This thesis reports the cell culture establishment and a somatic cell selection system optimized for the isolation of chlorsulfuron-resistant variants in asparagus (Asparagus officinalis L.). The development of this cell selection system benefited the isolation of chlorsulfuron-resistant variants from an elite asparagus genotype. A cell culture system, suitable for somatic cell selection, was established for asparagus genotype CRD 168. Friable callus was initiated from etiolated shoots in darkness and used to produce a high density of single cells in suspension. Cell density was estimated based on a linear relationship with settled cell volume. A mean plating efficiency of 0.19 % was recorded between 1-4x10⁵ cells/Petri dish. In vitro cell selection techniques were developed to identify mutant asparagus cells with resistance to a sulfonylurea herbicide, chlorsulfuron. A few key aspects were important to achieve this: a cell culture system for cell selection was initially established; a toxic concentration for the complete growth inhibition of the wild type asparagus cells was defined; rare, resistant cell colonies were isolated and characterized; and chlorsulfuron-resistant plants were regenerated. From about 50 million cells, 165 cell colonies were isolated in the presence of 8 nM chlorsulfuron. Characterization of these selected cell colonies yielded 24 escapes, 98 unstable variants, and 43 stable-resistant variants. Callus cultures from 34 of these stable variants retained resistance following 11 months growth in the absence of the selection agent. Plants were regenerated from 36 of these stable herbicide-resistant variants. Six of these chlorsulfuron-resistant variants were screened for their degree of resistance to chlorsulfuron, cross resistance to other acetohydroxyacid synthase (AHAS) inhibiting herbicides and AHAS enzyme activity. Cross resistance to imazamox was evident in four of the resistant variants, while lack of cross resistance to metsulfuron methyl was observed in all six resistant variants. A varying degree of resistance to chlorsulfuron was observed among the resistant variants. Both in the original and secondary callus, an uninhibited AHAS enzyme activity in all six resistant variants was recorded in the presence of high chlorsulfuron concentration (70-140 nM), compared to the total inhibition in the wild type. One chlorsulfuron-resistant variant, R-45, was used to compare the biochemical and physiological basis of resistance with the wild type. The AHAS enzyme activity in the tissue culture and greenhouse foliage of R-45 was significantly higher in the presence of up to 280 nM chlorsulfuron compared with the wild type. Chlorsulfuron retention was considerably higher due to the reduction of epicuticular wax deposits on the foliage of R-45, in comparison with the wild type. Consequently, the resistant line absorbed at least 1.6 fold more chlorsulfuron than the wild type plants. Therefore, foliar application of 15 g a.i./ha Glean (commercial formulation of chlorsulfuron) produced typical symptoms of chlorosis in R-45, similar to the wild type, in the greenhouse plants. Somatic cell selection was carried out using two elite asparagus genotypes, CRD 74 and Clone X. Of the 33 rare cell colonies isolated from Clone X, 22 unstable variants and 6 escapes were discarded. All five remaining resistant variants produced plants. One of the stable-resistant variants (Clone X-24) was evaluated for resistance to chlorsulfuron. Both in vitro shoot cultures and greenhouse-grown plants of Clone X-24 showed increased resistance to chlorsulfuron compared with the wild type. The AHAS enzyme activity in the foliar extracts also showed the presence of higher enzyme activity in Clone X-24.

