Browsing by Subject "HPLC"

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Analyzing resistance to ergot caused by Claviceps purpurea [Fr.] Tul. and alkaloid contamination in winter rye (Secale cereale L.)
(2022) Kodisch, Anna; Miedaner, Thomas
Ergot caused by Claviceps purpurea [Fr.] Tul. is one of the oldest well-known plant diseases leading already in medieval times to severe epidemic outbreaks. After the occurrence of honeydew, the characteristic ergot bodies called sclerotia are formed on the ear. These are containing toxic ergot alkaloids (EAs). Strict limits are set within the European Union. Rye (Secale cereale L.) as cross-pollinating crop is particularly vulnerable to ergot since the competitive situation of fungal spores and pollen during flowering. Nevertheless, even today the threat is real as agricultural practice is changing and screening studies revealed EAs in samples of the whole cereal value chain frequently. The aims were to establish a harmonized method to test ergot resistance and EA contamination in winter rye, to clarify major significant factors and their relevance and to reveal the suitability of one commercial ELISA test. Further, effort was paid to examine the covariation of ergot amount and EA content considering different factors because of prospective legislative changes. Genotypes showed significant variation for ergot severity and pollen-fertility restoration after natural infection as well as artificial inoculation whereas a high positive correlation could be found between both treatments. Additionally, variances of environment, general combining ability (GCA), specific combining ability (SCA), and interactions were significant. Although male pollen-fertility restoration was of utmost importance, the female component was also significant. This illustrates that apart from promising selection of high restoration ability the maternal restorability could be exploited in future breeding programs especially when a high pollen amount is already reached. A large-scale calibration study was performed to clarify the covariation of ergot severity, EA content (HPLC, ELISA) considering genotypes, locations, countries, years, and isolates. EA profile was rather stable across country-specific isolates although large differences regarding the EA content were detected. The moderate covariation between ergot severity and EA content (HPLC) indicates that a reliable prediction of the EA content based on ergot severity is not possible what can also not be explained by grouping effects of the factors. Further, EAs seem not to act as virulence factor in the infection process since EA content showed no relationship to disease severity. Additionally, the missing correlation of ELISA and HPLC leads to the conclusion that the ELISA is not an appropriate tool what can be used safely to screen samples regarding ergot in the daily life. The genetic variation of male-sterile CMS-single crosses was analysed in a special design without pollen in field and greenhouse to identify resistance mechanisms and to clarify whether ergot can be reduced in the female flower. At this, comparison of needle and spray inoculation revealed medium to high correlations illustrating that both methods were suitable for this research. Significant environment and genotype by environment interaction variances were detected. So, testing across several environments is necessary also without pollen. Further, small but significant genotypic variation and identification of one more ergot-resilient candidate revealed that selection of female lines could be promising to further reduce ergot. The EA content was lower for less susceptible genotypes. Thus, EA content can be considerably reduced by breeding. A strong positive correlation could be found for ergot severity and EA content when analysing 15 factorial single crosses. The male pollen-fertility restoration was also here the most relevant component but the female component contributed an obviously higher proportion for the EA content than for ergot severity. In conclusion, this thesis demonstrate that implementing of a high and environmental stable male fertility restoration ability via exotic Rf genes can effectively reduce ergot although also the female restorability enables great opportunities. The unpredictable covariation between ergot amount and EA content illustrates that both traits have to be assessed, in particular the EA content by a valid HPLC approach to guarantee food and feed safety.
