走査型電子顕微鏡による冠さび菌感染エンバク葉内部の観察 : 試料作製法の検討

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Present study was conducted to examine the procedures of sample preparation for three dimensional observations of internal structures of crown rust-infected oat leaves. Plant materials used were adult leaves of cultivar Pc 38 naturally infected with compatible race 226 of Puccinia coronata f. sp. avenae. Methods examined are as follows: (1) Non-fixation followed by freeze drying and gold-coating, (2) Glutaraldehyde fixation followed by freeze fracturing in isoamyl acetate, critical point drying and gold-coating, (3)Glutaraldehyde-OsO4 fixation followed by the same procedure as in method 2, (4)-(7) Glutaraldehyde-OsO4 fixation and treatment with tannic acid and OsO4 for electric conduction staining, (4) Freeze fractured in isoamyl acetate followed by critical point drying and gold-coating, (5) The same method as in 4 without gold-coating, (6) Freeze fractured in absolute ethanol followed by critical point drying and gold-coating, (7) The same method as in 6 without gold-coating, (8) Glutaraldehyde-OsO4 fixation followed by paraffin sectioning and gold-coating. The surface of intercellular hyphae and haustoria was smooth in non-fixed sample (method 1). In sample fixed by the methods 2-7, however, filamentous and cottony structures were observed on the surfaces of fungal organs and in host cytoplasm. In all samples observed, nucleus, chloroplast and mitochondoria were clearly recognized in methophyll cells, but other organelle such as endoplasmic reticulum and Golgi apparatus were hardly visible. Outer surface of mesophyll cells was smooth. Among eight procedures tested, the method 2 was the best for the fine structure of both hyphal organs and mesophyll cells in the sense of shrinkage and electric conductivity. The use of the method 1 is also recomened since this method provides the observations possible without elution of any soluble materials out of the internal surface.走査型電子顕微鏡を用いてエンパク冠さび菌感染葉内を立体的に観察するために試料の固定, 割断方法および導電法などについて検討した. 1. 葉肉細胞内を錯綜した細胞間菌糸の収縮は無固定-凍結乾燥試料では著しく, 固定試料では少なかった. しかし, いずれの試料作製法でも菌糸表面は滑沢であった. 吸器表面は無固定-凍結乾燥試料では滑沢であるが, 固定試料においては, その表面は葉肉細胞内で認められる繊維状あるいは綿屑状の構造物が付着していた. 葉肉細胞内で核, 葉緑体とミトコンドリアは明瞭に観察されるが, いずれの手法によっても小胞体, ゴルジ体などは識別できなかった. 葉肉細胞の表層は無固定-凍結乾燥および固定試料ともに滑沢であった. 2. 試験した8種類の方法のうち立体構造を観察するには, グルタールアルデヒドで固定後凍結割断し, 臨界点乾燥した試料作製(Method 2)が最も適していた. なお, 無固定-凍結乾燥試料で感染葉内を十分に観察できたことから, 葉肉細胞表層における水あるいは有機溶媒可溶性物質は保存されているものと推定される.
1982-10-20

走査型電子顕微鏡による冠さび菌感染エンバク葉内部の観察 : 試料作製法の検討

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