Transformation of opium poppy (Papaver somniferum L.) with Agrobacterium rhizogenes MAFF 03-01724

Summary Transformed cultures of opium poppy (Papaver somniferum L.) were established by infecting hypocotyl segments with Agrobacterium rhizogenes MAFF 03-01724. Undifferentiated calli formed on the infected site grew satisfactorily on phytohormone-free solid medium in the dark and produced opine, mikimopine, which could not be detected in a normal culture. Numerous adventitious shoots developed from transformed calli during subculture. The transformed shoots separated individually were cultured on phytohormone-free MS solid medium at 22 ° C under 14 h/day light. They displayed wider leaves and longer internodes than shoots established from seeds or non-transformed root culture. The content of morphinan alkaloids in the cultures and regenerated shoots were quantitatively analyzed by enzyme-linked immunosorbent assay and high performance liquid chromatography. HPLC analysis revealed that non-transformed shoots contained much more codeine (1310 gmg/g dry wt.) than morphine (50 g/g dry wt.), while the transformed shoot cultures did not contain morphine, although the level of morphinan alkaloids in the transformed shoots (213 g morphine equivalents/g fr. wt.) was comparable to that in non-transformed shoots (182 g morphine equivalents/g fr. wt.) by ELISA.Abbreviations MS Murashige-Skoog (Murashige and Skoog 1962) - 1/2 MS half strength MS - HF phytohormone-free - NAA 1-naphthaleneacetic acid - ELISA enzyme-linked immunosorbent assay - HPLC high performance liquid chromatography     

Structural analysis of free N-glycans occurring in soybean seedlings and dry seeds

The structures of unconjugated or free N-glycans in stems of soybean seedlings and dry seeds have been identified. The free N-glycans were extracted from the stems of seedlings or defatted dry seeds. After desalting by two kinds of ion-exchange chromatography and a gel filtration, the free N-glycans were coupled with 2-aminopyridine. The resulting fluorescence-labeled (PA-) N-glycans were purified by gel filtration, Con A affinity chromatography, reverse-phase HPLC, and size-fractionation HPLC. The structures of the PA-sugar chains purified were analyzed by the combination of two-dimensional sugar chain mapping, jack bean alpha-mannosidase digestion, alpha-1,2-mannosidase digestions, partial acetolysis, and ESI-MS/MS. The free N-glycan structures found showed that two categories of free N-glycans occur in the stems of soybean seedlings. One is a high-mannose type structure having one GlcNAc residue at the reducing end (Man 9 approximately 5 GlcNAc1, 93%), that would be derived by endo-GM (Kimura, Y. et al., Biochim. Biophys. Acta, 1381, 27-36 (1998)). The other small component is a xylose-containing type one having two GlcNAc residues at the reducing end (Man3Xyl1GlcNAc2, 7%), which would be derived by PNGase-GM (Kimura, Y. and Ohno, A., Biosci. Biotechnol. Biochem., 62, 412-418 (1998)). The detailed structural analysis of free glycans showed that high-mannose type free N-glycans (Man 9 approximately 5 GlcNAc1) in the soybean seedlings have a common core structural unit; Manalpha1-6(Man1-3)Manalpha1-6(Manalpha1-3)Ma nbeta1-4GlcNAc. Comparing the amount of free N-glycans in the seedling stems and dry seeds, the amount in the stems of seedlings was much higher than that in the dry seeds; approximately 700 pmol per one stem, 8 pmol in one dry seed. This fact suggested that free N-glycans in soybean seedlings could be produced by two kinds of N-glycan releasing enzymes during germination or seedling-development.     

Induction of in vitro flowering in Dendrobium Madame Thong-In (Orchidaceae) seedlings is associated with increase in endogenous N(6)-(Delta (2)-isopentenyl)-adenine (iP) and N (6)-(Delta (2)-isopentenyl)-adenosine (iPA) levels

