Made available in DSpace on 2017-07-10T17:37:12Z (GMT). No. of bitstreams: 1 Aline Kelly Pomini de Souza.pdf: 1804826 bytes, checksum: d87df5227a66ab496298a79ceeb8b614 (MD5) Previous issue date: 2016-02-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Produce wheat competitive and sustainable manner is one of the main challenges of agriculture, because the culture demands a high amount of nitrogen, this element with high cost of obtaining. The objective of this study was to evaluate the ability to promote growth of plant growth promoting bacteria (PGPB) previously isolated from forest areas with different types of soil inoculated by seeds, in wheat plants and the effect of the use of nitrogen fertilizers in the plant-bacteria association. The experiment was been conducted in a randomized block design in a factorial 3 x 7 with four replications. The first factor was composed of 3 doses of nitrogen fertilizer 0; 45 and 90 kg ha-1. The second factor was been formed by the absence of inoculation (control), and inoculation by seeds with the isolates: UFPRPALT2-24, Pseudomonas chlororaphis, UFPRPALM1-131, Ensifer adhaerens, Burkholderia ambifaria and standard strain AbV5 (Azospirillum brasilense), registered in the Ministry of Agriculture. During the experiment were measured the length of plants and chlorophyll content (SPAD). At the flowering stage were measured morphometric and physiological variables (gas exchange and SPAD), and in the end of the crop cycle the production components. Data were been subjected to analysis of variance and regression analysis used for the length of plants and the SPAD index. For the other variables, the averages were been compared by Tukey test at 5% probability of error. For the averages of morphometric analyzes performed at the stage of flowering and maturity was conducted Pearson's correlation and generated groupings of treatments by the methods of Unweighted Pair Group Method with Arithmetic Mean (UPGMA) and Tocher. The morphometric analyzes in the flowering stage was a significant difference in length of plants and number of viable tillers, to the isolated factor. Given the interaction of the factors tested, the significant interaction was observed for: mass of dry matter leaves, mass of dry matter stem, mass of dry matter ears, mass of dry matter of roots and length of ears. In the analysis of gas exchange there was a significant difference for internal CO2 concentration (Ci). Components of production, there was a significant interaction for: ear length, root - aerial part relationship, harvest index, number of grains and grain yield. Pearson's correlation were found 45 correlations, highlighting the relationship of SPAD index, ear length and harvest index, because the chlorophyll content be related to photosynthesis. The groups was observed that the treatments with no inoculation by both UPGMA, and Tocher remained in the same group, separate from treatments that stood out. Given the results exposed, it can being concluded that the bacteria used showed satisfactory results in the development of the wheat crop in combination with nitrogen and compared to Azospirillum brasilense, similar or higher behavior was observed / Produzir trigo de forma competitiva e sustentável tornou-se um dos principais desafios da agricultura, visto que a cultura demanda grande quantidade de nitrogênio, elemento este com alto custo de obtenção. O objetivo do presente estudo foi avaliar a capacidade de bactérias promotoras de crescimento de plantas (BPCP) previamente isoladas de áreas de mata com diferentes tipos de solo, inoculadas via sementes, em plantas de trigo e o efeito do uso de fertilizantes nitrogenados na associação planta-bactéria. O experimento foi conduzido em delineamento em blocos casualizados em esquema fatorial 3 x 7 com quatro repetições. O primeiro fator foi composto por 3 doses de fertilizante nitrogenado 0; 45 e 90 kg ha-1. O segundo fator foi formado pela ausência de inoculação (controle), e inoculação via sementes com os isolados: UFPRPALT2-24, Pseudomonas chlororaphis, UFPRPALM1-131, Ensifer adhaerens, Burkholderia ambifaria e a estirpe padrão AbV5 (Azospirillum brasilense), registrada no Ministério da Agricultura. Durante o experimento foram mensurados o comprimento de plantas e teor de clorofila (índice SPAD). No estádio de florescimento foram mensuradas variáveis morfométricas e fisiológicas (trocas gasosas e SPAD), e ao final do ciclo da cultura os componentes da produção. Os dados foram submetidos a análise de variância, sendo utilizada análise de regressão para o comprimento de plantas e índice SPAD. Para as demais variáveis, as médias foram comparadas pelo teste de Tukey a 5% de probabilidade de erro. Para as médias das análises morfométricas realizadas no estádio de florescimento e maturação foi realizada a correlação de Pearson e gerados agrupamentos dos tratamentos pelos métodos do Unweighted Pair Group Method with Arithmetic Mean (UPGMA) e Tocher. Das análises morfométricas realizadas no estádio de florescimento foi verificada diferença significativa para comprimento de plantas e número de perfilhos viáveis, para o fator isolado. Diante da interação dos fatores testados, a interação significativa foi verificada para: massa de matéria seca de folhas, massa de matéria seca de colmo, massa de matéria seca de espigas, massa de matéria seca de raízes e comprimento de espigas. Nas análises de trocas gasosas verificou-se diferença significativa para concentração interna de CO2 (Ci). Dos componentes da produção, houve interação significativa para: comprimento de espigas, relação raiz-parte aérea, índice de colheita, número de grãos e massa de grãos. Da correlação de Pearson foram verificadas 45 correlações, destacando a relação do índice SPAD, comprimento de espigas e índice de colheita, devido o teor de clorofila estar relacionado com a fotossíntese. Dos agrupamentos foi verificado que os tratamentos com ausência de inoculação tanto pelo UPGMA, quanto por Tocher permaneceram em um mesmo grupo, separados dos tratamentos que se destacaram. Diante dos resultados expostos, pode-se concluir que as bactérias utilizadas apresentaram resultados satisfatórios no desenvolvimento da cultura do trigo em associação com o nitrogênio e em comparação ao Azospirillum brasilense, foi verificado comportamento similar ou superior

