Production of aromatic three‐line hybrid rice using novel alleles of BADH2

Aroma is a key grain quality trait that directly influences the market price of rice globally. Loss of function of betaine aldehyde dehydrogenase 2 (OsBADH2) affects the biosynthesis of 2‐acetyl‐1‐pyrroline (2‐AP), which is responsible for aroma in fragrant rice. The current study was aimed at creating new alleles of BADH2 using CRISPR/Cas9 gene editing technology under the genetic background of the japonica Ningjing 1 (NJ1) and indica Huang Huazhan (HHZ) varieties. Sensory evaluation and analysis using headspace solid‐phase microextraction gas chromatography–mass spectrometry (HS‐SPME‐GC‐MS) showed that the grains of the four homozygous T₁ lines with new alleles of BADH2 (nj1‐cr ᴮᴬᴰᴴ²‐1, nj1‐cr ᴮᴬᴰᴴ²‐2, hhz‐cr ᴮᴬᴰᴴ²‐1 and hhz‐cr ᴮᴬᴰᴴ²‐2) produced moderate fragrance and had significantly increased 2‐AP content compared with wild‐types. Moreover, there were no significant differences in the amylose content and gelatinization temperature among the four lines with new alleles of BADH2 to the wild‐types. Thereafter, we crossed the HHZ background new alleles of BADH2 with CMS line Taonong 1A (TN1A) to produce a three‐line hybrid variety B‐Tao‐You‐Xiangzhan (BTYXZ) with increased grain aroma. The 2‐AP content in grains of the improved BTYXZ‐1 and BTYXZ‐2 reached at 26.16 and 18.74 μg/kg, and the gel consistency of BTYXZ‐1 and BTYXZ‐2 increased significantly by 9.1% and 6.5%, respectively, compared with the wild‐type Tao‐You‐Xiangzhan (TYXZ). However, the γ‐aminobutyric acid (GABA) content in the improved three‐line hybrid rice BTYXZ‐1 (5.6 mg/100 g) and BTYXZ‐2 (10.7 mg/100 g) was significantly lower than that of the TYXZ. These results demonstrated that CRISPR/Cas9 gene editing technology could be successfully utilized in improving aroma in non‐fragrant japonica and indica varieties. In addition, the newly developed BADH2 alleles provided important genetic resources for grain aroma improvement in three‐line hybrid rice.