Adaptation of the traditional arbuscular mycorrhizal staining technique for its study in bryophytes

SCERVINO, J.M.; MESSUTI, M.I.; COTTET, A.C.

Valdivia

Workshop; Symbiosis in the Southern of Cone of South America. International Workshop; 2017

Universidad Austral de Chile y otros

The most accepted traditional method for staining arbuscular mycorrhiza (AM) in vascular plants is the one proposed by Phillips & Hayman (1970). This method consists in fixing the root samples in FAA; later, clarifying them with 10% potassium hydroxide (KOH) (60 minutes, 90 °C); acidifying them with diluted hydrochloric acid (HCl); and staining with 0.05% Trypan-blue in lactophenol (5 minutes, room temperature). In particular, for the study of AM in plants included in bryophytes (s.l.), Anthocerophyta, Bryophyta (s.s.) and Marchantiophyta, some authors have introduced some modifications to this technique. For example, for Anthocerophyta, Schüßler (2000) clarifies the sample with 10% KOH (10 minutes, 121 °C) and acidifies with 3.7% HCl; for Bryophyta (s.s.), Zhang & Guo (2007) fix the material with 50% ethanol, clarify with 10% KOH (20 minutes, 90 °C), acidify with lactic acid (3 minutes, room temperature) and stain with 0.5% acid fuchsin (20 minutes, 92 °C); and for Marchantiophyta, Silvani et al. (2012) clarify with 15% KOH (48 hrs, 25 °C), acidify with 4% HCl and stain with trypan-blue in 0.1% lactic acid. Even though all these protocols stain AM hyphae, their main disadvantage is related to the result of maceration of the material by over-softening or completely destroying plant cells due to the high temperatures used, the reagents high concentrations or the long time exposure of the material to the chemicals. In order to optimize the results for the observation of arbuscular fungi in this group of plants, a new modification is presented to the techniques proposed above, using 70% ethanol to fix and as a first clarifier, 1% KOH (20 minutes, 80 °C) as a second clarifier; 1% HCl (10 minutes, 50 °C) as an acidifier and 0.05% Trypan-blue (20 minutes, 60 °C) for dyeing.