Screening genetically modified organisms using multiplex-PCR coupled with oligonucleotide microarray

In this research, we developed a multiplex polymerase chain reaction (multiplex-PCR) coupled with a DNA microarray system simultaneously aiming at many targets in a consecutive reaction to detect a genetically modified organism (GMO). There are a total of 20 probes for detecting a GMO in a DNA microarray which can be classified into three categories according to their purpose: the first for screening GMO from un-transgenic plants based on the common elements such as promoter, reporter and terminator genes; the second for specific gene confirmation based on the target gene sequences such as herbicide-resistance or insect-resistance genes; the third for species-specific genes which the sequences are unique for different plant species. To ensure the reliability of this method, different kinds of positive and negative controls were used in DNA microarray. Commercial GM soybean, maize, rapeseed and cotton were identified by means of this method and further confirmed by PCR analysis and sequencing. The results indicate that this method discriminates between the GMOs very quickly and in a cost-saving and more time efficient way. It can detect more than 95% of currently commercial GMO plants and the limits of detection are 0.5% for soybean and 1% for maize. This method is proved to be a new method for routine analysis of GMOs.     

High-efficiency transformation by biolistics of soybean, common bean and cotton transgenic plants

This protocol describes a method for high-frequency recovery of transgenic soybean, bean and cotton plants, by combining resistance to the herbicide imazapyr as a selectable marker, multiple shoot induction from embryonic axes of mature seeds and biolistics techniques. This protocol involves the following stages: plasmid design, preparation of soybean, common bean and cotton apical meristems for bombardment, microparticle-coated DNA bombardment of apical meristems and in vitro culture and selection of transgenic plants. The average frequencies (the total number of fertile transgenic plants divided by the total number of bombarded embryonic axes) of producing germline transgenic soybean and bean and cotton plants using this protocol are 9, 2.7 and 0.55%, respectively. This protocol is suitable for studies of gene function as well as the production of transgenic cultivars carrying different traits for breeding programs. This protocol can be completed in 7-10 months.     

Revisiting the Brazilian scenario of registry and protection of cultivars: an analysis of the period from 1998 to 2010, its dynamics and legal observations

During the last 20 years, the national production of grains has increased 156.1%; productivity increased 93.8% and there has been an increase of 29.1% in cultivated area. Currently, agribusiness is responsible for 40% of Brazilian exports. Nevertheless, there is little quantitative information on the main plant species of economic interest that have been registered and protected in the Agriculture, Fisheries and Food Supply Ministry (MAPA) by public and private companies, as well as by public-private partnerships. Consequently, we investigated the registry and protection of 27 species of economic interest, including the 15 that are the basis of the Brazilian diet, based on the information available on the site CultivarWeb, of MAPA, for the period from 1998 to August 30, 2010. We also examined the legislation that regulates registration and protection procedures and its implications for plant breeding and plant product development. It was found that the private sector controls 73.1% of the registrations and 53.56% of the protections, while 10.73% of the protections were of material developed overseas. Public-private partnerships contributed little to the development of new cultivars, with 0.5% of the registries and 3.61% of the protections. We conclude that plant protection directed private investment to development of wheat and rice varieties, with the greatest public investments directed to corn and sorghum. After the Cultivar Protection Law was implemented, there was restriction of access to germplasm banks, which could inhibit advances in Brazilian plant breeding programs, indicating a need for revision of this legal barrier.     

