Volume-based pollen size analysis: an advanced method to assess somatic and gametophytic ploidy in flowering plants

Abstract

Pollen size is often used as a biological parameter to estimate the ploidy and viability of mature pollen grains. In general, pollen size quantification is performed one- or two-dimensionally using image-based diameter measurements. As these approaches are elaborate and time consuming, alternative approaches that enable a quick, reliable analysis of pollen size are highly relevant for plant research. In this study, we present the volume-based particle size analysis technique as an alternative method to characterize mature pollen. Based on a comparative assay using different plant species (including tomato, oilseed rape, kiwifruit, clover, among others), we found that volume-based pollen size measurements are not biased by the pollen shape or position and substantially reduce non-biological variation, allowing a more accurate determination of the actual pollen size. As such, volume-based particle size techniques have a strong discriminative power in detecting pollen size differences caused by alterations in the gametophytic ploidy level and therefore allow for a quick and reliable estimation of the somatic ploidy level. Based on observations in Arabidopsis thaliana gametophytic mutants and differentially reproducing Boechera polyantha lines, we additionally found that volume-based pollen size analysis provides quantitative and qualitative data about alterations in male sporogenesis, including aneuploid and diploid gamete formation. Volume-based pollen size analysis therefore not only provides a quick and easy methodology to determine the somatic ploidy level of flowering plants, but can also be used to determine the mode of reproduction and to quantify the level of diplogamete formation.

Aberrant Classopollis pollen reveals evidence for unreduced (2 n ) pollen in the conifer family Cheirolepidiaceae during the Triassic–Jurassic transition

“…Partly, this is because the upper and lower tails of each subpopulation can overlap with one another. Such overlap has been observed in studies of pollen diameter from polyploid Arabidopsis plants, in which the right-hand tail of pollen from triploid (3n) plants overlaps with the left-hand tail of pollen from hexaploid (6n) plants [22]. Such overlap in the diameter of pollen from polyploids can be shown clearly in studies of modern plants because the ploidy of the parent plant is known.…”

“…Principally, the observed pattern can be ascribed to the formation of anucleate, haploid and unreduced 2n pollen. In modern plants, pollen size is related to the number of chromosomes with increasing pollen volume at higher ploidy levels [9,[19][20][21][22][32][33][34][35]. While the number of pollen openings has been reported to vary in some polyploidy angiosperms [36], in this study, the number of pores and the furrows of Classopollis remains constant.…”

“…As cell size correlates with the DNA content [1], stomatal size of living and fossil leaf remains has been used to infer ploidy history in angiosperms during the Caenozoic [17,18]. Similar to the stomatal size, pollen size and volume also varies with the ploidy level of a plant [9,10,[19][20][21][22]. In order to test our hypothesis, we examined the morphology of the fossil pollen Classopollis, which was produced by the Cheirolepidiaceae.…”

Cedrus atlantica pollen morphology and investigation of grain size variability using laser diffraction granulometry

“…Analysis showed that pollen grain size discriminated trees into different genetic and ecological groups, suggesting genetics influenced pollen size (Derridj et al 1991). The genetic control on pollen size may stem from genome size (Bennett 1972;De Storme et al 2013), in particular the effect of an increased number of chromosomes due to polyploidy (Kapadia & Gould 1964;Dyer et al 2013). Knight et al (2010) found a significant positive trend between pollen width and genome size across 464 species.…”

“…Grain size may be studied to improve the taxonomic resolution of pollen identification, for example with Pinus pollen (Desprat et al 2015), Poaceae pollen (Radaeski et al 2016), and indeed with Cedrus pollen (Fujiki et al 2003). Pollen size has also been correlated with genome size and may indicate polyploidy (three or more chromosome sets) in plants (Gould 1957;Kapadia & Gould 1964;Bennett 1972;Tate et al 2005;Knight et al 2010;De Storme et al 2013). Increasingly, geochemical studies utilising pollen, such as stable isotope analysis for palaeoclimate reconstructions (Amundson et al 1997;Loader & Hemming 2004;Nelson et al 2006Nelson et al , 2007King et al 2012;Nelson 2012;Bell et al 2017) and biomarker analysis for UV-B reconstructions (Rozema et al 2001(Rozema et al , 2002Fraser et al 2011;Willis et al 2011;Lomax et al 2012;, require detailed knowledge of grain size for developing techniques to isolate specific grains from fossil assemblages.…”