Agrobacterium rhizogenes-mediated transformation of opium poppy ...

Journal of Experimental Botany, Vol. 51, No. 347, pp. 1005–1016, June 2000

Agrobacterium rhizogenes-mediated transformation ofopium poppy, Papaver somniferum L., and Californiapoppy, Eschscholzia californica Cham., root cultures

Sang-Un Park and Peter J. Facchini1

Department of Biological Sciences, University of Calgary, Calgary, Alberta, T2N 1N4 Canada

Received 1 November 1999; Accepted 25 January 2000

Abstract cultures of opium poppy and California poppy are asimple, reliable and well-defined model system to

An efficient protocol for the establishment of trans-investigate the molecular and metabolic regulation of

genic opium poppy (Papaver somniferum L.) andbenzylisoquinoline alkaloid biosynthesis, and to evalu-

California poppy (Eschscholzia californica Cham.) rootate the genetic engineering potential of these import-

cultures using Agrobacterium rhizogenes is reported.ant medicinal plants.

Five strains of A. rhizogenes were tested for theirability to produce hairy roots on wounded opium poppy Key words: Agrobacterium rhizogenes, benzylisoquinolineseedlings and California poppy embryogenic calli. alkaloids, California poppy, Eschscholzia californica, hairyThree of the strains induced hairy root formation on root cultures, opium poppy, Papaver somniferum.both species, whereas two others either caused thegrowth of tumorigenic calli or produced no response.

IntroductionTo characterize the putative transgenic roots further,explant tissues were co-cultivated with the most Benzylisoquinoline alkaloids are a diverse group of nitro-effective A. rhizogenes strain (R1000) carrying the genous compounds with a restricted taxonomic distribu-pBI121 binary vector. Except for the co-cultivation tion in plants. In addition to various ecophysiologicalmedium, all formulations included 50 mg l−1 paromo- functions related to plant–pathogen and plant–herbivoremycin to select for transformants and 200 mg l−1 interactions, most benzylisoquinoline alkaloids exhibittimentin to eliminate the Agrobacterium. Four weeks potent pharmacological activity and many are used asafter infection, paromomycin-resistant roots appeared pharmaceuticals. Examples include the analgesics mor-on 92–98% of explants maintained on hormone-free phine and codeine, the antibiotic sanguinarine, themedium. Isolated hairy roots were propagated in liquid gout suppressant colchicine, and the muscle relaxantmedium containing 1.0 mg l−1 indole-3-acetic acid to (+)-tubocurarine. The total chemical synthesis of thesepromote rapid growth. Detection of the neomycin valuable compounds is difficult because of their structuralphosphotransferase gene, high levels of b-glucuronid- complexity; thus, wild or cultivated plants remain theirase (GUS) transcripts and enzyme activity, and GUS only commercial source.histochemical localization confirmed the integrative Benzylisoquinoline alkaloids are ubiquitous among thetransformation of root cultures. Transgenic roots grew Papaveraceae, which include the California poppyfaster than wild-type roots, and California poppy roots (Eschscholzia californica Cham.) and the opium poppygrew more rapidly than those of opium poppy. With (Papaver somniferum L.). The California poppy is athe exception of a less compact arrangement of epi- traditional medicinal plant of many indigenous peoplesdermal cells and more root hairs, transformed roots of in North America (Cheney, 1964). The remedial proper-both species displayed anatomical features and ties of this now common ornamental species result frombenzylisoquinoline alkaloid profiles that were virtually its ability to synthesize a variety of benzophenanthridine

alkaloids, which represent a subgroup of benzylisoquino-identical to those of wild-type roots. Transgenic root

1 To whom correspondence should be addressed. Fax: +1 403 289 9311. E-mail: [email protected]

© Oxford University Press 2000

1006 Park and Facchini

line alkaloids restricted in distribution to the and a variety of other secondary metabolites. Forexample, transformed root cultures derived from membersPapaveraceae and Fumariaceae. Sanguinarine is one of

the principal benzophenanthridine alkaloids found in of the Solanaceae have been used extensively to study theproduction of tropane alkaloids and nicotine (Aoki et al.,California poppy roots, and is used commercially as an

antiplaque agent in oral hygeine products due to its 1997; Hamill et al., 1990; Hashimoto et al., 1993;Jouhikainen et al., 1999; Robins et al., 1991; Sharp andpotent antimicrobial activity (Dzink and Socransky,

1985). Benzophenanthridine alkaloid biosynthesis has Doran, 1990). The Agrobacterium-mediated productionof hairy roots also creates a rapid and simple means tobeen studied extensively in California poppy cell suspen-

sion cultures ( Kutchan, 1998) because the pathway can introduce and express foreign genes in plant cells that arecapable of synthesizing specific secondary metabolites.be induced by the addition of fungal elicitors (Schumacher

et al., 1987) or methyl jasmonate (Blechert et al., 1995) For example, this approach has been used to alter theaccumulation of alkaloids normally produced in roots. Into the culture medium. Two cDNAs encoding alkaloid

biosynthetic enzymes in California poppy have been one study, a yeast cDNA encoding ornithine decarb-oxylase (ODC) was introduced into Nicotiana rustica L.cloned, and shown to be transcriptionally induced in

cultured cells in response to elicitor- or methyl jasmonate- hairy roots using an Agrobacterium-derived vector(Hamill et al., 1990). Although the hairy root culturestreatment (Dittrich and Kutchan, 1991; Pauli and

