A non-antibiotic marker for amplification of plant transformation vectors in E-coli

Concern over perceived risks from the presence of antibiotic resistance gen es in transgenic plants is leading to selection strategies which do not use antibiotic resistance genes as selectable markers. The concern regarding t he presence of antibiotic resistance genes has focused particularly on the bla gene for ampicillin resistance, which is used as a bacterial marker whe n pUC-based plasmids are amplified prior to microprojectile bombardment. Th e rd operon, which permits the use of ribitol as the sole carbon source, is present in Escherichia coli strain C but absent in all the K-12 laboratory strains commonly used for molecular biology. A ClaI fragment containing th e kinase, dehydrogenase and transporter components of the rd operon was iso lated from strain C and subsequently used to replace bla from two cloning v ectors, pBluescript and pMECA, to create pBluescript-R and pMECA-R. E. coli K-12 strain DH10B transformed with either plasmid acquired the ability to grow on ribitol as the sole carbon source, as long as pMECA-R was present w ithin the bacterial cell. Plasmid yields were evaluated and found to be com parable to those obtained from bacteria growing on LB medium supplemented w ith ampicillin. Hence, the use of genes from the rd operon might be an acce ptable alternative to the use of bla.