Memoria para optar al título profesional de: Ingeniero Agrónomo Mención Agroindustria / No autorizada por el autor para ser publicada a texto completo / En este estudio se evaluó el efecto de los agentes sanitizantes sobre la calidad funcional y microbiológica en los brotes de alfalfa (Medicago sativa L.). Los brotes fueron sumergidos durante 5 minutos en soluciones sanitizantes de hipoclorito de sodio (HS; 100 mg L-1), ácido peroxiacético (APA; 90 mg L-1), dióxido de cloro (DC; 10 mg L-1) y clorito de sodio acidificado (CSA; 500 mg L-1), que luego fueron envasados en bolsas plásticas (60 g) y almacenados a 5 °C durante 7 a 8 días. Los parámetros evaluados fueron: tasa respiratoria, atmósfera modificada, luminosidad (L), croma (C*), tono (Hab), fenoles totales, capacidad antioxidante, análisis microbiológicos y sensoriales. Los brotes de alfalfa lavados en las soluciones sanitizantes presentaron un aumento en la tasa respiratoria en todos los tratamientos. El hipoclorito de sodio, ácido peroxiacético y dióxido de cloro no afectaron el parámetro de color ni la calidad sensorial. Sin embargo, el clorito de sodio acidificado registró un leve pardeamiento y menores valores en calidad sensorial. El menor recuento microbiológico durante el tiempo de almacenamiento lo obtuvo el clorito de sodio acidificado, alcanzando una reducción promedio de 1,3 log UFC g-1. El contenido de fenoles totales y la capacidad antioxidante presentaron un leve aumento durante el almacenamiento a 5 °C, pero sin diferencias significativas entre los tratamientos.

The use of coated seed to establish crops is not a novel practice. Seed coatings have been used on small seeded crops to improve the handling ability by making the seed unit larger and heavier. Producers can therefore calibrate their sowing equipment more efficiently and wind will not cause as much drift at sowing. Seed coating can have an added benefit for leguminous crops if the symbiotic inoculant (Rhizobium) is added to the coating. This saves the producer time and allows peace of mind that inoculation was done by trained professionals. Other than the inoculation, there are some other constituents in the seed coating that can have benefits to the plant. It can, however, be theorized that added nutrients or pesticides will be beneficial to the plants only if these nutrients are deficient in the growth medium or when pests are present. This study evaluated the effect of seed coating on the life stages of germination, emergence and survival, seedling growth and ultimate yield of mature lucerne (Medicago sativa L.) plants, comparing the results with non-coated seed. Two cultivars were used in the evaluation, SA Standard and SuperCuf, to determine if the effects would be similar, or would genetic differences between the cultivar play a significant role. These life stages (germination, emergence and seedling growth) were chosen due to the importance of these stages to the success of establishment. Fast and uniform germination will result in a uniform stand with strong competition against weed infestation. The effect of growth medium on the emergence of seedlings and the interaction between the seed coating and the growth medium was important to determine to identify limitations in the use of seed coating. Changes in the growth of seedlings in terms of some physio-morphological characteristics will assist in identifying parameters influenced by the coating. It was, however, essential to not only do these trials under ideal agricultural conditions, but to identify if similar results would be obtained from stressed conditions, such as salinity, which is a growing concern for crop production areas. The question of whether seed coating will influence the ultimate production of the crop could then be answered. It was found that the method in which germination is tested can have a significant outcome for the results obtained. When the Jacobsen apparatus was compared with the use of petri dishes, using specification according to ISTA, it was found that the water movement in the Jacobsen apparatus overcomes concentrated nutrient conditions, especially for SuperCuf. Under saline conditions the coated SA Standard seed had higher germination than the non-coated seed, therefore overcoming inhibitions imposed by the salinity. It is clear that the coating influences germination of lucerne and the interaction with the seed environment is significant. It is also clear that the genetic differences between cultivars are significant and results should not be applied across all lucerne cultivars. When the emergence percentage had been determined in different growth media, namely a commercial growth media, a sandy loam soil and silica medium, it was found that the emergence was influenced by the media. Even though the emergence of seedlings are mostly determined by the nutrients in the cotyledons, some growing conditions did cause lower emergence for non-coated SA Standard seeds and was overcome by the use of coated seed. When the growing conditions were manipulated with saline irrigation it was found that coated SuperCuf had a higher emergence % than the non-coated treatments when irrigated with the 750 μS.cm-1 water. From the data collected from this trial, it can be concluded that, even though the use of seed coating does not always influence the emergence of lucerne seedlings, seed coating does have an influence on the emergence, but it is dependent on the growth medium quality in terms of nutrient composition and salinity. Similar to the germination trial, the genetic influence of the different cultivars was noticeable. To determine the differences caused by seed coating to the physio-morphological characteristics (stem height, leaf area etc.) of lucerne, a pot trial was conducted using different irrigation treatments, municipal water (180 μS.cm-1), 500 and 750 μS.cm-1 water, created with NaCl. It was found that the physio-morphological characteristics were highly correlated, i.e. the stem height, leaf area, number of leaves and dry matter production changed in relation to each other. There were, however, differences in this correlation coated and non-coated seed treatments, when irrigated with 500 μS.cm-1 water. For the seedlings grown from coated seed, the correlation