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 suspensions

of Dionaea muscipula and Drosera species
Ingrid L.I. Hook
Department 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 naphthoquinones: 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 only
in 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 contained
7-methyljuglone (0.33%) and differentiated into plants when placed onto hormone-free medium. Pink cultures
grown 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 biomass
production, cells excreted a mucin when cultured in both media (0.21 g dry mucin/g dry cells in McC) and (0.16 g
dry mucin/g dry cells in MS). Effects of the presence or absence of light during the growth period of 30 days showed
that there was no effect on biomass and only slight effects on mucin production and naphthoquinone contents.

Introduction
Increasing interest in the horticultural and medicinal
potential of carnivorous plants has resulted in overharvesting from natural sources. This, together with a
loss of their natural habitats has led to the protection of
many species. The result has been greater research into
their micropropagation and the use of in vitro-grown
plants as alternative sources of biomass.
Venus fly trap (Dionaea muscipula J. Ellis) is a
perennial plant indigenous to bogs in coastal areas of
North and South Carolina, USA, but has also been
introduced into Florida (Culham and Gornall, 1994).
Naturally grown plants contain the naphthoquinones
plumbagin, hydroplumbagin 4-O--glucopyranoside,
droserone, 3-chloroplumbagin (Kreher et al., 1990),
as well as flavonoids, phenol carboxylic acids and enzymes of the digestive glands. There appears to be
no documented evidence of the use of this plant as

a traditional herbal remedy. In contrast, of the more

than 100 species of Sundews (genus Drosera) which
exist, extracts of D. rotundifolia L. and other species
are traditional remedies for use against dry, irritating
coughs (Schilcher and Elzer, 1993). The constituents
of major importance present in the aerial structures are
also various 1,4-naphthoquinones, especially plumbagin (2-methyl-5-hydroxy-1,4-naphthoquinone) and
7-methyljuglone (syn. ramentaceone), in addition to
flavonoids, such as quercetin (Hager, 1973).
Intact plants can be readily cultured by in vitro
propagation techniques (Czany et al., 1992) and on
analysis have been found to produce the same naphthoquinones as naturally grown plants, e.g., with
Drosera spathulata (Budzianowski, 1995; Blehova et
al., 1995), D. rotundifolia (Bobak et al., 1995), D.
intermedia (Budzianowski, 1996) and D. communis
(Reichling et al., 1995). We previously reported on the
development and naphthoquinone content of in vitro

282
cultured plants and suspension cultures of D. capensis
(Hook et al., 1997) and report now on further work
dealing with plants and cultures developed from other
species of the family Droseraceae.

Materials and methods

Plant material and in vitro culture
In vitro cultured plants of Dionaea muscipula J. Ellis and Drosera capensis L. were originally purchased
from Carolina Biological Supply Co. (USA), while
D. rotundifolia was a gift from the University of
Vienna. Plant material of D. binata var. binata was obtained from Trinity College Botanic Gardens, Dublin
and sterilized using sodium hypochlorite solution and
sterile water rinses. Plants were cultivated in the laboratory as outlined in (Hook et al., 1997) and maintained
by periodic subdivision into a liquid or agar-solidified
medium containing Murashige and Skoog basal salts
(Murashige and Skoog, 1962), thiamine HCl (0.4 mg
l1 ), mesoinositol (100 mg l1 ) and sucrose (30 g l1 )
(= Medium 1). Plants grown in liquid culture were
in 150 ml medium in 250 ml conical flasks, maintained under light and agitation conditions as below,
and harvested after 45 months growth.
Suspension cultures
The following media were used in the initiation
and maintenance of cultures: Medium 2: Murashige
and Skoog basal salts, adenine hemisulphate (80 mg
l1 ), 6- - -dimethylallylaminopurine (2 mg l1 ),
thiamine HCl (0.4 mg l1 ), mesoinositol (100 mg
l1 ) and sucrose (30 g l1 ). Medium 3: Gamborgs B5 basal salts (Gamborg et al., 1968), 2,4dichlorophenoxyacetic acid (0.22 mg l1 ), naphthaleneacetic acid (0.18 mg l1 ), glycine (2 mg l1 ),
nicotinic acid (0.5 mg l1 ), pyridoxine HCl (0.5 mg
l1 ), thiamine HCl (0.4 mg l1 ), mesoinositol (100 mg
l1 ) and sucrose (30 g l1 ). Medium 4: McCowns
Woody Plant basal salts (Lloyd and McCown, 1980),
organic constituents as in Medium 3. Medium 5: Murashige and Skoog basal salts, organic constituents as
in Medium 3.
All cultures were agitated on an orbital shaker
set at 90 rpm, maintained at 25 , under cool-white
fluorescent lights delivering 85 mmol m2 s1 on an
18:6-h light : dark cycle. Suspensions were grown as
batch cultures in conical flasks (40% flask-fill). At

