Genetic engineering is an important tool in plant functional genomics research and crop improvement applications. During the production of transgenic plants, the regenerated plantlets (T₀) through transformation process will often mix with false-positive plants. In addition, the propagation of T₀ plants will generate a certain percentage of non-transgenic progenies through the median-segregation of the transgene. Rapid identification of the transformed transgenic plants from the non-transgenic plants is an essential step in the production of transgenic plants. The commonly used method for identifying the existence of transgene in plants is through the polymerase chain reaction (PCR), which uses a pair of the target gene-specific primers to amplify the target DNA fragment from the genomic DNA samples of the tested plants. The use of PCR methods in transgenic plant screening is prone to cross contamination, is time consuming, and is considerably expensive. Thus, the development of methods for identifying transgenic plants in a robust, precise manner is of great importance. In this study, we assessed different methods for identifying rapidly and precisely transgenic rice and soybean plants containing herbicide resistant genes, including phosphinothricin acetyl transferase gene (bar) and modified 5-enolpyruvylshikimate-3-phosphate synthase gene (EPSPS). The methods used for the research included leaf-painting of herbicides, leaf-incubation on the solid medium containing herbicides, and seed germination in presence of herbicides. For these experiments optimum concentrations of herbicides were determined. Results showed that all tested methods could be used for rapid screening of transgenic plants. Application of 300 mg/L Liberty^® could easily distinguish the bar-containing rice plants from the non-transgenic ones (Fig. 1), while Liberty^® at 135 mg/L was sufficient for the identification of transgenic soybean plants (Fig. 2). Similarly, EPSPS-containing rice and soybean plants were determined by leaf-painting of 1 % and 0.25 % roundup (active gradient of glyphosate was 41 %), respectively (Fig. 3, 4). Solid medium with 50 mg/L glyphosate was suitable to distinguish leaf samples of soybean plants containing EPSPS gene (Fig. 5). Seed germination was very sensitive to selective agents. The germination of the non-transgenic seeds was completely prohibited in water solution containing either of the herbicides at the concentration of 10 mg/L (Fig. 6-9). In conclusion, we establish three methods that can rapidly and precisely identify transgenic rice and soybean plants containing herbicide-resistant gene bar and EPSPS. Our results can be used in both transgenic research and safety assessment of transgenic crop production.

DNA transposable elements require the transposase from an autonomous parent element for transposition. The characteristic of MITEs are generally less than 500 bp with terminal inverted repeat (TIR) and target site duplication (TSD). MITEs often insert into insides of genes or regions near to genes, and therefore the markers based on TIRs of MITEs, termed inter-MITE polymorphism (IMP), are possibly lined to important agricultural traits and have been successfully used in barley, wheat, maize, oats etc. Extremely low polymorphism has been observed in tobacco by several molecular marker systems, such as RAPD, AFLP, ISSR, SRAP and SSR, due to its narrow genetic background during domestication. In this study, IMP markers were developed in tobacco using genomic sequences from the Tobacco Genome Initiative (TGI). Non-redundant tobacco genomic sequences from TGI were used to scan MITEs through a bioinformatic approach and 11 581 MITEs of unique TIRs were eventually identified and aligned (partial result was shown in Fig. 2). Two hundred and nineteen IMP primers were designed according to the alignments and then were testedamong nine representative tobacco varieties with different types (flue-cured, burley, oriental and cigar tobacco) (Table 1). In the 219 primers, 119 were successfully amplified by PCR, which demonstrated a success rate of 54.6 % (partial result was shown in Fig. 3). The 119 primers were further used in a doubled haploid (DH) population of a flue-cured tobacco, 29 of which showed polymorphism between the two parental lines (Honghuadajinyuan/Hicks Broadleaf). Of the 29, 21 were genotyped in the DH population and integrated into seven linkage groups with SSR markers (Fig. 5). Our results demonstrate that IMP markers are useful in tobacco such as genetic germplasm investigation and genetic map construction. But low development efficiency is also observed for this kind of marker system in tobacco. To improve the efficiency of IMP marker development, high-throughput detective/scanning platforms are needed in its genotyping process.

