1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)
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Item Molecular characterization of CRISPR/ Cas9 edited lines of rice for enhanced drought tolerance and yield(Department of plant biotechnology, college agriculture, Vellanikkara, 2023-10-09) Brindha, T M.; Rehna Augustine.Rice is an important stable and staple food around the world, providing essential nutrition to more than half of the population in more than a hundred countries, including China, India, Indonesia, Vietnam, and the Philippines. Being semi-aquatic annual plant, rice crop consume about 80% of the water used for irrigation. With expanding urbanization and industrialization processes, water is turning out to be scant, and dry season pressure might arise as an extraordinary test to farming everywhere. At the beginning of 2023, 27 of the 36 states and Union territories in India experienced either little or no precipitation, indicating that the dryness persisted throughout the month. Hence enhancing the yield and tolerance to drought remain as major breeding objectives in rice. Crop improvement through biotechnological interventions are imperative in this scenario. Out of various genome engineering tools, CRISPR/Cas9 is a third-generation genome editing tool that has proven to be one of the precise, more efficient and successful genome editing systems across a wide range of organisms, including plants given its ability to efficiently, inexpensively, rapidly, and accurately elicit desired changes. However, the Cas9 complex may bind to unintended regions and initiate cleavage leading to off-target effects, which is considered as the major disadvantage of this technology (Alkan et al., 2018). The current study was undertaken to characterize CRISPR/Cas9 meditated edited lines of rice previously developed in the lab against OsMADS26 (a negative regulator of drought tolerance), osa-miR1432 and osa-mi396b (both negatively regulates grain yield) by performing off-target analysis and gene expression studies. For off-target analysis, the off-target sites for each gRNAs used for developing the mutant lines of OsMADS26, osa-miR1432 and osa-mi396b were predicted using CRISPR Pv2.0 online tool and corresponding sequences were retrieved from Rice Genome Annotation Project database to design flanking primers. The primers were designed from the retrieved sequences, flanking the off-target sites, and evaluated using the ‘Oligoevaluator’ tool. The off-target sites were amplified using PCR, sequenced and analysed for the presence of any mutation. No off-target editing was observed in the edited lines for the evaluated off-target sites. The genome-edited lines of T1 generation were raised from seeds of T0 lines in the containment chamber. Around four T1 plants each from eight genome-edited events of OsMADS26 G1, four events of osa-miR1432 and two events of osa-miR396b were screened for the presence of hygR and Cas9 gene using PCR. The stability of mutations in the edited lines of OsMADS26, osa-miR1432 and osa-mi396b were analysed by amplifying the target region followed by sequencing and sequence analysis. A comparison of mutations observed between T0 and T1 generations were made which showed that mutations were stable. Selected edited lines from each construct were used for Real-time qRT-PCR analysis. Expression profiling of the candidate genes was performed using Real-time qRT PCR. In OsMADS26 G1 edited lines, OsMADS26 gene expression was found to be down regulated compared to the wild type. Expression of OsMADS26 downstream genes like CHI7, POX22.3, SALT, RAB21, and OsWRKY28, involved in various stress responses, was also analysed. In the osa-miR1432 edited lines, OsACOT gene, the target of osa-miR1432 was found to be upregulated. In osa-miR396b edited lines also showed higher expression of GRF6 & GRF8, targets of osa-miR396b.The phenotypic analysis of promising CRISPR/Cas9 edited lines identified in the study will be carried out in future for enhanced drought tolerance and yield.