title: A new double right border binary vector for producing marker-free transgenic plants.
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.
  
  
  RESULTS
  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.
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Matheka
authors.forename: Sylvester
authors.initials: S
authors.lastname: Anami
authors.forename: James
authors.initials: J
authors.lastname: Gethi
authors.forename: Rasha A
authors.initials: RA
authors.lastname: Omer
authors.forename: Amos
authors.initials: A
authors.lastname: Alakonya
authors.forename: Jesse
authors.initials: J
authors.lastname: Machuka
authors.affiliation: Biochemistry and Biotechnology Department, Kenyatta University, P, O, Box 43844, 00100 Nairobi, Kenya. runo.steve@ku.ac.ke.
authors.forename: Steven
authors.initials: S
authors.lastname: Runo
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics; D001681:Biological Assay; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics*; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D005819:Genetic Markers; D005822:Genetic Vectors / Q000737:chemistry*; D005966:Glucuronidase / Q000235:genetics; D030821:Plants, Genetically Modified; D014026:Nicotiana / Q000235:genetics*; D014170:Transformation, Genetic; D019076:Transgenes
chemical_list: D004269:DNA, Bacterial; D005819:Genetic Markers; C036483:T-DNA; D005966:Glucuronidase