Hiltunen, Raimo (Selkämerenkatu 10 B 31 Helsinki, FIN-00180, FI)
Teeri, Teemu Heikki (Porttitie 17 B Espoo, FIN-02180, FI)
Oksman-caldentey, Kirsi-marja (Pöytäalhontie 67 C 14 Järvenpää, FIN-04430, FI)
Hiltunen, Raimo (Selkämerenkatu 10 B 31 Helsinki, FIN-00180, FI)
Teeri, Teemu Heikki (Porttitie 17 B Espoo, FIN-02180, FI)
PROC. NATL. ACAD. SCI. U.S.A., Volume 89, December 1992, DAE-JIN YUN et al., "Metabolic Engineering of Medicinal Plants: Transgenic Atropa Belladonna with an Improved Alkaloid Compositon", pages 11799-11803.
|1.||A hairy root culture of a hyoscyamineaccumulating tis sue of Hyoscyamus plant producing enhanced amounts of sco polamine, wherein said hairy root culture is derived from a Hyoscyamus plant operably transformed by an h6h gene enco ding hyoscyamine 6ahydroxylase enzyme using Agrobacterium transformation.|
|2.||The hairy root culture according to claim 1, being ob tainable by infecting a Hyoscyamus plant with an Agrobacte rium rhizogenes strain carrying an h6h gene.|
|3.||The hairy root culture according to claim 1 or 2, whe rein the Hyoscyamus plant is Hyoscyamus muticus.|
|4.||The hairy root culture according to any one of claims 1 to 3, wherein the h6h gene is derived from Hyoscyamus ni ger.|
|5.||The hairy root culture according to any one of claims 1 to 4 having the deposit accession number ECACC 97062022.|
|6.||Use of the hairy root culture according to any one of claims 1 to 5 in production of scopolamine.|
|7.||A Hyoscyamus plant producing enhanced amounts of sco polamine, and overexpressing an h6h gene encoding hyoscy amine 6phydroxylase enzyme, wherein said plant has been operably transformed by said gene using Agrobacterium transformation.|
|8.||The Hyoscyamus plant according to claim 7, which plant is obtainable directly from the hairy root culture accor ding to any one of claims 1 to 5 or via protoplasts derived therefrom.|
|9.||The Hyoscyamus plant according to claim 7 or 8, which is Hyoscyamus muticus.|
|10.||The Hyoscyamus plant according to any one of claims 7 to 9, wherein the h6h gene is derived from Hyoscyamus ni ger.|
|11.||Use of the Hyoscyamus plant according to any one of claims 7 to 10 in production of scopolamine.|
|12.||A process for enhancing the production of scopolamine in a hyoscyamineaccumulating Hyoscyamus plant tissue, which process comprises transformation of an h6h gene into a suitable Agrobacte rium rhizogenes strain, infection of the leaves of a Hyoscyamus species with the transformed Agrobacterium strain obtained, and cultivation of hairy root clones obtained in a suitable growth medium.|
|13.||The process according to claim 12, which process furt her comprises development of plantlets directly derived from the hairy root culture according to any one of claims 1 to 5 or via protoplasts derived therefrom, and growing the plantlets into mature plants under suitable conditions.|
|14.||The process according to claim 12, wherein the hyoscy amineaccumulating tissue is derived from Hyoscyamus muti cus.|
BACKGROUND OF THE INVENTION Plants produce a large number of different known metaboli- tes which can be used as drugs, dyes, flavour, fragrances or pesticides. Today there are still several plant com- pounds, such as scopolamine, which have a chemical synt- hesis procedure too complicated to be commercially feasible or which cannot be synthesized chemically at all. Therefore these compounds are isolated from plants for the needs of industry.
Many solanaceous species, e. g. Hyoscyamus muticus, Egyptian henbane, produce pharmaceutically important secondary meta- bolites, tropane alkaloids. Hyoscyamine and scopolamine, the two major alkaloids, are anticholinergic drugs that act mainly on the parasymphathetic nervous system. They are used as antiemetics, e. g. via a transdermal patch, and against several gastrointestinal disorders (scopolamine-N- butylbromide) as well as in ophthalmology. Atropine, which is a rasemic mixture of 1-and d-hyoscyamines, is widely used as a pre-anesthetic drug. Worldwide market for scopol- amine is currently estimated to be about 10 times larger than that of hyoscyamine or atropine. In the wild type H.
muticus plants the scopolamine contents are relatively low, about one third of the hyoscyamine contents. However, with systematic selection the contents have increased to the level exceeding those found in Duboisia (Oksman-Caldentey et al., 1987a) which at the moment is the principal commer- cial scopolamine source.
