Agrobacterium-mediated transformation of tomato plants expressing defensin gene.

摘要

The aim of this study was to develop a protocol for transformation/regeneration of tomato plant (cv. Summer set) using a plant antifungal gene (defensin). Transformation was carried out using disarmed A. tumefaciens strain LBA4404 harbouring a binary vector pITB-AFP. Sequencing of plasmid DNA extracted from this strain indicated that it contains defesin gene (AFP), an antifungal protein-coding gene, under the control of a CaMV 35S promoter and Nopaline Synthase (NOS) terminator, hygromycin phosphotransferase (hpt) and beta -glucuronidase (GUS) genes, as selectable and marker genes, respectively. The factors that affect transformation/regeneration protocols were optimized in a series of experiments. Results indicated that exposure of cotyledonary explants to Agrobacterium inoculums of 0.8 O.D₆₀₀ for 30 min, selection on Hygromycin-containing medium (Hygromycin concentration of 25 mg L-1) and subsequent regeneration on MS medium supplemented with 2.5 mg L-1 BA as a cytokinin and 1.0 mg L-1 IAA as an auxin resulted in transformation efficiency of 7%. GUS expression was observed in transformed tomato shoots but never in the control plants. PCR amplification of DNA extracted from the transformed tissues demonstrated the generation of the expected amplicon, corresponding to AFP gene. This result strongly verifies the successful transformation of the tomato cultivar Summer set, an endeavour which is reported for the first time in Sudan. Moreover, this protocol paves the way for problem solving-applications encompassing other Sudanese crops of economic importance.