Secondary somatic embryogenesis in Chrysanthemum morifolium (Ramat.) cv. Borami

This is the first report describing culture conditions to induce secondary embryogenesis in Chrysanthemum morifolium 'Borami'. Somatic embryogenesis was induced from in vivo-grown leaf explants incubated on 1.0× Murashige and Skoog (MS) medium supplemented with a combination of 1.0 mg l^-1 Kinetin (Kn) and 3.0 mg l^-1 2,4-dichlorophenoxyacetic acid (2,4-D), which yielded the highest mean number of embryos (16.3) per explant after 5 weeks of culture. We evaluated the effects of MS basal medium, as well as various concentrations of sucrose and timentin on the proliferation of secondary somatic embryos. MS medium (1.0 ×) was observed to be more effective at promoting the proliferation of somatic embryos than 0.5 × MS. In addition, timentin was more efficient at inducing secondary embryogenesis than sucrose. Whole plantlets were obtained by culturing the secondary embryos on hormone-free, 1.0× MS medium. Plantlets were sub-cultured on 1.0× MS medium containing a combination of 0.1 mg l^-1 6-benzyladenine (BA) and 0.3 mg l^-1 α-naphthaleneacetic acid (NAA) and were successfully acclimatised in a greenhouse, with a survival rate of 90%. Timentin can be used for secondary somatic embryogenesis and the elimination of Agrobacterium tumefaciens following genetic transformation. Thus, the protocol described in this study should facilitate large-scale clonal propagation and genetic transformation of this cultivar of chrysanthemum.