High Efficient Procedure of Agrobacterium-mediated Transformation of Cultivated Tomato (Lycopersicon esculentum)

Abstract

Tomato is one of the most important crop species where the introduction of foreign genes is expected to have a major impact on agriculture. Several transformation methods exist that rely on the cocultivation of various tissue or organ explants. However, tomato is still considered more difficult to transform than species such as Petunia hybrida and Nicotiana tabacum and can show widely varying success rates. Using cotyledonous explants, we propose a highly efficient procedure of Agrobacterium-mediated transformation and regeneration of an agricultural cultivated tomato (L. esculentum cv. Summerset). Results showed that up to 90% of the cotyledons generated callus within 3 weeks (1 to 5 calli/cotyledon) and 50% of them regenerated shoots in another 3 weeks. Finally, it resulted in 50 to 100 independent transgenic plants per 100 inoculated explants within 10 weeks. These results are at least 40% more efficient than those of already published protocols. Moreover, up to 95% of the regenerated plants that form vigorous de novo roots under the antibiotic selection tested positive for the GUS assay. Screening by PCR for the presence of the T-DNA genes gave the predicted DNA fragment bands. This high efficiency procedure was mainly achieved by 1) an adequate optimization of the hormone composition and concentration of the successive culture media; 2) the fresh explant wounding before the Agrobacterium infection (important for optimal cell transformations); 3) the explant position, inside down for callus induction and coculture period, and upside down for the selection and organogenesis period (important for antibiotic selection).