高校生物の重複受精に関する授業への活用にむけた中性赤を用いた生体染色法による雄原細胞と精細胞の挙動の観察

抄録

Vital staining of germinating pollen grains with neutral red (NR) revealed that, in some members of the Liliaceae, Amaryllidaceae, and Iridaceae, a spindle-shaped, reddish area (NR-area) was observed within each pollen tube by about 10 hours after pollen dispersal on the surface of agar plate, which contained NR at a concentration of 10 μg/ml. The NR-area subsequently divided into two NR-areas. The resultant two NR-areas moved slowly within the pollen tube toward its growing tip. Each of the NR-areas contained a spherical or oblong, transparent region at its center. When some pollen tubes were stained with SYBR Green I, the transparent region emitted a green fluorescence characteristic of DNA- SYBR Green I complex, indicating its correspondence to the nucleus. In the oriental lily‘Siberia’, mitotic figures were observed within pollen tubes at the stage of the division of NR-area. These light and fluorescence microscope observations indicate that the NR-area and the resultant two NR-areas correspond to generative cell and sperm cells, respectively. Electron microscopy demonstrated that many small vacuoles, which were probably the entities responsible for the stainability of the generative cell with NR, were present within the cytoplasm of generative cell in the oriental lily‘Siberia’. The staining of germinating pollen grains with NR can readily be carried out and enable to chase the behavior of generative cell and sperm cells within pollen tubes under the conventional light microscope. The light microscope observation on the behavior of generative and sperm cells within pollen tubes employing the vital staining with NR may be applicable to the classes for teaching the role of pollen in double fertilization in high school biology.