In anise (Pimpinella anisum, family Apiaceae), callus-like embryogenic cells (embryogenic callus) are induced by culturing hypocotyl explants in 2,4-dichlorophenoxyacetic acid (2,4-D)-containing medium, and somatic embryos are formed from embryogenic callus transferred into 2,4-D-free medium. Anise somatic embryos are also induced even if embryogenic callus is continually cultured in 2,4-D-containing medium without subculturing. In this study, we aimed to clarify the dynamics of 2,4-D during anise cell culture. After culturing anise callus in 2,4-D-containing medium, 2,4-D in the medium was analyzed by thin-layer chromatography. In the medium, 2,4-D was decreased during anise callus culture, and fully abolished after 5-day culture. On the other hand, no decrease in 2,4-D was observed in the other Apiaceae species (carrot, fennel, dill, parsley, and coriander). After 7-day culture of anise callus, the medium was collected following removal of the cultured cells and 2,4-D was added to the collected medium. After 10 days of incubation and shaking, 2,4-D was markedly decreased in the medium. However, when the collected medium was heat-treated at 100°C, 2,4-D was detected after 20 days of incubation. Therefore, anise callus has a specific 2,4-D degradation system, in which heat-inactivated secreted molecules may participate.
