A ForcedVentilationMicropropagationSystem forPhotoautotrophicProduction ofSweetpotato PlugPlantlets in aScaled-up CultureVessel: I. Growthand Uniformity

Jeongwook Heo,1

Sandra B. Wilson,2 andToyoki Kozai3

ADDITIONAL INDEX WORDS. Air currents,air distribution system, culture vessel,Ipomoea batatas, plant tissue culture,photoautotrophic culture,photomixotrophic culture

SUMMARY. An improved forcedventilation micropropagation systemwas designed with air distributionpipes for uniform spatial distributionsof carbon dioxide (CO2) concentra-tion and other environmental factorsto enhance photoautotrophic growthand uniformity of plug plantlets.Single-node stem cuttings of sweetpo-tato [Ipomoea batatas (L.) Lam.‘Beniazuma’] were photoautotrophi-cally (no sugar in the culture medium)

cultured on a mixture of vermiculiteand cellulose fibers with half-strengthMurashige and Skoog basal salts in ascaled-up culture vessel with an insidevolume of 11 L (2.9 gal). CO2concentration of the supplied air andphotosynthetic photon flux on theculture shelf were maintained at 1500µmol·mol–1 and 150 µmol·m–2·s–1,respectively. Plantlets grown in forcedventilation systems were compared toplantlets grown in standard (naturalventilation rate) tissue culture vessels.The forced (F) ventilation treatmentswere designated high (FH), medium(FM), and low (FL), and corre-sponded to ventilation rates of 23mL·s–1 (1.40 inch3/s), 17 mL·s–1 (1.04inch3/s), and 10 mL·s–1 (0.61 inch3/s), respectively, on day 12. Thenatural (N) ventilation treatment wasextremely low (NE) at 0.4 mL·s–1

(0.02 inch3/s), relative to the forcedventilation treatments. On day 12, thephotoautotrophic growth of plantletswas nearly two times greater with theforced ventilation system than withthe natural ventilation system.Plantlet growth did not significantlydiffer among the forced ventilationrates tested. The uniformity of theplantlet growth in the scaled-upculture vessel was enhanced by use ofair distribution pipes that decreasedthe difference in CO2 concentrationbetween the air inlets and the airoutlet.

I n conventional micropropa-gation, chlorophyllous shoots andplantlets are cultured on a me-

dium containing sugar (i.e.,photomixotrophically) using a rela-tively small culture vessel (several hun-dred milliliter in volume), at a lowphotosynthetic photon flux (PPF). The

culture vessel closures are designed toprevent the entering of microbes, but,consequently, restrict the gas exchangebetween the inside and outside of theculture vessel (Desjardins, 1995). CO2concentration in the vessel containingplantlets decreases rapidly to 50 to 100µmol·mol–1 after 1 to 2 h of the start ofa photoperiod due to the limited gasexchange (Kozai, 1991). Fujiwara etal. (1987) showed that the low netphotosynthetic rate and poor growthof in vitro plantlets were due to the lowCO2 concentration inside the vesselduring the photoperiod. In addition,the air speed around in vitro plantlets inan airtight culture vessel is substantiallylower than that in the greenhouse orfield (Kitaya et al., 1997), which reducesthe photosynthesis and transpiration ofplantlets and thus their growth in vitro(Kozai et al., 1992).

Zobayed et al. (1999) engineereda forced ventilation system that suppliessterile nutrient solution throughout anextended culture period. They foundthat a stagnant nutrient solution underphotoautotrophic forced ventilationconditions significantly increased thegrowth and net photosynthetic perfor-mance of in vitro plantlets. Heo andKozai (1999) utilized a simplified, com-mercially available, forced ventilationmicropropagation system using a scaled-up [12.8 L (3.4 gal)] culture vessel thatwas 25 to 40 times larger than typicallyused. With forced ventilation using anair pump, the photoautotrophic growthand net photosynthetic rate of sweetpo-tato plug plantlets were remarkably in-

