Appropriate choice of antibiotics for plant regeneration and optimization of selective agents to be used in genetic transformation of chrysanthemum

Plant genetic transformation requires appropriate choice of antibiotics for regeneration of transformed tissues and elimination of Agrobacterium, and minimal concentration of selective agents for the selection of putative transformants during transformation. This study was conducted to examine the effects of three antibiotics (carbenicillin, cefotaxime, and Clavamox) on in vitro plant regeneration from leaf segments of Chrysanthemum morifolium (Ramat.) ‘Vivid Scarlet’, and percentages of shoot induction and number of shoots per explant were recorded after 5 weeks of culture. Carbenicillin and Clavamox had less inhibitory effects on number of shoots per explant than cefotaxime, but superior plant growth (i.e. number of leaves, number of roots, plant height, and fresh weight) was observed in shoots treated with 125 mgL Clavamox. Analysis of stomata size indicated that there was no variation in ploidy level between mother plants (controls) grown in the greenhouse and plants regenerated in vitro with 125 mgL Clavamox. These findings suggest that Clavamox can effectively replace carbenicillin or cefotaxime in Agrobacterium-mediated genetic transformation studies of ‘Vivid Scarlet’. In addition, screening of the selective agents revealed optimal concentrations of kanamycin 30 mgL and phosphinothricin 0.4 mgL, respectively, for the selection of putative transgenic ‘Vivid Scarlet’ plants.