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Agrobacterium-mediated transformation of round leaved sundew (Drosera rotundifolia L.)
Plant Science 162 (2002) 537 /542 www.elsevier.com/locate/plantsci
Agrobacterium-mediated transformation of round leaved sundew (Drosera rotundifolia L.) Merja Hirsikorpi a, Terttu Ka¨ma¨ra¨inen a, Teemu Teeri b, Anja Hohtola a,* a b
Department of Biology/Botany, University of Oulu, PO Box 3000, FIN-90014 Oulu, Finland Institute of Biotechnology, University of Helsinki, PO Box 56, FIN-00014 Helsinki, Finland
Received 14 May 2001; received in revised form 13 November 2001; accepted 3 December 2001
Abstract Agrobacterium tumefaciens -mediated genetic transformation method of the carnivorous medicinal plant round leaved sundew (Drosera rotundifolia L.) was developed. The micropropagation conditions of sundew aseptically germinated seeds were defined and the internal kanamycin resistance of sundew was tested. Transformation was made by cocultivation of micropropagated sundew leaves with A. tumefaciens strain C58C1 containing a cointegrate plasmid vector with neomycin phosphotransferase and luciferase genes. Transgenic sundews were selected for kanamycin resistance and viable, fully developed plantlets were further assayed by luciferase activity and by PCR and Southern analyses using luc -primers and a luc -probe. The transformation efficiency of D. rotundifolia L. was 17%. Sundew is an important source of pharmacologically active 1,4-naphthoquinones, and genetic transformation allows the engineering of its biochemical pathways. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Droseraceae ; Agrobacterium ; Luciferase; Genetic transformation; Naphthoquinones; Micropropagation
1. Introduction Drosera rotundifolia L. is a carnivorous perennial herb with a basal rosette of leaves. The upper surfaces of the leaf blades are covered with reddish, glandular hairs tipped with a sticky secretion that traps insects. Sundew compensates for the low availability of nutrients in its habitat by digesting caught insects with proteolytic enzymes. Sundew is a rich source of 1,4-naphthoquinones, the main ones being plumbagin (5-hydroxy-2-methyl-1,4naphthoquinone) and 7-methyljuglone (5-hydroxy-7methyl-1,4-naphthoquinone) [1]. The naphthoquinones of sundew have many physiological effects; for example juglone is toxic and an effective inhibitor of seed germination for many plants, it has shown to be inhibitory also to several insects and to be highly toxic to fungi as well as different fungal pathogens [2]. Sundew naphthoquinones have also important pharma-
* Corresponding author. Tel.: 358-8553-1540; fax: 358-85531500. E-mail address: [email protected] (A. Hohtola).
cological effects. Sundew leaves are collected from the wild and liquid extracts and tinctures of D. rotundifolia can be utilised as medicinal compounds in treating various respiratory diseases [3]. Naphthoquinones are phenolic compounds and they are formed through acetate /malonate and shikimic acid pathways. Naphthoquinones of sundew are derived from acetate, which is formed from L-tyrosine most likely by homogentisate ring-cleavage pathway [4]. The key enzyme of this ring-cleavage reaction is homogentisate oxidase [5]. Naphthoquinones are synthesised and accumulated also in plant tissue cultures of certain plant species one of which is sundew. Genetic modification of plants using e.g. Agrobacterium tumefaciens is today a routine procedure for a large number of plant species [6 /8]. The most important prerequisite for the method is the possibility to regenerate plants from tissue culture or explants. Genetic engineering by Agrobacterium has been used to improve horticultural and agricultural properties of crop plants [9], and to alter the content of secondary metabolites in medicinal plants [10]. For example, secondary metabolites of pharmacological interest were produced in high concentrations after transfection via Agrobacterium in
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Ginkgo biloba L. [11], Rubia peregrina L. [12] and Catharanthus roseus L. [13]. In order to improve naphthoquinone production in either tissue cultures or whole plants of Drosera , we have pursued the possibility to genetically engineer the plant. Here, we report a simple procedure for A. tumefaciens -mediated transformation and regeneration of leaf explants of D. rotundifolia .
2. Materials and methods 2.1. Plant material and micropropagation The plant material in the genetic transformation originated from in vitro cultivated D. rotundifolia L. from different locations in Finland. The cultivation of Drosera plants was started from seeds. Aseptically germinated seeds of five different origins were used as explants in the genetic transformation at the stage when the plants produced whole leaves. The seeds were germinated on half strength MS medium [14] supplemented with 6-benzylaminopurine (BAP) 0.1 mg l 1 and a-naphthaleneacetic acid (NAA) 0.05 mg l 1, 2% sucrose and 100 mg l 1 myoinositol. The pH of all media was adjusted to 5.7 prior to autoclaving and all media to this point were solidified with 0.65% agar (Ph. Eur., Telko). Multiplication of sundew leaves was also achieved on this medium but especially rapid multiplication was achieved by transferring the leaf cuttings to full strength MS medium containing BAP (2.25 mg l 1 or 4.5 mg l 1) alone or to the medium containing both BAP (0.45 mg l1) and NAA (0.372 mg l 1). Solidification of these media was made with 0.7% bacto-agar (Difco Laboratories). As a regeneration medium after Agrobacterium infection, we used half strength MS medium supplemented with hormones BAP 0.1 mg l 1 and NAA 0.05 mg l 1, 2% sucrose and 100 mg l1 myoinositol, with or without acetosyringone (0.1 mM). The shoot regeneration medium was half strength MS medium with BAP 0.1 mg l 1, NAA 0.05 mg l 1, 2% sucrose and 100 mg l 1 myoinositol supplemented with cefotaxime and/or kanamycin as described below. The media in the cocultivation and selection were solidified with 0.2% Phytagel (Sigma). 2.2. Bacterial strain The disarmed A. tumefaciens strain C58C1 rifR [15] carrying the cointegrate plasmid (pGV2260::pHTT204) was used as a vector system for genetic transformation. The plasmid pHTT204 (Fig. 1) is similar to pHTT370 [16], but carries between the Bam HI site the firefly luciferase cDNA from pDO432 [17]. pHTT204 is a cointegration vector and does not contain an origin of
Fig. 1. Plasmid pHTT204 used in the transformation of D. rotundifolia.
