a hydroponic culture system for growing is thaliana plantlets under sterile conditions

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A Hydroponic Culture System for GrowingArabidopsis thaliana Plantlets Under SterileConditions

BERNHARD SCHLESIER, FRÉDÉRIC BRÉTON* andHANS-PETER MOCK†

Institute of Plant Genetics and Crop Plant Research, Corrensstraße 3, 06466Gatersleben, Germany

Abstract. We developed a hydroponic cultivation system for growing Arabidopsisplantlets under sterile, controlled environmental conditions. The system consists of a pieceof stainless-steel wire cloth (125 µm mesh size) that is fixed between 2 flat rings and heldin place by 3 legs, placed in a commercially-available glass jar, and covered by the origi-nal glass lid or a sheet of sterilized cellophane. Sterilized seeds were distributed evenlyacross 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 mechan-ical damage. Proteome and metabolite analyses were performed on root and shoot tissuesand demonstrated excellent reproducibility, indicating that the system is advantageouswhen biological variation is minimized. Induction experiments can be performed by trans-ferring the apparatus (with plants) to a new jar containing a different nutrient solution. Theapparatus is reusable and can easily be sterilized by autoclaving or dry heat. The systemcan be adapted to other small-seed plants by varying the mesh size.

Key words: Arabidopsis, metabolic profile, proteome, roots, sterile hydroponic culture

Sterile hydroponics of Arabidopsis Schlesier et al.Introduction

Arabidopsis thaliana has been established as a model plant for many investiga-tions because of small genome size, mutant availability, a short generation cycle,and a size that allows growing many plants in parallel. Sequencing of the Ara-bidopsis genome has recently been completed (Arabidopsis Genome Initiative,2000) and has stimulated new research approaches, such as global analysis oftranscripts or protein patterns (Bevan, 2002).

Parallel investigation of a large number of transcripts or proteins is a cost-and labour-intensive process and requires standardization of plant growth to mini-mize the observed variation of the biological system being analysed. Whileestablishing techniques of proteome analysis by means of 2-dimensional (2-D) gel

Plant Molecular Biology Reporter 21: 449–456, December 2003© 2003 International Society for Plant Molecular Biology. Printed in Canada.

*Present address: CIRAD-CP, TA 80/02, Avenue Agropolis, F34398 Montpelliercedex 5, France.

†Author for correspondence: email: [email protected]; fax: ++49-39482-5139;ph: ++49-39482-5506.

electrophoresis, we became interested in separately harvesting and analysing rootand shoot tissue as a first step in reducing the proteome complexity. Harvestingplants grown on soil or agar resulted in poor recovery, root damage, and broadvariation in root tissue metabolite levels. Hydroponic culture allows for the har-vest of undamaged roots without soil or agar contamination. Several hydroponicsystems for Arabidopsis have been described (Arteca and Arteca, 2000; Gibeautet al., 1997; Huttner and Bar-Zvi, 2003; Siedlecka and Krupa, 2002; Tocquin etal., 2003; Toda et al., 1999), all of which use nonsterile conditions. We were in-terested in induction experiments with different sugars and needed a sterile cul-ture method to avoid contamination by algae or fungi.

We describe the development of a culture system for hydroponic growth ofArabidopsis under sterile, controlled environmental conditions. Examples demon-strating the usefulness of the system are given showing applications in proteomeand metabolite analysis.

Material and Methods

Plant material

A. thaliana Col-0 and Ws-2 ecotypes and different mutants and transgenic lineswere 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 µm mesh size) fixed

between 2 flat rings and held in place by 3 legs. Steel rings (80 mm outer diam-eter, 60 mm inner diameter) were cut from 1-mm-thick steel by means of laserablation. Wire cloth sheets (75 mm in diameter) were obtained from Drahtwe-berei Raguhn (Raguhn, Germany). Legs and handle with a screw thread weremade 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 meshto minimize illumination of the roots.

• Cellophane sheets were cut from dialysis tubing (Visking type 1-7/8ss, CarlRoth, Germany). A gas-permeable plastic foil (bioFOLIE; Vivascience, Ger-many) 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 reac-tion vessel by washing with 70% (v/v) ethanol for 2 min and sodium hypochloritesolution (7% available chlorine) containing 0.2% (v/v) Triton X-100 for 8 min.

450 Schlesier et al.

Seeds were then washed 3 times in sterile distilled water and stored in wet condi-tions for 3 d at 5°C for stratification.

Nutrient solution

The nutrient solution contained one-quarter-strength Murashige and Skoog me-dium, 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 ad-

just pH to 5.8 by using 0.1 M KOH.• Sterilize by autoclaving at 121°C for 15 min. The final solution pH is 5.9, and

the conductivity is 1.7 µS.

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 121°C for 15 min. Store aliquots of 1 mL at 5°C.

Preparing culture vessels

• Sterilize glass jars containing the carriers and covered by the original glass lidsat 160°C for 4 h.

The following steps are carried out under sterile conditions (clean bench):

Sterile hydroponics of Arabidopsis 451

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) Three-week-old plantlets grown by means of hydroponic culture under sterile conditions.

• Immediately before use, resuspend the surface-sterilized seeds in one reactionvessel with 100 µL of sterile agarose-sucrose solution and keep at 50°C on aheating block.

• Instantly distribute the seeds using a 200-µL pipette equipped with a cut1000-µL tip. Small droplets usually containing one seed are placed in equal dis-tance on the mesh (Figure 1B).

• After agarose droplets coagulate, fill the cultivation vessels with nutrient solu-tion up to the level of the steel mesh (~165 mL) and cover them with originalglass lids or sheets of autoclaved wet cellophane.

• Insert cultivation vessels in black protection pots to minimize the lighting ofroots. Grow plants under controlled environmental conditions.