Kutchan, 1998). produced more nicotine than wild-type roots, the increasein ODC activity was proportionately higher than theSanguinarine also accumulates constitutively in opium

poppy roots, and is induced in opium poppy cell suspen- increase in nicotine levels suggesting that ODC is not arate-determining step in the nicotine pathway. In anothersion cultures after treatment with fungal elicitors

(Facchini et al., 1996a). However, opium poppy also case, hyoscyamine-rich hairy roots of Atropa belladonnaL. (Hashimoto et al., 1993) and Hyoscyamus muticus L.produces an abundance of several other benzylisoquino-

line alkaloids including the putative anti-tumorigenic (Jouhikainen et al., 1999) were transformed with a genefrom Hyoscyamus niger L. encoding hyoscyamine-6b-agent noscapine (Ye et al., 1998) and morphine, both of

which accumulate in roots but are more abundant in the hydroxylase (H6H), which catalyses the final epoxidationstep in the conversion of hyoscyamine to scopolamine.latex of shoot organs. However, noscapine and morphine

are not produced in opium poppy cell suspension cultures As expected, the A. belladonna and H. muticus hairy rootscontained high levels of H6H activity and produced,due to an apparent, but poorly understood, requirement

for cell type-specific specialization; thus, de-differentiated almost exclusively, scopolamine.In this paper, the development of an efficient protocolcell cultures are of limited use to investigate the regulation

of most alkaloid pathways in opium poppy. Recently, to introduce foreign genes into transgenic opium poppyand California poppy hairy root cultures usingseveral cDNAs encoding alkaloid biosynthetic enzymes

in opium poppy have also been cloned and, as in Agrobacterium rhizogenes is described. With the exceptionof an increased number of root hairs and a less compactCalifornia poppy, shown to be transcriptionally regulated

(Facchini and De Luca, 1994; Facchini et al., 1996b; Yu arrangement of epidermal cells, it is shown that the hairyroots of both species display anatomical features andand Facchini, 2000; Unterlinner et al., 1999).

Attempts to understand the molecular mechanisms that benzylisoquinoline alkaloid profiles that are virtuallyidentical to those of wild-type roots. Therefore, theseregulate genes encoding benzylisoquinoline alkaloid

biosynthetic enzymes in opium poppy and California rapidly growing transformed hairy root cultures couldserve as a simple, reliable and well-defined model systempoppy have relied on transient expression systems using

microprojectile-bombarded cell suspension cultures to study the molecular regulation of genes encodingbenzylisoquinoline alkaloid biosynthetic enzymes, and to(Hauschild et al., 1998; Park et al., 1999). However, this

approach precludes the study of developmental, and many evaluate the potential to metabolically engineer theseimportant medicinal plants.inducible, mechanisms that control benzylisoquinoline

alkaloid pathways because the wound signal caused bythe entry of DNA-coated microcarriers into the cells

Materials and methodsactivates several of the relevant genes. A more completeapplication of advanced molecular and biochemical Seed sterilization and germinationapproaches to investigate the genetic and metabolic regu- Seeds of P. somniferum cv. Marianne and E. californica cv.

Aurantiaca were surface-sterilized with 70% (v/v) ethanol forlation of benzylisoquinoline alkaloid biosynthesis requires30 s and 2% (v/v) sodium hypochlorite solution for 10 min,the establishment of efficient protocols for the stablethen rinsed three times in sterilized water. Approximately 30transformation of opium poppy and California poppyseeds were placed on 25 ml of agar-solidified culture medium in

plants and differentiated tissue cultures. Petri dishes (100×15 mm). The basal medium consisted of B5Transgenic hairy root cultures have served as a useful salts and vitamins (Gamborg et al., 1968) solidified with 0.8%

(w/v) Phytagar (Gibco, Burlington, Canada). The medium wasmodel system to investigate the biosynthesis of alkaloids,

Transformed poppy root cultures 1007

adjusted to pH 5.8 before adding agar, and then sterilized by new tube and an equal volume of isopropanol was added. Thesample was incubated on ice for 5 min and then centrifuged forautoclaving 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 10 min at 13 000 rpm. The pellet was dried at 60 °C for 10 min,and then resuspended in 100 ml of TE buffer (10 mM TRIS-white fluorescent tubes (Sylvania Gros-Lux Wide Spectrum,

Mississauga, Canada) with a flux rate of 35 mmol s−1 m−2 and HCl, pH 7.4 and 1.0 mM EDTA). PCR was performedfor 30 thermal cycles (denaturation at 95 °C for 1 min, primera 16 h photoperiod.annealing at 55 °C for 1 min, and primer extension at 72 °Cfor 1 min) using primers specific to sequences found in thePreparation of Agrobacterium rhizogenesneomycin phosphotransferase (NTPII ) selectable markerThe binary vector pBI121 (Jefferson et al., 1987) was mobilizedgene (5∞-TATGTTATGTATGTGCAGATGATT-3∞ andby electroporation in the armed Agrobacterium rhizogenes5∞-GTCGACTCACCCGAAGAACTCGTC-3∞). Amplificationstrains 13333, 15834, C58C1, R1000, and R1200rolD.products were analysed on 1% (w/v) agarose gels.Transformed A. rhizogenes cultures were grown to mid-log

