Dissertations / Theses on the topic 'Plant transgenesis'

Orientador: Alessandra Alves de Souza
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: A produção do suco de laranja concentrado é uma das commodities mais importantes para o agronegócio brasileiro, entretanto, os constantes problemas fitossanitários que afetam a citricultura vem aumentando os custos de produção e consequentemente a rentabilidade econômica deste setor. É urgente a busca por alternativas para solucionar os problemas fitossanitários da citricultura, nesse sentido, a utilização de transgenia mostra-se uma ferramenta promissora, pois possibilita a obtenção de plantas com genes exógenos que conferem resistência a doenças. Uma das doenças que afetam a citricultura brasileira é a clorose variegada dos citros (CVC), causada pela bactéria Xylella fastidiosa, onde todas as variedades de Citrus sinensis mostram-se suscetíveis a doença. Uma estratégia que vem sendo utilizada para resistência a X. fastidiosa que afeta a cultura de uva na Califórnia-EUA envolve a chamada "confusão do patógeno" onde utiliza-se genes do próprio patógeno visando a alteração da sinalização molecular entre as células bacterianas, interferindo em sua patogenicidade. Neste trabalho foram abordadas as transformações de Nicotiana tabacum e Citrus sinensis com o gene rpfF de X. fastidiosa causadora da CVC, envolvido na síntese de uma molécula sinalizadora que regula a expressão de genes associados a patogenicidade dessa bactéria. Eventos dos cultivares de laranja doce Hamlin e Pineapple transformados com rpfF foram desafiados com X. fastidiosa e, após avaliações nas fases inicial e avançada de sintomas, foi observada uma redução na incidência e na severidade de sintomas de CVC. A movimentação bacteriana ao longo de tais plantas também foi comprometida, sendo que a população bacteriana em pontos distantes do ponto de inoculação foi maior em plantas do tipo selvagem quando comparadas as transgênicas. Esses resultados sugerem que as moléculas produzidas pelas plantas transgênicas foram capazes de alterar o comportamento da bactéria, reduzindo sua patogenicidade. Outro fitopatógeno que ataca pomares brasileiros é Xanthomonas citri subsp. citri, que também apresenta o gene rpfF, e assim como em X. fastidiosa, é responsável pela sinalização molecular. Eventos transgênicos de Carrizo e Pineapple também foram desafiados com X. citri. Nesse patógeno, a interrupção da sinalização mediada por DSF, Diffusible Signal Factor - uma molécula de ácido graxo, reduz sua virulência, e interessantemente, sintomas de cancro cítrico foram reduzidos nas plantas transgênicas. Em folhas transgênicas não foi observado o aparecimento de pústulas, e biofilmes formados na área da inoculação foram alterados. Genes modulados por DSF em bactérias isoladas de plantas transgênicas foram reprimidos, sugerindo que a sinalização foi comprometida. Por fim, seiva das plantas transgênicas não ativou a expressão do promotor engA::GFP em Xanthomonas campestris biosensoras, indicando que a molécula produzida por essas plantas foi capaz de alterar a sinalização. Por outro lado, a seiva das plantas transgênicas ativou a expressão do hxfA::PhoA em X. fastidiosa biosensoras, indicando a funcionalidade dessa molécula para esse fitopatógeno. Portanto, em ensaios com X. citri, o DSF das plantas transgênicas atuou como antagonista, diminuindo a virulência da bactéria através da alteração da sinalização molecular. Esses resultados mostram que as moléculas sinalizadoras produzidas por plantas transformadas com rpfF de X. fastidiosa são promissoras na tentativa de controle de CVC e cancro cítrico
Abstract: The production of concentrate orange juice is one of the most important commodities for Brazilian agribusiness, however, the constant phytosanitary problems affecting the citrus industry is increasing production costs and consequently the economic profitability of this sector. It is urgent to search for alternatives to solve citrus phytosanitary problems, in this sense, the use of transgenesis shows a promising tool because it enables the production of plants with exogenous genes that confer resistance to diseases. One of the diseases that affect Brazilian citrus industry is the citrus variegated chlorosis (CVC), caused by the bacterium Xylella fastidiosa, where all varieties of Citrus sinensis are susceptible to this disease. One strategy that has been used for X. fastidiosa resistance that affects grape cultures in California-USA involves the so-called "pathogen confusion" that is related to the usage of genes of the pathogen itself aiming to change the molecular signaling between bacterial cells by interfering in its pathogenicity. In this work we will discuss the transformation of Nicotiana tabacum and Citrus sinensis with the rpfF gene isolated from X. fastidiosa causing the CVC, involved in the synthesis of a signaling molecule that regulates the expression of genes associated with pathogenicity of these bacteria. Transgenic events of Hamlin and Pineapple transformed with rpfF were inoculated with X. fastidiosa and after evaluations in early and advanced