Efficient production of transgenic citrus plants using isopentenyl transferase positive selection and removal of the marker gene by site-specific recombination
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The presence of marker genes conferring antibiotic resistance in transgenic plants represents a serious obstacle for their public acceptance and future commercialization. In citrus, selection using the selectable marker gene nptII, that confers resistance to the antibiotic kanamycin, is in general very effective. An attractive alternative is offered by the MAT system (Multi-Auto-Transformation), which combines the ipt gene for positive selection with the recombinase system R/RS for removal of marker genes from transgenic cells after transformation. Transformation with a MAT vector has been attempted in two citrus genotypes, Pineapple sweet orange (Citrus sinensis L. Osb.) and Carrizo citrange (C. sinensis L. Osb. × Poncirus trifoliata L. Raf.). Results indicated that the IPT phenotype was clearly distinguishable in sweet orange but not in citrange, and that excision was not always efficient and precise. Nevertheless, the easy visual detection of the IPT phenotype combined with the higher transformation efficiency achieved in sweet orange using this system open interesting perspectives for the generation of marker-free transgenic citrus plants.
KeywordsCitrange Genetic transformation ipt Marker gene Marker-free transgenic plants Sweet orange
A. Ballester is recipient of a fellowship provided by the Instituto Nacional de Investigaciones Agrarias (INIA). We thank Dr. H. Ebinuma (Pulp and Paper Research Laboratory, Nippon Paper Industries, Tokio, Japan) for providing plasmid pEXMGFP1. This research was supported by grant AGL2003-01644 from the Ministry of Education and Science (Spain).
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