Timothy (Phleum pratense L.), a cool-season perennial, is the most important forage grass species in Nordic countries. Genetic diversity has been previously assessed  in a collection of 96 timothy accessions, of which 88 were of Nordic origin. Simple sequence repeat (SSR) markers revealed Nordic timothy accessions to be very polymorphic, having significant differences in the levels of diversity between countries, vegetation zones, and dif- ferent cultivar types. However, most of the variation (94 %) existed within accessions, and no clear clustering of accessions based on any grouping was observed. This lack of resolution may either reflect the outcrossing and hexaploid nature of timothy or that SSR markers are not suitable for resolving population structure in timothy.
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The objective of this study was to examine the effects of potassium fertilizer (standard (S) and high (H) levels) on water-soluble carbohydrate (WSC) content of timothy (Phleum pratense L.), si- lage fermentation, nutritive values, and nutrient intake. The silage treatments were as follows: S level without inoculant (SC), S level plus inoculant (SI), H level without inoculant (HC), and H level plus inoculant (HI). The K content was increased by 14.5% in timothy grown with the H level compared with the S level. The WSC contents of the S and H treatments were 75.9 and 66.1 g∙kg −1
Silage was prepared with (SI and HI) and without (SC and HC) an inoculant. In each treatment, the unwilted grass material was cut into 1 - 4 cm lengths and ensiled in 220 L FRP silos. Three replicates were prepared. For the inoculant, 11F25 (Lactobacillus plantarum and Enterococcus faecium) of Pioneer Hi-Bred Japan Co. Ltd. (Tokyo, Japan) was used. The preparation was added at 1 mg·kg −1 fresh grass, and the number of viable micro- organisms was 1.0 × 10 5 cfu·g −1 fresh grass. The silage was subjected to evaluation of the fermentative quality and feed and digestion trials after storage.
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Abstract: Resistance of common European grasses to diesel oil and petroleum pollution is not well-known. There- fore, this study aimed at determining the level of resistance of selected grasses to pollution by diesel and petroleum using the pot experiment. The achieved results were compared with those determined for grasses grown on the non- -polluted soil. Soil pollution with the tested products was found to significantly decrease the yield of all grasses, with the decrease being lower upon soil pollution with petroleum than with diesel oil. The most resistant to the pollution with diesel oil and petroleum were Phleum pratense L., Lolium perenne L. and Lolium × hybridum Hausskn. The de- gradation of particular groups of polycyclic aromatic hydrocarbons (PAHs) depended on their chemical properties, on the type of pollutant and grass species. The greatest degradation was determined in the case of BTEX, C 6 –C 12 benzines as well as 2- and 3-ring hydrocarbons, whereas the lowest in the case of 5-and 6-ring hydrocarbons and C 12 –C 25 oils. The most useful species in the remediation of soils polluted with diesel oil and petroleum turned out to be: Lolium perenne L., Lolium × hybridum Hausskn and Phleum pratense L., whereas the least useful appeared to be: Festuca rubra, Dactylis glomerata L. and Poa pratensis L.
Choke, caused by the ascomycete fungus Epi- chloë typhina (Pers.) Tul. & C. Tul., is a serious disease of grasses grown for seed. It has a wide host range and infects a number of cultivated and wild grasses (Mühle 1971). It is becoming economically important in the species grown for seed such as timothy (Phleum pratense L. and P. nodosum L.) and orchard grass (Dactylis glome- rata L.). The causal agent of the disease is an en- dophytic fungus which spends its life cycle inside the host. The fungus produces mycelial stromata that develop on emerging inflorescences which are suppressed completely or seed production on the infected fertile stem is partly prevented. Long-term observations of this disease in grasses grown for seed reveal an increasing tendency (Cagaš 2008, 2010). A stand with more than 5 infected plants per 100 m 2 was rejected until recently (Regulation No.
