Journal of Experimental Botany, Vol. 51, No. 347, pp. 1005-1016,
June 2000
© 2000 Oxford University Press
Agrobacterium rhizogenes-mediated transformation of opium poppy, Papaver somniferum L., and California poppy, Eschscholzia californica Cham., root cultures
Department of Biological Sciences, University of Calgary, Calgary, Alberta, T2N 1N4 Canada
Received 1 November 1999; Accepted 25 January 2000
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Abstract |
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An efficient protocol for the establishment of transgenic opium poppy (Papaver somniferum L.) and California poppy (Eschscholzia californica Cham.) root cultures using Agrobacterium rhizogenes is reported. Five strains of A. rhizogenes were tested for their ability to produce hairy roots on wounded opium poppy seedlings and California poppy embryogenic calli. Three of the strains induced hairy root formation on both species, whereas two others either caused the growth of tumorigenic calli or produced no response. To characterize the putative transgenic roots further, explant tissues were co-cultivated with the most effective A. rhizogenes strain (R1000) carrying the pBI121 binary vector. Except for the co-cultivation medium, all formulations included 50 mg l-1 paromomycin to select for transformants and 200 mg l-1 timentin to eliminate the Agrobacterium. Four weeks after infection, paromomycin-resistant roots appeared on 92–98% of explants maintained on hormone-free medium. Isolated hairy roots were propagated in liquid medium containing 1.0 mg l-1 indole-3-acetic acid to promote rapid growth. Detection of the neomycin phosphotransferase gene, high levels of ß-glucuronidase (GUS) transcripts and enzyme activity, and GUS histochemical localization confirmed the integrative transformation of root cultures. Transgenic roots grew faster than wild-type roots, and California poppy roots grew more rapidly than those of opium poppy. With the exception of a less compact arrangement of epidermal cells and more root hairs, transformed roots of both species displayed anatomical features and benzylisoquinoline alkaloid profiles that were virtually identical to those of wild-type roots. Transgenic root cultures of opium poppy and California poppy are a simple, reliable and well-defined model system to investigate the molecular and metabolic regulation of benzylisoquinoline alkaloid biosynthesis, and to evaluate the genetic engineering potential of these important medicinal plants.
Key words: Agrobacterium rhizogenes, benzylisoquinoline alkaloids, California poppy, Eschscholzia californica, hairy root cultures, opium poppy, Papaver somniferum.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Benzylisoquinoline alkaloids are a diverse group of nitrogenous compounds with a restricted taxonomic distribution in plants. In addition to various ecophysiological functions related to plant–pathogen and plant–herbivore interactions, most benzylisoquinoline alkaloids exhibit potent pharmacological activity and many are used as pharmaceuticals. Examples include the analgesics morphine and codeine, the antibiotic sanguinarine, the gout suppressant colchicine, and the muscle relaxant (+)-tubocurarine. The total chemical synthesis of these valuable compounds is difficult because of their structural complexity; thus, wild or cultivated plants remain their only commercial source.
Benzylisoquinoline alkaloids are ubiquitous among the Papaveraceae, which include the California poppy (Eschscholzia californica Cham.) and the opium poppy (Papaver somniferum L.). The California poppy is a traditional medicinal plant of many indigenous peoples in North America (Cheney, 1964
). The remedial properties of this now common ornamental species result from its ability to synthesize a variety of benzophenanthridine alkaloids, which represent a subgroup of benzylisoquinoline alkaloids restricted in distribution to the Papaveraceae and Fumariaceae. Sanguinarine is one of the principal benzophenanthridine alkaloids found in California poppy roots, and is used commercially as an antiplaque agent in oral hygeine products due to its potent antimicrobial activity (Dzink and Socransky, 1985). Benzophenanthridine alkaloid biosynthesis has been studied extensively in California poppy cell suspension cultures (Kutchan, 1998) because the pathway can be induced by the addition of fungal elicitors (Schumacher et al., 1987) or methyl jasmonate (Blechert et al., 1995) to the culture medium. Two cDNAs encoding alkaloid biosynthetic enzymes in California poppy have been cloned, and shown to be transcriptionally induced in cultured cells in response to elicitor- or methyl jasmonate-treatment (Dittrich and Kutchan, 1991; Pauli and Kutchan, 1998).
