青阳参组织培养及愈伤组织的成分分析

用青阳参(Cynanchum otophyllum)的嫩枝和芽在Ms 2．0mg／L2,4-D 0．1mg／L KIN的培养基上诱导愈伤组织。通过不同的培养基和激素配比实验,发现6,7-V 2．0mg／L2,4．D 0．3mg／LKIN最适合愈伤组织的生长。但在6,7-V 1．0mg／L2,4．D 0．1mg／L KIN培养基中的愈伤组织次生代谢物含量最高。愈伤组织的生长周期为27d,但在33d时次生代谢产物的含量最高。从愈伤组织中分离到7个化合物：(1)9,10,11-三羟基-十八碳-12(Z)-烯酸甲酯(methyl9,10,11-trihydroxy-12-octadecencate),(2)胡萝卜甙(daucosterol),(3)β-谷甾醇(β-sitoster01),(4)华木酸(betuliniic acid),(5)齐端果酸(oleamlic acid),(6)棕榈酸(hexadecanoic acid),(7)十八碳-9-烯酸(9-octadecenoic acid)。首次报道从植物愈伤组织中分离到多羟基十八碳烯酸,并讨论了化合物（1）对植物细胞生长的可能影响。     

青阳参组织培养及愈伤组织的成分分析

用青阳参（Cynanchum otophyllum）的嫩枝和芽在MS + 2.0 mg/L 2,4-D + 0.1 mg/L KIN的培养基上诱导愈伤组织。通过不同的培养基和激素配比实验,发现 6,7-V + 2.0 mg/L 2,4-D + 0.3 mg/L KIN 最适合愈伤组织的生长。但在6,7-V + 1.0 mg/L 2,4-D + 0.1 mg/L KIN 培养基中的愈伤组织次生代谢物含量最高。愈伤组织的生长周期为27 d,但在33 d时次生代谢产物的含量最高。从愈伤组织中分离到7个化合物：⑴9,10,11-三羟基-十八碳-12(Z)-烯酸甲酯 (methyl 9,10,11-trihydroxy-12-octadecenoate),(2) 胡萝卜甙 (daucosterol),(3)β 谷甾醇 (β sitosterol),（4） 华木酸 (betulinic acid),（5）齐端果酸 (oleanolic acid),（6）棕榈酸 (hexadecanoic acid),（7）十八碳-9-烯酸 (9-octadecenoic acid)。首次报道从植物愈伤组织中分离到多羟基十八碳烯酸,并讨论了化合物（1）对植物细胞生长的可能影响。     

Accumulation of anthraquinones in Morinda citrifolia cell suspensions

Cell suspensions of Morinda citrifolia were cultivated in a B5-medium containing 4% sucrose as the sole carbon source and 1 mg l^-1 naphthyl acetic acid (NAA) or 1 mg l^-1 2,4-dichloro-phenoxyacetic acid (2,4-D). Both auxins were able to support growth but only in the presence of NAA anthraquinone production was observed. 2,4-D inhibited the production in NAA cultures. Anthraquinone synthesis took place in the growth and the stationary phase and amounts of 0.2–0.4 mmol (about 100–200 mg) g^-1 dry weight could be reached.Under both growth conditions sucrose was hydrolyzed extracellularly by invertase. From the resulting monosaccharides, glucose was taken up preferentially and an appreciable uptake of fructose only took place when medium glucose was exhausted. Sugar uptake rates were similar when cells were grown in NAA and in 2,4-D medium but the intracellular sugar contents (expressed on a dry weight basis) differed considerably. The presence of sucrose, glucose and fructose was demonstrated under both growth conditions. The amounts of sucrose and glucose were much lower in the 2,4-D cells than in the NAA-cells especially during the growth phase. Fructose contents were low and comparable, while in NAA cells an unknown sugar (possibly the sugar moiety of the glycosylated anthraquinones) was observed especially at the end of the growth phase and in the stationary phase. The differences in sugar concentrations were even larger due to the lower water contents of the NAA cells.Respiration of 2,4-D cells was much higher than that of NAA cells during the growth phase. A sharp increase in sugar contents (mainly sucrose) occurred in the 2,4-D cells at the end of the growth phase and corresponded with the fall in respiratory activity.A possible correlation between the lack of production of anthraquinones in 2,4-D cells and a less efficient growth metabolism in these cells is discussed.Abbreviations AQ anthraquinones - 2,4-D 2,4-dichloro-phenoxy-acetic acid - DW dry weight - FW fresh weight - NAA naphthyl acetic acid - pCPO p-chloro-phenoxy-acetic acid     