The major forage grasses currently cultivated in Lithuania are common timothy (Phleum pratense L.), meadow fescue (Festuca pratensis Huds.), perennial ryegrass (Lolium perenne L.), cocksfoot (Dactylis glomerata L.), Festulolium hybrids and Kentucky blue grass (Poa pratensis L.). The productivity of these species is not stable. A very important property of forage grasses is their ability to give stable and high dry matter yields under different environmental conditions. The level of the produc- tivity and stability mostly depends on the genetic potential of the forage grass species (Černiauskas 1992, Chapman 1996, Moser et al. 1996).
was 0.41). In conclusion, common characteristics of the Trifolium species (Kocová and Mártonfi 2011; Kocová et al. 2014) and T. pratense cultivars are (i) EI of petals is always higher than EI of sepals and (ii) the highest EI is found in carpels in comparison with the remaining floral organs. Hence, these seem to be common features in the genus Trifolium. However, this rule should not be applied to species of other families (Barow and Meister 2003; Chen et al. 2011). In addition, endopolyploidy patterns differ in comparison with other Fabaceae species belong- ing to different genera such as Pisum sativum and Vicia faba, although the higher EI of petals over sepals corre- sponds to cases in Trifolium, carpels of the mentioned species were the ones with the lowest EI (Barow and Meister 2003). Putative inconsistency in endopolyploid patterns of the floral organs of different species does not result from coincidence, but is most probably determined by interplay between intrinsic (genetic, cytological, life form) and extrinsic (environmental) factors. It is well known that the endopolyploidy level of organs/tissues is mostly correlated with the phylogenetic placement of the species which is strongly associated with many pheno- typic traits (Bainard et al. 2012).
Within MASCOT, sunﬂ ower was cultivated as a part of a ﬁ ve-year stockless arable crop rotation (sugar beet-common wheat-sunﬂ ower-pigeon bean-durum wheat) comparing a conventional and an organic management system. Systems are replicated three times according to a Randomized Complete Block design, each experimental plot having a ﬁ eld size of 0.35 to 1 ha. In the organic system, red clover (Trifolium pratense) was interseeded in common and durum wheat and used as a green manure for sunﬂ ower or sugar beet. The MASCOT experiment was established on loamy soil, whose main characteristics are reported in Table 1.
The F 1 interspecific hybrids mostly display low lev- els of fertility and insufficient vigour. For this reason, efforts to improve seed productivity are focused on the repeated backcrossing of hybrids with cultivated species, as it has been shown that backcrossing is an effective way to increase both fertility and vigour. Indeed, in these experiments repeated open pol- lination helped to improve the level of fertility and vigour of the T. pratense × T. medium hybrids. Only those hybrids with higher chromosome numbers (42, 44, and more) were unsuccessful in establishing a population of fertile hybrids as a source for further selection of useful traits. In 2010, an application for Plant Breeders Rights to the variety Pramedi was filed at the Czech Plant Variety Office, which granted the rights in 2013 (variety number TPM14855 and variety code 5082339; Holders of Rights: Ing. Hana Jakešová, Breeding, Hladké Životice; Agricultural Research, Ltd.; Research Institute for Fodder Crops, Ltd. Troubsko; Masaryk University in Brno).
One of the aims in red clover (Trifolium pratense) breeding is to increase the polyphenol oxidase (PPO) activity, which may effectively reduce protein breakdown in silage and when cattle are fed by fresh clover. We analysed total PPO activity spectrophotometrically and on the level of gene expression using real-time quantitative PCR in single plants derived from an interspecific T. pratense × T. medium hybrid. Experiments were performed for two years and evaluated according to the general linear model with three factors (family, year, and cut). The analysis revealed considerable variability in total PPO activity between individuals and between families. Four families and two individuals with significantly higher PPO activity were selected. Their PPO activity ranged from 3.411 to 3.547 mkatal/min/g and from 4.041 to 5.731 mkatal/min/g, respectively, in comparison with the control variety Amos (2.370 mkatal/min/g). The majority of PPO transcripts were expressed by the two genes PPO1/5 and PPO2. In some genotypes, the PPO5 gene was expressed. Quantitative PCR confirmed the highest activity of PPO genes in seven hybrid plants with higher DNA contents corresponding to 30 chromosomes with 815 013 copies per plant. Our results indicate the suitability of combining two methods for improved selection: initial expression analysis to assess the PPO transcript level indicating gene activity and subsequent enzymatic assay. Keywords: expression analysis; polyphenolic enzyme; protein quality; Trifolium medium; Trifolium pratense