stages of symptoms, a reduction was observed in the incidence and severity of symptoms of CVC. Bacterial movement along these plants was also impaired, and the bacterial population analyzed far from the point of inoculation was higher in wild type plants compared to transgenic ones. These results suggest that the molecules produced by the transgenic plants were able to change the behavior of the bacteria, reducing its pathogenicity. Another pathogen that attacks Brazilian orchards is Xanthomonas citri subsp. citri, which also features rpfF gene, and as in X. fastidiosa is responsible for molecular signaling. Transformed plants of Carrizo and Pineapple were also challenged with X. citri. In this pathogen, the interruption of DSF-mediated signaling reduces its virulence, and interestingly, citrus canker symptoms were reduced in transgenic plants. In transgenic leaves there was no pustules development and alterations in biofilms formed in the area of inoculation were observed. Genes modulated by DSF in bacteria isolated from transgenic plants were repressed, suggesting that signaling was compromised. Finally, sap of transgenic plants did not activate the expression of the promoter engA::GFP in Xanthomonas campestris biosensors, indicating that the molecule produced by these plants was able to change the signaling. On the other hand, the sap of transgenic plants activated the expression of hxfA::PhoA in X. fastidiosa biosensors, indicating the functionality of this molecule for this pathogen. Therefore, in assays with X. citri, the DSF of transgenic plants acted as antagonist, decreasing the virulence of the bacteria by changing the molecular signaling. These results show that the signaling molecules produced by plants transformed with rpfF of X. fastidiosa are promising in the attempt to control CVC and citrus canker
Doutorado
Genetica Vegetal e Melhoramento
Doutora em Genética e Biologia Molecular

Dissertation (PhD) -- University of Stellenbosch, 2004.
ENGLISH ABSTRACT: In most plants the efficiency of sucrose production and the systemic distribution thereof are the major determinants of growth, development and yield. The factors governing sugar partitioning co-ordinate its distribution in response to intrinsic and environmental signals. These factors include sugar transporters and invertases as well as metabolites, including sucrose and glucose, which function as signalling molecules to modulate gene expression. The genetic transformation of plants and the subsequent development of transgenic lines with disturbed sugar metabolism have made an unprecedented impact on the study of sugar translocation and -partitioning. For instance, the transformation of plants with a yeast-derived invertase targeted to different subcellular compartments has led to the elucidation of several key aspects of sugar metabolism, including phloem loading mechanisms, the regulation of photosynthesis by sugars, the importance of sugar-metabolism compartmentation with regards to sucrose biosynthesis, storage and distribution, as well as the role of cell-wall invertase in phloem unloading and sink strength. In this study, a similar strategy of transgenic plant analysis was employed to expand our insight into the regulation of sugar partitioning. The yeast-invertase Suc2 gene, from Saccharomyces cere visiae , was overexpressed in either the cytosol, vacuole or apoplast of transgenic tobacco plants. These transgenic lines displayed varying increases in invertase activity, altered sugar levels and consequently disturbed sink-source interactions and sugar partitioning. Transgenic lines overproducing the yeast-derived invertase in either the vacuole (Vac-Inv) or apoplast (Apo-Inv) were utilised to analyse the effect of the altered sugar levels in sink and source organs on the expression of sugar transporters, as well as the endogenous cell wall invertase and inhibitors in these plants. Transcript levels of the sucrose transporter NtSUT1 and hexose transporter NtMST1 encoding genes increased significantly in the source leaves and roots of Vac-Inv lines, whereas increased NtMst1 transcript levels were also detected in the roots of Apo-Inv lines. The increased mRNA levels could be correlated to the altered invertase activities and sugar levels in these tissues. It is concluded that NtSUT1 and NtMST1 are differentially regulated by sucrose and/or hexose content on a transcriptional level. Furthermore, the regulatory effect of the altered sugar levels on transporter expression depended on the subcellular compartment in which the yeast invertase was expressed. It would seem that the subcellular compartmentation of sugar metabolism is also fundamental to the regulation of sugar partitioning. The transcription levels of the endogenous cell wall invertase (CWt) and cell wall invertase inhibitor (Cwi-Inh) genes were examined in the various tissues of Apo-Inv and Vac-Inv lines at both the vegetative and flowering growth stages. In comparison with the control lines, the various tissues of the Apo-Inv and Vac-Inv lines displayed altered Cwi and Cwi-Inh expression levels, depending on the