This paper is a portion of a dissertation submitted byJeongwook Heo in fulfilling a PhD degree require-ment. The authors are thankful to Chieri Kubota andSayed Zobayed for their critical reading of the manu-script. Appreciation is also extended to ChanghooChun for his beneficial advice during this experiment.The cost of publishing this paper was defrayed in partby the payment of page charges. Under postal regula-tions, this paper therefore must be hereby markedadvertisement solely to indicate this fact.1Principal researcher, Research Center for the Devel-opment of Advanced Horticultural Technology, De-partment of Horticulture, Chungbuk National Uni-versity, Cheongju, Chungbuk, 361-763, Korea.2Assistant professor, corresponding author, Depart-ment of Environmental Horticulture, Indian RiverResearch and Education Center, University of Florida,2199 South Rock Road, Fort Pierce, FL 34945.3Professor, Department of Bioproduction Science,Faculty of Horticulture, Chiba University, Matsudo,Chiba 271-8510, Japan.

Fig. 1. Component diagram of aforced ventilation micropropagationsystem.

creased, compared with those under theconventional, natural ventilation condi-tions. The plantlet growth, however,was not spatially uniform in the force-ventilated culture vessel, as growth wasgreater near the air inlet than near the airoutlet. Nonuniform spatial distributionsof air velocity and CO2 within the cul-ture vessel caused significant spatial varia-tions of the plantlet growth in thescaled-up culture vessel.

The objective of this study was todevelop an improved forced ventilationmicropropagation system (uniform airvelocity and CO2 distribution) for en-hancing the growth rate and final uni-formity of sweetpotato plug plantletsby using air distribution pipes in theculture vessel.

Materials and methodsPLANT MATERIAL AND CULTURE CON-

DITIONS. Single-node stem cuttings (ini-

tial fresh weight: 80 ± 23 mg), eachwith a single leaf excised from in vitrosweetpotato plantlets, were used asexplants in all treatments. A mixture ofvermiculite and cellulose fibers(Florialite, Nisshinbo Industries, Inc.,Tokyo, Japan) was used as supportingmaterial for plantlets; 30 mL (1.0 fl oz)per explant of half strength MS(Murashige and Skoog, 1962) basiccomponents, without sucrose, wasadded to each culture vessel. The pHof culture medium was adjusted to 5.7before autoclaving.

Culture vessels were placed for 12d in a culture room, where room airtemperature and relatively humiditywere maintained at 29/26 ± 0.5 oC(84/79 ± 2 oF) (photo/dark period)and 80% ± 5%, respectively. A photo-period of 16 h was provided by cool-white fluorescent lamps at 150µmol·m–2·s–1 PPF. Photosynthetic pho-

ton flux was measured at 20 locationson the empty culture shelf to ensurelight uniformity in all vessels. The at-mospheric CO2 concentration in theculture room was controlled at 1500µmol·mol–1 with an infrared CO2 con-troller (ZFP, Fuji Electric Co. Ltd.,Tokyo, Japan) and recorded by a com-puterized system (Greenkit 100, E.S.D.Co. Ltd., Tokyo, Japan).

FORCED VENTILATION MICROPROP-AGATION SYSTEM. The forced ventila-tion micropropagation system includedacrylic air distribution pipes within ascaled-up culture vessel (Bio-Safe Car-rier box, Nalge Co. Ltd.) 368 mm(14.5 inches) long by 178 mm (7inches) high by 170 mm (6.7 inches)wide, 11.14 L (2.94 gal) air volume),an air flow rate meter (Purge type: No.5S, Kofloc Co. Ltd., Tokyo, Japan)with a manual air valve (KT-6, IuchiSangyo Co. Ltd., Tokyo, Japan), mem-brane filter disks, and an air pump forsupplying the CO2-enriched air (Fig.1). The culture vessel contained a 4 by10-cell tray (Minoru Sangyo Co. Ltd.,Tokyo, Japan), three air inlets, andone air outlet for forced ventilation.Membrane filter disks (NipponMillipore Co. Ltd., Yonezawa, Japan,0.5 µm) were attached to each of theinlets and outlets to prevent microbesfrom entering. To distribute the venti-lated air evenly in the culture vessel,three acrylic air distribution pipes [3-mm (0.08-inch) inside diameter] with1-mm (0.04-inch) diameter holes wereset on the surface of the multicell traycontaining the supporting material(Fig. 2). To create uniform airflowalong the length of the pipes, the threedistribution pipes were designed ex-actly the same, whereby twice as manyholes were inserted near the air outletthan near the inlet. The end of each airdistribution pipe was sealed so that theventilation air entered the vesselthroughout the numerous pipe holesand exited the vessel through the out-let hole made on the vessel wall (oppo-site side of the inlet). The three air inletpipes and one air outlet in the culturevessel were located at opposite ends ofthe vessel at heights of 40 and 168 mm(1.6 and 6.6 inches) from the bottomof the vessel, respectively. The three-branch valve (Fig. 2B) was used todistribute the inlet air among the threepipes. The forced ventilation rate wasmeasured with an air flow rate meterand controlled with a flow rate con-troller. The average air speed was cal-

