Isolation of Male and Female Gametes of Rice

To improve the quality of rice (Oryza sativa L. ssp. japonica auct.) sperm cells for various types of studies, we developed a method for isolation involving the in vivo-in vitro method, in which the stigma was pollinated and pollen tubes were allowed to elongate in vivo for a period. Then styles were cut near the ovary and floated on the surface of a medium until pollen tubes emerged. Over 63% of the rice pollen grains opened and released their contents, including the two sperm cells that are present in pollen grains, during incubation in a medium containing 5 to 25% sucrose or 7.5 to 20% glucose. With 10% polyethylene glycol (PEG) (molecular weight 4500) added to the medium, >40% of the pollen grains germinated and formed short pollen tubes. After transfer of the pollen with short tubes to a bursting solution containing 8% mannitol, many tubes burst and released two sperm cells. Hundreds of living egg cells were also isolated from blooming flowers, and living zygotes were isolated from flowers at 6 h after anthesis using dissection or enzymatic maceration combined with micromanipulation. The successful isolation of these rice sperm and egg cells facilitates obtaining sufficient material to allow molecular studies of rice male and female gametes.