Douglas-fir (Pseudotsuga menziesii) is an economically important softwood that is clonally propagated for reforestation purposes by somatic embryogenesis. The molecular basis of embryogenesis in conifers is largely unknown and this prevents progress in somatic embryogenesis protocols. In angiosperms, the LEAFY COTYLEDON1 (LEC1) gene, encoding the HAP3 subunit of the eukaryotic CCAAT box-binding factor, is important in embryo formation, and necessary for somatic embryogenesis. A candidate gene strategy was employed to isolate the Douglas-fir LEC1 homologue, PmLEC1, via the polymerase chain reaction (PCR) with degenerate primers based on the Arabidopsis conserved domain, and the full-length cDNA sequence was obtained by rapid amplification of cDNA ends-PCR (RACE-PCR). The putative protein sequence shared high sequence identity with Arabidopsis LEC1. Northern analysis and quantitative real-time PCR indicate that this is an embryo-specific gene, expressed with the highest abundance during early embryogenesis. Antibodies were raised against a synthetic 18-amino acid PmLEC1 peptide, and in contrast to mRNA expression, Western blotting shows that PmLEC1 protein expression persists until the seedling stage. To gain insight into modulation of PmLEC1 expression and its inducibility in mature tissues, stress and hormone treatments were performed on mature seed and the promoter sequence was isolated by genome walking. Sorbitol, mannitol and 2,4-epibrassinolide were found to significantly up-regulate PmLEC1 expression. The PmLEC1 promoter contains a 5’ UTR intron with numerous enhancer elements, and factors that bind to these elements mediate responses to auxin, UV light and developmental cues, osmotic stress, biotic stress, and tissue culture. Some of the regulatory elements are binding sites for seed-specific transcription factors that are well known from angiosperms, providing new evidence that AGL15, ABI3 and VP1 proteins have a direct role on LEC1 expression. In investigating the embryogenic capacity of PmLEC1, ectopic expression of PmLEC1 in the embryo lethal Arabidopsis lec1-1 null mutant complemented the mutation and permitted the production of viable, desiccation tolerant seeds. In addition, transgenic seedlings produced embryo-like structures from vegetative organs and expressed seed-specific genes. In wild type plants, ectopic expression of PmLEC1 resulted in a bushy phenotype but expression of seed-specific genes was not observed. Taken together, these results show that PmLEC1 is an embryo-specific gene with an essential role throughout embryogenesis, and PmLEC1 expression may be induced in mature seeds by stress and hormone treatments. Because mature seeds show only trace amounts of PmLEC1 transcripts and Douglas-fir somatic embryogenesis can only be induced from immature embryos, this information provides useful insight into initiation of embryogenesis from vegetative tissues. The identification of binding sites for transcription factors known from angiosperms in the promoter region of PmLEC1 has revealed the identity of several genes which are expected to play pivotal roles in conifer embryogenesis.

A cana-de-açúcar é uma das principais culturas agroindustriais do Brasil, sendo amplamente cultivada para a produção de açúcar e etanol. Esta cultura se torna a cada dia mais importante no cenário mundial, devido à busca constante por fontes de energia alternativas e mais sustentáveis. Para atender a crescente demanda, é necessária a liberação frequente de novas variedades, mais adaptadas às regiões de cultivo e tolerantes às alterações ambientais. Assim, o estabelecimento da metodologia de transformação genética além de contribuir para o estudo funcional de genes de interesse é uma metodologia alternativa para obtenção de novas variedades. O processo de obtenção de transgênicos é dependente de um eficiente protocolo de regeneração de plantas in vitro, que geralmente envolve uma fase de formação de células indiferenciadas (calos). A indução e a manutenção dos calos são favoráveis ao aumento da atividade de elementos de transposição (ETs) os quais são muito freqüentes no genoma de cana e podem acarretar variabilidade no genoma vegetal pela alteração dos padrões e funções gênicas devido a essa mobilização, afrontando a fidelidade genética dos cultivares transgênicos obtidos. Baseando-se na importância de reduzir o período de cultura de tecidos e controlar a atividade dos ETs