Wounding by bombardment yields highly efficient Agrobacterium-mediated transformation of carnation (Dianthus caryophyllus L.)

Highly efficient Agrobacterium-mediated transformation of carnation (Dianth us caryophyllus L.) was obtained by first wounding stem explants via microp rojectile bombardment. When this was followed by cocultivation with disarme d Agrobacterium in the dark, the transformation frequency-based on transien t GUS expression-increased to over 10-fold that of explants wounded by othe r means and cocultivated under constant light. Two cycles of regeneration/s election on kanamycin were employed to generate stably transformed carnatio n plants and eliminate chimeras: first, plantlets were regenerated from ino culated stem explants and then leaves from these plantlets were used to gen erate transgenes in a second selection cycle of adventitious shoot regenera tion. Agrobacterium strain AGLO, carrying the binary vector pCGN7001 contai ning uidA and nptII genes, was used in the stable transformation experiment s. The combination of wounding via bombardment, cocultivation in the dark a nd two cycles of kanamycin selection yielded an overall transformation effi ciency of 1-2 transgenes per 10 stem explants for the three carnation varie ties analyzed. Histochemical and molecular analyses of marker genes in T-0 and T-1 generations confirmed the transgenic nature of the selected plants.