Browsing by Author "Pepper, Timothy Bryan."

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Development of novel antibacterial and antiviral transgene vectors and techniques for their application and analysis in sugarcane.
(2002) Pepper, Timothy Bryan.; Watt, Maria Paula Mousaco Deoliveira.; Rutherford, Richard Stuart.; Huckett, Barbara Isobel.
Sugarcane is challenged by a number of phytopathogenic bacteria and viruses that are best managed by the development of resistant varieties. Genetic engineering is a promising strategy in such breeding efforts, as it allows novel mechanisms of resistance not available in any parent germplasm to be introduced into the crop. DNA sequences encoding cystatin from papaya (Carica papaya), and pleurocidin from the winter flounder (Pleuronectes americanus) were envisaged as transgenes in this work due to their theoretical potential to increase sugarcane resistance to viruses and pathogenic or opportunistic bacteria, respectively. Cystatin is a cysteine proteinase inhibitor. Cysteine proteinases are used by potyviruses to cleave the polyprotein gene product, an essential step in the viral life cycle. Constitutive expression of cystatin may therefore lend the host plant resistance to a range of potyviruses, including the economically important pathogen sugarcane mosaic virus (SCMV). Pleurocidin is an amphipathic, α-helical, cationic peptide, with broadspectrum anti-bacterial activity at physiological pH. By binding to the cell membranes of both Gram positive and Gram negative bacteria, pleurocidin disrupts the membrane potential, causing it to become more permeable, especially to cations, leading to death of the bacterial cell. Initial microbiological bioassays showed that pleurocidin has inhibitory and bactericidal effects on the organisms which cause leaf scald (Xanthomonas albilineans), gumming disease (Xanthomonas campestris pv. vasculorum) and post-harvest sucrose conversion in sugarcane, as well as inhibitory effects against Leifsonia xyli ssp. xyli, which causes ratoon stunting disease (RSD). For transformation vector construction, the cystatin and pleurocidin coding sequences were altered so that their start codons were in the most favourable consensus context for expression in monocotyledonous plants. In the case of pleurocidin, an extracellular peroxidase signal sequence was attached. The prepared sequences were spliced into the vector pUBI510 in which the gene of interest is driven by the CaMV 35S promoter linked in tandem to a derivative of the maize ubiquitin promoter. The constructs generated were named pUBI510-cys3 and pUBI510-pleur08 respectively. The plasmid structures were confirmed using restriction endonuclease analysis and DNA sequencing. Since the transformation of sugarcane is known to be inefficient, two routes of morphogenesis for the production of somatic embryos were compared in the transformation procedure. These were (1) indirect embryo production via callus and (2) the direct and indirect production of embryos from transverse sections of leaf roll. Field grown sugarcane varieties N12 and NCo376 were the source of explant material. Plasmids pUBI510-cys3 and pUBI510-pleuro8 were respectively co-delivered by microprojectile bombardment with the antibiotic resistance selection plasmid pUBIKN containing the neomycin phosphotransferase gene (npt-II). Cultures were maintained in the dark on selection medium containing various concentrations of the antibiotic geneticin (G418) for several weeks before being allowed to regenerate in the light. Plantlets coming through selection were hardened off in the glasshouse when approximately 100mm high. Primer pairs for amplification of the cystatin insert were designed in various ways. The primer pair which ultimately proved most useful was designed to be complementary to the 5' and 3' ends of the papaya cystatin nucleotide sequence. Primer Premier analysis of a sorghum cystatin sequence provided additional possible primers. A further pair for potential future use was devised based on complementarity to conserved regions on maize cystatins 1 and 2, sorghum, rice, and papaya cystatins. The nucleotide sequence was constructed using the most common monocotyledon codon permutations for each amino acid. Pleurocidin primers were designed to be complementary to 5' and 3' regions of the nucleotide sequence encoding the pleurocidin pre-pro-protein. PCR and RT-PCR protocols for the detection of transgenes and transcript production in putative transgenic plants were developed using these primers. No plants survived selection via the callus route, although some were regenerated via direct embryogenesis. Putative transformed plants were analysed using PCR to test for the presence of integrated transgenes and Southern hybridization to determine transgene copy number. Both types of transgene were reproducibly detectable by PCR in DNA from some immature plants, but results were negative in DNA from those same plants when mature. Southern hybridization analysis detected the cystatin transgene in DNA from immature plants but no transgenes were detected in up to 20 µg DNA from mature plants. Single copy constructions of the transgenes in backgrounds of non-transformed DNA were detectable by both PCR and Southern hybridization analysis. Overall, PCR, RT-PCR and Southern hybridization results indicated that the plants regenerated fell into two categories: non-transformed plants that had survived selection (escapes) and chimaeric individuals with a component of both transformed and non-transformed cells, in which the transgene had probably become diluted during plant development under non-selective conditions. A method for extracting leaf exudates was tested, in conjunction with a cysteine proteinase assay to detect the presence of cystatin transgenes in the intracellular spaces of sugarcane leaves of confirmed transformants. Although it could not be applied within the scope of this project, this assay will prove useful in future work.