replication that would function in agrobacterium. The sequence between the ‘Sm/Sp’ and ‘RB’ originates from the plasmid pBR322 and is homologous to the pBR322 sequence engineered into pGV2260. The plasmid contains the neomycin phosphotransferase II gene (NPTII), driven by the nopaline synthase promoter, which was used as a selective marker as it provides the transgenic leaves resistance to kanamycin. The firefly luciferase gene (LUC), driven by the cauliflower mosaic virus (CaMV) 35S promoter, was used as a convenient reporter gene to verify gene transfer and expression in the plant. Bacteria were grown 2 days in LA (peptone 10 g l1, NaCl 6 g l1, yeast extract 6 g l 1, bacto agar 15 g l 1) plates at room temperature with the antibiotics rifampicin 100 mg l 1, streptomycin 300 mg l 1 and spectinomycin 100 mg l 1. For transformation of D. rotundifolia a single colony from the plate was picked and grown in 25 ml LB (peptone 10 g l1, NaCl 6 g l1, yeast extract 6 g l 1) medium for 2 days (40 h) at room temperature with shaking. The Agrobacterium suspension was diluted with 15 ml fresh LB medium and the bacteria were further grown for 4 h. 2.3. Establishing the selection conditions The internal resistance of D. rotundifolia L. to kanamycin was tested by growing the plants in half strength MS medium supplemented with different concentrations of kanamycin in order to find out the right selection conditions. The kanamycin concentrations used were 0, 10, 20, 50, 100, 150, 250, 350, 400 and 450 mg l 1. Drosera plants were observed after 8 weeks of culture for the number and colour of the growing leaves.
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2.4. Genetic transformation Micropropagated Drosera leaves of five different origins were wounded with a sterile needle and infected for 10 min in Agrobacterium suspension, blotted dry with filter paper and transferred to regeneration medium for cocultivation. To half of the regeneration media used in cocultivation, acetosyringone was added to a final concentration of 0.1 mM. After 72 h of cocultivation, the infected leaves were washed with 1/2 MS liquid medium supplemented with cefotaxime (200 mg l 1) and blotted dry. Then, the explants were transferred to shoot regeneration medium complemented with 400 mg l 1 kanamycin for selection and 200 mg l1 cefotaxime for inhibiting growth of residual Agrobacterium . The explants were transferred to a fresh selection medium every 3 weeks. After 2 months of selection, cefotaxime was excluded from the media and Drosera plants were cultivated on media containing only kanamycin (400 mg l 1). Transformation efficiency was estimated by counting the number of explants growing on the selection medium after 16 weeks from infection. This number was divided by the number of all infected explants and multiplied by 100. Statistical analyses were performed with the two-way ANOVA test. 2.5. Luciferase assay The enzymatic assay for firefly luciferase was conducted after 16 weeks of transformation with Promega’s reagents following the manufacturer’s instructions. For 4 weeks before the assay, the plants were growing on the medium where the antibiotics were excluded without visible bacterial growth. Three Drosera leaves were ground in an Eppendorf tube in 100 ml Lysis buffer (Promega) and the tubes were centrifuged briefly at 4 8C. Ten microliters of the supernatant was pipetted to 100 ml of Luciferase Assay Reagent (Promega) and the light quanta produced by luciferase activity were counted for 1 min in a liquid scintillation counter set up to count chemiluminescense (TRIATHLER Multilaber tester, HIDEX). 2.6. Isolation, PCR and southern analysis of DNA Genomic DNA of sundew explants was isolated by modifying the method of Sul and Korban [18]. Incubation in the extraction buffer was prolonged to 20 min in 65 8C and the ethanol precipitation was made at 70 8C for 30 min. The samples were centrifuged after the precipitation step for 15 and 5 min instead of 3 and 15 s mentioned in the original method. In PCR analysis, two primers of the luciferase gene were used: 5?-ACC TTG GCG ACC TCT CGT TG-3? and 5?-GAA TCT CAC GCA GGC AGT TCT AT-3?. Expected size of the fragment was 1.8 kb. The primers
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used were designed by us according to luciferase sequence and were constructed by Pharmacia Biotech. PCR amplification was carried out in 50 ml containing 0.3 ml of the DNA solution, 200 mM dNTPs, 5 ml 10 buffer (Finnzymes) 0.25 mM of each primer and one unit of Dynazyme DNA polymerase (Finnzymes). DNA was subjected to 30 cycles of 1 min at 94 8C, 1 min at 60 8C, and 3 min at 72 8C. Amplified DNA fragments were electrophoresed on 1% agarose gel with ethidium bromide and observed under ultraviolet light. About 10 mg of genomic DNA from transformed and non-transformed sundew was digested with Bam HI restriction enzyme for Southern hybridisation analysis. DNA was separated by electrophoresis through a 0.8% agarose gel and transferred onto a positively charged nylon membrane (Roche Molecular Biochemicals). Hybridization was made by the DIG System User’s Guide for Filter Hybridization (Roche Molecular Biochemicals). As hybridisation solution UltraHyb solution (Ambion) was used and hybridisation was done overnight in 42 8C. A 1.8 kb luciferase gene fragment isolated from pHTT204 plasmid was used as a probe. The probe was labelled with digoxigenin using a random-primed DIG DNA labelling kit (Roche Molecular Biochemicals, and 0.4 ng ml 1 of labelled probe was used in the hybridisation solution. A 1.8 kb luc fragment of Bam HI-digested plasmid pHTT204 was used as a positive control for hybridisation. Washing conditions were twice for 5 min in room temperature with 2 SSC, 0.1% SDS solution and twice for 15 min in 42 8C with 0.5 SSC, 0.1% SDS solution.
