No description available.

Thesis--Leyden. / In Periodical Room.

Cauliflower (Brassica oleracea var. botrytis) is described as a recalcitrant plant to genetic transformation processes especially Agrobacterium-mediated and as an extremely low frequency event then it requires a large amount of explants for this procedure to succeed. This thesis describes the development and refinement of a mass propagation system for cauliflower micropropagation and its use for overcoming recalcitrance to genetic transformation. Shoot meristematic tissue was taken from the curd of cauliflower and used to establish in-vitro cultures in liquid medium. Explants were cultured in a Murashige and Skoog (MS) medium containing various plant growth regulators combinations to induce shoot regeneration and which were optimised to be 2 mg L-1 (9.29 μM) kinetin and 1 mg L-1(4.9 μM) IBA. Shoots were cultured for 4–6 weeks to obtain rooted plants, which were then suitable for weaning and subsequently produce fully- developed in-vivo plants in pots in soil with a 95%+ success rate. A procedure for detection of the presence of insert DNA in recombinant plasmids in individual Agrobacterium tumefaciens strains was refined. Cauliflower was transformed using the EHA105 strain of A. tumefaciens harboring the binary vector pPRTL2 plasmid carrying the antioxidant gene Ascorbate peroxidase (APX) for increased stress resistance coupled with neomycin phosphotransferase II (nptII) for resistance to kanamycin and β-glucuronidase (GUS) as a marker gene. Selection was carried out in MS medium containing kanamycin (50 mg L-1), and surviving tissues were then tested by histochemical GUS assay.Agrobacterium-mediated plant genetic transformation requires a two-step process for its success: selection and regeneration of transformed tissues, and the elimination of the transformation vector (Agrobacterium). This study used carbenicillin and cefotaxime in MS media to eliminate A. tumefaciens, at selection levels of 25 and 50 mg L-1 kanamycin. Kanamycin severely reduced explant growth and regeneration of control cultures at concentrations as low as 10 mg L-1 and completely inhibited shoot organogenesis at 50 mg L-1. The integration of APX gene into putative transformant lines was confirmed using GUS and leaf disc assays. Genomic integration of the gene cassette was optimised using PCR analysis with primers flanking npt II and CaMV promoter regions. The stable integration of the APX gene in the putative transgenic plants was detected using PCR at 478bp. The result confirmed the first report of transformation with APX gene in Brassica oleracea. Thus, a protocol for effective Agrobacterium-mediated genetic transformation of cauliflower was optimized. Transformed and control lines were sub-cultured many times on maintenance medium over 2 years without any loss of the transgene and then tested for salt resistance as in-vitro and in-vivo plants using a leaf disc assay. Control plants had little or no NaCI resistance whilst transformed plants showed varying degrees of resistance. Analysis of APX gene expression under salt treatment showed that putative transgenic cauliflower survived salinity stress compared with control plants. Non-acclimated and acclimated in-vivo plants were also assessed for resistance to frost. Both non-acclimated and acclimated APX transformed lines showed improved frost resistance compared to controls. The results clearly confirmed that NaCI and frost resistance were stable traits attributable to improved APX expression.

Agrobacterium tumefaciens is a gram-negative soil bacterium that causes crown gall tumors on dicotyledenous plants. The transferred DNA (T-DNA) portion of the A. tumefaciens tumor-inducing (Ti) plasmid enters infected plant cells and integrates into plant nuclear DNA. The T-DNA is accompanied into plant cells by the VirD2 endonuclease covalently attached to its 5' end. VirE2, a cooperative, single-stranded DNA-binding protein is also transported into plant cells during infection by A. tumefaciens. VirD2 and VirE2 contain nuclear localization signals (NLSs) and are transported into the plant cell nucleus. The location of functional domains by the insertion of Xhol linker oligonucleotides throughout virE2 is reported. A ssDNA binding domain was located in the C-terminal half of VirE2, as well two domains involved in cooperative single-stranded DNA binding. Further, we isolated a mutation in the central region of VirE2 that decreased tumorigenicity, but did not affect ssDNA binding. / Graduation date: 1997

Würzburg, Universiẗat, Diss., 2008.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1940. / Typescript. Includes abstract and vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 45-47).

Thesis (Ph. D.)--University of Florida, 1984. / Description based on print version record. Typescript. Vita. Includes bibliographical references (leaves 120-133).

Thesis (Ph. D.)--University of Chicago, Dept. of Chemistry, Dec. 2002. / Includes bibliographical references (p. 185-196). Also available on the Internet.

No description available.

