1 ABSTRACT Kateřina Muráriková Influencing of production of plant tissue culture of Trifolium pratense L. I The aim of this thesis was to check the influence of cinnamic acid, as a precursor of the phenylpropanoids biosynthesis, on the flavonoids production of Trifolium pratense L. suspension culture, to compare two varieties of this plant, the DO-8 one and the Tempus one, in terms of the effect of this precursor, and to help achieve the successful increase of the production of secondary metabolites by the Trifolium pratense L. explant cultures. Concerning the DO-8 variety, the changes of the isoflavonoids production as a result of the cinnamic acid influence have been also tested. The suspension cultures were being cultivated on the Gamborg's nutrient medium with the addition of 2 mg.l-1 2,4-dichlorphenoxyacetic acid and 2 mg.l-1 6-benzylaminopurine. The cultivation was being realised on a slow moving roller at temperature of 25 řC and in the period of 16 hours of light and 8 hours of dark. The cinnamic acid was added in the four concentrations and its influence was observed after 6, 24, 48 and 168 hours. The flavonoids determination was being applied on the check samples and the samples with the addition of cinnamic acid by the spectrophotometric method in agreement with the Český lékopis 2009 and the...

1 Abstract Veronika Machová Effect of 2-benzylthiopyridine-4-carbothioamide derivatives on isoflavonoids and flavonoids accumulation in suspension culture of Trifolium pratense L. The paper examinate the effect of three concentration of 2-(2-fluoro-6- nitrobenzylsulfanyl) pyridine-4-carbothioamide on the production of flavonoids and isoflavonoids by the Trifolium patense L. (variety DO-8, variety Tempus). The culture were cultivated in the Gamborg nutrient media with addition of 2 mg·l-1 2,4- dichlorophenoxyacetic acid a 2 mg·l-1 6-benzylaminopurine, at the temperature of 25řC, 16-hr light/8-hr dark period. The elicited and the inspection samples underwent the photometric determination of flavonoids in accordance with the Czech Pharmacopoeia 2009 ant the determination of isoflavonoids via the HPLC method. The results show that the maximum content of flavonoids in suspension cultures of Trifolium pratense L. (variety DO-8) was caused by 48-hour elicitation by 100 µmol·l-1 solution of 2-(2-fluoro-6-nitrobenzylsulfanyl) pyridine-4-carbothioamide, when the a statistically significant increase in production was 93% compared to control cultures. The maximum content of flavonoids in variety Tempus was caused by 48-hour elicitation by 10 µmol·l-1 solution of 2-(2-fluoro-6-nitrobenzylsulfanyl) pyridine-4-...

Lucie Částková Influencing of production of plant tissue culture of Trifolium pratense L. II The aim of this thesis was to observe the effect of the abiotic elicitor vanadyl sulfate on the production of flavonoids in the suspension culture of Trifolium pratense L., variety DO-8 and variety Sprint. The effects on the production of isoflavonoids caused by the elicitor have been also studied. The cultures were cultivated at the temperature of 25 žC, 16 hour light/8 hour dark period, in the Gamborg's nutriet medium with the addition of 2 mg.l-1 of 2,4- dichlorophenoxyacetic acid and 2 mg.l-1 of 6-benzylaminopurine. Vanadyl sulfate was added in four concentrations and its influence was observed after 6, 24, 48 and 168 hours. The quantity of flavonoids was determined spectrophotometrically according to the Pharmacopoeia Bohemica 2009. The quantity of isoflavonoids was determined using HPLC. The maximal content of flavonoids, 0,459 %, was measured in the suspension culture of Trifolium pratense L., variety DO-8, taken after 48 hours of action of elicitor vanadyl sulfate, which concentration was 1 µmol.l-1 . The statistically significant increase in production was 106 % compared to the control. The production of flavonoids in variety Sprint was most influenced by the action of vanadyl sulfate, which...

