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Effective selection and regeneration of transgenic sugarcane plants using positive selection system

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Abstract

We compared the transformation frequency of a positive selection system, which employs an Escherichia coli-derived pmi (phosphomannose isomerase) gene as the selectable marker and mannose as the selective agent, to commonly used negative selection systems of bar (phosphinothricin acetyltransferase) /phosphinothricin (PPT), npt II (neomycin phosphotransferase) /G418 (geneticin), and hpt (hygromycin phosphotransferase) /Hygromycin. Transgenic sugarcane plants were obtained following biolistic transformation of callus with four different selectable marker genes. Under different selective agents, transformed callus was selectively proliferated on MS medium containing 13.57 μM of 2,4-D, regenerated on MS medium with 4.44 μM of BA, 9.29 μM of KT, and 0.89 μM of NAA, and rooted on half of MS medium with 13.38 μM of NAA and 0.44 μM of 6-BA. The results demonstrated that the transformation frequency of 4.2% scored in the pmi/mannose positive system was significantly higher than those in the negative selection systems (0.47% for bar/PPT, 1.38% for npt II/G418, and 0.63% for hpt/Hygromycin, respectively). Transgenic sugarcane plants using the pmi/mannose system were confirmed by polymerase chain reaction (PCR) analysis and chlorophenol red (CPR) assay. The CPR assay was found to efficiently and qualitatively detect the transgenic sugarcane from the nontransformed plants because medium color changes in the CPR assay were highly responsive to pmi gene expression and enzyme activity. Thus, a pmi/mannose positive selection system for sugarcane transformation was developed to obviate the use of negative selection systems based on herbicides and antibiotics.

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Acknowledgments

The authors are grateful to Dr. Jack C. Comstock (plant pathologist of Sugarcane Field Station at Canal Point, USDA-ARS), Dr. Yi Li (Professor of Plant Biotechnology, University of Connecticut), and Dr. Christopher Brooks (Bioscience Editing Solutions) for critically reading this paper and providing helpful suggestions. Financial support was provided by the National High Technology Research and Development Program of China (project 2008AA10Z114), Guangxi Natural Science Foundation (project 2014GXNSFFA118002) and Guangxi special funding for distinguished experts.

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The authors have declared no conflict of interest.

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Authors and Affiliations

  1. Guangxi University, Nanning, 530004, China

    Muqing Zhang & Jihua Wang

  2. Fujian Agriculture and Forestry University, Fuzhou, 350002, China

    Muqing Zhang, Xiaolei Zhuo, Yang Wu, Wei Yao & Rukai Chen

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  1. Muqing Zhang
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  2. Xiaolei Zhuo
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Correspondence to Muqing Zhang.

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Editor: John Forster

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Zhang, M., Zhuo, X., Wang, J. et al. Effective selection and regeneration of transgenic sugarcane plants using positive selection system. In Vitro Cell.Dev.Biol.-Plant 51, 52–61 (2015). https://doi.org/10.1007/s11627-014-9644-y

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  • DOI: https://doi.org/10.1007/s11627-014-9644-y

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