蝴蝶兰花器官冰冻切片技术探讨

A study on Phalaenopsis floral organ cryo-sectioning technique

通过对蝴蝶兰的子房、花瓣、唇瓣和合蕊柱进行切片,建立了蔗糖保护-液氮冷冻的冰冻切片技术体系。将蝴蝶兰花器官经过蔗糖磷酸缓冲液保护液处理后抽真空,再经过液氮速冻、固定、包埋、切片、展片观察、染色以及拍照,制作出蝴蝶兰花器官较完整的显微结构切片。研究结果表明,蝴蝶兰花器官冰冻切片最适条件为:(1)蔗糖磷酸缓冲液处理的最适质量分数为子房4%,花瓣8%,唇瓣4%,合蕊柱16%;(2)最适切片厚度为子房15μm,花瓣10μm,唇瓣20μm,合蕊柱10μm;(3)子房、花瓣和唇瓣最适染色方法均为番红单染色法,合蕊柱最适染色方法为番红-固绿双染色法,4种花器官均不适合单独使用固绿染色。

A sucrose protection-liquid nitrogen cryo-sectioning technology system was established by slicing the ovary,petal,lip and gynostemium of Phalaenopsis .That is designed to make the relatively complete microstructure sections of Phalaenopsis organ section through vacuum the phalaenopsis organs after being treated with sucrose phosphate buffer protection solution,and then freezed by the liquid nitrogen,fixing,embedding,film slicing,observation,staining and photographing.The results showed that the optimal conditions for cryo-sectioning of Phalaenopsis were as follows:(1) Optimum treatment of quality scoresucrose phosphate buffer was 4% sucrose in PBS for ovary,8% sucrose in PBS for petal,4% sucrose in PBS for lip,and 16% sucrose in PBS for gynostemium;(2) The optimum slice thickness is ovary 15 μm,petal 10 μm,lip flap 20 μm and gynostemium 10 μm;(3) The best staining solution was red stain for the three organs,ovary,petal and lip,whereas for the gynostemium was a red-solid green stain solution.These four floral organs were not suitable for solid green dyeing alone used.