durante o desenvolvimento in vitro, o objetivo desse trabalho foi buscar alternativas no controle e na redução do tempo para regeneração de plantas, inclusive com a aplicação de peptídeos hormonais, assim como de transformar geneticamente as variedades RB835089 e RB835486 com o gene Ddm1 de Arabidopsis, visando o silenciamento dos elementos de transposição em cana-de-açúcar. Para isso, foram analisados os meios de cultura MS3c e ML1G1 e o efeito da água de coco na indução e formação de calos como também na regeneração de plantas. Foram testados os meios de regeneração de plantas MSAc, SRM, ML1R3 e ML1R4, obtendo-se em média 5,2 plantas por explante no meio MSAc, que foi superior aos demais meios. Este meio foi utilizado para testar o efeito individual dos peptídeos hormonais CLV3 e PSK- em calos embriogênicos, os quais apresentaram acréscimo na regeneração de plantas para 9,3 plantas por explantes com doses de 30 µM de PSK-a. A transformação genética por biolística através da cotransformação dos genes neo e AtDdm1 resultou em 34 plantas transgênicas. O estudo da mobilização dos ETs durante o desenvolvimento in vitro foi realizado para quatro retrotransposons. A expressão heteróloga do gene AtDdm1 em cana-de-açúcar mostrou atuar no controle da expressão do retroelemento TE010. O estudo da mobilização dos retrotransposons e do gene Ddm1 endógeno de cana (SsDdm1) durante o desenvolvimento in vitro confirmou que o gene SsDdm1 foi chave no controle da expressão dos retroelementos. A transformação genética com o gene AtDdm1 aliada a rápida regeneração de plantas a partir de discos foliares possibilitam condições que minimizam a expressão dos ETs em cana-de-açúcar. / Sugarcane is one of the major agro-industrial crops of Brazil being widely cultivated for the production of sugar and ethanol. This culture has become increasingly more important on the world stage each day due to the constant search for alternative and sustainable energy sources. In order to meet growing demand, it is necessary to often release new varieties, better adapted to cultivated expansion area and tolerant to environmental changes. Thus, the establishing of genetic transformation methodology beyond of contributing to the functional study of genes of interest and it is an alternative method for obtaining new varieties. The process of obtaining transgenic plants is dependent of an efficient protocol for in vitro plant regeneration, which generally involves a phase of undifferentiated cells (callus). The induction and maintenance of callus are favorable to increase the activity of transposable elements (TEs) which are very frequent in the genome of sugarcane and may cause variability in the plant genome by altering patterns and gene functions due to this mobilization, confronting the genetic fidelity of the transgenic cultivars obtained. Based on the importance of reducing the period of tissue culture and control the activity of TEs during in vitro development, the objective of this work was to seek alternatives to control and reduce the time for plant regeneration, including the use of peptides hormone, as well as to genetically transform sugarcane varieties RB835089 and RB835486 with the Ddm1 Arabidopsis gene to silence the transposable elements in cane sugar. For this, we tested the culture media MS3c and ML1G1and the effect of coconut water in callus induction and growth as well as on plant regeneration. We tested the plant regeneration media MSAc, SRM, ML1R3 and ML1R4, obtaining an average of 5.2 plants per explants using MSAC, superior to other medium tested. It was used to test the individual effect of peptides hormones such as CLV3 and PSK- in embryogenic callus, which showed an increase in plant regeneration to 9.3 plants per explant with doses of 30µM PSK-a. Genetic co-transformation with the neo and AtDdm1 genes by biolistic resulted in 34 transgenic plants. A study of TEs during in vitro development was performed for four retrotransposons. Heterologous expression of the AtDdm1 gene in sugarcane showed to control the expression of the retroelement TE010. The study of mobilization of retrotransposons and the endogenous Ddm1 gene (SsDdm1) during in vitro development confirmed that SsDdm1 was key gene in controlling the expression of retrotransposons. Genetic transformation with the AtDdm1 gene and the fast regeneration of plants provide positive conditions to minimize the expression of ETs in sugarcane.

