Crispr cas9 mediated editing of rice sd1 gene for reducing height

Abstract

Crop domestication and improvement have been central to the advancement of human civilization. Among cereals, rice (Oryza sativa L.) holds exceptional significance as a staple food for more than half the global population. However, traditional rice varieties, especially aromatic landraces like Nua-kalajeera, although prized for their grain quality, aroma, and cultural value, often suffer from agronomic drawbacks such as tall plant height and lodging susceptibility. Lodging not only reduces yield but also impairs grain quality and complicates harvest. Conventional breeding efforts to introgress semi-dwarf traits into such landraces have largely failed due to challenges such as linkage drag and the prolonged breeding cycle. The Green Revolution addressed lodging issues through the introduction of the sd1 allele, a loss-of function variant of the OsSD1 gene encoding GA20-oxidase-2 an enzyme critical in the gibberellin biosynthesis pathway. While this allele has been widely used in breeding semi-dwarf varieties, traditional approaches are slow and imprecise. In contrast, CRISPR-Cas9-based genome editing offers a precise and efficient tool to generate targeted mutations without foreign DNA integration or linkage drag. This study aimed to develop semi-dwarf, lodging-resistant lines of the aromatic rice landrace Nua-kalajeera using CRISPR-Cas9-mediated targeted mutagenesis of the OsSD1 gene. Three primary research hypotheses were formulated: (1) that a reproducible and efficient in vitro regeneration protocol could be established for Nua-kalajeera; (2) that guide RNAs (gRNAs) designed for OsSD1 could be rapidly validated for cleavage efficiency using a protoplast-based transient assay; and (3) that targeted knockout of OsSD1 via CRISPR-Cas9 would result in semi-dwarf mutants with improved plant architecture without compromising yield or grain quality. To address the first objective, an optimized tissue culture protocol was established, achieving a high callus induction rate using MS medium supplemented with 2 mg/L 2,4-D