Abiotic stresses such as drought, salt and exposure to toxic metals adversely affect the growth and productivity of crop plants and are serious threats to agriculture. We are currently working with an Arabidopsis family of proteins known as Stress Associated Proteins (SAPs). There are a total of 14 proteins in the Arabidopsis SAP family whose members have been suggested to provide tolerance to abiotic stresses in plants. For this project, we aim to characterize AtSAP13, which codes for a protein of 249 amino acid residues. Through overexpression, we investigated the sensitivity or tolerances provided by the overexpressed protein in comparison to wild-type plants. Our preliminary results showed that Arabidopsis plants overexpressing SAP13 showed strong tolerance to arsenite, cadmium, and zinc. A semi-quantitative RT-PCR was performed to analyze SAP13’s mRNA levels in wild type plants exposed to the same set of stresses. Tissue specific expression was analyzed using a GUS histochemical assay. Sub-cellular localization of AtSAP13 was analyzed by creating an in-frame fusion of SAP13 and enhanced GFP (eGFP). We set out to optimize growth conditions for Crambe abyssinica in order to determine if Crambe could be grown as a biofuel crop in New England. We have determined that Crambe can be grown successfully in the New England climate. We tested three fertilizer application rates, two different cultivars, and two different soil types for changes in yield. In the end, we observed the greatest increase in yield when planted on well drained soil.

Flavonoids are plant secondary metabolites well known for many key roles in the life cycle of plants. They also can affect human health. Citrus paradisi is known to produce several glucosylated flavonoids and these compounds are glucosylated by enzymes known as glucosyltransferases (GTs). The focus of this research was to optimize the heterologous expression, enrichment, and biochemical characterization of grapefruit putative GT protein, PGT2, and to test the hypothesis that PGT2 is a flavonoid GT. Results showed detectable amounts of activity with quercetin, a flavonol; however, activity was lower than what would be expected if this enzyme were a flavonol-specific GT. In an additional aspect of this study, bioinformatics were used to test the hypothesis that additional putative GT clones could be identified using the harvEST database.

Genetic modification of plastids in the model plant tobacco (Nicotiana tabacum) has demonstrated that numerous foreign gene products can accumulate to high levels in this setting. Plastid biotechnology is maturing to encompass the improvement of food and feed species and the production of biopharmaceutical proteins for oral delivery necessitating development of stable transplastomic edible plants. In the interest of establishing an edible platform we have investigated the use of native and foreign regulatory elements in relation to foreign gene expression in plastids. Multiple sequence alignments of intergenic regions for 20 species of angiosperm showed that despite 95% identity in the coding region, identity in the psbA upstream region is 59% across all taxa examined, other gene coding regions displayed sequence identity of 80-97%, whereas the non-coding regions were 45-79% suggesting that our physical data can be extrapolated beyond the model presented. We found that by exchanging psbA untranslated regions (UTRs) between N. tabacum and lettuce (Lactuca sativa), the expression of the CTB-proinsulin (CTB-Pins) monocistronic transcript declined by 84% and foreign protein accumulation was reduced by as much as 97% in mature leaves. Polyribosome association assays suggest that ribosome-free transgenic transcripts are stabilized where the native UTR is employed. RNA EMSA revealed that binding proteins interacted with psbA 5' UTRs in a species specific manner and the half life of the L. sativa 5'UTR-CTB-Pins mRNA was reduced by 3.7 fold in N. tabacum stromal extracts. Our data indicate that the use of species-specific regulatory elements could lead to establishment of reproducible plastid transformation in desirable target species such as L. sativa. Using transplastomic L. sativa for oral delivery of bioencapsulated CTB-Pins we delayed the onset of diabetes in NOD mice when retinyl acetate supplement was provided compared to untouched mice. In this 30 week study we monitored blood glucose levels and evaluated the in vitro suppressive capacity of regulatory T cells isolated from diabetic mice. Whether delay or prevention was achieved appeared to be a function of antigen dose as high dose resulted in a nine week delay of onset while low dose reduced the incidence of diabetes by 36%. In addition we have evaluated metabolic engineering in the N. tabacum model where we generated cis-genic lines expressing nucleus-encoded methionine pathway enzymes in plastids. Transplastomic expression of Cystathionine gamma-Synthase led to a three-fold increase in enzyme activity and a doubling of methionine content in leaves without a deleterious phenotype. In exploring molecular mechanisms supporting gene expression in plastids and applying transplastomic technology to real human problems this work seeks address the potential of plastid biotechnology for improvement of commodity crops and production of biopharmaceuticals.

Nitrogen use efficient (NUE) wheat varieties have potential to reduce input costs for growers, limit N runoff into water ways, and increase wheat adaptability to warmer environments. Previous studies have done little to explain the genetic basis for NUE and components, nitrogen uptake efficiency (NUpE) and nitrogen utilization efficiency (NUtE). Four studies were conducted to 1) determine genotypic stability of NUE under high and low N regimes and under warming 2) determine effect of warming on NUE 3) indentify QTL associated with NUE components 4) assess the utility of canopy spectral reflectance (CSR) as a high-throughput phenotyping device for NUE. Genotypic response to N stress or warming varied. Uptake efficiency was found to be more important than utilization efficiency to genotypic performance under high and low N environments and under warming. Selection under low N for NUpE and under high N for NUtE most efficiently identified NUE varieties. Uptake and utilization were lower under warming due to quickened development. No strong correlations between the CSR indices and NUE existed. No QTL were found to be significantly associated with NUE components. Further research into the mechanisms controlling NUE and to reveal plant response to N stress and under warming is necessary.