O estudo da reprodução sexual de plantas e uma área de crescente interesse devido a importância de sementes e frutos em nossa dieta diária, ambos resultantes do desenvolvimento de partes do pistilo, apos fertilização. O objetivo deste trabalho foi caracterizar um novo gene F-box expresso no pistilo de N. tabacum. Proteínas F-box atuam na interação proteína-proteína, geralmente direcionando proteínas alvo para degradação pela via ubiquitina-proteassomo. Foram identificados cinco genes de função desconhecida que codificam putativas proteínas F-box, em duas bibliotecas de cDNAs de estigmas/estiletes de N. tabacum (DEPAOLI, 2006; QUIAPIM et al., 2009) previamente construídas em nosso laboratório. A expressão de cada um destes genes foi analisada nos diferentes órgãos de N. tabacum, por qRT-PCR. O clone 085H05 da biblioteca TOBEST (QUIAPIM et al., 2009) apresentou expressão preferencial nos órgãos florais. Este clone foi selecionado para uma caracterização funcional mais detalhada. O padrão de expressão deste gene foi avaliado no estigma/estilete durante os 12 estádios do desenvolvimento floral de N. tabacum (KOLTUNOW et al., 1990). O resultado revelou que sua expressão e regulada durante o desenvolvimento, atingindo o maior nível de expressão na antese (estádio 12). Isto sugere que este gene esteja envolvido no desenvolvimento do estigma/estilete. A sequência codificadora do gene correspondente a 085H05 foi determinada e, apos amplificação e clonagem, este gene foi denominado S/S_F-box (Stigma/Style_F-box). Para compreender a função da proteína de S/S_F-box, plantas transgênicas de superexpressao e de silenciamento (por RNAi) deste gene foram geradas. As plantas de RNAi apresentaram o estilete e o ovário reduzidos quando comparados ao controle SR1. Em concordância, as plantas de superexpressao produziram flores com o estilete mais alongado do que o controle, alem do estigma e do ovário de maior tamanho. Altas concentrações de exudato foram observadas na superfície do estigma destas plantas, a partir do estádio 7 tardio. No controle SR1, concentrações equivalentes apenas são observadas nos estádios finais do desenvolvimento. Os fenótipos observados nas plantas transgênicas sugerem que a proteína codificada por S/S_F-box esteja envolvida com o desenvolvimento do pistilo e com o controle do tamanho deste órgão. Adicionalmente, as plantas de RNAi apresentaram o fenótipo de perda da dominância apical. Os níveis de expressão do gene S/S_F-box foram avaliados em plantas que tiveram aumento na produção de auxina no estigma/estilete (plantas STIG1prom::iaaM), revelando que este gene não e regulado, a nível transcricional, por este hormônio. Experimentos de localização subcelular, realizados por expressão transitória da sequência de S/S_F-box fusionada a sequência dos genes repórteres GFP e YFP (S/S_F-box::GFP; S/S_F-box::YFP), indicaram que a proteína S/S_F-box esta localizada no citoplasma e no núcleo celular. Adicionalmente, foi realizado o screening de uma biblioteca de cDNAs de estigma/estilete, construída no sistema de duplo-hibrido, para investigar proteínas candidatas a interagirem com a proteína de S/S_F-box. Os resultados indicaram interação da proteína S/S_F-box com SKP1, confirmando a participação de S/S_F-box no complexo SCF, que promove a degradação de proteínas alvo pela via ubiquitina-proteassomo. Duas proteínas candidatas a alvo foram identificadas: os fatores de transcrição VOZ1 e SIP1, ambos envolvidos com a proliferação celular. Em suma, e possível propor que a proteína codificada por S/S_F-box tenha função relacionada a proliferação celular e ao desenvolvimento dos órgãos vegetais, incluindo o pistilo. / The study of sexual reproduction in plants is an area of increasing interest due to the importance of seeds and fruits in our daily diet, both resulting from the development of parts of the pistil, after fertilization. The aim of this study was to characterize a new F-box gene expressed in the N. tabacum pistil. F-box proteins act in protein-protein interactions, generally directing target proteins to degradation via ubiquitin-proteasome. Five genes of unknown function coding for putative F-box proteins were identified at two cDNAs libraries from N. tabacum stigmas/styles (DEPAOLI, 2006; QUIAPIM et al., 2009), previously constructed in our laboratory. The expression of each of these genes was analyzed in the different N. tabacum organs, by qRT-PCR. The 085H05 clone from the TOBEST library (QUIAPIM et al., 2009) showed preferential expression in floral organs. This clone was select for a more detailed functional characterization. The expression pattern of this gene was evaluated in the stigma/style during the 12 N. tabacum flower developmental stages (KOLTUNOW et al., 1990). The result revealed that its expression is regulated during development, reaching the highest expression level at anthesis (stage 12). It suggests that this gene is involved in the stigma/style development. The coding sequence of the gene corresponding to 085H05 was determined and, after amplification and cloning, the gene was named S/S_F-box (Stigma/Style_F-box). To understand the S/S_F-box protein function, transgenic plants either overexpressing or silencing (by RNAi) the S/S_F-box gene were generated. The RNAi plants showed reduced style and ovary when compared to the control SR1. In accordance, the overexpressing plants produced flowers with a style more elongated than the control, besides an ovary and a stigma of larger size. High concentrations of exudate were observed on the stigma surface of these plants, since the later stage 7. In the control SR1, equivalent concentrations are only observed at the later stages of development. The phenotypes observed in the transgenic plants suggest that the protein encoded by S/S_F-box is involved with pistil development and with the control of pistil size. Additionally, the RNAi plants showed the phenotype of loss of apical dominance. The expression levels of the S/S_F-box gene were evaluated in plants with increased auxin production in the stigma/style (plants STIG1prom::iaaM), showing that this gene is not transcriptionally regulated by this hormone. Subcellular localization experiments, carried out by transient expression of the S/S_F-box sequence fused to the reporter genes GFP and YFP V (S/S_F-box::GFP; S/S_F-box::YFP), showed that the S/S_F-box protein is localized in the cytoplasm and in the nucleus. Additionally, the screening of a stigma/style cDNA library constructed on the yeast two hybrid system was performed, to investigate candidate proteins for S/S_F-box protein interaction. The results indicated interaction between S/S_Fbox and the SKP1 protein, confirming the involvement of the S/S_F-box protein in the SCF complex, which promotes degradation of target proteins via ubiquitin-proteasome. Two candidates for target proteins were identified: the transcription factors VOZ1 and SIP1, both involved in cell proliferation. In summary, it is possible to propose that the protein encoded by S/S_F-box has functions related to cell proliferation and organ development, including the pistil.

