The Observation of Pollen and Pollen Tube of Cedrus deodara with Laser Scanning Confocal Microscope

Abstract: Aims:The aim of this study was to reveal the structure of pollen grain and demonstrated nuclei in the pollen and the artificially germinated pollen tube of Cedrus deodara. Methods: Following Carnoy fixation, the pollen and pollen tube samples were respectively treated with Ehrlich，s hematoxylin, eosin or Hoechst 33342 single-staining method and eosin/Hoechst33342 double-staining method, cleared in methyl salicylate and finally observed with Leica TCS SP2 laser scanning confocal microscope（LSCM）. Results: Different staining methods produced different results, but the eosin/Hoechst33342 double-staining method was believed to be the best. When the sample ,which was doubly stained with eosin and Hoechst33342, was excited by UV, the LSCM image showed three nuclei in one pollen grain clearly. When the sample was excited by 488nm laser, the LSCM image showed not only the pollen wall or pollen tube wall, but also the structure and spacial position of the tube cell, the body cell and the stalk cell in pollen grain or pollen tube. Conclusion: An ease and rapid procedure had been developed for the observation of pollen structure and the demonstration of nuclei in pollen and pollen tube with LSCM. Due to the critical features of confocal arrangement, serial optical scanning, advanced three-dimensional reconstruction and simultaneous multi-channel imaging, the LSCM could directly and visually display the three-dimensional architecture of the pollen and pollen tube with much more clarity than previously possible. The results of the present study suggested that the LSCM was an ideal tool to study the pollen biology.