The biosynthesis of tropane alkaloids has been characteri- zed to certain level (Hashimoto and Yamada, 1987; Leete, 1990; Robins et al., 1993). Scopolamine which is a 6,7-epo- xide of hyoscyamine is formed from hyoscyamine via 6ß-hyd- roxyhyoscyamine. Both reactions are catalyzed by hyoscy- amine 6p-hydroxylase (H6H, EC 126.96.36.199), as is illustra- ted in the following scheme: hyoscyamine N 13.. OR | hyoscyamine 6 ß-hydroxylase (H6H) H 6ß-hydroxyhyoscyamine | H6H H, OU ... H J H OSoR scopolamine The H6H enzyme that catalyzes the first reaction in the epoxide formation is a 2-oxoglutarate-dependent dioxygena- se. The enzyme was purified and characterized from cultured roots of H. niger and it is a monomer of 38 kDa, as deter- mined by SDS-PAGE (Yamada et al., 1990). H6H is encoded by the h6h gene. The nucleotide sequence of H6H cDNA has been reported (Matsuda et al., 1991).
Today it is possible to manipulate plant cell cultures at the genetic level. If the activity of the secondary metabo- lite pathway could be regulated by introducing structural genes which encode enzymes acting at the initial level of the biosynthetic pathway, or by manipulating some regula- tory genes controlling overall operation of the enzymes, the productivity of the desired product could be enhanced (Rhodes et al., 1990). Foreign genes can be introduced to the plant cell by several methods.
One of the most efficient indirect gene transfer methods is the transformation by Agrobacterium tumefaciens or A. rhi- zogenes. Virulent strains of the soil pathogenic bacteria A. rhizogenes cause hairy roots in the infected plant tis- sue. The DNA that is transformed to plant (T-DNA) contains four root locus genes, rolA, B, C and D, which are respons- ible for the induction of hairy roots in plants. Once the transformed DNA has been integrated into the plant DNA, its expression is required for hairy root development. When a hairy root has been formed, it can continue to grow even in the absence of agrobacteria. The formed tissue proliferates on sterile culture media without addition of plant hormones which mostly are necessary for the cultivation of non- transformed plant cells (Chilton et al., 1982).
The advantages of the Agrobacterium system are the simple transformation technique and a good and stable transforma- tion frequency with the capacity to transfer relatively long sequences (up to 50 kb). However, the greatest disad- vantage of this method is the limited host range, i. e. mainly only dicotyledonous plant species.
Protocols used for establishing hairy root cultures vary, as well as the susceptibility of plant species to infection by Agrobacterium (Toivonen et al., 1993, and references therein). Transformed hairy roots have been obtained from several dicotyledonous medicinal plant species belonging to
a number of families (Tepfer, 1990; Rhodes et al., 1990).
Transformed roots are fast growing in vitro, their growth is comparable to the suspension cell cultures, and they are genetically and biochemically stable. They are laterally highly branched, and they can be cultivated in hormone-free media because genes in the Ri (root inducing) T-DNA regula- te the balance of endogeneous hormones. From the pharma- ceutical point of view, hairy root cultures produce high levels of secondary metabolites characteristic of the host plant (Flores et al., 1987). Secondary product formation is highly dependent on the maintenance of root organization.
On the other hand, use of hairy roots is limited to those products which are synthesized in roots of the intact plants.
It is known that the Agrobacterium strain used for trans- formation has an influence on root morphology and the deg- ree of secondary metabolite accumulation in hairy root cultures (Vanhala et al., 1995). It has also been shown that it is possible, by systematic clone selection, to find high-yielding stable hairy root clones (Mano et al., 1989).
The cell culture techniques for H. muticus are well esta- blished (Oksman-Caldentey, 1987). Hairy roots are not as readily manipulated by altering culture conditions as sus- pension cultures. However, addition of chitosan as an eli- citor increased hyoscyamine content fivefold in the hairy roots of H. muticus (Sevon et al., 1992), and the addition of a fungal elicitor increased thiophene accumulation in hairy roots of Tagetes patula (Mukundan and Hjortso, 1990).
