Floral and apical meristems as a target for Agrobacterium-mediated transformation of Arabidopsis thaliana

Abstract The majority of transformation protocols for Arabidopsis thaliana are based on Agrobacterium -mediated transformation of female reproductive cells in vivo , or somatic cells of leaf, stem, hypocotyl, or root explants in vitro . Here, Agrobacterium -mediated transformation systems using in vitro meristematic floral buds or apical meristems from precultured seedlings are reported. Arabidopsis floral cultures were established from inflorescences of seed-derived plants grown in vitro . On cytokinin-containing medium, chopped inflorescence segments quickly produced fast-growing cultures consisting mainly of real de novo inflorescences. Cytokinin-containing medium was also used for 1-wk preculture of seedlings for induction of multiple meristems from shoot apical buds. NPTII and GUS were used as selectable and visual markers for transformation of in vitro floral cultures, and aadA and RUBY were used for transformation of precultured seedlings. Transformation frequency for floral culture was about 15% (calculated as the number of independent transformants per initial number of explants). Transformation frequency for precultured seedlings using RUBY was roughly 12% (calculated as the number of RUBY positive plants per number of seedlings inoculated). Molecular and phenotypic analysis of T 1 progeny confirmed that the transgenes were stably integrated and inherited in progeny.