Genetically modified (GM) crops are commercially cultivated worldwide but there are concerns on their possible negative impacts on soil biodiversity. A glasshouse study was conducted to determine effects of Bt maize residues on soil microbial diversity. Residues of Bt maize (PAN 6Q-308B) and non-Bt maize (PAN 6Q-121) were incorporated into the soil and corresponding maize seeds planted. The treatments were replicated three times. Fertilizer and water application were similar for both treatments. Rhizosphere and bulk soil was destructively sampled from each treatment and analyzed for microbial community level physiological profiles using Biolog plates with 31 different carbon substrates. Absorbance in the Biolog plates was recorded after 72 h of incubation at 20oC. Arbuscular mycorrhizal fungi spore counts were also determined. Field studies were conducted at the University of Free State and University of Fort Hare Research Farms to determine the effects of growing Bt maize on soil microbial diversity. One Bt maize cultivar (PAN6Q-308B) and non-Bt maize (PAN6Q-121) were grown in a paired experiment at University of Free State farm, while two Bt maize (DKC61-25B and PAN6Q-321B) and their near-isogenic non-Bt maize lines (DKC61-24 and PAN6777) were grown in a randomized complete block design with three replicates. Fertilization, weed control and water application, were similar for both Bt maize cultivars and their non-Bt maize counterparts. Rhizosphere soil samples were collected by uprooting whole plants and collecting the soil attached to the roots. The samples were analysed for microbial diversity and for arbuscular mycorrhizae fungal spore counts. Principal component analysis showed that soil microbial diversity was affected more by sampling time whereas genetic modification had minimal effects. Presence of residues also increased the diversity of microorganisms. Mycorrhizal fungal spores were not affected by the presence of Bt maize residues. Growing Bt maize had no effect on the soil microbial diversity in the rhizosphere.

O estudo da reprodução sexual de plantas e uma área de crescente interesse devido a importância de sementes e frutos em nossa dieta diária, ambos resultantes do desenvolvimento de partes do pistilo, apos fertilização. O objetivo deste trabalho foi caracterizar um novo gene F-box expresso no pistilo de N. tabacum. Proteínas F-box atuam na interação proteína-proteína, geralmente direcionando proteínas alvo para degradação pela via ubiquitina-proteassomo. Foram identificados cinco genes de função desconhecida que codificam putativas proteínas F-box, em duas bibliotecas de cDNAs de estigmas/estiletes de N. tabacum (DEPAOLI, 2006; QUIAPIM et al., 2009) previamente construídas em nosso laboratório. A expressão de cada um destes genes foi analisada nos diferentes órgãos de N. tabacum, por qRT-PCR. O clone 085H05 da biblioteca TOBEST (QUIAPIM et al., 2009) apresentou expressão preferencial nos órgãos florais. Este clone foi selecionado para uma caracterização funcional mais detalhada. O padrão de expressão deste gene foi avaliado no estigma/estilete durante os 12 estádios do desenvolvimento floral de N. tabacum (KOLTUNOW et al., 1990). O resultado revelou que sua expressão e regulada durante o desenvolvimento, atingindo o maior nível de expressão na antese (estádio 12). Isto sugere que este gene esteja envolvido no desenvolvimento do estigma/estilete. A sequência codificadora do gene correspondente a 085H05 foi determinada e, apos amplificação e clonagem, este gene foi denominado S/S_F-box (Stigma/Style_F-box). Para compreender a função da proteína de S/S_F-box, plantas transgênicas de superexpressao e de silenciamento (por RNAi) deste gene foram geradas. As plantas de RNAi apresentaram o estilete e o ovário reduzidos quando comparados ao controle SR1. Em concordância, as plantas de superexpressao produziram flores com o estilete mais alongado do que o controle, alem do estigma e do ovário de maior tamanho. Altas concentrações de exudato foram observadas na superfície do estigma destas plantas, a partir do estádio 7 tardio. No controle SR1, concentrações equivalentes apenas são observadas nos estádios finais do desenvolvimento. Os fenótipos observados nas plantas transgênicas sugerem que a proteína codificada por S/S_F-box esteja envolvida com o desenvolvimento do pistilo e com o controle do tamanho deste órgão. Adicionalmente, as plantas de RNAi apresentaram o fenótipo de perda da dominância apical. Os níveis de expressão do gene S/S_F-box foram avaliados em plantas que tiveram aumento na produção de