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The scientific monograph covers the botanical classification of the genus Helleborus L. and the Christmas rose species (Helleborus niger L.), which has undergone numerous changes throughout its botanical history. The Christmas rose is becoming increasingly important in the ornamental plant market. Its characteristic flowering in the coldest months of the year represents a great advantage over many other species of ornamental plants. As the Christmas rose does not require high temperatures to start flowering, its cultivation can be considered affordable and environmentally friendly. The supply of attractive varieties is likely to be one of the key factors that will affect its popularity in the future. The number of genetically improved (bred) varieties is limited on the market, so selective breeding of Christmas rose will become inevitable in the future. The presented results of pollination analyses, related to insect activity, are important for breeding. Observations indicate that the Christmas rose is an entomophilous and a predominantly allogamous species. Knowledge of plant ecology and cultivation technology will also be exceptionally important. In intensive cultivation, the production technology for Christmas roses is still to a large extent incomplete. The monograph includes scientific findings on the ecology and biology of the Christmas rose, its reproductive processes, and the production technology for potted plants. The monograph also presents the results of the study on the effects of beneficial microorganisms (bacteria and endomycorrhizal fungi) on the growth and development of seedlings from in vivo and in vitro environments. The results of the study indicate that biotisation is a useful technology and that inoculation with specific microorganisms has a beneficial effect on growth.

A new bufadienolide (1), two new bufadienolide glycosides (2 and 3), a new ecdysteroid (4), and four known compounds (5–8), were isolated from the whole plants of Helleborus niger L. (Ranunculaceae). The structures of the new compounds (1–4) were determined by spectroscopic analysis, including 2D NMR spectral data, and hydrolytic studies. Compounds 1–6 showed cytotoxicity against HL-60 human leukemia cells, A549 human lung adenocarcinoma cells, and SBC-3 human small-cell lung cancer cells, with IC50 values ranging from 0.0055 to 1.9 µM. HL-60 cells treated with either 3 or 4 showed apoptosis characteristics, such as nuclear chromatin condensation, accumulation of sub-G1 cells, and activation of caspase-3/7.

Malignant pleural mesothelioma (MPM) is a rare cancer with a dismal prognosis. Viscum album extracts (VAE) have strong immune stimulatory properties, cytotoxic effects, can downregulate cancer genes and inhibit angiogenesis. VAE are often used as an adjunct treatment in cancer patients but have rarely been investigated in MPM. Helleborus niger extracts (HNE) have been used in anticancer therapy since antiquity, and also show tumor specific cytotoxic effects. We present a case of a 64-year old woman with epithelioid MPM of the right chest with node involvement (T2N1M0, stage III). Deciding against the recommended radio-chemotherapy, surgery and pleurodesis, she opted for an integrative treatment approach and was treated with VAE and HNE. After 6 weeks’ treatment, the pleural and nodal MPM manifestations were reduced by about 15%. Subsequent tumor growth was slow, and the patient remained in good health, enabling her to remain physically active until shortly before her death 56 months after the initial diagnosis. This is a rare case of an MPM patient not receiving any standard anticancer treatment; it still shows an extraordinary long survival and good performance status. We presume that VAE and HNE might had an impact on this clinically relevant outcome and therefore should be further investigated in MPM.

Background: Helleborus niger L (Ranunculaceae) is used Ayurvedic and Unani systems and other herbal medicine systems. The roots of H. niger have a good medicinal value. Aims: To conduct a pharmacognostical and phytochemical study of H. niger. Materials and Methods: The pharmacognostical studies on roots including parameters such as taxonomical, macroscopic, microscopic characters, physico-chemical, ultra-violet analysis and phytochemical studies are established. Results: Macroscopically, the roots are brownish-black in colour, cylindrical in shape, feeble odour, slightly acrid taste with irregularly branched. Microscopically the root showed the presence of epidermis, air-chambers, fissure periderm, periderm, inner cortex, pith, phloem, xylem, vessels and xylem vessels. Microscopic examination of the powder showed the presence of parenchyma cells, parenchyma mass, periderm, cell inclusion, laticifer, lateral wall pith, perforation, xylem bundle and xylem elements. Ultra-violet and ordinary light analyses with different reagents were conducted to identify the drug in powder form. Physico-chemical evaluation established, Ash values - Total, acid insoluble, water soluble and sulphated ash values were 7.3%, 4.1%, 3.7% and 5.2%, respectively. Extractive values - Alcohol soluble, water soluble and ether soluble extractive values were 22.8%, 7.4% and 5.6%, respectively. Loss on drying was 3.3%. Preliminary phytochemical screening showed the presence of carbohydrate, glycoside, saponins, flavonoid, phytosterols, tannins and phenolic compounds. Conclusions: The results of the study can serve as a valuable resource of pharmacognostic and phytochemical information. This will serve as appropriate, standards for discovery of this plant material in future investigations and applications and also contribute towards establishing pharmacopoeial standards.

