Teratomas of Drosera capensis var. alba as a source of naphthoquinone: ramentaceone
Plant Cell, Tissue and Organ Culture (PCTOC) Journal of Plant Biotechnology ISSN 0167-6857 Volume 103 Number 3 Plant Cell Tiss Organ Cult (2010) 103:285-292 DOI 10.1007/ s11240-010-9778-5
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Author's personal copy Plant Cell Tiss Organ Cult (2010) 103:285–292 DOI 10.1007/s11240-010-9778-5
ORIGINAL PAPER
Teratomas of Drosera capensis var. alba as a source of naphthoquinone: ramentaceone Aleksandra Krolicka • Anna Szpitter • Krzysztof Stawujak Rafal Baranski • Anna Gwizdek-Wisniewska • Anita Skrzypczak • Marian Kaminski • Ewa Lojkowska
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Received: 19 January 2010 / Accepted: 1 June 2010 / Published online: 17 June 2010 Ó Springer Science+Business Media B.V. 2010
Abstract Plants belonging to genus Drosera (family Droseraceae) contain pharmacologically active naphthoquinones such as ramentaceone and plumbagin. Hairy root cultures obtained following Agrobacterium rhizogenesmediated 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 establish 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—teratomas 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
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:
[email protected] 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
rolB and rolC gene sequences. HPLC analysis of ramentaceone content indicated 60% higher level of this metabolite 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 productivity of ramentaceone in elicited teratoma cultures was about sevenfold higher than in liquid cultures of D. capensis 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
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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)
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-7methyl-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. Cultures of genetically transformed tissue (hairy roots or teratomas) 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 (Mahagamasekera and Doran 1998). Studies of Subroto et al. (1996) and Saito et al. (1989) have shown that the range of secondary metabolites synthesized by shooty teratomas is generally the same as those produced by non-transformed shoots. The commonly used method of increasing production of pharmacologically valuable secondary metabolites in plant in vitro cultures is the application of abiotic and biotic elicitors (Naha´lka et al. 1996; Hook 2001; Komaraiah et al. 2002). An earlier study indicated that jasmonic acid is the most efficient elicitor for naphthoquinone 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
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Plant material and bacterial strains D. capensis var. alba plantlets were obtained from the Botanical Garden of Wroclaw, Poland. The optimal conditions 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 Alimentaires, 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 sonication 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
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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 Erlenmeyer 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 untransformed 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, transferred 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.
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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 bactericidal 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 resuspended 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 ramentaceone (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 suspension (McFarland 3.6) of Pseudomonas aeruginosa K337 (ML 5087) kindly provided by Dr K. Poole, Queen’s University, 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 tetrahydrofuran (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 significantly 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 independent 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 1 þ b clone 2 þ c JA þ d Ch þ e Ps:a þ f N þ 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
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var. alba using A. rhizogenes ATCC 15834 strain. The transformed shoots were obtained only when explants (stems and leaves) were scarified with the needle containing A. rhizogenes, which was grown on media supplemented 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, transferred 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 compared 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 contaminating 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 [lanes 10–12]. 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 genome 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. rhizogenes—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 agropine 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. rhizogenes was cultured on medium with acetosyringone. Acetosyringone also improved the effectiveness of transformation 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 transformation of Drosera rotundifolia by the use of modified Agrobacterium tumefaciens strain with about 17% of efficiency. 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-benzylaminopurine and naphthaleneacetic acid which were recommended 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 aeruginosa (Krolicka et al. 2008, 2009). Application of the broth macrodilution method demonstrated that 150 lg ml-1 ramentaceone is the bactericidal concentration for A. rhizogenes (MBC). In case of chloroform extract of D. capensis 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 naphthoquinone present in their tissues.
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Hairy roots produced by transformation with A. rhizogenes 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 disadvantageous characteristics. D. capensis var. alba plants obtained 30 weeks after transformation from teratoma tissue 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. capensis 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 basilicum (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-transformed 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. (Pellegrineschi 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 abiotic). The presence of elicitors in the medium in most of the cases caused a moderate increase in the level of ramentaceone 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 cultures 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 naphthoquinones—ramentaceone and plumbagin in tissue cultures of D.
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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
Growth index (t30/t1)a
Content of ramentaceone lg/g DW
Growth index (t30/t1)a
Clone C21/25 Content of ramentaceone lg/g DW
Growth index (t30/t1)a
Content 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
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.
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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 (Naha´lka 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 negative (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 suspension of Plumbago rosea L (Komaraiah et al. 2002). Jung et al. (2003) used either fresh or autoclaved suspension of Staphylococcus aureus, Bacillus cereus and Ps. aeruginosa in order to increase the production of scopolamine 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 successful 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 nontransformed plants the productivity of this naphthoquinone in JA elicited culture was about seven times higher in comparison to non-transformed tissue.
Author's personal copy Plant Cell Tiss Organ Cult (2010) 103:285–292 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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