转基因棉花中糖类和游离氨基酸含量的变化对棉蚜泌蜜量及蜜露主要成分的影响

以3个转基因棉和2个亲本对照棉花品种为研究材料,分别测定了这5种棉花植株体内主要糖分与游离氨基酸含量;同时,分别用这5个棉花品种的叶片饲养棉蚜Aphis gossypii Glover并测定其蜜露分泌量及其主要营养成分。结果表明,转基因棉花“国抗22”叶片中葡萄糖、蔗糖、麦芽糖的平均含量及可溶性糖总量分别比亲本对照棉“泗棉3号”减少61.76%、 89.05%、77.86%和23.61%,转基因棉花“苏抗103”和“中抗310”叶片中葡萄糖、蔗糖、麦芽糖的平均含量及可溶性糖总量分别比亲本对照棉“苏棉12”下降14.15%、32.80%、92.22%、11.46% 和4 6.81%、93.19%、61.11%、43.91%,游离氨基酸总量及各种氨基酸、果糖、鼠李糖、海藻糖的含量在不同转基因棉与亲本对照棉花品种间也存在很大差异,其中一些处理间的差异达显著或极显著水平。这表明外源基因的导入已经影响到了转基因棉花品种中主要糖分与游离氨基酸的合成。棉蚜取食转基因棉花品种“国抗22”后,蜜露的日平均分泌量比取食对照品种“泗棉3号”减少40.54%,取食其他两个转基因棉花品种“苏抗103”和“中抗310”后蜜露的分泌量也比取食对照棉花品种“苏棉12”降低22.67%和30.0%,但棉蚜取食转基因棉花后蜜露中游离氨基酸的总量均高于对照棉花品种,蜜露中可溶性总糖、蔗糖和各种氨基酸含量在取食转基因棉和常规棉花品种间存在一定差异。     

Detecting un-authorized genetically modified organisms (GMOs) and derived materials

Genetically modified plants, in the following referred to as genetically modified organisms or GMOs, have been commercially grown for almost two decades. In 2010 approximately 10% of the total global crop acreage was planted with GMOs (James, 2011). More than 30 countries have been growing commercial GMOs, and many more have performed field trials. Although the majority of commercial GMOs both in terms of acreage and specific events belong to the four species: soybean, maize, cotton and rapeseed, there are another 20 + species where GMOs are commercialized or in the pipeline for commercialization. The number of GMOs cultivated in field trials or for commercial production has constantly increased during this time period. So have the number of species, the number of countries involved, the diversity of novel (added) genetic elements and the global trade. All of these factors contribute to the increasing complexity of detecting and correctly identifying GMO derived material. Many jurisdictions, including the European Union (EU), legally distinguish between authorized (and therefore legal) and un-authorized (and therefore illegal) GMOs. Information about the developments, field trials, authorizations, cultivation, trade and observations made in the official GMO control laboratories in different countries around the world is often limited, despite several attempts such as the OECD BioTrack for voluntary dissemination of data. This lack of information inevitably makes it challenging to detect and identify GMOs, especially the un-authorized GMOs. The present paper reviews the state of the art technologies and approaches in light of coverage, practicability, sensitivity and limitations. Emphasis is put on exemplifying practical detection of un-authorized GMOs. Although this paper has a European (EU) bias when examples are given, the contents have global relevance.     

主要农作物转基因研究现状和展望

近15年来,大豆、水稻、玉米、小麦等主要农作物转基因研究取得了较大进展,几乎各种遗传转化方法在这些作物上都取得了成功,尤其是农杆菌介导法,不仅在难转化的双子叶作物大豆上取得了成功,而且在单子叶作物水稻、玉米、小麦上先后取得了突破。同时,将一些与重要性状改良有关的外源基因转入了主要农作物,包括抗虫、抗病、抗除草剂、抗逆、品质改良、发育调控、营养吸收等。转基因大豆、玉米、棉花、油菜在生产上得到了大面积种植,产生了极大的经济效益,2004年全球转基因作物的种植面积达到了8100万公顷。本文对大豆、玉米、水稻和小麦等主要农作物转基因研究历史和产业化现状进行了综述,并对主要农作物转基因研究中存在的问题进行了分析。     

Dynamics and seasonal abundance of stink bugs (Heteroptera: Pentatomidae) in a cotton-soybean ecosystem