phase (A600=0.5) at 28 °C on a gyratory shaker at 180 rpm inAssay of b-glucuronidase activityliquid 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 Cultured roots were ground with extraction buffer (50 mMkanamycin. The bacterial cells were collected by centrifugation KPO4 buffer, pH 7.0, 1 mM EDTA, and 10 mMfor 10 min at 1500 rpm, and resuspended at a cell density of b-mercaptoethanol ) in an Eppendorf tube. 4-Methyl-A600=1.0 in liquid inoculation medium (B5 salts and vitamins umbelliferyl-b--glucuronide (MUG) was added to a finalcontaining 20 g l−1 sucrose). concentration of 0.44 mg ml−l to the b-glucuronidase (GUS)

flurometric assay buffer (50 mM NaPO4 buffer, pH 7.0, 10 mMProduction of transgenic root cultures b-mercaptoethanol, 10 mM EDTA, 0.1% [w/v] sodium lauryl

sarcosine, and 0.1% [w/v] Triton X-100). Assays were performedExcised shoots (i.e. with the roots removed) from 5-d-oldon 50 ml of tissue extract for 3 h at 37 °C and stopped with aopium poppy and California poppy seedlings, and embryogenic10× volume of 0.2 M Na2CO3. A fluorescence spectrophoto-callus cultures of California poppy, were used as the explantmeter (F-2000, Hitachi, Tokyo, Japan) was used to quantifymaterial for co-cultivation with A. rhizogenes. The embryogenicthe amount of 4-methylumbelliferone (MU ) cleaved fromcallus was induced from seed-derived primary callus ofMUG. Protein concentration was determined according toCalifornia poppy as described previously (Park and Facchini,Bradford (Bradford, 1976) with BSA as the standard.2000). Seedlings and embryogenic callus were randomly

wounded using a scalpel, immersed in an A. rhizogenes culturesuspended in liquid inoculation medium for 10–15 min, blotted RNA gel blot hybridizationdry on sterile filter paper, and incubated in the dark on Total RNA for gel blot hybridization analysis was isolatedPhytagar-solidified B5 medium. After 2 d of co-cultivation, the using the method of Logemann et al. (Logemann et al., 1987),explant tissues were transferred to hormone-free selection and 15 mg was fractionated on a 1.0% formaldehyde agarosemedium containing B5 salts and vitamins, 3% (w/v) sucrose, gel before transfer to nylon membrane (Sambrook et al., 1989).50 mg l−1 paromomycin, 200 mg l−1 timentin and 8 g l−1 Blots were hybridized with a random-primer 32P-labelledPhytagar. Within 4–5 weeks, numerous paromomycin-resistant (Feinberg and Vogelstein, 1984) full-length GUS probe.roots had emerged from the wound sites. The hairy roots were Hybridization was performed at 65 °C in 0.25 mM sodiumseparated from the explant tissue and sub-cultured in the dark phosphate buffer, pH 8.0, 7% (w/v) SDS, 1% (w/v) BSA, andat 25 °C on hormone-free selection medium. After repeated 1 mM EDTA. Blot was washed at 65 °C, twice with 2× SSCtransfer to fresh selection medium, rapidly growing hairy root and 0.1% (w/v) SDS and twice with 0.2× SSC and 0.1% (w/v)cultures were obtained. Isolated roots (0.5 g) were transferred SDS (Sambrook et al., 1989; 1× SSC=0.15 M NaCl andto 40 ml of B5 liquid medium, containing 3% (w/v) sucrose, in 0.015 M sodium citrate, pH 7.0), and autoradiographed with125 ml flasks. Wild-type root cultures were established by an intensifying screen at −80 °C for 24 h.inoculating hormone-free B5 liquid medium, containing 3%(w/v) sucrose, with excised roots from opium poppy or

b-Glucuronidase histochemical stainingCalifornia poppy seedlings grown in vitro. Root cultures wereHistochemical staining for GUS activity was performed asmaintained at 25 °C on a gyratory shaker (100 rpm) in a growthdescribed by Jefferson (Jefferson, 1987) with modificationschamber under standard cool white fluorescent tubes (Sylvaniarecommended by Kosugi et al. (Kosugi et al., 1990). RootsGros-Lux Wide Spectrum) with a flux rate of 35 mmol s−1 m−2were fixed in a 0.35% (v/v) formaldehyde solution containingand a 16 h photoperiod. Growth rates were determined by10 mM MES, pH 7.5, and 300 mM mannitol for 1 h at 20 °C,measuring the fresh weight of cultured roots at a 1-weekrinsed three times in 50 mM sodium phosphate, pH 7.5, andinterval. The addition to the culture medium of varioussubsequently incubated in 50 mM sodium phosphate, pH 7.5,concentrations of the auxin analogues indole-3-acetic acid10 mM EDTA, 300 mM mannitol, pH 7.0, and 1 mM 5-bromo-(IAA), indole-3-butyric acid (IBA) and 1-naphthaleneacetic4-chloro-3-indolyl-b--glucuronide cyclohexylammonium saltacid (NAA) was tested to promote the growth of hairy roots.for 6–12 h at 37 °C. Stained roots were rinsed extensively inAll experiments were conducted in triplicate and repeated at70% ethanol to remove residual phenolic compounds.least twice.