No description available.

Araucaria cunninghamii is an ancient tropical conifer with substantial value as a forestry species in Australia and Papua New Guinea, and has been subject to a genetic improvement program for more than 50 years. This study was undertaken to demonstrate the utility of quantitative genetic analysis in describing the genetic architecture of commercial traits in A. cunninghamii. Linkage maps were prepared using the pseudotestcross strategy in what was believed to be a wide interprovenance cross using microsatellites and AFLP. A very low rate of marker polymorphism and limited differentiation between the parental provenances was identified, resulting in low mapping efficiency. The population genetic structure of A. cunninghamii was assessed to establish the underlying causes for the limited differentiation and low marker heterozygosity and assess the implications for future analysis of quantitative traits. Despite the limited mapping efficiency, genetic maps were generated for both parents. The maternal map for individual H15 contained 14 linkage groups comprising of 51 AFLP and one microsatellite. The map covered 1290 cM, representing 89% of the estimated genome size. The paternal map for individual Gil24 was 633 cM, consisting of eight linkage groups. Genetic architecture of quantitative traits was examined with putative QTL identified for height, DBH and stem straightness; one was highly significant (p<0.01), three significant (0.01<p<0.05) and 13 suggestive (p<0.10). Significant QTL each accounted for 7-11% of the phenotypic variance with a high allele substitution effect (0.63-0.81). These QTL were likely to be associated with genes of moderate effect. The suggestive QTL each accounted for 3-6% of the phenotypic variance with an allele substitution effect of 0.40-0.63. Three genomic regions contributed to the expression of multiple traits at multiple ages. Stable QTL had decreasing phenotypic effects with increasing age. The population genetic survey characterised low levels of allelic diversity across the geographic range. Three broad regions were characterised; Papua New Guinea, Cape York and northern Queensland to NSW. There was limited differentiation between provenances within these regions, and high diversity within provenances. Limited genetic differentiation between provenances seems to be the result of genetic stability due to long overlapping generations, limited founder effects and a very low mutation rate. The latter may also contribute the low heterozygosity. Limited marker polymorphism and limited differentiation between provenances within broad regions are common features in A. cunninghamii. Therefore careful parental selection and alternative experimental approaches will be required before undertaking further analysis of quantitative traits.

Part 1: Visitation of potential pollinators to Cornus florida and C. kousa flowers was assessed in East Tennessee in 2008 and 2009. Data regarding insect visitation rates to multiple trees per location were gathered throughout the flowering period. Diurnal and seasonal variations in visitation were recorded. Pollen coverage was assessed on portions of captured insect exoskeletons that were most likely to contact the stigma. Eleven families in four insect orders were collected from C. florida and 26 families in five orders from C. kousa. The most important pollinators in eastern Tennessee were bees in the Andrenidae and Halictidae. The most common visitors to C. kousa flowers were scarab, cerambycid and cantharid beetles. Halictid bees were also frequent visitors to C. kousa. Part 2: Dispersion of flowering dogwood pollen in an orchard was evaluated by performing parentage analysis on open pollinated seedlings collected from a single maternal tree. Pollen source for 45 seedling trees were established using three polymorphic simple sequence repeat (SSR) loci. The expected leptokurtic distribution was not seen. Although the majority of paternal trees were within the 12 m radius of the study area, the trees most likely to donate pollen were some of the furthest away. Unusual pollen movement may be a product of lack of synchronization of flowering times between the mother tree and potential pollen donors in the area. Additionally relative proportions of certain pollinating insects (andrenid and halictid bees) may have moved pollen further than expected. Appendix 1: Pollen from 6 species in the genus Cornus was analyzed with Fourier Transform Infrared (FTIR) Spectroscopy to determine the utility of this tool to identify pollen grains to species. Although there were differences visible in the spectra, principal components analysis coupled with cluster analysis could not consistently identify pollen grains. Appendix 2: Floral volatile emissions from 6 species of Cornus were collected in a headspace chamber and analyzed with gas chromatography-mass spectrometry to determine if differences between emitted volatiles of C. florida and C. kousa explain differences in floral insect visitation. The relationship between floral volatile emission and the phylogeny of Cornus was also examined.

