Influence of methyl jasmonate and coniferyl alcohol on pinoresinol and matairesinol accumulation in a Forsythia × intermedia suspension culture

The accumulation of the lignans pinoresinol and matairesinol (both predominantly as glucosides) in a Forsythia 2 intermedia cell suspension culture was enhanced about three- and sevenfold, respectively, by the addition of methyl jasmonate to the cell culture medium. Cells accumulated 0.86ǂ.19 mg/g dry weight pinoresinol and 2.24ǃ.00 mg/g dry weight matairesinol. Feeding experiments with the precursor coniferyl alcohol resulted in a fast increase in the pinoresinol content, but matairesinol accumulation was not influenced. The racemic ratio of pinoresinol was 77dž% (+)-pinoresinol in methyl jasmonate-treated cells and 21Dž% (+)-pinoresinol in cells fed with coniferyl alcohol.     

Rhizogenesis in Forsythia×intermedia and Syringa vulgaris; application of a simple internode experimental system

 Differences in rhizogenesis between easy-to-root Forsythia×intermedia Zab. cv. Lynwood and difficult-to-root Syringa vulgaris L. cv. Madame Lemoine were measured in an experimental system based upon internodal stem sections excised from axillary shoot cultures. Root induction in Syringa was distinctly polar, responding best to distal application of indole-3-butyric acid (IBA), whereas Forsythia was equally responsive to IBA applied at either end. Root initiation in Syringa declined rapidly from 73% to 32% over 48 h when the application of a 24-h pulse of IBA was delayed following excision of the internode; in Forsythia a smaller decline (93–70%) occurred over 144 h. Forsythia internodes were the more responsive, and developed roots after distal or proximal application of 3 μM IBA, whereas Syringa required the distal application of 30 μM IBA. Received: 22 March 2000 / Revision received: 5 June 2000 / Accepted: 5 June 2000     

Regeneration and Agrobacterium-mediated transformation of Forsythia×intermedia "Spring Glory"

Internode explants ofin vitro plants ofForsythia x intermedia Spring Glory were transformed with thegus andnpt II genes after inoculation with theA. tumefaciens strain EHA 101 harbouring the plasmid pFAJ3000. Shoot organogenesis took place from callused edges of explants. The first transformed buds were detected 4 to 6 weeks after transfer on regeneration medium, containing 25 mg/l kanamycin as selective agent. An average of 1% of explants regenerated transgenic shoots.-glucuronidase assays and culture on kanamycin-containing medium provided the first indication of integration and expression of introduced genes in transformants. Southern blot and polymerase chain reaction amplification analyses gave molecular confirmation of genetic transformation. Transgenic plants were acclimatized in the greenhouse. Enzymatic assays on several organs of mature plants still showed -glucuronidase activity, thus confirming stable integration of T-DNA in the plant genome.Abbreviations BAP 6-benzyl-aminopurine - CaMV Cauliflower Mosaic Virus - GUS andgus -glucuronidase - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog - NOS nopaline synthase - NPT II andnpt II neomycin phosphotransferase II - PCR polymerase chain reaction - SDS sodium dodecyl sulphate - SSC sodium chloride-sodium citrate - X-Gluc 5-bromo-4-cbloro-3-indolyl glucuronide     

Enhancement of α-tocopherol content through transgenic and cell suspension culture systems in tobacco

