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Best practices in plant cytometry

Flow cytometry (FCM) and flow cytometric sorting (FCS) systems have developed as experimental tools of remarkable power and are enjoying an ever-increasing impact in the general field of biology. Application of these tools to plant biology has developed more slowly given that the natural form of plants infrequently resembles that of the single cell suspension, prototypically the hematopoietic system that drove the original development of FCM/FCS. Nevertheless, these systems have had a profound influence at all levels of plant biology, from the study of single cells and subcellular organelles, to the behavior of populations of plants, and ultimately to the performance of ecosystems. It is safe to say their impact has not plateaued, as further applications of this unique technology are increasingly developed by innovative scientists around the world to address questions both in the basic sciences, and to increasingly confront emerging problems in the applied sector. For example, in addressing the challenges of sustainable production of sufficient food resources based on plant breeding involving ploidy-based approaches (e.g., induction of polyploidy) for the needs of our future global citizens, FCM, and FCS systems will play central roles in this effort. The degree to which FCM and FCS systems have impacted plant biology and applied agricultural sciences must not be understated. The major applications of DNA FCM are ploidy level and genome size estimations, and cell cycle analysis/endoreplication (with the later included in a lower percentage of studies). Indeed, FCM is currently/ extensively and almost exclusively employed as the method of choice for measurement of plant genome sizes. Measurements of this type impact agriculture in terms of ploidy estimation, with applications ranging from plant biotechnology, breeding and seed quality testing to taxonomy and population biology. They also impact the fundamental plant sciences in terms of biosystematics, ecology, evolution, genomics, and conservation, among other applications. One of the most startling observations of the angiosperms is the bandwidth occupied by genome size, which spans almost 2400-fold. Flow sorting of higher plant chromosomes has provided invaluable information regarding the organization of DNA sequences within plant species. It has also greatly facilitated the process of wholegenome sequencing by permitting subdivision of large genomes into samples comprising entire chromosomes or chromosome arms. FCS methods applied to wall-less cells (protoplasts) expressing fluorescent proteins (FPs) in a cell type-specific manner have allowed elucidation of patterns of co-regulated gene expression and plant hormone gradients identification within organized tissues, such as roots. The trigger to develop this virtual issue came from the publication, in 2017, of an article entitled “Guidelines for the use of flow cytometry and cell sorting in immunological studies” in the European Journal of Immunology. As noted in that article, one of the advantages of FCM/FCS systems is that they are relatively simple to implement, with some qualifications, which coupled with the development of user-friendly devices and software during the last 15 years led to increasing applications in other areas, such as plant sciences. However, it is also simple to implement and operate the instruments inappropriately. This calls for a comprehensive and collective summary of the best practices when applying FCM/FCS to plants, as was done for immunology. The first consideration addresses the problem that plants, particularly the vascular plants, in their commonly recognized and utilized forms, exist not as single cell suspensions (typical of immunology) but as complex three-dimensional tissues comprising cells of irregular shapes, different types and functions, that collectively cooperate to produce the final plant form. Optimal methods for producing suspensions of cells, subcellular organelles and other components appropriate for FCM/FCS from these plant tissues and organs, are therefore one of the challenges discussed in this virtual issue. We are fully aware of the mantra that “junk in equals junk out” and having samples of the highest quality prior to FCM/FCS is a critical concern we also addressed here. The second consideration relates to the vast variety of different plant species found globally, and the recognition of the consummate ability of plants to produce secondary metabolites/products, affecting DNA staining and resulting fluorescence. Again, methods for recognizing and handling the different challenges provided to FCM/FCS methods by the biochemistries of the source samples are required. The third consideration focuses on the problem of addressing the non-critical application of FCM/FCS methods developed for mammalian cell systems (typically hematopoietic) to plants without careful consideration of their appropriateness. As it will be detailed in this virtual issue, application of FCM/FCS methods to mammalian cell systems almost exclusively occurs in the context of analysis of samples that comprise a majority, often close to 100%, of single cells in suspension. For plants, particularly when using these instruments and methods for the analysis of organelles in tissue homogenates, the objects of interest comprise a very minor subpopulation of the total particles passing through the instrument. Concepts such as placing initial gates around populations defined by forward scatter (FS) versus side scatter (SS), as routinely used to define leukocytes or other Received: 11 November 2020 Revised: 7 December 2020 Accepted: 14 December 2020