Brachiaria é um gênero de forrageiras da família Poaceae que apresenta plantas que se reproduzem por via sexual e assexualmente por apomixia,reprodução por sementes. A apomixia desperta interesse biológico e biotecnológico, pela perspectiva de levar esta característica de clonagem de plantas via sementes, a outras espécies. As cultivares plantadas de B. brizantha cv. Marandu e B. decumbens cv. Basilisk são poliplóides e reproduzem-se por apomixia, enquanto as plantas sexuais são diplóides,o que inviabiliza os cruzamentos, dificultando sobremaneira o melhoramento. A transformação genética é uma estratégia que vem sendo incorporada ao melhoramento genético. A natureza apomítica destasplantas pode permitir a clonagem e estabilidade das plantas transgênicas. Para transformação genética é necessário o desenvolvimento de um método eficiente de regeneraçãoin vitro. B. brizantha é considerada recalcitrante a cultura de tecidos, e métodos eficientes associados com os sistemas de transformação genética ainda não foram descritos na literatura. O arroz (Oryza sativa) é uma Poaceae modelo para estudos de genética inversa, no entanto, cultivares tropicais do grupo japônica são recalcitrantes a transformação genética, como é o caso da cultivar Primavera. O método direto de transformação genética mais amplamente utilizado é a biobalística, e vem sendo aplicado em espécies de monocotiledôneas, uma vez que essas não são hospedeiros naturais de Agrobacterium tumefaciens. No entanto, vários fatores tem sido testados no sentido de favorecer a interação e transferência de genes durante a cocultura para obtenção de transgênicos em diversas espécies de monocotiledôneas. Os objetivos deste estudo foram obter sistemas de regeneração in vitro deB. Brizanthae de arroz cultivar Primavera para transformação genética destas espécies. Em B. brachiaria foram obtidos sistema de micropropagação, organogênese, calos embriogênicos, unidades embriogênicas e suspensões celulares, e para a cultivar Primavera de arroz foram obtidas unidades embriogênicas, que foram caracterizadas morfo-anatomicamente e quanto as condições de indução, multiplicação e regeneração in vitro. Métodos de expressão transiente e estável de genes marcadores foram estabelecidos para B brizantha via biobalística e Agrobacterium tumefaciens. A natureza da transgenia foi confirmada por métodos histoquímico e molecular como PCR e Southern blot. Os sistemas de regeneração e transformação obtidos mostraram-se eficientes e irão contribuir para os estudos da apomixia e introdução de genes de interesse em braquiária / Brachiaria is a genus of Poaceae family forage grass that reproduces by sexual and asexually by apomixis. Apomixis is of biological and biotechnological interest awakened by the prospect of bringing this feature of cloning plants through seed to other species. B. brizantha cv. Marandu and B. decumbens cv. Basilisk are polyploid and reproduce by apomixis, while the sexual plants are diploid, which makes the crosses, greatly hindering the improvement. Genetic transformation is a strategy that is being incorporated into breeding programs. The nature of these apomictic plants may allow the cloning and the stability of transgenic plants. For genetic transformation is necessary to develop an efficient method of in vitro regeneration. B. brizantha is considered recalcitrant to tissue culture, and efficient methods associated with the genetic transformation systems have not been described in the literature. Rice (Oryza sativa) is a Poaceae model for studies of reverse genetics; however, tropical cultivars from japonica group are recalcitrant to genetic transformation, such as Primavera cultivar. Biolistic is the genetic transformation direct method most widely used, and has been applied to species of monocots, since these are not natural hosts of Agrobacterium tumefaciens. However, several factors have been tested in order to promote interaction and gene transfer during coculture for obtaining transgenics in several monocots species. The objectives of this study were to obtain in vitro regeneration and genetic transformation systems for these species. In B. Brachiaria systemsfor micropropagation, organogenesis, embryogenic units and embryogenic cell suspensions were obtained, and forrice Primavera cultivar embryogenic units were obtained, which was morpho-anatomical characterized and in vitro induction, proliferation and regeneration conditions established. Methods for transient and stable gene expression have been acquired for B. brizanthavia biolistic and Agrobacterium tumefaciens. The nature of the embryogenic callus and transgenic plants was confirmed by histochemical and molecular methods such as PCR and Southern blot. The regeneration and transformation systems showed to be effective and will contribute to apomixis studies and introduction of genes of interest in B. brizantha

