Plant DNA flow cytometry and estimation of nuclear genome size

Abstract

Background: DNA flow cytometry describes the use of flow cytometry for estimation of DNA quantity in cell nuclei. The method involves preparation of aqueous suspensions of intact nuclei whose DNA is stained using a DNA fluorochrome. The nuclei are classified according to their relative fluorescence intensity or DNA content. Because the sample preparation and analysis is convenient and rapid, DNA flow cytometry has become a popular method for ploidy screening, detection of mixoploidy and aneuploidy, cell cycle analysis, assessment of the degree of polysomaty, determination of reproductive pathway, and estimation of absolute DNA amount or genome size. While the former applications are relatively straightforward, estimation of absolute DNA amount requires special attention to possible errors in sample preparation and analysis.

Scope: The article reviews current procedures for estimation of absolute DNA amounts in plants using flow cytometry, with special emphasis on preparation of nuclei suspensions, stoichiometric DNA staining and the use of DNA reference standards. In addition, methodological pitfalls encountered in estimation of intraspecific variation in genome size are discussed as well as problems linked to the use of DNA flow cytometry for fieldwork.

Conclusions: Reliable estimation of absolute DNA amounts in plants using flow cytometry is not a trivial task. Although several well-proven protocols are available and some factors controlling the precision and reproducibility have been identified, several problems persist: (1) the need for fresh tissues complicates the transfer of samples from field to the laboratory and/or their storage; (2) the role of cytosolic compounds interfering with quantitative DNA staining is not well understood; and (3) the use of a set of internationally agreed DNA reference standards still remains an unrealized goal.

Fig. 1.

Estimation of absolute nuclear DNA amount (genome size) in Ensete gilletii. The histogram of relative DNA content was obtained after flow cytometric analysis of propidium iodide-stained nuclei of Ensete and soybean, which were isolated, stained and analysed simultaneously. Soybean (Glycine max ‘Polanka’, 2C = 2·50 pg DNA) served as internal reference standard. The gain of the cytometer was adjusted so that the G₁ peak of soybean was positioned on channel 200. The ratio of G₁ peak means (Ensete : soybean) was equal to 0·484 and hence the 2C DNA amount of E. gilletii was estimated as 1·210 pg. Note that a reliable estimation of genome size of a species requires that several randomly selected plants are analysed, each of them several times and on different days. The replicate measurements of the same plant facilitate the detection of variation in the procedure and estimation of variation between plants.