Two soybean varieties (early-maturing group V and late-maturing group VII) and two cotton varieties (conventional and transgenic (Bt) were grown in adjacent replicated large field plots (approximately 0.1 ha each) at two locations for 3 yr. The dynamics and relative abundance of phytophagous stink bugs within these two crops were observed. The most abundant pentatomid species in both crops for all 3 yr were Nezara viridula (L.), Acrosternum hilare (Say), and Euschistus servus (Say). Several other species also were commonly collected. This is the first record of Mormidea lugens (F.) on soybean and E. quadrator Rolston, E. obscurus (Palisot), Holcostethus limbolarius (St?l), and Oebalus pugnax (F.) on cotton. Stink bugs began arriving in soybean when plant growth stages ranged from pod formation to full seed development. Peak numbers of these insects were found in soybean from the time of full-size seeds in the pods until early maturity. The bugs were first attracted to the earlier maturing cultivar (group V), where they remained until plants began to mature (R7). The pentatomids then moved to the later-maturing cultivar (group VII) as it reached full pod to full seed. Stink bugs began arriving in cotton from the time of the earliest flowers until after the first bolls formed. Peak numbers in cotton occurred during the time when all stages of developing bolls were present. Stink bug numbers were much greater in soybean than in cotton over all three seasons. This preference for soybean over cotton indicates the potential use of soybean as a trap crop for attracting stink bugs away from cotton. Additionally, the coordinated use of early- and late-maturing soybean cultivars as a trap crop could minimize the area requiring insecticides, as well as the number of insecticide applications to cotton.     

Debate on GMOs health risks after statistical findings in regulatory tests

We summarize the major points of international debate on health risk studies for the main commercialized edible GMOs. These GMOs are soy, maize and oilseed rape designed to contain new pesticide residues since they have been modified to be herbicide-tolerant (mostly to Roundup) or to produce mutated Bt toxins. The debated alimentary chronic risks may come from unpredictable insertional mutagenesis effects, metabolic effects, or from the new pesticide residues. The most detailed regulatory tests on the GMOs are three-month long feeding trials of laboratory rats, which are biochemically assessed. The tests are not compulsory, and are not independently conducted. The test data and the corresponding results are kept in secret by the companies. Our previous analyses of regulatory raw data at these levels, taking the representative examples of three GM maize NK 603, MON 810, and MON 863 led us to conclude that hepatorenal toxicities were possible, and that longer testing was necessary. Our study was criticized by the company developing the GMOs in question and the regulatory bodies, mainly on the divergent biological interpretations of statistically significant biochemical and physiological effects. We present the scientific reasons for the crucially different biological interpretations and also highlight the shortcomings in the experimental protocols designed by the company. The debate implies an enormous responsibility towards public health and is essential due to nonexistent traceability or epidemiological studies in the GMO-producing countries.     

寄主植物对桃蛀螟生长发育及产卵选择行为的影响

为明确寄主植物对桃蛀螟生长发育及产卵选择行为的影响,利用实验种群生命表和二项产卵选择试验,研究了玉米、大豆、棉花和桃等4种寄主植物对桃蛀螟种群生长发育及产卵选择性的影响。结果表明:取食棉花的桃蛀螟幼虫存活率最低、幼虫历期最长,取食玉米的幼虫存活率最高、幼虫历期最短,取食桃和大豆的幼虫存活率和历期居于棉花处理组和玉米处理组之间;玉米处理组的桃蛀螟化蛹率、蛹重和蛹历期均为最高,棉花处理组为最低,大豆和桃处理组的这些参数均显著小于玉米处理组而大于棉花处理组;发育至成虫后,取食玉米的桃蛀螟羽化率显著高于其他3个处理组;取食桃的桃蛀螟成虫寿命(雌虫和雄虫)及个体发育历期均显著高于其他3种处理组;同时取食桃的桃蛀螟单雌产卵量最高,其次是玉米处理组,两者均显著高于大豆和棉花处理组。二项产卵选择试验结果显示,桃蛀螟雌蛾在棉花和玉米处理组、玉米和大豆处理组或棉花和大豆处理组间的落卵量差异不显著;但在包含桃的处理组中,桃蛀螟在棉花、玉米或大豆处理区的落卵量均显著高于桃处理区。上述结果表明,供试4种寄主植物中,桃蛀螟偏好在棉花、玉米和大豆上产卵,其中玉米对桃蛀螟的适合度相对较高,棉花对桃蛀螟的适合度相对较低...     

