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 con-
cerns 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 trans-
genic plants exhibited an enhanced resistance to Xan-
thomonas 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.