Differences in adventitious shoot regeneration capacity among Japanese chrysanthemum [Dendranthema grandiflorum (Ramat.) Kitamura] cultivars and the improved protocol for Agrobacterium-mediated genetic transformation

Stem segments of 23 chrysanthemum (Dendranthema grandiflorum (Ramat.) Kitamura) cultivars were cultivated on MS medium containing NAA 2.0 mg . liter(-1) and BA 0.2 mg liter(-1). Among which 11 cultivars had high adventitious shoot regeneration potentials. Three of these cultivars, 'Shuhonochikara', 'New Summer Yellow' and 'Yamabiko' which were selected for transformation experiments had their regeneration potential suppressed by kanamaycin at 7.5 similar to 10.0 mg . liter(-1). The stem segment explants were co-cultivated at 25 degrees C for 3 days with Agrobacterium tumefaciens strain LBA4404, containing pBI121 plasmid. After co-cultivation. the explants were placed on regeneration medium, containing 7.5 similar to 10.0 mg . liter(-1) kanamycin. The presence of the NPTII gene in the regenerated plants was confirmed by PCR-southern analysis. The transformation efficiency increased to 2.46% when the Agrobacterium suspension was diluted with a regeneration medium, containing 100 mu M acetosyringone.