Papaver Bracteatum Research Articles

Cytogenetic studies in “X-7”, a new line of Papaver bracteatum Lindl. with increased thebaine content

Papaver bracteatum Lindl. is of substantial medical and pharmaceutical interest as material for commercial production of codeine. During various experiments, some involvingcolchicine treatment of seeds, a diploid plant (called “X-7”) developed. This plant had a substantial increase in the amounts of thebaine, the main alkaloid produced in the capsules. By cloning this plant, a new series of bracteaturn plants were obtained, and the thebaine content of the new individuals vaned markedly from ca 3 % to about 17 % against normal variation from 1 70 to 3 70. Subsequent cytological analyses revealed the X-7 plants to be diploid (2n=14). Their karyotype may contain minor structural differences from the mother line, but these were difficult to characterize. Marked differences of meiotic behaviour occurred. The “X-7” line appears to have arisen through diploidization and chromosomal rearrangements from polyploid tissue.

Molecular Modeling and Site-Directed Mutagenesis Reveal the Benzylisoquinoline Binding Site of the Short-Chain Dehydrogenase/Reductase Salutaridine Reductase

Recently, the NADPH-dependent short-chain dehydrogenase/reductase (SDR) salutaridine reductase (E.C. 1.1.1.248) implicated in morphine biosynthesis was cloned from Papaver somniferum. In this report, a homology model of the Papaver bracteatum homolog was created based on the x-ray structure of human carbonyl reductase 1. The model shows the typical alpha/beta-folding pattern of SDRs, including the four additional helices alphaF'-1 to alphaF'-4 assumed to prevent the dimerization of the monomeric short-chain dehydrogenases/reductases. Site-directed mutagenesis of asparagine-152, serine-180, tyrosine-236, and lysine-240 resulted in enzyme variants with strongly reduced performance or inactive enzymes, showing the involvement of these residues in the proton transfer system for the reduction of salutaridine. The strong preference for NADPH over NADH could be abolished by replacement of arginine residues 44 and 48 by glutamic acid, confirming the interaction between the arginines and the 2'-phosphate group. Docking of salutaridine into the active site revealed nine amino acids presumably responsible for the high substrate specificity of salutaridine reductase. Some of these residues are arranged in the right position by an additional alphaE' helix, which is not present in SDRs analyzed so far. Enzyme kinetic data from mutagenic replacement emphasize the critical role of these residues in salutaridine binding and provide the first data on the molecular interaction of benzylisoquinoline alkaloids with enzymes.

Opportunities of Finding Novel Anti-Infective Agents from Plant Cell Cultures

Higher plants have evolved into efficient biochemical defense mechanisms, which comprised of a wide variety of secondary compounds including alkaloids, flavonoids, terpenoids and saponins. Induction of secondary plant metabolism in cells, tissues and organ cultures do provide a potential alternative to the use of the whole plant or total synthesis of the active components. In vitro manipulation of plant secondary products offers biotechnological techniques to exploit cultured plant cell metabolism. In spite of the fact that very few plant cell processes are operating commercially, the most successful commercial pharmaceuticals produced from undifferentiated cell cultures are antibiotic compounds. The naphthoquinone shikonin from Lithospermum erythrorhizon is an anti-inflammatory, the isoquinoline alkaloid berberine from Coptis japonica is an intestinal antiseptic and the oral antimicrobial benzo-phenanthridine alkaloid sanguinarine from Papaver bracteatum are well known examples. Moreover, intensive research studies is currently devoted to produce the anti-leukaemic alkaloids vinblastine and vincristine from Catharanthus roseus, the antimalaria artemisinin from Artemisia annua and the anti-ovarian cancer taxol from Taxus brevifolis. The scope of this review is to analyze the successful in vitro production examples of antimicrobial agents, to give examples of novel chemical structures produced by plant cell cultures and not reported from the corresponding parent plants and to highlight the poten tial of biotransformation, elicitation and transformation techniques for the production of antiinfective agents from plant cell, tissue and organ cultures. Keywords: antimicrobials, biotransformation, elicitation, hairy roots, plant cell culture

Using AFLP markers for species differentiation and assessment of genetic variability of in vitro-cultured Papaver bracteatum (section Oxytona)

