Protocol: optimising hydroponic growth systems for nutritional and physiological analysis of Arabidopsis thaliana and other plants
- Simon J Conn1,
- Bradleigh Hocking1, 2,
- Maclin Dayod1, 2,
- Bo Xu1, 2,
- Asmini Athman1, 2,
- Sam Henderson1, 2,
- Lucy Aukett1,
- Vanessa Conn1, 2,
- Monique K Shearer1, 3,
- Sigfredo Fuentes1,
- Stephen D Tyerman1, 2 and
- Matthew Gilliham1, 2Email author
© Conn et al.; licensee BioMed Central Ltd. 2013
Received: 20 November 2012
Accepted: 30 January 2013
Published: 5 February 2013
Hydroponic growth systems are a convenient platform for studying whole plant physiology. However, we found through trialling systems as they are described in the literature that our experiments were frequently confounded by factors that affected plant growth, including algal contamination and hypoxia. We also found the way in which the plants were grown made them poorly amenable to a number of common physiological assays.
The drivers for the development of this hydroponic system were: 1) the exclusion of light from the growth solution; 2) to simplify the handling of individual plants, and 3) the growth of the plant to allow easy implementation of multiple assays. These aims were all met by the use of pierced lids of black microcentrifuge tubes. Seed was germinated on a lid filled with an agar-containing germination media immersed in the same solution. Following germination, the liquid growth media was exchanged with the experimental solution, and after 14-21 days seedlings were transferred to larger tanks with aerated solution where they remained until experimentation. We provide details of the protocol including composition of the basal growth solution, and separate solutions with altered calcium, magnesium, potassium or sodium supply whilst maintaining the activity of the majority of other ions. We demonstrate the adaptability of this system for: gas exchange measurement on single leaves and whole plants; qRT-PCR to probe the transcriptional response of roots or shoots to altered nutrient composition in the growth solution (we demonstrate this using high and low calcium supply); producing highly competent mesophyll protoplasts; and, accelerating the screening of Arabidopsis transformants. This system is also ideal for manipulating plants for micropipette techniques such as electrophysiology or SiCSA.
We present an optimised plant hydroponic culture system that can be quickly and cheaply constructed, and produces plants with similar growth kinetics to soil-grown plants, but with the advantage of being a versatile platform for a myriad of physiological and molecular biological measurements on all plant tissues at all developmental stages. We present ‘tips and tricks’ for the easy adoption of this hydroponic culture system.
KeywordsHydroponics Plant nutrition Arabidopsis Gas exchange ACA2 CAX1 CAX2 VHA-α Transient transformation
Single cell sampling and analysis
Quantitative real-time RT-PCR
Green fluorescent protein
Upstream activation sequence
Basal nutrient solution
Low calcium solution
High calcium solution
Calcium proton exchanger.
Arabidopsis thaliana (L.) Heynh. (Arabidopsis) has been adopted as a model plant of choice in many laboratories for a variety of reasons. These include a brief life cycle, a small and well-annotated genome, its amenability to tissue culture, the limited cell-layers per cell type (for developing roots), the availability of natural diversity sets and targeted mutants, and the ease at which it can be genetically transformed . The diminutive stature and rosette growth habit of Arabidopsis also means that it does not require a large area to cultivate. At the same time, the size of Arabidopsis has presented considerable challenges for those wanting to perform physiological measurements on intact plants such as gas exchange, hydraulic conductance, or for obtaining single-cell parameters such as turgor pressure and membrane potential. To benefit from the vast molecular resources of Arabidopsis, physiologists have had to adapt measuring equipment and assays to the microscale; these technological challenges have curtailed the use of Arabidopsis as a tractable physiological model . In order to perform such assays whilst providing a flexible experimental platform for manipulation of both the shoot and root environment, the use of hydroponics for research purposes has become common.
Advantages and disadvantages between geoponics, agar plates and three distinct aggregate hydroponics methods for cultivating arabidopsis plants
Geoponics (i.e. soil/sand)
This system (Conn et al.)
