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Background  The ability to modulate levels of individual fatty acids within soybean oil has potential to increase shelf-life and frying stability and to improve nutritional characteristics. Commodity soybean oil contains high levels of polyunsaturated linoleic and linolenic acid, which contribute to oxidative instability – a problem that has been addressed through partial hydrogenation. However, partial hydrogenation increases levels of  trans -fatty acids, which have been associated with cardiovascular disease. Previously, we generated soybean lines with knockout mutations within fatty acid desaturase 2-1A ( FAD2-1A ) and  FAD2-1B  genes, resulting in oil with increased levels of monounsaturated oleic acid (18:1) and decreased levels of linoleic (18:2) and linolenic acid (18:3). Here, we stack mutations within  FAD2-1A  and  FAD2-1B  with mutations in fatty acid desaturase 3A ( FAD3A ) to further decrease levels of linolenic acid. Mutations were introduced into  FAD3A  by directly delivering TALENs into  fad2-1a fad2-1b  soybean plants.    Results  Oil from  fad2-1a fad2-1b fad3a  plants had significantly lower levels of linolenic acid (2.5 %), as compared to  fad2-1a fad2-1b  plants (4.7 %). Furthermore, oil had significantly lower levels of linoleic acid (2.7 % compared to 5.1 %) and significantly higher levels of oleic acid (82.2 % compared to 77.5 %). Transgene-free  fad2-1a fad2-1b fad3a  soybean lines were identified.    Conclusions  The methods presented here provide an efficient means for using sequence-specific nucleases to stack quality traits in soybean. The resulting product comprised oleic acid levels above 80 % and linoleic and linolenic acid levels below 3 %.    Electronic supplementary material  The online version of this article (doi:10.1186/s12870-016-0906-1) contains supplementary material, which is available to authorized users.

Direct stacking of sequence-specific nuclease-induced mutations to produce high oleic and low linolenic soybean oil