[Isolation of sperm cells from rice pollen tubes].

ABSTRACT

Isolation of sperm cells from higher plants is a basis for studying the mechanism of double fertilization. In this study, the isolation of rice sperm cells from pollen tube was conducted. When fresh pollen grains from nearly blooming flowers were put into a medium containing 20% sucrose, 10% polyethylene glycol 4500 (PEG 4500), 0.05% CaCl2, 0.01% boric acid, over 40% pollen grains germinated and formed a pollen tube. After pollen tubes were transformed into a broken solution containing 8% mannitol, the tubes broke and released tube cytoplasm including two sperm cells. However, both sperm cells were enrapt in the cytoplasm and could not be identified. When 0.5% cellulase and pectinase were added into the broken solution, two sperm cells were released from cytoplasm. Both sperm cells could be collected using micromanipulator. We also tried to isolate sperm cells using in vivo-in vitro method: styles were pollinated and pollen tubes were allowed to grow for 40 min in vivo. Then styles were cut near ovary and floated in the same medium above-mentioned for 1 h until tubes emerged from the cut end. The styles with pollen tube were transformed into the broken solution and released the content including two sperm cells.