Plant Cell, Tissue and Organ Culture 67: 281285, 2001. 2001 Kluwer Academic Publishers. Printed in the Netherlands. 281

Naphthoquinone contents of in vitro cultured plants and cell suspensionsof Dionaea muscipula and Drosera species

Ingrid L.I. HookDepartment of Pharmacognosy, School of Pharmacy, Trinity College, Dublin 2, Ireland (Fax: +353-1-6082804;E-mail: ihook@tcd.ie)

Received 9 January 2001; accepted in revised form 13 June 2001

Key words: in vitro plants, mucin, naphthoquinones, Sundews, suspensions, Venus flytrap

Abstract

In vitro cultured carnivorous plants were grown on a hormone-free medium. They produced the following naph-thoquinones: Dionaea muscipula (plumbagin: 5.3%), Drosera rotundifolia (7-methyljuglone: 0.6%), D. binata(plumbagin: 1.4%), and D. capensis (7-methyljuglone: 0.5%). A red, slow-growing suspension culture of D.muscipula was maintained in a modified McCowns Woody Plant (McC) medium and produced plumbagin (2.59%)after 30 days growth. A suspension culture of D. rotundifolia grew slowly as multicoloured small aggregates onlyin a modified Murashige and Skoog (MS) medium. No quantifiable amounts of naphthoquinones were produced.Several cell lines of D. capensis were developed. Green aggregates grown in a modified MS medium contained7-methyljuglone (0.33%) and differentiated into plants when placed onto hormone-free medium. Pink culturesgrown in modified McC medium contained 7-methyljuglone (1.24%), while dark red cultures produced ca. 1%in both modified McC and MS media. Though the latter medium was significantly better with regard to biomassproduction, cells excreted a mucin when cultured in both media (0.21 g dry mucin/g dry cells in McC) and (0.16 gdry mucin/g dry cells in MS). Effects of the presence or absence of light during the growth period of 30 days showedthat there was no effect on biomass and only slight effects on mucin production and naphthoquinone contents.

Introduction

Increasing interest in the horticultural and medicinalpotential of carnivorous plants has resulted in over-harvesting from natural sources. This, together with aloss of their natural habitats has led to the protection ofmany species. The result has been greater research intotheir micropropagation and the use of in vitro-grownplants as alternative sources of biomass.

Venus fly trap (Dionaea muscipula J. Ellis) is aperennial plant indigenous to bogs in coastal areas ofNorth and South Carolina, USA, but has also beenintroduced into Florida (Culham and Gornall, 1994).Naturally grown plants contain the naphthoquinonesplumbagin, hydroplumbagin 4-O--glucopyranoside,droserone, 3-chloroplumbagin (Kreher et al., 1990),as well as flavonoids, phenol carboxylic acids and en-zymes of the digestive glands. There appears to beno documented evidence of the use of this plant as

a traditional herbal remedy. In contrast, of the morethan 100 species of Sundews (genus Drosera) whichexist, extracts of D. rotundifolia L. and other speciesare traditional remedies for use against dry, irritatingcoughs (Schilcher and Elzer, 1993). The constituentsof major importance present in the aerial structures arealso various 1,4-naphthoquinones, especially plum-bagin (2-methyl-5-hydroxy-1,4-naphthoquinone) and7-methyljuglone (syn. ramentaceone), in addition toflavonoids, such as quercetin (Hager, 1973).

Intact plants can be readily cultured by in vitropropagation techniques (Czany et al., 1992) and onanalysis have been found to produce the same naph-thoquinones as naturally grown plants, e.g., withDrosera spathulata (Budzianowski, 1995; Blehova etal., 1995), D. rotundifolia (Bobak et al., 1995), D.intermedia (Budzianowski, 1996) and D. communis(Reichling et al., 1995). We previously reported on thedevelopment and naphthoquinone content of in vitro

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cultured plants and suspension cultures of D. capensis(Hook et al., 1997) and report now on further workdealing with plants and cultures developed from otherspecies of the family Droseraceae.

Materials and methods

Plant material and in vitro culture

In vitro cultured plants of Dionaea muscipula J. El-lis and Drosera capensis L. were originally purchasedfrom Carolina Biological Supply Co. (USA), whileD. rotundifolia was a gift from the University ofVienna. Plant material of D. binata var. binata was ob-tained from Trinity College Botanic Gardens, Dublinand sterilized using sodium hypochlorite solution andsterile water rinses. Plants were cultivated in the labor-atory as outlined in (Hook et al., 1997) and maintainedby periodic subdivision into a liquid or agar-solidifiedmedium containing Murashige and Skoog basal salts(Murashige and Skoog, 1962), thiamine HCl (0.4 mgl1), mesoinositol (100 mg l1) and sucrose (30 g l1)(= Medium 1). Plants grown in liquid culture werein 150 ml medium in 250 ml conical flasks, main-tained under light and agitation conditions as below,and harvested after 45 months growth.