Brachiaria é um gênero de forrageiras da família Poaceae que apresenta plantas que se reproduzem por via sexual e assexualmente por apomixia,reprodução por sementes. A apomixia desperta interesse biológico e biotecnológico, pela perspectiva de levar esta característica de clonagem de plantas via sementes, a outras espécies. As cultivares plantadas de B. brizantha cv. Marandu e B. decumbens cv. Basilisk são poliplóides e reproduzem-se por apomixia, enquanto as plantas sexuais são diplóides,o que inviabiliza os cruzamentos, dificultando sobremaneira o melhoramento. A transformação genética é uma estratégia que vem sendo incorporada ao melhoramento genético. A natureza apomítica destasplantas pode permitir a clonagem e estabilidade das plantas transgênicas. Para transformação genética é necessário o desenvolvimento de um método eficiente de regeneraçãoin vitro. B. brizantha é considerada recalcitrante a cultura de tecidos, e métodos eficientes associados com os sistemas de transformação genética ainda não foram descritos na literatura. O arroz (Oryza sativa) é uma Poaceae modelo para estudos de genética inversa, no entanto, cultivares tropicais do grupo japônica são recalcitrantes a transformação genética, como é o caso da cultivar Primavera. O método direto de transformação genética mais amplamente utilizado é a biobalística, e vem sendo aplicado em espécies de monocotiledôneas, uma vez que essas não são hospedeiros naturais de Agrobacterium tumefaciens. No entanto, vários fatores tem sido testados no sentido de favorecer a interação e transferência de genes durante a cocultura para obtenção de transgênicos em diversas espécies de monocotiledôneas. Os objetivos deste estudo foram obter sistemas de regeneração in vitro deB. Brizanthae de arroz cultivar Primavera para transformação genética destas espécies. Em B. brachiaria foram obtidos sistema de micropropagação, organogênese, calos embriogênicos, unidades embriogênicas e suspensões celulares, e para a cultivar Primavera de arroz foram obtidas unidades embriogênicas, que foram caracterizadas morfo-anatomicamente e quanto as condições de indução, multiplicação e regeneração in vitro. Métodos de expressão transiente e estável de genes marcadores foram estabelecidos para B brizantha via biobalística e Agrobacterium tumefaciens. A natureza da transgenia foi confirmada por métodos histoquímico e molecular como PCR e Southern blot. Os sistemas de regeneração e transformação obtidos mostraram-se eficientes e irão contribuir para os estudos da apomixia e introdução de genes de interesse em braquiária / Brachiaria is a genus of Poaceae family forage grass that reproduces by sexual and asexually by apomixis. Apomixis is of biological and biotechnological interest awakened by the prospect of bringing this feature of cloning plants through seed to other species. B. brizantha cv. Marandu and B. decumbens cv. Basilisk are polyploid and reproduce by apomixis, while the sexual plants are diploid, which makes the crosses, greatly hindering the improvement. Genetic transformation is a strategy that is being incorporated into breeding programs. The nature of these apomictic plants may allow the cloning and the stability of transgenic plants. For genetic transformation is necessary to develop an efficient method of in vitro regeneration. B. brizantha is considered recalcitrant to tissue culture, and efficient methods associated with the genetic transformation systems have not been described in the literature. Rice (Oryza sativa) is a Poaceae model for studies of reverse genetics; however, tropical cultivars from japonica group are recalcitrant to genetic transformation, such as Primavera cultivar. Biolistic is the genetic transformation direct method most widely used, and has been applied to species of monocots, since these are not natural hosts of Agrobacterium tumefaciens. However, several factors have been tested in order to promote interaction and gene transfer during coculture for obtaining transgenics in several monocots species. The objectives of this study were to obtain in vitro regeneration and genetic transformation systems for these species. In B. Brachiaria systemsfor micropropagation, organogenesis, embryogenic units and embryogenic cell suspensions were obtained, and forrice Primavera cultivar embryogenic units were obtained, which was morpho-anatomical characterized and in vitro induction, proliferation and regeneration conditions established. Methods for transient and stable gene expression have been acquired for B. brizanthavia biolistic and Agrobacterium tumefaciens. The nature of the embryogenic callus and transgenic plants was confirmed by histochemical and molecular methods such as PCR and Southern blot. The regeneration and transformation systems showed to be effective and will contribute to apomixis studies and introduction of genes of interest in B. brizantha

