O presente trabalho teve como principal objetivo obter plantas transgênicas, dos cultivares porta-enxerto limão \'Cravo\' e laranja azeda e de cultivares copa de laranja doce, expressando genes que possam influenciar no nível de resistência ao vírus da tristeza dos citros (CTV) e, possivelmente à morte súbita dos citros. Buscou-se ainda estudar a organogênese in vitro de espécies cítricas. Experimentos para a indução da organogênese in vitro foram realizados a partir de segmentos de epicótilos de plântulas germinadas in vitro de espécies cítricas avaliando-se: a resposta organogênica de três diferentes regiões do epicótilo, na presença (1,0 mg.L-1) ou ausência de BAP, em meio MT, e a regeneração em diferentes concentrações de BAP (0; 0,5; 1,0; 1,5 e 2,0 mg.L-1) adicionadas ao meio MT. Também avaliou-se a regeneração de segmentos internodais de limão ?Cravo? e laranja azeda em diferentes concentrações de BAP (0; 0,5; 1,0; 2,0 e 4,0 mg.L-1) em meio MT; e de laranja azeda em meio de cultura MT e DBA3, suplementados com diferentes concentrações de BAP (0; 1,0 e 2,0 mg.L-1) e ANA (0; 0,3 e 0,5 mg.L-1). Foi utilizado o método de transformação genética mediado por Agrobacterium tumefaciens utilizando-se tecido juvenil coletado de plantas cultivadas in vitro (segmentos de epicótilo) ou em casa de vegetação (segmentos internodais) como explantes. Utilizou-se a estirpe EHA105 de A. tumefaciens, contendo os plasmídeos: a) pCTV-CP: contendo o gene da capa protéica do CTV; b) pCTV-dsCP: contendo repetições invertidas do gene da capa protéica do CTV, interligadas pelo intron do gene da quitinase de citros; c) pCTVcons: contendo uma seqüência conservada antisense do CTV. As construções gênicas foram elaboradas a partir do plasmídeo pCAMBIA 2201, dirigidas pelo promotor 35S e o terminador NOS. Foram utilizados também os genes de seleção nptII e o repórter GUS. As gemas adventícias desenvolvidas foram identificadas como transgênicas pelo teste histoquímico GUS e por PCR e a confirmação da transformação genética foi feita por Southern Blot. A expressão da proteína do CTV foi verificada pelo teste serológico de PTA-ELISA. A resposta morfogênica em função da região do epicótilo e das concentrações da citocinina BAP é dependente do cultivar de citros. A suplementação do meio de cultura com a citocinina BAP proporcionou um maior número de gemas adventícias regeneradas por explante, tanto em segmentos de epicótilo como em segmentos internodais dos cultivares de citros estudados. A suplementação do meio de cultura com a citocinina BAP é essencial na regeneração de gemas adventícias em segmentos internodais de laranja azeda. Foi possível regenerar plantas transgênicas, pelo protocolo de transformação genética mediado por Agrobacterium tumefaciens de limão \'Cravo\' contendo o gene da capa protéica do vírus da tristeza dos citros (pCTV-CP) utilizando-se segmentos de epicótilo e segmentos internodais como fonte de explante, e com uma seqüência conservada antisense do CTV, utilizando-se segmentos internodias; e de laranja \'Hamlin\' contendo o gene da capa protéica do CTV, com as construções gênicas pCTV-dsCP e pCTV-CP, e com uma seqüência conservada antisense do CTV, utilizando-se segmentos de epicótilo como fonte de explante. / The objective of the present work was to obtain transgenic plants of rootstocks Rangpur lime and sour orange as well as sweet orange scion varieties, expressing genes that would possibly influence the levels of resistance to the Citrus tristeza virus (CTV) and Citrus sudden death disease. The in vitro organogenesis of Citrus species was also studied. In vitro organogenesis experiments were conducted with epicotyl segments obtained from in vitro germinated seedlings of different Citrus species in order to evaluate: organogenic response of three epicotyl regions, in the presence (1,0 mg.L-1) or absence of BAP, in MT medium, and the regeneration using different BAP concentrations (0; 0,5; 1,0; 1,5 e 2,0 mg.L-1) added to MT medium. The regeneration of internodal segments of Rangpur lime and sour orange was evaluated in MT medium with different BAP concentrations (0; 0,5; 1,0; 2,0 e 4,0 mg.L-1) as well as sour orange regeneration in MT and DBA3 mediums, supplemented