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Biology

High-throughput, Robust and Highly Time-flexible Method for Surface Sterilization of Arabidopsis Seeds

Published: October 4, 2021 doi: 10.3791/62893
Automatic Translation
1, 1, 1, 1
1Department of Biodiversity and Molecular Ecology, Research and Innovation Centre, Fondazione Edmund Mach

Summary

A high-throughput protocol for the surface sterilization of Arabidopsisthaliana (Arabidopsis) seeds is provided, optimizing the liquid handling steps with a simple suction device constructed with a vacuum pump. Hundreds of seed samples can be surface-sterilized in one day.

Abstract

Arabidopsis is by far the plant model species most widely used for functional studies. The surface sterilization of Arabidopsis seeds is a fundamental step required towards this end. Thus, it is paramount to establish high-throughput Arabidopsis seed surface sterilization methods to handle tens to hundreds of samples (e.g., transgenic lines, ecotypes, or mutants) at once. A seed surface sterilization method based on the efficient elimination of liquid in tubes with a homemade suction device constructed from a common vacuum pump is presented in this study. By dramatically reducing labor-intensive hands-on time with this method handling several hundreds of samples in one day is possible with little effort. Series time-course analyses further indicated a highly flexible time range of surface sterilization by maintaining high germination rates. This method could be easily adapted for surface sterilization of other kinds of small seeds with simple customization of the suction device according to the seed size, and the speed desired to eliminate the liquid.

Introduction

Arabidopsis is a diploid plant species belonging to the Brassicaceae family. Its relatively short life cycle (two months per generation under long-day growing conditions), small plant size, and self-pollination with the production of hundreds of seeds per plant have made it the first fundamental plant model species1,2. In addition, its genome was fully sequenced3, extensive reverse genetics tools (saturated T-DNA, transposon, and chemically mutagenized populations) are available4,5,6, and effective Agrobacterium-mediated transformation is well-established to obtain sufficient transgenic lines for further downstream work7. Thus, during the last two decades, great advances have been achieved using Arabidopsis as a model species to dissecting diverse aspects of plant biology at the molecular level, including natural, genetic and phenotypic variation8,9.

To functionally characterize genes of interest in Arabidopsis, seed surface sterilization to eliminate fungal and bacterial contaminants is the prerequisite step for many downstream protocols requiring axenic cultures. Genetic transformation for the overexpression10, knock-down (RNA-I11) or knock-out (genome editing12,13) of gene function, subcellular localization14, promoter activity15,16, protein-protein17 and protein-DNA interaction18, to cite only the most common applications, all necessitate a seed surface sterilization step. Thus, despite its relative simplicity, seed surface sterilization plays a fundamental role in many functional analyses.

So far, two major categories of seed surface sterilization methods have been developed based either on gas- or on liquid-phase sterilization19. While the throughput of gas-phase seed surface sterilization is medium to high, using the hazardous reagent chlorine gas as a surface sterilization agent has hindered its wide application. Methods based on liquid-phase sterilization, on the contrary, rely on milder chemicals like ethanol and bleach solutions for surface sterilization, and they are more widely used despite they have an inherently lower throughput than chlorine fumigation. In general, two different methods which use liquid reagents are commonly used. One largely used method is based on washing with ethanol and bleach at different concentrations for different duration of time20,21. Another method is based on the application of bleach only21,22. Both methods are mainly applied for small-scale seed surface sterilization. However, in many experiments, it is necessary to screen many Arabidopsis transgenic lines derived from one transformation15,23 or screen in parallel many transgenic lines generated from different transformations24,25. To the best of our knowledge, no liquid-based method for high-throughput seed surface sterilization has been published, which constitutes, although little-recognized, an important bottleneck for functional genomics approaches. Therefore, developing safe, robust, and high-throughput methods for seed surface sterilization is a necessary and critical step towards the success of the functional characterization of many genes at once.

To this end, in the current study, an improved method for surface sterilization of Arabidopsis seeds is presented. This method is safe, low cost, highly robust, and high-throughput, allowing handling 96 independent lines within one hour from the beginning of seed surface sterilization until the end of seed sowing in Petri dishes. The method demonstrated relies on widely available, basic laboratory instrumentation like a vacuum pump, consumable glassware, and plastic ware. This improved method provides the scientific community a safe, simple, and affordable approach to streamline seed surface sterilization with a throughput adequate to modern functional genomics approaches in Arabidopsis and other non-model plant species.

