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.

Centre for Functional Ecology Department of Life Sciences University of Coimbra Coimbra Portugal

Department of Botany Faculty of Science Charles University Prague Czech Republic

Department of Evolutionary Plant Biology Institute of Botany of the Czech Academy of Sciences Průhonice Czech Republic

Department of Integrative Biology University of Guelph Guelph Ontario Canada

Zobrazit více v PubMed

Brewbaker JL. The distribution and phylogenetic significance of binucleate and trinucleate pollen grains in the angiosperms. Am J Bot. 1967;54:1069-83.

Pacini E, Franchi GG, Ripaccioli M. Ripe pollen structure and histochemistry of some gymnosperms. Plant Syst Evol. 1999;217:81-99.

Hirano T, Hoshino Y. Detection of changes in the nuclear phase and evaluation of male germ units by flow cytometry during in vitro pollen tube growth in Alstroemeria aurea. J Plant Res. 2009;122(2):225-34. https://doi.org/10.1007/s10265-008-0208-2.

Kron P, Husband BC. Using flow cytometry to estimate pollen DNA content: improved methodology and applications. Ann Bot. 2012;110(5):1067-78. https://doi.org/10.1093/aob/mcs167.

Yan F, Zhang Z, Lin Y, Li Y, Zhang L, Peng J, et al. Subpollens delivery of Platanus acerifolia pollen allergen Pla a3 and nucleic acid into lungs and cells. Biochem Biophys Res Commun. 2019;513:767-e774.

Borges F, Gardner R, Lopes T, Calarco JP, Boavida LC, Slotkin RK, et al. FACS-based purification of Arabidopsis microspores, sperm cells and vegetative nuclei. Plant Methods. 2012;8(44):44. https://doi.org/10.1186/1746-4811-8-44.

Pauls KP, Chan J, Woronuk G, Schulze D, Brazolot J. When microspores decide to become embryos - cellular and molecular changes. Can J Bot. 2006;84(4):668-78. https://doi.org/10.1139/b06-064.

Schulze D, Pauls KP. Flow cytometric characterization of embryogenic and gametophytic development in Brassica napus microspore cultures. Plant Cell Physiol. 1998;39(2):226-34.

Santos MR, Bispo C, Becker JD. Isolation of Arabidopsis pollen, sperm cells, and vegetative nuclei by fluorescence-activated cell sorting (FACS). In: Schmidt A, editor. Plant germline development: methods and protocols. New York: Humana Press; 2017. p. 193-210. https://doi.org/10.1007/978-1-4939-7286-9_16.

Kreiner JM, Kron P, Husband BC. Frequency and maintenance of unreduced gametes in natural plant populations: associations with reproductive mode, life history and genome size. New Phytologist. 2017;214(2):879-89. https://doi.org/10.1111/nph.14423.

Kron P, Husband BC. Distinguishing 2N gamete nuclei from doublets in pollen using flow cytometry and pulse analysis. Cytometry A. 2015;87(10):943-57. https://doi.org/10.1002/cyto.a.22696.

Misra CS, Santos MR, Rafael-Fernandes M, Martins NP, Monteiro M, Becker JD. Transcriptomics of Arabidopsis sperm cells at single-cell resolution. Plant Reproduction. 2019;32:29-38. https://doi.org/10.1007/s00497-018-00355-4.

Bishop JW.(2015) Chromosome pairing and its relationship to fertility in cytotypes of prairie cordgrass (Spartina pectinata link). [MSc thesis], Urbana, Illinois.

Wu D, Ruban A, Fuchs J, Macas J, Novák P, Vaio M, et al. Nondisjunction and unequal spindle organization accompany the drive of Aegilops speltoides B chromosomes. New Phytol. 2019;223:1340-52. https://doi.org/10.1111/nph.15875.

Nikoloudakis N, Aissat A, Katsiotis A. Screening A. ventricosa populations for 2n gametes. Euphytica. 2018;214(2):34. https://doi.org/10.1007/s10681-017-2107-x.

