Preparation and assay of C-glucosyltransferase from roots of Pueraria lobata

C-glucosyltransferase (EC 2.4.1.X) is one of the key enzymes for the biosynthesis of puerarin. This paper describes the methodology in purification and assay of the enzyme for the first time in Puerarin lobata (Wild.) Ohwi. C-glucosyltransferase from roots of P. lobata was extracted and partially purified by (NH4)2SO4 saturation. The effects of pH, temperature, and substrate concentration on the activity of the enzyme were investigated. The properties of the puerarin produced by C-glucosyltransferase were studied by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). The peak activity of C-glucosyltransferase was detected in fraction of by 80% saturation of (NH4)2SO4 and the optimal conditions for enzymatic reaction were 35.5 micromol l(-1) of isoliquiritigenin and 560 micromol l(-1) of UDP-G at pH 8.1, 28 degrees C for 1 h. Mn2+ at 1 mmol l(-1) and Al3+ at 1 mmol l(-1) increased the enzyme activity, while Mg2+ inhibited its activity. The enzyme activity in Nicotiana tabacum and P. lobata were detected under the above assay conditions. Higher activity was found in roots than in leaves and stems of P. lobata, while no enzyme activity was detected in leaves of N. tabacum. It was the first time that activity of C-glucosyltransferase, which transforms isoliquiritigenin to puerarin, was detected in P. lobata.     

Nutrient consumption and production of isoflavones in bioreactor cultures of Pueraria lobata (Willd)

This paper reports the successful culture of Pueraria lobata (Willd.) suspension cells in a bioreactor. In vitro culture of this Chinese herb has potential as an alternative production method for industrial applications. Calli of P. lobata obtained from leaf explants were cultured in a 5.0 L bioreactor for two weeks. During this period, the pH of the medium declined from 5.8 to 4.5. By the end of the run about 70% of the sugars and reducing sugars and about 50% of nitrate was consumed. Almost 70% of inorganic phosphate and about 80% of the iron was exhausted. The bioreactor results indicated an isoflavone yield of 328.9 microg/ml, with an increase of about 1.77 fold. The yield of puerarin increased about 2.42 fold and reached 73.4 microg/ml in the bioreactor culture.     

In vitro studies of the submerged angiospermRuppia maritima: Auxin and cytokinin effects on plant growth and development

Axenic tissue cultures ofRuppia maritima L. were established and propagated clonally in vitro from terminal rhizome segments collected from Tampa Bay, Florida, USA. Cultures were maintained in a base medium consisting of synthetic seawater supplemented with half-strength Murashige and Skoog salts and 1% sucrose at pH 5.6. The effects of five cytokins [6-furfurylaminopurine (kinetin), 6-benzylaminopurine (BAP), 2-isopentyladenine (2iP), 6-(4-hydroxy-3-methyl-but-2-enylamino) purine (zeatin), andn-phenyl-n-1,2,3-thidiazol-5yl urea (thidiazuron)] and one auxin [napthalene acetic acid (NAA)] on explant growth and development were investigated. Cytokinin additions resulted in a 3- to 4-fold increase in nodal production, branching, and biomass ofR. maritima after 12 wk in culture. Cultures responded in a dose-dependent manner to 2iP but exhibited broad dose-response curves to kinetin, BAP, zeatin, and thidiazuron. NAA addition resulted in increased leaf and internodal lengths, but reduced the number of leaves per node and the rhizome biomass. The addition of NAA almost completely suppressed root growth in media without cytokinins and had an antagonistic effect on nodal production and branching in cytokinin-supplemented media.     

