Effect of ethylene on sanguinarine production from Papaver somniferum cell cultures

A Papaver somniferum cell line capable of producing sanguinarine equivalent to 3% of cell dry weight was used to determine if ethylene was involved in signalling the biosynthesis of this alkaloid. A 3.3-fold increase in ethylene emanation from these cell suspension cultures was observed 7 h after elicitation with a Botrytis fungal homogenate. The rate of ethylene release then decreased to near zero after 48 h, suggesting that a pulse of ethylene production may be involved in sanguinarine production. However, sanguinarine biosynthesis was not promoted when either the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), or the ethylene releasing agent, 2-chloroethylphosphonic acid (ethephon), was added to the culture. These results strongly suggest that ethylene is not intimately involved in the production of sanguinarine from Papaver somniferum cell cultures or in the transduction of the elicitation event.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid     

Semi-continuous production of sanguinarine and dihydrosanguinarine by Papaver somniferum L. cell suspension cultures treated with fungal homogenate

Papaver somniferum L. (opium poppy) cells were elicited with a Botrytis sp. homogenate and cultured by a semi-continuous process. Elicitation induced synthesis of sanguinarine and dihydrosanguinarine. Significant release of both alkaloids into the culture medium occurred. Medium exchange at 2-day intervals enabled product recovery from spent medium and maintained culture viability. Culture growth was not inhibited by elicitor treatment necessitating sub-culture prior to re-elicitation. Re-elicited cultures displayed an increasing sensitivity (reduced growth rate, higher alkaloid yield) to the elicitor with each successive treatment and did not survive a fourth elicitation.Abbreviations DW dry weight - FW fresh weight - 2,4-D 2,4-dichlorophenoxyacetic acid - SGE sanguinarine - DSGE dihydrosanguinarine Publication 29143 from the National Research Council of Canada     

Effect of polymeric adsorbents on the production of sanguinarine by Papaver somniferum cell cultures

The suitability of adsorbent polymeric resins, Amberlite XAD-4 and XAD-7 (Rohm and Hass, Inc.), was investigated for the accumulation of sanguinarine from Papaver somniferum cell cultures. The adsorption and desorption of sanguinarine from aqueous solution was most effective with XAD-7. In addition to sanguinarine, the resins were found to absorb growth regulators and vitamins from the culture medium. Growth inhibition was overcome by delaying for approximately 4 days resin addition after cell inoculation in fresh medium. Resin addition (5% wt/vol) to actively growing uneclicited cultures led to increases in sanguinarine production and release of 30% to 40% and 60%, respectively. The addition of resins to elicited cultures led to increases in alkaloid production of up to 50% to 85% with similar increases in alkaloid release as observed for nonelicited cells. Overall yield of sanguinarine increased from 21 mg . g biomass dry weight(-1) (dw) for elicited cultures to more than 39 mg . gdw(-1) when elicitation was combined with resin addition. Higher quantities of resin (10% to 20% wt/vol) increased marginally the release of sanguinarine into the medium, and on the resin, up to 85% of total production. The use of resin appears promesing for the development of a bioprocess for sanguinarine production by cultured plant cells. (c) 1992 John Wiley & Sons, Inc.     

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.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - EM electron microscopy - ER endoplasmic reticulum - HPLC high-pressure liquid chromatography - MRST Murashige and Skoog's revised tobacco medium     

Biotransformation of thebaine by cell suspension cultures of Papaver somniferum cv. Marianne

Thebaine is biotransformed to neopine by cell suspension cultures of Papaver somniferum cv. Marianne grown in O-B5 medium. Results of precursor stu     

Biotransformation of thebaine by cell cultures of Papaver somniferum and Mahonia nervosa

A Papaver somnifverum cell culture transforms thebaine into tetrahydrothebaine and thebainone while Mahonia nervosa transforms thebaine into oripavine     

Transformation of Papaver somniferum cell suspension cultures with sam1 from A. thaliana results in cell lines of different S-adenosyl-L-methionine synthetase activity