Made available in DSpace on 2017-07-10T17:37:20Z (GMT). No. of bitstreams: 1 Paulo_Evandro_Jandrey.pdf: 921971 bytes, checksum: d3457f5f5270b5d9b9a7612a24091ca0 (MD5) Previous issue date: 2013-05-17 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Knowledge of the degree of seed germination in the ear is useful both for crop condition as for breeding in wheat. The results of the different tests used to identify cultivars with improved tolerance of sprouting indicate that there is possibility to keep the grains with suitable properties for industry, enabling decrease the amount of losses occurring on the wheat production areas. Accordingly, the aim of this study was to evaluate the tolerance to sprouting and grain yield in wheat grown at two sowing dates. The field experiment was conducted in the area of the Center of Research in COODETEC Palotina-PR using randomized blocks with 15 x 2 factorial arrangement with 3 replications. The first factor refers to genotypes, 3 cultivars (Mirante, CD 150 and Frontana) and 12 lineages resulting from the backcross of cultivars CD 150 with Frontana (CD 12901, CD 12902, CD 12903, CD 12904, CD 12905, CD12906 , CD 12907, CD 12908, CD12 909, CD12 910, CD12 911 and CD 12912). The second factor relates to the two sowing dates (31/03 and 05/03/2012). Plots consisted of 6 rows of 6 meters in length, 0.17 meters spacing between lines, being regarded as one boundary line on each side and 0.50 meters from each end. Evaluations of sprouting, the experiments were conducted in the laboratory, using a randomized block design in 15 x 2 factorial arrangement with 4 replications, each replication was composed of 10 spikes. In the evaluations of sprouting was evaluated for germination in two distinct metologias (immersion in water and rainfall simulator). The variable number of days to silking, plant height, thousand grain weight and number of grains per spike were the characteristics that have greater variability among genotypes, and the sowing in 03/05 was the one that led to the highest for plant height, test weight, number of spikelets per spike and number of grains per spike. The cultivar Frontana showed high resistance to germination of the grain in the ear, regardless of sowing and methodology. Genotypes CD 12901, CD 12905, CD 12907 showed tolerance to the germination of the grain in the ear when evaluated by the method of rainfall simulator in sowing on 31/03. Highest grain yield was obtained for genotypes CD 12902, CD 12905, CD 12909, CD 12910 and CD 12911 in sowing in 03/05. Already Frontana to cultivate, cultivate greater number of days to silking, the highest yield was obtained in sowing on 31/03 / O conhecimento do grau de germinação da semente na espiga é de utilidade tanto para condição de lavoura como para trabalhos de melhoramento na cultura do trigo. Resultados de diferentes testes utilizados para identificar as cultivares com melhor tolerância à germinação na espiga indicam que existe possibilidade de manter os grãos com as propriedades adequadas para a indústria, possibilitando diminuir a quantidade de perdas ocorrentes nas regiões produtoras de trigo. Nesse sentido, objetivou-se com este trabalho avaliar a tolerância a germinação na espiga e a produtividade de grãos em genótipos de trigo cultivados em duas épocas de semeadura. O experimento a campo, foi conduzido na área experimental do Centro de Pesquisa da COODETEC em Palotina-PR utilizando o delineamento em blocos ao acaso em esquema fatorial 15 x 2, com 3 repetições. O primeiro fator refere-se aos genótipos, sendo 3 cultivares (Mirante, CD 150 e Frontana) e 12 linhagens resultantes do retrocruzamento das cultivares CD 150 com Frontana (CD 12901, CD 12902, CD 12903, CD 12904, CD 12905, CD 12906, CD 12907, CD 12908, CD 12909, CD 12910, CD 12911 e CD 12912). O segundo fator refere-se as duas épocas de semeadura (31/03 e 03/05/2012). As parcelas foram constituídas de 6 linhas de 6 metros de comprimento, com espaçamento de 0,17 metros entre linhas, sendo considerado como bordadura 1 linha de cada lado e 0,50 metros de cada extremidade. Para as avaliações de germinação na espiga, os experimentos foram conduzidos em laboratório, utilizando o delineamento de blocos ao acaso em esquema fatorial 15 x 2 com 4 repetições, sendo que cada repetição foi composta de 10 espigas. Nas avaliações de germinação na espiga, foi avaliado o índice de germinação em duas metodologias distintas (imersão em água e simulador de chuva). As variáveis número de dias para o espigamento, altura de plantas, massa de mil grãos e numero de grãos por espiga foram as características que apresentaram maior variabilidade entre os genótipos estudados, sendo que a semeadura realizada em 03/05 foi a que proporcionou maiores valores para altura de plantas, peso do hectolitro, número de espiguetas por espiga e número de grãos por espiga. O cultivar Frontana apresentou alta resistência ao processo germinativo do grão na espiga, independente da época de semeadura e metodologia utilizada. Os genótipos CD 12901, CD 12905, CD 12907 apresentaram tolerância ao processo germinativo do grão na espiga quando avaliados pelo método de simulador de chuva em semeadura realizada em 31/03. Maior rendimento de grãos foi obtido para os genótipos CD 12902, CD 12905, CD 12909, CD 12910 e CD 12911 na semeadura realizada em 03/05. Já para o cultivar Frontana, cultivar de maior numero de dias para o espigamento, o maior rendimento foi obtido na semeadura realizada em 31/03

