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We have developed a high-frequency method for Agrobacterium-mediated gene targeting by combining an efficient transformation system using rice suspension-cultured calli and a positive/negative selection system. Compared with the conventional transformation system using calli on solid medium, transformation using suspension-cultured calli resulted in a 5- to 10-fold increase in the number of resistant calli per weight of starting material after positive/negative selection. Homologous recombination occurred in about 1.5% of the positive/negative selected calli. To evaluate the efficacy of our method, we show in this report that knockout rice plants containing either a disrupted Waxy (granule-bound starch synthase) or a disrupted Xyl (β1,2-xylosyltransferase) gene can be easily obtained by homologous recombination. Study of gene function using homologous recombination in higher plants can now be considered routine work as a direct result of this technical advance.

plant and cell physiology/plant and cell physiology

Development of an Efficient Agrobacterium-Mediated Gene Targeting System for Rice and Analysis of Rice Knockouts Lacking Granule-Bound Starch Synthase (Waxy) and β1,2-Xylosyltransferase