Cytochrome P450 monooxygenases (P450s or CYPs) belong to a large family of hemoproteins which are widely distributed among various organisms ranging from eukaryotes to prokaryotes. P450s play a pivotal role in the biosynthesis of bioregulators such as prostaglandins, leucotrienes and steroid hormones, and participate in the metabolism of xenobiotic such as poisons, drugs, and environmental pollutants. In recent years, more and more researches reported that P450s were potentially useful in the environmental remediation. And bacterial CYPs have been found to be more promising than those from plants and animals because of more solubility and higher stability, not membrane-associated and higher reaction rates. However, the activity of wild type prokaryotic P450s towards recalcitrant contaminants has been less reported as compared to eukaryotic P450s. In our laboratory, a novel cytochrome P450 monooxygenase CYP102A16, which belonged to CYP102 family, was cloned from Bacillus sp. C3 and expressed in Escherichia coli. CYP102 family represents a bacterial P450 one naturally fused with cytochrome P450 and cytochrome P450 reductase, making them potentially more useful in biotechnological applications due to their self-sufficiency, while the reaction catalyzed by cytochrome P450 requires cytochrome P450 reductase for transfer electron. The recombinant CYP102A16 was then purified by Ni-NTA affinity chromatography. The effects of temperature, pH, organic solvents, surfactants, metal and non-metal ions on the activity and stability of purified CYP102A16 were investigated based on nicotinamide adenine dinucleotide phosphate (NADPH) oxidation assay. The results showed that the optimum pH for CYP102A16 was 6.5-7.5, with a highest NADPH consumption rate of (885.5 ± 11.7) U at pH 7.5 in Tris-HCl buffer (Fig. 1C). CYP102A16 was stable under a wide range of pH, retaining more than 80 % of its activity after incubated at pH 5.0-10.0 for 30 min (Fig. 1D). The enzyme reached a highest activity of (511.0 ± 20.5) U at 35 ºC, and kept stability at the temperatures below 45 ºC with residual activity of above 95 % (Fig. 1A, B). CYP102A16 was completely stable in 20 % dimethylsulfoxide (DMSO), 30 % methanol, 10 % ethanol and 20 % acetone, and could retain above 45 % activity in 10 % acetonitrile, n-propanol and isopropanol, but was almost inactivated in 10 % n-butanol (Table 1). The enzyme activity increased about 40 % and 60 % when low levels of cetylpyridine chloride (CPC) (0.003-0.02 g/L) and Triton X-100 (0.1-0.2 g/L) were added respectively, while non-significant effect was found with the addition of low concentration of sodium dodecyl sulphate (SDS) (0.004-0.008 g/L). The activity decreased gradually with increasing the surfactant concentration, and it was almost inactivated at 0.09 g/L CPC, 5.0 g/L Triton X-100 or 0.08 g/L SDS (Fig. 2). Addition of 1-20 mmol/L K⁺, 20-50 mmol/L Na⁺ and 0.05 mmol/L Cd²⁺ increased CYP102A16 activity slightly, while NH₄⁺, Ca²⁺, Mg²⁺, Fe³⁺, Co²⁺, Mn²⁺, Zn²⁺, Cu²⁺ and ethylene diamine tetraacetic acid (EDTA) exhibited negative effects on enzyme activity (Fig. 3). Inhibition at 1-100 mmol/L concentrations was observed by the order of Ca²⁺ > Mg²⁺ > NH₄⁺ > EDTA, and the effect was positively correlated with the concentration of metal and non-metal ions. Exploration of the in vitro catalytic conditions for CYP102A16 provides a reliable basis for constructing a bett

At present, flocculants are prevalent in the variety of industrial processes such as wastewater treatment, drinking water purification and downstream processing in fermentation processes. In general, flocculants are classified into three groups: inorganic flocculants, organic synthetic flocculants and naturally occurring flocculants. Bioflocculants have received considerable scientific and biotechnological attention in recent years because of their biodegradability, harmlessness and lack of secondary pollution of their degradation intermediates. Compared with inorganic flocculants and synthetic flocculants, bioflocculants have not yet been produced industrially because of their low flocculating capability and high cost. In order to reduce costs of production, research is needed to Bacillus megaterium MBFF6, and the strain producing bioflocculant B. megaterium MBFF6 was investigated by signal factor test. At the same time, thermal stability and chemical characteristics of bioflocculant were studied. The bioflocculant BF6 was purified from supernatant by ethanol precipitation. Whether containing α-amino acids and polysaccharide of the purified bioflocculant was analyzed by ninhydrin reaction and Molish and anthrone reaction. Proteins and nucleic acids were qualitatively determined by UV spectrophotometry. The chemical groups of purified bioflocculant were analyzed using a Fourier-transform infrared (FTIR) spectrophotometer (FTIR-8400S). was named as BF6. In this study, Optimization for culture conditions of strain MBFF6 and chemical characteristics of bioflocculant BF6 were studied. The effect of culture