Perakitan Padi Lokal Tinggi Sakuranetin Berbasis Genome Editing dengan Target Mutagenesis Gen Pengkode Udp-Glucosyltransferase yang Dimediasi Agrobacterium Tumefaciens LBA4404

Abstract

Assembly Of High-Yield Local Rice Variety Sakuranetin Using Genome Editing Targeting Mutagenesis UDP-Glucosyltransferase Genes Mediated By Agrobacterium Tumefaciens LBA4404; Putri Kusuma Wardani, 221510501012; 2026: 73 pages; Department of Agrotechnology, Faculty of Agriculture, University of Jember. UGTs (UDP-glucosyltransferases) are enzymes involved in the flavonoid biosynthetic pathway, particularly by catalyzing the conversion of naringenin into other compounds such as naringenin glucoside and apigenin glucoside. The activity of UGT enzymes can divert naringenin away from the sakuranetin biosynthetic pathway, thereby negatively affecting the accumulation of this phytoalexin. Therefore, increasing sakuranetin content can be achieved through the upregulation of the NOMT (naringenin 7-O-methyltransferase) gene and the silencing of UGT genes, preventing the conversion of naringenin into apigenin glucoside and naringenin glucoside. This study aimed to confirm the effect of UGT gene silencing using the pRGEB32 plasmid construct based on the CRISPR/Cas9 system and mediated by Agrobacterium tumefaciens on enhancing sakuranetin accumulation in local rice varieties through descriptive statistical analysis. The results showed that two Koshihikari plants, two Mentik Susu plants, and three Siam Epang plants were successfully transformed with the pRGEB32::UGTs.sgRNA1 construct. The transformation efficiencies were 0.003% for Koshihikari, 0.005% for Mentik Susu, and 0.01% for Siam Epang. Differences in transformation efficiency among varieties were attributed to genotype-specific responses to A. tumefaciens infection and T-DNA transfer processes. Successful integration of the pRGEB32::UGTs.sgRNA1 construct into the rice genome was confirmed by PCR analysis using the HPTII marker, which produced a 545 bp amplicon, and the Cas9 marker, which produced a 495 bp amplicon. These results verified that the regenerated plants were positive transformants carrying the CRISPR/Cas9-UGTs construct.