3. Results and discussion 3.1. Micropropagation Nearly all cultivations started from overwintering buds (hibernicula) of D. rotundifolia were contaminated (data not shown), but when started from seeds contamination was reduced significantly and axenic cultures were possible to maintain from all five sundew accessions. In vitro culture of Drosera explants has been difficult due to fungal and bacterial contaminants on the surface of the leaves as reported in the study by Crouch et al. [19]. In the in vitro propagation study done by Anthony [20] 95% of the D. rotundifolia cultures started from whole plants and whole leaves were also contaminated. Multiplication of D. rotundifolia was achieved on all nutrient media and growth regulator combinations tested (not shown). Best multiplication was achieved on MS medium supplemented with BAP 0.45 mg l 1 and NAA 0.372 mg l 1 (up to 20 /30 buds per leaf). Propagation was slower when lower concentrations of growth regulators were used. Boba´k et al. [21] achieved
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the best shoot organogenesis on growth regulator-free MS medium. Crouch et al. [19] produced an average of 20 buds on the Drosera leaf surface in culture by using 1/ 5 strength MS medium with 30 g l 1 sucrose added and supplemented with 0.1 mg l1 BAP and 0.05 mg l 1 NAA. Micropropagated sundew plants were produced using seeds as a starting material. The seeds germinated on the medium and formed shoots and roots, sometimes even flowers developed in vitro. The sundew plantlets were successfully transferred to grow also ex vitro in standard greenhouse conditions. The leaves of these micropropagated shoots were used in the genetic transformation of sundew. Plantlets originating from transformed explants were fully developed, equally viable and as large as the untransformed plantlets. Adventitious buds were visible on the transformed explants after 2 weeks from infection and transfer to the selection medium with antibiotics. It is about the same time, which is needed for untransformed explants on the medium without antibiotics. Untransformed leaves on the selection medium did not grow, but turned black and died. Multiplication rate varied from 10 to 30 buds per one green, alive leaf explant. Amount was dependent from the type of media, as stated above. Rooting took place on the same medium. Transgenic plantlets were not transferred to the field. The aim is to work only by in vitro material in the future, too.
3.2. Selection conditions The inherent antibiotic tolerance of sundew was tested in order to find the optimal kanamycin concentration for selection conditions. Levels of 0/20 mg l1 kanamycin generated dark green, normal looking plantlets. Levels of 50 mg l 1 and above kanamycin in the medium showed clear concentration-dependent bleaching of the newly formed leaves. After 8 weeks of cultivation, the non-transformed sundew plants growing on kanamycin concentration 250 mg ml 1 were still partly alive and light green but the concentration of 350 mg l 1 was lethal and all plants were white. Kanamycin concentration 400 mg l1 was chosen for selecting transformants in the subsequent experiments. The kanamycin concentration used for sundew selection is rather high compared with that of used by Elomaa et al. [16] (25 mg l1) for Gerbera hybrida where even 10 mg l 1 suppressed the growth of gerbera leaflets efficiently. In transformation of arctic bramble (Rubus arcticus L.) the kanamycin selection concentration was 50 mg l 1 [22] and Eucalyptus camaldulensis Dehnh. transformants were selected at the concentration of 40 mg l 1 kanamycin [23]. Thus, D. rotundifolia L. has a high internal resistance to kanamycin.
