The effects of wounding type, preculture, infection method and cocultivation temperature on the Agrobacterium-mediated gene transfer in tomatoes

Abstract

To reduce the complexity of Agrobacterium-mediated gene transfer in tomatoes, effects of various parameters, such as shoot regeneration medium (SRM), wounding type, infection method, preculture and cocultivation temperature, have been evaluated. Transformation frequency was analysed by the Agrobacterium strain LBA4404, harbouring a recombinant binary expression vector pIG121Hm-GS, which contained the glutathione synthetase gene under the control of the CaMV 35S promoter. The transformation frequency was highly dependent on the wound type of the explants, the infection method and the cocultivation temperature. On the other hand, a commonly used, preincubation method of the explants, on the preculture medium, did not show any significant improvement regarding transformation frequency. Optimal transformation frequency was observed when fresh perforated cotyledonary explants were directly infected with a bacterial solution A, followed by cocultivation at 24 degrees C in a coculture medium for 2 days and subsequent shoot regeneration on a selective SRM1. The presence of transgene, in putative transgenic plants, was confirmed by polymerase chain reaction (PCR) and Southern blot analyses. By using the most effective treatment from each category, an average of 20.7% kanamycin-resistant and PCR-positive shoots were recovered from the three tomato cultivars examined. The optimisation of these parameters may offer a simple, consistent, efficient and much less laborious protocol for tomato transformation.