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Application of protoplast technology to CRISPR/Cas9 mutagenesis: from single‐cell mutation detection to mutant plant regeneration |
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| Authors: | Choun‐Sea Lin Chen‐Tran Hsu Ling‐Hung Yang Lan‐Ying Lee Jin‐Yuan Fu Qiao‐Wei Cheng Fu‐Hui Wu Han C.‐W. Hsiao Yesheng Zhang Ru Zhang Wan‐Jung Chang Chen‐Ting Yu Wen Wang Li‐Jen Liao Stanton B. Gelvin Ming‐Che Shih |
| Affiliation: | 1. Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan;2. Department of Biological Sciences, Purdue University, West Lafayette, IN, USA;3. Department of Bioinformatics and Medical Engineering, Asia University, Taichung City, Taiwan;4. State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China;5. Institute of Life Science, National Kaohsiung Normal University, Kaohsiung, Taiwan |
| Abstract: | 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. |
| Keywords: | protoplast isolation CRISPR/Cas9 protoplast regeneration single‐cell analysis |