However, the basic nutrient requirements of each clone, even those derived from the same origin, may vary, and thus growth and production conditions should be optimized indi- vidually (Oksman-Caldentey et al., 1994).
Recently, plant regeneration of hairy roots has become possible, but very little information is yet available of their secondary metabolite production (Oksman-Caldentey and
Hiltunen, 1996). The characterization as well as tropane alkaloid production has been performed in such plants (Oks- man-Caldentey et al., 1991). Protoplasts can be isolated from hairy roots to obtain pure clones. Furthermore, these protoplasts can be regenerated to plants (Sevon et al., 1995). Scopolamine can readily be isolated from the scopol- amine-rich H. muticus hairy root clone or plant by recrys- tallization of the total alkaloid fraction from the leaf samples.
In the isolation of alkaloids the chemical characteristics of quaternary nitrogen is exploited. Tropa-alkaloids are obtained in water-soluble form as salts in acidic solu- tions, and under basic conditions alkaloids dissolve in or- ganic solvents. The material containing alkaloids is soni- cated in acidic solution and extracted into water. The water phase is made alkaline and the alkaloids can subse- quently be extracted into an organic solvent.
Since the transformed root cultures of H. muticus produce high contents of hyoscyamine but only small amounts of scopolamine, the expression of H6H in its cultures and re- generated plants is assumed to increase the scopolamine content drastically.
The hydroxylase gene under the control of the cauliflower mosaic virus 35S promoter has been introduced into Atropa belladonna via Agrobacterium transformation (Yun et al., 1992; Hashimoto et al., 1993). With this system it has been demonstrated that the metabolic engineering of medicinal plants is possible. To our knowledge, these are the only reports in which similar type of technique as in the pre- sent invention is applied to a medicinal plant. The en- gineered hairy roots contained up to 5 times more scopola- mine compared to the control. Their target plant Atropa belladonna belongs to the same family (Solanaceae) as Hyos-
cyamus muticus, but it behaves in a very different way in the cell and tissue cultures.
We have now developed via Agrobacterium transformation a hairy root culture of H. muticus which overexpresses theh6h gene encoding the H6H enzyme of H. niger and produces about a hundredfold amount of scopolamine compared to control cultures, and with very good growth rate. We have thus succeeded in enhancing significantly the conversion of hyoscyamine to scopolamine in transformed hairy root tis- sues. We have also developed a scopolamine-rich transgenic H. muticus plant overexpressing the h6h gene encoding the H6H enzyme of H. niger.
SUMMARY OF THE INVENTION In one aspect, the present invention thus provides a hairy root culture of a hyoscyamine-accumulating tissue of Hyos- cyamus plant overexpressing an h6h gene encoding hyoscy- amine 6p-hydroxylase enzyme, and producing enhanced amounts of scopolamine. Said hairy root culture is derived from a Hyoscyamus plant operably transformed by said gene using Agrobacterium transformation.
In its further aspect, the present invention provides a transformed Hyoscyamus plant overexpressing a h6h gene encoding hyoscyamine 6p-hydroxylase enzyme, and producing enhanced amounts of scopolamine. The overexpression has been achieved by operably transforming said plant by said gene using Agrobacterium transformation.
Thus the above-mentioned transformed hairy root culture or Hyoscyamus plant can be used in production of scopolamine.
In yet another aspect, the present invention provides a process for enhancing the production of scopolamine in a
hyoscyamine-accumulating Hyoscyamus plant tissue, which process comprises -transformation of a h6h gene into a suitable Agrobacte- rium rhizogenes strain, -infection of the leaves of a Hyoscyamus species with said transformed Agrobacterium strain, and -cultivation of tissue clones obtained in a suitable growth medium.
Further areas of applicability of the present invention will be apparent from the detailed description given here- inafter.
According to the present invention engineered H. muticus hairy roots contained markedly increased amounts, even 100- fold higher concentrations, of scopolamine than wild-type hairy roots. Such genetically engineered hairy roots are useful for enhancing scopolamine productivity in in vitro root culture systems.
BRIEF DESCRIPTION OF THE DRAWINGS The invention as well as other objects, features and advan- tages thereof will be understood more clearly and fully from the following detailed description, with reference to the accompanying drawings, in which: Figure 1 depicts the strategy of infecting Hyoscyamus plant leaves with Agrobacterium rhizogenes and cultivation of the hairy root clones obtained in liquid medium.
