Plant Molecular Biology Reporter 21: 449456, December 2003 2003 International Society for Plant Molecular Biology.

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Protocols

A Hydroponic Culture System for Growing Arabidopsis thaliana Plantlets Under Sterile Conditions
BERNHARD SCHLESIER, FRDRIC BRTON* and HANS-PETER MOCK
Institute of Plant Genetics and Crop Plant Research, Corrensstrae 3, 06466 Gatersleben, Germany Abstract. We developed a hydroponic cultivation system for growing Arabidopsis plantlets under sterile, controlled environmental conditions. The system consists of a piece of stainless-steel wire cloth (125 m mesh size) that is fixed between 2 flat rings and held in place by 3 legs, placed in a commercially-available glass jar, and covered by the original glass lid or a sheet of sterilized cellophane. Sterilized seeds were distributed evenly across the mesh piece, the size of which allowed root growth and kept the seeds in place. After 3 weeks of cultivation, shoot and root tissues were easily harvested without mechanical damage. Proteome and metabolite analyses were performed on root and shoot tissues and demonstrated excellent reproducibility, indicating that the system is advantageous when biological variation is minimized. Induction experiments can be performed by transferring the apparatus (with plants) to a new jar containing a different nutrient solution. The apparatus is reusable and can easily be sterilized by autoclaving or dry heat. The system can be adapted to other small-seed plants by varying the mesh size. Key words: Arabidopsis, metabolic profile, proteome, roots, sterile hydroponic culture

Introduction Sterile hydroponics of Arabidopsis

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Arabidopsis thaliana has been established as a model plant for many investigations because of small genome size, mutant availability, a short generation cycle, and a size that allows growing many plants in parallel. Sequencing of the Arabidopsis genome has recently been completed (Arabidopsis Genome Initiative, 2000) and has stimulated new research approaches, such as global analysis of transcripts or protein patterns (Bevan, 2002). Parallel investigation of a large number of transcripts or proteins is a costand labour-intensive process and requires standardization of plant growth to minimize the observed variation of the biological system being analysed. While establishing techniques of proteome analysis by means of 2-dimensional (2-D) gel
*

Present address: CIRAD-CP, TA 80/02, Avenue Agropolis, F34398 Montpellier cedex 5, France. Author for correspondence: email: mock@ipk-gatersleben.de; fax: ++49-39482-5139; ph: ++49-39482-5506.

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electrophoresis, we became interested in separately harvesting and analysing root and shoot tissue as a first step in reducing the proteome complexity. Harvesting plants grown on soil or agar resulted in poor recovery, root damage, and broad variation in root tissue metabolite levels. Hydroponic culture allows for the harvest of undamaged roots without soil or agar contamination. Several hydroponic systems for Arabidopsis have been described (Arteca and Arteca, 2000; Gibeaut et al., 1997; Huttner and Bar-Zvi, 2003; Siedlecka and Krupa, 2002; Tocquin et al., 2003; Toda et al., 1999), all of which use nonsterile conditions. We were interested in induction experiments with different sugars and needed a sterile culture method to avoid contamination by algae or fungi. We describe the development of a culture system for hydroponic growth of Arabidopsis under sterile, controlled environmental conditions. Examples demonstrating the usefulness of the system are given showing applications in proteome and metabolite analysis. Material and Methods Plant material A. thaliana Col-0 and Ws-2 ecotypes and different mutants and transgenic lines were used. Equipment and chemicals

Round-rim glass jars (1/4 L mold jar; Weck Company, Wehr, Germany) Plantlet carriers consist of a stainless-steel wire cloth (125 m mesh size) fixed between 2 flat rings and held in place by 3 legs. Steel rings (80 mm outer diameter, 60 mm inner diameter) were cut from 1-mm-thick steel by means of laser ablation. Wire cloth sheets (75 mm in diameter) were obtained from Drahtweberei Raguhn (Raguhn, Germany). Legs and handle with a screw thread were made from 5-mm rods. Drill-holes in the rings take up 3 legs and one handle, thereby fixing the mesh between the rings (Figure 1A). Light protective pots were cut from flowerpots to the height of the carrier mesh to minimize illumination of the roots. Cellophane sheets were cut from dialysis tubing (Visking type 1-7/8ss, Carl Roth, Germany). A gas-permeable plastic foil (bioFOLIE; Vivascience, Germany) was successfully used in place of the cellophane. Clean bench Heating block Murashige and Skoog medium micro and macro elements, including Gamborg B5 vitamins (DUCHEFA, The Netherlands) Sucrose Low-melt agarose (New Sieve GTG, FMC Bio Products, Rockland ME, USA)

