A Flow Cytometry Protocol for Measurement of Plant Genome Size Using Frozen Material

Abstract

Flow cytometry is widely applied to infer the ploidy and genome size (GS) of plant nuclei. The conventional approach of sample preparation, reliant on fresh plant material to release intact nuclei, often results in poor yields of nuclei in conditions when a plant material cannot be kept fresh due to logistical constraints. Previous attempts to use frozen plant material were mainly limited to ploidy analysis and relied on chopping methods, which restrict the material input and often result in poor nuclei yield, especially in frozen samples, due to incomplete disruption. Here, we present a modified protocol for GS estimation using frozen plant material that facilitates larger volumes of tissue to be processed while improving debris removal. Nuclei isolated from this protocol can also be used for DNA or RNA extraction. Genome size estimates from frozen material are similar to those from fresh material, with a reduction in error range, although not always significant (p > 0.05). In certain species, frozen samples can yield substantially more nuclei than fresh material. With the addition of specific debris compensation algorithms, coefficient of variation (CV%) can be maintained below 5%. This method has special value in estimating the GS of samples collected from remote locations and frozen for use in plant genome sequencing. Freezing preserves high-quality DNA and RNA, enabling the same sample to be used for both flow cytometry and genome sequencing.

Plant phenotyping relevance

凍結植物材料からフローサイトメトリーで植物ゲノムサイズを推定する改良プロトコルを開発・検証しており、植物形質取得法が研究の中心である。

Code and data availability

The paper deposits its raw flow cytometry fluorescence dataset (genome size estimation of fresh vs frozen plant material) in FlowRepository and its supplementary materials (ANOVA table, histogram/peak-modeling figures, microscopy images, protocol) in a Zenodo database. Both are paper-specific, publicly accessible, and