| Summary: | Soybean oil is a traditional edible oil. Increasing the oleic acid content is an important direction of soybean breeding. The soybean <i>FAD2</i> family consists of seven genes that regulate how oleic acid is converted into linoleic acid. Five genes of the soybean <i>FAD2</i> gene family, <i>GmFAD2-1B</i>, <i>GmFAD2-1A</i>, <i>GmFAD2-2B</i>, <i>GmFAD2-2C</i>, and <i>GmFAD2-2D</i>, were taken as target genes in this study. Firstly, a multivalent CRISPR/Cas9 gene-editing vector was constructed to regulate <i>FAD2</i> gene expression. Multiple knockout vectors were inserted into the soybean varieties JN38, T6098, and T7010 using <i>Agrobacterium</i>-mediated soybean cotyledon transformation. The functional analysis, agronomic character analysis, and comparison of the mutant lines of the offspring of different genotypes indicated that the JN38 mutant was significantly taller in terms of plant height than the receptor JN38. The fatty acid content of the three groups showed the same trend. The fatty acid contents of mutant plants were higher than those of recipient plants, and the linoleic acid contents of mutant plants were lower than those of recipient plants. The best-performing among the three groups was the JN38 mutant, whose oleic acid content increased from 18.58% to 54.07% and whose linoleic acid content decreased from 57.79% to 26.17%. In conclusion, the knockout expression of multiple <i>FAD2</i> genes increased the soybean oleic acid content and decreased the linoleic acid content in different receptors.
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