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

1. Reagents and media preparation
<ol>
	<li>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.</li>
	<li>Prepare 5% bleach solution: Add 5 mL of household bleach (containing ~3.5% of Sodium hypochlorite...

<ol>
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Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+a+large+dataset+indicates+that+internal+transcribed+spacer+(ITS)+should+be+incorporated+into+the+core+barcode+for+seed+plants.">Comparative analysis of a large dataset indicates that internal transcribed spacer (ITS) should be incorporated into the core barcode for seed plants.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (49), 19641-19646 (2011).</li><li>Mathur, J., Koncz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+maintenance+of+cell+suspension+cultures.+Arabidopsis+Protocols.">Establishment and maintenance of cell suspension cultures. Arabidopsis Protocols.</a> Methods in Molecular Biology. 82, 27-30 (1998).</li><li>Li, M., Cappellin, L., Xu, J., Biasioli, F., Varotto, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening+for+in+planta+characterization+of+VOC+biosynthetic+genes+by+PTR-ToF-MS.">High-throughput screening for in planta characterization of VOC biosynthetic genes by PTR-ToF-MS.</a> Journal of Plant Research. 133 (1), 123-131 (2020).</li><li>Li, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+planta+recapitulation+of+isoprene+synthase+evolution+from+ocimene+synthases.">In planta recapitulation of isoprene synthase evolution from ocimene synthases.</a> Molecular Biology and Evolution. 34 (10), 2583-2599 (2017).</li><li>Li, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+isoprene+emission+in+Arecaceae+(palms).">Evolution of isoprene emission in Arecaceae (palms).</a> Evolutionary Applications. 14, 902-914 (2020).</li><li>Murashige, T., Skoog, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+revised+medium+for+rapid+growth+and+bio+assays+with+tobacco+tissue+cultures.">A revised medium for rapid growth and bio assays with tobacco tissue cultures.</a> Physiologia Plantarum. 15 (3), 473-497 (1962).</li><li>Bent, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+thaliana+floral+dip+transformation+method.">Arabidopsis thaliana floral dip transformation method.</a> Methods in Molecular Biology. 343, 87-104 (2006).</li><li>Lundberg, D. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+arsenic+stress+response+by+ethylene+biosynthesis+and+signaling+in+Arabidopsis+thaliana.">Regulation of arsenic stress response by ethylene biosynthesis and signaling in Arabidopsis thaliana.</a> Environmental and Experimental Botany. 185, 104408 (2021).</li><li>Lindsey, B. E., Rivero, L., Calhoun, C. S., Grotewold, E., Brkljacic, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardized+method+for+high-throughput+sterilization+of+Arabidopsis+seeds.">Standardized method for high-throughput sterilization of Arabidopsis seeds.</a> Journal of Visualized Experiments: JOVE. 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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 ...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Aquarium valve</td>
			<td>Amazon</td>
			<td>B074CYC5SD</td>
			<td>Kit including 2 valves and thin-walled tubings. The valve prevents the liquids to go back to the sterile tip</td>
		</tr>
		<tr>
			<td>Arabidopsis Col-0 wild-type seeds</td>
			<td>Nottingham Arabidopsis Stock Center</td>
			<td>N1093</td>
			<td>Wild type seeds (sensitive to kanamycin)</td>
		</tr>
		<tr>
			<td>Arabidopsis transgenic line AdoIspS-79 seeds</td>
			<td>NA</td>
			<td>NA</td>
			<td>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&#8211;2599). Available on request from the authors</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Eppendorf</td>
			<td>EP022628188</td>
			<td>Benchtop microcentrifuge used for spinning down the seeds</td>
		</tr>
		<tr>
			<td>Murashige &#38; Skoog medium including vitamins</td>
			<td>Duchefa</td>
			<td>M0222</td>
			<td>Standard medium for plant sterile culture</td>
		</tr>
		<tr>
			<td>Pipette controller</td>
			<td>Brand</td>
			<td>26300</td>
			<td>Used to operate the serological pipette</td>
		</tr>
		<tr>
			<td>Polyethylene tube 1</td>
			<td>Roth</td>
			<td>9591.1</td>
			<td>Tube for connection from vacuum pump to decantation bottle (inner diameter: 7 mm; outer diameter: 9 mm)</td>
		</tr>
		<tr>
			<td>Polyethylene tube 2</td>
			<td>Roth</td>
			<td>9587.1</td>
			<td>Tube for connection from decantation bottle to the aquarium valve&#160; (inner diameter: 5 mm; outer diameter: 7 mm)</td>
		</tr>
		<tr>
			<td>Screw cap with connectors</td>
			<td>Roth</td>
			<td>PY86.1</td>
			<td>2-way dispenser screw cap GL45 in polypropylene for decanting bottle</td>
		</tr>
		<tr>
			<td>Serological pipette</td>
			<td>Brand</td>
			<td>27823</td>
			<td>Graduated glass (reusable) serological pipette. Disposable pipettes can be used instead</td>
		</tr>
		<tr>
			<td>Shakeret al.</td>
			<td>Qiagen</td>
			<td>85300</td>
			<td>TissueLyser II bead mill used normally for tissue homogenization. Without the addition of beads to the tubes it works as shaker.</td>
		</tr>
		<tr>
			<td>Technical ethanol</td>
			<td>ITW Reagents (Nova Chimica Srl)</td>
			<td>212800</td>
			<td>Ethanol 96% v/v partially denatured technical grade</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Merck Millipore</td>
			<td>655205</td>
			<td>Non-ionic detergent acting as surfactant</td>
		</tr>
		<tr>
			<td>Universal tubing connectors</td>
			<td>Roth</td>
			<td>Y523.1</td>
			<td>Can be used to improve/simplify tubing connections</td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>Merck Millipore</td>
			<td>WP6222050</td>
			<td>Used for making the suction device</td>
		</tr>
	</tbody>
</table>