auxina no estigma/estilete (plantas STIG1prom::iaaM), revelando que este gene não e regulado, a nível transcricional, por este hormônio. Experimentos de localização subcelular, realizados por expressão transitória da sequência de S/S_F-box fusionada a sequência dos genes repórteres GFP e YFP (S/S_F-box::GFP; S/S_F-box::YFP), indicaram que a proteína S/S_F-box esta localizada no citoplasma e no núcleo celular. Adicionalmente, foi realizado o screening de uma biblioteca de cDNAs de estigma/estilete, construída no sistema de duplo-hibrido, para investigar proteínas candidatas a interagirem com a proteína de S/S_F-box. Os resultados indicaram interação da proteína S/S_F-box com SKP1, confirmando a participação de S/S_F-box no complexo SCF, que promove a degradação de proteínas alvo pela via ubiquitina-proteassomo. Duas proteínas candidatas a alvo foram identificadas: os fatores de transcrição VOZ1 e SIP1, ambos envolvidos com a proliferação celular. Em suma, e possível propor que a proteína codificada por S/S_F-box tenha função relacionada a proliferação celular e ao desenvolvimento dos órgãos vegetais, incluindo o pistilo. / The study of sexual reproduction in plants is an area of increasing interest due to the importance of seeds and fruits in our daily diet, both resulting from the development of parts of the pistil, after fertilization. The aim of this study was to characterize a new F-box gene expressed in the N. tabacum pistil. F-box proteins act in protein-protein interactions, generally directing target proteins to degradation via ubiquitin-proteasome. Five genes of unknown function coding for putative F-box proteins were identified at two cDNAs libraries from N. tabacum stigmas/styles (DEPAOLI, 2006; QUIAPIM et al., 2009), previously constructed in our laboratory. The expression of each of these genes was analyzed in the different N. tabacum organs, by qRT-PCR. The 085H05 clone from the TOBEST library (QUIAPIM et al., 2009) showed preferential expression in floral organs. This clone was select for a more detailed functional characterization. The expression pattern of this gene was evaluated in the stigma/style during the 12 N. tabacum flower developmental stages (KOLTUNOW et al., 1990). The result revealed that its expression is regulated during development, reaching the highest expression level at anthesis (stage 12). It suggests that this gene is involved in the stigma/style development. The coding sequence of the gene corresponding to 085H05 was determined and, after amplification and cloning, the gene was named S/S_F-box (Stigma/Style_F-box). To understand the S/S_F-box protein function, transgenic plants either overexpressing or silencing (by RNAi) the S/S_F-box gene were generated. The RNAi plants showed reduced style and ovary when compared to the control SR1. In accordance, the overexpressing plants produced flowers with a style more elongated than the control, besides an ovary and a stigma of larger size. High concentrations of exudate were observed on the stigma surface of these plants, since the later stage 7. In the control SR1, equivalent concentrations are only observed at the later stages of development. The phenotypes observed in the transgenic plants suggest that the protein encoded by S/S_F-box is involved with pistil development and with the control of pistil size. Additionally, the RNAi plants showed the phenotype of loss of apical dominance. The expression levels of the S/S_F-box gene were evaluated in plants with increased auxin production in the stigma/style (plants STIG1prom::iaaM), showing that this gene is not transcriptionally regulated by this hormone. Subcellular localization experiments, carried out by transient expression of the S/S_F-box sequence fused to the reporter genes GFP and YFP V (S/S_F-box::GFP; S/S_F-box::YFP), showed that the S/S_F-box protein is localized in the cytoplasm and in the nucleus. Additionally, the screening of a stigma/style cDNA library constructed on the yeast two hybrid system was performed, to investigate candidate proteins for S/S_F-box protein interaction. The results indicated interaction between S/S_Fbox and the SKP1 protein, confirming the involvement of the S/S_F-box protein in the SCF complex, which promotes degradation of target proteins via ubiquitin-proteasome. Two candidates for target proteins were identified: the transcription factors VOZ1 and SIP1, both involved in cell proliferation. In summary, it is possible to propose that the protein encoded by S/S_F-box has functions related to cell proliferation and organ development, including the pistil.

