Rapid and massive protocol to disinfection of Arabidopsis seeds using gaseous chlorine

Rapid and massive protocol to disinfection of Arabidopsis seeds using gaseous chlorine

Sehom Rivera-Gutiérrez^{1, #}, Yemitzel Camarero-Arellano^{1, #, $}, María del Carmen Oliver-Salvador^1,, Jesus-Agustín Badillo-Corona¹ and Noé Durán-Figueroa^1, *

¹Instituto Politécnico Nacional, Unidad Profesional Interdisciplinaria de Biotecnología, Laboratorio de Biotecnología Molecular, CDMX     , México. 

^$Avenia Acueducto, La laguna Ticoman, Gustavo A. Madero, Zip Code 07340: Laboratorio de Biotecnología Molecular, Unidad Profesional Interdisciplinaria de Biotecnología, CDMX, México.

*For correspondence: nduranf@ipn.mx

^#Contributed equally to this work.

Abstract

Plant molecular biologist that use Arabidopsis thaliana as model plant require faster, efficient and cheaper seed disinfection protocols to improve selection of transgenic seeds. There are different methods to sterilize Arabidopsis seeds, the most common is using chloride solution, with this classical method the volume of seeds to be disifected is limited, coupled with potential risk of contamination. Here, we report a cheap and efficient protocol to disinfecting seeds with the use of commercial chlorine on seeds of Arabidosps thaliana ecotype Col-0. The procedure consist in expose of seeds to chlorine gas generated  by the reaction by the reaction between commercial sodium hypochlorite and hydrochloric acid. The gas acts by killing the existing microorganisms in the seed, which can take less than an hour of exposure to the gas, and for massive disinfection of seeds, a maximum of 4 hours of exposure is recommended. This protocol can be applied for a few hundred seeds or also massively.

Keywords: Sterilization of Arabidopsis seeds, Chlorine gas, Sodium hyoichlorite, Hydrochloric acid, disinfection of seeds

Background

Working groups around the world using Arabidopsis seeds require efficient, fast and cheap seed disinfection protocols where larger volumes of seed can be sterilized at once. One of the most used methods, washing seeds with a solution of 50% commercial sodium hypochlorite and 0.1% Tween 20 (Lindsey et al., 2017), where a wash is carried out with said solution and 6 washes with water, and at each wash, the tubes are centrifuged and vortexed. In this technique, it is not recommended to wash more than 12 tubes per batch, and each tube should not contain more than 500 seeds to achieve good disinfection, which limits the methods for treating large volumes of seeds. An alternative strategy is the gaseous chlorine disinfection protocol, where the number of seeds to be disinfected can exceed one million. This technique is more efficient and faster, and reduces the risk of seed contamination by repeatedly opening and closing the tube. The principle is to release molecular chlorine from sodium hypochlorite and hydrochloric acid; NaClO + 2HCl  Cl₂ + NaCl + H₂O (Tiburcio-Munive et al., 2020). Molecular chlorine will poison microorganisms on the seed surface, but it must be removed before it reaches the seed embryo. Gaseous chlorine is lethal for microorganisms (Al-Sa´ady et al., 2020); then, this protocol reported here guarantees the disinfection of the seeds. 

Materials and Reagents

1. Parafilm paper (PARAFILM M PM-992 of 2 inches was used, Country of origin: USA)
2. Commercial Sodium Hypochlorite (Cloralex brand manufactured and distributed by ALEN DEL NORTE S.A. de C.V. under license from Industrias Alen S.A. de C.V.; contains 4-6% Sodium Hypochlorite. The lot used was: Lot-P, 22/01/27, 21:21)
3. Concentrated Hydrochloric Acid (37%) (FERMONT brand was used, code 01245, CAS 7647-01-0)
4. Sterile distilled water.
5. Murashige and Skoog (MS) medium (Sigma brand, M5519-50L, Lot is used. #ALCG5376 ).
6. Commercial sodium bicarbonate (ARM & HAMMER the standard of purity brand was used).

