Open AccessApplication of protoplast technology to CRISPR/Cas9 mutagenesis: from single‐cell mutation detection to mutant plant regeneration
Author(s) -
Lin ChounSea,
Hsu ChenTran,
Yang LingHung,
Lee LanYing,
Fu JinYuan,
Cheng QiaoWei,
Wu FuHui,
Hsiao Han C.W.,
Zhang Yesheng,
Zhang Ru,
Chang WanJung,
Yu ChenTing,
Wang Wen,
Liao LiJen,
Gelvin Stanton B.,
Shih MingChe
Publication year - 2018
Publication title -
plant biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.525
H-Index - 115
eISSN - 1467-7652
pISSN - 1467-7644
DOI - 10.1111/pbi.12870
Subject(s) - biology , crispr , mutagenesis , protoplast , cas9 , mutant , genetics , nicotiana tabacum , dna , gene , insertional mutagenesis , phytoene desaturase , transfection , microbiology and biotechnology , biosynthesis
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 sg RNA s, 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 ( Nt PDS ) gene. Genotyping of albino regenerants indicated that all four Nt PDS alleles were mutated in amphidiploid tobacco, and no Cas9 DNA could be detected in most regenerated plants.