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, 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.</li>
	<li>Prepare half-strength Murashige and Skoog (&#189; MS) medium26.
	<ol>
		<li>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.</li>
		<li>After autoclaving, cool down the medium to 50-53 &#176;C in a water bath and pour it into Petri dishes under the laminar flow hood. To prepare the selective medium, add 1000 &#181;L/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 &#176;C) to the medium (50-53 &#176;C). Mix well by swirling, and pour into Petri dishes as mentioned before.</li>
	</ol>
	</li>
</ol>
2. Aspirator setup
NOTE: Instrument setup is summarized in Figure 1.
<ol>
	<li>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&#39;s junction tightly with a sealing film (Table of Materials) to ensure air-tight connection.</li>
	<li>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.</li>
	<li>Just before use, fit a sterile 200 &#181;L pipette tip to the inlet of the aquarium filter under the laminar flow hood.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62893/62893fig01.jpg" /> 
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 &#181;L pipette tip, and (E) the 1.5 mL microcentrifuge tube containing seeds and sterilization liquid. Arrows indicate the direction of the airflow. <a href="https://www.jove.com/files/ftp_upload/62893/62893fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
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.
<ol>
	<li>Label with a permanent marker two batches of 48 x 1.5 mL microcentrifuge tubes with progressive numbers.</li>
	<li>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).</li>
	<li>Aliquot around 1000 &#181;L 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).</li>
	<li>Shake the tubes at an oscillation frequency of 8.0 Hz for at least 3 min in a shaker.</li>
	<li>Remove the adapters from the shaker and transfer them into the basket of a benchtop microcentrifuge.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Fit a sterile 200 &#181;L yellow tip onto the aquarium valve inlet of the homemade aspirator under the laminar flow hood and switch on the pump.</li>
	<li>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.</li>
	<li>Aliquot into each tube around 1000 &#181;L of 5% bleach using a 10 mL sterile serological pipette inside the laminar flow hood.</li>
	<li>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.</li>
	<li>Quickly spin down the seeds using the pulse function of the benchtop centrifuge for the time necessary to reach 1880 x g (~15 s).</li>
	<li>Fit a new sterile 200 &#181;L yellow tip onto the aquarium valve connected to the vacuum pump under the laminar flow hood, and switch on the pump.</li>
	<li>Insert the yellow tip above the level of the seeds to avoid touching the seeds when sucking the bleach solution.</li>
	<li>Aliquot into each tube around 1000 &#181;L 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.</li>
	<li>Fit a new sterile 200 &#181;L yellow tip onto the aquarium valve connected to the vacuum pump under the laminar flow hood and switch on the pump.</li>
	<li>Insert the yellow tip just above the level of the seeds to avoid touching the seeds when sucking the H2O.</li>
	<li>Aliquot into each tube around 500 &#181;L 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 &#176;C overnight.</li>
	<li>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. 
	&#8203;NOTE: Autoclave the liquid to kill all the seeds inside the reservoir.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62893/62893fig02.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/62893/62893fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Plating and scoring of Arabidopsis on &#189; MS plates
<ol>
	<li>Transfer the seeds and 300-400 &#181;L of sterile H2O into a Petri dish by gentle pipetting with a 1000 &#181;L pipette.</li>
	<li>After transferring 10 tubes, pour into each plate around 1.5-2.0 mL of melted &#189; MS medium without antibiotics. 
	NOTE: Melt in advance and then keep the &#189; MS melted medium in a thermostatic bath set at 50-53 &#176;C to avoid solidification. Ensure that the temperature does not exceed 58 &#176;C to avoid decreasing the germinability of the seeds.</li>
	<li>Quickly swirl the plate to distribute the seeds inside it. Tape the plates on opposite sides.</li>
	<li>Wrap the plates in a plastic or aluminum foil and then place them in a fridge (4 &#176;C) for 3 days in the dark to obtain uniform germination.</li>
	<li>Transfer the plates into a growth chamber set at 23 &#176;C under long-day conditions (16 h light/8 h dark) with a light intensity of 100-120 &#181;mol&#183;m-2&#183;s-1 and 60% relative humidity.</li>
	<li>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.</li>
</ol>
5. Statistical analyses
NOTE: Here, Tukey&#39;s pairwise test was used for statistical analyses.
<ol>
	<li>Consider the P-values below 0.01 as statistically significant. Perform all the experiments at least with five biological replicates.</li>
</ol>

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All authors declare no conflicts of interest.

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><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&amp;ndash;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 &amp;amp; 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&amp;nbsp; (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...

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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 ...