As urease de plantas catalisam a hidrólise da ureia e apresentam efeitos tóxicos a fungos patogênicos e insetos fitófagos. Em soja [Glycine max L. Merrill] foram descritas duas ureases estruturais: a embrião-específica, codificada pelo gene Eu1, e a ubíqua, codificada pelo gene Eu4. Sabe-se que a urease embrião-específica purificada apresenta efeito inibitório sobre o crescimento in vitro de fungos filamentosos e desenvolvimento de insetos. A urease ubíqua é responsável pela reciclagem de toda a ureia proveniente do metabolismo, mas não há informações sobre seu envolvimento no sistema de defesa das plantas. A transformação genética é uma ferramenta importante em estudos de genômica funcional e, portanto, a disponibilidade de sistemas eficientes é um pré-requisito essencial. O objetivo deste trabalho foi a obtenção de plantas estavelmente transformadas a partir de embriões somáticos de soja submetidos ao sistema integrado bombardeamento/Agrobacterium, bem como a identificação e caracterização funcional dos genes que codificam as ureases estruturais de soja, especialmente a urease ubíqua em relação aos processos de resposta a fungos patogênicos. Inicialmente, testamos a eficiência de transformação de embriões somáticos secundários por um método que combina o bombardeamento de partículas livres de DNA com o sistema Agrobacterium. Plantas transgênicas férteis foram regeneradas de vários experimentos independentes de transformação utilizando diferentes plasmídios. Posteriormente, foi realizada a caracterização dos genes que codificam ureases presentes no genoma da soja. O gene Eu4 apresentou um padrão de expressão diferencial para genótipos suscetível e resistente ao longo do período de infecção por Phakopsora pachyrhizi, o agente etiológico da ferrugem asiática. Plantas transgênicas foram geradas visando a superexpressão de Eu4. Contudo, apenas uma planta apresentou níveis aumentados de expressão desse gene, enquanto que as demais plantas apresentaram o fenômeno de co-supressão dos genes endógeno e transgene. Avaliou-se o crescimento vegetativo dos fungos Rhizoctonia solani, Phomopsis sp., Fusarium solani, Colletotrichum gossypii e Penicillium herguei em meio de cultura contendo extrato protéico bruto de plantas transgênicas expressando maiores e menores níveis de urease e de plantas não-transgênicas. O crescimento dos fungos foi inversamente proporcional a quantidade da urease presente no extrato protéico das plantas. Quando infectadas por uredósporos de P. pachyrhizi, folhas destacadas das plantas co-suprimidas desenvolveram um número significativamente maior de lesões, pústulas e pústulas abetas do que folhas com níveis normais da enzima. Em conjunto estes resultados indicam um 15 importante envolvimento da urease ubíqua da soja na resposta à infecção da planta por fungos patogênicos. Além disso, um terceiro gene que codifica urease foi encontrado no banco de dados com a sequência completa do genoma da soja. O gene foi denominado Eu5 e seu produto SBU-III. A análise filogenética mostra que SBU-III está fortemente relacionada à isoforma embrião-específica. Apesar da grande similaridade na seqüência primária da proteína, SBU-III apresenta uma mutação em um aminoácido altamente conservado entre as ureases, sugerindo ausência da atividade ureolítica. O padrão de expressão do gene Eu5 em diferentes órgãos e estágios de desenvolvimento foi determinado por RT-qPCR. Transcritos foram detectados em sementes um dia após a quebra de dormência, em raízes de plantas jovens e em embriões em desenvolvimento. As evidências sugerem que SBU-III não está envolvida na disponibilização de nitrogênio para as plantas, mas esta pode ter função de defesa. / Plants ureases catalyze urea hydrolysis and display toxic effects against pathogenic fungi and phytophagous insects. For soybean [Glycine max L. Merrill] two structural ureases have been described: the embryo-specific, encoded by Eu1 gene, and the ubiquitous, encoded by Eu4 gene. The toxic property of purified embryo-specific urease against filamentous fungi and insects was demonstrated in vitro. The ubiquitous urease is responsible for recycling all metabolically-derived urea, but there were no information about its putative defense role. Plant genetic transformation offers significant advancement in functional genomics. Therefore an efficient transformation system is required. This study aims to obtain stable transformed plants derived from somatic embryos submitted to the integrated bombardment/ Agrobacterium system, as well as identify and functionally characterize the soybean structural urease-encoding genes, specially the ubiquitous urease gene response to fungi. First, the transformation of soybean proliferating somatic embryos by a procedure that combines DNA-free particle bombardment and Agrobacterium was evaluated. Transgenic fertile plants were recovered from many transformation experiments using different plasmids. After, a study of ureases enconding genes present in the soybean genome was carried out. In the present work, Eu4 gene showed a differential expression pattern in susceptible and resistant genotypes over the course of Phakopsora pachyrhizi infection, the Asian rust causal agent. Transgenic plants aiming Eu4 overexpression were obtained. However, a single transgenic plant exhibited Eu4 overexpression, whereas the other ones showed co-suppression of endogenous and transgenes urease genes. The growth of Rhizoctonia solani, Phomopsis sp., Fusarium solani, Colletotrichum gossypii and Penicillium herguei in media containing crude protein extract from either transgenic or non-transgenic leaves was evaluated. Fugal growth was inversely proportional to ubiquitous urease amount in plant crude extracts. When infected by P. pachyrhizi uredospores, detached leaves of co-suppressed plants developed a significantly higher number of lesions, pustules and erupted pustules than leaves containing normal levels of the enzyme. These results suggested an important role of soybean ubiquitous urease in plant response against fungal infection. Furthermore, by searching the completed soybean genome sequence, a third urease-encoding locus was identified. The gene was designated Eu5 and its product, SBU-III. Phylogenetic analysis shows that SBU-III is closely related to the embryo-specific isoform. Although a high similarity in amino acid sequence was observed, a mutation in a highly conserved residue suggests absence of ureolytic activity. Expression profile of Eu5 gene in different organs and developmental stages was determined by RT-qPCR. Transcripts were detected in seeds one day after dormancy break, roots of young plants and embryos of developing seeds. Evidences suggest that SBU-III may not be involved in nitrogen availability to plants, but a defense role was proposed.