Submitted to: Journal of Cereal Science
Publication Type: Peer reviewed journal
Publication Acceptance Date: 6/16/2005
Publication Date: 1/1/2006
Citation: Gadaleta, A., Giancaspro, A., Blechl, A.E., Blanco, A. 2006. Phosphomannose isomerase, pmi, as a selectable marker gene for durum wheat transformation. Journal of Cereal Science. 43: 31-37 Interpretive Summary: Biotechnology provides a powerful tool for improving plant characteristics that are difficult to modify through conventional breeding methods. Identification of transgenic plants requires the use of selectable marker genes, as transformation efficiencies are less than optimal for many important species, especially for cereals such as durum wheat (Triticum turgidum var durum). Many concerns have been expressed about the persistence of currently used marker genes in plants destined for field cultivation and commercial production. New efficient selection methods are needed. A recent development is the use of selective genes that give transformed cells a metabolic advantage (positive selection) compared to untransformed cells, which are slowly starved with a concomitant reduction in growth and viability. This selection strategy is in contrast to traditional negative selections during which the transgenic cells are able to survive on a selective medium while the non-transgenic cells are actively killed by the selective agent. In this study, we report that employing the Escherichia coli phosphomannose isomerase (pmi) gene as the selectable gene and mannose as a positive selective agent was 3x more efficient for identifying pasta wheat transformants than using the Streptomyces hygroscopicus bar gene to confer resistance to the herbicide bialaphos. This work will allow researchers to use a sugar rather than a herbicide to select wheat transformants.
Technical Abstract: The development of transgenic plants requires the use of selectable marker genes, because transformation efficiencies are less than optimal for many important species, especially for monocots such as durum wheat (Triticum turgidum var durum). To sustain further progress in this area, alternative efficient selection methods would be desirable. In the present study, we compared the “selection efficiency” of a commonly used negative selection method that employs the Streptomyces hygroscopicus bar gene to confer resistance to the herbicide bialaphos, to a positive selection employing the Escherichia coli phosphomannose isomerase (pmi) gene as the selectable gene and mannose as the selective agent. Calli derived from immature embryos of the durum wheat cultivar Svevo were bombarded separately with bar and pmi genes using the biolistic system. The integration and expression of the two genes in the T0 generation were confirmed by PCR analysis with specific primers for each gene and the chlorophenol red assay, respectively. The selection efficiency, calculated as the number of expressing plants divided by the number of total regenerants, was higher when pmi was used as the selectable marker gene (90.1%) than when bar was used (26.4%). Thus, an efficient selection method for durum wheat transformation was established that obviates the use of herbicide résistance genes.