Genetic engineering of rice: Agrobacterium tumefaciens-mediated transformation of rice and evaluation of a corn pollen-specific promoter using thegusA gene in transgenic rice
Abstract
Rice genetic engineering permits access to an unlimited gene pool through the transfer of desirable genes between any two species of interest, irrespective of their evolutionary or taxonomic relationships. However, there are major problems to be considered for a successful foreign gene introgression into rice. Two of the major problems were considered in this thesis: first is the development of an efficient transformation method for foreign gene transfer to rice, and the second is the identification of conditions that will allow expression of the transgene in the proper developmental and tissue-specific manner. Agrobacterium-mediated transformation of rice was studied following reports of its successes in transformation of dicots and its potential in transforming monocots. Conditions for successful Agrobacterium-mediated transformation of rice was studied based on transient GUS expression, including the type and age of explant, preinduction and co-cultivation conditions, acetosyringone concentrations, the media (co-cultivation and selection), the promoter and the Agrobacterium strains. A protocol was etablished which resulted in the production of stably transformed calli at high efficiency (36.1% for Nortai and 5.9% for TCS10) using immature embryos treated with At656 (pCNL56). Transgenic plants were not obtained which may be due to the long duration of exposure of inoculated embryos and calli to high concentrations of G418 during selection. Following reports of Hiei et al. (1994) in obtaining high efficiency of transformed rice plants using scutellar tissue infection with LBA4404 (pTOK233), immature embryos of indica (IR54, IR72, and TCS10) and a japonica variety (Radon) were infected with the same Agrobacterium strain, using the protocol established in this study with minor modifications based on Hiei et al. (1994). Transgenic plants were obtained after hygromycin selection at a high efficiency for Radon (27.0%), and at a lower but acceptable efficiency for TCS10 (0.5% to 1.2%) and IR72 (1.9% to 8.3%). Southern blot analysis of genomic DNA from transgenic Radon, TCS10, and IR72 plants showed very few integration events. Fertility of the T$\rm\sb{o}$ plants was comparable to the seed-derived plants. Progeny analyses of seeds from T$\rm\sb{o}$ plants exhibited a 3:1 ratio of GUS$\sp+$ to GUS$\sp-$ plants, indicative of gene integration at a single locus. The second major problem addressed was identification of conditions that would allow the expression of a transgene in the proper developmental and tissue-specific manner. This problem was dealt with using the promoter of a corn pollen-specific gene Zmg13 in order to develop a system to genetically engineer rice whose fertility and sterility can be controlled. Rice protoplasts were co-transformed with the plasmid pCPPGUS containing the Zmg13 promoter starting from the $-$260 position in front of the $\beta$-glucuronidase gene, and a bar gene-containing plasmid (p35SBarB) using polyethylene glycol-mediated transformation. Transgenic plants were obtained after selection with phosphinothricin, which produced high selection and co-transformation efficiencies. Transgenic plants exhibited GUS activity in a pollen-specific manner depending on the transgene copy number and the physiological state of the anther/pollen.
Degree
Ph.D.
Advisors
Hodges, Purdue University.
Subject Area
Botany|Plant propagation|Genetics
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