Overexpression of R2R3-MYB IbMYB1a induces anthocyanin pigmentation in soybean cotyledon

Key message This is the first report showing anthocyanin accumulation in the soybean cotyledon via genetic transformation of a single gene . Abstract Soybean [ Glycine max (L.) Merrill] contains valuable components, including anthocyanins. To enhance anthocyanin production in Korean soybean Kwangankong, we utilized the R2R3-type MYB gene (IbMYB1a ), known for inducing anthocyanin pigmentation in Arabidopsis . This gene was incorporated into constructs using two promoters: the CaMV 35S promoter (P35S) and the β-conglycinin promoter (Pβ-con). Kwangankong was transformed using Agrobacterium , and the presence of IbMYB1a and Bar transgenes in T 0 plants was confirmed through polymerase chain reaction (PCR), followed by gene expression validation. Visual inspection revealed that one P35S: IbMYB1a and three Pβ-con: IbMYB1a lines displayed seed color change. Pβ-con: IbMYB1a T 1 seeds accumulated anthocyanins in cotyledon outer layers, whereas P35S: IbMYB1a and non-transgenic black soybean (Cheongja 5 and Seum) accumulated anthocyanins in the seed coat. During the germination and growth phase, T 1 seedlings from Pβ-con: IbMYB1a lines exhibited anthocyanin pigmentation in cotyledons for up to 1 month without growth aberrations. High-performance liquid chromatography confirmed cyanidin-3- O -glucoside as the major anthocyanin in the Pβ-con: IbMYB1a line (#3). We analyzed the expression patterns of anthocyanin biosynthesis genes, chalcone synthase 7,8, chalcone isomerase 1A, flavanone 3-hydroxylase, flavanone 3’-hydroxylase, dihydroflavanol reductase 1, dihydroflavanol reductase 2, anthocyanidin synthase 2, anthocyanidin synthase 3, and UDP glucose flavonoid 3-O-glucosyltransferase in transgenic and control Kwangankong and black soybean (Cheongja 5 and Seum) seeds using quantitative real-time PCR. We conclude that the induction of gene expression in transgenic plants in comparison with Kwangankong was attributable to IbMYB1a transformation. Notably, flavanone 3-hydroxylase, flavanone 3’-hydroxylase, and dihydroflavanol reductase 1 were abundantly expressed in black soybean seed coat, distinguishing them from transgenic cotyledons.