Figure 2 shows the restriction map of plasmid pLAL1, which is obtained as a result of transformation of H. niger h6h gene into the plasmid pUC18.
Figure 3 shows the cloning of h6h gene containing plasmid pLAL1 into pD0432 vector having the cauliflower mosaic virus 35S promoter (35s).
Figure 4 shows the restriction map of plasmid pLAL20.
Figure 5 shows the construction of plasmid pLAL21. The fragment containing h6h gene and promoter is cut from plas- mid pLAL20 by digestion with restriction enzymes BglII and SacI. This fragment is cloned into plant vector pRD400 digested with BamHI and SacI, thereby obtaining the plasmid pLAL21."LB"means left border and"RB"right border se- quences.
Figure 6 shows the restriction map of plasmid pLAL21."LB" and"RB"are as above.
Figure 7 is a graph which shows the scopolamine and biomass productions of the best transformed H. muticus hairy root clone KB7 (13A7).
Figure 8 is a bar graph which shows a comparison of tropa- alkaloid production of the Hyoscyamus muticus control clone LBA-1S, the best transformed clone KB7 (13A7) and leaves from the H. muticus plant (late flowering) which was used for the transformation experiment (control plant).
DETAILED DESCRIPTION OF THE INVENTION The h6h gene of Hyoscyamus niger can be isolated in prin- ciple by following the methods of Yamada et al., 1990, and Matsuda et al., 1991, and inserted into suitable Agrobacte- rium strains. Briefly, total RNA can be isolated from the roots of the intact H. niger plant. Purified Poly (A) *RNA is copied to cDNA with reverse transcriptase. h6h is then mul- tiplied with PCR using specific primers. The desired frag-
ment is cloned to Escherichia coli with the help of a suit- able vector. Further, the h6h gene is cloned into a vector containing 35S promoter, and subsequently to a plant vec- tor. Finally, the plant vector containing the h6h gene is transferred conjugatively from E. coli to A. rhizogenes strains, whereby suitable binary vectors are obtained.
Those binary vectors may preferably contain both rifampicin and kanamycin resistance genes and therefore be cultivated on YMB medium containing said antibiotics.
Agrobacterium transformation can be carried out using the system described by Vanhala et al., 1995. Each hairy root obtained is preferably cultivated separately in liquid cul- ture, first in the presence of cefotaxime to remove the ex- cess of bacteria and then in a suitable medium, e. g. B50 medium, in erlenmeyer flasks. The first cultivation is repeated as many times as is necessary to get rid of the Agrobacterium, i. e. to obtain sterile clones. A schematic procedure depicting the formation of hairy root cultures is shown in Figure 1. The best clones can then be cultivated in mist spray bioreactor.
The hairy roots are preferably harvested in a late statio- nary growth stage, e. g. after 22 to 35 days of incubation.
Fresh and dry weights are determined, and dry e. g. lyophi- lized roots can be extracted e. g. with a lower alcohol.
Scopolamine and hyoscyamine determinations are performed directly on the extracts. Scopolamine content can be deter- mined by enzyme immunoassay. The assay is based on direct binding between scopolamine and nor-scopolamine-alkaline phosphatase conjugate tracer to the scopolamine antibody immobilized on the wells of the microtiter plates. The assay procedure is described in more detail in Example 1.
Hyoscyamine content can be determined by radioimmunoassay according to Oksman-Caldentey et al., 1987b.
Characterization of transferred genes in hairy root clones is preferably carried out by PCR according to Sevón et al., 1995.
Mature plants can be regenerated directly from A. rhizo- genes-transformed hairy root cultures of H. muticus or via protoplasts derived therefrom, e. g. according to Oksman- Caldentey et al., 1991, or Sévon et al., 1995. From 8 to 12 weeks are generally required for the development of small plantlets, which are subsequently grown in pots under greenhouse conditions into mature plants.
The following Examples will further illustrate the present invention.
EXAMPLE 1 Preparation of H6H expression vector The h6h gene of Hyoscyamus niger was isolated in principle following the methods of Yamada et al., 1990 and Matsuda et al., 1991, and inserted into two different Agrobacterium rhizogenes strains, LBA9402 and 15834 (provided by Prof.