sink-source status and growth stage. However, no obvious correlation between the Cwi and Cwi-Inh expression levels and soluble sugar content of these tissues was found. It is suggested that the post-transcriptional and post-translation control of these proteins by sugars might play an important role in their regulation. Analysis of the Cwi:Cwi-lnh mRNA ratio and growth observations of the various tissues of control as well as Apo-Inv and Vac-Inv lines indicated that this transcription ratio could be an accurate indicator of the sink strength of sink organs. In addition, the influence of sink-source interactions on sugar partitioning was investigated. Reciprocal grafting between Apo-Inv and control lines resulted in scions with an altered sucrose metabolism in either the sink or source organs. These scions were subjected to biomass distribution, soluble sugar quantification and C4C]- radiolabelling experiments. The latter revealed an unaltered state of sugar partitioning from the above-ground tissues of the Apo/GUS scions and a significant shift in sugar partitioning towards the roots of the GUS/Apo scions in comparison to the control GUS/GUS scions. Phenotypic changes, opposite to those observed in Apo-Inv lines expressing the heterologous invertase in both sink and source organs, could initially be observed in the GUS/Apo and Apo/GUS scions. However, no significant differences in phenotype or biomass distribution could be observed between the mature GUS/Apo, Apo/GUS and GUS/GUS scions seven weeks postgrafting. This inconsistency between phenotype and sugar partitioning might be explained by an increase in the respiration rate of the tissues as supported by the soluble sugar content. These results highlight the complexity and adaptability of sucrose metabolism and sugar partitioning. In addition, it confirms that sugar partitioning can be modulated by sink-source interactions and emphasise the importance of invertases in the regulation of sugar partitioning through its ability to alter sink strength. This study forms part of the rapidly expanding initiative to unravel the control mechanisms of sugar partitioning. The results obtained in this study confirmed again that the introduction and expression of a single heterologous gene in transgenic plants could provide significant insight into the regulation of this process. It was shown here that the expression of sugar transporters is closely regulated by sugar levels and therefore fulfils a vital function in sugar sensing and consequently the regulation of sugar partitioning. The data presented in this study also demonstrated the intricate and flexible nature of the relationship that exists between sugar metabolism, partitioning and growth phenomena.
AFRIKAANSE OPSOMMING: Die doeltreffendheid van sukroseproduksie, tesame met die sistemiese verspreiding daarvan, is die vernaamste faktore wat die groei, ontwikkeling en opbrengsvermoë van die meeste plante bepaal. Die faktore wat suikerverdeling beheer, funksioneer om suikerverspreiding te koordineer in reaksie op beide inherente- en omgewingsseine. Hierdie faktore sluit suikertransporters en invertases in, asook metaboliete soos sukrose en glukose wat funksioneer as seinmolekule in die modulering van geenuitdrukking. Die genetiese transformasie van plante en die gevolglike daarstelling van transgeniese lyne met veranderde suikermetabolismes het 'n beduidende inwerking op die bestudering van suikervervoer en -verdeling gehad. Byvoorbeeld, die transformasie van plante met 'n gis-invertase geteiken na verskillende sub-sellulêre kompartemente, het tot die toeligting van verskeie aspekte van suikermetabolisme gelei, insluitende dié van floëemladingsmeganismes, die regulering van fotosintese deur suikers, die belang van kompartementalisering ten opsigte van sukrosebiosintese, -opberging en -verspreiding, en die rol van selwand-invertases in floëemontlaaiing en swelgpuntkrag. In hierdie studie is van soortgelyke transgeniese plantontledings gebruik gemaak om 'n dieper insig tot die regulering van suikerverdeling te verkry. Die gis-invertase Suc2 geen, afkomstig van Saccharomyces cerevisiae, is ooruitgedruk in óf die sitosol, vakuool óf apoplastiese ruimte van transgeniese tabakplante. Hierdie transgeniese lyne het wisselende toenames in invertase-aktiwiteite en veranderde suikervlakke getoon, asook gevolglike versteurde bron-swelgpunt interaksies en suikerverdeling. Transgeniese lyne met ooruitdrukking van die gis-invertase in óf die vakuool (Vac-Inv) óf die apoplast (Apo-Inv) is gebruik om die gevolg van die veranderde suikervlakke in bron- en swelgpuntorgane op die uitdrukking van suikertransporters, asook die endogene selwand-invertase en invertase-inhibitor in hierdie plante te bepaal. Transkripsievlakke van die sukrosetransporter NtSut1 en die heksosetransporter, NtMst1, het beduidend toegeneem in die bron-blare en wortels van die Vac-Inv lyne; 'n toename in NtMst1 transkripsievlakke is ook in die wortels van Apo-Inv lyne bevestig. Die toenames in boodskapper RNA kon gekorreleer word met die veranderde invertase-aktiwiteite en suikervlakke in hierdie