Isolation of High Purity Tissues from Developing Barley Seeds
Isolation of High Purity Tissues from Developing Barley Seeds
复制标题
DOI：
10.3791/61681
复制
发表时间：
2020-10-01
影响因子：
1.2
通讯作者：
Pecinka, Ales
中科院分区：
文献类型：
--
作者：
Kovacik, Martin;Nowicka, Anna;Pecinka, Ales
了解谷类种子发育的调控机制对于植物育种和提高产量至关重要。然而，谷物种子的分析是具有挑战性的，由于微小的尺寸，一些组织的液体特性，以及紧密的组织间连接。在这里，我们展示了一个详细的协议解剖的胚胎，胚乳和种子母体组织在早期，中期和后期阶段的大麦种子发育。该方案基于使用细尖工具和双目显微镜进行手动组织解剖，然后进行基于倍性分析的纯度控制。种子的母体组织和胚为二倍体，胚乳为三倍体。这允许使用流式细胞术监测样品纯度。额外的测量揭示了从这些样品中分离的RNA的高质量及其用于高灵敏度分析的可用性。总之，该方案描述了如何从栽培大麦和潜在的其他谷物的发育中的谷粒中实际解剖纯组织。
Understanding the mechanisms regulating the development of cereal seeds is essential for plant breeding and increasing yield. However, the analysis of cereal seeds is challenging owing to the minute size, the liquid character of some tissues, and the tight inter-tissue connections. Here, we demonstrate a detailed protocol for dissection of the embryo, endosperm, and seed maternal tissues at early, middle, and late stages of barley seed development. The protocol is based on a manual tissue dissection using fine-pointed tools and a binocular microscope, followed by ploidy analysis-based purity control. Seed maternal tissues and embryos are diploid, while the endosperm is triploid tissue. This allows the monitoring of sample purity using flow cytometry. Additional measurements revealed the high quality of RNA isolated from such samples and their usability for high-sensitivity analysis. In conclusion, this protocol describes how to practically dissect pure tissues from developing grains of cultivated barley and potentially also other cereals.