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Protocol

1. Reagents and media preparation

  1. Prepare 70% ethanol solution: Add 737 mL of 95% technical ethanol to 263 mL of distilled water. Mix thoroughly.
    NOTE: Prepare 70% ethanol solution on a non-sterile working bench.
    CAUTION: Ethanol is highly flammable and can cause serious irritation to the eyes. Keep away from flames and heat sources. In case of contact with eyes, rinse with abundant water.
  2. Prepare 5% bleach solution: Add 5 mL of household bleach (containing ~3.5% of Sodium hypochlorite, NaClO) to 95 mL of sterile distilled water. Add a few drops of non-ionic detergent (e.g., Tween 20) and mix thoroughly.
    NOTE: Prepare 5% bleach solution inside the laminar hood.
    CAUTION: Sodium hypochlorite, the active component of bleach, is highly irritating. It is highly corrosive and can cause serious damages to the gastrointestinal tract. In case of contact, rinse immediately with abundant water. In case of ingestion, call the poison control center or a medical physician for treatment advice.
  3. Prepare half-strength Murashige and Skoog (½ MS) medium26.
    1. Add 2.2 g of MS medium powder (including vitamins) and 10 g of sucrose in 800 mL of distilled water. Adjust the pH of the solution using 1 M KOH and bring the volume up to 1 L using distilled water. Aliquot 500 mL into a 1 L bottle and add 4g of agar to prepare a solid medium. Autoclave the solution.
    2. After autoclaving, cool down the medium to 50-53 °C in a water bath and pour it into Petri dishes under the laminar flow hood. To prepare the selective medium, add 1000 µL/L of 50 mg/mL kanamycin stock solution (mix 500 mg of Kanamycin sulfate monohydrate in 10 mL of distilled water, filter sterilize and store at -20 °C) to the medium (50-53 °C). Mix well by swirling, and pour into Petri dishes as mentioned before.

2. Aspirator setup

NOTE: Instrument setup is summarized in Figure 1.

  1. Connect the vacuum pump inlet to one end of a polyethylene (PE) tube of suitable size. Connect the other end of the tube to the outlet of the two-way lid of the decantation bottle. Wrap the tubing's junction tightly with a sealing film (Table of Materials) to ensure air-tight connection.
  2. Connect a second PE tube to the inlet (the hole protruding to the inside of the bottle) of the screwcap on the decantation bottle. Fit the other side of the tube to the outlet of an aquarium valve. If necessary, wrap with a sealing film along the junction to eliminate air leakage.
  3. Just before use, fit a sterile 200 µL pipette tip to the inlet of the aquarium filter under the laminar flow hood.

Figure 1
Figure 1: Schematic drawing of the suction device for high-throughput removal of sterilization liquids. For clarity, the single parts are not drawn to scale. Letter (A) indicates the vacuum pump, (B) the reservoir bottle to collect liquids (ethanol, bleach, or sterile water), (C) the valve to avoid reflux of the liquids, (D) the sterile 200 µL pipette tip, and (E) the 1.5 mL microcentrifuge tube containing seeds and sterilization liquid. Arrows indicate the direction of the airflow. Please click here to view a larger version of this figure.

3. High-throughput liquid surface sterilization of seeds

NOTE: The overall procedure and minimal time required for surface sterilization of Arabidopsis thaliana (L.) Heynh wild-type (Col-0) (Arabidopsis) seeds with 96 independent samples are summarized in Figure 2.

