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Improved protocols for transformation of indica rice mediated by Agrobacterium tumefaciens

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Abstract

A highly efficient gene transfer method mediated by Agrobacterium tumefaciens was developed for Group I indica rice, which had been quite recalcitrant in tissue culture and transformation. Freshly isolated immature embryos from plants grown in a greenhouse were inoculated with A. tumefaciens LBA4404 that harbored super-binary vector pTOK233 or pSB134, which had a hygromycin-resistance gene and a GUS gene in the T-DNA. The efficiency of gene transfer varied with the kinds of gelling agents and the basic compositions of co-cultivation media. The highest activity of GUS after co-cultivation was observed when NB medium solidified with agarose was used. For the subsequent cultures, two types of media (modified NB and CC) were chosen to recover hygromycin-resistant cells efficiently. The transformation protocol thus developed worked very well in all of the varieties tested in this study, and the transformation frequency (number of independent hygromycin-resistant and GUS-positive plants per embryo) reached more than 30% in IR8, IR24, IR26, IR36, IR54, IR64, IR72, Xin Qing Ai 1, Nan Jin 11, and Suewon 258. Most of the transformants (T0) were normal in morphology and fertile. Stable integration, expression and inheritance of transgenes were demonstrated by molecular and genetic analysis of transformants in the T0 and T1 generations. For the recovery of multiple independent transgenic events from a single immature embryo, procedures were developed to section the embryo into as many as 30 pieces after non-selective cultures following co-cultivation. Transformants were then obtained from the pieces cultured on the selective media, and, in the highest case, more than seven independent transgenic plants per original embryo (transformation frequency of 738%) were produced. Thus, the efficiency of transformation was remarkably improved.

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Abbreviations

2,4-D:

2,4-dichlorophenoxyacetic acid

GUS:

β-glucuronidase

HPT:

hygromycin phosphotransferase

BA:

6-benzylaminopurine

NAA:

α-naphthaleneacetic acid

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Acknowledgements

The authors thank Dr T. Kubo for helpful discussions and advice. The skillful assistance of Ms S. Kuroda is gratefully acknowledged.

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Authors and Affiliations

  1. Plant Innovation Center, Japan Tobacco Inc., 700 Higashibara, Iwata, Shizuoka, 438-0802, Japan

    Yukoh Hiei & Toshihiko Komari

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  1. Yukoh Hiei
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  2. Toshihiko Komari
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Correspondence to Yukoh Hiei.

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Hiei, Y., Komari, T. Improved protocols for transformation of indica rice mediated by Agrobacterium tumefaciens . Plant Cell Tiss Organ Cult 85, 271–283 (2006). https://doi.org/10.1007/s11240-005-9069-8

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