Ovary-drip transformation: a simple method for directly generating vector- and marker-free transgenic maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) with a linear <Emphasis Type="Italic">GFP</Emphasis> cassette transformation |
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Authors: | Aifu Yang Qiao Su Lijia An |
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Institution: | (1) Department of Bioscience and Biotechnology, Dalian University of Technology, Dalian, 116024, People’s Republic of China |
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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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Keywords: | Linear GFP cassette Maize Ovary-drip method Vector- and selectable marker-free |
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