between shoot dry matter yield and the other parameters were low, while the seedlings growth from non-coated seed, leaf area was not correlated with the other parameters. It was concluded that the tolerance mechanism for salinity for plants irrigated with 500 μS.cm-1 water, caused more differences than the other water treatments. Taking into consideration that coating influences the germination and emergence of lucerne and that the two cultivars react differently to the seed coating, the yields obtained from field trials could then be interpreted if differences were observed. Three field trials were established, namely a trial established in autumn (established in 2009) and second trial established in spring (established in 2010) which were sown at 25 kg.ha-1, while a third trial established spring (established in 2010) was sown at 5 different sowing densities, namely 80%, 90%, 100%, 110% and 120% of recommended sowing density (25 kg.ha-1). It was found that the pasture stands established with SA Standard, did not show many differences between the coated and non-coated seed treatments and were mostly restricted to the second growing season, where the non-coated seed treatments had significantly higher dry matter yield than the coated seed treatments. Stands established with SuperCuf, displayed more variation between the seed treatments and the non-coated seed treatments had higher yields in the first season. It was, however, found that the stands established with coated SuperCuf seed had lower stem: leaf ratio’s, indicating that a better quality fodder can be produced from coated seed. The data from the sowing density trial showed very little difference between the yields of the stands established with coated and non-coated SA Standard seed. Differences were, however, observed between seed treatments at 90% and 120% of the recommended sowing density, where the stands established with non-coated seed had higher yields than the stands established with coated seed. It can be concluded that under these trial conditions, the use of coated seed had very little influence on the yield of the lucerne stands. The observed differences suggest that the lucerne growth under these few conditions, the stands established from non-coated seed had better yield, but the stands established from coated seed had better quality. It is, however, more likely that there will be no differences between the seed treatments. Data from the sowing density trial also led to the conclusion that stands established at 20% less seed will not result in lower yields if the stand establishment is successful. The similarity between the seed treatments and the sowing densities suggests that the number of plants per area were the same, caused by seedling mortality during the high growth rate in the early growing stage, or the morphological characteristics, such as number of stems per plant and number of leaves per stem, adapted to result in similar yields and quality. / Dissertation (MSc Agric)--University of Pretoria, 2013. / gm2014 / Plant Production and Soil Science / unrestricted

The aim of this diploma thesis is determination of alfalfa (Medicago sativa) infestation in Agricaltural company Kvasicko a. s. The monitoring of infestation was doing from 2013 to 2016 on land Odšenkovna, sawn on 29. 8. 2013, and on land Novina levá, sawn on 11. 4. 2014. Evaluation was made by counting method. The results of this evaluation were processed with DCA analysis and canonical corespondence analysis (CCA). It was found 11 species of weeds on observed lands during the monitoring. The most frequently weeds were: Capsella bursa-pastoris, Stellaria media, Taraxacum sec. Ruderalia and Veronica.

Alfalfa, Medicago sativa L., cultivars, breeding lines and germplasm releases (populations) and Rhizobium meliloti L. strains that exhibit nitrogen fixation efficiency and tolerance to salinity and drought stresses should enhance seedling establishment, increase yields, reduce nitrogen dependency on petroleum-based nitrogen fertilizers, and allow wider use of irrigated lands in semiarid and a rid regions. In vitro experiments were conducted to determine relative salt (sodium chloride -- NaCl) tolerance: l) during germination and early seedling growth of 229 alfalfa populations from North Africa, the Middle East and the United States, 2) survival and growth of 92 rhizobial strains obtained from different laboratories in the United States or isolated from host plants growing in saline and arid regions of the Intermountain west, 3) compatibility, nodulation and nitrogen fixation efficiency of the most salt-tolerant alfalfa populations and rhizobial strains, and 4) survival, nodulation and nitrogen fixation efficiency of the most salt-tolerant alfalfa populations and rhizobial strain tested under drought conditions. Results from screening of alfalfa at salinity levels of 0 to 3.2 S m-1 (0.1 S m-1 = 640 mg L-1 = 10 mM NaCl at 25° C) indicated 29 populations exhibited salt tolerance at 2.8 S m-1. At salinity levels of 0 to 7.2 S m-1, 14 rhizobial strains exhibited salt tolerance at 6.4 s m-1. Compatibility, nodulation and nitrogen fixation efficiency in the 29 alfalfa populations and the 14 rhizobial strains under controlled conditions showed that four of the rhizobial strains were highly compatible with all 29 populations. These 29 populations and four strains were then exposed to 0 to 1.6 S m-1 NaCl, with the result that nitrogen was fixed with highly compatible symbionts at 1.6 S m-1 NaCl. From these results, the six most highly compatible alfalfa populations and the best overall rhizobial strain were combined and tested under simulated drought stress. These populations were able to fix more nitrogen under drought stress (-0.76 MPa) (0.088 nmol seedling-1 s-1) than they did under salt stress (1.6 S m-1) (0.066 nmol seedling-1 s-1). The in vitro screening technique for acetylene reduction appears to be a useful tool for detecting physiological changes due to salinity and water stresses and for measuring seedling nitrogen fixation efficiency. Breeding for drought and salinity tolerance in conjunction with high nitrogen fix ing potential may be more realistic than breeding strictly for nitrogen fixation without regard for environmental adaptation.

Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2018-01-16T11:32:00Z No. of bitstreams: 1 texto completo.pdf: 1247418 bytes, checksum: e985fa1f1156f534c1dbec006984c2de (MD5) / Made available in DSpace on 2018-01-16T11:32:00Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1247418 bytes, checksum: e985fa1f1156f534c1dbec006984c2de (MD5) Previous issue date: 2017-07-18 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / O desenvolvimento de novos cultivares de alfafa está relacionado, além do potencial produtivo, com o valor nutritivo da forragem, ambos determinados por uma série de caracteres de controle genético complexo. Por isso, o melhoramento da cultura exige adoção de técnicas que maximizem a acurácia seletiva. Nesse contexto, a utilização das Redes Neurais Artificiais (RNAs) pode ser útil para otimizar a seleção simultânea de caracteres, que tem sido a estratégia mais utilizada no melhoramento da alfafa. Assim, o objetivo deste trabalho foi aplicar a abordagem de índices de seleção no contexto das redes neurais artificiais para auxiliar o melhoramento da cultura da alfafa, além de avaliar a eficiência das redes para realizar a seleção de caracteres complexos. Para isso, foram utilizados dados de 77 genótipos de alfafa avaliados em quatro cortes realizados ao longo das diferentes estações no ano, mensurados quanto a caracteres de produção e valor nutritivo. Primeiramente, foram estimados os ganhos por seleção direta, indireta e simultânea pelo índice de Taí para cada um dos grupos de caracteres. Para obtenção das redes para cada grupo, os genótipos foram subdivididos em classes segundo os escores do índice. A fim de garantir boa capacidade de generalização das redes, nas etapas de treinamento e validação foram utilizados dados ampliados de cada grupo de caracteres e, só então, as redes foram utilizadas para classificar os dados reais dos quatro cortes. Posteriormente, utilizou- se dados ampliados de três cortes para treinamento e validação e a rede, então estabelecida, foi empregada em dados reais, relativos a um dos cortes que não participou do processo de ampliação. De modo geral, as seleções diretas proporcionaram ganhos per se satisfatórios para os propósitos do melhoramento, mas sua utilização deve ser bastante criteriosa tendo em vista a existência de correlações desfavoráveis entre alguns caracteres estudados. Já as seleções indiretas apresentaram dificuldades para manter ganhos desejados em todos os caracteres no conjunto de genótipos. Os resultados da seleção simultânea utilizando o índice de Taí proporcionaram distribuição de ganhos mais equilibrada para os conjuntos de caracteres em todos os cortes. No contexto das redes neurais artificiais, as taxas de erros das redes de produção e valor nutritivo foram baixas, evidenciando a grande concordância destas com o índice. Entretanto, a limitação do conjunto de treinamento e teste das RNAs para ampliação de três cortes, levou tanto a altas taxas de erro ou a níveis muito baixos, indicativo de perda da habilidade de generalização. Quando se utilizou as redes no novo corte, as taxas de erro foram altas em todos os cenários. O baixo desempenho obtido pode ser explicado pela discrepância observada entre o conjunto de treinamento e o conjunto de teste. Dessa forma, percebe-se a importância da qualidade do conjunto de dados para treinamento de uma rede eficiente, pois a utilização desta é diretamente relacionada com a disponibilidade de bons exemplos e em grande número. O uso das RNAs será mais difundido à medida que haja mudança no paradigma da experimentação em que as decisões passarão a ser tomadas em bancos de dados, construídos ao longo do tempo de um programa de melhoramento, que constituirão de base para o processamento de dados. / Developing new alfalfa cultivars is related with productive potential besides to the nutritive value of the forage, which are determined by traits of complex genetic control. That is why alfalfa breeding requires adoption of techniques that maximize selective accuracy. In this context, using Artificial Neural Networks (ANNs) can be useful to optimize the simultaneous selection, which has been the strategy most used in alfalfa breeding. Thus, the aim of this work was to apply the selection index approach in the context of artificial neural networks to help in alfalfa breeding, as well as to evaluate the efficiency of the ANNs to perform the complex trait selection. The data from 77 alfalfa genotypes evaluated in four cuts were collected considering different seasons in the year. In each cut were measured production and nutritive value traits. Firstly, we estimated the gains by direct, indirect and simultaneous selection by the Taí index for each trait groups. To obtain the ANN for each group, the genotypes were subdivided into classes according to the index scores. In order to ensure good network generalization, in the training and validation stages we used amplificated data from each group of characters, later we used the ANNs to classify the real data of the four cuts. Subsequently, we used amplificated data from three cuts for training and validation, and the established ANNs were used in a new cut that did not participate in the amplification process. In general, the direct selections provided satisfactory individual gains for the breeding purposes, but their use must be very judicious since the existence of unfavorable correlations between some studied traits. Indirect selections, presented difficulties in maintaining desired gains in all traits in the genotype set. The results of simultaneous selection using the Taí index provided a more balanced distribution gains for the trait sets in all cuts. In the context of the artificial neural networks, the error rates of the production and nutritional value groups were low, evidencing the great agreement of these with the index. However, the limitation of the training set and test of ANNs for data of three cut amplification, led either to high error rates or to very low levels, indicating of loss of the generalization ability. When the networks were used in the new cut, error rates were high in all scenarios. The low performance obtained can be explained by the discrepancy between the training set and the test one. In this way, the importance of the quality of data set for training an efficient network is perceived, since its use is directly related to the availability of good examples and in large numbers. The use of ANNs will be more widespread with a change in the experimentation paradigm in which decisions will be made into databases built over time from a breeding program that will form the basis for data processing.