time, carbon source, nitrogen source, C/N ratio, pH, metal ions, culture temperature and inoculum size on bioflocculant production byidentify and select new bioflocculant-producing microorganisms and strains capable of utilizing low-cost substrates, and to learn how to optimize fermentation conditions to enhance bioflocculant activity. We had previously screened a bioflocculant-producing strain identified as The results indicated that the optimal medium for bioflocculant production was glucose 5 g/L, (NH₄)₂SO₄ 0.5 g/L, K₂HPO₄ 5 g/L, KH₂PO₄ 2 g/L, MgSO₄·7H₂O 0.2 g/L, pH 7; The optimal culture conditions were inoculum size 2 % (V/V), culture temperature 30 °C, and shaking speed 150 r/min for 24 h (Fig. 1-5). This bioflocculant was stable at 60-100 °C. The production of bioflocculant BF6 from fermentation broth was 1.28 g/L and it was a white powdery substance. The results of color reaction showed that the anthrone reaction produced blue-green compounds, and Molish reaction appeared a clear violet ring on the boundary of sulfuric acid and samples, and the ninhydrin reaction produced blue compounds, which indicated that the bioflocculant contained polysaccharide and protein. The results of UV spectrophotometry revealed that there was a faint characteristic absorption peak at 280 nm, but no absorption peak at 260 nm, confirming that the bioflocculant was primarily consisted of polysaccharide and proteins but no nucleic acids. Infrared spectrophotometry analysis revealed that this bioflocculant contained hydroxyl, amino, carbonyl, and methoxyl groups in its structure and these groups were the preferred ones for flocculation process (Fig. 6). The experimental result above shows that the bioflocculant-producing strain MBFF6 has such advantages as simple nutrition and short fermentation period, and it synthesises a bioflocculant with better thermal stability and high flocculating activity. It can be seen that the B. megaterium MBFF6 is a very broad application bioflocculant-producing strain. Bioflocculant BF6 may find possible application as a polymer for environmental b

Streptococcus pneumoniae is a conditional pathogenic bacteria which is associated with significant global morbidity and mortality. The main infection way is through mucosa system. Due to its variable serotype, there still have no effective method to prevent it. Secretory immunoglobulin A (SIgA) is the most important antibody secreted by plasmocytes and also the maximum in number, which can prevent bacterial adhesion to cells of host. Besides, secretory piece of SIgA can bind pneumococcal surface protein A, which help immune cells clear bacteria. Previous study showed that SIgA decreased when a host was infected with S. pneumoniae in the intestinal tract. SIgA could restrain bacteria being translocated to other organs and activate complement pathway, so when secretion decreased, host immune resistance become weakened, thus caused pathological changes. Corresponding to this research, increase of the antibody could help inhibit infection. This experiment aimed to investigate the expression condition of SIgA in intestinal mucosal immune system when rhesus monkeys were infected with S. pneumoniae spontaneously, and its role in the pathopoiesis of S. pneumoniae. Due to the high similarity of genes shared between human beings and rhesus monkeys, this experiment would also be helpful for the understanding of pneumonia happened on mankind. Three infected and three normal rhesus monkeys were blooded to death and obtained relative organs after anaesthesia. The changes of SIgA expression in esophago, jejunum, caecum, stomach and liver of rhesus monkeys were detected by immunohistochemistry and in situ hybridization on paraffin sections. Meanwhile, the images were collected, and the optical density value and positive cell area were detected by microscope. Immunohistochemistry results showed that the expression of SIgA in infected groups was smaller than that in normal ones, especially in jejunum where the expression was significantly reduced (P< 0.05) (Table 1 and Fig. 1). Similar results were obtained by in situ hybridization test. The total areas of positive cells were reduced in infected groups, and the differences were significant (P< 0.05) in jejunum, caecum, stomach (Table 2 and Fig. 2). Optical density values increased in infected groups, but the differences were not significant (P > 0.05) compared with normal ones. Most of the positive cells concentrated in the mucous layer and some in the blood vessel, while in the liver they were diffused. The positive cells were principally lymphocytes, hepatocytes and cells in blood vessels, still some glandular cells, endotheliocytes, non-cuticular epitheliums. The results above suggest that since SIgA plays a dominant role in the mucosal immune system, it possibly involves in the S. pneumoniae infection and may participate in the clearance of S. pneumoniae through humoral immunity, and the reduction of the antibody may be one of the reasons for S. pneumoniae infection.

