Rhizobium inoculation trials designed to support a tropical forage legume selection programme

Summary Three phases of Rhizobium inoculation trials were carried out as part of a programme to select forage legume germplasm adapted to acid, infertile Oxisols of tropical America. Firstly, a range of tropical forage legumes were evaluated for their response to N fertilization or inoculation with strains previously shown to be effective in Leonard jars, using cores of undisturbed soil or in the field at Carimagua, Meta, Colombia. In pure legume stands onlyCentrosema macrocarpum andC. pubescens showed increases in N yield due to both inoculation and N fertilization;C. brasilianum responded only to N fertilization;Zornia latifolia, Z. brasiliensis andStylosanthes capitata responded to neither treatment. Trials in cores and in grass-legume mixtures showed responses ofDesmodium ovalifolium, Pueraria phaseoloides andS. capitata to N fertilization but not to inoculation. In the second phase of experiments strains were screened in soil cores with 16 ecotypes ofDesmodium, Centrosema, Stylosanthes andPueraria spp. Significant increases in N yield due to inoculation occurred with at least one strain in all the legumes exceptS. guianensis tardio, and in some trials withS. capitata. In the third phase of trials the most effective strains were tested in the field. Significant response ofP. phaseoloides andC. macrocarpum to inoculation at two sites and in the second year after establishment were shown. Further screening trials and field trials at different sites are needed in order to provide better recommendations for inoculation of grazing trials being set up in the region under study.     

Management of kudzu by the bioherbicide,Myrothecium verrucaria,herbicides and integrated control programmes

Replicated field plots were established and monitored for two years to evaluate management practices for kudzu. The bioherbicidal plant pathogen, Myrothecium verrucaria, several herbicides and a variety of integrated control programmes achieved a high level of kudzu suppression, although no system tested reliably achieved eradication in this time frame.     

粉葛PtCHI基因的克隆、原核表达和亚细胞定位

为探究葛根品种间异黄酮类物质代谢关键酶基因PtCHI的分子机制差异，并揭示其品种间异黄酮物质含量差异的原因，该研究以野葛品种‘桂葛8号’和粉葛品种‘桂葛1号’为材料，经乙醇提取并通过高效液相色谱仪对野葛和粉葛中葛根素和总黄酮的含量进行测定，基于已报道的野葛CHI基因，通过同源克隆方法分离粉葛中PtCHI基因，并在体外进行蛋白表达，同时在拟南芥原生质体中研究PtCHI基因的定位。结果表明：(1)野葛中的葛根素含量显著高于粉葛的，野葛的总黄酮含量也高于粉葛但未达到显著水平。(2)成功分离到粉葛PtCHI基因，长度为742 bp且包含672 bp完整的ORF框，编码223个氨基酸，与野葛的CHI基因具有99%的同源性。(3)CHI基因在粉葛中的表达量为茎>根>叶子，在野葛中则为根>茎>叶子，除叶子外野葛中CHI基因的表达量均显著高于粉葛。(4)经预测，粉葛PtCHI蛋白为稳定的亲水性蛋白且大小为27.8 kD,二、三级结构以α-螺旋为主，具有25个磷酸化位点，与野葛、大豆和乌拉尔甘草的亲缘关系较近，与F3H2、F3H、4CL4、DFR2及CHS发生互作的可能性较大。(...     

Enzyme‐linked immunosorbent assay by enhanced chemiluminescence detection for the standardization of estrogenic miroestrol in Pueraria candollei Graham ex Benth

Miroestrol (ME) is a potent phytoestrogen from the P. candollei tuberous root. It has been approved for use in clinical trials due to its beneficial effect on disorders associated with estrogen deficiency. To ensure medical efficacy and safety, high performance analytical methods for ME analysis are required to standardize products from the P. candollei root. An enhanced chemiluminescence enzyme‐linked immunosorbent assay (ECL‐ELISA) was developed and validated using a polyclonal antibody against ME and a chemiluminescent system of luminol–H₂O₂–horseradish peroxidase‐4‐(1‐imidazolyl) phenol. The ECL‐ELISA system exhibited linearity over a concentration range of 0.31–10.00 ng mL^?1, for which the relative standard variation (%RSD) was less than 10% for both intra‐ and interplate determinations. The ECL‐ELISA is reliable for the determination of ME as reflected by the high recovery percentage (101.22–103.06%). As a comparative analysis, the ME content in each sample determined by ECL‐ELISA was correlated with high coefficients of determination with colorimetric ELISA (R² = 0.998) and high performance liquid chromatography (HPLC) (R² = 0.998) methods. The ECL‐ELISA method could be applied to all of the commercial products containing P. candollei root, when the products contain between 0.706 ± 0.046 and 13.123 ± 0.794 µg g^?1 dry wt. of ME. This method is useful as a high performance analytical method for the quantity control of ME in raw materials and end products at both the research and industrial levels. Copyright © 2014 John Wiley & Sons, Ltd.     

