Construction Of Rice Mutants By T-DNA Insertional Mutagenesis And Analysis Of Flanking Sequences

Rice is the staple food for more than one third of the world population. Because of its small genome size (430 Mb), relatively well developed genetic maps and easily to be transferred, rice is a model plant for functional genomics research. Currently, three methods, physical-chemical mutagenesis, transposon tagging and T-DNA tagging have been employed to create mutants. Agrobacterium-mediated T-DNA transformation is one of the most important technology for producing mutants. In this study, we have been obtained following results:1. Rice T-DNA tagging lines produced by Agrobacterium-mediated transformation of Oryza sativa L. cv. Zhonghua 11 with an activation tagging vector pSKI015.2. A short period of callus dehydration (24～48 h) after being cultured on the selective media is helpful for shoot differentiation from calli. After selection, calli treated with or without dehydration, the transformation frequency was 8.7% and 9.4%, respectively. However, the transformation frequency was just 0.3% if calli were directly transferred onto shoot differentiation media without selection on the selective media containing 30～90 ppm Basta or 3～5 ppm Bialaphos.3. Dot and Southern blotting analysis of transgenic individuals resistant to herbicide Basta indicated that all of these individuals contained T-DNA insertion. It is convenient and effective to screen transgenic plants with Basta. The recommended concentration of Basta is 500 ppm at seedling stage for killing the non-transgenic plants.4. The T₂ population of tagging lines was further screened by Basta. Segregation ratio of Basta-resistant and sensitive was 3:1, 15:1 and 63:1. Average copy number of insertion was 1.89.5. Flanking sequences at T-DNA insertion site of the tagging lines were obtained by plasmid rescue. Homologous comparison by BLASTn and BLASTx was conducted. 6. Phenotyping of T-DNA tagging lines showed that there were variations on agronomic traits such as early ripening, dwarf, upright leaf, albino, leaf mosaic, leaf color change, etc.Within two years, we have established a primary system for rice mutant induction and flanking sequence analysis, which is a foundation for further creation of a huge number of rice mutants and for functional genomics study in rice.