Amplified fragment length polymorphism (AFLP) markers were employed to deteet genetic variation among species of Papever (section Oxytona) and assess genetic fidelity between in vitro cell lines of Papaver bracteatum and mature plants derived from the propagation of their callus cultures. Regenerated plants exhibited morphological and phytochemical characteristics dissimilar to those of their source material. Thebaine, the dominant alkaloid produced by Papaver bracteatum, was not detected in capsules from mature regenerated accessions, indicating that there may have been a loss of genetic uniformity. Instead, the dominant alkaloid produced by the regenerated plant was shown to be isothebaine (by TLC and GC/MS), a metabolic characteristic of P. pseudo-orientale. A Neighbor-Joining tree constructed from AFLP fingerprints distinetly separates the three species of Oxytona while firmly grouping the in vitro-cultured plants with P. pseudo-orientale. Additionally phytochemical data and chromosome counts indicate that the seed used to initiate cultures was of hybrid origin and ihat the loss in genetic uniformity was not due to somaclonal variation occurring during the in vitro culture process. AFLP fingerprinting was therefore able to differentiate Oxytona species and invesgigate allopolyploidy in closely related papaver species.

AFLP Analysis of Opium Poppy

Amplified restriction fragment length polymorphic (AFLP) DNA analysis was performed on leaf samples of 40 accessions of opium poppy (Papaver somniferum L.) and two other control genera (Papaver bracteatum Lindley and Papaver setigerum DC.) from a commercial breeding collection held in Tasmania, Australia. A similarity dendrogram was produced on the basis of the analysis of all AFLP bands that ranged between 66 and 367 base pairs (bp) seen on an autoradiogram from denaturing polyacrylamide sequencing electrophoretic gels with three different primer pairs. It was necessary to combine the analysis of all three primer pairs into a single dendrogram to ensure that replicate analyses of related populations were grouped together. It was necessary to use more than one primer pair to resolve clearly the genetic relationship between the closely related plants described in this study; however, even a single primer pair could easily distinguish between populations of different poppy species. The application of AFLP DNA analysis for cultivar identification represents an efficient, reliable procedure for identifying opium poppy breeding lines in a definitive manner. This procedure required only a small amount of leaf material for analysis and the degree of the genetic relatedness was performed on autoradiographic banding patterns with commercially available software. These data were used to generate a similarity dendrogram depicting the predicted genetic relationship of the opium poppy cultivars. Opium poppy accessions sharing common parental lines could be distinguished unambiguously from poppy accessions of more distant genetic background. The banding patterns were reproducible, consistent within a genotype, and the DNA polymorphisms were frequent enough to be useful in characterizing genetic diversity in even closely related breeding lines.

Molecular Characterization of the Salutaridinol 7-O-Acetyltransferase Involved in Morphine Biosynthesis in Opium Poppy Papaver somniferum

Salutaridinol 7-O-acetyltransferase (EC ) catalyzes the conversion of the phenanthrene alkaloid salutaridinol to salutaridinol-7-O-acetate, the immediate precursor of thebaine along the morphine biosynthetic pathway. We have isolated a cDNA clone that corresponds to the internal amino acid sequences of the native enzyme purified from a cell suspension culture of opium poppy Papaver somniferum. The recombinant enzyme acetylated the 7-hydroxyl moiety of salutaridinol in the presence of acetyl-CoA. The apparent K(m) value for salutaridinol was determined to be 9 microm and 54 microm for acetyl-CoA. The gene transcript was detected in extracts from Papaver orientale and Papaver bracteatum in addition to P. somniferum. Genomic DNA gel blot analysis indicated that there is likely a single copy of this gene in the P. somniferum genome. The amino acid sequence of salutaridinol 7-O-acetyltransferase is most similar (37% identity) to that of deacetylvindoline acetyltransferase of Catharanthus roseus. Salutaridinol 7-O-acetyltransferase is the second enzyme specific to morphine biosynthesis for which we have isolated a cDNA. Taken together with the other cDNAs cloned encoding norcoclaurine 6-O-methyltransferase, (S)-N-methylcoclaurine 3'-hydroxylase, the cytochrome P-450 reductase, and codeinone reductase, significant progress has been made toward accumulating genes of this pathway to enable the end goal of a biotechnological production of morphinan alkaloids.

