Imaging arbuscular mycorrhizal structures in living roots of Nicotiana tabacum by light, epifluorescence, and confocal laser scanning microscopy

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Abstract
Light and epifluorescence (blue light excitation) microscopy was used to obtain micrographs of the same sections of unstained (living roots) and stained (dead) tobacco (Nicotiana tabacum L.) roots colonized by the arbuscular mycorrhizal fungus Glomus mosseae (Nicol. & Gerd.) Gerd. & Trappe. To visualize all mycorrhizal structures, roots were in situ stained with trypan blue. The metabolically active fungal tissue was determined by an in situ succinate dehydrogenase stain. A comparison of micrographs of unstained and stained mycorrhizal tobacco roots revealed that (i) finely branched arbuscules do not autofluoresce, but high autofluorescence was observed in clumped structures of collapsed arbuscules; and (ii) finely branched arbuscules are metabolically active, but no activity can be detected in autofluorescent collapsed arbuscules. Confocal laser scanning microscopy was used in combination with the two fluorochromes 5(6)-carboxyfluorescein diacetate or 5(6)-carboxy-seminaphthorhodafluor. Both fluorochromes administered to abraded tobacco leaves are transported via the phloem to the roots. Loading plants with 5(6)-carboxyfluorescein diacetate resulted in a fluorescence of root cells with highly branched arbuscules. After loading the phloem with 5(6)-carboxy-seminaphthorhodafluor, all fungal structures in the root (from relatively thick hyphae to finest branches of arbuscules) were clearly visible in the intact root. The transport route of compounds from the plants to arbuscular mycorrhizal fungi is discussed.Key words: Glomales, mycorrhiza, fluorescence, SDH, confocal, transport.