Dissertations / Theses on the topic 'Medicago sativa'

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Biotecnologia<br>The aim of this thesis is a survey of the toxicity of Quantum Dots (QD’s) in in vitro cultures of the legumes Medicago sativa and Medicago truncatula. Two samples of QD’s were used to assess toxicity: QD50_MPA tested in a concentration of [160 nM] and QD68_MPA with [40 nM]. The QD50_MPA sample was applied in the already available embryogenic cell suspension culture of M. truncatula (M9-10a) to evaluate the effect of QD’s in plant differentiation at somatic embryogenesis. Results showed no differences in embryo morphology or development time. A fine suspension culture of Medicago sativa (line M699) was established to evaluate the effect of QD68_MPA QD’s in cell growth, viability as well in oxidative stress by ROS production. It was concluded that no major differences were seen when cells were exposed to QD’s in a period of 4 hours, despite an overall oxidative stress was detected. After 24 hours of exposure of cell suspension cultures to QD’s viability of cells started to decrease and ROS production was intensified, also the morphology of cells had significant changes. Moreover, it was visualized the internalization of both QD´s samples after 72 h of cell suspension cultures exposure to QD’s. QD50_MPA sample was used in the two cell suspension cultures used: M. sativa and M. truncatula, and in both lines QD’s were visualized in the nucleus and in cytosol, with the exception of the vacuoles. QD68_MPA sample was found to be up-taked by M. sativa cells and located mainly in the hyaloplasm and organelles such as plasts, and similarly to the other sample, no internalization was observed in the vacuoles.

The synthesis of isoflavonoid phytoalexins in legumes is relatively well understood, but far less is known about how these phytotoxic compounds are metabolised by the plant when no longer required. In this project medicarpin, the major isoflavonoid phytoalexin in alfalfa, was prepared in radiolabelled form and fed to cell cultures and seedlings of alfalfa. The metabolism of the radioactive phytoalexin was then studied by characterising the radiolabelled metabolites formed. Uptake of radiolabelled phytoalexin by cells was faster in elicitor-treated cultures than in untreated cultures. However, there was little difference in pattern or speed of metabolism in treated or untreated cultures. Labelled medicarpin was rapidly metabolised to a complex range of extractable medicarpin products (MPs). A very small proportion of the dose was broken down to (^14)C02. A total of 8 MPs could be resolved as distinct metabolites by HPLC and TLC. However, as incubation time increased the radioactivity became associated with multiple minor components which could not be identified. The 8 MPs were characterised by UV and mass-spectrometry and where possible by co- chromatography with authentic standards by TLC and HPLC. Four MPs were unambiguously identified as medicarpin-3-0-glucoside-6"-O-malonate (MGM), the isoflavans vestitol and sativan and the pterocarpan 6a-hydroxy-3,4'- dimethoxypterocarpan (variabilin). In addition a hydroxylated derivative of medicarpin, termed pseudomedicarpin was also tentatively identified. Of the four remaining metabolites MPl had a relative molecular mass of 166 but remained unidentified. MP2 was formed from pseudomedicarpin, but could not be characterised due to its labile nature. Similarly MP3 and MP6 remained unidentified, though the evidence suggested that MPS was a demethylated product of medicarpin. The metabolism of medicarpin in seedlings resembled that of cell cultures with the exception that rather more of the medicarpin was conjugated to form MGM.

Backcross populations (BC) from Medicago sativa L. x M. dzhawakhetica Bordz. hybrids were analyzed for chromosome number, fertility and morphological characteristics. Previously obtained F1 hybrids were recovered when diploid (2n = 2x = 16) M. sativa was crossed with tetraploid (2n = 4x = 32) M. dzhawakhetica. Resulting F1 hybrids were triploid (2n = 3x = 24), completely male sterile and had low levels of female fertility. Subsequent populations were obtained by successive backcrossing to unrelated (4x) M. sativa clones. The BC1 plants were pentaploid (2n = 5x = 40) and both male and female fertile. BC2 populations had chromosome numbers ranging from 2n = 32 to 48, and most plants (94% were male and female fertile. BC3 populations were tetraploid (2n = 32) or near tetraploid (2n = 33) and were morphologically similar to M. sativa. Preliminary analysis of mitochondrial nucleic acids by agarose gel electrophoresis, indicated biparental inheritance of this organelle in the F1 hybrids; however, further analysis provided inconclusive results.

In this study, the responses and signal mechanisms were explored with a model system, concerning the interactions between lucerne suspension cells (<I>Medicago sativa</I> L., cv. Kabul) and elicitor either from avirulent (V2) or virulent (V1) isolate of <I>Verticillium, alboatrum</I>. V2 elicitor induced a two-phase of H<SUB>2</SUB>O<SUB>2</SUB> accumulation in the cell cultures. Activation of defence expression led to an increase in PAL activity, phytoalexin accumulation and deposition of phenolic polymers. However, activities of antioxidant enzymes, such as catalase, peroxidases, glucanase, glutathione reductase, did not show obvious increase within 24 h after treatment with the elicitor. Glutathione S-transferase activity increased after 1<SUP>st</SUP> oxidative burst. V1 elicitor induced similar defence responses as V2 did, but a stronger response was observed when the same concentration of elicitor was used, confirming that Kabul is a resistance cultivar to <I>V. albo-atrum</I>. Ca<SUP>2+</SUP> influx is necessary for oxidative burst, PAL activity and phytoalexin accumulation. Either blocking Ca<SUP>2+ </SUP>channel by La<SUP>3+</SUP> or reduction of extracellular Ca<SUP>2+</SUP> amount by EGTA, had an important inhibition on oxidative burst, PAL activity and phytoalexin accumulation. Intracellular Ca<SUP>2+</SUP> also played a role in downstream signalling. Intracellular Ca<SUP>2+</SUP> inhibitors. TBM-8 and Ruthenium red, strongly inhibited the PAL activity and phytoalexin accumulation. Oxidative burst has a relation with defence expression. An NADPH oxidase inhibitor, diphenyleneiodonium (DPI), which inhibited oxidative burst effectively, also inhibited PAL activity and phytoalexin accumulation. However, DCN, an inhibitor of peroxidase, also inhibited and oxidative burst, PAL activity and phytoalexin accumulated in micromolar range. Oxidative burst with superoxide-origin is related to defence activation. The H<SUB>2</SUB>O<SUB>2</SUB> itself did not stimulate an activation of PAL activity. Addition of superoxide dismutase (SOD) stimulated an increase in H<SUB>2</SUB>O<SUB>2</SUB> accumulation. Microsomal membranes are capable of superoxide synthesis when NADPH/NADH was used as electron donor, which was DPI-sensitive. This enzyme activity increased after treatment of the cell cultures with elicitor.

