S04 - Session P1 - New Tools I - Optimization of an efficient protocol for protoplast isolation, shoot regeneration, and transient transfection in Petunia hybrida cv. Mirage Rose

Authors: HyunHee Kang *, Suleimon Oluwaseun Adedeji, Aung Htay Naing, Chang Kil Kim

Editing of undesired genes using CRISPR/Cas9 ribonucleoprotein (RNP) complex system has been proven as a powerful tool in plant biotechnology. This system needs a success of shoot regeneration from protoplasts and delivery of the RNP complex to the protoplast. In this study, we investigated the factors involved in protoplast isolation and shoot regeneration in Petunia hybrida cv. Mirage Rose. In addition, we established an efficient protocol that supports the delivery of the RNP complex to the protoplast using green fluorescent protein (GFP). The following conditions were found to be most optimal for protoplast yield and viability: 0.6 M mannitol, 2.0% cellulase, and 6 h digestion time. A plating density of 10 × 10 4 protoplasts/mL under osmoticum condition (0.58 M mannitol) showed high microcolony viability in liquid culture. The Kao and Michayluk medium found to be appropriate for callus proliferation from micro calli under a 16-h light photoperiod. Calli cultured in Murashige and Skoog medium containing 1.0 mg/L 6-benzylaminopurine and 0.2 mg/L 3-indole butyric acid showed the highest shoot regeneration frequency and number of shoots obtained per explant. The transient transfection was investigated by optimizing different concentration of polyethylene glycol (PEG), protoplast density, plasmid DNA concentration, and transfection duration, respectively. We found that a high transfection efficiency was obtained when the protoplasts were incubated in the solution (40% PEG-CaCl2, MMg with 0.4M Mannitol, 2.5 x 10 5 protoplast/ml, 10 μg of plasmid DNA, 15min of transfection duration). We expect that this optimal protocol will provide advantageous to the editing of the genes in this petunia using CRISPR/Cas9 RNP system