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No. of bitstreams: 2 Dissertação_Francieli_Cima.pdf: 1564850 bytes, checksum: 1875a0cdb8f46c6a1880c860e06bdbe3 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-03-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / No Brasil, a cultura do trigo ocupa lugar de destaque nos sistemas de produção da Região Sul, representando uma opção muito importante como um cereal de inverno. O melhoramento genético das culturas autógamas tem contado com vários métodos para produzir cultivares mais recentes e superiores com características melhoradas. Um exemplo muito bem sucedido tem sido o método de produção de plantas duplohaploides usado para acelerar o desenvolvimento de uma nova cultivar. Em trigo, a cultura de micrósporos isolados tem sido usada para a obtenção de plantas duplohaploides. No entanto, esta técnica tem sido caracterizada como altamente genótipo-dependente e de produzir altas taxas de plantas albinas. Assim, o objetivo deste trabalho foi otimizar um protocolo através da cultura in vitro de micrósporos isolados utilizando-se genótipo de trigo com alta produção de plantas albinas. Para este estudo, plantas F1 oriundas do cruzamento de trigo entre Toropi x BRS 194 foram usadas como doadoras de micrósporos. Foram realizados três experimentos. No primeiro experimento, anteras foram removidas das espiguetas e tratadas em três soluções de pré-tratamentos à 4°C: a) manitol (62 g L-1) + 15 µM de sulfato de cobre (CuSO4.5H20); b) manitol (62 g L-1) e c) 15 µM de sulfato de cobre. No segundo experimento, foram testadas as mesmas soluções de pré-tratamento do experimento anterior, com a diferença de que neste experimento, espiguetas inteiras foram usadas para o pré-tratamento. No terceiro experimento, somente o pré- tratamento a frio foi aplicado nas espigas e houve uma modificação no meio de indução, quando foram testados nove tratamentos: controle (meio de indução padrão); sulfato de cobre (2,0 µM); prolina (10 mM); glutationa (2,0 µM); cefotaxima (100 mg L-1); sulfato de cobre (4,0 µM); sulfato de cobre (2,0 µM) + prolina (10 mM); sulfato de cobre (2,0 µM) + glutationa (2,0 µM); sulfato de cobre (2,0 µM) + cefotaxima (100 mg L-1). Os resultados obtidos nos experimentos 1 e 2 mostrou que a solução de pré-tratamento contendo 15 µM de sulfato de cobre foi mais eficiente para desencadear a embriogênese dos micrósporos e regenerar plantas verdes em trigo. No terceiro experimento, o meio de indução suplementado com 100 mg L-1 de cefotaxima mostrou um aumento na formação de embriões e também na regeneração de plantas verdes. / In Brazil, the wheat crop occupies an outstanding place in the production systems of the Southern Region, representing a very important option as a winter cereal. Genetic improvement of self-fertilizing crops has relied on several methods to produce newer and superior cultivars with enhanced traits. A very successful example has been the use as double haploids as a method to accelerate the development of a new cultivar. In wheat, isolated microspore culture has been used to obtain doubled haploid plants. However, this technique has been characterized as highly genotype dependent and producing high rates of albino plants. Thus, the objective of this work was to optimize a protocol through the in vitro culture of isolated microspores utilizing a wheat genotype with high production of albino plants. For this study, F1 plants originated from the wheat cross between Toropi x BRS 194 were used as microspore donor plants. Three experiments were carried out. In the first experiment, anthers were treated in three pre-treatment solutions, at 4°C: a) mannitol (62 g L-1) + 15 µM copper sulphate (CuSO4.5H20); b) mannitol (62 g L-1) and c) 15 µM copper sulphate. In the second experiment, the same pre-treatment solutions of the previous experiment were tested, with the difference that in this experiment, whole spikelets were used for the pre-treatment. In the third experiment, only cold pre-treatment was applied to the spikes, and there was a modification in the induction medium, when nine treatments were tested: control (standard induction medium); copper sulphate (2,0 µM); proline (10 mM); glutathione (2,0 µM); cefotaxime (100 mg L-1); copper sulphate (4,0 µM); copper sulphate (2,0 µM) + proline (10 mM); copper sulphate (2,0 µM) + glutathione (2,0 µM); copper sulphate (2,0 µM) + cefotaxime (100 mg L-1). Results obtained in experiments 1 and 2 showed that the pre-treatment solution containing 15 µM of copper sulphate was more efficient to trigger the embryogenesis of microspores and regenerate green plants in wheat. In the third experiment, the induction medium, supplemented with 100 mg L-1 of cefotaxime showed an increase in the formation of embryos and also in the regeneration of green plants.