Treatment of seeds For each culture vessel, 2.5 mg of seeds were surface-sterilized in a 1.5-mL reaction vessel by washing with 70% (v/v) ethanol for 2 min and sodium hypochlorite solution (7% available chlorine) containing 0.2% (v/v) Triton X-100 for 8 min.

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Figure 1. Culture system for Arabidopsis plantlets. (A) A carrier of the culture apparatus. (B) Dispensing of seeds on the wire cloth. (C) Vessels with plantlets in a culture room. (D) Threeweek-old plantlets grown by means of hydroponic culture under sterile conditions.

Seeds were then washed 3 times in sterile distilled water and stored in wet conditions for 3 d at 5C for stratification. Nutrient solution The nutrient solution contained one-quarter-strength Murashige and Skoog medium, including Gamborg B5 vitamins, and was supplemented with 0.5% (w/v) sucrose (or other concentrations if experimentally desired). Dissolve Murashige and Skoog medium and sucrose in distilled water and adjust pH to 5.8 by using 0.1 M KOH. Sterilize by autoclaving at 121C for 15 min. The final solution pH is 5.9, and the conductivity is 1.7 S. Agarose-sucrose solution to fix seeds on wire cloth

Prepare a solution with 2% low-melt agarose and 30% sucrose in water. Sterilize by autoclaving at 121C for 15 min. Store aliquots of 1 mL at 5C.

Preparing culture vessels Sterilize glass jars containing the carriers and covered by the original glass lids at 160C for 4 h. The following steps are carried out under sterile conditions (clean bench):

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Immediately before use, resuspend the surface-sterilized seeds in one reaction vessel with 100 L of sterile agarose-sucrose solution and keep at 50C on a heating block. Instantly distribute the seeds using a 200-L pipette equipped with a cut 1000-L tip. Small droplets usually containing one seed are placed in equal distance on the mesh (Figure 1B). After agarose droplets coagulate, fill the cultivation vessels with nutrient solution up to the level of the steel mesh (~165 mL) and cover them with original glass lids or sheets of autoclaved wet cellophane. Insert cultivation vessels in black protection pots to minimize the lighting of roots. Grow plants under controlled environmental conditions. In our laboratory, plantlets are routinely grown at 22C under a fluorescent lamp (universal white, NARVA, Germany) using a light period of 12 h per day with a light intensity of 120 E s-1 m-2 (Figure 1C) at leaf level.

Results We developed a culture system that allows Arabidopsis plantlet growth under sterile conditions. Shoot and root tissue were easily harvested without mechanical damage. Our hydroponic culture system is contained in a glass jar. The carrier mesh size was selected as follows: (A) mesh should be narrow enough to prevent seeds from passing into the liquid medium (resulting in submerse plantlet growth requiring time-consuming and laborious efforts to avoid root preparation contamination) and (B) the size should not restrict root growth into the liquid medium. Arabidopsis shows phenotypic variation in seed size (Alonso-Blanco et al., 1999). A 125-m mesh size wire cloth was selected to cultivate the Col-0 ecotype of Arabidopsis thaliana, ensuring that seeds were kept on the mesh and allowing root system development over 3 wk. Larger mesh sizes (250 m) resulted in a number of submerse growing plantlets because some seeds passed through the mesh. As shown in Figure 1A, the mesh is fixed between 2 stainless-steel rings; 3 legs and a handle are mounted so the apparatus can be placed in a cultivation jar. We also established a protocol to uniformly distribute single seeds on the mesh. Seeds were sterilized according to standard procedure, which includes a cold treatment to ensure uniform germination. After stratification, seeds were resuspended in a sterile solution of 2% (w/v) low-melting agarose and 30% (w/v) sucrose. This high-density solution kept seeds afloat and assisted in the even distribution of single seeds over the wire cloth. After the agarose gelled, seeds were fixed on the wire cloth and were not disturbed during transport of vessels from the clean bench to the culture room. Seeds were placed at 35-40 equal distance positions on a 28 cm2 net. Rosette leafs grew up to 2 cm over 3 wk. In addition to the primary root, plantlets developed many lateral roots up to 3 cm in length (Figure 1D). One vessel routinely yielded up to 200 mg fresh weight of roots and more than 2 g of leaf material. By collecting plant tissue from several jars, obtaining enough material for most purposes (e.g., biochemical analyses, isolation of organelles) is possible, even from limited root material. We performed analyses of protein and phenylpropanoid patterns for both roots and leafs.

