title: Direct stacking of sequence-specific nuclease-induced mutations to produce high oleic and low linolenic soybean oil.
abstract: 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.
  
  
  
  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.
  
  
  
  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 %.
authors.affiliation: Calyxt, Inc., 600 County Road D West Suite 8, New Brighton, MN, 55112, USA.
authors.forename: Zachary L
authors.initials: ZL
authors.lastname: Demorest
authors.affiliation: Calyxt, Inc., 600 County Road D West Suite 8, New Brighton, MN, 55112, USA.
authors.forename: Andrew
authors.initials: A
authors.lastname: Coffman
authors.affiliation: Calyxt, Inc., 600 County Road D West Suite 8, New Brighton, MN, 55112, USA.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Baltes
authors.affiliation: Calyxt, Inc., 600 County Road D West Suite 8, New Brighton, MN, 55112, USA.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Stoddard
authors.affiliation: Calyxt, Inc., 600 County Road D West Suite 8, New Brighton, MN, 55112, USA.
authors.forename: Benjamin M
authors.initials: BM
authors.lastname: Clasen
authors.affiliation: Calyxt, Inc., 600 County Road D West Suite 8, New Brighton, MN, 55112, USA.
authors.forename: Song
authors.initials: S
authors.lastname: Luo
authors.affiliation: Calyxt, Inc., 600 County Road D West Suite 8, New Brighton, MN, 55112, USA.
authors.forename: Adam
authors.initials: A
authors.lastname: Retterath
authors.affiliation: Calyxt, Inc., 600 County Road D West Suite 8, New Brighton, MN, 55112, USA.
authors.forename: Ann
authors.initials: A
authors.lastname: Yabandith
authors.affiliation: Calyxt, Inc., 600 County Road D West Suite 8, New Brighton, MN, 55112, USA.
authors.forename: Maria Elena
authors.initials: ME
authors.lastname: Gamo
authors.affiliation: Calyxt, Inc., 600 County Road D West Suite 8, New Brighton, MN, 55112, USA.
authors.forename: Jeff
authors.initials: J
authors.lastname: Bissen
authors.affiliation: Calyxt, Inc., 600 County Road D West Suite 8, New Brighton, MN, 55112, USA.
authors.forename: Luc
authors.initials: L
authors.lastname: Mathis
authors.affiliation: Calyxt, Inc., 600 County Road D West Suite 8, New Brighton, MN, 55112, USA.
authors.forename: Daniel F
authors.initials: DF
authors.lastname: Voytas
authors.affiliation: Calyxt, Inc., 600 County Road D West Suite 8, New Brighton, MN, 55112, USA. fzhang@calyxt.com.
authors.forename: Feng
authors.initials: F
authors.lastname: Zhang
mesh_terms: D000072669:Gene Editing; D009154:Mutation / Q000235:genetics; D019301:Oleic Acid / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D013024:Soybean Oil / Q000235:genetics* / Q000378:metabolism; D013025:Glycine max / Q000235:genetics / Q000378:metabolism*; D017962:alpha-Linolenic Acid / Q000235:genetics* / Q000378:metabolism
chemical_list: D010940:Plant Proteins; D017962:alpha-Linolenic Acid; D019301:Oleic Acid; D013024:Soybean Oil
keywords: Genome editing; High oleic acid; Low linolenic acid; Soybean; Soybean oil; TALEN; Targeted mutagenesis