Thesis (Ph. D.)--University of Oregon, 2001. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 121-147). Also available for download via the World Wide Web; free to University of Oregon users.

Brassica carinata (A.) Braun and Camelina sativa (L.) Crantz are two re-emerging oilseed crops of the Brassicaceae family that are being adapted for cultivation in western Canada. Both seeds of these species reportedly accumulate considerable amounts of sulfur-containing secondary metabolites called glucosinolates. The purpose of the current work was to gain knowledge of the occurrence and distribution of glucosinolates during primary processing of these oilseeds, including during pressing and dehulling. In the first study, a reversed phase HPLC method was developed for the analysis of sinigrin, the major glucosinolate in B. carinata. Both C18 columns selected were able to separate the compound with an isocratic eluent containing 100% tetramethylammonium bromide (10 mM, pH 5) delivered at 1 mL/min at a column temperature of 25oC. These chromatographic conditions were applied and sinigrin concentration of whole B.carinata seed was estimated to be 29 μg/mg. Average matrix effect was estimated to be 104% that was caused by other components in the B. carinata seed matrix. In the second study, high concentrations of glucosinolates were detected and identified in fractions of C. sativa seeds using HPLC-ESI-MS. Methods for extraction, isolation, and purification of three individual glucosinolates from these fractions are reported. Quantitation of total glucosinolates was performed on proton NMR using DMF as an internal standard. Quantitation of individual glucosinolates was achieved by using MS extracted ion chromatogram data. Total glucosinolates were found in C. sativa whole seed at a concentration of 14 μg/mg, and glucocamelinin, the major glucosinolate, constituted 65% of the total amount. In addition, a dehulling treatment was applied to C. sativa seeds, from which both oil content and crude protein content increased after dehulling of the seeds.

Temperature is a major abiotic stress limiting plant growth. Thermotolerance evaluation during germination and early growth may help identify adaptable genotypes of new crops. Two studies were conducted to evaluate temperature effects on 12 Brassica carinata genotypes during germination and early growth. During germination, genotype AX17004 was both the most cold- and heat-tolerant. During early-season growth (35 d after seeding), there were temperature and genotype effects on shoot, root, and physiological components. Cumulative low- and high-temperature response indices, and cumulative root and shoot response indices were related, indicating the importance of these traits. Genotype AX17006 was identified as heat tolerant, and AX17009 as cold tolerant during early-season growth. When genotypes were grouped according to breed types, hybrids generally had better responses than the inbred lines, and double haploids and the check responses were intermediate. These studies provided rapid results that will reduce the number of genotypes assessed in field studies.

This thesis examines the toxicity of the herbicide glyphosate (two formulations ¡V technical grade and Roundup Biactive RB) and the insecticide chlorpyrifos CPF to a model freshwater food chain of a producer and consumer. The importance of studying the toxicity of low (environmentally realistic) concentrations of pesticides to non-target organisms is highlighted. An extensive literature review on the toxicity of glyphosate and chlorpyrifos to aquatic organisms is provided. The requirements for the maintenance of algal (Chlorella vulgaris, Chlorella pyrenoidosa and Pseudokirchneriella subcapitata) and Daphnia carinata cultures are discussed. The effects of two formulations of the herbicide glyphosate (technical grade and Roundup Biactive„µ) and the insecticide chlorpyrifos on the growth of Chlorella pyrenoidosa and Pseudokirchneriella subcapitata were studied, and the EC50 values determined. Hormesis was observed when P. subcapitata was exposed to concentrations of Roundup equal to 7% and 4% of its EC50 respectively. When exposed to chlorpyrifos concentrations 0.3-5 ƒÝg/L, hormesis was observed for both algal species with a maximum at 0.06% of EC50. The effects of sublethal concentrations of chlorpyrifos on population characteristics of Daphnia carinata were investigated in multiple-generation toxicity testing using individual culture. Exposure to chlorpyrifos affected survival and fecundity of animals in the first generation. In the second generation the most affected endpoint was time to the first brood with an indication of hormesis. LC50 tests were then conducted using animals of the third generation from each of the exposures in individual tests. Results of testing the third generation showed a constant significant decline in LC50 in the order of control daphnids through to ¡¥0.1 LC50¡¦ pre-exposed daphnids. The same experimental protocol was used in testing of glyphosate (technical grade and Roundup Biactive). Glyphosate was tested in two different media: sea salt solution and M4 medium, while Roundup Biactive was tested in M4 medium. Results indicated that glyphosate and Roundup Biactive had low toxicity to Daphnia. Hormesis was evident in sea salt medium exposures in the first and second generations of daphnids with glyphosate. When exposed to glyphosate and Roundup Biactive in M4 medium animals showed no indication of hormesis. It is hypothesized that glyphosate may have compensated for the lack of microelements in the sea salt medium, and possible mechanisms discussed.The modifying effect of glyphosate on the toxicity of cadmium to Daphnia carinata was studied using the same experimental design. Low concentrations of Roundup Biactive reduced the toxicity of cadmium, and the performance of daphnia was enhanced in terms of animal size, survival, fecundity, and the rate of natural increase in both generations in the presence of glyphosate. However when the third generation was tested for their sensitivity to Cd in the 48-h LC50 experiments there was no difference between RB-free and RB-spiked treatments in pair wise comparisons, indicating that no adaptation mechanisms were involved in the enhancement. The implications of these observed effects for environmental freshwater food chains subjected to pesticide exposure are discussed and recommendations on modifying pesticide use are provided.

