CRISPR/Cas9-mediated editing of Wx and BADH2 genes created glutinous and aromatic two-line hybrid rice

Amylose content (AC) is one of the physicochemical indexes of rice quality, which is largely determined by the Waxy (Wx) gene. Fragrance in rice is favored because it adds good flavor and a faint scent. Loss of function of the BADH2 (FGR) gene promotes the biosynthesis of 2-acetyl-1-pyrroline (2AP), which is the main compound responsible for aroma in rice. Here, we used a CRISPR/Cas9 system to simultaneously knock out Wx and FGR genes in 1892S and M858, which are the parents of an indica two-line hybrid rice, Huiliangyou 858 (HLY858). Four T-DNA-free homozygous mutants (1892S-1, 1892S-2, M858-1, and M858-2) were obtained. The 1892S and M858 were crossed to generate double mutant hybrid lines HLY858-1 and HLY858-2. Size-exclusion chromatography (SEC) data indicated that true AC of the wx mutant starches ranged from 0.22 to 1.63%, much lower than those of the wild types (12.93 to 13.76%). However, the gelatinization temperature (GT) of the wx mutants in backgrounds of 1892S, M858, and HLY858 were still high, and showed no significant differences with the wild type controls. The aroma compounds 2AP content in grains of HLY858-1 and HLY858-2 were 153.0 μg/kg and 151.0 μg/kg, respectively. In contrast, 2AP was not detected in grains of HLY858. There were no significant differences in major agronomic traits between the mutants and HLY858. This study provides guidelines for cultivation of ideal glutinous and aromatic hybrid rice by gene editing.