PCR analysis of transformation HPLC analysis of benzylisoquinoline alkaloids

Alkaloids from wild-type and hairy roots of opium poppy andPlant genomic DNA for polymerase chain reaction (PCR)analysis was extracted as described by Edwards et al. (Edwards California poppy were extracted by grinding 0.5 g fresh weight

of tissue with 95% (v/v) methanol in Eppendorf tubes. Afteret al., 1991). Roots (50 mg fresh weight) were homogenized in200 ml of extraction buffer (0.5% [w/v] SDS, 250 mM NaCl, filtration to remove insoluble debris, the extracts were reduced

to dryness under vacuum, and re-dissolved in 50 ml of methanol.100 mM TRIS-HCl, pH 8.0, and 25 mM EDTA) and centrifugedat 13 000 rpm for 5 min. The supernatant was transferred to a Samples were analysed by HPLC on a liquid chromatography


system (System Gold 126, Beckman-Coulter, Mississauga, instead of hairy roots. Although the values for infectionCanada) and photodiode array detector (System Gold 168, frequency and number of hairy roots per seedling wereBeckman-Coulter), using a C18 reverse phase column

almost identical for strains 13333, R1000 and R1200rolD,(4.6×250 mm; Ultrasphere, Beckman-Coulter) at 1200 psi.hairy roots produced by infection with strain R1000 grewOpium poppy extracts were separated at a flow rate of

0.75 ml min−1 using a stepped gradient of methanol5water (654 faster than those produced by strains 13333 andfor 15 min, ramped to 951 over 5 min, maintained at 951 for R1200rolD (Table 1).10 min, ramped to 654 over 5 min, maintained at 654 for 5 min) Wounded California poppy seedlings were also suscept-containing 0.1% (v/v) triethylamine. California poppy extracts

ible to infection by A. rhizogenes strains 13333, R1000were separated at a flow rate of 0.75 ml min−1 using an isocraticand R1200rolD, and several paromomycin-resistant hairygradient of methanol5water (852) containing 0.1% (v/v)

triethylamine. Peaks for morphine, noscapine, and sanguinarine roots were produced from each explant (Table 2).were identified from their UV spectra and by comparison of However, wounded embryogenic callus of Californiatheir retention times to those of authentic standards. poppy was selected as the superior explant tissue for

co-cultivation with A. rhizogenes because it displayed aHistological preparationsgreater frequency of infection, and the resulting hairyRoots were fixed in 1.6% (v/v) paraformaldehyde and 2.5%roots grew more rapidly than those derived from seedlings(v/v) glutaraldehyde in 50 mM phosphate buffer, pH 6.8, for(Table 2). Strains R1000 and R1200rolD caused infec-24 h at 4 °C. After fixation, the roots were dehydrated in methyl

Cellosolve, followed by two changes of absolute ethanol, and tions on almost all exposed embryogenic calli, and eachembedded in Historesin (Leica, Toronto, Canada). Serial induced approximately six hairy root initials per callussections (3 mm) were cut with a glass knife on a 2040 Autocut within 7 d. In contrast to their effect on opium poppy(Reichert-Jung, Heidelberg, Germany) rotary microtome. The

seedlings, strains 15834 and C58C1 produced no responsesections were stained using the Periodic acid–Schiff ’s (PAS)on California poppy embryogenic calli (Table 2) or seed-reaction for total carbohydrates and counter-stained with amido

black 10B for proteins, or toluidine blue O for general lings. However, consistent with the opium poppy datahistological organization (Yeung, 1984). Root anatomy was was the determination that hairy roots of Californiaobserved and photographed using an Aristoplan (Leitz, poppy induced by strain R1000 grew more rapidly thanWillowdale, Canada) microscope.

those produced by strains 13333 and R1200rolD, despitesimilarities in infection frequency and the number of roots

Results per explant (Table 2). Based on these results, R1000 wasselected as the optimal strain for the induction of opiumEstablishment of hairy root culturespoppy and California poppy hairy roots.

Five different strains of A. rhizogenes were tested for their Except for the co-cultivation medium, all formulationsability to induce the formation of hairy roots on opium used in subsequent steps included the antibiotics paromo-poppy and California poppy explants. Wounded opium mycin for the selection of transformed plant tissues, andpoppy seedlings were highly susceptible to infection by timentin to eliminate the Agrobacterium after co-culture.each strain of A. rhizogenes, as shown by the percentage In preliminary experiments, the effects of two differentof seedlings from which paromomycin-resistant tissues aminoglycoside antibiotics, which are both inactivated byemerged (Table 1). Strains 13333, R1000 and R1200rolD the NPTII gene product, on the growth of untransformedinfected more than 90% of the seedlings and induced an opium poppy and California poppy root cultures wereaverage of three to four hairy root initials per seedling examined. At concentrations between 10 and 200 mg l−1,within 4 weeks. In contrast, strains 15834 and C58C1 kanamycin did not inhibit the growth of cultured rootsinfected 80–85% of exposed seedlings, but caused the from either species. In contrast, paromomycin progress-

ively inhibited root growth at concentrations fromformation of paromomycin-resistant tumorigenic calli

Table 1. Effect of different strains of Agrobacterium rhizogenes on the frequency of infection and the growth of opium poppy hairyroot cultures

Values represent the mean±SD of three independent measurements 28 d after inoculation. Approximately 50 seedlings were examined for eachmeasurement.

