Mutationen in Genen, die für Kernhüllproteine codieren sind mit einer stetig zunehmenden Anzahl menschlicher Erkrankungen verbunden, die als Envelopathien bezeichnet werden. Erstaunlicherweise betrifft die Pathologie dieser Krankheiten spezifische Gewebe und Organe, obwohl entsprechende Proteine meist ubiquitär exprimiert werden. So führen beispielsweise Defekte in Emerin, einem Protein der inneren Kernhülle, zur X-chromosomalen Emery- Dreifuss Muskeldystrophie (EDMD). Diese Krankheit ist durch Muskelschwäche oder – schwund gekennzeichnet. Defekte im Kernhüllprotein MAN1 sind dagegen mit Krankheiten verbunden, die Knochen- und Hautgewebe betreffen. Interessanterweise besitzen beide Proteine eine evolutionär hoch konservierte Domäne, die sog. LEM-Domäne. LEM-Domänen Proteine können mit der Kernlamina interagieren, ebenso mit dem sog. Barrier-to- Autointegration Factor (BAF) sowie mit zahlreichen Transkriptionsfaktoren. Dennoch ist die funktionelle Rolle der LEM-Domänen Proteine bis dato nicht vollständig aufgeklärt. In der vorliegenden Studie sollten daher die Funktionen von MAN1 und Emerin während der Frühentwicklung von Xenopus laevis untersucht werden. Vorangehende Untersuchungen zeigten, dass Mikroinjektionen von XMAN1- Antikörpern in Zwei-Zell-Stadien befruchteter Eizellen zu einem Arrest der Zellteilung in der injizierten Blastomere führten. Da dabei eine Störung der Kernhüllbildung spekuliert wurde, sollte durch Antikörper-vermittelter Inhibition von XMAN1 die Bildung von in vitro Kernen im Xenopus Eiextrakt untersucht werden. Dabei wurden Kerne beobachtet, die dekondensiertes Chromatin zeigten, bei denen jedoch eine Fusion von Membranvesikeln zu einer durchgehenden Kernhülle nicht stattgefunden hatte. Frühere Charakterisierungen von MAN1 und Emerin zeigten unterschiedliche Expressionsmuster während der Entwicklung von X. laevis. Da XMAN1 ubiquitär exprimiert und Xemerin jedoch erstmals ab Stadium 41 nachweisbar ist, war es mittels Mikroinjektion von Xemerin möglich zu zeigen, dass es in der Lage ist den Arrest der Zellteilung zu verhindern. Es wurde daher die These aufgestellt, dass MAN1 und Emerin während der Frühentwicklung von Xenopus überlappende Funktionen besitzen. Um diese These zu prüfen, wurde zunächst unter Verwendung des Proximity Ligation Assays untersucht, ob beide Proteine miteinander interagieren können. Mit Hilfe dieser Methode konnte gezeigt werden, dass Interaktionen beider Proteine innerhalb der Kernhülle lokalisieren. Die Interaktionen blieben während der Mitose bestehen und waren erst wieder zum Ende der Mitose in der Kernhülle nachweisbar. Diese Resultate deuten daher darauf hin, dass XMAN1/Xemerin-Interaktionen während der ... / Mutations in genes encoding for nuclear envelope proteins are linked to an increasing number of human diseases, called envelopathies. Interestingly, pathology of these diseases affects specific tissues and organs, even though the related proteins are expressed ubiquitous. Defects in the inner nuclear membrane protein emerin for example, are leading to X-linked Emery- Dreifuss muscular dystrophy (EDMD), characterized by muscle weakness or wasting. Conversely, defects in the nuclear envelope protein MAN1 are linked to bone and skin disorders. Both proteins share a highly conserved domain, called LEM-domain. LEM proteins are known to interact with the nuclear lamina, the so called Barrier-to-Autointegration Factor (BAF) and several transcription factors. Nevertheless, knowledge of the functional roles of LEM proteins is still unclear. For this reason, this study aimed to investigate the roles of MAN1 and emerin during early Xenopus laevis development and nuclear envelope assembly. ...

