nonrecirculating hydroponic system suitable studies at ...this report extends and clarifies an...

Plant Physiol. (1991) 95, 1125-11300032-0889/91/95/1125/06/$01 .00/0

Received for publication August 27, 1990Accepted December 11, 1990

Nonrecirculating Hydroponic System Suitable for UptakeStudies at Very Low Nutrient Concentrations'

Vincent P. Gutschick* and Lou Ellen KayDepartment of Biology, New Mexico State University, Las Cruces, New Mexico 88003

ABSTRACT

We describe the mechanical, electronic, hydraulic, and struc-tural design of a nonrecirculating hydroponic system. The systemis particularly suited to studies at very low nutrient concentra-tions, for which on-line concentration monitoring methods eitherdo not exist or are costly and limited to monitoring relatively fewindividual plants. Solutions are mixed automatically to chosenconcentrations, which can be set differently for every pump fedfrom a master supply of deionized water and nutrient concen-trates. Pumping rates can be varied over a 50-fold range, up to400 liters per day, which suffices to maintain a number of large,post-seedling plants in rapid growth at (sub)micromolar levels ofN and P. The outflow of each pump is divided among as many as12 separate root chambers. In each chamber one may monitoruptake by individual plant roots or segments thereof, by meas-uring nutrient depletion in batch samples of solution. The systemis constructed from nontoxic materials that do not adsorb nutrientions; no transient shifts of nitrate and phosphate concentrationsare observable at the submicromolar level. Nonrecirculation ofsolutions limits problems of pH shifts, microbial contamination,and cumulative imbalances in unmonitored nutrients. We noteseveral disadvantages, principally related to high consumption ofdeionized water and solutes. The reciprocating pumps can beconstructed inexpensively, particularly by the researcher. Wealso report previously unattainable control of passive temperaturerise of chambers exposed to full sunlight, by use of white epoxypaint.

Long-term solution culture at micromolar levels of nu-trients is required in studies of adaptations of nutrient uptakesystems and root growth under a variety of stresses. Fullexpression of growth adaptations, such as in root:shoot ratio,requires growth to considerable size (9). Large, vigorouslygrowing plants will deplete large volumes of nutrient solution.We take an example from our recent experiments and con-sider a plant having a dry mass m = 2 g, a tissue phosphoruscontent of 0.2% by dry weight (fractional contentf= 0.002),and a relative growth rate R of 0.15 d-'. Consider furtherproviding phosphorus (atomic weight M = 31 g molV') as

phosphate at a concentration of 1 mmol m-3. If one allows a

30% relative concentration depletion, Ac = 0.3 mmol m-3(0.3 uM), the required flow rate J is determined by solving theequation between plant uptake and solution depletion:

Mass of P taken up per unit time = mfR = JAcM. (1)

'Funding by the U. S. Department of Energy, Ecological Research.1125

With the given parameter choices, the required flow rate is65 L/d for every replicate plant. Most hydroponic systemscapable ofproviding such large volumes do so by recirculatingsolution from a large reservoir (2, 3), often with automatedmakeup of depleted nutrients to maintain concentrations andto measure uptake (e.g. 7, 13, 14). Recirculation can increaseproblems of microbial contamination. It also requires auto-mated controls for makeup of nutrient ions or of hydrogenions (pH balance) during nitrate or ammonium usage. Con-centration sensors for the controls are commonly based uponion-selective electrodes (4, 7) or, rarely, flow-injection analysis(5). Electrodes are adequately sensitive for nitrate down to 5,AM (4) but not down to (sub)micromolar levels at which onemay wish to maintain other nutrients, such as phosphate andthe micronutrients. Flow-injection systems are quite costlyand can monitor relatively few individual plants or groups ata time (for example, 8 plants, in the case of ref. 5, comparedwith the 28 to 84 plants we survey with our system). Typically,recirculating systems monitor relatively few nutrients and failto monitor most of the nutrients, particularly micronutrients.The requirement to oversupply the unmonitored nutrientsmight cause initial toxicity problems in multiweek experi-ments with nutrients such as copper.We have designed our system to mix, automatically on