Clonal plants, which are generated through both sexual reproduction and vegetative propagation (clonal growth) in nature, can affect species distribution patterns and dynamics, as well as the species composition and diversity of communities. Clonal plants with ability to adapt to the harsh environment have strong competence for survival, and thus play an important role in regeneration and revegetation of the forest dynamics. Based on the investigation of the root-sprouting situation of shrubs and woody vines with DBH (diameter at breast height) ≥ 1 cm or height ≥ 2 m and trees in Baishanzu permanent plot, the objective of this study was to research the sprouting ability of different species with different growth forms, the relationship between root-sprouting rate and DBH of parent-ramet and the association between sprouting rate and habitats. A 5-hm² plot in Baishanzu Mountain was divided into 2 000 quadrats (5 m × 5 m) to survey species identity, DBH, root-sprouting number and root-sprouting DBH / basal diameter for shrubs and woody vines with DBH ≥ 1 cm and all trees. Species in the plot were classified into canopy trees, sub-canopy trees, understory trees, shrubs and vines to survey the differences of their root-sprouting capacity. The relationship between root-sprouting rate and DBH of parent-ramet was analyzed based on six common species with the number of mother plant ≥ 400 and root-sprouting rate ≥ 1.0. According to the differences of slope, convexity and elevation among quadrates, the plot was divided into four habitats, including ridge, less-steep slope, steep slope and gully to analyze the association between sprouting rate and habitats. The results showed that: 1) The total parent-ramet and sprout in the plot were 36 769 and 34 786 respectively, thus the sprouting rate of the species in the plot was 0.94 (Table 1); 2) the sprouting rates of species with different growth forms were different with the descending order of canopy species, sub-canopy species, understory species, shrubs and vines (Table 1); 3) twelve species according with the mother plant numbers ≥ 200 and root-sprouting rate ≥ 0.50 belonged to the dominant species from canopy, sub-canopy, understory and shrub species (Table 2); 4) the sprouting rates were positively related to the DBH of the parent-ramet for all species together or six main species, respectively. At the same time, the maximum values of the sprouting rate were responded to the DBH values which were slightly larger than the intermediate values of DBH for all species (Fig. 2, 3); 5) the sprouting rates and sprout density in four habitats in the plot were different, and the sprouting rates and parent-ramet density were both descended with the order of ridge, less-steep slope, steep slope, gully (Table 3). In conclusion, the relatively high sprouting rate in the plot indicates that the clonal reproduction is an important reproduction strategy for woody plants in the communities; the generally high sprouting rate of the dominant species from canopy trees, sub-canopy trees, understory trees and shrubs indicates that the clonal reproduction plays an important role in the formation of forest community structure; the positive relationship between the parent-ramet DBH and root-sprouting rate reflects that the growth and ecesis of root-sprouting depend on resource supplement from the parent-ramet; meanwhile, the relationship between sprouting rates and habitat is consistent with the risk-sharing principle of the clonal plant genets.

Enrofloxacin is the first fluoroquinolone antimicrobial agent solely for veterinary use. The drug has a broad spectrum of activity, being active against both Gram-positive, Gram-negative bacteria and mycoplasmas. Now the traditional clinical dosage forms include powder, injection, solution and suspension, and so on. Some new dosage forms such as nanoparticles, microsphere, liposomes, β-cyclodextrin inclusion complex, sustained-release granules, microencapsulation and microcapsules have been also studied and reported accompanying with the development of the new preparation technique, but the research about enrofloxacin nanoemulsion has not yet been reported. The low water solubility is still an important limiting factor hampering enrofloxacin administration and the development of the novel preparation. So, we aimed to prepare enrofloxacin nanoemulsion with enrofloxacin as a model drug and nanoemulsion as a drug carrier by incorporating enrofloxacin into a nanoemulsion to improve its water solubility. In order to screen an optimal prescription for preparing enrofloxacin nanoemulsion, the oil phase was screened among isopropyl myristate (IPM), acetic ether, soybean oil and peanut oil; the surfactant was screened among polyoxyl 40 hydrogenated castor oil, polyoxyethylene castor oil (EL-40), Tween-80, lecithin and Span 80; the cosurfactant was screened among ethanol, 1, 2-propanediol, glycerin, acetic acid (HAc), lactic acid and propanoic acid; and the mass ratio (Km) of surfactant to cosurfactant was screened among 3∶1, 3∶2 and 3∶3, respectively. A pseudo-ternary phase diagram was established to investigate the effect of various compositions on the formation region of the nanoemulsion. The phase diagram was obtained by an aqueous titration method with one axis representing the mixture of surfactant and cosurfactant named as combined surfactant (S_mix), another representing the oil phase, and the third representing the water phase. Based on the nanoemulsion region, the optimal prescription was screened. Then, enrofloxacin nanoemulsion was prepared according to the screened prescription and its quality was evaluated. The results showed that the optimized formulation was IPM as oil phase (Table 1), EL-40 as surfactant (Fig. 1B), HAc as cosurfactant (Fig. 2A), the distilled water as the aqueous phase. In this screened prescription, Km was 3∶2 (Fig. 3B), and it contained enrofloxacin of 15 %, IPM of 3 %, Smix of 48 %, and distilled water of 34 %. According to the prescription, the prepared enrofloxacin nanoemulsion was with a transparent and uniform appearance, the structural type of oil-in-water, and the spherical droplet observed by transmission electron microscope (Fig. 4). The mean size of the nanoemulsion droplet was (22.45 ± 2.92) nm with Gaussian distribution verified by laser scattering analyzer (Fig. 5), and the value of pH was 4.63, and the minimal inhibitory concentration (MIC) against Escherichia coli ATCC₂₅₉₂₂ and Staphylococcus aureus ATCC₂₅₉₂₃ was both 1/2 times the MIC of the crude drug (Table 2). The nanoemulsion was not found the phenomenon of the delamination, demulsification and medicine leakage under the centrifugalization trial and the thermal test. The results above suggest that the optimal preparation procedure of enrofloxacin nanoemulsion is simple and practicable. Enrofloxacin nanoemulsion has a high solubility, strong antibacterial activity, good stability and benefit administration of group drinking water, and effectually overcome the defects such as a poor water-solubility and instability to preserve the traditional dosage form of enrofloxacin. It can be used as a new type of nano-antimicrobial drug for veterinary clinic.

