Moreno-Sanz P, D'Amato E, Nebish A, Costantini L, Grando M S
1Center Agriculture Food Environment (C3A), University of Trento, Via. E. Mach 1, 38010 San Michele all'Adige, Italy.
2Research and Innovation Centre, Fondazione Edmund Mach, Via E. Mach 1, 38010 San Michele all'Adige, Italy.
Plant Methods. 2020 May 1;16:61. doi: 10.1186/s13007-020-00604-6. eCollection 2020.
Reproductive success in seed plants depends on a healthy fruit and seed set. Normal seed development in the angiosperms requires the production of functional female gametophytes. This is particularly evident in seedless cultivars where defects during megagametophyte's developmental processes have been observed through cytohistological analysis. Several protocols for embryo sac histological analyses in grapevine are reported in literature, mainly based on resin- or paraffin-embedding approaches. However their description is not always fully exhaustive and sometimes they consist of long and laborious steps. The use of different stains is also documented, some of them, such as hematoxylin, requiring long oxidation periods of the dye-solution before using it (from 2 to 6 months) and/or with a differentiation step not easy to handle. Paraffin-embedding associated to examination with light microscope is the simplest methodology, and with less requirements in terms of expertise and costs, achieving a satisfactory resolution for basic histological observations. Safranin O and fast green FCF is an easy staining combination that has been applied in embryological studies of several plant species.
Here we describe in detail a paraffin-embedding method for the examination of grapevine ovules at different phenological stages. The histological sample preparation process takes 1 day and a half. Sections of 5 µm thickness can be obtained and good contrast is achieved with the safranin O and fast green FCF staining combination. The method allows the observation of megasporogenesis and megagametogenesis events in the different phenological stages examined.
The histological sample preparation process proposed here can be used as a routine procedure to obtain embedded ovaries or microscope slides that would require further steps for examination. We suggest the tested staining combination as a simple and viable technique for basic screenings about the presence in grapevine of a normally and fully developed ovule with embryo sac cells, which is therefore potentially functional.
种子植物的繁殖成功取决于健康的果实和种子形成。被子植物中正常的种子发育需要产生功能性的雌配子体。这在无核品种中尤为明显，通过细胞组织学分析已观察到雌配子体发育过程中的缺陷。文献中报道了几种用于葡萄胚囊组织学分析的方法，主要基于树脂或石蜡包埋法。然而，它们的描述并不总是详尽无遗的，有时还包括冗长且费力的步骤。不同染色剂的使用也有记录，其中一些，如苏木精，在使用前需要对染液进行长时间氧化（2至6个月），和/或需要一个不易操作的分化步骤。与光学显微镜检查相关的石蜡包埋是最简单的方法，在专业知识和成本方面要求较低，能为基本的组织学观察提供令人满意的分辨率。番红O和固绿FCF是一种简单的染色组合，已应用于几种植物的胚胎学研究。
在此，我们详细描述了一种石蜡包埋方法，用于检查不同物候期的葡萄胚珠。组织学样本制备过程需要一天半时间。可以获得5微米厚的切片，番红O和固绿FCF染色组合能实现良好的对比度。该方法可观察在所检查的不同物候期的大孢子发生和雌配子体发生事件。
这里提出的组织学样本制备过程可作为一种常规程序，用于获得需要进一步检查步骤的包埋子房或显微镜载玻片。我们建议将经过测试的染色组合作为一种简单可行的技术，用于对葡萄中是否存在具有胚囊细胞且正常发育完全因而可能具有功能的胚珠进行基本筛选。