demand, large volumes of fresh solution for single-pass flow.The contents of all nutrients at inputs to every plant arecontrolled by mixing ratios between three solutions. Theaverage concentrations at the plant root system are drawndown from input levels by nutrient uptake, but adequatepumping rates can hold all concentrations within relativelytight limits. The design addresses several other major practicalproblems, foremost that of providing inexpensive, well-regu-lated pumping for multiple treatments and replicates. (Thepumps may also be used to advantage in recirculating sys-tems.) We have designed and built positive-displacementpumps based on 140-mL plastic syringes, with electronicallycontrolled repetition rates. Simple hydraulic resistance divid-ers apportion flows reproducibly to replicate plants. Ourgrowth containers are divided into three separate chambersmeeting in the center, allowing two- and even three-waydivisions of root systems for differential treatment. We alsohave identified a coating that is highly reflective at shortwavelengths and high in thermal emissivity, allowing surfacesexposed to full sun to remain within 1°C of ambient airtemperature. The entire system is constructed of nontoxicmaterials (10) that are also low in capacity to release or adsorbnutrient ions.

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Plant Physiol. Vol. 95, 1991

This report extends and clarifies an earlier presentation(1 1).

MATERIALS AND METHODS

Figure 1 shows the full system schematically. Full mechan-ical and electronic plans are available from us.

Nutrient solutions are mixed automatically in two stages.First, the system mixes a base solution of all the elements notbe varied in concentration between experimental groups, thatis, all elements but N and P in our case. The invariantelements are provided as two 90x concentrates (A, B in TableI), designed to be soluble separately. The A and B concentratesare mixed on demand with DIW2 provided at nearly linepressure through two large mixed-bed deionizer tanks in series(each with commercial designation of 10,500-grain capacity[as CaCO3J, equivalent to 7 mol of divalent anions and 7 moldivalent cations). Outflows of both deionizer tanks are mon-itored for specific resistivity to give timely warning of needfor replacement. The mixed base solution is held in a 20-Lpolyethylene carboy suspended on a metal spring. When thecarboy is depleted to 7 L, it rises to trip a microswitch. Alatching relay then activates (via solid-state relays) severalsolenoid valves in order to: (a) empty the contents oftwo 150-mL reservoirs of A and B base concentrates into the base-solution carboy; it is important to maintain a 7-L reserve sothat incoming concentrates do not precipitate on contact; (b)shut off the gravity-fed refilling of the 150-mL concentratereservoirs; (c) turn on the DIW inflow and activate a magneticstirrer mounted on the carboy bottom; and (d) shut offoutflow from the carboy, so that continued consumption ofbase solution by pumps does not alter the concentration asmixing occurs over the 3-min refill time. A second basesolution carboy, mounted lower, provides solution for pumpswhile refilling of the first carboy proceeds. When the basecarboy refills to 20 L, it trips a second microswitch thatdeactivates the solenoids noted above. It also trips a secondlatching relay that opens inflow valves to refill the A and Bconcentrate reservoirs, by gravity flow from 20-L carboysmounted above. Inflow ceases when the liquid level in areservoir bridges two stainless steel pins, closing a low-currentrelay circuit that shuts off solenoid valves.The adjustable-concentration nutrients (e.g. N and P) are

held as 50-fold concentrates in 20-L carboys; they are mixedwith base solution at the pump. A reciprocating crank operatesthree syringes. The large syringe (Monoject3 140-mL plasticdisposable; Sherwood Medical, DeLand, FL) is driven with a12.5-cm stroke and delivers 95 mL of base solution per cycle.The Leur-Lock fitting is bored out to reduce pumping back-pressure, greatly prolonging syringe life to over 5000 cyclesbefore leakage becomes significant. Two glass syringes (Bec-ton-Dickinson, 5-mL) operate on a 1.8-cm stroke and eachdelivers 2 mL ofN or P concentrate per cycle. A single crankarm holds the glass syringes in a rubber pad to eliminate

2 Abbreviations: DIW, deionized water; FeEDTA, ferric ethylene-diaminetetracetic acid; VAC, volts-alternating current.

3The mention of a specific produce name is not a unique endorse-ment of this product. It does indicate satisfactory performance of thespecific product in long-term operation.

optional 1her

bOaE.2

Figure 1. Schematic diagram of hydroponic system. See text for fullexplanation. Solution flows along lines indicated in boldface. Dashedlines indicate electrical connections. Solenoid-controlled valves arenoted as either normally open (N.O.) or normally closed (N.C.). Systemis shown configured for seven separate pumps, as used by authors.

