Novel antibiotics regeneration and genetic transformation with RsMYB1 gene of recalcitrant chrysanthemum cv. Shinma

This research was conducted to develop genetic transformation of the recalcitrant chrysanthemum cv. Shinma by application of appropriate antibiotics and selective agents. Clavamox had the least inhibitory effect on shoot regeneration compared to timentin, carbenicillin, and cefotaxime. Clavamox, at a concentration of 125mgL(-1), was found to be the most suitable for shoot regeneration and production of quality shoots, suppressing the growth of Agrobacterium in explants infected with strains GV3101 or C58C1 for 3 and 4weeks, respectively. The concentration of phosphinothricin (PPT) was found to be 1.0mgL(-1) for screening of putative transgenic shoots. Moreover, transgenic chrysanthemums were obtained by culturing explants co-cultivated with A. tumefaciens strain GV3101 harboring an anthocyanin regulatory gene RsMYB1 isolated from radish (Raphanus sativus), which was placed under the control of cauliflower mosaic virus promoter (CaMV) 35S and petal-specific promoter InMYB1 isolated from the morning glory (Ipomoea nil), on shoot regeneration medium supplemented with recommended concentration of antibiotic and selective agent. Flow cytometry analysis revealed that there was no variation in ploidy level between transgenic plants and donor plants (non-transformants). To our knowledge, this is the first report of the use of Clavamox and MYB transcription factor for genetic transformation of this chrysanthemum.