with different concentrations of BAP (0; 1,0 e 2,0 mg.L-1) and NAA (0; 0,3 e 0,5 mg.L-1). The Agrobacterium tumefaciens Agrobacterium tumefaciens mediated genetic transformation method of juvenile tissue obtained from in vitro (epicotyls) or green house cultivated plants (internodal segments) was used in this work. The EHA 105 strain was used with the following plasmids: a) pCTV-CP: containing the coat protein gene from CTV; b) pCTV-dsCP: containing inverted repeats of the coat protein gene from CTV interconnected by the Citrus quitinase gene intron; c) pCTVcons: containing an antisense sequence of CTV untranslated region. The gene constructs were built from the pCAMBIA 2201 plasmid, and contained the 35S promoter and the NOS terminator. The nptII selection gene and the GUS reporter gene were also used. The adventitious buds developed were identified as transgenic by GUS histochemical test and PCR, these results were later confirmed by Southern Blot. The transgenic coat protein expression was detected by the serological test PTA-ELISA. The morphogenic response related to the epicotyl region and BAP cytokinin were dependent on the Citrus varieties. The addition of BAP cytokinin to the culture medium showed a greater number of adventitious buds regenerated per explant in both epicotyl and internodal segments of the Citrus varieties studied. The addition of BAP to the culture medium is essential for the regeneration of adventitious buds in sour orange internodal segments. Using the Agrobacterium mediated genetic transformation protocol it was possible to regenerate transgenic plants of different varieties and gene constructs: Rangpur lime containing the coat protein gene of CTV (pCTV-CP) using epicotyl and internodal segments as explant source, and an antisense sequence of CTV untranslated region using internodal segments as explant source; Hamlin sweet orange containing the coat protein gene of CTV in constructions pCTV-CP and pCTV-dsCP, and an antisense sequence of CTV untranslated region using epicotyl segments as explant source.

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.

Les agrobactéries établissent des relations à long terme avec les plantes et ce, dans deux styles de vie différents, rhizosphérique et pathogène (galle du collet). Dans ce mode de vie, les bactéries modifient génétiquement leur hôte et se créent ainsi une niche écologique spécifique (tumeur). La transition entre les deux styles de vie est déclenchée par la perception de signaux végétaux, parmi lesquels des acides hydroxycinnamiques (HCAs) comme l’acide férulique. Or dans l’espèce Agrobacterium fabrum, des gènes spécifiques permettent la dégradation des HCAs. Nous avons émis l’hypothèse que cette dégradation était un signal de proximité de la plante et influençait alors des fonctions importantes pour l’interaction avec celle-ci. Nous avons caractérisé la régulation de la dégradation des HCAs, évalué son rôle dans la valeur sélective d’A. fabrum, et suggéré son importance dans la transition entre les styles de vie rhizosphérique et pathogène. Nous avons montré que la dégradation des HCAs module le métabolisme carboné bactérien, notamment l’utilisation d’acide aminés et d’oligosaccharides de la famille du raffinose. Nous avons caractérisé la protéine MelB qui permet l’import de ces sucres, du mélibiose et du galactinol. Leur utilisation est importante pour la colonisation des plantes dès la germination. L’analyse de l’expression des gènes et du métabolisme bactérien en présence d'un composé signal de la plante, nous a révélé de nouveaux déterminants importants pour l’écologie de ce phytopathogène, notamment des facteurs de transcription. En outre, cette analyse a confirmé l’importance des échanges cellulaires et de déterminants impliqués dans la compétition bactérienne / Agrobacterium establish long term interactions with plants, either in a rhizosphere or pathogenic lifestyle. Pathogenic agrobacteria are causing the crown gall disease by genetically modifying the plant cells host, thus creating a specific ecological niche (tumor). The transition from the rhizosphere to the pathogenic lifestyle is triggered by bacterial perception of plant-derived signals, including hydroxycinnamic