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The Mutant Form of Acetolactate Synthase Genomic DNA from Rice is an Efficient Selectable Marker for Genetic Transformation

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Abstract

The proper use of a marker gene in a transformation process is critical for the production of transgenic plants. However, consumer concerns and regulatory requirements raise an objection to the presence of exogenous DNA in transgenic plants, especially antibiotic-resistant genes and promoters derived from viruses. One approach to overcome this problem is the elimination of marker genes from the plant genome by using several site-specific recombination systems. We propose an alternative method to solve this problem using a marker gene exclusively derived from the host plant DNA. We cloned a genomic DNA fragment containing regulatory and coding sequences of acetolactate synthase (ALS) gene from rice, and mutagenized the ALS gene into a herbicide-resistant form. After transfer of this construct to the rice genome, transgenic plants were efficiently selected with a herbicide, bispyribac-sodium salt, which inhibits the activity of wild type ALS. We also analyzed the regulatory feature of the rice ALS gene promoter with the gusA reporter gene and revealed that GUS expression was observed constitutively in aerial parts of rice seedlings and root tips. The marker system consisted exclusively of host plant DNA and enabled efficient selection in a monocot crop plant, rice. The selection system can potentially be applied to generate transgenic plants of other crop species and can be expected to be publicly acceptable.

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Abbreviations

4-MUG:

4-methylumbelliferyl β-d-glucuronide

CaMV35S:

Cauliflower Mosaic Virus 35S

GUS:

β-glucuronidase

X-gluc:

5-bromo-4-chloro-3-indolyl-β-d-glucuronide

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Authors and Affiliations

  1. Department of Plant Biotechnology, National Institute of Agrobiological Sciences, Kannondai 2-1-2, 305-8602, Tsukuba, Ibaraki, Japan

    Keishi Osakabe, Yaeko Nishizawa, Kazuko Ono, Kiyomi Abe, Hidemitsu Nakamura, Hiroaki Ichikawa & Seiichi Toki

  2. Graduate School of Life and Environmental Sciences, Tsukuba University, Tennoudai 1-1-1, Japan, 305-8572, Tsukuba, Ibaraki, Japan

    Masaki Endo & Shigeo Nishimura

  3. Life Science Research Institute, Kumiai Chemical Industry Co., Tamari 276, 436-0011, Kakegawa, Shizuoka, Japan

    Kiyoshi Kawai & Tsutomu Shimizu

  4. Graduate School of Life Sciences, Tohoku University, Katahira 2-1-1, 980-8577, Aoba-ku, Sendai, Japan

    Yuichi Ishikawa

Authors
  1. Keishi Osakabe
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  2. Masaki Endo
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  3. Kiyoshi Kawai
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  4. Yaeko Nishizawa
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  5. Kazuko Ono
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  6. Kiyomi Abe
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  7. Yuichi Ishikawa
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  8. Hidemitsu Nakamura
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  9. Hiroaki Ichikawa
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  10. Shigeo Nishimura
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  11. Tsutomu Shimizu
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  12. Seiichi Toki
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Correspondence to Seiichi Toki.

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Osakabe, K., Endo, M., Kawai, K. et al. The Mutant Form of Acetolactate Synthase Genomic DNA from Rice is an Efficient Selectable Marker for Genetic Transformation. Mol Breeding 16, 313–320 (2005). https://doi.org/10.1007/s11032-005-0999-y

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  • DOI: https://doi.org/10.1007/s11032-005-0999-y

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