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Okadaic acid and trifluoperazine enhance Agrobacterium-mediated transformation in eastern white pine

  • Physiology and Biochemistry
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Abstract

Mature zygotic embryos of recalcitrant Christmas tree species eastern white pine (Pinus strobus L.) were used as explants for Agrobacterium tumefaciens strain GV3101-mediated transformation using the uidA (β-Glucuronidase) gene as a reporter. Influence of the time of sonication and the concentrations of protein phosphatase inhibitor (okadaic acid) and kinase inhibitor (trifluoperazine) on Agrobacterium-mediated transformation have been evaluated. A high transformation frequency was obtained after embryos were sonicated for 45–50 s, or treated with 1.5–2.0 μM okadaic acid or treated with 100–200 μM trifluoperazine, respectively. Protein phosphatase and kinase inhibitors enhance Agrobacterium-mediated transformation in eastern white pine. A 2–3.5-fold higher rate of hygromycin-resistant callus was obtained with an addition of 2 μM okadaic acid or 150 μM trifluoperazine or sonicated embryos for 45 s. Stable integration of the uidA gene in the plant genome of eastern white pine was confirmed by polymerase chain reaction (PCR), Southern and northern blot analyses. These results demonstrated that a stable and enhanced transformation system has been established in eastern white pine and this system would provide an opportunity to transfer economically important genes into this Christmas tree species.

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Abbreviations

BA:

N6-benzyladenine

2,4-D:

2,4-dichlorophenoxyacetic acid

hpt :

Hygromycin phosphotransferase gene

uidA :

β-Glucuronidase

IBA:

Indole-3-butyric acid

PCR:

Polymerase chain reaction

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Acknowledgements

We would like to thank members of the Conifer Biotechnology Laboratory at East Carolina University for support and useful suggestions during this study. This work was supported by the Eastern North Carolina Christmas Tree Association.

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Authors and Affiliations

  1. Department of Biology, Howell Science Complex, East Carolina University, Greenville, NC, 27858-4353, USA

    Wei Tang & Ronald J. Newton

  2. Key Laboratory of Photosynthesis and Molecular Environment Physiology, Institute of Botany, The Chinese Academy of Sciences, Beijing, 100093, P.R. China

    Jinxing Lin

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  1. Wei Tang
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  2. Jinxing Lin
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  3. Ronald J. Newton
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Correspondence to Wei Tang.

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Communicated by W. H. Wu

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Tang, W., Lin, J. & Newton, R.J. Okadaic acid and trifluoperazine enhance Agrobacterium-mediated transformation in eastern white pine. Plant Cell Rep 26, 673–682 (2007). https://doi.org/10.1007/s00299-006-0270-y

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