The availability of complete plastid genomes has been playing an important role in resolving phylogenetic relationships among the major clades of land plants and in improving our understanding of the evolution of genomic organization. The increased availability of complete genome sequences has enabled researchers to build large multi-gene datasets for phylogenetic and molecular evolutionary studies. In chapter 2 of this thesis a web-based multiple sequence web viewer and alignment tool (MSWAT) is developed to handle large amount of data generated from complete genome sequences for phylogenetic and evolutionary analyses. We expect that MSWAT will be of general interest to biologists who are building large data matrices for evolutionary analyses. The third chapter presents the sequenced plastid genomes of three magnoliids, Drimys (Canellales), Liriodendron (Magnoliales), and Piper (Piperales). Data from these genomes, in combination with 32 other angiosperm plastid genomes, were used to assess phylogenetic relationships of magnoliids to other angiosperms and to examine patterns of variation of GC content. Evolutionary comparisons of three new magnoliid plastid genome sequences, combined with other published angiosperm genomes, confirm that GC content is unevenly distributed across the genome by location, codon position, and functional group. Furthermore, phylogenetic analyses provide the strongest support so far for the hypothesis that the magnoliids are sister to a large clade that includes both monocots and eudicots. The fourth chapter presents the Trifolium subterraneum plastid genome sequence, which is unusual in genome size and organization relative to other angiosperm plastid genomes. The Trifolium plastid genome is an excellent model system to examine mechanisms of rearrangements and the evolution of repeats and unique DNA. / text

xvii, 215 leaves : ill. ; 30 cm.0 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Agronomy, 1974