Evaluation of new open pollinating broccoli genotypes (Brassica oleracea convar. botrytis var. italica) specifically bred for organic farming conditions focusing on agronomic performance and glucosinolate content
(2018) Sahamishirazi, Samira; Graeff-Hönninger, Simone
Currently, a considerable share of varieties being used in the organic vegetable production are developed for conventional high-input production systems, and broccoli is no exception. In addition, F1 hybrids are cultivated in organic broccoli production to a great extent because of high quality and yield. Two main restrictions of cultivating the mentioned categories of varieties in organic farming are; 1) ban of using cytoplasmic male sterility (CMS) in organic agriculture for reproduction of F1 hybrids of broccoli and limitations of farmers to produce their own seeds, 2) absence of special traits of these varieties which result in weaker performance when being cultivated under organically low-input conditions. In contrast to hybrids, cultivation of open pollinating broccoli varieties gives the opportunity of reproducing seeds to organic farmers. Therefore, developing new open pollinating broccoli varieties, which have the same quality (agronomical, chemical and sensorial) as F1 hybrids, through organic breeding programs (on-farm breeding) would allow the organic broccoli farmers to replace the hybrids with varieties adapted to organic production conditions. With this in mind, the German Federal office for Agriculture and Food (BLE) initiated a project on “Breeding development of open pollinating cultivars of broccoli for organic farming in terms of agronomic characteristics, secondary and bioactive ingredients and sensory properties”. This was a joint project which was done through the cooperation of University of Hohenheim and Kultursaat e. V. (NGO of on-farm breeders) in two parts during six years (2011-2016). The present doctoral thesis, which was a part of the mentioned project, aims at 1) investigating the agronomic performance of the newly bred open pollinating genotypes of broccoli, 2) developing a Near-Infrared Spectroscopy (NIRS) method for fast analysis of total, indole, aliphatic and individual glucosinolates content of broccoli samples; and 3) determining the total and individual glucosinolate content of the newly bred open pollinating genotypes of broccoli. For investigations on agronomic performance, two field experiments were carried out by cultivating eleven newly bred open pollinating genotypes, two F1 hybrids and an open pollinating variety of broccoli over two growing seasons of fall 2015 and spring 2016. Evaluation of the effect of genotype, growing season and their interactions on agronomic parameters were targeted in this study. According to our findings, assessment of agronomic variables indicated that although there were distinctions in different parameters such as head firmness, head shape and total biomass fresh weight among the newly bred open pollinating genotypes, some genotypes performed similar to hybrid varieties in organic farming. However, most of the open pollinating genotypes had 16 % to 73 % lower yields compared to the hybrid varieties depending on growing season. Generally, the “marketable yield” of the genotypes was under the significant effect of “genotype × growing season interaction”. Head weight was significantly affected by growing season which resulted in significantly lower head weight of some genotypes in the spring compared to the fall season. Overall, cultivation of the genotypes in fall season led to significantly higher marketable yields, head weight and total biomass weight, as well as firmer heads in contrast to the spring season. Considering the performance of different agronomic parameters, we recommend genotypes “TH-CAN-SPB”, “Calinaro”, “CHE-GRE-G” for both fall and spring growing season. Other genotypes such as “CHE-GRE-A”, “CHE-BAL-A” and “CHE-MIC” and “Line 701” are also recommended for cultivation in spring growing season specifically due to the high marketable yield and share of marketable heads. In addition, this thesis aimed at testing a fast analytical technique for determination of glucosinolates content in order to help breeders to quickly test their most favorable genotypes during breeding procedures based on glucosinolates content. For this purpose, the accuracy of NIRS technic was tested, regardless of type of genotype, for fast analysis of the individual and total glucosinolates content of broccoli samples. NIRS calibration was developed by reference method of High Performance Liquid Chromatography (HPLC) based on modified partial least squares regression, to measure individual and total glucosinolates content of open pollinating genotypes of broccoli regardless of the type of genotype. The