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Plant Cell, Tissue and OrganCulture (PCTOC)Journal of Plant Biotechnology ISSN 0167-6857Volume 103Number 3 Plant Cell Tiss Organ Cult(2010) 103:285-292DOI 10.1007/s11240-010-9778-5

Teratomas of Drosera capensis var.alba as a source of naphthoquinone:ramentaceone

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ORIGINAL PAPER

Teratomas of Drosera capensis var. alba as a sourceof naphthoquinone: ramentaceone

Aleksandra Krolicka • Anna Szpitter • Krzysztof Stawujak •

Rafal Baranski • Anna Gwizdek-Wisniewska •

Anita Skrzypczak • Marian Kaminski • Ewa Lojkowska

Received: 19 January 2010 / Accepted: 1 June 2010 / Published online: 17 June 2010

� Springer Science+Business Media B.V. 2010

Abstract Plants belonging to genus Drosera (family

Droseraceae) contain pharmacologically active naphtho-

quinones such as ramentaceone and plumbagin. Hairy root

cultures obtained following Agrobacterium rhizogenes-

mediated transformation have been reported to produce

elevated levels of secondary compounds as well as exhibit

desirable rapid biomass accumulation in comparison to

untransformed plants. The aim of this study was to estab-

lish hairy root or teratoma cultures of Drosera capensis

var. alba and to increase the level of ramentaceone in

transformed tissue by application of abiotic and biotic

elicitors. The appearance of transformed tissues—terato-

mas but not hairy roots was observed 18 weeks after

transformation. The transformation efficiency was 10% and

all teratoma cultures displayed about 3 times higher growth

rate than non-transformed cultures of D. capesis. The

transformation was confirmed by PCR and Southern

hybridization using primers based on the A. rhizogenes

rolB and rolC gene sequences. HPLC analysis of ramen-

taceone content indicated 60% higher level of this metab-

olite in teratoma tissue in comparison to non-transformed

cultures. Among the elicitors tested jasmonic acid

(2.5 mg l-1) turned out to be the most effective. The pro-

ductivity of ramentaceone in elicited teratoma cultures was

about sevenfold higher than in liquid cultures of D. cap-

ensis var. alba and amounted to 2.264 and 0.321 mg

respectively during 4 weeks of cultivation. This is the first

report on the transformation of Drosera plant with

A. rhizogenes.

Keywords Agrobacterium rhizogenes �Naphthoquinone � Transformation �Drosera capensis var. alba

Abbreviations

CTAB Hexadecyltrimethylammoniumbromide

DW Dry weight

FW Fresh weight

JA Jasmonic acid

MBC Minimal Bactericidal Concentration

MIC Minimal Inhibitory Concentration

Introduction

Drosera capensis var. alba (Droseraceae) commonly

known as the Cape sundew (Fig. 1) is a carnivorous plant

native to the Cape in South Africa. The plants of Drosera

genus are a natural source of pharmacologically important

compounds (e.g. naphthoquinones, flavonoid glucosides,

flavonoids, phenolic acids) used for the production of

A. Krolicka (&) � A. Szpitter � A. Gwizdek-Wisniewska �E. Lojkowska

Department of Biotechnology, Laboratory of Plant Protection

and Biotechnology, Intercollegiate Faculty of Biotechnology,

University of Gdansk and Medical University of Gdansk, Kladki

24, 80-822 Gdansk, Poland

e-mail: krolicka@biotech.ug.gda.pl

K. Stawujak � R. Baranski

Department of Genetics, Plant Breeding and Seed Science,

University of Agriculture in Krakow, Al. 29 Listopada 54,

31-425 Krakow, Poland

A. Skrzypczak � M. Kaminski

Department of Analytical Chemistry, Gdansk University

of Technology, Gabriela Narutowicza 11/12, 80-952 Gdansk,

Poland

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DOI 10.1007/s11240-010-9778-5

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pharmaceuticals because of their interesting biological

activities like antimicrobial, antimycobacterial, antifungal

or anticancer (Caniato et al. 1989; Juniper et al. 1989;

