Thesis (M. Sc.)--University of Canterbury, 2008. / Typescript (photocopy). Includes bibliographical references (leaves 62-79). Also available via the World Wide Web.

Thesis (doctoral)--Helsinki University of Technology, 2008. / Includes bibliographical references. Also available on the World Wide Web.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 78-94).

Thesis (MSc)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: The genetic information of plants is found in the nucleus, the mitochondria, and the plastids. The DNA of plastids is comprised of multiple copies of a double-stranded, circular, prokaryoticallyderived genome of -150 kb. The genome equivalents of plastid organelles in higher plant cells are an attractive target for genetic engineering as high protein expression levels are readily obtained due to the high genome copy number per organelle. The resultant proteins are contained within the plastid organelle and the corresponding transgenes are inherited, in most crop plants, uniparentally, preventing pollen transmission of DNA. Plastid transformation involves the uniform modification of all the plastid genome copies, a process facilitated by homologous recombination and the non-Mendelian segregation of plastids upon cell division. The plastid genomes are in a continuous state of inter- and intra-molecular exchange due to their common genetic complement. This enables the site-specific integration of any piece of DNA flanked by plastid targeting sequences, via homologous recombination. The attainment of homoplasmy, where all genomes are transformed, requires the inclusion of a plastid-specific selectable marker. Selective pressure favouring the propagation of the transformed genome copies, as well as the random segregation of plastids upon cell division, make it feasible to acquire uniformity and hence genetic stability. From this, a complete transplastomie line is obtained where all plastid genome copies present are transgenic, having eliminated all wild-type genome copies. The prokaryotic nature of the chloroplast genetic system enables expression of multiple proteins from polycistronic mRNAs, allowing the introduction of entire operons in a single transformation. Expression cassettes in vectors thus include single regulatory elements of plastid origin, and harbour genes encoding selectable and screenable markers, as well as one or more genes of interest. Each coding region is preceded by an appropriate translation control region to ensure efficient translation from the polycistronic mRNA. The function of a plastid transformation vector is to enable transfer and stable integration of foreign genes into the chloroplast genomes of higher plants. The expression vector constructed in this research is specific for the transformation of the grape chloroplast genome. Vitis vinifera L., from the family, Vitaceae, is the choice species for the production of wine and therefore our target for plastid transformation. All chloroplast derived regulatory elements and sequences included in the vector thus originated from this species. / AFRIKAANSE OPSOMMING: Die genetiese inligting van plante word gevind in die kern, die mitochondria, en die plastiede. Die DNA van plastiede bestaan uit veelvuldige kopieë van 'n ~ 150 kb dubbelstring, sirkulêre genoom van prokariotiese oorsprong. Die genoomekwivalente van plastiede in hoër plante is 'n aantreklike teiken vir genetiese manipulering, aangesien die hoë genoom kopiegetal per organel dit moontlik maak om gereeld hoë vlakke van proteïenuitdrukking te verkry. Hierdie proteïene word tot die plastied beperk, en die ooreenstemmende transgene word in die meeste plante sitoplasmies oorgeërf, sonder die oordrag van DNA deur die stuifmeel. Plastied transformasie behels die uniforme modifikasie van al die plastied genoomkopieë, 'n proses wat deur homoloë rekombinasie en die nie-Mendeliese segregasie van plastiede tydens seldeling gefasiliteer word. As gevolg van die gemeenskaplike genetiese komplement, vind aanhoudende interen intra-molekulêre uitruiling van plastiedgenome plaas. Dit maak die setel-spesifieke integrasie, via homoloë rekombinasie, van enige stuk DNA wat deur plastied teikenvolgordes begrens word, moontlik. Vir die verkrying van homoplasmie, waar alle genome getransformeer is, word die insluiting van 'n plastiedspesifieke selekteerbare merker benodig. Seleksiedruk wat die vermeerdering van die getransformeerde genoomkopieë bevoordeel, en die lukrake segregasie van plastiede tydens seldeling, maak dit moontlik om genetiese stabiliteit en uniformiteit van die genoom te verkry. Dit kan op sy beurt tot die verkryging van 'n volledige transplastomiese lyn lei, waar alle aanwesige plastiedgenome transgenies is, en wilde tipe genoomkopieë geëlimineer is. Die prokariotiese aard van die chloroplas genetiese sisteem maak die uitdrukking van veelvuldige proteïene vanaf polisistroniese mRNAs moontlik, wat die toevoeging van volledige operons in 'n enkele transformasie toelaat. Uitdrukkingskassette in vektore bevat dus enkel regulatoriese elemente van plastied oorsprong, gene wat kodeer vir selekteerbare en sifbare merkers, asook een of meer gene van belang (teikengene). Voor elke koderingsstreek, is daar ook 'n toepaslike translasie beheerstreek om doeltreffende translasie vanaf die polisistroniese mRNA te verseker. Die funksie van 'n plastied transformasie vektor is om die oordrag en stabiele integrasie van transgene in chloroplasgenome van hoër plante moontlik te maak. Die uitdrukkingsvektor wat in hierdie studie gekonstrueer is, is spesifiek vir die transformasie van die druif chloroplasgenoom. Vitis vinifera L., van die familie Vitaceae, is die voorkeur species vir die produksie van wyn, en daarom die teiken vir plastied transformasie. Alle chloroplast-afgeleide regulatoriese elemente en volgordes wat in hierdie vektor ingesluit is, het huloorsprong vanaf VUis vinifera L.