acids (HCAs) such as ferulic acid. However, A. fabrum strains have species-specific genes that allow HCAs degradation.We hypothesized that in A. fabrum, the degradation of the HCAs is perceived as a plant signal which influences important functions involved in the interaction with plants. We characterized the regulation of HCAs degradation, evaluated its role in the fitness of A. fabrum, and suggested its importance for the transition between the rhizosphere and pathogenic lifestyles. Then, we showed that the degradation of HCAs modulates carbon metabolism, such as the use of amino acids and sugars belonging to the raffinose family oligosaccharides (RFO). We have demonstrated that besides these sugars, the MelB protein allows the import melibiose and galactinol. Their use is important for plant colonization, since seed germination. The analyzes of gene expression and bacterial metabolism in the presence of a plant signal compound, revealed new determinants important for A. fabrum ecology, including transcription factors. In addition, it confirmed the importance of cellular exchanges and bacterial competition for Agrobacterium fitness in planta

Les bactéries du genre Agrobacterium forment un ensemble taxonomiquement diversifié composé de nombreuses espèces, présent dans la plupart des sols et des rhizosphère. Les agrobactéries sont le plus souvent anodines voire stimulatrices de la croissance des plantes. Par contre, celles qui hébergent un plasmide Ti induisent la maladie de la galle du collet à de nombreuses plantes d'intérêt agronomique. Dans ce contexte, nous avons d'une part donné l'état actuel des connaissances sur la taxonomie du genre Agrobacterium, et nous avons fait une revue des méthodes d'isolement et de typage de ces bactéries. D'autre part, nous avons cherché à mettre au point des méthodes d'identification rapides et fiables des différentes espèces d'agrobactérie. La méthode de MALDI-TOF MS a permis d'identifier les espèces mais elle n'était pas assez résolutive pour typer des souches et encore moins la présence de plasmides Ti dans les isolats. Nous avons alors développé des amorces de PCR spécifiques de 17 espèces, du genre Agrobacterium et de la famille Rhizobiaceae. Ces amorces se sont révélées efficaces pour identifier les bactéries cultivées et aussi pour détecter leur présence dans des communautés microbiennes. Nous avons utilisé ces outils pour étudier la répartition des agrobactéries à l'échelle d'un pays, d'une station et entre sols nus et sols rhizosphériques en utilisant soit des isolats soit des ADN extraits des différents environnements. Enfin, nous avons montré que le clonage-séquençage ou le séquençage à haut débit d'amplicons obtenus à partir d’ADN de communautés microbiennes nous permettaient de connaître la diversité des populations d'agrobactéries / Agrobacterium are Alphaproteobacteria common in most soils that closely interact with plants in two respects. Firstly, they are rhizospheric bacteria saprophytically living in the rhizosphere of numerous plants and they are likely beneficial to plants. Secondly, when they harbor a dispensable Ti plasmid (i.e. tumor inducing plasmid), agrobacteria are plant pathogens able to cause the crown gall disease to most dicots and gymnosperms and some monocots. An epidemiological survey of crown gall thus also requires expert determination of the Agrobacterium taxonomy. In this thesis we evaluated the usefulness of MALDI-TOF MS technique as a high throughput tool to determine and classify agrobacteria. Then we set up a recA-based PCR method to accurately and exhaustively assess agrobacterial diversity either of isolated agrobacteria or directly in various biotopes. We applied standard biochemical, recA-based and Ti plasmid-based identification methods to study the prevalence of pathogenic and non-pathogenic agrobacteria at the country and local scales. Finally, we tested whether analyzing the internal composition of recA amplicons could be a way to directly assess the micro-diversity of agrobacterial populations using cloning sequencing or pyrosequencing approaches. The later methodology was applied to establish the actual field diversity of Agrobacterium and to evaluate whether plant genotypes differentially select agrobacteria in their root systems, providing first data upon biotic factors shaping the population structure of agrobacteria