Finnie and van Staden 1993). Ramentaceone (5-hydroxy-7-

methyl-1,4-naphthoquinone)—an isomer of plumbagin is

reported as the major quinone in D. capensis var. alba

(Juniper et al. 1989). It was proven that ramentaceone

exhibits antimicrobial activity towards human bacterial

pathogens (Krolicka et al. 2009) and cytotoxic activity

against tumor cell lines (Kawiak et al. 2006). Because all

plants from the Droseraceae family belong to endangered

species, tissue cultures seem to be a good source of plant

material. Hairy roots obtained after transformation of plant

tissue with Agrobacterium rhizogenes are considered as

fast growing cultures rich in secondary metabolites. Cul-

tures of genetically transformed tissue (hairy roots or ter-

atomas) are an efficient source of species and tissue

specific secondary metabolites (Giri and Narasu 2000). It is

worth underlining that the growth of shooty teratoma as

well as hairy root tissue cultures is independent of media

supplementation with plant growth regulators (Mahagam-

asekera and Doran 1998). Studies of Subroto et al. (1996)

and Saito et al. (1989) have shown that the range of sec-

ondary metabolites synthesized by shooty teratomas is

generally the same as those produced by non-transformed

shoots. The commonly used method of increasing pro-

duction of pharmacologically valuable secondary metabo-

lites in plant in vitro cultures is the application of abiotic

and biotic elicitors (Nahalka et al. 1996; Hook 2001;

Komaraiah et al. 2002). An earlier study indicated that

jasmonic acid is the most efficient elicitor for naphtho-

quinone production in cultures of Droseraceae plants

(Krolicka et al. 2008).

The goal of the presented study was to establish hairy

root or teratoma cultures of D. capensis var. alba and to

stimulate ramentaceone production in transformed tissue

by application of elicitors.

Materials and methods

Plant material and bacterial strains

D. capensis var. alba plantlets were obtained from the

Botanical Garden of Wroclaw, Poland. The optimal con-

ditions for micropropagation of carnivorous plants were

developed: liquid � MS medium (Murashige and Skoog

1962) with 3 g l-1 sucrose. The pH of the media was

adjusted to 5.6 prior to autoclaving. Experiments were

carried out in a 250 ml Erlenmeyer flasks containing 35 ml

of � MS medium. D. capensis plantlets were grown at a

temperature of 20 ± 2�C under white fluorescent light with

a 16 h photoperiod (White cool fluorescent light, Philips,

TLD 58 W/84o, 30–35 lmol m-2 s-1) on a rotary shaker

at 110 rpm, amplitude 9 for 4 weeks.

A. rhizogenes agropine strains: LBA 9402 (NCPPB

1855), A4 (ATCC 31798) [obtained from the Botanical

Garden of Wroclaw, Poland] and ATCC 15834 [obtained

from the Laboratoire Agronomie et Environnement, Ecole

Nationale Superieure d’Agronomie et des Industries Ali-

mentaires, Nancy, France] were grown on YEB agar

medium (Miller 1972) supplemented with 200 lM of

acetosyringone (Sigma), at 26�C in the dark on a gyratory

shaker at 260 rpm. For transformation 24 h old bacterial

cultures were used (OD600 0.6).

Establishment of teratoma cultures

About 500 explants of D. capensis (leaves and stems) were

inoculated with different A. rhizogenes strains using two

methods of inoculation: with preparative needle or soni-

cation of explants for 3 s with bacterial suspension—OD600

0.6. After inoculation explants were transferred to the �MS media supplemented with 7.5 g l-1 agar and 30 g l-1

sucrose. On the 3th day of culture in the darkness the

explants were transferred to fresh media (� MS, 7.5 g l-1

Fig. 1 Growth of teratomas Drosera capensis var. alba a. on � MS

medium ? 500 mg l-1 Claforan and 500 mg l-1 Carbenicillin in

darkness 10 weeks after transformation b. plants regenerated from

teratomas 30 weeks after transformation on � MS liquid medium in

photoperiod (16/8; day/night); c non-transformed D. capensis var.

alba in vitro plants on � MS liquid medium in photoperiod (16/8;

day/night)

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agar, 30 g l-1 sucrose) supplemented with claforan

(Hoechst, M. Roussel) and carbenicillin (Polfa, Tarchomin)

in a concentration of 500 mg l-1 each in order to eliminate

A. rhizogenes. After 4 weeks of growth in the darkness the

appearing transformed shoots were excised, transferred to

the same medium with antibiotics and cultured for 8 weeks

in the darkness. Axenic cultures derived from single shoots

were established after 3–5 subcultures in a 250 ml Erlen-

meyer flask containing 35 ml of � MS medium with

claforan and carbenicillin without plant growth regulators.

