Co-culturing on dry filter paper significantly increased the efficiency of Agrobacterium-mediated transformations of maize immature embryos
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In the Agrobacterium tumefaciens-mediated transformations of maize immature embryos (IEs), the common co-culturing media used are MS or N6-based (MC). Here, we used a novel co-culturing method in which maize ‘Qi319’ IEs inoculated with Agrobacterium-harboring target vector were placed on dry filter paper (DC) in a petri dish. To compare the effects of the DC and MC co-culturing methods on transformation efficiency, we designed three experiments: (1) A. tumefaciens strain AGL1 independently carrying two plasmids, pXQD12 and pXQD70; (2) two A. tumefaciens strains, AGL1 and EHA105, carrying pXQD12; and (3) strains AGL1 and EHA105 each independently inoculated with pXQD12 and pXQD70 for different infiltration periods, 5, 10, 15, 20 and 25 min. We used A. tumefaciens to inoculate IEs derived from maize ears 9–15 d after pollination, and then IEs were placed in petri dishes for co-culturing. The DC treatment significantly increased the percentage of IEs expressing green fluorescence protein (%GFP), indicating positive transformants. DC-treated IEs had ~ 3 to 4 times the %GFP compared with MC-treated IEs at 8 d after inoculation (3 d co-culture and 5 d restoration). The average regeneration frequency (%GFP positive regenerated calli of infected IEs) and stable transformation frequency (%GFP positive T0 plants of infected IEs) significantly increased with the DC treatment. Thus, the DC method may be used to develop a more efficient Agrobacterium-mediated transformation method for maize IEs.
KeywordsDry filter paper Co-culturing Transformation efficiency Agrobacterium Maize
This work was supported by the Ministry of Agriculture of China (Grant Number 2016ZX08009002), the National Natural Science Foundation of China (Grant Number 31301080) and the foundation of the Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations (Grant Number lzujbky-2016-bt05). We thank also LetPub (www.letpub.com) and Liwen Bianji (www.liwenbianji.cn/ac) for their linguistic assistance during the preparation of this manuscript.
XD contributed all reagents and materials used in the experiments. XD, LZ, JS, WL, WW and HA designed and performed the experiments. HA and XD analyzed the data and wrote the paper.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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