Dendryphion penicillatumandPleospora papaveracea, Destructive Seedborne Pathogens and Potential Mycoherbicides forPapaver somniferum

ABSTRACT Dendryphion penicillatum and Pleospora papaveracea were isolated from blighted Papaver somniferum and Papaver bracteatum plants grown in growth chambers and the field in Beltsville, MD. The etiology of the diseases was determined, and the fungi are being investigated as potential mycoherbicides to control the narcotic opium poppy plant. P. papaveracea is known to be a highly destructive seedborne pathogen of Papaver somniferum, causing seedling blight, leaf blight, crown rot, and capsule rot. Single conidia and ascospores were isolated and cultures established from naturally infested seed and diseased foliage and pods of opium poppy from Iran, Colombia, Venezuela, Sweden, India, and the United States (Maryland and Washington). Mycelia and conidia of P. papaveracea and D. penicillatum produced on necrotic leaf tissues appear morphologically similar, and the fungi were previously considered to be anamorph and teleomorph. However, no anamorph/teleomorph connection could be established, and the fungi appear to be distinct taxa. P. papaveracea produced conidia, mature pseudothecia, and chlamydospores in vitro and on infected stems. D. penicillatum produced conidia, microsclerotia, and macronematous conidiophores. Although both fungi were pathogenic to three poppy cultivars, conidial inoculum from P. papaveracea cultures was more virulent than conidial inoculum from D. penicillatum. Eight-week-old plants became necrotic and died 8 days after inoculation with a conidial suspension of P. papaveracea at 2 x 10(5) spores per ml. Disease severity was significantly enhanced by inoculum formulations that contained corn oil, by higher conidial inoculum concentrations, and by increased wetness periods. Symptoms on plants inoculated with either pathogen included leaf and stem necrosis, stem girdling, stunting, necrotic leaf spots, and foliar and pod blight. Inoculated seedlings exhibited wire stem, damping-off, and root rot. Conidia, and less frequently pseudothecia, of P. papaveracea and conidia of D. penicillatum were produced abundantly on inoculated, necrotic foliage, pods, and seedlings. Cultures from conidia or ascospores reisolated from these tissues consistently produced fungi whose morphologies were typical of the fungus from which the inoculum was derived.

Genetic and alkaloid analysis of Papaver species and their F1 hybrid by RAPD, HPLC and ELISA

Total DNA was extracted from the leaves of a F1 hybrid and its parents, Papaver bracteatum Lindle and P. pseudo-orientale Medw. Analysis of random-amplified polymorphic DNA (RAPD) using ten arbitrary oligonucleotide 10-mers, showed that F1 hybrid was confirmed to be genetically intermediate of both parental plants compared with the genetic distance between F1 hybrid and individual parents. Furthermore, the comparison of the band patterns between a F1 hybrid, P. bracteatum and P. pseudo-orientale clearly showed that part of the bands of both parents were induced into a F1 hybrid. The content of thebaine was determined by HPLC and ELISA used anti-thebaine monoclonal antibody.

Regeneration in cultures of Papaver bracteatum as influenced by growth hormones and temperature

Callus was induced on Papaver bracteatum Lindl. seedlings inoculated on Murashige & Skoog (1962) medium supplemented with α-naphthaleneacetic acid (NAA) (1.0 mg 1-1) and benzylamine purine (BA) (0.5 mg 1-1). Subculture resulted in excellent callus proliferation but no organogenesis. Shoots were regenerated in cultures grown on MS medium containing NAA (1.0 mg 1−1), BA (0.5 mg 1−1) and casein hyrdrolyzate (2.0 mg 1-1). The shoots developed into plantlets after 8 weeks of culture, and were induced to root on 1/2 MS without the addition of growth regulators. The rooted plantlets were transferred to soil after hardening.

Differential enhancement of benzophenanthridine alkaloid content in cell suspension cultures of Sanguinaria canadensis under conditions of combined hormonal deprivation and fungal elicitation.

