Study about factors affecting Agrobacterium-mediated transient expression by vacuum infiltration

Background: In the last decades, vacuum infiltration had been applied for the Agrobacterium-mediated transient expression of foreign gene in plants. However, the relevant influencing factors have not been fully studied yet. Aims: This study aimed to evaluate the effects of vacuum infiltration pressure, time of vacuum infiltration, concentration of Agrobacterium tumefaciens (A. tumefaciens), and incubation time post infiltration on the transient expression of foreign gene in leaves of Nicotiana benthamiana (N. benthamiana). Methods: Two A. tumefaciens strains, LBA4404 and EHA105, carrying a reporter gene of green fluorescent protein (GFP), were used to infiltrate leaves of N. benthamiana via vacuum infiltration. The changes of GFP expression with changes of vacuum infiltration pressure, time of vacuum infiltration, concentration of A. tumefaciens, and incubation time post infiltration were measured. Results: Increase of vacuum pressure from 0.03 to 0.07 MPa increased the GFP expression in the leaves infiltrated with either LBA4404 or EHA105, and the vacuum pressure at 0.07 MPa made almost all of area of the infiltrated leaf express GFP. The increase of the time of vacuum infiltration from 10 to 20 min significantly enhanced the GFP expression in either the LBA4404 or EHA105-infected leaves. Among the different concentrations of Agrobacterium (from OD600 0.1 to 0.9), the concentrations of LBA4404 strain suspension at OD600 0.5 and EHA105 strain suspension at OD600 0.3, respectively, were most effective concentrations for enhancing the GFP expression in the infected leaves. The increase of incubation time post infiltration from 2 to 4 days largely enhanced the intensity of GFP expression in the either LBA4404 or EHA105-infected leaves. However, further increase of incubation time post infiltration from 4 to 6 days decreased the GFP expression in the infected leaves. Conclusions: Vacuum infiltration pressure/time, concentration of A. tumefaciens, and the incubation time post infiltration were important factors affecting the level of transient expression by vacuum infiltration. Optimizing these factors is essential for improving the level of transient expression of foreign gene by vacuum infiltration.