Assegurando-se condições não limitantes de radiação incidente, água e nutrientes, acompanhou-se, em casa-de-vegetação, o desenvolvimento de plantas isoladas da cultivar Europe de alfafa, desde a emergência até o estádio primeiros legumes maduros. Medidas semanais permitiram quantificar o ritmo de formação de fitômeros da haste principal e suas ramificações primárias, assim como o surgimento de inflorescências e seu número de flores. Paralelamente, plantas mantidas nas mesmas condições foram amostradas para determinar a evolução e repartição da biomassa formada. Independentemente do tipo de haste, a indução floral provoca uma diminuição na velocidade de emissão de fitômeros concomitante com uma redução progressiva no tamanho da folha e no número de flores por inflorescência. As diferentes hastes (haste principal – HP e as ramificações – R1 a Rn) apresentam comportamento distinto quanto a todos os parâmetros estudados. Com base no filocrono, identificou-se um grupo de maior velocidade formado pela HP, e R1 a R4, enquanto as demais são mais lentas. A velocidade de desenvolvimento foi a variável mais pertinente para explicar a maior biomassa e o maior número de flores produzidas pelas hastes do primeiro grupo. Um modelo de produção de flores por planta baseado na temperatura média diária é proposto, integrando sub-modelos de desenvolvimento de cada grupo de hastes. Validado com dados independentes da mesma cultivar e da cultivar Cinna, o modelo mostrou-se adequado como referencial da produção potencial de flores por planta.

The objectives of this study were to describe demographic change in an alfalfa population and determine whether genetic changes were associated with stand loss in an alfalfa field during the year of establishment. A nondormant composite population (AZ-88NDC) was sown at 1944 seeds per square meter in Oct. 1988 at Tucson, AZ. Only 29% of the seeds sown germinated and emerged. Survivorship curve of the emerged seedling population had some characteristics of Deevey type III. Two heavy mortality periods were observed. Low temperatures might be the major cause of the first heavy stand loss. The second significant stand loss could be explained by increased density stress. Isozyme profiles were produced from 60 day survivors dug from the field, unselected greenhouse-grown AZ-88NDC (='Unsel') and 300-d progenies produced by intermating plants surviving 300 days. Significant differences were observed in overall level of population heterozygosity of 60-d and unselected populations based on fixation indices of six isozyme loci. This suggests that genetic changes may be associated with establishment of an alfalfa stand.

La creixent població mundial està augmentant la demanda de fonts de proteïnes vegetals dietètiques barates i més sostenibles per a complementar o fins i tot reemplaçar fonts cares i limitades de proteïna animal. L'alfals (Medicago sativa) és una important lleguminosa productiva farratgera, àmpliament conreada a tot el món. A causa de la seva alta qualitat nutricional i adaptabilitat, la proteïna de la fulla d'alfals s'usa principalment com un suplement alimentós, mentre que només juga un paper menor en el sector de l'alimentació humana. L'ús de concentrats de proteïnes vegetals derivats de l'alfals en l'alimentació humana està limitat per la seva qualitat negativa, com el color marró, el sabor amarg i els compostos antinutricionals. Després de la collita de les fulles d'alfals, les proteases endògenes de la fulla estan degradant la proteïna, mentre que els enzims polifenoloxidasa (PPO) i peroxidasa (POD) amb alta activitat inicial estan causant simultàniament l'ennegriment del teixit. El resultat d'aquestes accions és una proteïna fosca i parcialment degradada. Per a evitar aquests efectes negatius en la proteïna extreta, es va dur a terme un primer estudi sobre els efectes del blanqueig en vaporitzar les fulles d'alfals des del començament de la collita. El primer objectiu de la tesi va ser optimitzar la inactivació de PPO, POD i proteases de plantes en fulles d'alfals a través del procés de blanqueig de vapor. També es va investigar l'efecte d'aquest procés en l'índex d’enfosquiment, el color, el nitrogen no proteic i el pes molecular. Algunes de les propietats indesitjables de l'alfals poden deure's als nivells més alts de saponina com un factor antinutricional principal que es troba en l'alfals. La segona part de la tesi va tenir com a objectiu descobrir les condicions òptimes d'extracció assistida per ultrasò per a obtenir el major rendiment de soponines totals i la seva bioaccesibilitat per a la seva posterior aplicació en els aliments utilitzant la tècnica RSM. A més, s’ha emprat la irradiació UV per a la degradació de les saponines d'alfals a diferents temperatures i pH. En conseqüència, el procés d'extracció de la proteïna d'alfals com a aliment és extremadament important per aconseguir una proteïna d'alta qualitat sense cap problema de seguretat. Així, s’ha desenvolupat i utilitzat la tècnica de precipitació isoelèctrica alcalina assistida per ultrafiltració ultrasònica (UUAAIP) com un nou procés en l'extracció de proteïnes de les fulles d'alfals. Finalment, es van investigar la composició, les propietats fisicoquímiques i funcionals de la proteïna extreta i es van comparar amb els resultats d'altres mètodes comuns d'extracció. En cobrir tots els problemes esmentats, aquesta tesi tracta de proporcionar un aïllat de proteïna d'alfals d'alta qualitat sense cap problema de seguretat. Aquesta recerca suggereix que el blanqueig amb vapor de fulles d'alfals senceres fresques en condicions òptimes va ser útil per a evitar l'aparició del color fosc i la degradació de la proteïna extreta. Per tant, la combinació de mètodes d'extracció i purificació va crear un mètode nou i eficient, que pot millorar la puresa, la seguretat, la qualitat i les propietats funcionals de la proteïna d'alfals. La UUAAIP és una tècnica apropiada per a fabricar aïllats de proteïna d'alfals i podria resoldre les restriccions del consum humà de proteïna d'alfals.<br>La creciente población mundial está aumentando la demanda de fuentes de proteínas vegetales dietéticas baratas y más sostenibles para complementar o incluso reemplazar fuentes caras y limitadas de proteína animal. La alfalfa (Medicago sativa) es una importante leguminosa productiva forrajera, ampliamente cultivada en todo el mundo. Debido a su alta calidad nutricional y adaptabilidad, la proteína de la hoja de alfalfa se usa principalmente como un suplemento alimenticio, mientras que solo juega un papel menor en el sector de la alimentación humana. El uso de concentrados de proteínas vegetales derivados de la alfalfa en la alimentación humana está limitado por su calidad negativa, como el color pardo, el sabor amargo y los compuestos antinutricionales. Después de la cosecha de las hojas de alfalfa, las proteasas endógenas de la hoja están degradando la proteína, mientras que las enzimas polifenoloxidasa (PPO) y peroxidasa (POD) con alta actividad inicial están causando simultáneamente el pardeamiento del tejido. El resultado de estas acciones es una proteína oscura y parcialmente degradada. Para evitar estos efectos negativos en la proteína extraída, se llevó a cabo un primer estudio sobre los efectos del blanqueo al vaporizar las hojas de alfalfa desde el comienzo de la cosecha. El primer objetivo de la tesis fue optimizar la inactivación de PPO, POD y proteasas de plantas en hojas de alfalfa a través del proceso de blanqueo de vapor. También se investigó el efecto de este proceso en el índice de pardeamiento, el color, el nitrógeno no proteico y la masa molecular. Algunas de las propiedades indeseables de la alfalfa pueden deberse a los niveles más altos de saponina como un factor antinutricional principal que se encuentra en la alfalfa. La segunda parte de la tesis tuvo como objetivo hallar las condiciones óptimas de extracción asistida por ultrasonido para obtener el mayor rendimiento de saponinas totales y su bioaccesibilidad para su posterior aplicación en los alimentos utilizando la técnica RSM. Además, se ha utilizado la irradiación UV para la degradación de las saponinas de alfalfa a diferentes temperaturas y pH. En consecuencia, el proceso de extracción de la proteína de alfalfa como alimento es extremadamente importante para lograr una proteína de alta calidad sin ningún problema de seguridad. Así, se ha desarrollado y utilizado la técnica de precipitación isoeléctrica alcalina asistida por ultrafiltración ultrasónica (UUAAIP) como un nuevo proceso en la extracción de proteínas de las hojas de alfalfa. Finalmente, se investigaron la composición, las propiedades fisicoquímicas y funcionales de la proteína extraída y se compararon con los resultados de otros métodos comunes de extracción. Al cubrir todos los problemas mencionados, esta tesis trata de proporcionar un aislado de proteína de alfalfa de alta calidad sin ningún problema de seguridad. Esta investigación sugiere que el blanqueo con vapor de hojas de alfalfa enteras frescas en condiciones óptimas fue útil para evitar la aparición del color oscuro y la degradación de la proteína extraída. Por lo tanto, la combinación de métodos de extracción y purificación creó un método nuevo y eficiente, que puede mejorar la pureza, la seguridad, la calidad y las propiedades funcionales de la proteína de alfalfa. La UUAAIP es una técnica apropiada para obtener aislados de proteína de alfalfa y podría resolver las restricciones del consumo humano de proteína de alfalfa.<br>The world’s growing population is raising the demand for sources of cheap and more sustainable dietary plant proteins to supplement or even replace expensive and limited sources of animal protein. Alfalfa (Medicago sativa) is an important productive forage legume, widely cultivated around the world. Owing to its high nutritional quality and adaptability, the alfalfa leaf protein is mainly used as a feed supplement, while it only plays a minor role in the human food sector. The use of plant protein concentrates derived from alfalfa in human food is limited by their negative quality such as brown color, bitter taste, and anti-nutritional compounds. After harvesting of the alfalfa leaves, the endogenous proteases of the leaf are degrading the protein while the polyphenol oxidase (PPO) and peroxidase (POD) enzymes with high initial activity are simultaneously causing browning in the tissue. The result of these actions is a dark and a partially degraded protein. To avoid these negative effects in the extracted protein, the authors carried out a first study on the effects of blanching by steaming the leaves of alfalfa from the beginning of the harvest. The first aim of the thesis was to optimize the inactivation of PPO, POD and plant proteases in alfalfa leaves through the steam blanching process. The effect of this process on the browning index, color, non-protein nitrogen and molecular weight was also investigated. Some of undesirable properties of alfalfa may be due to the higher levels of saponin as a main anti-nutritional factor found in alfalfa. The second part of thesis aimed to find out the optimal conditions of ultrasound-assisted extraction for obtaining the highest yield of total saponins and their bioaccessibility for further application in the food using the RSM. Beside, UV irradiation has been employed for degradation of alfalfa saponins in different temperature and pH. Accordingly, the extraction process of alfalfa protein as a food is extremely important for achieving a high quality protein without any safety concerns. So the ultrasonic-ultrafiltration-assisted Alkaline Isoelectric precipitation (UUAAIP) technique as a new process was developed and employed in the alfalfa leaves’ protein extraction. Finally, the composition, physiochemical and functional properties of the extracted protein were investigated and compared with the results of other extraction common methods. By covering all the mentioned issues, this thesis tries to provide alfalfa protein isolate with high quality without any safety concerns. This research suggests that steam blanching of fresh whole alfalfa leaves under the optimum conditions was helpful for avoiding the appearance of the dark color appears and the degradation of the extracted protein. Thus, the combination of methods of extraction and purification created a new and efficient method, which is able to improve the purity, safety, quality and functional properties of alfalfa protein. UUAAIP is an appropriate technique for manufacturing alfalfa protein isolates and could resolve restrictions of human consumption of alfalfa protein.

