Abstract
Transgenic plants containing low copy transgene insertion free of vector backbone are highly desired for many biotechnological applications. We have investigated two different strategies for increasing the percentage of low copy events in Agrobacterium-mediated transformation experiments in maize. One of the strategies is to use a binary vector with two separate T-DNAs, one T-DNA containing an intact E.coli manA gene encoding phosphomannose isomerase (PMI) as selectable marker gene cassette and another T-DNA containing an RNAi cassette of PMI sequences. By using this strategy, low copy transgenic events containing the transgenes were increased from 43 to 60 % in maize. An alternate strategy is using selectable marker gene cassettes containing regulatory or coding sequences derived from essential plant genes such as 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) or MADS box transcription factor. In this paper we demonstrate that higher percentage of low copy transgenic events can be obtained in Agrobacterium-mediated maize transformation experiments using both strategies. We propose that the above two strategies can be used independently or in combination to increase transgenic events that contain low copy transgene insertion in Agrobacterium-mediated transformation experiments.
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Acknowledgments
The authors would like to acknowledge Vance Kramer, Jeremy Gould, Michael Schweiner, Eddie Lauer, Wenling Wang, Ryan Carlin and John Clark Jr. for their help in this project. We also thank other Syngenta colleagues for their enthusiasm and support of this work. The authors also would like to thank Drs. Liang Shi and Bruce Vrana for their support.
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Nucleotide sequence alignment of MADS-domain transcription factor region in OsMADS1 (constructs 20070, 21099 and 22230), OsMADS2 (construct 22096) and OsMADS16 (construct 22072) compared with maize ZmMADS9 (GenBank accession NM_001177865) and ZmMADS10 (GenBank accession EF552705) (TIFF 123 kb)
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Sivamani, E., Li, X., Nalapalli, S. et al. Strategies to improve low copy transgenic events in Agrobacterium-mediated transformation of maize. Transgenic Res 24, 1017–1027 (2015). https://doi.org/10.1007/s11248-015-9902-8
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DOI: https://doi.org/10.1007/s11248-015-9902-8