草铵膦对转基因抗草铵膦马铃薯田间杂草的防效及安全性评价

为了解草铵膦对转基因抗草铵膦马铃薯田间杂草的防效及对马铃薯和环境的安全性,本研究在转基因抗草铵膦马铃薯苗期向田间杂草和马铃薯茎叶定向喷施有效成分分别为0(G0)、847.5(G1)、1 271.25(G2)和1 695 g·hm^-2(G3)的草铵膦,系统比较了药后1、4、11和20 d时马铃薯叶片丙二醛(MDA)和脯氨酸(Pro)含量、超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性,药后30和45 d时马铃薯植株的平均株高、茎直径、根茎叶鲜干重和杂草株数,以及成熟期马铃薯块茎营养品质、单株产量、土壤和块茎中草铵膦残留在各处理间的差异。结果表明,药后不同时期,各处理间的马铃薯叶片MDA和Pro含量、SOD和CAT活性均无显著差异。药后30和45 d时,除各草铵膦处理区的根茎叶鲜干重显著高于清水对照外,各处理间的马铃薯株高、茎直径和成熟期块茎营养品质均无显著差异,说明试验剂量的草铵膦对马铃薯生长发育及品质无显著影响。与清水对照相比,各剂量的草铵膦对杂草均有明显的防除效果,且可以明显地提高马铃薯的单株产量,杂草的株、鲜重防效和成熟期马铃薯单株产量在各处理间的差异均依次表现为G3>G2>G1,但在土壤和块茎中均未检测到草铵膦残留。综上,采用有效成分1 695 g·hm^-2草铵膦可以有效防除杂草,对马铃薯安全,且在土壤和马铃薯块茎中未检出草铵膦残留。本研究结果为草铵膦的科学使用提供了理论依据。     

玉米光敏色素A1基因(ZmPHYA1)在棉花中的转化及分子鉴定

为评价玉米ZmPHYA1基因在棉花种质资源改良中的价值,本研究利用农杆菌介导法在陆地棉(Gossypium hirsutum)R15材料中进行了玉米ZmPHYA1基因的遗传转化。经过愈伤组织诱导、抗性愈伤筛选、体细胞分化诱导后获得棉花转基因再生植株。通过田间草铵膦除草剂筛选鉴定抗性植株,并利用PCR扩增其草铵膦抗性基因和目的基因ZmPHYA1进行分子鉴定,发现阳性植株对草铵膦除草剂具较好抗性,并可扩增到256 bp的草铵膦抗性基因和217 bp ZmPHYA1基因的特异条带。进一步通过免疫印迹检测表明,3个不同转基因株系中外源ZmPHYA1基因可正常表达约170 kD大小的蛋白,且在不同组织中该外源蛋白均可正常表达。此外,对转基因植株的不同农艺性状分析表明,转基因株系株高明显低于受体对照,而铃重和纤维长度等性状无明显差异。本研究成功获得具有草铵膦抗性和外源ZmPHYA1基因的棉花新种质材料,为进一步利用光敏色素基因创新种质资源提供了材料来源。     

Assessment of conditions affecting Agrobacterium-mediated soybean transformation using the cotyledonary node explant

Conditions affecting Agrobacterium-mediated transformation of soybean [Glycine max (L.) Merr.], including seed vigor of explant source, selection system, and cocultivation conditions, were investigated. A negative correlation between seed sterilization duration and seed vigor, and a positive correlation between seed vigor and regenerability of explants were observed in the study, suggesting that use of high vigor seed and minimum seed sterilization duration can further improve transformation efficiency. Selection schemes using glufosinate or bialaphos as selective agents in vitro were assessed. Glufosinate selection enhanced soybean transformation as compared to bialaphos. The use of 6 mg L^-1 glufosinate during shoot induction and shoot elongation stages yielded higher final transformation efficiency ranging from 2.0% to 6.3% while bialaphos at 4 to 6 mg L^-1 gave 0% to 2.1% efficiency. Including cysteine and DTT during cocultivation increased the transformation efficiency from 0.2–0.9% to 0.6–2.9%. This treatment also improved T-DNA transfer as indicated by enhanced transient GUS expression. Shoot regeneration and Agrobacterium infection were attained in twelve soybean cultivars belonging to maturity groups I-VI. These cultivars maybe amenable to genetic transformation and may provide a valuable tool in soybean improvement programs. This revised version was published online in July 2006 with corrections to the Cover Date.     

