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.

Surface sterilization for isolation of endophytes: Ensuring what (not) to grow

“…Similarly, in some reports, the efficiency of seed sterilization process was also enhanced by employing Tween 20 as a surfactant along with sterilant solution [31,64,65].…”

“…In the study, decreased microbes population is ascribed to the surfactant capacity in lowering the superficial tension of sterilant solution, thus allowing the interaction between sterilant and the fruit surface. Similarly, in some reports, the efficiency of seed sterilization process was also enhanced by employing Tween 20 as a surfactant along with sterilant solution [31,64,65].…”

“…In addition, pretreatment with surfactants like Triton X-100, Tween 80, Tween 20, and Teepol are also used to enhance efficiency [27][28][29]. Despite gigantic progress in endophyte research, detailed reviews on different aspects of surface sterilization protocols were meager, especially with respect to the type of surface sterilizing agents, their concentration, and exposure time [24,30,31]. Thus, an exhaustive compilation of surface sterilants, sterilization methods, and other aspects on endophyte isolation are thoroughly discussed in this review.…”

Application of Barley Tweaky Spike Mutants for the Study of Effects of Plant Immunity-Related Substances

“…A notable feature of the grain material used in the present study is the pesticide-free production mode, which was applied for many years. Under the background of high natural infection, the most effective means against the moulding of germinating grains is widely used grain surface sterilization [31][32][33], which was effective independently of the plant genotype and seed material from different years. However, a comparison of sterilized and unsterilized seed material appeared to be an effective strategy for the auxinpathogen relationship study.…”

Incorporation of engineered nanoparticles of biochar and fly ash against bacterial leaf spot of pepper

“…Capsicum annum L. seeds (Yolo Wonder) were purchased from the local seed market (Ghula Mandi, Lahore) and surface sterilized with 70% ethanol for 10 min followed by washing with 50% NaOHCl solution (100 mL of NaOHCl + 100 mL of distilled + 50 µL tween-20 detergent) and thrice washing with distilled deionized water 38 . These seeds were sown in clay pots ¾ filled with sterilized 20% leaf compost soil (The soil texture was a sandy loam (82.88% sand, 13.04% silt, and 4.08% clay) with a pH of 7.88 and an electrical conductivity (EC) of 1.55 dS/m (measured using a pH meter and an EC meter); organic matter content (OM) of 0.54%; containing 3% total N and 1.5% total C; having a C/N ratio of 0.5; and containing 12, 68, and 100 mg·kg −1 of Ca, P, and K, respectively).…”