Strategies to improve low copy transgenic events in Agrobacterium-mediated transformation of maize.
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.
Syngenta Crop Protection, LLC, Research Triangle Park, NC, USA. sivamani.elumalai@syngenta.com.
Elumalai
http://orcid.org/0000-0002-6239-2711
E
Sivamani
Syngenta Biotechnology China Co. Ltd, Beijing, People's Republic of China.
Xianggan
X
Li
Syngenta Crop Protection, LLC, Research Triangle Park, NC, USA.
Samson
S
Nalapalli
Syngenta Crop Protection, LLC, Research Triangle Park, NC, USA.
Yoshimi
Y
Barron
Syngenta Crop Protection, LLC, Research Triangle Park, NC, USA.
Anna
A
Prairie
Syngenta Crop Protection, LLC, Research Triangle Park, NC, USA.
David
D
Bradley
Syngenta Crop Protection, LLC, Research Triangle Park, NC, USA.
Michele
M
Doyle
Syngenta Crop Protection, LLC, Research Triangle Park, NC, USA.
Qiudeng
Q
Que
D016960:Agrobacterium tumefaciens / Q000235:genetics*; D056915:DNA Copy Number Variations; D004269:DNA, Bacterial / Q000235:genetics*; D017343:Genes, Plant; D005822:Genetic Vectors; D030821:Plants, Genetically Modified / Q000235:genetics* / Q000254:growth & development; D010957:Plasmids / Q000235:genetics*; D014170:Transformation, Genetic / Q000235:genetics*; D019076:Transgenes / Q000235:genetics*; D003313:Zea mays / Q000235:genetics* / Q000254:growth & development
D004269:DNA, Bacterial; C036483:T-DNA
Agrobacterium; Backbone free; Co-suppression; Low copy; Maize transformation; RNAi; Silencing; T-DNA; Transgenic events