Angl. resumé

[EN] Alfalfa (Medicago sativa L.), is a forage legume with a significant content of protein, being the most widely cultivated forage around the world. In this species, the protein content decreased during growth processes as well as other resources used by the plant during the flowering process. Alfalfa also contains a lower concentration of condensed tannins or proanthocyanidins (PAs), less than required to remedy the digestive disorder of ruminant livestock causing pasture bloat by production of greenhouse gases as result of the microbial fermentation and the excessive desamination of proteins in the rumen (by-pass effect). These defects on nutrition are reflected in the yield and production of ruminants. Both problematics are difficult to improve by conventional methods. The overall objective of this thesis is to develop molecular tools useful for genetic manipulation of certain traits of agronomic interest in alfalfa, to enhance their nutritional value. At a first step, we have developed an Agrobacterium tumefaciens-mediated transformation protocol via somatic embryogenesis applicable to various genotypes of alfalfa to produce viable embryos in the 50% of the inoculated explants. Chimaeric plants or scapes were almost not detected because most of the transgenic plants contained the T-DNA with all the transgenes incorporated, having thus the indispensable tool to perform the other two objectives. We have isolated two orthologs of the TERMINAL FLOWER 1 (TFL1) gene from alfalfa (MsTFL1c and MsTFL1a). Both genes were constitutively expressed in A. thaliana, and later on in alfalfa, to evaluate their possible use as biotechnological tool for the improvement of the nutritional value of this forage legume, producing a delay in the flowering time and thereby an increase of vegetative development. MsTFL1a performs the same functions as TFL1 in Arabidopsis y its overexpression in this plant produces a remarkable delay of flowering time. In alfalfa, its constitutive expression also produces a delay in flowering. However, this phenotype was not associated to an increase of vegetative growth, being plants of reduced size that appears tied to a limitation in cell proliferation. Moreover, the limitation in cell proliferation was associated with a reduction in the expression levels of certain cell division effector genes, as it is the case of cyclins. Our results support the hypothesis that TFL1-like genes could also participate in signaling pathways that regulate cell differentiation in plants. M. sativa could be a good model to study the possible range of biochemical diversification of these proteins. On the other hand, we have made a multigenic construct in the modular cloning system GoldenBraid 2.0 (35S::Del::Ros1::MtANR::MtLAR). In a first step, it was functionally validated by transient expression in N. benthamiana. In addition, this construct was introduced by stable transformation in two experimental models (A. thaliana and N. tabacum) and later on in M. sativa, where it has been studied the integration capacity and expression levels of the different transgenes. In N. tabacum it has been achieved the activation of both the anthocyanin and PAs biosynthetic pathways. However, it was not able to activate both routes in alfalfa. The TFs Delila and Rosea1 were not able to activate the genes involved in the regulation of these metabolic pathways in alfalfa, or were not sufficient levels of expression of both TFs for this purpose. Therefore, this multigenic construct does not resulted a good biotechnological tool for the activation of PAs biosynthesis in alfalfa. Our results suggest that the mechanisms of transcriptional control of both biosynthetic pathways could be different between plant species, and that additional specific genetic information on the anthocyanin and PAs biosynthesis in legumes is needed to efficiently activate both pathways in this species. / [ES] La alfalfa (Medicago sativa L.), es una leguminosa forrajera con un importante contenido en proteína, siendo la más cultivada a nivel mundial. En esta especie, los contenidos protéicos se ven mermados por los procesos de crecimiento y los recursos utilizados por la planta en el proceso de floración. Además, la alfalfa contiene una concentración de taninos condensados o proantocianidinas (PAs) muy inferior a la requerida para subsanar el desorden digestivo del ganado rumiante que genera hinchamiento por gases de efecto invernadero o meteorismo y la excesiva desaminación de las proteínas en el rumen producida por las fermentaciones de la flora microbiana ruminal. Ambas problemáticas son difíciles de abordar mediante métodos convencionales de mejora. El objetivo general de esta Tesis es desarrollar herramientas moleculares de utilidad para la manipulación genética de determinados caracteres de interés agronómico en la alfalfa, con objeto de