Phosphomannose isomerase,pmi, as a selectable marker gene for durum wheat transformation |
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| Institution: | 1. Genetics and Plant Breeding Section, Department of Environmental and Agro-Forestry Biology and Chemistry, University of Bari, Via Amendola 165/A, 70126 Bari, Italy;2. USDA-ARS Western Regional Research Center, Albany, CA 94710-1105, USA;1. Epidemiology Unit, Ministry of Health, Sri Lanka;2. Polio Eradication Department, World Health Organization, Geneva, Switzerland;3. Polio and Picornavirus Laboratory Branch, Centers for Disease Control and Prevention, Atlanta, GA, USA;1. Department of Entomology, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, OH, United States;2. Department of Entomology, The Ohio State University, Columbus, OH, United States;3. Department of Evolution, Ecology and Organismal Biology, The Ohio State University, Columbus, OH, United States;1. BIOMICs Research Group, Lascaray Research Center, University of the Basque Country UPV/EHU. Avda. Miguel de Unamuno, 3, 01006 Vitoria-Gasteiz, Spain;2. Laboratoire National De Référence, Université Mohammed IV des Sciences de la Santé, Boulevard Mohamed Taieb Naciri, Hay El Hassani, BP 82403, Casa Oumrabii, Casablanca, Morocco;3. Laboratoire de Génétique Humaine, Faculté de Médecine et de Pharmacie, Université Hassan II, Casablanca, Morocco;4. Inserm, UMR 1078, France, Université de Bretagne Occidentale, Brest, France, Etablissement Français du Sang, Bretagne, CHRU Brest, Hôpital Morvan, Laboratoire de Génétique Moléculaire et d’Histocompatibilité, Brest, France;5. Academic Unit of Neurology, Trinity Biomedical Sciences Institute, Dublin, 2, Ireland;6. Department of Neurology, Beaumont Hospital, Dublin, 9, Ireland;7. Delegação do Norte do Instituto Nacional de Medicina Legal, Jardim Carrilho Videira, 4050-167 Porto, Portugal |
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| Abstract: | Transgene technology provides a powerful tool for developing traits that are otherwise difficult to achieve through conventional methods. The development of transgenic plants requires the use of selectable marker genes, as the efficiency of plant transformation is less than optimal for many important species, especially for monocots such as durum wheat (Triticum turgidum var. durum). Many concerns have been expressed about the persistence of currently used marker genes in plants used for field cultivation. To sustain further progress in this area, alternative efficient selection methods are desirable. 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 whereas the non-transgenic cells are actively killed by the selective agent. 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 a 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. |
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