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

La bactérie Agrobacterium tumefaciens est bien connue pour son utilisation en génie génétique végétale où elle sert comme vecteur de gènes. A l’origine, cette bactérie ainsi que l’espèce voisine Agrobacterium rhizogenes sont des bactéries phytopathogènes qui induisent respectivement des tumeurs et des racines anormales sur des plantes sensibles telles que la vigne ou des arbres fruitiers. L’action pathogène résulte d’un transfert horizontal de gènes de la bactérie vers l’hôte végétal, à partir d’un plasmide, le pTi (plasmide inducteur de tumeurs) ou pRi (plasmide inducteur de racines). Mon travail de thèse concerne deux aspects particuliers de cette bactérie.1. Sa capacité à transformer durablement des espèces végétales dans la nature, donnant ainsi naissance à des plantes naturellement transformées, notamment dans le genre Nicotiana. Nous avons pu montrer par séquençage à haut débit du génome de N. tomentosiformis et par l’analyse d’autres séquences complètes de Nicotianées publiées récemment l’existence inattendue de 5 séquences venant d’Agrobacterium (cT-DNAs) avec une taille total de 65 kb, dont certaines portent des gènes intacts. Nous avons montré que deux de ces gènes (TB-mas2’ de N. tabacum et TE-6b de N. otophora) ont une activité biologique. Une étude comparative approfondie a permis de mieux comprendre l’évolution de ces cT-DNAs (Chen et al., 2014). Le gène mas2’ est bien connu, il code pour une enzyme qui catalyse la synthèse du désoxyfructosyl-glutamine (DFG) dans des tumeurs ou racines induites par Agrobacterium. Des résultats récents dans notre groupe portant sur le gène TB-mas2’ montrent que ce gène est exprimé de façon très active dans plusieurs cultivars de N. tabacum, et y donne naissance à l’apparition de quantités mesurables de DFG. Ce travail est présenté sous forme d’un manuscrit à soumettre.2. Une deuxième partie de la Thèse concerne les propriétés du gène T-6b, qui fait partie de l’ADN transféré par A. vitis souche Tm4 et provoque une croissance anormale caractérisée par l’apparition d’énations, sans que l’on connaisse son mode d’action. Le gène 6b fait partie de la famille des gènes plast (pour plasticité phénotypique), avec des effets différents et souvent remarquables sur la croissance des plantes. Le gène T-6b a été mis sous contrôle d’un promoteur inductible par le dexaméthasone, et des plantes de tabac transformées par cette construction ont été étudiées en détail, à différents moments après son induction. Un grand nombre de changements a été décrit incluant des analyses anatomiques montrant des modifications encore jamais décrites chez les plantes, comme par exemple l’apparition de méristèmes foliaires ectopiques à la base de trichomes, ou l’apparition de systèmes vasculaires ectopiques parallèles au système vasculaire normal avec un développement régulier menant à des structures complexes ordonnées (Chen and Otten, 2015). Le gène TE-6b de N. otophora a été mis sous contrôle d’un promoteur fort constitutif et introduit dans des plantes de tabac, où il provoque des changements de croissance différents de ce qui a été observé pour le gène T-6b. Ces derniers résultats préliminaires sont présentés en complément des observations sur le gène T-6b. Ils indiquent que le transfert horizontal du gène TE-6b vers l’ancêtre de N. otophora aurait pu contribuer à une modification de la croissance et ainsi à la création d’une nouvelle espèce. / The bacterium Agrobacterium tumefaciens is well-known for its utilisation in plant genetic engineering where it serves as a gene vector. This bacterium and the related species Agrobacterium rhizogenes are phytopathogens that induce tumors and hairy roots respectively on susceptible plants like grapevine or fruit trees. Their phytopathogenicity is due to horizontal transfer of bacterial genes to the plant host, from a plasmid called the Ti (tumor-inducing) or Ri (root-inducing) plasmid. The subject of my Thesis concerns two particular aspects of this bacterium.1. Their capacity to stably transform several