title: Microprojectile-DNA delivery in conifer species: factors affecting assessment of transient gene expression using the β-glucuronidase reporter gene.
abstract: The Biolistic(®) microprojectile DNA-delivery method was used to test the usefulness in conifers of eight gene constructs based on the 35S promoter, the AMV translational enhancer, and gene fusion between the P-glucuronidase and the neomycin phosphotransferase II genes. The evaluation was done with embryogenic cells of Picea glauca, where the relative strengths of the promoters were 35S-35S-AMVE>35S-AMVE>35S-35S>35S as evaluated by transient gene expression. The fusion gene of GUS and NPT II gave lower levels of transient gene expression than the unfused GUS gene as detected by X-GLU histochemical assays. Experiments comparing the EM promoter of wheat and the 35S-35S-AMVE promoter (with and without fusion between GUS and NPT II) were done in Picea rubens, P. mariana, P. glauca, and Larix x eurolepis. The unfused gene with the 35S-35S-AMVE promoter gave higher levels of transient gene expression than the fused GUS-NPT II gene. The fluorescent MUG assay was more sensitive than the histochemical X-GLU assay to detect the activity of the β-glucuronidase gene.
authors.affiliation: Molecular Genetics and Tissue Culture Group, Petawawa National Forestry Institute, Forestry Canada, K0J 1J0, Chalk River, Ontario, Canada.
authors.forename: P J
authors.initials: PJ
authors.lastname: Charest
authors.forename: N
authors.initials: N
authors.lastname: Caléro
authors.forename: D
authors.initials: D
authors.lastname: Lachance
authors.forename: R S
authors.initials: RS
authors.lastname: Datla
authors.forename: L C
authors.initials: LC
authors.lastname: Duchêsne
authors.forename: E W
authors.initials: EW
authors.lastname: Tsang