We analysed the endogenous cytokinin levels of Dendrobium Madame Thong-In seedlings grown in vitro during vegetative and flowering-inductive periods. HPLC was used to fractionate the extracts and radioimmunoassay (RIA) was used for assay of zeatin (Z), dihydrozeatin (DZ), N(6)-(Delta(2)-isopentenyl)-adenine (iP) and their derivatives. Coconut water used in experiments was found to contain high level (>136 pmol ml(-1)) of zeatin riboside (ZR). Protocorms and seedlings cultured in medium with coconut water were found to contain 0.5-3.9 pmol g(-1) FW of the cytokinins analysed. Seedlings (1.0-1.5 cm) cultured in flowering-inductive liquid medium containing 6-benzyladenine (BA, 4.4 muM) and coconut water (CW, 15%) contained up to 200 and 133 pmol g(-1) FW of iP and iPA, respectively. These levels were significantly higher than all other cytokinins analysed in seedlings of the same stage and were about 80- to 150-folds higher than seedlings cultured in non-inductive medium. During the transitional (vegetative to reproductive) stage, the endogenous levels of iP (178 pmol g(-1) FW) and iPA (63 pmol g(-1) FW) were also significantly higher than cytokinins in the zeatine (Z) and dihydrozeatin (DZ) families in the same seedlings. Seedlings that grew on inductive medium but remained vegetative contained lower levels of iPA. The importance of the profiles of iP and its derivatives in induction of in vitro flowering of D. Madame Thong-In is discussed.     

Quantification of indol-3-yl acetic acid in pea and maize seedlings by gas chromatography-mass spectrometry

A procedure is described for the identification and quantification of IAA in plant tissues by GC/MS analysis of the N-heptafluorobutyryl ethyl ester of IAA using [²H₅]IAA as an internal standard. The detection limit is ca 3 pmol IAA/tissue sample. By using this method, IAA levels of 30–90 pmol/g fr. wt were obtained for dark-grown Pisum sativum epicotyls and 71–199 pmol/g fr. wt for dark-grown Zea mays seedlings. When either methanol or ethanol was used as extraction solvent, some esterification of IAA during sample preparation was observed. No evidence for the natural occurrence of methyl or ethyl esters of IAA in Pisum sativum seedlings was found.     

Growth and rutin production in hairy root cultures of buckwheat (Fagopyrum esculentum M.)

Buckwheat (Fagopyrum esculentum Moench.) is a potentially important source of rutin, a natural flavonoid with antihyperglycemic, antihypertensive, and antioxidative properties. To examine in vitro production of rutin, we established a hairy root culture of buckwheat by infecting leaf explants with Agrobacterium rhizogenes R1000, and tested the growth conditions and rutin production rates of these cultures. Ten hairy root clones were established; their growth and rutin production rates ranged from 233 to 312 (mg dry wt per 30 mL flask, and 0.8 to 1.2 (mg/g dry wt), respectively. Clone H8, which had high growth and rutin production rates (312 mg dry wt per 30 mL flask and 1.2 mg/g dry wt, respectively), was selected for further experiments. H8 showed maximal growth and rutin content at 30 days in culture in MS medium. Of four tested culture media, half-strength MS medium was found to induce the highest levels of growth (378 mg dry wt per 30 mL flask) and rutin production (1.4 mg/g dry wt) by clone H8. In contrast, supplementation with auxins (0.1-1 mg/l IAA, IBA and NAA) increased the growth rate, but had no significant effect on rutin production by H8. Collectively, these findings indicate that hairy root cultures of buckwheat culture could be a valuable alternative approach for rutin production.     

Cyclic AMP,cyclic GMP,and bean rust uredospore germlings

Ten millimolar cyclic AMP (cAMP) or cyclic GMP (cGMP) induced bean rust uredospore germlings to undergo one round of mitosis and to form septa, processes normally associated with appressorium formation. To assess the possibility of cyclic nucleotide regulation of bean rust development, we used an 8-azido-[³²P]cAMP photoaffinity probe to identify three cyclic nucleotide binding peptides. The peptides bound either cAMP or cGMP. The phosphorylation of one peptide in uredospore germling extracts by [γ-³²P]ATP was stimulated by either 1 μM cAMP or cGMP, but only in the presence of 10 mM Na₂MoO₄, a phosphatase inhibitor. Uredospores contain about 1500 and 23 pmol cAMP and cGMP/g dry wt, respectively, as determined by radiobinding assays.     