Background The therapeutic use of Helleborus niger L. is manifold due to its specific phytochemical composition. Two compound groups, the ranunculin derivates including protoanemonin and the steroidal saponins, are also associated with toxicity (genotoxicity, disintegration of membrane structures). Therefore, in vitro investigations were performed on safety aspects of a Helleborus niger aqueous fermented extract (HNE). In addition its therapeutic potential against various cancer cell lines was assessed to gain insight into the respective mechanisms of action. Methods To evaluate the safe use of HNE, Ames and hemolytic tests were carried out. Two angiogenesis assays in 2D and 3D design were conducted to assess the anti-angiogenetic potential, for which human umbilical vein endothelial cells (HUVEC) were chosen. A panel of tumor cell lines was used in 2D and 3D proliferation assays as well in the migration- and invasion-assay. All investigations were performed with HNE compared to reference substances. The 2D proliferation assay was additionally performed with isolated compounds of HNE (characteristic steroidal saponins). Results HNE did not exhibit any genotoxic potential. Concentrations up to 10 μl/ml were classified as non-hemolytic. HNE exerted anti-angiogenetic effects in HUVEC and anti-proliferative effects in five cancer cell lines, whereas hellebosaponin A and D as well macranthosid I did not show comparable effects neither singly nor in combination. Due to the inherent instability of protoanemonin in isolated form, parallel investigations with protoanemonin could not be performed. HNE (600–1000 μg/ml) inhibited the migration of certain cancer cells by > 80% such as Caki-2, DLD-1 and SK-N-SH. Conclusion HNE exhibit neither genotoxic nor hemolytic potential. The present investigations verify the anti-angiogenetic effects on HUVEC, the anti-proliferative effects and migration-inhibiting properties on tumor cells. The lower effect of the relevant steroidal saponins compared to the whole extract underlines the fact that the latter is more effective than a blend of isolated pharmacologically active components. Electronic supplementary material The online version of this article (10.1186/s12906-019-2517-5) contains supplementary material, which is available to authorized users.

Raziskava v diplomskem delu je bila opravljena v letih 2016 in 2017 na treh sortah črnega teloha (Helleborus niger): ´Praecox´, ´Happy day´ in ´Happy day Royal´. Namen diplomskega dela je bil proučiti morfološke lastnosti teh treh sort, ki so selekcionirane za pridelavo v loncih. Rastline so bile posajene v substrat Klasmann Tonsubstrat in postavljene na plato za gojenje rastlin v loncih v delu, ki je bil dodatno senčen s senčilnimi mrežami. Ugotovili smo, da med proučevanimi sortami črnega teloha obstajajo statistično značilne razlike v morfoloških lastnostih. Povprečno število razvitih listov proučevanih sort se je statistično značilno razlikovalo le ob prvi in drugi meritvi, ob zadnji meritvi pa je bilo število listov enakomerno. Sorta ´Happy day Royal´ je razvila značilno večje liste in tvorila statistično značilno več cvetov v primerjavi s sortama ´Praecox´ in ´Happy day´. The research for this thesis was conducted in the years 2016 and 2017 on three different species of Christmas Rose (Helleborus niger): 'Praecox', 'Happy day' and 'Happy day Royal'. The purpose was to investigate morphological characteristics of three species of Christmas Rose which are selected for pot production. The plants were planted in Klasmann Tonsubstrat and placed on a crate for growing plants in pots that are additionally shadowed with shade nets. The research indicates, that there are statistically significant differences in morphological characteristics among the investigated species of Christmas Rose. The average number of developed leaves among the investigated species differentiated in a statistically significant manner only in the first and second measurements. During the last measurement, the number of leaves leveled. The species 'Happy day Royal' developed characteristically bigger leaves and produced, statistically significant, more flowers compared to the other two species, 'Praecox' and 'Happy day' respectively.

The Christmas rose is becoming a very important ornamental plant on the market. It is relatively new to intensive production technologies. The number of genetically-improved varieties on the market is limited, and many of the old cultivars are no longer available because of slow and economically unfeasible vegetative propagation. In the future, its genetic improvement will probably become inevitable. New cultivars will have to satisfy the specific requirements of the market and producers as well as strict environmental policy. In order to obtain new cultivars it will be necessary to evaluate the existing germplasm and apply an adequate genetic breeding approach. There are seven main possibilities of creating new varieties: the use of natural variation associated with seed germination, population approach based on recurrent selection, individual selection based on specific genetic combinations, the creation of inbred lines and the formation of hybrids, interspecific hybridisation, mutagenesis and genetic engineering. Molecular markers can be very helpful during the breeding process. They can be used in order to study genetic relationships amongst populations, ecotypes, varieties, and hybrids.

