Double-right-border (DRB) binary vectors for producing selectable marker-free transgenic rice

Abstract

The continued presence of selectable marker genes, especially antibiotic and herbicide resistance genes, in transgenic plants to be released into the environment is of increasing public concern. Techniques such as the Cre/LoxP system of P1 bacteriophage, the FLP/FRT system, or the use of super binary vectors with two sets of T-DNA border sequences have been employed by researchers to produce selectable marker-free (SMF) transgenic progenies. We have further extended the twin T-DNA concept by using a double-right-border (DRB) binary vector. The structure of the DRB binary vector is as follows: RB1-selectable marker-RB2-GOI (gene of interest)-LB (left border). The assumption here is that two types of inserts (RB1 to LB and RB2 to LB) will be delivered and integrated into the genome and that, in the subsequent progenies, the two will segregate, thus allowing selection of SMF transgenic plants (RB2 to LB insert). We have tested this concept using two selectable marker genes (hph and bar) and have successfully segregated the second selectable marker gene from the first. Using the DRB binary vector system, we have recovered SMF transgenic lines containing a rice ragged stunt virus-derived synthetic resistance gene in two rice cultivars (Jarrah and Xu Shui) among segregating T 1 progeny plants. The SMF progenies of four out of eight Jarrah transgenic lines contained RRSVS5AS transgenes at a frequency of 50–100%. We are now using this system to stack two more useful genes in these rice cultivars.