Sanguinarine also accumulates constitutively in opium poppy roots, and is induced in opium poppy cell suspension cultures after treatment with fungal elicitors (Facchini et al., 1996a). However, opium poppy also produces an abundance of several other benzylisoquinoline alkaloids including the putative anti-tumorigenic agent noscapine (Ye et al., 1998) and morphine, both of which accumulate in roots but are more abundant in the latex of shoot organs. However, noscapine and morphine are not produced in opium poppy cell suspension cultures due to an apparent, but poorly understood, requirement for cell type-specific specialization; thus, de-differentiated cell cultures are of limited use to investigate the regulation of most alkaloid pathways in opium poppy. Recently, several cDNAs encoding alkaloid biosynthetic enzymes in opium poppy have also been cloned and, as in California poppy, shown to be transcriptionally regulated (Facchini and De Luca, 1994; Facchini et al., 1996b; Yu and Facchini, 2000; Unterlinner et al., 1999).
Attempts to understand the molecular mechanisms that regulate genes encoding benzylisoquinoline alkaloid biosynthetic enzymes in opium poppy and California poppy have relied on transient expression systems using microprojectile-bombarded cell suspension cultures (Hauschild et al., 1998; Park et al., 1999). However, this approach precludes the study of developmental, and many inducible, mechanisms that control benzylisoquinoline alkaloid pathways because the wound signal caused by the entry of DNA-coated microcarriers into the cells activates several of the relevant genes. A more complete application of advanced molecular and biochemical approaches to investigate the genetic and metabolic regulation of benzylisoquinoline alkaloid biosynthesis requires the establishment of efficient protocols for the stable transformation of opium poppy and California poppy plants and differentiated tissue cultures.
Transgenic hairy root cultures have served as a useful model system to investigate the biosynthesis of alkaloids, and a variety of other secondary metabolites. For example, transformed root cultures derived from members of the Solanaceae have been used extensively to study the production of tropane alkaloids and nicotine (Aoki et al., 1997; Hamill et al., 1990; Hashimoto et al., 1993; Jouhikainen et al., 1999; Robins et al., 1991; Sharp and Doran, 1990). The Agrobacterium-mediated production of hairy roots also creates a rapid and simple means to introduce and express foreign genes in plant cells that are capable of synthesizing specific secondary metabolites. For example, this approach has been used to alter the accumulation of alkaloids normally produced in roots. In one study, a yeast cDNA encoding ornithine decarboxylase (ODC) was introduced into Nicotiana rustica L. hairy roots using an Agrobacterium-derived vector (Hamill et al., 1990). Although the hairy root cultures produced more nicotine than wild-type roots, the increase in ODC activity was proportionately higher than the increase in nicotine levels suggesting that ODC is not a rate-determining step in the nicotine pathway. In another case, hyoscyamine-rich hairy roots of Atropa belladonna L. (Hashimoto et al., 1993) and Hyoscyamus muticus L. (Jouhikainen et al., 1999) were transformed with a gene from Hyoscyamus niger L. encoding hyoscyamine-6ß-hydroxylase (H6H), which catalyses the final epoxidation step in the conversion of hyoscyamine to scopolamine. As expected, the A. belladonna and H. muticus hairy roots contained high levels of H6H activity and produced, almost exclusively, scopolamine.
In this paper, the development of an efficient protocol to introduce foreign genes into transgenic opium poppy and California poppy hairy root cultures using Agrobacterium rhizogenes is described. With the exception of an increased number of root hairs and a less compact arrangement of epidermal cells, it is shown that the hairy roots of both species display anatomical features and benzylisoquinoline alkaloid profiles that are virtually identical to those of wild-type roots. Therefore, these rapidly growing transformed hairy root cultures could serve as a simple, reliable and well-defined model system to study the molecular regulation of genes encoding benzylisoquinoline alkaloid biosynthetic enzymes, and to evaluate the potential to metabolically engineer these important medicinal plants.
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Materials and methods |
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Seed sterilization and germination
Seeds of P. somniferum cv. Marianne and E. californica cv. Aurantiaca were surface-sterilized with 70% (v/v) ethanol for 30 s and 2% (v/v) sodium hypochlorite solution for 10 min, then rinsed three times in sterilized water. Approximately 30 seeds were placed on 25 ml of agar-solidified culture medium in Petri dishes (100x15 mm). The basal medium consisted of B5 salts and vitamins (Gamborg et al., 1968
) solidified with 0.8% (w/v) Phytagar (Gibco, Burlington, Canada). The medium was adjusted to pH 5.8 before adding agar, and then sterilized by autoclaving at 1.1 kg cm-2 (121 °C) for 20 min. The seeds were germinated in a growth chamber at 25 °C under standard cool white fluorescent tubes (Sylvania Gros-Lux Wide Spectrum, Mississauga, Canada) with a flux rate of 35 µmol s-1 m-2 and a 16 h photoperiod.
