Self-incompatibility-induced programmed cell death in field poppy pollen involves dramatic acidification of the incompatible pollen tube cytosol.

Self-incompatibility (SI) is an important genetically controlled mechanism to prevent inbreeding in higher plants. SI involves highly specific interactions during pollination, resulting in the rejection of incompatible (self) pollen. Programmed cell death (PCD) is an important mechanism for destroying cells in a precisely regulated manner. SI in field poppy (Papaver rhoeas) triggers PCD in incompatible pollen. During SI-induced PCD, we previously observed a major acidification of the pollen cytosol. Here, we present measurements of temporal alterations in cytosolic pH ([pH]cyt); they were surprisingly rapid, reaching pH 6.4 within 10 min of SI induction and stabilizing by 60 min at pH 5.5. By manipulating the [pH]cyt of the pollen tubes in vivo, we show that [pH]cyt acidification is an integral and essential event for SI-induced PCD. Here, we provide evidence showing the physiological relevance of the cytosolic acidification and identify key targets of this major physiological alteration. A small drop in [pH]cyt inhibits the activity of a soluble inorganic pyrophosphatase required for pollen tube growth. We also show that [pH]cyt acidification is necessary and sufficient for triggering several key hallmark features of the SI PCD signaling pathway, notably activation of a DEVDase/caspase-3-like activity and formation of SI-induced punctate actin foci. Importantly, the actin binding proteins Cyclase-Associated Protein and Actin-Depolymerizing Factor are identified as key downstream targets. Thus, we have shown the biological relevance of an extreme but physiologically relevant alteration in [pH]cyt and its effect on several components in the context of SI-induced events and PCD.

Improved sanguinarine production via biotic and abiotic elicitations and precursor feeding in cell suspensions of latex-less variety of Papaver somniferum with their gene expression studies and upscaling in bioreactor.

Elicitors play an important role in challenging the plant defense system through plant-environment interaction and thus altering the secondary metabolite production. Culture filtrates of four endophytic fungi, namely, Chaetomium globosum, Aspergillus niveoglaucus, Paecilomyces lilacinus, and Trichoderma harzianum were tested on embryogenic cell suspensions of latex-less Papaver somniferum in dose-dependent kinetics. Besides this, abiotic elicitors salicylic acid, hydrogen peroxide, and carbon dioxide were also applied for improved sanguinarine production. Maximum biomass accumulation (growth index (GI) = 293.50 ± 14.82) and sanguinarine production (0.090 ± 0.008 % dry wt.) were registered by addition of 3.3 % v/v T. harzanium culture filtrate. Interestingly, it was further enhanced (GI = 323.40 ± 25.30; 0.105 ± 0.008 % dry wt.) when T. harzanium culture filtrate was employed along with 50 µM shikimate. This was also supported by real-time (RT) (qPCR), where 8-9-fold increase in cheilanthifoline synthase (CFS), stylopine synthase (STS), tetrahydroprotoberberine cis-N-methyltransferase (TNMT), and protopine 6-hydroxylase (P6H) transcripts was observed. Among abiotic elicitors, while hydrogen peroxide and carbon dioxide registered low level of sanguinarine accumulation, maximum sanguinarine content was detected by 250 µM salicylic acid (0.058 ± 0.003 % dry wt.; GI = 172.75 ± 13.40). RT (qPCR) also confirms the downregulation of sanguinarine pathway on CO2 supplementation. Various parameters ranging from agitation speed (70 rpm), impeller type (marine), media volume (2 l), inoculum weight (100 g), and culture duration (9 days) were optimized during upscaling in 5-l stirred tank bioreactor to obtain maximum sanguinarine production (GI = 434.00; 0.119 ± 0.070 % dry wt.). Addition of 3.3 % v/v T. harzanium culture filtrate and 50-µM shikimate was done on the 6th day of bioreactor run.

Morphine biosynthesis in opium poppy involves two cell types: sieve elements and laticifers.