Agrobacterium strain Infection frequencya Number of hairy roots Root length(%) (or tumours) (cm)

13333 90 3.3±1.4 2.6±0.315834 85 3.4±1.1b —C58C1 81 3.0±1.3b —R1000 92 4.1±1.7 3.2±0.2R1200rolD 91 3.5±1.3 2.8±0.3

aPercentage of seedlings from which paromomycin-resistant tissues emerged.bCaused the induction of tumours.


Table 2. Effect of different strains of Agrobacterium rhizogenes and explant tissues on the frequency of infection and the growth ofCalifornia poppy hairy root cultures

Values represent the mean±SD of three independent measurements 28 d after inoculation. Approximately 50 seedlings were examined for eachmeasurement.

Explant tissue Agrobacterium strain Infection frequencya Number of hairy roots Root length(%) (cm)

Embryogenic callus 13333 95 5.4±1.8 3.3±0.3Embryogenic callus 15834 0 — —Embryogenic callus C58C1 0 — —Embryogenic callus R1000 96 6.2±2.2 4.1±0.2Embryogenic callus R1200rolD 98 5.8±2.0 3.7±0.3Seedling R1000 93 4.6±1.7 3.6±0.6Seedling R1200rolD 92 4.2±1.4 3.4±0.4

aPercentage of calli from which paromomycin-resistant roots emerged.

5–50 mg l−1 in both species; thus, paromomycin was used (4) the level of GUS enzyme activity. PCR performedfor the selection of transformed hairy roots at a final using primers specific for sequences in the NTPII geneconcentration of 50 mg l−1. The ineffectiveness of kana- resulted in the amplification of a single amplicon with themycin, and the efficacy of paromomycin, for the selection expected size of 823 bp in 18 out of 20 paromomycin-of transformed opium poppy tissues has been reported resistant opium poppy hairy root cultures, and 17 out ofpreviously (Belny et al., 1997). The sensitivity of opium 20 paromomycin-resistant California poppy hairy rootpoppy and California poppy callus cultures to timentin, cultures, that were tested. Histochemical staining for GUSwhich is comprised of a combination of b-lactam and activity was performed to determine whether A. rhizogenesb-lactamase inhibitors, was also tested at concentrations strain R1000 produced complete root transformation.up to 400 mg l−1. The plant cells were unaffected by The cauliflower mosaic virus 35S promoter-GUS fusion400 mg l−1 timentin, and the antibiotic was effective at contained in the pBI 121 binary vector should result ineliminating Agrobacterium in hairy root cultures at a constitutive GUS activity in all cell types of paromomy-concentration of 200 mg l−1. cin-resistant tissues. Strong GUS staining was visible in

After 2 d of co-cultivation with A. rhizogenes strain the growing root tips and vascular tissues of youngR1000, explant tissues were transferred to agar-solidified, (Fig. 1G) and mature (Fig. 1H) NPTII-positive hairyhormone-free selection medium. Hairy root initials roots of opium poppy produced after co-cultivation ofemerged from wound sites on opium poppy seedlings wild-type seedlings with A. rhizogenes strain R1000. GUS(Fig. 1A) and California poppy embryogenic calli staining was also detected in cortical tissues of opium(Fig. 1D) within 5–7 d after inoculation. After 10–14 d, poppy hairy roots, but at much lower levels than thoseputative transgenic hairy roots of opium poppy (Fig. 1B) observed in the stele and meristematic regions. Thisand California poppy (Fig. 1E) began to grow more pattern of GUS staining was consistent in all NPTII-rapidly. About 4–5 weeks after co-cultivation with A. positive hairy roots of opium poppy. Strong GUS stainingrhizogenes, hairy roots from both species were excised occurred in all tissues of young (Fig. 1I ) and maturefrom the necrotic explant tissues and subcultured on fresh (Fig. 1J ) NPTII-positive hairy roots of California poppyagar-solidified selection medium (Fig. 1C, F). Mature produced after co-cultivation of wild-type embryogenicCalifornia poppy roots were generally thicker, exhibited calli with A. rhizogenes strain R1000. An abundance ofmore prolific branching and produced a greater abund- GUS-positive lateral root primordia were visible emergingance of root hairs compared to mature opium poppy from the pericycle of California poppy hairy rootsroots, as shown in Fig. 1C and F. After repeated transfer (Fig. 1J ). GUS staining was not detected in wild-typeto fresh selection medium for 2–3 months, rapidly grow- roots from either species.ing hairy root cultures of opium poppy and California Two randomly-selected, NTPII-positive and rapidlypoppy were transferred to liquid culture medium con- growing hairy root lines of each species were tested totaining 50 mg l−1 paromomycin and 200 mg l−1 timentin. confirm the presence of GUS transcripts (Fig. 2). RNA

gel blot hybridization analysis revealed high levels ofAnalysis of transformation GUS transcripts in each of the putative transgenic hairy

root cultures, although GUS mRNAs were more abund-The complete and stable transformation of paromomycin-ant in some hairy root lines than in others (Fig. 2). Noresistant root cultures was evaluated by determining (1)signal was detected with the GUS probe in wild-typethe histochemical localization of GUS activity in variousroots of either species. Ten independent, randomly-root tissues; (2) the integration of the NPTII gene into

the plant genome; (3) the presence of GUS mRNAs; and selected NTPII-positive hairy root cultures from both


