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. × 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 µM acetosyringone liquid medium for 6 h at 25 °C before used to infect citrus explants. We incubated epicotyl segments in an MS liquid medium containing 13.2 µM 6-BA, 4.5 µM 2,4-D, 0.5 µM NAA for 3 h at 25 °C prior to Agrobacterium infection. In the co-cultivation medium, we added 30 µM paclobutrazol and 10 µM 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.

Promoting initiation and biomass production of roots is significant for plant-based industries including using roots as bioreactors. We used two citrus genotypes, Carrizo and US-897, as model plants to test the effects of root-predominantly overexpressed the iaaM (indoleacetic acid-tryptophan monooxygenase) gene and a CKX (a cytokinin oxidase/dehydrogenase) gene. The iaaM transgenic lines exhibited markedly faster root initiation, more root numbers, and higher root biomass compared to their wild-type counterparts. The transgenic iaaM + CKXplants also exhibited similar phenotypes, albeit to a lesser extent than the iaaMplants. Molecular analysis revealed an auxin-responsive CsGH3.1 gene was up-regulated in the iaaM roots and iaaM + CKX roots, and a cytokinin-responsive gene CsARR5 gene was down-regulated in the iaaM + CKX roots. Our results demonstrate that root predominant overexpression of the iaaM or both the iaaM and CKX genes drastically enhances the initiation, growth and biomass production of roots. These results provide additional support that manipulation of auxin and cytokinin levels in roots via transgenic or gene-editing technologies may benefit production of high-value secondary metabolites using roots as bioreactors and also improve rooting of recalcitrant plant species.

Deciduous woody plants like poplar (Populus spp.) have seasonal bud dormancy. It has been challenging to simultaneously delay the onset of bud dormancy in the fall and advance bud break in the spring, as bud dormancy and bud break were thought to be controlled by different genetic factors. Here, we demonstrate that heterologous expression of the REVEILLE1 gene (named AaRVE1) from Agave (Agave americana) not only delays the onset of bud dormancy but also accelerates bud break in poplar in field trials. AaRVE1 heterologous expression increases poplar biomass yield by 166% in the greenhouse. Furthermore, we reveal that heterologous expression of AaRVE1 increases cytokinin contents, represses multiple dormancy-related genes, and up-regulates bud break-related genes, and that AaRVE1 functions as a transcriptional repressor and regulates the activity of the DORMANCY-ASSOCIATED PROTEIN 1 (DRM1) promoter. Our findings demonstrate that AaRVE1 appears to function as a regulator of bud dormancy and bud break, which has important implications for extending the growing season of deciduous trees in frost-free temperate and subtropical regions to increase crop yield.

Promoting initiation and biomass production of roots is significant for plant-based industries including using roots as bioreactors. Two citrus genotypes, Carrizo and US-897, were used as model plants to test the effects of root-predominantly overexpressed the iaaM (indoleacetic acid-tryptophan monooxygenase) gene and a CKX (a cytokinin oxidase/dehydrogenase) gene. The iaaM transgenic lines exhibited markedly faster root initiation, more root numbers, and higher root biomass compared to their wild-type counterparts. The transgenic iaaM + CKX plants also exhibited similar phenotypes, albeit to a lesser extent than the iaaM plants. Molecular analysis revealed an auxin-responsive CsGH3.1 gene was up-regulated in the iaaM roots and iaaM + CKX roots, and a cytokinin-responsive gene CsARR5 gene was down-regulated in the iaaM + CKX roots. Our results demonstrate that root predominant overexpression of the iaaM or both the iaaM and CKX genes drastically enhances the initiation, growth and biomass production of roots. These results provide additional support that manipulation of auxin and cytokinin levels in roots via transgenic or gene-editing technologies may benefit production of high-value secondary metabolites using roots as bioreactors and also improve rooting of recalcitrant plant species.