title: Genetic transformation and gene silencing mediated by multiple copies of a transgene in eastern white pine.
abstract: 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.
authors.affiliation: Department of Biology, Howell Science Complex, East Carolina University, Greenville, NC 27858-4353, USA. tangw@mail.ecu.edu
authors.forename: Wei
authors.initials: W
authors.lastname: Tang
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Newton
authors.forename: Douglas A
authors.initials: DA
authors.lastname: Weidner
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics; D032461:Chromosomes, Plant; D004269:DNA, Bacterial / Q000378:metabolism; D020868:Gene Silencing; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis; D017403:In Situ Hybridization; D018613:Microscopy, Confocal; D028223:Pinus / Q000235:genetics* / Q000254:growth & development; D018520:Plant Shoots / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000254:growth & development / Q000378:metabolism; D012987:Soil; D014170:Transformation, Genetic; D019076:Transgenes
chemical_list: D004269:DNA, Bacterial; D012987:Soil; C036483:T-DNA; D049452:Green Fluorescent Proteins