Preparation of Agrobacterium rhizogenes
The binary vector pBI121 (Jefferson et al., 1987) was mobilized by electroporation in the armed Agrobacterium rhizogenes strains 13333, 15834, C58C1, R1000, and R1200rolD. Transformed A. rhizogenes cultures were grown to mid-log phase (A600=0.5) at 28 °C on a gyratory shaker at 180 rpm in liquid Luria-Bertani medium (1% [w/v] tryptone, 0.5% [w/v] yeast extract, and 1% [w/v] NaCl, pH 7.0), containing 50 mg l-1 kanamycin. The bacterial cells were collected by centrifugation for 10 min at 1500 rpm, and resuspended at a cell density of A600=1.0 in liquid inoculation medium (B5 salts and vitamins containing 20 g l-1 sucrose).
Production of transgenic root cultures
Excised shoots (i.e. with the roots removed) from 5-d-old opium poppy and California poppy seedlings, and embryogenic callus cultures of California poppy, were used as the explant material for co-cultivation with A. rhizogenes. The embryogenic callus was induced from seed-derived primary callus of California poppy as described previously (Park and Facchini, 2000). Seedlings and embryogenic callus were randomly wounded using a scalpel, immersed in an A. rhizogenes culture suspended in liquid inoculation medium for 10–15 min, blotted dry on sterile filter paper, and incubated in the dark on Phytagar-solidified B5 medium. After 2 d of co-cultivation, the explant tissues were transferred to hormone-free selection medium containing B5 salts and vitamins, 3% (w/v) sucrose, 50 mg l-1 paromomycin, 200 mg l-1 timentin and 8 g l-1 Phytagar. Within 4–5 weeks, numerous paromomycin-resistant roots had emerged from the wound sites. The hairy roots were separated from the explant tissue and sub-cultured in the dark at 25 °C on hormone-free selection medium. After repeated transfer to fresh selection medium, rapidly growing hairy root cultures were obtained. Isolated roots (0.5 g) were transferred to 40 ml of B5 liquid medium, containing 3% (w/v) sucrose, in 125 ml flasks. Wild-type root cultures were established by inoculating hormone-free B5 liquid medium, containing 3% (w/v) sucrose, with excised roots from opium poppy or California poppy seedlings grown in vitro. Root cultures were maintained at 25 °C on a gyratory shaker (100 rpm) in a growth chamber under standard cool white fluorescent tubes (Sylvania Gros-Lux Wide Spectrum) with a flux rate of 35 µmol s-1 m-2 and a 16 h photoperiod. Growth rates were determined by measuring the fresh weight of cultured roots at a 1-week interval. The addition to the culture medium of various concentrations of the auxin analogues indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA) was tested to promote the growth of hairy roots. All experiments were conducted in triplicate and repeated at least twice.
PCR analysis of transformation
Plant genomic DNA for polymerase chain reaction (PCR) analysis was extracted as described by Edwards et al. (Edwards et al., 1991). Roots (50 mg fresh weight) were homogenized in 200 µl of extraction buffer (0.5% [w/v] SDS, 250 mM NaCl, 100 mM TRIS-HCl, pH 8.0, and 25 mM EDTA) and centrifuged at 13 000 rpm for 5 min. The supernatant was transferred to a new tube and an equal volume of isopropanol was added. The sample was incubated on ice for 5 min and then centrifuged for 10 min at 13 000 rpm. The pellet was dried at 60 °C for 10 min, and then resuspended in 100 µl of TE buffer (10 mM TRIS-HCl, pH 7.4 and 1.0 mM EDTA). PCR was performed for 30 thermal cycles (denaturation at 95 °C for 1 min, primer annealing at 55 °C for 1 min, and primer extension at 72 °C for 1 min) using primers specific to sequences found in the neomycin phosphotransferase (NTPII) selectable marker gene (5'-TATGTTATGTATGTGCAGATGATT-3' and 5'-GTCGACTCACCCGAAGAACTCGTC-3'). Amplification products were analysed on 1% (w/v) agarose gels.
