Abstract
The number of pollen grains is a critical part of the reproductive strategies in plants and varies greatly between and within species. In agriculture, pollen viability is important for crop breeding. It is a laborious work to count pollen tubes using a counting chamber under a microscope. Here, we present a method of counting the number of pollen grains using a cell counter. In this method, the counting step is shortened to 3 min per flower, which, in our setting, is more than five times faster than the counting chamber method. This technique is applicable to species with a lower and higher number of pollen grains, as it can count particles in a wide range, from 0 to 20,000 particles, in one measurement. The cell counter also estimates the size of the particles together with the number. Because aborted pollen shows abnormal membrane characteristics and/or a distorted or smaller shape, a cell counter can quantify the number of normal and aborted pollen separately. We explain how to count the number of pollen grains and measure pollen size in Arabidopsis thaliana, Arabidopsis kamchatica, and wheat (Triticum aestivum).
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Shimizu KK, Tsuchimatsu T (2015) Evolution of selfing: recurrent patterns in molecular adaptation. Annu Rev Ecol Evol Syst 46:593–622
Darwin C (1877) The different forms of flowers on plants of the same species. John Murray, London
Sicard A, Lenhard M (2011) The selfing syndrome: a model for studying the genetic and evolutionary basis of morphological adaptation in plants. Ann Bot 107:1433–1443
Oka H-I, Morishima H (1967) Variations in the breeding systems of a wild rice, Oryza perennis. Evolution 21:249–258
De Storme N, Geelen D (2014) The impact of environmental stress on male reproductive development in plants: biological processes and molecular mechanisms. Plant Cell Environ 37:1–18
Dolferus R, Ji X, Richards RA (2011) Abiotic stress and control of grain number in cereals. Plant Sci 181:331–341
Boeven PHG, Longin CFH, Leiser WL et al (2013) Genetic architecture of male floral traits required for hybrid wheat breeding. Theor Appl Genet 129:2343–2357
Wright SI, Kalisz S, Slotte T (2013) Evolutionary consequences of self-fertilization in plants. Proc R Soc B 280:20130133
Tsuchimatsu T, Kakui H, Yamazaki M et al (2020) Adaptive reduction of male gamete number in the selfing plant Arabidopsis thaliana. Nat Commun (provisionally accepted)
Willis JH (1999) The contribution of male-sterility mutations to inbreeding depression in Mimulus guttatus. Heredity 83:337–346
De Vries AP (1974) Some aspects of cross-pollination in wheat (Triticum aestivum L.). 3. Anther length and number of pollen grains per anther. Euphytica 23:11–19
Ortega R, Aresti M, Pereira I (2011) Implementation and evaluation of an image analysis system for determining viability of pollen grains in temperate rice. Chil J Agr Res 71:16–22
Costa CM, Yang S (2009) Counting pollen grains using readily available, free image processing and analysis software. Ann Bot 104:1005–1010
OLS OMNI Life Science (2015) CASY TT cell counter & analyzer; operators guide. https://cellcounting.de/downloads/OLS_CASY_TT-OperatorsGuide_2015.pdf
Heidmann I, Di Berardino M (2017) Impedance flow cytometry as a tool to analyze microspore and pollen quality. In: Schmidt A (ed) Plant germline development. Springer, New York, NY
Schindelin J, Arganda-Carreras I, Frise E et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682
R Core Team (2013) R foundation for statistical computing
Smyth DR, Bowman JL, Meyerowitz EM (1990) Early flower development in Arabidopsis. Plant Cell 2:755–767
Yew C-L, Kakui H, Shimizu KK (2017) Agrobacterium-mediated floral dip transformation of the model polyploid species Arabidopsis kamchatica. J Plant Res 9:1–10
Browne RG, Iacuone S, Li SF et al (2018) Anther morphological development and stage determination in Triticum aestivum. Front Plant Sci 9:1–13
Alexander MP (1969) Differential staining of aborted and nonaborted pollen. Stain Technol 44:117–122
Paape T, Briskine RV, Halstead-Nussloch G et al (2018) Patterns of polymorphism and selection in the subgenomes of the allopolyploid Arabidopsis kamchatica. Nat Commun 9:3909
Singla J, Lüthi L, Wicker T et al (2017) Characterization of Lr75: a partial, broad-spectrum leaf rust resistance gene in wheat. Theor Appl Genet 130:1–12
Acknowledgements
This study was supported by the Swiss National Science Foundation 31003A_182318, a JST CREST Grant Number JPMJCR16O3 and MEXT KAKENHI Grant Number 16H06469 to KKS, and a MEXT KAKENHI Grant Number 19K05976 to H.K. We thank Beat Keller for plant material and Reiko Akiyama and Toshiaki Tameshige for their valuable suggestions.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Kakui, H., Yamazaki, M., Hamaya, NB., Shimizu, K.K. (2020). Pollen Grain Counting Using a Cell Counter. In: Geitmann, A. (eds) Pollen and Pollen Tube Biology. Methods in Molecular Biology, vol 2160. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0672-8_1
Download citation
DOI: https://doi.org/10.1007/978-1-0716-0672-8_1
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0671-1
Online ISBN: 978-1-0716-0672-8
eBook Packages: Springer Protocols