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An efficient regeneration system and Agrobacterium-mediated transformation of Chinese upland rice cultivar Handao297

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Abstract

A highly efficient tissue culture system and Agrobacterium-mediated transformation protocol for Chinese upland rice cultivar Handao297 has been established with mature embryos as explants. Up to 81.2% of mature embryos were induced to regenerate good-quality calli on NB medium (a medium combining N6 macronutrient components and B5 micronutrient and organic components) containing 3 mg/l 2,4-dichlorophenoxyacetic acid in 10 days. More than 80% of the calli were morphogenic within 1 week and regenerated green plantlets within 1 month on Murashige and Skoog medium supplemented with 0.5 mg/l 6-benzyladenine, 0.5 mg/l kinetin, 1 mg/l zeatin, 0.5 mg/l thidizazuron (TDZ), 0.5 mg/l naphthaleneacetic acid, 0.15 mg/l indoleacetic acid, and 0.15 mg/l indolebutyric acid. This tissue culture system was suitable for Agrobacterium-mediated transformation of upland rice Handao297. Furthermore, some important factors affecting transformation frequency were investigated with Agrobacterium strain AGL1 containing the plasmid pCAMBIA1381. The addition of 30 mg/l hygromycin B followed by 60 mg/l hygromycin B to the selection induction medium facilitated the revival of calli from selection and reduced false positive calli. Hygromycin B at 10 mg/l was most effective in suppressing non-transgenic callus growth in the differentiation medium. The addition of TDZ to the differentiation medium promoted the morphogenesis of calli and facilitated the generation of adventitious shoots by five to tenfold in comparison to medium without TDZ.

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Abbreviations

AS:

Acetosyringone

BA:

6-Benzyladenine

2,4-D:

2,4-Dichlorophenoxyacetic acid

IAA:

Indole acetic acid

IBA:

Indole-3-butyric acid

KT:

6-Furifuryl-aminopurine

NAA:

α-Naphthalene acetic acid

PGR:

Plant growth regulator

TDZ:

Thidiazuron

ZT:

Trans-zeatin

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Acknowledgments

The authors thank Dr. Judy Harington for her critical reading of the manuscript. This work was supported by the Ministry of Agriculture of China for transgenic research (grant nos, 2008ZX08009-003 and 2008ZX08001-005) and Chinese Academy of Sciences (grant no. KSCX1-YW-03).

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Authors and Affiliations

  1. The State Key Laboratory of Plant Cell and Chromosome Engineering, National Centre for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, Beijing, 100101, China

    Weina Zhao, Shusong Zheng & Hong-Qing Ling

  2. Graduate University of the Chinese Academy of Sciences, Yuquan Road, Shijingshan District, Beijing, 100049, China

    Weina Zhao

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  1. Weina Zhao
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  2. Shusong Zheng
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  3. Hong-Qing Ling
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Correspondence to Hong-Qing Ling.

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Zhao, W., Zheng, S. & Ling, HQ. An efficient regeneration system and Agrobacterium-mediated transformation of Chinese upland rice cultivar Handao297. Plant Cell Tiss Organ Cult 106, 475–483 (2011). https://doi.org/10.1007/s11240-011-9946-2

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  • DOI: https://doi.org/10.1007/s11240-011-9946-2

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