Sora D, Kron P, Husband BC. Genetic and environmental determinants of unreduced gamete production in Brassica napus, Sinapis arvensis and their hybrids. Heredity. 2016;117:440-8. https://doi.org/10.1038/hdy.2016.69.

Lepší M, Koutecký P, Nosková J, Lepší P, Urfus T, Rich TCG. Versatility of reproductive modes and ploidy level interactions in Sorbus s.l. (Malinae, Rosaceae). Bot J Linn Soc. 2019;191:502-22. https://doi.org/10.1093/botlinnean/boz054.

Dewitte A, Eeckhaut T, Van Huylenbroeck J, Van Bockstaele E. Occurrence of viable unreduced pollen in a Begonia collection. Euphytica. 2009;168(1):81-94. https://doi.org/10.1007/s10681-009-9891-x.

Roberts AV. The use of bead beating to prepare suspensions of nuclei for flow cytometry from fresh leaves, herbarium leaves, petals and pollen. Cytometry A. 2007;71A(12):1039-44. https://doi.org/10.1002/cyto.a.20486.

Kuo LY, Huang YJ, Chang J, Chiou WL, Huang YM. Evaluating the spore genome sizes of ferns and lycophytes: a flow cytometry approach. New Phytol. 2017;213(4):1974-83. https://doi.org/10.1111/nph.14291.

Pan G, Zhou Y, Fowke LC, Wang H. An efficient method for flow cytometric analysis of pollen and detection of 2n nuclei in Brassica napus pollen. Plant Cell Rep. 2004;23(4):196-202. https://doi.org/10.1007/s00299-004-0830-y.

Schoft VK, Chumak N, Bindics J, Slusarz L, Twell D, Köhler C, et al. SYBR green-activated sorting of Arabidopsis pollen nuclei based on different DNA/RNA content. Plant Reproduction. 2015;28(1):61-72. https://doi.org/10.1007/s00497-015-0258-2.

Tennant RK, Jones RT, Brock F, Cook C, Turney CSM, Love J, et al. A new flow cytometry method enabling rapid purification of fossil pollen from terrestrial sediments for AMS radiocarbon dating. J Quat Sci. 2013;28(3):229-36. https://doi.org/10.1002/jqs.2606.

Mitsumoto K, Yabusaki K, Kobayashi K, Aoyagi H. Development of a novel real-time pollen-sorting counter using species-specific pollen autofluorescence. Aerobiologia. 2010;26(2):99-111. https://doi.org/10.1007/s10453-009-9147-1.

SRH Z, Brown TA, Hassel C, Heck J. Testing pollen sorted by flow cytometry as the basis for high-resolution lacustrine chronologies. Radiocarbon. 2019;61(1):359-74. https://doi.org/10.1017/RDC.2018.89.

Urbanczyk J, Casado MAF, Díaz TE, Heras P, Infante M, Borrego AG. Spectral fluorescence variation of pollen and spores from recent peat-forming plants. Int J Coal Geol. 2014;131:263-73.

Moon HS, Eda S, Saxton AM, Ow DW, Stewart CN Jr. An efficient and rapid transgenic pollen screening and detection method using flow cytometry. Biotechnol J. 2011;6(1):118-23. https://doi.org/10.1002/biot.201000258.

Negron A, DeLeon-Rodriguez N, Waters SM, Ziemba LD, Anderson B, Bergin M, et al. Using flow cytometry and light-induced fluorescence to characterize the variability and characteristics of bioaerosols in springtime in metro Atlanta, Georgia. Atmos Chem Phys. 2020;20:1817-38. https://doi.org/10.5194/acp-20-1817-2020.

Braun P, Winkelmann T. Flow cytometric analyses of somatic and pollen nuclei in midday flowers (Aizoaceae). Caryologia. 2016;69(4):303-14. https://doi.org/10.1080/00087114.2016.1188359.

Sharpless TK, Melamed MR. Estimation of cell size from pulse shape in flow cytofluorometry. J Histochem Cytochem. 1976;24(1):257-64. https://doi.org/10.1177/24.1.1254921.

