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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.
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*2018/11/19 10:30 AM
Dr. Chiao-Chun Liao (Dr.Shu-Mei Liang’s Lab)
The role of autophagy on the regulation of cells migration and ovarian cancer progression (ABRC Young Scientist Forum)
Auditorium A134, Agricultural Technology Building, Agricultural Biotechnology Research Center

*2018/12/03 10:30 AM
Dr. Choun-Sea Lin (Research Specialist, Agricultural Biotechnology Research Center, Academia Sinica, Taiwan)
2015-2018植物技術核心實驗室服務及研究進度報告
Auditorium A134, Agricultural Technology Building, Agricultural Biotechnology Research Center

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