Made available in DSpace on 2017-07-10T17:37:44Z (GMT). No. of bitstreams: 1 Adeline Neiverth.pdf: 1649092 bytes, checksum: d7d182e371115519785a39ebe6e95aee (MD5) Previous issue date: 2011-07-11 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Wheat is the most important staple food of the world. The increase in grains productivity and protein content is correlated to increase inorganic nitrogen absorption. Usually, urea is the most convenient source of N2, but, it causes the increase in crops costs beyond injuries to the environment. The BNF realized by diazotrophics bacteria is an alternative to supplement or replace the nitrogen fertilizers and promote plant growth. The objective of this study was to evaluate the performance of Brazilian wheat genotypes for PCV( plant promoter growth) and BNF by association between diazotrophic bacteria (H. seropedicae SmR1) and wheat cultivars under in vitro and greenhouse conditions. In in vitro experiment, eight wheat plantlets of 8 genotypes were tested in tubes with liquid culture medium and these were co-cultured for 7 days with 107 cells.mL-1. As control plantlets without inoculum under the same conditions were used. The experimental design was completely randomized in a 2 x 8 factorial with 3 repetitions. We analyzed the fresh mass of roots, fresh and dry weight of shoots, total nitrogen (TN) content of NH4 +, microbial counting (CFU), glutamine synthetase activity (GS), the morphology of the roots by microscopy and molecular analysis of epiphytic and endophytic bacteria recovered after co-cultivation. In the greenhouse, it was planted five wheat genotypes. The seeds were placed in pots with 4.5 kg of soil under four treatments: 1 - Control 2 - Addition of N as urea (142 kg ha-1 of N) 3 - Addition of inoculum containing H. seropedicae (106 cells.mL-1) (Hs) and 4 - Addition of inoculum containing urea + H. seropedicae (N + Hs). The experimental design was completely randomized in a factorial 5 x 4 with 5 repetitions. The fresh mass of roots, fresh and dry weight of shoots, NT, NH4 + content and GS activity were evaluated. As agronomic parameters were evaluated the whole plant mass at the end of the phenological cycle, yield per plant and weight of 100 seeds.As results in vitro, it was observed the presence of epiphytic bacteria on the roots of all genotypes and the presence of endophytic bacteria in genotypes CD 105, CD 108, CD 111, CD 117, CD 120. There was a sharp increase of root hairs in the genotypes CD 105, CD 117, CD 119 and CD120. The cultivars CD 105 and CD 120 by the presence of endophytic bacteria showed an increase of root hairs, probably it may be the most promising for a response of BNF. The levels of NH4 +, NT and GS in the roots were not decisive for in vitro plant growth promotion. The results obtained in the greenhouse showed significant interactions among parameters, although there were were not crucial for the definition of a specific genotype which answers to the interaction. However, there was a contribution to be further studied, where the CD 120 cultivar showed evidence of association with response to the bacterium and possible occurrence of the BNF. There was no negative effect of inoculation to plants. Additional studies are needed to get answers about the interactions of these genotypes with diazotrophic bacteria related to BNF and plant growth promotion / Os fertilizantes nitrogenados são fontes convenientes de nitrogênio para a cultura de trigo, porém geram altos custos e podem ser poluentes. A fixação biológica de nitrogênio (FBN) é uma fonte alternativa de nitrogênio por meio das bactérias diazotráficas. Assim, o objetivo deste trabalho foi avaliar o desempenho de genótipos de trigo brasileiros para a FBN e promoção do crescimento vegetal (PCV) associados com a bactéria diazotrófica H. seropedicae SmR1, sob condições in vitro e em casa de vegetação. Nas condições in vitro, colocou-se plântulas de 8 genótipos de trigo em tubos de ensaio com meio de cultura líquido, co-cultivadas durante 7 dias com 107células de bactéria.mL-1. O mesmo número de plântulas foi mantido nas mesmas condições, porém sem inóculo. O delineamento experimental foi inteiramente casualizado, num esquema fatorial 8 x 2 com 3 repetições. Analisou-se a massa fresca de raízes, massa fresca e seca de parte aérea, o nitrogênio total (NT), conteúdo de amônio (NH4+), contagem microbiana (UFC), Glutamina sintetase (GS), a morfologia das raízes por microscopia e análise molecular das bactérias endofíticas e epifíticas recuperadas após co-cultivo. Em casa de vegetação, foram avaliados 5 genótipos de trigo, onde o delineamento experimental foi inteiramente casualizado num esquema fatorial 5 x 4 com 5 repetições, onde: 1 Testemunha; 2 Adição de N na forma de ureia, 142 kg ha-1 de N; 3 Adição de inóculo contendo H. seropedicae, 106 celulas por semente (Hs) e 4 Adição de ureia + inóculo (N+Hs). Avaliaram-se a massa fresca de raízes, massa fresca e seca de parte aérea, o NT, conteúdo de NH4+ e a atividade da GS. Como parâmetros agronômicos avaliaram-se a massa da planta inteira no final do ciclo fenológico, produção por planta e massa de 100 grãos. Como resultados, observou-se in vitro a presença de bactérias epifíticas nas raízes de todos os genótipos e presença de bactérias endofíticas não foi verificada nos genótipos CD 104, CD 119 e CD 150. Verificou-se um aumento acentuado de pêlos radiculares nos genótipos CD 105, CD 117, CD 119 e CD120. As cultivares CD 105 e CD 120 pela presença da bactéria endofiticamente associado com o aumento de pêlos radiculares, podem ser as mais promissoras para uma resposta da FBN. As plântulas inoculadas apresentaram senescência precoce. Os níveis de NH4+, GS e NT nas raízes não foram determinantes para a promoção do crescimento vegetal. Os resultados obtidos em casa de vegetação demonstram que, apesar de ter havido interações significativas, os parâmetros não foram determinantes para a definição de um genótipo que apresentasse uma resposta conclusiva a respeito da interação benéfica entre genótipo COODETEC e H. seropedicae. No entanto, observou-se uma contribuição a ser mais bem estudada, onde a cultivar CD 120 mostrou indícios de resposta à associação com a bactéria e possível ocorrência da FBN. Não foi observado nenhum efeito negativo da inoculação às plantas. Estudos complementares são necessários para obter respostas quanto às interações destes genótipos com estas bactérias diazotróficas, no processo de colonização, da FBN e da PCV