Combined effects of an oxidative enzyme and dissolved humic substances on 13C-labelled 2,4-D herbicide as revealed by high-resolution 13C NMR spectroscopy

Phenoxyalkanoic acids are a widely used class of herbicides. This work employed high-resolution ¹³C NMR to study the structural changes induced by humic substances and horseradish perodixase on 2,4-dichorophenoxyacetic acid (2,4-D) ¹³C-labelled in the side chain. NMR spectra showed that humic substances chemically catalyze abiotic splitting of [¹³C]2,4-D into 2,4-dichlorophenol and [¹³C]acetic acid at pH 7 but not at pH 4.7. Peroxidase did not catalyze the oxidative degradation of [¹³C]2,4-D at any pH tested and inhibited the effect of humic substances. Catalytic degradation by humic substances was attributed to free-radical reactions enhanced by the stereochemical contribution of large conformational structures formed by heterogeneous humic molecules at neutral pHs. Inhibition of 2,4-D degradation when humic substances were combined with peroxidase was explained by modification of both chemical and conformational humic structure due to peroxidase-promoted oxidative cross-coupling among humic molecules. Our findings show for the first time that the abiotic degradation of 2,4-D is catalyzed by dissolved humic substances at neutral pH. Journal of Industrial Microbiology & Biotechnology (2001) 26, 70–76. Received 09 February 2000/ Accepted in revised form 22 May 2000     

Effects of ethanol on Saccharomyces cerevisiae as monitored by in vivo 31P and 13C nuclear magnetic resonance

Cell suspensions of a respiratory deficient mutant of Saccharomyces cerevisiae were monitored by in vivo ³¹P and ¹³C Nuclear Magnetic Resonance in order to evaluate the effect of ethanol in intracellular pH and metabolism. In the absence of an added energy source, ethanol caused acidification of the cytoplasm, as indicated by the shift to higher field of the resonance assigned to the cytoplasmic orthophosphate. Under the experimental conditions used this acidification was not a consequence of an increase in the passive influx of H⁺. With cells energized with glucose, a lower value for the cytoplasmic pH was also observed, when ethanol was added. Furthermore, lower levels of phosphomonoesters were detected in the presence of ethanol, indicating that an early event in glycolysis is an important target of the ethanol action. Acetic acid was identified as responsible for the acidification of the cytoplasm, in experiments where [¹³C]ethanol was added and formation of labeled acetic acid was detected. The intracellular and the extracellular concentrations of acetic acid were respectively, 30 mM and 2 mM when 0.5% (120 mM) [¹³C]ethanol was added.Abbreviations P_i inorganic phosphate - P_ic inorganic phosphate in the cytoplasm - P_iv inorganic phosphate in the vacuole - tP terminal phosphate in polyphosphate     

Physiological adaptations of anaerobic bacteria to low pH: metabolic control of proton motive force in Sarcina ventriculi.