Contamination (algal, bacterial)
< 3 week-old seedlings
One common and significant problem associated with aggregate hydroponics growth systems is the algal contamination of the culture medium . This can occur in the tank, and particularly on rockwool or agar-based plugs, or the plant roots and shoots, due to the use of non-sterile phosphorous-rich medium and the exposure of these components to light. This becomes a problem for physiological studies as algal growth can reduce root nutrient uptake efficiency, plant growth, perturb the composition of the growth solution (nutrients, pH) and induce significant changes to the plant global transcriptome and proteome [16–18]. For this reason alone it is important that hydroponic systems avoid illumination of the growth media if they are to be used in physiological studies.
A major driver for developing this hydroponics system was to be able to manipulate Arabidopsis plants for a variety of assays including single cell sampling and analysis (SiCSA), which requires live plants to be fixed to a flat, hard growth surface [19–22]. The following system allowed us to sample single cells easily for both molecular and ionomic interrogation (the methodology for molecular analysis is outlined in ); it would also be ideal for micropipette techniques such as turgor measurement and electrophysiology. After considerable iterative development we present this simple, inexpensive, flexible and robust hydroponics system for the cultivation of Arabidopsis (and other plants), which addresses the above considerations and streamlines the methodology to allow other laboratories to adopt this procedure. In addition, we document how to adapt physiological measuring equipment to this hydroponics system and present some analyses of Arabidopsis plants grown in this hydroponic set-up. These comparisons show that the hydroponic system produces plants with equivalent growth rates to soil-grown plants but provides more flexibility for applying many physiological and molecular analyses of the plant tissues.
Agar, plant cell culture tested (e.g. Sigma, A7921)
Nutrient solution stocks (see Additional file 1 for detailed description of growth solutions).
1.5 mL microcentrifuge tube, black(e.g. Bioplastics, B74010), 48
50 mL polypropylene conical centrifuge tube with flat top screw cap (e.g. BD Biosciences, 352070), 48
Leather punch, or 15-18G × 1 1/2" hypodermic needles (e.g. Terumo, NN-1838R), 1
13 L multistacking container (e.g. Nally, IH305), 1
24 well floater microtube rack, blue with hinged lid (Scientific Specialties, 5100-43), 2
Or, for large scale planting ( > 100 seeds) pizza crisper trays with 11 mm holes (e.g. Willow, heavy metal bakeware 34 cm family size) and pot saucer that fits the pizza tray making it light tight (e.g. Reko, 430 mm saucer, RSRSTD430.07), 1 each.
Plastic support for tubes in hydroponics container, plastic, 1
Aquarium air pump (e.g. Resun, AC9904), 1
Freshwater aquarium air stones, 2
Aquarium tubing, 1.5 m to fit aquarium pump (e.g. Aquaone, 4 mm internal diameter tube)
Plastic Y-connectors to fit tubing, and clamps to adjust airflow
Fluorescent lamps 36W/840 cool white (e.g. Osram, 4050300517872).
Mature plant tank
Remove the conical base from the 50 mL centrifuge tubes using a hacksaw or band saw, and smooth the cut edges with a metal file to prevent future root damage. Drill a hole in the centre of 50 mL centrifuge tube lid (11 mm diameter) to support the lip of the plant holder. Forty-eight tubes are required per tank.
Adhere four plastic strips (20 × 120 × 10 mm) to the inside of an opaque 13 L hydroponics growth container (320 mm × 415 mm × 110 mm) with silicon-based adhesive, 20 mm from the top to support the microcentrifuge tube lid.
Plastic lids can be made from a rectangular plastic sheet (290 mm × 390 mm × 5 mm). Using a hole-bit drill 48 holes (6 × 8 pattern) of 32 mm diameter to fit the cut 50 mL centrifuge tubes.
Aeration of each hydroponics tank is provided via a single tube from a 4-outlet aquarium pump (5 W, 540 L.h-1 maximum), with a Y-connector fitted inline to permit the use of two freshwater airstones (30 –100 mm) in each tank. These can be anchored onto the base of the tank with silicone adhesive. Use clamps to adjust airflow if necessary.
Plants in hydroponics tanks can be illuminated as required. For this setup we use 36W/840 cool white fluorescent lamps, 8 lamps per shelf (3 tanks per shelf). Plants are typically grown 210 mm beneath lamps.