<p>Due to overpopulation and resource-poor habitat structure, deer threaten the<a> future of oak and other browse-sensitive species in hardwood forests. </a>Appropriate tools must be used to ensure desirable, diverse, and ecologically stable regeneration of future forests and the sustainability of native plant communities. We performed two experiments and a review to examine the effectiveness of available methods for managing browse of hardwood seedlings and to discover how these interact with each other and other silvicultural methods. First, we examined how fencing interacts with controlled-release fertilization, seed source (genetically select and non-select), and site type (afforested and reforested sites) to enhance the regeneration of planted northern red oak (<i>Quercus rubra </i>L.), white oak (<i>Quercus alba</i>), black cherry (<i>Prunus serotina</i>), and black walnut (<i>Juglans nigra</i>) at five sites in Indiana. Fencing proved to be the greatest determinant of seedling growth, survival, and quality. Fertilizer enhanced the early growth of white oak and black cherry, though for black cherry this occurred only inside fences. Select seed sources grew better and showed greater quality; however, the survival of select seedlings was limited by deer browse in absence of fences. Trees at afforested sites had lower survival if left non-fenced. Secondly, we also investigated how fencing and invasive shrub removal affected natural regeneration, species richness, and ground-layer plant cover under closed-canopy forests. Honeysuckle (<i>Lonicera maackii</i>) removal had a variable effect depending on species and site. Positive effects were most common for shade-intolerant species, while negative effects occurred for a few shade-tolerant species at some sites. Deer fencing had a positive effect on cherry and hackberry seedling density, and a negative effect on elm seedling density. Honeysuckle and deer fencing interacted antagonistically in some instances. Fencing without honeysuckle removal resulted in lower elm abundance and herbaceous-layer cover. In the densest invasions, leaving honeysuckle intact resulted in a complete lack of recruitment into the sapling layer. Our experiment suggests that invasive shrub removal and fencing be done together. Finally, we synthesized the existing literature on browse management options for hardwood regeneration to evaluate their relative effectiveness. Fences, tree shelters, repellents, facilitation by neighboring plants, deer population control, timber harvest, and slash all had positive effects on height growth of regenerating seedlings under deer browse pressure. Fences were more effective at reducing browse than repellents, while fertilizers increased browse and had no effects on growth. </p>

<p> Despite the continuous breeding efforts towards improving yield, seed quality, and yield-related traits, there is still little understanding of several aspects of soybean breeding; however, crop breeding is ever-evolving, and plant breeding technologies offer immense potential for accelerating genetic improvement in soybeans. This thesis explores different frameworks to further characterize tradeoffs among seed quality traits, soybean maturity's genetic architecture, and selections for yield. We explored the interactions of carbohydrate traits with other seed traits, flowering, and maturity using data from a large panel of <em>G. max </em>accessions from the USDA soybean germplasm collection. We found a negative correlation between sucrose and protein and a negative correlation between protein and oil, representing a significant challenge for improving seed quality. In contrast to other well-documented correlations, such as protein and oil, correlations between raffinose and oil content seem more specific to populations and environments and are unlikely to generalize to the whole specie; however, the correlations of sucrose with protein and seed size appears to be more stable. In addition, we performed a genome-wide association analysis (GWA) to detect novel QTLs for flowering (R1) time, maturity (R8) time, and reproductive length (RL) using a soybean panel with the same genotype for major <em>E </em>genes (<em>e1-as/E2/E3). </em>While major maturity <em>E</em> genes are known to have pleiotropic effects on R1 and R8, we found two QTLs associated with R8 and RL that do not control R1, suggesting minor-effect, trait-specific loci are also involved in controlling R1 and R8. In addition, w<em>e identified six genes that may play essential roles in regulating R1, R8, and RL; however, further validation of the QTLs and f</em>ine mapping and map-based cloning studies of the candidate genes are necessary before they can be used in breeding programs. Lastly, we conducted a selection experiment in progeny row (PR) populations of four breeding programs to compare the agronomic performance of lines selected by breeders using their usual selection methods to lines selected through prediction of yield performance using new sources of data and information. Our results suggest that aerial average canopy coverage (ACC) used as a secondary trait in combination with field spatial variation adjustment is an efficient high throughput methodology to effectively select high-yielding lines from non-replicated experiments at the PR stage. </p>