Structural Analysis of Free N-Glycans Occurring in Soybean Seedlings and Dry Seeds

The structures of unconjugated or free N-glycans in stems of soybean seedlings and dry seeds have been identified. The free N-glycans were extracted from the stems of seedlings or defatted dry seeds. After desalting by two kinds of ion-exchange chromatography and a gel filtration, the free N-glycans were coupled with 2-aminopyridine. The resulting fluorescence-labeled (PA-) N-glycans were purified by gel filtration, Con A affinity chromatography, reverse-phase HPLC, and size-fractionation HPLC. The structures of the PA-sugar chains purified were analyzed by the combination of two-dimensional sugar chain mapping, jack bean α-mannosidase digestion, α-1,2-mannosidase digestions, partial acetolysis, and ESI-MS/MS. The free N-glycan structures found showed that two categories of free N-glycans occur in the stems of soybean seedlings. One is a high-mannose type structure having one GlcNAc residue at the reducing end (Man_9~5GlcNAc₁, 93%), that would be derived by endo-GM (Kimura, Y. et al., Biochim. Biophys. Acta, 1381, 27-36 (1998)). The other small component is a xylose-containing type one having two GlcNAc residues at the reducing end (Man₃Xyl₁GlcNAc₂, 7%), which would be derived by PNGase-GM (Kimura, Y. and Ohno, A., Biosci. Biotechnol. Biochem., 62, 412-418 (1998)). The detailed structural analysis of free glycans showed that high-mannose type free N-glycans (Man_9~5GlcNAc₁) in the soybean seedlings have a common core structural unit; Manα1- 6(Man1-3)Manα1-6(Manα1-3)Manβ1-4GlcNAc.

Comparing the amount of free N-glycans in the seedling stems and dry seeds, the amount in the stems of seedlings was much higher than that in the dry seeds; approximately 700 pmol per one stem, 8 pmol in one dry seed. This fact suggested that free N-glycans in soybean seedlings could be produced by two kinds of N-glycan releasing enzymes during germination or seedling-development.     

Identification of 3',5'-cyclic AMP in axenic cultures of Lemna paucicostata by high-performance liquid chromatography.

Nucleotides were purified from the extract of aseptically grown Lemna paucicostata 6746 by low-pressure column chromatography and thin-layer chromatography. In this partially purified sample, a compound has been identified, using a highly sensitive HPLC-fluorometric detection method, with chromatographic properties and substrate specificity for cyclic nucleotide phosphodiesterase similar to authentic 3',5'-cyclic AMP. The level of cAMP has been estimated to be 70-80 pmol/g fresh wt.     

Purification of glyoxysomal polypeptides

Summary A strategy for the rapid purification of proteins from glyoxysomes of castor bean (Ricinus communis cv. Hale) is described. The first step was to separate the proteins in the mixture on the basis of hydrophobicity by reversed phase high performance liquid chromatography using a gradient of increasing acetonitrile concentration. Individual protein peaks were collected and fractionated according to molecular mass by preparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purified polypeptides were used to produce monospecific, polyclonal antibodies. One of these, an anti-catalase antibody, has been employed to assess the subcellular distribution of catalase in endosperm of maturing seeds, dry seeds and seedlings. During seed maturation 45% of the catalase activity was associated with structures sedimenting at high isopycnic densities (1.21 g/cm³). However, in dry seeds, only 6% or less of the catalase activity was associated with these dense particles. In 4-day seedlings 80% of catalase activity was associated with glyoxysomes (1.24 g/cm³). A novel catalase 59 kDa subunit was found in the cytosol of 4-day seedlings and in isolated organelles from maturing and dry seed.Abbreviations AN acetonitrile - CBBR Coomassie brilliant blue R-250 - HPLC high performance liquid chromatography - SDS sodium dodecylsulfate - PAGE polyacrylamide gel electrophoresis     

Effects of sowing time and plant age on critical nitrogen concentrations in canola (Brassica napus L.)

Critical concentrations of NO₃-N in fresh petiole tissue and total N in the dried lamina were determined for the youngest mature leaf (YML) of field-grown canola. For dry matter yield of canola sown on 4 May, critical NO₃-N concentration in the YML petiole at the rosette stage (RS) was 1.46 mg/g fresh wt. At the flower-buds-visible stage (BV) it was 0.45 mg/g fresh wt. For seed yield the values were 1.72 and 0.53 mg/g fresh wt. Critical total N concentration in the YML lamina for dry matter yield were 69 mg/g dry wt. at RS and 57 at BV. For seed yield they were 71 and 59 mg/g dry wt. Critical NO₃-N concentrations in the YML petiole of canola sown on 30 May were reduced by 50%; critical total-N concentrations in the YML lamina were not reduced to the same extent. Despite the reductions in critical N concentrations in the YML, critical N fertilizer rates for vegetative growth and seed yield were unaffected by sowing date or plant growth stage.     

