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T-DNA transfer and T-DNA integration efficiencies upon Arabidopsis thaliana root explant cocultivation and floral dip transformation

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Abstract

T-DNA transfer and integration frequencies during Agrobacterium-mediated root explant cocultivation and floral dip transformations of Arabidopsis thaliana were analyzed with and without selection for transformation-competent cells. Based on the presence or absence of CRE recombinase activity without or with the CRE T-DNA being integrated, transient expression versus stable transformation was differentiated. During root explant cocultivation, continuous light enhanced the number of plant cells competent for interaction with Agrobacterium and thus the number of transient gene expression events. However, in transformation competent plant cells, continuous light did not further enhance cotransfer or cointegration frequencies. Upon selection for root transformants expressing a first T-DNA, 43–69 % of these transformants showed cotransfer of another non-selected T-DNA in two different light regimes. However, integration of the non-selected cotransferred T-DNA occurred only in 19–46 % of these transformants, indicating that T-DNA integration in regenerating root cells limits the transformation frequencies. After floral dip transformation, transient T-DNA expression without integration could not be detected, while stable T-DNA transformation occurred in 0.5–1.3 % of the T1 seedlings. Upon selection for floral dip transformants with a first T-DNA, 8–34 % of the transformants showed cotransfer of the other non-selected T-DNA and in 93–100 % of them, the T-DNA was also integrated. Therefore, a productive interaction between the agrobacteria and the female gametophyte, rather than the T-DNA integration process, restricts the floral dip transformation frequencies.

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Abbreviations

DSB:

Double-stranded break

GUS:

β-Glucuronidase

P35S:

Cauliflower mosaic virus promoter

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Acknowledgments

We thank Karolina Komorowska, Anni Jacobs, Marion Naudts and Jonah Nolf for practical assistance, Marnik Vuylsteke for statistical analysis, and Martine De Cock and Annick Bleys for help in preparing the manuscript. This work was supported by grants from the Flemish Community and the Research Foundation Flanders (G.021106). F.V.E. was indebted to the Agency for Innovation through Science and Technology in Flanders for a predoctoral fellowship.

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The authors declare that they have no conflict of interest.

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  1. Frédéric Van Ex

    Present address: Bayer CropScience NV, Technologiepark 38, 9052, Ghent, Belgium

Authors and Affiliations

  1. Department Plant Systems Biology, VIB, Ghent University, Technologiepark 927, 9052, Ghent, Belgium

    Rim Ghedira, Sylvie De Buck & Ann Depicker

  2. Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052, Ghent, Belgium

    Rim Ghedira, Sylvie De Buck & Ann Depicker

  3. Laboratory of Plant Genetics, Institute for Molecular Biology and Biotechnology, Vrije Universiteit Brussel (VUB), 1050, Brussel, Belgium

    Frédéric Van Ex & Geert Angenon

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  1. Rim Ghedira
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Correspondence to Ann Depicker.

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R. Ghedira and S. De Buck contributed equally to this work.

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Ghedira, R., De Buck, S., Van Ex, F. et al. T-DNA transfer and T-DNA integration efficiencies upon Arabidopsis thaliana root explant cocultivation and floral dip transformation. Planta 238, 1025–1037 (2013). https://doi.org/10.1007/s00425-013-1948-3

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