Equipment

1. Desiccator (Desiccator Simax CK GL36 of 4 liters. Gzech Republic).
2. Pasteur pipette previously labeled to a capacity of 3 ml.
3. 250 ml Beaker, PIREX No. 1000, made in Germany.
4. Rack for microcentrifuge tubes.
5. Microcentrifuge tubes.
6. Petri dishes.
7. 27x35 cm plastic bag (usually polyethylene are suitable).
8. 1000 µl micropipette tips.

Procedure

This protocol is divided into three stages. Before starting the experiment, it is necessary to read the entire protocol, since gaseous chlorine is extremely toxic. Wear vinyl or latex gloves, face shield, and lab coat. If you do not have a gas extraction hood, carry out the protocol in a widespread and ventilated place. It is recommended to do it outdoors.

1. Installation of the disinfection chamber.

1.Add a maximum of 5000 seeds (0.095 g) (it is strongly recommended to place only 1000 seeds equals to 0.019 g. A seed weighs ~19 µg) to each 1.5 µl tube, and place the open tubes in the rack, for massive disinfection use Petri dishes (Fig 1, all images are in the attached file). See Note 1.

2.Put the rack with the tubes into a bag of the appropriate size so that it can be closed quickly when the disinfection chamber is opened (Fig 2). In the case of Petri dishes, it is not necessary to use a bag.

3. Add 200 ml of commercial sodium hypochlorite in a beaker small enough so that the desiccator can be closed (Fig 3). See note 2

4. With a Pasteur pipette previously labeled to a volume of 3 ml, add HCl as quickly as possible. You will notice an instantaneous reaction, resembling a commercial sodium hypochlorite boil (Fig 4).

5. Quickly close the desiccator and seal with Parafilm. It is NOT necessary to add vaseline to the desiccator (Fig 5)

6. Wait for the gaseous chlorine to act for 4 hours. Alternatively, if you need to sterilize less than 300 seeds per tube, one hour of exposure will be enough for the chlorine to do its disinfecting job. Don't forget to label the time you sealed the desiccator on the Parafilm paper (Fig 6). See note 3.

B. Seed removal from desiccator.

1. In a ventilated place, remove the Parafilm paper.
2. Open the desiccator wide enough to fit your hand, and close the bag containing the rack quickly (Fig 7). See note 4.

3. With the bag closed, begin closing the tubes (Fig 8).

4. In a laminar flow hood (widely recommended), or in a burner, open the tubes so that the gas inside is purged. 15 minutes of ventilation is enough for the gaseous chlorine to escape (Fig 9). See note 5.

C. Planting of seeds in artificial media.

1. Add 1 ml of sterile distilled water to each tube, and close it (Fig. 10). See note 6.

2. Centrifuge the tubes for 15 seconds to loosen the possible seeds that are in the lid.

3. Sow the seeds in petri dishes. It is recommended to sow a volume of 250 µl, regardless of the number of seeds, and with the same pipette, distribute the seeds evenly throughout the dish (Fig 11). See note 7.

4. Seal the dish with Parafilm paper, wrap them in aluminum foil to prevent them from receiving light, and keep them refrigerated at 4 °C for 2 days (It is not recommended to store for more than a week.), or alternatively, incubate them directly at 25 °C. See note 8. After 10 days of incubation, your plants should be ready for transplanting (Fig 12).

Finally, to discard the reaction mixture contained in the beaker, it is necessary to neutralize the possible molecules that have not reacted. To do this, add 0.1 g of commercial sodium bicarbonate; HCl + NaHCO₃ - NaCl + H₂O + CO₂. If you see bubbling, appearing to be a carbonated drink, this is an indication that CO₂ is being released (Roser, 1999). In this case, add another 0.1 g of bicarbonate, repeat the operation until no bubbling is observed. If you used the solutions and concentrations recommended here, the reaction mixture will not generate CO₂. Once the solution is neutralized, dispose of the liquid directly into the drain.

