AgNO<sub>3</sub> Sterilizes Grains of Barley (<i>Hordeum vulgare</i>) without Inhibiting Germination—A Necessary Tool for Plant–Microbiome Research
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 ob...
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doaj-b0e623d01d3746b8956f3360b00833532020-11-25T02:32:09ZengMDPI AGPlants2223-77472020-03-019337210.3390/plants9030372plants9030372AgNO<sub>3</sub> Sterilizes Grains of Barley (<i>Hordeum vulgare</i>) without Inhibiting Germination—A Necessary Tool for Plant–Microbiome ResearchVictoria Munkager0Mette Vestergård1Anders Priemé2Andreas Altenburger3Eva de Visser4Jesper Liengaard Johansen5Flemming Ekelund6Department of Biology, University of Copenhagen, 2100 Copenhagen, DenmarkDepartment of Agroecology, Aarhus University, 4200 Slagelse, DenmarkDepartment of Biology, University of Copenhagen, 2100 Copenhagen, DenmarkThe Arctic University Museum of Norway, UiT—The Arctic University of Norway, 9006 Tromsø, NorwayDepartment of Biology, University of Copenhagen, 2100 Copenhagen, DenmarkDepartment of Biology, University of Copenhagen, 2100 Copenhagen, DenmarkDepartment of Biology, University of Copenhagen, 2100 Copenhagen, DenmarkTo 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<sub>2</sub>O<sub>2</sub>, NaClO, and AgNO<sub>3</sub> yielded insufficient sterilization. We therefore developed a sterilization protocol with AgNO<sub>3</sub> by testing several concentrations of AgNO<sub>3</sub> and added two additional steps: Soaking the grains in water before the sterilization and rinsing with salt water (1% (<i>w</i>/<i>w</i>) NaCl) after the sterilization. The most efficient sterilization protocol was to soak the grains, sterilize with 10% (<i>w</i>/<i>w</i>) AgNO<sub>3</sub>, 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<sub>3</sub> residue on the grain, maintained germination percentage similar to unsterilized grains, and plant biomass was unaltered. Hence, our protocol using AgNO<sub>3</sub> can be used successfully for experiments on plant−microbiome interactions.https://www.mdpi.com/2223-7747/9/3/372barley<i>hordeum vulgare</i>seed sterilizationgrain sterilizationagno<sub>3</sub>silver nitrateplant–microbiomeendophyte sterilization |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Victoria Munkager Mette Vestergård Anders Priemé Andreas Altenburger Eva de Visser Jesper Liengaard Johansen Flemming Ekelund |
spellingShingle |
Victoria Munkager Mette Vestergård Anders Priemé Andreas Altenburger Eva de Visser Jesper Liengaard Johansen Flemming Ekelund AgNO<sub>3</sub> Sterilizes Grains of Barley (<i>Hordeum vulgare</i>) without Inhibiting Germination—A Necessary Tool for Plant–Microbiome Research Plants barley <i>hordeum vulgare</i> seed sterilization grain sterilization agno<sub>3</sub> silver nitrate plant–microbiome endophyte sterilization |
author_facet |
Victoria Munkager Mette Vestergård Anders Priemé Andreas Altenburger Eva de Visser Jesper Liengaard Johansen Flemming Ekelund |
author_sort |
Victoria Munkager |
title |
AgNO<sub>3</sub> Sterilizes Grains of Barley (<i>Hordeum vulgare</i>) without Inhibiting Germination—A Necessary Tool for Plant–Microbiome Research |
title_short |
AgNO<sub>3</sub> Sterilizes Grains of Barley (<i>Hordeum vulgare</i>) without Inhibiting Germination—A Necessary Tool for Plant–Microbiome Research |
title_full |
AgNO<sub>3</sub> Sterilizes Grains of Barley (<i>Hordeum vulgare</i>) without Inhibiting Germination—A Necessary Tool for Plant–Microbiome Research |
title_fullStr |
AgNO<sub>3</sub> Sterilizes Grains of Barley (<i>Hordeum vulgare</i>) without Inhibiting Germination—A Necessary Tool for Plant–Microbiome Research |
title_full_unstemmed |
AgNO<sub>3</sub> Sterilizes Grains of Barley (<i>Hordeum vulgare</i>) without Inhibiting Germination—A Necessary Tool for Plant–Microbiome Research |
title_sort |
agno<sub>3</sub> sterilizes grains of barley (<i>hordeum vulgare</i>) without inhibiting germination—a necessary tool for plant–microbiome research |
publisher |
MDPI AG |
series |
Plants |
issn |
2223-7747 |
publishDate |
2020-03-01 |
description |
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<sub>2</sub>O<sub>2</sub>, NaClO, and AgNO<sub>3</sub> yielded insufficient sterilization. We therefore developed a sterilization protocol with AgNO<sub>3</sub> by testing several concentrations of AgNO<sub>3</sub> and added two additional steps: Soaking the grains in water before the sterilization and rinsing with salt water (1% (<i>w</i>/<i>w</i>) NaCl) after the sterilization. The most efficient sterilization protocol was to soak the grains, sterilize with 10% (<i>w</i>/<i>w</i>) AgNO<sub>3</sub>, 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<sub>3</sub> residue on the grain, maintained germination percentage similar to unsterilized grains, and plant biomass was unaltered. Hence, our protocol using AgNO<sub>3</sub> can be used successfully for experiments on plant−microbiome interactions. |
topic |
barley <i>hordeum vulgare</i> seed sterilization grain sterilization agno<sub>3</sub> silver nitrate plant–microbiome endophyte sterilization |
url |
https://www.mdpi.com/2223-7747/9/3/372 |
work_keys_str_mv |
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