Optimization of protocol for efficient protoplast isolation and transient gene expression in carnation

The optimal protocols for protoplast isolation and transfection in carnation cv. Scarlet were developed by optimizing the factors involved in protoplast isolation and transfection processes. The best protoplast yield and viability were achieved using 0.5 M mannitol, enzyme concentrations of 1.0% cellulase and 0.1% macerozyme, and digestion time of 8 h. In contrast, the best transfection efficiency was obtained using protoplast density of 2.5 × 10⁵, polyethylene glycol (PEG) concentration of 20%, and transfection time of 15 min. The protocols could produce reasonable protoplast yield and transfection efficiency in other carnations. The transient expression of enhanced green fluorescent protein (eGFP) in the nucleus of the transfected protoplasts of all carnations was confirmed using PCR analysis. To our knowledge, this is the first report on transient gene expression in carnation protoplasts. This transient gene expression system could be valuable for genome editing of unwanted genes in carnations using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 technology.