Generation of seed lipoxygenase-free soybean using CRISPR-Cas9
Jie Wang,
Huaqin Kuang,
Zhihui Zhang,
Yongqing Yang,
Long Yan,
Mengchen Zhang,
Shikui Song,
Yuefeng Guan
Affiliations
Jie Wang
College of Resources and Environment, Fujian Provincial Key Laboratory of Haixia Applied Plant Systems Biology, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China; FAFU-UCR Joint Center for Horticultural Plant Biology and Metabolomics, Haixia Institute of Science and Technology, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
Huaqin Kuang
FAFU-UCR Joint Center for Horticultural Plant Biology and Metabolomics, Haixia Institute of Science and Technology, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
Zhihui Zhang
College of Resources and Environment, Fujian Provincial Key Laboratory of Haixia Applied Plant Systems Biology, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China; FAFU-UCR Joint Center for Horticultural Plant Biology and Metabolomics, Haixia Institute of Science and Technology, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
Yongqing Yang
Root Biology Center, College of Resources and Environment, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
Long Yan
The Key Laboratory of Crop Genetics and Breeding of Hebei, Institute of Cereal and Oil Crops, Hebei Academy of Agricultural and Forestry Sciences, Shijiazhuang 050035, Hebei, China
Mengchen Zhang
The Key Laboratory of Crop Genetics and Breeding of Hebei, Institute of Cereal and Oil Crops, Hebei Academy of Agricultural and Forestry Sciences, Shijiazhuang 050035, Hebei, China
Shikui Song
FAFU-UCR Joint Center for Horticultural Plant Biology and Metabolomics, Haixia Institute of Science and Technology, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
Yuefeng Guan
FAFU-UCR Joint Center for Horticultural Plant Biology and Metabolomics, Haixia Institute of Science and Technology, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China; Corresponding author.
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.
