Image cytometry (ICM) has been used to measure DNA 2C-values by evaluating the optical density of Feulgen-stained nuclei. This optical measurement is carried out using three basic tools: microscopy, digital video camera, and image analysis software. Because ICM has been applied to plants, some authors have remarked that studies should be performed before this technique can be accepted as an accurate method for determination of plant genome size. Based on this, the 2C-value of eight plants, which are widely used as standards in DNA quantifications, was reassessed in a cascade-like manner, from A. thaliana through R. sativus, S. lycopersicum, Glycine max, Z. mays, P. sativum, V. faba, to A. cepa. The mean 2C-values of all plants were statistically compared to the values reported by other authors using flow cytometry and/or ICM. These analyses demonstrated that ICM is an accurate and reliable method for 2C-value measurement, representing an attractive alternative to flow cytometry. Statistical comparison of the results also indicated Glycine max ‘Polanka’ as the most adequate primary standard. However, distinct authors have been advised that 2C DNA content of the reference standard should be close to that of the sample. As three further approaches also revisited the 2C-value of these eight plants, we have thus proposed a mean 2C-value for each eight species.