Flow cytometric analysis of pollen and spores: An overview of applications and methodology

Abstract

Pollen grains are the male gametophytes in a seed-plant life cycle. Their small, particulate nature and crucial role in plant reproduction have made them an attractive object of study using flow cytometry (FCM), with a wide range of applications existing in the literature. While methodological considerations for many of these overlap with those for other tissue types (e.g., general considerations for the measurement of nuclear DNA content), the relative complexity of pollen compared to single cells presents some unique challenges. We consider these here in the context of both the identification and isolation of pollen and its subunits, and the types of research applications. While the discussion here mostly concerns pollen, the general principles described here can be extended to apply to spores in ferns, lycophytes, and bryophytes. In addition to recommendations provided in more general studies, some recurring and notable issues related specifically to pollen and spores are highlighted.

Pollen DNA metabarcoding and related methods in global change ecology: prospects, challenges, and progress

“…As more long-term studies are completed using DNA metabarcoding, these methods could be used to understand recent change. Under these scenarios, pollen DNA metabarcoding and related methods could eventually become more predominant, especially given the benefits in terms of high-throughput for large sample sizes, and in combination with other newer technologies, such as flow cytometry (Kron et al, 2021) and machine-learning classifications (Goncalves et al, 2016) .…”

“…Machine learning (Goncalves et al, 2016) or deep learning (Dunker et al, 2021) methods can be used to improve the identification of taxa from the data obtained with either morphological or chemical methods. In addition, flow cytometry has been successfully used to count pollen and sort grains for downstream analyses (Kron, Loureiro, Castro, & Čertner, 2021). Combining chemical and image analyses with flow cytometry and deep learning methods can yield fast and accurate taxon identification and quantification (Dunker et al, 2021).…”

Research Progress on Aerobiology in the Last 30 Years: A Focus on Methodology and Occupational Health

“…However, the rapid advances in the "omics" tools and the management of "big data", as well the development of single-cell omics applications [101], will disclose many opportunities in occupational aerobiology. Although molecular and innovative methodologies have been utilized in recent years for the evaluation of different biocomponents in aerosols [102][103][104][105][106][107][108][109][110], analytical techniques such as portable pollen samplers [109,111,112] and flow cytometry [113,114], should be improved in order to expand the development of aerobiology. In this regard, their application in occupational settings assumes great importance [10,115], and they could also be aimed at investigating the role of occupants with regard to the transport of bioparticles using a multidisciplinary approach.…”

“…Regarding the keywords network (Figure 6), the most represented keyword was "aerobiology" (count = 555), a very predictable result considering it was the search input in the databases, and thus it was not considered thoroughly for the interpretation of results. Among others, the most represented keywords were "pollen" (113), "allergy" (65), "phenology" (42), "airborne pollen" (27), "climate change" (27), "fungal spore" (24), "meteorological parameter" (23), "bioaerosol" (23), "asthma" (20), "aeroallergen" (15), "fungi" (12), "pollen allergy" (12), "pollen calendar" (13), "aerosol" (12) and "meteorology" (10). Regarding centrality, the highest values were found for "pollen" (0.55), "asthma" (0.40), "allergy" (0.35), "meteorology" (0.27), "fungal spore" (0.23) and "airborne pollen" (0.20).…”

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

“…The preparation of nuclei from pollen grains usually requires a specialized isolation method [55]. Due to their particulate nature and rigid wall, most pollen grains escape unopened when chopped with a razor blade.…”