Des épisodes climatiques de sècheresse et/ou de hautes températures peuvent engendrer de fortes pertes de rendement pour les cultures de céréales au champ. Un QTL associé au rendement et à ses composantes a été détecté dans quatre populations de blé (Triticum aestivum L.) sur le bras long du chromosome 3B « qYDH.3BL ». Deux populations d’haploïdes doublés (RAC875/Kukri et Excalibur/Kukri) et deux populations de lignées recombinantes (RAC875/Kukri et Gladius/Drysdale) ont été utilisées pour cartographier finement le QTL, au même titre que l’identification de gènes candidats. Ces quatre populations ont été testées sous des conditions environnementales variées, incluant des périodes de sécheresse et/ou hautes températures en Australie et au Mexique. Des modèles statistiques mixtes et linéaires décomposant les variations génétiques et non-génétiques ont été utilisés pour la détection de QTL en considérant dans un premier temps chaque environnement unique, puis en considérant les environnements multiples dans une analyse commune. Les allèles de RAC875, Drysdale et Excalibur à ce locus ont montré une hausse du rendement de 5 à 12.5 % comparées à celles de Gladius ou Kukri. Un total de trente-sept marqueurs moléculaires a été cartographié dans la région du QTL. Les marqueurs moléculaires ont été sélectionnés (i) par comparaison avec une carte génétique publiée du chromosome 3B, ou (ii) en désignant de nouveaux marqueurs moléculaires sur les séquences de BAC-end, de contig ou de gènes provenant du projet de séquençage du chromosome 3B (3BSEQ, http://urgi.versailles.inra.fr/, cv. Chinese Spring). Ceci a permis la construction d’une carte génétique consensus du locus qYDH.3BL . A ce jour, aucun QTL associé au rendement ou ses composantes en condition de sécheresse et/ou de hautes températures n’a encore été cloné positionellement chez le blé tendre. Les marqueurs moléculaires de la région d’intérêt ont été utilisés pour cartographier physiquement des contigs, soit par PCR, soit par comparaison de séquences in silico. La région du QTL inclus un total de huit contigs physiques comprenant 85 gènes annotés. L’utilisation de base de données de transcris biologiques publiques ou internes ont été utilisées pour détecter la présence de ces gènes, réduisant la liste à soixante-cinq gènes. Sur les contigs ayant une confiance élevée, aucun des vingt gènes n’a été exprimé différentiellement entre RAC875 et Kukri. Cependant, un gène présentant du polymorphisme dans sa séquence ainsi qu’une délétion/insertion d’un segment portant 12 gènes ont été découvert permettant ainsi de continuer à affiner la liste de gènes candidats. Les trois lignées parentales (RAC875, Drysdale et Excalibur) qui ont l’allèle liée au haut rendement ont le même haplotype pour ce gène, et la même délétion/insertion en opposition au deux autres lignées parentales Gladius et Kukri. Ainsi, dans ce travail de thèse nous avons pu confirmer la présence d’un QTL répondant aux stresses environnementaux sur le chromosome 3BL dans différentes populations et différents environnements, identifier des gènes candidats sous le QTL, et proposer une liste restreinte pour de futures analyses sur la base de données d’expression et de polymorphismes entre les parents des populations de cartographie. / Drought and heat can occur during the growth cycle of crops and severely reduce yield. A QTL associated with yield and yield-related component was found in four wheat populations (Triticum aestivum L.) on the long arm of chromosome 3B “qYDH.3BL”. The four populations were grown under various climatic conditions including drought, heat and combinations of both in a number of different areas (Australia and Mexico). Linear mixed models that partition and account for genetic and non-genetic or extraneous variation were used to detect loci in single-environment and/or multi-environment QTL analysis using ASReml-R. The alleles carried by RAC875, Excalibur or Drysdale improved grain yield by between 5% and 12.5%. Two doubled haploid populations (RAC875/Kukri and Excalibur/Kukri) and two recombinant inbred line populations (RAC875/Kukri and Gladius/Drysdale) were used to fine map qYDH.3BL and identify candidate gene(s). A total of thirty-seven molecular markers were mapped on one or both genetic maps of chromosome 3B enabling development of a consensus genetic map of the qYDH.3BL region. The markers were selected based on comparisons with a published “neighbour map” of chromosome 3B or designed using either BAC-end, contig or gene sequences from the chromosome 3B sequencing project; 3BSEQ http://urgi.versailles.inra.fr/ (cv. Chinese Spring). A positional cloning approach was used to identify candidate genes for qYDH.3BL. Molecular markers from the targeted region were assigned to physical contigs by screening the chromosome 3B BAC library experimentally using PCR or in silico by sequence comparison. A total of eight physical contigs containing 85 genes, were anchored to the qYDH.3BL region. Public and in-house resources of wheat transcript sequences were used to restrict the gene list to 65 expressed genes. Based on comparison of the 65 gene sequences to gene probes in a drought transcriptomic database, three genes were found to be differentially expressed between RAC875 and Kukri under drought conditions. Short genomic sequence reads (10× coverage) from each of the five parental lines (RAC875, Kukri, Excalibur, Gladius and Drysdale) were mapped against the 65 genes for polymorphism discovery. One gene exhibited sequence polymorphism between the drought tolerant parents (RAC875, Excalibur and Drysdale) and the drought-sensitive parents (Gladius and Kukri). In addition, presence/absence polymorphisms were consistently detected throughout a region containing 12 genes, indicating that the drought tolerant parents may have a deletion (or alien introgression) in this region. Thus, in this work, we confirmed the genetic effect of qYDH.3BL in multiple environments and multiple populations, saturated the target region with new molecular markers and defined a preliminary list of genes located in the qYDH.3BL region and selected candidate genes for further investigations.

