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An efficient and high-throughput protocol for Agrobacterium-mediated transformation based on phosphomannose isomerase positive selection in Japonica rice (Oryza sativa L.)

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Abstract

A number of Agrobacterium-mediated rice transformation systems have been developed and widely used in numerous laboratories and research institutes. However, those systems generally employ antibiotics like kanamycin and hygromycin, or herbicide as selectable agents, and are used for the small-scale experiments. To address high-throughput production of transgenic rice plants via Agrobacterium-mediated transformation, and to eliminate public concern on antibiotic markers, we developed a comprehensive efficient protocol, covering from explant preparation to the acquisition of low copy events by real-time PCR analysis before transplant to field, for high-throughput production of transgenic plants of Japonica rice varieties Wanjing97 and Nipponbare using Escherichia coli phosphomannose isomerase gene (pmi) as a selectable marker. The transformation frequencies (TF) of Wanjing97 and Nipponbare were achieved as high as 54.8 and 47.5 %, respectively, in one round of selection of 7.5 or 12.5 g/L mannose appended with 5 g/L sucrose. High-throughput transformation from inoculation to transplant of low copy events was accomplished within 55–60 days. Moreover, the Taqman assay data from a large number of transformants showed 45.2 % in Wanjing97 and 31.5 % in Nipponbare as a low copy rate, and the transformants are fertile and follow the Mendelian segregation ratio. This protocol facilitates us to perform genome-wide functional annotation of the open reading frames and utilization of the agronomically important genes in rice under a reduced public concern on selectable markers.

Key message We describe a comprehensive protocol for large scale production of transgenic Japonica rice plants using non-antibiotic selectable agent, at simplified, cost- and labor-saving manners.

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Acknowledgments

The authors thank Dr. Heng Zhong from Syngenta Biotechnology (China), Co., Ltd. and Dr. Pengcheng Wei from Rice Research Institute, Anhui Academy of Agricultural Sciences for critical reading of the manuscript.

Conflict of interest

The authors declare that they have no conflict of interest.

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Author notes

  1. Wanggen Zhang

    Present address: China Seed Life Science and Technology Center, Wuhan, 430070, Hubei, People’s Republic of China

Authors and Affiliations

  1. College of Life Sciences, Anhui Agricultural University, Hefei, 230036, Anhui, People’s Republic of China

    Yongbo Duan & Fengshun Song

  2. Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei, 230031, Anhui, People’s Republic of China

    Yongbo Duan, Hao Li, Juan Li, Dahu Ni, Fengshun Song, Li Li & Jianbo Yang

  3. Syngenta Biotechnology (China), Co., Ltd, Beijing, 102206, People’s Republic of China

    Chenguang Zhai, Wenqian Mei, Huaping Gui & Wanggen Zhang

  4. Institute of Technical Biology and Agriculture Engineering, Chinese Academy of Sciences, Hefei, 230031, Anhui, People’s Republic of China

    Hao Li, Juan Li & Dahu Ni

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  1. Yongbo Duan
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Correspondence to Jianbo Yang.

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Communicated by Y. Lu.

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Duan, Y., Zhai, C., Li, H. et al. An efficient and high-throughput protocol for Agrobacterium-mediated transformation based on phosphomannose isomerase positive selection in Japonica rice (Oryza sativa L.). Plant Cell Rep 31, 1611–1624 (2012). https://doi.org/10.1007/s00299-012-1275-3

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  • DOI: https://doi.org/10.1007/s00299-012-1275-3

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