged cool and wet conditions. Synoptic weather maps for these events indicate that all ten replicated false-ring events in the instrumental era occurred during the highly unseasonable penetration of a cold front into the region. However, none of the significant false-ring events occurred in the same year at both sites. These separate false-ring chronologies indicate that there may be phenological differences in the timing of radial growth in redcedar between Kansas and Oklahoma, and that the weather conditions responsible for false-ring formation often occur at the mesoscale and do not often impact central Kansas and northcentral Oklahoma simultaneously.