published_or_final_version / Microbiology / Master / Master of Philosophy

Typescript. / Digitized by Kansas Correctional Industries

No description available.

Arabidopsis has emerged as an important model system for molecular plant biology. The extensive resources available for Arabidopsis make it an attractive system to study the molecular mechanisms involved in early seed development. During the early stages of seed development Arabidopsis endosperm is syncytial and proliferates rapidly through repeated rounds of mitosis without cytokinesis. This stage of endosperm development is both important in determining final seed size and is a model for studying various aspects of cellular and molecular biology, such as the cell cycle and genomic imprinting. However, the small size of Arabidopsis seed, the syncytial nature of the proliferative endosperm, and the surrounding maternal tissues make high throughput molecular analysis of the early endosperm technically difficult. To get around this we used laser capture microdissection to enable transcript analysis of the early proliferative endosperm of Arabidopsis at 4 days after pollination (DAP). Microarray results identified several thousand genes with endosperm expression, including many that were endosperm preferred. A number of genes were validated by relative quantification PCR and were consistent with the findings of the microarray. Meta analysis of the endosperm transcriptome revealed a developmental program dominated by mitosis and under the influence of several phytohormones, predominated by cytokinin signaling. The list of endosperm-preferred genes included all characterised imprinted genes in Arabidopsis. Imprinting is an epigenetic phenomenon by which genes are expressed predominantly from either their paternal or their maternal allele and very few imprinted genes have been identified in plants. The mono-allelic expression of the characterised imprinted genes appears to be limited to the endosperm where they provide important regulatory controls for seed development via direct effects on endosperm development. Genes from the endosperm-preferred list were screened for mono-allelic expression using sequence polymorphisms between the Colombia and Landsberg erecta ecotypes. We generated PCR products that spanned the polymorphisms of 67 genes from template obtained by laser capture of endosperm tissue from hybrid seed. Sequence analysis revealed three genes which gave strong allelic bias toward the maternal allele (At2g32460, At1g55550 and At2g21420) and one biased for the paternal allele (At1g47840). In summary, laser capture microdissection has enabled high-resolution transcript analysis of the proliferative stage of Arabidopsis endosperm development. The data generated provides a useful resource providing novel insight into early seed development, facilitating both identification of endosperm expressed and novel imprinted genes.

Abstract The genetic engineering industry for ornamental fish refers to the industry that applies biotechnology to ornamental fish to develop new species of ornamental fish. This research has determined that the features of such industry include: high added values, long time for development, long earning period, R&D-oriented, under numerous restrictions and with less energy dependency. According to a literature review and profile of the company, the research found that Taiwan has achieved significant results in terms of genetic transformation and cloning. Taiwan is one of the best countries when it comes to research and development abilities. Glowing fish play an important role in the upgrading of the ornamental fish industry. Also, the government encourages investment in biotechnology industries. In all biotechnology industries, agricultural biotechnology has lower risks and faster output than other medical biotechnology products, and hence agricultural biotechnology has greater development potential. The research suggests that the ornamental fish dealers enhance the participation to industry ¡Vacademy cooperation, cooperating with Academia Sinica, Fisheries Research institute, and the technological platform of ornamental fish genetic engineering industry, to accelerate the schedule of the research and the development of new species. In addition, facing the doubts related to Living Modified Organisms (LMO) in the international community, ornamental fish producers need to develop new technologies to sterilize genetically modified ornamental fish and create a safety evaluation and certification system for genetic-transformation of aqua-cultural animals to avoid or minimize any possible damage of LMO to the environment. Keywords: Genetic Transformation, Glowing Fish, Ornamental Fish

The Best MPhil Thesis in the Faculties of Dentistry, Engineering, Medicine and Science (University of Hong Kong), Li Ka Shing Prize / published_or_final_version / Pathology / Master / Master of Philosophy

Regeneration and transformation protocols for 'Comet' red raspberry were optimized with the purpose of making the Agrobacterium-mediated gene transfer system efficient for this crop. Adventitious shoot regeneration from leaf discs was improved using explants 10 mm in diameter and transferring to fresh medium at the fourth week of incubation. Additions of liquid medium to solid medium during incubation decreased regeneration and attempts to release the suppressive influence of larger shoots on initials (apical dominance) did not succeed. The presence of claforan did not affect shoot regeneration, but inoculations with Agrobacterium and the presence of kanamycin decreased regeneration moderately or considerably, respectively. The threshold for kanamycin concentration for screening for kanamycin resistant transformed raspberry tissue was 30 to 40 mg l$ sp{-1}.$ The best co-incubation interval between wild-type Agrobacterium and 'Comet' leaf discs ranged from 2 days for highly virulent strains to 3 or more days for moderate to low virulent strains. Among several wild-type strains, C58 was chosen as the most appropriate partially because a disarmed form was commercially available for use as a non-oncogenic vector for transformation of red raspberry. / The binary plasmid pBI121 containing the marker genes NPTII and GUS encoding kanamycin resistance and $ beta$-glucuronidase activity, respectively, was successfully introduced into the Agrobacterium strain LBA4404, which is a disarmed C58 derivative. Transformation of 'Comet' red raspberry was apparently achieved by inoculating leaf disc explants with LBA4404 containing pBI121. The probable integration and expression of the foreign genes into the plant cells were confirmed by screening for kanamycin resistance, GUS assays and Southern blot analyses. This transformation system appears to be effective and may be useful in further studies on red raspberry for both introduction of genes for desirable agronomic traits and basic studies of gene expression.

Schwanniomyces occidentalis (Debaryomyces occidentalis ) is able to grow rapidly with high cell mass on cheap starch as a carbon source, produce strong amylolytic enzymes extracellularly and secrete large proteins without hyper-glycosylation and measurable extracellular proteases. Schw. occidentalis thus has a high potential as, a useful alternative to Saccharomyces cerevisiae in the production of heterologous proteins. However, the molecular study of Schw. occidentalis has been very limited due to the insufficient transformation system and lack of gene expression information. / A new transformation system of Schw. occidentalis has been developed. This system was based on vector YEp13 ( LEU2) and a stable leu auxotrophic mutant, Schw. occidentalis DW88, obtained by treating the yeast with 1-methyl-3-nitro-1-nitrosoguanidine. The transformation efficiency of YEp13 by spheroplast-mediating method was 103 transformants/mug DNA. The 2-mum replicon is proposed to be responsible for YEp13 replication in Schw. occidentalis. The YEp13 stability in Schw. occidentalis was low, but it kept its structure in the yeast, suggesting that Schw. occidentalis DW88 does not modify foreign DNA. / After analysis of 14 cloned Schw. occidentalis genes and comparison of associated genes from both Schw. occidentalis and S. cerevisiae, 25 codons were arbitrarily chosen as putative preferred codons for Schw. occidentalis. They are similar to those of S. cerevisiae, except for TTA for leucine, and AAA for lysine. Codon Bias Index (CBI), a criterion to evaluate gene expression, is calculated from preferred codons. A computer program (PCBI) which reads a gene containing introns was developed to quickly calculate CBI. / Schw. occidentalis DWSS should be a good host to produce and secrete heterologous proteins and the putative preferred codons and program PCBI can facilitate molecular study of Schw. occidentalis. (Abstract shortened by UMI.)

No description available.

Dissertation (PhD)--University of Stellenbosch, 2006. / Bibliography. Also available via the Internet.