weefsels. Die gevolgtrekking word gemaak dat NtSUT1 en NtMST1 differensieël gereguleer word op transkripsionele vlak deur die sukrose en/of heksose inhoud van weefsels. Meer nog, die regulerende effek van die veranderde suikervlakke op transporteruitdrukking het afgehang van die subsellulêre kompartement waarin die gis-invertase uitgedruk is. Dit wil dus voorkom dat die subsellulêre kompartementalisering van suikermetabolisme fundamenteel tot die deurgee en waarneming van suikerseine is, met In gevolglike eweneens belangrike rol in die regulering van suikerverdeling. Die transkripsievlakke van beide die endogene selwand-invertase (CWI) en die selwand-invertase-inhibitor (CWI-Inh) enkoderende gene is in verskeie weefsels van die Apo-Inv en Vac-Inv lyne, tydens beide die vegetatiewe- en blomstadia, bestudeer. Die onderskeie weefsels van die Apo-Inv en Vac-Inv lyne het, in vergelyking met die kontrole lyne, veranderde Cwi en Cwi-inh transkripsievlakke getoon wat bepaal is deur bron-swelgpunt status en groeistadium. Geen duidelike korrelasie kon tussen beide Cwi en Cwi-inh uitdrukkingsvlakke en oplosbare suiker inhoud gevind word nie. Daar word voorgestel dat post-transkripsionele en posttranslasionele beheer deur suikers 'n belangrike rol in die regulering van hierdie proteïne speel. Bestudering van die Cwi:Cwi-lnh mRNA verhouding, asook groei verskynsels van die onderskeie weefsels van kontrole en Apo-Inv en Vac-Inv lyne, dui daarop dat hierdie transkripsievlak-verhouding moontlik 'n akkurate aanwyser van die swelgpuntkrag van 'n swelgpuntorgaan kan wees. Voorts is die invloed van bron-swelgpuntorgaan interaksies op suikerverdeling ondersoek. Omgekeerde enting tussen Apo-Inv en kontrole lyne het entlote met gemodifiseerde suikermetabolisme in óf hul bron- óf hul swelgpuntorgane tot gevolg gehad. Hierdie entlote is aan biomassaverspreidings-, oplosbare suiker kwantifisering en C4C]-radiomerking eksperimente onderwerp. Hierdie resultate het gewys dat, in vergelyking met die kontrole (GUS/GUS) ente, daar geen verandering in die status van suikerverdeling vanaf die bogrondse plantdele in die Apo/GUS ente is nie, maar wel 'n beduidende verskuiwing in suikerverdeling na die wortels van die GUS/Apo ente. Fenotipiese veranderinge, wat teenoorgesteld van dié teenwoordig in die Apo- Inv lyne waar die heteroloë invertase in beide bron en swelgpuntorgane uitgedruk word, is aanvanklik in die GUS/Apo en Apo/GUS ente waargeneem. Geen verskille in fenotipe of biomassa-verspreiding kon egter sewe weke na die entings prosedures tussen die GUS/Apo, Apo/GUS and GUS/GUS ente gevind word nie. Dit mag verduidelik word deur 'n moontlike toename in respirasietempo in die betrokke weefsels; die oplosbare suikervlakke wat in die verskillende ente aangeteken is ondersteun dié moontlikheid. Hierdie resultate as geheelonderstreep die kompleksiteit en aanpasbaarheid van suikermetabolisme en -verdeling. Verder bevestig dit dat suikerverdeling beïnvloed kan word deur bron-swelgpunt interaksies, asook die belang van invertases in die regulering van suikerverdeling gegewe die vermoë om swelgpuntkrag te verander. Hierdie studie vorm deel van 'n vinnig groeiende inisiatief om die beheermeganismes van suikerverdeling te ontrafel. Die resultate verkry in hierdie studie bekragtig die belang van rekombinante DNA tegnologie in die bestudering van fundamentele plantprosesse. Die invoeging en uitdrukking van 'n geteikende gisinvertase in transgeniese plante het gelei tot veranderde suikervlakke en bronswelgpunt interaksies in hierdie lyne met die gevolglike ontginning van waardevolle inligting ten opsigte van die regulering van suikerverdeling in reaksie tot interne seine. Daar is aangetoon dat suikertransporters onlosmaakbaar gekoppel is aan die deurgee en waarneming van suikerseine, spesifiek op die vlak van transkripsionele regulering, en dus ook die regulering van suikerverdeling. Voorts wys die resultate op die komplekse en aanpasbare aard van die verhouding wat bestaan tussen suikermetabolisme, -verdeling en groeiverskynsels.

Amb la finalitat de garantir la seguretat dels consumidors i del medi ambient, a la UE els aliments modificats genèticament (MG) estan sotmesos a un rigorós procés d'autorització que inclou: caracterització molecular del transgèn i anàlisis comparatives a nivell nutricional i agronòmic. L'ús de tècniques de profiling per avaluar la possible existència d'efectes no esperats derivats de la inserció i/o expressió del transgèn s'ha proposat com a una eina complementària per l'avaluació de la seguretat alimentària. L'objectiu de la present tesis és avaluar la variabilitat associada a la inserció i expressió de transgens en plantes, utilitzant com a exemple el blat de moro MON810. Es pretén complementar les aproximacions ja existents basades en l'estudi de paràmetres concrets mitjançant les tècniques de profiling. Des del punt de vista transcriptòmic i proteòmic, les varietats de blat de moro MON810 semblen ser substancialment equivalents a les seves varietats comparables no-MG. En conseqüència, és possible la producció de plantes MG amb el mínim d'efectes no esperats.