München, Techn. Univ., Diss., 2002.

Made available in DSpace on 2017-07-25T19:30:52Z (GMT). No. of bitstreams: 1 Leticia Reis.pdf: 8581445 bytes, checksum: 82298b67980249e52bd02781fd0c08df (MD5) Previous issue date: 2014-02-05 / significant world production. The true fruits of the strawberry are dry and called achenes while the fleshy and edible portion actually it is the floral receptacle developed. This set of achene and receptacle features the non-climacteric fruit pattern ripening, characterized by not show a peak respiration and ethylene production during the ripening process and also for this fruits not being able to complete the ripening after harvesting. Due to the small size of the plant, rapid propagation and production of fruit, the strawberry became the model plant in studies of non- climacteric fruit ripening, particularly with regard to the ethylene role in this process. Fruit ripening the focused studies have revealed the existence of a family of protein receptors responsible for the plant ethylene perception. In strawberry, have been identified so far three ethylene receptors genes sequences, divided into two subfamilies (FaETR1 and FaERS1 - subfamily I and FaERS2 - subfamily II). Among these genes, based on the literature, we believe that FaETR1 gene, a member of the ethylene receptors subfamily I, can develop a greater role to the other receptors in the control of perception and ethylene signal transduction. To assess the involvement of FaETR1 ethylene receptor in the strawberry ripening and toward an understanding of the relationship between ethylene and nonclimacteric fruits ripening, we seek the silencing of this particular gene. Thus, the objective of this work was the construction of a binary plasmid structure in hairpin format (hpRNA) that induces the activation of RNA interference system (RNAi) on the translation of FaETR1 gene in ethylene receptor protein. Also, knowing the importance of an appropriate regeneration an plant transformed selection method, we seek also develop a proper in vitro regeneration protocol of Camino Real and Festival strawberries cultivar, as well as to assess the sensitivity of these plants to kanamycin (antibiotic) and glufosinate ammonium (herbicide) selective agents, whose genes are present in pCAMBIA 3301 and pCAMBIA2301 cloning vectors used in this study. / O morangueiro (Fragaria x ananassa Duch.) é uma cultura de elevado valor comercial e de expressiva produção mundial. Os frutos verdadeiros do morangueiro são secos e chamados de aquênios enquanto que a porção carnosa e comestível na verdade se trata do receptáculo floral desenvolvido. Este conjunto de aquênio e receptáculo apresenta padrão de maturação de frutos não-climatéricos, caracterizado por não apresentar um pico de respiração e produção de etileno durante o processo de maturação e também, por não serem capazes de completar a maturação após a colheita. Devido o pequeno tamanho da planta, rápida propagação e produção de frutos, o morangueiro se tornou planta modelo nos estudos sobre a maturação de frutos não-climatéricos, principalmente no que se refere ao papel do etileno neste processo.Estudos voltados à maturação de frutos revelaram a existência de uma família de receptores proteicos responsável pela percepção do etileno em plantas. Em morangueiro, foram identificadas até o momento três sequencias de genes de receptores de etileno, divididos em duas subfamílias (FaETR1 e FaERS1 – Subfamília I e FaERS2 – Subfamília II). Dentre estes genes, baseados na literatura, acreditamos que o gene FaETR1, membro da subfamília I dos receptores de etileno, possa desenvolver um papel superior aos demais no controle da percepção e transdução de sinal do etileno. No intuito de avaliar o envolvimento do receptor de etileno FaETR1 na maturação do morangueiro e visando o entendimento da relação entre etileno e maturação de frutos não-climatéricos, buscamos o silenciamento deste gene em específico. Para tanto, o objetivo deste trabalho foi a construção em plasmídeo binário de uma estrutura em formato de grampo (hpRNA) que induza a ativação do sistema de RNA de interferência (RNAi) sobre a tradução do gene FaETR1 em proteína receptora de etileno. Além disso, sabendo da importância de um apropriado método de regeneração e seleção de plantas transformadas, buscamos também desenvolver protocolo de regeneração in vitro de plantas de morangueiro das cultivares Camiño Real e Festival, bem como, avaliar a sensibilidade destas plantas aos agentes seletivos canamicina (antibiótico) e glufosinato de amônia (herbicida) cujos genes estão presentes nos vetores de clonagem pCAMBIA3301 e pCAMBIA2301 utilizados neste trabalho.

