Preparation and application of Dahlia protoplast

Abstract Dahlia (Dahlia sp.), a species of the Asteraceae family, is widely cultivated in China and exhibits seasonal blooming. The main challenges associated with this species are low cross-breeding efficiency and slow breeding. In this study, dahlia leaves and petals were employed as test materials to ascertain the optimal conditions for protoplast isolation, to refine the critical factors for transient transformation, and to develop a system for the isolation, purification and utilisation of dahlia protoplasts. The best procedure for isolation of dahlia leaves protoplasts was 4°C dark pretreatment for 12 h + 1.0% cellulase + 0.5% macerozyme + 0.4% pectinase + enzyme digestion time for 4 h + 0.8 mol/L mannitol, with a maximum yield of 6.13 × 106 protoplasts/mL and a maximum viability of 89.23%; and the best procedure for dahlia petal protoplasts was 1.0% cellulase + 0.5% macerozyme + 0.4% pectinase + enzyme digestion time 10 h + 1.0 mol/L mannitol, with a maximum yield of 5.46 × 106 protoplasts/mL and a maximum viability of 88.83%. The pGBin-EGFP vector was used to assess transient transformation rates in leaves and petals protoplasts. The rates exhibited considerable variation across the samples, with values ranging from 32.57–60.67%. The optimal conditions for gene transfer in dahlia protoplast were identified as 50 ng/µL plasmid, 20% PEG, and a 20-minute transformation time.