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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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*2018/10/08 10:30 AM
Dr. Bernhard Grimm (Dean of the Life Sciences Faculty, Professor (C4) and Head of Department of Plant Physiology at the Institute of Biology, Humboldt-Universität Berlin, Germany)
Green or red? Synthesis of chlorophyll and heme - Multiple posttranslational control of glutamyl-tRNA reductase, the rate limiting enzyme of tetrapyrrole biosynthesis
Auditorium A134, Agricultural Technology Building, Agricultural Biotechnology Research Center

*2018/10/29 10:30 AM
Dr. Ling Li (Assistant Professor, Department of Biological Sciences, Mississippi State University, USA)
A molecular tool to increase protein content and broad disease resistance in crops
Auditorium A134, Agricultural Technology Building, Agricultural Biotechnology Research Center

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