Nuclear DNA Content Analysis of Plant Seeds by Flow Cytometry

Abstract

Procedures describing the utilization of seeds or their parts for flow cytometric determination of plant ploidy and endopolyploidy, genome size, and cell cycle activity are presented. The methods have been developed for a single‐fluorescence‐parameter flow cytometer, equipped with light sources for 488‐nm and UV‐light illumination. The procedures presented in this unit utilize the two most widely used fluorochromes for plant DNA content analysis, propidium iodide (PI) and 4′,6‐diamidino‐2‐phenylindole (DAPI). These methods provide an alternative to estimation of DNA content based on the fluorescence of DNA in cell nuclei isolated from plant leaves. In some instances seeds are more suitable for analysis than leaves, e.g., when plant material must be transported for a long distances or stored for prolonged periods before flow cytometric analysis, or when leaves contain fluorochrome‐staining inhibitors. In addition, flow cytometric determination of nuclear replication stages in seeds gives information about their physiological status (e.g., maturity, advancement of germination), which is valuable to seed producers and technologists.

Plant material selection, collection, preservation, and storage for nuclear DNA content estimation

“…It is possible that dormant embryonic tissues with low water content also release less nucleases and secondary metabolites into nuclear homogenate than typical fresh tissues [15]. as it usually improves the quality of measurement (see, e.g., [76] for a detailed procedure). In addition, while the addition of RNase is optional for most tissues, it is necessary in the case of seeds [14].…”

Isolation of plant nuclei for estimation of nuclear DNA content: Overview and best practices

“…Therefore, a universally accepted method for the preparation of nuclear suspensions for DNA FCM was developed by Galbraith et al [6]. In this pro- [40], silica-dried samples [41], and seeds [42]. In the case of seeds, the same buffers as for leaf tissue can often be used, but in some species, other buffers perform better, such as the seed buffer [43] developed specifically for this purpose.…”

“…Variations of Galbraith's et al protocol have been introduced, which differ in the way a sample is homogenized and by the composition of nuclear isolation buffers. Although originally developed for fresh plant tissues, Galbraith et al protocol is also suitable for the estimation of relative nuclear DNA content in dry herbarium vouchers [40], silica‐dried samples [41], and seeds [42]. In the case of seeds, the same buffers as for leaf tissue can often be used, but in some species, other buffers perform better, such as the seed buffer [43] developed specifically for this purpose.…”

Nuclear DNA replication and seed quality

“…Thus, the ratio of endosperm to embryo nuclei or extent of endoreduplication in the endosperm provides information on seed maturity (Sliwinska, 2006). pea (Pisum sativum), bean, sugarbeet and other non-endospermic seeds], while in others, it is present after maturation is completed [e.g.…”