A novel high efficiency, low maintenance, hydroponic system for synchronous growth and flowering of Arabidopsis thaliana
- Pierre Tocquin†1Email author,
- Laurent Corbesier†1,
- Andrée Havelange1,
- Alexandra Pieltain1,
- Emile Kurtem1,
- Georges Bernier1 and
- Claire Périlleux1
© Tocquin et al; licensee BioMed Central Ltd. 2003
Received: 6 December 2002
Accepted: 30 January 2003
Published: 30 January 2003
Arabidopsis thaliana is now the model organism for genetic and molecular plant studies, but growing conditions may still impair the significance and reproducibility of the experimental strategies developed. Besides the use of phytotronic cabinets, controlling plant nutrition may be critical and could be achieved in hydroponics. The availability of such a system would also greatly facilitate studies dealing with root development. However, because of its small size and rosette growth habit, Arabidopsis is hardly grown in standard hydroponic devices and the systems described in the last years are still difficult to transpose at a large scale. Our aim was to design and optimize an up-scalable device that would be adaptable to any experimental conditions.
An hydroponic system was designed for Arabidopsis, which is based on two units: a seed-holder and a 1-L tank with its cover. The original agar-containing seed-holder allows the plants to grow from sowing to seed set, without transplanting step and with minimal waste. The optimum nitrate supply was determined for vegetative growth, and the flowering response to photoperiod and vernalization was characterized to show the feasibility and reproducibility of experiments extending over the whole life cycle. How this equipment allowed to overcome experimental problems is illustrated by the analysis of developmental effects of nitrate reductase deficiency in nia1nia2 mutants.
The hydroponic device described in this paper allows to drive small and large scale cultures of homogeneously growing Arabidopsis plants. Its major advantages are its flexibility, easy handling, fast maintenance and low cost. It should be suitable for many experimental purposes.
Despite its worldwide acknowledged advantages for genetic and molecular studies, Arabidopsis is still hardly amenable to physiological analyses. Its small size and its rosette growth habit somehow make manipulation of the plants ex vitro difficult. Among specific experiments, studies dealing with mineral nutrients and root development would be greatly facilitated by growing the plants on liquid medium. Several hydroponic systems have already been designed for Arabidopsis. While Koyama et al.  set up a system which use is limited to young seedlings, others systems allow the completion of the life cycle [2–5]. In most cases, seeds are sown on rockwool or sponge pieces (called hereafter 'seed-holders') soaked with the nutrient solution. As a consequence of their flooding, seeds germinate poorly and must be sown in large excess. Another disadvantage of these seed-holders is that the root system is partly inaccessible for observation or harvest. More technical problems arise if the hydroponic device has to be connected to air for oxygenation of the roots, and/or sterilized to prevent algae contamination. Because of their complexity, these systems were all used on a reduced scale while most physiological analyses – or mutant screens – require large populations of synchronously growing individuals. Synchronization not only means homogeneous growth, but also requires the control of the developmental phases of the plants. We therefore designed a large-scale hydroponic system that allows to control the transition to flowering. Arabidopsis is a facultative long-day (LD-) and vernalization-requiring plant, which means that 'autonomous' flowering may occur in any growth conditions but will be accelerated by environment-dependent 'promotion' pathways [6–8]. We previously showed that the photoperiodic control may be strengthened to such an extent that Arabidopsis becomes responsive to a one-cycle inductive treatment . This consists in growing vernalized plants in 8-h short days (SDs) under low light intensity (LL). On soil, 8-week old plants are then induced to flower by a single 22-h LD while SD-controls remain vegetative for about 3.5 months from sowing.
In this paper, we aimed to establish a simple, inexpensive, and low maintenance hydroponic system for growing Arabidopsis plants. This method is convenient for several purposes since: (i) plant growth can be optimized and synchronized; (ii) mineral nutrition can be easily monitored and manipulated; (iii) roots can be observed and harvested without damage; (iv) floral transition can be induced by a single or multiple inductive cycles.
Results and Discussion
Description of the hydroponic system
The seed-holders are small plastic cylinders filled with 0.65 % agar (Fig. 1a). The liquid medium containers are 1-L black plastic boxes covered with a pierced black-PVC plate (Fig. 1b). When the culture is set up, the bottom of the seed holders dip into the nutrient solution. Originally, the seed-holders were inserted into the holes of the container's cover together with a piece of mesh at their bottom to prevent agar coming out (Fig. 1c), and the results presented here were obtained with this system. Since this step was the most time-consuming, we recently designed one-piece seed-holders industrially produced by mould-injection of black plastic (Fig. 1d). They consist in a cylinder with a collar at the top. Two crosswise extensions at the bottom of the cylinder guide the roots and partially obstruct the seed-holder, so retaining the agar.