麻黄愈伤组织细胞的悬浮培养

用中麻黄幼苗的３种外植体进行了离体培养。不同外植体诱导愈伤组织的结果表明：最佳培养基为ＭＳ＋２ｍｇ／Ｌ２,４－Ｄ＋１ｍｇ／Ｌ６－ＢＡ;最佳外植体为下胚轴;用源于下胚轴的愈伤组织进行悬浮培养,从第３代开始建立起稳定的悬浮系。愈伤组织、悬浮培养细胞及培养液中都含有麻黄碱,悬浮培养液中的麻黄碱含量高于愈伤组织和悬浮培养细胞。表２参１２     

Soil mycofloral responses following the exposure to 2, 4-D

2, 4-dichlorophenoxyacetic acid (2,4-D) is a post emergence herbicide. The tests were conducted to study the toxicity of 2,4-D using EC50 value in four dilutions. 2,4-D was applied at concentration of 25, 50, 75, 100, 200 and 300 mg l(-1) in the potato Dextrose agar medium. The effect of this herbicide was evaluated as the colony forming unit (CFU). EC50 value for 10(-3) dilution of soil was 138 +/- 5.944 mg l(-1). Soil physico-chemical parameters and mycofloral properties were also evaluated. Qualitatively 10 genera of fungi were observed in untreated soil, whereas 4 genera were found in 2,4-D administered dose.     

发根农杆菌T—DNA基因对3种葛属植物毛状根形态和葛根素含量的影响

野葛、山葛和三裂叶葛的离体叶片与发展根农杆菌R1601直接感染诱导出毛状根并能够合成葛根素,毛状根离体培养后均出现两种形态：一种是有较高的葛根素含量的典型毛状根,另一种是葛根素含量低但生长速度较快的愈伤组织化毛状根,两类毛状根均有rol/B基因存在,表明rolB基因对所有毛状根的诱导和生长是必需的,aux1存在于所有愈伤组织化毛状根,而在典型毛状根中的检出率为20％－50％,表明aux1基因有诱导毛状根产生愈作组织的作用,含有ags基因的毛状根葛根素含量低于不含ags基因的毛状葛根素含量,表明ags基因不利于次生代谢合成,表3参12     

水浮莲(Pisia stratiotes Linn.)的体外再生与繁殖

建立了水生单子叶植物水浮连(Pistia stratiotes Linn.)通过器官发生途径的体久高效再生与繁殖方法.采用叶、茎节和匍匐茎为外植体诱导愈伤组织,只有茎节能够在添加2,4-D和6-BA的MS基本培养基上形成合愈伤组织,而叶和茎在含有不同组合植物激素的培养基上都不能够诱导愈伤产生.将愈伤组织转到添加6-BA和NAA的MS固体分化培养基可以在2wk内形成小苗,将小苗移至含NAA的MS固体生根培养基形成完整的植株.将生根苗转入无植物激素的不同基本液体培养基里比较其生长效果,其中含有2倍大量元素的SH培养基最适合其生长繁殖,在2wk内可由1个小苗每秒殖出10个新的植株.本研究是关于该植物体外再生的首次报道.水浮莲体外再生及繁殖系统的建立不公可以用于在无菌条件下进行基础生理生化研究,还可以用于该植物遗传达室转化系统的建立.由于该植物生长迅速且为无性繁殖,生产成本低,通过基因工程方法表达外源基因将可以用于重组药用蛋白的生产及污染水体的转基因植物修复.     

Toxicity and bioaccumulation of cadmium and lead in Salvinia cucullata

The toxicity and accumulation of heavy metals, cadmium (Cd) and lead (Pb) in aquatic fern, Salvinia cucullata were studied. Plants were cultured in Hoagland's medium which was supplemented with 0.5,1,2, and 4 mg/l of Cd and 5, 10, and 40 mg/l of Pb and were separately harvested after 2,4,6, and 8 days. The toxicity symptoms of Cd and Pb to S. cucullata showed chlorosis on leaves. There were significant derceases in the relative growth, biomass productivity and total chlorophyll content when the exposure time and concentration were increased. The accumulation study showed the significant increases of both metals when the exposure time and concentration were increased. The roots of S. cucullata had higher Cd and Pb contents than leaves suggesting that the metals were bound to the root cells and were partially transported to the leaves.     

