Efficient genetic transformation of rice using Agrobacterium with a codon-optimized chromoprotein reporter gene (ChromoP) and introducing an optimized iPCR method for transgene integration site detection

Rice ( Oryza sativa L.) is a vital staple food for a significant portion of the global population. Meeting the demand and enhancing productivity in the face of climate change and population growth necessitate innovative strategies, including molecular modification, genetic engineering, and genome editing. This study presents a simplified gene transformation protocol for the rice cultivar Hashemi using 4 to 7-day-old calluses. The method, based on mature rice seeds and gene transformation to a 4 to 7-day-old callus, achieves a maximum 40% efficiency, circumventing complications associated with using immature. Notably, a novel reporter gene, ChromoP (a codon-optimized chromoprotein), is introduced for the first time to facilitate the optimization of gene transformation. The transgenic calli containing ChromoP exhibit a distinctive pinkish-purple coloration, enabling easy visual identification. Furthermore, the study introduces a simple, fast, and cost-effective protocol, named semi-universal inverse PCR (SUN-iPCR), for detecting individual events and determining the insertion site of a transgene. In conclusion, this study successfully outlines a gene transformation protocol for rice, covering the process until the detection of an independent transformant. Furthermore, the reporter gene and SUN-iPCR methodology utilized in this study hold promising potential for optimizing gene transformation in various other plant species. Key Message This article outlines an effective genetic transformation method for rice using Agrobacterium and the incorporation of a visible reporter gene (ChromoP) to enhance gene transformation in plants. Additionally, it introduces the use of 2 the semi-universal iPCR (SUN-iPCR), an optimized iPCR method for the identification of distinct transgenic events and the determination of transgene integration sites.