Journal
PLANT BIOTECHNOLOGY JOURNAL
Volume 16, Issue 7, Pages 1295-1310Publisher
WILEY
DOI: 10.1111/pbi.12870
Keywords
protoplast isolation; CRISPR/Cas9; protoplast regeneration; single-cell analysis
Funding
- Agricultural Biotechnology Research Center, Academia Sinica of Taiwan
Ask authors/readers for more resources
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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available