Fusarium root rot (FRR) disease, caused by the fungus Fusarium solani f. sp. phaseoli (FSP), is an important soil-borne disease reducing common bean (Phaseolus vulgaris L.) yields, and hence food security, in Uganda and elsewhere in developing countries where the crop is grown without fungicides. The key aim of this study was to elucidate the significance of bean root rot (BRR), appraise methods for screening germplasm for resistance to FRR, determine the genotypic variability of resistance, and the inheritance of resistance to FRR in common bean. This information was deemed useful in devising an appropriate strategy for breeding FRR resistance in beans. A participatory rural appraisal (PRA) was conducted in south-western and eastern Uganda to ascertain farmers’ awareness of BRR and their influence on preferred bean varieties. Bean root rot is considered to be the most devastating and most recognised disease, especially in south-western Uganda. Control measures for BRR were very minimal, and in some cases, non-existent. Use of resistant varieties to control the disease was not evident, because the most popular varieties were susceptible to the disease. The resistant bean varieties currently available have undesirable characteristics such as small seed size, black seed and late maturity. Large-seeded bean varieties, even though cited as being more susceptible to BRR than the small-seeded varieties, are still very popular. The study highlighted the need for breeding FRR resistance in the large-seeded bean varieties that are highly preferred by farmers. Four isolates of FSP (FSP-1, FSP-2, FSP-3 and FSP-4) were tested for pathogenicity under screenhouse and laboratory conditions. In addition, three methods of storing and maintaining the viability of FSP isolates were appraised. The isolate FSP-3, was found to be the most pathogenic, resulting in 100% disease incidence on all bean varieties tested, with high severity scores. The potato dextrose agar (PDA) slants stored at 5oC were found to be the best method of storage for pathogenic isolates. The FSP-3 isolate was subsequently utilised for screening bean lines for resistance to FRR. The influence of soil composition, irrigation frequency, and inoculation technique on the severity of FRR was studied on six bean lines. Interactions of irrigation frequency, soil composition, and bean lines were not significant. The 50% swamp soil:50% forest soil composition and forest soil alone categorized the varieties most distinctly according to their reaction to FRR. Also, the best distinct classification for the varieties was obtained under treatments that were watered daily and once in a week. Based on economic considerations, the standard forest soil and daily irrigation were subsequently adopted for screening bean germplasm for resistance to FRR. It was also found that sorghum seed as a medium for pathogen inoculation was better than the agar slurry medium. One hundred and forty seven common bean varieties were evaluated for resistance to FRR (isolate FSP-3) under screenhouse conditions. In order to confirm this resistance, 46 common bean lines selected from the screenhouse trial were further evaluated using natural inoculum in a BRR-infested field. Forty-four varieties comprising ten large-seeded, four medium-seeded and 30 small-seeded varieties showed moderate resistance to FRR; but none were resistant or immune to the disease. Based on adaptability, eight moderately resistant varieties were selected for use as parents in the study of inheritance of resistance to FRR. A 12 x 12 diallel mating design was utilised to develop 66 F1 and F2 populations, plus their reciprocal crosses, with the aim of studying the mode of inheritance of resistance to FRR. The F1 and F2 progeny evaluations showed that FRR resistance was mainly governed by additive genes in most populations. However, there were a few crosses which displayed highly significant specific combining ability (SCA) effects, implying that dominant effects were important in some populations. Maternal effects were also highly significant at both the F1 and F2 generations, suggesting that resistance was modified by cytoplasmic genes. The non-maternal effects were also significant in some populations, suggesting that the cytoplasmic genes were interacting with nuclear genes. The number of genes governing resistance to FRR varied from two to nine among the eight sources of resistance. The allelism test of resistant x resistant populations, and the observation of continuous distributions of severity scores, suggested the presence of many loci governing FRR resistance in beans. Broad sense heritability of disease resistance varied from 0.22-0.69, while heritability in the narrow sense was estimated as 0.35-0.49 in the populations. These results suggested that selection and backcrossing to both parents would be the best breeding procedures for improving resistance in the popular large-seeded bean varieties in Uganda. However, there could be complications in breeding for resistance to FRR in beans, because resistance was modified by cytoplasmic gene effects and their interaction with nuclear genes in some of the populations. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.

Abstract not available. / Thesis (Ph.D)-University of KwaZulu-Natal, Pietermaritzburg, 2008.

<p>Here, we seek to address the importance of biodiversity in plant ecosystems. We examined the productivity-diversity relationship through the lens of the modern coexistence theory, using a combination of both experimentation and mathematical simulation. We did this by tracking and comparing the productivity of mixed and monoculture plots, analyzing the growth responses of individual trees at forest plots (Chapter 1), confirming the productivity-diversity relationship in a greenhouse experiment using local herbaceous plants (Chapter 2), and finally simulating the productivity response of monoculture vs polyculture plantations to specialist enemy attack (Chapter 3).