The Development and Standardization of Testing Methods for Genetically Modified Organisms and their Derived Products

As the worldwide commercialization of genetically modified organisms （GMOs） increases and consumers concern the safety of GMOs, many countries and regions are issuing labeling regulations on GMOs and their products. Analytical methods and their standardization for GM ingredients in foods and feed are essential for the implementation of labeling regulations. To date, the GMO testing methods are mainly based on the inserted DNA sequences and newly produced proteins in GMOs. This paper presents an overview of GMO testing methods as well as their standardization.

Public awareness,participation and attitude toward the national biosafety framework and genetically modified organisms in Ghana

Public engagement in the development,promotion,and utilization of innovation is an important part of any biosafety decision-making process.Under the Cartagena Protocol on Biosafety,the public is expected to be involved in the development and handling of genetically modified organisms(GMOs)and the implementation of a national biosafety framework(NBF),which governs and regulates the operations of modern biotechnology and GMOs.In this study,we explore the state of public knowledge and awareness regarding GMOs and attitudes toward the NBF in Ghana using a survey conducted in three elite communities in Accra,the capital of Ghana.We interviewed 130 people and found that while most of the respondents obtained information on GMOs through the media,academic papers,and agriculture awareness workshops,access to information on the technology and the NBF was often limited.Our results showed that despite the existence of GMOs and an NBF in Ghana for many years,awareness,understanding,and knowledge of GMOs and the NBF remain inadequate.We found that young,better-educated males are more likely to accept GMOs and be aware of the NBF.This suggests the need for more widespread public education,engagement,and awareness development regarding GMOs,the NBF,and governing institutions as a way of resolving the problems created by misinformation,distrust,and fear,and increasing public confidence in GMOs.

Detection of Genetically Modified Crops by Combination of Multiplex PCR and Low-density DNA Microarray

Objective To develop a technique for simultaneous detection of various target genes in Roundup Ready soybean by combining multiplex PCR and low-density DNA microarray. Methods Two sets of the multiplex PCR system were used to amplify the target genes in genetically modified （GM） soybean. Seventeen capture probes （PCR products） and 17 pairs of corresponding primers were designed according to the genetic characteristics of Rroundup Ready soybean （GTS40-3-2）, maize （MonS10, Nk603, GA21）, canola （T45, MS1/RF1）, and rice （SCK） in many identified GM crops. All of the probes were categorized and identified as species-specific probes. One negative probe and one positive control probe were used to assess the efficiency of all reactions, and therefore eliminate any false positive and negative results. After multiplex PCR reaction, amplicons were adulterated with Cy5-dUTP and hybridized with DNA microarray. The array was then scanned to display the specific hybridization signals of target genes. The assay was applied to the analysis of sample of certified transgenic soybean （Roundup Ready GTS40-3-2） and canola （MS1/RF1）. Results A combination technique of multiplex PCR and DNA microarray was successfully developed to identify multi-target genes in Roundup Ready soybean and MS 1/RF1 canola with a great specificity and reliability. Reliable identification of genetic characteristics of Roundup Ready of GM soybean from genetically modified crops was achieved at 0.5% transgenic events, indicating a high sensitivity. Conclusion A combination technique of multiplex PCR and low-density DNA microarray can reliably detect and identify the genetically modified crops.

Evaluation of genetically modified rice detection methods 2011/884/EU and 2008/289/EC proposed by the European Union

Increases in the number of cases of identified genetically modified （GM） rice contamination can be traced back to the first Rapid Alert System for Food and Feed （RASFF） in 2006. In response to the lack of reliable detection methods, Decision 2011/884/EU proposed that new screening methods replace Decision 2008/289/EC, to identify all possible GM rice products originating in China. However, the synergy brands （SYBR） Green real-time PCR assay proposed by Decision 2011/884/EU has been shown to lack conformity with other TaqMan methods currently in use. To evaluate the specificity and repeatability of the methods recommended in Decision 2011/884/EU and Decision 2008/289/EC, we collected 74 rice products originating from six countries or districts. The 74 rice samples were tested using the Decision 2011/884/EU and Decision 2008/289/ EC methods. The parallel use of different instruments and reagents were used for testing in parallel, and the results were analyzed statistically. To avoid the limitations of specific laboratories, eight GM organism detection laboratories in China participated in a collaborative trial. In our tests, 24.3% （18/74） of the samples tested were positive with the SYBR Green real-time PCR assay using the Decision 2011/884/EU method, but were negative with the TaqMan real-time PCR assay using the Decision 2011/884/EU and Decision 2008/289/EC methods. Sequencing the PCR-amplified CrylA（b/c） genes in three samples （6, 30 and 43） showed that the products consisted of primer dimers rather than the targeted sequence. The combined experimental results showed that testing for the nopaline synthase gene （NOS） of Agrobacterium tumefasciens terminator and CrylA（b/c） produced false-positive results when the Decision 2011/884/EU method was used. Because of the high rate of false-positive results, the Decision 2011/884/EU SYBR Green method to detect GM rice requires improvement.

