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Rapid separation of Arabidopsis male gametophyte developmental stages using a Percoll gradient

Nature Protocols volume 11, pages 1817–1832 (2016)Cite this article

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Abstract

Research investigating the dynamics of male gametophyte (MG) development has proven to be challenging for the plant science community. Here we describe our protocol for separating Arabidopsis MG developmental stages, which is based on the centrifugation of pollen through a discontinuous Percoll concentration gradient. This Percoll gradient can be formed using a pipette, and it does not require a gradient maker. The purity of the isolated developing spores is as high as 70%, and in most separations it is well above 80%. Using this protocol, we can separate four different stages of pollen development—uninucleate microspore (UNM), bicellular pollen (BCP), tricellular immature pollen (TCP) and mature pollen grain (MPG). The duration of the separation procedure, excluding the cutting of flower inflorescences, is 6 h. This is reduced to 4 h when using a vacuum cleaning method to remove the MPGs before the Percoll density separation.

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Figure 1: Flowchart of the Percoll step gradient separation procedure.
Figure 2: DAPI staining of cell nuclei for the purity assessment of individual male gametophyte spore fractions after Percoll separation.
Figure 3: Pollen viability was evaluated with cell-permeable esterase-substrate fluorescein diacetate (FDA) and the cell-impermeant nucleic acid stain propidium iodide (PI).
Figure 4: Pollen viability test by Alexander staining is applicable for each fraction that was obtained during Percoll separation procedures (A1, A2, A3, B1, B2 and B3), including MPG.
Figure 5: Harvesting and homogenization of A. thaliana Col-0 (wild-type) flower inflorescences.
Figure 6: Percoll step gradients.
Figure 7: Individual collected fractions from the Percoll separation procedure.

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Acknowledgements

We thank J. Fíla for his careful reading of the manuscript, J. Novotney for English editing and I. Jelínková for sowing and collection of plant material. N.D. thanks V. Čapková for her support. This work was supported by the Czech Science Foundation (grants 13-41444P to N.D. and P305/12/2611 to D.H.).

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Authors and Affiliations

  1. Laboratory of Pollen Biology, Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Prague, Czech Republic

    Nikoleta Dupl'áková, David Reňák & David Honys

  2. Laboratory of Hormonal Regulations in Plants, Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Prague,, Czech Republic

    Petre I Dobrev

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  1. Nikoleta Dupl'áková
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  2. Petre I Dobrev
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  4. David Honys
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Contributions

N.D., D.R. and D.H. designed the protocol. N.D. performed the experiments. P.I.D. performed the HPLC/MS analysis. N.D. and D.H. wrote the manuscript.

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Correspondence to Nikoleta Dupl'áková or David Honys.

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The authors declare no competing financial interests.

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Dupl'áková, N., Dobrev, P., Reňák, D. et al. Rapid separation of Arabidopsis male gametophyte developmental stages using a Percoll gradient. Nat Protoc 11, 1817–1832 (2016). https://doi.org/10.1038/nprot.2016.107

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