Presented as Thesis (Ph. D.)--University of Wisconsin--Madison, 1920. / Cover title. "Reprinted from the Transactions of the Wisconsin Academy of Sciences, Arts, and Letters, Vol. XXI." Includes bibliographical references (p. 376-377).

Inaug.-Diss.--Sofia. / Bibliography: p. [35]-38.

Made available in DSpace on 2014-08-13T14:50:40Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-05-16Bitstream added on 2014-08-13T18:01:28Z : No. of bitstreams: 1 000747183_20150516.pdf: 388655 bytes, checksum: 28bb4ca0fd35b64c1425eeff151db6dc (MD5) Bitstreams deleted on 2015-05-19T12:01:09Z: 000747183_20150516.pdf,Bitstream added on 2015-05-19T12:01:40Z : No. of bitstreams: 1 000747183.pdf: 1675711 bytes, checksum: 172952b37ea05e8baceeccb4e6e6ea8f (MD5) / A franca expansão das florestas de eucalipto observada nos últimos anos em diferentes estados brasileiros aumentou a dependência tecnológica da cultura, tornando necessária a sua inserção no contexto biotecnológico. Para tal, a disponibilidade de ferramentas moleculares que viabilizem a produção de árvores geneticamente modificadas se faz necessária. Nesse contexto, o presente trabalho visou validar funcionalmente dois promotores de Eucalyptus grandis, o primeiro relacionado a um gene que codifica uma Lacase (denominado EgLac) com expressão específica em caule (câmbio), e o segundo relacionado a uma actina (denominado EgAct) com expressão ubíqua. Um cassete de expressão contendo o promotor EgLac em fusão transcricional com o gene repórter GUS (previamente construído) foi inserido em plantas de Arabidopsis thaliana, e uma expressão vascular foi constatada em testes histoquímicos. Em cortes histológicos observou-se que a expressão de GUS ficou restrita ao floema de raízes e folhas. Em paralelo, o gene EgAct teve a sua expressão ubíqua validada em diferentes órgãos/tecidos de eucalipto, e a sua região promotora foi amplificada. Um cassete de expressão contendo o promotor EgAct em fusão ao repórter GUS foi igualmente inserido em plantas de A. thaliana. Análises histoquímicas revelaram uma expressão vascular do gene repórter distribuída ao longo de toda a planta. Por outro lado, a quantificação da expressão de GUS em plantas transformadas revelaram níveis variáveis de acumulação de transcrição de acordo com a idade e o órgão/tecido analisado / The rapid expansion of eucalyptus plantations observed in recent years in different Brazilian states increased the technological dependence of the culture, and its inclusion in the biotechnological context is an urgent need. For this, the availability of molecular tools that enable the production of genetically modified trees is necessary. In this context, the present work aimed to functionally characterize two promoters of Eucalyptus grandis, the first one related to a gene encoding a laccase (called EgLac) with specific expression in stem (cambium region), and the second one related to a gene encoding an ubiquitously expressed actin (called EgAct). An expression cassette containing the EgLac promoter transcriptionally fused to the GUS reporter gene was inserted into Arabidopsis thaliana, and a vascular expression pattern was observed in histochemical tests. Histological sections showed that GUS expression was restricted to the phloem of roots and leaves. In parallel, the ubiquitous expression of EgAct was validated using different eucalyptus organs/tissues, and its promoter region was amplified. An expression cassette containing the EgAct promoter fused to the GUS reporter was also inserted into A. thaliana. Histochemical analysis revealed a vascular expression of the reporter gene distributed throughout the plant. Moreover, quantification of GUS expression in transformed plants revealed variable levels of transcript accumulation according to the age and organ/tissue analyzed