Distribution and heterogeneity of immunoreactive cholecystokinin (CCK) in the mucosa of the porcine gastrointestinal tract

The concentration and molecular nature of cholecystokinin-like immunoreactivity (CCK-LI) in extracts of porcine intestinal mucosa were determined using sequence-specific radioimmunoassays. Highest CCK concentrations were measured in duodenal mucosa (258 +/- 60 pmol/g in the distal duodenum) followed by jejunal mucosa (204 +/- 36 pmol/g in the proximal jejunum) and pylorus (51 +/- 9 pmol/g). All other gastrointestinal regions proximal to the pylorus and distal to the jejunum contained less than 20 pmol/g. Pancreas contained less than 1 pmol/g. Gel chromatography in 6 M urea revealed four immunoreactive forms and this was confirmed by reverse-phase high-pressure liquid chromatography (HPLC). The predominant molecular form in acid extracts of duodenal mucosa resembled CCK-33 although high concentrations of the larger CCK form ('CCK-58') and of the form intermediate in size between CCK-33 and CCK-8 were measured. A molecular form resembling CCK-8 was the principal form in neutral extracts of the duodenum.     

Somite chondrogenesis: alterations in cyclic AMP levels and proteoglycan synthesis

Cyclic AMP (cAMP) levels have been shown to have a positive influence on chondrogenesis in limb buds and pelvic cartilage. In the present study the level of cAMP was measured during somite chondrogenesis in vitro and found to decrease from 1.38 pmol/micrograms DNA on day 0 to 0.9 pmol/micrograms DNA on day 6. Inclusion of notochord with somites caused a marked reduction, with levels decreasing from 1.41 pmol/micrograms DNA on day 0 to 0.36 pmol/micrograms DNA on day 6. Concurrently, the incorporation of radioactive sulfate into sulfated glycosaminoglycans increased from day 3 to day 6 by 38% in somite and 77% in somite-notochord explants. The aggregation of proteoglycans was analyzed by gel chromatography and found to increase with a corresponding decrease in cAMP levels. The results indicate that a decrease in cAMP levels may be necessary for chondrogenic expression in somites.     

Influence of culture density,pH, organic acids and divalent cations on the removal of nutrients and metals by immobilized Anabaena doliolum and Chlorella vulgaris

The potential of alginate-immobilized Anabaena doliolum and Chlorella vulgaris was assessed for removal of nutrients (NO _inf3 ^sup- and NH _inf4 ^sup+ ) and metals (Cr₂O _inf7 ^sup2- and Ni²⁺) at different biomass concentrations (0.05, 0.1, 0.25, 0.49 and 1.22 g dry wt l^-1) and pH values (4 to 10). Though uptake of all these substances was higher in concentrated algal beads (0.25, 0.49 and 1.22 g dry wt l^-1), their rate of uptake was significantly (P<0.001) lower than that of low (0.05 g dry wt l^-1) cell density beads. For A. doliolum, there was no significant difference in uptake rates for beads having densities of 0.05 and 0.1 g dry wt l^-1. Chlorella vulgaris, however, showed maximum efficiency at 0.1 g dry wt l^-1. Uptake of both the nutrients and the metals was maximal at pH 7 followed by pH 8, 6, 9, 10, 5 and 4. Of the different substances (organic acids and divalent cations) used, humic acid was most efficient in decreasing metal uptake. Mg²⁺ was, however, more efficient than Ca²⁺ in decreasing Ni²⁺ uptake. Immobilized algae with a cell density of 0.1 g dry wt l^-1 were the most efficient for nutrient and metal removal at pH 6 to 8.     

