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Efficient gene targeting by homologous recombination in rice

Nature Biotechnology volume 20, pages 1030–1034 (2002)Cite this article

Abstract

Modification of genes through homologous recombination, termed gene targeting, is the most direct method to characterize gene function. In higher plants, however, the method is far from a common practice. Here we describe an efficient and reproducible procedure with a strong positive/negative selection for gene targeting in rice, which feeds more than half of the world's population and is an important model plant. About 1% of selected calli and their regenerated fertile plants were heterozygous at the targeted locus, and only one copy of the selective marker used was found at the targeted site in their genomes. The procedure's applicability to other genes will make it feasible to obtain various gene-targeted lines of rice.

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Figure 1: Strategy for the targeted disruption of the Waxy locus and analysis of homologous recombination events.
Figure 2: Southern blot analysis of the Waxy region and integrated hpt sequence in the six T0 plants.
Figure 3: Segregation of the targeted waxy Hmr allele revealed by phenotypic and PCR analyses.
Figure 4: Southern blot analysis of the Waxy region in the T1 segregants from the targeted T0 heterozygous plant.

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Acknowledgements

We thank H. Asao and M. Matsumoto for technical assistance, B. Burr and F. Burr for reading the manuscript, R. Bilang, A. Gierl, S. Kawasaki, Y. Kishima, J. Leemans, H. Nagano, K. Nakamura, Y. Sano, H. Uchimiya, and T. Yagi for providing plasmids and DNAs used for constructing vectors, and H.-Q. Li, A. Hoshino, and Y. Kishima for discussion. This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology in Japan.

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  1. Yoshishige Inagaki

    Present address: Faculty of Agriculture, Okayama University, Okayama, 700-8530, Japan

Authors and Affiliations

  1. National Institute for Basic Biology, Okazaki, 444-8585, Japan

    Rie Terada, Hiroko Urawa, Yoshishige Inagaki, Kazuo Tsugane & Shigeru Iida

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  1. Rie Terada
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Correspondence to Shigeru Iida.

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Terada, R., Urawa, H., Inagaki, Y. et al. Efficient gene targeting by homologous recombination in rice. Nat Biotechnol 20, 1030–1034 (2002). https://doi.org/10.1038/nbt737

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