Immunofluorescence labeling and shotgun proteomics were used to establish the cell type-specific localization of morphine biosynthesis in opium poppy (Papaver somniferum). Polyclonal antibodies for each of six enzymes involved in converting (R)-reticuline to morphine detected corresponding antigens in sieve elements of the phloem, as described previously for all upstream enzymes transforming (S)-norcoclaurine to (S)-reticuline. Validated shotgun proteomics performed on whole-stem and latex total protein extracts generated 2031 and 830 distinct protein families, respectively. Proteins corresponding to nine morphine biosynthetic enzymes were represented in the whole stem, whereas only four of the final five pathway enzymes were detected in the latex. Salutaridine synthase was detected in the whole stem, but not in the latex subproteome. The final three enzymes converting thebaine to morphine were among the most abundant active latex proteins despite a limited occurrence in laticifers suggested by immunofluorescence labeling. Multiple charge isoforms of two key O-demethylases in the latex were revealed by two-dimensional immunoblot analysis. Salutaridine biosynthesis appears to occur only in sieve elements, whereas conversion of thebaine to morphine is predominant in adjacent laticifers, which contain morphine-rich latex. Complementary use of immunofluorescence labeling and shotgun proteomics has substantially resolved the cellular localization of morphine biosynthesis in opium poppy.

Alternate transcripts of a floral developmental regulator have both distinct and redundant functions in opium poppy.

BACKGROUND AND AIMS: The MADS-box transcription factor AGAMOUS (AG) is an important regulator of stamen and fruit identity as well as floral meristem determinacy in a number of core eudicots and monocots. However, its role outside of these groups has not been assessed explicitly. Examining its role in opium poppy, a basal eudicot, could uncover much about the evolution and development of flower and fruit development in the angiosperms. METHODS: AG orthologues were isolated by degenerate RT-PCR and the gene sequence and structure examined; gene expression was characterized using in situ hybridization and the function assessed using virus-induced gene silencing. KEY RESULTS: In opium poppy, a basal eudicot, the AGAMOUS orthologue is alternatively spliced to produce encoded products that vary at the C-terminus, termed PapsAG-1 and PapsAG-2. Both transcripts are expressed at high levels in stamens and carpels. The functional implications of this alternative transcription were examined using virus-induced gene silencing and the results show that PapsAG-1 has roles in stamen and carpel identity, reflecting those found for Arabidopsis AG. In contrast, PapsAG-2, while displaying redundancy in these functions, has a distinctive role in aspects of carpel development reflected in septae, ovule and stigma defects seen in the loss-of-function line generated. CONCLUSIONS: These results describe the first explicit functional analysis of an AG-clade gene in a basal eudicot; illustrate one of the few examples of the functional consequences of alternative splicing in transcription factors and reveal the importance of alternative transcription, as well as gene duplication, as a driving force in evolution.

Proteins implicated in mediating self-incompatibility-induced alterations to the actin cytoskeleton of Papaver pollen.

BACKGROUND AND AIMS: Sexual reproduction in angiosperms involves a network of signalling and interactions between pollen and pistil. To promote out-breeding, an additional layer of interactions, involving self-incompatibility (SI), is used to prevent self-fertilization. SI is generally controlled by the S-locus, and comprises allelic pollen and pistil S-determinants. This provides the basis of recognition, and consequent rejection, of incompatible pollen. In Papaver rhoeas, SI involves interaction of pistil PrsS and pollen PrpS, triggering a Ca(2+)-dependent signalling network. This results in rapid and distinctive alterations to both the actin and microtubule cytoskeleton being triggered in 'self' pollen. Some of these alterations are implicated in mediating programmed cell death, involving activation of several caspase-like proteases. SCOPE: Here we review and discuss our current understanding of the cytoskeletal alterations induced in incompatible pollen during SI and their relationship with programmed cell death. We focus on data relating to the formation of F-actin punctate foci, which have, to date, not been well characterized. The identification of two actin-binding proteins that interact with these structures are reviewed. Using an approach that enriched for F-actin from SI-induced pollen tubes using affinity purification followed by mass spectrometry, further proteins were identified as putative interactors with the F-actin foci in an SI situation. KEY RESULTS: Previously two important actin-binding proteins, CAP and ADF, had been identified whose localization altered with SI, both showing co-localization with the F-actin punctate foci based on immunolocalization studies. Further analysis has identified differences between proteins associated with F-actin from SI-induced pollen samples and those associated with F-actin in untreated pollen. This provides candidate proteins implicated in either the formation or stabilization of the punctate actin structures formed during SI. CONCLUSIONS: This review brings together for the first time, our current understanding of proteins and events involved in SI-induced signalling to the actin cytoskeleton in incompatible Papaver pollen.

Integration of deep transcriptome and proteome analyses reveals the components of alkaloid metabolism in opium poppy cell cultures.