Transformed tissue was passaged from eight to ten times

on media containing antibiotics. In order to test for the

presence of A. rhizogenes in teratomas plant tissues were

homogenised and the obtained suspensions were plated on

YEB agar and Luria agar media. Plates were incubated for

several days at 26�C.

Teratoma cultures free from bacteria were maintained in

a 250 ml flasks containing 35 ml of a liquid � MS medium

without antibiotics in a 16 h photoperiod at a temperature

of 20 ± 2�C, on a rotary shaker at 110 rpm, amplitude 9.

Subcultures were made every 4 weeks.

PCR and Southern hybridization

In order to confirm the transformation of D. capensis var.

alba on the molecular level, the presence of the T-DNA

fragment in teratoma tissue was determined by the use of

PCR and Southern hybridization. Due to the presence of

secondary metabolites in plant tissue DNA from untrans-

formed and transformed D. capensis was isolated using a

method with CTAB with some modifications (Bekesiova

et al. 1999). As a positive control plasmid DNA isolated

from A. rhizogenes cells was used. In this case DNA was

extracted from 24 h cultures (OD600 4.0) using alkaline

lysis (Maniatis et al. 1982). Oligonucleotide primers and

procedure described by Krolicka et al. (2001) were used for

PCR detection of the sequence homologous to bacterial

rolB and rolC genes (present in T-DNA) in the plant

genome. In order to confirm that teratomas are free of

A. rhizogenes cells the PCR with primers homologous to

the sequence of virG gene (gene present in Ri plasmid but

beyond the transferred T-DNA) was performed according

the procedure described by Sidwa-Gorycka et al. (2009).

Southern blot hybridization was performed essentially as

described earlier (Baranski et al. 2008). For this purpose,

EcoRI digested DNA was separated in agarose gel, trans-

ferred to a nylon membrane by overnight capillary transfer

and hybridized to DIG-dUTP labelled probes, which were

obtained in PCR using the primers specific to rol genes.

Detection of hybridized DNA fragments was performed

using DIG Luminescent Detection Kit (Roche Applied

Sci.) according to the manufacturer instruction.

Antibacterial activity determination

Minimal Bactericidal Concentration—MBC (Thornsberry

1991) was determined in order to check the antibacterial

activity of plant extracts against A. rhizogenes. The bac-

tericidal activity of plant extracts was compared with those

of ramentaceone (obtained from University of Pretoria,

Republic of South Africa).

Three agropine type strains of A. rhizogenes (stored in

Faculty of Biotechnology, UG & MUG, Poland) were used:

LBA 9402, A4 and ATCC 15834. All bacteria were grown

overnight on YEB agar medium at 26�C.

Chloroform extract obtained from 1 g FW (in a range of

150–500 lg ml-1 of ramentaceone) was dried and resus-

pended in MeOH before application in the wells of a

96-well plate. Then the solvent was evaporated since

MeOH is toxic for bacteria and might influence the results.

The residue was suspended in 100 ll YEB liquid medium

and aliquots of 100 ll of the bacterial suspension (106

cfu ml-1) in YEB liquid medium were added into wells.

The plates were incubated overnight at 26�C. In order to

establish the MBC value, 100 ll of the content of each well

that showed no visible growth of bacteria was plated out on

an agar plate, spread evenly with a sterile bent glass rod

and incubated for 24 h at 26�C. A similar procedure was

applied when the antibacterial activity of purified ramen-

taceone (ranges of concentration 5–250 lg ml-1) was

tested. The MBC was defined as the lowest concentration

of the compound that reduced the inoculum by 99.9%

within 24 h (Thornsberry 1991). All experiments were

performed in triplicate.