• In our laboratory, plantlets are routinely grown at 22°C under a fluorescentlamp (universal white, NARVA, Germany) using a light period of 12 h per daywith a light intensity of 120 µE s-1 m-2 (Figure 1C) at leaf level.

Results

We developed a culture system that allows Arabidopsis plantlet growth under ster-ile conditions. Shoot and root tissue were easily harvested without mechanicaldamage. Our hydroponic culture system is contained in a glass jar. The carriermesh size was selected as follows: (A) mesh should be narrow enough to preventseeds from passing into the liquid medium (resulting in submerse plantlet growthrequiring time-consuming and laborious efforts to avoid root preparation contami-nation) 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-µm mesh size wire cloth was selected to cultivate the Col-0 ecotypeof Arabidopsis thaliana, ensuring that seeds were kept on the mesh and allowingroot system development over 3 wk. Larger mesh sizes (250 µm) resulted in anumber of submerse growing plantlets because some seeds passed through themesh. 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 cultivationjar.

We also established a protocol to uniformly distribute single seeds on themesh. Seeds were sterilized according to standard procedure, which includes acold treatment to ensure uniform germination. After stratification, seeds were re-suspended 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 dis-tribution of single seeds over the wire cloth. After the agarose gelled, seeds werefixed on the wire cloth and were not disturbed during transport of vessels fromthe clean bench to the culture room. Seeds were placed at 35-40 equal distancepositions on a 28 cm2 net. Rosette leafs grew up to 2 cm over 3 wk. In addition tothe primary root, plantlets developed many lateral roots up to 3 cm in length (Fig-ure 1D). One vessel routinely yielded up to 200 mg fresh weight of roots andmore than 2 g of leaf material. By collecting plant tissue from several jars, obtain-ing enough material for most purposes (e.g., biochemical analyses, isolation oforganelles) is possible, even from limited root material. We performed analyses ofprotein and phenylpropanoid patterns for both roots and leafs.


Proteins were separated by means of 2-D gel electrophoresis. A typical pro-tein pattern from Arabidopsis roots is shown in Figure 2A. The standardized cul-ture of the Arabidopsis plantlets resulted in a high reproducibility of the 2-D gelprotein patterns when comparing 4 independent experiments (Figure 2B) asshown for a selected area of the gels.

We also used the culture system for metabolic analysis. Figure 3 shows theseparation of methanol-soluble compounds on an RP-18 column with detection at280 nm. On the basis of retention times and spectra and by using reference sub-stances (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 func-tional genomics because of the availability of large mutant collections and thecompleted genome sequence. However, the low biomass of Arabidopsis renders ita difficult species for biochemical analyses. Obtaining leaf material is not thatcritical, but harvesting roots is a challenge. Harvesting uncontaminated and un-damaged roots from solid media like soil, silica sand, rockwool, or agarose is al-most 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


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

of Gibeaut et al. (1997) and improved by Huttner and Bar-Zvi (2003), plants weregrown on rockwool in a well-aerated nutrient solution, allowing cultivation ofArabidopsis up to the flowering stage. However, 3-cm-high rockwool cylindersmake isolating roots from young seedlings difficult. This is also the case if seed-lings are grown on sponge (Arteca and Arteca, 2000). Siedlecka and Krupa(2002) transferred the plants to liquid nutrient medium after 5 weeks of growth insoil, making it impossible to harvest uncontaminated roots from young plants.This goal is better accomplished with the method of Toda et al. (1999), in whichseeds are spread on a nylon mesh floating on the culture solution by means of aplastic photo slide mount. This technique is suitable only for short culture times.Tocquin et al. (2003) developed a small agar-containing seed-holder, allowingplants to grow from sowing to seed set. All these methods work under nonsterileconditions. Occasionally, algae and fungi can grow in the nutrient solution and af-fect the experiment. For many experiments, sterile conditions are desirable, suchas induction experiments that include sugars in the nutrient solution. This require-ment is accomplished in our culture system. The glass jars and stainless-steel car-rier are reusable and can be easily sterilized by dry heat. Induction experimentscan be performed simply by transferring the carrier with plants to a new vesselwith a different nutrient solution (Schlesier and Mock, unpublished), making iteasy to test the influence of nutrients and effectors on root metabolism.

Previous reports on Arabidopsis hydroponics describe the need for aerationand mixing the solution (Gibeaut et al., 1997; Huttner and Bar-Zvi, 2003; Sied-lecka and Krupa, 2002). We found no simple way to fulfill this requirement under


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

sterile culture conditions. Arteca and Arteca (2000) and Tocquin et al. (2003) re-ported that aeration of the nutrient solution was not necessary in small tanks.Consistent with this report, we used our system without aeration. Use of othermesh sizes allows adaptation of the system to other small-seed plants, such as to-bacco, for which a mesh size of 250 µm is better suited because of larger seedsand developing roots (Schlesier and Mock, unpublished).

Future applications will include isolating organelles from roots, which isdifficult to achieve with soil-grown plants. Preliminary results indicate the suit-ability of the new cultivation system for the isolation of mitochondria fromArabidopsis roots (Braun and Mock, unpublished).

Acknowledgments

We thank M. Eisbrenner, B. Kettig, and A. Wolf for their excellent technicalassistance. Funding of the DFG to H.-P. Mock (Mo 479/4-1) is gratefully ac-knowledged.

References

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Damerval C, de Vienne D, Zivy M, and Thiellement H (1986) Technical improvements intwo-dimensional electrophoresis increase the level of genetic variation detected inwheat-seedling proteins. Electrophoresis 7: 52-4.

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a hydroponic culture system for growing is thaliana plantlets under sterile conditions

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