Ulf Nyman, Royal Danish School of Pharmacy, Copenhagen, Denmark). Briefly, total RNA was isolated from the roots of the intact H. niger plant. Purified Poly (A) +RNA was copied to cDNA with reverse transcriptase. h6h was then multiplied with PCR using specific primers (see chapter Polymerase chain reaction below). The fragment thus obtained was clo- ned to E. coli DH5a with the help of pUC18 vector (Phar- macia Biotech) to form plasmid pLALl (Figure 2). Further, the h6h gene was cloned to pD0432 vector containing cauli- flower mosaic virus 35S promoter to form plasmid pLAL20 (Figures 3 and 4) and subsequently to the plant vector pRD400 to form plasmid pLAL21 (Figures 5 and 6). Finally, pLAL21 was transferred conjugatively from E. coli to A.
rhizogenes strains LBA9402 and 15834. The resulting binary vectors are referred to as 15834-H6H-9 and LBA9402-H6H-13.
Gene transfer and establishment of transformed root cultures Hyoscyamus muticus L., strain Cairo, was grown from seeds in greenhouse. The seeds were from the same origin as those used in the reference Oksman-Caldentey et al., 1991. Agro- bacterium transformation of said H. muticus strain was car- ried out using the system described by Vanhala et al., 1995. Each hairy root was cultivated separately in liquid culture, first in the presence of cefotaxime to remove the excess of bacteria and then in modified B50 medium in 100 ml erlenmeyer flasks (Oksman-Caldentey et al., 1991).
One of the best clones, KB7 (13A7), was then cultivated in 5 1 mist spray bioreactor four times (Wadenswil, Switzer- land). The bioreactor was inoculated with 2.5 g of said clone, which was then cultivated for 22 days in normal daylight. The biomass productivity was 5.1-fold/day.
The hairy roots were harvested after 22 days of incubation.
Fresh and dry weights were determined, and lyophilized dry roots (50 mg samples) were extracted with 80 % MeOH (5 ml) in test tubes for 16 hours in the temperature of 60 °C.
Analytical methods Prior to the analysis, the samples were diluted (1: 1-1: 2 000) in order to achieve the concentration range of stan- dard curves.
Quantitative scopolamine and hyoscyamine determinations were performed directly on the methanol extracts. Scopol- amine content was determined by enzyme immunoassay descri-
bed below and hyoscyamine content by radioimmunoassay ac- cording to Oksman-Caldentey et al., 1987b.
The scopolamine assay system comprised the following solu- tions: (1) coating antibody (6 ug/ml of anti-mouse Ig anti- body from Boehringer-Mannheim in 50 mM Na-carbonate buffer, pH 9.4), (2) blocking solution (1% bovine serum albumin in 4-tert-butylphenyl salicylate (TBS) containing 50 mM Tris buffer, pH 7.5, and 150 mM NaCl), (3) wash solution (500 pl of Tween 20 in 1 1 of TBS), (4) scopolamine antibody solu- tion (hybridoma cell line SP-4-A23-H8 medium was provided by Prof. E. W. Weiler, University of Ruhr, Department of Plant Physiology, Bochum, Germany, and it was diluted 1: 50 with the TBS), (5) scopolamine hydrobromide (Sigma) stan- dards in TBS, (6) the nor-scopolamine propionic acid (NSP) -alkaline phosphatase tracer described above, and (7) substrate solution (1 mg/ml of disodium paranitrophenyl- phosphate dissolved in 1 ml of 1.25 M diethanolamine buf- fer, pH 9.8).
Microtiter plates (Nunc, Maxisorb) were coated with 200 pl of (1) by incubating for 1 h at room temperature (RT), the wells were washed for 15 s with 200 pl of (3) three times, blocked by incubating for 30 min at RT with 200 pl of (2), washed three times, and reacted with 100 pl of (4) for 1 h at RT and washed again. For the competitive assay reaction 50 ul of standard (5) or sample and 150 pi of the tracer (6) were pipetted into each well on an ice bath and the plates were transferred for 2 h to RT and washed again. The tracer binding was determined by adding 200 pi of the substrate (7) and incubated at 35"C. The absorbance at 405 nm was read after every 30 min in an Easy Reader 400 ATC microplate spectrometer until the absorbance values for zero samples (buffer instead of standard or sample) were about one. Non-specific binding was determined in wells
where (4) was replaced with pure TBS. The assays were per- formed with fourfold samples throughout. The results were processed using Microsoft Excel and Cricket Graph programs.