Elicitation of secondary metabolite using abiotic

and biotic elicitors

For elicitation of secondary metabolites 2.5 mg l-1 JA

(Sigma) as elicitor was added to � MS medium before

planting 4–6 week old cultures of teratomas. In the next

experiment � MS medium was modified by reducing the

amount of KNO3 and NH4NO3 [N(-) medium - � MS

with � KNO3 and without NH4NO3] (Krolicka et al. 2008).

As biotic elicitors either an autoclaved overnight sus-

pension (McFarland 3.6) of Pseudomonas aeruginosa K337

(ML 5087) kindly provided by Dr K. Poole, Queen’s Uni-

versity, Kingston, Canada or chitosan (Sigma) were added to

� MS medium to the final concentration of 2.5% (v/v) and

1 g/l, respectively. Cultures of Ps. aeruginosa were grown

in LB medium (Sambrook et al. 1989) at 37�C for 24 h. The

suspension of bacteria was treated with toluene (100:1) and

autoclaved (30 min, 1 atm). Before autoclaving samples

were left for 1 h for toluene evaporation.

The 4-week-old cultures of teratomas of either elicited

or control plants were collected and the extraction of

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secondary metabolites was performed using sonication

with chloroform as solvent.

Qualitative and quantitative analysis of secondary

metabolites

The accumulation of naphthoquinone in extracts from D.

capensis tissue was determined by using high performance

liquid chromatography (HPLC) technique. A LaChrom

(Merck-Hitachi) gradient liquid chromatograph with

UV-DAD detector was used in the investigations.

Dihydroksypropyl stationary phases, hexane and tetrahy-

drofuran (Merck, Darmstadt, Germany) mixture as eluents

and gradient elution were used as was previously described

by Krolicka et al. (2008). Each measurement was repeated

three times (for each HPLC sample) and the results are the

averages of at least three values differing by no more than

5% relative.

Statistical analysis

The stepwise regression method (backward removal

method) has been applied to establish variables signifi-

cantly influencing the productivity of ramentaceone. The

initial linear model contained all parameters: clones and

elicitors added during the growth of plants of D. capensis

var. alba that may influence the productivity. The inde-

pendent variables assume values 0 or 1 depending on the

absence or presence (at the given concentration) during the

growth of plant culture. The initial model:

Productivity of ramentaceone ¼ a � clone 1þ b � clone 2

þ c � JAþ d � Chþ e � Ps:aþ f � Nþ g

where a, b, c, d, e, f, g—searched parameters in regression

model clone 1 and clone 2 are respectively clone of

D. capensis var. alba teratomas C2/2 and C21/25 JA—

presence (1) or absence (0) of jasmonic acid; Ch—presence

(1) or absence (0) of chitosan Ps.a—presence (1) or

absence (0) of Ps. aeruginosa K337; N—presence (0) or

absence (1) of standard N salt level.

The statistically significant regression model obtained

after applying the backward removal method includes only

the statistically significant (at a = 0.05) independent

variables for clone C2/2, clone C21/25 and JA treatment.

The software STATISTICAf 8.0 (StatSoft Inc) has been

used.

Results and discussion

Teratomas (transformed shoots) were obtained 18 weeks

after transformation but only after inoculation of D. capensis

var. alba using A. rhizogenes ATCC 15834 strain. The

transformed shoots were obtained only when explants

(stems and leaves) were scarified with the needle contain-

ing A. rhizogenes, which was grown on media supple-

mented with acetosyringone and later cocultured on a solid

� MS medium. No hairy roots characteristic for plants

transformed with A. rhizogenes were observed. After

5 weeks of culture first callus appeared in the scarified

explants and later on multiple long and thin shoots grew

from it (Fig. 1a). These were subsequently cut off, trans-

ferred to the liquid � MS medium and cultured in the dark.

After about 13 weeks culture transformed shoots were

subcultured in photoperiod (16/8; day/night) and later on

fully developed plants with formed roots were observed

(Fig. 1b).

