Production of chrysanthemum periclinal chimeras through shoot regeneration from leaf explants

Periclinal chimeras play important roles in vegetatively propagated plants such as chrysanthemum (Chrysanthemum morifolium). For example, periclinal chimerism causes flower color variation in chrysanthemums. In this study, a method for periclinal chimera production in chrysanthemum was examined. A wild-type plant of chrysanthemum 'Taihei' and its transgenic plant carrying a yellowish-green fluorescent protein gene from the marine plankton Chiridius poppei (CpYGFP) were used as plant materials. The cut faces of the leaf explants of both materials were partially attached and then were detached for further culture. Mosaic calli consisted of transgenic and wild-type cells formed on the detached faces of the explants. We examined 996 regenerated shoots from 4,120 explants and found only a single chimeric shoot that appeared to show mericlinal chimerism. Repeated axillary bud elongation from the nodes of the mericlinal chimera produced one L1-fluorescent and one L3-fluorescent chimeric plant. The L1 chimera showed fluorescence in the epidermal cells and trichomes of leaf and stem. The L3 chimera showed fluorescence in the cells of the central parts of stem and leaf, as well as in the whole root tissues. In summary, we obtained chrysanthemum periclinal chimeras through regeneration from leaf explants using the fluorescent protein transgene as a selection marker.