Plant regeneration from mesophyll protoplasts ofDianthus superbus

Leaf mesophyll protoplasts ofDianthus superbus were cultured at a density of 5 × 10⁴ protoplasts/ml and divided at about 18% plating efficiency in MS liquid medium supplemented with 0.5 mg/L BAP, 2.0 mg/L NAA and 9% mannitol after 2 weeks. Protocolonies formed after 3 to 4 weeks of culture in the dark at 27°C. These colonies were transferred to continuous illumination (21.5 E m^–2 sec^–1) for 2 weeks where most of the colonies divided to form microcalli, about 2 mm in diameter. Subsequently, green microcalli were transferred to MS solidified medium with 2.0 mg/L 2,4-D that induced shoot-forming calli after 4 weeks. These calli were transferred onto N₆-2 medium containing 0.1 mg/L 2,4-D, 0.1 mg/L NAA, 2.0 mg/L kinetin and 2.0 g/L casein hydrolysate and were cultured under light. After 5 weeks the calli gave rise to multiple shoots (10 to 15 per callus). Upon transfer to MS medium containing 2.0 mg/L NAA, individual shoots were rooted in 4 weeks. The regenerants were successfully transplanted into potting soil.Abbreviations MS Murashige and Skoog - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - N₆ Chu basal salt mixture - MES 2-N-morpholinoethanesulfonic acid