A simple and rapid method for imaging male meiotic cells in anthers of model and non-model plant species

Key message

We describe a simple method to view meiotic cells in whole anthers from a range of plants. The method retains spatial organisation and enables simultaneous analysis of many meiotic cells.

Abstract

Understanding the process of male meiosis in flowering plants, and the role of genes involved in this process, offers potential for plant breeding, such as through increasing the level of genetic variation or the manipulation of ploidy levels in the gametes. A key to the characterisation of meiotic gene function and meiosis in non-model crop plants, is the analysis of cells undergoing meiosis, a task made difficult by the inaccessible nature of these cells. Here, we describe a simple and rapid method to analyse plant male meiosis in intact anthers in a range of plant species. This method allows analysis of numerous cells undergoing meiosis and, as meiotic cells stay within the anther, it retains information of the three-dimensional organisation and the location of organelles in meiotic cells. We show that the technique provides information on male meiosis by looking at the synchrony of meiotic progression between and within locules, and comparing wildtype and mutant plants through the chromosome separation stages in Arabidopsis thaliana. Additionally, we demonstrate that the protocol can be adopted to other plants with different floral morphology using Medicago truncatula as an example with small floral buds and the non-model plant kiwifruit (Actinidia chinensis) with larger buds and anthers.

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Acknowledgements

We would like to thank Shona Seymour at Plant & Food Research for the collection of kiwifruit budwood. This work was funded by the Department of Biochemistry, University of Otago, New Zealand and the Endeavour Fund from the Ministry of Business Innovation and Employment (Smart Idea UOOX1801).

Funding

Department of Biochemistry, University of Otago and Ministry of Business Innovation and Employment Endeavour Fund (Smart Idea UOOX1801).

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CR carried out research, analysed data, wrote the manuscript. LLL carried out research, analysed data, SP designed and supported research. LB designed and carried out research, analysed data, wrote the manuscript. All authors read and approved the manuscript.

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Correspondence to Lynette Brownfield.

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Communicated by Dolf Weijers.

Supplementary Information

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A z-series whole intact anther of Arabidopsis thaliana Ler. Meiocytes in two locules can be observed with the top locule contained meiocytes of telophase II and the bottom locule meiocytes at metaphase II. A single optical section of this anther is shown in Online Resource 1 (AVI 5233 kb)

A z-series whole intact anther of Arabidopsis thaliana Col-0. From left to right locules contain: meiotic products close to cytokinesis; telophase II with the two meiotic divisions are complete; metaphase I with homologous chromosomes aligned; Interphase between the two meiotic divisions at the bottom of the anther to metaphase II with two sets of sister chromatins aligned at the top. A single section of this anther is shown in Fig. 2a (AVI 7031 kb)

A z-series whole intact anther of Arabidopsis thaliana Col-0. From top to bottom locules contain: anaphase I, telophase II, telophase II and prometaphase/metaphase II (AVI 11,999 kb)

Z-series of part of a locule in an anther from a wild type  A. thaliana Col-0 plant with meiocytes in diakinesis. A z-projection of this region is shown in Fig. 3a (AVI 2023 kb)

Z-series of part of a locule in an anther from a ps1 A. thaliana Col-0 plant with meiocytes in diakinesis. A z-projection of this region is shown in Fig. 3b (AVI 556 kb)

Z-series of part of a locule in an anther from a jas A. thaliana Col-0 plant with meiocytes in diakinesis. A z-projection of this region is shown in Fig. 3 (AVI 936 kb)

Z-series of part of a locule in an anther from a wild type A. thaliana Col-0 plant with meiocytes in metaphase I. A z-projection of this region is shown in Fig. 3d (AVI 620 kb)

Z-series of part of a locule in an anther from a w ps1 A. thaliana Col-0 plant with meiocytes in metaphase I. A z-projection of this region is shown in Fig. 3e (AVI 570 kb)