Orientador: Ivan de Godoy / Banca: Celso Luis Marino / Banca: Márcio José da Silva / Resumo: A franca expansão das florestas de eucalipto observada nos últimos anos em diferentes estados brasileiros aumentou a dependência tecnológica da cultura, tornando necessária a sua inserção no contexto biotecnológico. Para tal, a disponibilidade de ferramentas moleculares que viabilizem a produção de árvores geneticamente modificadas se faz necessária. Nesse contexto, o presente trabalho visou validar funcionalmente dois promotores de Eucalyptus grandis, o primeiro relacionado a um gene que codifica uma Lacase (denominado EgLac) com expressão específica em caule (câmbio), e o segundo relacionado a uma actina (denominado EgAct) com expressão ubíqua. Um cassete de expressão contendo o promotor EgLac em fusão transcricional com o gene repórter GUS (previamente construído) foi inserido em plantas de Arabidopsis thaliana, e uma expressão vascular foi constatada em testes histoquímicos. Em cortes histológicos observou-se que a expressão de GUS ficou restrita ao floema de raízes e folhas. Em paralelo, o gene EgAct teve a sua expressão ubíqua validada em diferentes órgãos/tecidos de eucalipto, e a sua região promotora foi amplificada. Um cassete de expressão contendo o promotor EgAct em fusão ao repórter GUS foi igualmente inserido em plantas de A. thaliana. Análises histoquímicas revelaram uma expressão vascular do gene repórter distribuída ao longo de toda a planta. Por outro lado, a quantificação da expressão de GUS em plantas transformadas revelaram níveis variáveis de acumulação de transcrição de acordo com a idade e o órgão/tecido analisado / Abstract: The rapid expansion of eucalyptus plantations observed in recent years in different Brazilian states increased the technological dependence of the culture, and its inclusion in the biotechnological context is an urgent need. For this, the availability of molecular tools that enable the production of genetically modified trees is necessary. In this context, the present work aimed to functionally characterize two promoters of Eucalyptus grandis, the first one related to a gene encoding a laccase (called EgLac) with specific expression in stem (cambium region), and the second one related to a gene encoding an ubiquitously expressed actin (called EgAct). An expression cassette containing the EgLac promoter transcriptionally fused to the GUS reporter gene was inserted into Arabidopsis thaliana, and a vascular expression pattern was observed in histochemical tests. Histological sections showed that GUS expression was restricted to the phloem of roots and leaves. In parallel, the ubiquitous expression of EgAct was validated using different eucalyptus organs/tissues, and its promoter region was amplified. An expression cassette containing the EgAct promoter fused to the GUS reporter was also inserted into A. thaliana. Histochemical analysis revealed a vascular expression of the reporter gene distributed throughout the plant. Moreover, quantification of GUS expression in transformed plants revealed variable levels of transcript accumulation according to the age and organ/tissue analyzed / Mestre

Le rôle des lipides comme des acteurs moléculaires de la voie sécrétoire des plantes n'est pas encore complètement élucidé. Le Glucosylceramide (GlcCer) est synthétisé par la Glucosylceramide synthase (GCS) chez les plantes et constitue un des sphingolipides complexes clé dans les membranes, mais on connaît peu les exigences cellulaires pour le GlcCer. Cette étude repose sur le blocage de la biosynthèse de GlcCer, par l'utilisation d'inhibiteurs chez le tabac et l’arabidopsis, et la production de mutants d'Arabidopsis, pour déterminer l'impact de la biosynthèse du GlcCer sur la dynamique endomembranaire des cellules végétales. Dans une approche qui inclue la biochimie des lipides, l'imagerie de cellules vivantes, des études ultrastructurales par microscopie electronique à transmission et des études sur le développement de la plante entière, nous avons acquis une meilleure compréhension de l'impact de la synthèse du GlcCer dans les cellules végétales qui peut être résumée comme suit: (1) Sur la base d’un analyse théorique et de la microscopie de cellules vivantes on a déterminé que la GCS est situé dans le Réticulum Endoplasmique (au début de la voie sécrétoire) dans les cellules végétales, (2) PDMP est un inhibiteur spécifique de GCS chez les plantes, (3) La perturbation de la biosynthèse du GlcCer par le PDMP et par un approche génétique ont montré que le GlcCer est important pour le trafic normale des protéines et pour la dynamique des endomembranes, notamment dans le maintien de la structure de l’appareil de Golgi, (4) La régulation du trafic des protéines mediée par la synthèse du GlcCer pourrait être critique dans l’etablissement et le maintien de la polarité cellulaire, tel qu’il est suggéré par des changements dans la localisation des marqueurs polaires chez l’Arabidopsis traitée avec PDMP, et (5) Un bloc dans la synthèse du GlcCer peut conduire à des défauts importants dans le développement des plantes, comme le montrent des mutants d'Arabidopsis avec une croissance anormale des racines primaires et incapables à se développer jusqu’aux étapes reproductives. Les interactions potentielles entre GlcCer et les machineries de transport sont discutés, ainsi que les mécanismes cellulaires qui sont potentiellement déclenchés dans les cellules végétales pour compenser une perturbation de la biosynthèse du GlcCer. / The role of lipids as molecular actors in protein secretion is not well understood in plants. Glucosylceramide (GlcCer) is synthesized by Glucosylceramide Synthase (GCS) in plants and constitutes a key complex sphingolipid in membranes, but little is known about the plant cellular requirements for GlcCer. This study relied upon the block of GlcCer biosynthesis, by the use of inhibitors in tobacco and Arabidopsis, and the production of Arabidopsis mutants, to determine the impact of GlcCer biosynthesis on plant cell endomembrane dynamics. In a comprehensive approach that included lipid biochemistry, live cell imaging, ultrastructural studies by Transmission Electron Microscopy, and whole plant developmental studies, we have gained a better understanding of the impact of GlcCer in plant cells that can be summarised as follows: (1) Based on theoretical analysis and live-cell microscopy the GCS is located to the Endoplasmic Reticulum (at the beginning of the secretory pathway) in plant cells, (2) PDMP is a specific inhibitor of GCS in plants, (3) Disruption of GlcCer biosynthesis using PDMP and genetic approaches showed that GlcCer is important for normal protein trafficking and endomembrane dynamics, notably in the maintenance of Golgi structure, (4) The regulation of protein trafficking by the synthesis of GlcCer could be critical in the establishment and maintenance of cell polarity, as suggested by defects in the localisation of polar markers in Arabidopsis treated with PDMP, and (5) A block in GlcCer synthesis may be conducive to severe defects in plant development, as Arabidopsis mutants showed abnormal primary root growth and inability to develop to reproductive stages. Potential interactions between GlcCer and the transport machineries are discussed, as well as cellular mechanisms that may be set off following a disruption of GlcCer biosynthesis in plant cells.

