Foreign gene transfer into Pinus radiata cotyledons using Agrobacterium tumefaciens

A wild type Agrobacterium tumefaciens strain was used to produce tumours on young Pinus radiata D. Don stems in vitro, and stable expression of the uidA reporter gene in this tumour tissue was confirmed histochemically. The regenerative capacity of detached cotyledons from excised P. radiata zygotic embryos was assessed after different treatments involved in A. tumefaciens infection. Exposure to acetosyringone or the antibiotic timentin did not have a negative effect on tissue health and regeneration, whereas exposure to the antibiotic cefotaxime resulted in poor tissue health. Meristematic activity and shoot elongation from very small portions of cotyledons was demonstrated. A protocol using A. tumefaciens to transfer the uidA reporter gene into adventitious-shoot-forming meristems was developed and co-cultivation parameters were improved to maximise transient expression of this reporter gene