Within leaf variation is the largest source of variation in agroinfiltration of Nicotiana benthamiana

Background Transient gene expression utilizing syringe agroinfiltration offers a simple and efficient technique for different transgenic applications. Leaves of Nicotiana benthamiana show reliable and high transformation efficiency, but in quantitative assays also a certain degree of variation. We used a nested design in our agroinfiltration experiments to dissect the sources of this variation. Results An intron containing firefly luciferase gene was used as a reporter for agroinfiltration. A number of 6 week old tobacco plants were infiltrated for their top leaves, several samples were punched from the leaves after 2 days of transient expression, and protein extracts from the samples were repeatedly measured for luciferase activity. Interestingly, most of the variation was due to differences between the sampling spots in the leaves, the next important source being the different leaves on each plant. Variation between similar experiments, between plants and between repetitive measurements of the extracts could be easily minimized. Conclusions Efforts and expenditure of agroinfiltration experiments can be optimized when sources of variation are known. In summary, infiltrate more plants but less leaves, sample more positions on the leaf but run only few technical replicates. Electronic supplementary material The online version of this article (doi:10.1186/s13007-015-0091-5) contains supplementary material, which is available to authorized users.