Beany flavor induced by three lipoxygenases (LOXs, including LOX1, LOX2, and LOX3) restricts human consumption of soybean. It is desirable to generate lipoxygenase-free new mutant lines to improve the eating quality of soybean oil and protein products. In this study, a pooled clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) strategy targeting three GmLox genes (GmLox1, GmLox2, and GmLox3) was applied and 60 T₀ positive transgenic plants were generated, carrying combinations of sgRNAs and mutations. Among them, GmLox-28 and GmLox-60 were gmlox1gmlox2gmlox3 triple mutants and GmLox-40 was a gmlox1gmlox2 double mutant. Sequencing of T₁ mutant plants derived from GmLox-28, GmLox-60, and GmLox-40 showed that mutation in the GmLox gene was inherited by the next generation. Colorimetric assay revealed that plants carrying different combinations of mutations lost the corresponding lipoxygenase activities. Transgene-free mutants were obtained by screening the T₂ generation of lipoxygenase-free mutant lines (GmLox-28 and GmLox-60). These transgene- and lipoxygenase-free mutants could be used for soybean beany flavor reduction without restriction by regulatory frameworks governing transgenic organisms.

Soybean (Glycine max [L.] Merr.) is an important source of human dietary protein and vegetable oil. A strong negative correlation between protein and oil contents has hindered efforts to improve soybean seed quality. The metabolic and genetic bases of soybean seed composition remain elusive. We evaluated metabolic diversity in a soybean near-isogenic line (NIL) population derived from parents (JD12 and CMSD) with contrasting seed oil contents. Using GC-TOF/MS, we compared seed primary metabolites of high protein/low oil lines, low protein/high oil lines, and their parents. Principal-components analysis showed that metabolic profiles of all progeny lines could be discriminated based on protein and oil contents. Univariate analysis revealed wide variation and transgressive segregation of metabolites in the population. Twenty-eight annotated metabolites, in particular free asparagine, free 3-cyanoalanine, and L-malic acid, were correlated with seed protein content or seed oil content or seed protein and oil content. These results shed light on the metabolic and genetic basis of soybean seed composition.