</p><p>It is no surprise that biodiversity has been decreasing at an exponential rate on the global scale because of effects such as habitat fragmentation, invasive species, spreading pathogens, and anthropogenic influences. Ecologists often found that plants in more species rich locations often exhibited higher productivity and stability in the face of stress. One such phenomenon is known as the productivity diversity relationship that implies biodiversity is key to sustaining ecosystems. Notably, while efforts are being put forth to address ecosystem destruction, much of the current tree planting strategy in the USA is based on timber profit rather than forest productivity and species coexistence with tree biology often being a secondary consideration. These thought processes are in opposition with historical experiments that indicate polyculture communities create more biomass making them significantly more productive than monocultures. However, we also acknowledge that it is not simply biodiversity that must be taken into consideration for a productive ecosystem but also species interaction through coexistence indicate whether or not a community will persevere. These interactions can be addressed using the modern coexistence theory which depends on these complementarity and fitness similarities for species to coexist through time. Here, we seek to address the importance of biodiversity in plant ecosystems. We examined the productivity-diversity relationship through the lens of the modern coexistence theory, using a combination of both experimentation and mathematical simulation. We did this by tracking and comparing the productivity of mixed and monoculture plots, analyzing the growth responses of individual trees at forest plots (Chapter 1), confirming the productivity-diversity relationship in a greenhouse experiment using local herbaceous plants (Chapter 2), and finally simulating the productivity response of monoculture vs polyculture plantations to specialist enemy attack (Chapter 3). Our research across the combination of approaches used found that species with overlapping niches and very different finesses will exclude one another due to high competition. Further, the productivity diversity correlation is necessary for ecosystem growth, but it is not sufficient for species coexistence. However, species can maintain this positive relationship despite a lack of coexistence if they maintain niche complementarity. Lastly, using a theoretic game model we were able to identify the impacts of a specialist pest on polyculture and monoculture forest. These results showed that a polyculture forest was more productive than that of a monoculture forest regardless of the presence of a specialist enemy. The results of the multiple threads of evidence found from these combined experiments indicate that while the productivity diversity correlation is important to ecosystems it is likely due to the impacts of niche complementarity that determine whether or not species will be productive within an ecosystem.</p>

<p dir="ltr">Urban agriculture has grown in popularity in recent decades, due to its ability to provide access to healthy fruits and vegetables in urban zones, as well as its importance in fostering knowledge of agriculture within communities. However, urban agriculture may struggle with unique challenges due to its proximity to urban and industrial activities, such as food safety risks due to toxic heavy metals and metalloids which may be present in urban soils in high concentrations. Heavy metals and metalloids (HM) like arsenic, cadmium, and lead are absorbed by plants from the soil, and may accumulate in the plants’ edible tissues, which are consumed by humans. Carrot (<i>Daucus carota</i> L.), in particular, hyperaccumulates these toxic heavy metals in its edible taproots, leading to food safety risks on urban farms.</p><p dir="ltr">One potential way to help address this challenge is to breed carrot varieties with low uptake of HM. In recent years, researchers have identified lines with high and low uptake in greenhouse trials and single location breeding nurseries. However, to be viable, these lines must consistently vary in HM across sites despite differences in environmental and management factors that can also greatly influence HM bioavailability and uptake. Moreover, screening for differences in HM uptake is time-consuming and expensive, and breeders need new tools to select among segregating breeding populations. By using on-farm participatory research as well as advanced phenotyping technologies, we investigate the viability of breeding carrots for HM uptake and the potential of new tools to advance these efforts in order to mitigate the risks on urban farms.</p><p dir="ltr">In the summers of 2021 and 2022, participatory on-farm trials were conducted to determine the HM risks on Indiana urban farms and to investigate the consistency of differences in HM uptake between carrot breeding lines taken from breeding trials (Chapter 2). Results of these trials indicated that while carrot genotype had an effect, there was still significant variability in carrot uptake of arsenic, cadmium and lead between farm sites and years. Results indicated significant differences between site-years, and carrot HM concentrations that correlated strongly with soil concentrations for that particular element. However, there were some site-years with low soil HM content and other soil factors expected to reduce uptake such as pH and phytoavailable zinc concentrations (such as site-year H), that had high carrot HM content. There were significant differences in carrot cadmium (Cd) and arsenic (As) content between carrot breeding lines. For instance, breeding line 3271 had a high As average concentration but low Cd average concentration, while breeding lines 6220 and 2327 had low As and high Cd concentrations. We identify the possibility of other mediating factors, such as uptake of antagonistic micronutrients, or microbe-assisted HM uptake and amelioration that need further attention.</p><p dir="ltr">In the fall of 2022, a study was conducted to investigate the possibility of using phenotyping technologies such as RGB and hyperspectral imaging to detect Cd stress in carrot and attempt to predict uptake (Chapter 3). RGB (red green blue) is a digital color model in which cameras can capture important visual cues compiled from information about each pixel. Hyperspectral imaging uses cameras to capture wavelengths beyond the visible spectrum, which can detect plant stress indicators like increased anthocyanin content for specific environmental stresses. Results of this trial were useful, with some time points and indices noting differences between carrot lines. For instance, RGB factors hue and fluorescence as well as hyperspectral reflectance plots and vegetative indices swirNDVI and ANTH were the most diagnostic. Breeding lines 6636 and 8503 showed the greatest separation between Cd treated and control carrots in imaging indices. However, further studies will be needed to optimize this approach for breeding programs.