The theoretical part of the diploma thesis summarizes basic facts related to the global climate change and provides up-to-date knowledge on the issue of genetically modified plants more resistant to stress factors of environment, including specific examples of strategies regarding preparation of genetically modified plants resistant particularly to abiotic stress. Although genetically modified plants and global climate changes belong to very important and current issue which public and foremost young generation should be sufficiently aware of, contemporary biology books and high school framework educational program pay to the issue very little attention. Therefore, the goal of this thesis is not only to summarize the basic facts and current knowledge regarding this issue but mainly to handle the issue in didactic level in the form of activating learning tasks. There were 28 learning tasks created in the thesis that gradually in unconventional way familiarize students with the issue of genetically modified plants and with the impacts of climate changes. Students must actively acquire information from accompanying materials to address the tasks properly, think critically over them, utilizing acquired knowledge and experience from the past to some extent. Fourteen tasks were chosen from the file created which...

My dissertation research examined the effect of the cultivation of insect-resistant Bacillus thuringiensis (Bt) maize on the soil environment with a goal of understanding how to obtain a balance between technological advancement and maintenance of a healthy soil ecosystem. Although Bt plants may help to reduce pesticide use, conferring benefits to farm workers and the environment, there are still unresolved questions about how the cultivation of Bt plants affects soil organisms. For this dissertation project, I used 14 different genotypes of Bt maize and non-Bt maize (Zea mays) to investigate the effects of transgenic Bt plants on the colonization ability, abundance, and diversity of symbiotic arbuscular mycorrhizal fungi (AMF) in the soil ecosystem over time. My greenhouse studies demonstrated that Bt maize plants exhibited reduced AMF colonization across multiple Bt genotypes and that effects were most pronounced when fertilizer levels were limited and spore density was high. In addition, I found that although differences in AMF colonization between Bt and non-Bt maize were difficult to detect in the field, spore density was reduced in Bt field plots after just one growing season. When I tested the effect of plot history on AMF and plant growth, I found that Bt and non-Bt maize plants had higher leaf chlorophyll content when grown in plots previously cultivated with the same maize line as the previous year, indicative of a positive feedback effect. I also examined potential mechanisms contributing to the reduced AMF colonization observed in Bt maize in greenhouse studies and determined that follow-up experiments should continue to investigate differences in root apoplastic invertase activity and root permeability in Bt and non-Bt maize. Future investigations would also benefit from examining potential differences in root exudate profiles and volatile organic compounds between Bt and non-Bt cultivars. Taken together, my dissertation results suggest that, while difficult to detect in the field, reductions in AMF colonization in Bt maize roots may be ecologically significant as they could lead to a decrease in the abundance of AMF propagules in the soil over time, potentially impacting soil structure and function in areas where Bt crop cultivation is high.

No description available.

Introgression of genes from crops into ruderal populations is a multi-step process requiring sympatry, synchronous flowering, chromosomal compatibility, successful pollination and development of the zygote, germination, establishment and reproduction of hybrid progeny. The goal of this thesis was to generate data on as many steps in this process as possible and integrate them into a predictive statistical model to estimate the likelihood of successful introgression under a range of scenarios. Rape (Brassica napus) and wild turnip (B. rapa var. oleifera) were used as a model system. A homozygous dominant mutation in the rape genome conferring herbicide resistance provided a convenient marker for the study of introgression. Potential differences between wild turnip populations from a wide range of geographic locations in New Zealand were examined. Hand pollination established the genetic compatibility of rape and wild turnip and a high potential for gene introgression from rape to wild turnip. Interspecific hybrids were easily generated using wild turnip as the maternal plant, with some minor differences between wild turnip populations. The frequency of successful hybridisation between the two species was higher on the lower raceme. However, the upper raceme produced more dormant interspecific hybrid seed. Field trials, designed to imitate rare rape crop escapes into the ruderal environment, examined the ability of rare rape plants to pollinate wild turnip plants over four summers. At a ratio of 1 rape plant for every 400 wild turnip plants, the incidence of interspecific hybridisation was consistently low (<0.1 to 2.1 % of total seed on wild turnip plants). There was a significant year effect with the first season producing significantly more seed and a greater frequency of interspecific