Objetivo: Demostrar las propiedades estrogénicas del extracto hidroalcohólico de la especie Medicago sativa L (alfalfa) en ratas albinas ovariectomizadas (OVX). Método: Se emplearon 48 ratas albinas Sprague Dawley hembras de 200 a 250 g. de 8 semanas de edad de las cuales 40 fueron sometidas a extirpación quirúrgica de ambos ovarios (OVX) siguiendo la técnica de ovariectomía bilateral por el flanco. Después del post-operatorio las ratas se dividieron en seis grupos y se suministró el extracto hidroalcohólico de Medicago sativa (alfalfa) bajo el siguiente esquema de trabajo: Grupo 1:Control Negativo (OVX), vehículo, VO,2mL/kg; Grupo2: Control Positivo (OVX) ,Estradiol, SC, 3ug/kg; Grupo 3: (OVX)Extracto Alfalfa ,VO, 100 mg/kg; Grupo 4: (OVX),Extracto Alfalfa, VO, 500 mg/kg; Grupo 5: (OVX), Extracto Alfalfa, VO, 1000 mg/kg; Grupo 6: (No OVX),Control del procedimiento quirúrgico. El tratamiento duró 14 días. Los parámetros evaluados fueron peso del útero, peso corporal, cambios del ciclo estral mediante frotis vaginal y análisis del perfil hormonal. Resultados: Se observó aumento en el peso de útero en las dosis de 500mg/kg y 1000mg/kg.; además de la presencia de alcaloides, flavonoides y saponinas en el extracto hidroalcohólico de alfalfa en cantidad regular. En la técnica quirúrgica empleada la combinación anestésica xilazina (2mg/kg), ketamina (40mg/kg) indujo un plano quirúrgico óptimo (plano 2), sin complicaciones en el post-operatorio ni la muerte de los animales. Conclusiones: El extracto hidroalcohólico de Medicago sativa L (alfalfa) causa un efecto estrogénico al incrementar el peso de útero en ratas OVX en las dosis de 500mg/kg y 1000mg/kg. Además contiene saponinas en cantidad regular. La técnica quirúrgica ovariectomía bilateral por el flanco es un método práctico, confiable y menos traumático para los animales que permite realizar investigaciones sobre defiencia de estrógenos.<br>Objective: Demonstrate the estrogenic properties of hydroalcoholic extract of the species Medicago sativa L (alfalfa) in albino rats ovariectomized (OVX). Methods: We used 48 Sprague Dawley female albino rats of 200 to 250 g. 8 weeks of age, of which 40 underwent surgical removal of both ovaries (OVX) by means of bilateral ovariectomy on the flank. After post-operative rats were divided into six groups and provided the hydroalcoholic extract of Medicago sativa (alfalfa) under the following work schedule: Group 1: Negative Control (OVX), vehicle, VO 2 ml / kg, Group 2: Positive Control (OVX), estradiol, SC, 3ug/kg; Group 3 (OVX) Alfalfa extract, VO, 100 mg / kg, Group 4 (OVX), Alfalfa extract, VO, 500 mg / kg, Group 5: (OVX), Alfalfa extract, VO, 1000 mg / kg, Group 6: (No OVX), Control of the surgical procedure. The treatment lasted 14 days. The outcome measures were uterine weight, body weight, estrous cycle changes by vaginal smear and hormonal profile analysis. Results: The increase in uterine weight at doses of 500mg/kg and 1000mg/kg., Besides the presence of alkaloids, flavonoids and saponins in the hydroalcoholic extract of alfalfa in regular amount. In the surgical technique used anesthetic combination xylazine (2mg/kg), ketamine (40mg/kg) induced an optimal surgical plane (plane 2), without complications or postoperative death of animals. Conclusions: The hydroalcoholic extract of Medicago sativa L (alfalfa) causes an oestrogenic effect by increasing the uterus weight in OVX rats at doses of 500mg/kg and 1000mg/kg. It also contains saponins in regular amount. The surgical technique for bilateral ovariectomy flank is a practical, reliable and less traumatic for the animals that can do research on estrogen deficiency.<br>Tesis

