Production of transgenic citrus plants using isopentenyltransferase (ipt) positive selection and removal of the marker gene by site-specific recombination
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In any plant genetic transformation system, the use of a selectable marker gene is essential to promote the recovery of only transgenic plants. However, the presence of marker genes conferring antibiotic resistance in plants represents a serious obstacle for 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. Search of alternatives to nptII selection is not easy. An attractive possibility is offered by the MAT (Multi-Auto-Transformation) system, which combines the ipt gene for positive selection with the recombinase system R/RS for removal of marker genes from transgenic cells after transformation. Transgenic shoots for ipt gene lose apical dominance and show a characteristic phenotype. We are testing the efficiency of positive selection and excision by recombination with a MAT vector that comprises the ipt gene as selectable marker and the recombinase R gene, in citrus. Transformation with the MAT vector has been attempted in two citrus genotypes, Pineapple sweet orange (Citrus sinensis L. Osb.) and Carrizo citrange (C. sinensis L. Osb. X Poncirus trifoliata L. Raf.). Results indicated that the IPT phenotype was clearly shown 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.