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.
Hydroponiske systemer er blevet anvendt som en af de standardmetoder til plantebiologi forskning og anvendes også i kommerciel produktion i flere afgrøder, herunder salat og tomat. Inden anlægget forskningsverdenen, har mange hydroponiske systemer er designet til at studere plante reaktioner på biotiske og abiotiske påvirkninger. Her præsenterer vi en hydroponiske protokol, der let kan implementeres i laboratorier interesseret i at forfølge undersøgelser af anlæg mineral ernæring.
Denne protokol beskriver den hydroponiske system, der oprettes i detaljer og udarbejdelse af plantemateriale for vellykkede eksperimenter. De fleste af de i denne protokol materialer kan findes uden videnskabelige forsyningsselskaber, hvilket gør den sat op til hydroponiske eksperimenter billigere og bekvemt.
Anvendelsen af et hydroponiske vækst system er mest fordelagtig i situationer, hvor de næringsmedier skal velkontrolleret og når intakt roOTS skal høstes til efterfølgende anvendelser. Vi viser også, hvordan næringsstof koncentrationer kan ændres til at fremkalde plante svar på både essentielle næringsstoffer og giftige ikke-væsentlige bestemmelser.
Plants are among the few organisms that can synthesize all the required metabolites from inorganic ions, water and CO2 using the energy captured from the sun1. Hydroponics is a method of growing plants that takes advantage of this fact by providing all of the nutrients, in their inorganic form, in a liquid solution with or without solid media. Hydroponic systems have been extensively used by scientists for exploring nutrient requirements and also the toxicity of some elements in Arabidopsis and other plant species 2-5. For instance, Berezin et al.3, Conn et al.4, and Alatorre-Cobos et al.2 used hydroponic systems and several plant species including tomato and tobacco, to generate sufficient plant biomass for mineral analysis2-4. Industrial applications of hydroponics have also been developed for crops such as tomato and lettuce6. Here, we outline the use of hydroponics in the context of research, possible variations in available methods, and finally present a system that can be easily scalable and useful for research laboratories interested in studying plant mineral nutrition.
Hydroponic systems allow for easy separation of root tissue and precise control of nutrient availability
Hydroponics offers several advantages over soil-based systems. When removed from soil, root tissue is often mechanically sheared causing loss of tissue or damage. This is particularly true for fine root structures such as lateral roots and root hairs. Hydroponic systems that do not utilize an inert particulate media allow a less invasive separation of root and shoot tissues.
In soil systems, nutrient bioavailability changes throughout the soil matrix as nutrients bind to soil particles creating micro-environments within the soil. This heterogeneity could add an extra level of complexity in experiments needing a precise control on the external concentration of nutrients or other molecules. In contrast, the hydroponic solution is homogeneous and can be easily replaced throughout the course of the experiment.
Variants of hydroponic systems
All hydroponic cultures rely on a nutrient solution to deliver essential elements to the plant. In addition to the nutrients, the roots also need a steady supply of oxygen. When roots become anoxic they are unable to take up and transport metabolites to the rest of the plant body7. Hydroponic systems can be classified based on how they deliver oxygen and other nutrients to the roots: oxygen delivery by saturating the solution with air (classical hydroponics), by not submerging the roots at all times, or by allowing the roots to be completely exposed to the air (aeroponics)8. In hydroponics, nutrient solution can be saturated with air prior to its use and changed frequently, or air can be continuously supplied in the solution over the life cycle of the plant9. Alternatively, plants may also be grown on inert media (e.g., rockwool, vermiculite, or clay pellets) and subjected to wet-dry cycles by dripping solution through the media or periodically submerging the substrate in the nutrient solution10. In aeroponics, roots are sprayed with the nutrient solution to prevent desiccation.
Disadvantages of hydroponic systems
Although hydroponic cultures offer clear advantages over soil-based systems, there are some considerations that must be acknowledged when interpreting the data. For instance, hydroponic systems expose plants to conditions that may be seen as non-physiological. Therefore, phenotypes or plant responses detected using hydroponic systems may vary in magnitude when plants are grown in alternative systems (e.g., soil or agar-based media). These considerations are not unique for hydroponic systems; differential responses can also be observed if plants are grown in different types of soil 11,12.
The following protocol provides step-by-step instructions on how to set up a hydroponic system in a laboratory. This protocol has been optimized for Arabidopsis thaliana (Arabidopsis); however, similar or in some cases identical steps can be used to grow other species.
The health of seedlings used for hydroponics is one of the major factors contributing to the success of a hydroponic experiment. Sterilization of instruments, seeds, and culture media also play an important role in reducing the risk of contamination and provide a good start for the plants before they are transplanted into the hydroponic system. A working environment with facilities such as an autoclave, fume hood, cold-room (4 °C), and growth space with controlled conditions (light intensity and temperature) is necessary for a good experimental set up.
The freshness of the nutrient solution also determines the plant health and in turn determines the success of a hydroponic experiment. Since water evaporates faster under direct lighting, the concentration of salts will change due to a reduction of total solution volume; therefore it is best to change the hydroponic solution at least twice a week. However, if large, deep containers equipped with an air pump system are used it may not be necessary to replace the nutrient solution for experiments that are short in duration. Note that in the case of Arabidopsis we used Magenta vessels (77 mm width x 77 mm length x 97 mm height) but other, larger containers can also be used to accommodate larger plants.
