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The tobacco β-1,3-glucanase gene (GLU), alfalfa defensin gene alfAFP, and their bivalent gene GLU-AFP were introduced into tomato cv. Micro-Tom via Agrobacterium-mediated method. Transformants were obtained and confirmed by GUS histochemical staining, PCR, and Southern blotting. Northern blotting analysis with GLU and APF probes revealed a variation in the expression among these transformants at transcription level. One to three copies of the transgene were, respectively, integrated into the tomato nuclear genome. Performance test of resistance to Ralstonia solanacearum with T1 generation transgenic tomato lines showed that the transgenic lines exhibited higher resistance to the infected pathogens than nontransgenic plants, and the resistance levels were related to expression levels of the transgene, showing a dose effect. The transgenic tomato harboring GLU – AFP cassette showed the highest disease resistance, which suggested that the alfAFP and glucanase genes have synergistical effects on the resistance to R. solanacearum. Some independent lines with high disease resistance, low variability, and stable expression of transgenes could be selected for further studies and molecular breeding.