  1. Label with a permanent marker two batches of 48 x 1.5 mL microcentrifuge tubes with progressive numbers.
  2. Add 100-200 Arabidopsis seeds to each of the 96 sterile 1.5 mL microcentrifuge tubes (roughly 1-2 mm above the bottom of the conical end of the tube).
  3. Aliquot around 1000 µL of 70% ethanol into each tube using a 10 mL sterile serological pipette inside the laminar flow hood (seed batch one, 48 tubes) and carefully close the lids.
    NOTE: Dispensing the solutions does not need to be extremely accurate, as long as the dispensed volume is several times higher than the volume of seeds. Alternatively, perform this step outside of the laminar hood (non-sterile condition).
  4. Shake the tubes at an oscillation frequency of 8.0 Hz for at least 3 min in a shaker.
  5. Remove the adapters from the shaker and transfer them into the basket of a benchtop microcentrifuge.
  6. Quickly spin down the seeds using the pulse function (present in most benchtop centrifuges) to reach 1880 x g (~15 s).
    NOTE: Longer time or higher centrifugation forces negatively affects seed germination.
  7. Transfer the 48 tubes from the adapters to one rack and open all tubes under the laminar flow hood. Avoid contaminations by not touching the part of the lids fitting into the tubes. If lids are too close to each other, divide the tubes into two racks for easier handling.
  8. Fit a sterile 200 µL yellow tip onto the aquarium valve inlet of the homemade aspirator under the laminar flow hood and switch on the pump.
  9. Insert the yellow tip just above the level of the seeds to avoid touching the seeds when sucking the liquid. Alternatively, quickly position the tip at the bottom of the tube; if a seed blocks the suction of the liquid, eliminate the yellow tip and insert a new one.
  10. Aliquot into each tube around 1000 µL of 5% bleach using a 10 mL sterile serological pipette inside the laminar flow hood.
  11. Tightly close all the lids and put all the tubes back into the shaker adapters. Shake the tubes at an oscillation frequency of 8.0 Hz for at least 3 min in the shaker.
  12. Quickly spin down the seeds using the pulse function of the benchtop centrifuge for the time necessary to reach 1880 x g (~15 s).
  13. Fit a new sterile 200 µL yellow tip onto the aquarium valve connected to the vacuum pump under the laminar flow hood, and switch on the pump.
  14. Insert the yellow tip above the level of the seeds to avoid touching the seeds when sucking the bleach solution.
  15. Aliquot into each tube around 1000 µL of sterilized H2O using a 10 mL sterile serological pipette in the laminar flow hood.
    NOTE: Combine the two batches of seeds to minimize the operation time.
  16. Fit a new sterile 200 µL yellow tip onto the aquarium valve connected to the vacuum pump under the laminar flow hood and switch on the pump.
  17. Insert the yellow tip just above the level of the seeds to avoid touching the seeds when sucking the H2O.
  18. Aliquot into each tube around 500 µL of sterilized H2O using a 10 mL sterile serological pipette and close all the lids in the laminar flow hood. The seeds are ready to be sown. If needed, keep the tubes at room temperature for a few hours maximum or at 4 °C overnight.
  19. Fill the reservoir bottle used to collect liquid with an adequate amount of water and autoclave it. Afterward, discard the liquid into a normal sink.
    ​NOTE: Autoclave the liquid to kill all the seeds inside the reservoir.

Figure 2
Figure 2: Overview of the procedure and minimal time required for surface sterilization of Arabidopsis seeds with 96 independent samples. In the presented experiment, 96 independent samples are handled in two equal-sized batches. The entire procedure is the same for both batches, and they are processed in parallel, but batch two is processed with one step delay compared to batch one. Please click here to view a larger version of this figure.

4. Plating and scoring of Arabidopsis on ½ MS plates

  1. Transfer the seeds and 300-400 µL of sterile H2O into a Petri dish by gentle pipetting with a 1000 µL pipette.
  2. After transferring 10 tubes, pour into each plate around 1.5-2.0 mL of melted ½ MS medium without antibiotics.
    NOTE: Melt in advance and then keep the ½ MS melted medium in a thermostatic bath set at 50-53 °C to avoid solidification. Ensure that the temperature does not exceed 58 °C to avoid decreasing the germinability of the seeds.
  3. Quickly swirl the plate to distribute the seeds inside it. Tape the plates on opposite sides.
  4. Wrap the plates in a plastic or aluminum foil and then place them in a fridge (4 °C) for 3 days in the dark to obtain uniform germination.
  5. Transfer the plates into a growth chamber set at 23 °C under long-day conditions (16 h light/8 h dark) with a light intensity of 100-120 µmol·m-2·s-1 and 60% relative humidity.
  6. After two days, score the plants by the presence of radicles. Detect the radicle emergence and green cotyledon formation (full opening of the two cotyledons) to evaluate seed germination.

5. Statistical analyses

NOTE: Here, Tukey's pairwise test was used for statistical analyses.