L'OBJECTIF DE CE TRAVAIL A ETE DE COMPRENDRE LES BASES AGRONOMIQUES ET GENETIQUES DU RENDEMENT GRAINIER DE LA LUZERNE, EN DEVELOPPANT SUCCESSIVEMENT TROIS ETUDES. UNE PREMIERE ETUDE SUR L'EFFET DU CULTIVAR ET DE L'ENVIRONNEMENT, REALISEE EN COUVERTS PORTE-GRAINES, A MONTRE QUE LES PRINCIPALES SOURCES DE VARIATION ENTRE CULTIVARS POUR LE RENDEMENT GRAINIER ETAIENT L'INDICE DE RECOLTE ET LE POIDS DE GRAINES PAR INFLORESCENCE. ENTRE ENVIRONNEMENTS, LA SOURCE DE VARIATION A ETE LA BIOMASSE AERIENNE DISPONIBLE AU MOMENT DE LA RECOLTE DE GRAINES. L'ETUDE GENETIQUE A EVALUER LA VARIABILITE ET LE CONTROLE GENETIQUE POUR LES COMPOSANTES DU RENDEMENT GRAINIER MESUREES EN PLANTES ISOLEES. TOUTES LES COMPOSANTES, EVALUEES AU NIVEAU DE LA PLANTE, DE L'INFLORESCENCE OU DE LA GOUSSE, ONT MONTRE UNE LARGE VARIABILITE INTER-ET INTRA-POPULATION. L'APTITUDE GENERALE A LA COMBlNAISON EST LA PRlNCIPALE SOURCE DE VARIATION GENETIQUE DANS UN PLAN DIALLE 7X7. L'ANALYSE D'UN PLAN FACTORIEL 7X7 A MONTRE UNE HERITABILITE AU SENS STRICT PARTICULEREMENT ELEVEE POUR LE POIDS DE GRAINES PAR INFLORESCENCE. ENTRE VARIETES, LE POIDS DE GRAINES PAR INFLORESCENCE MESURE EN PLANTES ISOLEES ETAIT CORRELE A CELUI MESURE EN COUVERTS PORTE-GRAINES. UN SCHEMA DE SELECTION COMBINÉE INDIVIDU-FAMILLE, UTILISANT COMME CRITERE DE SELECTION LE POIDS DE GRAINES PAR INFLORESCENCE OU LE POIDS DE L'INFLORESCENCE, A ETE PROPOSE POUR LA CREATION DE VARIETES DE LUZERNE A FORT POTENTEL GRAINIER. L'ETUDE AGRONOMIQUE A PERMIS DE CONFIRMER LA RELATION ENTRE LA BIOMASSE AERIENNE ET LA PRODUCTION GRAINIERE. LES ENVIRONNEMENTS FAVORABLES A LA PRODUCTION DE BIOMASSE MAXIMISENT AUSSI LA PRODUCTION DES GRAINES. DES PRATIQUES AGRONOMIQUES POUR AUGMENTER LA PRODUCTION DE GRAINES ONT ETE PROPOSEES. LA REGRESSION ENTRE LA BIOMASSE AERIENNE ET LA PRODUCTION DE GRAINES PEUT SERVIR D'OUTIL DE DIAGNOSTIC DANS LES CULTURES PORTE-GRAINES.

Analyse des polypeptides obtenus par traduction in vitro d'arn messagers extraits de racines non nodulees, de nodosites fixatrices d'azote ou de nodosites non fixatrices. Etude de l'expression des genes codant pour les leghemoglobines et analyse de l'organisation de ces genes chez medicago sativa, des hybrides somatiques, et chez des legumineuses apparentees. Utilisation de sondes d'adn isolees d'autres especes de plantes pour suivre l'expression des genes codant pour des proteines connues pour jouer un role dans la symbiose (glutamine synthetase) ou les interactions plantes-microorganismes (phenylalanine ammonia-lyase, chalcone synthase, extensine)

La mutagenese par le transposon tn5 d'un fragment de 30 kb du megaplasmide psym de r. Meliloti, a permis de mettre en evidence 3 regions portant des genes de nodulation dont on etudie les proteines correspondantes. Le transfert a r. Trifolii d'un plasmide portant les genes nodfeg et nodh de r. Meliloti rend la souche hybride capable de noduler la luzerne mais inapte a noduler le trefle

Mestrado em Biotecnologia Alimentar<br>O molecular pharming permite a produção de proteinas terapêuticas recombinantes a larga escala, de forma segura e a baixo custo. No presente trabalho, é proposta a produção heteróloga de quatro péptidos inibidores da ACE em dois emergentes sistemas de expressão vegetal, Lactuca sativa (alface) e Medicago truncatula (luz cortada). A utilização da alface, uma planta comestível, pode proporcionar um meio para a administração oral de péptidos anti-hipertensivos, criando um novo alimento funcional. Por outro lado, a utilização de M. truncatula, uma leguminosa modelo, garante não só a facilidade de transformação mas também a extrapolação processual para outras leguminosas. No contexto actual de demanda por terapias alternativas para a hipertensão e de processos mais eficientes de produção de péptidos inibidores da ACE, este trabalho assume particular importância.<br>Molecular pharming is a cost-effective, scalable and safe system to produce high-quality and biologically active recombinant therapeutic proteins. In the present work the heterologous production of four ACE inhibitory peptides in two emerging plant expression hosts, Lactuca sativa (lettuce) and Medicago truncatula is proposed. The use of lettuce, an edible plant, can provide a means for oral delivery of antihypertensive peptides, thus creating a novel functional food. On another hand, the use of M. truncatula, a model legume, ensures not only the simple transformation process but also the procedural extrapolation to other legume species. In the current scenario of global demand for alternative hypertension therapies and easier ACE inhibitory peptide manufacturing processes, this work assumes particular importance.

The alfalfa harvest in Ecuador constitutes one of the most important activities, especially as a primary product for animal feeding. The crop is widely distributed in the mountain region of the country. It is important to have studies on the production systems, which facilitate a real and compete vision of those characteristics that interact among them in the agricultural production. With this knowledge it will be possible to look for the most adequate solutions to the conditions of the area where alfalfa is produced. The objectives of the study reported herein were: 1. To make an agricultural, social and economical diagnosis in the community of El Troje, San Luis Parish, Riobamba Canton, Province of Chimborazo. 2. To identify the production system used by the farmers to harvest alfalfa in El Troje community.

