H₂O₂-Based Method for Rapid Detection of Transgene-Free Rice Plants from Segregating CRISPR/Cas9 Genome-Edited Progenies

• Main Authors: Tsung-Meng Wu, Jian-Zhi Huang, Hui-Min Oung, Yi-Ting Hsu, Yu-Chang Tsai, Chwan-Yang Hong
• Format: Article
• Language: English
• Published: MDPI AG 2019-08-01
• Series: International Journal of Molecular Sciences
• Subjects: DAB (3,3′-diaminobenzidine); H₂O₂; hygromycin; hygromycin phosphotransferase; rice; CRISPR; reactive oxygen species
• Online Access: https://www.mdpi.com/1422-0067/20/16/3885

Genome-editing techniques such as CRISPR/Cas9 have been widely used in crop functional genomics and improvement. To efficiently deliver the guide RNA and Cas9, most studies still rely on <i>Agrobacterium</i>-mediated transformation, which involves a selection marker gene. However, several limiting factors may impede the efficiency of screening transgene-free genome-edited plants, including the time needed to produce each life cycle, the response to selection reagents, and the labor costs of PCR-based genotyping. To overcome these disadvantages, we developed a simple and high-throughput method based on visual detection of antibiotics-derived H₂O₂ to verify transgene-free genome-edited plants. In transgenic rice containing <i>hygromycin phosphotransferase</i> (<i>HPT</i>), H₂O₂ content did not change in the presence of hygromycin B (HyB). In contrast, in transgenic-free rice plants with 10-h HyB treatment, levels of H₂O₂ and malondialdehyde, indicators of oxidative stress, were elevated. Detection of H₂O₂ by 3,3&#8242;-diaminobenzidine (DAB) staining suggested that H₂O₂ could be a marker to efficiently distinguish transgenic and non-transgenic plants. Analysis of 24 segregating progenies of an <i>HPT</i>-containing rice plant by RT-PCR and DAB staining verified that DAB staining is a feasible method for detecting transformants and non-transformants. Transgene-free genome-edited plants were faithfully validated by both PCR and the H₂O₂-based method. Moreover, HyB induced overproduction of H₂O₂ in leaves of <i>Arabidopsis</i>, maize, tobacco, and tomato, which suggests the potential application of the DAB method for detecting transgenic events containing <i>HPT</i> in a wide range of plant species. Thus, visual detection of DAB provides a simple, cheap, and reliable way to efficiently identify transgene-free genome-edited and <i>HPT</i>-containing transgenic rice.