Production of allantoin, rabdosiin and rosmarinic acid in callus cultures of the seacoastal plant Mertensia maritima (Boraginaceae)

A callus culture of the extreme halophyte seacoastal plant Mertensia maritima (Boraginaceae) was established from apical shoots of the plant using a modified Murashige and Skoog medium supplemented with 6-benzyladenine (0.5?mg/L) and ??-naphthaleneacetic acid (2.0?mg/L). Three main compounds, (?)-R-allantoin, (+)-rabdosiin and rosmarinic acid, were isolated from extracts of M. maritima calli by liquid chromatography and identified by ¹H and ¹³C NMR, UV, ECD and HPLC?CMS. Quantitative HPLC analysis showed that the calli produce (+)-rabdosiin (0.14% dry wt), rosmarinic acid (0.74% dry wt) and (?)-R-allantoin (3.7% dry wt). Allantoin was detected in plant cell cultures for the first time. All of these metabolites were also present in lower quantities in different parts of the plant. The presence of rabdosiin and rosmarinic acid, in combination with the skin-conditioning agent (?)-R-allantoin, represents a potentially useful novel composition for skin protection.     

Linum mucronatum: organ to organ lignan variations

The percentage of podophyllotoxin (PTOX) and its congener lignans were measured by HPLC in Linum mucronatum ssp. mucronatum (Linaceae) fresh plant organs. The highest amounts of PTOX (0.595 +/- 0.060% g/g dry wt) and 6-methoxypodophyllotoxin (MPTOX) (1.491 +/- 0.125% g/g dry wt) were found in the plant sexual organs. Whereas, the highest levels of beta-peltatin, 5'-demethoxy-MPTOX and yatein were found in not developed buds, petals and sepals, respectively.     

Determination of mannitol in ectomycorrhizal fungi and ectomycorrhizas by enzymatic micro-assays

Two sensitive methods for the enzymatic determination of mannitol are described which were applied to fungal and mycorrhizal extracts. Both methods are based on the oxidation of mannitol by mannitol dehydrogenase from Agaricus hortensis and the fluorometric determination of the NADPH produced in this reaction. The detection limits are 125 pmol for the direct fluorometric assay and 100 fmol, when enzymatic cycling of NADPH is included. The levels of mannitol detected were 123 pmol/g dry wt (mycelia from Cenococcum geophilum, cultivated on malt medium), below 0.3 or about 2.4 pmol/g dry wt (mycelia from Amanita muscaria, dependent on carbon source in the cultivation medium), and between 1.9 and 5.2 pmol/g dry wt in mycorrhizal short roots of Picea abies/Amanita muscaria.     

Comparative analysis of astaxanthin and its esters in the mutant E1 of Haematococcus pluvialis and other green algae by HPLC with a C30 column

A gradient reversed-phase high-performance liquid chromatography (HPLC) method using a C30 col-umn was developed for the simultaneous determination of astaxanthin, astaxanthin monoesters and astaxanthin diesters in the green algae Chlorococcum sp., Chlorella zofingiensis, Haematococcus plu-vialis and the mutant E1, which was obtained from the mutagenesis of H. pluvialis by exposure to UV-irradiation and ethyl methanesulphonate (EMS) with subsequent screening using nicotine. The re-sults showed that the contents of total astaxanthins including free astaxanthin and astaxanthin esters ranged from 1.4 to 30.9 mg/g dry biomass in these green algae. The lower total astaxanthin levels (< 2 mg/g dry biomass) were detected in the green algae Chlorococcum sp. and C. zofingiensis. The higher total astaxanthin levels (>16 mg/g dry biomass) were found in the green alga H. pluvialis and its mutant E1. It is notable that the mutant E1 is found to have considerably higher amounts of total astaxanthin (30.9 mg/g) as compared to the wild strain of H. pluvialis (16.1 mg/g). This indicates that UV-irradiation and EMS compound mutagenesis with subsequent screening using nicotine is an effective method for breeding of a high-producing astaxanthin strain of H. pluvialis. In addition, the green alga C. zofingien-sis had a remarkably higher percentage of astaxanthin diesters (76.3% of total astaxanthins) and a re-markably lower percentage of astaxanthin monoesters (18.0% of total astaxanthins) in comparison with H. pluvialis (35.5% for diesters and 60.9% for monoesters), the mutant E1 (49.1% and 48.1%) and Chlorococcum sp. (18.0% and 58.6%).     