Development of melting temperature-based SYBR Green I polymerase chain reaction methods for multiplex genetically modified organism detection

Commercialization of several genetically modified crops has been approved worldwide to date. Uniplex polymerase chain reaction (PCR)-based methods to identify these different insertion events have been developed, but their use in the analysis of all commercially available genetically modified organisms (GMOs) is becoming progressively insufficient. These methods require a large number of assays to detect all possible GMOs present in the sample and thereby the development of multiplex PCR systems using combined probes and primers targeted to sequences specific to various GMOs is needed for detection of this increasing number of GMOs. Here we report on the development of a multiplex real-time PCR suitable for multiple GMO identification, based on the intercalating dye SYBR Green I and the analysis of the melting curves of the amplified products. Using this method, different amplification products specific for Maximizer 176, Bt11, MON810, and GA21 maize and for GTS 40-3-2 soybean were obtained and identified by their specific Tm. We have combined amplification of these products in a number of multiplex reactions and show the suitability of the methods for identification of GMOs with a sensitivity of 0.1% in duplex reactions. The described methods offer an economic and simple alternative to real-time PCR systems based on sequence-specific probes (i.e., TaqMan chemistry). These methods can be used as selection tests and further optimized for uniplex GMO quantification.     

Pod and Seed Mycoflora on Transgenic and Conventional Soybean [Glycine max (L.) Merrill] cultivars in Mississippi

A 2-year (1999-2000) study was conducted at Starkville and Stoneville, MS to determine if the occurrence of the mycoflora varied on Roundup Ready (transgenic) compared to conventional soybean (Glycine max) cultivars. A total of 7,658 fungal isolates were identified from the pod and seed tissues of four cultivars compared at growth stages R6 and R8. Ninety-nine percent of all fungi isolated were mitosporic fungi and ascomycetes. In both years, total fungal isolates from the two locations were greater from the pod (65%) than from seed (33%) tissues. Isolation frequency from conventional cultivars was 54% compared to 46% for the transgenic cultivars. The most common fungi identified that are reported pathogens of soybean included Alternaria, Cercospora, Cladosporium, Diaporthe, Fusarium and Verticillium spp. When main effects and interactions were compared among the frequency data for the fungal genera, significant differences occurred, but consistent trends were not noted. Isolation frequencies of Diaporthe spp. during the R6 growth stage, were significantly greater on the conventional than on the transgenic cultivars in both years of the study, but only at Starkville. Isolation frequencies from samples taken during the R8 growth stage were similar at both locations in 1999 and 2000. Fusarium spp. isolated at R6 and R8 growth stages from pod and seed tissues were significantly greater on conventional than on transgenic cultivars in 2000. Even though frequencies were often significantly different between the transgenic and conventional cultivars, the data was not consistent between locations, pod and seed tissues, or growth stages. The pod and seed mycoflora of transgenic and conventional soybean cultivars was, therefore, similar in Mississippi.     

我国转基因抗虫棉应用现状及建议

对１９９９年底前经过安全性评价或经过审定的转基因抗虫棉品种２３个以及未经评价或审定的品种３１个进行了调研。转基因抗虫棉发展迅速,１９９９年种植面积近９００万亩,在生产上总体表现良好,棉铃虫抗性强,减少化学农药治虫,受到了广大棉农的欢迎。但抗虫棉本身还有一些不足之处,种子管理及生产利用上也存在一些问题,针对当前抗虫棉的情况和发展要求,对我国转基因抗虫棉的发展提出了几点建议。     

外源基因导入对玉米叶片物理性状的影响

【目的】探讨外源基因导入对玉米叶片物理性状的影响,为转基因玉米的安全性评价提供基础资料,也为转基因玉米的科学、有效利用提供依据。【方法】在扬州大学实验农牧场种植大北农转基因(转Cry Ab和epsps基因)和大北农(对照)、IE09S034转基因(转Cry IE基因)和IE09S034(对照)、808-双抗-12-5转基因(转Cry Ab/cry2Aj和Gloevo-epsps基因)和808瑞丰-1(对照)3对玉米品种,室内测定了不同时期(苗期、穂期和花粒期)各品种叶片的蜡质含量、叶绿素含量、茸毛密度、维管束埋深及Si、K、Ca、S、P和Cl含量。【结果】转基因玉米的叶片中蜡质含量、叶绿素含量和维管束埋深较对应的常规亲本品种大,而叶片茸毛密度则较对应的常规亲本品种小。其中,穗期的大北农、IE09S034和808-双抗-12-5转基因品种叶片蜡质含量分别较对照高17.95%、48.30%和39.31%;IE09S034和808-双抗-12-5转基因品种穗期叶片的维管束埋深分别较对照高13.70%和9.21%,花粒期分别高10.81%和14.47%;IE09S034和808-双抗-12-5转基因品种穗期叶片的叶绿素含量分别较对照高18.11%和13.13%,花粒期分别高16.62%和14.61%;大北农、IE09S034和808-双抗-12-5转基因品种花粒期叶片茸毛密度分别较对照低17.70%、17.43%和17.78%。3个品种的转基因玉米叶表面元素含量均大于相应的对照。其中,与常规亲本相比,穗期大北农转基因品种叶片中Ca和S含量分别高64.71%和61.18%,IE09S034转基因品种叶片中Si、Ca、S、P和Cl含量分别高110.26%、16.67%、44.44%、46.32%和20.00%,808-双抗-12-5转基因品种叶片中Si、Ca、S和P含量分别高34.78%、50.52%、115.47%和20.41%。【结论】外源基因的导入会诱导玉米叶片相关物理性状的改变。     