Polygalacturonase-inhibiting proteins (PGIPs) are cell wall-associated plant proteins that inhibit endopolygalacturonases (endo-PGs) from phytopathogenic fungi. For proteins to confer resistance to invading plant pathogens, it is preferred that they are either associated with the plant cell wall or secreted into the intercellular spaces where they can act almost immediately upon pathogen attack. The bactericidal efficacy of the Hen Egg White Lysozyme (HEWL) has previously been unequivocally demonstrated in transgenic plants; however, most of the protein remains intracellular. It was hypothesized that bean PGIP1, that has previously been expressed correctly in transgenic tomato plants and was found to inhibit the endopolygalacturonase activity of Stenocarpella maydis in a reducing sugar assay, would deliver the HEWL protein to the intercellular spaces due to its inherent translocation to the plant cell wall by means of a translational fusion between bean pgip1 and hewl genes. In this study, the efficacy of such a translational fusion was determined. The bean pgip1-hewl fusion was inserted into the binary vector pCAMBIA2300 and transformed into Nicotiana tabacum cv. LA Burley 21 plants by Agrobacterium-mediated transformation. Phenotypically normal transgenic plants were produced. Stable transgene insertion into the transgenic N. tabacum genomes was verified by PCR and Southern blot analyses. To demonstrate the efficacy of the bean PGIP1-HEWL fusion, independent homogenate and intercellular fluid protein extracts were prepared from transgenic N. tabacum leaf material. Protein extracts prepared so as to enrich for PGIP activity were tested in vitro for inhibition of S. maydis endo-PGs whereas protein extracts for HEWL activity were tested for lysis of Micrococcus luteus cells. Biochemical assays showed that bean PGIP1-HEWL inhibited S. maydis endo-PGs and cleaved M. luteus cell walls sufficiently to suggest that the PGIP1- HEWL fusion was structurally and functionally stable. Total protein extracts from the PGIP-HEWL and HEWL transgenic plants showed similar levels of HEWL specific activity, whereas intercellular fluid samples from PGIP-HEWL transgenic plants showed high activity in contrast to HEWL plants. With the success of showing protein activity in vitro of HEWL in intercellular spaces, bean PGIP1 can be recommended as a vehicle for delivery of other proteins to cell surfaces. Copyright 2006, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. Please cite as follows: Feltman, NR 2006, Development of the polygalacturonase inhibiting protein (PGIP) for delivery of foreign proteins to the surfaces of plant cells, MSc dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-07152008-111131 / > E416/gm / Dissertation (MSc)--University of Pretoria, 2012. / Plant Science / unrestricted

Master of Science

Técnicas de cultivo in vitro, associadas à criopreservação, são ferramentas importantes para conservação ex situ de espécies recalcitrantes. O objetivo deste trabalho foi o estudo da criopreservação no sistema A. angustifolia. Um protocolo de criopreservação para culturas celulares da espécie foi desenvolvido e os níveis de PAs, ABA e ROS, ao longo do protocolo, foram avaliados. Embriões zigóticos foram utilizados como modelo para o estudo de diferentes aspectos biofísicos do processo de criopreservação como a permeabilidade dos crioprotetores, a capacidade de resposta osmótica e congelamento, presença de água nos tecidos e a análise histológica da organização e aspectos das estruturas celulares, comparados ao controle, não resfriado. Os resultados deste trabalho permitiram uma melhor compreensão dos diferentes aspectos fisiológicos, bioquímicos e biofísicos durante a criopreservação, em espécies recalcitrantes, além da perspectiva de estabelecimento de bancos de germoplasma ex situ de culturas celulares de A. angustifolia. / The aim of this work was the study of cryopreservation A. angustifolia plant systems. A protocol for cryopreservation of A. angustifolia cell cultures was developed based on optimum DMSO concentration. It was observed that cells exposed to cryoprotectants showed lower levels of PAs, ABA and ROS compared to cells that were not exposed to them. Two low molecular weight bands could be observed more intensely stained only in the cells that were exposed to the cryoprotectant. The zygotic embryos were used as a model to study different aspects of the biophysical process of cryopreservation such as the permeability of cryoprotectants, osmotic and response characteristics of water freezing in tissue. The results of this work led to a better understanding of different physiological, biochemical and biophysical factors during cryopreservation in recalcitrant species and open perspective to the establishment of the ex situ conservation of A. angustifolia.

