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Genetic engineering and genome editing strategies were employed to develop glyphosate-resistant rice, aiming to enhance water conservation in rice cultivation. Following bioinformatics analyses, the EPSPS gene was isolated from the Hashemi rice cultivar, sequenced, and validated. Amino acid modifications associated with herbicide tolerance were investigated, and target nucleotide sites were identified. Four distinct genetic engineering approaches were designed and introduced into rice via Agrobacterium-mediated transformation: (1) targeted replacement of the second exon of the rice EPSPS gene to introduce two amino acid substitutions, (2) expression of the native rice EPSPS gene modified by a single nucleotide change, (3) base-editing, and (4) prime-editing. From the second, third, and fourth approaches, 23, 19, and 2 transgenic events were generated, respectively. Molecular analyses confirmed the presence of transgenes and CRISPR components in these events. Glyphosate tolerance was validated in T2-generation plants derived from the second approach, and the selectable marker gene within the T-DNA successfully excised from the genome in segregating progeny of selected events. Further characterization of plants generated through these approaches including purification and bioassays, will be detailed in subsequent studies.
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