Table. 1. List of materials required for construction of the ultrasonic misting system.

zSources: 1 = De Novo, San Dimas, Calif; 2 = local hardware store; 3 = United States Plastic Corp., Lima,Ohio; 4 = local drug store.

Construction andUse of anInexpensive inVitro UltrasonicMisting System

Brent Tisserat, Daniel Jones,and Paul D. Galletta

Additional index words. aeroponics,asexual embryogenesis, carrot, planttissue culture, humidifier

Summary. An inexpensive user-con-structed ultrasonic misting system togrow plant tissues in vitro is pre-sented. This system is constructed bycoupling two commercially availableproducts, a culture chamber and anultrasonic humidifier. Plant culturesare grown within a culture chamberon a platform and bathed periodicallyby an ultrasonic nutrient mist. Carrotcultures were found to grow as muchas four to 10 times as great as thosegrown on agar medium.

W ithin the past few years, anumber of novel plant tis-sue culture items and sys-

tems have become available commer-cially, such as the Falcon CultuSAK(Becton Dickinson Labware, LincolnPark, N.J.), De Novo automatedmicropropagation system (De Novo,San Dimas, Calif. ), Vegbox plant con-tainers (Plant Production Systems B.V.,Helmond, The Netherlands), and theMistifier (Manostat, New York, N.Y.).These novel systems improve culturegrowth rates and/or decrease labor forreculturing activities. One of the mostinteresting newly developed productshas been a sterile aeroponics culturesystem (Fox, 1988; Weathers and Giles,1988 ). Nutrients and water are sup-plied to the cultures in a fine mist thatcompletely bathes the culture. TheMistifier is composed of a number ofmodular components: An ultrasonicmist generator module, an air pump, aculture chamber, a misting controller

Agricultural Research Service, U.S. Department of Ag-riculture, Fruit and Vegetable Chemistry Laboratory,Pasadena, CA 91106.

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unit, and a peristaltic pump. Unfortu-nately, this aeroponics unit is expen-sive, retailing for about $4000 each.The cost of this unit precludes any in-depth testing with the aeroponics sys-tem for the average plant tissue cultureresearcher. As an alternative, we reporton the construction of a considerablyless-expensive ultrasonic misting sys-tem (UMS). Comparison of growthresponses obtained from misting car-rot cultures with other tissue culturesystems are given herein.

Construction of theultrasonic misting system

A list of items to construct theUMS is given in Table 1. A Sunbeam1.2-gal ultrasonic humidifier (Model#66004; Sunbeam Appliance Co.,Milwaukee, Wis.), obtained from alocal drug store, provided the ultra-sonic misting element and power sup-ply. The humidifier was modified byremoving the unit’s ultrasonic mistingelement and connecting the unit’s liq-uid level sensor (LLS) so that it ispermanently closed. The humidifierAC cord was connected to anIntermatic Time-All Timer (IntermaticInc., Spring Grove, Ill.) equipped with24 on/off settings per day (Fig. 1A).The culture chamber was constructedof polycarbonate (180 mm height ×340 mm length × 170 mm width) andwas equipped with three air-vent filters(Fig. 1 A and B). The chamber was

yCulture chambers also can be entirely self-constructed0001) (Nalgene Co.j Rochester, N.Y.) fit with GelmaAnn Arbor, Mich.) and 1/8-27 NPT threaded polyproAlaska).

modified by mounting a polypropy-lene LLS and the ultrasonic mistingelement at the bottom end of thechamber. The LLS was mounted in a1/8-27 NPT tapped hole. The ultra-sonic misting element was mountedunderneath a 19.05-mm-diameterhole. Nylon bolts, washers, and nutsand Buns-N O-rings were employedto secure the misting element to thechamber for a liquid-tight fit via twoadditional 2.2-mm-diameter holes.The culture chamber was attached tothe medium reservoir with 60 cm ofsilicone tubing, 2.5 mm id. x 4.5 mmo.d. (Fig. 1A). The level of the me-dium in the growth chamber was keptat a constant 25 mm depth by connect-ing the LLS to the medium reservoirvia the unidirectional peristaltic pump(Fig. 1A).