1126 GUTSCHICK AND KAY



HYDROPONIC SYSTEM

Table I. Composition of Nutrient Solutions

From concentrate "A," diluted 1:90CaCI2,2.0 mM; Ca2, 2 mM; Cl, 4 mMFeNaEDTA- 1.5 H20, 3.53 mg L-1; Fe3 , 0.5 Ag/ml (=9 MM)

From concentrate "B," diluted 1:90K2SO4, 1.5 mM; K+, 3 mM; SO2-, 2.5 mMMgSO4.7H20, 1 mM; Mg2+, 1 mMMnSO4.7H20, 3.02 mg L-1; Mn2+, 0.6 ,g/ml (11 MM)H3B03, 3.42 mg L-1; B, 0.6 Mg/ml (z55 MM)ZnSO4-7H20, 1.32 mg L-1; Zn2 , 0.3 Mg/ml (-5 MM)Na2MoO4.2H20, 0.126 mg L-1; Mo, 0.05 ,g/ml (-0.5 AM)CuSO4.5H20, 0.078 mg L-'; Cu2+, 0.02 Mg/ml (=0.3 MM)

From concentrates for specific treatments, diluted 1:60Treatment

1 2 3 4 7 6 5

Input N03 [Mm, from Ca(NO3)] 10 10 10 100 150 10 3Input "PO4" [MM, from KH2PO4] 0.1 0.3 0.3 1 3 3 3

P-stress series -N-stressseries -.

breakage. Three pairs of stainless-steel check valves preventbackflow at both entry and exit ports of each syringe. Thevalves (NuPro 1/3-psi) are only lightly spring-loaded to keeprefilling drag low, and they have negligible void volume.Valves must be checked individually every several days forback-leakage; the Neoprene flap can be reseated. The fullcrankshaft is bearing-mounted on both ends of its axis and ismachined from brass. The crank and motor mounts and thebase are machined from '/2-inch- (12.7 mm) thick stainlesssteel for resistance to corrosion and stability against torques.Four posts, one at each corner ofthe pump base, fit into otherpump bases, allowing stacking up to four pumps in onefootprint area.The crankshaft is turned by a 3-rpm, high-torque, gear-

reduction motor (Jakel Motors model BG5P3K; Highland,IL) operating at 1 7 VAC. Pumping rate is adjusted by settingthe interval between full cycles, to any value between 20 and990 s in 10-s increments. This interval is controlled separatelyfor each pump by an integrated-circuit timer reading thesettings on a two-digit thumbwheel display. The timer maycontrol the entire revolution of a geared stepping motor(Superior Electric, Bristol, CT) or, as in our system, it maystart an asynchronous motor. Revolution time of the lattervaries slightly with load, so that we use a crankshaft detentand microswitch (details available) to provide power for anexact 360° rotation, irrespective of load.

Outflows of the three syringes pumping base, N, and Psolutions merge in Tygon tubing linked with rigid Y-connec-tors. The combined outflow entrains about 5 mL of air,derived from (reproducible) air leakage past the large syringe'spiston during filling and perhaps from cavitation. Because airbubbles can randomly plug the capillary-tube flow dividers(described shortly), the air is removed by a simple standpipe(of dark amber glass, to minimize chances of algal growth).The flow from any single pump is then divided between 4

to 12 separate root chambers, representing individual plants

or distinct sections of their root systems. Inflow to eachchamber goes through a uniform hydraulic resistance pro-vided by a 10-cm length of 1-mm-diameter glass capillarytubing. Only rapid flows such as provided by an intermittentreciprocating pump can be hydraulically divided accurately.Without flow division, a separate positive-displacement pumpwould be needed for each plant, at prohibitive cost or com-plexity of operation.One 2-L polystyrene beaker is divided into three root

chambers (Fig. 2) by acrylic plastic pieces held in place withsilicone sealant. The beaker lid of 1/2-in- (12.7-mm-)thickacrylic plastic is cut to hold either (a) one plant positioned atthe center, with two or three parts ofits root system in differentroot chambers, or (b) three plants, one in each root chamber.Each root chamber has its own lines for air inflow (aerationand stirring), solution inflow, and solution outflow. To reduceair flows to manageable levels, air fed from a regulated-pressure supply passes at each chamber through a membranefilter. All of the root chambers fed by a single pump must beplaced at the same height, within 5 mm, to prevent siphoningof solution from one chamber to another after pumpingceases. Plants are held by electronics-grade, unfilled (translu-cent), silicone putty around the stem, providing a strong,nondamaging seal that contains no detectable, leachable tracesof nitrogen- or phosphorous-containing compounds. Eachplant may be removed independently and quickly by remov-ing the machine screws holding the lid sections together.The entire three-chamber beaker and its lid are specially

painted to prevent light entry and algal growth and to keepthe temperature within 1°C of air temperature even in fullsunlight and still air. (In contrast, chambers with clean metal