Medicago is widely used as a forage crop. It is often susceptible to various pathogenic infections and exhibits low growth in drought and extreme climatic conditions. In the current study, a strategy was developed for over-expressing an “Osmotin-Chitinase” gene chimera in transgenic Medicago that could potentially confer resistance to different biotic and abiotic stresses. Seed germination of several cultivars of Medicago (M. sativa ssp. sativa, M. sativa ssp. falcata, M. sativa ssp. caerulea, M. truncatula, and M. Rugosa) was tested to determine the cultivars with good germination rates. Among these, M. sativa ssp. sativa showed an average of 80% germination over a period of one week and was subsequently selected for regeneration and transformation experiments. Different explants (cotyledons, hypocotyls, petioles) were tested for regeneration. Among these, hypocotyl explants showed highest (46.17 %) percent regeneration. Escherichia coli harboring Osmotin-Chitinase (OSM-CHI) gene chimera cloned into binary vector pBTEX with nptII as a selection marker was mobilized in Agrobacterium tumefaciens strain EHA105 which was employed in the transformation of hypocotyl explants of Medicago. Transformed calli were grown on callus inducing medium containing kanamycin for screening. Further screening of the positive transgenics was performed using PCR. Southern hybridization was carried out for further confirmation of successful transformation. Transformed shoots will be grown on the root inducing medium for developing into plantlets which would then be transferred to the green house and later tested for their degree of resistance to various biotic and abiotic stresses.

Memoria para optar al título profesional de Ingeniero Agrónomo Mención Agroindustria<br>El objetivo de este estudio fue evaluar el efecto de distintos sanitizantes como hipoclorito de sodio (NaClO 100 mg L-1), dióxido de cloro (ClO2 5 y 10 mg L-1), ácido peroxiacético (APA 50 y 90 mg L-1) y clorito de sodio acidificado (CSA 250 y 500 mg L-1) sobre la carga microbiana y calidad sensorial en brotes de alfalfa envasados en atmósfera modificada pasiva conservados a 5º C por 7 días. En dos ensayos independientes se emplearon las mismas dosis de sanitizantes, pero diferentes bolsas con diferente permeabilidad a los gases. Los parámetros evaluados fueron respiración, concentración de gases en el interior de las bolsas, color, carga microbiana y calidad sensorial. El uso de bolsas de baja permeabilidad utilizadas en el Ensayo I, junto con las dosis de sanitizantes no fue efectivo para mantener la calidad sensorial de los brotes de alfalfa. Las concentraciones alcanzadas al interior de las bolsas fueron de 18,2 a 27% de CO2 y 1 a 1,2% de O2 tras 7 días. En todos los tratamientos se obtuvo una reducción de los recuentos en aerobios mesófilos, psicrófilos y enterobacterias al utilizar los distintos sanitizantes. El CSA 250 y 500 presentaron los recuentos más bajos, sin embargo afectaron negativamente el color, y calidad sensorial de los brotes. En el Ensayo II, las concentraciones al interior de las bolsas fueron de 2,7 a 4,1% de CO2 y 3,6 a 6,7% de O2 y al igual que el Ensayo I, el CSA 500 fue el sanitizante que presentó los menores recuentos en aerobios mesófilos, enterobacterias y psicrófilos con 4,6, 4,8 y 5,5 log ufc g-1, respectivamente, seguido por CSA 250 con 5,4, 5,3 y 5,7 log ufc g-1, respectivamente, sin afectar las características sensoriales. En ambos ensayos fue el NaClO (Bp) el tratamiento que registró recuentos más altos en mesófilos y enterobacterias, lo que confirmaría que el envasado en AM mantiene mejor la calidad microbiológica que el envasado en contacto con el aire. Por tanto los brotes de alfalfa lavados con CSA 250 y 500 en combinación con un envasado en atmósfera modificada diminuyeron la carga microbiana sin afectar la calidad sensorial, manteniéndose dentro de la media aceptable durante los 7 días a 5º C.<br>The objective of this study was to evaluate the effect of different sanitizers as sodium hypochlorite (NaClO 100 mg L -1 ), chlorine dioxide (ClO2 5 and 10 mg L -1 ), peroxyacetic acid (APA 50 and 90 mg L -1 ) and acidified sodium chlorite (ASC 250 and 500 mg L -1 ) on the microbial and sensory quality in packaged alfalfa sprouts passive modified atmosphere maintained at 5° C for 7 days. In two independent assays the same doses of sanitizers, and bags with different permeability to gases were used. The parameters evaluated were breathing, gas concentration inside the bags, color, microbial load and sensory quality. The use of low permeability bags used in the assay I, along with doses of sanitizers was not effective to maintain the sensory quality of alfalfa sprouts. Concentrations achieved within the bags were 18.2 to 27% CO2 and 1 to 1.2% O2 after 7 days. In all treatments a reduction in aerobic plate counts, mesofilos, psychrofiles and enterobacteriaceae using different sanitizers was observed. The CSA 250 and 500 showed the lowest counts, but affected negatively the color and sensory quality of sprouts. In assay II, the concentrations inside the bags were from 2.7 to 4.1% CO2 and 3.6 to 6.7% O2 and just that the assay I, CSA 500 was the sanitizer that presented the lower aerobic plate counts, enterobacteriaceae and psychrophiles with 4.6, 4.8 and 5.5 log CFU g -1 respectively, followed by CSA 250 with 5.4, 5.3 and 5.7 log cfu g -1 respectively, without affecting the sensory characteristics. In both assays was the NaClO (BP) treatment to higher counts recorded plate and Enterobacteriaceae, which would confirm that the packaging AM maintains the microbiological quality better than the packaging in contact with air. So alfalfa sprouts washed with CSA 250 and 500 in combination with modified atmosphere packaging decreases the microbial load without affecting the sensory quality, while remaining within acceptable average for 7 days at 5º C.