hybrid progeny than the other years. The frequency of interspecific hybrid progeny increases when the ratio of rape: wild turnip plant numbers increases. The relative importance of anemophily and entomophily in the production of interspecific hybrids was examined. Wild turnip plants produced twice as many seeds with bee pollination relative to wind pollination. However, the frequency of interspecific hybrids under wind pollination was nearly twice that for bee pollination. Light reflectance patterns under UV light revealed a marked difference between wild turnip and rape flowers compared to near identical appearance under visible light. The data indicates that bees are able to distinguish between rape and wild turnip flowers and exhibit floral constancy when foraging among populations with these two species. Hybrid survival in the seed bank, germination and seedling establishment in the field are important components of fitness. Seed banks established in the soil after the field trials described above germinated in subsequent spring seasons. The predominantly brassica weed populations were screened for herbicide resistance and the numbers of interspecific hybrids germinating compared to the original frequency in the field trial results. Frequency of interspecific hybrids was reduced in the populations compared to the original seed deposit. Seed with a known frequency of interspecific hybrid seed was sown in a separate trial, and the frequency of interspecific hybrids compared at 0, 4, 6, and 8 weeks after sowing. Poor germination resulted limited competition between seedlings, however the frequency of interspecific hybrids declined over time indicating low plant fitness. There were no significant population effects on any parameters tested. Interspecific hybrids grown in a glasshouse were backcrossed to the parental species and selfed within the plant and within populations. Pollen from the interspecific hybrids was found to have markedly reduced fertility. Interspecific hybrid plants had low female fertility, with the majority (88%) of the pollinated flowers aborting the siliques. Of the remaining siliques, most (98%) had only one to three seeds per silique. Inheritance of the herbicide resistance gene was regular in backcrosses but highly skewed following self pollination with an excess of herbicide-sensitive progeny. Production of a stochastic predictive model integrated the information acquired over the practical work phase of this thesis and utilised the capabilities of @risk, a new application of a risk analysis tool. The three outputs examined were the number of flowering plants resulting from backcrosses to rape and wild turnip and self pollination of the interspecific hybrid progeny. Five scenarios were modelled and all demonstrated the high likelihood of introgression failure in this system. In all scenarios, >75% of simulations resulted in no interspecific hybrid progeny surviving to flowering in the third generation. In all scenarios, and for all three outputs, the seed set on the interspecific hybrids of the second generation was the major factor that limited the number interspecific hybrid progeny surviving to flowering in the third generation.

Commercialisées depuis 1996, les plantes génétiquement modifiées produisant une toxine insecticide (toxine Cry) dérivée de Bacillus thuringiensis et appelées plantes Bt ciblent certains Lépidoptères ou Coléoptères ravageurs. Au fil des ans, les surfaces cultivées en plantes Bt sont de plus en plus importantes et contrôlent de larges populations d'insectes. Pour limiter le risque de développement de populations résistantes, une stratégie agricole appelée 'Haute Dose / Zone Refuge' est actuellement recommandée aux Etats-Unis par l'Environmental Protection Agency. Cette stratégie préventive nécessite la plantation d'une 'zone refuge' composée de plantes non-Bt utilisables par le ravageur ciblé et plantée à proximité de la 'zone Bt' qui synthétise une haute dose de toxine Cry. <p><p>Mon projet de recherche s’inscrit dans le cadre de l’évaluation de l'efficacité de cette stratégie et s’articule en deux phases :une phase expérimentale et une phase théorique. La première se concentre sur la caractérisation en laboratoire de l'impact des toxines Cry sur la biologie d'un ravageur. Cette phase constitue un support au volet théorique :la mise au point d’un modèle mathématique évaluant l'efficacité de la stratégie HD/R. L'originalité de ce projet repose entre autre sur l'interactivité entre ces deux volets.<p><p>Volet expérimental. Impact des toxines Cry sur la biologie de Plodia interpunctella. Nous évaluons séparément l'impact d'une gamme de concentrations de deux toxines Cry (CryXX et CryYY) sur une série de paramètres comportementaux et biologiques d'un insecte commun des denrées stockées: Plodia interpunctella (Hübner) (Lepidoptera :Pyralidae). Ces paramètres sont sélectionnés car leur variation pourrait avoir un impact sur l'efficacité de la stratégie HD/R dans le contrôle de la résistance. Il est donc pertinent de les quantifier pour intégrer dans le modèle des ordres de grandeur réalistes et générer des résultats qui ne sont pas uniquement basés sur des spéculations théoriques.