J. Agronomy &: Crop Science 164, 168—173 (1990)© 1990 Paul Parey Scientific Publishers, Berlin and HamburgISSN 0931-2250

Fachbereicb Biologie der Universitat Kaiserslautern

Effect of Whole Season CO2 Enrichmenton the Cultivation of a Medicinal Plant, Digitalis

T. STUHLFAUTH and H. P. FOCK

Authors' address: Dr. THOMAS STUHLFAUTH and Prof. Dr. HEINRICH P. FoCK, Fachbereich Biologie,Universitat Kaiserslautern, Posifach 3049, D-6750 Kaiserslautern, Federal Republik of Germany.

With 5 figures

Received October 25, 1989; accepted December 17, 1989

Abstract

A versatile method was developed for the application of 1000 ppm CO? durtng the whole growth penod ofplants. Temperature controlled water coohng and ventilation of the greenhouse resulted in a monthly CO^enrichment time of 60 to 90% of the total light period. Digitalis lanata, grown in greenhouses with CO2enrichment during the whole growth phase from April to November, produced twice as much biomass asfield cultivated plants.

The relative yield of the glycoside digoxin per gram Digitalis drug dry weight was 0.4% in field grown and0.7% in greenhouse cultivated plants. The production of digoxin per hectare in the greenhouse at 1000 ppmCOi was almost 3.5-fold that by field cultivation. Drug yield and secondary metabohte production inD. lanata were remarkably influenced by increased temperature and elevated CO: partial pressure in thegreenhouse.

Key words: Carbon dioxide enrichment, Digitalis lanata, digoxin, greenhouse cultivation, greenhouse watercooling.

Introduction

Assimilated carbon can be utilized either inprimary metabolism or for the formation ofsecondary metabolites. Therefore, the enrich-ment of CO2 in the surrounding air is not onlycapable to improve plant biomass yield, butadditionally offers the potential to increasesecondary metabolite content. This is of spe-cial interest with regard to medicinal plants.

Digitalis lanata Ehrh., the woolly foxglove,is cultivated for the production of cardiac gly-cosides, indispensable substances for the treat-ment of cardiac insufficiency. In Europe, thebiennial plants arc grown in the field fromApril to November and are harvested at tbeend of this first and only vegetative phase(MASTENBROEK 1985). Digoxin, the relevantpharmaceutical glycoside, is produced by ex-

traction, deglucosylation and deacetylation ofleaf cardenolides, mainly of lanatosid C.

Generally, COi enrichment over the wholeseason has to overcome two major complica-tions (BELLAMY and KIMBALL 1986): Firstly,plants cultivated in greenhouses will be ex-posed to elevated temperatures, and photosyn-thesis may be depressed. Secondly, even shortperiods of sunny weather wili cause openingand ventilation of the house, inducing a com-plete loss of enriched CO2.

Using a culture system adjusted to the re-quirements of Digitalis lanata., we investigatedthe effect of 1000 ppm CO2 on the growth anddigoxin production in comparison with plantsgrown in ambient air (350 ppm CO2), either inthe greenhouse or in the field. By that ap-proach it was possible to distinguish between

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Whole Season CO, Enrichment 169

Fig. 1. Net CO; uptake (//molm" s"'} versus leaf tempera-ture (°C). Leaf photosynthesiswas estimated under similarconditions as during plantgrowth at 350 (O) or 1000 (•)ppm COi. Data points repre-sent the mean of 5 replicates.Point size > ± SE

the CO2 effect and variations accompanyinggreenhouse cultivation like higher tempera-tures and a reduction of irradiation.

Materials and Methods

Digitalis lanata Ehrh., a cuhivar of the BoehringerMannheim GmbH, was grown in plastic-coveredgreenhouses (Type RUBP, 6 x 8 m, Scharr, Stutt-gart-Vaihingen, Germany) from the beginning ofApril or in the field from the middle of April untilNovember.

Seeds were soaked for 3 days in light under run-ning tap water and then germinated in rock wool(Deutsche Rockwool Mineralwoll-GmbH, Glad-beck, Germany). After 6 weeks the plants were

transferred to 11 cm pots and 6 weeks later again to5 1 containers with garden soil (Einheitserde ED 73,Einheitserdenwerk Stangenberg, Hameln, Ger-many), mixed with 20% (v/v) sand. Pots were spaced40 cm apart, and only morphologically similar plant-lets were transferred. Plants were watered with acommercial nutrient solution (Compo Garten-dunger, Miinster, Germany).

Drug yield was estimated by drying leaves from atleast 10 randomly chosen plants at 50°C. Driedleaves were powdered, and an average sample (5 g)of the powder was analysed for digoxin, as previous-ly described (STUHLFAUTH et al. 1987).

