Protoplast isolation and shoot regeneration from protoplast-derived calli of Chrysanthemum cv. White ND

Key message The regeneration of chrysanthemum protoplasts into whole plants is difficult due to the recalcitrant nature of these plants and because of their genotype-dependent response. This study is appropriate for protoplast isolation and callus formation of Chrysanthemum cv. White ND, and it also achieved regenerating chrysanthemum protoplasts-derived calli into whole plants. In this study, we sought to optimize the isolation of protoplasts from chrysanthemums by manipulating the mannitol and cellulase levels, the incubation period, and the purification method, followed by the conversion of the protoplasts into calli and shoots. A high protoplast yield was achieved using 0.5 M mannitol, 1.5% cellulase, and a 4 h incubation period. Cell wall regeneration was observed after 3 days, with the first cell division occurring approximately 4-5 days after culturing. The addition of sucrose to the culture media was more beneficial than glucose; in sucrose media the protoplasts grew more rapidly and successfully reached the colony and microcalli stage. The addition of activated charcoal to the culture improved colony and microcalli formation. Greater proliferation of microcalli was also achieved using solid Murashige & Skoog (MS) media supplemented with 1 mg l(-1) 6-Benzylaminopurine (BA) and 2 mg l(-1) Naphthaleneacetic acid (NAA). The calli produced shoots THE on media supplemented with 2 mg l(- 1) BA and 0.5 mg l(-1) NAA. These findings could facilitate further chrysanthemum protoplast-based research.