In dieser Fallbeschreibung wird ein individuelles integratives Behandlungskonzept bei einer palliativen Patientin mit metastasiertem Mammakarzinom vorgestellt. Im Mittelpunkt steht dabei Helleborus niger (Christrose), eine in der komplementaren Onkologie bisher wenig beachtete Heilpflanze. Im vorliegenden Fall wurde Helleborus niger D12 in Form von taglichen Kaltinhalationen zunachst zur symptomatischen Behandlung von Dyspnoe und Reizhusten bei Pleuraerguss eingesetzt. Daruber hinaus verabreichten wir taglich subkutane Injektionen zur Immunmodulation wie auch zur Unterstutzung der Diurese. In Kombination mit anderen komplementaren Therapieprinzipien liesen sich zusatzlich gute Effekte hinsichtlich Kachexie, Muskelabbau, Schlaflosigkeit, Angst und Unruhe erzielen, die alle palliativmedizinisch relevant sind. Dabei waren weder Nebenwirkungen noch Wechselwirkungen mit chemischen oder pflanzlichen Stoffen zu verzeichnen. Nach 9-monatiger Therapie war klinisch wie sonografisch kein Pleuraerguss mehr nachweisbar. Auch die ubrigen Tumormanifestationen befanden sich in Regression, was mit einer deutlichen Stabilisierung der Patientin und Verbesserung ihrer Lebensqualitat einherging. Es lohnt daher, Helleborus niger in integrative Behandlungskonzepte aufzunehmen und durch breiteren klinischen Einsatz weitere Wirkmechanismen zu eruieren.

The influence of in vitro conditions including sucrose and nitrogen content in the culture medium as well as temperature on the pattern of chlorophyll a photochemical efficiency of photosystem II (PSII) of Helleborus niger L. plantlets upon transfer to ex vitro conditions were examined. In the experiment, the micro-shoots which were proliferated and rooted in vitro on the media with different levels of sucrose (10, 20, 30, 40, 50, 60, 70, and 80 g L−1) and nitrogen salts KNO3 and NH4NO3 (both 25, 50, 100 % according to MS medium), as well as temperature (15 and 20 °C) were used. The multiplication of axillary shoots were performed on modified MS medium supplemented with cytokinins (2iP, BAP and kinetin, each at a concentration of 1.0 mg L−1) and GA3 (2.5 mg L−1) added together. For the rooting of shoots, the medium was supplemented with IBA 1 mg L−1 and NAA 0.1 mg L−1 added together. The maximal photochemical efficiency of PSII immediately upon removal of microplants from in vitro culture ranged from 0.24 to 0.81 depending on the treatment. High concentration of sucrose (above 50 g L−1) and standard (100 %) or nitrogen salts decreased to 25 % in the MS medium as well as 20 °C temperature decrease the photochemical efficiency of microplants at transplanting time to ex vitro conditions as well as hinder acclimatisation and further growth out of culture. During acclimatisation to ex vitro conditions, a gradual increase of the maximal photochemical efficiency of in vitro-derived leaves was observed in all treatments. The maximal photochemical efficiency of PSII ranged from 0.79 to 0.83 after 4 weeks and from 0.81 to 0.83 after 20 weeks, indicating that H. niger plantlets gradually adapted to the greenhouse conditions and recovered their photosynthetic performance.