high-throughput, robust and highly time-flexible method for surface sterilization of arabidopsis seeds

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.

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 met...

Watch this Scientific Journal Video about High-throughput, Robust and Highly Time-flexible Method for Surface Sterilization of Arabidopsis Seeds at JoVE.com

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

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.

Arabidopsis is by far the plant model species most widely used for functional studies. The surface sterilization of Arabidopsis seeds is a fundamental ...

This protocol improve seed surface sterilization, a fundamental step in functional genomics of the model species Arabidopsisthaliana. The main advantages of this technique are ease and high throughput, allowing the processing of hundreds of samples per day. With some simple modifications, this method can be applied to many other model and non-model plant species.For first-time users, please pay attention to the temperature of the medium and the centrifugation speed as they are both critical for seed survival. To begin, prepare 70%ethanol by adding 95%technical ethanol to distilled water and mixing thoroughly. Next, prepare 5%bleach solution by adding five milliliters of household bleach to 95 milliliters of sterile distilled water.Then, add a few drops of nonionic detergent to the bleach solution, and mix thoroughly. Next, prepare half-strength Murashige and Skoog medium by adding 2.2 grams of MS medium powder including vitamins and 10 grams of sucrose in 800 milliliters of distilled water. Adjust the pH of the medium to 5.7 using one molar potassium hydroxide, then bring the volume up to one liter using distilled water.Aliquot 500 milliliters of the medium into a one-liter bottle, and add four grams of agar to prepare a solid medium. After autoclaving, allow the medium to cool down to 50 to 53 degrees Celsius in a water bath. Then pour it into Petri dishes under the laminar flow hood.To prepare selective medium, add kanamycin to the medium, and pour it into Petri dishes as demonstrated earlier. To set up the aspirator, connect the vacuum pump inlet to one end of a polyethylene tube of a suitable size. Then 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 to ensure airtight connection. Next, connect a second polyethylene tube to the inlet of the screw cap on the decantation bottle. Then connect the other side of the tube to the outlet of an aquarium valve fitted with a thin polyethylene tube.If necessary, wrap the junction with the sealing film to eliminate air leakage. After labeling two batches of 48 1.5-milliliter microcentrifuge tubes with progressive numbers, add 100 to 200 Arabidopsis seeds to each of the 96 sterile microcentrifuge tubes, roughly one to two millimeters above the bottom of the conical end of the tube. Next, using a 10-milliliter sterile serological pipette, add around one milliliter of 70%ethanol into each tube, and carefully close the lids.Shake the tubes at an oscillation frequency of eight hertz for three minutes in a shaker. Then remove the adapters from the shaker and transfer them into the basket of a benchtop microcentrifuge for spinning down the seeds using the pulse function. After transferring the tubes to a rack, open all tubes under the laminar flow hood.Do not touch the part of the lids fitting into the tubes to avoid contamination. Then, under the laminar flow hood, fit a sterile 200-microliter yellow tip onto the aquarium valve inlet of the homemade aspirator, and switch on the pump. Insert the tip just above the level of the