ANALYSIS OF STABLE EVENTS OF TRANSFORMATION IN WHEAT VIA PEG-MEDIATEDDNA UPTAKE INTO PROTOPLASTS

Protoplasts derived from an embryogenic suspension culture of hexaploi d wheat (Triticum aestivum L.) cv. Oderzo have been transformed via PE G-mediated DNA uptake. The chimaeric gene utilized for transformation was the neomycin phosphotransferase-II gene under the 35S CaMV promote r. The frequency of transformation was between 1 and 2.25 x 10(-6) tre ated protoplasts. Calliclones were selected on 100 mg/l kanamycin, and their resistance analysed at the cellular and molecular level. Cultur es originated from transformed protoclones were highly resistant to ka namycin, neomycin and geneticin (G418); their resistance was maintaine d at a stable condition in the absence of selective pressure. PCR anal yses showed that the selected protoclones growing on kanamycin contain ed the NPT-II gene and that the gene was active, as confirmed by NPT-I I enzymatic assay. Several kanamycin-resistant protoclones were analyz ed by Southern hybridization for the modality of gene integration. Var ious patterns of integration of the NPT-II gene were observed; in seve ral cases multiple insertions and rearrangements of the integrated gen e 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 v ariable number of copies and in different locations. The NPT-II activi ty detected in the calli analyzed was always high, independently from the copy number of the gene inserted.