BACKGROUND: Papaver somniferum (opium poppy) is the source for several pharmaceutical benzylisoquinoline alkaloids including morphine, the codeine and sanguinarine. In response to treatment with a fungal elicitor, the biosynthesis and accumulation of sanguinarine is induced along with other plant defense responses in opium poppy cell cultures. The transcriptional induction of alkaloid metabolism in cultured cells provides an opportunity to identify components of this process via the integration of deep transcriptome and proteome databases generated using next-generation technologies. RESULTS: A cDNA library was prepared for opium poppy cell cultures treated with a fungal elicitor for 10 h. Using 454 GS-FLX Titanium pyrosequencing, 427,369 expressed sequence tags (ESTs) with an average length of 462 bp were generated. Assembly of these sequences yielded 93,723 unigenes, of which 23,753 were assigned Gene Ontology annotations. Transcripts encoding all known sanguinarine biosynthetic enzymes were identified in the EST database, 5 of which were represented among the 50 most abundant transcripts. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of total protein extracts from cell cultures treated with a fungal elicitor for 50 h facilitated the identification of 1,004 proteins. Proteins were fractionated by one-dimensional SDS-PAGE and digested with trypsin prior to LC-MS/MS analysis. Query of an opium poppy-specific EST database substantially enhanced peptide identification. Eight out of 10 known sanguinarine biosynthetic enzymes and many relevant primary metabolic enzymes were represented in the peptide database. CONCLUSIONS: The integration of deep transcriptome and proteome analyses provides an effective platform to catalogue the components of secondary metabolism, and to identify genes encoding uncharacterized enzymes. The establishment of corresponding transcript and protein databases generated by next-generation technologies in a system with a well-defined metabolite profile facilitates an improved linkage between genes, enzymes, and pathway components. The proteome database represents the most relevant alkaloid-producing enzymes, compared with the much deeper and more complete transcriptome library. The transcript database contained full-length mRNAs encoding most alkaloid biosynthetic enzymes, which is a key requirement for the functional characterization of novel gene candidates.

Involvement of lipoxygenase in elicitor-stimulated sanguinarine accumulation in Papaver somniferum suspension cultures.

The involvement of lipoxygenase (LOX, EC 1.13.11.12) in elicitor-induced opium poppy defense response was investigated. Papaver somniferum L. suspension cultures were treated with abiotic elicitor methyl jasmonate (MJ), fungal elicitor (Botrytis cinerea homogenate) and phenidone (specific inhibitor of LOX) to determine the involvement of this enzyme in production of sanguinarine, the major secondary metabolite of opium poppy cultures. P. somniferum suspension cultures responded to elicitor treatment with strong and transient increase of LOX activity followed by sanguinarine accumulation. LOX activity increased in elicited cultures, reaching 9.8 times of the initial value at 10 h after MJ application and 2.9 times after B. cinerea application. Sanguinarine accumulated to maximal levels of 169.5 ± 12.5 µg g⁻¹ dry cell weight in MJ-elicited cultures and 288.0 ± 10.0 µg g⁻¹ dry cell weight in B. cinerea-elicited cultures. The treatment of cells with phenidone before elicitor addition, significantly reduced sanguinarine production. The relative molecular weight of P. somniferum LOX (83 kDa) was estimated by using immunobloting and its pH optimum was shown to be pH 6.5.

Plant defense responses in opium poppy cell cultures revealed by liquid chromatography-tandem mass spectrometry proteomics.

Opium poppy (Papaver somniferum) produces a diverse array of bioactive benzylisoquinoline alkaloids, including the narcotic analgesic morphine and the antimicrobial agent sanguinarine. In contrast to the plant, cell cultures of opium poppy do not accumulate alkaloids constitutively but produce sanguinarine in response to treatment with certain fungal-derived elicitors. The induction of sanguinarine biosynthesis provides a model platform to characterize the regulation of benzylisoquinoline alkaloid pathways and other defense responses. Proteome analysis of elicitor-treated opium poppy cell cultures by two-dimensional denaturing-polyacrylamide gel electrophoresis coupled with liquid chromatography-tandem mass spectrometry facilitated the identification of 219 of 340 protein spots based on peptide fragment fingerprint searches of a combination of databases. Of the 219 hits, 129 were identified through pre-existing plant proteome databases, 63 were identified by matching predicted translation products in opium poppy-expressed sequence tag databases, and the remainder shared evidence from both databases. Metabolic enzymes represented the largest category of proteins and included S-adenosylmethionine synthetase, several glycolytic, and a nearly complete set of tricarboxylic acid cycle enzymes, one alkaloid, and several other secondary metabolic enzymes. The abundance of chaperones, heat shock proteins, protein degradation factors, and pathogenesis-related proteins provided a comprehensive proteomics view on the coordination of plant defense responses. Qualitative comparison of protein abundance in control and elicitor-treated cell cultures allowed the separation of induced and constitutive or suppressed proteins. DNA microarrays were used to corroborate increases in protein abundance with a corresponding induction in cognate transcript levels.