Brassica carinata is being actively pursued as a new industrial oil crop platform for the Canadian Prairies. A genetic assessment of B. carinata was performed to elucidate its evolutionary origins and create a genetic map to assist in locating genes and traits of interest that would help in marker-assisted breeding. First, genetic analysis using simple sequence repeat (SSR) markers, previously tested on B. juncea and B. napus, was performed, to examine the genetic diversity of 37 B. carinata lines. SSR analysis revealed world accessions were more diverse than lines conditioned to grow in the prairies. Diversity analysis revealed that the parental lines of a double haploid (DH) population, 179 and 345, obtained from the John Innes Centre (JIC), were among the more genetically diverse lines, supporting the use of this population for linkage mapping. Genetic markers created from 3’ targeted SNP discovery between 179 and 345, were tested on the DH population resulting in the generation of a B. carinata genetic linkage map essentially with no prior sequence data knowledge. This genetic map contained 341 SNP and 86 SSR loci identifying eight linkage groups belonging to the B genome, nine belonging to the C genome and two unidentified groups spanning 2041 cM. Comparative mapping of polymorphic markers identified in the amphidiploid B. carinata indicated the orientation of B and C genomes coincide with that of other Brassica species, and the two genomes have remained essentially unaltered, with no major chromosomal rearrangements since the formation of B. carinata. A lesser number of polymorphic markers were detected in the C genome, which suggested the B genome is more genetically diverse in B. carinata. Limited field trials of the 179 x 345 DH population were performed during the 2011 and 2012 growing seasons. Preliminary quantitative trait loci (QTLs) for agronomic traits including flowering time (FT), plant height (PH), and seed quality were identified.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2015-11-10T07:53:00Z No. of bitstreams: 1 texto completo.pdf: 2611221 bytes, checksum: c8dea29167f7bd10e2a43b07daef4e97 (MD5) / Made available in DSpace on 2015-11-10T07:53:00Z (GMT). No. of bitstreams: 1 texto completo.pdf: 2611221 bytes, checksum: c8dea29167f7bd10e2a43b07daef4e97 (MD5) Previous issue date: 2014-02-21 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Os fatores ambientais podem causar variações na morfologia e anatomia das folhas. Representantes da família Bromeliaceae já se mostraram sensíveis às variações microclimáticas. O Parque Nacional do Caparaó (PNC) apresenta ambientes com condições adversas, como altas luminosidades e baixas temperaturas no inverno. Objetivou-se com este trabalho (1) avaliar quais variáveis ambientais atuam diretamente em cada característica analisada em espécies de Bromeliaceae ocorrentes no PNC e (2) identificar como interações entre os parâmetros ambientais alteram os padrões nos traços funcionais e na separação de nichos de espécies de Bromeliaceae ocorrentes no PNC. As hipóteses a serem testadas são que (1) variações ambientais atuam nas respostas diretas das espécies estudadas em relação à morfologia, anatomia e fisiologia e que (2) as variáveis ambientais determinam diferentes nichos através da relação com traços funcionais das espécies estudadas. Foram avaliadas folhas das espécies de Bromeliaceae Aechmea vanhoutteana, Quesnelia kautskyi e Pitcairnia carinata do PNC, que se encontravam em diferentes condições ambientais de luminosidade, umidade, velocidade do vento e temperatura. O material botânico coletado foi submetido às análises anatômicas para morfometria e diafanizações para contagem de estômatos e escamas, usando protocolos convencionais. Discos foliares foram coletados para as análises fisiológicas de conteúdo de pigmentos fotossintéticos (clorofila a e b e carotenoides) e fenóis. Foram realizadas medições de área da bainha e área foliar. Das variáveis ambientais avaliadas individualmente, a velocidade do vento foi a única variável que não influenciou diretamente nenhuma característica. A umidade do ar e a temperatura foram as que apresentaram maior relevância na alteração dos caracteres, apesar da luminosidade ser considerada um dos fatores principais para variações. A. vanhoutteana foi a espécie que menos apresentou variações estruturais (área da bainha); o aposto foi observado para Q. kautskyi, que apresentou uma diversidade maior de variações (área foliares, espessura do parênquima aquífero e clorofiliano, conteúdo de clorofilas, carotenoides e fenóis). Em P. carinata houve variações na área da bainha, espessura do parênquima clorofiliano, conteúdo de clorofila a e fenóis. A PCA com