The tocopherols are amphipathic antioxidant synthesized by photosynthetic organisms, which forms the essential component in the human diet. To increase the α-tocopherol content in tobacco, two approaches have been attempted in this study: (1) transgenic approach, by constitutive overexpression of the genes encoding Arabidopsis homogentisate phytyltransferase (HPT) and tocopherol cyclase (TC) through Agrobacterium-mediated genetic transformation; (2) non-transgenic approach, by supplementation of intermediates/precursors of vitamin E biosynthesis like tyrosine, p-hydroxyphenyl pyruvic acid, homogentisic acid (HGA) and phytol in different concentrations and combinations using cell suspension culture system. Molecular analyses by PCR, RT-PCR and Southern hybridization were carried out to confirm the HPT and TC expressing transgenic tobacco lines. The α-tocopherol content in transgenic plants expressing HPT and TC increase by 5.5 and 4.1, respectively, over the wild type. These results indicate that, HPT and TC activities are important in tobacco plants for enhancing the vitamin E content. In the second approach, the supplementation of precursor in cell suspension cultures, i.e., combination of 150 μM HGA + 100 μM phytol, showed the maximum enhancement of α-tocopherol, i.e., 36-fold. These findings clearly imply that enhancement of α-tocopherol levels in tobacco system is possible, if we could modulate the vitamin E metabolic pathway. This is a very useful finding for the large-scale production of natural Vitamin E. Among the two systems tested, cell suspension culture-based system is ideal over the transgenic technology due to its efficiency and no biosafety concerns.     

Hydroxylation and glycosylation of Δ9-tetrahydrocannabinol by Catharanthus roseus cell suspension culture

Δ⁹-tetrahydrocannabinol is the active constituent in Cannabis sativa, with reported analgesic, anti-emetic, anti-oxidative, neuroprotective, and anti-inflammatory activities. Δ⁹-THC has been used to treat a number of disease states including pain, anxiety, asthma, glaucoma, and hypertension. Poor water solubility of Δ⁹-THC greatly reduces its clinical effectiveness. Consequently, there is a need to modify the compound to increase its polarity and pharmaceutical efficacy. The aim of this study was to test the capability of Catharanthus roseus suspension cultured cells to convert Δ⁹-THC into more polar derivatives. The transformed metabolites were analyzed and isolated by HPLC. Structures of some new derivatives were proposed on the basis of molecular ion peaks and fragmentation patterns obtained from LC-MS and UV spectra obtained by HPLC, respectively. Δ⁹-THC was rapidly absorbed by Catharanthus roseus cultured cells and upon biotransformation new glycosylated and hydroxylated derivatives were isolated by preparative HPLC. In addition, cannabinol was detected as degradation product, including its glycosylated derivative. Based on these results, it is concluded that Catharanthus cultured cells have great potential to transform Δ⁹-THC into more polar derivatives and can be used for the large scale production of new cannabinoids, which can be a source of new compounds with interesting pharmacological profiles.     

Establishment and growth kinetics of the mutant Ehrlich-Lettré ascites cell strain HD33 in permanent suspension culture

Cells of a mutant in vivo subline of the Ehrlich-Lettré mouse ascites tumour (ELAT) were converted to growth in suspension culture. Kinetic analysis revealed the selective character of the conversion process; without a detectable adaptation period, a fraction of about 2 X 10(-5) of the explanted cells continued to grow in vitro. The resulting, mutant Ehrlich-Lettré ascites cell strain was designated HD33 and propagated uninterruptedly from 1974 on. The corresponding in vivo ELAT subline HD33 was derived from the HD33 ascites cell strain by intraperitoneal retransplantation. In HD33 cell suspension cultures, the population doubling time, the average intermitotic interval, as determined by videomonitoring, and the average duration of the cell cycle, as determined from percentage of labelled mitoses (PLM) data, were all measured at 15 hr. Cell loss and quiescent compartments were insignificant. The duration of the G1 phase was effectively zero. Both PLM data and [3H]/[14C] thymidine double-labelling measurements revealed an S-phase duration of between 11 and 12 hr. The G2 phase lasted 3-5 hr. The HD33 strain differs from comparable suspension strains of wild-type Ehrlich ascites cells in the insignificant role of density-dependent inhibition in growth, and the striking prolongation of the S phase which is associated with an excessive, cytoplasmic storage of glycogen by the mutant cells.     

Hydrogen peroxide. Ubiquitous in cell culture and in vivo?