Wersto RP, Chrest FJ, Leary JF, Morris C, Stetler-Stevenson M, Gabrielson E. Doublet discrimination in DNA cell-cycle analysis. Cytometry. 2001;46(5):296-306. https://doi.org/10.1002/cyto.1171.

O'Connor DJ, Iacopino D, Healy DA, O'Sullivan D, Sodeau JR. The intrinsic fluorescence spectra of selected pollen and fungal spores. Atmos Environ. 2011;45(35):6451-8. https://doi.org/10.1016/j.atmosenv.2011.07.044.

Parducci L, Bennett KD, Ficetola GF, Alsos IG, Suyama Y, Wood JR, et al. Ancient plant DNA in lake sediments. New Phytol. 2017;214(3):924-42. https://doi.org/10.1111/nph.14470.

Suda J, Kron P, Husband BC, Trávníček P. Flow cytometry and ploidy: applications in plant systematics, ecology and evolutionary biology. In: Doležel J, Greilhuber J, Suda J, editors. Flow Cytometry with plant cells. Weinheim: Wiley-VCH; 2007. p. 103-30.

Kellenberger RT, Byers KJRP, De Brito Francisco RM, Staedler YM, LaFountain AM, Schönenberger J, et al. Emergence of a floral colour polymorphism by pollinator-mediated overdominance. Nat Commun. 2019;10(1):63. https://doi.org/10.1038/s41467-018-07936-x.

Trávníček P, Ponert J, Urfus T, Jersáková J, Vrána J, Hřibová E, et al. Challenges of flow-cytometric estimation of nuclear genome size in orchids, a plant group with both whole-genome and progressively partial endoreplication. Cytometry A. 2015;87(10):958-66. https://doi.org/10.1002/cyto.a.22681.

Kesidis CE. 2019 The effect of formation pathway on allopolyploids between Brassica carinata, Brassica napus, Brassica juncea and Sinapis arvensis. [MSc. thesis] Carleton University, Ottawa

Bao S, Shen G, Li G, Liu Z, Arif M, Wei Q, et al. The Arabidopsis nucleoporin NUP1 is essential for megasporogenesis and early stages of pollen development. Plant Cell Rep. 2019;38(1):59-74. https://doi.org/10.1007/s00299-018-2349-7.

Mieulet D, Jolivet S, Rivard M, Cromer L, Vernet A, Mayonove P, et al. Turning rice meiosis into mitosis. Cell Res. 2016;26:1242-54. https://doi.org/10.1038/cr.2016.117.

Doležel J, Greilhuber J, Suda J. Estimation of nuclear DNA content in plants using flow cytometry. Nat Protoc. 2007;2(9):2233-44.

Pohlker C, Huffman JA, Poschl U. Autofluorescence of atmospheric bioaerosols - fluorescent biomolecules and potential interferences. Atmos Meas Tech. 2012;5:37-71. https://doi.org/10.5194/amt-5-37-2012.

Lo KH, Pauls KP. Plant growth environment effects on rapeseed microspore development and culture. Plant Physiol. 1992;99(2):468-72. https://doi.org/10.1104/pp.99.2.468.

Deng H. Relationship between nuclear DNA-content of sperm cells and the timing of events in the cell cycle of Brassica campestris L.(Brassicaceae). Int J Plant Biol. 2016;7(1):6336.

Heidmann I, Schade-Kampmann G, Lambalk J, Ottiger M, Di Berardino M. Impedance flow cytometry: a novel technique in pollen analysis. PLoS One. 2016;11(11):e0165531. https://doi.org/10.1371/journal.pone.0165531.

Yelina NE, Ziolkowski PA, Miller N, Zhao X, Kelly KA, Muñoz DF, et al. High-throughput analysis of meiotic crossover frequency and interference via flow cytometry of fluorescent pollen in Arabidopsis thaliana. Nat Protoc. 2013;8:2119-34. https://doi.org/10.1038/nprot.2013.131.

van Tuyl JM, de Vries JN, Bino RJ, Kwakkenbos TAM. Identification of 2n-pollen producing interspecific hybrids of Lilium using flow cytometry. Cytologia. 1989;54(4):737-45. https://doi.org/10.1508/cytologia.54.737.