[The floral meristem undetermination mutation in Papaver somniferum L.: spontaneous phenotypic variation in ontogeny].

A new morphogenetic mutation of the shoot, floral meristem undetermination, was found in Papaver somniferum L. with monocarpic shoot. The expression of the DFM (determination of floral meristem) gene, which limits the proliferative activity of stem cells in the floral meristem, was affected. The mutation displayed spontaneous phenotypic instability in ontogeny, variation in the mutant character expression on different flowers of the same plant in the same genotypic environment. The mutation phenotype varied from no expression or formation of individual phyllomes in the center of the primary ovary to formation of a new flower and a new capsule with viable seeds.

Quantitative 1H NMR metabolomics reveals extensive metabolic reprogramming of primary and secondary metabolism in elicitor-treated opium poppy cell cultures.

BACKGROUND: Opium poppy (Papaver somniferum) produces a diverse array of bioactive benzylisoquinoline alkaloids and has emerged as a model system to study plant alkaloid metabolism. The plant is cultivated as the only commercial source of the narcotic analgesics morphine and codeine, but also produces many other alkaloids including the antimicrobial agent sanguinarine. Modulations in plant secondary metabolism as a result of environmental perturbations are often associated with the altered regulation of other metabolic pathways. As a key component of our functional genomics platform for opium poppy we have used proton nuclear magnetic resonance (1H NMR) metabolomics to investigate the interplay between primary and secondary metabolism in cultured opium poppy cells treated with a fungal elicitor. RESULTS: Metabolite fingerprinting and compound-specific profiling showed the extensive reprogramming of primary metabolic pathways in association with the induction of alkaloid biosynthesis in response to elicitor treatment. Using Chenomx NMR Suite v. 4.6, a software package capable of identifying and quantifying individual compounds based on their respective signature spectra, the levels of 42 diverse metabolites were monitored over a 100-hour time course in control and elicitor-treated opium poppy cell cultures. Overall, detectable and dynamic changes in the metabolome of elicitor-treated cells, especially in cellular pools of carbohydrates, organic acids and non-protein amino acids were detected within 5 hours after elicitor treatment. The metabolome of control cultures also showed substantial modulations 80 hours after the start of the time course, particularly in the levels of amino acids and phospholipid pathway intermediates. Specific flux modulations were detected throughout primary metabolism, including glycolysis, the tricarboxylic acid cycle, nitrogen assimilation, phospholipid/fatty acid synthesis and the shikimate pathway, all of which generate secondary metabolic precursors. CONCLUSION: The response of cell cultures to elicitor treatment involves the extensive reprogramming of primary and secondary metabolism, and associated cofactor biosynthetic pathways. A high-resolution map of the extensive reprogramming of primary and secondary metabolism in elicitor-treated opium poppy cell cultures is provided.

Gene transcript and metabolite profiling of elicitor-induced opium poppy cell cultures reveals the coordinate regulation of primary and secondary metabolism.

Elicitor-induced sanguinarine accumulation in opium poppy (Papaver somniferum) cell cultures provides a responsive model system to profile modulations in gene transcripts and metabolites related to alkaloid biosynthesis. An annotated expressed sequence tag (EST) database was assembled from 10,224 random clones isolated from an elicitor-treated opium poppy cell culture cDNA library. The most abundant ESTs encoded defense proteins, and enzymes involved in alkaloid metabolism and S-adenosylmethionine-dependent methyl transfer. ESTs corresponding to 40 enzymes involved in the conversion of sucrose to sanguinarine were identified. A corresponding DNA microarray was probed with RNA from cell cultures collected at various time-points after elicitor treatment, and compared with RNA from control cells. Several diverse transcript populations were coordinately induced, with alkaloid biosynthetic enzyme and defense protein transcripts displaying the most rapid and substantial increases. In addition to all known sanguinarine biosynthetic gene transcripts, mRNAs encoding several upstream primary metabolic enzymes were coordinately induced. Fourier transform-ion cyclotron resonance-mass spectrometry was used to characterize the metabolite profiles of control and elicitor-treated cell cultures. Principle component analysis revealed a significant and dynamic separation in the metabolome, represented by 992 independent detected analytes, in response to elicitor treatment. Identified metabolites included sanguinarine, dihydrosanguinarine, and the methoxylated derivatives dihydrochelirubine and chelirubine, and the alkaloid pathway intermediates N-methylcoclaurine, N-methylstylopine, and protopine. Some of the detected analytes showed temporal changes in abundance consistent with modulations in the profiles of alkaloid biosynthetic gene transcripts.

