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Characterization of Agrobacterium-mediated co-transformation events in rice using green and red fluorescent proteins

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Abstract

Background

Biotechnologists seeking to develop marker-free transgenic plants have established co-transformation methods. For co-transformation using mixed Agrobacterium strains, the mix ratio of Agrobacterium strains and selection scheme may influence co-transformation frequency. This study used fluorescent GFP and RFP markers to compose different selection schemes for observation of the selective dynamics of transformed rice cells and to investigate the factors affecting co-transformation efficiency.

Methods and results

We utilized GFP and RFP markers in co-transformation and tested the combinations of an antibiotic-selectable vector (pGFP-HPT) and a single RFP vector (pRFP) and of two antibiotic-selectable vectors (pGFP-HPT and pRFP-HPT) in rice. The pGFP-HPT/pRFP combination resulted in 70.9% to 81.2% of co-transformation frequencies while lower frequencies (56.6% on average) were obtained with the pGFP-HPT/pRFP-HPT combination. Based on GFP/RFP segregation patterns, 55% of the pGFP-HPT/pRFP co-transformants contained unlinked T-DNAs and segregated single RFP progeny, which simulated the selection process of marker-free transgenic plants that carry an actual gene of interest. Transgene expression levels in the rice lines varied as revealed by RT-PCR, and tandem-linked T-DNAs were detected in co-transformants, suggesting that transgene expression might be affected by duplicated T-DNA structures.

Conclusion

Co-transformation via mixed Agrobacterium strains is feasible, and approximately 55% of the pGFP-HPT/pRFP co-transformants contained unlinked T-DNAs and segregated single RFP progeny. The pGFP-HPT/pRFP and the pGFP-HPT/pRFP-HPT vector combinations showed distinctive selective dynamics of transformed rice cells, suggesting that co-transformation efficiency depends on both vector system and selection scheme.

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Abbreviations

GFP:

Green fluorescent protein

RFP:

Red fluorescent protein

HPT:

Hygromycin phosphotransferase

Hyg:

Hygromycin

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Acknowledgements

The authors are grateful to Xiaowei Zou, Xiaoqing Gao, Haihe Yang, Haiyan Qu, and Suqin Zheng for their experimental assistance.

Funding

This work was financially supported by the State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products in Zhejiang Academy of Agricultural Sciences, and by the National Natural Science Foundation of China (Grant No. 31672016).

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Authors and Affiliations

  1. College of Plant Protection, Hunan Agricultural University, Changsha, 410128, China

    Lihua Li & Liangying Dai

  2. Institute of Virology and Biotechnology, Zhejiang Academy of Agricultural Sciences, Hangzhou, 310021, China

    Xudan Tian, Lanlan Wang, Jianhua Zhao, Jie Zhou, Haiyan He & Shaohong Qu

  3. College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua, 321004, China

    Xudan Tian & Jianhua Zhao

Authors
  1. Lihua Li
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  2. Xudan Tian
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  3. Lanlan Wang
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  4. Jianhua Zhao
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  5. Jie Zhou
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  6. Haiyan He
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  7. Liangying Dai
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Contributions

LL, XT, LW, JZ, JZ and HH conducted the experiments, collected and analyzed the data. LL and SQ wrote the manuscript. SQ and DL designed the research schemes and supervised the whole process. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Liangying Dai or Shaohong Qu.

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Li, L., Tian, X., Wang, L. et al. Characterization of Agrobacterium-mediated co-transformation events in rice using green and red fluorescent proteins. Mol Biol Rep 49, 9613–9622 (2022). https://doi.org/10.1007/s11033-022-07864-6

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  • DOI: https://doi.org/10.1007/s11033-022-07864-6

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