Perennial clovers are being evaluated for their potential to reduce groundwater levels in Australian cropping zones where many soils are considered too acidic for reliable lucerne nodulation. However, the release of effective inocula for perennial clovers into such areas where sub clover is the predominant legume, could potentially compromise nitrogen fixation from this valuable annual clover if the symbiosis between the new inoculants and sub clover is not optimal. Studies were therefore designed to increase our understanding of these symbiotic interactions to optimise the management of legume-rhizobia interactions to extend (rather than restrict) the use of legumes in new environments. To assist the understanding of interactions between clovers and their microsymbionts, a glasshouse-based study of the cross-inoculation characteristics of 38 strains of Rhizobium leguminosarum biovar trifolii (R. l. trifolii) associated with 38 genotypes of annual and perennial Trifolium spp. from world centres of diversity was undertaken. Rhizobial isolates and clovers were assembled from South and equatorial Africa, North and South America and the Euro-Mediterranean regions. There was substantial specificity amongst the African clovers for effective nodulation. No strain of rhizobia from the South American perennial T. polymorphum, or from the Ethiopian clovers, was able to nodulate sub clover effectively, whilst less than 33% of the 18 strains from these regions could form nodules with the less promiscuous Mediterranean annual T. glanduliferum. Seventy of 476 cross-inoculation treatments examined did not nodulate, whilst 81 treatments clearly demonstrated effective nodulation. The remainder of the crossinoculation pairings revealed only partially effective or ineffective nodulation. Two barriers to effective nodulation were identified from the cross- inoculation study: a geographic barrier representing the broad centres of clover diversity, across which few host- strain combinations were effective; and within each region, a significant barrier to effective nodulation between an isolate from an annual host on a perennial host, or vice versa. Clovers and their rhizobia from within the Euro-Mediterranean region of diversity were more able to overlap the annual/perennial barrier than genotypes from the other regions. The data indicate that it will be a substantial challenge to develop inocula for perennial clovers that do not adversely affect nitrogen fixation by sub clover and other annual clovers in commerce, especially if the perennial clovers originate from Africa or America. To investigate the management of legume-rhizobia interactions when introducing legumes into new environments, a study was initiated in Uruguay (Mediterranean annual clovers were introduced into a predominantly perennial clover setting) that could be considered opposite to the situation emerging within southern Australia (perennial clovers evaluated in a predominantly annual clover setting). The Uruguayan grasslands contain populations of indigenous R. l. trifolii that nodulate endemic T. polymorphum but form ineffective nodules on clovers originating from the Mediterranean region. Importantly in the Uruguayan setting, Government policy has facilitated the introduction of numerous varieties of annual Mediterranean clovers with the aim of improving overall winter production in their naturally managed grasslands. In an attempt to understand the rhizobial ecology of this scenario, a cross-row experiment was set-up in 1999 in a basaltic, acid soil in Glencoe, Uruguay, to follow the survival and symbiotic performance of nine exotic strains of R. l. trifolii. In this thesis I report on the ability of the introduced strains to compete for nodule occupancy of Mediterranean clover hosts and show the impacts of the introduced strains on the productivity of the indigenous Uruguayan clover, T. polymorphum. Of the introduced strains, WSM1325 was a superior inoculant and remained highly persistent and competitive in forming effective symbioses with the Mediterranean hosts, T. purpureum and T. repens, in the Uruguayan environment over a 3 year period. T. purpureum and T. repens, when inoculated with the introduced strains, did not nodulate with any indigenous R. l. trifolii as typed from nodules of T. polymorphum. Conversely, there were no nodules on the Uruguayan host T. polymorphum that contained the introduced R. l. trifolii. These results revealed that there were effective symbioses between strains of R. l. trifolii and clovers, even though the soil contained ineffective R. l. trifolii for all hosts. This represents the first reported example of selective nodulation for an effective symbiosis in situ with annual and perennial clovers in acid soils. This phenomenon raised the question of whether this was restricted to the particular edaphic scenario in Glencoe, Uruguay. Glasshouse-based experiments in Australia were conducted to further understand the selection phenomenon. Two strains were selected for comparisons; strain WSM1325 isolated from an annual clover in the Mediterranean and WSM2304 isolated from the perennial clover T. polymorphum in Uruguay, South America. Variables that may have been specific to Glencoe were investigated. Thus, the effect of cell density and strain ratio at the time of inoculation, as well as soil pH, were examined on the two hosts (T. purpureum and T. polymorphum). Each was exposed to the same effective and ineffective micro-symbionts. In co-inoculation experiments at a cell density of 104 cells mL-1, each host nodulated solely with its effective strain, even when this strain was out-numbered 100:1 by the ineffective strain. However, the selection process ceased when the effective strain was out-numbered 1000:1. At higher basal cell concentrations of 105 - 108 cells mL-1, selection for WSM1325 to form effective nodules on T. purpureum was evident, but was significantly reduced as the ratio of ineffective cells in the inoculum increased above 4-fold. These results indicate that the selection mechanism is highly dependent upon the basal rhizobial cell density. Soil pH did not significantly alter the process, which could not be simply explained by the rate of strain growth, or extent of nodulation. Greater precision was sought in the terminology applied to nodulation outcomes where legumes have a choice of micro-symbiotic partners from within the same species of root-nodule bacteria. The nominated preferred terms are “nonselective”, “exclusive”, and “selective” nodulation. In view of the difference in host range between WSM1325 and WSM2304 and the selective nodulation process, a preliminary investigation into the genetic backgrounds of WSM1325 and WSM2304 was conducted. A selected range of gene regions were amplified by PCR from each strain and sequenced. Comparative analysis of the nucleotide sequences revealed that although the 16S rRNA sequences were identical, the atpD, GSII and nodD sequences contained distinct differences revealing disparity between the pSym replicons and between the chromosomal replicons of these strains. Of the genes sequenced, the highest degree of divergence was noted for the symbiotic NodD protein products, which are known to be critical determinants in the nodulation of specific hosts. An examination of the nodD gene region of WSM1325 and WSM2304 revealed a further contrasting feature; the regulatory gene nodR was present in the nodD gene region of WSM1325 but absent in WSM2304. Since NodR is known to be required for adding highly unsaturated fatty acyl groups onto the Nod-factor backbone, I could now hypothesise that the nodulation incompatibility observed between Trifolium hosts and micro-symbionts obtained from different geographical locations may result from differences in Nod-factor decoration. With the full genome sequence of the two strains WSM1325 and WSM2304 soon to be available, the role of nodR and any link to the selection phenomenon described in this thesis can be addressed.

xvii, 215 leaves : ill. ; 30 cm.0 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Agronomy, 1974

Diss. (sammanfattning). Uppsala : Sveriges lantbruksuniv. / Härtill 4 uppsatser.

