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

Authors: COLLIER, RAY - University Of Wisconsin; Thomson, James - Jim; Thilmony, Roger

Submitted to: Plant Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/18/2018
Publication Date: 6/14/2018
Citation: Collier, R., Thomson, J.G., Thilmony, R.L. 2018. A versatile and robust Agrobacterium-based gene stacking system generates high quality transgenic Arabidopsis plants. Plant Journal. 95(4):573-583. https://doi.org/10.1111/tpj.13992.
DOI: https://doi.org/10.1111/tpj.13992

Interpretive Summary: The genetic improvement of crops is one of the most effective ways to increase their productivity in agriculture. One means of genetically improving plants is through the use of modern plant biotechnology, which has the capability of introducing one or more genes with known sequences and protein products into plant chromosomes. Until now, it has been difficult to predictably genetically engineer complex traits - like yield and nutritional improvements - that require the action of multiple genes. In this manuscript, a novel technology called GAANTRY (for Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technologY) is described that enables the efficient assembly of multiple genes in Agrobacterium and their transfer to plants as single units. The authors demonstrate that at least 10 different genes can be joined together via a simple, reliable and highly effective process. They show that 90% of the transformed plants express all the introduced traits as desired. This technological breakthrough will enable genetic engineers to effectively improve complex traits in a wide array of crops.

Technical Abstract: Plant biotechnology has great potential to continue to provide a means for the genetic improvement of crops. Although single genes have been important in engineering herbicide and pest tolerance traits, future improvements of complex traits like yield and nutritional quality will likely require the introduction 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). 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 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. Thus, GAANTRY is a powerful new tool for transgene stacking, plant synthetic biology and the generation of high quality genetically engineered plants.