A transgenic durum wheat line that is free of marker genes and expresses 1Dy10

As currently practiced, genetic engineering of monocots requires the use of selective agents, such as herbicides and antibiotics, and marker genes for resistance to favor the multiplication of the initially transformed cells. In the present paper we have used minimal gene cassettes and positive selection to generate transgenic durum wheat lines free of herbicide and antibiotic resistance marker genes. Two biolistic transformation experiments were carried out using three minimal gene cassettes consisting of linear DNA fragments each excised from the source plasmids. The targeted trait genes were two bread wheat sequences encoding the Dx5 and Dy10 high-molecular-weight (HMW) glutenin subunits, which have been associated with superior bread-making quality and which arc absent from durum wheats. The positive selectable marker was the Escherichia coli phosphomannose isomerase (pmi) gene, whose product catalyzes the reversible interconversion of mannose-6-phosphate and fructose-6-phosphate, allowing plant cells to utilize mannose as a carbon Source. PCR assays of genomic DNA from regenerated plants identified 15 T-o plants that contained the pmi marker gene for an overall transformation efficiency of 1.5%, which is similar to biolistic transformation efficiencies of durum wheat with intact circular plasmids. Line TC-52, which initially contained pmi, non-expressed ID,6, and expressed 1Dy10 HMW glutenin subunit transgenes, was further investigated. PCR was used to follow inheritance of the pmi marker gene and 1Dx5 from the T-1 to T-3 generations. Transgene expression was monitored by the chlorophenol-red assay for pmi and SDS-PAGE of seed proteins for 1Dy10. From these analyses, we observed that the 1Dy10, 1Dx5 and pmi transgenes were not linked, allowing us in the T-3 generation to identify 1Dy10 transgenic segregants that contained no marker or silent 1Dx5 transgenes. Homozygotes containing and expressing only the 1DY10 transgene were identified in the T-4 generation. These experiments show that it is possible to combine biolistic transformation by minimal gene cassettes with genetic segregation to make marker-free transgenic wheat plants with new traits. (C) 2007 Elsevier Ltd. All rights reserved.