In order to confirm transformation process the presence

of fragments of T-DNA from A. rhizogenes in teratoma

cells was determined using PCR method. Application of

primers for rolB and rolC genes allowed for amplification

of PCR product when DNA isolated from teratoma cultures

were used as a template (Fig. 2a). In order to confirm that

PCR products are complementary to the rolB and rolC

genes from A. rhizogenes they were sequenced and com-

pared with NCBI database with the use of BLAST

(www.ncbi.nlm.nih.gov/BLAST/). Sequence aligment

allowed to confirm 100% similarity between obtained

products and respective sequences of rolB and rolC genes

deposited in the database. In order to confirm that amplified

products do not come from A. rhizogenes cells contami-

nating teratoma tissues, a PCR reaction with primers for

the virG virulence gene, which is present on Ri plasmid

beyond the transferred T-DNA, was performed (Aoyama

et al. 1989). It was demonstrated that sequences

Fig. 2 a PCR analysis of Agrobacterium rhizogenes ATCC 15834

[lanes 1–3] and Drosea capensis var. alba teratomas transformed by

A. rhizogenes ATCC 15834 [lanes 4–6 (clone 1—C2/2); lanes 7–9(clone 2—C21/25)] and untransformed D. capensis var. alba [lanes10–12]. GeneRulerTM 100 bp DNA ladder (lane M). Arrows show

amplified fragments of rolB (423 bp; lanes 1, 4, 7, 10), rolC (626 bp;

lanes 2, 5, 8, 11) virG (273 bp; lanes 3, 6, 9, 12) genes. b Southern

hybridization with rolC probe: pRi15834 plasmid DNA (lane P),

untransformed control (lane C), clone 1 C2/2 [lane 1], clone 2—C21/

25 [lane 2] GeneRulerTM 1 kb DNA ladder [lane L]

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homologous to the virG gene (273 bp) were not amplified

when DNA isolated from transformed tissue (teratomas) of

D. capensis var. alba were used as a template (Fig. 2a).

Integration of the rol genes to D. capensis var. alba gen-

ome was also confirmed by Southern blot hybridization

(Fig. 2b). The hybridization signals were detected for DNA

isolated from transformed teratoma tissues as well as for

plasmid DNA used as a positive control. No signal was

obtained for untransformed control.

The PCR analysis confirmed that the transformation

process was successful in 10% of both explants used (stems

and leaves) but only when inoculation with A. rhizoge-

nes—agropine strain 15834 was performed. Also the work

of Porter (1991) indicated that A. rhizogenes strain 15834 is

the most effective and allowed for transformation of a

broad spectrum of plant species. It was proven that agro-

pine strains are characterized by a high virulence due to the

presence of two separate T-DNA segments in the plasmid:

the left one (TL-DNA) and the right one (TR-DNA) (de

Paolis et al. 1985). All successful transformations of

D. capensis var. alba were obtained only when A. rhizog-

enes was cultured on medium with acetosyringone.

Acetosyringone also improved the effectiveness of trans-

formation in the case of Atropa belladonna (Yan-Nong

et al. 1990) and Alhagi pseudoalhagi (Wang et al. 2001).

To our knowledge so far there are no reports in literature

concerning transformation of carnivorous plants using

A. rhizogenes. Hirsikorpi et al. (2002) described transfor-

mation of Drosera rotundifolia by the use of modified

Agrobacterium tumefaciens strain with about 17% of effi-

ciency. The described protocol was not successful for

D. capensis var. alba transformation due to the fact that the

regeneration of this species is sensitive to 6-benzylamino-

purine and naphthaleneacetic acid which were recom-

mended by Hirsikorpi et al. (2002). Difficulties in

obtaining hairy root cultures from carnivorous plants might

results from the fact that not all plant species are equally

susceptible to transformation caused by Agrobacterium

strains (Porter 1991). Moreover it was shown that plants

belonging to the Droseraceae family produce secondary

metabolites having strong antibacterial activity against

Enterococcus faecalis, Staphylococcus aureus, Klebsiella

pneumoniae, Escherichia coli and Pseudomonas aerugin-

osa (Krolicka et al. 2008, 2009). Application of the broth

macrodilution method demonstrated that 150 lg ml-1

ramentaceone is the bactericidal concentration for A. rhiz-

ogenes (MBC). In case of chloroform extract of D. cap-

ensis var. alba tissues 3.6 lg DW of extract/ml-1 inhibited

A. rhizogenes growth (MIC value), while MBC was 4.8 lg

DW ml-1. The obtained results show that difficulties in

obtaining transformed tissues of D. capensis var. alba may

be connected with the antimicrobial activity of naphtho-

quinone present in their tissues.