Assay of ß-glucuronidase activity
Cultured roots were ground with extraction buffer (50 mM KPO4 buffer, pH 7.0, 1 mM EDTA, and 10 mM ß-mercaptoethanol) in an Eppendorf tube. 4-Methylumbelliferyl-ß-D-glucuronide (MUG) was added to a final concentration of 0.44 mg ml-l to the ß-glucuronidase (GUS) flurometric assay buffer (50 mM NaPO4 buffer, pH 7.0, 10 mM ß-mercaptoethanol, 10 mM EDTA, 0.1% [w/v] sodium lauryl sarcosine, and 0.1% [w/v] Triton X-100). Assays were performed on 50 µl of tissue extract for 3 h at 37 °C and stopped with a 10x volume of 0.2 M Na2CO3. A fluorescence spectrophotometer (F-2000, Hitachi, Tokyo, Japan) was used to quantify the amount of 4-methylumbelliferone (MU) cleaved from MUG. Protein concentration was determined according to Bradford (Bradford, 1976) with BSA as the standard.
RNA gel blot hybridization
Total RNA for gel blot hybridization analysis was isolated using the method of Logemann et al. (Logemann et al., 1987), and 15 µg was fractionated on a 1.0% formaldehyde agarose gel before transfer to nylon membrane (Sambrook et al., 1989). Blots were hybridized with a random-primer 32P-labelled (Feinberg and Vogelstein, 1984) full-length GUS probe. Hybridization was performed at 65 °C in 0.25 mM sodium phosphate buffer, pH 8.0, 7% (w/v) SDS, 1% (w/v) BSA, and 1 mM EDTA. Blot was washed at 65 °C, twice with 2x SSC and 0.1% (w/v) SDS and twice with 0.2x SSC and 0.1% (w/v) SDS (Sambrook et al., 1989; 1x SSC=0.15 M NaCl and 0.015 M sodium citrate, pH 7.0), and autoradiographed with an intensifying screen at -80 °C for 24 h.
ß-Glucuronidase histochemical staining
Histochemical staining for GUS activity was performed as described by Jefferson (Jefferson, 1987) with modifications recommended by Kosugi et al. (Kosugi et al., 1990). Roots were fixed in a 0.35% (v/v) formaldehyde solution containing 10 mM MES, pH 7.5, and 300 mM mannitol for 1 h at 20 °C, rinsed three times in 50 mM sodium phosphate, pH 7.5, and subsequently incubated in 50 mM sodium phosphate, pH 7.5, 10 mM EDTA, 300 mM mannitol, pH 7.0, and 1 mM 5-bromo-4-chloro-3-indolyl-ß-D-glucuronide cyclohexylammonium salt for 6–12 h at 37 °C. Stained roots were rinsed extensively in 70% ethanol to remove residual phenolic compounds.
HPLC analysis of benzylisoquinoline alkaloids
Alkaloids from wild-type and hairy roots of opium poppy and California poppy were extracted by grinding 0.5 g fresh weight of tissue with 95% (v/v) methanol in Eppendorf tubes. After filtration to remove insoluble debris, the extracts were reduced to dryness under vacuum, and re-dissolved in 50 µl of methanol. Samples were analysed by HPLC on a liquid chromatography system (System Gold 126, Beckman-Coulter, Mississauga, Canada) and photodiode array detector (System Gold 168, Beckman-Coulter), using a C18 reverse phase column (4.6x250 mm; Ultrasphere, Beckman-Coulter) at 1200 psi. Opium poppy extracts were separated at a flow rate of 0.75 ml min-1 using a stepped gradient of methanol : water (6 : 4 for 15 min, ramped to 9 : 1 over 5 min, maintained at 9 : 1 for 10 min, ramped to 6 : 4 over 5 min, maintained at 6 : 4 for 5 min) containing 0.1% (v/v) triethylamine. California poppy extracts were separated at a flow rate of 0.75 ml min-1 using an isocratic gradient of methanol : water (8 : 2) containing 0.1% (v/v) triethylamine. Peaks for morphine, noscapine, and sanguinarine were identified from their UV spectra and by comparison of their retention times to those of authentic standards.
Histological preparations
Roots were fixed in 1.6% (v/v) paraformaldehyde and 2.5% (v/v) glutaraldehyde in 50 mM phosphate buffer, pH 6.8, for 24 h at 4 °C. After fixation, the roots were dehydrated in methyl Cellosolve, followed by two changes of absolute ethanol, and embedded in Historesin (Leica, Toronto, Canada). Serial sections (3 µm) were cut with a glass knife on a 2040 Autocut (Reichert-Jung, Heidelberg, Germany) rotary microtome. The sections were stained using the Periodic acid–Schiff's (PAS) reaction for total carbohydrates and counter-stained with amido black 10B for proteins, or toluidine blue O for general histological organization (Yeung, 1984). Root anatomy was observed and photographed using an Aristoplan (Leitz, Willowdale, Canada) microscope.