ResumenEn esta tesis se ha optimizado un protocolo de regeneración a partir de material varietal de 'Helena' y 'Canino'. Mediante el estudio de los diversos factores que afectan la transformación de material adulto, se ha establecido por primera vez un protocolo eficiente de transformación mediada por Agrobacterium tumefaciens de una variedad comercial de albaricoquero.El diseño de una estrategia de selección gradual con paromomicina ha permitido la regeneración de plántulas transformadas con los genes marcadores nptII y sgfp o gus, con las eficiencias más elevadas que se han publicado hasta el momento para transformar material varietal en especies del género Prunus, aunque la baja viabilidad de las yemas transformadas redujo el número final de plantas obtenidas.El protocolo establecido en esta tesis sienta las bases que permitirán la introducción de genes de interés agronómico y comercial, modificando de manera discreta variedades élite aceptadas y establecidas en el mercado. / In this thesis a protocol of regeneration has been optimized from leaf explants of the cultivars 'Helena' and 'Canino'. By means of the study of the diverse factors that affect the transformation of adult material, an efficient protocol of Agrobacterium tumefaciens-mediated transformation has been established for the first time for a commercial cultivar of apricot.The design of a gradual selection strategy with paromomycin has permitted the regeneration of transformed shoots with the marker genes nptII and sgfp or gus, with the highest efficiencies that have been published up to now from adult material in Prunus, although the low viability of the transformed buds reduced the final number of plants obtained. This protocol establishes the bases that will permit the introduction of agronomic and commercial interesting genes, modifying discreetly commercial cultivars accepted and established in the market.

Dans les génomes bactériens, les fréquents transferts horizontaux de gènes (HGT) introduisent des innovations génomiques qui peuvent entraîner la diversification des populations bactériennes. À l'inverse, la recombinaison homologue (RH) au sein des populations homogénéise leurs génotypes, et ainsi renforce leur cohésion. Ces processus d'échange génétique, et la fréquence à laquelle ils interviennent au sein et entre les populations, doivent avoir un grand impact sur la cladogénèse bactérienne. Au-delà de la configuration des échanges qui se sont réellement produits entre les bactéries, les traces de RH et de HGT que nous observons dans leurs génomes reflètent les événements qui ont été fixés tout au long de leur histoire. Ce processus de fixation peut être biaisé en ce qui concerne la nature des gènes ou allèles qui ont été introduits. La sélection naturelle peut notamment conduire à la fixation des gènes transférés qui apportent de nouvelles adaptations écologiques. En outre, des biais mécaniques dans le processus de recombinaison lui-même peuvent conduire à la fixation d'allèles non-adaptatifs. Nous avons cherché à caractériser certains de ces processus adaptatifs et non-adaptatifs qui façonnent les génomes bactériens. À cette fin, plusieurs aspects de l'évolution des génomes, comme les variations de leurs répertoires de gènes, de leur architecture et de leur composition en nucléotides ont été examinés à la lumière de leur histoire de transfert et de recombinaison / In bacterial genomes, the frequent horizontal gene transfers (HGT) introduce genomic novelties that can promote the diversification of bacterial populations. In opposition, homologous recombination (HR) within populations homogenizes their genotypes, enforcing their cohesion. These processes of genetic exchange, and their patterns of occurrence among and within lineages, must have a great impact on bacterial cladogenesis. Beyond the pattern of exchanges actually occurring between bacteria, the traces of HR and HGT we observe in their genomes reflect what events were fixed throughout their history. This fixation process can be biased regarding the nature of genes or alleles that were introduced. Notably, natural selection can drive the fixation of transferred genes that bring new ecological adaptations. In addition, some mechanical biases in the recombination process itself may lead to the fixation of non-adaptive alleles. We aimed to characterize such adaptive and non-adaptive processes that are shaping bacterial genomes. To this end, several aspects of genome evolution, such as variations of their gene repertoires, of their architecture and of their nucleotide composition were examined in the light of their history of transfer and recombination

Crown gall disease is an agricultural problem caused by the soil-borne bacterium, Agrobacterium tumefaciens. A. tumefaciens oncogenes cause transformed plant cells to overproduce the hormones, auxin and cytokinin. High hormone levels cause unorganized plant cell growth resulting in a gall. Control of crown gall disease is difficult because after plant cell transformation has occurred, the bacterium is no longer required for the disease to progress. Apple trees engineered to express double-stranded RNA of two A. tumefaciens oncogenes, ipt and iaaM, silenced the expression of the wild-type oncogenes and prevented crown gall disease. Only the iaaM oncogene was targeted for posttranscriptional gene silencing (PTGS) as measured by biological assays and by quantitative reverse-transcription polymerase chain reaction (q-RTPCR) on transgenic tissue. However, if the translation initiation sequence of the iaaM construction was eliminated, gall formation was not prevented, indicating that translatable RNA initiates silencing whereas untranslatable RNA does not. Other data indicate that the Arabidopsis thaliana micro-RNA pathway gene is involved in A. tumefaciens-mediated tumorigenesis. A. thaliana plants with a mutation in HEN1, a gene required for micro-RNA maturation, demonstrated a tenfold reduction in tumorigenesis upon A. tumefaciens infection compared to wild-type. The same plant line showed no difference in T-DNA transfer and nuclear uptake. / Graduation date: 2006

