Plant Genome Size Estimation by Flow Cytometry: Inter-laboratory Comparison

Abstract

Flow cytometry is a convenient and rapid method that has been used extensively for estn. of nuclear genome size in plants. In contrast to general expectations, results obtained in different labs. showed some striking discrepancies. The aim of this joint expt. was to test the reliability and reproducibility of methods. Care was taken to avoid a bias due to the quantity of DNA in the nucleus, the procedure for nuclei isolation or the type of instrument. Nuclear DNA content was estd. in nine plant species representing a typical range of genome size (2C = approx. 0·3-30 pg DNA). Each of the four labs. involved in this study used a different buffer and/or procedure for nuclei isolation. Two labs. used arc lamp-based instruments while the other two used laser-based instruments. The results obtained after nuclei staining with propidium iodide (a DNA intercalator) agreed well with those obtained using Feulgen densitometry. On the other hand, results obtained after staining with DAPI (binding preferentially to AT-rich regions) did not agree with those obtained using Feulgen densitometry. Small, but statistically significant, differences were found between data obtained with individual instruments. Differences between the same type of instruments were negligible, while larger differences were obsd. between lamp- and laser-based instruments. Ratios of fluorescence intensity obtained by laser instruments were higher than those obtained by lamp-based cytometers or by Feulgen densitometry. The results obtained in this study demonstrate that flow cytometry with DNA intercalators is a reliable method for estn. of nuclear genome size in plants. However, the study confirmed an urgent need for an agreement on stds. Given the small but systematic differences between different types of flow cytometers, anal. of very small differences in genome size should be made in the same lab. and using the same instrument.