The concentration levels and stability of protoanemonin, a characteristic constituent of Ranunculaceae species with antimicrobial and fungicidal properties, were studied for the first time in plant extracts prepared from Helleborus niger L. and Pulsatilla vulgaris Mill. using fermentative production processes. Protoanemonin levels quantified by HPLC-DAD analysis were 0.0345 and 0.0662 mg/g in two freshly prepared Helleborus (whole, flowering plant) extracts and 0.3875 mg/g and 0.4193 mg/g in Pulsatilla (flowers) extracts. Protoanemonin proved to be rather instable in aqueous-fermented extracts stored at 15 °C in the dark, and its concentration decreased rapidly over 12 months of storage independently of the plant species. The decrease was most pronounced when initial concentrations were high (decrease by about 70%). In contrast, low protoanemonin levels remained stable in solution for more than 12 months. Anemonin, the dimer of protoanemonin, was detected in increasing concentrations only in Pulsatilla samples, but its concentration only accounted for less than 50% of the theoretically expected amount. With respect to fermented extracts, both physical processes such as self-polymerization and adsorption/binding to other extract constituents as well as biodegradation were concluded to be responsible for protoanemonin decline. As opposed to plant extracts, both protoanemonin and anemonin levels decreased in 0.22 μm-filtered samples stored in vials. This may be explained by a reduced release from plant material in combination with physicochemically induced degradation. Reduction was most pronounced upon light exposure and elevated temperatures, clearly indicating that photochemical degradation is involved. Contents of protoanemonin in a set of extract batches were 0.0896 ± 0.0125 mg/g and 0.0618 ± 0.0180 mg/g in Helleborus and Pulsatilla extracts, and anemonin levels were 0.0230 ± 0.0076 mg/g and 0.0482 ± 0.0282 mg/g, respectively. Due to its antibiotic effects, but also its reactivity, protoanemonin is a therapeutically and toxicologically relevant constituent, and its concentration should therefore be carefully monitored.

[ohne Zusammenfassung], Der Palmengarten, Bd. 72 Nr. 2 (2008)

The Christmas rose is becoming a very important ornamental plant on the market. It is relatively new to intensive production technologies. The number of genetically-improved varieties on the market is limited, and many of the old cultivars are no longer available because of slow and economically unfeasible vegetative propagation. In the future, its genetic improvement will probably become inevitable. New cultivars will have to satisfy the specific requirements of the market and producers as well as strict environmental policy. In order to obtain new cultivars it will be necessary to evaluate the existing germplasm and apply an adequate genetic breeding approach. There are seven main possibilities of creating new varieties: the use of natural variation associated with seed germination, population approach based on recurrent selection, individual selection based on specific genetic combinations, the creation of inbred lines and the formation of hybrids, interspecific hybridisation, mutagenesis and genetic engineering. Molecular markers can be very helpful during the breeding process. They can be used in order to study genetic relationships amongst populations, ecotypes, varieties, and hybrids. Črni teloh postaja na trgu okrasnih rastlin zelo pomembna rastlinska vrsta, ki pa je relativno nova v intenzivni proizvodnji. Število novih požlahtnjenih sort je na trgu sorazmerno nizko. Veliko starih sort ni več na voljo zaradi počasnega in ekonomsko nerentabilnega vegetativnega razmnoževanje. V prihodnosti bo potrebno zagotoviti nove atraktivne sorte, ki bodo morale izpolnjevati posebne zahteve trga in proizvajalcev, kakor tudi stroge okoljske omejitve. Uspeh žlahtnjenja novih sort je odvisen predvsem od genetske variabilnosti razpoložljivih materialov v genskih kolekcijah in naravnih populacijah ter izbrane metode dela. Obstaja sedem različnih možnosti za oblikovanje novih sortnih materialov: uporaba naravne variabilnosti v povezavi z uporabo semen, populacijsko žlahtnjenje (ki temelji na rekurentni selekciji), neposredna odbira najboljših osebkov iz potomstev specifičnih genskih kombinacij (križanj ali samooploditev), ustvarjanje homozigotnih linij in oblikovanje hibridov, medvrstna hibridizacija, oblikovanje mutacij in genski inženiring. V procesu žlahtnjenja so lahko zelo koristni tudi molekulski markerji, ki se lahko prav tako uporabljajo za proučevanje genetske sorodnosti med populacijami, ekotipi, sortami in hibridi.

