Juvenile hormones (JHs) are key regulators of both metamorphosis and adult reproductive processes. The role of two key enzymes in the biosynthetic pathway of JH were examined: Farnesoic Acid O-Methyltransferase (FAMeT) and Juvenile Hormone Acid Methyltransferase (JHAMT). In crustaceans, FAMeT has been found to methylate farnesoic acid (FA), producing methyl farnesoate (MF) prior to epoxidation at the penultimate stage of JH biosynthesis. JHAMT was discovered more recently in the silkworm Bombyx mori and converts epoxidated FA (JHacids) to active JH through methylation using S-adenosyl-L-methionine (SAM). The aim of the proposed research is to examine the influence of a) decreasing the amount of FAMeT produced using an enhancer trapping P-element and b) increasing the levels of JHAMT and FAMeT in specific tissues using GAL4 overexpression techniques. Immunohistochemical analysis was used to confirm the presence of FAMeT in the CA of D. melanogaster ring glands. Analysis of MF, JHIII and JHB3 release in wild type and mutant stocks in the presence and absence of Drome AST (PISCF-type) suggest that Drosophila FAMeT has little if any effect on the sesquiterpenoid biosynthesis. Drome-AST appears to have a select effect on JHB3 biosynthesis and not MF or JHIII. Analysis of JHB3 release from larval and adult flies ubiquitously overexpressing JHAMT showed a significant increase when compared to wildtype (p<0.01 and p<0.0001 respectively). No significant difference was seen in JHB3 release in flies ubiquitously overexpressing FAMeT. A significant increase in hatching success was seen in flies overexpressing FAMeT in the larval ring gland and oocytes (p<0.05) whereas no significant decrease was seen in JHAMT-overexpressing flies during development. A significant extension of lifespan was also seen when FAMeT was overexpressed in the border and follicle cells of the oocyte (p<0.0001). The direct role of JHAMT in JHB3 synthesis has been demonstrated. The involvement of FAMeT and JHAMT in development and longevity may require other interacting proteins to elicit an effect, which is a limiting factor in overexpression experiments of the two enzymes. Additionally, this is the first example of AST action within D. melanogaster. / Thesis (Master, Biology) -- Queen's University, 2007-09-27 20:08:23.69

A cana-de-açúcar é uma monocotiledônea poliplóide, alógama que possui baixa taxa reprodutiva devido a dificuldade de florescimento. Devido estas características genéticas e fisiológicas os programas de melhoramento são longos e laboriosos. Alternativamente, modernas aplicações da biotecnologia visam contribuir com o desenvolvimento de novos cultivares. Neste trabalho estudou-se a metodologia de cultura de tecidos a partir de discos de folhas imaturas para o estabelecimento da cultura de calos embriogênicos e regeneração de plantas a partir dos calos embriogênicos e diretamente, a partir de folhas imaturas. O objetivo principal foi contribuir para o desenvolvimento de métodos eficientes para produção de plantas transgênicas a partir de calos e folhas imaturas, considerando-se a crescente necessidade de produção de novos cultivares com características agronômicas específicas. Diversas concentrações de 2,4-D e cinetina em meio MS foram testadas para o estabelecimento de calos altamente embriogênicos e para a indução da desdiferenciação celular nos discos foliares antecedendo a regeneração de plantas. Meios de cultura sem reguladores de crescimento (MS) e com a adição de BAP e ANA foram testados para a regeneração de plantas a partir de discos foliares. Calos embriogênicos com 12 a 20 semanas de cultivo produziram em média 3 a 5 plantas, em meio de regeneração MS. Folhas imaturas apresentaram elevado potencial de regeneração de plantas quando se utilizou 2,4-D em concentrações de 5 e 8 mg/L nos períodos de 5 e 8 dias no escuro. Houve indução a formação de embriões somáticos que resultaram em média 12 a 16 plantas por explante no período total de 7 a 10 semanas. Além disso, foi testado o pré-tratamento dos discos foliares em meio MS3K, contendo 2,4-D (3mg/L) e cinetina (0,1 mg/L), antes da transferência do discos para meio de regeneração MS. Os discos submetidos a