3.3. Transformation and luciferase assay Regeneration of putatively transformed plants was noticed by bud induction on the surface of the wounded leaves after 2 weeks of cultivation on the selection medium containing kanamycin. All five different strains of sundew were responsive to genetic transformation. Calculated as number of emerging transformants per explant, the D. rotundifolia transformation efficiency was 17% (Table 1). Transformation efficiency showed differences due to the origin of the Drosera plants (twoway ANOVA, F (4, 10) P 0.015). Of all 1200 treated explants 205 were able to proliferate on the selection medium. No signs of Agrobacterium growth were detected after excluding the antibiotic cefotaxime from the selection media 4 weeks before luciferase assay. Wounded tobacco plants produce naturally the phenolic compound acetosyringone that attracts and induces the virulence system of agrobacteria [24]. In cases where production of inducing phenolics by the plant is not known, acetosyringone can be added to the cocultivation system [25]. However, the use of acetosyringone did not increase the transformation efficiency of D. rotundifolia (two-way ANOVA, F (1, 10) P 0.784; Table 1). In the genetic transformation study of loblolly pine and Norway spruce by Wenck et al. [26] acetosyringone was essential for high transformation efficiency, but high concentrations (100 mM or greater) were not beneficial. Drosera cells seem to produce phenolic virulence inducing compounds sufficiently and adding acetosyringone into the cocultivation medium has no beneficial effects. The antibiotic resistance of transformed sundews was indicative of gene transfer, but luciferase activity was also measured to further confirm the T-DNA transfer. All tested transgenic lines were shown to express the transferred luciferase gene (Table 2). 3.4. PCR analysis and Southern blot of transgenic sundew DNA In addition to the ability to grow on the medium containing kanamycin and express the transferred luciferase gene, PCR and Southern hybridisation analysis were used to confirm at DNA level the integration of foreign genes into the genome of the sundew plants. Presence of the T-DNA in the plant genome was only detected by PCR and Southern analyses in one of the clones obtained due to difficulties in extracting DNA of suitable quality for molecular analysis from leaves of this species. Secondary metabolites of sundew interfere with the reagents in both DNA extraction and PCR as is the case in wild strawberry Fragaria vesca L. [27]. Studies to optimise the DNA extraction method by reducing the secondary metabolite concentration during the procedure are currently under way.
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Table 1 Efficiency of genetic transformation of D. rotundifolia Cocultivation medium
TE 1
TE 2
TE 3
TE 4
TE 5
Average TE%
(1/2 MS medium) (1/2 MSAS medium) Both media
7.5% 10.0%
17.5% 23.3%
20.8% 30.0%
7.5% 6.7%
35.8% 11.7%
17.8% 16.3% 17.0%
Sundew plants (strains 1 /5) were wounded with sterile needle and transformed with A. tumefaciens , cocultivated in 1/2-MS-media and 1/2-MS-media supplemented with acetosyringone (AS). The average transformation efficiency (TE) for round leaved sundew was 17%. Table 2 Luciferase activity measurement Sundew strain
CPS
1 2 3 4 5 Control 1 Control 2
207 330 342 000 577 000 1 579 000 1 284 000 140 2800
Three sundew leaves were ground in 100 ml of lysis buffer. After a short centrifugation 10 ml of supernatant was added to the assay reagent. Chemiluminescence was measured as counts per second (CPS) in liquid scintillation counter for 60 s. Two non-transgenic sundews were used as controls.
PCR amplification produced the expected 1.8 kb product from both the transgenic sundew DNA and plasmid DNA used as a positive control as shown in Fig. 2. No amplification of DNA was detected from non-transgenic sundew DNA. In Southern blot analysis, transgenic and non-transgenic sundew genomic DNA was digested with Bam HI and fractionated on a 0.8% agarose gel. As shown in Fig. 3, a 1.8 kb DIG labelled luc -probe hybridised to Bam HI-digested genomic DNA of transgenic plants (lane 1). No hybridisation was detected in the control non-transgenic D. rotundifolia Bam HI-digested genomic DNA (lane 2). As a positive control a 1.8 kb Bam HI plasmid pHTT204 fragment containing the luc -gene was used (lane C).
Fig. 3. Southern blot detection of gene transfer. D. rotundifolia plants were transformed with Agrobacterium containing plasmid (pGV2260::pHTT204) which contains NPT and LUC genes. Ten micrograms of Drosera genomic DNA was digested with Bam HI restriction enzyme, separated using an agarose gel and transferred to a nylon membrane. The probe used was DIG-labelled luc -gene. As a positive control Bam HI-digested plasmid pHTT204 luc -fragment was used (lane C). A 1.8 kb luc -fragment was detected from transformed Drosera DNA (lane 1) but not from untransformed Drosera DNA (lane 2).
3.5. Conclusions We have achieved genetic transformation of round leaved sundew, D. rotundifolia L., through an A. tumefaciens -mediated method. Conditions for micropropagation, infection and selection were defined. Agrobacterium -mediated genetic transformation efficiency for D. rotundifolia was 17%. All five strains of sundew used were transformable, although transformation efficiency showed differences due to the origin of sundew plants. The results presented here allow the genetic modification of secondary metabolite pathways of this pharmacologically important carnivorous plant, and facilitate the future goal of producing transgenic sundew plants with a higher naphthoquinone content or a modified naphthoquinone profile.
Acknowledgements Fig. 2. PCR analysis of transgenic sundew plants showing the presence of the expected 1.8 kb DNA fragment of luciferase gene in lanes 2 and 3. Lane 1: GeneRuler (MBI Fermentas) marker; Lane 2: Positive control (plasmid pHTT204); Lane 3: Transgenic Drosera and Lane 4: Negative control (untransformed sundew).
Paula Elomaa, PhD is acknowledged for the technical guidance. This study was a part of the project POHERIKA (Pohjoisen Erikoiskasvit, Northern Special Plants)
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and was financially supported by the grants from Tauno To¨nning Foundation, Finnish Cultural Fund (Kainuu district) and Finnish Natural Resources Foundation.