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.

Doctor of Philosophy / Polyphenol oxidase (PPO) catalyses undesirable darkening in wheat products such as Asian noodles. Genetic variation for PPO activity is characterized in bread wheat. Australian wheat breeding programmes recognize that reduced PPO activity is an important quality target. Despite this interest from breeders, no varieties possessing extremely low and null PPO activity exist. The development of null PPO wheat varieties is dependant on an understanding of the genetic control of the null phenotype. Knowledge of these factors will accelerate efforts to develop them. The inheritance of PPO activity was investigated in two populations that were derived from hybrids between a null PPO genotype and Australian wheat varieties Lang and QAlBis. Observed genetic ratios were consistent with two and three gene control, respectively in these populations. QTL mapping was performed in the QALBis x VAW08-A17 population. The Diversity Array Technology (DArT) approach was employed to genotype the QALBis x VAW08-A17 population. Three highly significant QTLs that control PPO activity were identified on chromosomes 2AL, 2BS and 2DL. Close associations between PPO activity and DArT marker loci wPt-7024, wPt-0094 and wPt-2544 were observed, respectively. Collectively, these loci explained 74% of the observed variation in PPO activity across seasons. Significant QTLs on chromosomes 1B and 3B were also identified that together explained an additional 17% of variation in PPO activity. The relationship between PPO activity and yellow alkaline noodles (YAN) colour stability parameters was investigated in a DM5637*B8 x H45 doubled haploid population. PPO activity and changes in YAN brightness (ΔL* 0-24h) and yellowness (Δb* 0-24h) in both seasons were analysed. Quantitative trait analyses of PPO activity, flour yellowness (b*) and YAN colour stability was also conducted in this population. QTL mapping of variation in PPO activity in the DM5637*B8 x H45 DH population identified a highly significant QTL on chromosome 2AL, which explained 52% of the observed variation across seasons. Regression analysis identified that wPt-7024 was highly significantly associated with PPO activity in this population. A highly significant association between this marker and PPO was also identified in the QALBis x VAW08-A17 population. Collectively, the three identified QTLs (on chromosomes 2AL, 7A and 7B) explained 71% of variation in PPO activity across seasons. A highly significant (P<0.001) QTL on chromosome 2B along with significant (P<0.01) QTLs on the chromosomes 1A, 3B, 4B and 5B were found to control flour yellowness. The QTLs on 2B, 4B and 5B were detected in both seasons analysed and accounted for 90% of variation in flour b* across seasons. The study on YAN colour stability located two highly significant (P<0.001) QTLs and two significant (P<0.01) QTLs that controlled the change in brightness of yellow alkaline noodle. The 2A QTL accounted for 64% of observed variation across seasons. It was in the same location as the PPO QTL and shared a common closest marker wPt-7024. Only one significant QTL for YAN a* (0-24h) was identified. It accounted for 12% of variation across seasons and was only detected in one season. One highly significant (P<0.001) QTL and two significant (P<0.01) QTLs were identified that controlled the change in yellowness of yellow alkaline noodle. The 2A QTL accounted for 68% of observed variation across seasons. The location of this QTL corresponded with that of 2A QTLs for PPO activity and L* of YAN in this study. Furthermore, wPt-7024 was also identified as the marker with the most significant association with L*. The identification of a correlation between the characters and a common location of a highly significant QTL for each of these characters indicates that it is likely that PPO activity is directly responsible for a large proportion of the changes in brightness and yellowness of YAN. QTLs for L* and b* of YAN were detected in a common location on chromosome 1A. However, no corresponding QTL was identified that controls PPO activity, highlighting the complexity of the relationship between these traits. Resistance to three rust pathogens (Puccinia graminis, Puccinia striiformis, and Puccinia triticina) was also investigated in the DM5637*B8 x H45 DH population because they are major yield limiting diseases in wheat. Disease response data at the seedling stage were converted to genotypic scores for rust genes Sr24/Lr24, Sr36, Lr13 and Yr7 to construct a genetic linkage map. No recombination was observed between rust resistance genes Sr36, Lr13 and Yr7 in this DH population. Therefore, these genes mapped in the same position on chromosome 2B. The Lr24/Sr24 locus was incorporated into the chromosome 3D map. Interval mapping analysis identified QTLs on chromosomes 2B, 3B, 4B and 5B that control adult plant resistance (APR) to stripe rust. Two QTLs on chromosomes 2B and 3D were identified that controlled APR to leaf rust in this DH population.