A mesofauna edáfica, compreendendo ácaros e colêmbolos, é sensível a alterações no solo, sendo considerada um indicador de qualidade, uma vez que realiza a fragmentação de matéria orgânica, controle populacional de determinados grupos da biota do solo e ingestão de microrganismos. A colonização de plantas por micro-organismos, como a de leguminosas por rizobios, pode levar a mudanças na rizosfera vegetal e influenciar a mesofauna. Este estudo teve por objetivo avaliar o efeito da inoculação de plantas de trevo vermelho com rizóbios sobre os ácaros e colêmbolos edáficos e avaliar se colêmbolos alimentados com rizóbios poderiam inocular plantas hospedeiras. Para isso, plantas de trevo vermelho, inoculadas com as estirpes SEMIA 2081, SEMIA 2082 e SEMIA 222, e plantas controle sem inoculação foram plantadas em tubos-armadilha e inseridas em campo. A seguir, a mesofauna atraída foi extraída e identificada. Outro experimento em laboratório utilizou plantas de trevo vermelho que foram colocadas em contato com colêmbolos da família Hypogastruridae e dos gêneros Proisotoma, Onychiurus e Orthonychiurus, os quais haviam sido alimentados com um substrato impregnado com o rizóbio da estirpe SEMIA 2081 As plantas inoculadas com as três estirpes de rizóbios atrairam ácaros edáficos mas não colêmbolos, quando comparadas com as plantas que não foram inoculadas nem receberam fonte de nitrogênio. Considerando os números totais de ácaros e colêmbolos, somente as plantas inoculadas com a estirpe SEMIA 222 apresentaram atratividade quando comparadas ao controle sem inoculação e sem fonte de nitrogênio. A atratividade de ácaros e colêmbolos observada no tratamento controle com nitrogênio foi similar aos tratamentos inoculados. No experimento de laboratório, os colêmbolos vivos e o macerado de colêmbolos da família Hypogastruridae, e gêneros Proisotoma e Orthonychiurus foram capazes de transferir os rizóbios que receberam pela alimentação às plantas, formando nódulos radiculares. As plantas que receberam o macerado de colêmbolos da família Hypogastruridae apresentaram maior número de nódulos comparadas às plantas que receberam os mesmos colêmbolos vivos. / The soil mesofauna comprising mites and springtails, is sensitive to changes in soil, and it is considered a quality indicator since it performs fragmentation of organic matter, population control of certain soil biota groups and ingestion of microorganisms. The plants colonized by microorganisms, such as legumes by rhizobia can lead to changes in plant rhizosphere and influence mesofauna. This study aimed to evaluate the effect of inoculation of red clover plants with rhizobia on edaphic mites and springtails and assess if springtails fed with rhizobia could inoculate host plants. For this, red clover plants inoculated with strains SEMIA 2081, SEMIA 2082 and SEMIA 222, and control plants without inoculation were planted in trap-tubes and inserted on field. Then, the attracted mesofauna was submitted to extraction and identification. Another laboratory experiment used red clover plants put in contact with springtails. These springtails of Hypogastruridae family and Proisotoma, Onychiurus and Orthonychiurus genera, had been fed with a substrate impregnated with rhizobia strain SEMIA 2081. Plants inoculated with three Rhizobia strains attracted edaphic mites, but not Collembola, when compared to uninoculated plants with no nitrogen source Considering the total numbers of mites and springtails, only plants inoculated with SEMIA 222 strain showed attractiveness when compared to the control without inoculation and no nitrogen source. The attractiveness of mites and springtails observed in the control treatment with nitrogen was similar to the inoculated treatments. In the laboratory experiment, the living springtails and the macerate of springtails of Hypogastruridae family, and Proisotoma and Orthonychiurus genera were able to transfer the rhizobia that they received by feeding to the plants, forming root nodules. The plants that received the macerate of springtails of the Hypogastruridae family had a higher number of nodules compared to the plants that received the same living springtails.

