Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Submitted by Jadson Francisco de Jesus SILVA (jadson@uefs.br) on 2018-01-30T22:51:06Z No. of bitstreams: 1 Tese Definitivo ZERGurgel.pdf: 2392487 bytes, checksum: 97870ae444449d037535f874c18b1d37 (MD5) / Made available in DSpace on 2018-01-30T22:51:06Z (GMT). No. of bitstreams: 1 Tese Definitivo ZERGurgel.pdf: 2392487 bytes, checksum: 97870ae444449d037535f874c18b1d37 (MD5) Previous issue date: 2017-07-24 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Comanthera mucugensis Giul. subsp. mucugensis, an endemic species of the municipality of Mucug?-BA is threatened with extinction. To reduce extractivism in natural populations, it is necessary to develop efficient multiplication protocols; In this sense somatic embryogenesis and organogenesis may be viable and preservation cryopreservation may be a strategy for its long-term. The objectives of this study were: (1) to analyze the embryogenic competence and the indirect organogenesis of C. mucugensis; (2) to evaluate the cryopreservation of seeds and different methods of cryopreservation of C. mucugensis plants and (3) to identify the best way to store seeds in the long term. Experiments were performed to induce somatic embryogenesis with different concentrations of 2,4-D X BAP and Picloran X BAP and for indirect organogenesis BAP and ANA were used. Seeds were cryopreserved for 0 (control), 1, 7, 30, 360 and 540 days and other seeds were stored at different temperatures to verify the best form of storage. Plants were cryopreserved with the technique of vitrification and encapsulation-desiccation. Plants from cryopreserved seeds were acclimatized in sand and vegetal soil (1: 1) for 60 days. The work showed that the plant regulators Picloran and 2,4-D are promising in the induction of callus with embryogenic potential and that the plant regulator ANA at 4.9 ?M is efficient in indirect organogenesis. The seeds of C. mucugensis can be cryopreserved without compromising their physiological quality, but the techniques for cryopreservation of plants have not been efficient. / Comanthera mucugensis Giul. subsp. mucugensis, possui flor de interesse comercial que devido ao extrativismo excessivo encontra-se amea?ada de extin??o. Para suprir a demanda do mercado e evitar o decl?nio populacional, faz-se necess?rio desenvolver protocolos para sua multiplica??o, podendo a embriog?nese som?tica e a organog?nese serem alternativas vi?veis. Al?m disso, ? importante investir em estudos de conserva??o a longo prazo como, por exemplo, a criopreserva??o. O trabalho teve como objetivos: realizar estudos para tornar a micropropaga??o mais eficiente e avaliar a conserva??o de C. mucugensis em diferentes temperaturas como estrat?gia para a conserva??o do seu germoplasma. Foram testados para a indu??o da embriog?nese som?tica 2,4-D x BAP e Picloram x BAP e para organog?nese indireta BAP e ANA. Na criopreserva??o foram avaliadas sementes mantidas em nitrog?nio l?quido (-196?C) por 0, 1, 7, 30, 360 e 540 dias e as plantas inteiras foram submetidas a duas t?cnicas: vitrifica??o e encapsulamento-desidrata??o. Para avaliar o armazenamento, as sementes foram mantidas em temperatura ambiente, 4?C, -20?C, -80?C e -196?C por 30, 90 e 180 dias. Visando observar se a criopreserva??o das sementes interfere no desenvolvimento, plantas oriundas da germina??o in vitro de sementes criopreservadas foram aclimatizadas em areia e terra vegetal (1:1) por 60 dias. O trabalho demonstrou que os reguladores vegetais Picloram e 2,4-D s?o promissores na indu??o de calos com potencial embriog?nico e que o regulador vegetal ANA (4,9 ?M) ? eficiente na organog?nese indireta. As sementes de C. mucugensis podem ser criopreservadas sem comprometer sua qualidade fisiol?gica, entretanto, n?o foram eficientes as t?cnicas para criopreserva??o de plantas inteiras.