For researchers interested in plant nutrients, hydroponic experiments provide a unique setting to test plant phenotypes and responses to different nutrient availability17. By manipulating the concentrations of the elements of interest, researchers can set up different experiments to test the effects of sufficiency, deficiency, or toxic concentrations of essential and non-essential nutrients. Compared to the soil-based system, the hydroponic system provides a more homogeneous nutrient medium to the plants with less risk of soil-borne diseases. In addition, both root and shoot tissues can be harvested and separated easily for further analyses on specific plant tissues.
In the representative section, we introduced two examples in which a simple hydroponic system was used for more detailed studies on plant nutrition. In the first example, by growing plants on a zinc concentration gradient, we were able to illustrate the level of control that can be achieved on nutrient composition using this hydroponic system. Plants grown with 7 µM Zn grew much more vigorously compared to plants grown in 50 µM Zn, while plants grown without extra Zn added were stunted compared to plants grown with 7 µM Zn. This was in part due to the length of time the plants were allowed to grow under sufficient conditions; earlier removal of Zn from the media is likely to induce stronger zinc-deficiency symptoms. Applying the same principle, we were able to induce toxicity using the non-essential metal, cadmium, which is known to impair plant growth.
In the second example, the elemental composition of Col-0 roots and shoots treated with 20 µM Cd for 72 hr was determined by ICP-OES. We found differences in all detected metals between roots and shoots. Macro-elements were found in higher concentrations in the shoots relative to the roots, while iron and zinc were found more abundant in roots. Cadmium followed a pattern similar to iron and zinc, being more concentrated in roots compared to shoots. These data reinforce the idea that leaves and roots provide different information about the ionome status of the plant and therefore both tissues need to be analyzed separately to understand mineral nutrition and composition at the whole plant level. Besides ICP-OES several spectroscopic methods such as Atomic Absorption Spectroscopy (AAS) or Inductively Coupled Plasma Mass Spectrometry (ICP-MS) can also be used to measure the elemental composition (ionome) of plant tissues18-20.
In a hydroponic experiment, the symptoms and phenotypes of plants responding to different nutrient conditions represent the beginning of what could be extended into more elaborated analyses such as gene expression (transcriptomics) and protein abundance (proteomics). These -omic techniques are keys to integrate plant metabolism by considering processes in a tissue-specific manner.
The authors have nothing to disclose.
This research was supported by the University of Missouri Research Board (Project CB000519) and the US National Science Foundation (IIA-1430428 to DMC). Nga T. Nguyen was supported by the Vietnam Education Foundation Training Program (Exchange visitor program No. G-3-10180). We also thank Roger Meissen (MU Bond Life Sciences Center) for his assistance and expertise during the video recording and editing sessions.
For seed sterilization | |||
Bleach | The Clorox Company | NA | The regular bleach |
www.cloroxprofessional.com | |||
Hydrochloric acid | Fisher Scientific | A144-500 | |
Desiccator body | Nalgene | D2797 SIGMA | Marketed by Sigma-Aldrich |
Desiccator plate | Nalgene | 5312-0230 | Marketed by Thermo Scientific |
For one quarter MS medium preparation | |||
MES | Acros Organics | 172591000 | 4-Morpholineethanesulfonic acid hydrate |
Murashige and Skoog (MS) | Sigma-Aldrich | M0404-10L | |
KOH | Fisher Scientific | P250-500 | |
Phytoagar | Duchefa Biochemie | P1003.1000 | |
Square plate | Fisher Scientific | 0875711A | Disposable Petri Dish With Grid |
For seed plating | |||
Filter paper | Whatman | 1004090 | |
Toothpick | Jarden Home Brands | NA | |
Aluminum foil | Reynolds Wrap | NA | Standard aluminum foil |
Micropore tape | 3M Health Care | 19-898-074 | Surgical tape; Marketed by Fisher Scientific |
For hydroponic solution preparation | |||
KNO3 | Fisher Scientific | BP368-500 | |
KH2PO4 | Fisher Scientific | P386-500 | |
MgSO4 | Fisher Scientific | M63-500 | |
Ca(NO3)2 | Acros Organics | A0314209 | |
H3BO3 | Sigma | B9645-500G | |
MnCl2 | Sigma-Aldrich | M7634-100G | |
ZnSO4 | Sigma | Z0251-100G | |
Na2MoO4 | Aldrich | 737-860-5G | |
NaCl2 | Fisher Scientific | S271-1 | |
CoCl | Sigma-Aldrich | 232696-5G | |
FeEDTA | Sigma | E6760-100G | |
“Stericup & Steritop” bottle | Milipore Corporation | SCGVU02RE | Micronutrient container |
For root wash buffer preparation | www.milipore.com | ||
EDTA | Acros Organics | A0305456 | |
Tris | Fisher Scientific | BP154-1 | |
For hydroponic set up | |||
Autoclavable foam tube plug | Jaece Industries Inc. | L800-A | Identi-Plugs fit to holes with 2R=6-13mm |
Foam Board | Styrofoam Brand Dow | ESR-2142 | Thickness is 1/2 inches |
Cork borer | Humboldt | H-9662 | Cork Borer Sets with Handles, , Plated Brass Set of 6, 3/16" to 1/2" OD Size |
Air pump | Aqua Culture | MK-1504 | |
Marketed by Wal-mart Stores, Inc. | |||
Airline tubing and aquarium bubble stones | Aqua Culture | Tubing: 928/25-S | |
Marketed by Wal-mart Stores, Inc. | Stone: ASC-1 | ||
Outro | |||
Ethanol | Fisher Scientific | A995-4 | Reagent Alcohol |
Cadmium Chloride (CdCl2) | Sigma-Aldrich | 10108-64-2 |