Cancer and hepatitis viruses are two of the leading causes of death worldwide. Recombinant Interferon alpha2b (IFNa2b) is used as an immunotherapeutic drug for cancers, hepatitis viruses and several other viral diseases. Interferons are produced in low quantity naturally and production cost for recombinant IFNa2b in E.coli is very high. Since prokaryotes cannot form disulfide bonds, additional techniques have to be employed to create a functional form of IFNa2b. The average cost per patient for treatment with recombinant IFNa2b is $26,000 per year. Around 800 million people in the world are infected with Hepatitis C virus and most of them cannot afford the treatment costs. Producing recombinant IFNa2b in tobacco chloroplasts will overcome these problems and make the drug affordable for many people. A recombinant IFNa2b chloroplast vector was introduced into the tobacco cultivars Petit Havana (model) and LAMD-609 (low nicotine hybrid plants) in the Daqjell lab by particle gun bombardment. In this research, second-generation transgenic plants with the IFNa2b gene are subjected to various experiments to study the levels of IFNa2b expression. The psbA regulatory sequences present in the chloroplast vector are known to enhance protein expression in the presence of light. To analyze this effect and to find optimal growth conditions for maximal IFN a2b production, continuous light studies were performed. These results can be vital for mass production of IFNa2b.

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Thesis (Ph.D.) -- University of Western Sydney, 2002. / "A thesis submitted in fulfilment of the requirement for the degree of Doctor of Philosophy" Bibliography: leaves 161-189.

A hibridação somática de citros tem sido extensivamente aplicada, favorecendo o desenvolvimento de híbridos somáticos em programas de melhoramento genético, como fonte de germoplasma ou como variedades copa e porta-enxerto. Neste contexto, este trabalho foi desenvolvido com o objetivo de selecionar plantas de toranja (Citrus grandis L. Osbeck) tolerantes à Phytophthora sp. e utilizá-las como parentais no processo de hibridação somática com outras espécies do gênero Citrus, a fim de produzir híbridos somáticos para o melhoramento de porta-enxertos. Plantas de 20 variedades de toranja tolerantes à Phytophthora spp. foram selecionadas, após serem cultivadas em solo infestado. Experimentos de fusão de protoplastos foram realizados envolvendo laranjas doces, tangerinas e o tangor ‘Murcote’, como parentais embriogênicos, e variedades de toranja selecionadas e plantas enxertadas de 12 variedades de toranja, como parentais não-embriogênicos, utilizando-se a técnica de fusão química, via polietilenoglicol (PEG). Microcolônias foram transferidas para meio de cultura MT semi-sólido, suplementado com 500 mg.l -1 de extrato de malte para indução da embriogênese somática. A confirmação da hibridação somática das plantas regeneradas e aclimatizadas foi realizada por meio de análises de morfologia foliar, de citologia, pela contagem do número de cromossomos, e moleculares, por marcadores do tipo RAPD. As metodologias utilizadas para seleção de plantas matrizes, fusão de protoplastos, regeneração de plantas e confirmação da hibridação somática foram adequadas, e permitiram a obtenção de híbridos somáticos de laranja ‘Hamlin’ com toranja enxertada ‘Indian Red’ e com ‘seedling’ selecionado de toranja ‘Singapura’, que apresentam potencial para serem incorporados em programas de melhoramento de porta-enxertos. / Citrus somatic hybridization has been extensively applied assisting the development of the somatic hybrids which can be used in improvement programs, indirectly as germoplasm source or directly as scion and rootstock varieties. In this context, this research was developed with the objective of selecting plants of pummelo (C. grandis L. Osb) tolerant to Phytophthora sp. and use these plants as parents in the somatic hybridization process with other species of Citrus. Plants of 20 pummelo varieties, tolerant to Phytophthora sp., were selected after being grown in infested soil. Protoplast fusion experiments were induced by chemical method, with polyethylene glicol (PEG), involving sweet oranges, mandarins and Murcott tangor, as embryogenic parents, selected pummelo varieties and grafted plants of 12 pummelo varieties, as non-embryogenic parents. Microcolonies were transferred to EME semi-solid MT containing 500 mg.l -1 of malt extract for somatic embryogenesis. Somatic hybridization was confirmed by analysis of leaf morphology, citology by chromosome counting and molecular analysis by RAPD markers. The protocols used to select plants to be used as protoplast source, protoplast fusion, plant regeneration and somatic hybridization confirmation were adequate, allowing to produce somatic hybrids of Hamlin sweet orange with Indian Red grafted pummelo and Singapura pummelo selected seedling, which may be used as rootstocks and incorporated in rootstocks improvement programs.