a interação das três espécies apresentou separação de nicho entre as três espécies. P. carinata devido à luminosidade, foi separada de A. vanhoutteana e Q. kautskyi. Nestas duas últimas espécies não pôde ser determinado o fator que levou à separação, possivelmente, pelo fato de não ter sido avaliado neste trabalho. Em Q. kautskyi, pôde-se observar que indivíduos epífitos foram funcionalmente diferentes dos terrestres, sendo a umidade responsável pela formação dos grupamentos. A incidência luminosa e o vento foram os fatores que melhor se ajustaram ao modelo de interação dos fatores ambientais para responder às variações nos traços funcionais. As características peculiares dos locais de coleta de P. carinata determinaram alterações significativas nos traços de seus indivíduos. Com esse estudo, comprova-se a sensibilidade da família Bromeliaceae em relação às mudanças ambientais, tanto na interação das espécies como individualmente. Devido à melhor distribuição nos ambientes e das variações observadas em Q. kautskyi, esta espécie se mostrou a mais plástica das estudadas. As mudanças nos padrões ambientais também contribuíram para a determinação de diferentes nichos nas espécies, que permite a coexistência destas no mesmo ambiente. / Environmental factors can cause variations in the morphology and anatomy of leaves. Representatives of the Bromeliaceae family has proved sensitive to microclimatic variations. The Caparaó National Park (PNC) presents environments with adverse conditions such as high solar irradiation and low temperatures in winter. The objective of this study was (1) to evaluate which environmental variables act directly on each characteristic analyzed in species of Bromeliaceae occurring in PNC and (2) identify how interactions between environmental parameters change the standards on functional traits and the separation of species niches of Bromeliaceae occurring in PNC. The hypothesis to be tested are (1) environmental variations operate in the direct responses of the studied species in relation to morphology, anatomy and physiology and (2) environmental variables determine different niches according to the relationship with functional traits of the studied species. Leaves of species of Bromeliaceae Aechmea vanhoutteana, Quesnelia kautskyi and Pitcairnia carinata occurring in PNC, which were in different environmental light conditions, humidity, wind speed and temperature were measured. The botanical material was subjected to anatomical analyzes for morphometric and diafanization to counting of stomata and scales, using conventional protocols. Leaf discs were collected for physiological analyzes of content of photosynthetic pigments (chlorophyll a and b and carotenoids) and phenols. Measurements of the sheath and leaf area were performed. Of all Environmental variables individually evaluated, the wind speed was the only one that does not directly affect any trait. The humidity and temperature showed the greatest relevance in changing of the characters, despite the high irradiation be considered one of the major factor for variations. A. vanhoutteana was the species that showed less structural variations (area of the sheath); the opposite was observed for Q. kautskyi, which showed a greater diversity of variations (leaf area, thickness of parenchymal aquifer and chlorenchyma, chlorophyll content, carotenoids and phenols). In P. carinata there were variations in the area of the sheath, thickness of chlorenchyma, content chlorophyll a of phenols. The full PCA showed niche separation among the three species. P. carinata due to light, was separated from Q. kautskyi and A. vanhoutteana. In these two latter species the factor that led to the break up could not be determined, possibly because it was not assessed in this study. In Q. kautskyi, it was possible to observe that epiphytic individuals were functionally different from the terricolous, with moisture responsible for the grouping formation. The light incidence and the wind were the factors that best fit the interaction of environmental factors to respond to changes in functional traits model. The peculiar characteristics of the locations of P. carinata gathering determined significant changes in the features of its subjects. This study proves the sensitivity of the Bromeliaceae family in relation to environmental changes, both in the interaction of species and as individuals. Due to the better distribution in the environment and the variations observed in Q. kautskyi, this species showed the most studied of plastic. Changes in environmental standards have also contributed to the determination of various niches in species, which allows the coexistence of both in the same environment.