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Research

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Biology

3.2K Views.  Fondazione Edmund Mach. Dit protocol verbetert de sterilisatie van het zaadoppervlak, een fundamentele stap in de functionele genomica van de modelsoort Arabidopsisthaliana. De belangrijkste voordelen van deze techniek zijn gemak en hoge doorvoer, waardoor de verwerking van honderden monsters per dag mogelijk is. Met enkele eenvoudige aanpassingen kan deze methode worden toegepast op vele andere model- en niet-modelplantensoorten.Let voor nieuwe gebruikers op de temperatuur van het medium en de centrifugeersn...

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

Adherence of Bacteria to Plant Surfaces Measured in the Laboratory

This manuscript describes a method to measure bacterial binding to axenic plant surfaces in the light microscope and through the use of viable cell counts. Plant materials used include roots, sprouts, leaves, and cut fruits. The methods described are inexpensive, easy, and suitable for small sample sizes. Binding is measured in the laboratory and a variety of incubation media and conditions can be used. The effect of inhibitors can be determined. Situations that promote and inhibit binding can also be assessed. In some cases it is possible to distinguish whether various conditions alter binding primarily due to their effects on the plant or on the bacteria.

An easy method for measuring and characterizing bacterial adhesion to plants, particularly roots and sprouts, is described in this article.

This manuscript describes a method to measure bacterial binding to axenic plant surfaces in the light microscope and through the use of viable cell ...

Environment

A Flexible Low Cost Hydroponic System for Assessing Plant Responses to Small Molecules in Sterile Conditions

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for assessing plant responses to chemicals and other substances of interest is presented. This system is highly efficient in obtaining homogeneous and healthy seedlings of the C3 and C4 model species Arabidopsis thaliana and Setaria viridis, respectively. The sterile cultivation avoids algae and microorganism contamination, which are known limiting factors for plant normal growth and development in hydroponics. In addition, this system is scalable, enabling the harvest of plant material on a large scale with minor mechanical damage, as well as the harvest of individual parts of a plant if desired. A detailed protocol demonstrating that this system has an easy and low-cost assembly, as it uses pipette racks as the main platform for growing plants, is provided. The feasibility of this system was validated using Arabidopsis seedlings to assess the effect of the drug AZD-8055, a chemical inhibitor of the target of rapamycin (TOR) kinase. TOR inhibition was efficiently detected as early as 30 min after an AZD-8055 treatment in roots and shoots. Furthermore, AZD-8055-treated plants displayed the expected starch-excess phenotype. We proposed this hydroponic system as an ideal method for plant researchers aiming to monitor the action of plant inducers or inhibitors, as well as to assess metabolic fluxes using isotope-labeling compounds which, in general, requires the use of expensive reagents.

A simple, versatile, and low-cost in vitro hydroponic system was successfully optimized, enabling large-scale experiments under sterile conditions. This system facilitates the application of chemicals in a solution and their efficient absorption by roots for molecular, biochemical, and physiological studies.

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for ...

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

Translating Ribosome Affinity Purification (TRAP) to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome affinity purification (TRAP) method and consecutive optimized low-input library preparation.
As starting material, we employ plant lines that express GFP-tagged ribosomal protein RPL18 in a cell type-specific manner by use of adequate promoters. Prior to immunopurification and RNA extraction, the tissue is snap frozen, which preserves tissue integrity and simultaneously allows execution of time series studies with high temporal resolution. Notably, cell wall structures remain intact, which is a major drawback in alternative procedures such as fluorescence-activated cell sorting-based approaches that rely on tissue protoplasting to isolate distinct cell populations. Additionally, no tissue fixation is necessary as in laser capture microdissection-based techniques, which allows high-quality RNA to be obtained.
However, sampling from subpopulations of cells and only isolating polysome-associated RNA severely limits RNA yields. It is, therefore, necessary to apply sufficiently sensitive library preparation methods for successful data acquisition by RNA-seq.
TRAP offers an ideal tool for plant research as many developmental processes involve cell wall-related and mechanical signaling pathways. The use of promoters to target specific cell populations is bridging the gap between organ and single-cell level that in turn suffer from little resolution or very high costs. Here, we apply TRAP to study cell-cell communication in lateral root formation.

Translating Ribosome Affinity Purification TRAP to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

Translating ribosome affinity purification (TRAP) offers the possibility to dissect developmental programs with minimal processing of organs and tissues. The protocol yields high-quality RNA from cells targeted with a green fluorescent protein (GFP)-labeled ribosomal subunit. Downstream analysis tools, such as qRT-PCR or RNA-seq, reveal tissue and cell type-specific expression profiles.

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome ...

