Co-culturing on dry filter paper significantly increased the efficiency of Agrobacterium-mediated transformations of maize immature embryos.

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.