Hatched area: One of 3 iseparate root chambers

Figure 2. Top view of lid covering 2-L beaker divided into threeseparate root chambers. Lid holds either three plants with undividedroot systems, at locations P1, P2, P3, or one plant with root systemdivided into two or three segments, at location PC. Dashed linesindicate structural features underneath the lid. Details are shown foronly one of the root chambers, for plant at P3.

.1

I

1127




or ordinary white-painted surfaces readily reach 5 to 10°Cabove ambient in these conditions). An epoxy-based paint(Koppers, Inc. item A1737, mil. spec. Mil C 81773, color17875 white; retail sales through Burbank Paint, Burbank,CA, and others) reflects shortwave radiation almost com-pletely, while its very high thermal-infrared emissivity allowsit to lose most of the gained energy. The polystyrene surfaceofthe breaker must be primed first by painting with a solvent/roughener (10% by volume powdered chlorinated polyethyl-ene [Dow Chemical CPE 4213 or 2552] mixed with 80%tetrahydrofuran and 10% 1,1,l-trichloroethane) and thenwith black primer (fast black polyamide epoxy A809C-66,color 17038 black).When measuring plant uptake rates or confirming solution

concentrations, we took 1-L outflow samples from each plantor treatment inflow, in high-density polyethylene bottles. Weacidified each sample ofpH 2 and refrigerated it until analysis.The concentration depletion times flow rate yielded our esti-mate of whole-plant uptake, converted as necessary to uptakerate per unit mass. We analyzed nitrate spectrophotometri-cally with an Autoanalyzer (cadmium reduction to nitrite andchromogenic reaction with aromatic reagents [1]). We cor-rected all peak heights, A, for second-order reaction kineticsand subsequent nonlinear relation to concentration, c, as A =bc/(l + ac2); a and b are empirical coefficients dependingupon run conditions (temperature, flow rate, cadmium re-ductor-column diameter). The parameters a and b are deter-mined using assays on dilution-series standards. We measuredphosphate spectrophotometrically by the phosphomolybdate-blue reaction, using ascorbic acid as reductant. For phosphateconcentrations of 1 to 20 Mm (or microformal, to reportproperly the summation over ionization states; ref. 11, p. 70),we extracted the reaction mixture with methyl isobutyl ketoneand read absorbance at 722 nm; this minimized interferencefrom iron chelates and from temperature shifts. For concen-trations below 1 MM, we (8) modified a method (6) of tripleextraction of the phosphomolybdate from the aqueous phasewith methyl isobutyl ketone. Only this method proved freefrom interference by chelated iron in nutrient solutions.

RESULTS AND DISCUSSION

Our system with seven pumps reliably delivered a varietyof controlled nutrient levels. Over several seasons we haveraised four replicate plants per pump to the stage of 20 g freshmass (9) (LE Kay, VP Gutschick, manuscript in preparation;we began with as many as 12 replicate plants per pump,sacrificing groups sequentially for physiological analyses.) Inthe treatment groups growing at very low nutrient concentra-tions (0.1 Mm phosphate, 3 Mm nitrate), yet at significantrelative growth rates of 0.08 to 0.10 d-', the nutrient flow raterequired was very high, 400 L d-', and the pumps operatedcontinuously. We observed neither mortality nor signs oftoxicity from materials (cfJ 10) or from xenobiota. Nutrientconcentrations at the inflow of root chambers held constantwithin 2% because the solution mixing ratios were stable.Concentrations in the root chamber also held constant in theshort term (before plant growth requires adjustment ofpump-ing rates). The 650-mL volume buffers the change in concen-tration between the existing root-chamber contents and the