The increased risk of thromboembolic events in patients undergoing therapy with SERM raloxifene limits its usage in elderly patients who are already prone to develop thrombotic effects due to advancing age. This indicates the necessity to look for other natural SERMs which will possess the osteoprotective functions of raloxifene but is devoid of its side effects. Trifolium pratense also called as red clover is a plant rich in phytoestrogens Formononetin and Biochanin A which are reported to possess osteoprotective properties and tissue specific estrogen receptor modulating properties (Suita and Pattanayak. 2011). Formononetin and Biochanin A could be potential alternatives to raloxifene in the management of osteoporosis but the fact that they are derived from Trifolium pratense necessitates investigations pertaining to their effects on blood clotting because of the increased thinning tendency of the blood in patients administered Trifolium pratense which is contributed to the presence of a class of anticoagulatory compound referred to as coumestans (Osoki.,m 2003).Prothrombin time, partial thromboplastic time, bleeding time and clotting time are simple parameters which will provide an idea about the effect of test compounds on the clotting cascade (Arber., 1990). Prothrombin is a serine protease which is the inactive precursor of the protein thrombin. Thrombin is essential for the final step of the clotting cascade- the conversion of fibrinogen to fibrin. Thromboplastic on the other hand is a protein which is required for the conversion of inactive prothrombin to active thrombin. Partial thromboplastic time implies the time required for the conversion of prothrombin to thrombin and consequently the time required to form the fibrin clot. Measurement of Bleeding time gives an idea about the time till which the blood can remain in a liquid state when it is made to ooze from a source (injured blood capillary/vessel or a deliberate prick) and measurement of clotting time provides an idea about the time required for the liquid blood to form the insoluble fibrin clot. All these parameters together provide an idea about the influence of a test compound as a procoagulatory or anticoagulatory agent.
The objective of present study is to develop and validate a simple, precise, rapid, specific and accurate reverse phase HPLC method for the simultaneous determination of daidzein, genistein, formononetin and biochanin A obtained from in vitro cultured cells of Trifolium pratense L. Chromatographic separation was achieved on reverse phase octadecylsilane C18 column with isocratic elution. The mobile phase consisted of a mixture of ammonium acetate and methanol (40:60) pumped at a flow rate of 1.0 ml. min -1 . The effluents were monitored at 254 nm with a UV detector at ambient temperature. Validation was carried out according to International Conference on Harmonisation (ICH) guidelines with respect to linearity, sensitivity, accuracy, precision, robustness and ruggedness. Good linearity was observed (r 2 > 0.998) over the test concentration range of the four isoflavones. Precision, accuracy, limit of detection and limit of quantification obtained were within acceptable range in each case. The method was robust and specific for the four isoflavones. The results indicate that the developed method is simple, precise, rapid, specific and accurate, and would be suitable for the quantification of daidzein, genistein, formononetin and biochanin A obtained from in vitro cultured cells of Trifolium pratense L.
2.4.2. Total Flavonoid Content. Total flavonoid content was ascertained based on the method by Zhishen et al. . In brief, 50 mg of each fraction was dissolved in 10 mL of 80% aqueous methanol and filtered through Whatman filter paper number 42 (125 mm). In a 10 mL test tube, 300 𝜇L of each extract, 3.4 mL of 30% methanol, 150 𝜇L of 0.5 M NaNO 2 , and 150 𝜇L of 0.3 M AlCl 3 ⋅6H 2 O were added and mixed, followed by 5 min incubation and the addition of 1 mL of NaOH (1 M). Absorbance was measured at 510 nm with a spectrophotometer. The standard curve for total flavonoids was made using rutin standard solution (0–100 mg/L) using the same aforementioned procedure. The total flavonoid content was shown as milligram of rutin equivalents (CTE) per gram dry matter of extract.