incrementar su valor nutricional. Para ello, hemos puesto a punto un protocolo de transformación genética mediada por Agrobacterium tumefaciens vía embriogénes somática aplicable a varios genotipos de alfalfa, que permite la producción de embriones viables capaces de germinar y producir plantas completas en el 50% de los explantes inoculados. Prácticamente no se detectó la presencia de quimeras o escapes ya que la mayoría de las plantas obtenidas contenían el T-DNA con todos los transgenes incorporados, disponiéndose por tanto de la herramienta imprescindible para la realización de los otros dos objetivos. Hemos aislado dos ortólogos del gen TERMINAL FLOWER 1 (TFL1) de alfalfa (MsTFL1c y MsTFL1a). Ambos genes se sobreexpresaron constitutivamente en A. thaliana y después en alfalfa para su posible uso como herramienta biotecnológica para producir un retraso en el tiempo de floración y con ello un posible aumento del desarrollo vegetativo. MsTFL1a extiende la fase vegetativa e inflorescente y retrasa la floración en Arabidopsis. En alfalfa su expresión constitutiva también produce retraso de la floración. Sin embargo, a este fenotipo no se le asocia un incremento del desarrollo vegetativo, presentando las plantas un tamaño más reducido que parece vinculado a una limitación en la proliferación celular. Hemos comprobado que este hecho se asocia a una reducción en los niveles de expresión de determinados genes efectores de la división celular, como es el caso de las ciclinas. Nuestros resultados apoyan la hipótesis de que los genes TFL1-like podrían participar adicionalmente en vías de señalización que regulan la diferenciación celular en plantas. También, hemos realizado una construcción multigénica mediante el sistema de clonaje por módulos GoldenBraid 2.0 (35S::Del::Ros1::MtANR::MtLAR). Se ha validado su funcionalidad mediante expresión transitoria en N. benthamiana y se ha introducido de manera estable en dos modelos experimentales: A. thaliana y N. tabacum, y en M. sativa, donde se ha estudiado su capacidad de integración, así como la de expresión de los transgenes. Hemos comprobado que esta construcción multigénica es capaz de activar la ruta de biosíntesis de antocianinas y de PAs en N. tabacum, pero no fue capaz de activar ambas rutas en alfalfa. Los factores transcripcionales Delila y Rosea1 no fueron capaces de activar los genes implicados en la regulación de ambas vías metabólicas en esta especie, o no fueron suficientes sus niveles de expresión para este fin. Por tanto, esta construcción no es una buena herramienta biotecnológica que procure la activación de la ruta de biosíntesis de PAs en la alfalfa. Nuestros resultados sugieren que los mecanismos de control transcripcional de la biosíntesis de antocianinas y PAs son diferentes entre las distintas especies, y que se necesita información genética adicional específica de la biosíntesis de antocianinas y PAs en leguminosas para poder diseñar eficientemente herrami / [CA] L'alfals (Medicago sativa L.), és una lleguminosa farratgera amb un important contingut en proteïna, sent la més cultivada a nivell mundial. En aquesta espècie, els continguts proteics es veuen desfavorits amb els processos de creixement i els requeriments utilitzats per la planta en el procés de floració. A més, l'alfals conté una concentració de tanins condensats o proantocianidines (PAs) molt inferior a la requerida per a esmenar el desorde digestiu del bestiar remugant que genera unflament per gasos o meteorisme per la producció de gasos d'efecte hivernacle, i l'excessiva desaminació de les proteïnes en el rumen produïda per les fermentacions de la flora microbiana. Ambdós problemàtiques són difícils d'abordar per mitjà de mètodes convencionals de millora. L'objectiu general d'aquesta Tesi és desenvolupar ferramentes moleculars d'utilitat per a la manipulació genètica de determinats caràcters d'interés agronòmic en l'alfals, a fi d'incrementar el seu valor nutricional. Per a això, hem posat a punt un protocol de transformació mediada per Agrobacterium tumefaciens via embriogènesi somàtica aplicable a diversos genotips d'alfals que permet la producció d'embrions viables capaços de germinar i produir plantes completes en el 50% dels explants inoculats. Pràcticament no es va detectar la presència de fugues, ja que la majoria de les plantes obtingudes contenien el T-DNA amb tots el transgens introduïts, tenint per tant la ferramenta imprescindible per a la realització dels altres dos objectius. Es van aïllar i sobreexpressar constitutivament dos ortòlegs del gen TERMINAL FLOWER 1 (TFL1) d'alfals (MsTFL1c i MsTFL1a) en A. thaliana i en alfals per al seu possible ús com a ferramenta biotecnològica, procurant un retard en el temps de floració i amb això un augment del desenvolupament vegetatiu. MsTFL1a estén la fase vegetativa i inflorescent i retarda la floració en Arabidopsis. En alfals