Alternative selectable marker genes for durum wheat genetic transformation

The production of transgenic plants relies on the delivery into the target tissues of selectable marker genes together with the useful genes, in order to allow the regeneration into a plant of the only cells that have integrated and express the foreign DNA. The most efficient selectable markers commonly employed in plant genetic transformation are bacterial genes conferring resistance to antibiotics or herbicides. However, the presence of such genes in crop plants grown in the field destined to human or animal feeding has become a matter of concern for the public opinion. Thus, the scientific community is being stimulated to test alternative selection systems based on new genes conferring resistance to chemicals other than antibiotics or herbicides. In the present work we show the application of two new alternative selection systems for durum wheat biolistic transformation. The first one is a “positive” selection based on the use of mannose as the selective agent and the Escherichia coli phosphomannose isomerase (pmi) gene as the selectable marker, conferring to the transformed plant cells the ability to grow on a medium containing mannose as the only carbon source. The second alternative marker is the hemL gene isolated from Synechococcus strain GR6, coding for a mutant form of the enzyme GSA-AT (glutamate-semialdehyde aminotransferase) which is insensitive to the phytotoxin gabaculine. The hemL gene was found very efficient in tobacco (Gough et al. 2000) and alfalfa (Rosellini et al. 2007) genetic transformation using gabaculine as selective substance. The hemL gene can be a good candidate for a safer selection system as it is present in all plant species and it is involved in one metabolic step only, so that unintended effects of its over-expression in plants are not probable. The GSA-AT cDNA from Medicago sativa was cloned and point-mutated to reproduce the gabaculine-resistance mutation of the Synechococcus hemL gene, and it has been shown to be an efficient marker in alfalfa and tobacco transformation (Rosellini et al., unpublished). In the present work both pmi and the hemL gene were compared to the conventional bar marker gene from S. Hygroscopicus conferring resistance to Bialaphos herbicide. Pmi gene showed a more efficient selectable marker than bar both when delivered in the form of traditional plasmid vector, and in the form of linear minimal expression cassettes (1.14% and 1.50% vs 0.99%). A co-transformation experiment was carried out on wheat calli by delivering plasmids pAHC20 and pAPCK-GSA carrying the bar and the mutant GSA-AT genes, respectively. After gene delivery the two marker genes were evaluated during all the selection phases, from callus regeneration to adult plant formation and compared for their transformation and selection efficiency. Transformation with pAPCK-GSA was more efficient than with the other plasmid.