Fig. 1. Development of hairy roots from opium poppy seedlings (A–C ) and California poppy embryogenic calli (D–F ) after inoculation withAgrobacterium rhizogenes strain R1000. (A, D) 7 d after inoculation; (B, E) 14 d after inoculation; (C, F) 28 d after inoculation. Histochemicalstaining of opium poppy (G, H) and California poppy (I, J ) hairy root tissue transformed with the GUS gene. (G, I ) Paromomycin-resistant rootsinduced on the surface of explant tissues 7 d after inoculation; (H, J ) Paromomycin-resistant roots 28 d after inoculation. The bars in all panels,except (C) and (F), represent 1 mm. In (C) and (F) the bars represent 0.5 cm.


gene copy number, the location of chromosomal insertion,or a variety of post-translational effects.

Optimization of culture medium for transgenic root growth

Various concentrations of different auxin analogues wereadded to the liquid culture medium to promote the rapidgrowth of opium poppy and California poppy transgenicroot cultures. In both species, auxin treatment increasedthe growth rate of hairy roots (Table 3). Although rapidgrowth rates were induced by 1.0 mg l−1 IBA or0.5–1.0 mg l−1 NAA, these conditions also caused theformation of callus tissue on the transgenic roots. Theaddition of 1.0 mg l−1 IAA, which was just as effectiveFig. 2. RNA gel blot hybridization analysis for the b-glucuronidase

(GUS) reporter gene in wild-type ( WT) and paromomycin-resistant (1, as IBA or NAA but did not induce callus formation, to2) opium poppy and California poppy agarose gel, transferred to a B5 liquid medium containing 3% (w/v) sucrose was usednylon membrane, and hybridized at high stringency with a 32P-labelled

to promote the growth rates of root cultures. Under thesefull-length probe for GUS. The gel was were stained with ethidiumbromide prior to blotting to ensure equal loading. conditions, transgenic roots of both species grew 25–30%

faster than wild-type roots, and California poppy rootsgrew approximately 130% more rapidly than opiumopium poppy and California poppy were also tested forpoppy roots (Fig. 4).GUS enzyme activity levels compared to wild-type root

cultures. Transgenic hairy roots contained much higherComparison of wild-type and transgenic rootsGUS activity levels than non-transformed roots, which

exhibited only background activity (Fig. 3). Individual The histologies of wild-type and transformed roots oftransformants expressed a wide range of GUS activities, opium poppy and California poppy were nearly identicalfrom 250–1220 MU min−1 mg−1 protein for opium with the exception of the arrangement and structure ofpoppy (Fig. 3A) and from 230–1450 MU min−1 mg−1 epidermal cells (Fig. 5). All roots possessed a central steleprotein for California poppy (Fig. 3B). The observed of vascular tissues, surrounded by a few layers of corticaldistribution in GUS transcript and enzyme activity levels cells, and an outer layer of epidermis. The epidermal cellsis a common phenomenon in transformed plant tissues of wild-type roots from both species were densely packeddue to a combination of several factors including trans- and produced relatively few root hair extensions (Fig. 5A,

C ). In contrast, epidermal cells from transformed rootswere more loosely organized and gave rise to a largenumber of root hair extensions (Fig. 5B, C ), comparedto wild-type roots.

The benzylisoquinoline alkaloid content of transformedroot cultures was compared to that of wild-type rootcultures. Although the only available authentic standards

Table 3. Effect of auxins on the growth of transgenic hairy rootsof opium poppy and California poppy in liquid culture

Values represent the mean±SD of three independent measurements sixweeks after inoculation. Flasks were inoculated with 0.5 g fr. wt.root tissue.

Auxins Concentration Opium poppy California poppy(mg l−1) (fr. wt. g−1 flask) (fr. wt. g−1 flask)

Control — 1.9±0.1 4.3±0.2IAA 0.1 2.1±0.2 4.6±0.2

0.5 2.3±0.2 5.3±0.41.0 2.6±0.3 5.9±0.3

IBA 0.1 2.3±0.2 4.8±0.10.5 2.5±0.2 5.4±0.31.0 2.7±0.2a 6.1±0.2a

NAA 0.1 2.5±0.2 5.3±0.30.5 2.8±0.11 6.1±0.21Fig. 3. GUS activity in wild-type (WT) and paromomycin-resistant1.0 2.9±0.11 6.6±0.21(1–10) opium poppy (A) and California poppy (B) hairy root cultures

using 4-methylumbelliferyl-b--glucuronide (MUG) as the substrate.Bars represent the mean±SD of three independent measurements. aCaused the formation of callus.