Submitted by (edna.saturno@ufrpe.br) on 2016-06-29T15:35:21Z No. of bitstreams: 1 Nise de Fatima Coutinho Souto.pdf: 727551 bytes, checksum: 95ee1193333767288a6ff7225ee4caf5 (MD5) / Made available in DSpace on 2016-06-29T15:35:21Z (GMT). No. of bitstreams: 1 Nise de Fatima Coutinho Souto.pdf: 727551 bytes, checksum: 95ee1193333767288a6ff7225ee4caf5 (MD5) Previous issue date: 2008-08-11 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The in vitro culture is a tool of great importance in the production of seedlings of the fitossanitary quality. The Tapeinochilos ananassae (Hassk) K. Schum is a tropical species that has a great acceptance in the world of floriculture market and presents, in the Brazilian Northeast, fitossanitary problems in which the Rhabdovirus is highlighted. In the present work, aiming to assess the establishment and development of various explants in the in vitro culture of T. ananassae, including the study of biochemical and anatomical variables. For this, lateral buds and mature zygotic embryos were inoculated in MS medium, full or reduced to its half ionic strength, using three sources of antioxidants: ascorbic acid, activated charcoal and PVP, in the concentrations 0,25g.L-1; 3,0g.L-1 and 0,5g. L-1. The ½MS culture medium supplemented with 3,0g. L-1 activated charcoal promoted a higher rate of success in the establishment of zygotic embryos. The plants formed from the culture of embryos were propagated by nodal segments in different concentrations of BAP and GA3: ½MS; ½MS + 0,5mg.L-1 GA3; ½MS + 1,0mg.L-1 GA3; ½MS + 0,5mg.L-1 BAP; ½MS + 0,5mg.L-1 BAP + 0,5mg.L-1 GA3; ½MS + 0,5mg.L-1 BAP + 1,0mg.L-1 GA3; and showed better development when cultivated in ½MS medium free from growth regulators. The lateral buds had not shown morfogenetic development under the conditions of study, besides a large phenolic oxidation had been seen, regardless of the culture medium that had been used, and a high level of microbial contamination, especially bacterial, when buds that had come from basal branches are used. In the enzymatic analysis of lateral buds that were cultivated in vitro in a process of oxidation, had been realized that the activity of peroxidase had declined through the time, suggesting the loss of organogenetic potential, but the activity of polyphenoloxidase had tended to increase during the oxidation process of explants. / O cultivo in vitro é uma ferramenta de grande importância na produção de mudas com qualidade fitossanitária. O Tapeinochilos ananassae (Hassk) K. Schum é uma espécie tropical com grande aceitação no mercado floricultor mundial. No Nordeste brasileiro, vem enfrentando sérios problemas fitossanitários causados principalmente pelo Rhabdovirus. O presente trabalho teve como objetivo avaliar o estabelecimento e desenvolvimento de diferentes explantes no cultivo in vitro de T. ananassae, incluindo o estudo de variáveis bioquímicas e anatômicas. Para isso, gemas laterais e embriões zigóticos maduros foram inoculados em meio MS completo ou reduzido à metade da força iônica, utilizando três fontes de antioxidantes: ácido ascórbico, carvão ativado e PVP, nas concentrações 0,25g L-1; 3,0g L-1 e 0,5g L-1. O meio de cultivo ½MS suplementado com 3,0g. L-1 de carvão ativado promoveu um maior índice de sucesso no estabelecimento de embriões zigóticos. As plantas formadas a partir da cultura de embriões foram propagadas por segmentos nodais em diferentes concentrações de BAP e GA3: ½MS; ½MS+0,5mg.L-1 GA3; ½MS+1,0mg. L-1 GA3; ½MS+0,5mg.L-1 BAP; ½MS+0,5mg.L-1 BAP+0,5mg.L-1 GA3; ½MS+0,5mg.L-1 BAP+1,0mg.L-1 GA3. O meio de cultivo constituído de ½ dos sais MS e isento de reguladores de crescimento mostrou-se mais adequado para o cultivo de segmentos nodais de T. ananassae. As gemas laterais não apresentaram desenvolvimento morfogênico nas condições de estudo deste trabalho. Além disso, foi observado grande oxidação fenólica, independentemente do meio de cultivo utilizado, e elevado índice de contaminação microbiana, sobretudo bacteriana, quando do uso de gemas advindas de ramos basais. Nas análises enzimáticas das gemas laterais cultivadas in vitro em processo de oxidação, percebeu-se que a atividade da peroxidase decaiu ao longo do tempo, sugerindo a perda do potencial organogenético; já a atividade da polifenoloxidase tendeu a aumentar ao longo do processo de oxidação dos explantes.

