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A large-scale Agrobacterium-mediated transformation procedure with a strong positive-negative selection for gene targeting in rice (Oryza sativa L.)

  • Genetic Transformation and Hybridization
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Abstract

A large-scale transformation procedure handling an adequate number of stable transformants with highly efficient positive-negative selection is a necessary prerequisite to successful gene targeting by homologous recombination, as the integration of a transgene by somatic homologous recombination in higher plants has been reported to be 10-3 to 10-5 compared with random integration by non-homologous end joining. We established an efficient and large-scale Agrobacterium-mediated rice transformation protocol that generated around 103 stable transformants routinely from 150 seeds and a strong positive-negative selection procedure that resulted in survivors at 10-2 using the gene for diphtheria toxin A fragment as a negative marker. The established transformation procedure provides a basis for efficient gene targeting in rice.

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Abbreviations

AS::

Acetosyringone

5-FU::

5-Fluorouracil

FW::

Fresh weight

GT::

Gene targeting

HR::

Homologous recombination

NHEJ::

Non-homologous end joining

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Acknowledgements

We thank Yoshishige Inagaki and Hong-Qing Li for their construction of the plasmids and for their helpful discussion. This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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Authors and Affiliations

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

    R. Terada, H. Asao & S. Iida

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  1. R. Terada
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  2. H. Asao
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  3. S. Iida
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Correspondence to S. Iida.

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Communicated by H. Ebinuma

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Terada, R., Asao, H. & Iida, S. A large-scale Agrobacterium-mediated transformation procedure with a strong positive-negative selection for gene targeting in rice (Oryza sativa L.). Plant Cell Rep 22, 653–659 (2004). https://doi.org/10.1007/s00299-003-0752-0

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  • DOI: https://doi.org/10.1007/s00299-003-0752-0

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