Z-series of part of a locule in an anther from a  jas A. thaliana Col-0 plant with meiocytes in metaphase I. A z-projection of this region is shown in Fig. 3f (AVI 369 kb)

Z-series of part of a locule in an anther from a wild type A. thaliana Col-0 plant with meiocytes in anaphase I. A z-projection of this region is shown in Fig. 3g (AVI 751 kb)

Z-series of part of a locule in an anther from a  ps1 A. thaliana Col-0 plant with meiocytes in anaphase I. A z-projection of this region is shown in Fig. 3h (AVI 638 kb)

Z-series of part of a locule in an anther from a jas A. thaliana Col-0 plant with meiocytes in anaphase I. A z-projection of this region is shown in Fig. 3i (AVI 710 kb)

Z-series of part of a locule in an anther from a wild type A. thaliana Col-0 planti with meiocytes in interphase. A z-projection of this region is shown in Fig. 3j (AVI 565 kb)

Z-series of part of a locule in an anther from a ps1 A. thaliana Col-0 plant with meiocytes in interphase. A z-projection of this region is shown in Fig. 3k (AVI 1171 kb)

Z-series of part of a locule in an anther from a jas A. thaliana Col-0 plant with meiocytes in interphase. A z-projection of this region is shown in Fig. 3l (AVI 723 kb)

Z-series of part of a locule in an anther from a wild type A. thaliana Col-0 plant with meiocytes in metaphase II. A z-projection of this region is shown in Fig. 3m (AVI 862 kb)

Z-series of part of a locule in an anther from a ps1 A. thaliana Col-0 plant with meiocytes in metaphase II. A z-projection of this region is shown in Fig. 3n (AVI 668 kb)

Z-series of part of a locule in an anther from a jas A. thaliana Col-0 plant with meiocytes in metaphase II. A z-projection of this region is shown in Fig. 3 (AVI 562 kb)

Z-series of part of a locule in an anther from a wild type A. thaliana Col-0 plant with meiocytes in anaphase II. A z-projection of this region is shown in Fig. 3p (AVI 1024 kb)

Z-series of part of a locule in an anther from a ps1 A. thaliana Col-0 plant with meiocytes in anaphase II. A z-projection of this region is shown in Fig. 3q (AVI 819 kb)

Z-series of part of a locule in an anther from a jas A. thaliana Col-0 plant with meiocytes in anaphase II. A z-projection of this region is shown in Fig. 3r (AVI 1117 kb)

A z-series of an intact anther of Medicago truncatula. Chromosomal DNA and organelles have been stained with DAPI. From left to right locules contain: metaphase II, metaphase I, prometaphase II and telophase II. A single section of this anther is shown in Fig. 4a (AVI 3448 kb)

A z-series of an intact anther of Medicago truncatula. Chromosomal DNA and organelles have been stained with DAPI. From left to right locules contain: metaphase I, interphase and telophase II. A single section of this anther is shown in Fig. 4b (AVI 4380 kb)

Online Resource 1

Single optical section from CLSM analysis of an anther from A. thaliana Ler. The anther was isolated from a flower bud, chromosomal and organelle DNA stained with DAPI and observed with CLSM. A z-series for this anther is shown in Online Resource 2. Two locules are visible, one with meiocytes of metaphase II (MII) and one at telophase II (TII). Not all nuclei are visible in the optical section. An arrow indicates a binucleate tapetal cell and an arrowhead indicates an organelle band between two chromosome groups in meiosis II. Scale, 50 µm (TIF 5538 kb)

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Rossig, C., Le Lievre, L., Pilkington, S.M. et al. A simple and rapid method for imaging male meiotic cells in anthers of model and non-model plant species. Plant Reprod 34, 37–46 (2021). https://doi.org/10.1007/s00497-021-00404-5

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Keywords

  • Male meiosis
  • Meiocytes
  • Anther
  • Confocal laser scanning microscopy
  • Fluorescent staining