Isolation and identification of a bioflocculant-producing strain and optimisation of cultural conditions via a response surface model

A bioflocculant-producing strain named MY6-2 was isolated from a mixed activated sludge by a nitrogen-free medium. Based on 16S rDNA sequence, biochemical and morphological characteristics, the strain MY6-2 was identified as Bacillus mucilaginosus. The chemical analysis indicated that the flocculant MY6-2 was mainly composed of extracellular polysaccharide. The result of the composition of the medium showed that MY6-2 was able to generate bioflocculant in a nutrient-poor medium that consisted of 5?g?L^?1 sucrose and no nitrogen source. Response surface methodology (RSM) was used to optimise the fermentation condition of MY6-2 for maximum flocculating activity by using central composite design (CCD). Based on the result, the optimum conditions were as follows: 100?mL of broth in a 250?mL Erlenmeyer flask, initial pH 8.0 and inoculum concentration 10%, respectively. The highest flocculating rate of 90% was achieved under these conditions by adding 0.5?mL fermentation supernatant to 95?mL of Kaolin suspension.     

Tolerance and accumulation of lead in Vetiveria zizanioides and its effect on oil production

Experiments were conducted to evaluate lead tolerance and accumulation in vetiver grass Vetiveria zizanioides (L.), grown in hydroponics and a pot study and to examine the effect of lead on vetiver oil production. Elevated concentrations of lead decreased the length of shoots and roots of plants. However, vetiver grown in highly contaminated soils showed no apparent phytotoxicity symptoms. Lead concentrations in the shoots and roots of vetiver plants grown in hydroponics were up to 144 and 19530 mg kg(-1) and those grown in soil were 38 and 629 mg kg(-1), respectively. Lead had an effect on vetiver oil production and composition by stimulating oil yield and the number of its constituents. Oil yield ranged from 0.4-1.3%; the highest yields were found in plants grown in nutrient solution with 100 mg Pb l(-1) for 5 weeks (1.29%) and 7 weeks (1.22%). The number of total constituents of vetiver oil also varied between 47-143 compounds when lead was presentin the growth medium. The highest number (143) was found in plants grown in soil spiked with 1000 mg Pb kg(-1). The predominant compound was khusimol (10.7-18.1%) followed by (E)-isovalencenol (10.3-15.6%). Our results indicated that lead could increase the oil production of vetiver.     

Study of fluoride content in groundwater of Nawa Tehsil in Nagaur, Rajasthan

There is a severe fluoride problem in Nawa tehsil of Nagaur district. Villagers are suffering from dental fluorosis and skeletal fluorosis. So an extensive geochemical study of 27 villages of eastern, south-eastern and southern zone of Nawa tehsil was done. Total 46 ground water samples were collected and analyzed for various physicochemical parameters as well as fluoride content. The ground water samples collected in clean polyethylene plastic containers were analyzed for pH, electrical conductivity, total dissolved salts, calcium, magnesium, total hardness, chloride and alkalinity. The fluoride concentration in the three different zones ranged from 0.64 to 14.62 mg l(-1) where 13.04% samples were found within permissible limit while 86.96% had fluoride beyond permissible limit (> 1.5 mg l(-1)). It was found that among the three different zones south-eastern zone was under serious fluoride contamination where fluoride concentration ranged between 1.10 to 14.62 mg l(-1). In the eastern zone fluoride concentration was recorded from 1.52 to 5.13 mg l(-1) whereas in the southern zone it was found between 0.64 to 3.63 mg l(-1).     

PP333在库拉索芦荟组培快繁中的应用研究

以MS 6-BA2.5mg/L NAA0．2mg/L为初代培养基，以MS 6-BA2.0mg/L NAA0．1mg/L为增殖培养基，1，3MS IBA0.5mg/L为生根培养基，添加不同浓度的PP333，其结果表明：初代培养基中添加0．5mg/LPP333有利于库拉索芦荟外植体出芽，增殖培养基中添加0．1-0．5mg/LPP333对库拉索芦荟试管苗增殖有效，而在生根培养基中添加0．5—1．0mg／LPP333有利于生根和移栽，其中以1／3MS IBA0．5mg/L PP3330．5mg/L培养基对库拉索芦荟试管苗生根和移栽有良好效应．     

TDZ和CPPU在虎杖组织培养中的应用

将高活性细胞分裂素类物质TDZ和CPPU应用于虎杖的组织培养研究.经实验筛选发现,虎杖茎段比叶柄、叶片更容易诱导愈伤组织的形成,其诱导愈伤组织较适的培养基组成为MS 0.5mg/LCPPU 0.2mg/LNAA,适宜的愈伤组织增殖培养基为SH 0.05mg/LTDZ 1.0mg/LNAA;诱导芽分化的适宜培养基为MS 0.05mg/LTDZ 0.5mg/LNAA;促进幼苗生长的培养基为MS 0.1mg/LCPPU 1.0mg/LNAA 0.5%活性炭;快速生根及壮苗的培养基为1/2MS 1.0mg/LNAA 0.5%活性炭.TDZ和CPPU在愈伤组织的诱导、不定芽的分化及组培苗的生长等方面表现出不同的效果.以此方式进行虎杖人工快速繁殖,繁殖系数可达到5～7,繁殖时间缩短为2～3wk.图4表2参12     

海莲红树林土壤CH4动态研究

对海南东寨港河港河口海莲红树林土壤CH4动态进行研究。CH4通量季节变化规律为春季>夏季>秋季>冬季,所有季节所有滩面其平均值为1.21 mg/(m2d)。不同季节CH4产生量平均值的大小顺序也为春季>夏季>秋季>冬季,所有季节所有滩面其平均值为8.28 mg/(m2d);土壤理化因子对河港海莲林土壤CH4产生率的重要性依次为全氮>NaCl>Na >Cl->SO42->含水量>总含盐量>有机质。暖季CH4氧化量高于冷季,平均值为7.06 mg/(m2d)。在所有季节所有滩面上,河港海莲林土壤CH4传输率的平均值为28.21%。     

In vitro mass multiplication of screw pines (<Emphasis Type="Italic">Pandanus</Emphasis> spp.) - an important costal bio- resource

An efficient in vitro mass multiplication protocol was developed for selected species of screw pine (Pandanus fascicularis Lam., P. furcatus Roxb.). The protocol could be successfully used for large scale production of planting materials leading to cultivation and making bio-fences or green belts in coastal areas, wetlands for protection and preventing soil erosion. This will facilitate the replenishment of the bio resource and also provide good breeding space for various fauna associated with it. For developing the tissue culture protocol, shoot tips and tillers from the mature plants were used as the explants. Surface sterilization with 0.1 % mercuric chloride for 1 min. Yielded good fraction (65 %) of contamination free explants. Explants inoculated in MS solid medium supplemented with 6-benzylaminopurine (BAP, 2.5 mg/l) and indole-3-acetic acid (IAA, 0.5 mg/l), induced 2–3 shoot buds in 5 weeks. These buds either individually or along with the explant portion, sub-cultured to fresh media containing the same hormonal combinations, resulted induction of 5–6 shoot buds in 4 weeks. Repeated subcultures of shoot buds in the same medium produced 8–10 shoot buds in every 4 weeks. Shoot elongation was achieved (~ 3 cm) by transferring the shoot clusters or individual shoots to basal medium, and the elongated shoots were rooted in vitro or ex vitro. Root induction in shoots (87.4 %) was achieved in MS medium supplemented with 0.1 mg/l indole-3-butyric acid (IBA). Rooted shoots were established in paper/polythene cups filled with fine sand, which subsequently recorded 85 % establishment under the shade-net house with proper irrigation. Plants grown in poly-bags were later successfully established in different field conditions and recorded 100 % survival.     

Effects of industrial waste water on heavy metal accumulation, growth and biochemical responses of lettuce (Lactuca sativa L.)

The waste water showed high values of total solid (TS), hardness and chloride with slightly alkaline pH along with high concentrations of Cr (2.03 mg l(-1)), Ni (1.59 mg l(-1)) and Zn (0.46 mg l(-1)). The concentration of Cu (0.21 mg l(-1)) and Zn in industrial waste water was low than Ni and Cr. The diluted (25 and 50%), undiluted (100%) waste water was used to irrigate the lettuce plants grown in alluvial soils. Plants accumulated heavy metals in their shoot (Ni, 13.65; Cr, 19.73; Zn, 21.6 and Cu 14.76 microg g(-1) dry weight) and root (Ni, 41.4; Cr, 31.6; Zn, 30.2 and Cu 15.85 microg g(-1) dry weight) in high concentrations after irrigation with undiluted industrial waste water. Maximum accumulation of heavy metals was found in the root than the shoot (13.65-21.60 microg g(-1) dry weight). Dry matter yield and biomolecules (Chlorophyll a, b and sugar contents) was found to increase with increase in concentration of waste water up to 50%, which declined at the exposure of undiluted waste water. Catalase activity was found to increase with increase in waste water concentrations up to 100%, while carotenoids content increased in plants only up to the 50% waste water irrigation. Use of industrial waste water in such form, on agricultural lands is not found suitable without proper treatment. It could be injurious to plants growth and may be a potential threat to food web.     

Toxizität und anreicherung von blei bei der miesmuschel Mytilus edulis im laborexperiment

In a laboratory experiment, conducted over 130 days, three batches of a total of 100 common mussels, Mytilus edulis, were maintained in media containing different lead concentrations. Two vessels served as controls. There was no acute injury to the mussels; but, over an extended period of time, a marked increase in mortality occurred which was related to the lead concentration in the medium. The median lethal time (LT₅₀) was computed to be 218 days for the control, 150 days with 0.5 mg Pb/l in the medium, 129 days with 1 mg/l, and 105 days with 5 mg/l. Quantitative analyses of the soft parts of M. edulis by flameless atomic-absorption-spectrophotometry revealed a distinct accumulation of lead. From a natural lead content of 8.4 μg Pb/g dry weight, the lead concentration increased to 12,840 μg/g at 0.5 mg/l, to 20,770 μg/g at 1 mg/l and to 39,830 μg/g at 5 mg/l. The ratio of the concentration of lead in the soft parts of M. edulis to the concentration in the medium remained in the same order of magnitude both under laboratory conditions using high lead concentrations and under in situ lead levels. Over a period of 130 days, lead uptake expressed as percentage of lead offered was 10.9% at 0.5 mg/l, 9.5% at 1 mg/l, and 3.4% at 5 mg/l, respectively.     

钒胁迫对水稻幼苗生理生化和富集特性的影响

水稻（Oryza.sativa L.）是我国最重要的粮食作物之一,水稻产量占粮食总产量的一半以上,一旦水稻受到重金属污染,将会影响水稻植株的正常生长和生理特性。目前关于钒胁迫对水稻植株生理特性指标的影响方面报道较少。通过水培实验,研究了不同钒（V）质量浓度（0、4、8、12、16、20 mg·L-1）对水稻幼苗（Oryza.sativa L）生理生化和富集特性的影响。结果表明：随着V胁迫浓度的增加,叶绿素含量、可溶性蛋白含量、过氧化氢酶（CAT）活性、过氧化物酶（POD）活性、超氧化物歧化酶（SOD）等均呈现先上升、后下降的变化趋势。当ρ（V）≤12 mg·L-1,与对照相比较,叶绿素含量、可溶性蛋白含量和酶活性增大了135.3%、104.2%、77.8%（CAT）、84.5%（POD）和273.2%（SOD）;当ρ（V）〉12 mg·L-1,则分别降低37.2%、39.4%、41.1%、24.1%和24.5%。随着 V 胁迫浓度的增加,丙二醛（MDA）含量和细胞膜透性逐渐增大,与对照相比,分别增加了38.5%~289.3%、21.2%~303.2%,根系活力下降了10.9%~82.2%。可见,低ρ（V）（≤12 mg·L-1）对水稻幼苗的生长有一定的刺激作用,水稻幼苗自身保护酶表现出较强的自我调节能力;高ρ（V）（〉12 mg·L-1）明显抑制叶绿素和蛋白合成、抗氧化酶活性和根系活力,伤害了细胞质膜系统,影响水稻幼苗的生长发育。不同V浓度胁迫下,水稻幼苗累积的V含量为：根〉茎叶。随着V胁迫浓度增加,水稻幼苗各器官V含量增大,其中根部增幅远大于茎叶,当ρ（V）从5 mg·L-1增加到40 mg·L-1,与对照相比较,根部增加了0.98~25.3倍,茎叶部增加了0.26~4.74倍。生物富集系数（BF）先增加后降低,最大值为2.8408;迁移系数（TF）下降,最低值为0.1170,说明水稻对V有较强的富集能力,但迁移能力较低,积累的V主要富集在根部,可减轻V对地上部植物的危害。     

