Successful floral-dipping transformation of post-anthesis lisianthus (Eustoma grandiflorum) flowers

The adaptation of the Agrobacterium-mediated floral-dipping technique is limited, to date, to a small number of plants. In this paper, we present the efficient transformation of one of the leading plants in the cut flower industry, lisianthus (Eustoma grandiflorum). This method is approximately 18 months shorter than the known tissue culture-based transformation. The Excalibur Pink cultivar and two additional breeding lines, X-1042 and X-2541, were transformed using three different marker genes (benzyl alcohol acetyltransferase (BEAT) originating from Clarkia breweri, the feedback-insensitive bacterial gene AroG*, and the empty pART27 vector expressing a kanamycin-resistance cassette (nptII)). Genomic transformation was successful in all tested cases with transformation efficiency ranked from 0.2 to 2.9%, which is well in the range of results from Arabidopsis studies. Unlike Arabidopsis, in which floral-dipping transformation was efficient only at a pre-anthesis stage before ovary sealing, lisianthus flowers were transformed when dipping occurred 4 days pre-anthesis or 3-5 days post-anthesis with 1.5 and 3.7% efficiencies, respectively. Post-anthesis transformation occurred when the flower ovaries were sealed. Flower dipping of Excalibur Pink flowers with fluorescent Agrobacterium containing a GFP marker gene demonstrated Agrobacterium entrance into the sealed flower ovary through the open stigma and style tube. In this study, we demonstrated floral-dipping transformation of a commercial plant, lisianthus Excalibur Pink, occurring after sealing of the ovaries, probably via the stigma and wide open style tunnel.