Hydrogen peroxide (H2O2) is widely regarded as a cytotoxic agent whose levels must be minimized by the action of antioxidant defence enzymes. In fact, H2O2 is poorly reactive in the absence of transition metal ions. Exposure of certain human tissues to H2O2 may be greater than is commonly supposed; levels of H2O2 in the human body may be controlled not only by catabolism but also by excretion, and H2O2 could play a role in the regulation of renal function and as an antibacterial agent in the urine. Cell culture is a widely used method for the investigation of "physiological" processes such as signal transduction and regulation of gene expression, but chemical reactions involving cell culture media are rarely considered. Addition of reducing agents to commonly used cell-culture media can lead to generation of substantial amounts of H2O2. Some or all of the reported effects of ascorbic acid and polyphenolic compounds (e.g., quercetin, catechin, epigallocatechin, epigallocatechin gallate) on cells in culture may be due to H2O2 generation by interaction of these compounds with cell culture media.     

CRF stimulates α-MSH secretion and cyclic AMP accumulation in rat pars intermedia cells

Ovine corticotropin-releasing factor (CRF) stimulates α-MSH release and cyclic AMP accumulation in rat pars intermedia cells in culture at ED₅₀ values of 1 and 6 nM, respectively. The stimulatory effect of CRF on both parameters is inhibited by the dopaminergic agonist 2-bromo-α-ergocryptine (CB-154). The present data show that CRF is a potent stimulator of peptide secretion in the intermediate lobe of the pituitary gland and suggest a role of this pituitary lobe in the response to stress. In addition, the present data clearly indicate a role of cyclic AMP as mediator of the action of CRF in pars cells.     

Effect of picloram and methyl jasmonate on growth and taxane accumulation in callus culture of Taxus × media var. Hatfieldii

We have analysed the effect of some culture conditions and media components on callus growth rate and production of taxanes in callus of Taxus × media var. Hatfieldii. For callus induction and maintenance a Gamborg B5 medium and a White - Rangaswamy medium (WR) with different modifications were used. On an improved WR medium (containing 10 μM picloram) the callus growth factor increased up to 5.8 fold (fresh weight). Picloram only enhanced the growth of callus, but not taxane production. On WR medium with (100 μM) methyl jasmonate the paclitaxel content increased from 2.37 μg g-1 to 90 μg g-1 and cephalomannine from 5.14 μg g-1 to 29.14 μg g-1 (dry weight), whereas growth of the cultures ceased. The presence of paclitaxel and cephalomannine was established by high performance liquid chromatography. This revised version was published online in June 2006 with corrections to the Cover Date.     

Artefacts in cell culture: α-Ketoglutarate can scavenge hydrogen peroxide generated by ascorbate and epigallocatechin gallate in cell culture media

Ascorbate and several phenolic compounds readily oxidise in cell culture media to generate hydrogen peroxide. However, addition of α-ketoglutarate, which is known to be released by several cell types, decreased the levels of H₂O₂, and the α-ketoglutarate was depleted and converted to succinate. These observations could account for previous reports of the protective effects of α-ketoglutarate in promoting the growth of cells in culture, and may contribute to explaining some of the variability in the literature in reported rates of H₂O₂ production from autoxidisable compounds in cell culture systems.     

Glutathionyl-biliverdin IXα, a new heme catabolite in a marine annelid: Sex and cell specific accumulation

We have isolated and characterized the green pigment accumulating in coelomic cells (eleocytes) of the common clam worm Nereis virens by means of RP-HPLC and ESI-tandem mass spectrometry. This pigment is a novel biliverdin-glutathione conjugate in which the glutathione is linked to the biliverdin-backbone via a thioether bond. The yolk precursor vitellogenin, a female-specific high-density lipoprotein (ρ = 1179 kg/m³) with a native molecular mass of ∼500 kDa and a subunit mass of ∼150 kDa, is capable of transporting this pigment as well as heme. The sex-independent large discoidal lipoprotein present in the coelomic fluid, which is sequestered by the eleocytes, could also be shown to transport heme. This renders both lipoproteins as heme-lipoproteins. As the vitellogenin is secreted by the eleocytes, this suggests the eleocytes as a metabolic hub linking the uptake of the potent pro-oxidant heme thought to arise from aged hemoglobin via the large discoidal lipoprotein and its conversion to a bile pigment-conjugate. The conjugate is exported from the eleocytes to the oocytes via vitellogenin leading to the accumulation of green yolk protein. In contrast, no such export route exists in male eleocytes resulting in an accumulation of the biliverdin-conjugate in these cells.     