L'acide rétinoïque (AR) est un morphogène dérivé de la vitamine A, qui intervient dans le contrôle de l'organogenèse, de la prolifération et de la différenciation cellulaires chez les Chordés. Dans ce contexte, nous avons étudié les régulations spatio-temporelles de la voie de signalisation de l’AR au cours du développement de l’amphioxus, en mettant l'accent sur l’espèce européenne Branchiostoma lanceolatum.Nous avons tout d'abord inhibé ou activé la voie de signalisation de l’AR lors du développement embryonnaire en traitant des embryons d’amphioxus à des doses variables de composés pharmacologiques interférant avec le métabolisme des rétinoïdes. Grâce à l’utilisation d’outils mathématiques spécifiques, nous avons établi un schéma détaillé des effets des traitements effectués sur le développement du système nerveux central (SNC) et du pharynx chez l’amphioxus en nous basant sur l’expression de gènes marqueurs de tissus spécifiques. À l’issue de cette première analyse, nous avons par la suite étudié les effets d’une perturbation de la signalisation de l’AR à des points clés du développement chez l’amphioxus lors de la régionalisation du SNC et du pharynx. Nous avons ainsi montré que la voie de signalisation de l’AR intervient dans la régionalisation de l’axe antéro-postérieur via le contrôle des gènes hox dès le stade gastrula et jusqu’aux stades larvaires. En outre, nous avons réalisé l'étude préliminaire du gène homologue chez l’amphioxus du gène aldh1a2 des Vertébrés, et avons démontré que la régulation du niveau de synthèse de l’AR au cour du développement est conservée entre l’amphioxus et les Vertébrés. Finalement, nous avons montré que la voie de l’AR participe également à la morphogenèse caudale chez l’amphioxus, et que le mécanisme impliqué semble différent de celui proposé chez les Vertébrés où l’AR contrôle la structuration de la nageoire caudale par le ciblage des tissus mésenchymateux. / Retinoic acid (RA) is an endogenous vitamin A-derived morphogen. In this context, we studied the spatiotemporal roles of RA signaling in amphioxus development, focusing on the European amphioxus species: Branchiostoma lanceolatum. We first created excess and insufficiency models of RA signaling by exposing amphioxus embryos to series of doses of different pharmacological compounds targeting either the RA receptors or the RA metabolism machinery. By introducing the important mathematical concept of a Cartesian coordinate system founded by René Descartes, we created detailed diagrams of the concentration-dependent defects caused by RA signaling in the central nervous system (CNS) and pharynx of amphioxus by evaluating the statistical significances of tissue-specific marker gene expression in labeled embryos. This analysis yielded a very detailed description of the sensitivities of the developing amphioxus CNS and pharynx to altered RA signaling levels. Following this initial challenge, we correlated the effects of altered RA signaling levels with key amphioxus developmental stages characterized by structural transitions in CNS and pharynx. We show that hox-mediated RA signaling in axial patterning is active beyond the gastrula stage and might be maintained until at least early larval stage, with possible roles in more regionalized axis formation and organ induction. In addition, we carried out a preliminary study on a RA synthesizing gene in amphioxus, called aldh1a, a possible homolog of the vertebrate aldh1a2 gene, demonstrating that the feedback between RA signaling and RA synthesizing levels has emerged before the split of the cephalochordate and vertebrate lineages. Moreover, we are able to show that RA signaling also participates in tail fin morphogenesis in amphioxus by a mechanism that is probably not comparable to that in vertebrates, where RA modulates caudal fin patterning through targeting mesenchymal derivatives.

Born and educated for the most part in the United States, I have enjoyed the luxury of excellent mentorship during my career thus far as an independent scientist in France. All these mentors have taken it on trust that my training for a Ph.D. also included the necessary tools for directing original research responsibly, at all levels. However, the habilitation is an obligate rite of passage for researchers in France, Germany, Sweden and a number of other European countries. It ensures both that I am competent to not only continue to conduct original research, and that I have a directive seam in my research interests over time that is sufficiently rich to support myself and those trainees who will learn from my experience and contribute their efforts by my side to advancing science. To demonstrate that the faith of these esteemed colleagues has been well-placed since my Ph.D., I hereby present, to the best of my ability, my acquired credentials and my near- to mid-term projects. The over-arching theme of my work has been to identify molecular hallmarks and improve the physiopathological understanding of congenital and progressive conditions implicating a highly plastic embryological cell population known as the neural crest. These neural crest cells (NCC) participate directly or indirectly in the formation of a stunning array of tissues organs during embryogenesis. When the genes regulating the differentiation, proliferation, or migratory and appropriately invasive behavior of NCC are muted, this can lead to associations of pediatric congenital malformations or tumorigenesis. I make use of avian and, more recently, murine models, as well as careful observations effected on tissues derived from normal human embryos, to tease apart those mechanisms.

