Agriculture and Food Systems - Theses

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    Regeneration and genetic transformation of Australian rice (Oryza sativa L.) varieties
    Azria, Diah ( 2001)
    The rice industry is a major contributor to Australia's agricultural production, which also puts Australia as one of the highest rice yield producing countries in the world. One of the priorities in Australian rice improvement program includes the generation of plants with useful qualitative and quantitative traits that affect agronomic performance and consumer preferences, which can be facilitated by genetic transformation techniques. An efficient tissue culture and regeneration system for four commercial varieties of Australian rice namely; Amaroo, Millin, Langi and Pelde are described. In this study, efficient plant regeneration via organogenesis was achieved in a short time, by optimising explant source as well as the composition of culture medium. MS medium containing BAP (2-4 mg/L) + NAA (1 mg/L) was found to be ideal for shoot initiation from mature embryo derived callus. Of the four varieties tested, Millin showed the best regeneration frequency followed by Amaroo, Pelde and Langi. The development of protocols for Agrobacterium-mediated transformation of Australian rice varieties was discussed. Using a binary vector, pIG121Hm, several parameters affecting Agrobacterium infection were optimised, including the choice of embryogenic calli; density of Agrobacterium; co-cultivation conditions including composition of medium, temperature and light, the presence of acetosyringone and the concentration of hygromycin in the medium. With the optimised protocol, transgenic rice plants were obtained in 3 to 4 months, with an average transformation efficiency of 11.6% and 2.0% for Amaroo and Millin, respectively. The plants grew normally and set seeds under glasshouse conditions. The integration of the transgenes was confirmed by Southern blot analysis. The optimised Agrobacterium-mediated transformation protocol was used to study expression of a pollen- and a generative cell-specific gene, Ory s 1 and LGC1, repectively in transgenic rice plants. Analysis of transgenic plants carrying Ory s 1- uidA (GUS) confirmed spatial and temporal expression of Ory s 1 in mature pollen. Deletion analysis (-405 bp and -812 bp) of promoter region of Ory s 1 gene (-1524 bp, full-length) showed that the 5' regulatory region contains enhancer/quantitative and pollen-specificity elements upstream of -405 bp and within the -405 bp regions, respectively. Further experiments using antisense construct resulted in reduction of Ory s 1 protein in two types of transgenic plants. Analysis of plants carrying LGC1- uidA (GUS) confirmed the spatial LGC] expression in generative cell of pollen. Whilst analysis of transgenic plants carrying each of the cytotoxic genes, barnase (Hartley, 1989) and diptheria toxin A (DT-A) (Greenfield et al., 1983) driven by LGC1 promoter showed generative cell-specific ablation caused by expression of barnase, resulting in arrested pollen development at late binucleate stage, reduced pollen size and lesser starch production, and 50-75% pollen sterility in transgenic plants. These studies showed that it is possible to transform Australian commercial varieties of rice. And the optimised transformation conditions can be used to introduce foreign genes for desired manipulation of important traits.