Efficient production of marker-free transgenic \'Tarocco\' blood orange (Citrus sinensis Osbeck) with enhanced resistance to citrus canker using a Cre/loxP site-recombination system

Marker-free transgenic plants alleviate concerns regarding the biosafety of genetically modified organisms and promote their commercialization. In this study, a transformation vector pLI35SAAT, harboring a Cre/loxP-mediated recombination system combined with the isopentenyl transferase (ipt) selectable marker gene and an anti-bacterial peptide gene AATCB, was used to produce marker-free transgenic \'Tarocco\' blood orange (Citrus sinensis Osbeck) with enhanced resistance to citrus canker. Using ipt positive selection, a transformation efficiency of 21.4 % was achieved. When the DNA between two loxP sites was excised, phenotypically normal shoots gradually appeared from 74.8 % of the transgenic ipt shoots. Their marker-free transgenic nature was confirmed using PCR and sequencing analyses. In vitro evaluations of citrus canker disease resistance revealed that marker-free transgenic plants exhibited an enhanced resistance to Xanthomonas axonopodis pv. citri. The marker-free transgenic plants appeared phenotypically normal under greenhouse conditions. Thus, marker-free transgenic citrus plants with targeted traits can be efficiently produced using a Cre/loxP-mediated recombination system combined with ipt positive selection.