Kwiatkowska Monika, Kadłuczka Dariusz, Wędzony Maria, Dedicova Beata, Grzebelus Ewa
1Department of Plant Cytology and Embryology, Institute of Botany, Jagiellonian University, Gronostajowa 9, 30-387 Kraków, Poland.
2Institute of Plant Biology and Biotechnology, Faculty of Biotechnology and Horticulture, University of Agriculture in Krakow, 29 Listopada 54, 31-425 Kraków, Poland.
Plant Methods. 2019 Jul 8;15:71. doi: 10.1186/s13007-019-0452-6. eCollection 2019.
Clearing methods allow relatively quick processing of plant material and examination of cellular structures by rendering tissues and organs translucent. They have been adapted for plant embryology, primarily to study ovule development, megasporogenesis, megagametogenesis and embryogenesis. Such clearing methods overcome several disadvantages of the conventional embedding-sectioning techniques that are arduous and time-consuming. Although numerous protocols with different clearing solutions have been described, there have been no reports to date proposing a reliable method to clear the crassinucellate ovules of the sugar beet ( L.), an economically important crop. Therefore, this study aims to find a suitable approach to improve the tissue transparency of sugar beet ovules at different developmental stages.
We established a methyl salicylate-based protocol that significantly improved the transparency of the ovule structures, which allowed us to observe the megagameto- and embryogenesis of that species. This was achieved by (1) chemical softening of the tissues; (2) vacuum pump-assisted infiltration step; (3) shaking-assisted incubation with clearing mixtures; and (4) manual removal of the chemically softened seed coat.
The effectiveness of our method is due to the strategy combining various approaches at different stages of the procedure aiming at increasing the accessibility of the internal ovule structures to the clearing solution. The results of this study may be applied in sugar beet breeding programs, and it will provide a basis for further investigation of numerous aspects of the species' embryology. Moreover, that unique approach may be easily adapted to other species developing crassinucellate ovules.
透明处理方法能够通过使组织和器官变得半透明，相对快速地处理植物材料并检查细胞结构。它们已被应用于植物胚胎学，主要用于研究胚珠发育、大孢子发生、雌配子体发生和胚胎发生。这种透明处理方法克服了传统包埋切片技术的几个缺点，传统技术既费力又耗时。尽管已经描述了许多使用不同透明溶液的方案，但迄今为止，尚未有报告提出一种可靠的方法来透明处理经济作物甜菜（Beta vulgaris L.）的厚珠心胚珠。因此，本研究旨在找到一种合适的方法来提高不同发育阶段甜菜胚珠的组织透明度。
我们建立了一种基于水杨酸甲酯的方案，该方案显著提高了胚珠结构的透明度，使我们能够观察该物种的雌配子体和胚胎发生。这是通过以下方式实现的：（1）组织的化学软化；（2）真空泵辅助渗透步骤；（3）与透明混合物振荡辅助孵育；（4）手动去除化学软化的种皮。
我们方法的有效性归因于在该过程的不同阶段结合各种方法的策略，旨在增加透明溶液对胚珠内部结构的可及性。本研究结果可应用于甜菜育种计划，并将为该物种胚胎学众多方面的进一步研究提供基础。此外，这种独特的方法可能很容易适用于其他发育厚珠心胚珠的物种。
