School of Life Sciences, Xiamen University, Xiamen, 361005, China.
School of Agricultural Science, Ningxia University, Yinchuan, 750021, China.
Plant Reprod. 2020 Mar;33(1):35-40. doi: 10.1007/s00497-020-00383-z. Epub 2020 Jan 29.
Sperm 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.
可以采用渗透压冲击的方法从药用野生稻（Oryza officinalis）成熟花粉粒中分离精子细胞，通过酶解和机械切割步骤分离有活力的卵母细胞。通过关联分析已知功能基因与目标性状表现，已经在自然品种和野生稻中鉴定出了适合水稻育种的有利等位基因。将这些基因从野生稻转移到栽培稻品种中是水稻育种者的重要目标之一。野生稻和栽培稻精子和卵子的分离是远缘栽培稻和野生稻系体外杂交的前提条件。在这里，我们提供了一种分离野生稻（Oryza officinalis）精子和卵子的技术方法。在该方法中，根据渗透压冲击法从药用野生稻（O. officinalis）成熟花粉粒中分离精子细胞。此外，还通过酶解和机械切割步骤分离有活力的 O. officinalis 卵母细胞。具体来说，将胚珠在含有果胶酶和纤维素酶的酶溶液中消化 30 分钟，然后将胚珠切成两半。通过轻轻向珠孔端施加压力，释放出三个卵器细胞。通常，从 20 个胚珠中可以在 60 分钟内分离出 6 到 7 个卵母细胞。同样的方法也用于分离授粉后 24 小时的花朵中的合子。这项技术解决了 O. officinalis 中分离精子和卵子的困难，并允许通过远缘野生和栽培稻系的体外杂交来结合分离的精子和卵子。
