Rapid generation of fragrant thermo-sensitive genic male sterile rice with enhanced disease resistance via CRISPR/Cas9.

MAIN CONCLUSION: The three, by mutagenesis produced genes OsPi21, OsXa5, and OsBADH2, generated novel lines exhibiting desired fragrance and improved resistance. Elite sterile lines are the basis for hybrid rice breeding, and rice quality and disease resistance become the focus of new sterile lines breeding. Since there are few sterile lines with fragrance and high resistance to blast and bacterial blight at the same time in hybrid rice production, we here integrated the simultaneous mutagenesis of three genes, OsPi21, OsXa5, and OsBADH2, into Zhi 5012S, an elite thermo-sensitive genic male sterile (TGMS) variety, using the CRISPR/Cas9 system, thus eventually generated novel sterile lines would exhibit desired popcorn-like fragrance and improved resistance to blast and bacterial blight but without a loss in major agricultural traits such as yield. Collectively, this study develops valuable germplasm resources for the development of two-line hybrid rice with disease resistance, which provides a way to rapid generation of novel TGMS lines with elite traits.

Creation of Two-Line Fragrant Glutinous Hybrid Rice by Editing the Wx and OsBADH2 Genes via the CRISPR/Cas9 System.

Global food security has benefited from the development and promotion of the two-line hybrid rice system. Excellent eating quality determines the market competitiveness of hybrid rice varieties based on achieving the fundamental requirements of high yield and good adaptability. Developing sterile and restorer lines with improved quality for two-line hybrid breeding by editing quality genes with clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 is an efficient and practical alternative to the lengthy and laborious process of conventional breeding to improve rice quality. We edited Wx and OsBADH2 using CRISPR/Cas9 technology to produce both homozygous male sterile mutant lines and homozygous restorer mutant lines with Cas9-free. These mutants have a much lower amylose content while having a significantly higher 2-acetyl-1-pyrroline aroma content. Based on this, a fragrant glutinous hybrid rice was developed without too much effect on most agronomic traits. This study demonstrates the use of CRISPR/Cas9 in creating two-line fragrant glutinous hybrid rice by editing the components of the male sterile and the restorative lines.

Impact of OsBadh2 Mutations on Salt Stress Response in Rice.

Mutations in the Betaine aldehyde dehydrogenase 2 (OsBadh2) gene resulted in aroma, which is a highly preferred grain quality attribute in rice. However, research on naturally occurring aromatic rice has revealed ambiguity and controversy regarding aroma emission, stress tolerance, and response to salinity. In this study, mutant lines of two non-aromatic varieties, Huaidao#5 (WT_HD) and Jiahua#1 (WT_JH), were generated by targeted mutagenesis of OsBadh2 using CRISPR/Cas9 technology. The mutant lines of both varieties became aromatic; however, WT_HD mutants exhibited an improved tolerance, while those of WT_JH showed a reduced tolerance to salt stress. To gain insight into the molecular mechanism leading to the opposite effects, comparative analyses of the physiological activities and expressions of aroma- and salinity-related genes were investigated. The WT_HD mutants had a lower mean increment rate of malondialdehyde, superoxide dismutase, glutamate, and proline content, with a higher mean increment rate of γ-aminobutyric acid, hydrogen peroxide, and catalase than the WT_JH mutants. Fluctuations were also detected in the salinity-related gene expression. Thus, the response mechanism of OsBadh2 mutants is complicated where the genetic makeup of the rice variety and interactions of several genes are involved, which requires more in-depth research to explore the possibility of producing highly tolerant aromatic rice genotypes.

RNAi-mediated down regulation of BADH2 gene for expression of 2-acetyl-1-pyrroline in non-scented indica rice IR-64 (Oryza sativa L.).

2-acetyl-1-pyrroline (2AP) is a principal aroma compound in scented rice and a mutation in betaine aldehyde dehydrogenase 2 (OsBADH2) is responsible aroma in scented rice. The present study was aimed at inducing 2AP production in non-scented indica rice cultivar IR-64 by silencing OsBADH2 via RNAi technique. A vector pBSK was used for the construction of RNAi cassette and pRI101ON as a binary vector. Agrobacterium (GV3101)-mediated transformation was done using embryogenic calli of IR-64. The resultant transgenic lines showed up to 14-fold reduction in expression of OsBADH2 gene and 50% inhibition in enzyme activity. Gas chromatography (GC-MS) analyses showed a significant amount of 2AP production in RNAi callus, leaves, and seeds of IR-64. A total 39 volatile compounds were identified from the control and RNAi seeds of IR-64. Among them, octanal and 2-pentylfuron were found to be increased (30-40%) in RNAi seeds of IR-64. The content of precursors, proline, and methylglyoxal increased substantially, whereas GABA content reduced up to 25% in transgenic IR-64 lines. The study demonstrated that RNAi approach could be successfully used for imparting pleasant aroma character in non-scented indica rice cultivars.