This hydroponic system is adapted to Arabidopsis growth habit, is simple, cheap, and requires low maintenance. As compared to other devices described in the literature [2, 4, 5], one of its specific advantages is the nature of the support used for germination and seedling growth. The 0.65 % agar that was used to fill the seed-holders ensures full success for germination and so avoids a 'pre-culture' step and a waste of plant material. Furthermore, agar is a soft support that allows fast growth of the seedling roots, as well as their complete and easy harvest. Seedling root growth through the agar usually took 7 to 10 days, irrespective of the growing conditions.
Another novelty of our system is the movable seed-holder that allows transfer of individual plants from one container to another and makes the system highly flexible. The covers may also be swapped for transfer of plant batches.
Since both the solution container and the cover are made of black plastic, algae growth is prevented; the solution remains clear and no sterilization is required. We also observed that aeration of the nutrient solution was not necessary in small tanks, as reported previously by Arteca & Arteca .
This experimental device has been set up in the general context of studying the regulation of flowering time in Arabidopsis. The need to synchronize plant development to focus physiological investigations implies a strict control of growing conditions. The quality of the substrate, so the mineral nutrition, was one of the aspects that turned out to be critical to discriminate flowering time and growth rate. Thus, after having designed the equipment, the next step was to make sure that growth was not limited by nutrients in our hydroponic system, and to analyse the floral behaviour. Because of its major role as mineral element, we optimised the N-supply.
Effects of N-supply on plant growth in 8-h SDs
The composition of the nutrient solution was based on Hoagland's, with macronutrients diluted 4-fold . Plant growth response to N-supply was recorded on 5-week old plants grown on a range of N-concentrations, from 220 μM to 21 mM NO3-. The NO3-:NH4+ ratio was kept constant at 27:1 and the nutrient solution was renewed weekly from the third week of growth.
Chemical composition of the Hoagland and our Arabidopsis 'standard' nutrient solutions
Hoagland solution (mol.l-1)
Standard solution (mol.l-1)
Under HL, the floral transition started after 6 weeks and all plants had formed visible floral buds 8 weeks after sowing. LL strongly delayed the process since the floral buds were visible in the earliest individuals after about 10 weeks. This delay was partly rescued by vernalization (2°C, 6 weeks) that advanced the floral transition by about 10 days (Fig. 4c). Note that all plants eventually formed flowers, even when non-vernalized.
Expectedly, flowering was much faster in 16-h LDs than in 8-h SDs (see Fig. 2): all plants had formed floral buds after 4 weeks (Fig. 4a), under both HL and LL. The entire life cycle was analysed under HL: non-vernalized plants reached anthesis about one month after the initiation of floral buds – i.e. two months after sowing – and produced an average of 473 ± 45 mg of seeds (2864 ± 555 seeds) per plant. In total, the generation time was about 2.5 months.
Flowering of nitrate reductase deficient mutants
Effect of daylength on total leaf number initiated before the first flower by vernalized WT and nia1nia2 plants. Plants were grown under a photon flux density of 120 μmol.m-2.s-1, on soil. Results of a typical experiment are given; standard deviation calculated on 12 plants
71.8 ± 3.4
26.3 ± 3.3
13.0 ± 1.6
13.3 ± 1.5
43.9 ± 4.0
23.3 ± 3.7
17.5 ± 3.1
Flowering response of vernalized WT and nia1nia2 plants submitted to one 22-h LD. Plants were grown in 8-h SDs / 50 μmol.m-2.s-1, then subjected to the LD treatment or not (SD-controls). On soil, WT and nia1nia2 plants were exposed to the LD when 8- and 10-week old, respectively. In hydroponics, plants were 6- and 8-week old, respectively.
% of flowering plants
In conclusion, this paper describes an original device for growing Arabidopsis hydroponically. Beside the general advantages of this culture system, such as control of mineral nutrition and access to the root system, we specifically paid attention to technical parameters ensuring homogeneity of the plant material, i.e. successful germination, synchronised growth and development. Containers and seed-holders were also designed for flexibility, easy setting up and low maintenance of the culture, so that this equipment will be suitable for many experimental purposes.
Set up of the culture
Seed holders were prepared by cutting off the cap and the conical end of 0.5 ml microfuge tubes (VWR International, Overijse, Belgium). Melted 0.65% agar was poured into the cylinders placed upside down on paper tape. Once cooled down, the seed-holders may be wrapped with plastic and stored at 4°C until used. The containers were black Polarcup boxes (18 × 13 × 6.5 cm, L × W × H; VariaPack, Leuven, Belgium) covered with a black PVC plate pierced of 9-mm diameter holes. Before sowing, 1 L of nutrient solution was placed in each container and the seed-holders were set up. To prevent leaching of agar in the nutrient solution, the bottom of the seed-holders was wrapped with a piece (15 × 40 mm) of grey mesh (1 mm) (Fig. 1c) that stuck to it when both parts were inserted together into the holes of the container cover. For sowing, seeds were suspended in 0.2 % agar and dropped individually with a pipette on the top of the agar-filled seed-holders.
