THE CLEAN GENE AS MOLECULAR TOOL FOR THE OBTAINMENT OF CISGENIC WHEAT PLANTS

Although biotechnology has been established as a very powerful and promising tool in the agricultural field, the application of genetic modification aimed to crop improvement is still associated with serious concerns about safety and environmental risks, due to the presence of foreign DNA. Ethical concerns and risk perceptions linked to biotech crops, are mainly due to the presence in GMOs of several genetic elements derived from non-compatible species, and containing selectable marker genes for antibiotics or herbicide resistance. Cisgenesis and intra-genesis technology have been developed as new tools for crop modification and plant breeding, alternative to conventional transgenesis. Cisgenesis consists in a genetic modification using a complete copy of natural genes with their flanking regulatory regions (native introns, promoter and terminator) belonging exclusively to the transformed plant itself, or isolated from a sexually compatible “donor”. In particular, the routine production of transgenic plants involves transformation with foreign DNA carried on plasmids, leading to the integration of vector backbone sequences into the host genome along with the transgenes. Plasmids can promote gene rearrangements and transgene silencing. “Clean gene” technology, intended as the use of minimal expression cassettes represented by linear DNA fragments containing only promoter, gene coding region and terminator/polyadenylation sites, has been showing significant advantages in reducing all these events in several crop species. In the present work, we carried out durum wheat biolistic transformation on several durum wheat cultivars by using either whole plasmids containing suitable gene constructs, or minimal gene cassettes which were linear DNA fragments lacking vector sequences excised from source plasmid. We delivered as the target genes, two sequences encoding the 1Dx5 and 1Dy10 HMW glutenin subunits from hexaploid wheat flanked by their native regulatory regions, and the phosphomannose isomerase (pmi) gene as the selectable marker in order to test its efficiency in several durum wheat cultivars.