Anther culture and chromosome reduction in wheat × Thinopyrum wide crosses

Callus cultures of Ambrosia tenuifolia were established from sub-apical leaves. The explants were grown on basal media MS (Murashige and Skoog) supplemented with 10 μM kinetin, 1 μM 2-4 dichlorophenoxyacetic, ascorbic acid and cysteine and cultivated either in light or darkness. To determine the effect of each individual salt component on the growing rate of the callus and the psilostachyinolides and altamisine production in the culture, the concentrations of each nutrient was tested at different levels, ranging from 50% above to 50% below the standard medium. Interestingly, increased concentration of boron in the medium, resulted in a four fold increase in the production of sesquiterpene lactones by the callus. However, positive production of psilostachynolides and altamisine was only detected when the basal concentrations of the others components of the basal media were used. In addition, production of altamisine was highly sensitive to the variations of nutrients. No statiscally effect on terpenoide production by the callus was detected by varying light exposure. These results indicates that conditions for the optimal production of these terpenoids can be enhanced by modifying nutrients of the MS basal media. This revised version was published online in June 2006 with corrections to the Cover Date.     

Pyrimidine degradation in tomato cell suspension cultures and in Euglena gracilis—localization of enzymes

• 1.1. Subcellular location of dihydropyrimidinase and NCβA-amidohydrolase2 was studied in a cell suspension culture of tomato (Lycopersicon esculentum cv. Lukullus) and in Euglena gracilis.
• 2.2. By differential centrifugation, crude extracts were separated into ten fractions. Activities of both enzymes were found mainly in cytosolic fractions marked by EDH (tomato) and glu-6-P-DH (E. gracilis).
• 3.3. A cytosolic location was also found by a 20–60% and a 17.5–30% sucrose density gradients.
• 4.4. Using mitochondrial marker enzymes such as fumarase, SDH, CS and MDH, a mitochondrial occurrence of both enzymes or their release from mitochondria can be excluded by sucrose gradient centrifugations. This can also be achieved using purified mitochondria prepared from tomato cells by two subsequent sucrose gradients.
• 5.5. A possible vacuolar location of dihydropyrimidinase and NCβA-amidohydrolase was excluded by comparing their activities in isolated protoplasts and purified vacuoles which were characterized by their marker enzyme α-mannosidase.
• 6.6. A nuclear location of both enzymes and/or their release from the nucleus during procedures used cannot be excluded.
• 7.7. The results are discussed in relation to subcellular location to other pyrimidine-metabolizing enzymes in plant cells.

     

Behaviour in culture of isolated protoplasts from “Paul's scarlet” rose suspension culture cells