Hairy roots produced by transformation with A. rhiz-

ogenes can, in some species, spontaneously regenerate to

whole plants (Tepfer 1984). In such a case regenerated

plants sometimes exhibit characteristic morphologic traits

such as wrinkled leaves or reduced apical dominance (hairy

root syndrome) (Tepfer 1990). In the case of D. capensis

var. alba the obtained teratomas show no such disadvan-

tageous characteristics. D. capensis var. alba plants

obtained 30 weeks after transformation from teratoma tis-

sue did not differ morphologically from non-transformed

tissue (Fig. 1b, c). The regeneration of shoots from single

hairy roots was also spontaneous in the case of Nicotiana

tabacum and Convolvulus arvensis while being induced by

somatic embryogenesis in hairy root of Daucus carota

(Tepfer 1984). It was suggested that the physiological

status of a transformed tissue is dependent on number of

copies, place of incorporation of T-DNA and specificity of

transformed explant. The result of N. tabacum leaves

transformation was the appearance of multiple small leaves

at the site of injury, whereas inoculating the stem tissue

resulted in the growth of typical hairy roots (Tepfer 1984).

The HPLC analysis of ramentaceone content in D. cap-

ensis var. alba showed a 26–60% increase in the level of

this naphthoquinone in the obtained teratoma tissues in

comparison to non-transformed cultures (Table 1). An

increased level of secondary metabolites in hairy roots of

Platycodon grandiflorum (Ahn et al. 1996), Ocimum ba-

silicum (Tada et al. 1996), Atropa belladonna (Bonhomme

et al. 2000), Panax ginseng (Mallol et al. 2001) and Ammi

majus (Krolicka et al. 2001) in comparison to non-trans-

formed roots were also observed. In addition there are

several reports on higher concentration of secondary

metabolites in plants regenerated from hairy roots in

comparison to non-transformed tissue in Ajuga reptans

(Tanaka and Matsumoto 1993), Pelargonium spp. (Pelle-

grineschi et al. 1994), Vinca minor (Tanaka et al. 1995).

In order to obtain higher level of ramentaceone in

D. capensis var. alba teratoma cultures, clones C2/2 and

C21/25 were treated with elicitors (two biotic and two abi-

otic). The presence of elicitors in the medium in most of the

cases caused a moderate increase in the level of ramentace-

one per g of DW (Table 1). Additionally, the presence of JA

in the medium resulted in the highest growth factor (t30/t1) in

teratoma cultures (clone C21/25) and positively influenced

productivity of ramentaceone which amounted to 2.267 and

0.321 mg in teratomas compared to non-transformed cul-

tures of D. capensis var. alba, respectively, during 4 weeks

per 4 flasks (Fig. 3). JA and its derivatives play an integral

role in the cascade of events that occur in the elicitation

process causing activation of the genes of secondary

metabolism (Gundlach et al. 1992). Earlier research showed

that elicitation with JA increases the level of naphthoqui-

nones—ramentaceone and plumbagin in tissue cultures of D.

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capensis ‘Broadleaf’ and Dionaea muscipula respectively

(Krolicka et al. 2008). JA was also an effective as an elicitor

of the production of tropane alkaloids in hairy roots of

Brugmansia candida (Spollansky et al. 2000). Based on

stepwise regression analysis the transformation is the major

factor increasing productivity (mg ramentaceone/4 weeks/4

flasks) of this naphthoquinone in D. capensis var. alba.

Productivity of ramentaceone = 897 (±97)�clone C2/2 ?