Doctor of Philosophy / Polyphenol oxidase (PPO) catalyses undesirable darkening in wheat products such as Asian noodles. Genetic variation for PPO activity is characterized in bread wheat. Australian wheat breeding programmes recognize that reduced PPO activity is an important quality target. Despite this interest from breeders, no varieties possessing extremely low and null PPO activity exist. The development of null PPO wheat varieties is dependant on an understanding of the genetic control of the null phenotype. Knowledge of these factors will accelerate efforts to develop them. The inheritance of PPO activity was investigated in two populations that were derived from hybrids between a null PPO genotype and Australian wheat varieties Lang and QAlBis. Observed genetic ratios were consistent with two and three gene control, respectively in these populations. QTL mapping was performed in the QALBis x VAW08-A17 population. The Diversity Array Technology (DArT) approach was employed to genotype the QALBis x VAW08-A17 population. Three highly significant QTLs that control PPO activity were identified on chromosomes 2AL, 2BS and 2DL. Close associations between PPO activity and DArT marker loci wPt-7024, wPt-0094 and wPt-2544 were observed, respectively. Collectively, these loci explained 74% of the observed variation in PPO activity across seasons. Significant QTLs on chromosomes 1B and 3B were also identified that together explained an additional 17% of variation in PPO activity. The relationship between PPO activity and yellow alkaline noodles (YAN) colour stability parameters was investigated in a DM5637*B8 x H45 doubled haploid population. PPO activity and changes in YAN brightness (ΔL* 0-24h) and yellowness (Δb* 0-24h) in both seasons were analysed. Quantitative trait analyses of PPO activity, flour yellowness (b*) and YAN colour stability was also conducted in this population. QTL mapping of variation in PPO activity in the DM5637*B8 x H45 DH population identified a highly significant QTL on chromosome 2AL, which explained 52% of the observed variation across seasons. Regression analysis identified that wPt-7024 was highly significantly associated with PPO activity in this population. A highly significant association between this marker and PPO was also identified in the QALBis x VAW08-A17 population. Collectively, the three identified QTLs (on chromosomes 2AL, 7A and 7B) explained 71% of variation in PPO activity across seasons. A highly significant (P<0.001) QTL on chromosome 2B along with significant (P<0.01) QTLs on the chromosomes 1A, 3B, 4B and 5B were found to control flour yellowness. The QTLs on 2B, 4B and 5B were detected in both seasons analysed and accounted for 90% of variation in flour b* across seasons. The study on YAN colour stability located two highly significant (P<0.001) QTLs and two significant (P<0.01) QTLs that controlled the change in brightness of yellow alkaline noodle. The 2A QTL accounted for 64% of observed variation across seasons. It was in the same location as the PPO QTL and shared a common closest marker wPt-7024. Only one significant QTL for YAN a* (0-24h) was identified. It accounted for 12% of variation across seasons and was only detected in one season. One highly significant (P<0.001) QTL and two significant (P<0.01) QTLs were identified that controlled the change in yellowness of yellow alkaline noodle. The 2A QTL accounted for 68% of observed variation across seasons. The location of this QTL corresponded with that of 2A QTLs for PPO activity and L* of YAN in this study. Furthermore, wPt-7024 was also identified as the marker with the most significant association with L*. The identification of a correlation between the characters and a common location of a highly significant QTL for each of these characters indicates that it is likely that PPO activity is directly responsible for a large proportion of the changes in brightness and yellowness of YAN. QTLs for L* and b* of YAN were detected in a common location on chromosome 1A. However, no corresponding QTL was identified that controls PPO activity, highlighting the complexity of the relationship between these traits. Resistance to three rust pathogens (Puccinia graminis, Puccinia striiformis, and Puccinia triticina) was also investigated in the DM5637*B8 x H45 DH population because they are major yield limiting diseases in wheat. Disease response data at the seedling stage were converted to genotypic scores for rust genes Sr24/Lr24, Sr36, Lr13 and Yr7 to construct a genetic linkage map. No recombination was observed between rust resistance genes Sr36, Lr13 and Yr7 in this DH population. Therefore, these genes mapped in the same position on chromosome 2B. The Lr24/Sr24 locus was incorporated into the chromosome 3D map. Interval mapping analysis identified QTLs on chromosomes 2B, 3B, 4B and 5B that control adult plant resistance (APR) to stripe rust. Two QTLs on chromosomes 2B and 3D were identified that controlled APR to leaf rust in this DH population.

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.

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.