[14]
[15]
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Agrobacterium-mediated transformation of round leaved sundew (Drosera rotundifolia L.) Merja Hirsikorpi a, Terttu Ka¨ma¨ra¨inen a, Teemu Teeri b, Anja Hohtola a,* a b
Department of Biology/Botany, University of Oulu, PO Box 3000, FIN-90014 Oulu, Finland Institute of Biotechnology, University of Helsinki, PO Box 56, FIN-00014 Helsinki, Finland
Received 14 May 2001; received in revised form 13 November 2001; accepted 3 December 2001
Abstract Agrobacterium tumefaciens -mediated genetic transformation method of the carnivorous medicinal plant round leaved sundew (Drosera rotundifolia L.) was developed. The micropropagation conditions of sundew aseptically germinated seeds were defined and the internal kanamycin resistance of sundew was tested. Transformation was made by cocultivation of micropropagated sundew leaves with A. tumefaciens strain C58C1 containing a cointegrate plasmid vector with neomycin phosphotransferase and luciferase genes. Transgenic sundews were selected for kanamycin resistance and viable, fully developed plantlets were further assayed by luciferase activity and by PCR and Southern analyses using luc -primers and a luc -probe. The transformation efficiency of D. rotundifolia L. was 17%. Sundew is an important source of pharmacologically active 1,4-naphthoquinones, and genetic transformation allows the engineering of its biochemical pathways. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Droseraceae ; Agrobacterium ; Luciferase; Genetic transformation; Naphthoquinones; Micropropagation
1. Introduction Drosera rotundifolia L. is a carnivorous perennial herb with a basal rosette of leaves. The upper surfaces of the leaf blades are covered with reddish, glandular hairs tipped with a sticky secretion that traps insects. Sundew compensates for the low availability of nutrients in its habitat by digesting caught insects with proteolytic enzymes. Sundew is a rich source of 1,4-naphthoquinones, the main ones being plumbagin (5-hydroxy-2-methyl-1,4naphthoquinone) and 7-methyljuglone (5-hydroxy-7methyl-1,4-naphthoquinone) [1]. The naphthoquinones of sundew have many physiological effects; for example juglone is toxic and an effective inhibitor of seed germination for many plants, it has shown to be inhibitory also to several insects and to be highly toxic to fungi as well as different fungal pathogens [2]. Sundew naphthoquinones have also important pharma-
* Corresponding author. Tel.: 358-8553-1540; fax: 358-85531500. E-mail address: [email protected] (A. Hohtola).
cological effects. Sundew leaves are collected from the wild and liquid extracts and tinctures of D. rotundifolia can be utilised as medicinal compounds in treating various respiratory diseases [3]. Naphthoquinones are phenolic compounds and they are formed through acetate /malonate and shikimic acid pathways. Naphthoquinones of sundew are derived from acetate, which is formed from L-tyrosine most likely by homogentisate ring-cleavage pathway [4]. The key enzyme of this ring-cleavage reaction is homogentisate oxidase [5]. Naphthoquinones are synthesised and accumulated also in plant tissue cultures of certain plant species one of which is sundew. Genetic modification of plants using e.g. Agrobacterium tumefaciens is today a routine procedure for a large number of plant species [6 /8]. The most important prerequisite for the method is the possibility to regenerate plants from tissue culture or explants. Genetic engineering by Agrobacterium has been used to improve horticultural and agricultural properties of crop plants [9], and to alter the content of secondary metabolites in medicinal plants [10]. For example, secondary metabolites of pharmacological interest were produced in high concentrations after transfection via Agrobacterium in
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Ginkgo biloba L. [11], Rubia peregrina L. [12] and Catharanthus roseus L. [13]. In order to improve naphthoquinone production in either tissue cultures or whole plants of Drosera , we have pursued the possibility to genetically engineer the plant. Here, we report a simple procedure for A. tumefaciens -mediated transformation and regeneration of leaf explants of D. rotundifolia .
2. Materials and methods 2.1. Plant material and micropropagation The plant material in the genetic transformation originated from in vitro cultivated D. rotundifolia L. from different locations in Finland. The cultivation of Drosera plants was started from seeds. Aseptically germinated seeds of five different origins were used as explants in the genetic transformation at the stage when the plants produced whole leaves. The seeds were germinated on half strength MS medium [14] supplemented with 6-benzylaminopurine (BAP) 0.1 mg l 1 and a-naphthaleneacetic acid (NAA) 0.05 mg l 1, 2% sucrose and 100 mg l 1 myoinositol. The pH of all media was adjusted to 5.7 prior to autoclaving and all media to this point were solidified with 0.65% agar (Ph. Eur., Telko). Multiplication of sundew leaves was also achieved on this medium but especially rapid multiplication was achieved by transferring the leaf cuttings to full strength MS medium containing BAP (2.25 mg l 1 or 4.5 mg l 1) alone or to the medium containing both BAP (0.45 mg l1) and NAA (0.372 mg l 1). Solidification of these media was made with 0.7% bacto-agar (Difco Laboratories). As a regeneration medium after Agrobacterium infection, we used half strength MS medium supplemented with hormones BAP 0.1 mg l 1 and NAA 0.05 mg l 1, 2% sucrose and 100 mg l1 myoinositol, with or without acetosyringone (0.1 mM). The shoot regeneration medium was half strength MS medium with BAP 0.1 mg l 1, NAA 0.05 mg l 1, 2% sucrose and 100 mg l 1 myoinositol supplemented with cefotaxime and/or kanamycin as described below. The media in the cocultivation and selection were solidified with 0.2% Phytagel (Sigma). 2.2. Bacterial strain The disarmed A. tumefaciens strain C58C1 rifR [15] carrying the cointegrate plasmid (pGV2260::pHTT204) was used as a vector system for genetic transformation. The plasmid pHTT204 (Fig. 1) is similar to pHTT370 [16], but carries between the Bam HI site the firefly luciferase cDNA from pDO432 [17]. pHTT204 is a cointegration vector and does not contain an origin of
Fig. 1. Plasmid pHTT204 used in the transformation of D. rotundifolia.