The general goal of this research was to understand the agronomic and physiological changes of a lucerne crop in distinct physical radiation environments and to verify the potential of lucerne to grow under shaded conditions. To achieve this, the research was conducted in four main steps: (i) firstly, experimental data collection in the field using two artificial shade materials (shade cloth and wooden slats) under inigated and non-irrigated conditions; (ii) a second experiment with data collection in a typical temperate dryland agroforestry area under non-irrigated conditions; (iii) generation of a light interception sub-model suitable for shaded crops and (iv) a linkage between the light interception sub-model and a canopy photosynthesis model for agroforestry use. In experiments 1 and 2, lucerne crop was exposed to 6 different light regimes: full sunlight (FS), shade cloth (FS+CL), wooden slats (FS+SL), trees (T), trees+cloth (T +CL) and trees+slats (T+SL). The FS+SL structure produced a physical radiation environment (radiation transmission, radiation periodicity and spectral composition) that was similar to that observed in the agroforestry site (f). The mean annual photosynthetic photon flux density (PPFD) was 41 % under the FS+CL, 44% under FS+SL and 48% under T compared with FS in clear sky conditions. Plants were exposed to an intermittent (sun/shade) regime under both FS+SL and T, whereas under FS+CL the shaded light regime was continuous. The red to far-red (RIFR) ratio measured during the shade period under the slats was 0.74 and under the trees was 0.64. However, R/FR ratio increased to 1.26 and 1.23 during the illuminated period under FS+SL and T, respectively, and these were equivalent to the ratio of 1.28 observed under the FS+CL and 1.31 in FS. The radiation use efficiency (RUE) of shoots increased under the 5 shaded treatments compared with full sunlight. The pattern of radiation interception was unchanged by radiation flux, periodicity and spectral composition and all treatments had a mean extinction coefficient of 0.82. However, the magnitude of the decrease in canopy growth was less than those in PPFD transmissivity. The mean lucerne annual dry matter (DM) yield was 17.5 t ha⁻¹ in FS and 10 t ha⁻¹ under the FS+CL, FS+SL and T regimes. This declined to 3.4 t DM ha⁻¹ under T+CL (22% PPFD transmissvity) and 4.1 t DM ha⁻¹ under T+SL (23% transmissivity). A similar pattern of response was observed for leaf net photosynthesis (Pn) rates under the shade treatments compared with full sun. In addition, spectral changes observed under the trees and slats affected plant motphology by increasing the number of long stems, stem height and internode length compared with full sunlight. Thus, there were two main explanations for the increase in RUE under shade compared with full sun: (i) preferential partition of assimilates to shoot rather than root growth and/or (ii) leaves under shade were still operating at an efficient part of the photosynthetic light curve. The changes proposed for the canopy Pn model were appropriate to simulate the radiation environment of an agroforestry system. However, the model underestimated DM yields under the continuous and intermittent shade regimes. These were considered to be mainly associated with plant factors, such as overestimation in maintenance respiration and partitioning between shoots and roots in shade and the intermittency light effect on leaf Pn rates. Further investigation in these topics must be addressed to accurately predict crop yield in agroforestry areas. Overall, the lucerne crop responded typically as a sun-adapted plant under shade. It was concluded that lucerne yield potential to grow under intermediate shade was superior to most of C3 pastures previously promoted in the literature.

A alfafa (Medicago sativa L.) é uma leguminosa com alto potencial produtivo, qualidade de forragem e flexibilidade de utilização. Quando bem manejada, é uma cultura capaz de fornecer forragem de qualidade e promover um aumento na produtividade dos rebanhos. Para a maior expansão desta forrageira no Brasil, torna-se necessário superar alguns entraves, como a falta de cultivares adaptadas às nossas condições de solo, baixa fertilidade do solo e inexistência de cultivares melhor adaptadas ao pastejo. O melhoramento genético de alfafa para tolerância a solos ácidos e aptidão ao pastejo, pode ser uma alternativa importante para a maior utilização desta cultura, contribuindo para a produção de carne e leite no país. O presente estudo tem como objetivo a seleção precoce de genótipos de alfafa para tolerância ao alumínio (Al) e aptidão ao pastejo e para isso, foram realizados dois experimentos. No experimento de seleção de plantas para tolerância ao Al tóxico foram avaliados nove genótipos (Crioula como testemunha, ECF1, Solo, Solução, Erechim, POA, Estrela e SJI) em solução nutritiva contendo diferentes concentrações de Al (0,0; 3,0; 6,0; 12; 24; 48 μMol/L AlCL3). O comprimento radicular (CR) foi mensurado, sendo selecionadas as 25 plântulas que apresentavam o maior comprimento de radicular. Foi realizado mais um ciclo de seleção e os resultados mostraram superioridade do crescimento radicular das populações SJI, Solução e POA, indicando uma maior tolerância ao Al tóxico. Já no experimento de seleção para aptidão ao pastejo, foram avaliados oito genótipos (ABT como testemunha, Erechim, POA, SJI, Estrela e as populações que já participam do Programa de Melhoramento da UFRGS para aptidão ao pastejo: E1C2, E1C3, E2C2 e E2C3. Foram utilizados os marcadores morfológicos comprimento do 1º e 2º entrenós (cm). Foram selecionadas as 25 plântulas, de cada população, que apresentaram o menor comprimento do 1º e do 2º entrenó, totalizando 50 plantas. Os genótipos SJI e as populações que já participam do programa de Melhoramento Genético da UFRGS apresentaram comportamento bastante semelhante à testemunha ABT, mostrando possuir maior aptidão ao pastejo. Além disso, o marcador morfológico do comprimento do 1º entrenó mostrou-se mais eficiente na seleção dos genótipos para aptidão ao pastejo. Portanto, os resultados mostraram que é possível obter-se progresso tanto na seleção para aptidão ao pastejo quanto para tolerância ao Al.<br>The alfalfa (Medicago sativa L.) is a legume that has many relevant characteristics such as high yield potential and forage quality and flexibility of use, and that when properly managed, a culture able to provide quality forage and promote an increase in the quality of herds. For the further expansion of this forage in Brazil, it is necessary to overcome certain obstacles such as lack of cultivars adapted to our climate and soil conditions, low soil fertility and inadequate management. Genetic improvement of alfalfa for tolerance to acid soils and suitability to grazing can be an important alternative for the development and establishment of this culture, contributing to the production of meat and milk in the country. The present study aims at the early selection of alfalfa genotypes for tolerance to aluminum (Al) and to grazing aptitude. For this, two experiments were conducted. In the experiment for selecting plants for Al tolerance were evaluated nine genotypes (Crioula as witness, ECF1, Soil, and Solution Erechim, POA, Estrela and SJI) in a nutrient solution containing different concentrations of Al (0.0, 3.0 , 6.0, 12, 24, 48 μMol / L AlCl3). Root length was measured, and selected twenty-five seedlings that had the greatest root length. There was one more cycle of selection and the results showed the superiority of root growth of populations SJI, Solution, and POA, indicating a higher tolerance to Al toxicity. In the experiment to select for grazing aptitude, were assessed eight genotypes (ABT as a check, Erechim, POA, SJI, Star, and the populations already on the Plant Breeding Program for grazing aptitude, UFRGS, (E1C2, E1C3, E2C2 and E2C3 ). The morphological markers used were the length of 1st and 2nd internode (cm). Were selected 25 seedlings from each population, which had the shortest length of 1st and 2nd internode, totaling fifty plants. The genotype SJI and the populations that already participate in the Plant Breeding Program behaved very similar to the witness (ABT), showing that have a higher grazing aptitude. In addition, the morphological marker of the 1st internode length was more efficient in the selection of genotypes for grazing aptitude. Therefore, the results showed that it is possible to make progress for grazing aptitude as well as for Al tolerance.