To ensure the safety of consumers and the environment, genetically modified (GM) food and feed are submitted to strict legislation. The EU establishes an authorisation procedure requiring: molecular characterisation of the transgene and compositional and agronomic comparative analysis. The use of profiling approaches to evaluate the possible occurrence of unintended effects derived from the insertion and/or expression of the transgene has been proposed as complementary tool for safety assessment. The objective of this thesis was to evaluate the variability associated to the insertion and expression of transgenes in plants, using as example MON810 maize. We aim to complement the existing targeted approaches by providing more unbiased information on the basis of profiling techniques. From the transcriptomics and proteomics perspectives, MON810 maize varieties seem to be substantially equivalent to their non-GM comparators. Thus, the production of GM plants with minimal unexpected effects is possible.

A hybrid between a highly regenerative diploid clone (BARD 1-3) of Solanum phureja and haploid inducer IVP 101 was transformed with Agrobacterium tumefaciens strain 4404 containing plasmid pHB2892 with genes for green florescent protein (GFP) and kanamycin resistance. Hemizygous primary transformants (To) were produced from three leaf discs: 17 diploid plants from one leaf disc, three and nine tetraploids from the other two leaf discs. GFP expression was observed qualitatively under fluorescence microscopes and quantitatively with a GFP meter. Anther culture of tetraploids produced 29 plants, none with high levels of GFP. Segregation ratios for tetraploid T1 seedlings fit models for single duplex insertions (35 transgenic: 1 non) or double simplex insertions (15 transgenic: 1 non). Diploid T1 seedlings segregated for deleterious traits: dwarfed size and curled leaves, as well as the GFP transgene. Similar segregation patterns in diploid families implied that all diploids may have been from the same transformation event. The cumulative segregation showed the dwarfed and curled plants fit a single recessive gene ratio (3 normal: 1 mutant), and GFP fit a double-copy insertion ratio (15 transgenic: 1 non). There was substantial GFP silencing evidenced by the loss of expression in plants that had originally been selected for high GFP. However, six selections were found to be free of deleterious traits, consistently high expressers of GFP, and producers of stainable pollen with less 2n than IVP 101.
Master of Science

Genetically modified crops are submitted to strict regulation to ensure the safety of consumers and the environment. To complement the comparison between GM plants and their counterparts, in the present Thesis, we evaluated the possible unexpected effects of the transgene on the host plant, by means of transcriptomic technologies. More exactly, we studied three pathogen-resistant GM rice lines: S-afp, expressing constitutively the antifungal protein AFP; and S-bp217 and S-bp213, expressing undecapeptide BP100 derivatives, which were developed in the UdG in the context of this Thesis. Although the high phytotoxicity of the BP100 derivatives on the host plant the transcriptional changes observed in S-afp, S-bp217 and S-bp213 compared to the conventional line Senia were similar that those observed in other GM crops, of other species and with different transgenes, and only the half of them was attributed to the insertion and/or expression of the transgene.
Les plantes modificades genèticament (MG) destinades a comercialització estan sotmeses a estricta legislació per garantir la seguretat del consumidor i del medi ambient. Per complementar la comparativa entre plantes MG i convencionals, en aquesta tesi s’ha abordat l’avaluació dels possibles efectes no esperats del transgèn sobre la planta hoste, mitjançant tècniques de transcriptòmica. Concretament s’han estudiat línies d'arròs MG que presenten fenotips de resistència a patògens: S-afp, que expressa constitutivament la proteïna antifúngica AFP, i S-bp213 i S-bp217, que expressen derivats de l’undecapèptid BP100, desenvolupat a la UdG, que s’han obtingut també en el marc d’aquesta tesi. Malgrat l’elevada fitotoxicitat dels derivats de BP100 enfront la planta hoste, els canvis transcripcionals de S-afp, S-bp213 i S-bp217 respecte la línia convencional Senia són similars als observats en altres events MG, de diferents espècies i amb diferents transgens; i només la meitat d’ells s’ha atribuit a la presència o expressió del transgèn.

Diante do atual cenário de expansão dos plantios geneticamente modificados e da carência de metodologias no Brasil para prever os seus impactos foi desenvolvimento um método intitulado Impactos-PGM para Avaliação de Impactos Ambientais e Alimentares de Plantas Geneticamente Modificadas utilizando como ferramenta um Software de aplicação geral adequado e modificado com esta finalidade. Para tanto, foi inicialmente realizado o levantamento de indicadores de impactos a partir da literatura. Esta consulta foi realizada pela aplicação de questionário de acordo com a técnica delphi. Posteriormente, os especialistas foram entrevistados presencialmente: foram apresentados os indicadores mais representativos para a avaliação dos impactos das PGMs, possibilitando a elaboração do método Impactos-PGM por meio da adequação do Software Impactos.
