title: Molecular breeding of a novel herbicide-tolerant rice by gene targeting.
abstract: We have previously reported the production of a rice cell line tolerant to the acetolactate synthase (ALS)-inhibiting herbicide bispyribac (BS), and demonstrated that the BS-tolerant phenotype was due to a double mutation in the rice ALS gene. We further indicated that while changing either of the two amino acids (W548 L or S627I) individually resulted in a BS-tolerant phenotype, conversion of both amino acids simultaneously conferred increased tolerance to BS. As the BS-tolerant cell line had lost the ability to regenerate during two years of tissue culture selection, we attempted to introduce these two point mutations into the rice ALS gene via gene targeting (GT). Using our highly efficient Agrobacterium-mediated transformation system in rice, we were able to regenerate 66 independent GT rice plants from 1500 calli. Furthermore, two-thirds of these plants harbored the two point mutations exclusively, without any insertion of foreign DNA such as border sequences of T-DNA. The GT plants obtained in the present study are therefore equivalent to non-GM herbicide-tolerant rice plants generated by conventional breeding approaches that depend on spontaneous mutations. Surprisingly, GT rice homozygous for the modified ALS locus showed hyper-tolerance to BS when compared to BS-tolerant plants produced by a conventional transgenic system; ALS enzymatic activity in plants homozygous for the mutated ALS gene was inhibited only by extremely high concentrations of BS. These results indicate that our GT method has successfully created novel herbicide-tolerant rice plants that are superior to those produced by conventional mutation breeding protocols or transgenic technology.
authors: Plant Genetic Engineering Research Unit, Division of Plant Sciences, National Institute of Agrobiological Sciences, 2-1-2, Kannondai, Tsukuba, Ibaraki 305-8602, Japan.
authors: Masaki
authors: M
authors: Endo
authors: Keishi
authors: K
authors: Osakabe
authors: Kazuko
authors: K
authors: Ono
authors: Hirokazu
authors: H
authors: Handa
authors: Tsutomu
authors: T
authors: Shimizu
authors: Seiichi
authors: S
authors: Toki
mesh_terms: D000095:Acetolactate Synthase / Q000235:genetics; D000222:Adaptation, Physiological / Q000235:genetics*; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D017931:DNA Primers; D018390:Gene Targeting; D006540:Herbicides / Q000494:pharmacology*; D012275:Oryza / Q000235:genetics* / Q000502:physiology; D017354:Point Mutation; D016133:Polymerase Chain Reaction
chemical_list: D017931:DNA Primers; D006540:Herbicides; D000095:Acetolactate Synthase