Improved Plant Synthetic Biology Using a Versatile and Robust Agrobacterium-Based Gene Stacking System

Plant synthetic biology provides a means for the rapid genetic improvement of crops and will enable future improvements of complex traits like yield and nutritional quality through the introduction and coordinated expression of multiple genes. GAANTRY (Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technologY) is a flexible and effective system for stably stacking multiple genes within an Agrobacterium virulence plasmid Transfer-DNA (T-DNA) (Collier et al. The Plant Journal 2018). The system utilizes unidirectional site-specific recombinases in vivo 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 kilobase pair T-DNA, which was used to generate hundreds of transgenic Arabidopsis events. Approximately 90% of the events identified using the 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. Research is also underway to demonstrate that the GAANTRY system can successfully genetically engineer rice, as well as other important crop species. Thus, GAANTRY is a powerful, yet simple to use, new tool for transgene stacking and plant synthetic biology.