Rice (Oryza sativa L.) as a photophilous crop, often encounters low-light environment during the growth stage in rice-producing areas particularly in Sichuan. Low-light stress seriously affects the morphology, physiology, grain yield and quality of rice. However, the response of rice root system to low-light stress is not so far clear. Therefore, we seeks to examine the responses of five rice varieties to low-light stress at different growth stages through pot experiments under artificially-controlled shading condition using white cotton yarn screen. By a completely randomized design, the pot experiments were carried out on the experimental farm of Sichuan Agricultural University in 2009. In experiment 1, one-layer and two-layer white cotton yarn screens, which shaded about 53 % and 73 % of the full light intensity respectively, were covered on the top of rice canopy of Gangyou 906 at tillering-elongation stage, elongation-booting stage, booting-heading stage, and heading-maturing stage respectively. In experiment 2, the response of root system of Ⅱ you 498, Gangyou 188, Gangyou 527 and Chuanxiang 9838 to shading was studied at tillering-elongation stage and booting-heading stage by covering with one-layer white cotton yarn screen. The screens were more than 2.0 m above the ground to ensure good ventilation and were large enough to fully cover the shaded plots. Plants without covers were set as control (CK). Root/shoot ratio, root volume, maximal root length, root total absorbing area, root active absorbing area and root α-naphthylamine oxidizing activity were analyzed at the end of shading treatment. The results were as follows: 1) Root volume, root total absorbing area and root active absorbing area of Gangyou 906 were decreased with the increase of shading degree before filling stage, resulting in root growth retard (Fig. 1, 2). At tillering-elongation stage and elongation-booting stage, shading greatly (P < 0.05) reduced root volume, and the decline was severe with the increase of low-light stress. As compared with the control, root volume was significantly decreased (P < 0.05), but the decrement with treatment of 53 % shading was greater than that of 73 % shading at booting-heading stage. Root total absorbing area and active absorbing area significantly (P < 0.05) decreased with the increase of shading intensity at elongation-booting stage and booting-heading stage. After heading stage, the root system growth was promoted, while the root senescence was delayed under 53 % shading treatment. Meanwhile, the root volume, root total absorbing area and active absorbing area increased by 14.6 %, 17.5 % and 39.5 % respectively with 53 % shading treatment. However, 73 % shading treatment had negative effects on root volume, root total absorbing area, root active absorbing area and α-naphthylamine oxidizing activity in roots (Fig. 1-3). 2) The responses of different varieties to shading treatment were different. Shading significantly (P < 0.05) reduced root α-naphthylamine oxidizing activity, root absorbing area and active absorbing area of Ⅱyou 498, Gangyou 188 and Gangyou 527 (Table 3, 4), leading to the hindrance of the formation and capacity of moisture and mineral nutrients of root system. But for Gangyou 906, the root α-naphthylamine oxidizing activity with 53% and 73% shading treatment at tillering-elongation stage and booting-heading stage were 104.7 %, 35.5 %, 83.2 %, and 0.5 % respectively higher than those of non-shading treatment. It can be concluded that different degrees of shading have different influences on root system characteristics of rice at each growth stage, and strong shade-tolerant variety such as Gangyou 906, can reduce the disadvantaged effect on grain yield of rice by improving the vigor of root system. It also suggests that the root α-naphthylamine oxidizing acti

Aluminum (Al) is considered as the major factor constraining crop performance when soil pH is lower than 5.5. The most easily recognized symptom of A1 toxicity is the inhibition of root growth, and this has become a widely accepted measure of A1 stress in plants. In simple nutrient solutions, micromolar concentrations of A1 can begin to inhibit root growth within 60 min. Kinetin (KT), one of the cytokins, is a unique plant hormone that promotes cell division. KT has been widely used to alleviate the toxicityin plants caused by abiotic stresses, for example, cadmium, lead, and salt stress. However, little attention has been paid to study the alleviating effects of KT on Al toxicity to plants, especially for barley, a major winter crop in South China. In the present study, hydroponic experiments were 6-kinetin (6-KT) on barley seedlings under Al stress by investigating the