Different expression of uidA gene in transformant tobacco leaves

Abstract

Agrobacterium-mediated transformation of tobacco leaf disks with two Agrobacterium tumefaciens strains, LBA4404 and EHA105, and four plasmids, pCAMBIA1201, pCAMBIA1301, pCAMBIA2301 and pCAMBIA3201, was used for introduction of an uidA reporter gene and corresponding plant selection genes (hpt, npt or bar gene) into tobacco (Nicotiana tabacum L. cv. Havana 38). Expression of the uidA gene was evaluated by histochemical analysis of ?-glucuronidase (GUS) activity in transformed tobacco plants that successfully grew on appropriate selection media. GUS staining also revealed differences in both intensity and pattern of blue coloration in plants transformed with the same plasmid and bacteria strain. Molecular analysis of blue coloured plants by the PCR method confirmed the integration of the reporter gene and corresponding selection genes. Moreover, PCR revealed the incorporation of reporter and selection genes into some plants that grew on selection media, but exhibited no uidA gene expression. Lack of uidA gene expression could be due to a random incorporation of foreign DNA into transcription inactive parts of chromosomes or due to a phenomenon called gene silencing, which appears to be a fairly frequent problem in establishing successful transformation protocols.