Vikal, YogeshNavneet Kaur2022-11-122022-11-122022Navneet Kaur (2022). Conversion of non-aromatic rice to aromatic rice by editing Betaine Aldehyde Dehydrogenase (BADH2) gene (Unpublished M.Sc. thesis). Punjab Agricultural University, Ludhiana, Punjab, India.https://krishikosh.egranth.ac.in/handle/1/5810189834Aroma in rice is a highly appreciated trait and acts as determining factor for market price. Basmati rice varieties possess characteristic aroma, however, are usually low yielding. Attempts for transfer of aroma to high-yielding rice varieties through conventional breeding approaches have not been much successful. CRISPR/Cas9 technology has been established as a revolutionary crop improvement technique. The ribonucleoprotein (RNP) mediated delivery of the CRISPR/Cas9 complex is showing immense potential for transgene-free genome editing in rice. The current research was carried out for editing of OsBADH2 gene through CRISPR/Cas9-RNP complex in PR114, high yielding non-aromatic rice variety. A total of five gRNAs were designed through CRISPR P 2.0 for generation of OsBADH2 mutants. Primers were designed for the assembly of gRNA-DNA template for two gRNA selected and transcribed to gRNA utilizing “GeneArtTM Precision gRNA synthesis Kit”. The cleavage efficiency of RNP complex was detected through in vitro cleavage detection. The gRNA were incubated with Cas9 nuclease enzyme to form RNP complexes and coated onto gold particles for the biolistic delivery into the explants. Embryos excised from mature seeds and calli were used as explants for transformation. A variety of media were tested for callus induction and regeneration in PR114. Approximately, 2000 mature embryos and 381 calli were transformed using gRNA-Cas9 complex delivered through gene gun. A total of 35 plants, created through transformation of gRNA-RNP complex targeting exon 7, were characterized through MSBSPPCR technique. The plants showing positive results were proceeded for Sanger’s sequencing. The sequencing results were analysed through FinchTV software for quality of sequencing. The sequences were aligned using Clustal Omega and NCBI-BLASTn online tools. An addition of nucleotide “A” was found in one plant. The nucleotide sequence was translated through Expasy tool. Through insilico analysis of the amino-acid sequence of mutant frameshift mutation was observed. The mutation resulted in alteration in the last four aminoacids of the peptide chain. The T1 progeny of this mutant plant didn’t show presence of the mutation. Rest of the transformed T0 plants are being screened through restriction digestion assay. The mutants so found will be further analyzed through molecular and biochemical techniquesEnglishThesis