An efficient marker-free vector for clean gene transfer into plants

Abstract

A marker-free vector, pBINMF, for clean gene transfer was constructed based on the binary vector pBINPLUS. Vector pBINMF, carrying only a multiple cloning site (MCS) between the left and the right T-DNA border, was suitable to directly generate marker-free transgenic plants (MFTPs) without any vector sequences inserted into plant genomes. Transformation efficiency of pBINMF was tested by transferring gusA reporter gene and nptII gene into tomato, respectively. The results from three experiments show that 4.4% of regenerated shoots were transgenic by polymerase chain reaction (PCR) test, and the phenotype expression percentage of PCR positive transformants was 57.4% as tested by beta-glucuronidase (GUS) assay. Moreover, 24 out of 27 phenotype positive transformants were homogeneous as confirmed by blue staining over whole leaves and roots in GUS staining. The vector might also be used in tobacco marker-free gene transfer as was confirmed by transient expression assay of gusA. The results indicate that pBINMF was an efficient and reliable vector to directly generate complete MFTPs in plant species, and might be especially suitable for vegetatively propagating species.

Key words: Marker-free vector, GUS assay, tomato, tobacco.