Rozprawy doktorskie na temat „Lotus corniculatus”

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Físicas e Matemáticas. Programa de Pós-Graduação em Química.
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Um estudo fitoquímico de L. corniculatus foi realizado e biomonitorado através de bioensaios de atividade antioxidante, antibacteriana e anti-inflamatória. O estudo fitoquimico foi efetivado através de sucessivas cromatografias em coluna de sílica gel e monitoradas por cromatografia em camada delgada, sendo as estruturas elucidadas através de técnicas de IV, RMN de 1H e 13C e COSY. A atividade antioxidante in vitro foi avaliada nos modelos como a capacidade sequestrante DPPH, potencial redutor e peroxidação lipídica. O efeito antibacteriano foi avaliado através do método de microdiluição o qual produz a Concentração Inibitória Mínima e a Concentração Bactericida Mínima. Finalmente, o efeito anti-inflamatório de Lotus corniculatus foi analisado no modelo in vivo da pleurisia induzida pela carragenina em camundongos, que é um modelo de inflamação aguda. Foram isolados três compostos: dois terpenos, o ?-sitosterol e o ácido oleanólico e um flavonóide glicosilado canferitrina. Após realizados os ensaios de atividades biológicas, nossos resultados demonstraram que Lotus corniculatus cv. São Gabriel possui importante propriedade antibacteriana e anti-inflamatória. Estes efeitos podem ser atribuídos aos compostos isolados como o ácido oleanolico, ?-sitosterol e principalmente ao canferitrina o qual demonstrou o melhor efeito farmacológico.

A utilização de pastagens capazes de tolerar o alumínio em níveis elevados apresenta-se como uma alternativa viável para contornar um dos principais problemas existentes em áreas de cultivo com solos ácidos. Entre as leguminosas forrageiras, o cornichão (Lotus corniculatus L.) destaca-se pela sua qualidade nutricional e versatilidade de adaptação. Em função disso, esse trabalho foi desenvolvido com o objetivo de selecionar plantas de L. corniculatus tolerantes ao alumínio, bem como analisar a diversidade genética existente entre estes materiais com o auxílio de marcadores moleculares do tipo microssatélites. Foram utilizados três genótipos de cornichão (Draco, São Gabriel e UFRGS) em solução nutritiva, contendo 200µmol/L de cálcio (CaCl2.2H2O), 100 µmol/L de alumínio (AlCl3) e pH controlado em uma faixa de 4,1 - 4,3. Realizaram-se dois ciclos de seleção, sendo as plântulas selecionadas pelo comprimento final das radículas. Posteriormente, testou-se o progresso alcançado pelos ciclos de seleção através de um experimento em solução nutritiva, semelhante ao utilizado nas seleções, com as populações originais e melhoradas, em quatro concentrações de alumínio (0, 50, 100 e 150 µmol/L) (AlCl3). Foram avaliados o comprimento inicial, final e o crescimento das radículas. Após o término do segundo ciclo de seleção, realizou-se a análise da diversidade genética existente entre os genótipos de cornichão estudados. Foram utilizados 18 pares de primers desenvolvidos para Lotus japonicus (Regel) Larsen e Trifolium repens L. As similaridades genéticas, com base no coeficiente de Jaccard, foram utilizadas para fazer o agrupamento dos genótipos pelo método UPGMA, através do programa NTSYS pc 2.1. O número de alelos e o conteúdo de informação de polimorfismo (PIC) também foram calculados para cada loco. A seleção em solução nutritiva mostra-se eficiente na seleção de plântulas de cornichão tolerantes ao alumínio. Os materiais analisados apresentam diferenças em relação a esta característica, destacando-se como o genótipo mais tolerante UFRGS F2, proveniente de dois ciclos de seleção. Os marcadores microssatélites revelam-se adequados para acessar a variabilidade genética entre os genótipos de L. corniculatus.
The use of grasslands capable to tolerate high levels of aluminum is a viable alternative to bypass one of the main problems existing in cultivated areas with acid soils. Among the forage legumes, the birdsfoot trefoil (Lotus corniculatus L.) stands out for its nutritional quality and versatility of adaptation. Given the importance, this work was carried out to select plants of L. corniculatus tolerant to aluminum, and analyze the genetic diversity between these materials with the aid of microsatellite molecular markers. Three genotypes of birdsfoot trefoil were used (Draco, São Gabriel and UFRGS) in nutrient solution containing 200 µmol/L of calcium (CaCl2.2H2O), 100 µmol/L of aluminum (AlCl3) and controlled in a pH range of 4.1 - 4.3. Two selection cycles were made, where the seedlings were selected by the root length. Subsequently, the progress achieved by cycles of selection was tested in an experiment in nutrient solution, similar to that used in the selections, with the original and improved populations, in four concentrations of aluminum (0, 50, 100 and 150 µmol/L) (AlCl3). The initial, final and the growth of the radicle were evaluated. After the end of the second cycle of selection it was carried out the analysis of genetic diversity among genotypes of birdsfoot trefoil studied. Eighteen pairs of primers developed for Lotus japonicus (Regel) Larsen and Trifolium repens L. were used. The genetic similarities based on Jaccard coefficient was used to make the grouping of genotypes by the UPGMA method, using the program NTSYS pc 2.1. The number of alleles and the polymorphism information content (PIC) were calculated for each locus. The selection in nutrient solution was efficient in the selection of seedlings of birdsfoot trefoil tolerant to aluminum. The materials analyzed differ for this characteristic, with the genotype UFRGS F2, being the most tolerant. The microsatellite markers are suitable for accessing the genetic variability among genotypes of L. corniculatus.

Lotus corniculatus L. (Legtuninosae), is a perennial herb common throughout Britain. Its main pollinators are bumblebees (Bombus spp., Apidae: Hymenoptera). This is a study of the ecological factors which are important to flowering and fruiting in the species, and some of their evolutionary implications. The work was carried out at Wytham Estate, Oxfordshire, U.K., mainly in an ex-arable field (Upper Seeds) and a more established grassland (Lower Seeds Reserve). The literature on self-incompatibility in L. corniculatus is reviewed; there are conflicting reports, but wild material is fundamentally self-incompatible. Plants in Upper Seeds are larger than in Lower Seeds Reserve. Comparative data on soil nutrients in the two sites suggests that the cause is the persistence of phosphorus from inorganic fertiliser. There is a positive, linear relationship between plant size, flower production and fruit production. The species regulates investment in flowers mainly at the level of the whole inflorescence, rather than altering number of flowers per inflorescence. Within individuals, there are no consistent trade-offs between number of fruit per infructescence, numbers of seeds per fruit and seed weight. Weather patterns only partially explain the flowering phenology of L. corniculatus. Timing of first flowering and peak flowering are correlated but are variable between individuals, and between years for the same individuals. They are not correlated with flowering synchrony. An individual's flowering pattern does not consistently affect fruit-set; the overriding determinant of fruit production is plant size. Selection is therefore unlikely to be acting on flowering time in this species. The production of large numbers of self-incompatible flowers does not seem to reduce fruit-set; pollinators do not visit enough flowers per foraging trip (perhaps because nectar production is low) for geitonogamy to become a problem. Seed predation by larvae of a chalcid wasp, a weevil and a moth differs between individual plants, but not consistently so between years. Seed predation is not consistently correlated with plant size, mean flowers per inflorescence, number of seeds per fruit or seed size. There is no evidence for selection acting on these traits through seed predation. Partially predated seeds are often viable, which may have implications for seedling demography. Seed predation and flowering phenology are not defmitively linked, strengthening the argument that flowering time is not adaptive in this species.

Birdsfoot Trefoil (Lotus corniculatus L. cv. Leo), is a perennial forage legume that has difficulty in establishing stands due to slow seedling growth, weed competition and herbicide sensitivity. The development of herbicide resistant cultivars would be of economic importance. Resistant lines were isolated after sequential selection at the callus, shoot and whole plant levels to the sulfonylurea (SU) herbicides Harmony (DPX-M6316, code name for methyl 3- ( ( ((4-methoxy-6-methyl-1,3,5,triazine-2-yl)amino) carbonyl) amino) sulfonyl-2-thiophenecarboxylate), or Classic (DPX-F6025, code name for 2(( (4-chloro-6-methoxypyrimidine-yl)amino carbonyl) aminosulfonyl)benzoic acid ethyl ester. In field and growth chamber tests the Harmony regenerant lines displayed an increased tolerance as compared to control plants from tissue culture, and from seed. Results of the evaluation of callus cultures of regenerated mutant lines signify stability of the resistance. Outcrossed seeds collected from field trials, and tested in vitro for herbicide resistance, indicate that the trait is heritable and that resistance may be due to reduced sensitivity of acetolactate synthase to SU inhibition. It is concluded that herbicide resistant birdsfoot trefoil cultivars can be isolated using in vitro selection.

Protoplast fusion of Lolium perenne and Lotus corniculatus for gene introgression by Sanjeev V. Raikar Lolium perenne is one of the most important forage crops globally and in New Zealand. Lotus corniculatus is a dicotyledonous forage that contains valuable traits such as high levels of condensed tannins, increased digestibility, and high nitrogen fixing abilities. However, conventional breeding between these two forage crops is impossible due to their markedly different taxonomic origin. Protoplast fusion (somatic hybridisation) provides an opportunity for gene introgression between these two species. This thesis describes the somatic hybridisation, the regeneration and the molecular analysis of the putative somatic hybrid plants obtained between L. perenne and L. corniculatus. Callus and cell suspensions of different cultivars of L. perenne were established from immature embryos and plants were regenerated from the callus. Of the 10 cultivars screened, cultivars Bronsyn and Canon had the highest percentage of callus induction at 36% each on 5 mg/L 2,4-D. Removal of the palea and lemma which form the seed coat was found to increase callus induction ability of the embryos. Plant regeneration from the callus was achieved when the callus was plated on LS medium supplemented with plant growth regulators at different concentrations. Variable responses to shoot regeneration was observed between the different cultivars with the cv Kingston having the lowest frequency of shoot formation (12%). Different factors affecting the protoplast isolation of L. perenne were investigated. The highest protoplast yield of 10×106 g-1FW was obtained when cell suspensions were used as the tissue source, with enzyme combination ‘A’ (Cellulase Onozuka RS 2%, Macerozyme R-10 1%, Driselase 0.5%, Pectolyase 0.2%), for 6 h incubation period in 0.6 M mannitol. Development of microcolonies was only achieved when protoplasts were plated on nitrocellulose membrane with a L. perenne feeder layer on PEL medium. All the shoots regenerated from the protoplast-derived calli were albino shoots. The highest protoplast yield (7×106 g-1FW) of L. corniculatus was achieved from cotyledons also with enzyme combination ‘A’ (Cellulase Onozuka RS 2%, Macerozyme R-10 1%, Driselase 0.5%, Pectolyase 0.2%), for 6 h incubation period in 0.6 M mannitol. The highest plating efficiency for L. corniculatus of 1.57 % was achieved when protoplasts were plated on nitrocellulose membrane with a L. perenne feeder layer on PEL medium. The highest frequency of shoot regeneration (46%) was achieved when calli were plated on LS medium with NAA (0.1 mg/L) and BA (0.1 mg/L). Protoplast fusion between L. perenne and L. corniculatus was performed using the asymmetric somatic hybridisation technique using PEG as the fusogen. L. perenne protoplasts were treated with 0.1 mM IOA for 15 min and L. corniculatus protoplasts were treated with UV at 0.15 J/cm2 for 10 min. Various parameters affecting the fusion percentage were investigated. Successful fusions were obtained when the fusions were conducted on a plastic surface with 35% PEG (3350 MW) for 25 min duration, followed by 100 mM calcium chloride treatment for 25 min. A total of 14 putative fusion colonies were recovered. Shoots were regenerated from 8 fusion colonies. Unexpectedly, the regenerated putative hybrid plants resembled L. corniculatus plants. The flow cytometric profile of the putative somatic hybrids resembled that of L. corniculatus. Molecular analysis using SD-AFLP, SCARs and Lolium specific chloroplast microsatellite markers suggest that the putative somatic hybrids could be L. corniculatus escapes from the asymmetric protoplast fusion process. This thesis details a novel Whole Genome Amplification technique for plants using Strand Displacement Amplification technique.