  1. Consider the P-values below 0.01 as statistically significant. Perform all the experiments at least with five biological replicates.

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Representative Results

In order to assess the time required for the entire seed sterilization procedure, the time differences for liquid handling 96 samples in the current protocol were calculated and compared with traditional pipetting methods. The result indicates that the current protocol saves time, cutting the liquid handling time to one-fourth of that with the traditional protocols (Table 1). The table further highlights that the liquid removal time in the current protocol saves more time than that of the traditional methods, with an overall eight-fold reduction.

Selection of the time range for seed sterilization
Longer sterilization steps minimize contamination rates but can negatively affect seed germination. To determine the best time range for seed sterilization with the highest germination rates without contamination, different durations of each sterilization step were tested, assessing both seed germination and green cotyledon emergence rates. The germination analyses performed from day 2 to day 4 and at day 7 indicated no significant differences among the time range from 10 min to 40 min of 70% ethanol sterilization. However, from 40 min of treatment with 70% ethanol, the germination rates declined (Figure 3). Correspondingly, green cotyledon emergence rates decreased (Figure 4). As shorter sterilization times can increase the throughput but increase the contamination rates, the minimal time needed to sterilize 96 different seed samples in two batches of 48 samples was assessed. In addition, given the preferred setup with shaking performed in a shaker, we tested the minimal time needed for seed sterilization by handling 96 samples in two batches of 48 samples each. The minimal time required to handle 48 samples at once was 3 min, thus allowing the second set of 48 samples to be processed immediately after the first set without any waiting time. Thus, 3 min for 70% ethanol and 3 min for 5% bleach were applied as the minimum time to sterilize the seeds (point (a) in Figure 3). These analyses resulted in germination rates similar to the highest ones without contamination and loss of seed vitality (Figure 3).

In summary, the suitable time ranges for maintaining the highest percentage of seed germination without contamination derived from sufficient elimination of microorganisms is between 3-30 min for 70% ethanol and between 3-22.5 min for 5% bleach.

In order to demonstrate that the seeds we used originally were contaminated with microorganisms, the non-sterile seeds were sown directly on MS plates. Fungi appeared on the plates after two-day sowing and spread all over the plates after seven-day germination (Supplemental Figure 1).

Evaluation of cross-contamination between different genotypes of seeds
To check whether the use of a single sterile pipette tip to process different seed samples could result in cross-contamination and to assess the amount of such cross-contamination, two different genotypes of seeds (Col-0 wild-type, sensitive to kanamycin, and Arabidopsis transgenic line AdoIspS-79, resistant to kanamycin24) were alternated during the sterilization procedure. After standard seed sterilization, they were sown in half-strength MS solid plates supplemented without or with 50 mg/L kanamycin. The experiments were replicated five times. These analyses indicated that around 96% of the seeds germinated in MS plates containing kanamycin, but no green cotyledons were observed on the 7th day of germination in any plates sown with the Col-0 genotype (Figure 5). In parallel, Col-0 seeds sown in MS plates showed around 94% germination, and all cotyledons were green after 7-day germination. These results indicate no carry-over of contamination between samples despite using a single pipette tip to remove the sterile solution.

Figure 3
Figure 3: Seed germination percentage after 2-, 3-, 4- and 7-day Arabidopsis seed sowing with different sterilization times indicated with different letters. a: 3 min of 70% ethanol and 3 min of 5% bleach, b: 10 min of 70% ethanol and 7.5 min of 5% bleach, c: 20 min of 70% ethanol and 15 min of 5% bleach, d: 30 min of 70% ethanol and 22.5 min of 5% bleach, e: 40 min of 70% ethanol and 30 min of 5% bleach, f: 50 min of 70% ethanol and 37.5 min of 5% bleach, g: 70 min of 70% ethanol and 52.5 min of 5% bleach. Two stars indicate significant differences according to Tukey's pairwise test (p < 0.01). Please click here to view a larger version of this figure.

