[Generation of vector backbone-free and selectable marker-free transgenic maize (Zea mays L.) via ovary-drip method].
The presence of vector backbone sequences and selectable marker genes in transgenic plants has been the key concern for biosafety. A direct solution is to totally avoid the use of vector backbone sequences and selectable marker genes from the beginning of transgenic plant generation. In this study, the ovary-drip method was established and optimized. The key features of this method focused on the complete removal of the whole styles, and the subsequent application of a DNA solution directly to the ovaries. A vector backbone-free and selectable marker-free linear GFP cassette (Ubi-GFP -nos) was transformed into maize via the ovary-drip method. PCR analysis showed that suitable maize variety was 9818 and optimal transformation time was 18-20 h after pollination, which produced the highest PCR positive frequency (3.01%). Southern blotting analysis showed that the transgenic plants had simple integration patterns (1-2 bands). GFP transcription was de-tected by RT-PCR analysis. Green fluorescence was observed in roots and immature embryos of transgenic plants by a fluorescence microscopy.
Yang
Ai-Fu
AF
Department of Bioscience and Biotechnology, Dalian University of Technology, Dalian 116024, China. yangaifu@yahoo.com.cn
Su
Qiao
Q
An
Li-Jia
LJ
D015139:Blotting, Southern; D035264:Flowers / Q000235:genetics*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics*; D008856:Microscopy, Fluorescence; D030821:Plants, Genetically Modified / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012641:Selection, Genetic; D003313:Zea mays / Q000235:genetics* / Q000378:metabolism