Isolation of the sperm and egg cells in wild rice (Oryza officinalis) as a mechanism for crop improvement.

key messageSperm cells can be isolated from the mature pollen grains of medicinal wild rice (Oryza officinalis) using an osmotic shock method, and the viable egg cells can be isolated by enzymatic digestion and mechanical dissection steps. Favorable alleles for rice breeding have been identified in natural cultivars and wild rice by association analysis of known functional genes with target trait performance. Transferring these genes from wild rice into cultivated rice varieties is one of the important objectives for rice breeders. The isolation of the sperm and egg cells of wild and cultivated rice is a prerequisite for the in vitro hybridization of distantly related cultivated rice and wild rice lines. Here, we provide a technical approach for isolating the sperm and egg cells of wild rice (Oryza officinalis). In this method, sperm cells were isolated from the mature pollen grains of medicinal wild rice (O. officinalis) according to an osmotic shock method. Additionally, viable O. officinalis egg cells were isolated following enzymatic digestion and mechanical dissection steps. Specifically, ovules were digested in an enzymatic solution containing pectinase and cellulase for 30 min, after which the ovule was cut into two halves. Three egg apparatus cells were released by gently applying pressure to the micropylar end. Generally, six or seven egg cells could be isolated from 20 ovules in 60 min. The same method was used to isolate zygotes from flowers at 24 h after pollination. This technology solved the difficulty of isolating sperm and egg cells in O. officinalis and allowed the isolated sperm and egg cells to be combined by in vitro hybridization of distantly related wild and cultivated rice lines.