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Results |
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Establishment of hairy root cultures
Five different strains of A. rhizogenes were tested for their ability to induce the formation of hairy roots on opium poppy and California poppy explants. Wounded opium poppy seedlings were highly susceptible to infection by each strain of A. rhizogenes, as shown by the percentage of seedlings from which paromomycin-resistant tissues emerged (Table 1
). Strains 13333, R1000 and R1200rolD infected more than 90% of the seedlings and induced an average of three to four hairy root initials per seedling within 4 weeks. In contrast, strains 15834 and C58C1 infected 80–85% of exposed seedlings, but caused the formation of paromomycin-resistant tumorigenic calli instead of hairy roots. Although the values for infection frequency and number of hairy roots per seedling were almost identical for strains 13333, R1000 and R1200rolD, hairy roots produced by infection with strain R1000 grew faster than those produced by strains 13333 and R1200rolD (Table 1).
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Wounded California poppy seedlings were also susceptible to infection by A. rhizogenes strains 13333, R1000 and R1200rolD, and several paromomycin-resistant hairy roots were produced from each explant (Table 2
). However, wounded embryogenic callus of California poppy was selected as the superior explant tissue for co-cultivation with A. rhizogenes because it displayed a greater frequency of infection, and the resulting hairy roots grew more rapidly than those derived from seedlings (Table 2). Strains R1000 and R1200rolD caused infections on almost all exposed embryogenic calli, and each induced approximately six hairy root initials per callus within 7 d. In contrast to their effect on opium poppy seedlings, strains 15834 and C58C1 produced no response on California poppy embryogenic calli (Table 2) or seedlings. However, consistent with the opium poppy data was the determination that hairy roots of California poppy induced by strain R1000 grew more rapidly than those produced by strains 13333 and R1200rolD, despite similarities in infection frequency and the number of roots per explant (Table 2). Based on these results, R1000 was selected as the optimal strain for the induction of opium poppy and California poppy hairy roots.
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Except for the co-cultivation medium, all formulations used in subsequent steps included the antibiotics paromomycin for the selection of transformed plant tissues, and timentin to eliminate the Agrobacterium after co-culture. In preliminary experiments, the effects of two different aminoglycoside antibiotics, which are both inactivated by the NPTII gene product, on the growth of untransformed opium poppy and California poppy root cultures were examined. At concentrations between 10 and 200 mg l-1, kanamycin did not inhibit the growth of cultured roots from either species. In contrast, paromomycin progressively inhibited root growth at concentrations from 5–50 mg l-1 in both species; thus, paromomycin was used for the selection of transformed hairy roots at a final concentration of 50 mg l-1. The ineffectiveness of kanamycin, and the efficacy of paromomycin, for the selection of transformed opium poppy tissues has been reported previously (Belny et al., 1997
). The sensitivity of opium poppy and California poppy callus cultures to timentin, which is comprised of a combination of ß-lactam and ß-lactamase inhibitors, was also tested at concentrations up to 400 mg l-1. The plant cells were unaffected by 400 mg l-1 timentin, and the antibiotic was effective at eliminating Agrobacterium in hairy root cultures at a concentration of 200 mg l-1.
After 2 d of co-cultivation with A. rhizogenes strain R1000, explant tissues were transferred to agar-solidified, hormone-free selection medium. Hairy root initials emerged from wound sites on opium poppy seedlings (Fig. 1A) and California poppy embryogenic calli (Fig. 1D) within 5–7 d after inoculation. After 10–14 d, putative transgenic hairy roots of opium poppy (Fig. 1B) and California poppy (Fig. 1E) began to grow more rapidly. About 4–5 weeks after co-cultivation with A. rhizogenes, hairy roots from both species were excised from the necrotic explant tissues and subcultured on fresh agar-solidified selection medium (Fig. 1C, F). Mature California poppy roots were generally thicker, exhibited more prolific branching and produced a greater abundance of root hairs compared to mature opium poppy roots, as shown in Fig. 1C and F. After repeated transfer to fresh selection medium for 2–3 months, rapidly growing hairy root cultures of opium poppy and California poppy were transferred to liquid culture medium containing 50 mg l-1 paromomycin and 200 mg l-1 timentin.
