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

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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.

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Acknowledgments

We would like to thank Susan Armstrong (University of Birmingham) for demonstrating the meiotic spreading protocol. Gratitude to Tim Bekaert (University of Ghent, Civil Engineering) for helping with the MATLAB-based image analysis and particle sizing software. Many thanks to Rob Dirks (Rijk Zwaan Breeding B. V.), Raphael Mercier (INRA, Versailles), Rod Scott (University of Bath), and Filip Debersaques (University College Ghent) for providing plant material. This research is supported by an aspirant fellowship to Nico De Storme and research grant G006709N offered by the Flemish Funding Agency for Scientific Research (FWO). Collaborations and travel were supported by the COST action FA0903.

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  1. Department of Plant Production, Faculty of Bioscience Engineering, University of Ghent, Coupure Links 653, 9000, Ghent, Belgium

    Nico De Storme, Linda Zamariola & Danny Geelen

  2. Leibniz Institut für Pflanzengenetik und Kulturpflanzenforschung, Corrensstraße 3, 06466, Stadt Seeland, OT Gatersleben, Germany

    Martin Mau & Timothy F. Sharbel

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  1. Nico De Storme
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  2. Linda Zamariola
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  4. Timothy F. Sharbel
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  5. Danny Geelen
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Correspondence to Danny Geelen.

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Communicated by Scott Russell.

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497_2012_209_MOESM1_ESM.pptx

Suppl. Fig. 1 Volume-based diameter distribution and representative images of mature pollen harvested from kiwifruit (a), poppy (b), lily (c), and lesser bindweed (d); scale bar, 25 µm Supplementary material 1 (PPTX 980 kb)

497_2012_209_MOESM2_ESM.pptx

Suppl. Fig. 2 Size distribution and overlay of pollen isolated from diploid, tetraploid, and octaploid Arabidopsis plants acquired by image-based one-and two-dimensional pollen size analysis (minor and major axis length and transsectional area, respectively) Supplementary material 2 (PPTX 316 kb)

497_2012_209_MOESM3_ESM.pptx

Suppl. Fig. 3 Chromosome spreads of male meiocytes at the start of the second meiotic division in diploid, tetraploid, and triploid Arabidopsis plants Supplementary material 3 (PPTX 888 kb)

497_2012_209_MOESM4_ESM.pptx

Suppl. Table 1 Volume-based pollen size characteristics for a somatic ploidy series in Arabidopsis thaliana Supplementary material 4 (PPTX 46 kb)

497_2012_209_MOESM5_ESM.m

Suppl. File MATLAB-based image analysis software program for the analysis of pollen grain size Supplementary material 5 (M 2 kb)

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De Storme, N., Zamariola, L., Mau, M. et al. Volume-based pollen size analysis: an advanced method to assess somatic and gametophytic ploidy in flowering plants. Plant Reprod 26, 65–81 (2013). https://doi.org/10.1007/s00497-012-0209-0

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