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

Abstract

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.

Creation of aromatic maize by CRISPR/Cas

“…The Indian Basmati rice and Thai Jasmine rice are not only popular in Asia but also in America and Europe (Okpala et al, 2019). The popularity and strong desire for aromatic grains expressed by consumers is motivating more breeders to develop aromatic rice cultivars by conventional breeding and molecular breeding, using marker‐assisted selection or genome editing (Okpala et al, 2019; Shan et al, 2015; Ashokkumar et al, 2020). Such aromatic characteristics are also preferred by consumers of soybean, and aromatic soybeans are more popular than nonaromatic cultivars.…”

“…Aromatic rice varieties with loss‐of‐function badh2 alleles are broadly used to improve the fragrance characteristics of rice in conventional breeding programs. However, breeding efforts can now be dramatically shortened by harnessing genome editing with systems based on transcription activator‐like effector nucleases (TALLENs) or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)–CRISPR associated endonuclease (Cas) (Shan et al, 2015; Ashokkumar et al, 2020).…”

Rice Aroma: Biochemical, Genetics and Molecular Aspects and Its Extraction and Quantification Methods

“…Therefore, insights into extraction and quantification methods and various factors affecting the quality of aroma are essential, and also modern biotechnological advances like Transcription Activator Like Effector Nuclease (TALENs), Zinc Finger Nuclease (ZFNs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated endonuclease Cas9 (CRISPR/Cas9) are being entrusted in improving the rice aroma content and quality. Researchers succeeded in editing Badh2 gene and generating high yielding fragrance rice varieties by using TALENs [128] and CRISPR/CAS9 [129][130][131] technologies, which led to increased accumulation of 2AP.…”

Application of CRISPR/Cas9 in Crop Quality Improvement

“…The first fragrant rice was created by targeting the OsBADH2 gene using TALENs in 2015 [ 70 ]. More recently, researchers have made a breakthrough in creating novel alleles of OsBADH2 through CRISPR/Cas9, which successfully converted an unscented rice variety, ASD16, into a novel aromatic rice [ 71 ].…”