Current advances of biocontainment strategy in synthetic biology

Synthetic biotechnology has led to the widespread application of genetically modified organisms(GMOs)in biochemistry, bioenergy, and therapy. However, the uncontrolled spread of GMOs may lead to genetic contamination by horizontal gene transfer, resulting in unpredictable biosafety risks. To deal with these challenges, many effective methods have been developed for biocontainment. In this article, we summarize and discuss recent advances in biocontainment strategies from three aspects: DNA replication, transcriptional regulation, and protein translation. We also briefly introduce the efforts in the biocontainment convention, such as the recent publication of the Tianjin Biosecurity Guidelines for the Code of Conduct for Scientists.

Construction of the Plasmid Reference Molecule for Detection of Transgenic Soybean MON89788

[Objective] The aim was to construct a plasmid reference molecule (PRM) for detection of transgenic soybean MON89788. [Method] the lectin gene sequence,3'-junction and 5'-junction sequence between host plant DNA integrated DNA of MON89788 soybean were amplified independently,and the three fragments were cloned into the cloning vector pMD18-T in order through molecular manipulation method to construct pMD-LM3M5,the applicability of the constructed novel PRM was tested. [Result] Sequencing confirmation result showed that the PRM was 3 700 bp in length,containing 1 029 bp of recombined DNA fragment. The limits of qualitative detection of the PRM were 10 copies. [Conclusion] The PRM constructed in this study was suitable for the identification of MON89788 event.

An efficient method to extract DNA from refined rapeseed oil

Genetically modified oilseeds were used as processing raw material for edible oils. The protection of consumer rights to information as well as the genetically modified orgamisms （GMO） labe-ling polily required analytical methods to determine whether the oils contained genetically modified ingre-dient or not. Polymerase chain reaction （PCR） - based method was used commonly to determine the presence of GMOs. Adulteration attracted peoples concern also. Thus it was crucial that enough DNA ex-tracts can be obtained successfully from oil samples. For the purpose, three DNA extraction methods （modified emulsification method, the kits Wizard Magnetic DNA purification system for food and Nucleospin Food） ,were applied to 3 different grades of rapeseed oil samples. Those methods were compared by the amplification of Brassica napus reference gene CruA using real - time PCR. The results demonstra-ted that both the modified emulsification and the Nucleospin methods were able to extract enough DNA from refined oils. The modified emulsification method was superior to the kit of Nucleospin food due to smaller volume of required sample.

Comparison of GMO Safety Management Policies in Major Countries and Regions

With the rapid development of genetic technology,the application of genetically modified crops has brought revolutionary changes to the global agricultural production and agricultural trade patterns,and has played an important role in ensuring human health,environmental safety and sustainable agricultural development.In order to strengthen genetically modified organisms(GMO)safety management,countries around the world have formulated GMO safety management policies adapted to their own national conditions with reference to internationally accepted practices.In this study,we carried out comparative studies among the United States,the European Union,Japan and other developed countries or regions and China from the aspects of regulations and management institutions,safety evaluation systems,and label management,so as to provide references for the improvement of China's GMO safety management policies.

Effects of GMO Agricultural Products on Living Things

By obtaining changes on gene sequences of living things with the applied biotechnological methods;The idea of“Genetically Modified Organisms(GMO)”,which aims to bring the living creature in question the original gene combinations with the desired characteristics,came to life in the late twentieth century.Despite the high probability that hunger problems may increase with the increasing world population;It is thought that plant breeding with classical farming methods will be insufficient in solving these problems.With various GMO applications developed all over the world,it aims to produce solutions to these problems.With the presence of GMO,it was possible to increase the shelf life of qualitative and quantitative values of the existing foods.In addition,decreases in agricultural use of pesticides used in agricultural struggle and threatening human health with GMO production are noteworthy.However,some concerns about anomalies that may occur in living things fed GMO products remain on the agenda.Because,in the long term,there is no clear and precise information that GMO will not have negative effects on living things;There are many recorded incidents showing their negative effects.

Constructing the barley model for genetic transformation in Triticeae

Barley（Hordeum vulgare L.） is one of the oldest domesticated crops, showing dramatic adaptation to various climate and environmental conditions. As a major cereal crop, barley ranks the 4th after wheat, maize and rice in terms of planting area and production all over the world. Due to its diploid nature, the cultivated barley is considered as an ideal model to study the polyploid wheat and other Triticeae species. Here, we reviewed the development, optimization, and application of transgenic approaches in barley. The most efficient and robust genetic transformation has been built on the Agrobacterium-mediated transfer in conjunction with the immature embryo-based regeneration. We then discussed future considerations of using more practical technologies in barley transformation, such as the T-DNA/transposon tagging and the genome editing. As a cereal crop amenable to genetic transformation, barley will serve as the most valuable carrier for global functional genomics in Triticeae and is becoming the most practical model for generating value-added products.

