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

Jonathan M Matheka, Sylvester Anami, James Gethi

  • 1Biochemistry and Biotechnology Department, Kenyatta University, P, O, Box 43844, 00100 Nairobi, Kenya. runo.steve@ku.ac.ke.

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A new double right border (DRB) vector enables the production of marker-free transgenic plants. This system facilitates the separation of genes of interest from selectable marker genes (SMGs), addressing biosafety concerns.

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Area of Science:

  • Plant Biotechnology
  • Molecular Biology
  • Genetics

Background:

  • Selectable marker genes (SMGs) are essential for developing transgenic plants but become unnecessary post-development.
  • Continued presence of SMGs can lead to unintended effects and raise biosafety concerns.
  • Existing SMG removal methods are often patented or prohibitively expensive.

Purpose of the Study:

  • To develop a novel, cost-effective vector system for producing marker-free transgenic plants.
  • To create a practical method for separating genes of interest from SMGs.

Main Methods:

  • Adaptation of a standard binary vector into a double right border (DRB) design using conventional cloning.
  • Construction of the pMarkfree5.0 vector with the bar gene flanked by two right border sequences and SMGs (gus, nptII) followed by a left border.
  • Agrobacterium-mediated transformation of tobacco using the developed vector.

Main Results:

  • The DRB vector system successfully generated 55.6% kanamycin-resistant transgenic tobacco plants.
  • Co-transformation of the gene of interest (bar) and SMGs (nptII) occurred at a frequency of 66.7%.
  • Independent segregation of transgenes was confirmed, enabling the separation of the gene of interest from SMGs.

Conclusions:

  • The developed DRB system offers a practical and effective strategy for producing marker-free transgenic plants.
  • This approach facilitates the creation of "clean" plants with desirable agronomic traits.
  • The system addresses the need for accessible methods to remove unnecessary SMGs in plant biotechnology.