Somatic embryogenesis in the endemic black iris

Abstract

Somatic embryogenesis was achieved from callus, cell suspension and protoplast culture systems in the endemic black iris (Iris nigricans). Subculture of friable callus fragments on embryogenesis induction medium (EIM) containing 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 μM kinetin, 4.5 μM 1-naphthaleneacetic acid (NAA) and 300 mg l-1 proline in the dark was necessary before transfer to regeneration medium (RM). Regeneration was studied by transferring friable callus fragments from EIM to RM containing (0.0, 4.5, 9.0, 13.5 μM) of either 6-benzyladenine (BA), 2-isopentenyladenine (2iP), zeatin or thidiazuron (TDZ) in combination with 0.49 μM indole-3-butyric acid (IBA), 0.45 μM 2,4-D. Maximum embryogenesis was obtained at 4.5 μM BA while zeatin and TDZ were not effective and embryogenesis did not occur with these treatments. Sucrose at 0.2 M was more effective for embryogenesis when compared to glucose or fructose. Growing cells in suspension culture on EIM containing 4.5 μM 2,4-D in combination with 0.2 M sucrose for four weeks and transferring cells to RM (containing 4.5 μM BA) gave significant embryogenesis with maximum number of embryos (3568 embryos/g cells). Using 4.5 μM 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% converted to rooted plantlets. Produced plantlets gave 95% survival ex vitro. Plantlets developed to whole plants in the greenhouse and flowered.