Eucalyptus trees are a significant source of fuelwood, timber and raw material for the paper and pulp industry. In South Africa, Eucalyptus grandis and its hybrids are in high demand due to their fast growth and suitability of their timber for a wide range of products. Breeding and selection for superior quality eucalypts could sustain this high demand through selection and subsequent multiplication of superior genotypes and their use in controlled crosses. However, for this to be successful, a wide genepool must be available and maintained. Germplasm conservation of both vegetative and sexual material is therefore an integral part of such activities. However, in the case of trees, in vivo conservation practices are expensive and hazardous. The aim of this project was therefore to establish alternative conservation strategies for short-to-medium and long-term use of Eucalyptus spp. to facilitate on-going breeding and clonal programmes. For short-to-medium term storage, shoot cultures were subjected to various minimal growth conditions. Of the investigated treatments, reducing nutrients was the best storage method and shoots were maintained for 10 months with multiplication rates of 13.75 ± 7.05 shoots/explant. Encapsulated axillary buds were stored in jars at 10 °C or 28 °C. Of these two treatments, viability was sustained for longer (6 months) at 4 °C. Before establishing pollen storage regimes, viability assessment methods were evaluated and these consisted of in vitro germination on a BK (Brewbaker and Kwack, 1963) medium for 24 hours (26 ± 3.0%) and staining with two tetrazolium salts. Medium-term storage of pollen was best achieved by maintenance in the fridge (4 °C) without any desiccating substance (8 months at 6.73 ± 1.21%). Cryopreservation protocols were investigated for axillary buds and pollen. Buds that were 2 mm long were pretreated with chemical cryoprotectants, and a mixture of DMSO (dimethylsulphoxide) and glycerol was found to induce high survival rates (63%) after washing with MS (Murashige and Skoog, 1962) and 4 g.l-1 sucrose solution. Explants precultured in 1M sucrose showed increased tolerance (explant retained high survival rates of 80%) when desiccated to 20% moisture content fresh weight basis (fwb). Although pretreatments were successfully established, explants did not survive storage in liquid nitrogen indicating the need for further optimization of protocols. Pollen was successfully cryopreserved for 12 months with 23% survival rates. Applications and future research strategies of the developed protocols to Eucalyptus breeding programmes are discussed. / Thesis (M.Sc.)-University of Natal, Durban, 1998.

Técnicas de cultivo in vitro, associadas à criopreservação, são ferramentas importantes para conservação ex situ de espécies recalcitrantes. O objetivo deste trabalho foi o estudo da criopreservação no sistema A. angustifolia. Um protocolo de criopreservação para culturas celulares da espécie foi desenvolvido e os níveis de PAs, ABA e ROS, ao longo do protocolo, foram avaliados. Embriões zigóticos foram utilizados como modelo para o estudo de diferentes aspectos biofísicos do processo de criopreservação como a permeabilidade dos crioprotetores, a capacidade de resposta osmótica e congelamento, presença de água nos tecidos e a análise histológica da organização e aspectos das estruturas celulares, comparados ao controle, não resfriado. Os resultados deste trabalho permitiram uma melhor compreensão dos diferentes aspectos fisiológicos, bioquímicos e biofísicos durante a criopreservação, em espécies recalcitrantes, além da perspectiva de estabelecimento de bancos de germoplasma ex situ de culturas celulares de A. angustifolia. / The aim of this work was the study of cryopreservation A. angustifolia plant systems. A protocol for cryopreservation of A. angustifolia cell cultures was developed based on optimum DMSO concentration. It was observed that cells exposed to cryoprotectants showed lower levels of PAs, ABA and ROS compared to cells that were not exposed to them. Two low molecular weight bands could be observed more intensely stained only in the cells that were exposed to the cryoprotectant. The zygotic embryos were used as a model to study different aspects of the biophysical process of cryopreservation such as the permeability of cryoprotectants, osmotic and response characteristics of water freezing in tissue. The results of this work led to a better understanding of different physiological, biochemical and biophysical factors during cryopreservation in recalcitrant species and open perspective to the establishment of the ex situ conservation of A. angustifolia.