The study is based on documentation and analysis of the major visitors and pollinators of the Christmas rose (Helleborus niger L.) flowers in Slovenian naturally occurring populations. The emphasis was put on 5 groups of insects: bees, bumblebees, large flies, small dipterous flies and pollinators of minor importance. Systematic observations took place in March 2003, at two different locations: on the valley of Bohinjska Bela and on the Peca Mountain. The first location was not far from a rural area, while the second one was in an isolated area, completely in the wild. The analysis of insect activity showed that there were obvious differences in frequency of visits among the insect groups investigated, locations and time of day. In the population of Bohinjska Bela, bees were the most important pollinators, with the highest activity occurred between 10 a.m. and 11 a.m. In the isolated population in Peca the most frequent visitors were small dipterous flies. The study suggests that the Christmas rose is probably an entomophylous (the most important pollinators are insects, such as bees and flies) and predominantly cross-pollinating species. The entomophylous nature appears to be closely associated with the specific botanic characteristics of flowers. Another pollinating agent is probably wind. Raziskava je bila opravljena na črnem telohu (Helleborus niger L.) in obsega dokumentiranje in analizo glavnih opraševalcev črnega teloha v naravnih populacijah. Osredotočili smo se na 5 skupin insektov: čebele, čmrlje, muhe, majhne dvokrilne insekte in opraševalce manjšega pomena. Sistematično opazovanje je potekalo v marcu 2003 na dveh lokacijah: v dolini Bohinjska Bela in na planini Peca. Prva lokacija je bila blizu naselja, medtem ko je bila druga lokacija daleč od ruralnega okolja v naravi. Analize opraševanja, povezane z aktivnostjo žuželk, nakazujejo, da obstajajo očitne razlike v pogostnosti obiska med proučevanimi skupinami žuželk, lokacijo in dnevnim časom. V populaciji Bohinjska Bela so bili najpomembnejši opraševalci čebele z največjo aktivnostjo med 10 in 11 uro zjutraj. V populaciji Peca so bili najpogostejši obiskovalci majhni dvokrilni insekti. Opazovanja kažejo, da je črni teloh verjetno prevladujoče tujeprašna, entomofilna vrsta (najpomembnejši opraševalci so insekti, predvsem čebele in muhe). Entomofilna narava je ozko povezana z botaničnimi lastnostmi cveta. Med možne opraševalce spada tudi veter.

RATIONALE: Isolation and extensive nuclear magnetic resonance (NMR) analyses revealed polyhydroxy steroid saponins to be characteristic constituents in Helleborus niger L. roots. A comprehensive study including various multistage mass spectrometry (MS n ) experiments provided first solid chromatographic and mass spectrometric information facilitating future analysis and structural assessment of polyhydroxy saponins by LC/MS n techniques without isolation and NMR analyses. METHODS: The polyhydroxy saponins were analyzed by direct syringe injection or chromatographically separated on a capillary high-performance liquid chromatography (HPLC) system coupled to an electrospray ionization (ESI) source. MS n spectra were recorded on an ion trap mass spectrometer including up to four fragmentation stages (LC/ESI-MS/ MS). Additionally, high-resolution mass spectra were recorded on an Orbitrap Fourier transform (FT) mass spectrometer equipped with a nanospray-ESI interface. RESULTS: The polyhydroxy hellebosaponins A and D were discovered to be significant constituents from H. niger roots. Extensive study of their MS n data revealed that they readily fragmented in the positive ion mode providing diagnostic fragments for elucidation of the steroidal character and number of OH groups. The negative ion mode yielded valuable information on the [M–H] – ion, number and location of acetyl groups and sugar units. Additionally, fragmentation pathways for positive and negative ion modes were proposed. CONCLUSIONS: These results not only extend the knowledge about H. niger saponins, but also provide a facilitated approach to the analysis of polyhydroxy saponins by LC/MS n without prior isolation and extensive NMR identification. Additionally, proposed fragmentation pathways for positive and negative ionization modes provide a solid complementary database for further, more detailed MS n studies. Copyright © 2014 John Wiley & Sons, Ltd.

The aerial parts of the medicinal plant Helleborus niger L. comprise a substantial number of constituents with only few of them identified so far. To expand the knowledge of its secondary metabolite profile, extracts from H. niger leaves and stems were investigated by liquid chromatography/tandem mass spectrometry (LC/MS(n) ). Specific identification strategies using LC/MS are established and discussed in detail. The leaves turned out to contain acylated and non-acylated quercetin and kaempferol oligoglycosides, protoanemonin and its precursor ranunculin, β-ecdysone, and a variety of steroidal saponins, mainly in the furostanol form. The sapogenins were elucidated as of sarsasapogenyl, diosgenyl, and macranthogenyl structures, and confirmed by comparison with the respective reference compounds. The secondary metabolite profiles were almost identical in both plant parts except that the stems lacked kaempferol derivatives and some saponins. The ranunculin derivatives and β-ecdysone were found in both plant parts. Correlations between the location of the compound groups and the plant's defense strategies are proposed. Additionally, the role of the detected secondary metabolites as protective substances against exogenic stress and as a defense against herbivores is discussed.

Morphological changes and phenolic patterns of developing hellebore sepals and the effects of pistil removal on these parameters were studied by comparing six flower stages of Helleborus niger. Color changes were evaluated colorimetrically, chlorophyll content was measured spectrophotometrically, and anthocyanins and flavonols were identified and quantified with HPLC-MS. Pistil removal not only altered the morphological development of hellebore flower resulting in smaller flower and significant color changes but also lead to several biochemical modifications. Five cyanidin glycosides have been identified from the group of anthocyanins in hellebore. Individual and total anthocyanin content increased from bud to subsequent developmental stages. Moreover, significantly higher content levels of individual and total anthocyanins have been measured in non-pollinated flower sepals compared to sepals of pollinated flowers. From the group of flavonols eight quercetin and kaempferol compounds have been quantified in hellebore sepals. Flavonol content significantly decreased during flower development with lowest levels recorded in sepals of non-pollinated and senescent pollinated hellebore flowers. Sepals of pollinated flowers contained highest levels of chlorophyll and significantly lower amounts of chlorophyll were measured in non-pollinated flowers and in sepals of senescent stage.

