苹果退绿叶斑病毒组织印迹的免疫法检测与定位

Immunological Detection and Localization of CLSV -infected Fuji Apple by Tissue Blotting

用组织培养技术将富士苹果茎尖外植体建立在ＭＳ培养基中，形成试管苗。取４—６周龄的茎与愈伤组织，徒手切取约１ｍｍ厚的横切面，印迹在硝化纤维膜上。印迹组织经封闭后，与ＣＬＳＶ碱性磷酸酶标抗体反应。对反应结果进行显色。感染ＣＬＳＶ的印迹组织呈紫色反应，正常组织无显色反应。结果表明，该方法十分容易检测印迹组织中的ＣＬＳＶ。带毒愈伤组织较茎的显色反应敏感，显色反应时间仅为茎的一半（１５ｍｉｎ）。ＣＬＳＶ在愈伤组织中有两种分布情况：一是所有组织均有显色反应；二是呈不均匀分布。ＣＬＳＶ在茎组织中有三种分布情况：第一，除髓部外，表皮、皮层及维管系统中均有分布；第二，不均匀分布，ＣＬＳＶ集中分布于表度组织中，在皮层及韧皮部仅有少量分布；第三，“嵌合”分布，即同一种组织中部分组织带毒，部分为正常组织。

Shoot-tip explants of Fujiapple were established and micropropagated on MS medium. Callus and mi-croshoot with 4 6 weeks old were used for the present study. Sections(lmm)were cut from fresh tissuesby hand using razor blade,Tissue printings were made by pressing the freshly cut tissue surface on nitrocel-lulose membrances. After tissue blottings were blocked,CLSV antigen in tissue blottings were reacted withCISV-specific alkaline-phosphatase-labelled antibody. Then, the reacting results were coloured. The posi-tive reaction on tissue blottings showed a purple colour, while the negetive one produced no colour.Resultsshowed that CLSV in both shoots and calli was readily detected on tissue bolttings. Colour reaction of CLSV-infected callus was more sensitive than that of CLSV-infected shoot.There exsited two types of CLSV distri-bution in callus,a throughout and an uneven distribution. CLSV distribution in shoot was found in threetypes.Firstly, CLSV was located in epidermis,cortex and Vascular svstem, but never in pith.Secondly,CLSV was unevenly distributed with a main concentration in epidermis and only a quite few in cortex andphloem. Thirdly, CLSV distribution was like a' chimerd',i.e. a part of the same tissue was CLSV-infectedand the other were healthy, The suecessful development of tissue blotting technique for CLSV detection andlocalization opened a new way for studies on fruit tree viruses.