Preparing germination lids
Prepare 100 mL of germination medium (GM) (recipe Additional file 1) in a autoclavable bottle and add 0.7 g agar (final conc. 0.7% w/v), autoclave and cool slightly. The solution can also be microwaved to dissolve the agar as aseptic culture is not required.
Using a leather punch or hypodermic needle, bore a single 1.2 – 1.8mm diameter hole into the centre of a black microcentrifuge tube lid.
Cut lids from the microcentrifuge tube base, retaining 1 – 2mm of the hinge, invert lid onto clingfilm or adhesive tape. Once all lids have been prepared, fill each with ~250-300 μL germination medium agar and leave to solidify for 15 min.
CRITICAL POINT: The hinge of the microcentrifuge lid can be used for easy manipulation of plants with tweezers.
CRITICAL POINT: Ensure the lids are filled such that there is a dome of GM-agar for each lid, but avoid overflowing as this may cause the lids to sit askew in the germination tank. If the solution escapes through the lid hole, either allow media to cool (55-60°C is ideal) or supplement with additional media.
Invert lids into floating racks with the agar plug in contact with liquid GM to create the functional seed/seedling/plant holder.
NOTE: Prefabricated 34 cm diameter pizza crisper trays, containing over three hundred holes of 11 mm in diameter, can be used to hold larger batches of plants. We found it essential that each of the microcentrifuge tube lids sat snugly enough in the holding tray to prevent light penetration into the GM, but loosely enough so they could be easily removed and transferred to another container when required.
Using a moistened toothpick, place up to three seeds in the hole of the lid on the agar surface to maximise chances of seed germination. Then, cover the entire container with plastic clingfilm to enhance humidity, leaving at least 10 mm above the plant for growth. Stratify seeds in the dark at 4°C for at least 48 h.
Transfer the germination tank into growth cabinets under a 8:16 h, light:dark cycle, with 55% atmospheric humidity, at 22°C and an irradiance of 150 μmol photons m-2.s-1 at the plant level. Under these conditions, the roots of these seedlings emerge from the agar plug after 4-7 days.
At this stage thin down to a single plant per lid and replace the bath solution incrementally from GM to a standard growth solution (in our case, a modified ¼ Hoagland's solution, hereafter referred to as BNS, refer to Additional file 1 for recipe). On day 1 of the solution change, 1/3 of the GM was replaced with BNS. On day 2, 50% of the existing solution was exchanged with BNS and on day three the entire solution was replaced with BNS.
After day 14, puncture holes in the clingfilm to decrease humidity and then remove completely after day 17.
CRITICAL POINT: Do not allow agar plugs to dry out at this stage, this is rarely a problem if using floating racks but it is extremely important to keep the solution level topped up if using pizza or equivalent trays to germinate the seedlings.
When the roots are 40–50 mm in length, approximately 21 days post-germination, plants are the appropriate size to survive transfer into an aerated hydroponics tank. Transfer plants in lids to the modified 50 mL centrifuge tubes, passing the roots through the 11 mm diameter hole drilled in its lid to support the lip of the seedling holder. Then insert this unit into the lid of the tank containing 10 L of growth solution and continue until all 48 positions are filled.
CRITICAL POINT: These holders permit access for the roots to the whole growth media but prevent root entanglement for up to ~7 weeks when grown under short (8 h:16h) photoperiod (Figure 1).
NOTE: If not all 48 plant tubes are filled with plants, unused holes must be covered to exclude light from the growth solution. Use 50 mL centrifuge tube lids without holes or place an intact lid or base of a black microcentrifuge tube within the 11 mm hole if present, or use large pieces of aluminium foil wrapped in plastic clingfilm to cover multiple holes simultaneously.
NOTE: Plants can remain in these 13 L tanks, each holding 48 plants, with weekly solution changes until analysis. After ~3 days in these larger tanks the agar plug dries to form a thin film that separates itself from the root system. This occurs because the agar plug no longer is in contact with the growth solution when the plant holder is placed in the hydroponic tank. As such, this permits full access to the whole of the shoot and/or root system. The plant holder provides a useful handling tool for transferring the seedling to experimental chambers or different solutions, whilst limiting mechanical stress, but could be removed from the plant by cutting the plastic lid in half. This is particularly useful for imaging whole plants for reporter localisation studies.