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Red clover (Trifolium pratense L.) is a legume forage abundant in phenolic compounds. It tends to brown when cut for hay, due to oxidation of phenolic compounds catalyzed by polyphenol oxidase (PPO), and subsequent binding to proteins. Selecting for a greener hay may provide information about the relationship of browning, PPO, and phenolics to each other. The red clover Kenland cul- tivar was selected over eight breeding cycles for decreased browning after being cut and dried 48 h in the field. Expression of PPO1 and PPO3, in Kenland and three of the eight cycles, was com- pared by real-time quantitative PCR. Phenolic compounds in Kenland and Cycle 8, collected 0, 24, and 48 h after cutting, were quantified by high-performance liquid chromatography (HPLC). Visual browning scores decreased 12% between Kenland and Cycle 8 (P = 0.02). PPO1 and PPO3 gene expression were not affected by selection. Clovamide decreased 26% in Cycle 8 relative to Kenland (P = 0.016). Sissotrin decreased 10% in Cycle 8 (P = 0.043). Neither total formononetin nor total biochanin A was affected by selection (P = 0.63 and 0.45, respectively). These results suggest that when selecting clover for decreased postharvest browning, a decrease occurs in a phenolic com- pound that can bind protein independently of PPO. However, PPO1 and PPO3 gene expression, and the major red clover isoflavones, are minimally affected.
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ways within the limits established by Italian legislation . Melissopalynological analysis revealed that 94% of samples were wildflower honeys and only 6% were monofloral Castanea honeys; it indicates the existence of several spectra of vegetation for the different sites in the Parks corresponding to various seasons. Results obtained from this study showed the presence of a lot of botanical species in Gran Sasso Natural Park and “Monti della Laga”. The most common floral varieties were: Robinia pseudoacacia L. and Castanea sativa Mill. for woody species; Paliurus spina-christi Mill., Sambucus nigra L. and Cornus sanguinea L. for shrubby species; Echinacea spp., Satureja montana L., Echium italicum L., Trifolium
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The experiment was conducted in north-western Croatia, during three years (1995–1998), at two Faculty of Agriculture experimental fields: Zagreb – alluvium-colluvium’s brown soil (123 m altitude – lowland, 836 mm average annual precipitation, 10.3°C mean annual temperature) and Medvednica – brown acid soil (650 m altitude – mountain re- gion, 1230 mm average annual precipitation, 6.6°C mean annual temperature). Five diploid Trifolium pratense L. cultivars (Croatia, K-17, Reichersberger, Marino, Viola) and one tetraploid cultivar (Nada) were sown on May 1995. At the time of seeding the experimental areas received N, P and K at the rate of 40, 130 and 130 kg/ha, respectively. No further N was applied and the fertilizer application over next two years was 130 kg P/ha and 130 kg K/ha. The seeding rate for all cultivars was 18 kg/ha. The experimental design was a randomised complete block with six replications. The plot size was 5 × 2.5 m with an effective harvest area of 4 × 1.5 m after allowing for discarded borders. The plots were cut three times in the first year, four times in the second, and once in the third year at a height of 4 cm, when red clover plants reached 10% flowering. Before each
Genistin transport assay. The pellets of vacuoles were diluted in test tubes with 8.0 mL of stand- ard solutions that contained the tested substances (3.0 mmol/L MgATP; 0.03 mmol/L bafilomycin A1 – Baf; 0.5 mmol/L N,N'-dicyclohexylcarbodiimide – DCCD) with or without Gin (0.1 mmol/L); the other solutions contained only Gin or water as a blank sample. Immediately after preparation, 2.0 mL of the suspension was removed from each mixture of solutions and centrifuged (10 min, 4°C, 2650 g). The supernatant was mixed with 1.0 mL of 80% metha- nol and transferred to a vial. The pellet of vacuoles was dispersed in 2.0 mL of 80% methanol using the Labdancer for a duration of 15 s. The extract was filtered through a microfilter (0.2 µm) into vials using an injection syringe. Sampling and processing were carried out in the same manner after 2, 4 and 24 h.
