Generation of Excellent Soybean Germplasm with High Oleic Acid and Low Lipoxygenase by Multigene Editing

Soybean is a major source of plant oil and protein. The content of unsaturated fatty acid is the main factor to affect quanlity of plant oil. Oleic acid is one of the most abundant ingredient in unsaturated fatty acid, it has strong oxidation stability and thermal stability. Increasing the content of oleic acid can enhance the storability of soybean oil, it is also beneficial to human health. GmFAD2-1A and GmFAD2-1B are key genes to encode fatty acid desaturase, regulate the conversion of oleic acid to linoleic acid. Lipoxygenase is one of the antinutritional factors in soybean seeds. It can produce beany flavor under the condition of enzymatic oxidation, which 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. GmLOX genes（including GmLOX1, GmLOX2 and GmLOX3） encode soybean lipoxygenase. It can also catalyze the oxidation of linoleic acid to produce lipid hydroperoxides, and then form a beany flavor. Therefore, reducing the content of linoleic acid is helpful to reduce the formation of bean flavor. In this study, CRISPR/Cas9 was used to edit GmFAD2-1A, GmFAD2-1B, GmLOX1, GmLOX2 and GmLOX3 in Zhongji 602. Seven homozygous five-gene mutants with different alleles were isolated in T₂ generation by Agrobacterium-mediated genetic transformation of soybean. Compared with Zhongji 602, the activity of lipoxygenase was significantly decreased, and the content of oleic acid was increased to 81.7%-83.5% in the mutants seeds. These mutants could be used for beany flavor reduction and creation of high oleic acid soybeans to provide a framework to efficiently develop soybean germplasm to meet changing market demands.