Endogenous auxin is an important regulator of in vivo organ development, but its role in in vitro organogenesis is unclear. It has been observed that the basal end of epicotyl cuttings of juvenile citrus seedlings produces fewer shoots than the apical end. Here, we report that elevated endogenous auxin levels in the basal end of citrus epicotyl cuttings are inhibitory for in vitro shoot organogenesis. Using transgenic citrus plants expressing an auxin-inducible GUS reporter gene, we have observed elevated levels of auxin at the basal end of stem cuttings that are mediated by polar auxin transport. Depleting endogenous auxin or blocking polar auxin transport enhances shoot organogenesis. An auxin transport inhibitor, N-1-naphthylphthalamic acid (NPA), can also enhance shoot organogenesis independent of its action on polar auxin transport. Finally, we demonstrate that the promotional effects of depleting endogenous auxin or blocking polar auxin transport on shoot organogenesis are cytokinin-dependent. Our study thus provides meaningful insights into possible roles of endogenous auxin and polar auxin transport, as well as auxin–cytokinin interactions, in in vitro shoot organogenesis. Meanwhile, our results may also provide practical strategies for improving in vitro shoot organogenesis for citrus and many other plant species.

The effects of a maize knotted1 (kn1) gene under the control of the cauliflower mosaic virus 35S promoter on genetic transformation efficiencies of six citrus genotypes were tested. The kn1 gene construct was used to transform 'Pineapple', 'Hamlin', 'Sucarri' andOur results demonstrate that expression of the kn1 gene enhances transformation efficiencies from 3 to 15 fold compared to a control vector, 3-11 fold relative to the highest transformation efficiencies reported for these citrus genotypes. Stable incorporations of T-DNA into the citrus genome have been confirmed with both histochemical staining of GUS activity and molecular analyses. The majority of kn1 over-expressing citrus plants grow and develop normally at young seedling stages, similar to those of the wild type plants. With all six genotypes of citrus tested including Eureka lemon, a cultivar difficult to be transformed, our results demonstrate that the kn1 gene may provide an effective molecular tool to enhance genetic transformation efficiencies of various citrus varieties. High transformation efficiency of citrus is of great importance for large scale characterization of gene functions and also cultivar development via transgenic and genome editing technologies.