Split GFP assay to show chloroplast targeting ofAgrobacteriumVirD2 protein

Regenerating fertile Arabidopsis thaliana plants from tissue culture cells with transformed plastid genomes is difficult, because of somaclonal variation in tissue culture cells. For nuclear gene transformation, tissue culture limitations were overcome in Arabidopsis by direct transformation of the female gametes using the floral dip protocol and identification of transgenic events in the seed progeny. During Agrobacterium transformation the VirD2 protein guides the T-complex, consisting of single stranded transferred-DNA (T-DNA) coated with VirE2 proteins, to the plant nucleus. To enable floral dip transformation of the plastid DNA, we retargeted VirD2 to chloroplasts. We show plastid targeting of VirD2 in a split GFP assay, where VirD2-GFP 11 complements GFP 1-10 in chloroplasts. Floral dip transformation of plastids will avoid tissue culture altogether, making plastid transformation readily available for the research community.