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Analysis of transformation
The complete and stable transformation of paromomycin-resistant root cultures was evaluated by determining (1) the histochemical localization of GUS activity in various root tissues; (2) the integration of the NPTII gene into the plant genome; (3) the presence of GUS mRNAs; and (4) the level of GUS enzyme activity. PCR performed using primers specific for sequences in the NTPII gene resulted in the amplification of a single amplicon with the expected size of 823 bp in 18 out of 20 paromomycin-resistant opium poppy hairy root cultures, and 17 out of 20 paromomycin-resistant California poppy hairy root cultures, that were tested. Histochemical staining for GUS activity was performed to determine whether A. rhizogenes strain R1000 produced complete root transformation. The cauliflower mosaic virus 35S promoter-GUS fusion contained in the pBI 121 binary vector should result in constitutive GUS activity in all cell types of paromomycin-resistant tissues. Strong GUS staining was visible in the growing root tips and vascular tissues of young (Fig. 1G
) and mature (Fig. 1H) NPTII-positive hairy roots of opium poppy produced after co-cultivation of wild-type seedlings with A. rhizogenes strain R1000. GUS staining was also detected in cortical tissues of opium poppy hairy roots, but at much lower levels than those observed in the stele and meristematic regions. This pattern of GUS staining was consistent in all NPTII-positive hairy roots of opium poppy. Strong GUS staining occurred in all tissues of young (Fig. 1I) and mature (Fig. 1J) NPTII-positive hairy roots of California poppy produced after co-cultivation of wild-type embryogenic calli with A. rhizogenes strain R1000. An abundance of GUS-positive lateral root primordia were visible emerging from the pericycle of California poppy hairy roots (Fig. 1J). GUS staining was not detected in wild-type roots from either species.
Two randomly-selected, NTPII-positive and rapidly growing hairy root lines of each species were tested to confirm the presence of GUS transcripts (Fig. 2). RNA gel blot hybridization analysis revealed high levels of GUS transcripts in each of the putative transgenic hairy root cultures, although GUS mRNAs were more abundant in some hairy root lines than in others (Fig. 2). No signal was detected with the GUS probe in wild-type roots of either species. Ten independent, randomly-selected NTPII-positive hairy root cultures from both opium poppy and California poppy were also tested for GUS enzyme activity levels compared to wild-type root cultures. Transgenic hairy roots contained much higher GUS activity levels than non-transformed roots, which exhibited only background activity (Fig. 3). Individual transformants expressed a wide range of GUS activities, from 250–1220 MU min-1 mg-1 protein for opium poppy (Fig. 3A) and from 230–1450 MU min-1 mg-1 protein for California poppy (Fig. 3B). The observed distribution in GUS transcript and enzyme activity levels is a common phenomenon in transformed plant tissues due to a combination of several factors including transgene copy number, the location of chromosomal insertion, or a variety of post-translational effects.
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Optimization of culture medium for transgenic root growth
Various concentrations of different auxin analogues were added to the liquid culture medium to promote the rapid growth of opium poppy and California poppy transgenic root cultures. In both species, auxin treatment increased the growth rate of hairy roots (Table 3). Although rapid growth rates were induced by 1.0 mg l-1 IBA or 0.5–1.0 mg l-1 NAA, these conditions also caused the formation of callus tissue on the transgenic roots. The addition of 1.0 mg l-1 IAA, which was just as effective as IBA or NAA but did not induce callus formation, to B5 liquid medium containing 3% (w/v) sucrose was used to promote the growth rates of root cultures. Under these conditions, transgenic roots of both species grew 25–30% faster than wild-type roots, and California poppy roots grew approximately 130% more rapidly than opium poppy roots (Fig. 4).
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Comparison of wild-type and transgenic roots
The histologies of wild-type and transformed roots of opium poppy and California poppy were nearly identical with the exception of the arrangement and structure of epidermal cells (Fig. 5). All roots possessed a central stele of vascular tissues, surrounded by a few layers of cortical cells, and an outer layer of epidermis. The epidermal cells of wild-type roots from both species were densely packed and produced relatively few root hair extensions (Fig. 5A, C). In contrast, epidermal cells from transformed roots were more loosely organized and gave rise to a large number of root hair extensions (Fig. 5B, C), compared to wild-type roots.
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The benzylisoquinoline alkaloid content of transformed root cultures was compared to that of wild-type root cultures. Although the only available authentic standards were morphine (peak a in Fig. 6A
, B), noscapine (peak c in Fig. 6A, B), and sanguinarine (peak f in Fig. 6A, B and peak i in Fig. 6C, D), several other chromatographic peaks displayed UV spectra with characteristic benzylisoquinoline and benzophenanthridine signatures. HPLC analysis showed that the relative concentration of morphine, noscapine, sanguinarine, and other putative alkaloids were virtually identical in wild-type (Fig. 6A) and transgenic roots (Fig. 6B) of opium poppy. Similarly, the relative concentrations of sanguinarine and other putative benzophenanthridine alkaloids were also nearly identical in wild-type (Fig. 6C) and transformed roots (Fig. 6D) of California poppy. The ‘normal’ alkaloid profile of the opium poppy and California poppy hairy roots shows that this study's transformation protocol could serve as a valuable tool to study the regulation of genes encoding alkaloid biosynthetic enzymes, and the network architecture of the pathway via genetically engineered alterations in the activity of individual enzymes.