Melampus is a seer-healer of Greek myth attributed with having healed the young princesses of Argos of madness. Analysis of this legend and its sources sheds light on the early stages of the “medicalizing” shift in the history of ancient Greek medicine. Retrospective psychological diagnosis suggests that the descriptions of the youths’ madness rose from actual observation of behavioral and mental disorders. Melampus is credited with having healed them by administering hellebore. Pharmacological analysis of botanical specimens proves that Helleborus niger features actual neurological properties effective in the treatment of mental disorders. The discussion aims at examining the rational aspects of the treatment of mental conditions in Greco-Roman antiquity.

The increased horticultural interest in Helleborus requires the development of techniques for flowering control. Therefore, we have determined flower initiation and differentiation under natural conditions for Helleborus niger L. and the hybrid Helleborus x ericsmithii B. Mathew. Once flower primordia were present in the underground buds of the plants, the effect of a cold treatment (336, 504 or 672 chilling units (CU)) and a supplemental gibberellic acid (GA 3 ) application (500 mg L −1 ) on pre-cooled plants was examined. The cold treatment resulted in a progressive decrease of the time to flower. Moreover, when pre-cooled plants received a supplemental GA 3 application early flowering was further enhanced and these plants had a higher number of flowers with longer flower stems and larger flowers.

Containerized seedlings of Helleborus foetidus L. (stinking hellebore), H. niger L. (Christmas rose), and H. ×hybridus L. (Lenten rose) were grown under long-day conditions in controlled-environment chambers for 95 days with 9-hr days of 14, 18, 22, 26, or 30C (57, 64, 72, 79, or 86F) in factorial combination with 15-hr nights of 10, 14, 18, 22, or 26C (50, 57, 64, 72, or 79F). Long-day conditions were provided by a 3-hr night interruption. Growth of each species responded differently to day and night temperatures. Calculated maximum root, top, and total dry weight, and leaf area of H. foetidus occurred with days/nights of 20/15, 18/13, 19/14, and 18/15C (68/59, 65/55, 66/57, and 65/59F), respectively. While night temperature (NT) had no effect on root:top ratio [RTR (root dry weight ÷ top dry weight)], RTR was greatest (0.65) with days of 22C (72F). Helleborus niger had calculated maximum root dry weight and total dry weight with days of 14C (57F) and nights of 16 and 13C (60 and 55F), respectively. Top growth of H. niger decreased linearly as NTs increased for days of 14 or 22C (57 or 72F). Day temperatures (DTs) had no effect on RTR, whereas RTR responded quadratically as NT increased with a calculated maximum RTR at nights of 19C (66F). Leaf area was maximized at days/nights of 14/10C (57/50F). At days of 22 or 26C (72 or 79F), top growth of H. ×hybridus responded quadratically as NT increased with maxima occurring at nights of 18 or 17C (64 or 63F). Root dry weight responded quadratically at days of 14, 22, or 26C (57, 72, or 79F) and calculated maxima occurred with nights of 18C (64F). At days of 22 or 26C (72 or 79F), there were quadratic responses in total dry weight with calculated maximum growth of H. ×hybridus at nights of 18 or 17C (64 or 63F), respectively. For days of 14, 22, or 30C (57, 72, or 86F), there were quadratic responses in RTR with greatest RTR calculated at nights of 15, 18, or 16C (59, 64, or 60F), respectively. There were quadratic responses at days of 22 or 26C (72 or 79F) for leaf area with calculated maxima at nights of 18 or 17C (64 or 63F), respectively. As DTs increased from 14 to 30C (57 to 86F) net CO2 assimilation (PN) of H. ×hybridus also increased linearly whereas increased NTs had no effect on PN. In contrast, stomatal conductance was not impacted by DT or NT.

Hellbori nigri rhizoma is a drug that is difficult to distinguish from other species of the genus Helleborus. In this communication we present a DNA-based identification by high-resolution melting analysis (HRM) that is able to differentiate between Helleborus niger and other species of the genus. HRM is a very specific, time- and labour-saving method for identifying DNA sequence variations and is ideally suitable for routine PCR analysis. The HRM assay developed is specific for the genus Helleborus. This method not only detects the presence of the target species H. niger but also, to a certain extent, identifies other Helleborus species by their different melting curve shapes. Markers were developed based on the trnL-trnF intergenic spacer and on the matK sequence. For an unambiguous identification of Helleborus niger, melting curves of both markers should be used.