Both greenhouses were built with their major axesin a north-south direction. The internal temperaturesin both houses never differed more than 2°C. Attemperatures between 35 and 38°C inside the

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Fig. 2. The monthly CO2 en-richment time expressed as per-cent of the total light phase dur-ing the cultivation period1987 (April to November)

in

170 STUI-ILFAUTH and FoCK

Fig. 3. Drug dr> weight (DW)versus cultivation time (weeks),calculated from the first week ofApril. The plants were culti-vated in the field (•) or in thegreenhouse at 350 (O) or 1000(•) ppm COi. A representativesample from the dried leaves ofat least 10 plants was analyzedfor each data point

houses, well-water was circulated through 100 m ofcopper pipe and was subsequently sprayed over thetop of the outside roof. Above 38^C, water coolingwas stopped and the ventilation of both houses wassimultaneously opened.

A 1000 ppm CO; level was maintained in onegreenhouse by a CO; cylinder equipped with amagnetic valve. The opening of the valve was con-trolled by an infrared CO; analyzer (DGT CO;-Scanner, Dietrich, Sprendlingen, Germany), and theCO2 enrichment was stopped as long as the ventila-tion remained open. About 10 kg CO; 100 m"'week"' were necessary for enrichment.

CO; enrichment, humidity, air and leaf tempera-ture were permanently recorded throughout the veg-etation period. Plant gas exchange was determined inan open gas exchange system at lOOOjtimol photonsm"V, according to STUHLFAUTH et al. (19S8).

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Results and Discussion

D. lanata was grown at temperatures up toabout 40°C in the greenhouses at either 350 or1000 ppm CO2. The adapted plants exhibited abroad temperature optimum of photosyn-thesis, ranging from 27 to about 4C°C (Fig. 1).Over the temperature range measured, netphotosynthesis at 1000 ppm CO2 was higherthan at 350 ppm CO2. At a leaf temperature of44*^0, the maximum temperature ever meas-ured in the greenhouses, the 350 ppm plantsshowed only a slight decrease of net CO2assimilation, whereas the photosynthesis of1000 ppm plants was still increased. Therefore,the growth of D, lanata was not inhibited byhigh temperatures during greenhouse cultiva-

Fig. 4. Digoxin yield as %o of drugdry weight versus cultivation time(weeks), calculated from the firstweek of April. The plants were cul-tivated in the field (•) or in thegreenhouse at 350 (O) or 1000 (•)ppm COi. A representative samplefrom the dried leaf drug of at leastten plants was analyzed at each datapoint of the cui"ves

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Whole Season CO. Enrichment 171

Fig. 5. Digoxin yield (kg/ha)versus cultivation time (weeks),calculated from the first week ofApril. The plants were culti-vated in the field (•) or in thegreenhouse at 350 (O) or 1000(•) ppm CO> A representativesample from the dried leaf drugof at least ten plants was analy-zed at each data point of the

curves

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a

10

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tion. The enhanced net pbotosynthetic rate at1000 ppm CO2 might be attributed to a di-minished O2 inhibition at higher temperatures(MoEand MORTENSEN 1986).

The temperature resistance of D. lanatamade it possible to fix the opening signal forthe ventilation of the greenhouses at about38°C. The number and the total length ofventilation periods could thus be reduced andrestricted to sunny weather conditions. AsCO2 losses remained high on days when theweather often changed between sunny andcloudy periods and concomitantly led to ahigher frequency of ventilation openings, acooling device was installed and connected tothe temperature control system. As the tem-perature of the closed greenhouse exceeded35°C, water cooling was initiated. Water cool-ing effectively delayed tbe temperature rise inthe greenhouses by increased heat exchange.

At frequently changing weather conditions,water cooling delayed ventilation long enoughto reach the next cloudy period, so that watercooling stopped below 35 °C, and the enrichedCO2 was preserved. On bright, sunny dayswben the temperature of the houses perma-nently exceeded 38°C, ventilation replaced wa-ter cooling and CO2 enrichment was stopped.

Even durmg summer, the monthly CO2 en-richment time always exceeded 60% of thetotal light period (Fig. 2). As expected for thecolder seasons, the CO2 enrichment was moreefficient in spring and autumn, with the excep-

tion of the relatively wet August and the dryApril in 1987 (Fig. 2). The COi enrichmentmethod described here proved more economi-cal than previously described methods for C O Tenrichment in unventilated houses (KIMBALL

and MITCHELL 1979, WiLLiTset al. 1981, FULLER

and SALE 1983).The drug yield increased with cultivation

time (Fig. 3). At the end of the vegetationperiod, the dry weight of plants cultivated in1000 ppm CO2 was almost twice as high asthat of field grown controls (Fig. 3). Thoughgrown at different temperatures, plants grownin the field and in the 350 ppm CO2 green-house showed similar results (Fig. 3). Thus,differences in cultivation temperature hardlyaffected biomass production.

