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

Abstract

Arbuscular mycorrhizal fungi (AMF) establish symbiotic interactions with the roots of vascular plants, including grapevines. Verifying AMF colonisation routinely requires establishing the presence of hyphae, arbuscules and vesicles. Clearing roots with potassium hydroxide (KOH) followed by staining with trypan blue has been used previously to visualise fungal structures, however visualisation is difficult with darkly pigmented roots, such as those of grapevines so additional steps are required to ensure clear visualisation. Three fixing and clearing processes were evaluated prior to staining with trypan blue: 1) fixing grapevine roots in 70% v/v ethanol overnight; 2) clearing by heating the roots in either 2% or 10% w/v KOH; and 3) clearing the roots in 3% v/v hydrogen peroxide for 10 min. Roots were examined under a compound light microscope for the presence of AMF. A combination of fixing grapevine roots in 70% ethanol overnight and clearing by autoclaving in 10% KOH produced the greatest enhancement in subsequent staining of grapevine roots with trypan blue overnight. The best method tested enabled the discrimination of arbuscular mycorrhizal structures in fresh roots of grapevines without the use of toxic chemical fixatives.