In order to establish protocols for embryo rescue techniques, in a first step this study aimed at the identification of culture conditions for ovules from intraspecific crosses. The effects of dissection date after pollination and temperature during ovule culture were examined in intraspecific crossings of Helleborus niger, H. argutifolius, H. × hybridus and H. foetidus. Ovaries were harvested 3-6 weeks after pollination, surface disinfected in 70% ethanol for 30 s, 2% sodium hypochlorite with one drop Tween for 10 min and rinsed in sterilised water three times. Ovules were dissected from ovaries and cultured on medium based on MS solidified with 0.4% Gelrite at a pH of 5.8. Two media supplemented with 2.5 or 5% sucrose were compared. Ovules were cultured in darkness at 24±1 or 16±1°C for 12 weeks. Thereafter, ovules of each temperature treatment were split and one half was incubated at 6±1°C for 11 weeks, while the other half remained in the initial temperature. Afterwards the ovules were placed back to their initial temperature. On average of all species, 2% of the ovules dissected 3 weeks and 1% dissected 4 weeks after pollination germinated, while preparation after 5 weeks resulted in 4% and after 6 weeks in 8% germination, respectively. The intermediate cold treatment with 6°C turned out to be very beneficial for later germination. Furthermore, no effect of the media supplemented with either 2.5 or 5% sucrose was observed.

Leaf spots were found on Christmas rose (Helleborus niger) in Yamagata Prefecture, Japan, in October 2006. The morphology of the causal fungus was very close to that of Colletotrichum truncatum. Classifying the species from the sequences of the internal transcribed spacer regions of ribosomal DNA was inconclusive, and the isolates were identified only as Colletotrichum sp. Artificial inoculation confirmed the pathogenicity of isolates to the host plant and some legumes. We propose the name anthracnose of Christmas rose for this disease by Colletotrichum sp.

Helleborus species are members of the family of the Ranunculaceae. These popular perennials are all diploids (2n = 2x = 32). This study investigates polyploidy induction by different antimitotic agents. Colchicine, oryzalin and trifluralin were tested in vitro on shoots of Helleborus niger, H. orientalis and H. × nigercors. Furthermore the effect of the antimitotic agents on the viability and the multiplication rate of cultured plantlets were analyzed. Flow cytometry demonstrated that polyploidisation was genotype dependent: using H. niger, tetraploids were obtained using either oryzalin (3 μM) or trifluralin (3 or 10 μM), whereas for H. × nigercors only trifluralin (3 or 10 μM) induced polyploidisation. For H. orientalis neither treatment was effective to produce tetraploids or mixoploids. For these three species, colchicine (100 μM) was ineffective. The polyploidisation events in H. niger and H. × nigercors were confirmed by chromosome counts of mounted nuclei derived from root tips (2n = 4x = 64).

Razvite države se vse bolj zavzemajo za kakovostnejše okolje, zato se uveljavljajo obnovljivi viri energije. Velik pomen dosega fotovoltaika. Uporaba sončne energije ima številne prednosti, vendar je na voljo premalo informacij o dejanskih izračunih uporabnosti solarnih tehnologij za bodoče uporabnike. Zato smo se v diplomskem delu osredotočili na možnosti uporabe solarne tehnologije na različnih lokacijah v Sloveniji. Obenem smo prikazali izračun proizvedene električne energije s pomočjo fotovoltaike, in približno oceno stroškov za lokacijo Ptuj. Rezultat izračuna predvidene letne proizvodnje in stroškov za malo sončno elektrarno na Ptuju je bil izjemno ugoden. Na razpoložljivi površini strehe, 98 m2, bi potrebovali 13 kWp nazivne moči. Začetni stroški investicije bi se povrnili predvidoma v 9 letih. Na podlagi pridobljenih podatkov, o fotovoltaični tehnologiji, smo mnenja, da je fotovoltaika ena boljšim alternativnih možnosti pridobivanja električne, ki bi veliko prispevala k ohranjanju naravnega okolja, v katerem živimo. Many developed countries have become more concerned about the environmental issues and as a consequence renewable sources of energy are widely used. Photovoltaic is currently gaining its importance. The use of solar energy has a lot of advantages since the solar energy is plentiful, but there is not enough informations of actual calculations. We have decided in my thesis to focus on the possibilities of solar energy usage in different locations in Slovenia for production of electricity. At the same time we presented, by means of photovoltaic, the calculations of the produced electricity, and also the estimated costs of investment in Ptuj. Results of calculation of anticipated annual productions and of costs for small sunny power station on Ptuj were exceptionally favourable. On available surface of roof, 98 m2, 13 kWp power would be required,. Initial costs of investment would be probably refunded within 9 years. Based on the data acquired about the photovoltaic technology we have come to the conclusion that this is definitely one of the best alternative possibilities, which could contribute to a great extent to the conservation of the environment we live in.