Cultures were incubated on apolypropylene culture tray consistingof a rectangular perforated polypropy-lene sheet (300 mm length × 130 mmwide x 5 mm thickness with 4.75-mm-diameter holes staggered on 7.94-mmcenters) mounted on four corner-po-sitioned 45-mm-long x 15-mm-diam-eter Deb-in rods (Fig. 1 B ). Polypropy-lene netting (63 x 75 strands/cm) wasplaced on the surface of the tray toprovide support for tissues. It is impor-tant that the misting element’s bub-bling pathway be free of obstructionsto obtain the best misting performance;therefore, an opening was made in the

by using Nalgene Bio-Safe carriers (model #7135-n Bacterial Air Vents (#4210) (Gelman Sciences,pylene couplings (Ark-Plas Products, Inc., Flippin,

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Fig. 1. Schematic views of components used in the ultrasonic misting system. (A) Overall view ofsystem. Ultrasonic mist is produced directly in the culture chamber by the misting element. (B)Culture growth chamber used. The perforated culture tray supports the plant tissue cultures abovethe nutrient medium level, which is monitored by the liquid level sensor. The misting elementgenerates the nutrient mist to provide nutrients for culture growth.

culture tray. The culture chamber withattached LLS and misting element,containing the culture tray, siliconetubing, and reservoir, were autoclavetogether to sterilize.

Following culture-planting on thesupport tray, the electrical lines wereconnected, allowing for automatic fill-ing of the culture chamber via the LLSand peristaltic pump. The ultrasonicmisting element was then activated.Within 60 see, the entire chamber wasfilled with a white media cloud. Cul-tures were misted for 15-rein periodsat ten equally spaced times per day.

Construction of anautomated culture system

An automated culture system(ACS) was used that introduced nutr-ent medium into the culture chamber,bathing the cultures. The medium wasthen evacuated automatically (DeNovo, San Dimas, Calif.) (Tisserat andVandercook, 1985; 1986; Tisserat,1990). This system consists of thesame components used in the UMSminus the LLS, misting element, cul-ture tray, and power supply. Using theACS, medium was introduced andevacuated from the culture chamberfour equally spaced times per day, andcultures were allowed to soak for 5-min periods.

Cultures and media‘Danvers Half Long’ carrots

(Daucus carota L.) (Los Angeles SeedCo., Los Angeles) petioles and leafletswere obtained from sterile, germinatedseedlings. To initiate callus, 1-cm-longpieces of 3-week-old shootlets consist-ing of petioles and leaflets were cul-tured on a basal nutrient medium (BM)contained the following (in mg/li-ters): KNO3, 500; Ca(NO3)2•3H2O,250; NH4N O3, 500; H2B O3, 5.5;CaCl2•2H20,70;CuSO4•5H2O,0.05;FeS04•7H2O,2.8;MgCl 2•6H2O,100;MgSO4•7H2O,350;MnSO 4•H2O,25;CoCl2•6H2O, 0.03; KI, 1; KH2PO4,150; Na2MnO4•2H2O, 0.28; EDTA,40.9; ZnSO4•7H2O, 25; 2,4-dichloro-phenoxyacetic acid (2,4-D), 0.1; thia-mine•HCl, 0.5; i-inositol, 120; su-crose, 30,000; and agar, 8000. ThepH value was adjusted to 5.7 ± 0.1with 0.1 N HCl or NaOH before theaddition of agar. In some cases, cul-tures were grown in liquid BM ± 2,4-D with and without agitation at 25rpm on a gyrotary shaker. The me-dium was dispensed in 50-ml aliquots

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in 275-ml polycarbonate containersand capped with translucent polypro-pylene lids. When the UMS or theACS was employed, 1.5-liter BM wassterilized in a 2-liter polycarbonatereservoir bottle. All media were steril-ized for 15 min at 1.05 kg•cm-1 and121C. Experiments were conductedwith carrots to test the ability of thevarious culture systems to induce: 1 )petioles and leaflets to produce em-bryogenetic callus, and 2) callus toproduce asexual embryos and plant-lets. A 0.5-cm2 piece of embryogeneticcarrot callus or three 1-cm-long peti-ole–leaf explants obtained from a 3-week-old sterile germinated seedlingwere cultured on BM and BM –2,4-D,

respectively, using the: 1) UMS, 2)ACS, 3) agar medium, 4) stationaryliquid medium, and 5) shaken liquidmedium. Following 4 weeks of incu-bation, data on morphogenetic re-sponses, including culture fresh weightand number of somatic embryos, wererecorded. Nine replicates per treat-ment were planted, and experimentswere repeated at least twice. Cultureswere incubated at a constant 26 ± 1Cunder a 16-hr daily exposure to 2.2W/m’ cool-white fluorescent lamps.

