微波快速石蜡切片法观察谷子幼嫩叶片组织技术的优化

Optimization of Microwave Paraffin Techniques for Clear Observation of Cell Structures of Millet(Setaria italica) Young Leaves

C4植物发育的研究是近年植物学研究的一个热点,对于探明C4高光效机理及培育C4水稻至关重要。而C4幼嫩叶片的细胞形态学观察是研究C4发育的基本手段之一。谷子是研究C4光合发育的模式植物之一,且作为禾本科植物,谷子幼嫩叶片硅质含量高,组织硅质化,固定液透过表皮进入组织困难。同时幼嫩叶片细胞含水量高,如利用常规方法短时间内脱水剂不容易将细胞组织液彻底替换,这就给谷子幼叶发育的组织学观察带来一定的难度。微波是一种高频电磁波,可以使试剂液体与组织细胞间渗透和交换效率显著提高。本试验以谷子幼嫩叶片为材料,对以往的微波快速石蜡切片法进行优化,将微波快速石蜡切片法与梯度酒精脱水法相结合,并使用叔丁醇代替二甲苯作为脱水剂和透明剂,改变脱水时间,摸索出一套制备完整的谷子幼嫩叶片组织切片的快捷、有效的方法。

It is a hot spot on plant sciences to study on C4 leaf development which is crucial for the discovery on the molecular mechanisms of its high photosynthetic capacity and for the final breeding of C4 rice. As a basic approach to study C4 development, it is usually necessary to observe cell morphology of C4 young leaves. Millet(Setaria italica) is a modern plant to study C4 photosynthetic development, however, as a grass, the content of siliceous materials in leaves is high even at young stage, resulting in silicification of tissues. When conducting conventional paraffin sectioning, it is difficult for fixative solution to permeat through epidermal cells.Furthermore, in young leaves of millet, water content of cells is also very high, making it difficult for thorough exchange between dehydrants and tissue fluid. These bring certain difficulties for histological obervation of its leaves. Microwave is a kind of high frequent electric magnetic wave which can significantly increase permeability and exchange rate between the chemical reagents and cellular fluid, therefore it was widely used for paraffin section making recently. In this study, microwave paraffin techniques were further optimized in order for clear observation of cell structures of millet young leaves. By combining quick microwave paraffin section methods and dehydration using grading ethanol, and substituting paraxylene for tert-butanol as dehydrating and clearing agent,complete and clear tissue sections of millet young leaves were obtained.