Key message

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.

Abstract

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.

中文翻译：

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分离野生稻（Oryza officinalis）中的精子和卵细胞，作为改善作物的机制。

关键信息

可以使用渗透压休克方法从药用野生稻（Oryza officinalis）的成熟花粉粒中分离出精子细胞，并可以通过酶消化和机械解剖步骤分离出活的卵细胞。

抽象

通过对已知功能基因与靶性状表现的关联分析，已经在自然栽培品种和野生稻中鉴定了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育种者的重要目标之一。野生稻和栽培稻的精子和卵细胞的分离是远距离栽培稻和野生稻品系体外杂交的先决条件。在这里，我们提供了一种分离野生稻（Oryza officinalis）精子和卵细胞的技术方法。在这种方法中，根据渗透休克法从药用野生稻（O. officinalis）的成熟花粉粒中分离出精子细胞。另外，可行的O. officinalis酶消化和机械解剖步骤分离出卵细胞。具体而言，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。通过轻轻地向毛孔末端施加压力来释放三个卵器细胞。通常，在60分钟内可以从20个胚珠中分离出六个或七个卵细胞。授粉后24小时，用相同的方法从花中分离受精卵。该技术解决了在分离精子和卵细胞的难度O.巴戟并使其通过远亲野生和栽培稻的行体外杂交组合的分离的精子和卵细胞。