In face of the current expansion scenario of genetically modified crops and the lack of methodologies in Brazil to predict its impacts, the present work has been proposed to develop a method entitled Impactos-GMP for \"Food and Environmental Assessment of Genetically Modified Plants (GMPs) Impacts using as tool a software of general application suitable and modified for this purpose. Thus, the survey was first conducted by identifying impact indicators from the literature. This survey was held through the application of a questionnaire according to the delphi technique. Subsequently, the researchers were interviewed in person to whom were presented the most representative indicators for GMPs assessing impacts, enabling the development of the method Impactos-GMP with the adequacy of the iImpacts software.

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.

碩士
國立中興大學
生物科技學研究所
102
Infectious bursal disease virus (IBDV) is an acute and highly contagious virus which is main cause of infectious bursal disease. It primarily occurrs in 3-6 weeks chickens and seriously affects the economy loss of poultry industry. Currently, inactivated and attenuated vaccines are two main strategies for chicken IBD prevention. In recent years, the safer and low-cost vaccines are produced by using plants as a platform. In this study, we constructed Bamboo mosaic virus (BaMV), Tobacco mosaic virus (TMV), Foxtail mosaic virus (FoMV), and Potato virus X (PVX) as plant viral vectors. The coat protein genes of viral vectors are replaced with IBDV VP2 gene and fused with His-tag in the C-terminal, to generate BaMVdC-VP2His, TMVdC-VP2His, FoMVdC-VP2His, and PVXdC-VP2His. To test the VP2 production, these four recombinant viruses were individually transfected into Nicotiana benthamiana leaves by agro-infiltration. The results showed that among four viral vectors, BaMV vector expressed the most high-level accumulation of VP2 protein. In order to stably express VP2 protein in plants, we generated BaMVdC-VP2His transgenic N. benthamiana plants by Agrobacterium- mediated transformation. Western blot analysis confirmed the VP2 protein could be expressed in homologous transgenic lines. The virus like particles (VLP) of VP2 approximately 20-30 nm in diameter were purified and observed by transmission electron microscopy. Taking into account the environmental safety and high expression of VP2His protein, different gene silencing suppressors replace BaMV TGB or co-expression with BaMVdC-VP2His. Furthermore, complementation coat protein function of BaMVdC-VP2His by co-expression of BaMV CP. The results indicate VP2 protein dose not significantly increase. To develop edible vaccine, the transgenic barley (Hordeum vulgare) were generated. The results showed that BaMVdC-VP2His transgenic barley accumulated low VP2 protein. However, we have successfully established tissue culture and plant regeneration system in barley. We will use VP2-VLP to immuning chicken and to test the neutralizing antibodies production. We hope in the future, to develop subunit vaccine against IBDV by the BaMV based vector system.

Please read the abstract in the section, 00front, of this document
Dissertation (MSc (Agric))--University of Pretoria, 2006.
Agricultural Economics, Extension and Rural Development
unrestricted

by Fong Man Kim.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2001.
Includes bibliographical references (leaves 127-137).
Abstracts in English and Chinese.
Thesis committee --- p.i
Acknowledgements --- p.ii
Abstract --- p.iii
List of figures --- p.viii
List of tables --- p.xi
Abbreviations --- p.xii
Table of contents --- p.xiv
Chapter CHAPTER 1 --- GENERAL INTRODUCTION --- p.1
Chapter CHAPTER 2 --- LITERATURE REVIEW --- p.3
Chapter 2.1 --- MIH from Penaeus vannamei --- p.3
Chapter 2.1.1 --- General Introduction to P. vannamei --- p.3
Chapter 2.1.1.1 --- Morphology --- p.3
Chapter 2.1.1.2 --- Geographical distribution --- p.5
Chapter 2.1.1.3 --- Economic value --- p.5
Chapter 2.1.2 --- Physiology of Molting in Crustacean --- p.7
Chapter 2.1.2.1 --- The molt cycle --- p.7
Chapter 2.1.2.2 --- Physiological effects of ecdysone --- p.8
Chapter 2.1.2.3 --- Regulation of the secretion of ecdysone --- p.9
Chapter 2.1.2.4 --- Physiological effects of Molt-inhibiting hormone --- p.10
Chapter 2.1.3 --- Cloning of MIH cDNA from P. vannamei --- p.14
Chapter 2.1.3.1 --- Molecular identity of MIH --- p.14
Chapter 2.1.3.2 --- Cloning of MIH cDNA --- p.15
Chapter 2.1.3.3 --- Comparison of the cloned MIH-like cDNA with the CHH/MIH/VIH peptide family --- p.16
Chapter 2.2 --- Plants as Bioreactors --- p.20
Chapter 2.2.1 --- Principles & Techniques --- p.20
