Transformation of blueberry without antibiotic selection

Transformation of the blueberry cultivar North Country (Vaccinium corymbosum x V. angustifolium) was achieved using the disarmed Agrobacterium tumefaciens isolate LBA4404, containing a binary vector with an intron-containing beta-glucuronidase (GUS) marker gene. Plant regeneration was carried out in the absence of antibiotics due to their toxicity to blueberry explants. Agrobacterium contamination was controlled by dipping the explants in antibiotics and rinsing in sterile distilled water. Once regeneration had been achieved, the plantlets were placed on to medium containing the antibiotic ticaricillin at 250 mg litre(-1) to control and try to eliminate any remaining Agrobacterium. Selection of regenerating explants expressing GUS was achieved by growing the plant material for 2 days on a medium containing 4-methyl umbelliferyl glucuronide (MUG), and examining the medium under UV light to detect fluorescent activity. From the 19 explants showing signs of regeneration, seven produced fluorescent patches on MUG medium. From the selected explants, five plantlets were found to express the GUS gene as detected by fluorometric and histochemical analysis. PCR was used to confirm the presence of the GUS and/or NPTII marker genes after 2 years in culture. Bacterial contamination isolated from plant material (which appeared free of contamination) was examined for GUS activity and analysed using PCR with GUS and NPTII specific primers, but no positive results were obtained.