No description available.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

The ratio of auxin to cytokinin determines de novo organogenesis in plants. Relatively little is known about the effect of genetically altered auxin signaling on in vitro organogenesis. Here, callusogenesis, shoot, and root formation were studied in loss- (LOF) and gain-of-function (GOF) alleles in two phylogenetically related Auxin Response Factors (ARFs), MONOPTEROS (MP/ARF5) and NON-PHOTOTROPHIC HYPOCOTYL 4 (NPH4/ARF7). Reduced MP activity greatly diminished shoot regeneration, and partially diminished callusogenesis and root formation. LOF in NPH4 strongly decreased callusogenesis, and mildly decreased shoot and root regeneration in particular categories of explants. By contrast, organogenesis responses were strongly increased in aerial explants carrying the GOF transgene dMP. Thus, both MP and NPH4 seem to act as positive regulators of certain organogenesis processes and the GOF dMP transgene may be of interest for stimulating organogenesis in plant species with poor regeneration properties. Also, organogenesis in vitro may reveal unknown developmental ARF functions.

The ratio of auxin to cytokinin determines de novo organogenesis in plants. Relatively little is known about the effect of genetically altered auxin signaling on in vitro organogenesis. Here, callusogenesis, shoot, and root formation were studied in loss- (LOF) and gain-of-function (GOF) alleles in two phylogenetically related Auxin Response Factors (ARFs), MONOPTEROS (MP/ARF5) and NON-PHOTOTROPHIC HYPOCOTYL 4 (NPH4/ARF7). Reduced MP activity greatly diminished shoot regeneration, and partially diminished callusogenesis and root formation. LOF in NPH4 strongly decreased callusogenesis, and mildly decreased shoot and root regeneration in particular categories of explants. By contrast, organogenesis responses were strongly increased in aerial explants carrying the GOF transgene dMP. Thus, both MP and NPH4 seem to act as positive regulators of certain organogenesis processes and the GOF dMP transgene may be of interest for stimulating organogenesis in plant species with poor regeneration properties. Also, organogenesis in vitro may reveal unknown developmental ARF functions.

Made available in DSpace on 2014-08-20T13:59:12Z (GMT). No. of bitstreams: 1 dissertacao_ilda_castro_silva.pdf: 4362339 bytes, checksum: bf7e02c52f488cce4b322ad0f90ed17c (MD5) Previous issue date: 2010-03-25 / Quinces (Cydonia oblonga Mill.) are good alternative for pear tree rootstock diversification, due the interest to control the plants development to obtain fast frutification, plants uniformity and higher fruit quality. Their propagation through the tissue culture allows the plant production in a large scale, with high sanitary in a short period of time. Another application of this technique is the regeneration or in vitro morphogenesis that consists in the organs induction by somatic embryogenesis or organogenesis, which is a pre-requirement for genetic transformation. The current work aimed at optimizing the multiplication and in vitro regeneration of Cydonia oblonga Mill. rootstocks, cultivars Adams and MC. For the multiplication experiments were used apicals shoots with excised tip (1,5cm), and inoculated in MS½ and MS¾ or MS medium modified (¾ of the normal concentration of NH4NO3 and KNO3 and EDTA-Ferric), containing different BAP concentrations or interaction among BAP and AG3 concentrations. The explants were maintained in growth chamber with 16h photoperiod, 48 μmol m-2 s-1 density photon flux and 25±2ºC temperature, for 40 days. The variables analyzed were shoots number per explants, shoots length, node number per shoot and percentage of hyperhydric shoots. In the regeneration experiments it was used as initial explants entire leaves or third basal leaves, young or adult leaves, with or without petiole, inoculated in MS medium, supplemented with TDZ (0, 1, 2, 3 and/or 4mg dm-3) and ANA (0,1mg dm-3), maintained in darkness for 40 days. Later, they