Lolium perenne is one of the most important forage crops globally and in New Zealand. Lotus corniculatus is a dicotyledonous forage that contains valuable traits such as high levels of condensed tannins, increased digestibility, and high nitrogen fixing abilities. However, conventional breeding between these two forage crops is impossible due to their markedly different taxonomic origin. Protoplast fusion (somatic hybridisation) provides an opportunity for gene introgression between these two species. This thesis describes the somatic hybridisation, the regeneration and the molecular analysis of the putative somatic hybrid plants obtained between L. perenne and L. corniculatus. Callus and cell suspensions of different cultivars of L. perenne were established from immature embryos and plants were regenerated from the callus. Of the 10 cultivars screened, cultivars Bronsyn and Canon had the highest percentage of callus induction at 36% each on 5 mg/L 2,4-D. Removal of the palea and lemma which form the seed coat was found to increase callus induction ability of the embryos. Plant regeneration from the callus was achieved when the callus was plated on LS medium supplemented with plant growth regulators at different concentrations. Variable responses to shoot regeneration was observed between the different cultivars with the cv Kingston having the lowest frequency of shoot formation (12%). Different factors affecting the protoplast isolation of L. perenne were investigated. The highest protoplast yield of 10×10⁶ g⁻¹FW was obtained when cell suspensions were used as the tissue source, with enzyme combination 'A' (Cellulase Onozuka RS 2%, Macerozyme R-10 1%, Driselase 0.5%, Pectolyase 0.2%), for 6 h incubation period in 0.6 M mannitol. Development of microcolonies was only achieved when protoplasts were plated on nitrocellulose membrane with a L. perenne feeder layer on PEL medium. All the shoots regenerated from the protoplast-derived calli were albino shoots. The highest protoplast yield (7×10⁶ g⁻¹FW) of L. corniculatus was achieved from cotyledons also with enzyme combination 'A' (Cellulase Onozuka RS 2%, Macerozyme R-10 1%, Driselase 0.5%, Pectolyase 0.2%), for 6 h incubation period in 0.6 M mannitol. The highest plating efficiency for L. corniculatus of 1.57 % was achieved when protoplasts were plated on nitrocellulose membrane with a L. perenne feeder layer on PEL medium. The highest frequency of shoot regeneration (46%) was achieved when calli were plated on LS medium with NAA (0.1 mg/L) and BA (0.1 mg/L). Protoplast fusion between L. perenne and L. corniculatus was performed using the asymmetric somatic hybridisation technique using PEG as the fusogen. L. perenne protoplasts were treated with 0.1 mM IOA for 15 min and L. corniculatus protoplasts were treated with UV at 0.15 J/cm² for 10 min. Various parameters affecting the fusion percentage were investigated. Successful fusions were obtained when the fusions were conducted on a plastic surface with 35% PEG (3350 MW) for 25 min duration, followed by 100 mM calcium chloride treatment for 25 min. A total of 14 putative fusion colonies were recovered. Shoots were regenerated from 8 fusion colonies. Unexpectedly, the regenerated putative hybrid plants resembled L. corniculatus plants. The flow cytometric profile of the putative somatic hybrids resembled that of L. corniculatus. Molecular analysis using SD-AFLP, SCARs and Lolium specific chloroplast microsatellite markers suggest that the putative somatic hybrids could be L. corniculatus escapes from the asymmetric protoplast fusion process. This thesis details a novel Whole Genome Amplification technique for plants using Strand Displacement Amplification technique.

Solos ácidos com toxidez por Al são comuns, sendo geralmente destinados à pecuária. A utilização de leguminosas forrageiras adaptadas a esta situação minimizaria o uso de correção de acidez e ainda possibilitaria um aporte de N ao sistema via fixação biológica. A caracterização e a identificação de mecanismos de tolerância ao Al tóxico são os passos iniciais em um programa de seleção e melhoramento visando maior adaptação a esta condição de estresse. Este trabalho objetivou: (i) caracterizar genótipos de cornichão (L. corniculatus), espécies diplóides, inclusive da espécie modelo L. japonicus e linhas endogâmicas recombinantes (LER) de Lotus, quanto à tolerância ao Al tóxico; (ii) identificar o acúmulo de Al e exsudação radicular de ácidos orgânicos nas espécies cultivadas; e (iii) selecionar genótipos com respostas contrastantes ao Al tóxico, comparando-os através de caracteristicas agronômicas e moleculares. Os resultados da caracterização mostraram que as espécies modelo diplóides MG-20 e GIFU, a população UFRGS, e as cvs. San Gabriel e São Gabriel foram as mais produtivas de maneira geral. A população UFRGS apresentou ainda uma grande superioridade em situações de moderada acidez do solo (22,2% de Al), demonstrando um potencial de utilização em regiões onde se realize correção parcial do solo. Das 180 LERs testadas, 24 foram superiores e 39 foram inferiores a espécie modelo GIFU. Com relação à exsudação radicular de ácidos orgânicos, na ausência de Al observaram-se baixos níveis de exudação para todos os genótipos. Já na presença do Al, os resultados mostraram que o genótipo UFRGS, selecionado para tolerância ao Al (UF-T2), apresentou aumento significativo (pelo menos 50% superior) da exudação de ácido oxálico em relação aos demais genótipos, assim como a população UFRGS mostrou-se superior à cv. Draco em todas as avaliações. O resultado de dois ciclos de seleção massal visando tolerância ao Al mostrou incrementos na produção de matéria seca em todas as concentrações de Al testadas, enquanto que a seleção para sensibilidade ao Al pareceu estar relacionada com o baixo vigor das plantas, uma vez que na ausência de Al estes genótipos apresentaram menor acúmulo de matéria seca em relação às populações originais. A grande diversidade observada nos genótipos avaliados pode indicar que mecanismos de tolerância ao Al possam atuar em diferentes intensidades. A exudação de ácido oxálico parece ser um mecanismo que permite a manutenção do crescimento do cornichão em condições de Al tóxico, sendo que a seleção de um genótipo mais tolerante ao Al proporcionou aumentos significativos neste mecanismo de defesa.
Acid soils with aluminum (Al) toxicity are common, being generally destined for cattle livestock. The utilization of forage legumes adapted to this situation would minimize the use of agricultural liming materials and still allow a nitrogen input to the system via biological fixation. The characterization and identification of tolerance mechanisms to toxic Al are the initial steps in a selection and breeding program aiming a greater adaptation to this stress condition. This work was aimed to: (i) characterize cultivated populations of birdsfoot trefoil (tetraploid), model species (diploid) and recombinant inbred lines (RIL) of Lotus, regarding toxic Al tolerance; (ii) identify the accumulation of Al and root exudation of organic acids in the cultivated species; and (iii) select genotypes with contrasting responses to the toxic Al, comparing their morphological and molecular characteristics. The results of the characterization showed that the diploid genotypes, MG-20 and GIFU, and the cultivated UFRGS genotypes, San Gabriel and São Gabriel were the most productive in a general matter. The UFRGS genotype also presented a great superiority in situations of moderate soil acidity (22,2% of Al), demonstrating a utilization potential in regions where partial soil amelioration is done. Of the180 RILs tested, 24 were superior and 39 were inferior to the GIFU genotype. Regarding root exudation of organic acids, in the absence of aluminum, low levels of exudation were observed for all genotypes. However, in the presence of aluminum, the results showed that the UFRGS genotype selected for Al tolerance presented a significant increase (at least 50% higher) of oxalic acid exudation compared to the other genotypes, and the UFRGS genotype proved superior to the Draco genotype in all evaluations. The result of two mass selection cycles aiming Al tolerance showed increments in dry matter production in all of the toxic aluminum concentrations tested, while the selection for Al sensibility seemed to be related to the plants’ low vigor, since in the absence of Al these genotypes presented a lower accumulation of dry matter compared to the original populations. The great diversity observed in the evaluated genotypes may indicate that the Al tolerance mechanisms may act in different intensities. The exudation of oxalic acid is apparently a mechanism that permits the maintenance of the birdsfoot trefoil growth in different conditions of toxic Al, as well as the selection of a more tolerant genotype to aluminum permitted a significant increase in this defense mechanism.

The usefulness of RAPDs (Random Amplified Polymorphic DNA) to distinguish among different taxa of Lotus was evaluated. The following species were included: L. corniculatus, L. tenuis, L. alpinus, L. japonicus, and L. uliginosus. Several accessions for each species were studied. Following DNA extraction, amplification reactions were performed in a Hybaid DNA Thermal Cycler, and the product visualized according to a standard procedure. Twenty primers were used for each species/accession. Clear bands and several polymorphisms were obtained for all primers. A phenogram was drawn based on the genetic distance among the species. L. alpinus appears as the most distant species from L. corniculatus, followed by L. uliginosus, L. tenuis, and L. japonicus. With the exception of L. alpinus, these findings are in agreement with previous experimental studies in the L. corniculatus group. The use of a greater number of primers and increased number of species may provide a greater resolution of the systematics of these taxa.

A method was developed for rapid plant regeneration from protoplasts of birdsfoot trefoil (Lotus corniculatus cv. Leo) using in vitro cotyledons and a new enzyme formula. Protoplasts of a transgenic Nicotiana tabacum cv. Xanthi line KCR were isolated from leaves, in vitro shoots, and calli by an enzyme formula consisting of 0.5 % Cellulase R-10 and 0.05 % Pectolyase Y23 with either 0.4 M sucrose or 0.5 M mannitol as the osmoticum. Nine asymmetric fusion experiments were conducted between iodoacetate inactivated birdsfoot trefoil and irradiated transgenic tobacco protoplasts. The fusion products underwent several divisions but no visible colonies were obtained. Resistance analyses revealed the inactivation of the herbicide resistant gene in the transgenic tobacco calli. A putative chlorsulfuron resistant line has been obtained by in vitro selection from birdsfoot trefoil cotyledonous protoplasts. Their regenerants had normal ploidy, and expressed improved tolerance when sprayed with chlorsulfuron at the rate of 30 g/ha.

Recentes estudos têm mostrado que os rizóbios podem colonizar raízes, caules e folhas de gramíneas, promovendo o crescimento da planta. A produção de fito-hormônios, principalmente ácido indol acético (AIA) é, possivelmente, o principal mecanismo de promoção de crescimento de gramíneas por rizóbios. O objetivo do trabalho foi quantificar a produção de AIA por rizóbios e avaliar o efeito da inoculação destes rizóbios na germinação e no crescimento vegetal de azevém, braquiária, capim Tanzânia e pensacola. Foram estudados sete rizóbios nativos de solos do Rio Grande do Sul, isolados de nódulos de Lotus corniculatus e a estirpe SEMIA816, recomendada para L. corniculatus. Para quantificação da produção de AIA, os rizóbios foram cultivados em meio levedura-manitol (LM), com e sem adição de triptofano. Foram conduzidos experimentos em laboratório e em casa de vegetação, para se avaliar a capacidade das bactérias em promover crescimento vegetal. Todos os rizóbios testados foram capazes de produzir AIA. Todos os rizóbios foram capazes de promover crescimento vegetal em ao menos uma das gramíneas estudadas. Em laboratório, a germinação do azevém foi antecipada em todos os tratamentos inoculados. O capim Tanzânia e pensacola foram as espécies mais responsivas à inoculação com os rizóbios estudados nos experimentos em casa de vegetação, parecendo haver certa compatibilidade na interação gramínea/rizóbio. Há baixa similaridade genética entre os rizóbios estudados, os quais pertencem aos gêneros Mesorhizobium e Bradyrhizobium.
Recent studies have shown that rhizobia can colonize roots, stems and leaves of grasses, promoting plant growth. The production of phyto-hormones, mainly indole acetic acid (IAA) is possibly the main mechanism of growth promotion of grasses by rhizobia.The objective of this work was to quantify the production of IAA by rhizobia, and evaluate the effect of rhizobia inoculation on germination and plant growth of ryegrass, Brachiaria, Tanzânia grass and Pensacola. In this work were studied seven rhizobia native from Rio Grande do Sul soils, isolated from nodules of Lotus corniculatus and the strain SEMIA816 recommended for L. corniculatus. To quantify the production of IAA, the strains were cultured in yeast-mannitol (LM) medium, with and without tryptophan addition. Experiments were carried out in laboratory and under greenhouse conditions to evaluate the capacity of these bacteria to promote plant growth promotion. All rhizobia tested were able to produce IAA. All strains were able to promote plant growth in at least one of the grasses studied. In laboratory conditions, the ryegrass seed germination was anticipated in all inoculated treatments. Tanzania and pensacola grasses were the most responsive to rhizobial inoculation in greenhouse experiments, there appears to be certain specificity in interaction graminea / rhizobia. There is low genetic similarity between the rhizobia studied, which belong to the genera Mesorhizobium and Bradyrhizobium.

Glutamine synthetase (GS), the enzyme responsible for the first step in the assimilation of ammonium in higher plants, generally exists in a number of isoforms associated with different tissues or cellular compartments within the plant. This study has investigated the GS isoenzyme composition of roots and N-fixing root nodules of the temperate legume, Lotus comiculatus, and has used a novel transgenic approach to manipulate the spatial distribution of the enzyme within these tissues. The isoforms of GS were studied using ion-exchange chromatography and western blotting, and a nodule-specific isoform was identified. Nitrate treatment of the N2-fixing plants had a marked effect on the nodule isoform, converting it to a form that was indistinguishable by ion-exchange chromatography from the root isoenzyme. To allow tissue-specific manipulation of GS activity, a chimaeric gene was constructed consisting of a translational fusion between the pat and uidA genes coding for phosphinothricin acetyl transferase (PAT) and β-glucuronidase (GUS), respectively. The PAT enzyme detoxifies the GS inhibitor, phosphinothricin, while GUS is a readily assayable marker enzyme that allowed the localisation of PAT activity within the plant tissues to be inferred. The pat::uidA gene was shown to encode a bifunctional enzyme when expressed in E. coli and in transgenic L. comiculatus plants. Transgenic lines carrying a nodule-specific promoter fused to pat::uidA were resistant to PPT only when nodulated. In some of these lines, nodule GS activity was completely resistant to soil applications of PPT under conditions where root GS was 100% inhibited. After long-term PPT treatment, one line (12E) showed a two-fold increase in nitrogenase activity, a four-fold increase in GS activity, and a 50% increase in dry matter production. Possible explanations for how specific inhibition of root GS led to these effects are discussed.