Phospholipid hydroperoxide accumulation in liver of rats intoxicated with carbon tetrachloride and its inhibition by dietary alpha-tocopherol

The formation and accumulation of phospholipid hydroperoxides, especially of phosphatidylcholine hydroperoxide (PCOOH), a primary peroxidation product of phosphatidylcholine (PC), in livers of carbon tetrachloride-intoxicated rats was investigated. PCOOH in liver and blood plasma was measured by a chemiluminescence-high-performance liquid chromatography procedure originally developed by Miyazawa et al. (Anal. Lett. 20, 915, 1987; Free Radical Biol. Med. 7, 209, 1989). Male Sprague-Dawley rats (120 g body wt., 5 weeks of age) were used in the experiments. The amount of PCOOH in the liver of control rats (CCl4-untreated) was 160 +/- 20 pmol/100 mg protein (mean +/- SD) and the PCOOH/PC molar ratio was 1.1 +/- 0.1 X 10(-5). In CCl4 (0.1 ml/100 g body wt.)-dosed rats, the liver PCOOH was 289 +/- 65 pmol/100 mg protein (PCOOH/PC = 2.4 +/- 0.4 X 10(-5], 764 +/- 271 pmol/100 mg protein (PCOOH/PC = 5.2 +/- 1.7 X 10(-5], and 856 +/- 165 pmol/100 mg protien (PCOOH/PC = 6.0 +/- 0.8 X 10(-5] at 6 h, 24 h, and 1 week after the dose, respectively. Under such conditions, the liver phosphatidylethanolamine hydroperoxide (PEOOH) level was not altered and the concentration was less than 100 pmol/100 mg protein even after the dose. The increments of liver PCOOH were suppressed 56% by the oral supplementation of DL-alpha-tocopherol (5 mg/100 g body wt./day) for a week before CCl4 administration. A relatively larger amount of PEOOH was found after stimulation of PC hydroperoxidation in the liver of rats with a large amount of CCl4 (0.25 ml/100 g body wt.) rather than with the small amount of CCl4 (0.1 ml/100 g body wt.).(ABSTRACT TRUNCATED AT 250 WORDS)     

The life-cycle and productivity of Tibifex tubifex (Oligochaeta; Tibificidae) in the Moat-Feeder Stream, Cardiff, South Wales

A population of Tubifex tubifex in an organically rich stream was found to have an annual life-cycle with a prolonged period of reproductive activity throughout the winter and spring. Cocoons were produced mainly during the late winter and early spring. No cocoons were found during August and September, and there were few mature worms at this time.
The population density ranged between 5420 m⁻² in mid-September and 613000 m ⁻² in mid-May. The maximum population biomass (Bmax) recorded was 106 g dry wt m⁻² (March) and the minimum was 10 g dry wt m⁻² (September). Total annual production (P) was 139 g dry wt m⁻² and the average annual biomass ( B ) was 46 g dry wt m⁻² giving an annual P/ B ratio of 3.0, and a P/Bmax ratio of 1.3.     

Submergence(Fermentation)Culture of Anisodus acutanguhts Cells and Identification of Hyoscyamine and Scopolamine in the Cultured Ceils

The growth rate of Anisodus acutangulus cells in the submergence culture was 1.5 g dry wt/l/day, a rate 3 times as that in the suspension culture and more than 10 times over that of the solid static culture: However, the contents of hyoscyamine (0.203 mg/g dry wt) and scopolamine (0.178 mg/g dry. wt) in submergence culture were only slightly higher than those in the two other cultures. But when the 12-day-old submergence culture was supplemented with phenylalanine (5m mol/l) and kinetin (0.1mg/l), it was observed that not only the cell growth rate was increased but also the cellular content of hyoscyamine was raised to a level of 0.217 mg/g dry wt., and that scopolamine to 0.412 mg/g dry wt. The contents of these two alkaloids represented 1.1 and 2.3 times respectively the value of the culture without phenylalanine and kinetin supplements. The optimum date for harvesting the A. acutangulus culture Was on the 14th day of the culture. The monomers of hyoscyamine and scopolamine isolated from the cultured cells were purified and recrystalized, and then identifited as the two compounds in question by the thin-layer chromatography, melting point determination, and ultraviolet, infra-red and nuclear magnetic resonance spectra. In this paper, we also summarize and discuss the results of A. acutangulus culture experiments performed in the past 8 years. Our finding seems to inclieate that following a pilot production trial, the tissue culture method could well be employed to produce hyoscyamine and scopolamine from A. acutangulus cells on an industrial scale.