Actin depolymerization is sufficient to induce programmed cell death in self-incompatible pollen.

Self-incompatibility (SI) prevents inbreeding through specific recognition and rejection of incompatible pollen. In incompatible Papaver rhoeas pollen, SI triggers a Ca2+ signaling cascade, resulting in the inhibition of tip growth, actin depolymerization, and programmed cell death (PCD). We investigated whether actin dynamics were implicated in regulating PCD. Using the actin-stabilizing and depolymerizing drugs jasplakinolide (Jasp) and latrunculin B, we demonstrate that changes in actin filament levels or dynamics play a functional role in initiating PCD in P. rhoeas pollen, triggering a caspase-3-like activity. Significantly, SI-induced PCD in incompatible pollen was alleviated by pretreatment with Jasp. This represents the first account of a specific causal link between actin polymerization status and initiation of PCD in a plant cell and significantly advances our understanding of the mechanisms involved in SI.

Self-incompatibility triggers programmed cell death in Papaver pollen.

Sexual reproduction in many angiosperm plants involves self-incompatibility (SI), which is one of the most important mechanisms to prevent inbreeding. SI is genetically controlled by the S-locus, and involves highly specific interactions during pollination between pollen and the pistil on which it lands. This results in the rejection of incompatible ('self') pollen, whereas compatible ('non-self') pollen is allowed to fertilize the plant. In Papaver rhoeas, S-proteins encoded by the stigma component of the S-locus interact with incompatible pollen, triggering a Ca2+-dependent signalling network, resulting in the inhibition of pollen-tube growth. Programmed cell death (PCD) is a mechanism used by many organisms to destroy unwanted cells in a precisely regulated manner. Here we show that PCD is triggered by SI in an S-specific manner in incompatible pollen. This provides a demonstration of a SI system using PCD, revealing a novel mechanism to prevent self-fertilization. Furthermore, our data reveal that the response is biphasic; rapid inhibition of pollen-tube growth is followed by PCD, which is involved in a later 'decision-making' phase, making inhibition irreversible.

The self-incompatibility response in Papaver rhoeas pollen causes early and striking alterations to organelles.

Self-incompatibility (SI) in Papaver rhoeas is accompanied by a cascade of signalling events that result in the rapid arrest and eventual death of the pollen tube. We have used rapid freeze fixation, freeze substitution and transmission electron microscopy to provide the first description of changes to pollen at the ultrastructural level during SI in this species. Our studies reveal that dramatic alterations to the morphology of mitochondria, Golgi bodies and ER occur within 1 h of SI induction. Similar symptoms have also been observed during programmed cell death (PCD) in some cell types. These include: the conspicuous condensation of the vegetative and generative nuclei, the swelling and loss of cristae in mitochondria and the disappearance of Golgi bodies. Some of the early alterations to the mitochondria and Golgi bodies observed at 1 h, almost certainly occur when cells are still alive. Other events, such as nuclear condensation, occur later and coincide with DNA fragmentation and the loss of cell viability. Our observations suggest that the SI response in P. rhoeas pollen may potentially involve a type of PCD.

[Biosynthesis of poppy isoquinoline alkaloids in nature and in vitro culture. 2. Bracteum poppy (Papaver bracteatum Lindl.)]. / Biosintez izokhinolinovykh alkaloidov maka v prirode i v kul'ture in vitro. 2. Mak pritsvetnikovyi (Papaver bracteatum Lindl.).

Literature data and the data of the author's investigations on production of isoquinoline alkaloids by Papaver bracteatum Lindl. have been analyzed. Information on the methods of regulation and cell localization of morphine and sanguinarine biosynthesis is presented. The works studying differentiation processes in tissue cultures of bracteum poppy and relationship thereof with thebaine biosynthesis have been analyzed. Possible mechanism determining the induction of somatic embryos development and thebaine biosynthesis in the culture in vitro are proposed.

A tale of three cell types: alkaloid biosynthesis is localized to sieve elements in opium poppy.