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Discussion |
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Soil-borne pathogens of the genus Agrobacterium are able to transfer part of their DNA, the T-DNA carried on a large plasmid, to the genome of a host plant cell. Agrobacterium rhizogenes is the causal agent of ‘hairy root’ diseases in plants, and has been used for the production of hairy root cultures from a multitude of species. Over the last decade, transformed root cultures from plants have attracted considerable attention because of their genetic and biochemical stability, rapid growth rate and ability to synthesize secondary products at levels comparable to wild-type roots. An efficient A. rhizogenes-mediated protocol has been developed for the establishment of transgenic opium poppy and California poppy hairy root cultures. Of five A. rhizogenes strains tested, R1000 was found to be the most virulent and caused the formation of hairy roots exhibiting the most rapid growth rates (Tables 1
, 2). Two commonly used strains, 15834 and C58C1, were highly virulent only on opium poppy, but resulted in the formation of tumorigenic calli rather than roots. The growth rates of transformed roots of both species was improved by the addition of auxin to the liquid culture medium (Table 3). Exogenous application of auxin was also reported to stimulate growth in hairy root cultures of Lippia dulcis Trev. (Sauerwein et al., 1991). The minimum doubling times of approximately 2 weeks for California poppy and 3–4 weeks for opium poppy are comparable to hairy root cultures of other species (Loyola-Vargas and Miranda-Ham, 1995).
Several other studies have also reported the differential efficiency of various A. rhizogenes strains in promoting the formation and growth of hairy roots. For example, in addition to their variable ability to induce hairy root development, different A. rhizogenes strains also affected growth rate, saponin production and the ratio of different astragalosides in transgenic root cultures of Astragalus mongholicus Bge. (Ionkova et al., 1997). Strain 15834 was among the most effective at promoting hairy root growth and saponin synthesis. The strain of Agrobacterium also influenced development, growth rate and hyoscyamine production in transformed root cultures of H. muticus (Vanhala et al., 1995). C58C1 was among the most virulent strains, and also resulted in root cultures with the highest alkaloid content. In contrast, strain 15834 was the least effective for the induction of hairy roots of H. muticus. Transgenic root formation in pea (Pisum sativum L.) was dependent on both the strain of A. rhizogenes and the genotype of the host plant (Nicoll et al., 1995). In pea, strains R1000nal, which is derived from R1000, and 15834 were most effective for the promotion of hairy root formation. Strain 15834 was also the most virulent and efficient for hairy root development in Catharanthus roseus G. Don. (Brillanceau et al., 1989). Clearly, the selection of an effective Agrobacterium strain for the production of transformed root cultures is highly dependent on the plant species, and must be determined empirically. The differences in virulence, morphology and growth rate are at least partially related to the variety of plasmids contained within each bacterial strain.
The combined demonstration that in paromomycin-resistant roots, the NPTII gene was integrated into plant genomic DNA, high GUS transcript and enzyme activity levels were detected (Figs 2, 3), and the GUS protein was ubiquitous in hairy root tissues (Fig. 1) provides unequivocal proof that the stable and integrative transformation of opium poppy and California poppy root cultures has indeed been achieved. This represents, to the authors' knowledge, the first reported protocol for the transformation of root cultures not only from any member of the Papaveraceae, but from any benzylisoquinoline alkaloid-producing plant. Previously, de-differentiated transgenic cell cultures of opium poppy have been produced using A. tumefaciens (Belny et al., 1997) and A. rhizogenes (Yoshimatsu and Shimomura, 1992; Williams and Ellis, 1993). Infection of opium poppy hypocotyls with A. rhizogenes MAFF 03–01724 (Yoshimatsu and Shimomura, 1992) and an unidentified strain (Williams and Ellis, 1993) led to the formation of tumorigenic calli, rather than hairy roots, as observed with strains 15834 and C58C1 (Tables 1, 2). In one study, numerous adventitious shoots developed from the transformed callus, but the transgenic shoots were morphologically different from wild-type shoots (Yoshimatsu and Shimomura 1992). Moreover, the relative content of morphine and codeine was altered in transgenic shoots compared to non-transformed shoots. In contrast, transgenic suspension cultures derived via A. rhizogenes-mediated transformation did not exhibit differences in growth or alkaloid accumulation, with sanguinarine as the major product (Williams and Ellis, 1993).
