N2-fixation by ammonium-excreting Azospirillum brasilense in auxine-induced root tumours of wheat (Triticum aestivum L.)

Summary Wheat seedlings, treated with the auxine 2,4-dichlor-phenoxy acetic acid (2,4-D) during germination developed only a residual root system. Root elongation was extremely restricted and root tips were deformed to thick club-shaped tumours. When 2,4-D was added in a later stage of plant growth the plants developed additional nodule-like knots along primary roots. Root and shoot dry-matter production was slightly repressed in all 2,4-D treatments and N translocation from roots to shoots was repressed as well. When transferred to an auxine-free growth medium, the 2,4-D-affected roots were not capable of complete recovery. In plants inoculated gnotobiotically with Azospirillum brasilense, either with the wild type or with the NH ₄ ⁺ -excreting mutant strain C3, a 2,4-D addition increased rhizosphere acetylene-reduction activity at pO₂ 1.5 kPa. The O₂ sensitivity of root-associated nitrogenase activity tended to be reduced. The number of root-colonizing bacteria, at approximately 10⁸ colony-forming units (cfu) per g dry root, was similar in the 2,4-D treatments and untreated controls. Plant treatment with high concentrations of the chemical isomer 3,5-dichlor-phenoxy acetic acid (3,5-D) did not have comparable effects, either on plant development or on rhizosphere-associated nitrogenase activity. Root-tumour tissue inhabited by A. brasilense showed purple staining when subjected to a tetrazolium chloride solution, which may indicate intensive local nitrogenase activity in this tissue. Exposed to an ¹⁵N₂-enriched atmosphere, plants treated with 2,4-D and with A. brasilense incorporated significantly higher amounts of ¹⁵N than untreated controls. In all cases the highest values of ¹⁵N enrichment were found following inoculation with the NH ₄ ⁺ -excreting mutant strain C3. Present address: F. A. Janssens Laboratory of Genetics, Catholic University of Leuven, Willem de Croylan 42, B-3001 Heverlee, Belgium     

Measurement of phytase activity using tethered phytic acid as an artificial substrate: Methods development

The artificial chromophoric substrate analog of phytic acid, 5-O-[6-(benzoylamino)hexyl]-d-myo-inositol-1,2,3,4,6-pentakisphosphate (T-IP₅), may prove useful in measuring soil phytase activity. This chemical probe allows for direct measurement of phytase-catalyzed dephosphorylation (i.e., hydrolysis of the phosphoester bond) using high-performance liquid chromatography with UV detection. Before T-IP₅ can be used to measure phytase activity in environmental samples (soil, stream sediment, manure) refinement of the T-IP₅ probe methodology is required. Using ³¹P nuclear magnetic resonance (NMR) spectroscopy, we identified 5-O-[6-(benzoylamino)hexyl]-d-myo-inositol-trisphosphate (T-IP₃) as the key intermediate that accumulates during phytase-catalyzed dephosphorylation of T-IP₅. An improved HPLC method for separation of reactants is also presented.     

Quantitative effects of field water deficits on N2(C2H2) fixation in selected legumes grown in the Indian desert

Summary The nitrogenase activity of irrigated and rainfed plants of mung bean, cluster bean and moth bean was studied throughout the growth period in order to estimate the reduction in the potential nitrogen fixation (C₂H₂ reduction) rate due to field water deficits. Nitrogenase activity followed a similar trend in all crops and was dependent on both plant ontogeny and soil moisture levels. The loss of activity due to water deficits varied from 13% to 100% at different growth stages and was related to the plant water potential. The specific activity was directly correlated with the plant water potential under both the treatments. The average loss of nitrogen fixation rate during the season did not differ markedly among crops. There was an accumulation of ureides in the nodules with increasing field moisture stress in mung bean and moth bean while no such effect was found in cluster bean. The significance of these results is discussed in the N-economy of these legumes grown in the drought-prone areas of the Indian desert.     

Differentiation between bioavailable and total hazard potential of sediment-induced DNA fragmentation as measured by the comet assay with Zebrafish embryos

Goals, Scope and Background  While water quality strongly improved over decades in the Rhine River, sediments still reflect elapsed contaminations of organic pollutants and heavy metals. In comparing genotoxic effects induced by both sediment extracts and whole sediments, a ratio of bioavailable toxicity and total extractable toxicity is obtained. Since contaminated sites whose contaminants are toxic and as well bioavailable present an elevated risk to the ecosystem, such ratios may be used as a warning signal to identify sites of primary concern. Methods  Accordingly, two different exposure scenarios were compared to reveal the genotoxic potential of 18 sediment samples derived from 9 sample sites along the River Rhine. For assessment of effects on genome integrity, DNA fragmentation was measured using the comet assay with primary cells isolated from zebrafish embryos previously exposed to either organic sediment extracts or freeze-dried sediments at sublethal concentrations. Additionally, chemical data were used to determine responsible pollutants and correlate them with biological effects. Results  Whereas 17 out of 18 sediment extracts caused significant DNA damage to the embryo cells, only 4 native sediments showed a genotoxic potential. Thus, under field-like exposure conditions, a major part of potentially genotoxic compounds seem to remain particle-bound and ineffective, as shown for whole sediment exposure. Conversely, the organic extracts seem to contain enriched concentrations even of hardly soluble substances. Hence, organic extracts may be used as a screening tool to address potentially polluted sites, even though the relevance of these results for the field situation may be questionable. Investigations on native sediments determined few sites with bioavailable and therefore ecologically most relevant genotoxic sediment compounds. Discussion  However, these results may underestimate the total hazard potential of sample sites with hardly bioavailable substances. Chemical data revealed a variety of anthropogenic pollutants, ranging from PAHs to heavy metals. Nevertheless, chemical data on the measured priority pollutants did not fully explain the pollution pattern of the bioassays but clearly determined substances of concern (e.g., HCB, heavy metals) in particular sample sites. Conclusions  There is a striking advantage in assessing the genotoxicity by means of different exposure scenarios that focus on either bioavailable or extractable fractions, as the combination of the results allows obtaining information on specific properties of the genotoxicants and their bioavailability. An additional correlation with chemical data should be required to identify priority pollutants, as long as the responsible contaminant is known a priori. As many studies revealed inherent failures of such a correlation, an effect-driven analysis of pollutants is recommended as a promising tool to identify even non-priority pollutants by means of their ecotoxicological effectiveness.     

抗猪瘟兔化弱毒单克隆抗体杂交瘤细胞株的建立

用猪瘟兔化弱毒（ＳＦＶ—Ｃ）牛睾丸细胞培养上清液，经ＰＥＧ—２００００沉淀和蔗糖密度梯度离心提纯制备ＳＦＶ—Ｃ抗原，腹腔免疫Ｂａｌｂ／ｃ小鼠，取抗体阳性的免疫小鼠脾细胞，在５０％（Ｗ／Ｖ）ＰＥＧ—４０００作用下，与ＳＰ２／０－Ａｇ１４小鼠骨髓瘤细胞进行融合，产生杂交瘤细胞。用间接ＥＬＩＳＡ法筛选阳性杂交瘤细胞株。通过有限稀释进行３次克隆，获得了２株分泌抗ＳＦＶ—Ｃ的单克隆抗体杂交瘤细胞株，并对其染色体形态和数目进行分析鉴定。     

草莓伪温和黄边病毒抗血清制备及血清学检测技术研究

将提纯的草莓伪温和黄边病毒（ＳｔｒａｗｂｅｒｒｙｐｓｅｕｄｏｍｉｌｄｙｅｌｌｏｗｅｄｇｅｖｉｒｕｓＳＰＭＹＥＶ）作抗原免疫家兔，获得效价较高的抗血清，经ＳＤＳ－对流免疫电泳测其效价为１：１２８．四点疫吸附固定法检测ＳＰＭＹＥＶ效果甚理想，可检测到微量病素，提纯病毒浓度低至０．０５μｇ／ｍｌ仍有效果．酶联免疫吸附测定法检出灵敏度也相当高，当病毒浓度为０．１μｇ／ｍｌ时及病叶汁液稀释至１０２４倍时，仍呈阳性反应．免疫电镜技术检视，ＳＰＭＹＥＶ抗血清能清晰地”捕获”同源病毒，并且有装饰作用，抗血清以稀释为１：５００时“捕获”病毒粒子最多．     

基因枪转化甘蔗愈伤组织体系的优化

以生产上广泛应用的甘蔗商业品种新台糖16号的愈伤组织为受体材料,以含有GUS基因的植物表达载体pBI121为外源DNA,利用基因枪PDS 1000/He,设定不同的参数,将pBI121轰击新台糖16号愈伤组织.经GUS染色,结果发现在发射距离1/4in、可裂膜压力1100psi、轰击距离6cm时转化效率最高.     

荧光定糖法测定纤维素酶活力的条件研究

研究结果表明 ,荧光定糖法测定纤维素酶活力的反应最佳条件 :反应液浓度为46.7mg·g-1,反应时间为10min,反应温度为150℃。将荧光定糖法与滤纸酶活测定法相结合应用于纤维素酶蛋白组分及土壤纤维素酶活力的研究与测定。