Dans le cadre d’étude de l’amélioration de la teneur en Mg dans le grain par la sélection du blé (Triticum aestivum) afin de renforcer l’apport alimentaire du Mg, les objectifs de cette thèse étaient (i) de mettre en évidence les caractéristiques agro-physiologiques de la plante associées à la teneur en Mg, (ii) d’étudier l’effet de variants génétiques affectant la dynamique du Mg dans la plante et l’accumulation du Mg dans le grain, (iii) d’identifier les éléments minéraux influençant l’absorption et la répartition du Mg, et (iv) d’étudier par approche protéomique le métabolisme associé au Mg influençant la quantité du Mg dans le grain. A travers trois séries d’essai multifactoriel au champ conduites dans le but de sélectionner des génotypes de blé à haut rendement en Mg des grains, les suivis agro-physiologiques ont permis de préciser l’effet négatif du rendement, du poids de mille grains et du nombre de grains par épi sur la teneur en Mg dans le grain. Une différence génétique dans la dynamique du Mg de la plante au cours du développement du grain a été montrée avec l’implication de la translocation du Mg des racines ainsi que de la remobilisation du Mg des tissus végétatifs aériens. La réserve du Mg à préfloraison dans les tissus végétatifs influe sur la quantité de Mg dans le grain. Enfin, l’absorption du Mg est positivement associée avec celle des éléments Mn, P et Zn, et la teneur en Mg est positivement associée avec celle des éléments Zn et P. L’étude protéomique sur les couches périphériques du grain a mis évidence que le Mg intervient dans la division cellulaire, le repliement des protéines et le métabolisme d’énergie, et donc influence l’accumulation de la matière sèche du grain en déterminant la taille potentielle et le remplissage du grain. En conclusion, l’accumulation du Mg dans la plante influence la matière sèche du grain qui a un effet négatif sur la teneur en Mg. / In order to increase the Mg content in the grain of wheat (Triticum aestivum) through plant breeding to improve the dietary Mg intake, the objectives of the present study is (i) to identify the plant traits which are associated with the grain Mg concentration, (ii) to study genotypic variation in Mg dynamic in plant and its effect on Mg accumulation in grain, (iii) identify the minerals interacted with Mg in absorption and distribution, and (iv) study the Mg-related metabolisms influencing the amount of Mg in grain by proteomic approach. The study of 22 genotypes in a multiple-sites and -years field trial showed that there was negative effect of yield, thousand grain weight and number of grains per spike on the grain Mg concentration. The genetic difference was observed in the Mg dynamic during grain development. Mg in the grain depended on both Mg translocation from roots and remobilization from aboveground vegetative tissues, and could be affected by the Mg reserve in vegetative tissues. The study of correlation among different minerals showed that the Mg concentration in grain was positively associated with those of Zn and P, and the Mg content in aboveground was positively associated with those of Mn, P and Zn. Proteomic study of grain peripheral layers showed that Mg is involved in cell division, protein folding and energy metabolism, and hence influenced the grain dry matter accumulation by determining the potential size and grain filling. In conclusion, the accumulation of Mg in the plant influences the grain dry matter which has a negative effect on the Mg content.

O trigo (Triticum aestivum L.) é uma importante cultura no Brasil. Poucas cultivares são recomendadas para produção do tipo sequeiro no Bioma Cerrado onde a escassez de água limita o rendimento de grãos. Aqui reportamos uma análise de transcriptoma do MGS1 Aliança (cultivar de trigo adaptada ao Cerrado) sob estresse de seca. Um grupo de 4.422 transcritos diferencialmente expressos foi encontrado em raízes e folhas. O número de transcritos reprimidos em raiz (1.102) foi menor que os transcritos induzidos (1.706), enquanto o oposto ocorreu em folhas (1,017 induzidos e 647 reprimidos). O número de transcritos comuns entre ambos órgaõs foi 1.249, enquanto 2.124 foram específicos para raíz e 1.049 específicos para folhas. Análises de RT-qPCR de 35 transcritos selecionados ao acaso revelou uma correlação de 0,78 com os dados de transcriptoma. Os transcritos diferencialmente expressos foram distribuídos por todos os cromossomos e componentes do genoma. O número de transcritos no genoma B foi maior do que nos genomas A e D. Ainda, um grande número de transcritos relacionados à seca foi mapeado nos cromossomos 3B, 5B e 2B. Quando consideramos ambos órgãos, 116 diferentes rotas metabólicas foram alteradas. Uma rota em comum, entre as três mais alteradas em ambos órgãos, foi o metabolismo do amido e da sacarose. A comparação de transcritos derivados de raiz e de folha permite a identificação de transcritos importantes relacionados à respota ao estresse de seca em cada um destes órgãos. Os dados obtidos, também, abrem caminho para o desenvolvimento de futuros marcadores e seleção de genes candidatos ligados à característica. Estes resultados são úteis para o entendimento de rotas metabólicas envolvidas na tolerância à seca em trigo. A informação gerada será usada, a mais longo prazo, para propósitos de transgenia. Para isto, a metodologia de duplo-haploides é desejável e uma primeira investigação sobre a eficiência de protocolo se mostrou necessária. Micrósporos são células gaméticas com capacidade de dar origem a uma nova planta via embriogênese in vitro. Plantas duplo-haploides geradas pela cultura de micrósporos isolados são completamente homozigotas e representam uma importante ferramenta para estudos genéticos e melhoramento de plantas O processo androgenético é desencadeado por diferentes pré-tratamentos de estresse, os quais são empregados para mudar os micrósporos da rota gametofítica para a rota esporofítica. Embora a cultura de micrósporos isolados tenha inúmeras vantagens, importantes limitações tem impedido sua apliação em larga escala. Diferenças genotípicas na resposta androgenética e na formação de plantas albinas ainda constituem desafios. Embora o albinismo seja principalmente uma característica genética, pré-tratamentos e meios de cultura apropriados podem evitar este fenômeno até certo ponto. A resposta androgenética de cinco genótipos de trigo brasileiro foi avaliada no presente estudo. Dois pré-tratamentos foram testados: frio (4°C) e ácido 2-hidroxinicotinico (100 mg/L). O frio foi melhor que o pré-tratamento químico, produzindo mais plantas verdes em quatro de cinco genótipos. Somente dois genótipos brasileiros tratados com ácido 2-hidroxinicotinico produziram plantas, e um deles apenas uma única planta albina. Nossos reultados mostram, também, que o meio semilíquido (contendo 10% de Ficoll) promoveu uma maior resposta androgenética que o meio líquido, aumentando o número de embriões e plantas regeneradas. / Wheat (Triticum aestivum L.) is an important crop cultivated in Brazil. Few cultivars are recommended for rainfed production in the Cerrado Biome where water scarcity limits grain yield. Here we report a transcriptome analysis of MGS1 Aliança (a wheat cultivar adapted to the Cerrado) under drought stress. A set of 4,422 differentially expressed transcripts