Name: a flexible low cost hydroponic system for assessing plant responses to small molecules in sterile conditions
Uploaded: 2018-08-25T13:30:00
Description: Watch this Scientific Journal Video about A Flexible Low Cost Hydroponic System for Assessing Plant Responses to Small Molecules in Sterile Conditions at JoVE.com

This work was supported by the S&#227;o Paulo Research Foundation (FAPESP; Grant 12/19561-0) and the Max Planck Society. Elias F. Ara&#250;jo (FAPEMIG 14/30594), Carolina C. Monte-Bello (FAPESP; Grant 14/10407-3), Val&#233;ria Mafra (FAPESP; Grant 14/07918-6), and Viviane C. H. da Silva (CAPES/CNPEM 24/2013) are grateful for the fellowships. The authors thank Christian Meyer from the Institut Jean Pierre Bourgin (INRA, Versailles, France) for generously providing antibodies against RPS6. The authors thank RTV UNICAMP and Ed Paulo Aparecido de Souza Manoel for their technical support during the audio recording.

1. Preparation of Liquid and Solid Culture Media
<ol>
	<li>Prepare a liquid medium using half-strength Murashige and Skoog (MS) medium with vitamins [0.0125 mg/L of cobalt(II) chloride pentahydrate, 0.0125 mg/L of copper(II) sulfate pentahydrate, 18.35 mg/L of ethylenediaminetetraacetate ferric sodium, 3.10 mg/L of boric acid, 0.415 mg/L of potassium iodide, 8.45 mg/L of manganese sulfate monohydrate, 0.125 mg/L of sodium molybdate dihydrate, 4.30 mg/L of zinc sulfate heptahydrate, 166.01 mg/L of calcium chloride, 85 m...

This optimized hydroponic structure enables the successful in vitro culture of plants. Seeds germinate well on the solid medium at the pipette tip flat surface, a considerable gain in comparison to systems where seeds are soaked with the nutrient solution. A great advantage of this system is that during the seedling development, roots get directly in contact with the liquid medium without the need of transference. Moreover, chemical treatment can be easily applied in the liquid medium in a reduced volume. Humidi...

The advantages of growing plants using hydroponics have been widely recognized in the production of large and uniform plants, enabling reproducible experiments1,2,3. In this system, the composition of the nutritive solution can be properly controlled and recycled along all stages of plant growth and development. Furthermore, roots are not subjected to abiotic stresses, as can happen in soil-grown plants, such as nutrient starvation and water deficiency4. As plants grown hydroponically present morphological and physiological traits fairly similar to the ones cultured in soil, this system has been broadly employed in research because it allows the monitoring of root/shoot growth and their harvesting without injuries2,5.
Due to the possibility of changing the composition and concentration of the nutritive solution, most of the research using hydroponic conditions has been performed to characterize the functions of micro- and macronutrients1,3,6,7,8. However, this system has proved to be very useful to a broad range of applications in plant biology, such as to elucidate the functions of hormones and chemicals in plants. For instance, the discovery of strigolactones as a new class of hormones9 and the accelerated growth phenotype triggered by brassinosteroid application10 were performed under hydroponic conditions. Moreover, this system enables experiments with labeled isotopes (e.g., 14N/15N and 13CO2)11,12 to evaluate their incorporation into proteins and metabolites by mass spectrometry.
Considering the importance of this system in plant research, a high number of hydroponic cultures has been designed in the last few years, including systems that use (i) the transference of seedlings from plates to hydroponic containers3,13; (ii) rockwool that limits access to the early stages of root development2,14,15; (iii) polyethylene granulate as the floating body, which makes the homogeneous application of small molecules/treatments difficult16; or (iv) a reduced number of plants9,17. The volume of hydroponic tanks described in many of those protocols are usually large (small volumes ranging from 1 - 5 L, up to 32 L)18, which makes the application of chemicals extremely expensive. Although few studies do describe a hydroponic cultivation under aseptic conditions8,19, the assembly of the system is usually quite laborious, consisting of the perfect adjustment of nylon meshes into plastic or glass containers5,8,17,20.
Due to the importance of Arabidopsis thaliana as a model plant, the majority of hydroponics systems were designed for this species1,2,8,14,18,19,20. Nevertheless, there are a few studies reporting the hydroponic growth features of other plant species with a pretreatment of seeds to improve their germination and synchronization rates in vitro8,16. In order to work on a large scale, we developed a protocol for setting up a simple and low-cost maintenance hydroponic system that enables sterile conditions for growing plants, including A. thaliana and other species, such as the grass Setaria viridis. The method described here is suitable for different experiments, as the seedling growth can be maximized, synchronized, and easily monitored. Furthermore, this system has many advantages as: (i) its assembly is straightforward and its components can be reused; (ii) it allows the easy application of different chemicals into the liquid medium; (iii) the seedlings germinate and grow directly in the culture medium without the need of transference to the hydroponics system; (iv) the shoot and root development/growth can be closely supervised and the seedlings are harvested without damages; and (v) it makes it possible to work on a large scale, maintaining physiological conditions.