Les deux clones tétraploïdes (2n=32) MSR12 (Medicago sativa ssp. Sativa) et 3182 (Medicago sativa ssp. Falcata) et leurs hybrides sexués se différencient nettement à l'aide de 8 variables décrivant la morphologie foliaire, 2 variables de l'appareil stomatique foliaire. 19 variables concernant la répartition des trichomes foliaires, 17 variables décrivant la répartition des trichomes sur les sépales et 27 pics représentant en chromatographie les flavonoïdes floraux. Des plantes issues de fusions somatiques (E. TEOULE, 1983) sont ainsi décrites et leur origine est discutée en fonction des caractéristiques de leur caryotype. Deux plantes octoploïdes sont des hybrides somatiques totaux des caractères originaux par rapport aux parents et à l’hybride fertilité est discutée la suite de l'étude de leur méiose. Un hybride somatique l’origine octoploïde est devenu hexaploïde acquérant des caractères originaux ; sa méiose est très anormale et ses gamètes ont des nombres chromosomiques variés. Le phénomène d'élimination génomique est discuté. Deux plantes octoploïdes résultant d'autofusions de protoplastes d'un seul parent sont distinctes de celui-ci selon des caractères morphologiques. L'origine des autres plantes, ressemblant toutes à leur parent falcata, est recherchée : Une plante aneuploïde (2n = 33) est très peu distincte, et de fertilité normale i plusieurs hypothèses sont émises sur l'origine du chromosome supplémentaire. Une plante octoploïde devenue tétraploïde et à fertilité réduite, a subi des remaniements caryologiques. Deux autres variants, moins perturbés, sont observés tous sont distincts les uns des autres. Les autres plantes semblent être issues de protoplastes non fusionnés du parent falcata et leurs caractères plus ou moins originaux sont imputés au mode de régénération et non l l'influence du deuxième génotype.

Les tiges constituent l'élément fondamental du peuplement de luzerne et la partie aérienne limitant la digestibilité. Au cours de la croissance les tissus de soutien et de transport d'eau se mettent en place dans la tige. La proportion de ces tissus et des éléments constitutifs pourrait jouer un rôle déterminant dans leur dégradabilité. L'objectif de la thèse est de déterminer les bases génétiques des variations de la structure histologique des tiges de luzerne et d'établir la relation avec la digestibilité des tiges. Sur deux génotypes contrastés en digestibilité, différents caractères histologiques ont été quantifiés par analyse d'images, les teneurs en parois et en lignine mesurées et leur profil d'évolution observé le long de tiges caractérisées pour leur morphologie. La variabilité génétique pour ces caractères histologiques a été recherchée au sein et à l'intérieur de cultivars choisis pour leur différence de digestibilité. Enfin, la recherche de marqueurs associés à la variation de ces caractères a été initiée grâce au marquage d'une population F1 autotétraploïde issue du croisement entre deux plantes hétérozygotes. Les corrélations génétiques entre les différents caractères ont été calculées. L'analyse le long des tiges montre que la hauteur joue un rôle primordial dans la variation des caractères, et qu'ils évoluent similairement quel que soit le génotype. L'augmentation des teneurs en parois et en lignine en bas des tiges est associée à la mise en place du cambium responsable du développement de nouvelles assises cellulaires dont les parois se lignifient (xylème secondaire). Peu ou pas de variabilité entre cultivars mais une forte variabilité intra cultivar pour tous les caractères mesurés ont été mises en évidence. Une variabilité génétique importante au sein de la population F1 a permis de rechercher des marqueurs associés aux caractères histologiques, morphologiques et à la solubilité enzymatique des tiges. Cette étude a mis en évidence des marqueurs significativement associés à tous les caractères, expliquant de 6,5 % de la variation observée pour la proportion de parenchyme médullaire dans la tige à 40 % pour la hauteur des tiges. Nous avons également pu mettre en évidence des colocalisations entre la hauteur des tiges et le rapport feuilles/tiges, entre la hauteur des tiges, leur solubilité enzymatique, et la proportion d'écorce et la densité surfacique des parois du xylème. D'autre part l'analyse des corrélations a montré une relation négative entre la proportion de xylème, l'épaisseur et la densité surfacique des parois cellulaires du xylème, et la solubilité enzymatique des tiges; et une relation positive entre la solubilité enzymatique et la proportion des tissus non lignifiés. Cette thèse a montré qu'il est possible de sélectionner des luzernes de meilleure digestibilité en retenant comme critère de sélection des caractères liés à la proportion des différents tissus des tiges, et de rechercher des QTLs impliqués dans leur variation chez une espèce autotétraploïde. La saturation des cartes et la disponibilité d'un logiciel permettant de localiser des QTLs chez les espèces autotétraploïdes pourraient permettre d'envisager d'améliorer la digestibilité du fourrage grâce à la sélection assistée par marqueur. Une voie possible pour améliorer la digestibilité du fourrage serait de diminuer les proportions de xylème en introgressant les QTLs responsables de la diminution de ce caractère, et de l'épaisseur de ses parois en prenant garde à ne pas affecter la rigidité des tiges.

Memoria para optar al título profesional de: Ingeniero Agrónomo Mención Agroindustria<br>No autorizada por el autor para ser publicada a texto completo<br>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.

[Truncated abstract] In southern Western Australia the replacement of deep-rooted native vegetation with annual species has resulted in rising water tables and increased salinity due to insufficient water use. The area has a Mediterranean-type climate where rainfall during summer is generally low but variable resulting in limited plant growth. However, if rainfall does occur it potentially can contribute to to the increased water excess or drainage by increasing the soil water content before the main drainage period in winter. The first study investigated factors controlling soil water content changes during the fallow (December to May) in annual farming systems. This was achieved by examining variation in available soil water storage to a depth of 1.0-1.5 m at three sites within 13 seasons. Reasons for the variation were examined using the Agricultural Production Systems Simulator (APSIM). This study also investigated the relationship between soil water content at the end of the fallow period (1 May) and the amount of drainage below 2.5 m by using APSIM coupled to historical weather records at three locations. At the end of the fallow a mean of 24 mm (or 25%) of rainfall during the fallow was retained in the soil. Losses of soil water during the fallow were due to evaporation (mean of 60 mm), transpiration from plant cover (mean of 12 mm) and drainage below the root zone and run off (combined mean of 13 mm). Soil water accumulation during the fallow period had a significant impact on simulated drainage under wheat in the following growing season. Every 1 mm increase in soil wetness at the end of the fallow resulted in a 0.7-1 mm increase in simulated drainage during the growing season. ... Variation in the water excess due to variation in rainfall was greater than the reduction in water excess due to lucerne. This makes the decisions about when to grow lucerne to reduce water excess difficult if livestock enterprises are less profitable than cropping enterprises. The findings of this PhD indicate that lucerne does have a place in Mediterranean-type environments because of its greater water use than current farming practices. However, its use needs to be strategic and the strategy will vary from region to region. For example, in the low rainfall region lucerne sowings need to be matched with high soil water contents and phase length will generally be short (2-3 years). In comparison at high rainfall regions lucerne will need to be grown for longer or combined with other strategies to increase water use.

