CRISPR (clustered regularly interspaced short palindromic repeats) gene-edited (GEd) crops have demonstrated significant potential to enhance global food security in the face of escalating climate challenges and rapid population growth. Since 2019, for regulatory purposes, the United States (U.S.) and several other countries have recognized transgene-free, genome-edited lines as equivalent to conventionally bred varieties. Notably, the first genome-edited food product, Calyno™ soybean oil, was commercialized in the U.S. and marketed as a non-genetically modified organism (GMO) item. Recently, regulatory frameworks, such as the enactment of the Precision Breeding Law in the United Kingdom, the European Union’s New Genomic Techniques (NGT) legislation, and the repeal of the SECURE Rule in the United States, have further established guidelines permitting the use of genome-edited lines in agriculture similar to with conventionally bred crops, provided that these lines are free of transgenic elements. In Korea, researchers and policymakers are actively engaging in discussions to establish a preliminary review committee for GEd crops to align regulatory practices with international trade standards. Thus, this study aimed to evaluate two gene-edited rice lines for generational stability in terms of molecular characteristics, focusing on edited nucleotide sequences, gene expression, target phenotypes, the presence of transgene elements, and potential off-target effects across multiple generations. Additionally, several technical challenges in nucleotide editing tracing emerged during the evaluation process that warrant further attention. The findings presented in this study are expected to offer valuable insights for shaping the regulatory framework in Korea for CRISPR-based gene-edited crops.
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Gene editing technologies such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) systems have been developed to create targeted DNA mutagenesis in many crop plants. This report describes application of the TALEN system to generate
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With the continual development of genetically modified (GM) crops, it has become necessary to develop detailed and effective molecular characterization methods to select candidate events from a large pool of transformation events. Relative to traditional molecular analysis methods such as the polymerase chain reaction (PCR) and Southern blot hybridization, next generation sequencing (NGS) technology for whole-genome sequencing of complex crop genomes had proven comparatively useful for in-depth molecular characterization. In this study, four transformation events, including one in
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Beta-carotene, a defense chemical, is synthesized by the carotenoid biosynthesis pathway. In the present study, a transgenic soybean line, with a single copy insertion of
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A tissue-specific and developmentally expressed gene was isolated from Chinese cabbage (
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Confirmation of T-DNA inheritance and stability requires testing of several parameters such as phenotypic performance of transgenic lines, transgene integrity structure and expression stability for at least three consecutive generations, and Mendelian inheritance analysis, which are very important to transgenic breeding and commercialization of genetically modified crops (GMOs). In the present study, we measured nine agronomic and yield-related traits of Iksan526 and confirmed stable phenotypic performance comparing to the donor variety Dongjin over three years. Transgene integrity structure, flanking sequence, functional element copy numbers, absence of backbone sequence and transgene expression were all verified in Iksan526 over three consecutive generations, indicating a stable inheritance of the transgene. In addition, phenotypic and genotypic segregation were analyzed on F2 and F3 populations derived from a cross of Dongjin/Iksan526, and results complied with Mendel’s law of segregation based on single transgene locus. This study demonstrated detailed analyses and confirmation of transgene event Iksan526 which is crucial for commercialization of GMO.
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Resveratrol, a plant phenolic compound, has potential therapeutic benefits due to its antioxidant properties. This is substantiated by previous studies that show that resveratrol derived from rice grains is an effective treatment agent for metabolic syndrome. Here, we characterized the T-DNA sequence, inserted T-DNA structure, copy number, integrity of the transgene locus, resveratrol synthase gene expression and resveratrol contents in the grains of two resveratrol transgenic rice lines, Iksan515 and Iksan526. The T-DNA transformation vector contained two expression cassettes of the resveratrol synthase gene under the control of the ubiquitin promoter and the bar selection marker gene under the control of the CaMV35S promoter. Flanking sequence analysis indicated that the T-DNAs were inserted into intergenic regions of chromosome 4 for Iksan515 and chromosome 12 for Iksan526. Two T-DNAs connected in an inverted repeat structure at a single locus of the rice genome were identified by whole genome sequencing and Southern blot hybridization in both Iksan515 and Iksan526. No novel open reading frames (ORFs) around insertion sites, sequences encoding allergenic or toxic protein, or other unintended effects by T-DNA insertion were found in either case. In addition, resveratrol synthase gene expression in leaves and resveratrol detection in brown rice grains suggested the successful expression of the inserted foreign resveratrol synthase gene in two transgenic rice lines.
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Resveratrol is a stilbenoid and phytoalexin produced in response to stresses, such as wounding, and pathogen attacks by bacteria or fungi. Two resveratrol rice lines, Iksan515 and Iksan526, were used to examine resistance against Korean pathogen races for bacterial blight, leaf blast, and brown leaf spot. The screening test for bacterial blight demonstrated an increased susceptibility of both transgenic lines to K1 race, and a more susceptible Iksan515 to K2. Phenotypic evaluation for resistance to brown leaf spot also revealed the susceptibility of Iksan526 to the disease which did not significantly differ from the isogenic variety ‘Dongjin’, and a slightly more susceptible Iksan515 to the disease compared to check. When the lines were screened with three races (KJ301, KJ101 and KJ133) of leaf blasts in the field, both transgenic lines exhibited resistance but at the same level with ‘Dongjin’. Our overall findings suggest that foreign phytoalexin resveratrol production in transgenic rice lines transformed with
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