Organ formation is an important step during development of an organism that combines different scales from the molecular to the tissue level. Many organogenesis phenomena involve epithelial morphogenesis, where sheets of cells undergo rearrangements to form complex architectures – organ precursors, which subsequently develop into mature organs. Timely development of the characteristic architectures of the organ precursors is crucial for successful organogenesis and is determined by the choice of epithelial rearrangements that organise the constituent cells in space and time. However, for many organogenesis events the cellular dynamics underlying such epithelial rearrangements remain elusive. In the work presented here, I investigated the morphogenesis of the hemispherical retinal neuroepithelium (RNE), that serves as an organ precursor of the neural retina. Formation of RNE is an important event in vertebrates that shapes the optic cup and sets the stage for subsequent eye development. I investigated RNE morphogenesis in the developing zebrafish embryo by visualising and investigating the cellular dynamics of the process in vivo. My findings show that the zebrafish RNE is shaped by the combined action of two different epithelial rearrangements – basal shrinkage of the neuroepithelial cells and involution of cells at the rim of the developing optic cup. The basal shrinkage of the neuroepithelial cells bends the neuroepithelial sheet and starts the process of invagination. However, my results show that the major player in RNE morphogenesis is rim involution. Rim involution translocates prospective RNE cells to their designated location in the invaginating layer and contributes to RNE invagination. My work unravelled the so far unknown mechanism of rim involution. I show that the rim cells involute by collective epithelial migration using directed membrane protrusions and dynamic cell-matrix contacts. If rim migration is perturbed, the prospective RNE cells cannot reach the invaginating layer. As a result, these migration-defective cells attain the RNE fate at an ectopic location and disrupt the tissue architecture. Therefore, rim migration coordinates the cellular location with the timing of RNE fate determination and orchestrates RNE morphogenesis in space and time. Overall, my work highlights how morphogenetic processes shape the organ precursor architecture and ensure timely organ formation. These findings provide important insights not only for eye development but also for epithelial morphogenesis and organogenesis in many other systems. / Für die Entwicklung eines Organismus ist die Bildung von Organen (Organogenese) von zentraler Bedeutung. Organogenese umfasst Prozesse auf allen Ebenen der Längenskala: von der molekularen Ebene, der Gewebeebene, bis hin zur Ebene des ganzen Organismus. Viele Phänomene der Organogenese beinhalten dabei Veränderungen von Epithelien, bei der sich Schichten von Zellen zu komplexen Strukturen - Organvorläufern - umwandeln. Diese entwickeln sich später zu vollständigen Organen. Die rechtzeitige Entwicklung der charakteristischen Architektur der Organvorläufer ist entscheidend für eine erfolgreiche Organogenese und wird durch die Wahl der epithelialen Umwandlungsprozessen bestimmt, welche die Zellen in Raum und Zeit koordinieren müssen. Für viele dieser Prozesse ist jedoch genau diese zugrundeliegende Zelldynamik unklar. In der hier vorgestellten Arbeit untersuchte ich die Bildung des hemisphärischen retinalen Neuropepithels (RNE). Das RNE ist der Organvorläufer der neuralen Retina, weshalb dessen korrekte Bildung die Voraussetzung für die