Application of protoplast technology to CRISPR/Cas9 mutagenesis: from single‐cell mutation detection to mutant plant regeneration. Issue 7 (10th January 2018)

• Record Type: Journal Article
• Authors: Lin, Choun‐Sea; Hsu, Chen‐Tran; Yang, Ling‐Hung; Lee, Lan‐Ying; Fu, Jin‐Yuan; Cheng, Qiao‐Wei; Wu, Fu‐Hui; Hsiao, Han C.‐W.; Zhang, Yesheng; Zhang, Ru; Chang, Wan‐Jung; Yu, Chen‐Ting; Wang, Wen; Liao, Li‐Jen; Gelvin, Stanton B.; Shih, Ming‐Che
• Is Part Of: Plant biotechnology journal. Volume 16:Issue 7(2018)
• Page Start: 1295
• Page End: 1310
• Publication Date: 2018-01-10
• Subjects: protoplast isolation -- CRISPR/Cas9 -- protoplast regeneration -- single‐cell analysis; Plant biotechnology -- Periodicals; Plant genetic engineering -- Periodicals; 630.272
• DOI: 10.1111/pbi.12870
• Languages: English
• ISSNs: 1467-7644

Abstract

Summary: 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.