Gene transfer vectors based on the Agrobacterium tumefaciens Ti plasmid were used to develop a successful disarmed Agrobacterium tumefaciens-mediated transformation method for Lotus comiculatus. A binary vector construct, pJIT73, was used during the development of the Agrobacterium tumefaciens transformation system due to its selectable (Aph IV, nos- neo) and scorable markers. The effects of the antibiotics geneticin (G-418) and hygromycin B were studied. Use of kill curves and selection delay experiments allowed potentially suitable selection pressure parameters to be proposed. Using such selection during transformation experiments led to further optimisation of this stage of transformation. The influence of plant hormones on the regeneration of Lotus comiculatus explants was investigated and a modification of an established protocol using leaf explants was introduced as an attempt to reduce the overall time of regeneration. Various explants were used but leaf pieces were chosen as the most suitable explant on which to focus research. So, through alteration of various stages, including length of cocultivation and subsequent decontamination within the transformation process, a successful method was developed. Experiments indicated the optimum Agrobacterium tumefaciens strain to be used with Lotus comiculatus was the disarmed Ach5 type, LBA4404(pAL4404). Transgenic Lotus comiculatus plants were produced which expressed the scorable marker β-Glucuronidase gene (GUS) and the selectable marker for hygromycin B resistance, AphIV. Gene transfer was confirmed by Southern blotting. The new Agrobacterium tumefaciens-mediated vector system was used to introduce the cowpea trypsin inhibitor gene (CpTi) into Lotus comiculatus. However, although there was evidence for transformed callus development, no shoots were induced. By the use of previously established Agrobacterium rhizogenes-mediated system, an attempt was made to introduce the pea lectin gene (psl) into Lotus corniculatus. Hairy root regenerants were produced but genetic transfer was unconfirmed and attempted investigation of the plant - Rhizobium symbiosis involving Lotus corniculatus was not fulfilled.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde. Programa de Pós-Graduação em Farmácia.
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A Lotus corniculatus (Fabaceae) é distribuída em muitas regiões do mundo e possui grande valor agronômico por seus constituintes: proantocianidinas, flavonoides e ácido oleanólico. Esta planta é utilizada como alimento para o gado proporcionando o aumento no ganho de peso e na produção de leite, além do controle de infecções intestinais parasitárias nos animais. O objetivo deste trabalho foi avaliar o efeito anti-inflamatório e antibacteriano do extrato bruto (CE) da Lotus corniculatus v. São Gabriel, frações (hexano (HEX), acetato de etila (AcOEt), butanólica (BuOH) e resíduo aquoso (AF)), e compostos isolados (canferitrin, ácido oleanólico e ß-sitosterol). As partes aéreas da Lotus corniculatus foram secas a temperatura ambiente, e a partir desse material (620 g), foi extraído o extrato bruto (CE) por maceração com etanol 96% por um mês. O solvente foi evaporado resultando em 78 g de extrato bruto (CE). O extrato bruto foi fracionado por extração líquido-líquido utilizando solventes em ordem crescente de polaridade resultando nas frações hexano (HEX), acetato de etila (AcOEt), butanólica (BuOH) e resíduo aquoso (AF). A partir da fração HEX foram isolados o ácido oleanólico e o ß-sitosterol, e a partir da fração AcOEt o canferitrin. Para a avaliação da atividade anti-inflamatória foi utilizado o modelo de pleurisia induzida pela carragenina, em camundongos, e foram avaliados os seguintes parâmetros inflamatórios: concentrações de leucócitos, exsudação, atividade da mieloperoxidase (MPO) e da adenosina-deaminase (ADA), além das concentrações de nitrito/nitrato (NOx) e interleucina-1 beta (IL-1 ). Nesse protocolo experimental, foram utilizados camundongos albinos suíços, os quais foram distribuídos em diferentes grupos e tratados com CE (100 - 400 mg/kg), HEX (50 - 200 mg/kg), AcOEt (100 - 400 mg/kg), BuOH (50 - 200 mg/kg), AF (25 - 200 mg/kg), canferitrin (50 - 100 mg/kg), ácido oleanólico (10 - 100 mg/kg) ou ß-sitosterol (10 - 100 mg/kg) 0,5 h antes da administração da carragenina (1%, i.pl.). Os parâmetros inflamatórios foram avaliados 4 h após. Todos os animais, exceto nos experimentos que foram analisadas as atividades enzimáticas, foi administrado previamente (10 min.) Azul de Evans (25 mg/kg, i.v.) a fim de avaliar a exsudação. Dexametasona e indometacina foram utilizados como fármacos anti-inflamatórios de referência. Para a avaliação da atividade antibacteriana, foi utilizada a técnica de microdiluição em caldo para a determinação da concentração inibitória mínima (CIM) e da concentração bactericida mínima (CBM). Neste protocolo, o material vegetal, o extrato bruto (CE), frações e compostos isolados, foram dissolvidos em dimetilsulfóxido (DMSO) e transferidos para uma placa em diluição seriada e, a seguir, o inóculo bacteriano foi adicionado. Gentamicina foi utilizada como fármaco antibacteriano de referência. Diferenças estatísticas entre os grupos, para os parâmetros inflamatórios, foram determinadas pela análise dos testes t de Student ou de variância (ANOVA). Valores de P < 0,05 foram considerados significativos. O extrato bruto da Lotus corniculatus (200 - 400 mg/kg) e frações (50 - 200 mg/kg), assim como os compostos isolados (25 - 100 mg/kg) inibiram: leucócitos, neutrófilos, exsudação, a atividade da MPO e da ADA, além das concentrações de NO e IL-1 (P < 0,05). A indometacina e a dexametasona inibiram todos os parâmetros inflamatórios estudados (P < 0,05). Em relação à atividade antibacteriana, somente as frações HEX e AcOEt, e os compostos isolados ácido oleanólico e canferitrin, revelaram atividade bactericida e/ou bacteriostática. A fração HEX demonstrou moderada atividade antibacteriana (CIM = 100 µg mL-1) para a bactéria Bacillus cereus, e fraca atividade antibacteriana (CIM = 600 a 1000 µg mL-1) para as bactérias Enterococcus faecalis, Staphylococcus aureus, Listeria monocytogenes, Staphylococcus epidermidis, Providencia alcalifaciens e Acinetobacter calcoaceticus. A fração AcOEt demonstrou fraca atividade antibacteriana (CIM = 800 µg mL-1) para as bactérias Enterococcus faecalis, Bacillus cereus e Acinetobacter calcoaceticus. O ácido oleanólico revelou boa atividade antibacteriana para Staphylococcus aureus meticilina resistente (CIM = 100 µg mL-1),Listeria monocytogenes e Bacillus cereus (CIM = 25 µg mL1), e o canferitrin demonstrou boa atividade antibacteriana para as bactérias: Staphylococcus epidermidis, Shigella flexinerii, Salmonella typhimurium e Acinetobacter calcoaceticus (CIM = 100 µg mL-1). Este composto demonstrou excelente atividade antibacteriana para as bactérias Enterococcus faecalis (CIM = 3.9 µg mL-1) e Bacillus cereus (CIM = 8.5 µg mL-1). O extrato bruto, frações e compostos isolados da Lotus corniculatus v. São Gabriel demonstraram importante atividade anti-inflamatória e antibacteriana. Os compostos isolados: canferitrin, ácido oleanólico e ß-sitosterol podem ser responsáveis por estes efeitos anti-inflamatório e/ou antibacteriano. The Lotus corniculatus (Fabaceae) is distributed in many regions of the world and has a high agronomic value for its constituted: proanthocyanidins, flavonoids and oleanolic acid. This plant is used as food for cattle providing the enhancement of the weight and of milk production beyond the control of intestinal parasitic infections in the animals. The aim of this study was to evaluate the anti-inflammatory and anti-bacterial effects of crude extract (CE), fractions (hexane (HEX), ethyl acetate (AcOEt), n-butanol (BuOH) and aquous fraction (AF)), and isolated compounds (kaempferitrin, oleanolic acid and ß-sitosterol) from Lotus corniculatus v. São Gabriel. The aerial parts of Lotus corniculatus were dried at room temperature and this material (620 g) the crude extract (CE) was extracted by maceration with ethanol 96% for one month. The solvent was evaporated resulting in 78 g of crude extract (CE). The crude extract was fractionated by liquid-liquid extraction using solvents in growing order of polarity resulting in hexane (HEX), ethyl acetate (AcOEt), n-butanol (BuOH) and aqueous fraction (AF). From the HEX fraction it was isolated the oleanolic acid and the ß-sitosterol and from AcOEt fraction the kaempferitrin. To evaluate the anti-inflamatory activity, it was used the mouse model of pleurisy induced by carrageenan and the following inflammatory parameters were evaluated: leukocytes, exudation, myeloperoxidase (MPO) and adenosine-deaminase (ADA) activities, as well as, nitrite/nitrate (NOx) and interleukin-1 beta (IL-1ß) levels. In this experimental protocol, swiss mice were used in the in vivo experiments, which were distributed in different groups and they were treated with CE (100 - 400 mg/kg), HEX (50 - 200 mg/kg), AcOEt (100 - 400 mg/kg), BuOH (50 - 200 mg/kg), RA (25 - 200 mg/kg), kaempferitrin (50 - 100 mg/kg), oleanolic acid (10 - 100 mg/kg) or ß-sitosterol (10 - 100 mg/kg) 0,5 h before carrageenan (1%,i.pl.). The inflammatory parameters were evaluated 4 h after. All animals, except in the experiments that were analyzed enzymatic activities, the animals were previously pretreated (10 min.) with Evans blue dye (25 mg/kg, i.v.) in order to evaluate the exudation. Dexamethasone and indomethacin were used as anti-inflammatory reference drugs. To evaluate the anti-bacterial activity, it was used the microdilution in broth technical to determine the minimal inhibitory concentraton (MIC) and the minimal bactericidal concentration (MBC) methodology. In this protocol the crude extract (CE), fractions and isolated compounds were dissolved in dimethylsulfoxide (DMSO) and transferred to plate in serial dilution and, after, the bacterial inoculum were added. The gentamicin was used as the anti-bacterial reference drug. To the anti-inflammatory parameters, statistical differences between groups were determined by Student's t test and analysis of variance (ANOVA). Values of P < 0.05 were considered significant. The crude extract of Lotus corniculatus (200 - 400 mg/kg) and fractions (50 - 200 mg/kg), as well as its isolated compound (25 - 100 mg/kg) inhibited: lekocytes, neutrophils and exudation, the MPO and ADA activities, as well as NO and IL-1 levels (P < 0.05). Indomethacin and dexamethasone inhibited all the studied inflammatory parameters (P < 0.05). In regard to the anti-bacterial activity, only the HEX and AcOEt fractions and the isolated compounds, oleanolic acid and kaempferitrin, revealed anti-bacterial and/or bacteriostatic activities. The hexane fraction showed moderate anti-bacterial effect (MIC = 100 µg mL-1) on Bacillus cereus and weak anti-bacterial effect (MIC = 600 to 1000 µg mL-1) on Enterococcus faecalis, Staphylococcus aureus, Listeria monocytogenes, Staphylococcus epidermidis, Providencia alcalifaciens and Acinetobacter calcoaceticus. The AcOEt fraction demonstrated a weak anti-bacterial activity (MIC = 800 µg mL-1) on Enterococcus faecalis, Bacillus cereus and Acinetobacter calcoaceticus.The Oleanolic acid, isolated from hexane fraction, exhibited a good anti-bacterial activity on methycillin-resistant Staphylococcus aureus (MIC = 100 µg mL-1), Listeria monocytogenes and Bacillus cereus (MIC = 25 µg mL-1) and the kaempferitrin, a compound isolated from this fraction, demonstrated a good anti-bacterial effect on: Staphylococcus epidermidis, Shigella flexinerii, Salmonella typhimurium and Acinetobacter calcoaceticus (MIC = 100 µg mL-1). This compound showed an excellent anti-bacterial activity on Enterococcus faecalis (MIC = 3.9 µg mL-1) and Bacillus cereus (MIC = 8.5 µg mL-1). The crude extract, fraction and isolated compound from Lotus corniculatus v. São Gabriel, demonstrated important anti-inflammatory and anti-bacterial activities. The isolated compounds: kampferitrin, oleanolic acid and ß-sitosterol can be responsible for these anti-inflammatory and/or anti-bacterial effects.