We have developed a procedure for opium poppy ( Papaver somniferum ) transformation using Agrobacterium tumefaciens -mediated gene delivery. Hypocotyl-derived cell suspension cultures of P. somniferum were cocultivated with A. tumefaciens strain GV3101(pMP90) harbouring either the binary vector pTHW136 or the binary vector pO35SSAM. The former contained the uidA reporter gene and the nptII selectable gene, encoding the enzymes β -glucuronidase and neomycin phosphotransferase II, respectively. The latter contained the sam1 gene encoding the enzyme S -adenosyl-L-methionine (SAM) synthetase and the nptII gene. Putatively transformed cell lines were selected on media supplemented with paromomycin. Integration of the foreign genes was confirmed by Southern blot analysis with and without PCR amplification prior to hybridization. SAM synthetase activity was measured in extracts of 5 transformed cell lines. One of them expressed a significant overactivity while two others had a lower activity than the control cell line, leading us to question the possible partial cosuppression of both the resident and the foreign sam genes. To our knowledge, this is the first report of Agrobacterium tumefaciens -mediated transformation of Papaver somniferum cell suspension cultures.     

Laticifers in stamens of Papaver somniferum L

Summary Articulated anastomosing laticifers were identified at both light and electron microscopic levels in the stamens of Papaver somniferum L. They were observed associated with the phloem forming a continuous system from the filament into the anther of the stamen. Laticifers, which were comparable in structure to laticifers found elsewhere in the plant, possessed numerous vesicles of different sizes within the protoplast.     

Somatic embryogenesis of tissue cultures of Papaver somniferum and Papaver orientale and its relationship to alkaloid and lipid metabolism

Transfer from complete to 2,4-D free Gamborg's B5-medium efficiently induced somatic embryogenesis in Papaver tissue cultures (P. somniferum and P. orientale). Embryogenesis was preceded by a strong temporary accumulation of triacylglycerols. In both tissue cultures large amounts of sanguinarine type alkaloids were present, which disappeared during regeneration in the P. orientale cultures but persisted in the P. somniferum cultures. In the P. somniferum cultures protopine and morphine type alkaloids (morphine, codeine, thebaine) appeared about 45 days after exchanging the medium. Thebaine was the main alkaloid in the P. somniferum embryoids accumulating up to 0.2 % of dry weight.     

Oxidation of tyrosine by Papaver somniferum latex

The polyphenolase complex isolated from the organelles which sedimented at 1000 g from the latex of Papaver somniferum was found to be composed of soluble and mernbrane-bound fractions. Partial purification resolved two polyphenolases, only one of which utilized tyrosine, a probable precursor of the alkaloid morphine. Activity of these two polyphenolase fractions was shown to change during the development of the capsule.     

Enhanced codeine and morphine production in suspended Papaver somniferum cultures after removal of exogenous hormones

Summary Morphine and codeine accumulation in Papaver somniferum suspension cultures increased markedly after removal of hormones from the medium. Cultures developed hormone self-sufficiency without organogenesis or development of meristemoids; enhanced synthesis of morphinan alkaloids was not dependent on formation of shoots, roots or embryos. Without exogenous hormones, maximum codeine and morphine concentrations were 3.0 mg g^–1 dry weight and 2.5 mg g^–1 dry weight respectively, up to three times higher than in cultures supplied with hormones. Hormone-deprived cells produced a higher ratio of codeine:morphine than cultures supplied with auxin and cytokinin. Improved alkaloid production was correlated with slower overall growth rate.     

Biotransformation of (RS)-reticuline and morphinan alkaloids by cell cultures of Papaver somniferum

(RS)-Reticuline was stereospecifically converted to (—)-(S)-scoulerine and (—)-(S)-cheilanthifoline by cell cultures of Papaver somniferum and (—)-(R)-reticuline was recovered as an optical pure compound by racemic resolution. (—)-Codeinone was converted in high yield to (—)-codeine in both cell culture and enzyme preparation, but the other morphinans, thebaine, codeine and morphine, were not metabolized.     