An elicitation protocol, resulting in the accumulation of sanguinarine in suspension cultures of Papaver bracteatum, was assessed for induction of the same alkaloid in Sanguinaria canadensis. Although only a trace constituent of P. bracteatum plants, sanguinarine is a major alkaloid (1-3% dry wt) of S. canadensis rhizomes. By combining hormonal deprivation for various intervals and a 3-day fungal (Verticillium dahliae) elicitation, benzophenanthridine alkaloid accumulation was induced in S. canadensis cell suspensions. Chelirubine content increased (0.1-1.3% dry wt) consistently in elicited cell cultures while chelerythrine (0.01-0.10% dry wt) and sanguinarine (0-0.02% dry wt) levels were considerably less. Alkaloid accumulation always occurred upon removal of hormone but only at certain time intervals in the log phase upon fungal elicitation. Levels of dopamine, a precursor of the alkaloids, fluctuated over the incubation period, but displayed a 2- to 6-fold increase in cell suspensions grown without hormone. In some experiments dopamine accumulated to levels > 20% dry wt, and these increases were enhanced by the addition of fungal elicitor. Although the same fungal elicitor induces benzophenanthridines in taxonomically related S. canadensis and P. bracteatum, it did not elicit the accumulation of the same alkaloid in the two different plant cultures.

Enhancement of Dihydrosanguinarine Production in Suspension Cultures of Papaver, bracteatum, I. Medium Modifications

A suspension culture of Papaver bracteatum was developed, which initially yielded high levels (>2.55% of dry crt) of the benzophenanthridine alkaloid dihydrosanguinarine {1}. This productivity has been lost on repeated subculture. In an attempt to improve alkaloid yields modifications in the concentrations of various minor constituents present in the culture medium were evaluated

SalutaridineN-Oxide from the Capsules ofPapaver bracteatum

A novel compound, salutaridine N-oxide ( 1) has been isolated from PAPAVER BRACTEATUM Lindl (Papaveraceae) in which the major alkaloids were salutaridine and thebaine. Semisynthetic 1 was prepared for structure confirmation. Other components of the alkaloidal fraction were isothebaine, oripavine, neopine, and thebaine N-oxide.

Некоторые цито- и морфогенетические эффекты пара-фторфенилаланина в культуре тканей Nicotiana tabacum и Papaver вracteatum

Некоторые цито- и морфогенетические эффекты пара-фторфенилаланина в культуре тканей Nicotiana tabacum и Papaver вracteatum

Inheritance of morphological and chemical characters in interspecific hybrids between Papaver bracteatum and Papaver pseudo-orientale

The alkaloid profiles and morphological traits of the capsules of Papaver bracteatum, P. pseudo-orientale, and their hybrids were studied. Dominance of the hexaploid parent P. pseudo-orientale was observed for various characters. A genetic model assuming allelic additive effects and polysomic inheritance was elaborated for the control of isothebaine content in the capsules. The distribution of thebaine content in the segregating generations, F2 and BCF1 was evidence of the transfer of genes from the diploid parent P. bracteatum in the gametes of the interspecific hybrid and their expression in its progenies. These findings indicate the potential use of inter-specific hybrids between the Oxytona species in the breeding of cultivars for industrial or ornamental purposes.

Genetic evidence for the conversion of the morphinan alkaloid thebaine to oripavine in interspecific hybrids between Papaver bracteatum and Papaver orientale

The alkaloid profiles of ripe capsules of Papaver bracteatum, P. orientale and their interspecific hybrids (F1 and BCF1) were determined and compared. In the hybrids the thebaine content declined, while that of oripavine was higher than in the parental species. The results provided genetic evidence for the biosynthetic linkage between those alkaloids. A genetic model, assuming dosage effect interaction between the genomes of the two species was elaborated. The significance of the results for breeding purposes was considered.

Biotransformation of linalyl acetate by suspension cultures of Papaver bracteatum

Abstract Suspension cultures of Papaver bracteatum biotransform the acyclic monoterpene, linalyl acetate, into linalool, geraniol and the monocyclic alcohol, α-terpineol. Total metabolism of linalyl acetate occurs within eight days of substrate addition, with 40% of the substrate being transformed within the first 36 hr. No regiospecific hydroxylation of the 3-methyl-2-butenyl group is observed.

Ultrastructural changes associated with the accumulation and secretion of sanguinarine in Papaver bracteatum suspension cultures treated with fungal elicitor

