Abstract
We investigated the potential of a novel double T-DNA vector for generating marker-free transgenic plants. Co-transformation methods using a double T-DNA vector or using mixture of two Agrobacterium tumefaciens strains were compared, and showed that the double T-DNA vector method could produce marker-free transgenic tobacco (Nicotiana tabacum L.) plants more efficiently. A dual marker double T-DNA vector was then constructed by assembling the green fluorescent protein (GFP) gene mgfp5 and the neomycin phosphotransferase gene nptII into the same T-DNA. The frequency of co-transformants produced by this vector was 56.3%. Co-expression of mgfp5 and nptII was found in 28 out of 29 T1 lines, and segregation of the reporter β-glucuronidase gene, gusA, from mgfp5 to nptII was found in 12 out of 29 T1 lines. Therefore, GFP could be used as a vital marker to improve the transformation efficiency and to easily monitor the segregation of marker genes, thus facilitating screening of marker-free progeny.
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Acknowledgements
Financial support from the High Technology Research and Development (863) Program of China (2001AA212041, 2002AA212031), the State Key Fundamental Research and Development Plan of China (2001CB10901), the National Special Program for Research and Industrialization of Transgenic Plants (JY03-B-18-03) and the Rockefeller Foundation’s International Program on Rice Biotechnology (RF97042#14) is acknowledged. We are grateful to Jim Haseloff for kindly providing the mgfp5-ER construct.
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Communicated by W.A. Parrott
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Chen, S., Li, X., Liu, X. et al. Green fluorescent protein as a vital elimination marker to easily screen marker-free transgenic progeny derived from plants co-transformed with a double T-DNA binary vector system. Plant Cell Rep 23, 625–631 (2005). https://doi.org/10.1007/s00299-004-0853-4
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DOI: https://doi.org/10.1007/s00299-004-0853-4