An efficient system for or 'Agrobacterium'-mediated transformation of <i>Brassica carinata</i> was used together with a promoterless <i> gus</i>::<i>nptII</i> gene fusion to isolate putative promoter sequences. Cotyledonary petioles were transformed using the promoterless gene fusion construct. Only transformation events in which the promoterless gene fusion had integrated downstream from plant regulatory sequences were expected to produce viable tissue under kanamycin selection. Forty-two transgenic plants were recovered. Transformation efficiency was approximately 0.6%. Regenerated plants were screened for GUS expression in different tissues and organs by histological and fluorometric assays. Tissue-specific GUS expression was detected (stigmas, seed coat, leaf edges and vascular tissue) in some plants, while strong constitutive GUS expression was detected in others (based on GUS histological assays). Using subgenomic libraries, putative promoter fragments were isolated from the plants which exhibited GUS expression in stigmas, leaf edges and constitutively. A putative promoter fragment from a plant which exhibited GUS expression only in the stigma was fused with the gus gene and reintroduced by <i>Agrobacterium </i> -mediated transformation into <i>B. napus, B. carinata, Arabidopsis' and tobacco </i>. GUS expression was observed in the stigma of <i>B. napus </i> but not in ' B. carinata'. In <i>Arabidopsis </i> and tobacco GUS expression. was not tissue specific (weakly constitutive or restricted to two or more tissues). The 3' DNA sequence (15 kb) flanking the <i> gus</i>::<i>nptII </i> insert in the plant with GUS expression in the stigma was also isolated using a subgenomic library. A gene for a cytochrome P450 like protein was discovered on the minus DNA strand of the 3' sequence with a start codon approximately 6.5 kb from the T-DNA left border.

Uncertainty is pervasive in risk assessment. In ecotoxicological risk assessments, it arises from such sources as a lack of data, the simplification and abstraction of complex situations, and ambiguities in assessment endpoints (Burgman 2005; Suter 1993). When evaluating and managing risks, uncertainty needs to be explicitly considered in order to avoid erroneous decisions and to be able to make statements about the confidence that we can place in risk estimates. Although informative, previous approaches to dealing with uncertainty in ecotoxicological modelling have been found to be limited, inconsistent and often based on assumptions that may be false (Ferson & Ginzburg 1996; Suter 1998; Suter et al. 2002; van der Hoeven 2004; van Straalen 2002a; Verdonck et al. 2003a). In this thesis a Generalised Linear Modelling approach is proposed as an alternative, congruous framework for the analysis and prediction of a wide range of ecotoxicological effects. This approach was used to investigate the results of toxicity experiments on the effect of 2,4-Dichlorophenoxyacetic Acid (2,4-D) formulations and 4-Chlorophenol (4-CP, an associated breakdown product) on Daphnia carinata. Differences between frequentist Maximum Likelihood (ML) and Bayesian Markov-Chain Monte-Carlo (MCMC) approaches to statistical reasoning and model estimation were also investigated. These approaches are inferentially disparate and place different emphasis on aleatory and epistemic uncertainty (O'Hagan 2004). Bayesian MCMC and Probability Bounds Analysis methods for propagating uncertainty in risk models are also compared for the first time. For simple models, Bayesian and frequentist approaches to Generalised Linear Model (GLM) estimation were found to produce very similar results when non-informative prior distributions were used for the Bayesian models. Potency estimates and regression parameters were found to be similar for identical models, signifying that Bayesian MCMC techniques are at least a suitable and objective replacement for frequentist ML for the analysis of exposureresponse data. Applications of these techniques demonstrated that Amicide formulations of 2,4-D are more toxic to Daphnia than their unformulated, Technical Acid parent. Different results were obtained from Bayesian MCMC and ML methods when more complex models and data structures were considered. In the analysis of 4-CP toxicity, the treatment of 2 different factors as fixed or random in standard and Mixed-Effect models was found to affect variance estimates to the degree that different conclusions would be drawn from the same model, fit to the same data. Associated discrepancies in the treatment of overdispersion between ML and Bayesian MCMC analyses were also found to affect results. Bayesian MCMC techniques were found to be superior to the ML ones employed for the analysis of complex models because they enabled the correct formulation of hierarchical (nested) datastructures within a binomial logistic GLM. Application of these techniques to the