Detailed physiological studies were done to compare the influence of environmental pH and fermentation end product formation on metabolism, growth, and proton motive force in Sarcina ventriculi. The kinetics of end product formation during glucose fermentation in unbuffered batch cultures shifted from hydrogen-acetate production to ethanol production as the medium pH dropped from 7.0 to 3.3. At a constant pH of 3.0, the production of acetate ceased when the accumulation of acetate in the medium reached 40 mmol/liter. At a constant pH of 7.0, acetate production continued throughout the entire growth time course. The in vivo hydrogenase activity was much higher in cells grown at pH 7.0 than at pH 3.0. The magnitude of the proton motive force increased in relation to a decrease of the medium pH from 7.5 to 3.0. When the organism was grown at pH 3.0, the cytoplasmic pH was 4.25 and the organism was unable to exclude acetic acid or butyric acid from the cytoplasm. Addition of acetic acid, but not hydrogen or ethanol, inhibited growth and resulted in proton motive force dissipation and the accumulation of acetic acid in the cytoplasm. The results indicate that S. ventriculi is an acidophile that can continue to produce ethanol at low cytoplasmic pH values. Both the ability to shift to ethanol production and the ability to continue to ferment glucose while cytoplasmic pH values are low adapt S. ventriculi for growth at low pH.     

Metabolism of 2,4-dichlorophenoxyacetic acid in cell suspension cultures of soybean (Glycine max L.) and wheat (Triticum aestivum L.)

Cell suspension cultures of soybean (Glycine max L.) and wheat (Triticum aestivum L.) incorporated 2,4-dichlorophenoxyacetic acid (2,4-D) into a metabolite fraction which was insoluble in ethanol, water, and hot sodium dodecylsulphate. Further treatment with hot dimethylformamide solubilized a material which by the following criteria appeared to consist of 2,4-D derivatives covalently bound to lignin: i) co-chromatography of radioactivity and of UV-absorbing material upon gel permeation chromatography; ii) spectral similarity with authentic lignins (IR- and UV-spectra, phloroglucinol reaction), 2,4-D appeared to be incorporated as the intact molecule, as shown by comparison of ring- and sidechain-labeled 2,4-D and by detection of monohydroxylated and intact 2,4-D as the major radioactive products of acid hydrolysis. The same compounds were released from the metabolite material which could not be solubilized in dimethylformamide. The incorporation of xenobiotics or their metabolites into lignin, followed by deposition in the cell wall, is suggested as a general pathway for local excretion and detoxification by plant cells.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 4-OH-2,5-D 4-hydroxy-2,5-dichlorophenoxyacetic acid - SDS sodium dodecylsulphate - DMF dimethylformamide     

Auxin, actin and growth of the Arabidopsis thaliana primary root

To understand how auxin regulates root growth, we quantified cell division and elemental elongation, and examined actin organization in the primary root of Arabidopsis thaliana. In treatments for 48 h that inhibited root elongation rate by 50%, we find that auxins and auxin-transport inhibitors can be divided into two classes based on their effects on cell division, elongation and actin organization. Indole acetic acid (IAA), 1-naphthalene acetic acid (NAA) and tri-iodobenzoic acid (TIBA) inhibit root growth primarily through reducing the length of the growth zone rather than the maximal rate of elemental elongation and they do not reduce cell production rate. These three compounds have little effect on the extent of filamentous actin, as imaged in living cells or by chemical fixation and immuno-cytochemistry, but tend to increase actin bundling. In contrast, 2,4-dichlorophenoxy-acetic acid (2,4-D) and naphthylphthalamic acid (NPA) inhibit root growth primarily by reducing cell production rate. These compounds remove actin and slow down cytoplasmic streaming, but do not lead to mislocalization of the auxin-efflux proteins, PIN1 or PIN2. The effects of 2,4-D and NPA were mimicked by the actin inhibitor, latrunculin B. The effects of these compounds on actin were also elicited by a 2 h treatment at higher concentration but were not seen in two mutants, eir1-1 and aux1-7, with deficient auxin transport. Our results show that IAA regulates the size of the root elongation zone whereas 2,4-D affects cell production and actin-dependent processes; and, further, that elemental elongation and localization of PINs are appreciably independent of actin.     