玉米田两种阔叶杂草苍耳和藜对草铵膦敏感性测定

苍耳Xanthium sibiricum和藜Chenopodium ablum是我国北方及黄淮海玉米产区主要杂草，其发生和生长会严重影响玉米产量。为明确我国北方及黄淮海玉米主产区的藜和苍耳对草铵膦的敏感性水平，本试验采用整株生物测定法检测苍耳和藜种群对草铵膦的敏感性，以期为转基因抗草铵膦玉米田杂草防控提供数据支持。结果显示，6个苍耳种群对GR₅₀有效剂量为37.54～111.93 g a.i./ha，平均值为64.25 g a.i./ha，表明苍耳对草铵膦敏感。29个藜种群的GR₅₀有效剂量为24.55～106.75 g a.i./ha，平均值为60.43 g a.i./ha，GR₅₀值均低于405 g a.i./ha（田间推荐高剂量的1/2X剂量），表明藜对草铵膦也很敏感。采自北方玉米产区的藜对草铵膦的GR₅₀均值为66.93 g a.i./ha，高于采自黄淮海玉米产区的53.47 g a.i./ha。     

抗草丁膦转基因玉米不同DNA提取方法及PCR检测

以改良PEX,CTAB和SDS方法从转基因玉米材料中提取基因组DNA,并以外源CaMV35S启动子、bar基因及内源基因Zein的序列设计引物,用PCR方法扩增预期大小的DNA片段。结果显示,3种方法提取的DNA用以PCR检测都获得了预期大小的扩增片段,但改良PEX提取方法更省时省力。研究认为,改良PEX提取方法是一种简捷、快速和高效的植物DNA提取方法。     

Phytotoxic Effects of Residual Glufosinate on the Nitrogen Assimilation of Transplanted Rapeseed Seedlings

Herbicide application is an efficient method to control weed growth in modern agriculture production, but there is concern about the ecological impact of unwanted herbicide residues in the soil. Rapeseed varieties ZS11 and D148 were used to evaluate the phytotoxic effects of residual glufosinate on the assimilation of nitrogen (N) in rapeseed seedlings transplanted to untreated [0 g hm^?2 glufosinate] or treated [450 g hm^?2 and 900 g hm^?2 glufosinate] soils. Glutamine synthetase (GS) and glutamate dehydrogenase (GDH) activities, the contents of ammonium (NH₄⁺), free amino acids (FAA), and soluble protein (SP), and seedling dry weight (DW) were determined at 5, 8, 11, 20, 40, and 70 d post-transplant. Both concentrations of glufosinate induced physiological phytotoxicity on the N assimilation of transplanted seedlings of both varieties, as their leaves and roots presented reduced GS activities and SP contents, and increased GDH activities, and NH₄⁺ and FAA content. Glufosinate phytotoxicity on the N assimilation of transplanted seedlings reached a plateau at 11 to 20 d. further, GDH in roots and GS in leaves were still significantly different at 70 d. Meanwhile, ZS11 might be more sensitive to glufosinate than D148 since ZS11 had more variation than D148 at the same treatment, and the overdose of glufosinate more strongly inhibited N assimilation than the recommended dose. Therefore, it is essential to apply a suitable glufosinate dose to the transplanted variety, to minimize adverse effects on crops and the environment.

Abbreviations: N, Nitrogen; GS, Glutamine synthetase; GDH, glutamate dehydrogenase; NH₄⁺, ammonium; FAA, free amino acids; SP, soluble proteins; DW, Dry weight; ANOVA, one-way analysis of variance; NO₃^?, nitrate; OECD, Organisation for Economic Co-operation and Development; PPT, phosphinothricin; USEPA, United States Environmental Protection Agency     

The mode of action of glufosinate in algae: The role of uptake and nitrogen assimilation pathways

The purpose of this work was to investigate the in-vivo mode of action of glufosinate in comparison with that of the known glutamine synthetase inhibitor, methionine sulfoxmine, in photoautotrophic microorganisms. Using the eukaryotic green alga Chlorella fusca (Shih. & Krauss) and the cyanobacterium Anacystis nidulans (Kratz & Myers), currently developed [¹⁵N]NMR pulselabelling techniques have been employed to study the inhibition of ammonia assimilation. The results show that, while methionine sulfoximine immediately blocks glutamine synthesis, glufosinate action requires an induction process in both organisms investigated, possibly indicating the de-novo synthesis of an amino acid membrane carrier. In addition, the observed ability of C. fusca to incorporate nitrogen into glutamate under glutamine synthetase-inhibiting conditions is explicable by an anabolic function of a glutamate dehydrogenase in this organism. This might explain the large differences in observed species sensitivity to glufosinate exposure.     