Plant material and growing conditions
After stratification of the seeds at 2°C for 5 days or vernalization at 2°C for 6 weeks on wet filter paper and in darkness, plants of Arabidopsis (Columbia) were grown in 8-h SDs or 16-h LDs. The photon flux density was 120 (HL) or 50 μmol.m-2.s-1 (LL) PAR (Very High Output fluorescent tubes; Sylvania, Zaventem, Belgium) and the temperature was 20°C. Relative humidity – which is critical for the success of germination and seedling establishment – was set optimally at 70%.
Flowering time was recorded in 8-h SDs and 16-h LDs by regular observation of the plants under the dissecting microscope. A plant was recorded as 'floral' when at least one floral bud was visible at the apex, and a '% of flowering plants' was calculated for each batch examined.
For the analysis of the flowering response to the single cycle treatments, plants were grown for 5, 6, 7, or 8 weeks in 8-h SDs under LL before being exposed to a single 22-h LD, as described previously . The floral response to one LD was more deeply investigated for 7-week old plants which were exposed to a single LD of different durations. This allowed to establish the 'induction curve' and so determine the minimal duration of the inductive LD or 'critical photoperiod' . In these experiments aiming at synchronous induction of flowering, the floral response was evaluated 18 days after the photoperiodic treatment, by dissecting the plants under the dissecting microscope and evaluating the '% of flowering plants' as explained above.
List of abbreviations
inductively coupled plasma mass spectrometry
L.C. is grateful to the F.N.R.S. and P.T. to the F.R.I.A. for the award of research fellowships. This research was supported by grants from the European Community Contract BI04 CT97-2231; the Interuniversity Poles of Attraction Programme (Belgian State, Prime Minister's Office – Federal Office for Scientific, Technical and Cultural Affairs; P4/15); the 'Action de Recherches Concertées' 98/03-219 and the University of Liège (Fonds Spéciaux). We thank Dr. S. Melzer for critical reading of the manuscript. We wish to thank A. Schmitz and D. Libion for taking care of the plants.
- Koyama H, Toda T, Yokota S, Dawair Z, Hara T: Effects of aluminium and pH on root growth and cell viability in Arabidopsis thaliana strain Landsberg in hydroponic culture. Plant Cell Physiol. 1995, 36: 201-205.Google Scholar
- Delhaize E, Randall PJ: Characterization of a phosphate-accumulator mutant of Arabidopsis thaliana. Plant Physiol. 1995, 107: 207-213.PubMedPubMed CentralGoogle Scholar
- Hirai MY, Fujiwara T, Chino M, Naito S: Effects of sulfate concentrations on the expression of a soybean seed storage protein gene and its reversibility in transgenic Arabidopsis thaliana. Plant Cell Physiol. 1995, 36: 1331-1339.PubMedGoogle Scholar
- Gibeaut DM, Hulett J, Cramer GR, Seemann JR: Maximal biomass of Arabidopsis thaliana using a simple, low-maintenance hydroponic method and favorable environmental conditions. Plant Physiol. 1997, 115: 317-319. 10.1104/pp.115.2.317.PubMedPubMed CentralView ArticleGoogle Scholar
- Arteca RN, Arteca JM: A novel method for growing Arabidopsis thaliana plants hydroponically. Physiol Plant. 2000, 108: 188-193. 10.1034/j.1399-3054.2000.108002188.x.View ArticleGoogle Scholar
- Koornneef M, Alonso-Blanco C, Peeters AJM, Soppe W: Genetic control of flowering time in Arabidopsis. Annu Rev Plant Physiol Plant Mol Biol. 1998, 49: 345-370. 10.1146/annurev.arplant.49.1.345.PubMedView ArticleGoogle Scholar
- Levy YY, Dean C: The transition to flowering. Plant Cell. 1998, 10: 1973-1989. 10.1105/tpc.10.12.1973.PubMedPubMed CentralView ArticleGoogle Scholar
- Périlleux C, Bernier G: The control of flowering: do genetical and physiological approaches converge?. In Plant Reproduction. Annual Plant Reviews. Edited by: O'Neill SD, Roberts JA. 2002, Sheffield Academic Press, Sheffield, 6: 1-32.Google Scholar
- Corbesier L, Gadisseur I, Silvestre G, Jacqmard A, Bernier G: Design in Arabidopsis thaliana of a synchronous system of floral induction by one long day. Plant J. 1996, 9: 947-952. 10.1046/j.1365-313X.1996.9060947.x.PubMedView ArticleGoogle Scholar
- Wilkinson JQ, Crawford NM: Identification and characterization of a chlorate-resistant mutant of Arabidopsis thaliana with mutations in both nitrate reductase structural genes NIA1 and NIA2. Mol Gen Genet. 1993, 239: 289-297.PubMedGoogle Scholar
- Lang A: Physiology of flower initiation. In Encyclopedia of Plant Physiology. Edited by: Ruhland W. Berlin: Springer-Verlag, 1965:1379-1536.Google Scholar
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