Analysis of stable events of transformation in wheat via PEG-mediated DNA uptake into protoplasts

Protoplasts derived from an embryogenic suspension culture of hexaploid wheat (Triticum aestivum L.) cv. Oderzo have been transformed via PEG-mediated DNA uptake. The chimaeric gene utilized for transformation was the neomycin phosphotransferase-ll gene under the 35S CaMV promoter. The frequency of transformation was between 1 and 2.25 x 10 -6 treated protoplasts. Calliclones were selected on 100 mg/1 kanamycin, and their resistance analysed at the cellular and molecular level. Cultures originated from transformed protoclones were highly resistant to kanamycin, neomycin and geneticin (G418); their resistance was maintained at a stable condition in the absence of selective pressure. PCR analyses showed that the selected protoclones growing on kanamycin contained the NPT-II gene and that the gene was active, as confirmed by NPT-II enzymatic assay. Several kanamycin-resistant protoclones were analyzed by Southern hybridization for the modality of gene integration. Various patterns of integration of the NPT-II gene were observed; in several cases multiple insertions and rearrangements of the integrated gene were observed. In most cases 35S CaMV promoter and the NPT-II coding region were linked on the same restriction fragment. Either complete or partial chimaeric genes were inserted into the genomic DNAs, in a variable number of copies and in different locations. The NPT-II activity detected in the calli analyzed was always high, independently from the copy number of the gene inserted.