plant species in nature, thereby yielding naturally transformed plants, especially in the genus Nicotiana. We have shown by deep sequencing of the Nicotiana tomentosiformis genome and by analysis of other recently published Nicotiana sequences the presence of five different Agrobacterium-derived sequences (cT-DNAs), totalling 65 kb, some of which carry intact genes. We have shown that two of them (TB-mas2’ from N. tabacum and TE-6b from N. otophora) have biological activity. A detailed comparative study has allowed us to better understand the evolution of these cT-DNAs (Chen et al., 2014). The mas2’ gene is well-known, it codes for the synthesis of desoxyfructosyl-glutamine (DFG) in tumors or roots induced by Agrobacterium. Recent work in our group has shown that the TB-mas2’ gene is highly expressed in some N. tabacum cultivars and leads to the accumulation of detectable amounts of DFG. This work is presented as a manuscript to be submitted.2. A second part of the Thesis describes new properties of the T-6b gene, which is part of the DNA transferred by A. vitis strain Tm4 and leads to abnormal growth caracterized by the appearance of enations, so far the mode of action of this gene is unknown. The 6b gene is part of the so called plast family (for phenotypic plasticity), with different and often remarkable growth effects on plants. The T-6b gene wasearlier placed under control of a dexamethasone-inducible promoter, and tobacco plants transformed with this construct have now been studied in detail, at different times after the start of induction. A large number of changes was analyzed, both at the morphological and anatomical level, these include various unprecedented morphological changes, like for example the appearance of shoot primordia at the base of trichomes, or the appearance of ectopic vascular strands parallel to the normal strands with a regular development leading to complex but predictable structures (Chen and Otten, 2015). The TE-6b gene from N. otophora was placed under strong and constitutive promoter control and introduced into tobacco, where it was found to cause new types of morphological change, different from those observed for T-6b. The latter results are preliminary and will be presented as a complement to the work on T-6b. They indicate that the introduction of the TE-6b gene in the N. otophora ancestor could have caused a change in growth pattern, and might have favored the appearance of a new species.

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

Medicago is widely used as a forage crop. It is often susceptible to various pathogenic infections and exhibits low growth in drought and extreme climatic conditions. In the current study, a strategy was developed for over-expressing an “Osmotin-Chitinase” gene chimera in transgenic Medicago that could potentially confer resistance to different biotic and abiotic stresses. Seed germination of several cultivars of Medicago (M. sativa ssp. sativa, M. sativa ssp. falcata, M. sativa ssp. caerulea, M. truncatula, and M. Rugosa) was tested to determine the cultivars with good germination rates. Among these, M. sativa ssp. sativa showed an average of 80% germination over a period of one week and was subsequently selected for regeneration and transformation experiments. Different explants (cotyledons, hypocotyls, petioles) were tested for regeneration. Among these, hypocotyl explants showed highest (46.17 %) percent regeneration. Escherichia coli harboring Osmotin-Chitinase (OSM-CHI) gene chimera cloned into binary vector pBTEX with nptII as a selection marker was mobilized in Agrobacterium tumefaciens strain EHA105 which was employed in the transformation of hypocotyl explants of Medicago. Transformed calli were grown on callus inducing medium containing kanamycin for screening. Further screening of the positive transgenics was performed using PCR. Southern hybridization was carried out for further confirmation of successful transformation. Transformed shoots will be grown on the root inducing medium for developing into plantlets which would then be transferred to the green house and later tested for their degree of resistance to various biotic and abiotic stresses.

No description available.