la seua expressió constitutiva també produïx retard de la floració. No obstant això, a aquest fenotip no se li associa un increment del desenvolupament vegetatiu, sent les plantes d'una grandària més reduït que pareix vinculat a una limitació en la proliferació cel·lular. Hem comprovat que la limitació en la proliferació cel·lular s'associa a una reducció en els nivells d'expressió de determinats gens efectors de la divisió cel·lular, com és el cas de les ciclines. Els nostres resultats recolzen la hipòtesi que els gens TFL1-like podrien participar adicionalment en vies de senyalització que regulen la diferenciació cel·lular en plantes. Hem realitzat una construcció multigénica per mitjà del sistema de clonatge per mòduls GoldenBraid 2.0 (35S::Del::Ros1::MtANR::MtLAR). S'ha validat funcionalment per mitjà d'expressió transitòria en N. benthamiana. A més, s'ha introduït de manera estable en dos models experimentals (A. thaliana amb N. tabacum), i finalment en M. sativa, en els que s'ha estudiat la seua capacitat d'integració, així com la d'expressió dels transgens. Hem mostrat que aquesta construcció multigénica és capaç d'activar la ruta de biosíntesi d'antocianines i de PAs en N. tabacum. No obstant això, no van ser capaços d'activar la ruta de biosíntesi d'antocianines i per tant, la de PAs, en alfals. Els factors transcripcionals Delila i Rosea1 no són capaços d'activar els gens implicats en la regulació d'ambdues vies metabòliques en aquesta espècie, o alternativament no van ser prou els seus nivells d'expressió per a este fi. Per tant, aquesta construcció multigénica no és una bona ferramenta biotecnològica per activar la ruta de biosíntesi de PAs en l'alfals. Els nostres resultats suggerixen que els mecanismes de control transcripcional de la biosíntesi d'antocianines i PAs són diferents entre les distintes espècies, i que es necessita informació genètica addicional específica de la biosíntesi d'antocianines i PAs en lleguminoses per a / Fresquet Corrales, S. (2015). Diseño y validación de herramientas biotecnológicas para la mejora del valor nutricional de la alfalfa (Medicago sativa L.) [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/59240

Nous nous sommes intéressés à évaluer l'influence du changement climatique sur les patrons nets de réponse des plantes aux agents pathogènes. Dans ce travail, nous avons étudié les effets de l’augmentation de la température (20°, 25° et 28°C) sur le phénotype précoces (symptômes de maladie) et sur la fitness en fin de cycle de différentes accessions et mutants de nodulation de la plante légumineuse modèle Medicago truncatula, inoculées par des souches d’agent pathogène racinaire Verticillium adaptées à des différentes températures. Le comportement des variétés de Luzerne cultivée (Medicago sativa) dans ces conditions a été également analysé. Le travail a été divisé en 3 parties. La première partie nous a permis d’identifier parmi 12 souches de Verticillium spp., une souche froide (VA1) et une souche tempérée (V31.2), avec une température optimale de 20°C et 25°C respectivement pour la croissance, la sporulation et l'agressivité sur M. truncatula. Par contre, notre collection des souches ne renfermant pas de souches adaptées à des températures plus élevées. Nous avons obtenu par mutagénèse UV de la souche V31.2 une troisième souche (AS38) chaude qui est agressive à 28°C. Dans la deuxième partie nous avons observé les symptômes de maladie pour sept accessions naturelles de M. truncatula, inoculées par ces trois souches d’agent pathogène, à trois températures 20°, 25° et 28°C et en présence de la souche Sinorhizobium meliloti RCR2011. De faibles symptômes ont été relevés pour deux accessions A17 et DZA315.16 inoculées par VA1 à 20°C. Nous avons observé une sensibilité maximale pour trois accessions (F83005.5, DZA315.16 et L321) inoculées par V31.2 à 25°C, et pour quatre accessions (F83005.5, DZA315.16, L321 et L198) inoculées par AS38 à 28°C. Les résultats des symptômes de maladie ont été confirmés par une quantification moléculaire de l’ADN fongique (qPCR) et par ré-isolement à partir des tissus aériens de la plante. L’effet de VA1 et V31.2 sur trois caractères de fitness (nombre et poids de gousse par plante, ainsi que biomasse aérienne) de M. truncatula a été étudié. L’effet de VA1 s’observe uniquement à 20°C sur l'accession A17. Par contre, V31.2 a montré un impact sur les trois caractères de fitness qui diminuent chez les accessions sensibles, ainsi que sur le nombre de gousse pour l’accession résistante L198. Dans la troisième partie nous avons analysé de la même façon pour quatre mutants de nodulation dans le fond génétique A17. Les mutants ont montré un niveau de résistance à la souche VA1 plus élevé qu’A17, quelle que soit la température étudiée. Vis à vis de la souche V31.2, à 20°C les mutants skl et hcl ont montré le même taux de symptômes qu’A17 