山西市场动物饲料中转基因成分的检测

为了评估转基因玉米和大豆在山西动物饲料市场的占有率和标识情况,采用改良十六烷基三甲基溴化铵法 (Hexadecyltrimethy ammonium bromide, CTAB) 提取山西市场抽取的30份鸡和猪饲料,通过定性PCR打包筛查,对检测阳性结果打包饲料拆包并检测CaMV 35S启动子、NOS终止子、玉米内标zSSIIb、大豆内标Lectin和CryIA (b)基因。同时检测玉米和大豆转化体事件MON810和GTS40-3-2。结果表明,83.3%的饲料含有转基因成分。所抽取的玉米、大豆、猪饲料和鸡饲料转基因成分阳性率分别为6.67%、100%、93.3%和73.3%。实时荧光定量PCR检测结果与定性PCR一致。结果提示,鸡和猪饲料中转基因成分在山西市场的占有率较高。     

Effects of transgenic soybean on growth and phosphorus acquisition in mixed culture system

Transgenic soybean plants overexpressing the Arabidopsis purple acid phosphatase gene AtPAP15 (OXp) or the soybean expansin gene GmEXPB2 (OXe) can improve phosphorous (P) efficiency in pure culture by increasing Apase secretion or changing root morphology. In this study, soybean‐soybean mixed cultures were employed to illuminate P acquisition among plants in mixed stands of transgenic and wild‐type soybean. Our results showed that transgenic soybean plants were much more competitive, and had greater growth and P uptake than wild‐type soybean in mixed culture in both low P calcareous and acid soils. Furthermore, OXe plants had an advantage in calcareous soils when mixed with OXp, whereas the latter performed much better in acid soils. In soybean‐maize mixed culture, transgenic soybean had no impact on maize growth compared to controls in both acid and calcareous soils with different P conditions. As for soybean in mixed culture, OXp plants had no significant advantages regardless of P availability or soil type, while P efficiency improved in OXe in calcareous soils compared to controls. These results imply that physiological traits could be easily affected by the mixed maize. Transgenic soybean plants with enhanced root traits had more competitive advantages than those with improved root physiology in mixed culture.     

麦草畏室内生测方法建立及大豆对麦草畏耐受性分析

室内生物测定是植物对除草剂等化学物质耐受性鉴定的一种常用筛选方法,已广泛应用于大豆、稗草、棉花等植物对草甘膦、氯嘧磺隆等除草剂的耐受性研究,但麦草畏的室内生物测定方法和大豆对麦草畏耐受性相关研究尚未见报道。本研究以麦草畏对催芽大豆下胚轴伸长抑制率为评价指标,结合回归方程曲线分析和抑制中浓度分析,建立了大豆对麦草畏耐受性室内生物测定方法,确定以300μg/L麦草畏筛选浓度作为大豆室内生物测定临界筛选浓度。利用该方法对35份源自微核心种质的大豆品种进行鉴定,结果表明,随麦草畏浓度增加,不同品种对麦草畏的耐受性存在显著差异,大豆品种对麦草畏的耐受性降低,从中筛选出对麦草畏耐受性较高的大黄豆-1和什邡螺丝豆。本研究结果为培育抗麦草畏品种的亲本选配以及后代选择提供了理论依据、材料和技术支撑。     

