Plant regeneration systems from mesophyll- and cell suspension-derived protoplasts were established in Dianthus acicularis (2n=90), a species with resistance to Burkholderia caryophylli (Pseudomonas caryophylli). Protoplasts were isolated from both leaves of in vitro-grown plants and cell suspension cultures established from the calluses originated from leaves of in vitro-grown plants. Protoplasts isolated from both sources showed about the same response to the type and concentration of cytokinins, and gave the highest frequencies of cell division and colony formation in 0.1% (w/v) Gelrite^®-solidified Murashige and Skoog (MS) medium supplemented with 0.5 M glucose, 1.0 mg l⁻¹ (4.53 μM) 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.5 mg l⁻¹ (2.28 μM) zeatin. Numerous plantlets were regenerated after transfer of the colonies to 0.8% (w/v) agar-solidified half-strength MS medium supplemented with 0.5 mg l⁻¹ (2.28 μM) zeatin. Most plantlets exhibited normal phenotypes, but some showed variations, such as abnormal morphology with reduced chromosome number, precocious flowering, and vigorous growth with a tetraploid chromosome number. Possible mechanisms responsible for the observed somaclonal variation are discussed.