</p><p dir="ltr">This research demonstrates that growing carrots on most urban farms in Indiana is safe. The studies also provide further evidence that it will be possible to help lower food safety risks by selecting carrot varieties with low HM uptake, and phenotyping can help to advance these efforts. At the same time, new research to understand how soil factors such as microbiomes influence HM bioavailability and uptake on urban farms are also needed to further reduce potential risks. In the meantime, farmers should continue to test their soil for HM and take appropriate actions to reduce risks such as using raised beds and soil amendments that can bind metals like biochar. Consumers should also continue to wash and peel their carrots before consumption, as well as consume a balanced diet with a diverse set of vegetables and other crops.</p>

<div>Anthropogenically driven global change is disrupting ecosystems and habitats of many plant species, straining the ability of native species to survive and reproduce. The overarching goal of this research was to holistically work towards restoration of a threatened tree species by connecting research from different disciplines. In order to do so, the threatened butternut tree (<i>Juglans cinerea</i>) and its hybrids were used as a case study. Hybridization can incorporate stress tolerance in plants and could be a potential restoration tool. Evidence in some wild butternut populations indicates that naturalized hybrids of butternut with Japanese walnut (<i>Juglans ailantifolia</i>) may be more tolerant to butternut canker disease (BCD) than butternut, but this has not been formally tested. Thus, chapter 2 examined potential BCD tolerance within and between unadmixed and hybrid butternut inoculated with two BCD fungal isolates. Differences in canker growth were observed by fungal isolate, which could help to explain some differences in BCD severity found among butternut populations. Smaller and fewer cankers and greater genetic gains were detected in hybrid families, demonstrating that hybrids warrant further evaluation as a possible breeding tool for developing BCD-resistant butternut trees.</div><div>However, even with increased disease tolerance, hybrids must possess similar ecophysiological tolerances to their native progenitor to be an effective replacement. Butternut is extremely cold hardy, but Japanese walnuts are native to a warmer ecosystem, indicating potential disparities in extreme temperature tolerances between the two species and their hybrids. Thus, samples from mature trees were subjected to cold and heat treatments to compare relative extreme temperature tolerances within butternut and between butternut, Japanese walnut, and their hybrids. Within butternut, trees from colder areas exhibited less cold damage than those from warmer areas. Differences in heat damage among provenances occurred but did not follow a clear trend. Butternut exhibited greatest cold tolerance, Japanese walnut exhibited greatest heat tolerance, and hybrids were intermediate. Thus, the utility of hybrids for restoration could be limited at the extremes of the species’ distributions.</div><div>A second, but different type of freeze test was conducted for chapter 4 using seedlings to gain a more nuanced understanding of cold tolerance within butternut and between butternut and its hybrids. No survival or damage differences were detected in butternut provenances, although seedlings from the coldest provenances experienced more delayed budbreak at the two warmest treatments than those from warmer provenances. Interspecific differences were not observed in dieback but were detected in survival and budbreak. The hybrids had greater survival than butternut from warmer provenances at the lowest temperature treatment (-38 °C), but given that temperatures that low are extremely unlikely to occur in those provenances, it is not anticipated to give the hybrids an advantage if planted in those areas. However, the hybrids’ earlier budbreak could limit the success of restoration with these hybrids in the coldest extents of butternut’s range. </div><div>If hybrids, as well as genetically modified (GM) trees, are successfully developed for effective disease tolerance and to serve as an ecologically suitable replacement, success of restoration using hybrids will ultimately depend on those directly responsible for replanting efforts. A survey was administered to land managers in 46 organizations in Indiana to gauge perceptions of hybrid and GM trees, as well as current use of hybrid trees. Land managers had stronger concern for ecological, rather than economic, issues. Agreement was highest for using hybrid and GM trees for “conservation and restoration of at-risk species”, “timber production”, and “non-timber products (fruit, syrup, etc.)”. However, perceptions varied by characteristics, such as concern type, age, and the type of land they managed. Ecological concern and the type of land being managed most strongly predicted current hybrid use. Overall, results indicate the majority of land managers in Indiana would likely be agreeable to recommendations towards using hybrids. However, most nonetheless had strong ecological concerns about their suitability as a native replacement. It is important to note, though, that consistent with the results of previous studies, great variation was seen within the performance and characteristics of the butternut hybrids in chapters 2-4. Thus, it may be possible with careful selection and breeding to harness this variation to develop disease tolerant and ecologically similar hybrids acceptable to land managers.</div>