Elimination of the nptII marker gene in transgenic apple and pear with a chemically inducible R/Rs recombinase

The efficient production of marker-free transgenic plants is still a challenge in most fruit species even though such plants are a necessary component of many “new breeding technologies”, particularly cis- and intragenesis. Marker-free plant production is also necessary for the successive stacking of genes in an elite fruit transgenic line. Here, we used a R/Rs site-specific recombinase that is post-translationally regulated by dexamethasone through fusion with a ligand-binding domain for this hormone, and a bi-functional selectable marker gene coding for a cytosine deaminase/neomycin transferase (codA–nptII) protein; this enabled a first step of positive kanamycin selection, followed by a second step of negative 5-fluorocytosine selection. The aim of our study was to optimize this system on the apple cv. Galaxy and on the pear cv. Conference by conducting a detailed study of the effects of dexamethasone and 5-fluorocytosine treatments, and by comparing an early versus a delayed selection strategy. We were able to produce marker-free transgenic pear plants for the first time, and confirm the feasibility of producing marker-free transgenic apple plants using a chemically inducible recombinase system. We recommend the use of an early selection strategy for the pear cv. Conference and a delayed selection strategy for the apple cv. Galaxy.