Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2008. / Härtill 4 uppsatser.

Thèse (M.Sc.)--Université Laval, 2003. / Bibliogr.: f. 81-94. Publié aussi en version électronique.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21821号 / 農博第2334号 / 新制||農||1067(附属図書館) / 学位論文||H31||N5193(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 小川 順, 教授 植田 充美, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Anther dehiscence is an important process taking place at the end of the plant life cycle. This process consists of various follow-up steps which result in anther opening and pollen grains exposure. Good timing of the anther dehiscence must be synchronized with pollen grains maturation and flower opening. Atmospheric precipitation is a high-risk factor for the anther dehiscence. Male fitness of plants can be reduced when anthers open during poor weather conditions. The aim of this study was to investigate the effect of atmospheric precipitation, rain and dew, on Arabidopsis arenosa anther dehiscence. We observed that rain and dew led to a postponed final stage of the anther dehiscence. This caused delayed pollen release. The effect of aqueous and nonaqueous environment on the anther dehiscence was also tested. Experiments with transformation of A. arenosa using Agrobacterium tumefaciens were performed. Key words: anther dehiscence, flower opening, rain, dew, Arabidopsis arenosa, Agrobacterium tumefaciens, transformation

Septoria musiva (téléomorphe Mycosphaerella populorum) est un Ascomycète responsable de chancres sur tronc et jeunes branches en peupleraies et de taches foliaires en peupleraies et forêts. S. populicola (M. populicola) est responsable de taches foliaires sur peupliers. L’objectif de mon projet consiste à mettre en place un système de transformation à l’aide d’Agrobacterium tumefaciens pour ces deux champignons. A. tumefaciens est la bactérie causant la galle du collet, mais qui est utilisée couramment lors de transformation de végétaux et de champignons. Une première expérience a permis d’obtenir des transformants pour S. musiva avec le plasmide pPT1, mais aucun transformant n’a été obtenu pour S. populicola. Par la suite, un autre plasmide a été utilisé : pPL1. Cependant aucune souche transformée n’a pu être obtenue pour S. musiva, et S. populicola, malgré la vérification de différents paramètres. L’absence de transformant chez S. populicola peut être attribuée à un nombre insuffisant de conidies, tandis que pour S. musiva, un nombre insuffisant de bactéries ou un protocole de transformation non optimal pourrait expliquer les échecs successifs. / Seporia musiva (Mycosphaerella populorum) is an Ascomycota which causes canker and leafspot on hybrid poplar. S. populicola (M. populicola) only causes leafspot. The aim of my project was to transform these two fungi with Agrobacterium tumefaciens, a bacterium which causes Crown gall, and is commonly used to transform plant and fungi in laboratory. In a first experiment, transformation using plasmid pPT1 were successful for S. musiva, but not for S. populicola. However, no transformants were obtained from either S. musiva and S. populicola in subsequent experiments in which plasmid pPL1 was used and different parameters were tested. A possible explanation for the inhability to obtain transformants from S. populicola is the low number of conidia available for transformation experiments. In the case of S. musiva, low number of bacterial cells and non optimal protocols might explain negative results observed.

