**2. Estimation of Genome Size**

Two experimental approaches have been developed to estimate nuclear DNA amounts: biochemical and cytometric. The former seeks to quantify the DNA harbored within a known mass of plant tissue [4]; its weakness lies in the errors inherent in the estimation of the number of nuclei present in the sample, in the unknown proportion of nuclei present at each of the various different cell cycle stages and the non-estimable proportion of endo-reduplicated nuclei present. As a result, cytometry-based estimations tend to be preferred, since these are designed to quantify the DNA present in a population of nuclei at a known cell cycle stage [5]. The attempt by [6] to derive relative nuclear DNA amounts present in several plant species using Feulgen micro-densitometry led to the development of the now universally understood C-value terminology, where un-replicated haploid nuclei contain a 1C DNA amount; the terminology has been refined in recent years [7]. Feulgen microdensitometry was phased out during the 1980s as a result of the throughput benefits offered by flow cytometry, which offers the possibility of analyzing large numbers of isolated nuclei in a short time [5].

It is important to note that flow cytometry does not quantify nuclear DNA directly, but rather achieves this by capturing the signal emitted from fluorochrome-stained nuclei. In order to determine a nuclear DNA amount in absolute units, the fluorescence of an unknown sample has to be compared with that of a reference standard of known genome size [8]. To avoid errors due to non-linearity, an ideal reference standard should not differ in size by more than two or three-fold from the test sample, implying that a set of reference standards is needed in order to cope with the large range of genome size encountered among higher organisms. The question then becomes how to calibrate these reference standards if none of the candidate species has itself been completely sequenced.
