Abstract
Surface sterilization of seeds is a key step in providing microorganisms-free seeds for numerous applications like understanding the role of seed-borne microorganisms in plant development, studying microbial cells-plant interactions by inoculating model microorganisms in a simplified system or selective cultivation of seed endobionts. However applying efficient treatment for surface sterilization of seeds without affecting the plant growth is not an easy task. In this study we aimed to provide an efficient surface sterilization treatment for maize seeds using i) hydrogen peroxide (HP), ii) sodium hypochlorite (SH) and iii) ethanol-sodium hypochlorite (EtOH-SH) under stirring (st) and vacuum-stirring (va-st) conditions. We used fluorescence microscopy and ultra-high resolution Helium Ion Microscopy (HIM) as powerful imaging approaches in combination with macroscopic techniques to visualize, quantify and evaluate the efficiency of seed sterilization, quality of root germination, seedlings and root hair development as well as the presence or absence of microorganisms on the root surface. Our results showed a strong reduction in microbial cell numbers of 4 orders of magnitude after the EtOH-SH treatments. Moreover, seeds exposed to EtOH-SH treatments displayed the lowest percentage of microbial growth (50%) and the highest percentage of germinated seeds (100%) compared to other sterilization treatments. HIM imaging proved the absence of microbial cells on the roots grown from seeds exposed to EtOH-SH treatments. Moreover, root hair development seemed not to be affected by any of the sterilization treatments. Our findings demonstrated that EtOH-SH treatments are significantly reducing the abundance of microbial cells from the surface of maize seeds and can be used with high confidence in future studies.
Highlights
Seeds are the starting point of a growing plant and seed-borne microorganisms are primary inoculum source of the plant associated microbial community [1]
This result suggests that the EtOH-SH treatment is efficient in removing seed associated microorganisms
The quantitative and qualitative results of our study showed that EtOH-SH is the most efficient treatment for maize seed sterilization, drastically reducing the number of microbial cells on seeds surfaces, up to four orders of magnitude in comparison with HP and SH treatments
Summary
Seeds are the starting point of a growing plant and seed-borne microorganisms are primary inoculum source of the plant associated microbial community [1]. The surface of seeds is normally colonized by microorganisms passed on from their mother plant (early colonizers) and via seed contact with fruit microorganisms (late colonizers) [1]. Another source of microorganisms colonizing seeds is soil in which the seeds are growing [2, 3]. High resolution microscopy of surface sterilization of maize seeds de/). This project was carried out in the framework of the priority program 2089 “Rhizosphere spatiotemporal organization—a key to rhizosphere functions” funded by DFG.
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