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			<td>Ethanol</td>
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			<td>100983</td>
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			<td>Sodium hypochlorite solution</td>
			<td>Sigma-Aldrich</td>
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			<td>Polysorbate 20&#160;&#160;</td>
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			<td>Murashige and Skoog (MS) medium including vitamins&#160;</td>
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			<td>M0222</td>
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			<td>2-(N-morpholino)ethanesulfonic acid (MES) monohydrate</td>
			<td>Duchefa Biochemie</td>
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			<td>Sigma-Aldrich</td>
			<td>484016</td>
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			<td>Laminar flow hood</td>
			<td>Telstar</td>
			<td>BH-100</td>
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			<td>Hotplate</td>
			<td>AREC</td>
			<td>F20510011</td>
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			<td>Growth chamber</td>
			<td>Weiss Technik</td>
			<td>HGC 1514</td>
			<td></td>
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			<td>Glass Petri dish (150 mm x 25 mm)</td>
			<td>Uniglass</td>
			<td>189.006</td>
			<td></td>
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			<td>200 &#956;L pipette tip racks&#160;</td>
			<td>Kasvi</td>
			<td>K8-200-5 *</td>
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			<td>300 &#956;L multichannel pipette</td>
			<td>Eppendorf</td>
			<td>3122000060</td>
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			<td>300 &#956;L pipette tips</td>
			<td>Eppendorf</td>
			<td>30073088</td>
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			<td>200 &#956;L pipette&#160;</td>
			<td>Eppendorf</td>
			<td>3120000054</td>
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			<td>200 &#956;L pipette tips</td>
			<td>Eppendorf</td>
			<td>30000870</td>
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			<td>Scissors</td>
			<td>Tramontina</td>
			<td>25912/108</td>
			<td></td>
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			<td>Tweezer</td>
			<td>ABC Instrumentos</td>
			<td>702915</td>
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			<td>Scalpel blade</td>
			<td>Sigma-Aldrich</td>
			<td>S2771</td>
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			<td>Adhesive transparent tape (45mm x 50m)</td>
			<td>Scotch 3M</td>
			<td>5803</td>
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			<td>Disposable plastic boxes, external dimensions: 353 mm (L)x 178 mm (W) x 121mm (H)</td>
			<td>Maxipac</td>
			<td>32771</td>
			<td></td>
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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.

The TOR kinase is a major regulator that integrates nutrient and energy signaling to promote cell proliferation and growth in all eukaryotes. Efforts to elucidate TOR functions in plants include the generation of Arabidopsis transgenic lines containing TOR conditional repression through RNA interference or artificial microRNA28,30,31, given the embryo lethal phenotype of TOR knockout pla...

Watch this Scientific Journal Video about A Flexible Low Cost Hydroponic System for Assessing Plant Responses to Small Molecules in Sterile Conditions at JoVE.com

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

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 ...

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