este pré-tratamento durante 14, 21 e 28 dias apresentaram aumento significativo na eficiência de regeneração de plantas, variando em média de 41 a 50 plantas por disco foliar nas variedades RB835089 e RB855156. A redução no tempo para obtenção de plantas aliado ao aumento na média de plantas obtidas é a base para aumentar a eficiência de transformação genética de plantas. Experimentos de cotransformação dos genes neo e comt(AS), foram realizados por biolística. Em plantas regeneradas a partir de folhas imaturas da variedade RB835486, as análises de PCR confirmaram a incorporação do gene marcador neo em 57 e 90% das plantas em meio seletivo com geneticina (30 mg/L), sendo que a maior eficiência de regeneração de transgênicos (90%) foi obtida no pré-tratamento com o meio MS3K. Das plantas transgênicas para o gene neo, 7 e 38% também foram confirmadas para a incorporação do gene comt(AS). Nas plantas regeneradas a partir de calos embriogênicos em meio seletivo, as análises de PCR detectaram somente a incorporação do gene neo, o que ocorreu em 52% das plantas analisadas. Os resultados obtidos mostram que a cultura de discos de folhas imaturas para o processo de transformação genética por biolística é uma metodologia viável, rápida e menos onerosa, quando comparada com a cultura de calos embriogênicos. / Sugarcane is a polyploidy monocot and allogamous species that has low reproductive rate due to the difficulty of flowering. Because of these genetic and physiological characteristics breeding program takes long time and demand hard labor. Alternatively, modern biotechnology approaches contribute to the development of new cultivars. In this work we studied the methodology of plant tissue culture from immature leaf discs to establish callus culture and plant regeneration from those calli and from immature leaves, directly. The main objective was to contribute to the development of efficient methods to produce transgenic plants from callus and immature leaves, due to the growing need to produce new cultivars with specific agronomics traits. MS medium with different concentrations of 2,4-D and kinetin were tested to obtain highly embryogenic calli and to induce cellular dedifferentiation in the immature leaf discs prior to plant regeneration. Culture media without growth regulators (MS) and with the addition of BAP and NAA were tested for plant regeneration from leaf discs. Callus culture with 12 to 20 weeks resulted on average 3 to 5 plants on regeneration medium designed as MS. Immature leaves showed a high potential for plant regeneration when 2,4-D at concentrations of 5 and 8 mg/L in periods of 5 and 8 days in the dark. There were inducing of somatic embryos that resulted in average 12 to 16 plants per explant in the total period of 7 to 10 weeks. In addition, we tested the pre-treatment of leaf discs in MS3K medium which contain 2,4-D (3 mg/L) and kinetin (0.1 mg/L) before transfering to plant regeneration MS medium . The discs submitted to this pretreatment for 14, 21 and 28 days showed significant increase in the efficiency of plant regeneration, with on average of 41 to 50 plants per leaf disc in varieties RB835089 and RB855156. The reduction of time to obtain plants coupled with the increase of plants obtained is the basis for increasing the efficiency of plant genetic transformation. Co-transformation with genes neo and comt(AS), were performed by biolistics. Plants regenerated from immature leaves of the variety RB835486, PCR analysis confirmed the incorporation of the neo selection marker gene in 57 and 90% of the plants on selective medium with geneticin (30 mg/L), the higher efficiency of transgenic plants (90%) was obtained on pre-treatment in MS3K medium. Transgenic plants for the neo gene, 7 and 38% were also confirmed for the incorporation of comt (AS). PCR analysis of candidates transgenic plants from callus growing on selective medium, revelled only the insertion of the neo gene, which occurred in 52% of the analyzed plants. The results of this work showed that the approach of using immature leaf discs to obtain plant genetic transformation by biolistics methodology is a viable, cheaper and faster than using embryogenic callus.

