Ovary-drip transformation: a simple method for directly generating vector- and marker-free transgenic maize (Zea mays L.) with a linear GFP cassette transformation.
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 T(1) 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.
Yang
Aifu
A
Department of Bioscience and Biotechnology, Dalian University of Technology, Dalian, 116024, People's Republic of China. yangaifu@yahoo.com.cn
Su
Qiao
Q
An
Lijia
L
D015139:Blotting, Southern; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D035264:Flowers / Q000235:genetics* / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D005818:Genetic Engineering / Q000379:methods; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D030821:Plants, Genetically Modified / Q000235:genetics* / Q000378:metabolism; D053205:Pollen Tube / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D013997:Time Factors; D014170:Transformation, Genetic; D003313:Zea mays / Q000235:genetics* / Q000378:metabolism
D049452:Green Fluorescent Proteins; D004247:DNA; D016650:Fluorescein-5-isothiocyanate