Biochemical Characterization of Orange-Colored Rice Calli Induced by Target Mutagenesis of OsOr Gene

We generated an orange-colored (OC) rice callus line by targeted mutagenesis of the orange gene (OsOr) using the CRISPR-Cas9 system. The OC line accumulated more lutein, beta-carotene, and two beta-carotene isomers compared to the WT callus line. We also analyzed the expression levels of carotenoid biosynthesis genes by qRT-PCR. Among the genes encoding carotenoid metabolic pathway enzymes, the number of transcripts of the PSY2, PSY3, PDS, ZDS and beta-LCY genes were higher in the OC line than in the WT line. In contrast, transcription of the epsilon-LCY gene was downregulated in the OC line compared to the WT line. In addition, we detected increases in the transcript levels of two genes involved in carotenoid oxidation in the OC lines. The developed OC lines also showed increased tolerance to salt stress. Collectively, these findings indicate that targeted mutagenesis of the OsOr gene via CRISPR/Cas9-mediated genome editing results in beta-carotene accumulation in rice calli. Accordingly, we believe that this type of genome-editing technology could represent an effective alternative approach for enhancing the beta-carotene content of plants.

Automated summary

We generated an orange-colored rice callus line by targeted mutagenesis of the orange gene (OsOr) using the CRISPR-Cas9 system. The OC line accumulated higher levels of lutein, beta-carotene, and two beta-carotene isomers compared to the WT line. The expression levels of carotenoid biosynthesis genes were also altered in the OC line.