Chapter 2.2.2 --- Advantages of plant bioreactors --- p.21
Chapter 2.2.3 --- Tobacco expression system --- p.22
Chapter 2.2.3.1 --- Tobacco as model plants --- p.22
Chapter 2.2.3.2 --- Transformation methods --- p.23
Chapter 2.2.4 --- Phaseolin --- p.26
Chapter CHAPTER 3 --- EXPRESSION OF MIH IN TRANSGENIC TOBACCO --- p.28
Chapter 3.1 --- Introduction --- p.28
Chapter 3.2 --- Materials & Methods --- p.29
Chapter 3.2.1 --- Chemicals --- p.29
Chapter 3.2.2 --- Plant materials --- p.29
Chapter 3.2.3 --- Bacterial strains and plasmid vectors --- p.30
Chapter 3.2.4 --- Construction of chimeric genes - --- p.30
Chapter 3.2.4.1 --- PCR amplification of MIH --- p.30
Chapter 3.2.4.2 --- Cloning of PCR-amplified MIH into vector pET --- p.31
Chapter 3.2.4.3 --- Cloning of MIH into vector pBK/Phas-sp and pTZ/Phas --- p.31
Chapter 3.2.4.4 --- Cloning of MIH into binary vector pBI121 --- p.32
Chapter 3.2.5 --- Transformation of Agrobacterium with pBI121/Phas-sp-MIH and pBI121 /Phas-MIH by electroporation --- p.39
Chapter 3.2.6 --- Transformation of tobacco --- p.40
Chapter 3.2.7 --- Selection of transgenic plants --- p.41
Chapter 3.2.8 --- GUS assay --- p.42
Chapter 3.2.9 --- Extraction of leaf genomic DNA --- p.43
Chapter 3.2.10 --- Extraction of total RNA from developing seeds --- p.44
Chapter 3.2.11 --- Synthesis of DIG-labeled DNA and RNA probes --- p.45
Chapter 3.2.12 --- Southern blot analysis of genomic DNA --- p.47
Chapter 3.2.13 --- Reverse transcriptase - polymerase chain reaction (RT-PCR) --- p.47
Chapter 3.2.14 --- Northern blot analysis of total RNA --- p.48
Chapter 3.2.15 --- Protein extraction and tricine-SDS-PAGE --- p.49
Chapter 3.2.16 --- Purification of 6xHis-tag proteins --- p.50
Chapter 3.2.17 --- Western blot analysis --- p.50
Chapter 3.2.18 --- In vitro transcription & translation --- p.52
Chapter 3.2.18.1 --- Construction of transcription vector containing the chimeric MIH gene --- p.52
Chapter 3.2.18.2 --- In vitro transcription --- p.56
Chapter 3.2.18.3 --- In vitro translation --- p.56
Chapter 3.2.19 --- Particle bombardment --- p.57
Chapter 3.2.19.1 --- Construction of MIH-GUSN fusion chimeric genes --- p.57
Chapter 3.2.19.2 --- Conditions of particle bombardment --- p.63
Chapter 3.2.20 --- Codon modification of MIH gene --- p.63
Chapter 3.3 --- Results --- p.73
Chapter 3.3.1 --- Construction of chimeric MIH genes --- p.73
Chapter 3.3.2 --- "Tobacco transformation, selection and regeneration" --- p.73
Chapter 3.3.3 --- Detection of GUS activity --- p.74
Chapter 3.3.4 --- Southern blot analysis --- p.79
Chapter 3.3.5 --- Detection of MIH transcript in transgenic tobacco --- p.83
Chapter 3.3.5.1 --- RT-PCR --- p.83
Chapter 3.3.5.2 --- Northern blot analysis --- p.86
Chapter 3.3.6 --- Detection of MIH protein by Tricine-SDS-PAGE --- p.86
Chapter 3.3.7 --- Detection of MIH protein by western blot analysis --- p.88
Chapter 3.3.7.1 --- Western blot analysis using Anti-MIH antibody --- p.88
Chapter 3.3.7.2 --- Western blot analysis using Anti-His antibody --- p.90
Chapter 3.3.7.3 --- Western blot analysis using Anti-MIHA & Anti-MIHB antibodies --- p.90
Chapter 3.3.8 --- Purification of 6xHis-tag proteins by Ni-NTA column --- p.94
Chapter 3.3.8.1 --- Western blot analysis of proteins purified by Ni-NTA column --- p.97
Chapter 3.3.9 --- In vitro transcription and translation --- p.100
Chapter 3.3.9.1 --- In vitro transcription --- p.100
Chapter 3.3.9.2 --- In vitro translation --- p.100
Chapter 3.3.10 --- Particle bombardments --- p.103
Chapter 3.3.10.1 --- Transient expression of MIH in soybean & tobacco leaves --- p.103
Chapter CHAPTER 4 --- DISCUSSION --- p.107
Chapter 4.1 --- Transient expression of MIH genes --- p.109
Chapter 4.1.1 --- In vitro transcription and translation --- p.109
Chapter 4.1.2 --- Particle bombardments --- p.220
Chapter 4.2 --- Post-transcriptional gene silencing (PTGS) --- p.114
Chapter 4.2.1 --- Post-transcriptional cis-inactivation --- p.114
Chapter 4.2.2 --- Post-transcriptional trans-inactivation --- p.116
Chapter 4.2.3 --- MIH gene and PTGS --- p.118
Chapter 4.3 --- Codon usage --- p.119
Chapter 4.3.1 --- Codon usage of MIH in plants --- p.120
Chapter 4.3.2 --- Codon modification of MIH and further study on MIH expression in plants --- p.122
Chapter 4.4 --- Post-translational protein degradation --- p.123
Chapter 4.4.1 --- Construction of LRP-MIH fusion proteins --- p.123
CONCLUSION --- p.125
REFERENCES --- p.127