A hairy root clone LBA-1S was used as a control clone, which did not contain transformed h6h gene. Also the tropa- alkaloid content of leaves from the H. muticus parent plant (late flowering) used for transformation was analysed.
Polymerase chain reaction Characterization of transferred genes rolA, B, C derived from A. rhizogenes (Sevon et al., 1995) and h6h in hairy root clones was carried out by PCR. The 32-mer oligonucleotide primers designed to amplify h6h were 5'-CCG GAA TTC GGA TCC CAA CGT ATA GAT TCT TC-3'and 5'-CGG GAA TTC GGA TCC CAA ACC ATC ACT GCA AT-3'. The conditions for amplifica- tion of h6h were the following: initial denaturation at 94 °C for 5 min, annealing at 53 °C for 5 min and extension at 72 °C for 5 min for the first cycle, followed by 30 cycles of denaturation at 94 °C for 1 min 15 s annealing at 53 °C for 2 min, extension at 72 °C for 5 min. The amplified samples were analysed on a 2% agarose gel for rol genes and 1% agarose gel for h6h genes.
Results Altogether 68 hairy root clones were obtained after trans- formation with the two different Agrobacterium rhizogenes strains both carrying the h6h gene. There were great dif- ferences in their growth pattern, morphology and alkaloid production (Jouhikainen, 1997). 42 clones were analysed for their hyoscyamine and scopolamine production. Most of the clones showed improved scopolamine production compared to the control cultures whereas the hyoscyamine content re- mained the same as in the controls (Table I). Nine best scopolamine producing clones were grown for a period of 28
days in identical conditions as in the first time. The scopolamine production in some clones was slightly (about 10 %) higher than in the first run, most probably due to the better root morphology of the clones (less callus in the roots). Also hyoscyamine production remained the same.
Certain fluctuation of the contents always exists between different passages in various plant cell and tissue cultu- res, although long-term production is considered stable.
The transformation with several Agrobacterium strains has been performed earlier, and the high hyoscyamine-producing hairy root clones have been stably cultured over six years (results will be published in Sevon et al., 1997).
TABLE I Tropane alkaloid production in transformed hairy root clones of Hyoscyamus muticus. (DW = dry weight) Clone Passage Scopolamine Hyoscyamine mg/ll) mg/g mg/l1) mg/g (DW) (DW) KA3 (9A3) 8 6.63 0.41 111.3 7.15 KA4 (9A4) 2 1.46 0.09 83.5 5.59 KA18 (9A18) 8 3.49 0.20 135.1 8.00 KA21 (9A21) 8 3.99 0.22 168.8 9.99 KA22 (9A22) 8 6.84 0.40 121.5 7.21 KA23 (9A23) 8 1.96 0.11 148.7 8.90 KA24 (9A24) 4 1.26 0.07 153.4 8.89 KB5 (13A5) 8 8.87 0.54 76.4 4.54 KB7 (13A7) 8 14.41 0.78 125.4 7.40 Control 0.12 0.01 120.02) 9.02 Plant leaf 0.20 4.41 1) mg/l = mg scopolamine/1 of used liquid growth medium 2) Oksman-Caldentey et al., 1994 The growth (biomass production) and scopolamine production of the best clone KB7 (13A7) is shown in Figure 7. The scopolamine production of this clone was 14.4 mg/1. It is about 140 times more than the scopolamine content produced by the control culture (0.12 mg/1). The PCR studies confir-
med the presence of h6h gene as well as the rol genes from the T-DNA of A. rhizogenes. The clone KB7 (the original designation: 13A7) was deposited according to the Budapest Treaty at the depository European Collection of Cell Cultu- res (ECACC), Centre for Applied Microbiology & Research, Salisbury, Wiltshire SP4 OJG, United Kingdom, on June 20, 1997 with the deposit accession number ECACC 97062022.
The production, which usually is given as mg/1 of used liquid growth medium, takes well into account the biomass production. However, it is possible to express the results as mg/g dry weight in order to be able to compare the re- sults to the intact plant also. The scopolamine content for new clone KB7 (13A7), control hairy root clone and non- transformed plant were 0.78,0.01 and 0.20 mg/g, respecti- vely. When the results are expressed in this way, the new clone still produces about a hundredfold amount of scopol- amine compared to the control hairy root clone and about a fourfold amount compared to the mother plant. The tropa- alkaloid production of the best transformed hairy root clo- ne KB7 (13A7) compared to that of the control clone LBA-1S and intact control plant is shown in Figure 8.
