A versatile and robust Agrobacterium‐based gene stacking system generates high‐quality transgenic Arabidopsis plants

: Biotechnology provides a means for the rapid genetic improvement of plants. Although single genes have been important in engineering herbicide and pest tolerance traits in crops, future improvements of complex traits like yield and nutritional quality will likely require the introduction of multiple genes. This research reports a system (GAANTRY; Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technologY) for the flexible, in vivo stacking of multiple genes within an Agrobacterium virulence plasmid Transfer-DNA (T-DNA). The GAANTRY system utilizes in vivo transient expression of unidirectional site-specific recombinases and an alternating selection scheme to sequentially assemble multiple genes into a single transformation construct. To demonstrate GAANTRY's capabilities, 10 cargo sequences were sequentially stacked together to produce a 28.5-kbp T-DNA, which was used to generate hundreds of transgenic events. Approximately 90% of the events identified using a dual antibiotic selection screen exhibited all of the introduced traits. A total of 68% of the tested lines carried a single copy of the selection marker transgene located near the T-DNA left border, and only 8% contained sequence from outside the T-DNA. The GAANTRY system can be modified to easily accommodate any method of DNA assembly and generate high-quality transgenic plants, making it a powerful, yet simple to use tool for plant genetic engineering.