Suspension cultures

The following media were used in the initiationand maintenance of cultures: Medium 2: Murashigeand Skoog basal salts, adenine hemisulphate (80 mgl1), 6- - -dimethylallylaminopurine (2 mg l1),thiamine HCl (0.4 mg l1), mesoinositol (100 mgl1) and sucrose (30 g l1). Medium 3: Gam-borgs B5 basal salts (Gamborg et al., 1968), 2,4-dichlorophenoxyacetic acid (0.22 mg l1), naphthale-neacetic acid (0.18 mg l1), glycine (2 mg l1),nicotinic acid (0.5 mg l1), pyridoxine HCl (0.5 mgl1), thiamine HCl (0.4 mg l1), mesoinositol (100 mgl1) and sucrose (30 g l1). Medium 4: McCownsWoody Plant basal salts (Lloyd and McCown, 1980),organic constituents as in Medium 3. Medium 5: Mur-ashige and Skoog basal salts, organic constituents asin Medium 3.

All cultures were agitated on an orbital shakerset at 90 rpm, maintained at 25, under cool-whitefluorescent lights delivering 85 mmol m2 s1 on an18:6-h light : dark cycle. Suspensions were grown asbatch cultures in conical flasks (40% flask-fill). At

monthly intervals cells from one flask were aseptic-ally separated by suction filtration and a known weightsubcultured into fresh medium. Cells from replicateflasks were harvested by filtration (= fresh wt (g)) anddried at < 40 (= dry wt (g)). Growth index was cal-culated from culture fresh wt at harvest inoculumfresh wt at subculture.

Callus of Dionaea muscipula was originally de-veloped in 1997 from sterile in vitro plants growingon agar-solidified Medium 3 and was used to initiatesuspension cultures maintained in Media 4 and 5.

Callus of D. rotundifolia originally developed fromsterile in vitro grown plants on agar-solidified Medium3 in 1996, was used to initiate suspension culturesmaintained in Medium 5.

Callus of D. capensis-5 was originally developedfrom sterile in vitro grown plants placed on agar-solidified Medium 2 in 1996. Suspension cultureswere initiated in 1997 and maintained in Media 4and 5.

Suspension cultures of D. capensis-6 were de-veloped in 1997 from callus originally initiated on anin vitro plant growing on agar-solidified Medium 4.

Suspension cultures of New D. capensis-6 weredeveloped in 1998 from a selected culture of D.capensis-6 (above) and maintained on Media 4 and 5.

Naphthoquinone isolation and determinations

Naphthoquinones were extracted using the bi-phasicmethod previously reported (Hook et al., 1997) andwhich was found by Krenn et al. (1998) to be com-parable to steam distillation as a method of isolation.Determination of naphthoquinone content was car-ried out by GC using a WCOT fused silica capillarycolumn (50 m 0.22 mm i.d.) coated with cyan-opropyl (equiv.) polysilphenylene-siloxane (70%) at210C with the detector and injector at 280C. Usingthese conditions Rt for plumbagin was 12.11 min (s.d.0.05) and for 7-methyljuglone 13.35 min (S.D. 0.04).Quantitation of naphthoquinones was carried out on adaily basis by reference to a calibration curve preparedwith pure compounds. 7-Methyljuglone was isolatedand purified from bulked pet. ether extracts of D.capensis-6 (Hook et al., 1997), while plumbagin waspurchased (Sigma). Each extraction and determinationwas replicated (2). Identities of naphthoquinoneswas confirmed by GCMS.

Mucin separation

A volume of absolute ethanol equal to the volume of

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Table 1. Naphthoquinone content of in vitro grown plants

Species Naphthoquinone Content Culture medium(% of dry wt)

Dionaea Plumbagin 5.3% Agar-solidified 1muscipulaDrosera Plumbagin 1.4% Agar-solidified 1binataDrosera Plumbagin 2.6% Liquid-Medium 1binataDroserarotundifolia 7-Methyljuglone 0.6% Liquid-Medium 1Droseracapensis 7-Methyljuglone 0.5% Liquid-Medium 1

culture medium was added. The stringy/slimy super-natant scum which immediately formed (= mucin) wascollected by stirring with a glass rod, to which the mu-cin adhered (Baldwin and Bell, 1955). It was separatedand dried in an oven at < 35C.

Statistical analyses

Analyses were performed using InStat statisticalsoftware (Graph Pad, San Diego, CA).

Results and discussion

In vitro grown plants

The naphthoquinone contents of in vitro grown plantsare shown in Table 1. All Drosera species grew well onboth liquid and solid Medium 1, but Dionaea failed togrow in liquid culture. These results confirm the valueof in vitro culture as a means of propagating largenumbers of carnivorous plants. The naphthoquinonecontent of our plants, though appearing higher thanin other references (Wawrosch et al., 1996), could berelated to the changed extraction protocol (Hook et al.,1997; Krenn et al., 1998). All the naphthoquinonespresent as major metabolites were as in the nativeintact plants (Culham and Gornall, 1994).

