A Simple and Efficient Protocol for Plant Regeneration and Genetic Transformation of Tomato cv. Micro-Tom from Leaf Explants

摘要

A simplified protocol to obtain transgenic tomato plants was established. The effects of culture media composition and Agrobacterium concentration were evaluated. The highest shoot-forming capacity index (5.6) was observed when leaf explants were cultured for 6 weeks with 2 mg.L(-1) zeatin, 0.1 mg.L(-1) indoleacetic acid, and 300 mg.L(-1) timentin. Shoot elongation and root formation were performed in one step on growth regulator-free media. The highest percentage (82%) of fully developed plantlets was obtained when shoots were cultured for 4 weeks with 0.5x Murashige and Skoog (MS) media and 15 g.L(-1) sucrose. A 100% of plant survival rate was observed after 4 weeks of being transplanted to ex vitro conditions followed by fruit production (15 fruits/plant) after 2 more weeks. Transient expression of beta-glucuronidase was visualized in 100% of the leaf explants infected with Agrobacterium at an OD(600) = 0.5 and cocultured for 48 h with 2 mg.L(-1) benzylaminopurine, 0.1 mg.L(-1) naphthaleneacetic acid, and 100 mu M acetosyringone. Stable transformation was confirmed by histochemical glucuronidase assay and polymerase chain reaction (PCR) analysis with a total efficiency of 19.1%. The complete protocol, from shoot induction to fruit production of soil-adapted transgenic plants can be accomplished in only 4 months, and it seems to be very useful for both micropropagation and genetic transformation purposes.