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Rapid and accurate early-stage detection of T-DNA/plant flanking sequences of resistant kumquats

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Abstract

Agrobacterium-mediated genetic transformation is a widely applied tool in plant biotechnology. In this process, genes of interest are integrated into plant genomes via T-DNAs present on plasmids in Agrobacteria. Classical and standard methods for screening transformants, such as Southern blot, are inconvenient for most woodland plants because of extremely low transformation efficiency. For the purpose of identifying transgenic woody lines at early selection stages, a right-border T-DNA/plant conjunction sequence analysis was carried out. By analyzing these sequences, 15 out of 17 kanamycin-resistant kumquats were found to be integrated with foreign genes, and two or more copies were present in 33.3% of the transgenic lines, which is completely concordant with Southern blots. Moreover, T-DNA integration into plant nuclear DNA was random without any sequence hotspots, and cleavage sites are any base of the sequence ‘TGAC’. These results showed that this screening method could not only detect resistant woodland plants rapidly at the early selection stage, but unequivocally detect copy numbers. Compared with other screening technique, this method could save time and effort for conducting genetic transformation in woody plants, and also provides accurate integration information for transgenic plants.

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Abbreviations

DIG:

Digoxigenin

DSB:

Double-strand break

LB:

Left border

NCBI:

National Center for Biotechnology Information

npt II:

Neomycin phosphotransferase II

PCR:

Polymerase Chain Reaction

RB:

Right border

Ri:

Root-inducing

T-DNA:

Transfer DNA

Ti:

Tumor inducing

Vir :

Virulence

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Acknowledgements

This study was supported by the Natural Science Foundation of Zhejiang Province (LY16C150001 and LY14C150001).

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Authors and Affiliations

  1. College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua, 321004, People’s Republic of China

    Li Yang, Manman Chen, Nan Shao, Shulei Shan, Weidong Guo & Changchun Wang

  2. Department of Horticulture, Zhejiang University, Hangzhou, 310029, People’s Republic of China

    Changjie Xu

Authors
  1. Li Yang
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  2. Manman Chen
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  3. Nan Shao
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  4. Shulei Shan
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  5. Changjie Xu
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  6. Weidong Guo
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  7. Changchun Wang
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Corresponding author

Correspondence to Changchun Wang.

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Yang, L., Chen, M., Shao, N. et al. Rapid and accurate early-stage detection of T-DNA/plant flanking sequences of resistant kumquats. Plant Cell Tiss Organ Cult 129, 261–269 (2017). https://doi.org/10.1007/s11240-017-1174-y

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  • DOI: https://doi.org/10.1007/s11240-017-1174-y

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