1310 (±97)�clone C21/25 ? 316 (±99)�JA ? 374 (±71) ±

217 [N = 30, F (3, 26) = 66.8 (P \ 0.05), R = 0.94,

R2 = 0.88].

The application of other elicitors did not increase the

productivity of ramentaceone in teratoma cultures (Fig. 3).

Depletion of nitrogen content in culture media caused

elevation in the level of ramentaceone in D. capensis var.

alba teratomas culture in comparison to the non-elicited

control (Table 1) but the productivity was not higher than

in non-treated teratomas (Fig. 3). The elicitation of another

naphthoquinone: shikonin in cultures of Lithospermum

erythrorizon (Mizukami et al. 1977) and plumbagin in

cultures of Drosopyllum lusitanicum (Nahalka et al. 1996)

by depletion of nitrogen in the culture medium has been

reported.

Addition of chitosan and Ps. aeruginosa K337 lysate to

teratoma culture of D. capensis var. alba had either nega-

tive (clone 2/2) or neutral (clone 21/25) effect on the

productivity of ramentaceone (Fig. 3). Chitosan turned out

to be an effective elicitor of naphthoquinones in cell sus-

pension of Plumbago rosea L (Komaraiah et al. 2002).

Jung et al. (2003) used either fresh or autoclaved suspen-

sion of Staphylococcus aureus, Bacillus cereus and

Ps. aeruginosa in order to increase the production of sco-

polamine in hairy roots of Scopolia parviflora. Only the

preparations containing living bacteria had activity as

elicitor but at the same time they exerted a strong effect on

the growth and vitality of the culture (Jung et al. 2003).

Conclusion

In the presented work we report for the first time a suc-

cessful transformation of a carnivorous plant—D. capensis

var. alba with A. rhizogenes. Due to 60% higher level of

ramentaceone per g of DW and about three times higher

growth rate of teratoma cultures in comparison to non-

transformed plants the productivity of this naphthoquinone

in JA elicited culture was about seven times higher in

comparison to non-transformed tissue.

Table 1 Comparison of growth index and content of ramentaceone in non-transformed tissue and teratomas of D. capensis var. alba

Elicitors Non-transformed tissue Teratoma culture

Clone C2/2 Clone C21/25

Growth

index

(t30/t1)a

Content

of ramentaceone

lg/g DW

Growth

index

(t30/t1)a

Content

of ramentaceone

lg/g DW

Growth index

(t30/t1)aContent of

ramentaceone

lg/g DW

Non-elicited 1.3 247.0 ± 5.6 4.1 310.0 ± 2.8 4.8 410.0 ± 11.3

JA 1.6 281.0 ± 4.2 4.9 322.0 ± 5.6 5.1 444.0 ± 8.4

Chitosan 2.0 290.0 ± 5.6 4.3 341.0 ± 7.1 3.9 353.0 ± 4.2

Ps. aeruginosa 1.8 235.0 ± 7.1 3.7 340.0 ± 4.2 4.8 294.0 ± 5.6

N(-) 1.5 274.5 ± 6.3 2.9 385.0 ± 8.4 4.0 428.0 ± 9.9

Cultures were grown 30 days on � MS medium under white fluorescent light (16/8 photoperiod) at temperature 20–22�C, on a rotating (orbital)

shaker—110 rpm, amplitude 9. Cultures were elicited with 2.5 mg/l jasmonic acid [JA], 1% chitosan [chitosan], 2.5% culture lysate from Ps.aeruginosa K337 (ML 5087) [Ps. aeruginosa] or nitrogen deficiency by culture on � MS media with reduced amount of nitrogen salts [N(-)]a The growth index was established by a ratio of fresh weight of 30 days old cultures and initial weight of cultures

Fig. 3 Comparison of productivity of ramentaceone in transformed

and untransformed tissue of Drosera capensis var. alba. The plants

were grown on � MS medium [control] or subjected to treatment

with elicitors. For explanations see Table 1

290 Plant Cell Tiss Organ Cult (2010) 103:285–292

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Acknowledgments This work was supported by DS/0051-4-0010-9

and the Foundation for Polish Science grant START for Anna

Szpitter.

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