Revised cocultivation conditions produce effective Agrobacterium-mediated genetic transformation of carnation ( Dianthus caryophyllus L.)

Abstract

The cocultivation medium is one of the most important factors for the successful genetic transformation of carnation node explants. High genetic transformation efficiency of node explants cocultured by Agrobacterium tumefaciens strain AGL0 with pKT3 plasmid (containing β-glucuronidase (GUS) and neomycin phosphotransferase (NPTII)) was achieved when the explants were cocultivated on filter paper soaked with water or water and acetosyringone (AS). Conversely, no genetic transformation of node explants occurred when they were cocultivated on a phytohormone and nutrient-rich medium, regardless of whether AS was used. Scanning electron microscopic (SEM) observations revealed many bacteria clustered on the explants cocultivated on filter paper soaked with water or water and AS. The bacterium clusters were compact and showed a polar orientation with few cellulose fibrils. On the other hand, explants cocultivated on phytohormone-rich MS medium had few clusters, which were not congested and produced numerous cellulose fibrils. Explants precultured for 3 and 5 days before inoculation showed higher transient and stable GUS expression than those precultured for 7 days. The second and third nodes produce more GUS-positive shoots than the first and fourth nodes. However, the transformation efficiency still varied, depending on the cultivar, i.e., cultivar Otome was more sensitive to genetic transformation than cultivars Killer, Master, Tanga and Laurella. Successful genetic transformation of harvested shoots was confirmed by PCR and Southern blot analysis. Transformed plants were true-to-type with high gene expression in a greenhouse.