RAPID PRODUCTION OF MULTIPLE INDEPENDENT LINES OF FERTILE TRANSGENIC WHEAT (TRITICUM-AESTIVUM)

Improvement of wheat (Triticum aestivum) by biotechnological approache s is currently limited by a lack of efficient and reliable transformat ion methodology. In this report, we detail a protocol for transformati on of a highly embryogenic wheat cultivar, Bob-white. Calli derived fr om immature embryos, 0.5 to 1 mm long, were bombarded with microprojec tiles coated with DNA containing as marker genes the bar gene, encodin g phosphinothricin-resistance, and the gene encoding beta-glucuronidas e (GUS), each under control of a maize ubiquitin promoter. The bombard ment was performed 5 d after embryo excision, just after initiation of callus proliferation. The ability of plantlets to root in the presenc e of 1 or 3 mg/L of bialaphos was the most reliable selection criteria used to identify transformed plants. Stable transformation was confir med by marker gene expression assays and the presence of the bar seque nces in high molecular weight chromosomal DNA of the resultant plants. Nine independent lines of fertile transgenic wheat plants have been o btained thus far, at a frequency of 1 to 2 per 1000 embryos bombarded. On average, 168 d elapsed between embryo excision for bombardment and anthesis of the T0 plants. The transmission of both the resistance ph enotype and bar DNA to the T1 generation verified that germline transf ormation had occurred.