Isoflavone and Mineral Content in Conventional and Transgenic Soybean Cultivars

The objective of this study was to evaluate the differences in composition among six brands of conventional soybean and six genetically modified cultivars (GM). We focused on the isoflavones profile and mineral content questioning the substantial equivalence between conventional and GM organisms. The statement of compliance label for conventional grains was verified for the presence of genetic modified genes by real time polymerase chain reaction (PCR). We did not detect the presence of the 35S promoter in commercial samples, indicating the absence of transgene insertion. For mineral analysis, we used the method of inductively coupled plasma-optical emission spectrometry (ICP-OES). Isoflavones quantification was performed by high performance liquid chromatography (HPLC). The results showed no statistical difference between the conventional and transgenic soybean groups concerning isoflavone content and mineral composition. The concentration of potassium, the main mineral component of soy, was the highest in conventional soybeans compared to that in GM soy, while GM samples presented the highest concentrations of iron.

Regulation of gene-edited plants in Europe:from the valley of tears into the shining sun?

Some 20 years ago,the EU introduced complex regulatory rules for the growth of transgenic crops,which resulted in a de facto ban to grow these plants in fields within most European countries.With the rise of novel genome editing technologies,it has become possible to improve crops genetically in a directed way without the need for incorporation of foreign genes.Unfortunately,in 2018,the European Court of Justice ruled that such gene-edited plants are to be regulated like transgenic plants.Since then,European scientists and breeders have challenged this decision and requested a revision of this outdated law.Finally,after 5 years,the European Commission has now published a proposal on how,in the future,to regulate crops produced by new breeding technologies.The proposal tries to find a balance between the different interest groups in Europe.On one side,genetically modified plants,which cannot be discerned from their natural counterparts,will exclusively be used for food and feed and are-besides a registration step-not to be regulated at all.On the other side,plants expressing herbicide resistance are to be excluded from this regulation,a concession to the strong environmental associations and NGOs in Europe.Moreover,edited crops are to be excluded from organic farming to protect the business interests of the strong organic sector in Europe.Nevertheless,if this law passes European parliament and council,unchanged,it will present a big step forward toward establishing a more sustainable European agricultural system.Thus,it might soon be possible to develop and grow crops that are more adapted to global warming and whose cultivation will require lower amounts of pesticides.However,there is still a long way to go until the law is passed.Too often,the storm of arguments raised by the opponents,based on irrational fears of mutations and a naive understanding of nature,has fallen on fruitful ground in Europe.

The evolution of China’s regulation of agricultural biotechnology

To ensure safe use of genetically modified organisms(GMOs),since 1993,China has made great efforts to establish and improve the safety regulatory system for GMOs.Here,we summarize and analyze the regulatory framework of agricultural GMOs,and the progress in regulatory approval of GM crops in China.In general,the development of GMO safety regulations underwent four stages:exploration(1993–2000),development(2001–2010),improvement(2011–2020)and current(2021-present)stage.The first formal regulation was promulgated in 1993,which provided a basis for further development of the regulations,during the exploration stage,when insect-resistant GM cotton,expressing genes from Bacillus thuringiensis(Bt),was approved for cultivation.During the development stage,the Chinese government issued a series of administrative measures,which covered almost all the fields relative to GMO safety when the basic regulatory system was established.Along with the controversy over GMO safety,the regulations have been further,and greatly improved,during improvement stage.From 2021,a few additional revisions have been made,and meanwhile,the new regulation on gene-edited crops was introduced with the development of biotechnology,forming a relative complete regulation and law system for China.The well-developed GMO regulations establishes a firm basis for safe use of GM crops in China.Currently,GM cotton and GM papaya have been widely grown on a large scale in China that have brought great economic and ecological benefits.In addition,12 corn events,3 soybean events,and 2 rice events have also obtained biosafety certification,but presently,these lines have yet to enter commercial production.However,several GM soybean and corn events have entered pilot industrialization,and can soon be expected to be commercially grown in China.In addition to planting,six GM crops,including soybean,corn,cotton,canola,papaya and sugar beet,with a total of 64 events,have been approved for import as processing material in China.

Limited ecological risk of insect-resistance transgene flow from cultivated rice to its wild ancestor based on life-cycle fitness assessment

Ecological impact caused by transgene flow from genetically engineered （GE） crops to their wild rela- tives is largely determined by the fitness effect brought by a transgene. To estimate such impact is critical for the eco- logical risk assessment prior to the commercialization of GE crops. We produced F1 and F2 hybrid descendants from crosses of two insect-resistant GE rice lines （Bt, Bt/CpT1） and their non-GE rice parent with a wild rice （Oryza ruff- pogon） population to estimate the transgenic fitness. Insect damages and life-cycle fitness of GE and non-GE crop- wild hybrid descendants as well as their wild parent were examined in a common-garden experiment. No significant differences in insect damages were observed between the wild rice parent and GE hybrid descendants under high- insect pressure. The wild parent showed significantly greater relative survival-regeneration ratios than its GE and non-GE hybrid descendants under both high- and low-in- sect pressure. However, more seeds were produced in GE hybrid descendants than their non-GE counterparts under high-insect pressure. Given that the introduction of Bt and Bt/CpT1 transgenes did not provide greater insect resistance to crop-wild hybrid descendants than their wild parent, we predict that transgene flow from GE insect-resistant rice to wild rice populations may not cause considerable ecolog- ical risks.