replication that would function in agrobacterium. The sequence between the ‘Sm/Sp’ and ‘RB’ originates from the plasmid pBR322 and is homologous to the pBR322 sequence engineered into pGV2260. The plasmid contains the neomycin phosphotransferase II gene (NPTII), driven by the nopaline synthase promoter, which was used as a selective marker as it provides the transgenic leaves resistance to kanamycin. The firefly luciferase gene (LUC), driven by the cauliflower mosaic virus (CaMV) 35S promoter, was used as a convenient reporter gene to verify gene transfer and expression in the plant. Bacteria were grown 2 days in LA (peptone 10 g l1, NaCl 6 g l1, yeast extract 6 g l 1, bacto agar 15 g l 1) plates at room temperature with the antibiotics rifampicin 100 mg l 1, streptomycin 300 mg l 1 and spectinomycin 100 mg l 1. For transformation of D. rotundifolia a single colony from the plate was picked and grown in 25 ml LB (peptone 10 g l1, NaCl 6 g l1, yeast extract 6 g l 1) medium for 2 days (40 h) at room temperature with shaking. The Agrobacterium suspension was diluted with 15 ml fresh LB medium and the bacteria were further grown for 4 h. 2.3. Establishing the selection conditions The internal resistance of D. rotundifolia L. to kanamycin was tested by growing the plants in half strength MS medium supplemented with different concentrations of kanamycin in order to find out the right selection conditions. The kanamycin concentrations used were 0, 10, 20, 50, 100, 150, 250, 350, 400 and 450 mg l 1. Drosera plants were observed after 8 weeks of culture for the number and colour of the growing leaves.
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2.4. Genetic transformation Micropropagated Drosera leaves of five different origins were wounded with a sterile needle and infected for 10 min in Agrobacterium suspension, blotted dry with filter paper and transferred to regeneration medium for cocultivation. To half of the regeneration media used in cocultivation, acetosyringone was added to a final concentration of 0.1 mM. After 72 h of cocultivation, the infected leaves were washed with 1/2 MS liquid medium supplemented with cefotaxime (200 mg l 1) and blotted dry. Then, the explants were transferred to shoot regeneration medium complemented with 400 mg l 1 kanamycin for selection and 200 mg l1 cefotaxime for inhibiting growth of residual Agrobacterium . The explants were transferred to a fresh selection medium every 3 weeks. After 2 months of selection, cefotaxime was excluded from the media and Drosera plants were cultivated on media containing only kanamycin (400 mg l 1). Transformation efficiency was estimated by counting the number of explants growing on the selection medium after 16 weeks from infection. This number was divided by the number of all infected explants and multiplied by 100. Statistical analyses were performed with the two-way ANOVA test. 2.5. Luciferase assay The enzymatic assay for firefly luciferase was conducted after 16 weeks of transformation with Promega’s reagents following the manufacturer’s instructions. For 4 weeks before the assay, the plants were growing on the medium where the antibiotics were excluded without visible bacterial growth. Three Drosera leaves were ground in an Eppendorf tube in 100 ml Lysis buffer (Promega) and the tubes were centrifuged briefly at 4 8C. Ten microliters of the supernatant was pipetted to 100 ml of Luciferase Assay Reagent (Promega) and the light quanta produced by luciferase activity were counted for 1 min in a liquid scintillation counter set up to count chemiluminescense (TRIATHLER Multilaber tester, HIDEX). 2.6. Isolation, PCR and southern analysis of DNA Genomic DNA of sundew explants was isolated by modifying the method of Sul and Korban [18]. Incubation in the extraction buffer was prolonged to 20 min in 65 8C and the ethanol precipitation was made at 70 8C for 30 min. The samples were centrifuged after the precipitation step for 15 and 5 min instead of 3 and 15 s mentioned in the original method. In PCR analysis, two primers of the luciferase gene were used: 5?-ACC TTG GCG ACC TCT CGT TG-3? and 5?-GAA TCT CAC GCA GGC AGT TCT AT-3?. Expected size of the fragment was 1.8 kb. The primers
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used were designed by us according to luciferase sequence and were constructed by Pharmacia Biotech. PCR amplification was carried out in 50 ml containing 0.3 ml of the DNA solution, 200 mM dNTPs, 5 ml 10 buffer (Finnzymes) 0.25 mM of each primer and one unit of Dynazyme DNA polymerase (Finnzymes). DNA was subjected to 30 cycles of 1 min at 94 8C, 1 min at 60 8C, and 3 min at 72 8C. Amplified DNA fragments were electrophoresed on 1% agarose gel with ethidium bromide and observed under ultraviolet light. About 10 mg of genomic DNA from transformed and non-transformed sundew was digested with Bam HI restriction enzyme for Southern hybridisation analysis. DNA was separated by electrophoresis through a 0.8% agarose gel and transferred onto a positively charged nylon membrane (Roche Molecular Biochemicals). Hybridization was made by the DIG System User’s Guide for Filter Hybridization (Roche Molecular Biochemicals). As hybridisation solution UltraHyb solution (Ambion) was used and hybridisation was done overnight in 42 8C. A 1.8 kb luciferase gene fragment isolated from pHTT204 plasmid was used as a probe. The probe was labelled with digoxigenin using a random-primed DIG DNA labelling kit (Roche Molecular Biochemicals, and 0.4 ng ml 1 of labelled probe was used in the hybridisation solution. A 1.8 kb luc fragment of Bam HI-digested plasmid pHTT204 was used as a positive control for hybridisation. Washing conditions were twice for 5 min in room temperature with 2 SSC, 0.1% SDS solution and twice for 15 min in 42 8C with 0.5 SSC, 0.1% SDS solution.
