Efficient gene stacking in rice using the GAANTRY system

Authors: HATHWAIK, LEYLA - Former ARS Employee; Horstman, James; Thomson, James - Jim; Thilmony, Roger

Submitted to: Rice
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/28/2021
Publication Date: 2/6/2021
Publication URL: https://handle.nal.usda.gov/10113/7265761
Citation: Hathwaik, L.T., Horstman, J.D., Thomson, J.G., Thilmony, R.L. 2021. Efficient gene stacking in rice using the GAANTRY system. Rice. 14. Article 17 (2021). https://doi.org/10.1186/s12284-021-00460-5.
DOI: https://doi.org/10.1186/s12284-021-00460-5

Interpretive Summary: Crop biotechnology is an effective way to genetically improve crops increasing their agricultural productivity. Previously, it has been challenging to predictably genetically engineer complex traits like yield and nutritional improvements in crops that require the action of multiple genes. A technology called GAANTRY (for Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technologY) is described that enables the efficient assembly of eleven cargo sequences and their transfer to rice plants. The results demonstrate that the GAANTRY system provides a simple, reliable and highly effective process for the assembly of large transformation constructs that can be effectively introduced into rice plants. More than half of the recovered rice plants carried all of the expected sequences and expressed the desired introduced traits. This technology will enable researchers to effectively improve complex multi-gene traits in rice and potentially other cereal grain crops in the future.

Technical Abstract: Genetic engineering of rice provides a means for improving rice grain quality and yield, and the introduction and expression of multiple genes can produce new traits that would otherwise be difficult to obtain through conventional breeding. GAANTRY (Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technologY) was previously shown to be a precise and robust system to stably stack ten genes (28 kilobases (kb)) within an Agrobacterium virulence plasmid Transfer-DNA (T-DNA) and obtain high-quality Arabidopsis and potato transgenic events. To determine whether the GAANTRY system can be used to engineer a monocotyledonous crop, two new T-DNA constructs, carrying five (16.9 kb)) or eleven (37.1 kb) cargo sequences were assembled and transformed into rice. Characterization of 53 independent transgenic events demonstrated that more than 50% of the plants carried all of the desired cargo sequences. Additionally, more than 18% of the lines were high-quality events containing a single copy of the introduced transgenes, and were free of sequences from outside of the T-DNA. Therefore, GAANTRY provides a simple, precise and versatile tool for transgene stacking in rice and potentially other cereal grain crops.