was found in roots and leaves. The number of down-regulated transcripts in roots (1,102) was lower than the up-regulated transcripts (1,706), while the opposite occurred in leaves (1,017 induced and 647 repressed). The number of common transcripts between the two tissues was 1,249, while 2,124 were specific to roots and 1,049 specific to leaves. Quantitative RT-PCR analysis of 35 randomly selected transcripts revealed a 0.78 correlation with the transcriptome data. The differentially expressed transcripts were distributed across all chromosomes and component genomes. The number of transcripts on the B genome was greater than on the A and D genomes. Additionally, a greater number of drought related transcripts was mapped on chromosomes 3B, 5B and 5D. When considering both tissues, 116 different metabolic pathways were changed. One common pathway, among the top three changed pathways in both tissues, was starch and sucrose metabolism. The comparison of root- and leaf-derived transcripts allows the identification of important transcripts related to water stress response in each of these tissues. It also paves the way for future marker development and selection of candidate genes linked to that trait. These results are useful for understanding the metabolic pathways involved in wheat drought response. The information generated will be used for transgenic wheat purposes. For this the doubled-haploid method is desirable and an investigation about the protocol eficiency is needed. Microspores are gametic cells with capacity to give rise to a new plant via in vitro embryogenesis. Doubled haploid plants generated by isolated microspore culture are completely homozygous and represent an important tool for plant genetics and breeding research. This process is triggered by different stress pretreatments, which are employed to switch microspores from gametophytic to a sporophytic pathway. Although isolated microspore culture has innumerous advantages, important limitations have prevented its application on a large scale. Genotypic differences in androgenic response and the formation of albino plants remain great challenges. Although albinism is a major genetic characteristic, appropriated pretreatments and culture medium can avoid this phenomenon to some extent. The androgenic response of five Brazilian wheat genotypes was evaluated in the present study. Two pretreatments were tested: cold (4°C) and 2-hydroxynicotinic acid (100 mg/L). Cold was better than chemical pretreatment, producing more green plants in four out of five genotypes. Only two Brazilian genotypes treated with 2-hydroxynicotinic acid produced plants, and one of them produced a single albino plant. Our results also show that semi-liquid medium (containing 10% Ficoll) promoted a higher androgenic response than did liquid medium, increasing the number of embryos and regenerated plants.

Le rendement en grains (Rdt) et la teneur en protéines (%Prot) sont deux cibles majeures dans les programmes de sélection variétale chez le blé car ces caractères contribuent à la valeur économique de cette culture. Malheureusement, leur amélioration simultanée est empêchée par la relation négative %Prot-Rdt. Il a été montré que l’écart à cette relation (“Grain Protein Deviation”, GPD) est déterminé en partie génétiquement et serait utile pour modifier cette relation négative mais ses bases biologiques restent mal comprises à ce jour. Nous avons montré que le GPD est principalement relié à la variabilité génétique pour l’absorption d’azote post-floraison (ABSN) dans les conditions agro-climatiques du Nord-Ouest de l’Europe. Nous proposons que la variabilité génétique pour l’accès à l’azote du sol (architecture et fonctionnement racinaire) ou pour la régulation de ABSN par le statut azoté (transport et assimilation de l’azote) pourrait expliquer le GPD. Etant donné que le retardement de la sénescence durant la période post-floraison peut résulter en une augmentation de ABSN, nous avons analysé les déterminants génétique des relations entre durée de sénescence des feuilles après floraison et Rdt ou %Prot, observées au niveau phénotypique, en utilisant des données acquises sur une population de cartographie de blé cultivée au sein d’un large réseau expérimental. Une association positive entre durée de sénescence des feuilles après floraison et %Prot ou Rdt a été observée selon les environnements étudiés. Nous faisons l’hypothèse que l’impact d’un retardement de la sénescence des feuilles après floraison pourrait être modulé selon la disponibilité en azote durant cette période, ce qui conduirait à modifier la relation %Prot-Rdt selon les environnements étudiés. Enfin, des données obtenues sur trois populations de cartographie cultivées dans un large réseau expérimental ont permis de suggérer, après méta-analyse de QTL, des régions génomiques potentiellement utiles en sélection pour améliorer la %Prot sans diminuer le Rdt. Ceci a permis de mettre en avant des régions situées sur les chromosomes 2A et 3B. En particulier, la région située sur le 2A pourrait être reliée à la présence d’un gène codant pour une glutamine synthétase chloroplastique qui a été associée à la variabilité génétique pour %Prot chez le blé tendre dans une étude antérieure. / Grain yield (GY) and grain protein concentration (GPC) are two major targets in wheat breeding programs as these traits contribute to the economic value of the wheat crop. Unfortunately, their simultaneous improvement is hampered by the genetic negative GPC-GY relationship. It has been shown that the deviation to this relationship (“Grain Protein Deviation”, GPD) has a genetic basis and might be useful to shift this negative relationship but its biological bases remain unclear. GPD was shown to be mainly related to the genetic variability for post-anthesis nitrogen (N) uptake (PANU) in the North-West European agro-climatic conditions. We proposed that the genetic variability for the access to N in the soil (root architecture and functioning) or for the regulation of PANU by the plant N status (N transport and assimilation) could explain GPD. As delaying leaf senescence during the post-anthesis period might result in increasing PANU, we analysed the genetic determinants of the phenotypic relationships between leaf senescence duration after anthesis and GPC or GY using data obtained on a wheat mapping population grown in a large mutli-environment trial network. A positive association was found between leaf senescence duration and GPC or GY depending onthe environment. We suggested that the impact of delaying leaf senescence after anthesis on GY or GPC might be modulated by the N availability during the post-anthesis period and would lead to modify the GPC-GY relationship depending on the considered environments. Finally, data obtained on three connected mapping populations grown in a large mutli-environment trial network were used to suggest by meta-QTL analysis potential genomic regions possibly useful in wheat breeding to improve GPC without reducing GY. This put forward genomic regions located on the 2A and 3B chromosomes as potentially interesting targets to improve GPC. In particular, the region on the 2A might be related to a chloroplastic glutamine synthetase gene previously shown to be associated with genetic variability for GPC in bread wheat.

