Choun-Sea Lin*,Chen-Tran Hsu, Ling-Hung Yang, Lan-Ying Lee, Jin-Yuan Fu, Qiao-Wei Cheng, Fu-Hui Wu, Han Chen-Wei Hsiao, Yesheng Zhang, Ru Zhang, Wan-Jung Chang, Chen-Ting Yu, Wen Wang, Li-Jen Liao, Stanton B. Gelvin*,Ming-Che Shih*. (2018) Application of protoplast technology to CRISPR/Cas9 mutagenesis: From single cell mutation detection to mutant plant regeneration. Plant Biotechnol J. 2017 Dec 12. doi: 10.1111/pbi.12870
Plant protoplasts are useful for assessing the efficiency of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR Associated Protein 9 (Cas9) mutagenesis. We improved the process of protoplast isolation and transfection of several plant species. We also developed a method to isolate and regenerate single mutagenized Nicotianna tabacum protoplasts into mature plants. Following transfection of protoplasts with constructs encoding Cas9 and sgRNAs, target gene DNA could be amplified for further analysis to determine mutagenesis efficiency. We investigated N. tabacum protoplasts and derived regenerated plants for targeted mutagenesis of the phytoene desaturase (NtPDS) gene. Genotyping of albino regenerants indicated that all four NtPDS alleles were mutated in amphidiploid tobacco, and no Cas9 DNA could be detected in most regenerated plants.
2018/05/01 10:00 AM
Dr. K. J. Senthil Kumar (Post-Doctoral Fellow, Department of Forestry, National Chung Hsing University, Taiwan)
Auditorium A134, Agricultural Technology Building, Agricultural Biotechnology Research Center 2018/05/07 10:30 AM
Dr. Sophie A. Lelièvre (Professor of Basic Medical Sciences with courtesy appointment in Nutrition Science; coleader, Drug Discovery & Molecular Sensing NCI-designated Purdue Center for Cancer Research; Scientific Director, 3D Cell Culture Core (3D3C) Facility, Birck Nanotechnology Center, Discovery Park, Purdue University, USA)
Environmental Epigenetics for the Primary Prevention of Cancer
Auditorium A134, Agricultural Technology Building, Agricultural Biotechnology Research Center