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A number of methods for the transfer of genes to plants are assessed in this work. The potential of Agrobacterium tumefaciens infection of germinating pollen tubes in vitro as a method of gene transfer was investigated and evidence presented for an essential pre-requisite of infection, that of attachment of Agrobacteria to the pollen tube wall, with both a dicot and a monocot species. In addition, the possibility of direct uptake of DNA molecules by germinating pollen tubes was suggested by in vitro uptake studies. Microinjection of DNA molecules into the loculus of ovaries, with the aim of facilitating in vivo DNA uptake by the male and/or female gametes, was investigated with Salpiglossis sinuata ovaries. Evidence was presented for the persistence of DNA molecules in the ovary loculus and gene transfer using a non-oncogenic Agrobacterium plasmid vector was attempted and the resulting progeny were screened for transformation: The techniques of Agrobacterium-infection of leaf discs and direct DNA uptake by protoplasts were applied to Nicotiana tabacum. DNA transformation vectors containing a kanamycin resistance marker gene and a chimeric pea seed storage protein gene were constructed for use in this study. The seed-specific promoter of the pea legumin A gene was replaced with the nopaline synthase promoter that is expressed in a more constitutive manner in plant tissues. Kanamycin resistant transgenic plants were regenerated following both transformation techniques and the presence and structure of inserted foreign DNA was determined by Southern blot hybridizations. The transmission of kanamycin resistance to transformant progeny after self-fertilization demonstrated characteristic Mendelian inheritance. The expression of the inserted legumin gene in leaf tissue of a number of Agrobacterium-denved transformants was assessed by Northern blot hybridization; legumin transcripts were detected, although the protein immuno-detection procedures of Western blotting and ELISA did not detect legumin in the seeds or leaf tissue of transgenic plants derived from either transformation technique.

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Starch is an important component of a wheat grain, comprising 50-70% of its dry weight. Its biosynthesis involves a complex pathway mediated by several enzymes, each of which is encoded by genes that have more than one family member. To better understand starch synthesis in wheat grains, this study characterized the sucrose-starch metabolic pathway using physiological, molecular, biochemical and metabolic approaches. These analyses led to the identification of genes that appear to have predominant expression during grain development in wheat including, TaSUT1, TaSuSy2, AGPL1, SSI, SSIIIa and SBEIIa, suggesting that these genes play a regulatory role in starch accumulation. This was further confirmed by comparative analyses of starch synthesis between cultivars with contrasting thousand kernel weights, which revealed a closer association of the expression of the same set of genes with starch accumulation in developing wheat grains. The effect on starch yield of one of the candidate genes identified, AGPase, was examined through a transgenic approach, which involved expression of a gene encoding modified version of maize AGPase large subunit, designated as Sh2r6hs, in wheat under the control of maize’s constitutive Ubiquitin1 promoter. This manipulation of the wheat AGPase activity produced wheat lines with increased AGPase activity, grain weight and grain starch level, suggesting that the wheat grain size can be enhanced through increasing the capacity of starch synthesis both in the source and sink tissues. The study also identified and characterized a partial fragment of wheat rbcS promoter, and indicated that the promoter fragment can potentially be used as a tool for targeting the expression of genes of interest in photosynthetic source tissues. / October 2014

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Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Roman Reddy R. Ganta / Ehrlichia chaffeensis, an emerging tick-borne pathogen, causes human monocytic ehrlichiosis (HME). The relationship between E. chaffeensis and its target cells in ticks and vertebrates is critical as the organism must persist in them. We hypothesize that E. chaffeensis alters gene expression in support of adapting to dual hosts. In support of testing this hypothesis, we developed an ORF-based microarray and performed global transcriptional analysis on the pathogen grown in macrophage and tick cells. The analysis revealed the expression of about 30% of all the predicted E. chaffeensis genes, in macrophages or tick cell. Two-thirds of the transcribed genes are common for both host cell backgrounds. About 20% of the commonly expressed genes also varied in expression levels which ranged from two to five fold. Microarray data was verified by RT-PCR for a subset of randomly selected genes. Together, this is the first report describing the global host cell-specific gene expression patterns in E. chaffeensis. Differential gene expression may be an important adaptive mechanism used by E. chaffeensis for its continued survival in dual hosts. To test this hypothesis, we established many basic protocols and tools needed for performing mutational analysis in E. chaffeensis. Four antibiotic selection markers; gentamicin, chloramphenicol, spectinomycin and rifampin, and two promoters constitutively expressed in E. chaffeensis, genes rpsL and tr, were identified. Two regions of the genome were also identified for performing initial mutational analysis. Several plasmid constructs were also made. The optimal conditions for introducing these plasmids into host cell-free viable E. chaffeensis organisms were also established. The molecular evaluation of several E. chaffeensis transformants using these plasmids suggested that the plasmids gained entry, but failed to get integrated into the genome or remain in the bacteria for longer periods of time. In summary, we demonstrated global host cell-specific differential gene expression in E. chaffeensis by employing microarray analysis. Numerous host-specific expressed genes will be important for studies leading to effective methods of control. We also established several basic protocols and tools needed for performing mutational analysis useful in evaluating the impact of the loss of expression of uniquely expressed genes.

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