analysis of results from 4-CP toxicity testing on two strains of Daphnia carinata found that between-experiment variability was greater than that within-experiments or between-strains. Perhaps surprisingly, this indicated that long-term laboratory culture had not significantly affected the sensitivity of one strain when compared to cultures of another strain that had recently been established from field populations. The results from this analysis highlighted the need for repetition of experiments, proper model formulation in complex analyses and careful consideration of the effects of pooling data on characterising variability and uncertainty. The GLM framework was used to develop three dimensional surface models of the effects of different length pulse exposures, and subsequent delayed toxicity, of 4-CP on Daphnia. These models described the relationship between exposure duration and intensity (concentration) on toxicity, and were constructed for both pulse and delayed effects. Statistical analysis of these models found that significant delayed effects occurred following the full range of pulse exposure durations, and that both exposure duration and intensity interacted significantly and concurrently with the delayed effect. These results indicated that failure to consider delayed toxicity could lead to significant underestimation of the effects of pulse exposure, and therefore increase uncertainty in risk assessments. A number of new approaches to modelling ecotoxicological risk and to propagating uncertainty were also developed and applied in this thesis. In the first of these, a method for describing and propagating uncertainty in conventional Species Sensitivity Distribution (SSD) models was described. This utilised Probability Bounds Analysis to construct a nonparametric 'probability box' on an SSD based on EC05 estimates and their confidence intervals. Predictions from this uncertain SSD and the confidence interval extrapolation methods described by Aldenberg and colleagues (2000; 2002a) were compared. It was found that the extrapolation techniques underestimated the width of uncertainty (confidence) intervals by 63% and the upper bound by 65%, when compared to the Probability Bounds (P3 Bounds) approach, which was based on actual confidence estimates derived from the original data. An alternative approach to formulating ecotoxicological risk modelling was also proposed and was based on a Binomial GLM. In this formulation, the model is first fit to the available data in order to derive mean and uncertainty estimates for the parameters. This 'uncertain' GLM model is then used to predict the risk of effect from possible or observed exposure distributions. This risk is described as a whole distribution, with a central tendency and uncertainty bounds derived from the original data and the exposure distribution (if this is also 'uncertain'). Bayesian and P-Bounds approaches to propagating uncertainty in this model were compared using an example of the risk of exposure to a hypothetical (uncertain) distribution of 4-CP for the two Daphnia strains studied. This comparison found that the Bayesian and P-Bounds approaches produced very similar mean and uncertainty estimates, with the P-bounds intervals always being wider than the Bayesian ones. This difference is due to the different methods for dealing with dependencies between model parameters by the two approaches, and is confirmation that the P-bounds approach is better suited to situations where data and knowledge are scarce. The advantages of the Bayesian risk assessment and uncertainty propagation method developed are that it allows calculation of the likelihood of any effect occurring, not just the (probability)bounds, and that the same software (WinBugs) and model construction may be used to fit regression models and predict risks simultaneously. The GLM risk modelling approaches developed here are able to explain a wide range of response shapes (including hormesis) and underlying (non-normal) distributions, and do not involve expression of the exposure-response as a probability distribution, hence solving a number of problems found with previous formulations of ecotoxicological risk. The approaches developed can also be easily extended to describe communities, include modifying factors, mixed-effects, population growth, carrying capacity and a range of other variables of interest in ecotoxicological risk assessments. While the lack of data on the toxicological effects of chemicals is the most significant source of uncertainty in ecotoxicological risk assessments today, methods such as those described here can assist by quantifying that uncertainty so that it can be communicated to stakeholders and decision makers. As new information becomes available, these techniques can be used to develop more complex models that will help to bridge the gap between the bioassay and the ecosystem.