An Improved Procedure for Agrobacterium-Mediated Transformation of 'Carrizo' Citrange

Although several protocols for genetic transformation of citrus have been published, it is highly desirable to further improve its efficiency. Here we report treatments of Agrobacterium cells and citrus explants prior to and during co-cultivation process to enhance transformation efficiency using a commercially used rootstock 'Carrizo' citrange [Citrus sinensis (L.) Osb. x Poncirius trifoliata (L.) Raf.] as a model plant. We found explants from light-grown seedlings exhibited higher transformation efficiency than those from etiolated seedlings. We pre-cultured Agrobacterium cells in a 1/10 MS, 0.5 g/L 2-(N-morpholino) ethanesulfonic acid (MES) and 100 mu M acetosyringone liquid medium for 6 h at 25 degrees C before used to infect citrus explants. We incubated epicotyl segments in an MS liquid medium containing 13.2 mu M 6-BA, 4.5 mu M 2,4-D, 0.5 mu M NAA for 3 h at 25 degrees C prior to Agrobacterium infection. In the co-cultivation medium, we added 30 mu M paclobutrazol and 10 mu M lipoic acid. Each of these treatments significantly increased the efficiencies of transformation up to 30.4% (treating Agrobacterium with acetosyringone), 31.8% (treating explants with cytokinin and auxin), 34.9% (paclobutrazol) and 38.6% (lipoic acid), respectively. When the three treatments were combined, we observed that the transformation efficiency was enhanced from 11.5% to 52.3%. The improvement of genetic transformation efficiency mediated by these three simple treatments may facilitate more efficient applications of transgenic and gene editing technologies for functional characterization of citrus genes and for genetic improvement of citrus cultivars.