calibration was analyzed using coefficient of determination in prediction (R2) and ratio of preference of determination (RPD). Large variation occurred in the calibrations, R2 and RPD due to the variability of the samples. Derived calibrations for total glucosinolates (RPD = 1.36), aliphatic glucosinolates (RPD = 1.65), glucoraphanin (RPD = 1.63) and 4-methoxyglucobrassicin (RPD = 1.11) were quantitative with a high accuracy, while for indole glucosinolates (RPD = 0.95), glucosinigrin (RPD = 0.62), glucoiberin (RPD = 0.67), glucobrassicin (RPD = 0.81) and neoglucobrassicin (RPD = 0.56) they were more qualitative. Overall, the results showed a good potential of NIRS in determination of different glucosinolates in a large sample pool of broccoli quantitatively and qualitatively. The achieved calibration equations were used to measure glucosinolates content of the broccoli samples of following years. To evaluate the health beneficial value of the open pollinating genotypes, the glucosinolates content of them were determined. The determination was done by the tested NIRS technic. Six individual glucosinolates were detected in the broccoli samples similar to findings of the previous chapter. Glucoraphanin (1.44-1.69 µmol g-1 DW), glucobrassicin (0.63-0.77 µmol g-1 DW) and neoglucobrassicin (0.38-0.74 µmol g-1 DW) had the highest share and were the main individual glucosinolates. Total glucosinolates content ranged from 3.46 to 3.60 µmol g-1 DW across both growing season. Significant effect of genotype and growing season existed on the total glucosinolates content of broccoli samples. All individual glucosinolates were affected by genotype. The effect of growing season was significant on all individual glucosinolates, except for glucobrassicin. The interaction of genotype × growing season was significant on all indole glucosinolates, glucoraphanin and glucoiberin. Generally, the glucosinolates content of the samples were higher when broccoli genotypes were cultivated in the fall growing season, however the difference in the level of glucosinolates contents across seasons was significant only for glucoraphanin, neoglucobrassicin, 4-methoxyglucobrassicin and glucoiberin. The open pollinating genotypes showed a similar range of glucosinolates compared to the tested hybrids and performed as good as the hybrids. Since total glucosinolates were nearly similar in all open pollinating genotypes across seasons, all are recommended for cultivation in both growing seasons. It is important to note that this study only focused on a single health beneficial compound (glucosinolate) in broccoli heads. To provide a full insight into the nutritive and health benefiting compounds of broccoli such as vitamins and polyphenols, supplementary studies will have to be conducted. All in all, releasing new open pollinating broccoli varieties out of this pool of genotypes and replacing the present varieties with them seemed beneficial due to the well adapted agronomic performance and high health value with regard to glucosinolates content under organic farming conditions.
Generation of high oleic acid sunflower lines using gamma radiation mutagenesis and high-throughput fatty acid profiling
(2023) Rozhon, Wilfried; Ramirez, Veronica E.; Wieckhorst, Silke; Hahn, Volker; Poppenberger, Brigitte
Sunflower (Helianthus annuus L.) is the second most important oil seed crop in Europe. The seeds are used as confection seeds and, more importantly, to generate an edible vegetable oil, which in normal varieties is rich in the polyunsaturated fatty acid linoleic acid. Linoleic acid is biosynthesized from oleic acid through activity of the oleate desaturase FATTY ACID DESATURASE 2 (FAD2), which in seeds is encoded by FAD2-1, a gene that’s present in single copy in sunflowers. Defective FAD2-1 expression enriches oleic acid, yielding the high oleic (HO) acid trait, which is of great interest in oil seed crops, since HO oil bears benefits for both food and non-food applications. Chemical mutagenesis has previously been used to generate sunflower mutants with reduced FAD2-1 expression and here it was aimed to produce further genetic material in which FAD2-1 activity is lost and the HO trait is stably expressed. For this purpose, a sunflower mutant population was created using gamma irradiation and screened for fad2-1 mutants with a newly developed HPLC-based fatty-acid profiling system that’s suitable for high-throughput analyses. With this approach fad2-1 knock-out mutants could be isolated, which stably hyper-accumulate oleic acid in concentrations of 85-90% of the total fatty acid pool. The genetic nature of these new sunflower lines was characterized and will facilitate marker development, for the rapid introgression of the trait into elite sunflower breeding material.