ABSTRACT Triticum sphaerococcum (AABBDD, 2n = 6x = 42) is a land race of wheat known from the Indian subcontinent. It has several favourable characters including short and strong culms, hemispherical grains with a shallow crease (that may increase the yield of white flour), higher protein content compared to bread wheat (T. aestivum), and resistance to drought, and yellow rust caused by Puccinia striiformis. However, an unfavourable characteristic of T. sphaerococcum is its lower yield compared to bread wheat. Being a land race, the sphaerococcum wheat is poorly studied. This study was undertaken to increase knowledge of the physiology and genetics of this land race and determine if it may be possible to separate the favourable characters of T. sphaerococcum from its unfavourable characters. Plant height in bread wheat is controlled by many genes. ‘Reduced Height’ (Rht) genes which differ in their response to externally applied gibberellic acid (GA3) are responsible for the short stature of modern bread wheat varieties. Therefore, GA3 was used to probe the relationship between the semidwarf sphaerococcum phenotype and the Rht gene. T. sphaerococcum variety Sp5 showed a unique “seedling response” to externally applied GA3 when compared with T. aestivum varieties harbouring Rht1, Rht2, Rht8, Rht12, Rht13 or Rht18 alleles. A mapping population of doubled haploids was generated through wide hybridisation of F1 (Sp5 x Otane) with Zea mays. A genome-wide scan of Sp5 and Otane (parents) using 348 microsatellite (SSR) markers showed that only 169 of these markers (49%) were polymorphic between the parents. A DArT profiling yielded 348 markers that were polymorphic between the parents. Microsatellite markers and DArT markers were used to create a genetic map. The mapping population was phenotyped and a quantitative trait loci (QTL) analysis was performed for component traits of the complex sphaerococcum trait including plant height, spike length, awn length, yield, grain shape and crease size. Results of the QTL analysis indicated that it may be difficult to separate the favourable characters of T. sphaerococcum from its unfavourable characters through mutation because the component traits of the complex sphaerococcum trait may be under pleiotropic control of the Sp gene. The hypothesis that T. sphaerococcum originated through a mutation in T. aestivum was tested through induced mutation using gamma rays. Mutants from sphaerococcum-type to aestivum-type were isolated and phenotyped. Sphaerococcum-type mutants also were isolated and characterised from mutated aestivum-type wheat suggesting a possible origin of T. sphaerococcum through a mutation in T. aestivum.

ABSTRACT Triticum sphaerococcum (AABBDD, 2n = 6x = 42) is a land race of wheat known from the Indian subcontinent. It has several favourable characters including short and strong culms, hemispherical grains with a shallow crease (that may increase the yield of white flour), higher protein content compared to bread wheat (T. aestivum), and resistance to drought, and yellow rust caused by Puccinia striiformis. However, an unfavourable characteristic of T. sphaerococcum is its lower yield compared to bread wheat. Being a land race, the sphaerococcum wheat is poorly studied. This study was undertaken to increase knowledge of the physiology and genetics of this land race and determine if it may be possible to separate the favourable characters of T. sphaerococcum from its unfavourable characters. Plant height in bread wheat is controlled by many genes. ‘Reduced Height’ (Rht) genes which differ in their response to externally applied gibberellic acid (GA3) are responsible for the short stature of modern bread wheat varieties. Therefore, GA3 was used to probe the relationship between the semidwarf sphaerococcum phenotype and the Rht gene. T. sphaerococcum variety Sp5 showed a unique “seedling response” to externally applied GA3 when compared with T. aestivum varieties harbouring Rht1, Rht2, Rht8, Rht12, Rht13 or Rht18 alleles. A mapping population of doubled haploids was generated through wide hybridisation of F1 (Sp5 x Otane) with Zea mays. A genome-wide scan of Sp5 and Otane (parents) using 348 microsatellite (SSR) markers showed that only 169 of these markers (49%) were polymorphic between the parents. A DArT profiling yielded 348 markers that were polymorphic between the parents. Microsatellite markers and DArT markers were used to create a genetic map. The mapping population was phenotyped and a quantitative trait loci (QTL) analysis was performed for component traits of the complex sphaerococcum trait including plant height, spike length, awn length, yield, grain shape and crease size. Results of the QTL analysis indicated that it may be difficult to separate the favourable characters of T. sphaerococcum from its unfavourable characters through mutation because the component traits of the complex sphaerococcum trait may be under pleiotropic control of the Sp gene. The hypothesis that T. sphaerococcum originated through a mutation in T. aestivum was tested through induced mutation using gamma rays. Mutants from sphaerococcum-type to aestivum-type were isolated and phenotyped. Sphaerococcum-type mutants also were isolated and characterised from mutated aestivum-type wheat suggesting a possible origin of T. sphaerococcum through a mutation in T. aestivum.