Transient gene expression of microprojectile-introduced DNA in Douglas-fir cotyledons

Plasmid DNA containing the reporter gene uidA encoding β-glucuronidase (GUS), driven by the cauliflower mosaic virus 35S promoter, was introduced on high-velocity microprojectiles into cultured cotyledons of Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco]. Transient gene expression was measured by counting the number of distinct loci of GUS activity per cotyledon. Contrary to published results on angiosperms, repeated bombardments did not increase expression in Douglas-fir. Expression varied significantly among cotyledons from different seedlings. The amount of time between DNA delivery and treatment of cotyledons with auxins and cytokinins strongly affected GUS expression. The optimal cytokinin pretreatment produced an average of 20 loci per cotyledon. In several experiments, more than 95% of the treated cotyledons exhibited at least some transient expression. Expression remained constant up to three days following DNA delivery into cotyledons.