species. Over the last decade, transformed root culturesfrom plants have attracted considerable attention becauseof their genetic and biochemical stability, rapid growthrate and ability to synthesize secondary products at levelscomparable to wild-type roots. An efficient A. rhizogenes-mediated protocol has been developed for the establish-ment of transgenic opium poppy and California poppyhairy root cultures. Of five A. rhizogenes strains tested,R1000 was found to be the most virulent and caused theformation of hairy roots exhibiting the most rapid growthrates (Tables 1, 2). Two commonly used strains, 15834and C58C1, were highly virulent only on opium poppy,but resulted in the formation of tumorigenic calli ratherthan roots. The growth rates of transformed roots ofboth species was improved by the addition of auxin tothe liquid culture medium (Table 3). Exogenous applica-tion of auxin was also reported to stimulate growth inhairy root cultures of Lippia dulcis Trev. (Sauerwein et al.,1991). The minimum doubling times of approximately 2weeks for California poppy and 3–4 weeks for opiumpoppy are comparable to hairy root cultures of otherspecies (Loyola-Vargas and Miranda-Ham, 1995).Fig. 4. Time-course for the growth of opium poppy (A) and California

poppy (B) wild-type root cultures (open circles) and transgenic root Several other studies have also reported the differentialcultures (closed circles). In each case, cultures were inoculated with efficiency of various A. rhizogenes strains in promoting0.5 g of tissue. Values represent the mean±SD of three independent

the formation and growth of hairy roots. For example,measurements.in addition to their variable ability to induce hairy rootdevelopment, different A. rhizogenes strains also affectedgrowth rate, saponin production and the ratio of differentwere morphine (peak a in Fig. 6A, B), noscapine (peak

c in Fig. 6A, B), and sanguinarine (peak f in Fig. 6A, B astragalosides in transgenic root cultures of Astragalusmongholicus Bge. (Ionkova et al., 1997). Strain 15834and peak i in Fig. 6C, D), several other chromatographic

peaks displayed UV spectra with characteristic benzyliso- was among the most effective at promoting hairy rootgrowth and saponin synthesis. The strain ofquinoline and benzophenanthridine signatures. HPLC

analysis showed that the relative concentration of mor- Agrobacterium also influenced development, growth rateand hyoscyamine production in transformed root culturesphine, noscapine, sanguinarine, and other putative alkal-

oids were virtually identical in wild-type (Fig. 6A) and of H. muticus (Vanhala et al., 1995). C58C1 was amongthe most virulent strains, and also resulted in root culturestransgenic roots (Fig. 6B) of opium poppy. Similarly, the

relative concentrations of sanguinarine and other putative with the highest alkaloid content. In contrast, strain15834 was the least effective for the induction of hairybenzophenanthridine alkaloids were also nearly identical

in wild-type (Fig. 6C) and transformed roots (Fig. 6D) roots of H. muticus. Transgenic root formation in pea(Pisum sativum L.) was dependent on both the strain ofof California poppy. The ‘normal’ alkaloid profile of the

opium poppy and California poppy hairy roots shows A. rhizogenes and the genotype of the host plant (Nicollet al., 1995). In pea, strains R1000nal, which is derivedthat this study’s transformation protocol could serve as

a valuable tool to study the regulation of genes encoding from R1000, and 15834 were most effective for thepromotion of hairy root formation. Strain 15834 was alsoalkaloid biosynthetic enzymes, and the network architec-

ture of the pathway via genetically engineered alterations the most virulent and efficient for hairy root developmentin Catharanthus roseus G. Don. (Brillanceau et al., 1989).in the activity of individual enzymes.Clearly, the selection of an effective Agrobacterium strainfor the production of transformed root cultures is highlyDiscussiondependent on the plant species, and must be determinedempirically. The differences in virulence, morphology andSoil-borne pathogens of the genus Agrobacterium are able

to transfer part of their DNA, the T-DNA carried on a growth rate are at least partially related to the variety ofplasmids contained within each bacterial strain.large plasmid, to the genome of a host plant cell.

Agrobacterium rhizogenes is the causal agent of ‘hairy The combined demonstration that in paromomycin-resistant roots, the NPTII gene was integrated into plantroot’ diseases in plants, and has been used for the

production of hairy root cultures from a multitude of genomic DNA, high GUS transcript and enzyme activity


Fig. 5. Cross-section of 6-week-old wild-type and transgenic hairy roots of opium poppy and California poppy. (A) Wild-type opium poppy root;(B) transgenic opium poppy hairy root; (C) wild-type California poppy hairy root; (D) transgenic California poppy hairy root. The bar in eachpanel represents 100 mm.

levels were detected (Figs 2, 3), and the GUS protein transformed shoots. In contrast, transgenic suspensioncultures derived via A. rhizogenes-mediated transforma-was ubiquitous in hairy root tissues (Fig. 1) provides

unequivocal proof that the stable and integrative trans- tion did not exhibit differences in growth or alkaloidaccumulation, with sanguinarine as the major productformation of opium poppy and California poppy root

cultures has indeed been achieved. This represents, to the ( Williams and Ellis, 1993).The quantity and ratio of different benzylisoquinolineauthors’ knowledge, the first reported protocol for the

transformation of root cultures not only from any member alkaloids produced by opium poppy and California poppywere virtually identical in transformed and wild-type rootsof the Papaveraceae, but from any benzylisoquinoline

alkaloid-producing plant. Previously, de-differentiated (Fig. 6). Transformed root cultures of several otherspecies have been evaluated for their content of alkaloidstransgenic cell cultures of opium poppy have been pro-

duced using A. tumefaciens (Belny et al., 1997) and A. or other secondary metabolites relative to wild-type roots.Although the profile of secondary products is oftenrhizogenes (Yoshimatsu and Shimomura, 1992; Williams

and Ellis, 1993). Infection of opium poppy hypocotyls conserved, the concentration of specific compounds issometimes altered by transformation with armed A. rhi-with A. rhizogenes MAFF 03–01724 (Yoshimatsu and