Raziskava je bila narejena na črnem telohu (Helleborus niger L.) v letih 2006 in 2007. V naravni populaciji v kraju Doblatina smo proučevali možnost samooprašitve teloha in kaljivost semen samooprašenih rastlin. Analizirali smo semenitev samooprašenih in hibridiziranih rastlin. Z metodo barvanja z acetokarminom smo proučevali vitalnosti peloda glede na dolžino skladiščenja pri temperaturi -9 oC. Za analizo samooprašnosti smo v naravni populaciji izolirali 41 matičnih rastlin. Rastline, ki smo jih ročno samoopraševali, so semenile. Povprečna kaljivost je bila 9,17 %. Največje število semen je bilo 105, najmanjše pa 2 semeni na posamezno obravnavano rastlino. Pri primerjavi semenitve rastlin je bilo ugotovljeno, da hibridizirane rastline tvorijo povprečno težja semena kot samooprašene rastline. Z analizo je bilo ugotovljeno, da obstajajo statistično značilne razlike v viabilnosti peloda, ki smo ga različno dolgo skladiščili. The research work involved Christmas rose (Helleborus niger L.) plant material from naturally grown local population in Doblatina, during the years 2006 and 2007. We studied the possibilities of self-pollination and germination of seeds of self-pollinated plants. We compared seed development of self-pollinated and hybridized plants. With the method of colouring by aceto-carmine, we studied the pollen viability depending on the duration of storage at -9 oC. For the purposes of the self-pollination analysis, we isolated 41 parental plants in their natural populations. Plants that were manually self-pollinated frequently developed seeds. The average germination was 9.17 %. The highest number of seeds per individual plant, considered in the analysis, was 105, and the lowest number was 2. The comparison of seed development showed that, on average, hybridized plants formed heavier seeds than self-pollinated plants. The results of the analysis showed that there were statistically significant differences in viability of pollen with different duration of storage.

Raziskava je bila opravljena na črnem telohu (Helleborus niger L.) in obsega v prvem delu dokumentiranje in analizo glavnih opraševalcev črnega teloha v naravni populaciji v drugem pa analizo kalivosti peloda. Osredotočili smo se na pet skupin insektov: čebele, čmrlje in ose, velike dvokrilne insekte (muhe), majhne dvokrilne insekte in druge opraševalce, ki so majhnega pomena. Sistematično opazovanje je potekalo marca 2009 v Žičah pri Slovenskih Konjicah. Analize opraševanja povezane z aktivnostjo žuželk nakazujejo, da obstajajo očitne razlike v pogostosti obiska med proučevanimi skupinami žuželk in dnevnim časom. Najpogostejši obiskovalci so bile majhne mušice. Opazovanja kažejo, da je črni teloh verjetno prevladujoča tujeprašna, entomofilna vrsta. Pri analizi živosti peloda smo ugotavljali optimalne pogoje skladiščenja. Živost peloda smo vrednotili s tehniko in vitro kalitve. Pelod je statistično značilno bolje kalil na gojišču z višjim pH in nižjo koncentracijo sladkorja. Živost peloda se je ohranila tudi po 5 mesecih, če smo ga skladiščili v zamrzovalniku pri -75ºC. The research was done on Christmas rose (Helleborus niger L.) and constitutes of two parts in first we documented and analysed main natural pollinators of the flower in its natural environment and in second we analysed the pollen germination. The research was focused on five groups of insects: bees, bumblebees and wasps, large dipterous insects, small dipterous flies and other pollinators of minor importance. Systematic observation was performed in march 2009 in Žiče pri Slovenskih Konjicah. Pollination analysis with regard to frequency of insect visits indicate that visit frequency is strongly dependant on time of the day. The most frequent visitors were small dipterous flies. Our observations indicate that the Christmas rose is a cross-pollinating species and endomophylous. In pollen viability we examined the optimal conditions for pollen storation. We examined the pollen viability with in vitro germination technique. Pollen germinated better on a media with a higher pH and a lower concentration of sugar. The viability of pollen was preserved even after 5 months if kept on a temperature of -75 degrees Celsium.