monthly intervals cells from one flask were aseptically separated by suction filtration and a known weight
subcultured into fresh medium. Cells from replicate
flasks were harvested by filtration (= fresh wt (g)) and
dried at < 40 (= dry wt (g)). Growth index was calculated from culture fresh wt at harvest inoculum
fresh wt at subculture.
Callus of Dionaea muscipula was originally developed in 1997 from sterile in vitro plants growing
on agar-solidified Medium 3 and was used to initiate
suspension cultures maintained in Media 4 and 5.
Callus of D. rotundifolia originally developed from
sterile in vitro grown plants on agar-solidified Medium
3 in 1996, was used to initiate suspension cultures
maintained in Medium 5.
Callus of D. capensis-5 was originally developed
from sterile in vitro grown plants placed on agarsolidified Medium 2 in 1996. Suspension cultures
were initiated in 1997 and maintained in Media 4
and 5.
Suspension cultures of D. capensis-6 were developed in 1997 from callus originally initiated on an
in vitro plant growing on agar-solidified Medium 4.
Suspension cultures of New D. capensis-6 were
developed 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-phasic
method previously reported (Hook et al., 1997) and
which was found by Krenn et al. (1998) to be comparable to steam distillation as a method of isolation.
Determination of naphthoquinone content was carried out by GC using a WCOT fused silica capillary
column (50 m 0.22 mm i.d.) coated with cyanopropyl (equiv.) polysilphenylene-siloxane (70%) at
210C with the detector and injector at 280C. Using
these 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 a
daily basis by reference to a calibration curve prepared
with pure compounds. 7-Methyljuglone was isolated
and purified from bulked pet. ether extracts of D.
capensis-6 (Hook et al., 1997), while plumbagin was
purchased (Sigma). Each extraction and determination
was replicated (2). Identities of naphthoquinones
was confirmed by GCMS.
Mucin separation
A volume of absolute ethanol equal to the volume of

283
Table 1. Naphthoquinone content of in vitro grown plants
Species

Naphthoquinone Content
Culture medium
(% of dry wt)

Dionaea
muscipula
Drosera
binata
Drosera
binata
Drosera
rotundifolia
Drosera
capensis

Plumbagin

5.3%

Agar-solidified 1

Plumbagin

1.4%

Agar-solidified 1

Plumbagin

2.6%

Liquid-Medium 1

7-Methyljuglone 0.6%

Liquid-Medium 1

7-Methyljuglone 0.5%

Liquid-Medium 1

culture medium was added. The stringy/slimy supernatant scum which immediately formed (= mucin) was
collected by stirring with a glass rod, to which the mucin adhered (Baldwin and Bell, 1955). It was separated
and dried in an oven at < 35 C.
Statistical analyses
Analyses were performed using InStat
statistical
software (Graph Pad, San Diego, CA).