korrekte Entwicklung der Augen ist. Ich untersuchte die RNE-Morphogenese in sich entwickelnden Zebrafisch-Embryos durch Visualisierung und Untersuchung der zellulären Dynamik der beteiligten Prozesse in vivo. Meine Ergebnisse zeigen, dass das RNE in Zebrafischen durch die kombinierte Umwandlung von zwei verschiedenen Epithelien geformt wird. Zum einen findet eine Verkleinerung des basalen Prozesses der neuroepithelialen Zellen statt, zum anderen die Involution von Randzellen. Die basale Verkleinerung der neuroepithelialen Zellen verbiegt die neuroepitheliale Schicht und führt zur Einstülpung des RNE. Meine Ergebnisse zeigten allerdings, dass Involution von Randzellen noch bedeutsamer für die RNE-Morphogenese ist. Die involution von Randzellen transportiert potenzielle RNE-Zellen in das Neuroepithel und trägt zur RNE-Einstülpung bei. Die Bedeutung meiner Arbeit liegt darin, den bisher unbekannten Mechanismus der Randzell-Involution entdeckt zu haben. Ich zeigte, dass die Randzellen sich aktiv durch kollektive epitheliale Migration bewegen indem sie gerichtete Membranforsätze und dynamische Zell zu Matrix Kontakte etablieren. Wird die Migration der Randzellen inhibiert, so führt dies dazu, dass diese Zellen die eingestülpte RNE Schicht nicht erreichen. Sie landen dann an den falschen Positionen, wo sie die Gewerbearchitektur stören können. Daher koordiniert die Randzellmigration die Position der Zellen und orchestriert die RNE-Morphogenese in Raum und Zeit. Insgesamt zeigt meine Arbeit, wie morphogenetische Prozesse die Organvorläuferarchitektur prägen und eine rechtzeitige Organbildung sicherstellen. Diese Erkenntnisse sind sowohl für das Verständnis der Augenentwicklung, als auch für das der epithelialen Morphogenese und Organogenese in anderen Systemen von großer Bedeutung.

Organ formation is an important step during development of an organism that combines different scales from the molecular to the tissue level. Many organogenesis phenomena involve epithelial morphogenesis, where sheets of cells undergo rearrangements to form complex architectures – organ precursors, which subsequently develop into mature organs. Timely development of the characteristic architectures of the organ precursors is crucial for successful organogenesis and is determined by the choice of epithelial rearrangements that organise the constituent cells in space and time. However, for many organogenesis events the cellular dynamics underlying such epithelial rearrangements remain elusive. In the work presented here, I investigated the morphogenesis of the hemispherical retinal neuroepithelium (RNE), that serves as an organ precursor of the neural retina. Formation of RNE is an important event in vertebrates that shapes the optic cup and sets the stage for subsequent eye development. I investigated RNE morphogenesis in the developing zebrafish embryo by visualising and investigating the cellular dynamics of the process in vivo. My findings show that the zebrafish RNE is shaped by the combined action of two different epithelial rearrangements – basal shrinkage of the neuroepithelial cells and involution of cells at the rim of the developing optic cup. The basal shrinkage of the neuroepithelial cells bends the neuroepithelial sheet and starts the process of invagination. However, my results show that the major player in RNE morphogenesis is rim involution. Rim involution translocates prospective RNE cells to their designated location in the invaginating layer and contributes to RNE invagination. My work unravelled the so far unknown mechanism of rim involution. I show that the rim cells involute by