Gabaculine selection using bacterial and plant marker genes (GSA-AT) in durum wheat transformation

Selectable marker genes are widely used for the efficient transformation of crop plants. In most cases, selection is based on antibiotic or herbicide resistance genes because they tend to be most efficient. The Synechococcus hemL gene has been successfully employed as a selectable marker for tobacco and alfalfa genetic transformation, by using gabaculine as the selective agent. The gene conferring gabaculine resistance is a mutant form of the hemL gene from Synechococcus PCC6301, strain GR6, encoding a gabaculine insensitive form of the glutamate1- semialdehyde aminotransferase (GSA) enzyme. In the present study we compared the transformation and selection efficiency of the common selection method based on the Streptomyces hygroscopicus bar gene conferring resistance to Bialaphos, with both the Synechococcus hemL gene and a Medicago sativa mutated GSA gene (MsGSAgr) conferring resistance to phytotoxin gabaculine. Callus derived from immature embryos of the durum wheat cultivar Varano were simultaneously co-bombarded with bar/hemL and bar/MsGSAgr genes. After gene delivery, the marker genes were individually evaluated through all the selection phases from callus regeneration to adult plant formation, and compared for their transformation and selection efficiency. The integration of the three genes in the T0 generation was confirmed by PCR analysis with specific primers for each gene and southern blot analysis. Both Synechococcus hemL and MsGSA were more efficient than bar for biolistic transformation (2.8% vs. 1.8% and 1.1% vs. 0.5%) and selection (79% vs. 43% and 87% vs. 50%). Thus, an efficient selection method for durum wheat transformation was established that obviates the use of herbicide resistance genes.