Results and discussion

In vitro grown plants
The naphthoquinone contents of in vitro grown plants
are shown in Table 1. All Drosera species grew well on
both liquid and solid Medium 1, but Dionaea failed to
grow in liquid culture. These results confirm the value
of in vitro culture as a means of propagating large
numbers of carnivorous plants. The naphthoquinone
content of our plants, though appearing higher than
in other references (Wawrosch et al., 1996), could be
related to the changed extraction protocol (Hook et al.,
1997; Krenn et al., 1998). All the naphthoquinones
present as major metabolites were as in the native
intact plants (Culham and Gornall, 1994).
Suspension cultures
Suspension cultures of Dionaea muscipula grew as
dark-pink aggregates. Although originally maintained
in both Medium 4 and 5, cells grown in Medium 5 lost
viability after a few months. Only results relating to

culture in Medium 4 are presented (Figure 1). Growth

was slow (growth index 3.85 , S.D. 1.49, n = 44) and
cells were found to produce only plumbagin as naphthoquinone (2.59%, S.D. 0.37, n = 11), the same as the
parent plants (Culham and Gornall, 1994).
Suspension cultures of Drosera rotundifolia failed
to grow on Medium 4. They were originally developed
from callus initiated on plants growing on Medium 5
and were maintained in this medium, growing slowly
(growth index 3.56, S.D. 0.95, n = 20) as multicoloured aggregates (Figure 1). Although native D. rotundifolia plants are supposed to contain both plumbagin
and 7-methyljuglone (Culham and Gornall, 1994) and
the parent plants of these cultures were found to
produce up to 0.6% of 7-methyljuglone, suspension
cultures were devoid of quantifiable naphthoquinones.
Suspension cultures of D. capensis were originally developed in 1995 and were found to produce
7-methyljuglone, ca. 0.9%, depending on the formulation of the culture medium (Hook et al., 1997). This
cell line was also found to excrete a mucin into the
medium (Hook and Paper, 1996), a capacity which
was subsequently lost. A series of new cell lines were
therefore initiated from in vitro cultured plants. Cell
line-5 grew as green aggregates in Medium 5 (growth
index, 3.83, S.D. 0.62, n = 22) and grey aggregates
in Medium 4 (growth index, 2.67, S.D. 0.57, n = 15)
(Figure 1). Medium 5 proved to be statistically significantly better as a culture medium with regard to
growth, biomass and naphthoquinone production. 7methyljuglone 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 aggregates
were placed onto agar-solidified or liquid Medium 1,
they eventually differentiated into plantlets containing
0.80% of 7-methyljuglone. This cell line proved to be
a rapid source of large numbers of D. capensis plants.
Cultures designated D. capensis-6 were also initiated and grew as salmon-pink aggregates. Growth was
best in Medium 4 (growth index, 4.90, S.D. 1.57, n =
26, Biomass 9.72, S.D. 2.71, n = 22) and contents of
7-methyljuglone were high (1.24%, S.D. 0.34, n = 10).
Mucin was initially excreted into the culture medium
and chemical analysis found it to be an acidic polysaccharide rich in galactose units (Hook and Paper, 1996).
This secretory capacity was again lost, and led to the
selection of New D. capensis-6 from a flask showing
a highly viscous medium.
This cell line was maintained in both Medium 4
and 5 where growth was as dark red aggregates, although single pink-coloured cells were sloughed off

284

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

by agitation during the growth cycle. Growth in both

media was good (see Figure 1) but biomass production was considered signicantly superior in Medium
5 (p<0.0005). There was no significant difference in
the 7-methyljuglone content of the cells cultured in the
two media, both producing ca. 1% of dry cell weight.
Mucin production (calculated as dry mucin weight

per g dry cell weight) was found to be significantly

better with culture in Medium 4 (0.21 g/g, S.D. 0.13,
n = 47), compared to Medium 5 (0.16 g/g, S.D. 0.08, n
= 61). Results from experiments comparing the effect
on mucin production of the absence of light during the
culture period are shown in Figure 2. These showed
that there was no significant difference with regard to

285
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Figure 2. Effect of presence or absence of illumination during the

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Conclusion
The different nutritional requirements of carnivorous
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