Developmental Biology

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of dormant seeds or that of nondormant seeds irradiated by a far red (FR) light pulse. In order to further gain insight into the molecular genetic mechanisms underlying the germination repressive activity exerted by the endosperm, a &#34;seed coat bedding&#34; assay (SCBA) was devised. The SCBA is a dissection procedure physically separating seed coats and embryos from seeds, which allows monitoring the growth of embryos on an underlying layer of seed coats. Remarkably, the SCBA reconstitutes the germination repressive activities of the seed coat in the context of seed dormancy and FR-dependent control of seed germination. Since the SCBA allows the combinatorial use of dormant, nondormant and genetically modified seed coat and embryonic materials, the genetic pathways controlling germination and specifically operating in the endosperm and embryo can be dissected. Here we detail the procedure to assemble a SCBA.

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

We present the procedure to assemble a seed coat bedding assay (SCBA) from Arabidopsis thaliana seeds. The SCBA was shown to be a powerful tool to explore genetically and in vitro how the endosperm controls seed germination in dormant seeds and in response to light cues. The SCBA is in principle applicable under any situation where the endosperm is suspected to influence embryonic growth.

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of ...

Lateral Root Inducible System in Arabidopsis and Maize

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is however tedious, because it occurs only in a few cells inside the root and in an unpredictable manner. To circumvent this problem, a Lateral Root Inducible System (LRIS) has been developed. By treating seedlings consecutively with an auxin transport inhibitor and a synthetic auxin, highly controlled lateral root initiation occurs synchronously in the primary root, allowing abundant sampling of a desired developmental stage. The LRIS has first been developed for Arabidopsis thaliana, but can be applied to other plants as well. Accordingly, it has been adapted for use in maize (Zea mays). A detailed overview of the different steps of the LRIS in both plants is given. The combination of this system with comparative transcriptomics made it possible to identify functional homologs of Arabidopsis lateral root initiation genes in other species as illustrated here for the CYCLIN B1;1 (CYCB1;1) cell cycle gene in maize. Finally, the principles that need to be taken into account when an LRIS is developed for other plant species are discussed.

Lateral Root Inducible System in Arabidopsis and Maize

The Lateral Root Inducible System (LRIS) allows for synchronous induction of lateral roots and is presented for Arabidopsis thaliana and maize.

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is ...

Hydroponics: A Versatile System to Study Nutrient Allocation and Plant Responses to Nutrient Availability and Exposure to Toxic Elements

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several crops, including lettuce and tomato. Within the plant research community, numerous hydroponic systems have been designed to study plant responses to biotic and abiotic stresses. Here we present a hydroponic protocol that can be easily implemented in laboratories interested in pursuing studies on plant mineral nutrition.
This protocol describes the hydroponic system set up in detail and the preparation of plant material for successful experiments. Most of the materials described in this protocol can be found outside scientific supply companies, making the set up for hydroponic experiments less expensive and convenient.
The use of a hydroponic growth system is most advantageous in situations where the nutrient media need to be well controlled and when intact roots need to be harvested for downstream applications. We also demonstrate how nutrient concentrations can be modified to induce plant responses to both essential nutrients and toxic non-essential elements.

Here, we present an easy-to-follow protocol to establish a successful hydroponic system for plant nutrition studies. This protocol has been extensively tested in Arabidopsis and can easily be adapted to other plant species to study specific nutritional requirements or the effect of non-essential elements on plant growth and development.

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several ...

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of microbial contaminants present on the seed surface. Currently, Arabidopsis seeds are sterilized using two distinct sterilization techniques in conditions that differ slightly between labs and have not been standardized, often resulting in only partially effective sterilization or in excessive seed mortality. Most of these methods are also not easily scalable to a large number of seed lines of diverse genotypes. As technologies for high-throughput analysis of Arabidopsis continue to proliferate, standardized techniques for sterilizing large numbers of seeds of different genotypes are becoming essential for conducting these types of experiments. The response of a number of Arabidopsis lines to two different sterilization techniques was evaluated based on seed germination rate and the level of seed contamination with microbes and other pathogens. The treatments included different concentrations of sterilizing agents and times of exposure, combined to determine optimal conditions for Arabidopsis seed sterilization. Optimized protocols have been developed for two different sterilization methods: bleach (liquid-phase) and chlorine (Cl2) gas (vapor-phase), both resulting in high seed germination rates and minimal microbial contamination. The utility of these protocols was illustrated through the testing of both wild type and mutant seeds with a range of germination potentials. Our results show that seeds can be effectively sterilized using either method without excessive seed mortality, although detrimental effects of sterilization were observed for seeds with lower than optimal germination potential. In addition, an equation was developed to enable researchers to apply the standardized chlorine gas sterilization conditions to airtight containers of different sizes. The protocols described here allow easy, efficient, and inexpensive seed sterilization for a large number of Arabidopsis lines.

The objective of this study was to determine the effects of bleach and chlorine gas sterilization on seed germination of a range of Arabidopsis genotypes grown on sterile media. Optimized sterilization protocols have been developed to prevent the growth of microbial contaminants while providing satisfactory seed survival.

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of ...

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

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