Plant Cell Reports

, Volume 23, Issue 4, pp 196–202

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

Authors

  • Gang Pan
    • Department of BiochemistryUniversity of Saskatchewan
    • National Key Laboratory of Crop Genetic ImprovementHuazhong Agricultural University
  • Yongming Zhou
    • National Key Laboratory of Crop Genetic ImprovementHuazhong Agricultural University
    • Department of BiologyUniversity of Saskatchewan
  • Larry C. Fowke
    • Department of BiologyUniversity of Saskatchewan
    • Department of BiochemistryUniversity of Saskatchewan
Cell Biology and Morphogenesis

DOI: 10.1007/s00299-004-0830-y

Cite this article as:
Pan, G., Zhou, Y., Fowke, L.C. et al. Plant Cell Rep (2004) 23: 196. doi:10.1007/s00299-004-0830-y

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.

Keywords

Flow cytometryPollen nucleiDNA contentBrassica napusTriticum aestivum

Abbreviations

DAPI

4′,6-Diamidino-2-phenylindole

NIB

Nuclear isolation buffer

Copyright information

© Springer-Verlag 2004