The eﬀ ect of the combined grazing and cutting management on the phytocenological characteristics was examined at the submountain paddock in the South Bohemia. The botanical scans were sampled during the ﬁ ve-years study (2006–2010) starting a er the sowing the pasture sward in the originally arable ﬁ eld and 0–2 years a er the beginning of the grazing (paddock A and paddock B, respectively). The paddock A was grazed all year round, whereas the paddock B was grazed in spring and autumn and cut in summer for hay. At both paddocks, Lolium perenne, Trifolium repens and Taraxacum sect. Ruderalia dominated the community of total 43 and 47 species (paddock A and B, respectively). Among the sowing species, Lolium perenne, Festuca pratensis, Poa pratensis, Festuca rubra and Trifolium repens increased its cover on both paddocks, while Phleum pratense increased its cover only at paddock B. Lolium multiﬂ orum decreased it cover at both paddocks. Most of arable ﬁ eld weeds disappeared (paddock A) or decreased its cover (paddock B). The cover of herb layer was higher at paddock A than at paddock B, whereas the number of species (N), the diversity (H) and the equitability (J) was higher at paddock B than at paddock A. The cover of herb layer increased during the study at both the paddocks, while the number of species declined at paddock A and increased at paddock B.
The potential for reclaiming soils contaminated by Ba is as yet poorly developed. One of the tested methods is phytoremediation (Ribeiro et al., 2018). The special ability of some plants to uptake and accumulate metallic contami- nants via the root system and store them in various plant compartments is the basis for this method (Tangahu et al., 2011). There are only a limited number of reports concern- ing Ba accumulation by plants. A significant potential for Ba accumulation, expressed in the form of high bioconcen- tration factors (ratio of Ba concentration in plant biomass and soil) ranging from 2-20 was reported for tomatoes and soybeans (Choudhury and Carey, 2009). Li et al. (2011) found that the common reed (Phragmites australis) has a high potential as a plant which may be used in the phy- toremediation of Ba contaminated soil. The study also found significant Ba removal capabilities in silvergrass Miscanthus floridulus. Barium accumulation was also observed in the biomass of southern cattail (Typha domin- gensis), papyrus sedge (Cyperus papyrus) (Ribeiro et al., 2018), Indigofera cordifolia (Raghu, 2001), Brazil nuts (Bertholletia excels), different mushrooms (Harbison et al., 2015) and some legumes (Myrvang et al., 2016). The latter observed that bird velch (Vicia cracca L.) and white clover (Trifolium repens L.) examined in a pot experiment car- ried out on sandy soil accumulated a higher amount of Ba than barley, tall fescue, carrot, radish and spinach. Whelan (1993) showed that an increase in the Ba concentration of the soil due to its fertilization with barium selenate (the source of selenium for plants used as an animal forage) did not cause an increase in Ba concentration in subterranean clover (Trifolium subterraneum), even after three years of fertilization.
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Tři metody šlechtění jetele lučního (Trifolium pratense L.) byly porovnávány na výnos píce ve dvou sklizňových letech na třech lokalitách v Bredelokke (Dánsko), Hladké Životice (Česká republika) a Les Alleuds (Francie). Byly srovnávány tři typy 46 novošlechtění, které byly získány A) rekurentní selek- cí v následných generacích (37 novošlechtění, rozdělených na rané a pozdní), B) polycrossem (čtyři novošlechtění) a C) geno-fenotypovou selekcí (pět novošlechtění). Pokusy byly založeny v roce 2005 a v každém sklizňovém roce (2006 a 2007) byly provedeny tři seče. Pokusy byly hodnoceny na zá- kladě celkového výnosu suché píce. Novošlechtění šlechtěná pomocí polycrossu a geno-fenotypo- vé selekce měla významně vyšší výnos než novošlechtění pocházející z rekurentní selekce. Novo- šlechtění šlechtěné pomocí metody B a C se mezi sebou významně nelišila. Novošlechtění šlechtěná pomocí těchto metod byla vhodnější pro výnosnější a sušší prostředí v Hladkých Životicích (kde byl nejvyšší výnos, i když průměrné roční srážky byly nižší v porovnání s ostatními lokalitami (o 73 mm a 113 mm); zde byl průměrný výnos vyšší o 19 % a 13 % pro B a C metody v porovnání s A metodou. Byly zjištěny významné interakce mezi novošlechtěními a lokalitami. Závěrem lze konstatovat, že od- růdy určené pro určitá prostředí by měly být šlechtěny pomocí metod B a C; a také výchozí rodiče by měli zde být vybíráni.