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An Agrobacterium-mediated non-antibiotic selection-based transformation system for rice (Oryza sativa ssp. indica) cultivar “93-11” successfully produces TAC1-silenced transgenic plants

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Abstract

The rice (Oryza sativa L. ssp. indica) cultivar “93-11” is a reference genotype used in many studies; however, its recalcitrance during transformation and regeneration greatly limits functional genomics and breeding research. In this study, we developed an efficient Agrobacterium tumefaciens–mediated transformation system for “93-11”, based on the phosphor mannose isomerase (PMI) positive selection system. Calli of “93-11” were transformed with the Agrobacterium strain EHA105 harboring a binary vector, containing the PMI gene and an RNAi sequence targeting TILLER ANGLE CONTROLLING 1 (TAC1). We also developed a method for removing Agrobacterium from the callus following co-cultivation and determined the optimal conditions for PMI selection and callus differentiation. Compared with the hygromycin phosphotransferase (HPT) selection system, newly generated calli were recovered at higher rates on the PMI selection medium, with “93-11” transformation frequency reaching 7.50%. Notably, the down-regulation of TAC1 in the “93-11” genetic background led to a more compact plant architecture. Overall, this work presents a genetic transformation system suitable for the reference indica variety “93-11” using a non-antibiotic selectable agent. This advance will facilitate functional genomic research and the improvement of agronomic traits for indica varieties recalcitrant to transformation.

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Funding

This project was supported by the Ministry of Agriculture of China (2016ZX08001-004) and the Innovation Group for the Fujian Academy of Agricultural Sciences.

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Author notes

  1. Jingjing Hou and Hao Chen contributed equally to this work.

Authors and Affiliations

  1. MOE Key Laboratory of Crop Heterosis and Utilization, National Center for Evaluation of Agricultural Wild Plants (Rice), Beijing Key Laboratory of Crop Genetic Improvement, Department of Plant Genetics and Breeding, China Agricultural University, Beijing, 100193, China

    Jingjing Hou, Yuzhen Fang, Ying Zhu, Bing Han, Chuanqing Sun & Yongcai Fu

  2. Institute of Crop Sciences, Fujian Engineering Research Center for Characteristic Upland Crops Breeding, Fujian Engineering Laboratory of Crop Molecular Breeding, Fujian Academy of Agricultural Sciences, Fuzhou, 350013, China

    Hao Chen

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  1. Jingjing Hou
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  2. Hao Chen
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  3. Yuzhen Fang
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  4. Ying Zhu
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  5. Bing Han
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Contributions

J.H. performed the experiments, analyzed the data, and wrote the paper. H.C. analyzed the data and wrote the paper. Y.F., Y.Z., and B.H. helped to perform the experiments. C.S. revised the paper. Y.-C.F. designed the research, analyzed the data, and revised the paper.

Corresponding author

Correspondence to Yongcai Fu.

Additional information

Editor: Yong Eui Choi

Supplementary information

Supplementary Fig. S1

Conditions of “93-11” calli after a treatment with the PMI or HPT selection system. Scale bars = 2 cm. (PNG 2608 kb)

High Resolution (TIF 7363 kb)

Supplementary Table S1

Composition of the media and solutions used in the experiments. (PDF 44 kb)

Supplementary Table S2

Comparison of agronomic traits between negative transgenic plants and TAC1-RNAi-9311 transgenic plants. (PDF 94 kb)

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Hou, J., Chen, H., Fang, Y. et al. An Agrobacterium-mediated non-antibiotic selection-based transformation system for rice (Oryza sativa ssp. indica) cultivar “93-11” successfully produces TAC1-silenced transgenic plants. In Vitro Cell.Dev.Biol.-Plant 57, 786–795 (2021). https://doi.org/10.1007/s11627-021-10202-3

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