Development of an amenable system for site-specific addition to a maize chromosome

Currently, transgenic maize is produced by random integration of a transgenes into the plant. This works for single genes, but not as well for multiple traits. Identifying plants that contain several transgenes becomes a very difficult task. Gene stacking at a single location in the genome would make combining multiple transgenes into plants a simpler process. This project focused on the development of a system would allow for transgenes to be sequentially added to a specific site in the maize genome. The system utilizes two recombinases, Cre recombinase and _C31 Integrase, to remove a selectable marker and to integrate transgenes. An initial construct containing a selectable marker, flanked by LoxP sites, which are acted upon by Cre recombinase, and an attP site, were transformed. The selectable marker was then removed from the integrated transgene by exposure to Cre recombinase. Two amendment constructs enable modification of the integrated construct by utilizing complementary attP and attB sites, which are acted upon by _C31 Integrase. The amendment constructs contain cargo and a promoterless selectable marker which, upon successful recombination with the target site, restores expression of the selectable marker. Successful demonstration of this system simplifies generation of multi-transgene plants, and the assembly of multi-gene pathways in plants.