AgNO3 Sterilizes Grains of Barley (Hordeum vulgare) without Inhibiting Germination—A Necessary Tool for Plant–Microbiome Research

Abstract

To understand and manipulate the interactions between plants and microorganisms, sterile seeds are a necessity. The seed microbiome (inside and surface microorganisms) is unknown for most plant species and seed-borne microorganisms can persist and transfer to the seedling and rhizosphere, thereby obscuring the effects that purposely introduced microorganisms have on plants. This necessitates that these unidentified, seed-borne microorganisms are removed before seeds are used for studies on plant-microbiome interactions. Unfortunately, there is no single, standardized protocol for seed sterilization, hampering progress in experimental plant growth promotion and our study shows that commonly applied sterilization protocols for barley grains using H 2 O 2 , NaClO, and AgNO 3 yielded insufficient sterilization. We therefore developed a sterilization protocol with AgNO 3 by testing several concentrations of AgNO 3 and added two additional steps: Soaking the grains in water before the sterilization and rinsing with salt water (1% (w/w) NaCl) after the sterilization. The most efficient sterilization protocol was to soak the grains, sterilize with 10% (w/w) AgNO 3 , and to rinse with salt water. By following those three steps, 97% of the grains had no culturable, viable microorganism after 21 days based on microscopic inspection. The protocol left small quantities of AgNO 3 residue on the grain, maintained germination percentage similar to unsterilized grains, and plant biomass was unaltered. Hence, our protocol using AgNO 3 can be used successfully for experiments on plant-microbiome interactions.

Soil microorganisms decrease barley biomass uniformly across contrasting nitrogen availability

“…Evergreen is a high-yielding (87.3 hkg/ha), mlo-resistant cultivar suited for organic agriculture. We sterilized 200 undamaged grains within the weight range of 55±5 mg in 250 ml 1% AgNO3 (w/w) for five minutes and rinsed in 30 ml sterile ddH2O [46], five consecutive times. Sterilized grains were germinated in the dark (15 °C, 48 hours) on Potato Dextrose Agar plates (39 g L -1 ddH2O, Sigma) with ten grains per plate.…”

“…Sterilized grains were germinated in the dark (15 °C, 48 hours) on Potato Dextrose Agar plates (39 g L -1 ddH2O, Sigma) with ten grains per plate. The sterilization procedure should result in 88% sterility [46], so we only used grains from non-contaminated plates. A single grain was placed in each microcosm in the middle of the largest pipe.…”