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

Summary

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.

Abstract

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.

Introduction

The advantages of growing plants using hydroponics have been widely recognized in the production of large and uniform plants, enabling reproducible experiments^1,2,3. 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....

Protocol

1. Preparation of Liquid and Solid Culture Media

1. 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 mg/L of potassium dihydrogen phosphate, 950 mg/L of potassium nitrate, 90.27 mg/L of magnesium sulfate, 825 mg/L of ammonium nitrate, 1 mg/L of glycine....

Results

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 microRNA^28,30,31, given the embryo lethal phenotype of TOR knockout pla.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ethanol	Merck	100983
Sodium hypochlorite solution	Sigma-Aldrich	425044
Polysorbate 20	Sigma-Aldrich	P2287
Murashige and Skoog (MS) medium including vitamins	Duchefa Biochemie	M0222
2-(N-morpholino)ethanesulfonic acid (MES) monohydrate	Duchefa Biochemie	M1503
Agar	Sigma-Aldrich	A7921
Potassium hydroxide	Sigma-Aldrich	484016
Laminar flow hood	Telstar	BH-100
Hotplate	AREC	F20510011
Growth chamber	Weiss Technik	HGC 1514
Glass Petri dish (150 mm x 25 mm)	Uniglass	189.006
200 μL pipette tip racks	Kasvi	K8-200-5 *
300 μL multichannel pipette	Eppendorf	3122000060
300 μL pipette tips	Eppendorf	30073088
200 μL pipette	Eppendorf	3120000054
200 μL pipette tips	Eppendorf	30000870
Scissors	Tramontina	25912/108
Tweezer	ABC Instrumentos	702915
Scalpel blade	Sigma-Aldrich	S2771
Adhesive transparent tape (45mm x 50m)	Scotch 3M	5803
Disposable plastic boxes, external dimensions: 353 mm (L)x 178 mm (W) x 121mm (H)	Maxipac	32771