Abstract
Robust genome editing technologies are becoming part of the crop breeding toolbox. Currently, genome editing is usually conducted either at a single locus, or multiple loci, in a variety at one time. Massively parallel genomics platforms, multifaceted genome editing capabilities, and flexible transformation systems enable targeted variation at nearly any locus, across the spectrum of genotypes within a species. We demonstrate here the simultaneous transformation and editing of many genotypes, by targeting mixed seed embryo explants with genome editing machinery, followed by re-identification through genotyping after plant regeneration. Transformation and Editing of Mixed Lines (TREDMIL) produced transformed individuals representing 101 of 104 (97%) mixed elite genotypes in soybean; and 22 of 40 (55%) and 9 of 36 (25%) mixed maize female and male elite inbred genotypes, respectively. Characterization of edited genotypes for the regenerated individuals identified over 800 distinct edits at the Determinate1 (Dt1) locus in samples from 101 soybean genotypes and 95 distinct Brown midrib3 (Bm3) edits in samples from 17 maize genotypes. These results illustrate how TREDMIL can help accelerate the development and deployment of customized crop varieties for future precision breeding. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42994-024-00173-5.