High resolution microscopy to evaluate the efficiency of surface sterilization of Zea Mays seeds

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.

Fig 1. Experimental design and the applied methods.

Fig 2. Seeds germination and seedling development after exposure to different sterilization treatments.

Images depicting seeds exposed to different sterilization treatments and grown on filter paper for 3 days (a-f) and 7 days (h-m) as well as untreated seeds (g, n). Scale bar in (a) is applicable for (h, g, n). Scale bar in (b) is applicable for (c, d, e, f, i, j, k, l, m).

Fig 3. Microbial growth on seeds exposed to different sterilization treatments.

Macroscopic images showing microbial growth on seeds exposed to sterilization treatments and grown on PDA for 3 days (a-f) and 7 days (h-m) as well as untreated seeds (g, n). Black arrows show first sign of microbial growth after 3 days. Scale bar in (a) is applicable for all panels.

Fig 4. Seeds germination vs microbial growth after sterilization treatments.

Germination (blue bars) and microbial growth (red line-dot) depicted as % and calculated after 7 days growth on filter paper (for germination) and on PDA (for microbial growth) of seeds sterilized by different treatments.

Fig 5. Turbidity of the PDB medium as indicator for microbial growth over time.

Images show sterilized seeds by various treatments incubated in PDB medium for 2 days (a, b) and 7 days (c, d) as well as untreated seeds (e, g) and control medium (f, h). The corresponding turbidity development suggesting microbial growth. Scale bar in (a) is applicable for all panels.

Fig 6. Representative fluorescence microscopy images of DAPI stained PDB medium filtrate after 2 days of incubation.

Fluorescence microscopy micrographs showing high abundance of microbial cells after HP treatments (a, d) and very low number of microbial cells after SH (b, e) and EtOH-SH treatments (c, f). Scale bar represents 10μm for all images.

Fig 7. Representative fluorescence microscopy images of DAPI stained PDB medium filtrate after 7 days of incubation.

Fluorescence microscopy micrographs showing high abundance of microbial cells after HP treatments (a, d), SH-va-st (b) and EtOH-SH-st (f) and very low abundance of microbial cells after SH-st (e) and EtOH-SH-va-st (c) treatments. Scale bar represents 10μm for all images.

Fig 8. Representative fluorescence microscopy images of DAPI stained PDB medium of untreated seeds and control medium.

Fluorescence microscopy micrographs showing high abundance of microbial cells after 2 days (a) and 7 days (c) incubations of the untreated seeds and control medium without seeds (b, d) incubated in the same conditions. Scale bar represents 10μm for all images.

Fig 9. Graphical representation of total DAPI counts for various sterilization treatments.

Fig 10. Selected fluorescence microscopy images of DAPI stained roots after 7 days of root development.

Root fragments developed from seeds exposed to HP, SH and EtOH-SH sterilization treatments showing the presence of microbial cells. Scale bar 50μm applies to all images.

Fig 11. Representative HIM images showing microbial cells on root surface after different sterilization treatments.

HIM micrographs show the presence of microorganisms (red arrows and circles) on roots grown from seeds exposed to HP (a, d) and SH (b, e) similar with the untreated seeds (g) while no microbial cells were observed after EtOH-SH treatments (c, f).

Fig 12. Representative HIM images showing root hair development after different sterilization treatments.

HIM images showing no apparent negative effect on root hair development after sterilization treatments. Scale bar 100μm applies to all images.