3. Results and discussion 3.1. Micropropagation Nearly all cultivations started from overwintering buds (hibernicula) of D. rotundifolia were contaminated (data not shown), but when started from seeds contamination was reduced significantly and axenic cultures were possible to maintain from all five sundew accessions. In vitro culture of Drosera explants has been difficult due to fungal and bacterial contaminants on the surface of the leaves as reported in the study by Crouch et al. [19]. In the in vitro propagation study done by Anthony [20] 95% of the D. rotundifolia cultures started from whole plants and whole leaves were also contaminated. Multiplication of D. rotundifolia was achieved on all nutrient media and growth regulator combinations tested (not shown). Best multiplication was achieved on MS medium supplemented with BAP 0.45 mg l 1 and NAA 0.372 mg l 1 (up to 20 /30 buds per leaf). Propagation was slower when lower concentrations of growth regulators were used. Boba´k et al. [21] achieved
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the best shoot organogenesis on growth regulator-free MS medium. Crouch et al. [19] produced an average of 20 buds on the Drosera leaf surface in culture by using 1/ 5 strength MS medium with 30 g l 1 sucrose added and supplemented with 0.1 mg l1 BAP and 0.05 mg l 1 NAA. Micropropagated sundew plants were produced using seeds as a starting material. The seeds germinated on the medium and formed shoots and roots, sometimes even flowers developed in vitro. The sundew plantlets were successfully transferred to grow also ex vitro in standard greenhouse conditions. The leaves of these micropropagated shoots were used in the genetic transformation of sundew. Plantlets originating from transformed explants were fully developed, equally viable and as large as the untransformed plantlets. Adventitious buds were visible on the transformed explants after 2 weeks from infection and transfer to the selection medium with antibiotics. It is about the same time, which is needed for untransformed explants on the medium without antibiotics. Untransformed leaves on the selection medium did not grow, but turned black and died. Multiplication rate varied from 10 to 30 buds per one green, alive leaf explant. Amount was dependent from the type of media, as stated above. Rooting took place on the same medium. Transgenic plantlets were not transferred to the field. The aim is to work only by in vitro material in the future, too.
3.2. Selection conditions The inherent antibiotic tolerance of sundew was tested in order to find the optimal kanamycin concentration for selection conditions. Levels of 0/20 mg l1 kanamycin generated dark green, normal looking plantlets. Levels of 50 mg l 1 and above kanamycin in the medium showed clear concentration-dependent bleaching of the newly formed leaves. After 8 weeks of cultivation, the non-transformed sundew plants growing on kanamycin concentration 250 mg ml 1 were still partly alive and light green but the concentration of 350 mg l 1 was lethal and all plants were white. Kanamycin concentration 400 mg l1 was chosen for selecting transformants in the subsequent experiments. The kanamycin concentration used for sundew selection is rather high compared with that of used by Elomaa et al. [16] (25 mg l1) for Gerbera hybrida where even 10 mg l 1 suppressed the growth of gerbera leaflets efficiently. In transformation of arctic bramble (Rubus arcticus L.) the kanamycin selection concentration was 50 mg l 1 [22] and Eucalyptus camaldulensis Dehnh. transformants were selected at the concentration of 40 mg l 1 kanamycin [23]. Thus, D. rotundifolia L. has a high internal resistance to kanamycin.
