Aroma is an important grain quality trait in rice, which influences the rice price directly. While the consumer demand for aromatic and superior grain quality rice increases, its supply remains low. Driven by its desirability, rice breeders put greater effort into developing novel high yielding fragrant rice cultivars. Compared with conventional breeding methods via gene transfer or exploiting allelic variation, gene editing technology edits genomes more precisely and efficiently and therefore serves as a promising approach to enhance germplasm and develop cultivar.

Recently, the rice quality improvement research group from China National Rice Research Institute (CNRRI) bred three-line hybrid rice using BADH2 novel alleles generated through CRISPR/Cas9 technology. In the three-line hybrid system, the breeding period is long because of a highly restricted restoration and maintaining relationship. Using CRISPR/Cas9-edited male parent line Huang Huazhan (HHZ), CNRRI researchers successfully bred three-line hybrid with improved aromatic trait, providing a new pathway for grain aroma improvement in three-line hybrid rice.

Previous studies have revealed 2-acetyl-1-pyrroline (2-AP) as the major compound contributing to popcorn-like fragrance in rice, whose biosynthesis is affected by betaine aldehyde dehydrogenase 2 (OsBADH2). The researchers of this study firstly created new alleles of BADH2 using CRISPR/Cas9 system under the genetic background of the japonica Ningjing 1 (NJ1) and indica HHZ varieties. Using methods such as sensory evaluation and analysis using headspace solid-phase microextraction gas chromatography–mass spectrometry (HS-SPME-GC-MS), they found 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 to wild-type (WT) varieties. Analysis of the untargeted volatiles in mature grains also verified that among volatile compounds, 2-AP compound was the main difference between the allelic lines of BADH2 and their corresponding WT plants. The researchers then conducted comprehensive evaluation of grain quality and agronomic traits among the four mutant lines. 

After confirming that the hhz-cr^BADH2-1 and hhz-cr^BADH2-2 allele lines can significantly increase the content of 2-AP in the grains, the researchers crossed each of these two new alleles of BADH2 lines with a three-line fragrance sterile line, TN1A, to obtain BTYXZ hybrids with improved fragrance. Further analysis on grains of the above BTYXZ hybrids showed significantly increased 2-AP content, extremely significantly increased gel consistency, significantly reduced hardness, and no significant change in the comprehensive rice taste scores compared with TYXZ plants. Lastly, the researchers quantified GABA in both BTYXZ and TYXZ grains using High Performance Liquid Chromatography (HPLC) method and analyzed its relevance to 2-AP, providing reference for the development of quality aromatic rice cultivars rich in GABA.

The study entitled “Production of aromatic three-line hybrid rice using novel alleles of BADH2” was recently published in Plant Biotechnology Journal. HUI Suozhen, CNRRI graduate with a master's degree, and CNRRI Ph.D. candidates LI Huijuan and Amos Musyoki Mawia are the co-first authors. Prof. HU Peisong, Prof. HU Shikai and Prof. WEI Xiangjin from CNRRI are the co-corresponding authors.

This work was supported by grants from the National Natural Science Foundation of China (31872861, 32071991 and 31972961), National S&T Major Project of China (2016ZX08001006), the Key Research and Development Program of Zhejiang Province (2021C02056-1), Agricultural Sciences and Technologies Innovation Program of Chinese Academy of Agricultural Sciences (CAAS), China National Rice Research Institute Key Research and Development Project (CNRRI-2020-01), and the Central Public-interest Scientific Institution Basal Research Fund (No.CPSIBRFCNRRI-202103).

Paper link: https://onlinelibrary.wiley.com/doi/10.1111/pbi.13695

Figure Determination of 2-AP and GABA content in BTYXZ hybrid lines