Auxin and nutritional stress coupled somatic embryogenesis in <Emphasis Type="Italic">Oldenlandia umbellata</Emphasis> L.

Somatic embryos were induced from internodal segment derived callus of Oldenlandia umbellata L., in MS medium supplemented with different concentrations of 2,4-Dichlorophenoxy acetic acid (2,4-D). Initially calli were developed from internodes of microshoots inoculated in 2.5 µM NAA supplemented medium. Then calli were transferred to 2,4-D added medium for somatic embryogenesis. Nutritional stress coupled with higher concentration of 2,4-D triggered somatic embryogenesis. Nutritional stress was induced by culturing callus in a fixed amount of medium for a period up to 20 weeks without any external supply of nutrients. Addition of 2.5 µM 2,4-D gave 100% embryogenesis within 16 weeks of incubation. Callus mass bearing somatic embryos were transferred to germination medium facilitated production of in vitro plantlets. MS medium supplemented with 2.5 µM benzyl adenine and 0.5 µM α-naphthalene acetic acid produced 15.33 plants per culture within 4 weeks of culture. Somatic embryo germinated plants were then hardened and transferred to green house.     

Higher extracellular pH suppresses tracheary element differentiation by affecting auxin uptake

In an optimized liquid medium containing auxin and cytokinin, mesophyll cells isolated from Zinnia elegans L. seedlings can be induced to differentiate into tracheary elements (TEs) at high frequency. However, it is known that buffering the medium at neutral pH severely suppresses TE differentiation. In the process of modifying the medium, we found that excessive administration of auxin restored the suppression. Based on this finding, we physiologically characterized auxin actions involved in TE differentiation by focusing on the influence of extracellular pH. First, dose/response relationships between auxin [1-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D)] concentrations and differentiated cell ratios were determined under various extracellular pH conditions. Secondly, intracellular concentrations of free forms and metabolites of auxin species were determined by analyzing extracts from cells cultured with radiolabeled NAA and 2,4-D under different extracellular pH conditions with liquid scintillation counting and thin-layer chromatography autoradiograms. Higher extracellular pH was found to reduce both the auxin potency for inducing TE differentiation and intracellular auxin accumulation. Reduction levels correlatively varied depending on the auxin species. These results suggest that the weakening in auxin potency at higher extracellular pH is ascribed to lower auxin uptake, which leads to decreased intracellular perception of the auxin signal. A model to predict auxin action that considers membrane transport, metabolism, and the perception of auxin is also presented.     

Assessment of the changes of 2,4-dichlorophenoxyacetic acid concentrations in plant tissue culture media in the presence of activated charcoal

The rate of adsorption of 2,4-dichlorophenoxyacetic acid (2,4-D) by activated charcoal (AC) from liquid and semi-solid tissue culture media was determined using 2-[¹⁴C]-2,4-D. In liquid medium 99.5% of the added 2,4-D (10^-4 M) was adsorbed by AC (2.5 gl^-1) within 5 days of preparation of the medium. Higher 2,4-D levels of reduced AC concentrations increased the level of available 2,4-D in the medium and extended the period necessary for the level of 2,4-D in the medium to become stabilized. In semi-solid medium the rate of adsorption of 2,4-D by AC was considerably reduced. A stable ratio of gel/2,4-D:AC/2,4-D was only reached after 10 to 20 days, depending on the 2,4-D concentration used. Low pH levels and maintenance of the medium at higher temperatures (20–30°C) accelerated the adsorption of 2,4-D by AC. In vitro tissue cultures of coconut palm showed marked differences in their growth response according to the age of the medium used and the associated variations in 2,4-D concentrations.Abbreviations AC activated charcoal - BAP 6-benzylamino-purine - 2,4-D 2,4-dichlorophenoxyacetic acid     

Metabolism of 2,4-Dichlorophenoxyacetic Acid (2,4-D) in Soybean Root Callus : EVIDENCE FOR THE CONVERSION OF 2,4-D AMINO ACID CONJUGATES TO FREE 2,4-D

An auxin-requiring soybean root callus metabolized [1-¹⁴C]-2,4-dichlorophenoxyacetic acid (2,4-D) to diethyl ether-soluble amino acid conjugates and water-soluble metabolites. The uptake in tissue varied with incubation time, concentration, and amount of tissue. Uptake was essentially complete (80%) after a 24-hour incubation and the percentage of free 2,4-D in the tissue fell to its lowest point at this time. At later times, the percentage of free 2,4-D increased and the percentage of amino acid conjugates decreased, whereas the percentage of water-soluble metabolites increased only slightly. Similar trends were seen if the tissue was incubated for 24 hours in radioactive 2,4-D, followed by incubation in media without 2,4-D for 24 hours. Inclusion of nonlabeled 2,4-D during the 24-hour chase period did not reduce amino acid conjugate disappearance but did reduce the percentage of free [1-¹⁴C]2,4-D. Thus, an external supply of 2,4-D does not directly prevent amino acid conjugate metabolism in this tissue. It is concluded that 2,4-D amino acid conjugates were actively metabolized by this tissue to free 2,4-D and water-soluble metabolites.     

Enhanced production of the polysaccharide arabinogalactan using immobilized cultures of Tinospora cordifolia by elicitation and in situ adsorption

Immobilized callus cultures of Tinospora cordifolia (Willd) Miers ex Hooks and Thoms were investigated to find out the combined effect of elicitation, cell permeabilization with chitosan and in situ product recovery by polymeric neutral resin-like Diaion HP 20. In this study, callus cultures of T. cordifolia were immobilized using sodium alginate and calcium chloride and the beads were cultured in Murashige and Skoog's basal medium along with benzyl adenine (BA), 2,4-dichlorophenoxy acetic acid (2,4-D) and 3% sucrose. The immobilized cultures, when subjected to elicitation and cell permeabilization with chitosan and in situ removal of the secondary metabolites by addition of resin, showed a 10-fold increase in production of arabinogalactan (0.490% dry weight) as compared to respective controls devoid of resin and chitosan. This indicates that in situ adsorption may have reduced the feedback inhibition caused by accumulation of secondary metabolites in the media, while the dual effect of elicitation and cell permeabilization by chitosan may have released the intracellular (secreted) berberine and the polysaccharide arabinogalactan, respectively.     

Modulation of carrier-mediated uptake of abscisic acid by methyl jasmonate in Phaseolus coccineus L

The effects of methyl jasmonate and jasmonic acid on uptake of abscisic acid (ABA) by suspension-cultured runner-bean cells and subapical runner-bean root segments have been investigated. Increasing concentrations of methyl jasmonate inhibit ABA uptake by the cultured cells with a K _i of 22±3 M. This is not due to cytoplasmic acidification or to effects on metabolism of ABA, and is not additive with inhibition of radioactive ABA uptake by nonradioactive ABA. Uptake of indol-3-yl acetic acid (IAA) is unaffected by methyl jasmonate. The maximum effect of nonradioactive ABA in inhibiting uptake of radioactive ABA, previously shown to reflect saturation of an ABA carrier, is generally greater than the effect of maximally inhibitory concentrations of methyl jasmonate. Similar results were obtained with root segments, but longer incubation times were necessary to observe inhibitory effects of methyl jasmonate. Demethylation of methyl jasmonate to jasmonic acid does not appear to be required since similar concentrations of jasmonic acid had no observable direct effect on ABA uptake other than that attributable to cytoplasmic acidification. Histidine reagents, a proton ionophore and acidic external pH all affect in parallel the inhibition by methyl jasmonate and nonradioactive ABA of uptake of radioactive ABA by the cultured cells. There is no effect of ABA or nonradioactive methyl jasmonate on uptake of radioactive methyl jasmonate by the cultured cells. It is proposed that methyl jasmonate interacts with the ABA carrier. Various models for this interaction are discussed.Abbreviations ABA abscisic acid - DMO 5,5-dimethyloxazolidine-2,4-dione - IAA indol-3-yl acetic acid     

Soluble polyvinylpyrrolidine and bovine serum albumin adsorb polyphenols from soybean suspension cultures

Soybean (Glycine max [L.] Merrill, cv. Dare) suspension cultures grown in Gamborg B5 medium became discolored and the cells began aggregating after 1 week in culture, especially in the absence of 2,4-dichlorophenoxyacetic acid (2,4-D). The addition of either soluble polyvinylpyrrolidine (PVP) or bovine serum albumin (BSA) to cultures grown in Gamborg B5 medium with 2,4-D prevents discoloration and cell aggregation by adsorbing excess polyphenols from the cells. Transfer of the PVP-treated cultures to fresh medium without 2,4-D stimulated the recurrence of excess polyphenols. Cultures pretreated with BSA did not develop excess polyphenols when transferred to fresh 2,4-D-free medium. Addition of either PVP or BSA to cultures grown in the absence of 2,4-D was found to inhibit growth.     

A cell suspension ofLinum flavum (L.) in phosphate limited continuous culture

A cell suspension ofLinum flavum was grown in phosphate limited continuous culture at two different growth rates. Energy metabolism (respiration), coniferin and lignin production and overall biomass composition were analysed, in order to establish the relations between growth, maintenance and secondary metabolism. The ATP turnover rate was higher in the faster growing cultures, corresponding with a higher energy requirement. The coniferin production was not directly correlated with the growth rate, indicating the possibility of high production at high growth rates. Steady states grown under identical conditions showed different characteristics that may have evolved during pre-culture time.     

Effect of pH and acetic acid on growth and 2,3-butanediol production of Enterobacter aerogenes in continuous culture

Summary The effect of pH and acetic acid on growth and 2,3-butanediol production of Enterobacter aerogenes from glucose was investigated in a microaerobic continuous culture. At a dilution rate of 0.20 h^–1 and a fixed oxygen uptake rate (OUR) of 31.5 mmol l^–1 h^–1 the biomass concentration increased with pH ranging from 5.0 to 7.0, while the specific ATP requirement of the cells decreased. In the pH range 5.5–6.5 the product concentration (butanediol + acetoin) was maximal and nearly constant. However, the specific production continuously declined with increasing pH. Experiments with addition of acetic acid showed that the various effects of pH are due to inhibition of the by-product acetic acid on cell growth. The strength of the acetic and inhibition depended only on the concentration of its undissociated form [HAc]. The biomass concentration and the specific OUR were also only functions of [HAc], irrespective of the pH. Although the specific ATP requirement (q ATP) strongly depended on the pH, [HAc] at constant pH. Offprint requests to: W.-D. Deckwer     

Plant regeneration from callus and protoplasts of Brassica nigra (IC 257) through somatic embryogenesis

A method to obtain plants from embryogenic callus of Brassica nigra and protoplasts of hypocotyl expiants is described. Callus was initiated on Murashige and Skoog medium containing kinetin (kn) and 2,4-dichlorophenoxy acetic acid (2,4-D). Lowering of auxin induced embryo formation. Supplementation with gibberellic acid (GA₃) enhanced embryogenic response tenfold. Passage through liquid medium devoid of growth regulators was essential for the growth of embryos. Secondary embryos were produced on transfer to solid basal medium. Embryogenic callus retained its morphogenic ability even after 12 subcultures. Both primary and secondary embryos produced fertile plants. Hypocotyl-derived protoplasts were also regenerated to plants following the same protocol. The survival of plants on transfer to soil was about 80%. The seeds from plants derived from callus and protoplasts were viable.Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid - NAA naphthalene acetic acid - IAA indole acetic acid - kn kinetin - GA₃ gibberellic acid