Fig. 2. Photograph (A) and top-view schematic diagram (B) of the culturevessel with air distribution pipes in the forced ventilation micropropagationsystem; 25.4 mm = 1.0 inch.

culated by dividing the forced ventila-tion rate by the cross sectional area ofthe vessel.

NATURAL VENTILATION MICROPROPA-GATION SYSTEM. Three cylindrical poly-carbonate boxes with dimensions of10 cm (3.9 inches) high by 8 cm (3.2inches) wide and inside volume of 480mL (29 inches3) (Biopot, Nicca Chemi-cal Co. Ltd., Japan) were used as cul-ture vessels for the natural ventilation(more conventional) treatment. Toincrease the number of natural air ex-changes of the culture vessel, a mem-brane filter disk (Milli-Seal, MilliporeCo. Ltd., Japan, pore size: 0.5 µm) wasattached over each of three 10-mm(0.39-inch) diameter holes on the lidof the vessel. The air exchange rate ofthe culture vessels was estimated to be3.0/h according to the method de-scribed by Kozai et al. (1986). Conse-quently the ventilation rate wasapproximately 0.4 mL·s–1.

TREATMENTS. Ventilation treat-ments were designed to supply forcedair with a controlled amount of CO2 atvarying rates to determine the bestspatial uniformity of gas distributionwithin the vessels. Plantlets grown inforced ventilation systems were alsocompared to plantlets grown in stan-dard (natural ventilation rate) tissueculture vessels (Table 1). The forced(F) ventilation treatments were desig-nated high (FH), medium (FM), andlow (FL). The natural (N) ventilationtreatment was extremely low (NE),relative to the forced ventilation treat-ments. In FH, FM, and FL treatments,the forced ventilation rates in the ves-sels were gradually increased with pas-sage of days during the culture period.The ventilation rates on day 12 wereset at 23, 17, and 10 mL·s–1, respec-tively. Forty explants were planted ineach tray of FH, FM and FL treat-ments. Because of space limitations,only one tray was used per treatment.

In the NE treatment, the standardpractice of planting four explants ineach of the three cylindrical, polycar-bonate culture vessels was used.

VISUALIZATION OF AIR CURRENT IN A

SCALED-UP CULTURE VESSEL. The air cur-rent inside the culture vessel with sweet-potato plantlets was visualized at theventilation rate of 13 mL·s–1 (0.8 inch3/s) using fine particles of metaldehyde((C2H4O)4) as tracers and a high reso-lution and contrast camera (Super-eyeC2874, Hamamatsu Photonics, To-kyo, Japan) as described by Kitaya et al.(1997).

MEASUREMENTS OF THE PLANTLET

GROWTH AND CO2 CONCENTRATION. Sweet-

potato plantlets were harvested, anddestructive measurements were takenon fresh and dry weights on day 12.Leaf area per plantlet was estimated bya leaf area meter (Fujitsu Kyusyu Sys-tem Engineering Co. Ltd., Tokyo,Japan).