The seed germination process during sprout production provides suitable environmental conditions for the growth of pathogenic bacteria, such as Listeria monocytogenes. A potential way to control this bacterial growth is through the use of bacteriophages, which are naturally occurring viruses that specifically attack bacterial targets and have been shown to be effective antimicrobials in some foods. Therefore, the objective of this study was to evaluate the antimicrobial susceptibility of L. monocytogenes to bacteriophage on alfalfa sprouts during seed germination and subsequent refrigerated storage at 4 °C. Alfalfa sprout seeds were dip-inoculated with 5.5 x 105 CFU/ml L. monocytogenes serogroups 1 and 4. This was followed by treatment with the commercial bacteriophage LISTEX™ P100 at a concentration of 5.3 x 107 PFU/ml. The seeds were then soaked and germinated for 80 h using the glass jar method. The concentration of L. monocytogenes was determined every 24 h using PALCAM agar plated in triplicate. When compared to the spiked, untreated control, treatment of sprout seeds with LISTEX™ P100 resulted in a statistically significant (p < 0.05) reduction of 1.6 log10 CFU/g L. monocytogenes after the initial 24 h of germination. However, the bacteriophage did not show a lasting inhibitory effect, with no statistically significant reductions in L. monocytogenes growth as compared to the control at subsequent time points. The bacteriophage remained stable over the entire germination and storage period. Although biocontrol of Listeria with bacteriophages has high potential to serve as an alternative strategy to control foodborne illnesses, factors such as phage delivery and dose optimization in sprouts need to be further investigated.

Chez les legumineuses, les flavonoides ont une double fonction de composes antimicrobiens (phytoalexines) et d'inducteurs de la transcription des genes nod des bacteries symbiotiques de type rhizobium. L'expression chez la luzerne des genes chalcone synthase (chs), chalcone reductase (chr), chalcone isomerase (chi) et isoflavone reductase (ifr), impliques dans la biosynthese de la phytoalexine medicarpine, a ete etudiee lors de reactions de defense et de symbiose. Plusieurs adnc codant pour la chr ont ete isoles et leur expression caracterisee comparativement a celle de la chs. L'infection des feuilles par pseudomonas syringae pv. Pisi (interaction incompatible) provoque une induction rapide des genes chs, chr, chi et ifr dans les zones infiltrees et non infiltrees, avec un maximum entre 3 et 6 h apres l'infection. Trois flavonoides majeurs sont accumules des 24 h mais uniquement dans la zone infectee: la 2,4,4 -trihydroxychalcone, la 4,7-dihydroxyflavanone et la 4,7-dihydroxyflavone. Apres infection par xanthomonas campestris pv. Alfalfae (interaction compatible), les 4 genes sont induits moins rapidement et plus faiblement. Cependant aucune accumulation de flavonoide n'a pu etre detectee. De plus, l'acide salicylique induit ces 4 genes alors qu'aucun flavonoide est accumule. L'absence de correlation absolue entre l'induction de ces genes et la synthese des flavonoides demontre le role fondamental de la regulation post-transcriptionnelle dans la biosynthese des flavonoides. Par ailleurs, la carence en azote provoque dans les racines une accumulation de flavonoides et de messagers chs et ifr. Enfin, le facteur nodrmiv (c16:2, s) module egalement l'expression des genes de la voie de biosynthese des flavonoides

Le genre Medicago appartient à la famille des Légumineuses; il comprend plus de 50 espèces largement réparties dans le monde. Une faible part de richesse génétique est exploitée. En effet l’espèce M. Sativa et dans une moindre mesure l’espèce M. Falcata constituent la base génétique de l’espèce cultivée. La sélection classique a donné beaucoup de résultats mais actuellement certains problèmes tels l’amélioration du rendement sont difficiles à résoudre. C’est pourquoi un travail sur l’augmentation de la variabilité génétique au sein de l’espèce cultivée a été entrepris. L’objectif était l’exploitation des espèces sauvages, la plupart de celles-ci ne se croisant pas avec M. Sativa, l’hybridation somatique a été choisie comme outil de travail. Une collection d’espèces sauvages a été testée pour l’aptitude à la culture de protoplastes. Des régénérations de plantes ont ainsi été obtenues sur des espèces proches de M. Sativa (M. Varia, M. Tianshanica, M. Falcata, M. Hemicycla and M. Coerulea). Sur les espèces plus éloignées, les résultats vont de l’absence de divisions à la formation de cals. Chez M. Sativa plusieurs variétés sont aptes à la culture de protoplastes. Des hybridations somatiques ont été tentées pour 47 combinaisons entre des types sativa (ou proches) et des types sauvages. Des plantes ont été obtenues à partir de 6 combinaisons de fusion différentes: M. Sativa et M. Arborea. M. Sativa et M. Polymorpha, M. Sativa et M. Jupulina, M. Sativa et M. Romanica. M. Sativa et M. Falcata. L’analyse de marqueurs morphologique et d’isoenzymes ainsi que les critères de fertilité n’ont pas permis de conclure à la nature hybride de ces plantes. Toutefois de fortes perturbations morphologiques et chromosomiques (polyploïdie) ont été observées. Des hybrides somatiques M. Sativa + M. Falcata précédemment obtenus ont été observées en champ et en serre. Il ressort de ces études un comportement particulier des hybrides au niveau somatique (instabilités phénotypiques ponctuelles) et au niveau reproducteur (difficultés de croisement). Par ailleurs un caractère d’intérêt agronomique (production de stolons) absent chez les 2 parents et chez l’hybride sexué est apparu chez un des hybrides somatiques. Une réflexion sur l’intégration de l’hybridation somatique en amélioration des plantes est ensuite envisagée en tenant compte de l’ensemble de ces résultats<br>Genus Medicago belongs to leguminous tribe; it includes more than 50 species widely scattered all over the world. A small part of this genetic wealth is used. For the species M. Sativa and ln a lower part M. Falcata compose the genetic basis of the cultivated species. Classical plant breeding has given a lot of results but presently some problems like yield improvement are difficult to solve. It is the reason why a work dealing with genetic variability increase within the cultivated species has been under­ taken. The main aim is utilization of species, most of them don't cross sexually with M. Sativa, so somatic hybridization is the tool we used. Wild species of a collection were tested for protoplast culture. Plant regeneration has been obtained with some species akin to M. Sativa (M. Varia, M. Tianshanica, M. Falcata, M. Hemicycla and M. Coerulea). With remote species we obtained callus, or just some divisions or even no divisions. Plant regeneration from protoplast can be obtained with several cultivars of M. Sativa. Somatic hybridization has been tried on 47 combinations of saliva types and wild types. Plants have been obtained from 6 different ones: M. Sativa and M. Arborea. M. Sativa and M. Polymorpha, M. Sativa and M. Jupulina, M. Sativa and M. Romanica. M. Sativa and M. Falcata. Morphological markers, isoenzymes were analysed either was fertility: nothing allowed us to identify somatic hybrids. Yet we noticed great disorders in morphology and ploïdy level. Somatic hybrids we got previously have been studied in field and in greenhouse. Their behaviour is quite special on somatic level (punctual phenotypic instabilities) and on reproductive level (troubles in crossing). Otherwise an interesting agronomy feature, (stolons production), has appeared on one of the somatic hybrids although missing on the 2 parents an on the sexual hybrid. Consideration about introducing somatic hybridization ln plant breeding is done lacking ail these results into account

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.<br>Dissertation (MSc Agric)--University of Pretoria, 2013.<br>gm2014<br>Plant Production and Soil Science<br>unrestricted