were transferred to MS medium containing 1mg dm-3 TDZ and maintained in the light for 30 days more. After that period, the percentage of regenerate explants, the shoots number for regenerate explant and type of organogenesis formed was evaluated. Both cultivars present potential for in vitro propagation, however 'Adams' is more responsive, because it was obtained 6,3 shoots per explant, with 2,8mg dm-3 BAP, while ' MC' presents larger sensibility to this plant growth regulator and to develop the hyperhydric explants, however it demonstrated to be more efficient to the in vitro regeneration, presenting 26% of regenerating explants (with 1mg dm-3 TDZ). It was verified for both cultivars, the entire explant leaves were more responsive for the regeneration than basal thirds and that this happens mainly in the basal area of the explant, through direct and 9 indirect organogenesis. Based on the multiplication and the regeneration results, it was concluded that is possible the in vitro multiplication of these cultivars in large scale, as well as to improve the regeneration rates for future plant breeding works for characteristics of interest, through genetic transformation. / Os marmeleiros (Cydonia oblonga Mill.) são excelente alternativa de diversificação de porta-enxertos para pereiras, devido ao interesse por plantas com porte reduzido, rápida frutificação, uniformidade nos pomares e boa qualidade das frutas. A sua propagação por meio da cultura de tecidos permite a produção de plantas homogêneas, em larga escala, com alta qualidade sanitária e num curto espaço de tempo. Outra aplicação desta técnica é a regeneração ou morfogênese in vitro, que consiste na formação de órgãos, seja por meio de embriogênese somática ou organogênese, sendo esta um pré-requisito para a transformação genética. O presente trabalho teve como objetivo otimizar a multiplicação e regeneração in vitro dos porta-enxertos de Cydonia oblonga Mill., cultivares Adams e MC. Para os experimentos de multiplicação foram utilizadas brotações apicais com ápice excisado (1,5cm), inoculadas em meio MS½ e MS¾ ou MS modificado (¾ da concentração normal de NH4NO3 e KNO3 e Ferro na forma de EDTA-Férrico), contendo diferentes concentrações de BAP ou interação entre BAP e AG3, mantidas em câmara de crescimento com fotoperíodo de 16 horas, 48 μmol m-2 s-1 e 25±2ºC de temperatura por 40 dias. Após, analisaram-se as variáveis: número de brotações por explante, comprimento das brotações, número de nós por brotação e percentagem de explantes com hiperidricidade. Nos experimentos de regeneração utilizou-se como explante inicial folhas inteiras ou terços basais, jovens ou adultas, com ou sem pecíolo, inoculados em meio MS, suplementados com TDZ (0, 1, 2, 3 e/ou 4mg dm-3) e ANA (0,1mg dm-3), permanecendo no escuro por 40 dias. Posteriormente, foram transferidos para meio MS contendo 1mg dm-3 de TDZ e mantidos na luz por mais 30 dias. Após esse período, avaliou-se a percentagem de explantes regenerados, o número de brotações por explante regenerante e tipo de organogênese formada. As duas cultivares estudadas apresentam potencial para a propagação in vitro, porém Adams foi mais responsivo, pois obteve-se 6,3 brotações por explante, com 2,8mg dm-3 de BAP, enquanto MC apresenta maior sensibilidade a altas concentrações deste regulador de crescimento e à vitrificação, porém demonstrou ser mais eficiente durante a regeneração in vitro, apresentando 7 valores aproximados de 26% de explantes regenerantes (utilizando 1,0mg dm-3 de TDZ). Para ambas cultivares, o explante folha inteira é mais responsivo à regeneração do que terços basais e que esta ocorre principalmente na região basal do explante, via organogênese direta ou indireta. Baseado nos resultados obtidos tanto na fase de multiplicação quanto na de regeneração, verifica-se que é possível multiplicar in vitro estas cultivares em larga escala, bem como melhorar as taxas de regeneração para futuros trabalhos de melhoramento para características de interesse, via transformação genética.