Figure 4
Figure 4: Green cotyledon percentage after 7-day Arabidopsis seed sowing with different sterilization times indicated with different letters. a: 3 min of 70% ethanol and 3 min of 5% bleach, b: 10 min of 70% ethanol and 7.5 min of 5% bleach, c: 20 min of 70% ethanol and 15 min of 5% bleach, d: 30 min of 70% ethanol and 22.5 min of 5% bleach, e: 40 min of 70% ethanol and 30 min of 5% bleach, f: 50 min of 70% ethanol and 37.5 min of 5% bleach, g: 70 min of 70% ethanol and 52.5 min of 5% bleach. Two stars indicate significant differences according to Tukey's pairwise test (p < 0.01). Please click here to view a larger version of this figure.

Figure 5
Figure 5: Cross-contamination assay between Col-0 wild-type and AdoIspS-79 seeds. Col-0 germination in half-strength MS plates (left), Col-0 in half-strength MS plate supplemented with 50 mg/L kanamycin (middle) and AdoIspS-79 in half-strength MS plate supplemented with 50 mg/L kanamycin (right), scalebar = 1 cm. A representative image of one of the five replicates is shown in the figure. Please click here to view a larger version of this figure.

Procedure Current protocol (min) Traditional pipetting (min)
Adding liquid 1 12 24
Removing liquid 1 6 48
TOTAL 18 72

Table 1: Summary of the minimal time required for liquid handling during sterilization of 96 seed samples. The table lists the total time (min) required to add and remove the liquid throughout the main steps of seed surface sterilization using the current protocol and a protocol where traditional pipetting is used.

Supplemental Figure 1: Contamination detection after 7-day Arabidopsis seed sowing. The image shows the degree of contamination of non-sterile seeds (rinsed with water; left) and sterile seeds (subjected to surface sterilization as described in the main text; right). Scalebar = 1 cm. Please click here to download this File.

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Discussion

Sterilization of seeds is the fundamental step for functional studies in Arabidopsis. Although it is frequently carried out for many different purposes, limited studies on high-throughput seed surface sterilization in Arabidopsis are available.

So far, one of the methods with the highest throughput is using chlorine gas generated by mixing bleach with concentrated HCl. Although this method requires limited hands-on time, it uses a gas highly toxic to human beings27. In addition, the number of seeds to be surface-sterilized and the duration of surface seed sterilization must be carefully controlled. If the seeds are too many, the dosage of chlorine gas would not be sufficient to completely kill the microorganisms present on the seed coat, resulting in widespread contamination of the seed batches. On the other hand, if the duration of surface sterilization is too long, the seeds will be killed. Thus, despite some advantages, fumigation is not the favorite method for seed surface sterilization in Arabidopsis.

By contrast, there are a number of liquid-based methods for surface sterilization of Arabidopsis seeds available28,29,30. However, the main limitation of these methods is that they can be utilized exclusively for a low number of samples, and they are not optimized for throughput. To our knowledge, the only study on high-throughput surface sterilization of Arabidopsis seeds was described by Lindsey, B. E. et al.31. In this study, the authors proposed two different methods. In addition to one approach with chlorine gas mentioned above, the authors proposed a liquid-based surface sterilization method using a concentrated bleach solution applied for 5-10 min sterilization. Although this method provided the first systematic study for surface seed sterilization with different concentrations of bleach for different times and analyzed the corresponding output, the protocol could still be improved. First of all, this method used highly concentrated bleach, which can be harmful to the operator and the environment. Second, due to the utilization of highly concentrated bleach, the best seed surface sterilization timing is only between 5-10 min, thus affording a narrow time window for completion of the protocol. In addition, to eliminate the bleach, many washing steps were necessary, making the protocol both time- and work-intensive and limiting the throughput in such a short time range of the surface sterilization step.

In order to overcome all these limitations and to combine the best aspects of previous works, the method described here was carried out with 70% technical ethanol followed by a further step with a low percentage of bleach (5%) which reduced the toxicity and the labor-intensive washing steps. Additionally, the systematic analyses of seed germination provided a wider time range between 3-30 min for seed surface sterilization without affecting the germination rates. This allows higher flexibility to organize the workflow, allowing to regulate the surface sterilization time according to concurrent work. More importantly, the labor-intensive pipetting procedure was replaced by multiple dispensing with a serological pipette for adding the liquids and, most importantly, by a very fast aspiration method assisted by a simple though performant homemade suction device not requiring tip exchange between samples. Given the presence of a reservoir bottle, no cross-contamination among lines was experimentally observed, as seeds that may be inadvertently aspired with the pipette tip are decanted into the bottle. The valve used as an adapter between the sterile tip and the tubing was specifically selected to guarantee that the liquid is aspirated into the collection bottle without flowing back to the tube containing seeds. Thus, this method dramatically improved the seed surface sterilization procedure with easy handling and time-saving.

