A chemical-inducible Cre-LoxP system allows for elimination of selection marker genes in transgenic apricot

Abstract

Transgenic plant development relies on the introduction of marker genes along with the gene(s) of interest to select and/or identify transgenic regenerants. Due to public concerns and regulatory issues, it would be advantageous to eliminate these marker genes once they are no longer needed. The chemical-inducible Cre-LoxP system is especially suitable for clonally-propagated plants, such as fruit trees, as no sexual crosses or rounds of transformation are required for marker-gene elimination. In this study, four transgenic pX6-GFP apricot (Prunus armeniaca L.) (cv. Helena) lines, carrying the gfp reporter gene encoding for the green fluorescent protein, were obtained following Agrobacterium tumefaciens-mediated transformation of leaf explants. The DNA site-specific recombination was precise and tightly controlled by the inducer β-estradiol. Expression of the gfp gene was only detected when 3 μM β-estradiol was added to the medium. When nodal explants were incubated on a meristem development medium supplemented with 3 μM β-estradiol, marker gene elimination was observed in buds of all four transgenic lines, at an average frequency of 11.3 %, based on GFP expression. Further molecular analyses of four GFP-positive shoots, a single shoot from each transgenic line, revealed that DNA recombination was complete in two of shoots, but incomplete in the other two shoots.