Mineral Nutrition and Morphine Production in Papaver somniferum

Mineral nutrition of poppy (Paparer somniferum L.) was studied in its effects on morphine production. Hydroponic cultures were carried out with nutritive solutions percolating over sand. The anion NO_3- is the most efficient form of nitrogen for the production of fresh matter, dry matter or total morphine; in the latter respect, it rates higher than the NH₄NO₃ form which, on the other hand, gives higher alkaloid contents, while both cation NH₄⁺ and urea have depressive effects. Phosphates have apparently little effect on the growth of the poppy, but solutions enriched with assimilable phosphate do stimulate flower proliferation and fruit development, without increasing markedly the total morphine output. Mg²⁺ and Ca²⁺ are important factors; Mg deficiencies will bring about a marked elongation of stems and early flowering, without any notable decrease in morphine outputs; conversely, Ca deficiencies will cause a drop in alkaloid production, while more calcium in the solution will give stronger elongation, a larger number of capsules and a marked increase in the weight of dry matter and morphine outputs, without any marked change in content. Sodium will favour poppy development (flowers and capsules) and will increase both the content and output of morphine. Na⁺ should therefore be introduced whenever possible in the fertilizing of poppy crops.     

Regulatory role of elements in the formation and accumulation of alkaloids in Papaver somniferum L. seedlings

The effects of 12 elements on the formation and accumulation of isoquinolines were studied in opium poppy seedlings (Papaver somniferum L.). Three types of concentration dependences of the effects of the elements under study were determined. The elements served as activators (Co, Mo, W, Cr, and Cu) or inhibitors of these processes (B, Fe, V, Mn, and Ca). The molecular mechanisms of action of these regulators are discussed. The optimum concentrations of Co and Mo were tested under combined treatment conditions with these elements.     

Regulatory role of elements in the formation and accumulation of alkaloids in Papaver somniferum L. seedlings

The effects of 12 elements on the formation and accumulation of isoquinolines were studied in opium poppy seedlings (Papaver somniferum L.). Three types of concentration dependences of the effects of the elements under study were determined. The elements served as activators (Co, Mo, W, Cr, and Cu) or inhibitors of these processes (B, Fe, V, Mn, and Ca). The molecular mechanisms of action of these regulators are discussed. The optimum concentrations of Co and Mo were tested under combined treatment conditions with these elements.     

Somatic embryogenesis in the opium poppy, Papaver somniferum

A procedure is described for the induction of somatic embryos in the opium poppy. Papaver somniferum L. Callus was obtained from seedling hypocotyls on an agar solidified medium [Murashige and Skoog (1962) Physiol. Plant. 15: 473–497] containing 0.25 mg/l (1.2 μ M ) kinetin and 2.0 mg/l (10.7 μ M ) naphtalene acetic acid (NAA). Suspension cultures were initiated from callus using a liquid medium in which 2.0 mg/l (9.0 μ M ) 2,4-dichlorophenoxyacetic acid was substituted for NAA. Meristemoids, spheres of closely packed cells, developed in suspensions and on the surface of a few callus cultures. Differentiation of meristemoids into somatic embryos was accomplished by removing growth regulators from the liquid medium. Embryoids appeared morphologically normal and similar to torpedo stage embryos, however, they possessed mature tracheary elements and laticifers in areas that should have contained only procambium. Whole plants have been obtained by placing embryos in the light on solid medium that also lacked growth regulators.     

Bound morphine and codeine in the capsule of Papaver somniferum

The polysaccharide fraction of the capsule of Papaver somniferum contained bound morphine and codeine. The alkaloids appear to be bound to the polymer by two different types of linkage.     

Variability of flavonol contents during floral morphogenesis in Papaver somniferum L

We studied the contents of flavonols (kaempferol and quercetin) in the meristem of vegetative and generative apices of the main plant shoot in floral Papaver somniferum mutants, as well as in the normal plants at successive stages of flower development. Five stages of flower development were distinguished. Flavonols (kaempferol and quercetin) were present in all flower organs at all stages of floral morphogenesis we studied. However, their contents and distribution in different organs and at different stages of flower development markedly varied. No significant differences were found in the contents of flavonols in the meristems of vegetative and generative apices of the main shoot in the lines of floral mutants, as well as between the lines with different amounts of vegetative phytomeres. In the plants with normal flower structure, the contents of flavonols (kaempferol + quercetin) sharply increased with the beginning of differentiation of flower organs, i.e. from stage 3, to reach a maximum in the open flower, when gametogenesis is terminated and fertilization takes place. The level of flavonol contents in the petals (upper part) and stamen was at a maximum at all stages of flower development, while that in the gynaecium was at a minimum. The kaempferol: quercetin ratio shifted towards quercetin at successive stages of flower development, most significantly in the stamens. The involvement of flavonols in the regulation of floral morphogenesis at stages of flower organs differentiation and functioning is discussed.