Abstract:
Background: Once a transgenic plant is developed, the selectable marker gene (SMG) becomes unnecessary in the
plant. In fact, the continued presence of the SMG in the transgenic plant may cause unexpected pleiotropic effects
as well as environmental or biosafety issues. Several methods for removal of SMGs that have been reported remain
inaccessible due to protection by patents, while development of new ones is expensive and cost prohibitive. Here,
we describe the development of a new vector for producing marker-free plants by simply adapting an ordinary binary
vector to the double right border (DRB) vector design using conventional cloning procedures.
Findings: We developed the DRB vector pMarkfree5.0 by placing the bar gene (representing genes of interest) between
two copies of T-DNA right border sequences. The β-glucuronidase (gus) and nptII genes (representing the selectable
marker gene) were cloned next followed by one copy of the left border sequence. When tested in a model species
(tobacco), this vector system enabled the generation of 55.6% kanamycin-resistant plants by Agrobacterium-mediated
transformation. The frequency of cotransformation of the nptII and bar transgenes using the vector was 66.7%. Using
the leaf bleach and Basta assays, we confirmed that the nptII and bar transgenes were coexpressed and segregated
independently in the transgenic plants. This enable separation of the transgenes in plants cotransformed using
pMarkfree5.0.
Conclusions: The results suggest that the DRB system developed here is a practical and effective approach for
separation of gene(s) of interest from a SMG and production of SMG-free plants. Therefore this system could be
instrumental in production of “clean” plants containing genes of agronomic importance.