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Figure 2. Two-dimensional gel electropherogram of proteins from Arabidopsis roots. Plant material was ground in liquid nitrogen immediately after harvesting and precipitated by TCA/acetone according to Damerval et al. (1986). Proteins were redissolved in 8 M urea, 2% CHAPS, 20 mM DTT, and 0.5% IPG buffer. (A) Using rehydration, 100 g of protein was applied on 13-cm IPG strips with immobilized pH gradients of 4-7 and separated on an IPGPhor unit (Amersham Biosciences, Freiburg, Germany). For the second dimension, proteins were resolved on SDS-PAGE with stacking gel. Proteins were detected by staining with Coomassie G250. (B) Reproducibility of protein pattern over 4 independent culture experiments for the selected area (marked with a rectangle in A).

Proteins were separated by means of 2-D gel electrophoresis. A typical protein pattern from Arabidopsis roots is shown in Figure 2A. The standardized culture of the Arabidopsis plantlets resulted in a high reproducibility of the 2-D gel protein patterns when comparing 4 independent experiments (Figure 2B) as shown for a selected area of the gels. We also used the culture system for metabolic analysis. Figure 3 shows the separation of methanol-soluble compounds on an RP-18 column with detection at 280 nm. On the basis of retention times and spectra and by using reference substances (Mock et al., 1992), the main peaks were identified as sinapoylglucose (RT 18.2 min) and sinapoylmalate (RT 29.1 min). Discussion Arabidopsis has become the model organism for plant genetics and plant functional genomics because of the availability of large mutant collections and the completed genome sequence. However, the low biomass of Arabidopsis renders it a difficult species for biochemical analyses. Obtaining leaf material is not that critical, but harvesting roots is a challenge. Harvesting uncontaminated and undamaged roots from solid media like soil, silica sand, rockwool, or agarose is almost impossible. Intact roots are a prerequisite for the unbiased analysis of, e.g., undisturbed patterns of proteins and metabolites such as secondary compounds. Therefore, hydroponic methods are preferred for such experiments. In the system

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Figure 3. Phenylpropanoid pattern of Arabidopis leaves. Methanolic leaf extracts were prepared and subjected to HPLC analysis as described by Mock et al. (1999). Chromatograms are shown for plants grown under sterile conditions (A) or on soil (B). Main peaks were identified as sinapoylglucose and sinapoylmalate by co-chromatography with authentic standards.

of Gibeaut et al. (1997) and improved by Huttner and Bar-Zvi (2003), plants were grown on rockwool in a well-aerated nutrient solution, allowing cultivation of Arabidopsis up to the flowering stage. However, 3-cm-high rockwool cylinders make isolating roots from young seedlings difficult. This is also the case if seedlings are grown on sponge (Arteca and Arteca, 2000). Siedlecka and Krupa (2002) transferred the plants to liquid nutrient medium after 5 weeks of growth in soil, making it impossible to harvest uncontaminated roots from young plants. This goal is better accomplished with the method of Toda et al. (1999), in which seeds are spread on a nylon mesh floating on the culture solution by means of a plastic photo slide mount. This technique is suitable only for short culture times. Tocquin et al. (2003) developed a small agar-containing seed-holder, allowing plants to grow from sowing to seed set. All these methods work under nonsterile conditions. Occasionally, algae and fungi can grow in the nutrient solution and affect the experiment. For many experiments, sterile conditions are desirable, such as induction experiments that include sugars in the nutrient solution. This requirement is accomplished in our culture system. The glass jars and stainless-steel carrier are reusable and can be easily sterilized by dry heat. Induction experiments can be performed simply by transferring the carrier with plants to a new vessel with a different nutrient solution (Schlesier and Mock, unpublished), making it easy to test the influence of nutrients and effectors on root metabolism. Previous reports on Arabidopsis hydroponics describe the need for aeration and mixing the solution (Gibeaut et al., 1997; Huttner and Bar-Zvi, 2003; Siedlecka and Krupa, 2002). We found no simple way to fulfill this requirement under

