整体透明观察技术是植物形态发育研究的基础手段之一, 是无需制作切片直接观察植物体内部形态结构的有效方法。该技术采用高折射率介质降低光在样品中的散射, 提高光通量, 增加视野深度, 从而实现组织样品透明观察。然而透明剂能改变透明液的渗透势和pH值, 从而对细胞形态保持产生负面影响。目前, 针对植物叶片和胚珠已建立了相对成熟的整体透明观察体系, 但根尖由于细胞壁较薄, 现有的整体透明方法常导致细胞形态改变, 不确定性增加(如根尖整体形态改变和细胞发生严重的质壁分离)。该研究以拟南芥(Arabidopsis thaliana)幼苗为实验材料, 通过检测根尖形态、细胞质壁分离情况和细胞清晰度, 对常用的透明液组分、pH值和透明时间进行优化, 旨在建立一种适用于根尖等较脆弱组织材料的整体透明方法。

Whole mount clearing is a routine method in morphological study, which allows observation of plant internal structure without section. Using high refractive index materials as medium, clearing techniques reduce light scattering, acquire enhancive light quantity and increase depth of field and vertical planes in a particular focal plane, to facilitate the samples transparency for observation. Nevertheless, clearing materials may disturb the osmosis and pH of sample medium, which is adverse to cells morphology. So far, the effective clearing techniques have been widely used in several studies with ovule and leaf. However, the current protocol is not reliable enough for root tip clearing, because the thin cell wall is vulnerable under the treatment of clearing solutions, resulting in abnormal root tips and cells plasmolysis. To achieve a stable and optimized clearing method for root tip, we established a standard protocol via evaluation of root tip morphology, plasmolysis and cells clarity in Arabidopsis thaliana. With these improved clearing methods, we developed an optimized clearing observation system (including clearing time, pH and composition) for root tip, which could provide a reliable technique for vulnerable tissues clearing.