new 95-mL slug of solution delivered on each pump stroke.One can readily calculate that, if the solution is depleted 30%from inflow concentration on the average, then an inflow of1/7 of the chamber volume causes an instantaneous jump of6% in nutrient concentration, or only 3% above the mean.Even at system startup, we observed no transient depressionor elevation of nutrient concentrations, such as adsorption ordesorption from surfaces might cause. For reference, note thata monolayer of ions on binding sites 0.5 nm apart on thesurface of one root chamber would be equivalent to about 0.3Mm in the chamber contents of 0.65 L. We attribute thissuccess partly to materials choice, principally polyethylene,polystyrene, and surgical-grade Tygon tubing, with limitedexposure of solutions to glass, stainless steel, silicone andNeoprene rubbers. Furthermore, we cleaned all componentsthoroughly with phosphate-free detergents, following this withacid rinses and final DIW rinses. The solution pH was stablewithin 0.3 unit at most, despite variable degrees of nitrateuptake. Nonrecirculation of solutions aids such pH stability,while vigorous aeration with air of normal CO2 contentprovides good buffering by the carbonic acid-bicarbonatecouple. The flow rates to individual chambers fed by a singlepump were stable individually, while varying as much as 30%between chambers.

Consequently, we could accurately resolve the time coursesof physiological adaptations in the same plant (LE Kay, VPGutschick, manuscript in preparation). These results are re-ported in preliminary form (9) and will be presented in fullin our companion study, in preparation. Figure 3 shows

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2.5

2.0

1.5

1.0

0.5

0.0+10.0 15.0 20.0 25.0

Days After Germination30.0

Figure 3. Relationship of root:shoot ratio on a fresh-weight basis toplant age in Helianthus annuus L. (cv Giant Grey Stripe) as a functionof nominal nutrient levels maintained continuously after day 10 (Mmnitrate [N] and phosphate [P]). Experiment began with 12 replicateplants per nutrient treatment group. All plants were germinated insaturated CaSO4 solution. On d 10 they had attained an initialroot:shoot ratio of 0.39 ± 0.07 on a fresh-weight basis. They were

randomly assigned to one of seven different nutrient treatmentsprovided by the hydroponic system described herein and grown in anaturally lit greenhouse with mean day/night temperatures approxi-mating 320C/200C. Four plants per treatment were harvested on d17, 22, and 27.




HYDROPONIC SYSTEM

root:shoot ratio as an example, with (N, P) treatments clearlydifferentiated (reanalysis of data of ref. 9, in terms of drymass). We also resolved modest diurnal cycling in uptakerates (Fig. 4), more so at high nutrient concentrations. Weinterpolated these uptake rates and verified that uptake inte-grated over the whole life ofthe plant agreed within 10% withnutrient concentrations in plants.

In addition to the generic advantages (and disadvantages)of hydroponics, especially allowing repeated, nondestructiveaccess to functioning roots in situ, our system has severalspecific advantages. The nonrecirculation of solutions limitsproblems of microbial activity, pH shifts, and cumulativeimbalances in unmonitored nutrients. The system enablesstudies of plant performance at very low concentrations,maintained in steady state or varied according to a chosenprogram. We have used 0.07 ,M phosphate and 2.5 lsM nitrate,at which levels there are no automated on-line methods tocontrol concentration and calculate uptake, other than costlyflow-injection analysis that can handle relatively few replicateplants. For example, below several gM, most or perhaps allhigh-resolution phosphate assays are batch methods usingmultiple solvent extraction of phosphomolybdate assay prod-

1.25

1:3- 1.000

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;., 0.75

> 0.50

co

cu

025

CZ 0.25

10.0 12.5 15.0 17.5 20.0 22.5 25.0Time After Midnight, 11 October [h]

Figure 4. Instantaneous rate of phosphate uptake per unit freshmass of plant, as a function of time in diurnal cycle, for plants at endof experiment (8) that generated the data in Figure 1. Uptake ratesare resolved by nutrient treatment during growth and for individualreplicate plants (lines connect data points for individual).

uct (6). At least 12 replicate plants within any treatment groupcan be monitored for uptake while total equipment cost iskept modest, by virtue of the flow-division system. The costof the entire system is modest compared to that of systemsusing peristaltic pumps and automatic monitoring andmakeup of nutrients. The cost is low particularly if the re-searcher can machine the components, using a milling ma-chine, drill press, and welder. The reciprocating pumps arecost-effective in recirculating systems also. One can also main-tain disjoint nutrient concentrations on two or three separateroot system segments of individual plants while monitoringtheir uptake rates via grab-samples of inflow and outflowsolutions. Finally, we note the excellent control of heat gainfrom shortwave radiation absorption, afforded by the specialwhite epoxy paint. This paint is also useful in shielding airtemperature sensors and the like.

