Tomato serves as an important model organism for Solanaceae in both molecular and agronomic research. With whole genome sequencing in progress, there is a need to study functional genetics through mutant lines that exceed the practical limitations imposed by the popular research cultivar, Micro-Tom. This project utilized Agrobacterium transformation and the transposon tagging construct, Ac-DsATag-Bar_gosGFP, to produce activation tagged and knockout mutants in the processing tomato variety, M82. The construct contained hygromycin resistance (hyg), green fluorescent protein (GFP), and maize transposase (TPase) on the stable Ac element, along with a 35S enhancer tetramer and glufosinate herbicide resistance (BAR) on the mobile Ds element. An in vitro propagation strategy was used to produce a population of 25 T0 plants from a single transformed plant regenerated in tissue culture. A T1 population of 10,568 selfed and M82 backcross progeny was produced from the functional T0 line. This population was screened by spraying with 0.05% Liberty® herbicide, followed by a 100 mg/L hygromycin leaf painting procedure to select for Ds only (herbicide tolerant and hygromycin sensitive) individuals. The T-DNA genotype of Ds only plants was confirmed through multiplex PCR and the location of insertions within the genome was determined through TAIL-PCR. Resulting product sequences were blasted against the pre-publication tomato genome browser to determine insertion sites. A population of 309 independent transposants dispersed to all twelve chromosomes from the original insertion site on chromosome five has been developed. The transposon tagged lines are currently being immortalized in seed stocks. / Master of Science

Vitamin C (ascorbic acid, AsA), an important primary metabolite of plants, functions as an antioxidant, an enzyme cofactor, and a cell-signaling modulator in a wide array of crucial physiological processes including biosynthesis of the cell wall, secondary metabolites and phytohormones, stress resistance, photoprotection, cell division, senescence, and growth. To identify genes that may regulate vitamin C levels in plants, about 3000 activation-tagged Arabidopsis lines were treated with ozone, which is a power oxidizing agent. Two mutants were selected for identification of potential genes involved in the regulation of vitamin C synthesis. A putative F-box gene, VCF1, and a purple acid phosphatase, AtPAP15, were identified for further characterization. Two homozygous SALK T-DNA knockouts in the open reading frame (ORF) of VCF1 exhibited high tolerance to ozone when treated with 450 ppb for 3 hours and the AsA levels of these mutants were 2 to 3 fold higher than wild-type (wt) plants. Developmental studies, using RT-PCR, indicated that foliar expression of the VCF1 gene increased with plant age from 1 to 5 weeks, whereas AsA decreased during this same period. The expression of VCF1 was higher under a low-light condition in which AsA was reduced considerably. The AsA levels in two VCF1 overexpressing lines were only 50 to 70% of wt plants. These results suggested that the putative F-box gene functions as a negative regulator of leaf ascorbate content. Overexpression of AtPAP15 with the CaMV 35S promoter resulted in up to 3-fold higher AsA levels than wt plants, where two independent SALK T-DNA insertion mutants in AtPAP15 had 50% less AsA than wt plants. Enzyme activity of bacterially expressed GST:AtPAP15 was greatest with phytate as a substrate indicating that AtPAP15 is a phytase. Phytase catalyzes hydrolysis of phytate (myo-inositol hexakisphosphate) to yield myo-inositol and free phosphate. Thus, AtPAP15 may regulate AsA levels by controlling the input of myo-inositol into this branch of AsA biosynthesis in Arabidopsis. AtPAP15 was expressed in all tested organs in wt plants and suggests that the enzyme may have functions other than phytate degradation during seed germination. / Ph. D.

As a warm season grass native to the North American continent, switchgrass is considered as one of the most promising biofuel crops in the USA. It is a C4 plant that makes it energy efficient. Switchgrass has a deep root system that allows it to grow on marginal land with low water and nutrient input. Switchgrass has been used as a forage crop and its use for biofuel will not affect food security. Biofuels are more environment-friendly than fossil fuels as they do not produce net greenhouse gases. However, the problem of high cost of production per unit for biofuel has to be overcome if we want to replace fossil fuels with biofuels. One of the major factors related to the high cost of biofuel are the expensive cellulase enzymes used in the pretreatment of feedstock. Endoglucanase is the key enzyme used for breaking down cellulose before fermentation. Currently, endoglucanase is produced from engineered E. coli or yeast strains, which is still expensive for enzyme production and purification of industrial scales. Expression of endoglucanase in plants has been previously reported. However, there are no reports of transgenic switchgrass producing cellulase enzyme. In this study, the catalytic domain of beta-endoglucanase gene was codon-optimized and synthesized based on the cDNA cloned from Hypocrea jecorina. Rice RuBisCO small subunit targeting signal peptide was fused to the N-terminus of the beta-endoglucanase gene, which was expected to target the fusion protein to chloroplast. This subcellular compartment targeting could minimize negative effects on cell function and plant development. The endoglucanase gene was cloned with maize ubiquitin promoter in a modified binary vector pCambia 1305-2 and transformed into switchgrass genotype HR8 by using Agrobacterium tumefaciens. In this study, I generated five independent transgenic switchgrass lines and they were confirmed by growing on the selection agent hygromycin, GUS assay, PCR amplification, southern blotting hybridization, for the presence of hygromycin and endoglucanase genes. However, based on RT-PCR analysis, only two transgenic lines were confirmed to produce mRNAs of the endoglucanase gene. These two transgenic lines were further characterized for their agronomic traits and the chlorophyll contents. Our results suggested that expression of endoglucanase in switchgrass could reduce chlorophyll content and affect plant development. Nevertheless, in this study, we demonstrated that a fungal endoglucanase gene could be expressed in switchgrass transgenic plants, though the gene expression level and the subcellular localization need to be carefully regulated in order to minimize the toxic effect of endoglucanase on plant cells. / Master of Science