Les modifications post-traductionnelles des protéines constituent un niveau crucial de régulation de l’expression des gènes. Parmi elles, la conjugaison peptidique impliquant l’ubiquitine intervient entre autre dans la régulation de la stabilité protéique. La fixation de ce peptide de 76 acides aminés, extrêmement conservé, sous forme de chaîne de polyubiquitine, nécessite l’intervention de trois enzymes (E1, E2 et E3) et constitue un signal de dégradation de la protéine ainsi modifiée. Cette voie de régulation intervient dans de très nombreux processus biologiques. Les complexes SCF sont impliqués dans la voie de protéolyse ciblée. Ils représentent l' une des classes les plus fréquentes d'ubiquitine ligase E3 et ils sont composés de quatre sous-unités (Rbx, Cullin, SKP1, et F-box). La structure et la fonction des complexes SCF, ont été étudiées chez la levure, l’Homme et la plante modèle A. thaliana. Cependant, peu de travaux ont été réalisés chez des plantes cultivées, en particulier les céréales, telles que le blé. Cinq gènes codant pour la sous-unité Skp1 (TSK1, TSK3, TSK6, TSK11 et TSK16), cinq gènes codant pour la sous-unité F-box (ZTL, ATFBL5, EBF, TIR1 et ABA-T), un gène codant pour la sous-unité Cullin1 et un gène codant pour la protéine RBX du complexe SCF du blé, ont été isolés et clonés. Les différents tests d’interaction entre les quatre sous-unités du complexe SCF ont été réalisés par la méthode du double-hybride dans la levure en utilisant la technologie Gateway. Ces études ont montré que les deux protéines, TSK1 et TSK3, fixent spécifiquement différentes sous-unités F-box. Parallèlement, nous avons montré que la protéine TSK11 représente une structure particulière. Des études d’insertion/délétion sur la protéine TSK11 ont permis d’identifier un nouveau domaine indispensable à l’interaction. Les analyses par PCR semi-quantitative des différents gènes codant pour la sous-unité Skp1, dans trois tissus différents (feuille tige et racine), ont mis en évidence une expression constitutive des gènes TSK3, TSK6 et TSK11. Tandis que les gènes TSK1 et TSK16 sont exprimés préférentiellement dans les racines. Les analyses par PCR semi-quantitative sur des plantules de blé à différents stades de développement, ont mis en évidence une surexpression du gène TSK11 au moment de la floraison. Ce qui suggère que TSK11 est probablement un équivalent fonctionnel d’ASK1 chez Arabidopsis thaliana. / The selective degradation of proteins is an important means of regulating gene expression and plays crucial roles in the control of various cellular processes. The Ubiquitin (Ub)–Proteasome System (UPS) is the principal non-lysosomal proteolytic pathway in eukaryotic cells and is required for the degradation of key regulatory proteins. Ubiquitin is a 76-residue protein that can be attached covalently to target proteins through an enzymatic conjugation cascade involving three enzymes denoted, E1, E2 and E3.The SCF complex is a type of ubiquitin-protein ligase (E3) that acts as the specific factor responsible for substrate recognition and ubiquitination. Some polyubiquitinated proteins are then targeted to the 26S proteasome for degradation. The SCF complex consists of four components including SKP1, Cullin1, Rbx1 and a large gene family of F-box proteins. Twenty one SKP1-related genes have been described in the Arabidopsis genome and some of these genes have been analyzed genetically. By contrast, little is known about the function and structure of SKP1 homologues in wheat. Some of the Triticum SKP1-related protein (TSKs) have been characterized in this study. Five complete sequences of SKP1 (TSK1, TSK3, TSK6, TSK11 and TSK16), five F-box (ZTL, ATFBL5, EBF, TIR1 and ABA-T), one Cullin1 and one Rbx, were successfully cloned and biochemically characterized. Yeast two-hybrid analysis showed that TSK1 and TSK3 are capable of interacting with different F-box proteins. Furthermore, TSK11 contains an additional domain that changed its interaction capabilities. In vitro analysis using a chimeric protein showed that this additional domain could modify the interaction between a SKP-like protein and two F-box proteins. Expression analyses revealed that TSK1 and TSK16 were expressed predominantly in roots. While, TSK3, TSK6 and TSK11 were expressed in several wheat organs. In addition, the TSK11 was up-regulated in the leaves at the flowering stage.