growth, chlorophyll and proline contents, as well as the activities of antioxidant enzymes in the leaves. conducted to focus on the alleviative effect of spraying Uniform barley seedlings were selected 10 days after sowing and transplanted onto a 5-L pot containing 4.5 L nutrient solution, which was covered with a polystyrol-plate with seven evenly spaced holes, and placed in a greenhouse with the photoperiod of 8 h light/16 h dark and the temperature of (21 ± 2) °C throughout the experiment. One week after transplanting to the basic solution culture, Al as AlCl₃·6H₂O was added to the nutrient solution. Meanwhile, different concentrations (100, 200, 400, and 800 μmol/L) of 6-KT were sprayed on barley leaves exposed to Al stress solution every other day. No Al and 6-KT but deionized water was sprayed on the leaves growing in control solution to following six treatments. The nutrient solution in the growth container was continuously aerated with pumps and replaced with fresh solution every five days. The experimental results showed that, spraying 100-400 μmol/L 6-KT under Al stress improved plant growth (Table 1), increased chlorophyll contents (Fig. 1), but decreased the contents of free proline (Fig. 4) and malondialdehyde (MDA) (Fig. 2), and decreased the activities of superoxide dismutase (SOD), peroxidase (POD) (Fig.3). Compared with spraying 100 μmol/L 6-KT, the catalase (CAT) activity decreased when the spraying concentrations were 200 and 400 μmol/L (Fig. 3). Furthermore, the optimal alleviation effect was at a concentration of 200 μmol/L 6-KT. At this spraying concentration, the chlorophyll content was increased by 27.5 %, while the MDA content was decreased by 29.5 %, compared with the Al individual treatment. However, when spraying concentration of 6-KT was 800 μmol/L, the plant growth was decreased, while the proline content and antioxidase activities were increased, in addition, the chlorophyll contents were decreased by 4.5 %, and MDA contents were increased by 3.0 %, as compared to the Al individual treatment. Moreover, the experiment demonstrated that free proline content accumulated in barley plants was a toxic symptom but not a resistance mechanism to Al stress. In conclusion, appropriate concentration of exogenous 6-KT can alleviate growth inhibition and oxidative stress caused by Al toxicity in barley seedlings

Fresh fruit juice has become more and more popular, since it has high nutritional value, thus, more attention is paid to its quality. However, traditional test methods are often time-consuming and cumbersome, can not realize real-time detection and obtain accurate and overall information only with single test results. Electronic nose (e-nose) and electronic tongue (e-tongue), which simulate human nose and tongue system, are new rapid detection instruments. It is found that both e-nose and e-tongue system can achieve rapid detection to get the overall information, with high sensitivity, reliability and repeatability. At present, research in fresh fruit juice using e-nose and e-tongue is still in the preliminary stage, and they have not been found in the fresh strawberry juice evaluation. In this study, an e-nose and an e-tongue were employed to detect the fresh juice of strawberries at different degrees of ripeness. Data were analyzed with three pattern recognition techniques: principal component analysis (PCA), linear discriminant analysis (LDA) and partial least square (PLS). The results showed that the fresh juice of strawberries at different degrees of ripeness could well be discriminated by PCA and LDA, and the result from e-tongue and e-nose was proved to be superior to that of e-nose only for classifying the samples. And the combination of e-tongue and e-nose had the similar level compared with e-tongue (Fig. 7-9). PLS was used to establish the relationship between response signals of e-nose and e-tongue and physicochemical indexes of fresh strawberry juice. Analysis of PLS showed that response signals of e-tongue could predict the value of soluble solids content, Vc content and pH value, but total acid content was recorded with the correction and prediction coefficient (R²) of determination 0.876 and 0.793, respectively (Fig. 10). However, the combination of e-nose and e-tongue with PLS could more efficiently predict soluble solids content, Vc content, total acid content and pH value (Fig. 11), among which the correction and prediction coefficient of determination of total acid content increased to 0.965 and 0.908, respectively. In conclusion, the combination of e-nose and e-tongue reveals better abilities to discriminate and predict fresh juice, which is confirmed to be a general detection for the comprehensive smell and taste information.