Les opines sont des molécules produites dans les cellules végétales transformées par l'ADN-T d'A. tumefaciens. Ces opines peuvent être utilisées comme nutriments par le phytopathogène et certaines d'entre elles agissent comme signaux moléculaires contrôlant la dissémination du plasmide de virulence Ti via la signalisation quorum-sensing. Le présent travail vise à une compréhension élargie du rôle des opines au cours des interactions A. tumefaciens-plantes hôtes. En se basant d'abord sur des analyses transcriptomiques, le régulon AccR d'A. tumefaciens C58, contrôlé par les opines agrocinopines, a été défini : celui-ci inclut des fonctions associées (i) à l'assimilation des agrocinopines, (ii) à l'assimilation de la nopaline, (iii) à la signalisation quorum-sensing et la conjugaison du plasmide Ti, (iv) à la conjugaison du plasmide At. La corrélation entre la co-régulation des conjugaisons des plasmides Ti et At et le co-transfert des deux réplicons a en outre été mise en évidence. Dans un second temps, associant des approches de génétique fonctionnelle à des travaux collaboratifs en biologie structurale, l'avantage sélectif conféré par les opines nopaline et octopine à A. tumefaciens au sein des tumeurs végétales a été quantifié. Les bases moléculaires sous-jacentes à cet avantage sélectif, notamment celles associées à la perception et l'importation des deux opines dans le cytoplasme bactérien, ont été décrites. Enfin, en combinant des approches de métabolomique et de génétique inverse avec des tests de conjugaison in planta, les effets opposés de la signalisation GABA d'une part et des signalisations opine et quorum-sensing d'autre part sur la dissémination du plasmide Ti ont été démontrés. En conclusion, nos résultats révèlent l'intrication des signalisations opine, quorum-sensing et GABA au cours de l'interaction A. tumefaciens-plantes hôtes. Ils soulignent en particulier les impacts de cette intrication sur la colonisation de l'hôte ainsi que sur la dissémination des gènes de virulence et d'adaptation à l'environnement tumeur portés par les plasmides Ti et At.

In this study, optimization of a transformation and regeneration system via indirect and direct organogenesis in cotyledon, hypocotyl, petiole, leaf and shoot base tissues of sugar beet (Beta vulgaris L. cv. ELK 345 and 1195) was investigated. Two different germination, three different callus induction and shoot induction medium was used for indirect organogenesis of sugar beet cultivar ELK 345. Except cotyledon, other explants (hypocotyl, petiole and leaf) produced callus. However no shoot development was observed from callus of these explants. Shoot base tissue of sugar beet cultivar 1195 was employed for direct organogenesis. Shoot development was achieved via direct organogenesis using 0.1 mg/L IBA and 0.25 mg/L BA. Root development and high acclimatization rate were accomplished from shoot base tissue. Different concentrations of kanamycin and PPT were applied to leaf blade explants to find out optimum dose for selection of transformants. Kanamycin at 150 mg/L and PPT at 3 mg/L totally inhibited shoot development from leaf blades. Moreover, an Agrobacterium mediated transformation procedure for leaf explants of ELK 345 was also optimized by monitoring transient uidA expression 3rd days after transformation. Effects of different parameters (vacuum infiltration, bacterial growth medium, inoculation time with bacteria, Agrobacterium strains and L-cysteine application in co-cultivation medium) were investigated to improve transformation procedure. Vacuum infiltration and Agrobacterium strains were significantly improved transformation procedure. Percentage of GUS expressing areas on leaves increased three folds from the beginning of the study.

The objective of this study was to transform sweet pepper ferredoxin-like protein (PFLP) gene, which has antimicrobial properties, to tobacco and investigate the disease resistance abilities of transgenic tobacco. This protein interacts with another protein, harpin that is produced by the bacteria which is invading the plant tissues, and stimulates hypersensitivity response in plants, thus the spreading of disease is limited. Gene transfer was achieved to tobacco by Agrobacterium- mediated method and with indirect organogenesis / the explants were grown on selective media and then transferred to jars and pots respectively. Molecular and genetic analyses such as PCR, RT-PCR, Sequence Analysis and Northern Blot, were performed with plants which their seeds survived and grew on selective medium and also gave positive reactions for GUS histochemical assay. Finally, with putative transgenic plants, some hypersensitive response assays were carried out with Pseudomonas syringae and it was observed that the recovered plants showed hypersensitive response (HR) in the preliminary tests. These results indicated that putative transgenic tobacco plants which carry pflp transgene, can be used in disease resistance studies.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv. / Härtill 4 uppsatser.

Diss. (sammanfattning) Alnarp : Sveriges lantbruksuniv. / Härtill 4 uppsatser.