Le blé tendre, Triticum aestivum, est une des céréales les plus cultivées au monde et est d’une importance considérable pour l’alimentation humaine, fournissant environ un cinquième des calories consommées par l’Homme. Le rendement en grain chez les céréales dépend majoritairement du nombre et de la taille des grains. Chez le riz (Oryza sativa), le gène GW2 a été isolé dans un locus à effet quantitatif majeur pour la taille et le poids du grain. Ce gène code pour une enzyme E3 ligase de type RING, qui régule négativement la taille et le poids du grain de riz. L’homologue de GW2 chez le blé tendre, le gène TaGW2, est exprimé par trois copies TaGW2-A,TaGW2-B et TaGW2-D, portées par chacun des génomes homéologues A, B et D. Les trois copies présentent des profils d’expression distincts au cours du développement du grain. TaGW2-A a été cartographié dans une région de QTLs pour le rendement, sur le chromosome 6AS ; et du polymorphisme dans sa séquence promotrice et intronique a été retrouvé associé au poids de 1000-grains dans une core collection mondiale de blé tendre. Afin de rechercher la fonction de TaGW2, l’extinction stable des trois copies TaGW2 a été entreprise par ARN interférence. De manière surprenante, les plantes transgéniques montrent des réductions significatives des dimensions et du poids du grain de blé (- 22,5 et - 30% du volume et de la masse du grain, respectivement), ainsi que du nombre de cellules de l’albumen (- 25%), comparé aux plantes témoins dans nos conditions ; suggérant que TaGW2 est un régulateur positif de la taille finale du grain chez le blé tendre. La protéine TaGW2-A a été caractérisée aux niveaux moléculaire et biochimique : elle est une E3 ubiquitine ligase fonctionnelle in vitro, et s’accumule dans la cellule au niveau du nucléole, du nucléoplasme et du cytoplasme. Sa fonction E3 ligase semble notamment influencer sa localisation subcellulaire. Afin de déterminer la ou les voie(s) de signalisation dans la(es)quelle(s) intervient TaGW2, une banque ADNc de grains de blé a été construite et criblée par double-hybride avec 320 acides aminés de la protéine TaGW2-A. Les premiers interacteurs potentiels identifiés suggèrent d’une part un rôle de TaGW2 dans la régulation de la division cellulaire, et d’autre part une fonction E3 Nedd8 ligase, en plus de son activité E3 ligase. / Wheat, Triticum aestivum, is one of the world’s major cereal crops and is of considerable importance to human nutrition, supplying one-fifth of the calories consumed by humans. For important food crops such as wheat, rice and maize, grain yield mainly depends on grain number and size. In rice (Oryza sativa), GW2 was isolated from a major quantitative trait locus for grain size and weight, and encodes an E3 RING ligase that negatively regulates these yield components. Wheat has TaGW2 homologs in A, B and D genomes; and copies show distinct expression pattern during whole grain development in wheat. TaGW2-A was mapped in a genomic region on 6AS, encompassing previous reported QTLs for yield; and polymorphisms in TaGW2-A (promoter and intron 7) were associated with thousand-grain weight, in a worldwide wheat core collection. To investigate TaGW2 function, RNA interference was used to down-regulate TaGW2 transcripts levels. Surprisingly, transgenic wheat lines significantly showed decreased grain weight and size-related dimensions, and endosperm cell number compared to controls. The present study thus suggests that TaGW2 is a positive regulator of the final grain size in wheat, conversely to GW2 in rice. Biochemical and molecular analyses of the protein TaGW2-A revealed that 1) TaGW2-A is a functional E3 ubiquitine ligase in vitro, 2) TaGW2-A accumulates in the nucleolus, the nucleoplasm, and the cytosol, 3) E3 ubiquitine ligase activity seems to impact TaGW2-A subcellular localization. To investigate the TaGW2 signalling pathway(s), cDNA library from whole wheat grains was built and screened with the bait protein TaGW2(1-320). Preliminary results from the interactomic study suggest that TaGW2 may regulate cell division. Moreover, TaGW2 may also function as an E3 Nedd8 ligase, besides its E3 ubiquitin ligase function.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The use of chemical fertilizers as well as providing greater productivity, is costly. The use of bacteria of the genus Azospirillum brasilense represent a potential tool to reduce production costs while improving productive potential of crops using environmental resources. It is also known that the search for an increasingly sustainable and productive agriculture is increasingly discussed, and within that reality, we seek new technologies to improve the producer's profit margin. Within this context, the objective of this research was to evaluate the wheat yield, inoculated or not with A. brasilense, subjected to N rates, seed treatment and physiological quality of the seeds. Chapter I aimed to evaluate the compatibility of Azospirillum brasilense bacteria fungicide (difenoconazole) and insecticide (thiamethoxan), and physiological quality in wheat seeds. The wheat seed retains its quality when it checks the germination, vigor and independent accelerated aging grow, use or not of fungicide, insecticide and Azospirillum brasilense. The insecticide thiamethoxam increased the length of shoot and root and provides compatibility with Azospirillum brasilense and fungicide inhibits the length of shoot and root and is antagonistic bacterium A. brasilense. In Chapter II objective was to evaluate the agronomic performance of wheat cultivars submitted to nitrogen doses and inoculation of Azospirillum brasilense in seed and foliar application. Inoculation foliar alone or jointly via seed treatment increased grain yield and other yield components of all cultivars. Among the cultivars tested the TBIO Sinuelo stood out, with inoculation response in the two years of study, confirming the good potential of the leaf inoculation. / O uso de fertilizantes químicos além de proporcionar maior produtividade, é oneroso. A utilização de bactérias do gênero Azospirillum brasilense representam uma ferramenta potencial para reduzir os custos de produção, e ao mesmo tempo melhorar o potencial produtivo das culturas utilizando os recursos do meio. Sabe-se ainda que a busca por uma agricultura cada vez mais sustentável e produtiva é cada vez mais discutida, e dentro dessa realidade, busca-se novas tecnologias para melhorar a margem de lucro do produtor. Dentro deste contexto, o objetivo dessa pesquisa foi avaliar a produtividade do trigo, inoculada ou não com A. brasilense, submetido a doses de N, tratamento de sementes e qualidade fisiológica de suas sementes. O capítulo I teve por objetivo avaliar a compatibilidade da bactéria Azospirillum brasilense com fungicida (difenoconazole) e inseticida (thiamethoxan), e a qualidade fisiológica, em sementes de trigo. A semente de trigo mantém a sua qualidade quando se verifica a germinação, vigor e o envelhecimento acelerado independente da cultivar, utilização ou não de fungicida, inseticida e Azospirillum brasilense. O inseticida thiamethoxam aumenta o comprimento da parte aérea e raiz e apresenta compatibilidade com o Azospirillum brasilense e o fungicida inibe o comprimento da parte aérea e raiz e é antagônico a bactéria A. brasilense. No capítulo II objetivo foi avaliar o desempenho agronômico de cultivares de trigo submetidas a doses de N e inoculação de Azospirillum brasilense em sementes e aplicação foliar. A inoculação via foliar de forma isolada ou de forma conjunta via tratamento de sementes, aumentaram a produtividade de grãos e os demais componentes de produtividade de todas as cultivares. Dentre as cultivares testadas a TBIO Sinuelo se destacou, apresentando resposta de inoculação nos dois anos de estudos, confirmando o bom potencial da inoculação foliar.