Agrobacterium-mediated transient expression assays are a convenient alternative to stable expression because they are simple,easy to perform,and achieve gene expression rapidly.This study investigated the factors affecting transient gene expression efficiency in citrus by observing the cryo-sectioning of leaf samples under a laser confocal microscope.These factors included the composition of the infiltration buffer,the Agrobacterium cell density,the leaf development stage,the incubation temperature,and plant genotype.The highest transient expression level of yellow fluorescent protein(YFP)was detected in Mexican lime(Citrus aurantifolia)on the third day after the intermediate-aged leaves were infiltrated with the improved infiltration buffer 1(15 mmol L~(-1) 2-(N-morpholino)ethanesulfonic acid,10 mmol L~(-1) MgCl_2,and 200μmol L~(-1)acetosyringone),which had an optical density of 0.8 and was incubated at 22°C.Additionally,this transient expression assay was applied to other citrus genotypes.Of note,trifoliate orange(Poncirus trifoliata)and kumquat(Fortunella obovate)had higher expression efficiency than other six genotypes of the Citrus genus.Our study provides research basis for the selection of optimization strategies in transient gene expression and improves the method for available genome investigation in citrus. Agrobacterium-mediated transient expression assays are a convenient alternative to stable expression because they are simple, easy to perform, and achieve gene expression rapidly. This study investigates the factors that transient gene expression efficiency in citrus by observing the cryo-sectioning of leaf samples under a laser confocal microscope. The factors include the composition of the infiltration buffer, the Agrobacterium cell density, the leaf development stage, the incubation temperature, and plant genotype. The highest transient expression level of yellow fluorescent protein (YFP) was detected in Mexican lime (Citrus aurantifolia) on the third day after the intermediate-aged leaves were infiltrated with the improved infiltration buffer 1 (15 mmol L -1 2- (N-morpholino) ethanesulfonic acid, 10 mmol L -1 MgCl 2 , and 200 μmol L -1 acetosyringone, which had an optical density of 0.8 and was incubated at 22 ° C. Additionally, this transient expression assay was applied to other citrus ge notypes.Of note, trifoliate orange (Poncirus trifoliata) and kumquat (Fortunella obovate) had higher expression efficiency than other six genotypes of the Citrus genus. Our study provides research basis for the selection of optimization strategies in transient gene expression and improves the method for available genome investigation in citrus.