Somatic embryogenesis in the endemic black iris

Somatic embryogenesis was achieved from callus, cell suspension and protopl ast culture systems in the endemic black iris (Iris nigricans). Subculture of friable callus fragments on embryogenesis induction medium (EIM) contain ing 4.5 mu M 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 mu M kinetin, 4.5 mu M 1-naphthaleneacetic acid (NAA) and 300 mg l(-1) proline in the dark wa s necessary before transfer to regeneration medium (RM). Regeneration was s tudied by transferring friable callus fragments from EIM to RM containing ( 0.0, 4.5, 9.0, 13.5 mu M) of either 6-benzyladenine (BA), 2-isopentenyladen ine (2iP), zeatin or thidiazuron (TDZ) in combination with 0.49 mu M indole -3-butyric acid (IBA), 0.45 mu M 2,4-D. Maximum embryogenesis was obtained at 4.5 mu M BA while zeatin and TDZ were not effective and embryogenesis di d not occur with these treatments. Sucrose at 0.2 M was more effective for embryogenesis when compared to glucose or fructose. Growing cells in suspen sion culture on EIM containing 4.5 mu M 2,4-D in combination with 0.2 M suc rose for four weeks and transferring cells to RM (containing 4.5 mu M BA) g ave significant embryogenesis with maximum number of embryos (3568 embryos/ g cells). Using 4.5 mu M 2,4-D in protoplast culture was necessary for the best protoplast division and colony formation. In all experiments, embryos developed on RM were transferred to hormone-free medium (HFM) and 90% conve rted to rooted plantlets. Produced plantlets gave 95% survival ex vitro. Pl antlets developed to whole plants in the greenhouse and flowered.