The GAANTRY system consists of four plasmid vectors in E. coli and a recipient Agrobacterium strain (ArPORT1). The plasmid vectors include P donor and B donor, which can be constructed to carry a cargo sequence of the researcher's choice, and P helper and B helper, which carry operons for site-specific recombinases. The system relies on site-specific recombinases and toggling antibiotic selection to allow researchers to sequentially introduce different cargo genes into the disarmed ArPORT1 virulence plasmid. In the 2018 proof of concept paper,1 10 gene sequences of measurable functional traits like herbicide resistance or luciferase expression were successfully stacked in ArPORT1, resulting in a large (28.5 kb) transgene construct that was consistently maintained in all tested bacterial colonies. The construct was then introduced into Arabidopsis, resulting in 89% of the transformed plants exhibiting all the expected engineered traits and only 8% containing contaminating vector backbone DNA sequences. Theoretically, the sequential recombinase-mediated transgene construction can accommodate the stacking of many more cargo sequences.
In subsequent papers, the GAANTRY system was demonstrated to work in transforming rice and potato plants.2,3 The same stack of 10 genes was used in potatoes, which use a different tissue culture transformation method than Arabidopsis; although transformation rates were lower and a higher frequency of plants lacked the complete construct than in the model system, plant lines exhibiting all of the desired traits and lacking backbone contamination were nevertheless recovered. In rice, the GAANTRY system was used to introduce a much larger transgene construct, with the majority of the transformed plants showing all of the expected herbicide resistance and reporter fluorescence traits.
These flexible molecular tools are a valuable resource to facilitate the engineering of complex traits in staple crops. By distributing the GAANTRY kit, ATCC is allowing materials developed by the US Agricultural Research Service to be utilized by a wide array of researchers and innovators.
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The GAANTRY system can be conveniently ordered as a kit from ATCC.
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MoreReferences
- Collier R, Thomson JG, Thilmony R. A versatile and robust Agrobacterium-based gene stacking system generates high-quality transgenic Arabidopsis plants. Plant J 95: 573-583, 2018. PubMed: 29901840
- Hathwaik LT, et al. Efficient Gene Stacking in Rice Using the GAANTRY System. Rice (N Y) 14(1): 17, 2021. PubMed: 33547973
- McCue KF, et al. Transgene stacking in potato using the GAANTRY system. BMC Res Notes 12(1): 457, 2019. PubMed: 31345264