Development of a regeneration and genetic transformation protocol for tomato (solanum lycopersicum l.) cv. Rutgers

Tomato (Solanum lycopersicum) is important in genetic engineering because it is a model for fruit postharvest studies. A requirement for genetic transformation is a protocol to carry out the organogenesis of transformed explants. However, current protocols of tomato organogenesis are slow, expensive and time demanding. The objective of this study was to develop an easy organogenesis protocol for tomato cv. Rutgers. The hypothesis was that the organogenesis of tomato using hypocotyl in combination with an appropriate media and transformation method, reduces the regeneration time to obtain full developed transgenic plants. The experimental design included the evaluation of the efficiency of five different regeneration media and tested in two types of explant. Organogenesis response of callus, root and shoot tissues were measured in all treatments. Tomato explants were genetically transformed by Agrobacterium tumefaciens strain GV3101 infection and biolistic gun at helium pressure of 1200 psi (8.27 MPa), with 900 psi (6.20 MPa) shoot pressure as well as 6 and 9 cm of shooting distance using the vector pBI121. The optimal treatment was the utilization of 9 cm distance along with the other parameters. Integration of the binary vector was confirmed by PCR amplification of the npt-II gene and histochemical GUS staining. The highest percentage (41.6%) of tomato plants regeneration was found in the AGK-1 medium: MS 4.3 gL1 salt, 30 gL1sucrose, 0.5 mgmL1 IAA, 0.5 mg mL1 kinetin, 0.1 mgmL1thiamine and 8 gL1 gelrite, tested in hypocotyl explant. In this medium, shoots and roots were developed at the same time. We obtained a large percentage of complete tomato transgenic plants by direct organogenesis from hypocotyls in 10 weeks, which is a rather short time as compared with previous tomato organogenesis protocols. These results will be useful for genetic transformations experiments to obtain stable transgenic tomato lines