为更好地观察枸杞(Lycium barbarum)根系内生丛枝菌根真菌(arbuscular mycorrhiza fungi,AMF)和深色有隔内生真菌(dark seprate endophytes,DSE)侵染定殖特征,建立了一种枸杞根系内生真菌适宜的染色方法.将健康枸杞根样置于100 g/L的KOH溶液中90 ℃水浴90 min,接着加入质量分数30%的H2O2漂白5 min,室温下乳酸酸化5 min后,用5种染色剂(醋酸墨水、台盼蓝、酸性品红、苏丹红Ⅳ和苯胺蓝)于室温染色3~5 min,脱色剂浸泡脱色后镜检.结果表明: 醋酸Quink牌墨水和台盼蓝的染色效果较好,根系皮层细胞内AMF菌丝、泡囊和DSE菌丝、微菌核等结构清晰可见,能准确分辨出AMF、DSE和其他真菌.综合考虑操作难易程度和安全性, 醋酸Quink牌墨水因操作简单、安全无毒、染色效果好,更适用于枸杞根系内生真菌的染色和制片观察.
In order to better observe the infection and colonization characteristics of endophytic arbuscular mycorrhizal fungi(AMF)and dark seprate endophytes(DSE)in Lycium barbarum roots, an optimal staining method of endophytic fungi in L. barbarum roots was established. The healthy L. barbarum roots were placed in 100 g/L KOH solution at 90 ℃ for 90 min, then bleached with 30% H2O2 for 5 min, acidified with lactic acid for 5 min, and dyed with vinegar and ink, trypan blue, acid fuchsin, Sudan Ⅳ and aniline blue for 3—5 min, root samples were soaked in decolorant and then examined under micriscope. The results showed that vinegar and Quink ink and trypan blue had the best staining effect. The hyphae and vesicles of AMF and the hyphae and microsclerotia of DSE in the cortical cells of roots can be clearly seen, which can accurately distinguish AMF, DSE and other fungi. However, considering the difficulty and safety of operation, vinegar and Quink ink is more suitable for staining and observation of endophytic fungi in L. barbarum roots because of its simple operation, safety, non-toxicity and good staining effect.
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