Mechanisms of frost adaptation and freeze damage in grapevine buds
(2002) Badulescu Valle, Radu Virgil; Blaich, Rolf
Mechanisms of frost hardening in compound (latent) buds of the grapevine cultivar ?Bacchus? were tested with different methods during three winters. The investigated parameters were LTE/HTE (low temperature exotherm/high temperature exotherm), water content, starch, sugar- and anions combination and bud histology. Water content from wood and buds was determined regularly every 2 weeks from March 1998 until Mai 2000. The lowest water content in wood and buds (about 40 %) was found between November and February. In general shoot sections and buds from the apical shoot area contained less water than in the basal area. Sugars and anions were analyzed with HPLC. The highest concentrations of soluble sugars were found in basal buds of the shoot, the lowest concentration in buds of the apical shoot area. Sucrose was the predominant soluble sugar, it was accompanied by glucose, fructose, sucrose, raffinose, and also stacchyose which was hitherto not described for grapevine buds. The concentration of soluble sugars increased during autumn and reached its maximum (around 150 mg/g dry matter) in November/December until the beginning of January then it decreased again to around 30 mg/g at bud burst. The predominant anion was sulphate while chloride could be detected only in traces. The anions reached their maximum at the beginning of January and in mid April. To evaluate the exotherm measuring method, model experiments were carried out with water drops (1µl) on filter paper and with small plant parts (leaf, stems, flower parts). Both the plant parts and the destilled water on the cellulose fiber freeze mainly between ?8 and ?15°C (an influence of the low osmotic value of the plant sap could not be found). After the first freezing the specimen were thawed and freezing repeated. The freezing points of the first and the second freezing cycle were significantly correlated. This shows that freezing does not occur at random, but is determined by ice nucleation sites characteristic for each sample. These sites even survive the physical destruction of the cells by the ice cristals. Further model experiments were carried out to get indications on possible barriers to ice cristal growth in plant tissue. Exotherm analysis was used to determine the freezing point of grapevine buds which is accompanied by a transient temperature rise called exotherm. The grapevine buds show 2 or more exotherms, one or two HTEs (high temperature exotherms) between ? 5 °C and ?10°C and the LTE (low temperature exotherm, sometimes more than one ) between ?10°C and ?25°C depending on the frost adaption of the buds. The HTEs are assumed to indicate the freezing of surface water or apoplastic water in the subtending tissue (bud pad), whereas the LTE (or LTEs) seem to be caused by freezing of the primary (and secondary) buds (shoot primordiy of the compound bud). The temperature minimum of the LTEs (down to ? 25 °C) is reached in January/February and is not influenced by humidity which, however, changes the THE values occuring usually around ? 10 ° and ? 4 °C, which are influenced by water in the bud scales. The LTEs of the buds in the lower area of the shoot were higher as compared to the buds in the middle and upper area of the shoot. The LTE analysis clearly shows the frost adaptation of the latent buds which usually reaches a maximum by the end of January but a clear relation to the changing air temperatures could not be established. Histological and cytological analyses were used to test for frost damage in bud parts and for changes during the cold adaptation. A modified staining method was developed to differentiate the cells. During automn and winter the buds contained a lot of starch grains which dissolved at bud burst. A permeability barrier between bud pad and shoot primordia could not be found, however it could be directly shown, that a HTE causes no cell damage in the buds, while after the appearence of the LTE(s) a disintegration of protoplasts in primary and secondary buds could be found. This is a direct evidence that LTEs indicates the death of the eyes in the complex grapevine bud. If after the appearance of the HTE the buds were held one day at this temperature before further cooling, no LTEs would appear. This and similar observations during the frost storage of grapevine cuttings is discussed in terms of the (harmless) ice formation in the bud base at moderate minus temperatures which would result in a freeze drying effect due to the lower water potential of the bud pad (in comparison to the non frozen eyes) and a further increase of the frost resistance of the growing points. If frost adapted grapevine shoots from the field were kept at 20°C deacclimation occurred after about 10 days. Accidentally wetted buds showed exotherms above ?4°C. In these buds and the watering water ice nucleating bacteria (Pseudomonas fluorescens) could be found.