Plant growth and seed collection
Transient protoplast transformation
A number of studies on promoter responsiveness, cellular localisation and protein-protein interactions can be undertaken in Arabidopsis protoplasts, rather than using the whole plants. Yoo et al.  presented a technique for transient expression of genes in protoplasts isolated from Arabidopsis mesophyll cells. We trialled a modified version of this protocol on protoplasts isolated from plants grown in soil or our hydroponics system, to detect expression of a cytosolic sGFP encoded on both a small vector (4 kb) and a large vector (12 kb), and quantifying the proportion of GFP-positive cells. The transfection efficiency of protoplasts derived from hydroponically-grown leaves was consistently higher than that of those derived from soil-grown plants, at least 2-fold higher for the 12 kb vector and 8–26% higher for the smaller vector, depending on DNA input (Figure 2B). Furthermore, as expected, we observed that the transformation efficiency of the larger vector was lower regardless of growth regime. However, for the hydroponically grown plants at least, the rate was sufficiently high at 14–20% to be used as a screening tool for specific applications like subcellular localisation of large membrane transporters. No difference was observed in the average size of protoplasts between methods, or the intracellular localisation of sGFP, yet the total yield of mesophyll protoplasts was consistently 15% above those from soil-grown plants, in part due to more uniform growth enabling the harvest of healthy leaves at consistent stages of development. Combining this higher yield and higher transformation rate, this constitutes an optimised approach for the study of many processes in protoplasts.
Plant nutrition and transcriptional response
Comparative ionomics of soil-grown and hydroponically-grown plants
1,608 ± 219
1,808 ± 120
9,402 ± 845
9,876 ± 492
8,449 ± 602
8,225 ± 204
34,214 ± 1874
37,747 ± 1542
44,314 ± 3005
38,821 ± 1603
64 ± 35
116 ± 42
85 ± 36
64 ± 20
3 ± 0.6
1 ± 0.2
1 ± 0.5
1.3 ± 0.4
65 ± 28
360 ± 108
2 ± 0.4
3 ± 1.1
The ability to isolate the entire root and shoot tissues of plants also enabled quantification of the transcriptional response to altered elemental concentrations in the growth media. We adjusted the calcium ion activity (aCa2+) to 3 levels; 1 mM (BNS), 0.025 mM (Low Calcium Solution, LCS) and 5 mM (High Calcium Solution, HCS) (Additional file 1), whilst keeping the activity of all other ions (except Cl–) similar. We quantified the transcriptional response of roots and shoots to these solution changes within the epidermal enhancer trap line, KC464 (Columbia-0 background) of: known tonoplast Ca2+/H+ exchangers (AtCAX1, AtCAX2) and endoplasmic reticulum-localised autoinhibited Ca2+-ATPase (AtACA2) calcium transporter; and vacuolar H+-ATP synthase subunits (AtVHA-a2, AtVHA-a3) (Figure 2).
Gas exchange measurement of hydroponically grown plants
To be able to perform these measurements we found it necessary to make all components of the Arabidopsis whole plant chamber airtight – without this, moisture from the hydroponics media compromised the gas exchange measurements. As detailed in Additional file 2, we sealed the plant holding lid into the centrifuge tube lid using teflon air-tight sealing tape. The plant, now held within a centrifuge tube lid, was transferred into an intact centrifuge tube base containing the treatment solution of interest. The centrifuge tube was then sealed into the LiCOR ‘cone-tainer’ using a 30 mm OD rubber O-ring. This system would allow exclusive measurement of rosette gas exchange for at least 3 h for 6-week old Arabidopsis plants without any detectable reduction in photosynthetic rate during the middle of the photoperiod (Figure 4). Gas exchange measurements were adjusted on the basis of the leaf area contained within: i) the extended reach chamber (LiCOR) estimated by taking a scaled photograph and analysis of the percentage of the leaf within the chamber window using ImageJ (National Institute of Health, NIH) as detailed in  or, ii) the whole Arabidopsis plant chamber (LiCOR) by estimating the rosette size using a customised code developed in MATLAB® 2010b (Mathworks Inc., Natick, MA, USA) and the Image Analysis Toolbox® to process scaled photographs semi-automatically. Two codes were used, a semi-automated and an automated code. The latter recognises by colour contrast the Arabidopsis rosette to obtain automatically the cover area. The semi-automated code was used in pictures where this contrast was not detected by the automation algorithm. In this case, a tool was developed to select a region of interest (ROI) corresponding to the rosette manually to extract the cover area. See Additional files 4, 5, 6 for further details of the code and method.