Pollution level in distillery effluent and its phytotoxic effect on seed germination and early growth of maize and rice

The effluent from a Lucknow- based distillery (Mohan Meakin Distillery) was analyzed for physico-chemical and biological parameters of pollution and concentration of potentially toxic heavy metals (Cd, Cr, Ni and Zn) and the effect of the distillery effluent, as such and on 50% dilution with tap water was studied on seed germination and seedling growth of maize (Zea mays L.) and rice (Oryza sativa L.). The effluent was wine red in colour and highly acidic (pH approximately 55) and possessed decaying alcoholic smell. The effluent contained high values of different pollution parameters, particularly total solids, 3450 mgl(-1) (soluble plus suspended solids), alkalinity 1500 mgl(-1), biological oxygen demand (BOD, 1649 mgl(-1)) and chemical oxygen demand (COD, 2036 mgl(-1)). It had very low values of dissolved oxygen (DO, 0.34 mgl(-1)). The heavy metals (Cd, Cr, Ni and Zn) content, particularly the nickel concentration (0.029 mg l(-1)) was high. Use of the distillery effluent, even on 1:1 dilution with tap water inhibited germination and early seedling growth of maize and rice. In both maize and rice, more so in the former germination % of seeds, length of radicle and plumule and the fresh and dry weight of the seedlings were significantly reduced. The emerging leaves of the seedlings also developed visible effects of toxicity some of which resembled the symptoms of nickel toxicity. Our observations suggest that the effluent, as discharged from the distillery carry a heavy load of pollutants. Its discharge into the river Gomti poses a potential threat to the aquatic life, perticularly during the summer months when the water flow in the river is drastically reduced. The distillery effluentis also harmful for irrigating crops grown along the drain carrying it.     

EDTA and citric acid mediated phytoextraction of Zn, Cu, Pb and Cd through marigold (Tagetes erecta)

Phytoextraction is an emerging cost-effective solution for remediation of contaminated soils which involves the removal of toxins, especially heavy metals and metalloids, by the roots of the plants with subsequent transport to aerial plant organs. The aim of the present investigation is to study the effects of EDTA and citric acid on accumulation potential of marigold (Tagetes erecta) to Zn, Cu, Pb, and Cd and also to evaluate the impacts of these chelators (EDTA and citric acid) in combination with all the four heavy metals on the growth of marigold. The plants were grown in pots and treated with Zn (7.3 mg l(-1)), Cu (7.5 mg I(-1)), Pb (3.7 mg l(-1)) and Cd (0.2 mg l(-1)) alone and in combination with different doses of EDTA i.e., 10, 20 and 30 mg l(-1). All the three doses of EDTA i.e., 10, 20 and 30 mg l(-1) significantly increased the accumulation of Zn, Cu, Pb and Cd by roots, stems and leaves as compared to control treatments. The 30 mg l(-1) concentration of citric acid showed reduced accumulation of these metals by root, stem and leaves as compared to lower doses i.e., 10 and 20 mg l(-1). Among the four heavy metals, Zn accumulated in the great amount (526.34 mg kg(-1) DW) followed by Cu (443.14 mg kg(-1) DW), Pb (393.16 mg kg(-1) DW) and Cd (333.62 mg kg(-1) DW) in leaves with 30 mg l(-1) EDTA treatment. The highest concentration of EDTA and citric acid (30 mg l(-1)) caused significant reduction in growth of marigold in terms of plant height, fresh weight of plant, total chlorophyll, carbohydrate content and protein content. Thus EDTA and citric acid efficiently increased the phytoextractability of marigold which can be used to remediate the soil contaminated with these metals.