An improved staining protocol for the assessment of arbuscular mycorrhizal in bryophytes

Abstract

The most accepted method for staining arbuscular mycorrhiza (AM) in vascular plants has been one proposed by Phillips & Hayman in 1970. In particular, for the study of AM in bryophytes (s.l.) [Anthocerotophyta, Bryophyta (s.s.), Marchantiophyta] some authors have introduced modifications to this technique. Even though all these protocols stain AM, their main disadvantage is related to the result of material maceration (e.g. over-softening or completely destroying plant cells due to the high temperatures used, the high concentrations of reagents or the long-term exposure to aggressive chemicals). In order to optimise the results for the observation of AM in this group of plants, a modification is presented to the traditional staining technique. In the protocol here proposed, 70% ethanol is used as fixative and first clarifier, 1% potassium hydroxide (KOH) (80 °C, 20 min) as a second clarifier; 1% hydrochloric acid (HCl) (50 °C, 10 min) as an acidifier and 0.05% trypan blue (60 °C, 20 min) for dyeing. This improved protocol is not destructive, it is fast to perform and it is of wide application since it allows staining the AM in bryophytes.

An improved clearing and staining protocol for evaluation of arbuscular mycorrhizal colonisation in darkly pigmented woody roots

“…Higher concentrations of H 2 O 2 may have increased transparency but were not tested in the current study, as it has been reported by Vierheilig et al (2005) to cause the disintegration of fungal hyphae in the tissue. In order to clear the background from any phenolic-like materials, the roots were immersed in 70% ethanol overnight for fixation prior to the clearing process as this has been reported to cause a partial decolourisation that dehydrates the cells which allow a greater penetration of the KOH (Cottet et al 2018). Incubating the roots in ethanol increased the subsequent penetration of the root tissue by potassium hydroxide and eliminated all unwanted background staining resulting in AMF structures being clearly visible to enable quantification.…”

“…For those treatments that included a fixing step, roots were covered with 70% v/v ethanol (~20 mL) as recommended by Fonseca et al (2014) and Cottet et al (2018) and left overnight at room temperature then decanted prior to clearing.…”

“…The concentration of trypan blue has been reduced from 1% to 0.05% over recent years to limit its toxicity. Recently, Cottet et al (2018) in their review of staining methods using trypan blue as a chemical stain suggested that optimisation of specific steps such fixation and clearing could affect the visibility of fungal structures in the roots.…”

“…Potassium hydroxide (KOH) has been widely used as a root-clearing method with various concentrations, temperatures and periods of time employed for different plant species. For example, Kobae & Ohtomo (2016) boiled soybean roots in 10% (w/v) KOH for 15 min, and other variations are detailed by Cottet et al (2018). Fungal structures contained within the root cells, can be deliberately damaged using a heat treatment to allow trypan blue to enter (Wilkes et al 2019).…”

Nuevo registro de hongos micorrícicos arbusculares en Nothoceros fuegiensis (Dendrocerotaceae, Anthocerotophyta)

“…Para observar la colonización por HMA, los gametofitos fueron lavados con agua corriente y preservados en etanol al 70% durante 24 horas a temperatura ambiente; posteriormente se colocaron en cajas de Petri a 50 °C sobre un agitador magnético de calentamiento (VELP Scientifica, Usmate, Velate, Italia), con placa caliente hasta la evaporación total del líquido. Posteriormente, fueron clarificados en hidróxido de potasio al 1% durante 20 minutos a 80 °C; acidificados con ácido clorhídrico al 1% durante 10 minutos a 50 °C y teñidos con azul de tripán al 0.05% durante 20 minutos a 60 °C (Cottet et al, 2018;Cottet y Messuti, 2019). El porcentaje de colonización por HMA se estimó utilizando la presencia o ausencia de las estructuras características de estos hongos con un aumento de 400× en todo el talo (47 talos).…”