tandis que les mutants nfp et sunn ont de taux de symptômes supérieur à celui d'A17. Ces mutants ont tous une sensibilité plus élevé à 25°C. Les résultats des symptômes de maladie ont été confirmés par le test de ré-isolement. Pour ces mutants nous montrons pour la première fois, que seul le mutant sunn (hypernodulant) à la même productivité qu’A17, quelle que soit la condition (contrôle ou inoculées) et la souche (VA1 ou V31.2) étudiée ; alors que le mutant skl (hypernodulant également) a une productivité plus faible. Les deux autres mutants déficients dans la nodulation nfp et hcl ont montré une productivité plus faible qu’A17 quelle que soit la souche et la température étudiée. Enfin une bonne similitude a été trouvée entre la réponse phénotypique précoce (symptômes de maladie) de M. truncatula et de M. sativa inoculées par Verticillium spp. Dans cette thèse, on n’est pas trouvé la corrélation positive entre la capacité de la nodulation et la protection contre la maladie, mais la symbiose augmente la fitness pour certaines de ces plantes. Les résultats suggèrent aussi que l'augmentation de la température pourrait contribuer à faire apparaître une souche adaptée à 28°C (AS38) qui est plus agressive et plus virulente que V31.2 sur M. truncatula. / We were interested to evaluate the influence of climate change on net patterns of plants responses to pathogens. In this work, we studied the effects of temperature increase (20 °, 25 ° and 28 ° C) on early phenotype (symptoms of disease) and on fitness at the end of growth cycle on different accessions and nodulation mutants of the legume model plant Medicago truncatula, inoculated by the root pathogen Verticillium adapted to different temperatures. The behavior of cultivated varieties of alfalfa (Medicago sativa) in these conditions was also analyzed. The work is divided into 3 parts. In the first part, we identified among 12 strains of Verticillium spp., a cold strain (VA1) and a temperate strain (V31.2) with an optimum temperature of growth, sporulation and aggressiveness to M. truncatula of 20°C and 25°C respectively. Since our strain collection doesn’t contain strains adapted to higher temperatures, we have obtained by UV mutagenesis of strain V31.2 a third strain (AS38), considered as a ‘hot’ strain, which is aggressive at 28°C. In the second part, we observed the symptoms of disease on seven natural accessions of M. truncatula, inoculated by the three strains of the pathogen at three temperatures 20°C, 25°C and 28°C in the presence of Sinorhizobium meliloti RCR2011. Mild symptoms were observed for two accessions A17 and DZA315.16 inoculated with VA1 at 20°C. We observed a maximal sensitivity for three accessions (F83005.5, DZA315.16 and L321) inoculated with V31.2 at 25 ° C, and for four accessions (F83005.5, DZA315.16, L321 and L198) inoculated with AS38 at 28 ° C. The results of phenotypic disease symptoms were confirmed by molecular quantification of fungal DNA (qPCR), and re-isolation of the fungus from aerial plant tissues. The effect of strains VA1 and V31.2 on three fitness traits (number and weight of pods per plant and aerial biomass) was studied. The effect negative of VA1 was observed only at 20°C on the A17 accession. In contrast, V31.2 showed an impact on the three fitness traits, which decrease in susceptible accessions, as well as on pod number of the resistant accession L198. In the third part, a similar analysis was made for four nodulation mutants in A17 genetic background. Nodulation mutants showed a higher level of resistance to VA1 than A17, at different studied temperatures. Towards strain V31.2 at 20°C the mutants skl and hcl showed the same symptom scores as A17 whereas nfp and sunn mutants had more susceptible. Mutants showed a higher sensitivity at 25°C to V31.2 fungal strain. The results of phenotypic disease symptoms were confirmed by re-isolation experiments. For the nodulation mutants we showed, for the first time, that only the sunn mutant (hypernodulant) has the same productivity as A17, regardless of the condition (inoculated or control) and the studied strain (VA1 or V31.2); while the skl mutant (hypernodulant also) has a lower productivity. The other two mutants defective in nodulation (nfp and hcl) showed lower productivity than A17 regardless of the strain (VA1 or V31.2) and the temperature studied. Finally, a strong similarity was found between the early phenotypic response symptoms disease in M. truncatula and M. sativa inoculated by Verticillium spp. In this thesis, we didn’t find a positive correlation between the ability of nodulation and protection against the disease, however the symbiosis increases the fitness of some of these plants. The results also suggest that increasing temperatures could favour appearance a strain adapted to 28°C (AS38), which is more aggressive and more virulent than V31.2 on M. truncatula.