A cana-de-açúcar é uma monocotiledônea poliplóide, alógama que possui baixa taxa reprodutiva devido a dificuldade de florescimento. Devido estas características genéticas e fisiológicas os programas de melhoramento são longos e laboriosos. Alternativamente, modernas aplicações da biotecnologia visam contribuir com o desenvolvimento de novos cultivares. Neste trabalho estudou-se a metodologia de cultura de tecidos a partir de discos de folhas imaturas para o estabelecimento da cultura de calos embriogênicos e regeneração de plantas a partir dos calos embriogênicos e diretamente, a partir de folhas imaturas. O objetivo principal foi contribuir para o desenvolvimento de métodos eficientes para produção de plantas transgênicas a partir de calos e folhas imaturas, considerando-se a crescente necessidade de produção de novos cultivares com características agronômicas específicas. Diversas concentrações de 2,4-D e cinetina em meio MS foram testadas para o estabelecimento de calos altamente embriogênicos e para a indução da desdiferenciação celular nos discos foliares antecedendo a regeneração de plantas. Meios de cultura sem reguladores de crescimento (MS) e com a adição de BAP e ANA foram testados para a regeneração de plantas a partir de discos foliares. Calos embriogênicos com 12 a 20 semanas de cultivo produziram em média 3 a 5 plantas, em meio de regeneração MS. Folhas imaturas apresentaram elevado potencial de regeneração de plantas quando se utilizou 2,4-D em concentrações de 5 e 8 mg/L nos períodos de 5 e 8 dias no escuro. Houve indução a formação de embriões somáticos que resultaram em média 12 a 16 plantas por explante no período total de 7 a 10 semanas. Além disso, foi testado o pré-tratamento dos discos foliares em meio MS3K, contendo 2,4-D (3mg/L) e cinetina (0,1 mg/L), antes da transferência do discos para meio de regeneração MS. Os discos submetidos a este pré-tratamento durante 14, 21 e 28 dias apresentaram aumento significativo na eficiência de regeneração de plantas, variando em média de 41 a 50 plantas por disco foliar nas variedades RB835089 e RB855156. A redução no tempo para obtenção de plantas aliado ao aumento na média de plantas obtidas é a base para aumentar a eficiência de transformação genética de plantas. Experimentos de cotransformação dos genes neo e comt(AS), foram realizados por biolística. Em plantas regeneradas a partir de folhas imaturas da variedade RB835486, as análises de PCR confirmaram a incorporação do gene marcador neo em 57 e 90% das plantas em meio seletivo com geneticina (30 mg/L), sendo que a maior eficiência de regeneração de transgênicos (90%) foi obtida no pré-tratamento com o meio MS3K. Das plantas transgênicas para o gene neo, 7 e 38% também foram confirmadas para a incorporação do gene comt(AS). Nas plantas regeneradas a partir de calos embriogênicos em meio seletivo, as análises de PCR detectaram somente a incorporação do gene neo, o que ocorreu em 52% das plantas analisadas. Os resultados obtidos mostram que a cultura de discos de folhas imaturas para o processo de transformação genética por biolística é uma metodologia viável, rápida e menos onerosa, quando comparada com a cultura de calos embriogênicos. / Sugarcane is a polyploidy monocot and allogamous species that has low reproductive rate due to the difficulty of flowering. Because of these genetic and physiological characteristics breeding program takes long time and demand hard labor. Alternatively, modern biotechnology approaches contribute to the development of new cultivars. In this work we studied the methodology of plant tissue culture from immature leaf discs to establish callus culture and plant regeneration from those calli and from immature leaves, directly. The main objective was to contribute to the development of efficient methods to produce transgenic plants from callus and immature leaves, due to the growing need to produce new cultivars with specific agronomics traits. MS medium with different concentrations of 2,4-D and kinetin were tested to obtain highly embryogenic calli and to induce cellular dedifferentiation in the immature leaf discs prior to plant regeneration. Culture media without growth regulators (MS) and with the addition of BAP and NAA were tested for plant regeneration from leaf discs. Callus culture with 12 to 20 weeks resulted on average 3 to 5 plants on regeneration medium designed as MS. Immature leaves showed a high potential for plant regeneration when 2,4-D at concentrations of 5 and 8 mg/L in periods of 5 and 8 days in the dark. There were inducing of somatic embryos that resulted in