In conclusion, this protocol provides the scientific community with a safe, highly time flexible, low-labor, and time-saving approach for surface sterilization of Arabidopsis seeds, which is inexpensive and easily set up in any lab. Furthermore, the method could also be applied to surface sterilization of any other type of small seeds by eventually changing the sterile tip, adapter, and tubing according to the seed sizes. In particular, this method was successfully used without modification for several Brassicaceae species (e.g., Cardamine impatiens L., Berteroa incana (L.) DC., Capsella bursa-pastoris (L.) Medicus, Alliaria petiolata (M. Bieberstein) Cavara et Grande, etc.), as well as for Nicotiana benthamiana Domin and Nicotiana tabacum L. seeds. The method could also be applied to even smaller seeds, like those of orchids, as long as precise determination of speed/time of centrifugation and length of surface sterilization treatment can be established32.

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Disclosures

All authors declare no conflicts of interest.

Acknowledgments

This research was funded by the Autonomous Province of Trento through core funding of the Ecogenomics group of Fondazione E. Mach.

Materials

Name Company Catalog Number Comments
Aquarium valve Amazon B074CYC5SD Kit including 2 valves and thin-walled tubings. The valve prevents the liquids to go back to the sterile tip
Arabidopsis Col-0 wild-type seeds Nottingham Arabidopsis Stock Center N1093 Wild type seeds (sensitive to kanamycin)
Arabidopsis transgenic line AdoIspS-79 seeds NA NA Transgenic line overexpressing an isoprene synthase gene from Arundo donax transformed in the Col-0 background, resistant to kanamycin (Li et al. (2017) Mol. Biol. Evol., 34, 2583–2599). Available on request from the authors
Microcentrifuge Eppendorf EP022628188 Benchtop microcentrifuge used for spinning down the seeds
Murashige & Skoog medium including vitamins Duchefa M0222 Standard medium for plant sterile culture
Pipette controller Brand 26300 Used to operate the serological pipette
Polyethylene tube 1 Roth 9591.1 Tube for connection from vacuum pump to decantation bottle (inner diameter: 7 mm; outer diameter: 9 mm)
Polyethylene tube 2 Roth 9587.1 Tube for connection from decantation bottle to the aquarium valve  (inner diameter: 5 mm; outer diameter: 7 mm)
Screw cap with connectors Roth PY86.1 2-way dispenser screw cap GL45 in polypropylene for decanting bottle
Serological pipette Brand 27823 Graduated glass (reusable) serological pipette. Disposable pipettes can be used instead
Shakeret al. Qiagen 85300 TissueLyser II bead mill used normally for tissue homogenization. Without the addition of beads to the tubes it works as shaker.
Technical ethanol ITW Reagents (Nova Chimica Srl) 212800 Ethanol 96% v/v partially denatured technical grade
Tween 20 Merck Millipore 655205 Non-ionic detergent acting as surfactant
Universal tubing connectors Roth Y523.1 Can be used to improve/simplify tubing connections
Vacuum pump Merck Millipore WP6222050 Used for making the suction device

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References

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High-throughput Robust Time-flexible Surface Sterilization Arabidopsis Seeds Functional Genomics Model Species Ease High Throughput Hundreds Of Samples Per Day Modifications Model And Non-model Plant Species Temperature Medium Centrifugation Speed Seed Survival 70% Ethanol 5% Bleach Solution Nonionic Detergent Half-strength Murashige And Skoog Medium MS Medium Powder Sucrose PH Adjustment Potassium Hydroxide Distilled Water Autoclaving Solid Medium Petri Dishes
High-throughput, Robust and Highly Time-flexible Method for Surface Sterilization of Arabidopsis Seeds
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Cite this Article

Li, M., Yu, J., Barbaro, E.,… More

Li, M., Yu, J., Barbaro, E., Varotto, C. High-throughput, Robust and Highly Time-flexible Method for Surface Sterilization of Arabidopsis Seeds. J. Vis. Exp. (176), e62893, doi:10.3791/62893 (2021).

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