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Atualmente o Rio Grande do Sul tem a alimentação pecuária baseada quase que exclusivamente em pastagens de elevado teor forrageiro. O cornichão (Lotus corniculatus L.) é uma forrageira que tem se destacado na região da campanha devido sua grande resistência ao frio e às geadas, porém, é uma semente muito pequena e leve, o que dificulta o uso e regulagem de tecnologias agrícolas. O recobrimento de sementes é uma técnica de beneficiamento que tem se apresentado promissora para corrigir falhas e melhorar as características de lotes de sementes, remover contaminantes, entre outros. O leito de jorro é um equipamento que pode ser utilizado no recobrimento de sementes pois seu movimento cíclico aliado com a atomização proporciona uma boa adesão da suspensão à partícula e uma secagem adequada resultando em uma cobertura regular e uniforme. Com isso, o presente trabalho apresentou como objetivo realizar o estudo do recobrimento de sementes de cornichão em leito de jorro. Para a realização deste trabalho foram utilizadas sementes de cornichão cedidas pela Embrapa Pecuária Sul (Bagé, RS). A metodologia consistiu em caracterizar física, fisiológica e morfologicamente as sementes; estudar diferentes formulações baseadas em componentes poliméricos para a suspensão de recobrimento; estudar a fluidodinâmica das sementes em leito de jorro; estudar o processo de recobrimento das sementes em leito de jorro e avaliar a qualidade do produto final. Para avaliar o recobrimento foi utilizado um planejamento experimental do tipo composto central rotacional (DCCR), tendo como variáveis independentes a temperatura do ar e a vazão da suspensão e como respostas a eficiência do processo, o crescimento das partículas e o peso de mil sementes (PMS). Os resultados obtidos para a fluidodinâmica mostraram que a carga mássica que melhor se adequou ao processo foi 500 g de sementes; os filmes poliméricos formados após o recobrimento em leito de jorro proporcionaram a obtenção de uma fina película sobre as sementes; os resultados da eficiência do processo foram entre 6 a 50 %, para o crescimento da partícula entre 0,3 a 2,9 % e para o PMS entre 1,10 a 1,52 g. A avaliação estatística permitiu verificar o efeito das condições experimentais (temperatura do ar e vazão da suspensão) sobre as respostas avaliadas. Considerou-se como mais satisfatório o experimento que proporcionou maiores valores para a eficiência do processo (50,36 %) e crescimento das partículas (2,91 %), provenientes do recobrimento com temperatura de 75 °C e vazão da suspensão de 13,4 mL/min. Através das análises realizadas para o produto final verificou-se que houve um incremento no tamanho e no peso das sementes. Também observou-se que as sementes não foram prejudicadas fisiologicamente pois não foram danificadas suas reservas de substâncias e estrutura justificando a realização deste trabalho.
Nowadays, Rio Grande do Sul has livestock feed based almost exclusively on high forage pastures. The birds foot trefoil (Lotus corniculatus L.) is a forage that has stood out in the region of the campaign due to its great resistance to the cold and the frosts, but it is a very small and light seed, which makes difficult the use and regulation of agricultural technologies. The seed coating is a beneficiation technique that has been promising to correct faults and to improve the characteristics of seed lots, to remove contaminants, among others. The spouted bed is an equipment that can be used in the coating of seeds because its cyclic movement combined with the atomization provides a good adhesion of the suspension to the particle and a suitable drying resulting in a regular and uniform coverage. Thus, the present study presented aimed at making the study of coating birdsfoot trefoil seeds in spouted bed. For the accomplishment of this work we used seeds of birds foot trefoil ceded by Embrapa Pecuária Sul (Bagé, RS). The methodology consisted in the physical, physiological and morphological characterization of the seeds; studying different formulations based on polymer components for the coating suspension; to study the fluid dynamics of the seeds in the spouted bed; to study the process of coating the seeds in the spouted bed and to evaluate the quality of the final product. In order to evaluate the coating, an experimental design of the central rotational compound type (DCCR) was used, having as independent variables the air temperature and the suspension flow rate and as responses to process efficiency, particle growth and the weight of one thousand seeds (PMS). The results obtained for the fluid dynamics showed that the mass load that best suited the process was 500 g of seeds; the polymer films formed after the spouted bed coating provided a thin film on the seeds; the results of the process efficiency were between 6 to 50 %, for particle growth between 0,3 to 2,9 % and for PMS between 1,10 to 1,52 g. The statistical evaluation allowed to verify the effect of the experimental conditions (air temperature and suspension flow) on the evaluated responses. It was considered as more satisfactory the experiment that provided higher values for the process efficiency (50,36 %) and particle growth (2,91 %), from the coating with a temperature of 75 °C and a flow rate of the suspension of 13,4 mL/min. Through the analyzes performed for the final product it was verified that there was an increase in the size and the weight of the seeds. It was also observed that the seeds were not harmed physiologically because their reserves of substances and structure were not damaged justifying the accomplishment of this work.

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A crescente necessidade da utilização de espécies forrageiras de estação fria na região sul do país vem determinando uma grande demanda por sementes de qualidade. Entre as leguminosas se destaca o cornichão (Lotus corniculatus L.), que é uma leguminosa forrageira oriundo da Europa e da região mediterrânea. Esta leguminosa forrageira tem na versatilidade a sua maior vantagem, visto que apresenta produção de forragem na primavera, no verão e no outono, possui resistência à seca e ao encharcamento temporário do solo e apresenta boa persistência da massa verde. Além disso, as sementes deste tipo de espécie podem ser misturadas com espécies de inverno, o que aumenta o rendimento da matéria seca, dá uma melhor distribuição da forragem ao longo do ano e agrega uma melhora na qualidade das pastagens. Apesar dessas vantagens, as sementes de cornichão comercializadas possuem ainda uma baixa qualidade. Esse fato é decorrente de um sistema de fiscalização ineficiente, associado à dificuldade de produção e beneficiamento do produto. Outro problema que o produtor de sementes desta espécie enfrenta é a dormência tegumentar, isto é, o revestimento da semente (tegumento) é impermeável e não permite que a água necessária para a germinação chegue ao interior da semente. Na literatura, diversos trabalhos têm relatado que cada vez mais há um aumento na demanda de sementes forrageiras em função do melhoramento do campo nativo e das pastagens cultivadas. Diante de um mercado tão promissor, a obtenção de sementes de melhor qualidade ainda apresenta dificuldades que vão desde a fase de produção no campo, onde as sementes são colhidas precocemente em relação ao ponto de maturidade fisiológica devido ao processo de perda natural, até seu beneficiamento, onde o processo de secagem apresenta-se como uma etapa limitante que necessita de uma atenção muito especial para evitar perdas fisiológicas no produto final. De um modo geral, a secagem é definida como a remoção, voluntária ou não, total ou parcial, de uma fase líquida ou gasosa de qualquer material através da transferência de calor e de massa. A secagem tem em sua essência a finalidade de assegurar a conservação de um produto por meio da redução do seu teor de água. No caso de materiais orgânicos, como é o caso de sementes, a redução no teor de água deve ser efetuada até o ponto em que a concentração de açúcares, ácidos, sais e outros componentes, sejam suficientemente elevados para reduzir a atividade de água e inibir, portanto, o desenvolvimento de micro-organismos. Na tecnologia de produção de sementes de alta qualidade a secagem é tida com uma das etapas de maior importância, pois permite a redução do teor de água em níveis adequados para o armazenamento e comercialização (12 a 15 % de umidade), preserva as sementes de alterações físicas e químicas induzidas pelo excesso de umidade, torna possível a manutenção da qualidade inicial durante o armazenamento e possibilita colheitas próximas da maturação fisiológica. Entretanto, o processo de secagem, seja ele qual for, deve acontecer de forma controlada e de maneira uniforme a fim de evitar elevados gradientes de umidade e temperatura no interior do material que podem provocar a perda da qualidade do produto final. De acordo com o que foi exposto, o presente trabalho de mestrado teve como objetivo realizar um estudo sobre o processo de secagem de sementes de cornichão (Lotus corniculatus L.) em um secador de leito fixo com fluxo de ar paralelo. Para alcançar tal objetivo, este trabalho foi dividido nas seguintes etapas: caracterização física e fisiológica das sementes, análise do processo de reumidificação das sementes, estudo das isotermas de dessorção das sementes reumidificadas e avaliação dos efeitos da variação da temperatura, da velocidade do ar e da altura da bandeja sobre a cinética de secagem e qualidade fisiológica do produto final. Os dados experimentais mostraram que os resultados obtidos para a umidade em base úmida das sementes secas ficaram na faixa de 5 % para as secagens realizadas a 60 °C e em aproximadamente 8 % para as secagens realizadas a 40 °C. No que se refere à germinação, os valores encontrados estão na ordem de 44 a 59 % para as secagens realizadas neste estudo. Dentre as secagens, a que obteve o maior valor de germinação, com valor de 59 %, foi a realizada na condição de T = 40 °C, v = 1,0 m/s e h = 1,0 cm.
The growing necessity for the use of cool-season forage species in the southern region of the country has indicated a high demand for quality seeds. Among the legumes, the birdsfoot trefoil (Lotus corniculatus L.) is a forage legume which is originated in Europe and in the Mediterranean region. This versatility in the forage legume has its biggest advantage, since it presents forage production in the spring, summer and autumn, is resistant to drought and temporary waterlogging and shows good persistence of green mass. Furthermore, seeds of such kind can be mixed with winter species, which increases the yield of dry matter, gives a better distribution of the material throughout the year and adds an improvement in the quality of pastures. Despite these advantages, the seeds of birdsfoot marketed still have a poor quality. This fact is a result of an inefficient system of monitoring and difficulties associated to production and processing of the product. Another problem is that the seed producer of this species is facing cutaneous numbness, that is, the seed coat (tegument) is impermeable and does not allow the water, necessary for germination to reache the interior of the seed. In the literature, several studies have reported that there is an increasing demand for an increase in forage seed due to the improvement of native grassland and cultivated pastures. Faced with such a promising market, obtaining better quality seed still presents difficulties ranging from the production phase of the field, where the seeds are harvested early in relation to physiological maturation due to the natural loss process until its processing where the drying process is presented as a limiting step that requires particular attention to avoid losses in physiological final product. In general, drying is defined as the removal voluntary or not, full or partial, of a liquid or gaseous phase of any material by the transfer of heat and mass. Drying has its essence in order to ensure the preservation of a product through reduction of water content. In the case of organic materials, such as seeds, reduction in water content must be performed to the extent that the concentration of sugars, acids, salts and other components are sufficiently high to reduce the water activity and inhibit, therefore, the growth of microorganisms. In the production of high quality seeds the drying technology is seen with one of the most important steps, since it allows reducing the water content suitable for storage and marketing levels (12 to 15% moisture), preserves the seeds from physical changing and chemical induced excess moisture, makes it possible to maintain the initial quality during storage and enables the next harvest physiological maturity. However, the drying process, whatever it may be, should occurs in a controlled and uniform manner in order to avoid high temperature and humidity gradients within the material that could result in loss of quality of the final product. Towards to it, this paper aimed to conduct a study on the drying of seeds of birdsfoot trefoil (Lotus corniculatus L.) in a fixed bed dryer with parallel air flow. To accomplish this goal, this study was divided into the following steps: physical and physiological seed characterization, analysis of rewetting seed process, study of desorption isotherms of the rewetted seeds and evaluation the effects of temperature, air velocity and the height of the tray on the drying kinetics and physiological quality of the final product. The experimental data showed that the results for the dry seeds of UBU were in the range of 5 % to drying carried out at 60 ° C and about 8% to drying carried out at 40 ° C. With regard to seed germination, the values are found in a range of 44 to 59% for the drying carried out in this study. The drying condition whom presented the highest germination value about 59 % was performed on T = 40 ° C, v = 1,0 m/s and h = 1,0 cm.

Three studies applicable to organic management and cultivation of birdsfoot trefoil (BFT, Lotus corniculatus L.) are presented here. The first is a histological analysis of lignification in BFT stems that supports recommendations to harvest BFT at approximately 6 weeks of regrowth, or early bloom. Lignification decreases digestibility and is correlated with high shear force required to break BFT stems. The sixth internodes from the base of 10 BFT and two alfalfa plants were sampled during 15 weeks of midsummer regrowth. The lignified xylary ring reached its maximum radial width by 7.5 weeks of regrowth, and full bloom was reached by 6.5 weeks of regrowth. The second study evaluated establishment of BFT, which competes poorly with weeds and produces low yields during establishment. At an organic, irrigated site in northern Utah the effects of autumn vs. spring seeding, seeding rates of 3, 7 20, and 34 kg pure live seed (PLS) ha-1 and use of a companion crop on 3 years of annual yields and foliar cover of mature stands were determined. First year yields increased linearly with seeding rate (P≤0.05). Use of a companion crop reduced first year yields, and did not reduce weed cover. Autumn seeding of BFT alone, following harvest of a summer crop, is recommended for irrigated production. The final study evaluated BFT establishment on five organic dairy farms in souther Idaho and northern Utah. Participating producers broadcast seeded 4-ha BFT pastures in the fall at a rate of 25 kg PLS ha-1. All farms achieved high BFT density, but only two farms had higher BFT than weed density. These two farms also had high BFT cover the spring following autumn seeding, and their pastures produced 6000 to 7600 kg of dry matter ha-1 by 20 June 2012 and supported grazing for the remainder of the summer. Establishment was enhanced by crop rotation and sprinkler irrigation.

A commercial solid matric priming method and an osmotic priming method were used to measure seed priming responses of birdsfoot trefoil, kura clover, and sericea lespedeza. Differences were not observed using standard germination tests, but both priming methods show potential for increased germination rate (P>0.05). Conflicting results for matric and osmotic priming were found in terms of seed storage potential after priming, with matric primed seeds showing higher (P<0.05) germination after accelerated aging and osmotic primed seeds showing significant lower germination(P<0.01). Birdsfoot trefoil benefited from priming, but responses varied by priming treatment, while kura clover showed less response to both priming treatments. In a field study comparing matric primed vs. unprimed seedling emergence, matric priming effects were small and these data suggest that solid matrix priming may be unlikely to improve the field establishment of either species.

Aqueous smoke solutions were also tested for effect on seed germination. Differences in final germination percent due to solution type (after exposure to liquid smoke solutions for 10- or 45-min) were not observed. Highest concentration of the 10-min solution treatment reduced (P<0.05) birdsfoot trefoil germination. Greater germination was observed only for 'Perfect Fit' kura clover treated with low or intermediate concentrations of either solution. High concentrations of 10-min smoke water increased time to 50% germination (T50) for all seeds, but some reduction in T50 occurred for kura clovers treated with low (5%) solution concentrations. The 45-min treatments had little effect on germination rates. Applying aqueous smoke solution to seeds at germination did not improve germination responses of these forage legume species.