一株耐酸野生葛藤(Pueraria lobata)根瘤菌的筛选与耐酸机理初步研究

从重庆市北碚区缙云山酸性黄壤(pH4.6)上生长的葛藤根瘤中分离到一株耐酸葛藤根瘤菌PR389,能在pH4.3的YMA培养基上正常生长,而一般根瘤菌最适生长pH值为6.5~7.5,说明PR389为一株耐酸葛藤根瘤菌。通过质子通量试验发现,与不耐酸的菌株相比,PR389的细胞膜能阻止过量的H 进入细胞,表明PR389具有某种能力使之在低酸性环境下不受伤害。在耐酸性试验中,PR389在加氯霉素的强酸性(pH3.8)YMA培养液中表现出来的耐酸性被氯霉素抑制,推测胞内特异蛋白质的诱导合成是PR389具有耐酸性的原因。     

Scale up production of isoflavonoids in cell suspension cultures of Pueraria tuberosa grown in shake flasks and bioreactor

Cell cultures of Pueraria tuberosa were grown in vessels of different sizes and 2L stirred tank bioreactor containing modified MS medium with morphactin (0.1 mg l^?1) and 2iP (5.0 mg l^?1) and 20% inoculum. Stable growth and total isoflavonoid yield of 76.6 mg l^?1 were recorded in the cultures during scale up. This was in concordance with the persistent yield of the individual isoflavonoids regardless of the vessel size.     

粉葛采收与加工的研究

本文利用紫外分光光度法,高压液相色谱法对粉葛总黄酮及5种异黄酮进行跟踪含量测定,对其采收时间与加工方法进行详细的研究。认为采收时间从1月底到2月初为宜,加工方法以不去皮为好。     

广西葛根内生细菌的分离鉴定及其促生特性

【背景】植物内生菌长期与宿主共生,对宿主生长发育产生影响。葛根作为重要的药食两用作物,葛根内生菌的研究具有重要实践意义。【目的】对广西葛根根部内生细菌进行分离、鉴定及促植物生长特性分析,旨在了解该药食同源植物内生细菌种群结构及其促生特性,为分析内生菌群体在药食同源植物产量和品质形成的作用及其内生细菌资源的开发利用提供参考。【方法】采用6种不同的培养基从广西葛根的根瘤、根系和根愈伤组织分离内生细菌,16S rRNA基因测序和系统发育分析内生细菌的分布特征和遗传多样性,采用生理生化方法测定分离菌株的固氮活性、溶磷特性、产生嗜铁素、分泌吲哚乙酸(indole-3-aceticacid,IAA)等促生特性。【结果】从葛根根瘤、根系和根部愈伤组织中共分离得到223个菌株,16S rRNA基因测序鉴定这些菌株隶属于2门4纲10科19属,其中芽孢杆菌属、假单胞菌属、土壤杆菌属、肠杆菌属为葛根优势菌群;内生细菌数量和群落组成存在明显的组织特异性,其数量表现为根瘤>根系>根愈伤组织,但其种群多样性表现为根愈伤组织>根系>根瘤。不同培养基分离出的细菌种群丰富度有差异。从供试菌株中筛...     

中国葛属（Pueraria DC．）的研究

本文对国产葛属植物９种２变种进行了全面整理．对其中的主要类群进行了深入的研究．通过野外考察,腊叶标本的研究和细胞染色体的计数．使对中国葛属植物有一个较为全面的了解。     

The Genetic Transformation of Pueraria as Medicinal Plant by Agrobacterium rhizogenes

Pueraria radix (the dried root of Pueraria plant) is known as a traditional Chinese drug. Hairy roots of Pueraria lobata (Willd.) Ohwi, P.lobata var. montana and P. phaseoloides (Roxb.) Benth. transformed by Agrobacterium rhizogenes (Riker et al .) Conn R1601 were developed directly from the surface of sterile leaves in vitro . The transformation frequency was 16.6%, 16.2% and 26.6%, respectively. All hairy roots in the three species displayed the typical phenotypes of rapid growth, highly branched and plagiotropism, and also exhibited hormone autotrophy and resistance to kanamycin.The genetic transformations were confirmed by opine paper electrophoretic analysis, rol gene PCR amplification and molecular hybridization.