Summary Protoplasts were enzymatically isolated from Paul's scarlet rose suspension culture cells. They were cultured in medium similar to that used to culture the cells from which they were isolated with the addition of sucrose as an osmotic stabiliser. They were studied by light and electron microscopy and their changes in size and number per culture were recorded. Expansion was greater when the protoplasts were cultured in medium plus 12% sucrose than with 24% sucrose. Budding was observed. In medium plus 12% sucrose about 45% of the protoplasts divided but in medium plus 24% sucrose far fewer divided. Cytokinesis was abnormal: the phragmoplast disappeared soon after cytokinesis began and the cell plate became a groove and then a fibril-lined or filled tongue which progressed across the vacuole, unconnected by strands to other parts of the protoplast. The wall regenerated after several days culture in medium plus 12% sucrose fluoresced with calcofluor. The wall regenerated in medium with 24% sucrose fluoresced usually only after several weeks culture. Cytokinesis hastened formation of a wall fluorescing with calcofluor. In the electron microscope the wall was seen to contain fibrils and non-fibrillar material. The latter was the minor component in medium plus 12% sucrose but was usually the major component in medium plus 24% sucrose. The growth in plasmolysing and nonplasmolysing medium of the cells from which protoplasts are isolated was also studied.It appears that loss of the wall alters the potential of protoplasts to expand and possibly also to regenerate a wall and to divide. Wall regeneration is initially linked with expansion and cytokinesis. Osmotic pressure of the external medium is also an important factor.This work formed part of a thesis by one of us (R.S.P.) approved for the degree of Ph. D. in the University of Nottingham. The work was supported by the Agricultural Research Council.     

Zinc accumulation,photosynthetic gas exchange,and chlorophyll a fluorescence in Zn-stressed Miscanthus × giganteus plants

Photosynthetica - Accumulation and distribution of zinc within Miscanthus × giganteus plants grown on elevated Zn concentrations and their photosynthetic performance were investigated. High...     

Cell suspension culture of Rhizoma zedoariae in a two-stage perfusion bioreactor system for β-elemene production

We presented a two-stage combined bioreactor system consisting of a stir-tank and an airlift column, and challenged with Rhizoma zedoariae cell suspensions for β-elemene production. Two-stage culture was initiated when the cell concentration in both vessels was maintained at an appropriate density. The cells were proliferated in stirred-tank with the maximal growth rate of 0.17 d⁻¹ to present enough cells for β-elemene synthesis. In the airlift column, continuous cell separation from culture medium was achieved by using a cell retention device based on centrifugal and gravity settling when the system was performed in perfusion mode. The results indicated that additives can efficiently promote the accumulation of β-elemene in R. zedoariae cells. In addition, the β-elemene content showed higher levels in cell lines of overexpressing 3-hydroxy-3-methylglutaryl coenzyme-A reductase, Farnesyldi phosphate synthase, and ST02C genes.

     

Agrobacterium-mediated transformation of embryogenic cell suspension cultures and plant regeneration in Lilium tenuifolium oriental × trumpet ‘Robina’

A method has been developed for embryogenic cell suspension cultures, plant regeneration and transformation of the important ornamental lily genotype (Lilium tenuifolium oriental × trumpet ‘Robina’). Bulb scales, filaments, ovaries and stem axis tissues were used as explants for callus induction in Murashige and Skoog (MS) medium with additions of growth regulators: picloram on its own, or in combination with 1-naphthaleneacetic acid (NAA), and thidiazuron (TDZ). The results show that the optimum medium for callus induction in bulb scale and filament tissue is MS + picloram 1.0 mg L^?1, and for the ovary, it is MS + picloram 1.5 mg L^?1. The stem axis had the highest rate (89.2 %) of callus induction with MS + NAA 2.2 mg L^?1 + TDZ 0.1 mg L^?1. The suspension cultures were established with the combination of NAA and TDZ with 2–5 mm cell clusters. These took a long time compared with suspension cultures established by picloram with 1–3 mm cell clusters. In three suspension cultures induced by picloram, the best callus from the point of view of proliferation and regeneration was derived from filaments. For plant regeneration, the growth rate of suspension cultures from the stem axis was higher than from the other three suspension culture induced by picloram. Vector pCAMBIA1301 with the β-glucuronidase (GUS) gene as reporter was transformed by Agrobacterium mediation into suspension cultures initiated from filament and stem axis material. After co-cultivation, the numbers of blue spots in material from the two sources were 26.8 ± 4.3 and 24.0 ± 4.7, respectively (difference not significant). Hygromycin-resistant callus was successfully regenerated into plantlets on plant growth regulator-free MS medium. Transgenic plants were also confirmed by the GUS histochemical assay, polymerase chain reaction.