EXAMPLE 2 Plant regeneration via protoplasts Mature plants are regenerated via protoplasts from Agro- bacterium rhizogenes transformed hairy root cultures of Hyoscyamus muticus according to the procedure described by Sévon et al., 1995. Protoplasts are isolated directly from the transformed hairy root culture obtained in Example 1 using an enzyme mixture comprising 1 % macerozyme R-10 and 2 % cellulase"Onozuka"R-10, both from Yakult, Japan, in an osmoticum consisting of 0.2 M CaCl2 and 0.6 M mannitol.
Protoplasts are first cultured in liquid NT/PRO I medium and subsequently on semi-solid NT/PRO II agar medium (as to
the media, see Oksman-Caldentey and Strauss, 1986). This procedure permits a highly efficient colony formation. The formed small individual colonies regenerate easily into shoots and roots.
From 8 to 12 weeks are required for the development of small plantlets from protoplasts. The gradual adaptation of the plantlets to greenhouse conditions is a crucial step, particularly during autumn and winter. The plantlets are then grown in pots under greenhouse conditions into mature plants. Transformed plants show strong phenotypic differen- ces from clone to clone due to the rolA, B and C genes they contain.
Direct plant regeneration Mature plants are obtained directly from A. rhizogenes transformed hairy root cultures of H. muticus according to the procedure of Oksman-Caldentey et al., 1991. The hairy root clones obtained in Example 1 are subcultured, and roots showing shoot differentiation and/or callus producti- on are transferred onto agar-solidified LSO medium (Lins- maier and Skoog, 1965) and subcultured every 3 weeks. After one to three subculturing passages the calli are transfer- red onto two media, LSO and modified B50 (Oksman-Caldentey et al., 1991). Shoots which have differentiated directly from hairy roots or from callus-producing hairy roots are excised and cultured subsequently on several rooting media (for further details, see Oksman-Caldentey et al., 1991).
Regenerated, about 6 to 8 weeks old, small, vigorously gro- wing plantlets are transferred from sterile conditions into a mixture of soil and Vermiculite@ (1: 1) and incubated for the first 2 weeks in a growth chamber to maintain high humidity. Subsequently the plants are transferred to soil in the greenhouse. Also in this case the resulting plants differ phenotypically from normal plants and from each
other. Plants have e. g. wrinkled, narrowed leaves, reduced apical dominance and abnormal flowering.
In both cases the alkaloid contents of regenerated plants are determined from leaves by radioimmunoassay and enzyme immunoassay as described earlier. The scopolamine content is the highest in the leaves during late flowering. Hyoscy- amine content is the highest during flowering (Oksman-Cal- dentey et al., 1987b).
DEPOSITED CULTURE The following hairy root culture was deposited according to the Budapest Treaty at European Collection of Cell Cultures (ECACC), Centre for Applied Microbiology & Research, Salis- bury, Wiltshire SP4 OJG, United Kingdom, on June 20,1997 with the deposit accession number 97062022.
Deposited culture Accession number Deposit date Hyoscyamus muticus hairy root clone 13A7 ECACC 97062022 June 20,1997 (new designation: KB7)
REFERENCES Chilton MD, Tepfer DA, Petit A, David C, Casse-Delbart F and Tempe J (1982): Agrobacterium rhizogenes inserts T-DNA into the genomes of the host plant root cells. Nature 295: 432-434.
Flores HE, Hoy MW and Pickcard JJ (1987): Secondary metabo- lites from root cultures. Trends Biotechnol 5: 6468.
Hashimoto T and Yamada Y (1987): Effects of culture condi- tions on tropane alkaloid formation in Hyoscyamus niger suspension cultures. Agric Biol Chem 51: 2769-2774.
Hashimoto T, Yun D-1 and Yamada Y (1993): Production of tropane alkaloids in genetically engineered root cultures.
Phytochemistry 32: 713-718.
Jouhikainen K (1997): Somaclonal variation and enhancement of scopolamine production in hairy root cultures of Hyoscy- amus muticus containing h6h transgene. (In Finnish), M. Sc.
(Pharm.) thesis, Helsinki, Finland 100 p + app.
Leete E (1990): Recent developments in the biosynthesis of the tropane alkaloids. Planta Med 56: 339-352.
Linsmaier EM and Skoog F (1965): Organic growth factor requirements of tobacco tissue cultures. Physiol Plant 18: 100-127.