collective epithelial migration using directed membrane protrusions and dynamic cell-matrix contacts. If rim migration is perturbed, the prospective RNE cells cannot reach the invaginating layer. As a result, these migration-defective cells attain the RNE fate at an ectopic location and disrupt the tissue architecture. Therefore, rim migration coordinates the cellular location with the timing of RNE fate determination and orchestrates RNE morphogenesis in space and time. Overall, my work highlights how morphogenetic processes shape the organ precursor architecture and ensure timely organ formation. These findings provide important insights not only for eye development but also for epithelial morphogenesis and organogenesis in many other systems. / Für die Entwicklung eines Organismus ist die Bildung von Organen (Organogenese) von zentraler Bedeutung. Organogenese umfasst Prozesse auf allen Ebenen der Längenskala: von der molekularen Ebene, der Gewebeebene, bis hin zur Ebene des ganzen Organismus. Viele Phänomene der Organogenese beinhalten dabei Veränderungen von Epithelien, bei der sich Schichten von Zellen zu komplexen Strukturen - Organvorläufern - umwandeln. Diese entwickeln sich später zu vollständigen Organen. Die rechtzeitige Entwicklung der charakteristischen Architektur der Organvorläufer ist entscheidend für eine erfolgreiche Organogenese und wird durch die Wahl der epithelialen Umwandlungsprozessen bestimmt, welche die Zellen in Raum und Zeit koordinieren müssen. Für viele dieser Prozesse ist jedoch genau diese zugrundeliegende Zelldynamik unklar. In der hier vorgestellten Arbeit untersuchte ich die Bildung des hemisphärischen retinalen Neuropepithels (RNE). Das RNE ist der Organvorläufer der neuralen Retina, weshalb dessen korrekte Bildung die Voraussetzung für die korrekte Entwicklung der Augen ist. Ich untersuchte die RNE-Morphogenese in sich entwickelnden Zebrafisch-Embryos durch Visualisierung und Untersuchung der zellulären Dynamik der beteiligten Prozesse in vivo. Meine Ergebnisse zeigen, dass das RNE in Zebrafischen durch die kombinierte Umwandlung von zwei verschiedenen Epithelien geformt wird. Zum einen findet eine Verkleinerung des basalen Prozesses der neuroepithelialen Zellen statt, zum anderen die Involution von Randzellen. Die basale Verkleinerung der neuroepithelialen Zellen verbiegt die neuroepitheliale Schicht und führt zur Einstülpung des RNE. Meine Ergebnisse zeigten allerdings, dass Involution von Randzellen noch bedeutsamer für die RNE-Morphogenese ist. Die involution von Randzellen transportiert potenzielle RNE-Zellen in das Neuroepithel und trägt zur RNE-Einstülpung bei. Die Bedeutung meiner Arbeit liegt darin, den bisher unbekannten Mechanismus der Randzell-Involution entdeckt zu haben. Ich zeigte, dass die Randzellen sich aktiv durch kollektive epitheliale Migration bewegen indem sie gerichtete Membranforsätze und dynamische Zell zu Matrix Kontakte etablieren. Wird die Migration der Randzellen inhibiert, so führt dies dazu, dass diese Zellen die eingestülpte RNE Schicht nicht erreichen. Sie landen dann an den falschen Positionen, wo sie die Gewerbearchitektur stören können. Daher koordiniert die Randzellmigration die Position der Zellen und orchestriert die RNE-Morphogenese in Raum und Zeit. Insgesamt zeigt meine Arbeit, wie morphogenetische Prozesse die Organvorläuferarchitektur prägen und eine rechtzeitige Organbildung sicherstellen. Diese Erkenntnisse sind sowohl für das Verständnis der Augenentwicklung, als auch für das der epithelialen Morphogenese und Organogenese in anderen Systemen von großer Bedeutung.

No description available.

