CRISPR/Cas9-mediated editing of BADH2 and Wx genes for the development of novel aromatic and soft-textured black and red rice.

Abstract

Black and red rice are known for their rich nutritional content, yet most varieties suffer from a firm texture and insufficient fragrance. In this study, we aimed to develop a fragrant and soft-textured black and red rice variety using the CRISPR/Cas9 technology to knock out the OsWx gene, which is associated with amylose content (AC), and the OsBADH2 gene, responsible for rice aroma. Our results showed that, compared to wild-type, CRISPR lines of XHZ, HM, NWZ, and PGZ targeting OsWx and OsBADH2 exhibited a reduction in AC content, altered gel consistency, and a more than 50% increase in gel consistency. Headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS) analysis revealed that the 2-acetyl-1-pyrroline (2-AP) content in the grains of xhz-c^{BADH2 Wx} and hm-c^{BADH2 Wx} reached 189.04 μg kg^-1 and 309.03 μg kg^-1, respectively. Furthermore, we observed a slight increase in anthocyanins and proanthocyanidins in these co-edited lines, without significant effects on their agronomic traits. Furthermore, to investigate the genes involved in the quality formation of black and red rice for the knockout of OsBADH2 and OsWx, we conducted RNA-seq analysis. The results indicated that knockout of OsBADH2 and OsWx affected the expression of genes involved in carotenoid biosynthesis, multiple amino acid metabolism genes, and endosperm starch and sucrose metabolic pathways. These findings suggest that the CRISPR/Cas9 technology can effectively target OsBADH2 and OsWx to develop high-quality black and red rice varieties with enhanced aroma and softer texture, providing a new strategy for the improvement of colored rice.