Medicago is widely used as a forage crop. It is often susceptible to various pathogenic infections and exhibits low growth in drought and extreme climatic conditions. In the current study, a strategy was developed for over-expressing an “Osmotin-Chitinase” gene chimera in transgenic Medicago that could potentially confer resistance to different biotic and abiotic stresses. Seed germination of several cultivars of Medicago (M. sativa ssp. sativa, M. sativa ssp. falcata, M. sativa ssp. caerulea, M. truncatula, and M. Rugosa) was tested to determine the cultivars with good germination rates. Among these, M. sativa ssp. sativa showed an average of 80% germination over a period of one week and was subsequently selected for regeneration and transformation experiments. Different explants (cotyledons, hypocotyls, petioles) were tested for regeneration. Among these, hypocotyl explants showed highest (46.17 %) percent regeneration. Escherichia coli harboring Osmotin-Chitinase (OSM-CHI) gene chimera cloned into binary vector pBTEX with nptII as a selection marker was mobilized in Agrobacterium tumefaciens strain EHA105 which was employed in the transformation of hypocotyl explants of Medicago. Transformed calli were grown on callus inducing medium containing kanamycin for screening. Further screening of the positive transgenics was performed using PCR. Southern hybridization was carried out for further confirmation of successful transformation. Transformed shoots will be grown on the root inducing medium for developing into plantlets which would then be transferred to the green house and later tested for their degree of resistance to various biotic and abiotic stresses.

Part 1: Visitation of potential pollinators to Cornus florida and C. kousa flowers was assessed in East Tennessee in 2008 and 2009. Data regarding insect visitation rates to multiple trees per location were gathered throughout the flowering period. Diurnal and seasonal variations in visitation were recorded. Pollen coverage was assessed on portions of captured insect exoskeletons that were most likely to contact the stigma. Eleven families in four insect orders were collected from C. florida and 26 families in five orders from C. kousa. The most important pollinators in eastern Tennessee were bees in the Andrenidae and Halictidae. The most common visitors to C. kousa flowers were scarab, cerambycid and cantharid beetles. Halictid bees were also frequent visitors to C. kousa.Part 2: Dispersion of flowering dogwood pollen in an orchard was evaluated by performing parentage analysis on open pollinated seedlings collected from a single maternal tree. Pollen source for 45 seedling trees were established using three polymorphic simple sequence repeat (SSR) loci. The expected leptokurtic distribution was not seen. Although the majority of paternal trees were within the 12 m radius of the study area, the trees most likely to donate pollen were some of the furthest away. Unusual pollen movement may be a product of lack of synchronization of flowering times between the mother tree and potential pollen donors in the area. Additionally relative proportions of certain pollinating insects (andrenid and halictid bees) may have moved pollen further than expected.Appendix 1: Pollen from 6 species in the genus Cornus was analyzed with Fourier Transform Infrared (FTIR) Spectroscopy to determine the utility of this tool to identify pollen grains to species. Although there were differences visible in the spectra, principal components analysis coupled with cluster analysis could not consistently identify pollen grains.Appendix 2: Floral volatile emissions from 6 species of Cornus were collected in a headspace chamber and analyzed with gas chromatography-mass spectrometry to determine if differences between emitted volatiles of C. florida and C. kousa explain differences in floral insect visitation. The relationship between floral volatile emission and the phylogeny of Cornus was also examined.

Fusarium head blight (FHB), caused by Fusarium graminearum Schwabe [telomorph: Gibberella zeae Schwein.(Petch)], is recognized as one of the most destructive diseases of wheat (Triticum aestivum L. and T. durum L.) and barley (Hordeum vulgare L.) worldwide. Breeding for FHB resistance must be accompanied by selection for desirable agronomic traits. Donor parents with two FHB resistance quantitative trait loci (QTL) Fhb1 (chromosome 3BS) and QFhs.nau-2DL (chromosome 2DL) were crossed to four adapted SRW wheat lines to generate backcross and forward cross progeny. F2 individuals were genotyped and assigned to 4 different groups according to presence/ absence of one or both QTL. The effectiveness of these QTL in reducing FHB in F2 derived lines was assessed in a misted, inoculated scab nursery. Resistance alleles and the interaction among FHB resistance QTL have distinct behavior in different genetic backgrounds in wheat. Fhb1 showed an average disease reduction of 12%, however it did not result in significant improvement of FHB resistance in all populations. In general, for the four backgrounds studied, the QFhs.nau-2DL QTL as more effective reducing FHB (19% average reduction). The combination of Fhb1 and QFhs.nau-2DL is not necessary, but recommended and it improved resistance in all populations. Backcross derived (BC) progeny from four genetic backgrounds were planted in replicated plots (2011 and 2012) and in the scab nursery in 2012. Population 2 had its progeny characterized by 961 DArT markers distributed throughout the genome. Several high-quality polymorphic markers were identified and listed as good predictors of phenotypic traits like disease resistance, and improved agronomic and quality characteristics. Backcross and forward cross derived progenies were tested for FHB resistance and agronomic and baking quality performance for 4 different populations sharing the same donor parent for resistance QTL. The results confirmed that F2 populations were effective indicators of expression levels of QTL prior to extensive backcrossing. The QTL Fhb1 and QFhs.nau-2DL increased FHB resistance without detriments on agronomic and quality traits on wheat populations investigated. BC populations were assessed as breeding populations and established as being rewarding tools for derivation of inbred lines in a breeding program, being BC2 the most recommended from our results.