Distribution and metabolism of D/L-, L- and D-glufosinate in transgenic, glufosinate-tolerant crops of maize (Zea mays L ssp mays) and oilseed rape (Brassica napus L var napus)

The aim of the present study was to determine whether post-emergence application of glufosinate to transgenic crops could lead to an increase in residues or to the formation of new, hitherto unknown metabolites. Transgenic oilseed rape and maize plants were treated separately with L-glufosinate, D-glufosinate or the racemic mixture. Whereas about 90% of the applied D-glufosinate was washed off by rain and only 5-6% was metabolised, 13-35% of the applied L-glufosinate remained in the form of metabolites and unchanged herbicide in both transgenic maize and oilseed rape. The main metabolite was N-acetyl-L-glufosinate with total residues of 91% in oilseed rape and 67% in maize, together with small amounts, of 5% in oilseed rape and 28% in maize, of different methylphosphinyl fatty acids. These metabolites were probably formed from L-glufosinate by deamination and subsequent decarboxylation. The residues were distributed in all fractions of the plants, with the highest contents in treated leaves and the lowest in the grains (0.07-0.3% in maize and 0.4-0.6% in oilseed rape). There was no indication of an accumulation of total residues or of residue levels above the official tolerances for glufosinate.     

Weed Control in Sugar Beet using Genetically Modified Herbicide-tolerant Varieties – A Review of the Economics for Cultivation in Europe

Genetically modified sugar beet varieties have been developed to be tolerant to glyphosate and glufosinate. To date, research regarding other active ingredients did not result in additional herbicide‐tolerant varieties and the approval of glufosinate‐tolerant varieties for market access has been withdrawn by the applicant. Therefore, only glyphosate‐tolerant varieties could be introduced for cultivation in the short run. Results concerning efficacy and cost of weed control using these varieties and the complementary herbicides were extensively reported in various contributions, mostly on a national level. Based on these results, the economics of weed control for sugar beet production in Europe were reviewed and aspects of integrated control, risk management, and issues of sustainable development of crop production are discussed. Efficient weed control is possible in almost any field situation with glyphosate at about 2 kg a.i. ha^?1, compared with conventional herbicides at 6 kg ha^?1 or higher, depending on weed infestation level. Cost savings for weed control with glyphosate would amount to an average of €150 ha^?1, without any great deviation across different sites and states. A technology fee of about €40 ha^?1 is assumed. The high selectivity of glyphosate may result in a 1–3 % higher yield performance of the crop. All assumptions being considered, total cost savings of €180 × 10⁶ year^?1 were calculated for the area of 1.7 × 10⁶ ha in the main EU sugar beet‐growing countries. A risk management by implementing a monitoring programme is compulsory, and systems of identity preservation or quality assurance are needed in order to enable the production of conventional and genetically modified sugar beet in coexistence. To date, costs are unknown for these measures. Because of the favourable ecotoxicological behaviour of glyphosate and the possibility of threshold‐based weed control, this new technology could provide an excellent option towards sustainable development of the crop. However, political reasons and the lack of acceptance of genetically modified varieties by the consumer have prevented the market entry of GMHT sugar beet to date, so that conventional herbicides will continuously be used in the future.     

彗星电泳检测草胺磷对蚯蚓体腔细胞 DNA 的损伤

【目的】探讨草胺磷对土壤动物的影响,以蚯蚓为受试动物,在实验室环境下,采用彗星电泳技术检测不同浓度草胺磷胁迫对蚯蚓体腔细胞DNA损伤的程度。【方法】草胺磷质量浓度设定为0、37.5、75、150、300 mg/L,胁迫时间24 h,取头部DNA含量、尾部DNA含量、尾长、尾矩、Olive尾矩5个参数分析DNA损伤程度。【结果】电泳结果显示,与空白对照比较,5个参数在4个草胺磷处理中都有显著差异;随草胺磷浓度升高,尾部DNA含量、尾长、尾矩、Olive尾矩随之升高,头部DNA含量随之降低,草胺磷胁迫引起了蚯蚓体腔细胞DNA的损伤,草胺磷浓度越高,DNA损伤越严重。随后降低草胺磷浓度,延长胁迫时间至96 h,得到相似结果。【结论】草胺磷会引起土壤动物蚯蚓体腔细胞DNA的损伤,草胺磷浓度与蚯蚓体腔细胞DNA损伤程度两者间存在显著的剂量-效应关系。