Abstract
Salinization is one of the leading causes of arable land shrinkage and rice yield decline, recently. Therefore, developing and utilizing salt-tolerant rice varieties have been seen as a crucial and urgent strategy to reduce the effects of saline intrusion and protect food security worldwide. In the current study, the CRISPR/Cas9 system was utilized to induce targeted mutations in the coding sequence of the OsDSG1, a gene involved in the ubiquitination pathway and the regulation of biochemical reactions in rice. The CRISPR/Cas9-induced mutations of the OsDSG1 were generated in a local rice cultivar and the mutant inheritance was validated at different generations. The OsDSG1 mutant lines showed an enhancement in salt tolerance compared to wild type plants at both germination and seedling stages indicated by increases in plant height, root length, and total fresh weight as well as the total chlorophyll and relative water contents under the salt stress condition. In addition, lower proline and MDA contents were observed in mutant rice as compared to wild type plants in the presence of salt stress. Importantly, no effect on seed germination and plant growth parameters was recorded in the CRISRP/Cas9-induced mutant rice under the normal condition. This study again indicates the involvement of the OsDSG1 gene in the salt resistant mechanism in rice and provides a potential strategy to enhance the tolerance of local rice varieties to the salt stress.
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No datasets were generated or analysed during the current study.
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This research was supported by the National Funded Independent Project coded ĐTĐL.CN.51/19 “Research and application of CRISPR/Cas9 technology to improve salinity tolerance in rice”.
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PD, LL, and NP conceived and designed the study; NL designed and constructed the CRISPR/Cas vectors; TB and LN performed rice transformation; LL conducted genotyping and phenotyping; LK, LHL, and TH performed salt tests; PQ assisted greenhouse performance; LL and TH analysed the data and prepared the manuscript; PD and HC revised and proof-read the manuscript. All authors read and approved the final manuscript.
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10142_2024_1347_MOESM1_ESM.pdf
Supplementary file1 Additional file 1: Fig. S1 Agrobacterium-mediated genetic transformation of KD18 cultivar. A Callus induction. B Pre-culture. C Co-culture. D Callus development on resting medium. E Calli on the selection medium. F Regenerated rice plants on the selection medium. Fig. S2 Sequencing chromatograms of the WT and mutant lines at the T0 generation. Target region 1 is marked by a red outlined rectangle, whereas target region 2 is represented by a blue outlined rectangle. Fig. S3 Sequencing chromatograms of the both strands (+Ve and –Ve) of the WT and homozygous mutant lines (D3.4-4 and D13.2-10). Target region 1 is marked by a red outlined rectangle, whereas target region 2 is represented by a blue outlined rectangle. Fig. S4 The transcription abundance of the OsDSG1,OsABI3 and OsABI5 genes in WT and mutant rice lines under the normal and salt stress conditions. The OsGAPDH gene was used as the normalizing reference. Values are represented as the mean ± SE (n = 3). Different letters (a, b, c) indicate significant differences (p < 0.05) using ANOVA and DMRT tests. Fig. S5 Rice morphology of WT and OsDSG1 mutant lines under greenhouse conditions in the T2 generation. A Plant height with scale bar = 10 cm. B Rice panicle with scale bar = 5 cm. C Seed morphologies with scale bar = 3 cm. KD18: WT plant; D3.4-4, D13.2-10: T2 plants from T1 lines D3.4-4 and D13.2-10, respectively. Fig. S6 The rice seedling phenotypes at 2 DAS under the salt treatments. A Seedling morphology of WT and OsDSG1 mutant lines. Scale bar = 3 cm. B Shoot length.C Root length. KD18: WT plant; D3.4-4, D13.2-10: T2 plants from T1 lines D3.4-4 and D13.2-10, respectively. Values are represented as the mean ± SE (n = 60), different letters (a-g) indicate significant differences (p < 0.05) using ANOVA and DMRT tests. Experiment was done with three independent replicates. (PDF 1675 KB)
10142_2024_1347_MOESM2_ESM.docx
Supplementary file2 Additional file 2: Table S1 List of primers used in this study. Table S2 The stable inheritance of mutations from T1 mutant lines to the T2 generation. (DOCX 17 KB)
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Ly, L.K., Ho, T.M., Bui, T.P. et al. CRISPR/Cas9 targeted mutations of OsDSG1 gene enhanced salt tolerance in rice. Funct Integr Genomics 24, 70 (2024). https://doi.org/10.1007/s10142-024-01347-6
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DOI: https://doi.org/10.1007/s10142-024-01347-6