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A rare transgenic event of rice with Agrobacterium binary vector backbone integration at the right T-DNA border junction

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Abstract

Agrobacterium tumefaciens precisely transfers the transferred DNA (T-DNA) between the right border (RB) and the left border (LB) and integrates it in the plant genome. However, transfer of the binary vector backbone (BVB) at the LB junction has been frequently reported. Here, we describe a complex T-DNA integration in the rice transgenic event CG27 with BVB at the RB junction. Amplification of an RB-plant DNA junction fragment by genome walking and its sequencing revealed that the T-DNA portion ended at the 1st bp of RB, but is followed by a binary vector backbone (BVB) along with an RB-flanking T-DNA portion, both in an inverse orientation. This is followed by a 5-bp filler sequence and the rice chromosome 1 sequence. The configuration of the BVB integration at the RB junction was independently confirmed by PCR. Disruption of native chromosome 1 sequence was confirmed by PCR with chromosome 1-specific primers designed from either side of the T-DNA integration site. BVB sequence close to the RB is transferred and integrated into the CG27 plant genome by a mechanism that does not involve LB-skipping and RB read-through. Therefore, a check for BVB sequences at both LB and RB junctions in a transgenic crop is important in biosafety evaluation.

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Abbreviations

T-DNA:

Transferred DNA

RB:

Right border

LB:

Left border

BVB:

Binary vector backbone

MS:

Murashige and Skoog medium

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Acknowledgements

This work was funded by the Department of Biotechnology, Ministry of Science and Technology, Government of India [Project entitled “Development of sheath blight disease-resistant transgenic rice: Resistance tests in PR-protein-expressing transgenic rice and discovery of new RNA silencing strategies”, Project number: F.No: BT/PR6466/COE/34/16/2012]. Indian National Science Academy, New Delhi is acknowledged for the Senior Scientist Fellowship to K.V. Dr. P. Sundaresan, Aravind Medical Research Foundation, Madurai is thanked for DNA Sequencing Service.

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Authors and Affiliations

  1. Department of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Madurai, 625 021, India

    Veeraputhiran Rajapriya, Pachamuthu Kannan, Ganapathi Sridevi & Karuppannan Veluthambi

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  1. Veeraputhiran Rajapriya
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  2. Pachamuthu Kannan
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  3. Ganapathi Sridevi
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  4. Karuppannan Veluthambi
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Contributions

KV and VR conceived and designed all experiments, analyzed the data and wrote the manuscript. GS generated the rice transgenic event CG27. PK did a part of genome walking experiments and nucleotide sequence analysis. All authors read and approved the final manuscript.

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Correspondence to Karuppannan Veluthambi.

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The authors declare that there are no potential conflicts of interest regarding the publication of this paper. All authors have read and approved this version of the article and due care has been taken to ensure the correctness of the data reported in the paper.

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Rajapriya, V., Kannan, P., Sridevi, G. et al. A rare transgenic event of rice with Agrobacterium binary vector backbone integration at the right T-DNA border junction. J. Plant Biochem. Biotechnol. 30, 421–428 (2021). https://doi.org/10.1007/s13562-021-00647-3

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