Growth responses of calluson BM - 2,4-D

Culturing carrot callus grown onthe UMS produced the highest fresh

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Fig. 2. Examples of culture growth for carrot leaf/petiole culture using the ultrasonic mistingsystem on basal medium. (A) Comparison of carrot callus grown in various culture systems. Notethat the greatest growth occurs on the misting system. Bar in upper right corner represents 10 mm.(B) Somatic embryos produced on the surface of carrot leaflets. Note the co-occurrence of minutecallus-like clusters, from which somatic embryos are protruding Bar in the upper right cornerrepresents 50 µm.

weights compared to cultures grownin other treatments (Table 2). Fourtimes as much culture fresh weight wasproduced in the UMS compared togrowth on agar medium. The ACSinduced higher fresh weights than allother culture treatments except theUMS (Table 2). Growth in agar andliquid systems was similar. The num-ber of somatic embryos produced inthe ACS and agar treatment were sub-

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stantially fewer compared to that pro-duced in the UMS (Table 2). Nosomatic embryos were produced in theliquid treatments. Also, the UMS al-lowed for production of larger plant-lets derived from somatic embryoscompared to any other system tested.In the UMS, some plantlets as long as10 to 15 cm were produced, while inthe other tested systems plantlets aver-aged about 2 to 5 cm in length.

Growth responses ofshootlets to BM

Petiole and leaflet sections en-larged considerably within the first 2weeks when cultured in the UMS (Fig.2A). By the end of 4 weeks, the origi-nal cultures had enlarged to as much asfive times their original size. A puz-zling, unexpected phenomenon thatoccurred from cultures in the UMSwas the direct induction of asexualembryoids from the enlarged leafletsurfaces (Fig. 2A). This phenomenonwas not present in other treatments.Callus formation was minimal in theUMS treatments from explants whenviewed with the unaided eye com-pared to that obtained in the agartreatment (Fig. 2A). However, whenthe UMS cultures were observed withthe dissecting microscope at ×25, itwas noted that minute callus and/orsomatic embryogenetic clusters wereproduced frequently on the surface ofthe leaflets. Also, microscopic exami-nation of the surface of leaves andpetioles cultured in the UMS suggeststhat direct somatic embryogenesiswithout prior callus involvement maybe occurring, although this remains tobe verified by histological documenta-tion (Fig. 2B). Culture fresh weightwas also greatest for the carrot peti-ole/leaflets cultures in the UMS com-pared to the other tested systems (Table1). Fresh weight increases in the UMSwere due to the enormous enlarge-ment of the original petiole and leaftissues and not to callus initiation andproliferation.

Weathers and Giles (1988) re-ported that plant cultures grew as wellin an aeroponics bioreactor comparedto growth on agar medium with aproduction cost savings of 65%. Theirmisting bioreactor (i.e., culture cham-ber) was constructed of Plexiglas andsterilized by successive rinses of hy-pochlorite, ethanol, and sterile water.A sterile aerosol spray that bathed thecultures uniformly was produced by acompressed air cylinder and a spraynozzle. Our study supports their find-ings, except that we obtained evenhigher yields in our UMS compared tothat obtained with the agar system. Wedemonstrate how a novel technologycan be implemented inexpensively toachieve greater growth rates of plantcultures than using the conventional

Summary

7 7

Table 2. Morphogenetic responses of carrot Cultures to various culture treatments.

zTreatment means sharing the same letter within columns are not significantly different using Student–Neuman-Keuls multiple vane test (P < 0.1).

YComparison of fresh weight of different treatments cultured on the same medium with the ultrasonictreatment.

techniques, vessels, and systems.Aeroponics requires further study todetermine the merits of using this un-usual system to tissue-culture plantsroutinely. We devised a simple, unso-phisticated UMS that employs an ul-trasonic misting element directly inthe culture chamber to study this tech-nology, thereby eliminating many com-ponents. Our system cost about $320to construct, which compares favor-ably to the commercial system—theMistifier. It should be noted that itrequires about 2 to 3 h to constructthis system. One drawback was foundwith our UMS: after four to five uses,the misting element usually requiredreplacement due to debris build-upand/or its deterioration. Fortunately,replacements were performed easily.However, for commercial purposes, werecommend that a more heavy-dutyultrasonic misting element be used.

AcknowledgementMention of a trademark name or

proprietary product does not consti-tute a guarantee or warranty of the

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product by the U.S. Department ofAgriculture and does not imply itsapproval to the exclusion of other prod-ucts that may be suitable. We thankL.J. Kutz, G.C. Sharma, and P.J.Weathers for critically reading this pa-per.

Literature CitedFox, J.L. 1988. Plants thrive in ultrasonicnutrient mists. Biotechnology 6:361.

Tisserat, B. and C.E. Vandercook. 1985.Development of an automated plant tissueculture system. Plant Cell and Organ Cult.5:107-117.

Tisserat, B. and C.E. Vandevcook. 1986.Computerized long-term tissue culture fororchids. Amer. Orchid Soc. Bul. 55: 35–42.

Tisserat, B. 1990. Micromachines to auto-mate plant tissue culture. Methods Mol.Biol. 6:563-569.

Weathers, P.J. and K.L. Giles. 1988. Re-generation of plants using nutrient mistculture. In Vitro Cell. and Dev. Biol.24:121–732.

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