Abstract
In the ongoing process of developing Brachypodium distachyon as a model plant for temperate cereals and forage grasses, we have developed a high-throughput Agrobacterium-mediated transformation system for a diploid accession. Embryogenic callus, derived from immature embryos of the accession BDR018, were transformed with Agrobacterium tumefaciens strain AGL1 carrying two T-DNA plasmids, pDM805 and pWBV-Ds-Ubi-bar-Ds. Transient and stable transformation efficiencies were optimised by varying the pre-cultivation period, which had a strong effect on stable transformation efficiency. On average 55% of 17-day-old calli co-inoculated with Agrobacterium regenerated stable transgenic plants. Stable transformation frequencies of up to 80%, which to our knowledge is the highest transformation efficiency reported in graminaceous species, were observed. In a study of 177 transgenic lines transformed with pDM805, all of the regenerated transgenic lines were resistant to BASTA®, while the gusA gene was expressed in 88% of the transgenic lines. Southern blot analysis revealed that 35% of the tested plants had a single T-DNA integration. Segregation analysis performed on progenies of ten selected T0 plants indicated simple Mendelian inheritance of the two transgenes. Furthermore, the presence of two selection marker genes, bar and hpt, on the T-DNA of pWBV-Ds-Ubi-bar-Ds allowed us to characterize the developed transformation protocol with respect to full-length integration rate. Even when not selected for, full-length integration occurred in 97% of the transformants when using bialaphos as selection agent.
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Abbreviations
- 2,4-D:
-
2,4-Dichlorophenoxyacetic acid
- GUS:
-
Glucuronidase
- MG/L liquid medium:
-
Garfinkel and Nester (1980) medium
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Acknowledgements
We thank Dr. Thomas Koprek for providing the vector pWBV-Ds-Ubi-bar-Ds, and Drs. Marianne Folling, Caixia Gao and Dale Godfrey for critical reading of the manuscript. We are grateful to Dr. Niels Roulund for advice on statistical analysis. This research was financially supported by a Marie Curie Training Site (HPMT-CT-2000-00194) with HTC under the EU-FP5 in collaboration with DLF-TRIFOLIUM A/S.
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An erratum to this article can be found at http://dx.doi.org/10.1007/s11248-008-9195-2
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Păcurar, D.I., Thordal-Christensen, H., Nielsen, K.K. et al. A high-throughput Agrobacterium-mediated transformation system for the grass model species Brachypodium distachyon L.. Transgenic Res 17, 965–975 (2008). https://doi.org/10.1007/s11248-007-9159-y
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DOI: https://doi.org/10.1007/s11248-007-9159-y