Master of Science

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O estado do Rio Grande do Sul tem grande destaque em atividades pecuarista como economia regional. Isto implica em uma crescente necessidade da utilização de espécies de sementes forrageiras de estação fria nesta região. Dentre as sementes forrageiras mais adaptáveis ao clima frio tem-se o cornichão (Lotus corniculatus L.), uma leguminosa forrageira hibernal, muito versátil, com alta tolerância a acidez do solo a qual é utilizada em ampla escala no Rio Grande do Sul. A semente é o veículo que leva ao agricultor todo o potencial genético de uma cultivar com características superiores, e em seu caminho, pequenas quantidades de sementes são multiplicadas até que sejam alcançados grandes volumes de produção, no decorrer do qual a qualidade dessas sementes estará sujeita a uma série de fatores capazes de causarem perda de todo potencial genético. Diante de um mercado tão promissor, a obtenção de sementes de melhor qualidade ainda apresenta dificuldades que vão desde a fase de produção no campo, onde as sementes são colhidas precocemente em relação ao ponto de maturidade fisiológica devido ao processo de perda natural, até seu beneficiamento, onde o processo de secagem apresenta-se como uma etapa limitante que necessita de uma atenção muito especial para evitar perdas fisiológicas no produto final. A operação de secagem aplicada a sementes tem a finalidade de assegurar a conservação deste produto por meio da redução do seu teor de água. Neste caso, onde trata-se de materiais orgânicos, a redução no teor de água deve ser efetuada de maneira bem controlada, necessitando um estudo e analise da operação aplicada ao material, já que a mesma se for realizada de maneira descontrolada pode acarretar na perda do material. Na tecnologia de produção de sementes de alta qualidade a secagem é tida com uma das etapas de maior importância, pois permite a redução do teor de água em níveis adequados para o armazenamento e comercialização (12 a 15 % de umidade), preserva as sementes de alterações físicas e químicas induzidas pelo excesso de umidade, torna possível a manutenção da qualidade inicial durante o armazenamento e possibilita colheitas próximas da maturação fisiológica. A utilização do secador de leito fluidizado para aplicação da operação de secagem vem sendo objeto de investigações cientificas, pois este secador apresenta já reconhecidas vantagens que vão desde alta capacidade de secagem pela eficiente transferência de massa e de energia, até redução de tempo de secagem pela rapidez na remoção da umidade e a facilidade de controles. Diante do exposto, o presente trabalho teve como objetivo uma modelagem matemática e aplicação de simulação da secagem em leito fluidizado, seguida de testes aplicados experimentalmente através de ensaios realizados em um secador de leito fluidizado confeccionado para este trabalho. Foram realizados estudos de caracterização física e fisiológica das sementes; modelagem e simulação da secagem de sementes de cornichão (Lotus corniculatus L.) em leito fluidizado; confecção do aparato experimental de leito fluidizado que foi utilizado no processo de secagem das sementes de cornichão; caracterização fluidodinâmica do leito fluidizado; secagem das sementes de cornichão em secador de leito fluidizado; estudo do comportamento do modelo simulado e ajustes com a experimentação realizada; analise de sensibilidade paramétrica do modelo proposto, e estudo da influência dos parâmetros do processo de secagem, umidade final e coeficiente global de transferência de massa, sobre a germinação das sementes de cornichão. Os resultados do estudo de secagem ficaram na faixa de 9,00 a 10,22 % em relação a umidade final em base úmida. No que se refere a germinação os valores encontrados ficaram na ordem de 72 a 78 %. Para estas duas respostas a temperatura foi a variável de maior influência. O estudo da modelagem e simulação apresentou-se eficaz para a secagem das sementes de cornichão em leito fluidizado. O coeficiente global de transferência de massa influencia de maneira significativa os índices de germinação das sementes, sendo seu aumento prejudicial a fisiologia das sementes de cornichão (Lotus coniculatus L.).
The Rio Grande do Sul state has great prominence in pastoralist activities as a regional economy. This implies a growing need to use species of fodder seeds cool season in this region. Among the most adaptable forage seeds to the cold weather has the birdsfoot trefoil (Lotus corniculatus L.) a Winter forage legume, very versatile, with high tolerance to acid which is used in large scale in Rio Grande do Sul. The seed is the vehicle that leads to the farmer all the genetic potential of a cultivar with superior characteristics, and on their way, small amounts of seeds are multiplied until they are achieved high volume production, during which the quality of the seeds will be subject to a number factors capable of causing loss of the entire genetic potential. Faced with such a promising market, obtaining better quality seeds still has difficulties ranging from the production phase in the countryside where the seeds are harvested early in relation to the physiological maturation due to the natural loss process until its processing where the drying process is presented as a limiting step that requires particular attention to avoid losses in physiological final product. The drying operation applied to seeds aims to ensure the conservation of this product through reduction of water content. In this case where these are organic materials, reduction in water content must be performed in a well controlled manner requiring a study and analysis of operation applied to the material, since the same if performed in an uncontrolled manner may result in loss of material. In high quality drying seed production technology is taken with one of the most important steps, since it allows reducing the water content at appropriate levels for storage and marketing (12 to 15% humidity), preserves the seeds of physical and chemical changes induced by excess moisture, makes it possible to maintain the initial quality during storage and enables next harvest physiological ripeness. The use of fluidized bed dryer for implementation of the drying operation has been the subject of scientific investigation since this dryer has been recognized advantages ranging from high drying capacity for efficient mass transfer and energy until drying time reducing the speed of moisture removal and ease of controls. Given the above, this study aimed to mathematical modeling and drying simulation application in fluidized bed, followed by tests applied experimentally by tests carried out in a fluidized bed dryer made for this job. physical and physiological characterization studies were performed seeds; Modeling and Simulation of drying seeds birdsfoot trefoil (Lotus corniculatus L.) fluidized bed; making the fluidized bed experimental apparatus that is used in the drying process of the seed birdsfoot trefoil (Lotus corniculatus L.); fluid dynamic characterization of the fluidized bed; Drying of the seeds birdsfoot trefoil (Lotus corniculatus L.) in a fluidized bed dryer; model behavior study simulated and adjustments to the trial held; analysis of parametric sensitivity of the model; study of the influence of the parameters of the drying process, the final moisture and overall mass transfer coefficient on the germination of seeds birdsfoot trefoil (Lotus corniculatus L.). Drying study results were in the range from 9.00 to 10.22% over the final moisture on a wet basis. As regards the germination values found were in the range 72-78%. For these two answers the temperature was the most influential variable. The study of modeling and simulation showed to be effective in drying the seeds birdsfoot trefoil fluidized bed. The overall mass transfer coefficient influences significantly the levels of seed germination and increase its adverse physiology of seed birdsfoot trefoil (Lotus coniculatus L.).

Les opines synthétisées par les cellules transformées des plantes atteintes de crown gall, sont catabolisées par la souche d'agrobacterium qui a provoqué la maladie. Ces opines favorisent la croissance et le maintien d'agrobacterium dans la tumeur de la plante infectée. Est-ce que dans une rhizosphère l'émission d'une substance particulière par les racines peut favoriser l'établissement d'une bactérie capable de consommer cette substance? Pour répondre à cette question, nous avons transposé le système agrobacterium-opine-dicotyledone en modèle azospirillum brasilense-lotier (lotus cornicalatus l. ) Transformé par agrobacterium rhizogenes 8196. Les gènes de catabolisme des opines mannityliques clonés dans un vecteur à large spectre d'hôte ont été transfères à trois souches d'a. Brasilense (sp7, bb#1, bb#2). Les transconjugants sont devenus capables d'utiliser la mannopine pour leur croissance. Deux d'entre eux bb#1(pydh208) et bb#2(pydh208) ont été inoculés aux lotiers, normaux ou transformés, en mélange avec la souche sauvage correspondante. Le plasmide catabolique phdh208 étant instable dans la souche bb#1, celle-ci ne s'est pas montrée compétitive. Pour la souche bb#2(pydh208) qui se maintient bien sur les lotiers, un avantage sélectif a été décelé sur les lotiers transformés. Cet effet est ponctuel. La très faible utilisation des exsudats du lotier par a. Brasilense n'a pas permis de mettre en évidence l'avantage sélectif net de la mannopine pour l'association a. Brasilense-lotier transforme

[Truncated abstract] Salinity and waterlogging interact to reduce the growth of most crop and pasture species. Species that are productive on saline-waterlogging land are needed for Australian farming systems. One option is Lotus tenuis, a perennial legume widely grown for pasture in the flood-prone and salt-affected Pampa region of Argentina. To identify mechanisms responsible for the adverse interaction between salinity and waterlogging, Lotus tenuis with a reputation for tolerance was compared with L. corniculatus, the most widely cultivated Lotus species. The physiology of salt and waterlogging tolerance in L. tenuis (4 cultivars) was evaluated, and compared with L. corniculatus (3 cultivars). Overall, L. tenuis cultivars accumulated less Na+ and Cl-, and more K+ in shoots than L. corniculatus cultivars, when exposed to 200 mM NaCl for 28 d in aerated or in anoxic (stagnant agar) solutions. In a NaCl dose response experiment (0 to 400 mM NaCl in aerated solution), Lotus tenuis (cv. Chaja) accumulated half as much Cl- in its shoots than L. corniculatus (cv. San Gabriel) at all external NaCl concentrations, and about 30% less shoot Na+ in treatments above 250 mM NaCl. Ion distributions in shoots were determined for plants at 200 mM NaCl; L. tenuis (cv. Chaja) accumulated about half as much Cl- in old leaves, young leaves and stems, compared with concentrations in L. corniculatus (cv. San Gabriel). There were not, however, significant differences between the two species for Na+ concentrations in the various shoot tissues under aerated NaCl treatment. '...' Therefore, during early stages of exposure to salinity, L. tenuis accumulated a higher proportion of total Na+ in the roots under combined stagnant-plus-NaCl treatment (55% versus 39% for L. corniculatus). Na+ transporters, particularly those relying on H+ gradients across membranes, which in turn require adequate ATP levels, could be impaired under O2 deficits that inhibit respiration. To study the effect of O2 deficiency on a Na+ transporter, an NHX1-like gene was cloned from L. tenuis and identity established via sequencing and yeast complementation studies. Real-time qPCR showed expression of NHX1 in L. tenuis roots increased under stagnant-plus-NaCl treatment, whereas it was reduced in L. corniculatus. Thus, maintaining O2 transport to roots, together with up-regulation of an NHX1-like gene for Na+ accumulation in vacuoles, contributes to tolerance of L. tenuis to combined salinity and waterlogging stresses. This study highlights the importance of minimising Cl- transport to shoots as a mechanism of salt tolerance and has identified a CCC-like gene in L. tenuis as a candidate for mediating root-to-shoot Cl- transport. Under combined stagnant-plus-NaCl treatment, control of Na+ transport is another mechanism contributing to tolerance in these Lotus species. Enhanced root aeration in L. tenuis maintains root Na+ transport processes, such as accumulation in vacuoles via NHX1-like genes, to diminish xylem loading to the shoot. Overall, this thesis has contributed new knowledge on the potential of Lotus tenuis as a saltland pasture and has significantly enhanced current understanding on the mechanisms of salinity and waterlogging tolerance in plants.

O presente trabalho foi realizado com o objetivo de avaliar e selecionar genótipos de alfafa crioula e de cornichão cultivar São Gabriel para aptidão ao pastejo. Neste trabalho, foi proposto o termo aptidão ao pastejo em substituição à resistência ao pastejo. O procedimento padrão utilizado no processo de seleção e de caracterização dos genótipos constou de experimentos nos quais as plantas eram submetidas a uma pressão de pastejo elevada e contínua (3-5 cm de altura do resíduo). Houve diferença (P<0,05) na sobrevivência das diferentes populações de alfafa crioula, evidenciando variabilidade para a aptidão ao pastejo, o que não se evidenciou de forma consistente para o cornichão, que, no entanto, apresentou uma maior porcentagem de sobrevivência em comparação com a alfafa. A avaliação das características morfofisiológicas em alfafa não evidenciou diferenças (P>0,05) na contração das coroas, área da coroa e da raiz, área foliar residual e específica, número de hastes por planta, tipo de haste predominante (basilar ou axilar) e reservas orgânicas. Foram evidenciadas diferenças (P<0,05) no comprimento do entrenó e no índice de gemas específicas, proposto neste trabalho como descritor funcional da aptidão ao pastejo. Ensaios em casa-de-vegetação identificaram um marcador morfológico (altura do primeiro nó) nas plântulas de alfafa e de cornichão, capaz de separar precocemente os genótipos contrastantes quanto à aptidão ao pastejo. Foi comparada a efetividade das estirpes de rizóbio recomendadas para o cornichão com a de novas estirpes isoladas, demonstrando a possibilidade de obtenção de estirpes mais eficientes.

