| Abstract: | Recently several DNA-binding fluotochromes have been used for demonstrating pollennuclei. However, the autofluorescence of pollen wall often obscured the fluorescence of nuclei, thus limited the use of this method. Methyl salicylate (MS) as a clearing agent has shownexcellent effect for observing embryo sac in whole-mounted ovules. This aroused me to trya combination of fluorescent staining with MS clearing in orded to make a better demonstration of the pollen nuclei. Mature 2-celled or 3-celled pollen of several angiosperm species stained with Hoechst 33258(H33258) and cleared (via ethanol dehydration) with MS showed clearcut fluorescence oftheir generative or sperm nuclei and vegetative nucleus. MS greatly decreased the wall fluorescence and increased the transparency of the pollen contents, meanwhile maintained the H33258stained fluorescence, consequently made the nuclei brighter under a darkened background. For example, in sunflower pollen a pair of elongated and winding sperm nuclei whichcould not be identified after simple H33258 staining were quite visible after MS clearing, inartificially germinated pollen tubes, the locomotion of nuclei from pollen grain into the tube,the sequence of generative and vegetative nucle travelling along the tube and the division of generative nucleus into two sperm nuclei could be well followed by this method. The present technique may be adoptable for observations on the processes of microsporogenesis and male gametophyte development, and rogenesis in cultured anthers, and also possiblyfor tracing the nuclear events during pollination-fertilization. |