Patchouli is an aromatic species of Asian origin that has been cultivated in various parts of the world for the extraction of essential oil of its leaves. The essential oil is used by the perfume, cosmetic and food industries. Patchouli is an economically important species need to have its genetic material preserved. In this context, the aim of this study was to develop protocols for micropropagation and in vitro conservation of three accessions of patchouli. For the experiments of micropropagation MS medium supplemented with different concentrations of kinetin and IAA or NAA were tested. For the acclimatization assays different mixtures of coconut coir and vermiculite supplemented with NPK fertilizer and limestone were tested. For callus induction, different doses of 2,4-D were combined with BAP and kinetin. For the in vitro conservation assays different growth temperatures, osmotic regulators and/or inhibitors of growth were tested. The different accessions showed shoot regeneration via direct organogenesis. Accession POG002 can be propagated using 0.5 mg.L-1 kinetin and 0.1 mg.L-1 NAA, accession POG014 using 2.0 mg L-1 kinetin and 0.1 mg.L-1 NAA, and accession POG021 using 1.0 mg.L-1 kinetin and 0.5 mg.L-1 IAA. The acclimatization of accessions POG002, POG014 and POG021 can be performed with the substrate coconut coir + NPK (3-12-6) fertilizer (12 g.L-1) + limestone (1 g.L-1). The larger sizes of calluses were obtained using: 0.022 mg.L-1 2,4-D and 0.113 mg.L-1 BAP, 0.022 mg.L-1 2,4-D and 0.225 mg.L-1 BAP and 0.110 mg.L-1 2,4-D and 0.113 mg.L-1 BAP for accession POG014; 0.022 mg.L-1 2,4-D and 0.113 mg.L -1 BAP, 0.022 mg.L-1 2,4-D and 0.225 mg.L-1 BAP for POG021; and 0.022 mg.L-1 2,4-D and 0.225 mg.L-1 BAP for POG002. The conservation of accession POG002 can be realized using 0.5 mg.L-1 abscisic acid for three months at 18°C; accession POG014 can be conservated for three months using 0.5 mg.L-1 of abscisic acid at 18°C; and POG021 can be maintained for six months using sucrose 10 g.L-1 and sorbitol 5 g.L-1 at 25°C. / O patchouli é uma espécie aromática de origem asiática que tem sido cultivada em diversas partes do mundo para extração de óleo essencial de suas folhas. Este óleo essencial é empregado nas indústrias de perfumes, cosmética e alimentícia. Por se tratar de uma espécie de importância econômica local e mundial o patchouli necessita ter seu material genético preservado. Neste contexto, o objetivo geral deste trabalho foi desenvolver protocolos de micropropagação e conservação in vitro de três acessos de patchouli. Para os experimentos de micropropagação foi testado meio MS suplementado com diferentes concentrações de cinetina e as auxinas AIA ou ANA. Nos ensaios de aclimatização testou-se diferentes misturas de pó de coco e vermiculita suplementado com adubo NPK formulado e calcário. Para indução de calos, doses distintas de 2,4-D foram combinadas com as citocininas BAP e cinetina. Nos ensaios de conservação in vitro testou-se diferentes temperaturas de cultivo, reguladores osmóticos e/ou inibidores de crescimento. Os diferentes acessos apresentaram regeneração de brotos via organogênese direta. O acesso POG002 pode ser propagado na presença de 0,5 mg.L-1 de cinetina e 0,1 mg.L-1 de ANA; o acesso POG014 com 2,0 mg.L-1 de cinetina e 0,1 mg.L-1 de ANA; e o acesso POG021 com o emprego de 1,0 mg.L-1 de cinetina e 0,5 mg.L-1 de AIA. A aclimatização dos acessos POG002, POG014 e POG021 pode ser realizada com o substrato pó de coco + NPK (3-12-6) (12 g.L-1) + calcário (1 g.L-1). Os maiores tamanhos de calos foram obtidos nos seguintes meios: 0,022 mg.L-1 de 2,4-D e 0,113 mg.L-1 de BAP, 0,022 mg.L-1 de 2,4-D e 0,225 mg.L-1 de BAP, e 0,110 mg.L-1 de 2,4-D e 0,113 mg.L-1 de BAP para o acesso POG014; 0,022 mg.L-1 de 2,4-D e 0,113 mg.L-1 de BAP, 0,022 mg.L-1 de 2,4-D e 0,225 mg.L-1 de BAP para o POG021; e 0,022 mg.L-1 de 2,4-D e 0,225 mg.L-1 de BAP para o POG002. A conservação do acesso POG002 pode ser realizada com 0,5 mg.L-1 ácido abscísico por três meses a 18°C; o acesso POG014 pode ser conservado por três meses empregando 0,5 mg.L-1 de ácido abscísico a 18°C; e o acesso POG021 pode ser mantido por seis meses com uso de sacarose 10 g.L-1 e sorbitol 5 g.L-1 a 25°C.