<p><p>Volet théorique A. Efficacité de la stratégie HD/R pour des plantes Bt synthétisant une ou deux toxines simultanément. La stratégie 'HD/R' a été développée pour prévenir la résistance envers les plantes Bt synthétisant une seule toxine. Or, depuis 2003, de nouvelles variétés de coton Bt synthétisant simultanément deux toxines Cry sont commercialisées (BollgardII® et WidestrikeTM). Nous évaluons, grâce au modèle que nous avons développé, l'efficacité de cette stratégie lors d'une utilisation exclusive de plantes Bt synthétisant une ou deux toxines.<p><p>Volet théorique B. Impact du ralentissement du développement des insectes sur les plantes Bt sur l'efficacité de la stratégie HD/R. Le volet expérimental met en évidence un allongement de la durée du développement des larves se nourrissant sur une diète contaminée en toxine Cry. Ce ralentissement induit une séparation temporelle entre l'émergence des adultes de la zone Bt et de la zone refuge et perturbe une hypothèse principale de la stratégie HD/R: le croisement aléatoire entre adultes, indépendamment du génotype et de la zone d'origine. Dans ce troisième chapitre, nous étudions l'impact de la perturbation du croisement aléatoire sur l'efficacité de la stratégie HD/R. Nous testons également deux options pour optimiser la stratégie en cas d'asynchronie: l'utilisation de plantes Bt synthétisant une faible concentration en toxine (atténuant le décalage entre l'émergence des adultes) ou l'augmentation de la taille de la zone refuge (favorisant la survie des individus porteurs d'allèle de sensibilité et donc optimisant la dilution de la résistance à la génération suivante). <p><p>Ce travail s'intègre dans une problématique actuelle et utilise des outils de biologie théorique (théories de la dynamique et de la génétique des populations) ainsi que le développement d'un modèle mathématique. Il apporte des éléments de réponse et de réflexion sur l'optimisation de la gestion de la résistance des insectes mais c'est aussi une illustration de la complémentarité entre la biologie expérimentale et théorique.<p><p><p>/<p><p>On the market since 1996, genetically modified plants synthesizing an insecticidal toxin (Cry toxin) stemmed from Bacillus thuringiensis, called Bt plants, target several insect pests (Lepidoptera or Coleoptera). Bt crops cover increasingly larger areas and control important pest populations The Insect Resistance Management Strategy (IRM) strategy currently recommended in the U.S.A. to limit the development of resistant populations is the High Dose / Refuge zone (HD/R) strategy. This pre-emptive strategy requires a refuge zone composed by non-Bt plants, usable by the target insect and in close proximity of the Bt zone synthesizing a high toxin concentration.<p><p>My research project contributes to the effectiveness assessment of this HD/R strategy. It is structured on two main parts: an experimental, and a theoretical section. The first part characterizes the impact of Cry toxins on the biology of an insect pest. It is the basis of the theoretical part: the implementation of a mathematical model, which evaluates the effectiveness of the HD/R strategy.<p>The originality of this project is based on the interactivity of these two components.<p><p>Experimental section. Impact of the Cry toxins on the biology of Plodia interpunctella. We assess the impact of a range of concentrations of two Cry toxins (CryXX et CryYY) on several behavioural and biological parameters of a common pest of stored products: Plodia interpunctella (Hübner) (Lepidoptera :Pyralidae). These parameters are selected because their variation could influence the effectiveness of a HD/R strategy. So, it is important to quantify these parameters so that realistic values can be integrated in our model. The results of the model are thus not based on theoretical assumptions alone.<p> <p>Theoretical section A. Effectiveness of a HD/R strategy with Bt plants synthesizing one or two toxins. Initially, the HD/R strategy has been developed to limit the resistance towards Bt plants synthesizing one toxin. However, since 2003, new Bt cotton varieties synthesize two toxins simultaneously (BollgardII® et WidestrikeTM). We assess, with our model, the effectiveness of this strategy for Bt plants synthesizing one or two toxins.<p><p>Theoretical section B. Impact of the slowing down of the insect development reared on Bt plants on the effectiveness of the HD/R strategy. The experimental part demonstrates that larvae reared on a Bt diet have a protracted development duration. The consequence of this is a temporal separation between adult emergence in the two zones (Bt zone and refuge zone). This could affect the main assumption of the HD/R strategy, i. e. random mating independently of the genotype and of the native zone. In this third chapter, we study the impact of random mating disruption on the effectiveness of a HD/R strategy. We test two options to optimise the strategy in case of asynchrony: the use of Bt plants synthesizing a lower toxin concentration (limiting emergence asynchrony) or increasing the refuge zone size (favouring the survival of insect carrying one or two susceptible allele and thus optimising the dilution of resistance at the next generation). <p><p>This work is applied to a current issue. It uses some of the tools of theoretical biology (theories of population dynamics and population genetics) and develops a mathematical model. It provides some responses and some elements of thought about insect resistance management. It is also an illustration of the complementarity between experimental and theoretical biology.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished