Alkaloid formation by habituated and tumorous cell suspension cultures of Catharanthus roseus

Habituated and tumorous Catharanthus roseus cells grown in the absence of hormones accumulated indole alkaloids. Total alkaloids and alkaloid pattern were the same when cells were cultured in medium without hormones or in alkaloid production medium with and without indole acetic acid. Treatment of cells with Pythium homogenate as elicitor did not increase total alkaloids or change the pattern of alkaloids produced. When either habituated or tumorous cells were grown in 1B5 medium after Gamborg et al (1968) containing 2,4-dichlorophenoxyacetic acid (2,4-D), their capacity to accumulate alkaloids decreased with time. The levels of tryptophan decarboxylase (TDC) and strictosidine synthase (SS) specific activities were constant throughout growth except when cells were exposed to 2,4-D in 1B5 medium, where enzyme activities declined in step with the decrease in alkaloid accumulation. Neither habituated nor tumorous cell suspension cultures accumulated vindoline, nor could they be induced to produce this alkaloid by any of the given treatments.NRCC No. 27514     

Habituation of plant cells does not mean insensitivity to plant growth regulators

Summary Fully habituated organogenic and nonorganogenic sugarbeet calluses reacted to application of the synthetic auxin [3-benzo(b) selenienyl] acetic acid by changes in growth and ethylene production. Treatment of fully habituated cells of periwinkle with 2,4-dichlorophenoxyacetic acid led to the decrease of free cytokinin contents (isopentenyl adenine, zeatin riboside, and zeatin) during the late exponential phase of growth. The polyamine contents were also modified and the capacity to biotransform secologanin into ajmalicine was decreased. Treatment of the habituated periwinkle cells with zeatin greatly increased the amount of a polypeptide of 16 kDa; this response was more marked than that displayed by the auxin-dependent line. These data show that hormone-independent calluses and cell suspensions can retain some sensitivity to growth hormones. However, differences of responses were observed between the auxin-dependent lines and the habituated lines.     

Leucobacter chromiireducens sp. nov, and Leucobacter aridicollis sp. nov., two new species isolated from a chromium contaminated environment

Two strains designated strains L-1^T and L-9^T were isolated from activated sludge of a treatment plant that receives wastewater from the tannery industry contaminated with chromium. Phylogenetic analysis showed that the organisms represented two new species of the genus Leucobacter. Strains L-1^T and L-9^T could be distinguished from the type strain of L. komagatae and from the type strain of “L. albus” by the B-type peptidoglycan composition, fatty acid composition, several phenotypic and physiological characteristics. The major fatty acids of the organisms were iso- and anteiso-branched C15:0 and C17:0, straight-chain C16:0 was also found in relatively high proportions. The organisms were halotolerant, grew in medium containing 9% NaCl, and all strains, including the type strain of L. komagatae grew in medium containing 5 mM Cr(VI). On the basis of the distinct peptidoglycan composition, 16S ribosomal DNA sequence analysis, percentage of DNA-DNA reassociation values, and phenotypic characteristics we are of the opinion that strain L-1^T represents a new species of the genus Leucobacter for which we propose the name Leucobacter chromiireducens and that strain L-9^T represents an additional new species of the same genus for which we propose the name Leucobacter aridicollis.     

Auxin-polyamine interaction with the calcium-controlled secretion of peroxidase by sugarbeet cells

Spermidine and ornithine given to normal auxin-requiring cell suspensions of sugarbeet inhibited peroxidase secretion in the absence of Ca²⁺. Habituated (organogenic or not) cells did not respond. Both compounds counteracted the Ca²⁺ - promoted enzyme secretion by three cell lines. Auxins (2,4-D and BSAA) did not modify the extracellular level of peroxidase activity in the absence of Ca²⁺ When Ca²⁺ was added, auxins increased its effect in normal cells and had practically no effect in habituated cells. The inhibitory effect of spermidine and ornithine was somewhat reduced by auxins in normal cells and increased in habituated cells. It was hypothesized that the effect of auxins did not involve the mediation of polyamines and that both types of compounds directly interacted with Ca²⁺ at the membrane level.Abbreviations BSAA [benzo(b)selenienyl-3]acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - HNO habituated non-organogenic - HO habituated organogenic - NNO normal non-organogenic     

Ethylene production in habituated and auxin-requiring tobacco callus cultures. Does ethylene play a role in the habituation?

Ethylene production of habituated and auxin-requiring tobacco ( Nicotiana tabacum L. cv. Xanthi) callus cultures were compared. More ethylene was produced by auxinrequiring i.e. auxin-heterotrophic cultures than by habituated ones. Treatment with 2,4-dichlorophenoxyacetic acid increased the ethylene evolution of habituated cultures over the range 10⁻⁷ to 10⁻⁴ M , which suggests that the higher ethylene production of auxin-dependent callus is caused by the 2,4-D in the medium. The IAA levels depended on the age of both types of callus cultures.     

yhcZ基因在苏云金芽胞杆菌生长中的功能研究

在枯草芽胞杆菌和蜡样芽胞杆菌中,yhcZ基因和yhcY基因组成双组分系统调控细菌生长,但yhcZ基因在苏云金芽胞杆菌中发挥的生物学功能尚未明确。本研究通过基因功能注释、上下游基因排列分析和氨基酸序列比对,证实苏云金芽胞杆菌库斯塔克亚种HD73中HD73_5824基因为yhcZ基因,推测其与HD73_5825基因(yhcY基因)共同组成双组份系统调控细菌生长。利用同源重组技术敲除HD73菌株中的yhcZ基因获得缺失突变体HD (ΔyhcZ),其在LB和SSM培养基中生长均慢于野生型HD73,而互补菌株HD(ΔyhcZ::yhcZ)菌株则能够部分恢复生长,表明yhcZ基因的缺失影响了该菌株细胞的生长。在以0.4%葡萄糖为唯一碳源的M9培养基中,HD (ΔyhcZ)生长速度快于HD73,表明yhcZ基因在该菌株吸收利用葡萄糖的过程中发挥重要作用。Biolog实验显示HD (ΔyhcZ)的单孔颜色变化率低于HD73,且对D/L-丝氨酸、甲酸、D-葡糖酸、L-组胺,D-乳酸甲酯以及柠檬酸等的吸收利用能力低于HD73,表明yhcZ基因能显著影响HD73菌株对碳源的利用。同时,HD(ΔyhcZ)对8% NaCl的耐受能力弱于HD73,表明该基因可能参与细菌细胞应力响应相关基因的表达与调控。以上结果表明yhcZ基因在HD73菌株生长过程中对葡萄糖及其他碳源的利用具有重要的促进作用。本研究结果为解析yhcZ基因调控葡萄糖及碳源利用的分子机制奠定基础,且为进一步研究细菌生长及发酵提供参考。     

Partial Synchronization of Carrot Cell Culture by Auxin Deprivation

A strain of carrot cells (Daucus carota cv. Kintoki) grew exponentially in the presence of 2,4-dichlorophenoxyacetic acid (2,4-D, 1 mg/1) with a doubling time of about 2 days. When those cells were transferred to a medium lacking 2,4-D, they continued to grow at almost the same rate for about a week. When the cells were again transferred to the auxin-free medium, the rate of cell division gradually decreased. After the cell division had ceased, cells were returned to the ordinary 2,4-D medium. A burst of cell divisions occurred after about 2 days. Timing of DNA synthesis and of mitosis suggested that the cells had been arrested at G₁ phase. In a medium containing indoleacetic acid instead of 2,4-D, the auxin was rapidly degraded and the culture was similarly synchronized as in the auxin-omitted medium.     

Simultaneous production of anthocyanin and triterpenoids in suspension cultures of Perilla frutescens

When cultivated in Murashige & Skoog medium supplemented with 0.2 mg l⁻¹ 2,4-dichlorophenoxy acetic acid and 0.5 mg l⁻¹ 6-benzyladenine, Perilla frutescens cells in suspension culture grew rapidly reaching about 13.6 g dry wt l⁻¹ after 12 days. The cell line produced both anthocyanin 0.9 g l⁻¹ and triterpenoids: 16 mg l⁻¹ oleanolic acid (OA), 25 mg l⁻¹ ursolic acid (UA) and 14 mg l⁻¹ tormentic acid (TA). When P. frutescens cells of 7-day-old cultures were exposed to a yeast elicitor at 0.5–5% (v/v) for 7 days, it was found that anthocyanin content peaked at 10.2% of dry weight with yeast elicitor at 1% (v/v) whereas the maximum production of oleanolic acid and ursolic acid in cultures treated with 2% (v/v) yeast elicitor was 19 and 27 mg l⁻¹, a 46 and 24% increase over the control, respectively. This is the first report of simultaneous production of both anthocyanin and triterpenoids in a single culture system.     

Influence of environmental factors on 2,4-dichlorophenoxyacetic acid degradation by Pseudomonas cepacia isolated from peat

A Pseudomonas cepacia, designated strain BRI6001, was isolated from peat by enrichment culture using 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole carbon source. BRI6001 grew at up to 13 mM 2,4-D, and degraded 1 mM 2,4-D at an average starting population density as low as 1.5 cells/ml. Degradation was optimal at acidic pH, but could also be inhibited at low pH, associated with chloride release from the substrate, and the limited buffering capacity of the growth medium. The only metabolite detected during growth on 2,4-D was 2,4-dichlorophenol (2,4-DCP), and degradation of the aromatic nucleus was by intradiol cleavage. Growth lag times prior to the on-set of degradation, and the total time required for degradation, were linearly related to the starting population density and the initial 2,4-D concentration. BRI6001, grown on 2,4-D, oxidized a variety of structurally similar chlorinated aromatic compounds accompanied by stoichiometric chloride release.     

Aerobic Degradation of Dinitrotoluenes and Pathway for Bacterial Degradation of 2,6-Dinitrotoluene

An oxidative pathway for the mineralization of 2,4-dinitrotoluene (2,4-DNT) by Burkholderia sp. strain DNT has been reported previously. We report here the isolation of additional strains with the ability to mineralize 2,4-DNT by the same pathway and the isolation and characterization of bacterial strains that mineralize 2,6-dinitrotoluene (2,6-DNT) by a different pathway. Burkholderia cepacia strain JS850 and Hydrogenophaga palleronii strain JS863 grew on 2,6-DNT as the sole source of carbon and nitrogen. The initial steps in the pathway for degradation of 2,6-DNT were determined by simultaneous induction, enzyme assays, and identification of metabolites through mass spectroscopy and nuclear magnetic resonance. 2,6-DNT was converted to 3-methyl-4-nitrocatechol by a dioxygenation reaction accompanied by the release of nitrite. 3-Methyl-4-nitrocatechol was the substrate for extradiol ring cleavage yielding 2-hydroxy-5-nitro-6-oxohepta-2,4-dienoic acid, which was converted to 2-hydroxy-5-nitropenta-2,4-dienoic acid. 2,4-DNT-degrading strains also converted 2,6-DNT to 3-methyl-4-nitrocatechol but did not metabolize the 3-methyl-4-nitrocatechol. Although 2,6-DNT prevented the degradation of 2,4-DNT by 2,4-DNT-degrading strains, the effect was not the result of inhibition of 2,4-DNT dioxygenase by 2,6-DNT or of 4-methyl-5-nitrocatechol monooxygenase by 3-methyl-4-nitrocatechol.     

Immunocytochemical characterization of the cell walls of bean cell suspensions during habituation and dehabituation to dichlobenil

The effects of the cellulose inhibitor dichlobenil on the cell wall composition and structure during the habituation/dehabituation process of suspension‐cultured bean cells were assessed. A range of techniques were used including cell wall fractionation, sugar analysis, immunofluorescence and fluorochrome labelling of resin‐embedded sections, and immunodot assays (IDAs) of cell wall fractions. The cell walls from bean cell suspensions with initial levels of habituation to dichlobenil had decreased levels of cellulose, but this effect lessened with increasing numbers of subcultures. All cell walls analysed showed calcofluor‐stained appositions. However, in habituated and dehabituated cells, appositions were not recognized by an anticallose antibody. This finding suggested the accumulation of an extracellular polysaccharide different to callose, probably a 1,4‐β‐glucan in these cell lines. Appositions in habituated cells also contained homogalacturonan (HG) with a high degree of methyl esterification (DE), rhamnogalacturonan (RG) and xyloglucan. Habituated cell walls were also enriched in pectins, particularly HG, with a low DE, and RG. The levels of extensin epitope that colocalized with RG in habituated cells also diminished with the increasing number of subcultures. Habituated cells also liberated less extensin into the medium. In habituated cells, a decrease in the cell wall arabinogalactan protein (AGP) labelling was observed both in cell walls and in the culture medium. The increase in the number of subcultures in 0.3 µM dichlobenil was accompanied by an increment in some pectic epitopes (JIM5 and LM5) and a decrease in other pectic and in protein epitopes (JIM7, PAM1, LM6, LM2 and MAC207), indicating a re‐structuring of cell walls throughout the habituation procedure. Dehabituated cells showed an overall composition similar to that of non‐habituated cells, with exception of an increase in glucose in hemicellulosic fractions tightly bound to cellulose. However, these cells also showed reduced levels of extensin and AGP labelling. These differences could be related to the high tolerance to dichlobenil observed in dehabituated cells.     

Isolation and characterization of a stable 2,4-dichlorophenoxyacetic acid degrading bacterium, Variovorax paradoxus, using chemostat culture

A strain of Variovorax paradoxus degrading 2,4-dichlorophenoxyacetic acid (2,4-D) was isolated from the Dijon area (France) using continuous chemostat culture. This strain, designated TV1, grew on up to 5 mM 2,4-D and efficiently degraded the herbicide as sole carbon source as well as in presence of soil extracts. It also degraded phenol and 2-methyl, 4-chlorophenoxyacetic acid at 3 mM and 2,4-dichlorophenol at 1 mM. This organism contained a stable 200 kb plasmid, designated pTV1, which showed no similarity in its restriction pattern with the archetypal 2,4-D catabolic plasmid pJP4. However, pTV1 contained an 11 kb BamHI fragment which hybridized at low stringency with the 2,4-D degradative genes tfdA, tfdB and tfdR from pJP4. PTV1 partial tfdA sequence showed 77 % similarity with the archetypal tfdA gene sequence from Ralstonia eutropha JMP134. Tn5 mutagenesis confirmed the involvement of this gene in the 2,4-D catabolic pathway. © Rapid Science Ltd. 1998     

The Effects of Agitated Liquid Medium on in vitro Cultures of Hevea brasiliensis

The initiation and prolonged growth of callus, from stem explants of young plants of Hevea brasilienies on solid medium yielded a heterogeneous callus, with areas which are the result of compact growth interspersed with brown necrotic tissue and soft white tissue formations. Subculturing this callus (O callus) to agitated liquid medium and returning it to solid medium resulted in the production of a homogeneous friable and rapidly growing callus (Rl callus) The two established lines O and Rl have remained stable over one year in culture and differ in gross morphology, anatomy, growth and auxin content. Both were maintained on Murashige and Skoog's medium, with 2 mg/1 2,4-D and 0.5 mg/I kinetin. R 1 but not O showed enhanced growth at the lower 2,4-D level of 0.2 mg/l: both lines failed to continue growing when 2,4-D was omitted. It is suggested that the changes resulting from subculture in agitated liquid medium are related to those undergone by callus cultures which become habituated. Thus the Rl callus line is regarded as partially habituated. Subculture in agitated liquid medium also resulted in the production of large numberr of polyploid cells but these did not persist over the long periods of subsequent growth on agar medium, Enhanced auxin production by the establihed Rl callus line was thus observed in the absence of a detectable level of polyploidy.     

Plant regeneration from intergeneric somatic hybridization between Triticum aestivum L. and Leymus chinensis (Trin.) Tzvel

The suspension derived protoplasts of wheat (Triticum aestivum) cv. Jinan 177 were used as a recipient to fuse with the protoplasts of the ⁶⁰Co gamma-ray irradiated calli of Legmus chinensis. The wheat suspension cells and their protoplasts were not capable of differentiating to whole plants. The irradiated calli of L. chinensis were also the same. The protoplasts originated from the treated or untreated calli were both unable to divide under the conditions of this experiment. However, the fusion products grew and developed to whole plants which were identified as hybrids according to the analysis of chromosome, isozyme and morphology. The above result revealed that the lost regeneration capacity of both parents could be complementarily restored through somatic hybridization. This phenomenon also occurred with our work on Triticum aestivum (+) Haynaldia villosa, T. aestivum (+) Agropyron elongatum and T. aestivum (+) Psathyrostachys juncea.     

Growth and Plating of Cell Suspension Cultures of Datura innoxia

Suspension cultures of Datura innoxia Mill, were successfully grown on a modified Murashige and Skoog medium with 2,4–D, NAA or BAP as growth substances, provided the micronutrient levels were reduced to 1/10. Normal amounts of micronutrients were toxic. Attempts to identify the toxic elements did not succeed. Cultures grew exponentially on a shaker at 27°C in the light. Their doubling times varied from 1.1 days on 2,4–D (10^–6M) or NAA (10^?5M)+ 1 g/1 casein hydrolysate to 2.7 days on BAP (3 × 10^?7M) and 5.1 days on supraoptimal levels of 2,4-D (10^?5M). Cultures grew on NH₄⁺-N alone (from ammonium malate) or on NO₃^?-N alone. Dry weight yield was proportional to the amount of nitrate-N added (47 mg/mg N). Filtered suspension cultures containing single cells (plating cultures) could be grown in agar in petri dishes when NAA or 2,4-D were used as growth substances. Cells grew at densities above 500 units/ml in the agar. Most colonies grew from cell aggregates but division in single cells was observed. The highest plating efficiency was about 50% on 10^?6 M 2,4-D + 1 g/1 casein hydrolysate.     

Suspension culture of sugarbeet (beta vulgaris L.). induction and habituation of dedifferentiated and self-regenerating cell lines

A systematic procedure is described to obtain habituated cell suspensions from different explant sources of sugarbeet (Beta vulgaris L.).Cytokinin habituation occurred spontaneously. Auxin habituation could be induced by successive lowering of the external hormone concentration. Habituation was reversible and independent of the cell type or explant source. The habituated nature of the cells was lost during regeneration and had to be reinduced by adequate treatments.A method for initiating habituated self-regenerating lines was established. These cultures were less stable. Total dedifferentiation caused the loss of the regeneration capacity. Reculturing of the regenerated plants did not result in a higher percentage of primary regeneration.Abbreviations BAP benzylaminopurine - NAA naphtalene acetic acid - 2,4-D 2,4-dichlorophenoxy acetic acid - PGo medium see Negrutiu et al. 1975     

发根农杆菌介导的菠菜毛状根遗传转化体系的建立

发根农杆菌(Agrobacterium rhizogenes)侵染植物后可诱导植物产生毛状根。菠菜(Spinacia oleracea)是常见的食用蔬菜, 目前尚未见菠菜毛状根的研究报道。经筛选得到适合诱导菠菜毛状根的发根农杆菌菌株LBA9402, LBA9402侵染菠菜外植体茎后, 毛状根的诱导率最高可达16%。菠菜毛状根呈白色, 具有丰富的根毛, 能在无外源激素的固体培养基上快速增殖生长。通过诱导菠菜毛状根产生愈伤组织并进行分化, 获得了菠菜毛状根的再生植株, 再生率为8%。此外, LBA9402可将含有Ri质粒的T-DNA和携带外源GFP基因的Ti质粒T-DNA共同导入外植体中。PCR检测和荧光显微观察结果显示, rolB及GFP基因在菠菜毛状根基因组中稳定表达, 共转化频率为50%。     

发根农杆菌介导的菠菜毛状根遗传转化体系的建立

发根农杆菌(Agrobacterium rhizogenes)侵染植物后可诱导植物产生毛状根。菠菜(Spinacia oleracea)是常见的食用蔬菜, 目前尚未见菠菜毛状根的研究报道。经筛选得到适合诱导菠菜毛状根的发根农杆菌菌株LBA9402, LBA9402侵染菠菜外植体茎后, 毛状根的诱导率最高可达16%。菠菜毛状根呈白色, 具有丰富的根毛, 能在无外源激素的固体培养基上快速增殖生长。通过诱导菠菜毛状根产生愈伤组织并进行分化, 获得了菠菜毛状根的再生植株, 再生率为8%。此外, LBA9402可将含有Ri质粒的T-DNA和携带外源GFP基因的Ti质粒T-DNA共同导入外植体中。PCR检测和荧光显微观察结果显示, rolB及GFP基因在菠菜毛状根基因组中稳定表达, 共转化频率为50%。     

Photochemical reactions of transition metal organyl complexes with olefins Part 10. Light-induced formal [5+2] cycloadditions at manganese

Tricarbonyl-η⁵-2,4-dimethyl-2,4-pentadien-1-yl-manganese (1) forms upon UV irradiation in THF at 208 K solvent stabilized dicarbonyl-η⁵-2,4-dimethyl-2,4-pentadien-1-yl-tetrahydrofurane-manganese (2). With butynedioic acid dimethyl ester (3) and diphenylacetylene (5) complex 2 yields tricarbonyl-η⁵-1,2-dimethoxycarbonyl-4,6-dimethyl- cyclohepta-2,4-dien-1-yl-manganese (4) and tricarbonyl-η-4,6-dimethyl-1,2-diphenyl-cyclohepta-2,4-dien-1-yl- manganese (6) in a formal [5+2] cycloaddition. Addition of carbon monoxide and a 1,4-H shift completes the reaction. Propynoic acid methyl ester (7) forms the 2:1 adduct dicarbonyl-η^5:2-1,3-dimethyl-6-methoxycarbonyl-6- (E-2′-methoxycarbonylvinyl)-cyclohepta-2,4-dien-1-yl-manganese (8). The crystal and molecular structure of 8 was determined by X-ray structure analysis. The molecular structures of the complexes 4 and 6 were established by IR and NMR spectroscopy. Formation mechanisms of 4, 6 and 8 are discussed. Crystal data for 8: monoclinic space group P2₁/c, a=802.6(3), b=1136.6(1), c=8872.3(3) pm, β=93.14(2)°, V=1.705 nm³, Z=4.     

Aerobic degradation of dinitrotoluenes and pathway for bacterial degradation of 2,6-dinitrotoluene

An oxidative pathway for the mineralization of 2,4-dinitrotoluene (2, 4-DNT) by Burkholderia sp. strain DNT has been reported previously. We report here the isolation of additional strains with the ability to mineralize 2,4-DNT by the same pathway and the isolation and characterization of bacterial strains that mineralize 2, 6-dinitrotoluene (2,6-DNT) by a different pathway. Burkholderia cepacia strain JS850 and Hydrogenophaga palleronii strain JS863 grew on 2,6-DNT as the sole source of carbon and nitrogen. The initial steps in the pathway for degradation of 2,6-DNT were determined by simultaneous induction, enzyme assays, and identification of metabolites through mass spectroscopy and nuclear magnetic resonance. 2,6-DNT was converted to 3-methyl-4-nitrocatechol by a dioxygenation reaction accompanied by the release of nitrite. 3-Methyl-4-nitrocatechol was the substrate for extradiol ring cleavage yielding 2-hydroxy-5-nitro-6-oxohepta-2,4-dienoic acid, which was converted to 2-hydroxy-5-nitropenta-2,4-dienoic acid. 2, 4-DNT-degrading strains also converted 2,6-DNT to 3-methyl-4-nitrocatechol but did not metabolize the 3-methyl-4-nitrocatechol. Although 2,6-DNT prevented the degradation of 2,4-DNT by 2,4-DNT-degrading strains, the effect was not the result of inhibition of 2,4-DNT dioxygenase by 2,6-DNT or of 4-methyl-5-nitrocatechol monooxygenase by 3-methyl-4-nitrocatechol.