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.
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.
National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki, Japan. ozawa@affrc.go.jp
Kenjirou
K
Ozawa
Yuhya
Y
Wakasa
Yuko
Y
Ogo
Kouki
K
Matsuo
Hiroyuki
H
Kawahigashi
Fumio
F
Takaiwa
D060054:Agrobacterium / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods; D012275:Oryza / Q000201:enzymology / Q000235:genetics*; D010430:Pentosyltransferases / Q000235:genetics*; D010940:Plant Proteins / Q000235:genetics*; D030821:Plants, Genetically Modified / Q000201:enzymology* / Q000235:genetics*; D013214:Starch Synthase / Q000235:genetics*; D014170:Transformation, Genetic / Q000235:genetics; D000097608:UDP Xylose-Protein Xylosyltransferase
D010430:Pentosyltransferases; D010940:Plant Proteins; D013214:Starch Synthase; D000097608:UDP Xylose-Protein Xylosyltransferase