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     

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.     

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     

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     

Alkaloid production by plant cell suspension cultures of Holarrhena antidysenterica: I. Effect of major nutrients

The effect of major nutrients on growth and alkaloid production by plant cell culture of Holarrhena antidysenterica was studied with a view to increasing the yield of the alkaloid conessine, a therapeutic drug used for treatment of dysentery and helminthic disorders. The studies resulted in development of a modified Murashige and Skoog (MS) medium that contained 60 mM total nitrogen with a NH(4) (+)-to-NO(3) (-) ratio of 5:1, 0.25 mM phosphate, and 40 g/L sucrose. The growth regulators 2,4-dichlorophenoxy acetic acid (2,4-D) and kinetin (Kn) were also found to affect the synthesis of alkaloid. Using an optimal level of inoculum (3 g/L), the modified medium resulted in alkaloid synthesis of 0.66 g/100 g dry cell weight, which represented a 4.25-fold increase over that obtained in standard MS medium.     

A rapid high performance liquid chromatographic method for the separation of the alkaloid precursor L-tyrosine and six tetrahydroisoquinoline alkaloids of Papaver somniferum

A high performance liquid chromatographic (HPLC) method was developed for the qualitative and quantitative determination of L-tyrosine and six common tetrahydroisoquinoline alkaloids (papaverine, noscapine, sanguinarine, morphine, codeine and thebaine) of Papaver somniferum. The reversed phase HPLC method yields baseline separation of the alkaloids in 20 min and is achieved using a simple H₂O: MeOH linear gradient. Silanol effects commonly associated with the separation of such strongly basic compounds were minimized by the addition of the amine modifier, triethylamine, to the mobile phase.     

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     

Effects of alginate and immobilization by entrapment in alginate on benzophenanthridine alkaloid production in cell suspension cultures of Eschscholtzia californica

The production of benzophenanthridine alkaloids (sanguinarine, chelerythrine and macarpine) in cells of Eschscholtzia californica is enhanced by sodium alginate and by entrapment in Ca2+-alginate. Tyrosine decarboxylase, a key enzyme of alkaloid biosynthesis, is induced by the treatments. Alginate- entrapped cells are elicited over an extended period of time which leads to increased alkaloid biosynthesis (up to 800-fold enhancement). A major portion of alkaloids produced are released into the growth medium.     

Seed Dormancy in Acer: An assessment of the Rôle of the Structures Covering the Embryo

Cells of a seven year old strain of Papaver somniferum L. when cultured for 2 weeks and incubated with substances known to elicit the formation of phytoalexins, responded by turning reddish brown within 6 h and accumulating sanguinarine. Morphinan alkaloids were not detected. Media (100 ml) containing 1 ml of Botrytis spec. preparations raised the level of sanguinarine in the cells 26 times over the maximum level found in controls. Over a culture period of 79 h the cells achieved a sanguinarine concentration of 2.9% of dry weight. Media (100 ml) with 1 ml of Rhodotorula rubra preparation, 15 mg arachidonic acid, 1 mg actinomycin, 0.5 ml of Helminthosporium gramineum, Sclerotinia sclerotiorum, or 5 ml Colletotrichum gloeosporoides preparation elicited a considerable, but relatively weaker response. Sanguinarine accumulation was also found to occur in the medium and reached a concentration of 43% of total sanguinarine per culture when cells were cultured in 100 ml medium with 5 ml Colletotrichum preparation for 24 h. Young poppy cell cultures initiated 9 months ago responded to the presence of Botrytis material as did 7-year-old cultures.     

Spatial and temporal distribution of the alkaloid sanguinarine in Sanguinaria canadensis L. (Bloodroot)

Acclimated and non-acclimated potted plants of Sanguinaria canadensis L. were harvested at early and late dormancy, anthesis, and immature and mature fruiting stages. Sanguinarine content and concentration were determined for rhizomes (distal, proximal, and middle sections), roots, leaves, flower, and fruit. Rhizomes had highest sanguinarine content and concentrations, and exhibited decreasing concentration gradients from the distal to proximal third. Concentrations in roots were a tenth of rhizome concentration. Concentrations in leaves, flowers, and fruit were one-thousandth of rhizome Sanguinarine content in whole acclimated plants was constant. Content in whole nonacclimated plants increased as the plant became physiologically active, but was constant during fruit maturation: content in roots, leaves, and fruit did not change. The substantial increase in whole-plant dry weight coupled with the unchanging sanguinarine content during fruit maturation suggests either a shift in photosynthate allocation from defense to growth, or a constant turnover of sanguinarine.     