CO2 effects on growth and biomass produc-tion of different plant species are welldocumented (STRAIN and SIONIT 1982, KIMBALL

1983) and could be demonstrated also forD. lanata cultivated under controlled condi-tions in growth chambers (STUHLFAUTH et al.1987).

CO2 enrichment stimulates the primary me-tabolism in two ways: it leads to a stimulationof ribulose-bisphosphate carboxylase/oxygen-ase and to a suppression of pbotorespiration(BRAVDO 1986).

The relative digoxin yield (percent of drugdry weight) increased to a maximum (Fig. 4).In comparison witb the sigmold curve of drugyield (Fig. 3), digoxin yield increased most

172 STUHLFAUTH and FOCK

during August and produced an optimum firstm the field culture, followed by the optima ofthe greenhouse cultures (Fig. 4). The curve forthe CO2 enriched plants exhibited a narrowpeak, whereas the others had broad optima. Incomparison to field grown plants, the relativedigoxin yield of the plants cultivated in thegreenhouse was almost 2-fold higher.

This 2-fold increase in digoxin content wasmore likely the result of warmer greenhousetemperatures than of CO? enrichment, as mdi-cated by the relatively high values reached bythe 350 ppm CO2 greenhouse culture. Thisresult is consistent with earlier findings(DAFERT et al. 1935) and indicates that gly-coside production is enhanced following warmand sunny periods (e.g. in July). Due to addi-tional absorbance and reflectance, quantumflux in the greenhouses was about 25 % lowerthan in the field; therefore, the enhancement ofglycoside accumulation was triggered by high-er temperatures and not by higher lrradiance aspreviously assumed (WEILER and WESTEKEMPER

1979).Field cultivation leads to a sigmoidal curve

of digoxin yield, whereas the greenhousegrown plants exhibit a distinct optimum(Fig. 5). Greenhouse cultivation combmedwith CO2 enrichment resulted in an almost4-fold increase in digoxin yield in comparisonwith field cultivated plants. This remarkableincrease in secondary metabolite accumulationwas the consequence of an enhanced biomassproduction on one hand and of a pronounceddigoxin accumulation on the other, as can beseen from the individual effects (Figs. 3 and 4).

From the beginning of September, digoxinaccounted for almost two-thirds of total car-diac glycosides in D. lanata leaves. In thisdevelopmental phase, digitoxin, the metabolicprecursor of digoxin, formed about 20—30%of total cardenolides. Differences in the digito-xin production may be explained by variationsin the digitoxin biosynthesis from steroid pre-cursors and the hydroxylation of digitoxin todigoxin.

The results of our investigations offer newpotentials in the cultivation of medicinal plantslike D. lanata. With the described CO2 enrich-ment, more of the secondary metabolite can besynthesized using less cultivation area. Addi-tionally, a higher concentration of the productin the drug lowers extraction costs.

To utilize these advantages to full extent,harvest of D. lanata within the optimum yieldrange should be accomplished. CO2 enrichedplants do show relatively short periods of opti-mal digoxin yield. Fortunately, greenhousecultivation at 1000 ppm CO2 produced a con-siderable increase in biomass and digoxinyield, even if the optimal harvest date was notmet.

In summary, the described method of CO2enrichment during the warm and temperateseasons of the year can successfully be utilizedto increase biomass and content of secondarymetabolites in D. lanata and may also be ap-plicable to other plants.

Zusammenfassung

Auswirkungen der ganzsaisonalen CO2-An-reicherung auf die Kultivierung einer Heil-pflanze. Digitalis lanata

Eine vielfaltig nutzbare Methode zur Begasungvon Pflanzen mit 1000 ppm CO2 wahrend dergesamten Vegetationsperiode wurde entwik-kelt. Temperaturkontrollierte Wasserkiihlungund Liiftung des Gewachshauses ermoghchteeine monatliche CO2-Anreicherungszeit zwi-schen 60 und 90% der gesamten Tageslicht-periode. Die wahrend der Wachstumsphasevon April bis November im Gewachshaus beiGO2-Anreicherung gezogenen Digitalis lanataPflanzen lieferten doppelt so viel Biomasse wieim Freiland kultivierte Pflanzen.

Der relative Ertrag des Glykosides Digoxinje Gramm Digitalis Drogentrockengewicht be-trug 0,4% im freilandkultivierten und 0,7% inGewachshauspflanzen. Die Produktion vonDigoxin je Hektar erreichte im Gewachshausbei 1000 ppm CO2 das 3,5fache der Freiland-kultur. Drogenertrag und Sekundarstoffpro-duktion von D. lanata wurden durch zuneh-mende Temperatur und Erhohung des CO2-Partialdrucks im Gewachshaus deutlich beein-

Acknowledgements

We thank Dr. T. BUCKHOUT for critically readingthe manuscript.

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