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Open Access Highly Accessed Technical Note

A new double right border binary vector for producing marker-free transgenic plants

Jonathan M Matheka1, Sylvester Anami2, James Gethi3, Rasha A Omer4, Amos Alakonya2, Jesse Machuka1 and Steven Runo1*

Author Affiliations

1 Biochemistry and Biotechnology Department, Kenyatta University, P. O. Box 43844, 00100 Nairobi, Kenya

2 Institute for Biotechnology Research, Jomo Kenyatta University of Agriculture and Technology, P.O. Box 62000–00100, Nairobi, Kenya

3 Kenya Agricultural Research Institute, P.O. Box 340–90100, Machakos, Kenya

4 Biosafety and Biotechnology Research Center, Agricultural Research Corporation, P.O. Box 126, Wad Medani, Sudan

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BMC Research Notes 2013, 6:448  doi:10.1186/1756-0500-6-448

Published: 8 November 2013

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.

Keywords:
Cotransformation; Double right border; Selectable marker gene free; Removal of SMG