This research assesses the importance of using ecologically-similar rather than geographically-local seed in grassland restoration projects, with particular reference to herbivorous invertebrates, including pollinators. Seed from Lotus corniculatus L. (bird’s-foot trefoil) populations at nine sites across south-west England were collected to represent ecotypes potentially adapted to a range of soil types (calcareous loam, neutral loam and calcareous sand (referred to as ‘sand’)) and management regimes (grazed, cut with aftermath grazing (referred to as ‘cut’) and unmanaged). From October 2011 the ecotypes were planted within three different treatment soils (calcareous loam, neutral loam and calcareous sand (“sand”), and two management treatments (grazed [simulated] or unmanaged [neither cut or grazed]). Differences in plant morphology and phenology under these treatments were recorded at four-weekly intervals and immediately prior to harvest on 16th July 2012. Fresh and dry biomass were recorded and leaf-nitrogen and leaf-hydrogen cyanide (HCN) levels determined. Treatments were maintained and plants grown for a further 15 months [after harvest]. Bee preference for ecotypes grown under treatment combinations was also recorded during peak flowering periods of 2012 and 2013. Data were tested using Kruskall-wallis and ANOVA. A Generalized Linear Mixed Model (GLMM) was built to test all ecotype and treatment differences including interactions. A separate Non-linear Mixed Effects model (NLME) was created to investigate spatial autocorrelation between ecotype sites. The standard chosen in the models was Cockey Down (a calcareous loam, grazed ecotype) grown in matching treatments. The phenotypic traits retained were most pronounced in ecotypes from home-sites of sand soil type and cut management, which were considered to be the more stressed environments of the study, requiring rapid adaptation. By harvest, ecotypes from sand home-sites produced significantly greater number of stems per plant, greater leaflet number per main stem and lower HCN. Model results for sand ecotypes additionally identified delayed seed pod formation, increased hirsuteness and higher leaf-HCN compared to the standard. Significant differences found within ecotypes from cut home-sites included fewer stems per plant, fewer leaflets per main stem, more seeds per pod, greater leaf-HCN and shorter time to first flower. The model also found this ecotype to be less hirsute with fewer seed pods (in unmanaged treatment) than the standard. Ecotypic traits shown in plants from the less stressed home-sites included calcareous loam ecotypes having two clear flowering peaks in both years and highest leaf-HCN, and unmanaged ecotypes having lower leaf-HCN. Three significant interactions indicated additive character factor effects: neutral loam ecotypes grown in neutral loam treatment soil had earlier pod formation than the standard; and, sand ecotypes grown in sand treatment soil and unmanaged ecotypes receiving unmanaged treatment had greater flower number (over both years) than all other ecotypes, treatments and combinations. Results from the bee ecotype preference study showed no preference for ecotypes geographically close to the test foraging area. However significant differences were shown by bees in terms of ecotype preference, with avoidance of plants containing highest leaf-HCN. Plants grown in calcareous soil treatment were preferred which suggests nectar of plants are of most value to bees when grown in optimum [for L. corniculatus growth] soil. Ecotypic differences in herbivory defence [leaf-HCN and hirsuteness induced by home-site soil and management], would be of importance to receptor site invertebrate herbivores/pollinators. Pollinators could also find difficulties with the ecotypic differences in flowering asynchronicity. Both home-site soil type and management could also influence the viability of the plant population through reducing fecundity. Delayed seed pod formation in sand ecotypes (compared to the standard) indicates an adaptation to summer temperatures or low-nutrient availability. Calcareous loam ecotypes lack such adaptation and if introduced to a sand sites and could fail due to poorly timed germination, deep seed burial [from shifting sand] or poorly allocated energy. Cut ecotypes also produced significantly fewer seed pods (than the standard) with significantly greater seed numbers per pod suggesting an energy allocation adaptation to produce fewer, larger pods before defoliation rather than continuous pod formation throughout the season, a potentially critical adaptation for seed return. Findings from this study are of national relevance, and Natural England should adopt new recommendations on seed provenance in agri-environment schemes. Instead of recommending strictly geographically local seed, the management regime (particularly details of intensity and timing of management operations) should ideally be similar between the donor and receptor sites. Soil types, especially pH and clay/organic matter content should also be matched as far as possible as these were the greatest limiting factors within this study. Suitable donor sites may be local sites of similar habitat. If no such sites are available then recommendations from this study should be followed in seeking suitable sowing material. If seed suppliers are used, then they should provide greater detail on donor site conditions to aid land managers.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde, Programa de Pós-Graduação em Farmácia, Florianópolis, 2011
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O objetivo deste estudo foi avaliar o efeito anti-inflamatório da Lotus corniculatus no modelo de pleurisia induzida por bradicinina, em camundongos. Materiais e métodos: As partes aéreas da L. corniculatus var. São Gabriel foram secas ao ar e cortadas. Ao material obtido foi adicionado etanol 96% para obter o extrato bruto hidroálcoólico (CE). Este foi fracionado por partição líquido-líquido utilizando hexano, acetato de etila, n-butanol e água, em ordem crescente de polaridade, resultando nas frações hexano (HEX), acetato de etila (AcOEt), butanol (BuOH), e aquosa (Aq). Os compostos 1 e 2 foram isolados das frações HEX e AcOEt por meio de coluna cromatográfica de sílica gel, e identificados como ácido oleanólico e canferitrina. Camundongos albinos suíços foram utilizados nos experimentos e a pleurisia foi induzida por bradicinina (BK), de acordo com metodologia descrita por Saleh et al. (1997). Inicialmente, diferentes grupos de animais foram tratados com diferentes doses de CE (25-200 mg/kg), HEX (5-25 mg/kg), AcOEt (5-25 mg/kg), BuOH (5-25 mg/kg), Aq (5-25 mg/kg), ácido oleanólico (1-5 mg/kg), ou canferitrina (1-10 mg/kg), 0,5 h antes da indução por BK. Os parâmetros inflamatórios (leucócitos, exsudação, atividade das enzimas mieloperoxidase (MPO) e adenosina-deaminase (ADA), e concentrações de nitrato/nitrito (NOx) e interleucina-17A (IL-17A)) foram avaliados 4 h após a indução da pleurisia. Diferenças estatísticas entre os grupos foram determinadas pela analise de variância (ANOVA), complementadas quando necessário, com Dunnett ou teste t de Student. Valores de p < 0,05 foram considerados significantes. Resultados: o CE, suas frações e compostos isolados inibiram significativamente os leucócitos, a exsudação, a atividade das enzimas MPO e ADA, assim como as concentrações de NOx e IL-17A (p < 0,05). Conclusão: A L. corniculatus apresentou importante efeito anti-inflamatório, principalmente pela inibição do influxo de leucócitos e exsudação causada pela BK. Este efeito parece ser em parte mediado pela inibição das enzimas MPO e ADA, e dos mediadores NO e IL-17A. Além disso, os compostos ácido oleanólico e canferitrina parecem ser responsáveis, em parte, pelo efeito anti-inflamatório observado.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Agrárias, Programa de Pós-Graduação em Agroecossistemas, Florianópolis, 1998
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Pressupondo que o manejo influi na composição botânica, introduziu-se por sobre-semeadura o Lotus corniculatus em pastagem naturalizada perene de verão, manejada em dois tempos de repouso em Pastoreio Racional Voisin, com o objetivo de verificar os seus efeitos sobre a flutuação estacional das principais espécies e a possibilidade do melhoramento da pastagem.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
O objetivo deste trabalho foi avaliar a viabilidade, o vigor da semente e o crescimento de plantas de cornichão e trevo vermelho, provenientes de áreas da Embrapa Pecuária Sul, submetidas a diferentes doses de herbicidas pré e pós emergentes (diclosulam, bentazona e iodosulfurom-metílico), com a finalidade de identificar os herbicidas seletivos à estas forrageiras. Na primeira etapa do experimento foi avaliado o efeito do herbicida pré-emergente diclosulam no potencial fisiológico das sementes e crescimento inicial das plântulas. As sementes das forrageiras, foram semeadas em substrato previamente umedecido com solução de diclosulam nas concentrações de 0; 0,5; 1,0 e 1,5 vezes a dose registrada do produto pelo fabricante. Na segunda etapa, após o cultivo em casa de vegetação por 107 dias, foi realizada a aplicação dos pósemergentes, por meio de pulverizador costal de pressão constante em condições atmosféricas de 23º C de temperatura e 71% de umidade relativa do ar (UR). Foram utilizadas concentrações do herbicida referentes a zero; 0,5; 1,0 e 1,5 vezes a dose registrada pelo fabricante. Os resultados da primeira etapa, demostraram que as variáveis comprimento da parte aérea, comprimento de raiz e massa seca total foram alteradas negativamente pela exposição às diferentes doses de diclosulam, enquanto o potencial germinativo e a capacidade de emergir não foram prejudicadas, em ambas as espécies estudadas. Na segunda etapa os resultados obtidos mostraram maior sensibilidade das plantas de cornichão ao herbicida bentazona e das plantas de trevo vermelho ao herbicida iodosulfurom-metílico. Portanto é possível concluir que, as diferentes doses de diclosulam não comprometem o potencial germinativo, nem a capacidade de emergir das sementes de cornichão e trevo vermelho, porém, interferem negativamente no seu desenvolvimento. Já com a aplicação dos pósemergentes, o cornichão apresentou sensibilidade ao herbicida bentazona e características de tolerância ao herbicida iodosulfurom-metílico, enquanto o trevo vermelho respondeu de forma contrária, sendo mais sensível a aplicação do iodosulfurom-metílico e tolerante ao bentazona, ativando mecanismos de adaptação e permitindo o desenvolvimento da planta.
The objective of this research was to evaluate viability, seed vigor and growth of birdsfoot trefoil and red clover, plants from Embrapa Pecuária Sul, was submitted to different doses of pre and post emergent herbicides (diclosulam, bentazon and iodosulfuron-methyl), with purpose to identify herbicides selective to these forages. In the first stage of the experiment the effect of the pre-emergent herbicide diclosulam in physiological potential of seeds and initial growth of seedlings was evaluated. The seeds of this forages were sown on a substrate previously moistened with diclosulam solution at concentrations 0; 0,5; 1,0 and 1,5 times the dose registred by manufacturer. In second stage, after the cultivation in the greenhouse for 107 days, the postemergence application was carried out by a costal sprayer with constant pressure at atmospheric conditions of 23 ºC and 71% relative humidity). Herbicide concentrations used were zero; 0,5; 1,0 and 1,5 times the dose registred by manufacturer. The results of the first stage showed that the variables shoot length, root length and total dry mass were negatively altered by exposure to different doses of diclosulam, while germination potential and emergence capacity were not impaired in both species studied. In the second stage, the results obtained showed greater sensitivity of birdsfoot trefoil plants to herbicide bentazon and the red clover plants to iodosulfuron-methyl herbicide. Therefore, it is possible to conclude that, different doses of diclosulam do not compromise germination potential, neither ability to emerge from birdsfoot trefoil and red clover seeds, but negatively interfere in its development. With the post-emergence application, the birdsfoot trefoil showed sensitivity to herbicide bentazon and characteristics of tolerance to herbicide iodosulfuron-methyl, while the red clover responded in a contrary way, being more sensitive the application of the herbicide iodosulfuron-methyl and tolerant to bentazon, activating mechanisms of adaptation and allowing plant development.

Deux légumineuses (Anthyllis vulneraria et Lotus corniculatus) adaptées aux métaux lourds constituent un matériel d’intérêt pour la phytostabilisation de sites miniers. Leur fonction de fixatrices biologiques d’azote grâce à leur symbiose avec des bactéries symbiotiques permet l’établissement efficace d’une couverture végétale durable limitant la dispersion des métaux dans l’environnement. Nos objectifs ont été d’étudier les effets des métaux lourds et de la plante hôte sur les populations symbiotiques naturellement associées à ces légumineuses en analysant (i) les populations symbiotiques associées à A. vulneraria sur 8 sites contaminés ou non (ii) les populations de rhizobia associées à L. corniculatus qui ont été comparées à celles d’Anthyllis. La distribution des souches de Mesorhizobium isolées de nodosités d’A. vulneraria et provenant de plusieurs sites contaminés ou non dépend des fortes teneurs en métaux lourds des sols qui sélectionnent fortement les souches symbiotiques résistantes et influencent leur composition taxonomique. Les souches appartenant à l’espèce M. metallidurans ont été retrouvées seulement dans les sites fortement contaminés. Deux nouvelles espèces potentielles et résistantes aux métaux semblent exister chacune sur un site minier distinct. L’une d’elle est proche de M. ciceri et de M. loti et tous ses membres présentent la particularité de ne pas posséder de gène cadA, un gène impliqué dans la tolérance aux métaux chez M. metallidurans. Par contre, les sites non contaminés révèlent une diversité taxonomique différente avec la présence de nouvelles espèces de Mesorhizobium sensibles aux métaux lourds. Quatre de ces nouvelles espèces ont été définies. A. vulneraria et L. corniculatus partagent la même diversité taxonomique dans les sites contaminés testés. Par contre, les propriétés symbiotiques des souches varient selon la plante hôte utilisée pour le piégeage. Les souches appartiennent soit au symbiovar (sv.) anthyllidis soit au sv. loti selon le site géographique d’origine et ceci indépendamment des teneurs en métaux lourds dans le sol. A. vulneraria s’associe avec les souches possédant les sv. anthyllidis ou sv. loti. En revanche, L. corniculatus ne s’associe qu’avec des souches du sv. loti. Dans tous les sols qu’ils soient contaminés ou non, A. vulneraria nodule préférentiellement avec le sv. anthyllidis. En conclusion, A. vulneraria et L. corniculatus établissent des symbioses avec les mêmes espèces de Mesorhizobium et s’associent préférentiellement avec un sv. Les taxons retrouvés dépendent fortement des sites d’isolement, ce qui pourrait traduire des adaptations particulières aux conditions environnementales. L’utilisation des ressources biologiques locales est une stratégie que nous recommandons pour la végétalisation d’anciens sites miniers
Two legumes (Anthyllis vulneraria and Lotus corniculatus) adapted to heavy metals form an interesting material for phytostabilisation strategy in mining sites. As biological nitrogen fixators, these legumes associated with compatible symbiotic bacteria provide an efficient establishment of a sustainable cover vegetation limiting metal dispersion in the environment. Our objectives were to study the effects of heavy metals and the host plant on symbiotic populations naturally associated with these legumes by analyzing (i) symbiotic populations associated with A. vulneraria on 8 contaminated and uncontaminated sites (ii) rhizobial populations associated with L. corniculatus that were compared with those of Anthyllis. The distribution of mesorhizobial strains isolated from A. vulneraria root-nodules from several contaminated and uncontaminated sites depends on high levels of heavy metals in soils by selecting highly resistant strains and impacting the taxonomic composition. Strains belonging to M. metallidurans were only found in highly contaminated sites. Two new potential metal-tolerant species were detected in two distinct mines. One of them was closely related to M. ciceri and M. loti and its members had the feature of not -possessing the cadA gene, a gene involved in metal-tolerance among M. metallidurans strains. By contrast, uncontaminated sites revealed a different taxonomic diversity with new species sensitive to heavy metals. Four of these new species were defined. A. vulneraria and L. corniculatus share the same taxonomic diversity in the contaminated sites tested. By contrast, symbiotic properties of the strains vary depending on the host plant used for trapping. Strains belong either to symbiovar (sv.) anthyllidis or to sv. loti according to geographic origins and independently of heavy metal levels in soils. A. vulneraria associated with strains of sv. anthyllidis or sv. loti. In contrast, L. corniculatus only associated with strains of sv. loti. In contaminated or uncontaminated soils, A. vulneraria was preferentially nodulated by sv. anthyllidis. In conclusion, A. vulneraria and L. corniculatus established symbiotic relationships with the same taxonomic groups of Mesorhizobium but associated with different symbiovars. The finding of taxonomic groups strongly depends on geographical sites, suggesting special adaptations to environmental conditions. Use of local biological resources is the strategy we recommend for revegetation of old mines