Shimomura, 1992) and an unidentified strain ( Williams zogenes strains. For example, hairy roots of ginseng(Panax ginseng Meyer) produced the same saponins andand Ellis, 1993) led to the formation of tumorigenic calli,

rather than hairy roots, as observed with strains 15834 ginsenosides as wild-type roots, but in quantities thatwere 2-fold higher on a dry weight basis (Yoshikawa andand C58C1 (Tables 1, 2). In one study, numerous adventi-

tious shoots developed from the transformed callus, but Furuya, 1987). Similarly, Korean balloon flower(Platycodon grandiflorum A. DC.) hairy roots producedthe transgenic shoots were morphologically different from

wild-type shoots (Yoshimatsu and Shimomura 1992). the polyacetylenes lobetyolin and lobetyolinin at levels160- and 2.6-fold higher, respectively, than those foundMoreover, the relative content of morphine and codeine

was altered in transgenic shoots compared to non- in wild-type roots (Ahn et al., 1996). In contrast, the


roots of Lotus corniculatus L. caused an increase in thecontent, and an alteration in the structure, of condensedtannins in a manner consistent with the substrate specifi-city of the transgene product (Bavage et al., 1997). Inanother study, the efficiency of the maize Sn gene, whichtransactivates the anthocyanin pathway in various tissues,at regulating anthocyanin biosynthesis in several dicotyle-donous species was tested using transformed root cultures(Damiani et al., 1998). The Sn gene was capable ofinducing anthocyanin biosynthesis in some heterologousroots, such as alfalfa (Medicago sativa L.) and lotus (Lotusangustissimus L.), but not in others, such as petunia(Petunia hybrida L.). A third interesting example involvesthe introduction of a heterologous tryptophan decarb-oxylase gene into hairy root cultures of Peganum harmalaL., which accumulate two biosynthetically related, trypta-mine-derived secondary metabolites: serotonin andb-carboline alkaloids (Berlin et al., 1993). Although sero-tonin accumulation in transgenic root cultures with elev-ated TDC activity was higher than in control cultures,b-carboline alkaloid levels were not affected; thus, trypta-mine supply was shown to be limiting for serotonin, butnot for b-carboline alkaloid, biosynthesis. Finally, trans-formed L. erythrorhizon root cultures expressing a bac-terial gene for chorismate pyruvate-lyase, which convertschorismate to the shikonin precursor 4-hydroxybenzoate(4HB), did not produce higher levels of shikonin (SommerFig. 6. HPLC elution profiles of methanol extracts from 6-week-old

wild-type and transgenic hairy root cultures of opium poppy and et al., 1999). These results suggest that the availability ofCalifornia poppy. (A) Wild-type opium poppy root culture; (B) 4HB does not normally limit the biosynthesis of shikonin.transgenic opium poppy hairy root culture; (C) wild-type California

The availability of an expanding collection of genespoppy hairy root culture; (D) transgenic California poppy hairy rootculture. In (A) and (B), peak a is morphine, peak c is noscapine, and encoding benzylisoquinoline alkaloid biosyntheticpeak f is sanguinarine. In (C) and (D), peak i is sanguinarine. Other enzymes, coupled with a protocol for the production ofpeaks identified with letters exhibit UV spectra with classic benzylisoqui-

rapidly growing transgenic root cultures of opium poppynoline alkaloid signatures.and California poppy, provide a powerful and versatilemodel system similar to those described above to investi-gate the molecular regulation of benzylisoquinoline alkal-productivity of the naphthoquinone shikonin in hairyoid biosynthesis, and to evaluate the potential toroot cultures of Lithospermum erythrorhizon Sieb. et Zucc.metabolically engineer these important medicinal plants.was similar to that of wild-type cell cultures, and displayed

the same light-dependent control of biosynthesis (Yazakiet al., 1998). Similarly, A. belladonna root cultures have Acknowledgementsbeen shown to accumulate hyoscyamine at levels similar

We thank Victor Loyola-Vargas and Felipe Vazquez-Flotato those of wild-type roots (Sharp and Doran, 1990), but(Centro de Investigacion Cientifica de Yucatan, Mexico) forwere also reported to synthesize the unusual tropaneproviding the Agrobacterium rhizogenes strains used in thisalkaloid littorine which is not found in non-transformed work, and Ed Yeung (University of Calgary) for his assistance

roots (Aoki et al., 1997). Moreover, hairy root cultures with the microtechniques and photomicroscopy. This researchof C. roseus accumulate the monoterpenoid indole alkal- was funded by a Natural Sciences and Engineering Research

Council of Canada grant to PJF. SUP was supported, in part,oids catharathine and ajmalicine at levels that are alsoby Midland-Walwyn, Nesbitt-Burns, and Bettina Bahlsensimilar to those found in wild-type roots (BrillanceauMemorial Graduate Scholarships awarded through the

et al., 1989; Toivonen et al., 1989; Vazquez-Flota et al., University of Calgary.1994).

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