average 12 to 16 plants per explant in the total period of 7 to 10 weeks. In addition, we tested the pre-treatment of leaf discs in MS3K medium which contain 2,4-D (3 mg/L) and kinetin (0.1 mg/L) before transfering to plant regeneration MS medium . The discs submitted to this pretreatment for 14, 21 and 28 days showed significant increase in the efficiency of plant regeneration, with on average of 41 to 50 plants per leaf disc in varieties RB835089 and RB855156. The reduction of time to obtain plants coupled with the increase of plants obtained is the basis for increasing the efficiency of plant genetic transformation. Co-transformation with genes neo and comt(AS), were performed by biolistics. Plants regenerated from immature leaves of the variety RB835486, PCR analysis confirmed the incorporation of the neo selection marker gene in 57 and 90% of the plants on selective medium with geneticin (30 mg/L), the higher efficiency of transgenic plants (90%) was obtained on pre-treatment in MS3K medium. Transgenic plants for the neo gene, 7 and 38% were also confirmed for the incorporation of comt (AS). PCR analysis of candidates transgenic plants from callus growing on selective medium, revelled only the insertion of the neo gene, which occurred in 52% of the analyzed plants. The results of this work showed that the approach of using immature leaf discs to obtain plant genetic transformation by biolistics methodology is a viable, cheaper and faster than using embryogenic callus.

La récente définition de Brachypodium distachyon comme modèle des graminées en fait un organisme de choix pour l’étude de leur paroi cellulaire, en particulier dans le cadre de leur utilisation comme matière première renouvelable pour le bioéthanol de seconde génération. Les lignines, dont les trois unités (H, G et S) proviennent de la polymérisation des monolignols, sont associées aux acides hydroxycinnamiques dans la paroi des céréales et représentent l’obstacle majeur à l’exploitation industrielle de la biomasse lignocellulosique. L’acquisition de connaissances sur les mécanismes dirigeant leur mise en place et leur organisation permettrait d’identifier des facteurs modulant les rendements de production qui y sont associés. Quatre familles de gènes ont été étudiées et l’implication dans la voie de biosynthèse des monolignols de trois gènes a été montrée : BdF5H2 possède une activité férulate-5-hydroxylase permettant la synthèse des précurseurs des unités S des lignines, BdCOMT3 est l’isoforme principale des acide cafféique O-Méthyltransférases et sa perte partielle de fonction cause une diminution de la quantité de lignine, la modification du rapport S/G et une baisse de quantité d’acide p-coumarique dans deux lignées mutantes indépendantes. Enfin, BdCAD1 est l’isoforme principale des alcools cinnamylique déshydrogénases : sa perte de fonction dans deux lignées indépendantes cause la diminution de la quantité globale de lignine et d’acide p-coumarique, une baisse du rapport S/G ainsi que l’accumulation de sinapaldéhyde. Par ailleurs ces deux lignées présentent des rendements de saccharification augmentés de plus d’un quart par rapport au sauvage. / Brachypodium distachyon was recently adopted as an experimental model for grass species. As such, it is used to study grass cell wall, in particular in the context of their use as renewable feedstock for the production of second generation bioethanol. Lignins are polymers of three main units (H, G and S) originating from the polymerization of monolignols, and are linked to hydroxycinnamic acids in grasses. They constitute the main bottleneck to industrial processes targeting lignocellulosic biomass and improving the understanding of the mechanisms directing their structure and deposition could lead to the identification of the factors modulating associated production yields. Four gene families were studied and the involvement of three genes in the monolignols biosynthetic pathway was shown: BdF5H2 displays a ferulate-5-hydroxylase activity enabling the synthesis of the S lignin units, BdCOMT3 is the main caffeic acid O-methyltransferase and its partial loss of function in two independent mutant lines leads to the reduction of lignin content, the modification of the S/G units ratio and a decrease in p-coumaric acid accumulation. BdCAD1 is the main cinnamyl alcohol dehydrogenase isoform: its loss of function in two independent mutant lines results in a decrease in lignin content and of the S/G ratio and the accumulation of sinapaldehyde. Moreover, these two lines display significatively increased saccharification yields.