There is no information available on water management of birdsfoot trefoil grown for seed. Information is also not available describing how to minimize seed losses due to shattering in order to obtain consistently high birdsfoot trefoil seed yields. The objectives of this research are to: (i) quantify crop water use, effects of soil-water availability and optimal water management conditions for birdsfoot trefoil grown for seed, (ii) determine the effects of irrigation timing and amount on flower production, seed yield, and yield components, and (iii) quantify the effects of soil-water availability on seed shattering and determine optimal harvest time to reduce seed yield losses due to shattering. Five supplemental irrigations treatments and a non-irrigated control were applied in 1994 and 1995; in 1996, only treatments low stress and non irrigated control were investigated, near Corvallis, OR on a Woodburn silt loam soil (fine-silty, mixed, mesic Aquultic Argixeroll). Increasing amounts of applied water increased seasonal ET[subscript c] with low stressed plants having the greatest ET[subscript c] and non-irrigated control plants the least. The fraction of available soil-water used was primarily dependent upon the irrigation depletion percentage and secondarily dependent upon irrigation replacement amount. Soil-water conditions favorable for vegetative development and seed yield water use efficiency are opposite. Birdsfoot trefoil grown for seed requires minimal or no supplemental irrigation. For non-irrigated conditions, the crop water requirement ranges from 240 to 255 mm. In the first year of production, plants under low-stress conditions sustained flowering longer than with limited or no irrigation applications. Flowering was not affected by irrigation in subsequent years of production. Total above-ground phytomass production was correlated with the amount of applied irrigation water (r=0.92). Umbel density and number of seeds per pod are the primary determinants of total seed yield (r=0.77 and 0.92, respectively). Manipulation of the reproductive development pattern by different water application times and amounts does not affect peak seed shattering events. Crop-water stress status affects the percentage of total shattered seeds shattered at harvest time (r=-0.76). Increasing amounts of applied water increase the percentage of potential shatter losses that will shatter by harvest time (r=0.65). Seed shatter losses fluctuate during the reproductive development period but are not influenced by the water application treatments. Climatic variables as measured in this experiment cannot be used to predict the time of peak seed shatter events. A total of 109 heat units are needed from the time from initial pod dehiscence until rapid shattering occurs.
Graduation date: 1998

Birdsfoot trefoil (Lotus corniculatus L.) is a popular perennial, non-bloating forage legume used for pasture, hay and silage throughout the temperate regions of Europe, Asia Minor, North Africa and North and South America. It is regarded as the most morphologically and biochemically variable species in the genus. Research investigating the relationships of morphological, ecological and genetic characteristics describing birdsfoot trefoil germplasm has not been done. This research was conducted to investigate if the geographic and ecological origins of birdsfoot trefoil genotypes are related to differences in: (i) crossing compatibility among diverse genotypes, (ii) morphological traits, (iii) PCR-RAPD banding patterns, and (iv) temperature response of chlorophyll Photosystem II variable fluorescence. The 28 genotypes were classified by morphological characteristics, 130 polymorphic random amplified polymorphic DNA bands, and eight ecological characteristics of the original collection sites. The ease of introgressing 27 exotic genotypes into other germplasm backgrounds was determined by using bidirectional crosses with a domestic and exotic genotype tester. The chlorophyll fluorescence transients ratios (FTR) were determined from eight genotypes that were selected by their ecological diversity with measurements made from 10 to 40��C in 5��C increments for 33 minutes from the time of initial dark adaptation in 3 minute increments. Morphological similarities among genotypes were related to the general geographic proximities of their collection sites and their genetic similarity based on RAPD markers. Utilizing genetic, morphological and ecological descriptions revealed combinations of variation among genotypes that would not be observed with single measurements. Incompatibility among crosses was expressed as either an inability of plants to set pods or F��� progeny resulting from crosses producing inviable pollen. Reproductive barriers were environmentally neutral and randomly distributed through the among the genotypes. Intermediate crosses could be identified to bridge any combination of genotypes that were incompatible. The eight genotypes differed in their FTR responses and were grouped into two classes. However, no associations were found between genotype similarities by FTR with genetic or ecologic similarities.
Graduation date: 1998

Documentos apresentados no âmbito do reconhecimento de graus e diplomas estrangeiros
A crescente necessidade da utilização de espécies forrageiras de estação fria na região sul do país vem determinando uma grande demanda por sementes de qualidade. Entre as leguminosas se destaca o cornichão (Lotus corniculatus L.), que é uma leguminosa forrageira oriundo da Europa e da região mediterrânea. Esta leguminosa forrageira tem na versatilidade a sua maior vantagem, visto que apresenta produção de forragem na primavera, no verão e no outono, possui resistência à seca e ao encharcamento temporário do solo e apresenta boa persistência da massa verde. Além disso, as sementes deste tipo de espécie podem ser misturadas com espécies de inverno, o que aumenta o rendimento da matéria seca, dá uma melhor distribuição da forragem ao longo do ano e agrega uma melhora na qualidade das pastagens. Apesar dessas vantagens, as sementes de cornichão comercializadas possuem ainda uma baixa qualidade. Esse fato é decorrente de um sistema de fiscalização ineficiente, associado à dificuldade de produção e beneficiamento do produto. Outro problema que o produtor de sementes desta espécie enfrenta é a dormência tegumentar, isto é, o revestimento da semente (tegumento) é impermeável e não permite que a água necessária para a germinação chegue ao interior da semente. Na literatura, diversos trabalhos têm relatado que cada vez mais há um aumento na demanda de sementes forrageiras em função do melhoramento do campo nativo e das pastagens cultivadas. Diante de um mercado tão promissor, a obtenção de sementes de melhor qualidade ainda apresenta dificuldades que vão desde a fase de produção no campo, onde as sementes são colhidas precocemente em relação ao ponto de maturidade fisiológica devido ao processo de perda natural, até seu beneficiamento, onde o processo de secagem apresenta-se como uma etapa limitante que necessita de uma atenção muito especial para evitar perdas fisiológicas no produto final. De um modo geral, a secagem é definida como a remoção, voluntária ou não, total ou parcial, de uma fase líquida ou gasosa de qualquer material através da transferência de calor e de massa. A secagem tem em sua essência a finalidade de assegurar a conservação de um produto por meio da redução do seu teor de água. No caso de materiais orgânicos, como é o caso de sementes, a redução no teor de água deve ser efetuada até o ponto em que a concentração de açúcares, ácidos, sais e outros componentes, sejam suficientemente elevados para reduzir a atividade de água e inibir, portanto, o desenvolvimento de micro-organismos. Na tecnologia de produção de sementes de alta qualidade a secagem é tida com uma das etapas de maior importância, pois permite a redução do teor de água em níveis adequados para o armazenamento e comercialização (12 a 15 % de umidade), preserva as sementes de alterações físicas e químicas induzidas pelo excesso de umidade, torna possível a manutenção da qualidade inicial durante o armazenamento e possibilita colheitas próximas da maturação fisiológica. Entretanto, o processo de secagem, seja ele qual for, deve acontecer de forma controlada e de maneira uniforme a fim de evitar elevados gradientes de umidade e temperatura no interior do material que podem provocar a perda da qualidade do produto final. De acordo com o que foi exposto, o presente trabalho de mestrado teve como objetivo realizar um estudo sobre o processo de secagem de sementes de cornichão (Lotus corniculatus L.) em um secador de leito fixo com fluxo de ar paralelo. Para alcançar tal objetivo, este trabalho foi dividido nas seguintes etapas: caracterização física e fisiológica das sementes, análise do processo de reumidificação das sementes, estudo das isotermas de dessorção das sementes reumidificadas e avaliação dos efeitos da variação da temperatura, da velocidade do ar e da altura da bandeja sobre a cinética de secagem e qualidade fisiológica do produto final. Os dados experimentais mostraram que os resultados obtidos para a umidade em base úmida das sementes secas ficaram na faixa de 5 % para as secagens realizadas a 60 °C e em aproximadamente 8 % para as secagens realizadas a 40 °C. No que se refere à germinação, os valores encontrados estão na ordem de 44 a 59 % para as secagens realizadas neste estudo. Dentre as secagens, a que obteve o maior valor de germinação, com valor de 59 %, foi a realizada na condição de T = 40 °C, v = 1,0 m/s e h = 1,0 cm.

Grünland mit hoher Produktivität und Futterqualität bildet die Grundlage der Wiederkäuerernährung. In Grünlandbeständen mit ausbleibender oder geringer Stickstoffdüngung sind Leguminosen unerlässlich für Produktivität und Futterqualität, was auf die Fähigkeit von Leguminosen Luftstickstoff zu binden zurückzuführen ist. Gegenwärtig ist Trifolium repens L. eine der wichtigsten Futterleguminosen im Grünland der gemäßigten Zonen Europas. Es ist allerdings bekannt, dass T. repens eine gute Wasserversorgung benötigt, um einen hohen Ertrag zu erzielen. Verringerte Niederschlagsmengen in der Vegetationsperiode, die unter Klimawandelbedingungen vorausgesagt werden, könnten somit die Ertragsleistung von T. repens verringern. In Zukunft steigt dadurch möglicherweise auch die Bedeutung anderer Futterleguminosen, die besser an trockenere Bedingungen angepasst sind und somit als Alternative für T. repens dienen könnten. Da die Kenntnisse über das agronomische Potenzial solcher möglichen alternativen Leguminosen begrenzt sind, haben wir in dieser Studie fünf vielversprechende und wahrscheinlich besser an Trockenheit angepasste Leguminosen untersucht. Für unsere Versuche haben wir Lotus corniculatus L., L. uliginosus Schkuhr, Medicago lupulina L., M. falcate L. und Onobrychis viciifolia Scop. ausgewählt. In einem ersten Schritt wurde das agronomische Potenzial der Leguminosen im Hinblick auf Etablierung und frühe Ertragsentwicklung mit nicht limitierter Wasserversorgung getestet. Weiterhin wurden der Ertrag und die Ertragsstabilität sowie die Wassernutzung der alternativen Leguminosen bei temporärer Trockenheit untersucht und mit der von T. repens verglichen. Der Einfluss von Trockenstress auf wichtige Futterwert bestimmende Inhaltsstoffe der Leguminosen (Rohprotein, neutrale Detergenzienfasern, saure Detergenzienfasen und wasserlösliche Kohlenhydrate) war überdies Gegenstand der Betrachtungen. Um die oben genannten Parameter zu untersuchen, wurde von 2009 (Einsaatjahr) bis 2011 ein Experiment in Großgefäßen in einer Vegetationshalle durchgeführt. In diesem Versuch wurden alle Leguminosen sowohl in Monokultur als auch in einer praxisüblichen Mischung mit Lolium perenne L. angesät. Im Versuchszeitraum folgten die klimatischen Bedingungen in der Vegetationshalle einem normalen jahreszeitlichen Verlauf, der Frost im Winter und höhere Temperaturen im Sommer umfasste. Der für den Versuch notwendige Trockenstress wurde in drei Aufwüchsen innerhalb von zwei Jahren durch temporären Bewässerungsstopp erzeugt. Dabei wurde im Frühjahr 2010 (April/Mai) ein moderater und im Sommer 2010 (Juli/August) sowie im Frühjahr 2011 (April/Mai) je ein starker Trockenstress induziert. Mit Ausnahme von M. falcata, welches eine verzögerte Anfangsentwicklung zeigte, waren die Keimung und die Etablierung von allen alternativen Leguminosen vergleichbar mit der von T. repens. Die Ertragsleistung von M. lupulina und L. corniculatus in Monokultur war ähnlich hoch wie die von T. repens. In Mischung zeigten beide alternativen Leguminosen zwar Potenzial, aber die Ertragsleistungen waren dennoch geringer als die der T. repens/L. perenne Mischung. In unserem Versuch führte Trockenstress zu verringertem Ertrag und er beeinflusste auch die agronomische Wassernutzungseffizienz (Verhältnis von Ertrag zu Wasserverbrauch). Ausschlaggebend waren dabei die Stärke und die Dauer des Trockenstresses. Besonders starker, aber auch bereits moderater Trockenstress führten bei T. repens zu erheblichen Ertragsverlusten von bis zu 56%. Demgegenüber zeigten vor allem M. lupulina, aber auch L. corniculatus und M. falcata lediglich marginale Ertragsverluste bei moderatem Trockenstress und meist geringere Ertragsverluste als T. repens bei starkem Stress. Die agronomische Wassernutzungseffizienz war bei moderatem Stress verhältnismäßig stabil, wohingegen starker Stress im Vergleich zur Kontrolle meist zu einer geringeren agronomischen Wassernutzungseffizienz führte. Sowohl unter Kontroll- als auch unter Stressbedingungen zeigte speziell M. lupulina in Monokultur eine ähnliche oder sogar eine höhere agronomische Wassernutzugseffizienz als T. repens. Zudem war die agronomische Wassernutzungseffizienz der Mischungen mit M. lupulina, L. corniculatus und M. falcata weniger negativ von starkem Trockenstress betroffen als die Mischung mit T. repens. Dies bestätigte zum einen die Trockenheitsempfindlichkeit von T. repens und zum anderen die bessere Anpassung der alternativen Leguminosen an trockenere Bedingungen. Wir beobachteten, dass eine Änderung in der Stickstofffixierungsleistung der Leguminosen eine gute Erklärungsgröße für Änderungen des Ertrags und der agronomischen Wassernutzungseffizienz darstellt. Hohe Stickstofffixierungsleistung geht dabei meist mit höherem Ertrag und höherer agronomischer Wassernutzungseffizienz einher. Die intrinsische Wassernutzungseffizienz (Verhältnis von assimiliertem CO2 und stomatärer Leitfähigkeit), gemessen als 13C, war ein schlechterer Indikator für die agronomische Wassernutzungseffizienz: Obwohl die intrinsische Wassernutzungseffizienz unter starkem Trockenstress zunahm, sank die agronomische Wassernutzungseffizienz meist ab. Dennoch besitzt die Erhöhung der intrinsischen Wassernutzungseffizienz ein gewisses Potenzial als Anpassung an trockenere Bedingungen Der Trockenstresseffekt auf die Futterqualität war in unserer Studie generell deutlich geringer als der Effekt auf den Ertrag. Besonders moderater Stress hatte wenig Einfluss auf die Futterqualität, während sich die Effekte bei starkem Stress verstärkten. Starker Trockenstress führte meist zu einer Verringerung des Rohprotein- und Fasergehalts (neutrale und saure Detergenzienfasern), wohingegen sich der Gehalt an wasserlöslichen Kohlenhydraten erhöhte. Dies könnte ein Hinweis darauf sein, dass sich die Futterqualität bei Trockenstress sogar verbessert. Nichtsdestotrotz hatten in unserem Versuch die Leguminosenart und die Einsaat als Monokultur oder Mischung einen größeren Einfluss auf die Futterqualität als der Trockenstress. Der Einfluss von Trockenstress auf die Futterqualität ist deshalb bei der Wahl einer geeigneten Futterleguminose weniger von Bedeutung als andere agronomische Eigenschaften. Zusammenfassend ist zu sagen, dass besonders M. lupulina und in geringerem Maße auch L. corniculatus und M. falcata Potenzial als Alternative für T. repens bei Trockenstress zeigen. Nach ausreichender Etablierungszeit entwickeln sich besonders M. lupulina aber auch L. corniculatus und M. falcata stabiler und können sogar höhere Erträge als T. repens bei Trockenstress produzieren. Bezüglich der Futterqualität sind oben genannte alternative Leguminosen ebenfalls vergleichbar mit T. repens.