A transformação genética possibilita a introdução de genes de interesse agronômico no genoma das plantas e pode ser empregada na tentativa de obter plantas resistentes a doenças. No entanto, para se obter uma planta transgênica é necessário primeiramente estabelecer um procotolo eficiente de regeneração de plantas in vitro. Assim, o objetivo desse trabalho foi estudar a organogênese in vitro e a transformação genética de limão Volkameriano e laranja azeda com um fragmento do gene da capa protéica do CTV. Para a organogênese in vitro utilizou-se, como explante, segmento internodal, obtido de planta cultivada em casa-de-vegetação, segmento de epicótilo, coletado de plântula cultivada in vitro e segmento de cotilédone associado ao hipocótilo obtido de semente introduzida in vitro. Esses explantes foram mantidos em meio de cultura EME suplementado com 6-benzilaminopurina (BAP 0,0; 0,5; 1,0; 1,5; 2,0 mg L- 1), sendo incubados sob fotoperíodo de 16 h de luz ou em condições de escuro por 30 dias e então transferidos para fotoperíodo de 16 h de luz. A avaliação foi realizada após 45 dias de cultivo, determinando-se o número de explantes responsivos e o número de gemas por explante. A caracterização anatômica do processo de regeneração foi realizada por meio de cortes histológicos. Pela análise dos dados foi possível verificar que a organogênese in vitro ocorreu a partir dos três tipos de explantes testados, sendo que, nas duas espécies em estudo, os melhores resultados foram obtidos com o cultivo de segmento de cotilédone associado ao hipocótilo. As concentrações de BAP que estimularam as melhores taxas de regeneração foram de 1,0 e 1,5 mg L-1, para limão Volkameriano, e 0,5 e 1,0 mg L-1 para laranja azeda. A incubação dos explantes em ausência de luz favoreceu a regeneração in vitro. Pela análise histológica foi possível observar que o processo de regeneração, a partir dos três tipos de explantes testados, ocorreu por meio de organogênese indireta. O protocolo de desenvolvimento estabelecido durante os experimentos de organogênese in vitro foi utilizado para a transformação genética dessas espécies via Agrobacterium, contendo o plasmídeo pCAMBIA 2201, com um fragmento do gene da capa protéida do CTV, em uma construção gênica do tipo hairpin. As gemas de limão Volkameriano e laranja azeda identificadas como transgênicas pelo teste histoquímico GUS foram enxertadas in vitro em citrange Carrizo. A confirmação da transformação genética foi realizada pela análise de PCR, a qual mostrou a amplificação de um fragmento de 671 pb, correspondente a parte do gene amplificada. / Genetic transformation permits the introduction of agronomically important genes in plant genome and can be utilized in order to produce disease resistant plants. However for the recovery of transgenic plants is required to establish an efficient in vitro plant regeneration protocol. In this work the aim was to study an in vitro organogenesis and the genetic transformation of Volkamer lemon and sour orange with a sequence of the CTV coat protein gene. For in vitro organogenesis explant internodal segments collected from plants cultivated in greenhouse, epicotyl segments obtained from in vitro cultivated seedlings and cotyledon fragment with hypocotyl attached obtained from in vitro germinated seed were used as explant. These explants were cultured in EME medium supplemented with benzilaminopurine (BAP 0,0; 0,5; 1,0; 1,5; 2,0 mg L-1). Cultures were maintained under a 16 h photoperiod or in the dark for 10 d and then transferred to a 16 h photoperiod. The evaluation was performed 45 d after the incubation determining the number of responsive explant and the number of buds per explant. The anatomical characterization of in vitro regeneration process was carried out through histological analyses. The in vitro organogenesis occurred in the three types of explant tested, however cotyledon fragment with hypocotyl attached showed higher morphogenetic potential in both species. The best responses of regeneration were obtained when the medium was supplementation with 1,0 e 1,5 mg L-1 BAP for the Volkamer lemon and 0,5 e 1,0 mg L-1 BAP for the sour orange. The incubation in darkness favored the in vitro regeneration. The histological analyses showed that the regeneration process occurred through indirect organogenesis in the three types of explants tested. The developed protocol was use for genetic transformation of Volkamer lemon and sour orange with Agrobacterium, containing pCAMBIA 2201 plasmid with a sequence of CTV coat protein gene, in a hairpin construction. Volkamer lemon and sour orange shoots identified as transgenic by histochemical test GUS were micrografted into Carrizo citrange. PCR analysis were performed after micrografted showing the presence of the 671 pb fragment of the transgene.

No description available.

A transformação genética possibilita a introdução de genes de interesse agronômico no genoma das plantas e pode ser empregada na tentativa de obter plantas resistentes a doenças. No entanto, para se obter uma planta transgênica é necessário primeiramente estabelecer um procotolo eficiente de regeneração de plantas in vitro. Assim, o objetivo desse trabalho foi estudar a organogênese in vitro e a transformação genética de limão Volkameriano e laranja azeda com um fragmento do gene da capa protéica do CTV. Para a organogênese in vitro utilizou-se, como explante, segmento internodal, obtido de planta cultivada em casa-de-vegetação, segmento de epicótilo, coletado de plântula cultivada in vitro e segmento de cotilédone associado ao hipocótilo obtido de semente introduzida in vitro. Esses explantes foram mantidos em meio de cultura EME suplementado com 6-benzilaminopurina (BAP 0,0; 0,5; 1,0; 1,5; 2,0 mg L- 1), sendo incubados sob fotoperíodo de 16 h de luz ou em condições de escuro por 30 dias e então transferidos para fotoperíodo de 16 h de luz. A avaliação foi realizada após 45 dias de cultivo, determinando-se o número de explantes responsivos e o número de gemas por explante. A caracterização anatômica do processo de regeneração foi realizada por meio de cortes histológicos. Pela análise dos dados foi possível verificar que a organogênese in vitro ocorreu a partir dos três tipos de explantes testados, sendo que, nas duas espécies em estudo, os melhores resultados foram obtidos com o cultivo de segmento de cotilédone associado ao hipocótilo. As concentrações de BAP que estimularam as melhores taxas de regeneração foram de 1,0 e 1,5 mg L-1, para limão Volkameriano, e 0,5 e 1,0 mg L-1 para laranja azeda. A incubação dos explantes em ausência de luz favoreceu a regeneração in vitro. Pela análise histológica foi possível observar que o processo de regeneração, a partir dos três tipos de explantes testados, ocorreu por meio de organogênese indireta. O protocolo de desenvolvimento estabelecido durante os experimentos de organogênese in vitro foi utilizado para a transformação genética dessas espécies via Agrobacterium, contendo o plasmídeo pCAMBIA 2201, com um fragmento do gene da capa protéida do CTV, em uma construção gênica do tipo hairpin. As gemas de limão Volkameriano e laranja azeda identificadas como transgênicas pelo teste histoquímico GUS foram enxertadas in vitro em citrange Carrizo. A confirmação da transformação genética foi realizada pela análise de PCR, a qual mostrou a amplificação de um fragmento de 671 pb, correspondente a parte do gene amplificada. / Genetic transformation permits the introduction of agronomically important genes in plant genome and can be utilized in order to produce disease resistant plants. However for the recovery of transgenic plants is required to establish an efficient in vitro plant regeneration protocol. In this work the aim was to study an in vitro organogenesis and the genetic transformation of Volkamer lemon and sour orange with a sequence of the CTV coat protein gene. For in vitro organogenesis explant internodal segments collected from plants cultivated in greenhouse, epicotyl segments obtained from in vitro cultivated seedlings and cotyledon fragment with hypocotyl attached obtained from in vitro germinated seed were used as explant. These explants were cultured in EME medium supplemented with benzilaminopurine (BAP 0,0; 0,5; 1,0; 1,5; 2,0 mg L-1). Cultures were maintained under a 16 h photoperiod or in the dark for 10 d and then transferred to a 16 h photoperiod. The evaluation was performed 45 d after the incubation determining the number of responsive explant and the number of buds per explant. The anatomical characterization of in vitro regeneration process was carried out through histological analyses. The in vitro organogenesis occurred in the three types of explant tested, however cotyledon fragment with hypocotyl attached showed higher morphogenetic potential in both species. The best responses of regeneration were obtained when the medium was supplementation with 1,0 e 1,5 mg L-1 BAP for the Volkamer lemon and 0,5 e 1,0 mg L-1 BAP for the sour orange. The incubation in darkness favored the in vitro regeneration. The histological analyses showed that the regeneration process occurred through indirect organogenesis in the three types of explants tested. The developed protocol was use for genetic transformation of Volkamer lemon and sour orange with Agrobacterium, containing pCAMBIA 2201 plasmid with a sequence of CTV coat protein gene, in a hairpin construction. Volkamer lemon and sour orange shoots identified as transgenic by histochemical test GUS were micrografted into Carrizo citrange. PCR analysis were performed after micrografted showing the presence of the 671 pb fragment of the transgene.