Opium poppy produces a diverse array of pharmaceutical alkaloids, including the narcotic analgesics morphine and codeine. The benzylisoquinoline alkaloids of opium poppy accumulate in the cytoplasm, or latex, of specialized laticifers that accompany vascular tissues throughout the plant. However, immunofluorescence labeling using affinity-purified antibodies showed that three key enzymes, (S)-N-methylcoclaurine 3'-hydroxylase (CYP80B1), berberine bridge enzyme (BBE), and codeinone reductase (COR), involved in the biosynthesis of morphine and the related antimicrobial alkaloid sanguinarine, are restricted to the parietal region of sieve elements adjacent or proximal to laticifers. The localization of laticifers was demonstrated using antibodies specific to the major latex protein (MLP), which is characteristic of the cell type. In situ hybridization showed that CYP80B1, BBE, and COR gene transcripts were found in the companion cell paired with each sieve element, whereas MLP transcripts were restricted to laticifers. The biosynthesis and accumulation of alkaloids in opium poppy involves cell types not implicated previously in plant secondary metabolism and dramatically extends the function of sieve elements beyond the transport of solutes and information macromolecules in plants.

Immunocytological localization of two enzymes involved in berberine biosynthesis.

Using post-embedding immunogold techniques the cytological localization of the two branchpoint enzymes of isoquinoline biosynthesis, berberine bridge enzyme (BBE) and (S)-tetrahydroprotoberberine oxidase (STOX), was demonstrated. Electron-microscopic examination revealed their exclusive compartmentation within vesicles. After these vesicles have fused with the central vacuole, they release their contents, resulting in a characteristic electron-dense precipitate at the tonoplast. Vesicles of similar structure could be identified in young meristematic tissues of roots or shoots from different Berberis species and Papaver somniferum L. The appearance of electron-dense osmiophilic material is strictly correlated with the alkaloid content of the tissue. Immunocytological staining of P. somniferum tissue with antibodies directed against BBE led to a characteristic labeling of electron-dense aggregates in idioblasts that are not connected to the laticifer system. This localization demonstrates the strictly cytological separation of benzophenanthridine and morphine biosyntheses within this plant.

Alkaloid biosynthesis in Papaver sp. cells in culture and during organogenesis.

In vitro cell cultures of two Papaver species, P. somniferum and P. bracteatum initiated from mature seeds were screened for their ability to produce alkaloids. Protocols for callus induction, somatic embryogenesis and organogenesis were established. The alkaloid contents were analysed by high-performance-liquid chromatography, thin-layer chromatography and spectrophotometric assays. Undifferentiated callus produced small amounts of sanguinarine, which increased with the degree of tissue differentiation. Embryogenic calli were maintained in culture for more than 2 years, retaining a high regeneration capability. Thin-layer chromatography analysis revealed variations in alkaloid spectrum between parallel cell lines. The morphinan alkaloid, thebaine, was found to be accumulated exclusively in morphogenous strains of P. bracteatum, and morphine was the major alkaloid in the spectrum of P. somniferum dedifferentiated callus. Regenerant plants synthesized thebaine and sanguinarine at the same level as juvenile plants grown from P. bracteatum seeds. We revealed differences in the ability to produce different types of alkaloids: seed-derived plants were able to accumulate thebaine while undifferentiated primary cell cultures produced only sanguinarine. The production of either sanguinarine and morphinan alkaloids are found in regenerants showing that both metabolic pathways were active in young plantlets.

Isoquinoline alkaloid production by transformed cultures of Papaver somniferum.

Three clones of transformed cultures of opium poppy (Papaver somniferum L.) were established by infection with Agrobacterium rhizogenes MAFF 03-01724. MAFF clone 1 being capable of forming somatic embryos was selected and its growth and isoquinoline alkaloid production was investigated. The illumination, temperature and nutrient medium composition greatly affected growth, cell morphology and alkaloid accumulation. The MAFF clone 1 cultured in Root Culture medium in the dark at 22 degrees C accumulated a high quantity of sanguinarine (652 micrograms/g dry weight) though the growth was poor (4.4 fold as fresh weight basis after 2 months of culture). The MAFF clone 1 cultured in a quarter macro salt strength Woody Plant medium under 14 h/day light at 22 degrees C developed into plantlets and accumulated significant quantity of codeine (648 micrograms/g dry wt) together with papaverine, noscapine, and sanguinarine. This clone was applied to a rotating drum fermenter (2 L working volume), and ca. 0.3 mg codeine and 0.06 mg sanguinarine were obtained after 4 weeks of culture. One quarter of the codeine produced was found in the culture medium.