The processive plant cellulose synthase (CESA) synthesizes (1→4)-β-D-glucans. CESAs assemble into a six-fold symmetrical cellulose synthase complex (CSC), with an unknown symmetry and number of CESA isomers. The CSC synthesizes a cellulose microfibril as the fundamental scaffolding unit of the plant cell wall. CESAs are approximately 110 kDa glycosyltransferases with an N-terminal RING-type zinc finger domain (ZnF), seven transmembrane α-helices (TMHs) and a cytoplasmic catalytic domain (CatD). In the CatD, the uridine diphosphate glucose (UDP-Glc) substrate is synthesized into (1→4)-β-D-glucans. The ZnF is likely to facilitate dimers in the CSC. Recombinant class-specific region (CSR), a plant specific insertion to the C-terminal end of the CatD is also known to form dimers in vitro. The CSR sequence is the primary source of distinction between CESA isoforms and class structure. Also within the CESA CatD is a 125-amino acid insertion known as the plant-conserved region (P-CR), whose molecular structure was unknown. The function of the P-CR is still unclear, especially in the context of complete CESA and CSC structures. Thus, one major knowledge gap is understanding how multimeric CSCs synthesize multiple chains of (1→4)-β-D-glucans that coalesce to form microfibrils. The specific number of CESAs in a CSC and how interactions of individual CESA isoforms contribute to the CSC are not known. Elucidating the structure-function relationships of the P-CR domain, and with the consideration of the ability of CSR and ZnF domains to dimerize, it is possible to more completely model the structure of the CSC.

Recombinantly expressed rice (Oryza sativa) secondary cell wall OsCESA8 P-CR domain purifies as a monomer and shows distinct α-helical secondary structure by circular dichroism analysis. A molecular envelope of the P-CR was derived by small angle X-ray scattering (SAXS). The P-CR was crystallized and structure solved to 2.4 Å resolution revealing an anti-parallel coiled-coiled domain. Connecting the coiled-coil α-helices is an ordered loop that bends back towards the coiled-coils. The P-CR crystal structure fits the molecular envelope derived by SAXS, which in turn fits into the CatD molecular envelope. The best fit places the P-CR between the membrane and substrate entry portal. In depth analysis of structural similarity to other proteins, and 3D-surface structure of the P-CR, leads to hypotheses that it could function in protein-protein interactions as a dimer, trimer or tetramer in the CSC, that it could form protein-protein interactions with CESA-interacting proteins, and/or modulate substrate entry through its N- and/or C-terminus. From modeling, hypothetically important residues within the P-CR or related to the P-CR through potential protein contacts were mutated in Arabidopsis thaliana AtCESA1 constructs. These constructs were expressed in the temperature-sensitive radial swelling (rsw) rsw1-1 mutant of AtCESA1 to test for complementation of growth phenotypes at restrictive temperatures. Preliminary experiments indicate that some mutated CESA1 sequences fail to complement the rsw1-1 phenotype, suggesting that specific functions of individual amino can be tested using this system.

In early 2003, a persistent drought threatened about 15 million people in the Southern African region (SADC) with starvation as farmers in this region were not able to produce enough food. A similar threat was experienced in the United States of America (USA). The Americans responded by introducing GM technology, which thankfully stabilised corn production and food security. It was against this backdrop that the South African government legalised and supported GM technology in the farming industry. However, the technology became a contentious issue amongst scholars, politicians and policy makers as well as farmers. Therefore, this study analysed the perceptions of small-scale and large-scale farmers, located in Paarl, Western Cape, South Africa, on the adoption of GM technology. This qualitative study, using a case study design, collected primary data from thirty (30) farmers: fifteen (15) small-scale and fifteen (15) large-scale farmers. The findings revealed complex factors influencing farmers’ adoption decisions and that Adopter perception (AP) and Consumer perception (CP) play a key role in their adoption of GM technology. These commercially and profit-driven farmers avoid using GM technology because public opinion and the markets weigh heavily against it. It was concluded that the farmers regarded GM technology as just one of many agricultural technologies and not as an exception. It was also considered unaffordable and detrimental to the environment, the economy and their livelihoods.The study recommends that the government should fully investigate public perceptions with regard to the adoption of any new agricultural innovation prior to making policy decisions.
Development Studies
M.A. (Development Studies)