Embora desfrute de inegável importância econômica, os citros estão sujeitos a muitos problemas sanitários sendo alguns, limitantes para o cultivo como é o caso das doenças causadas por vírus. A morte súbita dos citros é uma doença relacionada à combinação copa/ porta-enxerto e manifesta sintomas na região da enxertia sobre porta-enxertos intolerantes. Embora sua etiologia não tenha sido determinada, há indicações que a causa da MSC esteja relacionada a uma estirpe do vírus da tristeza dos citros (CTV), a um vírus do gênero Marafivirus, ou a uma associação entre eles. Uma vez que a transformação genética têm sido considerada como uma ferramenta auxiliar a programas de melhoramento de citros, o objetivo deste trabalho foi obter plantas transgênicas de limão \'Cravo\' e laranja \'Valência\' contendo o gene da replicase do Marafivirus e estudar a regeneração e obtenção de plantas in vitro de laranja azeda, via organogênese, visando futuros trabalhos de transformação genética. Experimentos para indução da organogênese in vitro foram realizados avaliando-se citocininas (BAP, TDZ e CIN), em diferentes concentrações, isoladamente ou em combinação com ANA, condições de luminosidade (fotoperíodo de 16 h e escuro por 30 dias), meios de cultivo e explantes (provenientes de plantas germinadas in vitro e de plantas mantidas em estufa). Além disso, avaliou-se o enraizamento dos brotos regenerados. Para a transformação genética, explantes de limão \'Cravo\' e laranja \'Valência\' foram inoculados e co-cultivados com a estirpe EHA 105 de Agrobacterium tumefaciens contendo o gene da replicase do Marafivirus (em seqüência sense e antisense interligadas por um íntron). A construção gênica foi elaborada a partir do plasmídeo pCAMBIA 2201, dirigidas pelo promotor 35S e terminador NOS, contendo ainda o gene de seleção nptII. A transformação foi confirmada por análises de PCR e \'Southern blot\'. A transcrição do gene foi avaliada por RT-PCR e \'northern blot\'. A adição de BAP, combinada ou não com ANA, e em combinações com CIN ao meio de cultivo, assegurou maior formação de gemas adventícias em segmentos de epicótilo de laranja azeda. Entretanto, TDZ não se mostrou favorável a essa resposta, que também é afetada pela ausência de luz. Os explantes provenientes do cultivo in vitro mostraram-se mais favoráveis à resposta organogênica. O enraizamento das brotações de laranja azeda regeneradas foi obtido no meio MT com metade da concentração de sais, sem ou com auxinas. Foi possível obter plantas transgênicas de limão \'Cravo\' e de laranja \'Valência\' contendo o gene da replicase do Marafivirus utilizando-se segmentos internodais como explantes. A análise de \'Southern blot\' confirmou a integração de um a quatro eventos de inserção do transgene no genoma das plantas. A transcrição do gene da replicase do Marafivirus e do gene nptII foi observada por RT-PCR. / In spite of great economic importance, the citrus industry is affected by many phytopathological problems some, limiting its cultivation such as virus-caused diseases. The citrus sudden death disease is related to scion/rootstock combinations and manifests symptoms in the grafting area of intolerant rootstocks. Although its etiology has not been determined, there are indications that the cause of MSC might be related to a strain of the Citrus tristeza virus (CTV), to a virus of the Marafivirus group, or to an association of both viruses. Since the genetic transformation has been considered as an auxiliary tool to programs of citrus improvement, the objectives of this work were to obtain transgenic plants of the Rangpur lime and Valencia sweet orange containing the Marafivirus replicase gene and study the in vitro regeneration of sour orange plants through organogenesis, aiming for future work in genetic transformation. Experiments for induction of in vitro organogenesis were carried out evaluating citocinins (BAP, TDZ and KIN), in different concentrations, separately or in combination with NAA, lighting conditions (photoperiod of 16 hours and darkness for 30 days), cultivation media and explants (coming from in vitro germinated plants and from green house cultivated plants). Besides this, rooting of the regenerated shoots was evaluated. For the genetic transformation, Rangpur lime and Valencia sweet orange explants were inoculated and co-cultivated with the EHA-105 Agrobacterium tumefaciens strain containing the Marafivirus replicase gene (in sense and antisense sequence linked by an intron). The genetic construct used derived from the pCAMBIA 2201 plasmid, driven by the 35S promoter and NOS terminator, containing the selection nptII gene. The genetic transformation was confirmed by PCR and Southern blot analysis. The gene transcription was evaluated by RT-PCR and northern blot. The addition of BAP to the culture medium, combined or not with NAA, and in combinations with KIN, assured a greater formation of adventitious buds in sour orange epicotyl segments. However, TDZ was not favorable to this response, that is also affected by the absence of light. Explants coming from in vitro cultivation were more favorable to the organogenic response. Rooting of sour orange regenerated shoots was obtained in MT medium with half the salt concentration, with or without auxin. It was possible to obtain transgenic Rangpur lime and Valencia sweet orange plants containing the Marafivirus replicase gene using internodal segments as explants. The Southern blot analysis confirmed the integration of one to four copies of the transgene in the plant genome. The transcription of the Marafivirus replicase gene and the nptII gene was observed by RT-PCR.

Rift Valley Fever (RVF) is an emerging infectious disease found in both livestock and humans. RVF is associated with high abortion and mortality rates in livestock and can be fatal in humans. As such, RVF is economically and socially significant to affected smallholder and subsistence farmers, those infected, and national livestock industries. However, Rift Valley Fever virus (RVFV) vaccines are not commercially available outside of endemic areas or for humans, and current vaccines are limited in their safety and efficacy. A plant-based, viral nanoparticle vaccine offers a more affordable alternative to conventional vaccines that is safe, rapidly producible, and easily scalable, better meeting the needs of impacted communities. This project focuses on assessing the potential of using a Nicotiana benthamiana plant expression system to generate recombinant tobacco mosaic virus (TMV) nanoparticles displaying RVFV glycoprotein epitopes. Eight TMV-RVFV glycoprotein constructs were designed. Five TMV-RVFV constructs were successfully cloned, and four recombinant TMV constructs were successfully expressed in planta. The antigenicity of these constructs was examined for their possible use in RVFV vaccine development.

Potato is one of the main targets for genetic improvement by gene transfer. The aim of this study was to compare the efficacy of genetic transformation of potato, cultivar Bintje, using two methods: Agrobacterium tumefaciens mediated transformation and microprojectile bombardment. The same plasmid p35SGUSint, which cosists of 35S CaMV promoter, gus and nptII genes, was used for both transformations of internodal potato explants. Kamamycin selection, transient and stable expressions of {$\beta$}-glucuronidase and PCR amplification of gus and nptII transgenes were used for transgenic plant selection, identification and analysis.

Embora desfrute de inegável importância econômica, os citros estão sujeitos a muitos problemas sanitários sendo alguns, limitantes para o cultivo como é o caso das doenças causadas por vírus. A morte súbita dos citros é uma doença relacionada à combinação copa/ porta-enxerto e manifesta sintomas na região da enxertia sobre porta-enxertos intolerantes. Embora sua etiologia não tenha sido determinada, há indicações que a causa da MSC esteja relacionada a uma estirpe do vírus da tristeza dos citros (CTV), a um vírus do gênero Marafivirus, ou a uma associação entre eles. Uma vez que a transformação genética têm sido considerada como uma ferramenta auxiliar a programas de melhoramento de citros, o objetivo deste trabalho foi obter plantas transgênicas de limão \'Cravo\' e laranja \'Valência\' contendo o gene da replicase do Marafivirus e estudar a regeneração e obtenção de plantas in vitro de laranja azeda, via organogênese, visando futuros trabalhos de transformação genética. Experimentos para indução da organogênese in vitro foram realizados avaliando-se citocininas (BAP, TDZ e CIN), em diferentes concentrações, isoladamente ou em combinação com ANA, condições de luminosidade (fotoperíodo de 16 h e escuro por 30 dias), meios de cultivo e explantes (provenientes de plantas germinadas in vitro e de plantas mantidas em estufa). Além disso, avaliou-se o enraizamento dos brotos regenerados. Para a transformação genética, explantes de limão \'Cravo\' e laranja \'Valência\' foram inoculados e co-cultivados com a estirpe EHA 105 de Agrobacterium tumefaciens contendo o gene da replicase do Marafivirus (em seqüência sense e antisense interligadas por um íntron). A construção gênica foi elaborada a partir do plasmídeo pCAMBIA 2201, dirigidas pelo promotor 35S e terminador NOS, contendo ainda o gene de seleção nptII. A transformação foi confirmada por análises de PCR e \'Southern blot\'. A transcrição do gene foi avaliada por RT-PCR e \'northern blot\'. A adição de BAP, combinada ou não com ANA, e em combinações com CIN ao meio de cultivo, assegurou maior formação de gemas adventícias em segmentos de epicótilo de laranja azeda. Entretanto, TDZ não se mostrou favorável a essa resposta, que também é afetada pela ausência de luz. Os explantes provenientes do cultivo in vitro mostraram-se mais favoráveis à resposta organogênica. O enraizamento das brotações de laranja azeda regeneradas foi obtido no meio MT com metade da concentração de sais, sem ou com auxinas. Foi possível obter plantas transgênicas de limão \'Cravo\' e de laranja \'Valência\' contendo o gene da replicase do Marafivirus utilizando-se segmentos internodais como explantes. A análise de \'Southern blot\' confirmou a integração de um a quatro eventos de inserção do transgene no genoma das plantas. A transcrição do gene da replicase do Marafivirus e do gene nptII foi observada por RT-PCR. / In spite of great economic importance, the citrus industry is affected by many phytopathological problems some, limiting its cultivation such as virus-caused diseases. The citrus sudden death disease is related to scion/rootstock combinations and manifests symptoms in the grafting area of intolerant rootstocks. Although its etiology has not been determined, there are indications that the cause of MSC might be related to a strain of the Citrus tristeza virus (CTV), to a virus of the Marafivirus group, or to an association of both viruses. Since the genetic transformation has been considered as an auxiliary tool to programs of citrus improvement, the objectives of this work were to obtain transgenic plants of the Rangpur lime and Valencia sweet orange containing the Marafivirus replicase gene and study the in vitro regeneration of sour orange plants through organogenesis, aiming for future work in genetic transformation. Experiments for induction of in vitro organogenesis were carried out evaluating citocinins (BAP, TDZ and KIN), in different concentrations, separately or in combination with NAA, lighting conditions (photoperiod of 16 hours and darkness for 30 days), cultivation media and explants (coming from in vitro germinated plants and from green house cultivated plants). Besides this, rooting of the regenerated shoots was evaluated. For the genetic transformation, Rangpur lime and Valencia sweet orange explants were inoculated and co-cultivated with the EHA-105 Agrobacterium tumefaciens strain containing the Marafivirus replicase gene (in sense and antisense sequence linked by an intron). The genetic construct used derived from the pCAMBIA 2201 plasmid, driven by the 35S promoter and NOS terminator, containing the selection nptII gene. The genetic transformation was confirmed by PCR and Southern blot analysis. The gene transcription was evaluated by RT-PCR and northern blot. The addition of BAP to the culture medium, combined or not with NAA, and in combinations with KIN, assured a greater formation of adventitious buds in sour orange epicotyl segments. However, TDZ was not favorable to this response, that is also affected by the absence of light. Explants coming from in vitro cultivation were more favorable to the organogenic response. Rooting of sour orange regenerated shoots was obtained in MT medium with half the salt concentration, with or without auxin. It was possible to obtain transgenic Rangpur lime and Valencia sweet orange plants containing the Marafivirus replicase gene using internodal segments as explants. The Southern blot analysis confirmed the integration of one to four copies of the transgene in the plant genome. The transcription of the Marafivirus replicase gene and the nptII gene was observed by RT-PCR.

The subject of my thesis was to genetically modify a potato for increased resistance against its pathogens and pests. In developing a resistant plant, it is quite common to use the same type of molecules that plants use themselves in their defense reactions. In this work I used the gene SPI-2 originating from a honeycomb moth (Galleria mellonella). The protein SPI-2 is a member of serine protease inhibitors. Since the previous attempts of the team to detect the protein in transformed plants haven't been successful, the basic form of the gene was modified by adding a Kozak sequence near the start codon, which should have increased the translation initiation and hence increase the level of the protein. Two constructs were prepared for the transformation: SPI-2-T a SPI-2-Y. They differ by one amino acid, which slightly changes their inhibitory activity. First, the construct SPI-2-T was used for a transient transformation of tobacco Nicotiana benthamiana by agroinfiltration of its leaves. Then both constructs were used for a stable transformation of Solanum tuberosum cv. Desireé. The detection of the protein has not been successful, although the inserted gene was transcribed and his sequence was verified by sequencing. It is therefore most likely that the protein has a low stability in the cytoplasm....