Several disadvantages accrue from nonrecirculation of thesolutions. The high volume of DIW and nutrient use incursa significant operational expense. We used 15,000 L in one2-week experiment on 84 plants, 28 of which grew to 14 gaverage fresh mass. (Recirculating systems also require dis-carding of large reservoirs after an experiment, as much as2000 L [3]). Such expense also precludes the use of PEG forosmotic stress studies. The large volumes for disposal maypreclude most uses of radioactive tracers, although we used33P at very low levels within radiation-safety guidelines. Theresearcher must monitor pump rates and pump functionfrequently. One must also run off-line solution analyses everyseveral days to assure that pumping rates are sufficient.

ACKNOWLEDGMENTS

We wish to thank a number of people at Los Alamos NationalLaboratory for their help. Faustin Trujillo and Dan Talley helped inthe design and initial machining of the pump components. MichaelA. Wolf and David Waechter helped in the design and construction,respectively, of the electronic controls. Consuelo Montoya helped usoperate the system at times. James Steger, Caroline Reynolds, andRalph Franklin (U.S. Department of Energy; currently at ClemsonUniversity) provided critical administrative support.

LITERATURE CITED

1. Armstrong FAJ, Sterns CR, Strickland JDH (1967) The meas-urement of upwelling and subsequent biological processes bymeans of the Technicon AutoAnalyzer and associated equip-ment. Deep-Sea Res 14: 381-389

2. Asher CJ, Edwards DG (1983) Modern solution culture tech-niques. In A Lauchli, RL Bieleski, eds, Encyclopedia of PlantPhysiology (New Series, Vol 15A). Springer-Verlag, Berlin, pp95-1 18

3. Asher CJ, Ozanne PG, Loneragan JF (1965) A method forcontrolling the ionic environment of plant roots. Soil Sci 100:149-156

4. Bloom AJ (1989) Continuous and steady-state nutrient absorp-tion by intact plants. In JG Torrey, LW Winship, eds, Appli-cations of Continuous and Steady-State Methods to RootBiology. Kluwer Academic, Dordrecht, pp 147-163

5. Breeze VG, Canaway RJ, Wild A, Hopper MJ, Jones LHP(1982) The uptake of phosphate by plants from flowing nu-trient solution. I. Control of phosphate concentration in solu-tion. J Exp Bot 33: 183-189

6. Gibson AR, Bailey JM, Giltrap DJ (1976) Determination oftrace amounts of phosphate in water extracts of soil. CommunSoil Sci Plant Anal 7: 427-436

/ .p \ ..._ / /~ N\..o :f

// /// N\ : '/ /~ '' '''''/ // ' '' \

// // ~ \'-

N-N 25-

_ / /N10P.N

/ N/ -N40P.

-N.....P.8

Pb~~~ .0 .O.R.Ov.

1129




7. Glass ADM, Siddiqi MY, Deane-Drummond CE (1983) A mul-tichannel microcomputer-based system for continuously meas-uring and recording ion activities of uptake solutions duringion absorption by roots of intact plants. Plant Cell Environ 6:247-253

8. Gutschick VP (1985) Occurrence of consistent nM levels ofphosphate in demineralized and demineralized-distilled water.Talanta 32: 93-94

9. Gutschick VP, Kay LE (1986) Root adaptations at stress levelsof nitrate, phosphate, or both simultaneously. In P Martin-Prevel, ed, Proceedings of the Sixth International Colloquiumon the Optimization of Plant Nutrition, Vol 3. AIONP/GER-DAT, Montpellier, pp 941-947

10. Hannay JW, Millar DJ (1986) Phytotoxicity of phthalate plas-


ticisers. 1. Diagnosis and commercial implications. J Exp Bot37: 883-897

11. Kay LE, Gutschick VP (1986) Solution culture method forstudying nutrient uptake and stress. In P Martin-Prevel, ed,Proceedings of the Sixth International Colloquium on theOptimization of Plant Nutrition, Vol 3. AIONP/GERDAT,Montpellier, pp 1003-1007

12. Kennedy JH (1990) Analtytical Chemistry: Principles, Ed 2. WB Saunders, New York

13. Koch GW, Winner WE, Nardone A, Mooney HA (1987) Asystem for controlling the root and shoot environment forplant growth studies. Environ Exp Bot 27: 365-377

14. Lynch J, Epstein E, Lauchli A, Weigt GI (1990) An automatedgreenhouse sand culture system suitable for studies of P nutri-tion. Plant Cell Environ 13: 547-554



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