A method for Agrobacterium-mediated transformation of hybrid poplar (Populus alba x P. grandidentata) suspension cultures and regeneration of transformed plants is described. The best protocol was one in which suspension cultures were inoculated with Agrobacterium tumefaciens to a density of 10⁷ cfu's/ml, cocultivated for 48 hours, plated to cellulose acetate filters at a density of 14 colonies/mm², and cultured on medium containing 1 mg/1 2,4-D. Although cefotaxime inhibited callus growth, it was used in the plating medium to suppress proliferation of Agrobacterium. Selection appeared to be more reliable using hygromycin as compared to kanamycin or geneticin (G418). Transgenic plants were regenerated by culturing the calli on media containing thidiazuron, but no shoots could be regenerated using BA. / Graduation date: 1992

The purpose of this study was to investigate variation in selected anatomical, physical and mechanical properties of three Populus x euramericana (Dode) Guinier hybrid clones grown on two dissimilar sites in Hungary. Six 15 years old trees from three clones on one site and six 10 years old trees from two clones on the other site were sampled at breast height. Anatomical properties, including vessel lumen diameter, area and shape factor, number of vessels per unit area, fiber lumen diameter and area, fiber length, ray area, and cell wall area percent were measured by an image analyzer. Site, clone and/or their interaction significantly affected one or more of these properties except fiber length. Variations were significant among trees within clone and site for all variables except vessel lumen diameter. However, most of the variations was within tree as a result of the effect of age. Statistically significant correlations were found between anatomical properties. For specific gravity, there was no significant differences between sites but there was a significant clone effect with a repeatability of 0.51 indicating genetic control on this property. There were no significant differences among clones for modulus of rupture, crushing strength, maximum tensile strength, and tension modulus of elasticity. Strength properties were significantly higher near the bark than close to the pith except for maximum tensile strength. Specific gravity was not the single most important factor affecting strength properties of the clones. The effect of age and growth rate on specific gravity and anatomical properties were also investigated. Differences among clones for growth ring widths were significant in the "good" years only. Specific gravity was high near the pith, then each of the clones exhibited a different radial pattern. Based on segmented regression, a quadratic model with a plateau proved to be useful for estimating the demarcation between juvenile and mature anatomical characteristics. The ages of maturation were not the same for all properties; however, the order of maturation was the same on both sites. No consistent relationships between growth rate and specific gravity and anatomical properties were found when growth rings of the same age were compared. / Ph. D.

Les protéines possédant un domaine RING (REALLY INTERESTING NEW GENE) avec une activité E3 ligase sont largement présentes chez les plantes, jouent des rôles importants dans la régulation de plusieurs processus par la reconnaissance d’une protéine cible pour l’ubiquitination. Auparavant, il a été montré que l'expression de PtaRHE1, codant une protéine contenant un domaine RING-H2 avec une activité E3 ligase, est associée à la mise en place de la croissance secondaire chez le peuplier. Dans le cadre de cette thèse, nous avons démontré que PtaRHE1 est mono-ubiquitiné in vitro en présence de l’E2 du peuplier PtaUbC5a. Par hydridation in situ et Western blot, nous montrons que PtaRHRE1 et la protéine correspondante sont exprimés dans les tissus vasculaires de la tige, c'est-à-dire le phloème, le cambium et le xylème. Par comparaison avec les plantes de type sauvage, la sous-expression de PtaRHE1 suite à l’expression d’un microARN (i) a donné lieu à une modification dans la morphologie des fibres secondaire de phloème avec une plus forte densité cellulaire et des paroi de fibres plus mince, (ii) à une modification de la qualité de lignine avec moins d’unité S au niveau des tissus de l’écorce, ces résultats suggèrent un rôle de PtaRHE1 dans la formation et / ou la maturation des fibres. La sur-expression de PtaRHE1 chez les peupliers engendre un phénotype pléiotropique caractérisé par l’enroulement des feuilles et une inhibition du développement racinaire. L’expression du gène codant pour le facteur de transcription WRKY23 est positivement corrélée à celui de PtaRHE1 dans le xylème de la tige des lignées sur-exprimant ou sous-exprimant PtaRHE1. Sur base d’un modèle, nous suggérons un rôle pour le couple PtaRHE1/PtaWRKY23 dans le développement des tissus vasculaires. De plus, nous avons montré que l'expression de PtaRHE1 et l'accumulation de la protéine correspondante sont modulées par l'humidité de l’air et du sol ainsi que par l'acide abscissique. Les informations présentées dans ce travail indiquent un rôle de PtaRHE1 au cours de développement de la plante ainsi que pendant la réponse au stress biotique et abiotique / Doctorat en Sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished

Vegetative propagation allows the amplification of selected genotypes for research, breeding, and commercial planting. However, efficient in vitro regeneration and genetic transformation remains a major obstacle to research and commercial application in many plant species. Our aims are to improve knowledge of gene regulatory circuits important to meristem organization, and to identify genes that might be useful for improving the efficiency of in vitro regeneration. In this thesis, we have approached these goals in two ways. First, we analyzed gene expression during poplar (Populus) regeneration using an AffymetrixGeneChip® array representing over 56,000 poplar transcripts. We have produced a catalog of regulated genes that can be used to inform studies of gene function and biotechnology. Second, we developed a GUS reporter system for monitoring meristem initiation using promoters of poplar homologs to the meristem-active regulatory genes WUSCHEL (WUS) and SHOOTMERISTEMLESS (STM). This provides plant materials whose developmental state can be assayed with improved speed and sensitivity. For the microarray study, we hybridized cDNAs derived from tissues of a female hybrid poplar clone (INRA 717-1 B4, Populus tremula x P. alba) at five sequential time points during organogenesis. Samples were taken from stems prior to callus induction, at 3 days and 5 days after callus induction, and at 3 and 8 days after the start of shoot induction. Approximately 15% of the monitored genes were significantly up-or down-regulated based on both Extraction and Analysis of Differentially Expressed Gene Expression (EDGE) and Linear Models for Microarray Data (LIMMA, FDR<0.01). Of these, over 3,000 genes had a 5-fold or greater change in expression. We found a very strong and rapid change in gene expression at the first time point after callus induction, prior to detectable morphological changes. Subsequent changes in gene expression at later regeneration stages were more than an order of magnitude smaller. A total of 588 transcription factors that were distributed in 45 gene families were differentially regulated. Genes that showed strong differential expression encoded proteins active in auxin and cytokinin signaling, cell division, and plastid development. When compared with data on in vitro callogenesis from root explants in Arabidopsis, 25% (1,260) of up-regulated and 22% (748) of down- regulated genes were in common with the genes that we found regulated in poplar during callus induction. When ~3kb of the 5' flanking regions of close homologs were used to drive expression of the GUSPlus gene, 50 to 60% of the transgenic events showed expression in apical and axillary meristems. However, expression was also common in other organs, including in leaf veins (40% and 46% of WUS and STM transgenic events, respectively) and hydathodes (56% of WUS transgenic events). Histochemical GUS staining of explants during callogenesis and shoot regeneration using in vitro stems as explants showed that expression was detectable prior to visible shoot development, starting 3 to 15 days after explants were placed onto callus inducing medium. Based on microarray gene expression data, a paralog of poplar WUS was detectably up-regulated during shoot initiation, but the other paralog was not. Surprisingly, both paralogs of poplar STM were down-regulated 3- to 6-fold during early callus initiation, a possible consequence of its stronger expression in the secondary meristem (cambium) than in shoot tissues. We identified 15 to 35 copies of cytokinin response regulator binding motifs (ARR1AT) and one copy of the auxin response element (AuxRE) in both promoters. Several of the WUS and STM transgenic events produced should be useful for monitoring the timing and location of meristem development during natural and in vitro shoot regeneration. / Graduation date: 2008