Notes

1. For a massive disinfection version, use Petri dishes with a volume of seeds whose height does not exceed 5 mm. You can use as many dishes as necessary and fit in the desiccator, as long as each dish does not exceed 5 mm in height of seeds. As data, to a 9 cm diameter dish, you can add up to 10 g of seeds, the equivalent of ~500,000 seeds.
2. Before moving on to the next step, make sure you have the parafilm ready and at hand. It will require handling in less than 3 seconds. This step must be done quickly to prevent gaseous chlorine from escaping.
3. You could leave up to 5 hours disinfecting your seeds, although it is not recommended, and for no reason exceed 6 hours, otherwise, the germination yield of your seeds may drop by more than 70% or more.
4. When opening the desiccator, exhale air while closing the bag. Once closed, withdraw from the place at least 4 meters away. The toxic cloud will take about 10-15 seconds to dissipate. In the case of Petri dishes, it is recommended to seal with Parafilm paper, to transport the dish to the laminar flow hood. This will avoid risks of contamination in the transfer of the Petri dish.
5. If you are disinfecting 5,000 seeds, you can optionally close the tube and invert it two or three times, to remove any traces of chlorine gas trapped at the bottom of the tube. For volumes less than a thousand seeds, it is not necessary to carry out this operation. If you want to store the disinfected seeds for more than a day, you must first remove the gaseous chlorine from the tubes, otherwise the seeds will die. Once the chlorine has been purged, seal the tube or Petri dish with Parafilm and store the seeds.
6. Do not allow the tip of the pipette to touch the tubes for any reason, if this happens, immediately discard the tip and take a new one. Similarly, if you accidentally touch the bottle of water with a contaminated tip, or with your hands, immediately discard that bottle and use a new one. If the wind of your laminar flow hood is vertical (it comes out from above), tilt both the bottle with water and the pipette and in the same way, add the guide in an inclined way. But if the wind from the laminar flow hood is horizontal (comes out from the front), you can work with the water and the pipette vertically. Remember that if the wind from the laminar flow hood is vertical, never pass your hands over open boxes, tubes or water bottles. Omitting these recommendations greatly increases the risk of contamination in your seeds. For massive disinfection in a Petri dish, add 20 ml of water to a 9 cm diameter dish, and pipette as many times as necessary, until the seeds settle and can be transferred to a falcon tube where they can be handled more easily, or sow directly into petri dishes. You can store the falcon tube at 4°C. If you decide to store it, it is necessary to remove all the water with the pipette, and every time you want to sow, add sterile water to resuspend the seeds and sow them. Don't forget to cover the tube from light, and try not to store it for more than three weeks, otherwise you run the risk that your seeds will germinate in the tube.
7. It is very important that you suck up and discharge the seeds quickly, before they settle on the tip of the pipette, otherwise the pipette will clog. Carry out this operation at a moderate speed, because if you suck too fast, you run the risk of the seeds touching the pipette and contaminating them, and if you discharge the seeds too fast, they will be ejected from the Petri dish. For antibiotic resistance experiments, it is not recommended to sow more than a thousand seeds per dish (9 cm diameter dishes).
8. If your experiment requires it, incubate the seeded dishes directly, otherwise, refrigerate for 2 days at 4 °C, preventing the seeds from receiving light. After two days, incubate the dishes at 25 °C, and after 10 to 12 days, your seedlings will be ready to be transplanted into soil.

Acknowledgments

Project to NVDF No. 20221934 from Secretría de Investigación y Posgrado of Instituto Politécnico Nacional. 

Competing interests

1. The authors declare non-financial competing interest.

References

Al­Saady A. T., Nahar H. S. and Saffah F. F. (2020). Antibacterial activities of chlorine gas and chlorine diaoxide gas against some pathogenic bacteria. EurAsia Journal of BioSciences 17: 3875­3882

Lindsey B. E., Rivero L., Calhoun C. S., Grotewold E. and Brkljacic J. (2017). Standardized method for high-throughput sterilization of Arabidopsis seeds. Journal of Visualized Experiments 128: e56587. doi: 10.3791/56587

Roser C. E. (1999). Pressure and Stoichiometry. Journal od Chemical  Education 76(5): 638.639

Tiburcio-Munive G, Salazar-Campoy M. M., Valenzuela-García J. L., Hernández-Negrete O.and Vázquez-Vázquez V. (2020). Dissolution of silver and gold with sodium hypochlorite and hydrochloric acid in refractory minerals (Mangano-Argentiferous). Mining, Metallurgy & Exploration. doi:10.1007/s42461-020-00216-7