Vašková D, Kolarčik V. Breeding systems in diploid and polyploid hawthorns (Crataegus): evidence from experimental pollinations of C. monogyna, C. subsphaerica, and natural hybrids. Forests. 2019;10:1059. https://doi.org/10.3390/f10121059.

Boluarte, T. 1999.Bulk segregant analysis for anther culture response and leptine content in backcross families of diploid potato. [PhD Thesis], Virginia Polytechnic Institute and State University, Virginia.

Caldeira CF, Abranches CB, Gastauer M, Ramos SJ, Guimarães JTF, Pereira JBS, et al. Sporeling regeneration and ex situ growth of Isoëtes cangae (Isoetaceae): initial steps towards the conservation of a rare Amazonian quillwort. Aquat Bot. 2019;152:51-8. https://doi.org/10.1016/j.aquabot.2018.10.001.

Zhang G, Campenot MK, McGann LE, Cass DD. Flow cytometric characteristics of sperm cells isolated from pollen of Zea mays L. Plant Physiol. 1992;99(1):54-9.

Błocka-Wandas M, Sliwinska E, Grabowska-Joachimiak A, Musial K, Joachimiak AJ. Male gametophyte development and two different DNA classes of pollen grains in Rumex acetosa L., a plant with an XX/XY1Y2 sex chromosome system and a female-biased sex ratio. Sex Plant Reprod. 2007;20(4):171-80. https://doi.org/10.1007/s00497-007-0053-9.

Stehlik I, Kron P, Barrett SCH, Husband BC. Sexing pollen reveals female bias in a dioecious plant. New Phytol. 2007;175(1):185-94. https://doi.org/10.1111/j.1469-8137.2007.02093.x.

Luria G, Rutley N, Lazar I, Harper JF, Miller G. Direct analysis of pollen fitness by flow cytometry: implications for pollen response to stress. Plant J. 2019;98(5):942-52. https://doi.org/10.1111/tpj.14286.

Ren R, Jiang X, Di W, Li Z, Li B, Xu J, et al. HSP70 improves the viability of cryopreserved Paeonia lactiflora pollen by regulating oxidative stress and apoptosis-like programmed cell death events. Plant Cell Tissue Organ Cult. 2019;139:53-64. https://doi.org/10.1007/s11240-019-01661-z.

Impe D, Reitz J, Köpnick C, Rolletschek H, Börner A, Senula A, et al. Assessment of pollen viability for wheat. Front Plant Sci. 2020;10:1588. https://doi.org/10.3389/fpls.2019.01588.

Kwok A, Husband BC, Kron P. Flow cytometric analysis of pollen grains collected from individual bees provides information about pollen load composition and foraging behaviour. Ann Bot. 2013;113(1):191-7. https://doi.org/10.1093/aob/mct257.

Laport RG, Minckley RL, Pilson D. Pollinator assemblage and pollen load differences on sympatric diploid and tetraploid cytotypes of the desert-dominant Larrea tridentata. Am J Bot. 2021;108(2):1-12. https://doi.org/10.1002/ajb2.1605.

Kleiber A, Ramoji A, Mayer G, Neugebauer U, Popp J, Henkel T. 3-step flow focusing enables multidirectional imaging of bioparticles for imaging flow cytometry. Lab Chip. 2020;20(9):1676-86.

Sauvageat E, Zeder Y, Auderset K, Calpini B, Clot B, Crouzy B, et al. Real-time pollen monitoring using digital holography. Atmos Meas Tech. 2020;13:1539-50. https://doi.org/10.5194/amt-13-1539-2020.

Bell KL, de Vere N, Keller A, Richardson RT, Gous A, Burgess KS, et al. Pollen DNA barcoding: current applications and future prospects. Genome. 2016;59:629-40. https://doi.org/10.1139/gen-2015-0200.