seeds to avoid touching the seeds when sucking the liquid.Next, using a 10-milliliter sterile serological pipette, aliquot one milliliter of 5%bleach solution into each tube. Close the lids before placing the tubes back into the shaker adapters, then shake the tubes as demonstrated previously. After spinning down the seeds using the pulse function of a benchtop centrifuge, fit a new sterile 200-microliter yellow tip onto the aquarium valve, and switch on the pump.Insert the tip above the level of the seeds to avoid touching the seeds when sucking the bleach solution. Next, using a 10-milliliter sterile serological pipette, aliquot one milliliter of sterilized water into each tube. After fitting a new sterile 200-microliter yellow tip onto the aquarium valve, switch on the pump.Insert the tip just above the level of the seeds to avoid touching the seeds when sucking the water. Finally, aliquot 500 microliters of sterilized water into each tube, and close all the lids in the laminar flow hood. The seeds are now ready to be sown.If required, keep the tubes at room temperature for up to a few hours or at four degrees Celsius overnight. Using a one-milliliter pipette, transfer the seeds with 300 to 400 microliters of sterile water into a Petri dish. After transferring 10 tubes, pour 1.5 to two milliliters of melted half-strength Murashige and Skoog medium without antibiotics into each plate.Quickly swirl the plate to distribute the seeds, and tape the plates on opposite sides. Wrap the plates in plastic or aluminum foil, and place them in a refrigerator for three days in the dark to obtain uniform germination. Germination analyses performed at days two, three, four, and seven indicated no significant differences among 10 to 40 minutes of sterilization time with 70%ethanol.However, when the sterilization time exceeded 40 minutes, the germination rates declined. Correspondingly, green cotyledon emergence rates also decreased. Based on the study, three minutes for 70%ethanol and three minutes for 5%bleach were selected as the minimum time to sterilize the seeds.To demonstrate that the seeds used originally were contaminated with microorganisms, non-sterile seeds were sown directly on the plates. Compared to sterile seeds, non-sterile seeds showed a fungal appearance after two days, which spread all over the plates after a seven-day germination. A cross-contamination assay performed using a single sterile pipette tip to process different seed samples showed that no green cotyledons were observed in the Columbia-0 genotype seeds sown in plates with kanamycin.In parallel, in Columbia-0 seeds sown in plates without kanamycin, all cotyledons appeared green after germination. These results indicate no carryover of contamination between samples despite using a single pipette tip to remove the sterile solution. This procedure is the basis for many other functional genomics methods like gene overexpression, genome editing, subcellular localization, promoter activity, and protein-protein and protein-DNA interactions.

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Research

JoVE Journal

Biology

3.0K vistas.  Fondazione Edmund Mach. Este protocolo mejora la esterilización de la superficie de las semillas, un paso fundamental en la genómica funcional de la especie modelo Arabidopsisthaliana. Las principales ventajas de esta técnica son la facilidad y el alto rendimiento, lo que permite el procesamiento de cientos de muestras por día. Con algunas modificaciones simples, este método se puede aplicar a muchas otras especies de plantas modelo y no modelo.Para los usuarios primerizos, preste atención a la temperat...