A mesofauna edáfica, compreendendo ácaros e colêmbolos, é sensível a alterações no solo, sendo considerada um indicador de qualidade, uma vez que realiza a fragmentação de matéria orgânica, controle populacional de determinados grupos da biota do solo e ingestão de microrganismos. A colonização de plantas por micro-organismos, como a de leguminosas por rizobios, pode levar a mudanças na rizosfera vegetal e influenciar a mesofauna. Este estudo teve por objetivo avaliar o efeito da inoculação de plantas de trevo vermelho com rizóbios sobre os ácaros e colêmbolos edáficos e avaliar se colêmbolos alimentados com rizóbios poderiam inocular plantas hospedeiras. Para isso, plantas de trevo vermelho, inoculadas com as estirpes SEMIA 2081, SEMIA 2082 e SEMIA 222, e plantas controle sem inoculação foram plantadas em tubos-armadilha e inseridas em campo. A seguir, a mesofauna atraída foi extraída e identificada. Outro experimento em laboratório utilizou plantas de trevo vermelho que foram colocadas em contato com colêmbolos da família Hypogastruridae e dos gêneros Proisotoma, Onychiurus e Orthonychiurus, os quais haviam sido alimentados com um substrato impregnado com o rizóbio da estirpe SEMIA 2081 As plantas inoculadas com as três estirpes de rizóbios atrairam ácaros edáficos mas não colêmbolos, quando comparadas com as plantas que não foram inoculadas nem receberam fonte de nitrogênio. Considerando os números totais de ácaros e colêmbolos, somente as plantas inoculadas com a estirpe SEMIA 222 apresentaram atratividade quando comparadas ao controle sem inoculação e sem fonte de nitrogênio. A atratividade de ácaros e colêmbolos observada no tratamento controle com nitrogênio foi similar aos tratamentos inoculados. No experimento de laboratório, os colêmbolos vivos e o macerado de colêmbolos da família Hypogastruridae, e gêneros Proisotoma e Orthonychiurus foram capazes de transferir os rizóbios que receberam pela alimentação às plantas, formando nódulos radiculares. As plantas que receberam o macerado de colêmbolos da família Hypogastruridae apresentaram maior número de nódulos comparadas às plantas que receberam os mesmos colêmbolos vivos. / The soil mesofauna comprising mites and springtails, is sensitive to changes in soil, and it is considered a quality indicator since it performs fragmentation of organic matter, population control of certain soil biota groups and ingestion of microorganisms. The plants colonized by microorganisms, such as legumes by rhizobia can lead to changes in plant rhizosphere and influence mesofauna. This study aimed to evaluate the effect of inoculation of red clover plants with rhizobia on edaphic mites and springtails and assess if springtails fed with rhizobia could inoculate host plants. For this, red clover plants inoculated with strains SEMIA 2081, SEMIA 2082 and SEMIA 222, and control plants without inoculation were planted in trap-tubes and inserted on field. Then, the attracted mesofauna was submitted to extraction and identification. Another laboratory experiment used red clover plants put in contact with springtails. These springtails of Hypogastruridae family and Proisotoma, Onychiurus and Orthonychiurus genera, had been fed with a substrate impregnated with rhizobia strain SEMIA 2081. Plants inoculated with three Rhizobia strains attracted edaphic mites, but not Collembola, when compared to uninoculated plants with no nitrogen source Considering the total numbers of mites and springtails, only plants inoculated with SEMIA 222 strain showed attractiveness when compared to the control without inoculation and no nitrogen source. The attractiveness of mites and springtails observed in the control treatment with nitrogen was similar to the inoculated treatments. In the laboratory experiment, the living springtails and the macerate of springtails of Hypogastruridae family, and Proisotoma and Orthonychiurus genera were able to transfer the rhizobia that they received by feeding to the plants, forming root nodules. The plants that received the macerate of springtails of the Hypogastruridae family had a higher number of nodules compared to the plants that received the same living springtails.