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

Victoria Munkager,Eva De Visser,Andreas Altenburger,Mette Vestergård,Anders Priemé,Flemming Ekelund,Jesper Liengaard Johansen

doi:10.3390/plants9030372

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 H2O2, NaClO, and AgNO3 yielded insufficient sterilization. We therefore developed a sterilization protocol with AgNO3 by testing several concentrations of AgNO3 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) AgNO3, 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 AgNO3 residue on the grain, maintained germination percentage similar to unsterilized grains, and plant biomass was unaltered. Hence, our protocol using AgNO3 can be used successfully for experiments on plant–microbiome interactions.

Highlights

Seeds are inhabited by a diverse array of viable microorganisms, which can affect subsequent plant development [1]
The term sterility will be used to describe conditions where no microorganisms on or near the grain or seedling were identified with a dissecting microscope after 21 days
The results from Step 1 (Section 4.2), aimed at finding the best sterilization agent to proceed with, revealed that 9% (w/w) NaClO and 3% (w/w) H2 O2 resulted in only 6.7% and 0% sterility respectively, rendering 1% (w/w) AgNO3, with 83.3% sterility (Figure 1) the most promising candidate for developing a protocol for sterilizing barley grains

Summary

Introduction

Seeds are inhabited by a diverse array of viable microorganisms, which can affect subsequent plant development [1]. To identify the effects of specific, purposely introduced microorganisms on plant growth requires that unidentified seed-borne microorganisms are removed. Seed-borne microorganisms are located on the seed surface (epiphytes) or inside the seed (endophytes) [2]. They belong primarily to the phyla Proteobacteria, Actinobacteria, Firmicutes, and to a lesser extend. Bacteroidetes [1,6,7,8]. This dominance of the phyla Proteobacteria, Actinobacteria, and Firmicutes as grain endophytes, has been shown for barley [9,10]. Fungal endophytes mostly belong to either of the two classes Dothideomycetes and Tremellomycetes [2]

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Conclusion