Mano Y, Okhawa H and Yamada Y (1989): Production of tropane alkaloids by hairy root cultures of Duboisia leichhardtii transformed by Agrobacterium rhizogenes. Plant Sci 59: 191-201.
Matsuda J, Okabe S, Hashimoto T and Yamada Y (1991): Mole- cular cloning of hyoscyamine 6a-hydroxylase, a 2-oxogluta- rate-dependent dioxygenase, from cultured roots of Hyoscy- amus niger. J Biol Chem 266: 9460-9464.
Mukundan U and Hjortso MA (1990): Effect of fungal elicitor on thiophene production in hairy root cultures of Tagetes patula. Appl Microbiol Biotechnol 33: 145-147.
Oksman-Caldentey K-M (1987): Scopolamine and hyoscyamine production by plants and cell cultures of Hyoscyamus muti- cus. Ph. D. (Pharm) thesis, Helsinki, Finland.
Oksman-Caldentey K-M and Strauss A (1986): Somaclonal vari- ation of scopolamine content in protoplast-derived cell culture clones of Hyoscyamus muticus. Planta Med 52: 6-12.
Oksman-Caldentey K-M, Vuorela H, Isenegger M, Strauss A and Hiltunen R (1987a): Selection for high tropane alkaloid
content in Hyoscyamus muticus plants. Plant Breeding 99: 318-326.
Oksman-Caldentey K-M, Vuorela H, Strauss A and Hiltunen R (1987b): Variation in tropane alkaloid content of Hyoscy- amus muticus plants and cell culture clones. Planta Med 53: 349-354.
Oksman-Caldentey K-M, Kivela O and Hiltunen R (1991): Spon- taneous shoot organogenesis and plant regeneration from hairy root cultures of Hyoscyamus muticus. Plant Sci 78: 129-136.
Oksman-Caldentey K-M, Sevón N, Vanhala L and Hiltunen R (1994): Effect of nitrogen and sucrose on the primary and secondary metabolism of transformed root cultures of Hy- oscyamus muticus. Plant Cell Tiss Org Cult 38: 263-272.
Oksman-Caldentey K-M and Hiltunen R (1996): Transgenic crops for improved pharmaceutical products. Field Crops Res 45: 57-69.
Robins RJ et al. (1993): Tropane alkaloid biosynthesis in root cultures. In: Plant Tissue Culture Manual G1, Kluwer Academic Publishers, Dordrecht, pp 1-17.
Rhodes MJC, Robins RJ, Hamill JD, Parr AJ, Hilton MG and Walton NJ (1990): Properties of transformed root cultures.
In: Secondary Products from Plant Tissue Culture. Charlwood BV and Rhodes MJC (eds), pp 201-225.
Sevón N, Hiltunen R and Oksman-Caldentey K-M (1992): Chi- tosan increases hyoscyamine content in hairy root cultures of Hyoscyamus muticus. Pharm Pharmacol Lett 2: 96-99.
Sevón N, Hiltunen R and Oksman-Caldentey K-M (1997): Soma- clonal variation in Agrobacterium transformed roots and protoplast-derived hairy root clones of Hyoscyamus muticus.
Planta Med (in press).
Sevón N, Oksman-Caldentey K-M and Hiltunen R (1995): Effi- cient plant regeneration from hairy root-derived proto- plasts of Hyoscyamus muticus. Plant Cell Rep 14: 738-742.
Tepfer D (1990): Genetic transformation using Agrobacterium rhizogenes. Physiol Plant. 79: 140-146.
Toivonen L (1993): Utilization of hairy root cultures for production of secondary metabolites. Biotechnol Prog 9: 12-20.
Vanhala L, Hiltunen R and Oksman-Caldentey KM (1995): Viru- lence of different Agrobacterium strains on hairy root formation of Hyoscyamus muticus. Plant Cell Rep 14: 236- 240.
Yamada Y, Okabe S and Hashimoto T (1990): Homogeneous hyos- cyamine 6 hydroxylase from cultured roots of Hyoscyamus niger. Proc Jpn Acad Ser B Phys Biol Sci 66: 73-76.
Yun D-J, Hashimoto T and Yamada Y (1992): Metabolic en- gineering of medicinal plants: Transgenic Atropa belladonna with an improved alkaloid composition. Proc Natl Acad Sci USA 89: 11799-11803.