The leaf gas exchange measurements were not significantly different for the hydroponics system using either the LiCOR whole Arabidopsis plant chamber or the extended leaf chamber (Figure 4). However, it was evident that the whole plant chamber took more consistent readings presumably due to the ability to sum the reading over a larger area and avoiding the need to seal the chamber directly onto the leaf tissue, which can confound results through improper sealing and/or leaf damage. We found that consistent results could be achieved with the extended leaf chamber when leaves were large enough, but the dimensions of the leaf and petiole made the clamping of a large amount of leaf area in the chamber a challenge unless the plant was older than 6 weeks. In contrast plants could be assayed in the whole Arabidopsis chamber from weeks 3-8. It is clear that this system offers potential to be widely used to study leaf gas exchange in a highly controlled manner throughout the majority of Arabidopsis development.
In our hands
Given the importance of aeration of hydroponics systems for adequate growth , several aeration systems were trialled. The media within the tank was aerated either using a standard 4-outlet aquarium pump that constantly bubbled air through airstones or by using an ebb-and-flow system that pumped media between the tank containing the plants and a solution holding reservoir every 60 min. Both systems produced plants that at least qualitatively resembled each other, however, the former technique was markedly simpler to construct and maintain so it was used for all further studies. Oxygenation levels in the constantly aerated plants were sufficient to avoid increased expression of known hypoxia induced genes, AtWRKY40 and AtNIP2;1[32, 33], in contrast, transcription of both genes were induced when the media was non-aerated for 7 days (Additional file 7).
Profiling of transgenic plants
The desire to accelerate the analysis of transgenic Arabidopsis plants has led to the design of a number of rapid approaches for selection of transformants. The method commonly used to select transformed Arabidopsis seedlings is by spreading the seeds on suitable growth media such as soil or agar. Soil is commonly used if the selection marker gene is phosphothrinocin, whereas agar is used if the marker gene is kanamycin or hygromycin. Thereafter, the putative transformants are usually transferred into soil for seed collection. The main problem with this method is the potential damage to the fragile root systems of the selected seedlings, which consequently affects their survival rate. We demonstrate that our hydroponic system can be used as an alternative to soil growth for cultivation of transformants selected on agar plates (using phosphothrinocin, kanamycin or hygromycin) as per Harrison et al. . Over 95% of transformants survived transfer using this method, with the collected seed displaying a high germination rate (Additional file 8). However, the real advantage lies in the ability to reliably analyse mature first generation transformants, particularly for root cellular localisation studies and root phenotypes that are impossible with soil- and agar plate- based selection methods.
Adapting the system for other plants
The improved hydroponic system we highlight here can be easily adapted for use with other plants with changes to the diameter of the hole produced in the lid of the microcentrifuge tube. We made holes of up to 6 mm in diameter (suitable for up to 4-week old cucumber and 6 week old tobacco ), and also grew Lotus spp. seedlings (data not shown). Furthermore, we also adapted the system for use with cereals using the 1.5 mL black microcentrifuge tubes with the bottom 7 mm cut off, this was sufficient to hold the seed, roots and shoots in place and removed the need for agar .
We demonstrate the quality and versatility of our hydroponics system by profiling and comparing with soil-grown plants and previous hydroponics reports many parameters throughout the growth of Arabidopsis, including biomass, ionomics and transcriptomics. We present this hydroponics growth system as an adaptable system for characterising the entire Arabidopsis plant and other plants by a variety of physiological and molecular biological methods, superior to and more inexpensive than many techniques currently in use.
We would like to acknowledge the invaluable assistance of Joern Nevermann for the manufacture of plastic components for this system. GFP plasmids were gifts from Tsuyoshi Nakagawa and Jen Sheen. This work was supported by funding awarded to MG, SDT, Brent Kaiser and Roger Leigh from the Australian Research Council including Discovery Project (DP0774063) and the Centre of Excellence in Plant Energy Biology, and the University of Adelaide.
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