A high-frequency cyclic secondary somatic embryogenesis system for Cyclamen persicum Mill

Abstract

In this study, a high frequency cyclic secondary somatic embryogenesis system was established for Cyclamen persicum. Moreover, the influence of primary somatic embryos (PSEs) at different stages of development, number of passages, abscisic acid (ABA), and sucrose concentrations on secondary somatic embryogenesis were investigated. Primary somatic embryos, at different stages of development, were incubated on an induction medium consisting of half-strength Murashige and Skoog (MS) (Physiol Plant 15:473–497, 1962), 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.2 mg/l 6-benzyladenine (BA). These initial PSE cultures along with calli were transferred to a plant growth regulator (PGR)-free medium to promote secondary somatic embryo (SSE) development. Embryogenic calli were induced at a frequency >90% from all PSE cultures. Both morphology and size of PSEs influenced embryogenic competence. Large-sized globular embryos (GEs) yielded the highest number of SSEs followed by small- and large-sized torpedo-stage embryos (TEs). Passages of induced calli obtained from large-sized GEs had no effects on frequency of secondary embryogenesis; however, they significantly influenced SSE production. Calli from the first passage exhibited the highest competence for secondary embryogenesis, 100% frequency of SSEs, producing 3,306 and 1,296 SSEs per gram fresh weight callus and embryo, respectively. Incubating embryogenic calli derived from large-sized primary GEs in the first passage on a medium containing 30 g/l sucrose, and supplemented with either 0.5 or 1.0 mg/l abscisic acid (ABA) increased the frequency of TEs, consequently enhancing normal embryo development of SSEs. Transfer of such embryogenic calli to a PGR-free medium containing 60 g/l sucrose was optimal for SSE induction, producing 3,204 SSEs per gram callus and 8% TEs.