外源基因导入技术在主要农作物育种上的应用进展

以国内科技期刊上发表的文献研究结果为依据,综述了农杆菌介导技术、基因枪导入技术、花粉管通道技术与激光微束穿刺技术在小麦、水稻、棉花、大豆、油菜等主要农作物育种上的应用进展。同时,还简要介绍了其中主要外源基因导入技术的基本原理,并评价了其优缺点。     

Future prospects for transgenic crops

Transgenic (genetically modified) crops are grown at present on more than 40 million hectares in 13 countries around the world. These `first generation' products principally comprise soybean, maize (corn) and cotton resistant to herbicides and/or insects. This review considers the wide range of `second generation' products under development and testing in many commercial and academic laboratories. Such products include examples for a variety of food, medical, veterinary and industrial purposes. In addition the review assesses the present state of public acceptance of transgenic products.     

Anthocyanin accumulation enhanced in <Emphasis Type="Italic">Lc</Emphasis>-transgenic cotton under light and increased resistance to bollworm

Breeding of naturally colored cotton fiber has been hampered by the limited germplasm, an alternative way is to use transgenic approach to create more germplasm for breeding. Here, we report our effort to engineer anthocyanin production in cotton. The maize Lc gene, under the control of the constitutive 35S promoter, was introduced into cotton through genetic transformation. Our data showed that the expression of the Lc gene alone is sufficient to trigger the accumulation of anthocyanin in a variety of cell types including fiber cells in cotton. However, the accumulation of colored anthocyanin in cotton fibers requires the participation of light signaling. These data indicate that it is feasible to engineer colored fibers through transgenic approach in cotton. Furthermore, we showed that the Lc-transgenic cotton plants are resistant to cotton bollworm. These transgenic plants are, therefore, potentially useful for cotton breeding against cotton bollworm.     

The contribution of nitrogen by promiscuous soybeans to maize based cropping the moist savanna of Nigeria

Agronomic results indicate that maize grain yields generally are higher when the crop is planted following soybean than in continuous maize cultivation in the moist savanna agroecological zones of West Africa. Many factors have been hypothesized to explain this phenomenon, including enhanced N availability and the so-called `rotational effect'. There is, however, hardly any quantitative information on the residual N benefits of promiscuous soybeans to subsequent cereal crops grown in rotation with soybean. Three IITA promiscuous soybean breeding lines and two Brazilian soybean lines were grown in 1994 and 1995 at Mokwa in the southern Guinea savanna, Nigeria, to quantify the nitrogen contribution by soybeans to a succeeding crop of maize grown in rotation with soybean for two consecutive years, 1996 and 1997 using two methods of introducing ¹⁵N into soil (fresh ¹⁵N labelling and its residual ¹⁵N) and three maize cultivars (including one cultivar with high N use efficiency) used as reference plants. The nodulating soybeans fixed between 44 and 103 kg N ha^–1 of their total N and had an estimated net N balance input from fixation following grain harvest ranging from –8 to 43 kg N ha^–1. Results in 1996 and in 1997 showed that maize growing after soybean had significantly higher grain yield (1.2 – 2.3-fold increase compared to maize control) except for maize cultivar Oba super 2 (8644-27) (a N-efficient hybrid). The ¹⁵N isotope dilution method was able to estimate N contribution by promiscuous soybeans to maize only in the first succeeding maize crop grown in 1996 but not in the second maize crop in 1997. The first crop of maize grown after soybean accumulated an average between 10 and 22 kg N ha^–1 from soybean residue, representing 17–33% of the soybean total N ha^–1. The percentage ¹⁵N derived from residue recovery in maize grown after maize was influenced by the maize cultivars. Maize crop grown after the N-efficient hybrid cultivar Oba Super 2 (844-27) had similar ¹⁵N values similar to maize grown after soybeans, confirming the ability of this cultivar to use N efficiently in low N soil due to an efficient N translocation ability. The maize crop in 1997 grown after maize had lower ¹⁵N enrichment than that grown in soybean plots, suggesting that soybean residues contributed a little to soil available N and to crop N uptake by the second maize crop. The differential mineralization and immobilization turnover of maize and soybean residues in these soils may be important and N contribution estimates in longer term rotation involving legumes and cereals may be difficult to quantify using the ¹⁵N labelling approaches. Therefore alternative methods are required to measure N release from organic residues in these cropping systems.