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sterile culture conditions. Arteca and Arteca (2000) and Tocquin et al. (2003) reported that aeration of the nutrient solution was not necessary in small tanks. Consistent with this report, we used our system without aeration. Use of other mesh sizes allows adaptation of the system to other small-seed plants, such as tobacco, for which a mesh size of 250 m is better suited because of larger seeds and developing roots (Schlesier and Mock, unpublished). Future applications will include isolating organelles from roots, which is difficult to achieve with soil-grown plants. Preliminary results indicate the suitability of the new cultivation system for the isolation of mitochondria from Arabidopsis roots (Braun and Mock, unpublished). Acknowledgments We thank M. Eisbrenner, B. Kettig, and A. Wolf for their excellent technical assistance. Funding of the DFG to H.-P. Mock (Mo 479/4-1) is gratefully acknowledged. References
Alonso-Blanco C, Blankestijn-De Vries H, Hanhart CJ, and Koornneef M (1999) Natural allelic variation at seed size loci in relation to other life history traits of Arabidopsis thaliana. Proc Natl Acad Sci 96: 4710-7. Arabidopsis genome initiative (2000) Sequence and analysis of the flowering plant Arabidopsis thaliana. Nature 408: 796-815. Arteca RN and Arteca JM (2000) A novel method for growing Arabidopsis thaliana plants hydroponically. Physiologia Plantarum 108: 188-93. Bevan M (2002) Genomics and plant cells: application of genomics strategies to Arabidopsis cell biology. Philos Trans R Soc Lond B Biol Sci 357: 731-6. Damerval C, de Vienne D, Zivy M, and Thiellement H (1986) Technical improvements in two-dimensional electrophoresis increase the level of genetic variation detected in wheat-seedling proteins. Electrophoresis 7: 52-4. Gibeaut DM, Hulett J, Cramer GR, and Seemann JR (1997) Maximale biomass of Arabidopsis thaliana using a simple, low maintenance hydroponic method and favorable environmental conditions. Plant Physiol 115: 317-9. Huttner D and Bar-Zvi D (2003) An improved, simple, hydroponic method for growing Arabidopsis thaliana. Plant Mol Biol Rep 21: 59-63. Mock H-P, Heller W, Molina A, Neubohn B, Sandermann H Jr, and Grimm B (1999) Expression of uroporphyrinogen decarboxylase or coproporphyrinogen oxidase antisense RNA in tobacco induces pathogen defense responses conferring increased resistance to TMV. J Biol Chem 274: 4231-8. Mock H-P, Vogt T, and Strack D (1992) Sinapoylglucose: malate sinapoyltransferase activity in Arabidopsis thaliana and Brassica rapa. Z. Naturforsch. 47c: 680-2. Siedlecka A and Krupa Z (2002) Simple method of Arabidopsis thaliana cultivation in liquid nutrient medium. Acta Physiol Plant 24: 163-166. Tocquin P, Corbesier L, Havelange A, Pieltain A, Kurtem E, Bernier G, and Perilleux C (2003) A novel high efficiency, low maintenance, hydroponic system for synchronous growth and flowering of Arabidopsis thaliana. BMC Plant Biology 3: 2. 25 Jan. 2004. <http://www.biomedcentral.com/1471-2229/3/2>.

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Toda T, Koyama H, and Hara T (1999) A simple hydroponic culture method for the development of a highly viable root system in Arabidopsis thaliana. Biosci Biotechnol Biochem 63: 210-212.