Suspension cultures of Papaver bracteatum Arya II Lindl., grown without hormone in the presence of conidial extracts of Verticillium dahliae Kleb., accumulate millimolar quantities of the benzophenanthridine alkaloid, sanguinarine. Under the fluorescence microscope, the elicitor-treated cells display an orange-yellow fluorescence characteristic of sanguinarine, primarily near the periphery of the cells. Electron-microscopic inspection showed the presence of slightly dilated endoplasmic reticulum and of electron-dense protuberances on the tonoplast of large central vacuoles. These osmiophilic aggregates lining the tonoplast bud into spherical bodies, appear to become detached from the membrane and are released into the vacuole. Upon subcellular fractionation of elicited cells on Renografin step gradients, sanguinarine was found to be distributed in all bands but with 86% concentrated in the gradient pellet. Analysis of the pellet by electron microscopy showed that it contained electron-dense fragments similar to the osmiophilic bodies observed on the tonoplast of intact elicited cells. In elicited cell cultures, most of the sanguinarine was recovered from medium in a 100·g sedimenting, cell-free, particulate fraction accounting for as much as 85% of the media sanguinarine and 62% of the total sanguinarine. The sanguinarine-rich 100·g media pellet was determined to be two-thirds protein, one-third RNA and was essentially devoid of phenolics, phospholipid and DNA. The pellet consisted of electrondense material and cytoplasmic remnants resembling those found in the Renografin pellet and tonoplast aggregates of intact cells. When placed under hypotonic conditions or extracted with aqueous buffer, pH 3-11, the pellet did not release sanguinarine. These observations provide evidence for storage of sanguinarine at electron-dense deposits which occur on the tonoplast and as freely floating bodies in vacuoles.

Accumulation and Distribution of Thebaine in the Roots ofPapaver bracteatumDuring Plant Development

The thebaine-yield components in the roots of PAPAVER BRACTEATUM were studied at various stages of plant development. The maximum concentration of thebaine was obtained at the start of flowering. However the dry weight of the roots and the thebaine yield increased until full flowering. Significant differences in the thebaine and dry matter distribution between various parts of the roots were found; the lower part had a higher concentration of the alkaloid than the upper ones. The high thebaine yield (2.5 g/m (2)) obtained during the first growing season is most promising for the exploitation of the roots as raw material for thebaine production.

Stimulation of Sanguinarine Production by Combined Fungal Elicitation and Hormonal Deprivation in Cell Suspension Cultures of Papaver bracteatum

Fungal elicitor preparations from either homogenized mycelia of Dendryphion penicillatum (Cda.) Fr., a specific pathogen of Papaver species, or conidia of Verticillium dahliae Kleb., a general pathogen, were added to 14-day-old suspension cultures of Papaver bracteatum. Plant tissue cultures were grown either in the presence or absence of 0.1 milligram of 2,4-dichlorophenoxyacetic acid per liter and 0.5 milligram of 6-benzylam-inopurine per liter. Dendryphion extracts elicited an accumulation of the benzophenanthridine alkaloid, sanguinarine, which was not greatly influenced by hormone deprivation. Millimolar concentrations of dopamine were detected under all conditions. Thebaine was found when cells were cultured in hormone-free media, but it was not elicitor dose dependent. Verticillium-elicited cultures accumulated sanguinarine in an elicitor-dose-dependent manner only under conditions of hormonal deprivation, resulting in an elevation of sanguinarine levels 5- to 500-fold greater than controls (2-10% dry weight). Most of the sanguinarine accumulated in the medium (23 milligrams per liter), with 85% of the alkaloid associated with a 100g sedimenting fraction that, upon light microscopic inspection, proved to be devoid of cells. In bioassays, sanguinarine showed significant biological activity at concentrations as low as 5 to 10 micrograms per milliliter against three general plant pathogens, Verticillium dahliae, Botrytis cinerea Pers. ex Fr., and Rhizoctonia solani Kuehn. Dendryphion was less affected by sanguinarine addition and displayed an ability to metabolize the alkaloid as evidenced by its loss from the media, subsequent accumulation in the mycelia, and ultimate disappearance over a 48-hour period. By comparison, dopamine and thebaine were less toxic to the general plant pathogens.

Alkaloids of cell cultures derived from strains of Papaver bracteatum

Growth and alkaloid production were studied in callus and cell suspension cultures derived from seeds of three strains of Papaver bracteatum. Traces of oxysanguinarine and sanguinarine were identified in the cultures. Dihydrosanguinarine was the major alkaloid produced by all cell cultures and its crystalline intracellular accumulation was observed using fluorescence microscopy. Suspension cultures developed from one seed strain produced more than 1% of dihydrosanguinarine on a dry weight basis. A time course analysis showed its maximum content (178 mg/1 culture) on day 21; alkaloid synthesis appeared to be growth associated. This is the first report of a high dihydrosanguinarine-producing cell culture of P. bracteatum.