The quantity and ratio of different benzylisoquinoline alkaloids produced by opium poppy and California poppy were virtually identical in transformed and wild-type roots (Fig. 6). Transformed root cultures of several other species have been evaluated for their content of alkaloids or other secondary metabolites relative to wild-type roots. Although the profile of secondary products is often conserved, the concentration of specific compounds is sometimes altered by transformation with armed A. rhizogenes strains. For example, hairy roots of ginseng (Panax ginseng Meyer) produced the same saponins and ginsenosides as wild-type roots, but in quantities that were 2-fold higher on a dry weight basis (Yoshikawa and Furuya, 1987). Similarly, Korean balloon flower (Platycodon grandiflorum A. DC.) hairy roots produced the polyacetylenes lobetyolin and lobetyolinin at levels 160- and 2.6-fold higher, respectively, than those found in wild-type roots (Ahn et al., 1996). In contrast, the productivity of the naphthoquinone shikonin in hairy root cultures of Lithospermum erythrorhizon Sieb. et Zucc. was similar to that of wild-type cell cultures, and displayed the same light-dependent control of biosynthesis (Yazaki et al., 1998). Similarly, A. belladonna root cultures have been shown to accumulate hyoscyamine at levels similar to those of wild-type roots (Sharp and Doran, 1990), but were also reported to synthesize the unusual tropane alkaloid littorine which is not found in non-transformed roots (Aoki et al., 1997). Moreover, hairy root cultures of C. roseus accumulate the monoterpenoid indole alkaloids catharathine and ajmalicine at levels that are also similar to those found in wild-type roots (Brillanceau et al., 1989; Toivonen et al., 1989; Vázquez-Flota et al., 1994).
Genetically engineered root cultures have been used as a model system to study various aspects of the metabolic and molecular regulation of several natural product pathways. For example, expression of a cDNA encoding Antirrhinum majus L. dihydroflavonol reductase in hairy roots of Lotus corniculatus L. caused an increase in the content, and an alteration in the structure, of condensed tannins in a manner consistent with the substrate specificity of the transgene product (Bavage et al., 1997). In another study, the efficiency of the maize Sn gene, which transactivates the anthocyanin pathway in various tissues, at regulating anthocyanin biosynthesis in several dicotyledonous species was tested using transformed root cultures (Damiani et al., 1998). The Sn gene was capable of inducing anthocyanin biosynthesis in some heterologous roots, such as alfalfa (Medicago sativa L.) and lotus (Lotus angustissimus L.), but not in others, such as petunia (Petunia hybrida L.). A third interesting example involves the introduction of a heterologous tryptophan decarboxylase gene into hairy root cultures of Peganum harmala L., which accumulate two biosynthetically related, tryptamine-derived secondary metabolites: serotonin and ß-carboline alkaloids (Berlin et al., 1993). Although serotonin accumulation in transgenic root cultures with elevated TDC activity was higher than in control cultures, ß-carboline alkaloid levels were not affected; thus, tryptamine supply was shown to be limiting for serotonin, but not for ß-carboline alkaloid, biosynthesis. Finally, transformed L. erythrorhizon root cultures expressing a bacterial gene for chorismate pyruvate-lyase, which converts chorismate to the shikonin precursor 4-hydroxybenzoate (4HB), did not produce higher levels of shikonin (Sommer et al., 1999). These results suggest that the availability of 4HB does not normally limit the biosynthesis of shikonin.
The availability of an expanding collection of genes encoding benzylisoquinoline alkaloid biosynthetic enzymes, coupled with a protocol for the production of rapidly growing transgenic root cultures of opium poppy and California poppy, provide a powerful and versatile model system similar to those described above to investigate the molecular regulation of benzylisoquinoline alkaloid biosynthesis, and to evaluate the potential to metabolically engineer these important medicinal plants.
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We thank Victor Loyola-Vargas and Felipe Vázquez-Flota (Centro de Investigación Cientifica de Yucatán, México) for providing the Agrobacterium rhizogenes strains used in this work, and Ed Yeung (University of Calgary) for his assistance with the microtechniques and photomicroscopy. This research was funded by a Natural Sciences and Engineering Research Council of Canada grant to PJF. SUP was supported, in part, by Midland-Walwyn, Nesbitt-Burns, and Bettina Bahlsen Memorial Graduate Scholarships awarded through the University of Calgary.
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1 To whom correspondence should be addressed. Fax: +1 403 289 9311. E-mail: pfacchin{at}ucalgary.ca
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