Two holes (2 mm diameter) weremade at the side-wall of the scaled-upculture vessel for measuring the CO2concentrations near the air inlets andoutlets. The holes were located at 90and 278 mm (3.5 and 10.9 inches),respectively, from the air inlets alongthe longitudinal direction of the cul-ture vessel. The CO2 concentrationwas measured in each treatment dur-ing the photoperiod when CO2 con-centration between inside and outsideof the culture vessel was stable overtime. The CO2 concentration mea-surement was conducted by using agas chromatograph (GC9A, ShimadzuCorporation, Kyoto, Japan).

STATISTICAL ANALYSIS. The natureof the experimental design did notallow for randomized vessels or anequal number of samples between treat-ments, therefore, standard deviations(σ) were calculated among treatmentsand used to detect differences betweentreatments at 95% confidence.

Results and discussionGROWTH OF SWEETPOTATO PLUG

PLANTLETS. Photographs of sweetpo-tato plantlets on day 12 are shown inFig. 3. The fresh weight per plantlet onday 12 was more than 1.2 times greaterin FH, FM, and FL treatments than inNE treatment (Table 2). The dryweight per plantlet was 1.4 to 1.5times greater in the forced ventilatedtreatments than in the NE. Final plant-let fresh and dry weights were similarwithin FH, FM, or FL ventilation treat-ments.

Leaf area per plantlet was 1.7times greater in FH and FM than inNE. There was no difference in leafarea per plantlet among the FH, FMand FL treatments. It has been re-ported earlier that leaf area per plantletof sweetpotato was significantly greaterin a forced ventilation micropropa-gation system, under high CO2 con-centration and high PPF cultureconditions, than in a natural ventila-tion micropropagation system (Heoand Kozai, 1999). A similar result hasbeen shown for sweetpotato plantletscultured photoautotrophically in vitrousing vermiculite in the culture vesselwith a high number of natural airexchanges (7.4/h) (Kozai et al., 1996).The benefit of forced ventilation forpromotion in photosynthesis andgrowth also has been shown by De etal. (1993). They showed that thegrowth of shoot and root in geraniumplantlets was two-three times greaterunder forced ventilation [air flow rateof 6 L·h–1 (1.6 gal/h)] than under theno ventilation condition. The thick-ness of the boundary layer around theleaves may have decreased by increas-ing the air current speed (Yabuki andMiyakawa, 1970), thereby reducingthe resistance to CO2 diffusion fromair to leaf surface and promotinggrowth.

SPATIAL VARIATIONS IN CO2 CONCEN-

TRATION AND PLANTLET GROWTH INSIDE

THE CULTURE VESSEL. Throughout theculture period, the CO2 concentrationinside the culture vessel during thephotoperiod was more than 350µmol·mol–1 lower in NE than in FH,FM, and FL treatments (Fig. 4). Thiscorrelated with a net photosyntheticrate (NPR) per plantlet on day 12 thatwas almost five times higher in the FHtreatment than in the NE treatment(Wilson et al., 2000). There were nosignificant differences between the FH

Table 1. Ventilation rate and average air speed on day 12 within forced ventila-tion and natural ventilation systems; FH = high forced ventilation, FM =medium forced ventilation, FL = low forced ventilation, and NE = extremelylow natural ventilation.

Treatment code Ventilation rate (mL·s–1)z Average air speed (cm·s–1)y

FH (forced, high) 23.0 0.08FM (forced, medium) 17.0 0.06FL (forced, low) 10.0 0.03NE (natural, extremely low) 0.4 not availablez16.4 mL·s–1 = 1.0 inch3/s.y1.00 cm·s–1 = 0.39 inch/s.

and FM treatmentsin CO2 concentra-tions between the airinlets and the airoutlet (Fig. 4). Inthe FL treatment,however, CO2 con-centration at the airoutlet was about200 µmol·m–2·s–1

lower than that atthe air inlets.