Suspension cultures

Suspension cultures of Dionaea muscipula grew asdark-pink aggregates. Although originally maintainedin both Medium 4 and 5, cells grown in Medium 5 lostviability after a few months. Only results relating to

culture in Medium 4 are presented (Figure 1). Growthwas slow (growth index 3.85 , S.D. 1.49, n = 44) andcells were found to produce only plumbagin as naph-thoquinone (2.59%, S.D. 0.37, n = 11), the same as theparent plants (Culham and Gornall, 1994).

Suspension cultures of Drosera rotundifolia failedto grow on Medium 4. They were originally developedfrom callus initiated on plants growing on Medium 5and were maintained in this medium, growing slowly(growth index 3.56, S.D. 0.95, n = 20) as multicol-oured aggregates (Figure 1). Although native D. rotun-difolia plants are supposed to contain both plumbaginand 7-methyljuglone (Culham and Gornall, 1994) andthe parent plants of these cultures were found toproduce up to 0.6% of 7-methyljuglone, suspensioncultures were devoid of quantifiable naphthoquinones.

Suspension cultures of D. capensis were origin-ally developed in 1995 and were found to produce7-methyljuglone, ca. 0.9%, depending on the formu-lation of the culture medium (Hook et al., 1997). Thiscell line was also found to excrete a mucin into themedium (Hook and Paper, 1996), a capacity whichwas subsequently lost. A series of new cell lines weretherefore initiated from in vitro cultured plants. Cellline-5 grew as green aggregates in Medium 5 (growthindex, 3.83, S.D. 0.62, n = 22) and grey aggregatesin Medium 4 (growth index, 2.67, S.D. 0.57, n = 15)(Figure 1). Medium 5 proved to be statistically sig-nificantly better as a culture medium with regard togrowth, biomass and naphthoquinone production. 7-methyljuglone was present at a concentration of 0.19%(S.D. 0.032) in cells grown in Medium 4 and 0.33%(S.D. 0.11) in Medium 5. When green aggregateswere placed onto agar-solidified or liquid Medium 1,they eventually differentiated into plantlets containing0.80% of 7-methyljuglone. This cell line proved to bea rapid source of large numbers of D. capensis plants.

Cultures designated D. capensis-6 were also initi-ated and grew as salmon-pink aggregates. Growth wasbest in Medium 4 (growth index, 4.90, S.D. 1.57, n =26, Biomass 9.72, S.D. 2.71, n = 22) and contents of7-methyljuglone were high (1.24%, S.D. 0.34, n = 10).Mucin was initially excreted into the culture mediumand chemical analysis found it to be an acidic polysac-charide rich in galactose units (Hook and Paper, 1996).This secretory capacity was again lost, and led to theselection of New D. capensis-6 from a flask showinga highly viscous medium.

This cell line was maintained in both Medium 4and 5 where growth was as dark red aggregates, al-though single pink-coloured cells were sloughed off

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Figure 1. Growth, biomass production and naphthoquinone contents of suspension cultures of (a) Dionaea muscipula, (b) Drosera rotundifoliaand (ce) Drosera capensis.

by agitation during the growth cycle. Growth in bothmedia was good (see Figure 1) but biomass produc-tion was considered signicantly superior in Medium5 (p

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Figure 2. Effect of presence or absence of illumination during theculture period (30 days) of New Drosera capensis-6 suspensionculture (Medium 4; n=9; Medium 5; n=10)

biomass between culture of the cells in the presence orabsence of light. Mucin production appeared to be op-timal with cells cultured in Medium 4 in the presenceof light (0.15 g/g, S.D. 0.04, n = 9). A preliminaryexperiment examining the effect on naphthoquinoneproduction, found that there was no significant dif-ference in Medium 5, between cells cultured underlight conditions (1.39%, S.D. 0.31) and those grownin the dark (1.25%, S.D. 0.56.). In all cases cultureof cells for longer than 30 days or at high biomasslevels resulted in blackening of the cells resulting intheir death and decomposition of 1,4-naphthoquinone(7-methyljuglone< 0.1%).

Conclusion

The different nutritional requirements of carnivorousplants and their in vitro cultures are not known. How-ever the presence and concentration in the culturemedium of ammonium nitrogen is known to affectnaphthoquinone formation. In filamentous fungi forexample, substitution of nitrate for ammonium ni-trogen can lead to the inhibition of naphthoquinonebiosynthesis (Medentsev and Akimenko, 1998), whilein suspension cultures of Lithospermum erythrorhizon,high levels of ammonium ions, (as for example inLinsmaier and Skoog medium where the basal saltcomposition is the same as in Murashige and Skoog),repressed the formation of the naphthoquinone pig-ment shikonin (Fukui et al., 1983). This repressioncould be removed by the addition of agaropectin,an acidic polysaccharide fraction of the polygalactanagar.

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