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论文类型：期刊论文

发表时间：2009-03-01

发表刊物：PLANTA

收录刊物：SCIE、PubMed、Scopus

卷号：229

期号：4

页面范围：793-801

ISSN号：0032-0935

关键字：Linear GFP cassette; Maize; Ovary-drip method; Vector- and selectable marker-free

摘要：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.