Chlorophyll plays an important role in our everyday life. It is used not only as an annexing agent in medicinal and calleidic products but also as an unartificial food coloring agent. Chlorophyll and its ramifications have antioxidant and anti-mutation properties. China has abundant mulberry resources. The mulberry leaf is an excellent Chinese herb from ancient times to the present day and contains many kinds of bioactive components, in which the chlorophyll content is an important physiological index. However, the optimization of chlorophyll extraction from mulberry leaf by response surface methodology (RSM) has not been reported so far. Response surface methodology (RSM) is an effectively statistical technique for optimizing complex processes. The greatest advantage of RSM is that we need fewer experimental trials. It is widely used in optimizing variables of the extraction process, such as polysaccharides, alkaloids, phenolic compounds and protein from various materials. Box-Behnken design (BBD) is one of RSM, which only has three-levels (low, medium and high, coded as −1, 0 and +1), and needs fewer experiments. It’s more efficient and easier to arrange and interpret experiments compared with others. This study focused on the optimal conditions of extracting mulberry leaf chlorophyll using RSM. The mixture of acetone and ethanol was used as extraction solvent. The chlorophyll content was calculated by Arnon’s method. Extraction time, temperature, ratio of liquor to material and volume ratio of acetone to ethanol in extraction buffer were selected as the experiment factors according to the results of single factor experiments. The extracting parameters were optimized by RSM. Under the optimal extraction conditions described above, the confirmatory experiment was conducted. The single factor experiment results indicated that extraction time of 5 h (Fig. 2) , extraction temperature of 50 °C (Fig. 3), ratio of liquor to material of 100∶1 (Fig. 4) and volume ratio of acetone to ethanol of 3∶1 (Fig. 1) were adopted for following experiment. Three independent variables were extraction time (C), extraction temperature (B) and ratio of liquor to material (A), according to the influence∶1. Under the optimal extraction condition, the chlorophyll productivity was 5.376 mg/g, which was in consistent with the predicted value of 5.451 mg/g.s of the four factors on the chlorophyll productivity. The statistical analysis indicated that the three variables (Table 4) and the quadratic of extraction time (C) and ratio of liquor to material (A) (Fig. 6) had significant effects on the yields, and there had the significant interaction effects between the variables of extraction temperature (B) and extraction time (C) (Fig. 7). A high determination co-efficient mathematical model was gained that could be used to optimize chlorophyll extraction. Design Expert 7.1.6 software showed the optimal extraction condition was as follows: extraction time of 5.25 h, extraction temperature of 56.5 °C, ratio of liquor to material of 103 In conclusion, the optimization of chlorophyll extraction by RSM is convenient, feasible and highly efficient

As one of the largest mollisol area in the world, the black soil in northeast of China is commonly thought to be fertile and productive. But recently, dozens of blood heavy metal exceeding events were caused by heavy metal polluted agricultural production through food chain. Soil quality of some farmland near a mining site is getting worse and the content of heavy metal has already exceeded the third level of environmental quality standard for soil in China (GB15618-1995). Crops harvested from these areas had high level of heavy metal and brought a further health problem to human. In order to decrease the adverse effect of heavy metal accumulation in soil, the phytoremediation technology, which is efficient, costless and non-secondary pollution compared with traditional method, has become a hot research point. In many researches, it is a promising way to enhance heavy metal absorption from soil through increasing plants biomass, improving plants resistant to heavy metal and raising the concentration of heavy metal in plants. As many studies proved, inoculation of plant growth-promoting microbes can not only increase plant biomass and nutrition through a number of mechanisms such as nitrogen fixation, production of phytohormones and siderophores, and transformation of nutrient elements, but also can enhance tolerance and accumulation of metals in plants. This experiment was carried out to investigate the effect of bacterium application on the performance of pakchoi (Brassica rapa L. ssp. chinensis) and Pb removal from a contaminated soil. One Pseudomonas aeruginosa strain M2, originally isolated from mining area contaminated with Pb, was examined for its effect on the pakchoi growth, Pb accumulation and rhizospheric microorganism in soil spiked with Pb(NO₃)₂ in a pot experiment. The strain was cultivated in nutrient medium at 30 ℃ for 48 h, whose cells were collected in the exponential phase by centrifugation at 6 000 r/min for 10 min, and were inoculated to soil pretreated with Pb(NO₃)₂ with 10⁸ CFU per gram fresh soil, and the non-inoculated pots were used as controls. The results showed that the biomass of pakchoi was increased by 20.9 % - 31.3 %, and the Pb concentrations in the above- and below-ground plant parts were increased by 15.0 % - 25.4 % and 15.6 % - 36.8 %, respectively, compared with the control pots of non-inoculated strain M2 (Table 1). Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis showed that inoculation with strain M2 changed significantly the microbial community structures of the soils treated with Pb(NO₃)₂ of different concentration (Fig. 1). Moreover, cluster analysis and principal component analysis (PCA) indicated that the number of bacteria, fungi in inoculated soils increased by 17.8 % - 29.6 % and 7.2 % - 15.4 % respectively, however the number of actinomycetes was not significantly affected, as compared with the control under the same Pb(NO₃)₂ concentration (Fig. 2, 3). The DGGE analysis showed that the number of bacterial species in soil increased by 50.0 % - 60.0 % after inoculation with strain M2 (Fig. 1B). It can be concluded from the above findings that P. aeruginosa strain M2 combined with pakchoi not only can remediate Pb polluted soil but also improve the growth of pakchoi by sustaining microbial species diversity.