Increased anthraquinone production in Galium vernum cell cultures induced by polymeric adsorbents

Anthraquinones produced by suspension cultures of Galium vernum are completely retained intracellularly. Surprisingly, in the presence of some polymeric adsorbents anthraquinones are partially released into the culture medium. The secretion and in situ removal stimulates anthraquinone production in cell cultures of Galium vernum. Best results were obtained with Wofatit ES and Amberlite XAD-2.Abbreviations DW dry weight - MS Murashige & Skoog[7]medium - NAA 1-naphthaleneacetic acid     

Modeling linear and variable growth in phosphate limited suspension cultures of Opium poppy

In examining the growth kinetics of cell suspensions of opium poppy (Papaver somniferum), the increase in biomass with time was observed to be linear over the entire batch growth period of up to 20 days. Although batch growth profiles were reproducible utilizing the same inoculum, growth rates varied tremendously when experiments were inoculated with cells from different flasks. Both of these phenomena are difficult to explain with conventional batch growth models. In a series of a experiments, phosphate was determined to be the growth-rate-limiting substrate. By expressing growth rate in terms of the intracellular reserves of phosphorus, a growth model which expresses kinetics in terms of the intracellular phosphorus contents of the cells is shown to predict both linear growth character and inoculum dependent variability in growth. The stationary phase phosphate content of seven plant suspension cultures of different plant species was found to be comparable to phosphorus levels of phosphate-starved poppy cells, which suggests that phosphate limitation may be common for plant tissue culture. The applicability of this model to other biological systems which display similar batch growth patterns when subjected to inorganic nutrient deprivation is discussed.     

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.     

Effect of 2-chloroethylphosphonic acid on capsule formation and alkaloid content in Papaver somniferum

Application of different concentrations of ethephon (2-chloroethylphosphonic acid) to Papaver somniferum L. at the times of stem elongation, bud, and capsule formation produced different effects. Ethephon (10^-2 M ) retarded growth of the plant and inhibited capsule formation during stem elongation, significantly reduced capsule size during the flowering period, but did not alter capsule development during capsule formation. When applied during the period of stem elongation, ethephon (10^-3 M and 10^-4 M ) reduced capsule size; alkaloid accumulation was reduced by ethephon at a concentration of 10^-3 M , but slightly increased by 10^-4 M . Ethephon (10^-3 M and 10^-4 M ) did not alter capsule development or alkaloid content significantly when applied during bud formation, but stimulated capsule size and alkaloid content when applied during capsule formation. Pretreating the plants with Ag⁺ (silver nitrate) did not reverse the ethephon effect. The results suggest that capsule maturation and alkaloid accumulation in P. somniferum are modified by ethylene, which is produced as a result of exogenous ethephon treatment.     

In situ extraction strategy affects benzophenanthridine alkaloid production fluxes in suspension cultures of Eschscholtzia californica

The effect of contact between cells and extractive phase on secondary metabolite production was investigated in two-phase suspension cultures of Eschscholtzia californica. A system was designed to extract benzophenanthridine alkaloids from the cell culture, without contact between XAD-7 resins and the cells: only medium was recirculated through a column packed with the extractive phase. This strategy was compared to the classic method of addition of resins directly into the cell suspension. Removal of the product directly from the medium enabled important increases in production of alkaloids, namely a 20-fold increase in sanguinarine production and a 10-fold increase in chelerythrine, with high recovery in the resin. The recirculation strategy greatly simplified the production process since the resins are easily recovered from the cell culture and enable harvest of product without termination of culture. However, due to limited flow rate, the recirculation strategy was slightly less effective than direct addition of resins into the cell suspension. In addition to enabling increased production, removal of secondary metabolites from the medium changed metabolic flux distribution, testifying to a complex control mechanism of production.     

Two-phase airlift fermentor operation with elicitation for the enhanced production of enzophenanthridine alkaloids in cell suspensions of Escherichia californica

Approaches to increasing the productivity of benzophenanthridine alkaloids in suspension cultures in Escherichia californica were made in an airlift fermentor under different culture conditions. Elicitation with yeast extract elicitor reduced the time required to obtain a certain amount of alkaloid production. In a two-phase airlift fermentor with compounded silicone fluid, total alkaloid concentration in silicone fluid was 153.1 mg/L and that in the aqueous cellular phase was 8.2 mg/L at day 21 from inoculation. The large accumulation capacity of silicone fluid made it possible to store correspondingly large amounts of total alkaloid and increased the alkaloid production. Act day 21 from inoculation, the volumetric alkaloid productivity and the netproduction in a two-phase airlift fermentor were 1.4 and 1.5 times higher than those of normal airlift fermentor operation. This performance was furthermore enhanced by elicitation. Elicitation in two-phase airlift fermentor operation increased the volumetric productivity and the new production 3.3- and 3.5-fold compared to those of normal airlift fermentor operation. (c) 1994 John Wiley & Sons, Inc.