3.3. Transformation and luciferase assay Regeneration of putatively transformed plants was noticed by bud induction on the surface of the wounded leaves after 2 weeks of cultivation on the selection medium containing kanamycin. All five different strains of sundew were responsive to genetic transformation. Calculated as number of emerging transformants per explant, the D. rotundifolia transformation efficiency was 17% (Table 1). Transformation efficiency showed differences due to the origin of the Drosera plants (twoway ANOVA, F (4, 10) P 0.015). Of all 1200 treated explants 205 were able to proliferate on the selection medium. No signs of Agrobacterium growth were detected after excluding the antibiotic cefotaxime from the selection media 4 weeks before luciferase assay. Wounded tobacco plants produce naturally the phenolic compound acetosyringone that attracts and induces the virulence system of agrobacteria [24]. In cases where production of inducing phenolics by the plant is not known, acetosyringone can be added to the cocultivation system [25]. However, the use of acetosyringone did not increase the transformation efficiency of D. rotundifolia (two-way ANOVA, F (1, 10) P 0.784; Table 1). In the genetic transformation study of loblolly pine and Norway spruce by Wenck et al. [26] acetosyringone was essential for high transformation efficiency, but high concentrations (100 mM or greater) were not beneficial. Drosera cells seem to produce phenolic virulence inducing compounds sufficiently and adding acetosyringone into the cocultivation medium has no beneficial effects. The antibiotic resistance of transformed sundews was indicative of gene transfer, but luciferase activity was also measured to further confirm the T-DNA transfer. All tested transgenic lines were shown to express the transferred luciferase gene (Table 2). 3.4. PCR analysis and Southern blot of transgenic sundew DNA In addition to the ability to grow on the medium containing kanamycin and express the transferred luciferase gene, PCR and Southern hybridisation analysis were used to confirm at DNA level the integration of foreign genes into the genome of the sundew plants. Presence of the T-DNA in the plant genome was only detected by PCR and Southern analyses in one of the clones obtained due to difficulties in extracting DNA of suitable quality for molecular analysis from leaves of this species. Secondary metabolites of sundew interfere with the reagents in both DNA extraction and PCR as is the case in wild strawberry Fragaria vesca L. [27]. Studies to optimise the DNA extraction method by reducing the secondary metabolite concentration during the procedure are currently under way.
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Table 1 Efficiency of genetic transformation of D. rotundifolia Cocultivation medium
TE 1
TE 2
TE 3
TE 4
TE 5
Average TE%
(1/2 MS medium) (1/2 MSAS medium) Both media
7.5% 10.0%
17.5% 23.3%
20.8% 30.0%
7.5% 6.7%
35.8% 11.7%
17.8% 16.3% 17.0%
Sundew plants (strains 1 /5) were wounded with sterile needle and transformed with A. tumefaciens , cocultivated in 1/2-MS-media and 1/2-MS-media supplemented with acetosyringone (AS). The average transformation efficiency (TE) for round leaved sundew was 17%. Table 2 Luciferase activity measurement Sundew strain
CPS
1 2 3 4 5 Control 1 Control 2
207 330 342 000 577 000 1 579 000 1 284 000 140 2800
Three sundew leaves were ground in 100 ml of lysis buffer. After a short centrifugation 10 ml of supernatant was added to the assay reagent. Chemiluminescence was measured as counts per second (CPS) in liquid scintillation counter for 60 s. Two non-transgenic sundews were used as controls.
PCR amplification produced the expected 1.8 kb product from both the transgenic sundew DNA and plasmid DNA used as a positive control as shown in Fig. 2. No amplification of DNA was detected from non-transgenic sundew DNA. In Southern blot analysis, transgenic and non-transgenic sundew genomic DNA was digested with Bam HI and fractionated on a 0.8% agarose gel. As shown in Fig. 3, a 1.8 kb DIG labelled luc -probe hybridised to Bam HI-digested genomic DNA of transgenic plants (lane 1). No hybridisation was detected in the control non-transgenic D. rotundifolia Bam HI-digested genomic DNA (lane 2). As a positive control a 1.8 kb Bam HI plasmid pHTT204 fragment containing the luc -gene was used (lane C).
Fig. 3. Southern blot detection of gene transfer. D. rotundifolia plants were transformed with Agrobacterium containing plasmid (pGV2260::pHTT204) which contains NPT and LUC genes. Ten micrograms of Drosera genomic DNA was digested with Bam HI restriction enzyme, separated using an agarose gel and transferred to a nylon membrane. The probe used was DIG-labelled luc -gene. As a positive control Bam HI-digested plasmid pHTT204 luc -fragment was used (lane C). A 1.8 kb luc -fragment was detected from transformed Drosera DNA (lane 1) but not from untransformed Drosera DNA (lane 2).
3.5. Conclusions We have achieved genetic transformation of round leaved sundew, D. rotundifolia L., through an A. tumefaciens -mediated method. Conditions for micropropagation, infection and selection were defined. Agrobacterium -mediated genetic transformation efficiency for D. rotundifolia was 17%. All five strains of sundew used were transformable, although transformation efficiency showed differences due to the origin of sundew plants. The results presented here allow the genetic modification of secondary metabolite pathways of this pharmacologically important carnivorous plant, and facilitate the future goal of producing transgenic sundew plants with a higher naphthoquinone content or a modified naphthoquinone profile.
Acknowledgements Fig. 2. PCR analysis of transgenic sundew plants showing the presence of the expected 1.8 kb DNA fragment of luciferase gene in lanes 2 and 3. Lane 1: GeneRuler (MBI Fermentas) marker; Lane 2: Positive control (plasmid pHTT204); Lane 3: Transgenic Drosera and Lane 4: Negative control (untransformed sundew).
Paula Elomaa, PhD is acknowledged for the technical guidance. This study was a part of the project POHERIKA (Pohjoisen Erikoiskasvit, Northern Special Plants)
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and was financially supported by the grants from Tauno To¨nning Foundation, Finnish Cultural Fund (Kainuu district) and Finnish Natural Resources Foundation.
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