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An efficient method for flow cytometric analysis of pollen and detection of 2n nuclei in Brassica napus pollen

  • Cell Biology and Morphogenesis
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Abstract

A simple and reliable method was developed for isolating pollen nuclei from Brassica napus and Triticum aestivum for DNA analysis using flow cytometry. The nuclei were released from pollen by ultrasonic treatment. The isolated nuclei following filtration through nylon mesh and a purification procedure were suitable for flow cytometric analysis as well as for isolating genomic DNA. Ultrasonic treatment time was optimized for B. napus pollen at different developmental stages. The method is effective and suitable for the preparation of many samples. We analyzed the nuclear DNA levels in pollen of B. napus at three major developmental stages as well as in mature wheat pollen. Only a single 1C peak representing the haploid DNA level was detected in the nuclei isolated from Brassica uninucleate microspores as well as in mature Triticum pollen. Interestingly, diploid nuclei were detected in both binucleate and mature pollen of B. napus. The possible origins of the diploid nuclei are discussed.

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Fig. 1a–c
Fig. 2a,b
Fig. 3a–e
Fig. 4a–d

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Abbreviations

DAPI:

4′,6-Diamidino-2-phenylindole

NIB:

Nuclear isolation buffer

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Acknowledgements

Financial support from the Natural Sciences and Engineering Research Council of Canada (HW, LF) and funding from Canada Foundation for Innovation (HW) for the benchtop flow cytometer is gratefully acknowledged. PG was financially supported in part by a National Natural Science Foundation of China grant (30070479) to YZ.

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Authors and Affiliations

  1. Department of Biochemistry, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK S7N 5E5, Canada

    Gang Pan & Hong Wang

  2. National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, 430070, China

    Gang Pan & Yongming Zhou

  3. Department of Biology, University of Saskatchewan, Saskatoon, SK S7N 5E2, Canada

    Yongming Zhou & Larry C. Fowke

Authors
  1. Gang Pan
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  2. Yongming Zhou
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  3. Larry C. Fowke
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  4. Hong Wang
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Correspondence to Hong Wang.

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Communicated by S. Gleddie

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

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  • DOI: https://doi.org/10.1007/s00299-004-0830-y

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