Creation of novel alleles of fragrance gene OsBADH2 in rice through CRISPR/Cas9 mediated gene editing

Fragrance in rice grains is a key quality trait determining its acceptability and marketability. Intensive research on rice aroma identified mutations in betaine aldehyde dehydrogenase ( OsBADH2 ) leading to production of aroma in rice. Gene editing technologies like CRISPR/Cas9 system has opened new avenues for accelerated improvement of rice grain quality through targeted mutagenesis. In this study, we have employed CRISPR/Cas9 tool to create novel alleles of OsBADH2 leading to introduction of aroma into an elite non-aromatic rice variety ASD16. PCR analysis of putative transformants using primers targeting the flanking regions of sgRNA in the 7 th exon of OsBADH2 identified 37.5% potential multi-allelic mutations in T 0 generation. Sensory evaluation test in the leaves of T 0 lines identified thirteen lines belonging to five independent events producing aroma. Sequence analysis of these aromatic T 0 lines identified 22 different types of mutations located within -17 bp to +15bp of sgRNA region. The -1/-2 bp deletion in the line # 8–19 and -8/-5 bp deletion in the line # 2–16 produced strong aroma and the phenotype was stably inherited in the T 1 generation. Comparative volatile profiling detected novel aromatic compounds viz., pyrrolidine, pyridine, pyrazine, pyradazine and pyrozole in the grains of T 1 progenies of line # 8–19. This study has demonstrated the use of CRISPR/Cas9 in creating novel alleles of OsBADH2 to introduce aroma into any non-aromatic rice varieties.