The spatialvariations in plant-let growth betweenthe air inlets and theair outlets were lessuniform in FL thanin FH and FM (datanot presented) dueto the differences in

the forced ventilation rate, air speedand resulting uneven distribution ofCO2 concentration inside the culturevessel during the photoperiod. Thespatial variations in plantlet growth inthe culture vessel were reduced byincreasing the forced ventilation rates

and utilizing air distribution pipes.In research by Ohyama and Kozai

(1997), CO2 concentrations in a testtube culture vessel (25 mm (1 inch)diameter, 120 mm (5 inches) height;commercially used in plant tissue cul-ture) were measured under four levelsof PPF during the photoperiod. It wasshown that the CO2 concentrationinside the test tube containing a sweet-potato plantlet decreased linearly withincreasing distances from the lid, indi-cating that the vertical distributions ofCO2 concentration and relative hu-midity were not uniform inside the testtube, which is relatively long and nar-row in shape. Likewise, on a largerscale, nonuniform spatial distributionsof CO2 within a culture vessel cancause spatial variations of plantletgrowth (Heo and Kozai, 1999). Byusing the present forced ventilationmicro-propagation system with con-trollable ventilation and air distribu-tion pipes, spatial variations in gaseousconcentrations can be decreased sig-nificantly.

AIR CURRENT PATTERN INSIDE THE

CULTURE VESSEL. The air current pat-tern inside the culture vessel in the FHtreatment on day 6 is shown in Fig. 5.Upward and downward air currentswere observed in the center and bothsides of the culture vessel, respectively(Fig. 5A). The upward air current speedinside the culture vessel was approxi-mately 2 cm·s–1 (0.8 inch/s). The aircurrents throughout the culture vesselwith the air distribution pipes wererelatively uniform, compared with thevessel without air distribution pipes(Fig. 5A and B). Increasing the forcedventilation rate during the culture pe-riod with the air distribution pipes inthe culture vessel resulted in uniformspatial distributions of air current andCO2 concentration near the air outletwhich, subsequently, increased plant-let growth uniformity.

ConclusionsThe growth of sweetpotato plug

plantlets was improved in the forcedventilation micropropagation systemwith air distribution pipes at a CO2concentration of 1500 µmol·mol–1, aPPF of 150 µmol·m–2·s–1, and withoutsugar in the culture medium, as com-pared with plantlet growth in the stan-dard, natural ventilation system.

Uniformity and promotion ofplantlet growth was achieved by ar-

Fig. 3. Photograph of sweetpotatoplug plantlets on day 12; FH = highforced ventilation, FM = mediumforced ventilation, FL = low forcedventilation, and NE = extremely lownatural ventilation; 2.54 cm = 1.0inch.

Table 2. Fresh weight, dry weight, and leaf area per sweetpotato plantlet on day12. Means ±standard deviations are shown; FH = high forced ventilation, FM =medium forced ventilation, FL = low forced ventilation, and NE = extremelylow natural ventilation.

Treatment Fresh wt Dry wt Leaf areacode (mg)z (mg) (cm2)z

FH 511 ± 85 46.6 ± 8.0 9.1 ± 1.8FM 563 ± 106 42.1 ± 8.6 9.0 ± 2.1FL 559 ± 123 47.0 ± 12.9 7.9 ± 2.1NE 415 ± 41 30.2 ± 0.8 5.3 ± 0.5z28,350 mg = 1.0 oz.y6.5 cm2 = 1.0 inch2.

Fig. 4. LongitudinalCO2 concentrationinside the culturevessel on day 12.Vertical linesrepresent standarddeviations (±2), n =8; FH = high forcedventilation, FM =medium forcedventilation, FL =low forced ventila-tion, and NE =extremely lownatural ventilation;2.54 cm = 1.0 inch.

ranging the air distribution pipes inthe forced ventilated culture vessel tominimize spatial variations in the dis-tributions of air current and CO2 con-centration. Commercialization of thissystem will require the development ofan environmental control system tomaximize photosynthetic (photoau-totrophic) growth of plantlets by con-trolling CO2 concentration, ventilationrate, and PPF; as well as relative hu-midity according to the growth stageof plantlets. Improvements of the sys-tem are currently underway.

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Fig. 5. Two dimensional patterns of air currents in the culture vessel with (A)and without (B) air distribution pipes under the forced ventilation. The forcedventilation rate was 13 mL·s–1 (0.8 inch3/s) in both figures. The arrow lengthand its direction represent the air current speed and the direction of aircurrent, respectively; 2.54 cm = 1.0 inch, 1 cm·s–1 = 0.4 inch/s.

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