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Ovary-drip transformation: a simple method for directly generating vector- and marker-free transgenic maize (Zea mays L.) with a linear GFP cassette transformation

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Abstract

The presence of selectable marker genes and vector backbone sequences has affected the safe assessment of transgenic plants. In this study, the ovary-drip method for directly generating vector- and selectable marker-free transgenic plants was described, by which maize was transformed with a linear GFP cassette (Ubi-GFP-nos). The key features of this method center on the complete removal of the styles and the subsequent application of a DNA solution directly to the ovaries. The movement of the exogenous DNA was monitored using fluorescein isothiocyanate-labeled DNA, which showed that the time taken by the exogenous DNA to enter the ovaries was shortened compared to that of the pollen-tube pathway. This led to an improved transformation frequency of 3.38% compared to 0.86% for the pollen-tube pathway as determined by PCR analysis. The use of 0.05% surfactant Silwet L-77 + 5% sucrose as a transformation solution further increased the transformation frequency to 6.47%. Southern blot analysis showed that the transgenic plants had low transgene copy number and simple integration pattern. Green fluorescence was observed in roots and immature embryos of transgenic plants by fluorescence microscopy. Progeny analysis showed that GFP insertions were inherited in T1 generation. The ovary-drip method would become a favorable choice for directly generating vector- and marker-free transgenic maize expressing functional genes of agronomic interest.

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Abbreviations

FITC:

Fluorescein isothiocyanate

GFP:

Green fluorescent protein

Ubi promoter:

Ubiquitin promoter

Nos terminator:

Nopaline synthase terminator

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Acknowledgments

This work was supported by grants from the Key Projects of Science and Technology Council of Liaoning Province (no. 2006208001). The authors specially thank Dr. Alan K. Chang (Dalian University of Technology) for critically reviewing this manuscript and helpful discussions.

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  1. Department of Bioscience and Biotechnology, Dalian University of Technology, Dalian, 116024, People’s Republic of China

    Aifu Yang, Qiao Su & Lijia An

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  1. Aifu Yang
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  2. Qiao Su
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  3. Lijia An
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Correspondence to Qiao Su or Lijia An.

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Yang, A., Su, Q. & An, L. Ovary-drip transformation: a simple method for directly generating vector- and marker-free transgenic maize (Zea mays L.) with a linear GFP cassette transformation. Planta 229, 793–801 (2009). https://doi.org/10.1007/s00425-008-0871-5

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  • DOI: https://doi.org/10.1007/s00425-008-0871-5

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