Generation of vector backbone-free and selectable marker-free transgenic maize (Zea mays L.) via ovary-drip method

Abstract

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.