Five rotational grazing experiments were carried out at Massey University's Riverside farm, in the Wairarapa, on the East Coast of the Southern North Island, New Zealand, to compare the effects of feeding Lotus corniculatus L. (birdsfoot trefoil; cv. Grasslands Goldie) or perennial ryegrass (Lolium perenne)/white clover (Trifolium repens) dominant pasture upon sheep year round productivity. These studies also investigated under grazing, seasonal and annual net herbage accumulation rate and seasonal dynamics of undisturbed (i.e. non-grazed) net herbage accumulation rate of L. corniculatus relative to that of grass-dominant pasture. Aspects of in vivo digestibility of dry matter (DMD), organic matter (OMD), digestible organic matter in the dry matter (DOMD) and metabolisable energy (ME) concentration of L. corniculatus at different stages of maturity over the spring, summer and autumn were investigated in three indoor digestion trials. 1. Two field experiments (Chapter 2) were conducted during spring to assess the effects of grazing mixed age undrenched ewes on L. corniculatus (n = 50) or pasture (n = 50) and their lambs (mainly twins) on live weight (LW), wool production, faecal nematode egg count (FEC) and dag score. In Experiment 1 (18 October 2000 to 21 January 2001) and Experiment 2 (3 October 2001 to 2 January 2002) both forages were fed ad libitum. Total condensed tannin (CT) concentration in the diet selected was 24 to 27 g CT/kg DM for L. corniculatus and 1.4 to 1.5 g CT/kg DM for pasture. The LW gain, weaning LW and wool production were consistently greater (P < 0.001) for lambs grazing L. corniculatus, in either Experiment 1 (258 vs. 189 g/day; 36.1 vs. 30.1kg; 1.17 vs. 0.98 kg) and in Experiment 2 (247 vs. 162 g/day; 31.8 vs. 24.1kg; 1.17 vs. 0.81 kg), respectively. Ewe and lamb dag scores were strongly and positively correlated with dag weight (P < 0.001) and generally increased with time in sheep grazing pasture, whilst grazing on L. corniculatus consistently reduced dag score. FEC in ewes grazing pasture showed a post-parturient rise (PPR) following lambing, whilst ewes grazing L. corniculatus had a reduced PPR in FEC. Up to day 70, FEC in lambs grazing L. corniculatus was lower than that for lambs grazing pasture, but between day 70 and the end of both experiments (approximately day 90), FEC in lambs grazing L. corniculatus increased to similar values as for pasture-fed lambs. FEC was not correlated with dag score or dag weight in ewes or lambs grazing pasture, but these indices were weakly and positively correlated in ewes and lambs grazing L. corniculatus, suggesting that lowering FEC on L. corniculatus also reduced dag formation. It was concluded that under dryland farming conditions, the use of L. corniculatus during the spring/early summer lactation period can increase lamb LW and wool production, whilst eliminating the need for pre-lambing anthelmintic drenching and probably reducing the amount of insecticide needed to control flystrike. These effects compared to pasture are probably due to higher digestibilty, higher ME concentration, higher voluntary feed intake (VFI), and to the effect of CT in reducing rumen protein degradability and controlling internal parasites in sheep grazing L. corniculatus. The absence of endophyte in L. corniculatus may have also have contributed to these effects. 2. During 2001 and 2002 (Chapter 3), grazing trials from February to November were conducted for 279 days (Experiments 1) and 285 days (Experiment 2), to compare the effects of grazing shorn mixed age Romney ewes in light condition on L. corniculatus versus pasture during the mating period (9 weeks, Experiment 1) and 11 weeks (Experiment 2). In Experiment 2, the length of time (days) that ewes need to graze L. corniculatus before mating to maximise reproductive performance was also investigated. Common objectives in both Experiments were to measure forage feeding effects on ewe wool production and LW of their lambs at weaning. In Experiment 1, groups of ewes (n = 100) were fed on either L. corniculatus or pasture at a herbage allowance of 1.8 kg green DM/ewe/day for the first three weeks of feeding and increased to ad libitum (2.3 kg green DM/ewe/day) during the mating period for two cycles. In Experiment 2, groups of 75 ewes grazed L. corniculatus for 42, 21, 10 and 0 days before a synchronised oestrus, with pasture being grazed for the balance of the 42 days. All L. corniculatus groups continued grazing L. corniculatus for a further 5 weeks. Feed allowance was initially 2.0 kg green DM/ewe/day, increased to 2.3 kg green DM/ewe/day during the mating period over the two cycles. At the end of L. corniculatus feeding in both experiments the groups were combined and grazed on pasture until weaning. Total CT concentration in the diet selected was 18 to 29 g CT/kg DM for L. corniculatus, with only trace amounts in pasture. In Experiment 1 mating ewes on L. corniculatus compared to pasture increased number of lambs born and lambs weaned per ewe lambing by 16 and 32% units respectively (P < 0.05), due to more multiple and less single births (P = 0.06) and to reduced lamb mortality (P < 0.05) between birth and weaning. In Experiment 2, increasing the numbers of days of grazing L. corniculatus before ovulation (0, 10, 21, 42 days) linearly increased ovulation rate (P < 0.05), lambs born and lamb weaned by up to 16% units, but had no effect upon lamb mortality. Mating ewes on L. corniculatus increased wool production (P < 0.01) and fibre length (P < 0.05) in Experiment 1 but not in Experiment 2. Grazing L. corniculatus had no effect on lamb birth weight and only small positive effects on weaning LW. It was concluded that, under commercial dryland farming conditions, the use of L. corniculatus during the mating season in late summer/autumn can be used to increase reproductive efficiency and wool production, with the largest responses in years with exceptionally dry autumn periods. These effects are probably due to the higher digestibility and ME concentration of L. corniculatus than pasture and to the CT in L. corniculatus reducing rumen protein degradability and leading to greater essential amino acid (EAA) absorption from the small intestine. Effects of forage CT upon the uterine microenvironment at the time of conception, implantation and early foetal growth, need to be investigated in future studies. It is also suggested that effects of mating on L. corniculatus upon lamb mortality between birth and weaning should be further investigated with ewe numbers/treatment increased from 100 to 350. 3. During the summer of 2002/2003, another grazing trial (Chapter 4: 95 days) compared the effects of grazing L. corniculatus and pasture on LW and the dynamics of nematode parasite infection in Suffolk x Romney weaned lambs fed ad libitum. Half of the lambs (n = 30) grazing either L. corniculatus or pasture received oral anthelmintic at the start and at monthly intervals (regular-drenched groups), whilst the remaining 30 lambs in each treatment only received oral anthelmintic when mean faecal nematode egg counts (FECs) exceed 1,000 eggs/g wet faeces (trigger-drenched groups), which occurred on day 58 only for both groups. Trigger and regular-drench lambs grazed separate areas. Total CT concentration in the diet selected was 40 to 31 g CT/kg DM for L. corniculatus, with only trace amounts in pasture. Regular-drenched lambs grazing L. corniculatus had significantly higher LW gain (298 g/day) and carcass weight gain (133 g/day) than all the other groups, whilst trigger-drenched lambs grazing L. corniculatus had significantly greater LW gain (228 g/day) and carcass gain (99 g/day) than regular-drenched (200; 66 g/day) and trigger-drenched (187; 63 g/day) lambs grazing pasture. Carcass fatness was significantly lower for trigger-drenched lambs than for regular-drenched lambs, when fed either L. corniculatus or pasture. Dag score was consistently lower for regular-drenched lambs grazing L. corniculatus than pasture; trigger-drenched lambs showed similar effects up to day 48, with no differences between the two groups thereafter. Regular anthelmintic treatment maintained FECs at low values, while parasitised lambs on L. corniculatus tended to have higher FECs than pasture-fed lambs. Relative to trigger-drenched lambs that grazed pasture, grazing trigger-drenched lambs on L. corniculatus had significantly reduced worm burdens of Haemochus contortus, Teladosargia spp., Nematodirus spp. and Cooperia spp. at slaughter, but greater burdens of Trichostrongylus spp., Chabertia ovina, Oesophagostonum spp. and Trichuris ovis were present in L. corniculatus-fed lambs. It was concluded that grazing L. corniculatus under dryland farming conditions compared to pasture can increase LW gain of weaned lambs, whilst reducing reliance on anthelmintic drenches to control parasites. These effects are probably due to increased protein supply from the action of CT enabling the lambs to have a higher LW gain when carrying a parasite burden, and to L. corniculatus better maintaining its high ME value under drought conditions. Using L. corniculatus to finish weaned lambs without anthelmintic drenches for a seven-week period is proposed. 4. A three-year study (Chapter 5; November 2000 to October 2003) was conducted to compare, under grazing conditions, seasonal and annual grazed net herbage accumulation rate and seasonal dynamics of undisturbed (i.e. non-grazed) net herbage accumulation rate of L. corniculatus relative to grass-dominant pasture. Prediction equations to estimate standing DM in L. corniculatus and pasture from the rising plate meter (RPM) and sward surface height were also generated. L. corniculatus and pasture growing in a moderate fertility and low-pH soil (pH 5.35) accumulated similar total herbage masses (24.3 vs. 24.1 t DM/ha) over the 3-year period, with the DM production being greater for L. corniculatus than for pasture during 2000-2001, producing more DM during summer/autumn drought conditions. The net herbage accumulation rate from undisturbed areas of L. corniculatus and pasture were similar in spring, summer and autumn. Seasonal variation in the calibration regressions fitted to estimate herbage mass of L. corniculatus non-destructively, suggested a combination of destructive and nondestructive methods are needed to assess herbage mass. It was concluded that L. corniculatus has the potential to increase the performance of a pasture-based sheep dryland farming system due to its ability to grow in acidic soils, its tolerance of drought conditions during summer/autumn and its seasonality of feed supply. 5. Three digestion experiments involving cryptorchid weaned lambs were conducted for 14 days over the spring, summer and autumn to determine changes in in vivo digestibility of DM, OM, digestible OM in the DM and ME concentration of L. corniculatus at different stages of maturity. In vivo digestibility samples were then used as standards to investigate if the enzymatic in vitro system of Roughan and Holland (1977) could predict OMD and DOMD of CT-containing L. corniculatus. Digestibility of L. corniculatus declined as it matured, but the rate of decline was much less than occurs for temperate grasses and for white clover. It was concluded that the in vitro enzymatic system of Roughan and Holland (1977) can be used to predict OMD and DOMD of L. corniculatus, provided a standard curve involving in vivo data generated with L. corniculatus is used. Using a standard curve with in vivo data from pasture led to bias which increased at lower OMD values. Reasons for the consistent differences between L. corniculatus and pasture standard curves are discussed, including possible effects of residual bound CT in lowering in vitro digestibility. From this series of experiments, this study is the first to report that relative to conventional perennial ryegrass/white clover, mating ewes on L. corniculatus under grazing conditions may reduce post-natal lamb mortality. It is also the first study to show that grazing sheep on L. corniculatus can maintain productivity during spring and summer with reduced dependence on anthelmintic drench input. It is concluded that whole farm modelling, mechanical harvesting and conservation strategies, selection of L. corniculatus germplasm for creeping-type plants more suited to grazing and the integration of new crops containing secondary compounds, such as chicory, should be considered to support major advances in sustainable dryland sheep farming systems.