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JXB Advance Access originally published online on December 6, 2006
Journal of Experimental Botany 2007 58(3):545-554; doi:10.1093/jxb/erl228
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© The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

RESEARCH PAPER

Genetic transformation and gene silencing mediated by multiple copies of a transgene in eastern white pine

Wei Tang1,*, Ronald J. Newton1 and Douglas A. Weidner2

1Department of Biology, Howell Science Complex, East Carolina University, Greenville, NC 27858-4353, USA
2Department of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, NC 27858-4354, USA

* To whom correspondence should be addressed. E-mail: tangw{at}mail.ecu.edu

An efficient transgenic eastern white pine (Pinus strobus L.) plant regeneration system has been established using Agrobacterium tumefaciens strain GV3850-mediated transformation and the green fluorescent protein (gfp) gene as a reporter in this investigation. Stable integration of transgenes in the plant genome of pine was confirmed by polymerase chain reaction (PCR), Southern blot, and northern blot analyses. Transgene expression was analysed in pine T-DNA transformants carrying different numbers of copies of T-DNA insertions. Post-transcriptional gene silencing (PTGS) was mostly obtained in transgenic lines with more than three copies of T-DNA, but not in transgenic lines with one copy of T-DNA. In situ hybridization chromosome analysis of transgenic lines demonstrated that silenced transgenic lines had two or more T-DNA insertions in the same chromosome. These results suggest that two or more T-DNA insertions in the same chromosome facilitate efficient gene silencing in transgenic pine cells expressing green fluorescent protein. There were no differences in shoot differentiation and development between transgenic lines with multiple T-DNA copies and transgenic lines with one or two T-DNA copies.

Key words: Agrobacterium tumefaciens, gene silencing, green fluorescent protein, Pinus strobus L., transgene

Received 15 August 2006; Revised 5 October 2006 Accepted 10 October 2006


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