Microwave-assisted technology for the clearing and staining of arbuscular mycorrhizal fungi in roots

Abstract

The use of microwave irradiation as a source of energy to clear and stain intra-radical arbuscular mycorrhizal fungi propagules has been tested on a variety of indigenous and cultivated herbaceous plants. The aim of the study was to evaluate the efficiency of microwave irradiation on root softening, fungi tissue staining, and preservation of DNA integrity for subsequent molecular analyses. The proposed methodology has been adapted from the standard procedures used to detect and quantify mycorrhizal root colonization levels. Using a domestic microwave oven, tissue clearing and staining required together between 30 s and 1.5 min of microwave treatment to be completed, depending the diameter size of the roots. The well-performing chemical stains tested were acid fuchsin, trypan blue, and aniline blue. The acid fuchsin clearing and staining processes, as performed, were also demonstrated to preserve DNA integrity for further molecular analyses. Irradiation by microwaves has been used with success in our laboratory within the frame of several studies. It offers considerable time saving over traditional method, reducing processing times from several hours to a few minutes while decreasing considerably the amount of chemicals and energy required to perform analyses.

Arbuscular Mycorrhizal Fungal Assemblages Significantly Shifted upon Bacterial Inoculation in Non-Contaminated and Petroleum-Contaminated Environments

“…Composite root samples from each plant cluster were surface rinsed in tap water to remove any remaining soil debris and cut in fragments 1 cm long. They were then cleared in a 10% KOH solution, acidified using acetic acid, and stained with trypan blue, following a microwave-assisted protocol [42]. Root fragments were mounted onto microscope slides in lactoglycerol medium (300 mL lactic acid, 300 mL glycerol, 400 mL double-distilled water), and colonization percentages were evaluated using the magnified intersections method [43], with 150 intersects examined per plant cluster for the presence of mycorrhizal structures.…”

Soil and seasons affect arbuscular mycorrhizal fungi associated with Tamarix rhizosphere in arid and semi-arid steppes

“…Tamarix roots required 5 min H 2 O 2 bath to remove pigments. A microwave modified staining procedure was used to bleach and stain roots (Phillips and Hayman, 1970;Dalpé and Seguin, 2013). Roots were bleached in KOH 5%, 25 s in microwave oven (800 W), rinsed three times in water and acidified 5 min in HCl 1 N. Half of the roots were stained in Trypan blue (5%) in lactic acid glycerol water and half in Fuschsin acid (4%) in lactic acid glycerol water (1:1:1, v:v:v) for 45 s in microwave (800 W).…”

Growth Behavior of Coleus (Plectranthus scutellarioides L.) Cuttings In Relation To Light Emitting Diodes (LED) Intensities

“…Willink in 1851 and due to its rapid growth rate and superior performance in the landscape, coleus quickly gained popularity as a bedding plant [1]. In Papua New Guinea, it is used as a Food additive, while in Southeast Asia it is considered a medicinal plant and used to treat a variety of ailments including dyspepsia, ophthalmia, and wound infections [1,3].…”