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Callus tissue induction and analysis of GUS reporter gene expression in tomato (Solanum lycopersicum L.) transformed with Agrobacterium tumefaciens

Abstract:

Tomato is one of the most important crops of Solanaceae familyand one of the largest products consumed in Ecuador. The productivity of this important crop has been affected due to the involvement of some abiotic and biotic factors. For this crop the application of genetic improvement techniquesinvolves as a first step the production of dedifferentiated in vitrotissue culture. The objective of this research is to generate callus formation from tomato leaves, and a protocol for the analysis of GUSgene expression by histochemical tests for further investigation regarding the genetic transformation with various applications for this important crop. The sterilization procedure consisted in using 15% w/v detergent and sodium hypochlorite (0.5% v/v and 0.8% v/v) and for the callus induction the explants were inoculated on MS medium with 2,4-D (0.25 to 3 mgL<sup>-1</sup>) and KIN (0.25 and 0.5 mgL<sup>-1</sup>). The transfer of the reporter gene was performed through tomato callus co-culture with Agrobacterium tumefaciens(O.D<inf>600nm</inf> 0.2 to 1) for 10 to 20 min, the plasmid used for transformation being pCAMBIA1305.2. Temporal expression in transgeniccells after 3 and 7 days of co-culture showed significant differences between the treatments applied. Treatment with O.D<inf>600nm</inf> 0.8, inoculation for 20 min and 5 days co-cultured with A. tumefaciens provided necessary conditions for T-DNA insertion into genome cells, being the most promising protocol for further important genetic transformation issues of this species.