Made available in DSpace on 2014-06-11T19:31:38Z (GMT). No. of bitstreams: 0 Previous issue date: 2001-06-27Bitstream added on 2014-06-13T20:02:33Z : No. of bitstreams: 1 campos_at_dr_botfca.pdf: 1232453 bytes, checksum: 23f42c8e84fcf5f7b59eb8f0187628d3 (MD5) / O objetivo deste trabalho foi proceder ao levantamento e caracterização do consumo de energia nas atividades relativas à implantação, condução, processos de fenação e armazenamento, assim como ao balanço energético global de duas forrageiras diferentes, sendo uma gramínea, o coast-cross, e a outra uma leguminosa, a alfafa, inseridas em um Sistema Intensivo de Produção de Leite, localizado na região de Coronel Pacheco, MG. Para o desenvolvimento do estudo, foram utilizados dados coletados pela Embrapa Gado de Leite, em uma média de sete anos, considerando a produção de um hectare para cada cultura, relacionando o consumo dos diversos insumos envolvidos nos processos produtivos e o emprego de máquinas, equipamentos e instalações necessários à condução tecnificada da atividade. Para o levantamento dos diversos fatores componentes da matriz, foram utilizados coeficientes energéticos apresentados por autores especializados. Para ambas culturas utilizadas para estudo, coast-cross e alfafa, a energia direta, relativa aos insumos, teve maior participação que a indireta, sendo os percentuais encontrados de 94,64 e 5,31, respectivamente, para o coast-cross e 93,05 e 6,95, respectivamente, para a alfafa. O principal consumidor de energia indireta, para ambas forrageiras, foi o trator, que aciona todos implementos, seguido pelo equipamento de irrigação. O galpão de feno utilizado em conjunto pelas culturas, apesar do elevado consumo energético desprendido em sua construção, possui uma elevada vida útil, com o armazenamento considerado em 24 horas por dia, o que faz com que sua participação ponderada pela Demanda Específica de Energia seja reduzida drasticamente. O processo de fenação do coast-cross apresentou maior eficiência energética que o da alfafa, com valores de 4,28 para o primeiro e 3,19 para a segunda... / The aim of this work was to carry out a survey and characterization of the energy comsumption in the activities related to introduce, development, hay processing and storage, as well as global energy inputs of two different forage, one of them is grass, the coast-cross, and the other is leguminous, alfalfa, inserted in an Intensive System of Milk production, located in the Coronel Pacheco MG-Brasil region. In order to develop the study it was used data colected by Embrapa Gado de Leite, an average of seven years, considering the production of 1 ha of each culture, linking the consumption of various inputs involved in the productive process and the use of machines, equipments and building that are necessary to conduct the activity in a technique way. To carry on the survey and the various components factors of the matrix, it was used energetic coefficient introduced by specialized researchers. For both cultures used in the study, coast-cross and alfalfa, the direct energy, related to inputs had bigger participation, being the percentages found 94.64 and 5.31 respectively, to the coast-cross, and 93.05 and 6.95, respectively, to the alfalfa. The main consumer of indirect energy, for both forage, was the tractor, which turns on all the implements, followed by an irrigation system. The warehouse hay used together by the cultures, despite the high energetic consuption used in its construction, the warehouse hay has a long useful life, so what concerns to the storage, considered within 24 h per day, what makes its participation slightly ponderous by the specific demand of energy, and this process reduces its participation in a drastic way. The hay process coast-cross presented energetic efficiency as much bigger as the alfalfa, values such as 4.28 to the first one and 3.19 to the second one. Most of the energy used was that one derived from fossil resources, diesel...(Complete abstract click electronic access below)

Two experiments were conducted with sheep to determine the nutritional value of 'Lathco' flatpea (Lathyrus sylvestris) hay. In experiment 1, a finishing trial was conducted with 50 group fed crossbred lambs (25 ewes and 25 wethers) fed diets consisting of 70% chopped forage and 30% ground corn grain for 70 d. Five pens of five wethers and five pens of five ewes were selffed diets in which the forage consisted of 100:0, 75:25, 50:50, 25:75, and 0:100 proportions of alfalfa (Medicago sativa L.) and flatpea hay. Feed efficiencies decreased linearly (P<.05) as flatpea increased in the diet. Linear decreases (P<.05) were obtained in kidney and pelvic fat, backfat, leg conformation, and yield grade with increased flatpea in the diet. Blood urea-N (BUN), ruminal pH and NH3-N increased (P<.005) as proportion of flatpea increased. Total volatile fatty acid (VFA) and acetate concentration decreased linearly (P<.005) and propionate, isovalerate, and valerate levels increased linearly (P<.01) with increased proportions of flatpea hay. In experiment 2, 30 wether lambs were fed diets consisting of 100:0, 75:25, 50:50, 25:75 and 0:100 proportions of chopped alfalfa and flatpea hays in a digestion trial. Neutral detergent fiber (NOE), acid detergent fiber (ADE), cellulose, hemicellulose and energy digestibilities decreased linearly (P<.01) with increased levels of flatpea hay. Nitrogen retention was not different between sheep fed the different diets. Apparent absorbtion and retention of Ca decreased linearly (P<.05) with increased level of flatpea hay Ruminal pH and NHa-N, and BUN increased linearly (P<.OOl) as flatpea was increased in the diet. Ruminal total VEA, acetate and acetate to propionate ratio decreased linearly (P<.001) and propionate, isobutyrate, isovalerate, and valerate levels increased linearly (P<.001) as level of flatpea increased. / Master of Science