Establishment of an efficient root mediated genetic transformation method for gene function verification in citrus

Although several methods of citrus genetic transformation have been studied and published, it is still necessary to further improve the efficiency of genetic transformation and increase the varieties used for citrus genetic transformation. Here, we report a simple, rapid and efficient root transformation technique for citrus to facilitate gene function studies. We found that infestation of Agrobacterium rhizogenes into citrus seedlings of five different species could produce stable transgenic roots in a short period after comparing and optimizing the selection of citrus species, bacterium concentration, cutting time and infection method. PCR and fluorescence observations showed a positive root transformation efficiency of approximately 13%-36% in the five tested citrus species. To demonstrate the utility of this method in the functional verification of citrus genes, a bimolecular fluorescence complementation (BiFC) assay was used to prove the interaction between CrCIPK6/9 and CrCBL5/6 in Citrus reticulata. Additionally, the PCR results showed that CrFT-RFP was transferred from the transgenic positive root to the stem in Citrus reticulata. Finally, overexpression of CjCAD from Citrus junos promoted lignin accumulation in transgenic citrus roots. This method could not only be used to study genes in roots but also various genes involved in plant growth and development. Taken together, we demonstrated that this method allows fast study of gene function in a wide range of citrus species, which would facilitate the understanding of novel genes in citrus.