Fluoride is major raw material of chemical industry and is also the main pernicious composition in industrial wastewater, which leads to serious environmental pollution. Anaerobic biological treatment technology is a kind of low cost wastewater treatment technology which combines wastewater treatment with energy recovery. The prospects for the anaerobic wastewater treatment are extremely promising. However, the bioactivity of anaerobic microorganism, especially the methanogens can be hampered seriously by toxic and harmful substances, such as nitrophenol positional isomers, PCP (pentachlorophenol), polycyclic aromatic hydrocarbon, fluoride-containing effluents, etc. Furthermore, the vailable information about the effects of fluoride-containing effluents on the methane production and biodegradation process of anaerobic microbe in biological wastewater treatment is very limited at present. The purpose of this study is to evaluate the inhibitory effect of fluoride against the main microbial populations expressing in the methanogenic activity and the recovery of the biological activity. To attain this goal, a short-term batch anaerobic toxicity test (ATA) was conducted. The batch test was consisted of five parts, namely, the first, the second and the third anaerobic toxicity assay, as well as the first and the second anaerobic resuming assay, respectively. The results indicated that the inhibitory effects of the low fluoride concentration of 25 mg/L against the activity of methanogenic bacteria were a bit slight. The inhibitory impact was recorded severe when the concentration of fluoride rose from 25 mg/L to 100 mg/L, dropping the methane production from 96 mL to 64 mL. However, the effect was not very obvious at the range of fluoride concentrations from 100 mg/Lto 400 mg/L; as a result, the methane production reduced from 64 mL to 60 mL (Fig. 1A). The inhibitory effects were enhanced during the subsequent addition of fluoride (Fig. 1B, C). The methanogenic activity recovery type was dependent on the dose of fluoride. The recovery type was metabolic at low fluoride concentration (≤ 25 mg/L), while it was physiological at high fluoride concentration (100-400 mg/L) (Fig. 2). The substrate biodegradation was decreased with an increase of fluoride concentration (Table 2, 3). Three main conclusions can be drawn from the results. First, the half inhibitory concentration (IC50) of methanogenic activity is 367.12 mg/L F⁻; second, the anaerobic resuming assay exhibits a considerable difference in the toxic effect of various fluoride concentrations on anaerobic granular sludge; third, the biodegradability of wastewater will be affected when the F⁻ concentration increases to above 100 mg/L.

With the development and progress of society, the problem of water environment pollution has become one of the topics which are cared and taken seriously by people. Higher requirements are brought forward to come up with the frequent and various water pollution events. The current used monitoring systems are difficult to meet the higher requirements referred before. How to monitor water quality effectively and to analyze the concentration of contaminants in water qualitatively during or after water pollution events have become arduous tasks for a water quality monitoring system. As it is known to all, there are lots of monitoring parameters in water quality detection, monitoring and information management system, and the evolution principles and changing trends of water quality are difficult to be obtained. The accuracy of water quality analysis and early-warning is hard to guarantee. Aiming to treat these problems, a new method for a water monitoring system is needed to study on. In this study, the rough set and Dempster-Shafer (D-S) evidence theories were researched, and the water quality analysis and early-warning algorithm based on rough set and D-S evidence theories were presented. The original monitoring water quality data was given (Table 1). The discretization in this paper was based on GB3838-2002, and the result of discretization was also given (Table 3). After discretization, the incomplete data and the conflicting data were deleted (Table 3). Moreover, in order to solve the problem of redundancy parameters, the rough set was used. After these steps, a minimal data list was acquired. Then, the data in the list was recovered to take the place of discrete value. Based on the function of basic probability assignment and the D-S rule of combination, the probability was assigned and the data was fused (Table 4), and the result of data fusion could reflect the water quality or the concentration of contaminants in the water qualitatively. Refer to the national standard, the value of COD_Mn and the water quality in different time points were acquired (Fig. 4, 5). The analysis results which were based on multistage function fitting and rough set theory were also given so as to compare with the result based on rough set and D-S evidence theories (Fig. 2-3). Through the results of analysis, the method based on rough set and D-S evidence theories was proved to be feasible and well. Comparing to the result based on rough set and D-S evidence theories, the result based on multistage function fitting had larger deviation with the passage of time, and the result based on rough set theory had the case which could not be inferred by historical data. In conclusion, the method based on rough set and D-S evidence theories can reduce the influence of historical water data losses, conflicts and redundancy, and overcome the data shortage in practical systems. The reduction of water monitoring parameters which is based on rough set theory also reduces the calculation cost dramatically