A dual-RPA based lateral flow strip for sensitive,on-site detection of CP4-EPSPS and Cry1Ab/Ac genes in genetically modified crops

Traditional transgenic detection methods require high test conditions and struggle to be both sensitive and efficient.In this study,a one-tube dual recombinase polymerase amplification(RPA)reaction system for CP4-EPSPS and Cry1Ab/Ac was proposed and combined with a lateral flow immunochromatographic assay,named“Dual-RPA-LFD”,to visualize the dual detection of genetically modified(GM)crops.In which,the herbicide tolerance gene CP4-EPSPS and the insect resistance gene Cry1Ab/Ac were selected as targets taking into account the current status of the most widespread application of insect resistance and herbicide tolerance traits and their stacked traits.Gradient diluted plasmids,transgenic standards,and actual samples were used as templates to conduct sensitivity,specificity,and practicality assays,respectively.The constructed method achieved the visual detection of plasmid at levels as low as 100 copies,demonstrating its high sensitivity.In addition,good applicability to transgenic samples was observed,with no cross-interference between two test lines and no influence from other genes.In conclusion,this strategy achieved the expected purpose of simultaneous detection of the two popular targets in GM crops within 20 min at 37°C in a rapid,equipmentfree field manner,providing a new alternative for rapid screening for transgenic assays in the field.

Effect of Bt traits on transgenic rice's growth and weed competitiveness

Transgene escape could lead to genetically modified rice establishing wild populations in the natural environment and competing for survival space with weeds.However,whether the expression of the Bacillus thuringiensis(Bt)gene in rice will alter the relationship between transgene plants and weeds and induce undesirable environmental consequences are poorly understood.Thus,field experiments were conducted to investigate the weed competitiveness and assess the ecological risk of transgenic Bt rice under herbicide-free and lepidopterous pest-controlled environments.Results showed that weed–rice competition in the direct-sowing(DS)field was earlier and more severe than that in the transplanting(TP)field,which resulted in a significant decrease in biomass and yield in DS.However,conventional Bt and non-Bt rice yield was not significantly different.The weed number,weed coverage ratio,and weed diversity of conventional Bt rice were significantly higher than those of non-Bt rice at the early growth and mature stages,especially in DS plots,suggesting that Bt traits did not increase the weed competitiveness of transgenic rice and had no negative effect on weed diversity.Grain yield and weed number varied between different hybrid rice lines,but those differences were insignificant between Bt and non-Bt rice.The number of insects increased with the increase of weeds in hybrid rice plots,whereas the insect number and diversity did not display a significant difference between Bt and non-Bt rice.Therefore,the ecological risk of transgenic Bt rice is comparable to non-Bt rice.

Prey-mediated effects of mCry51Aa2-producing cotton on the predatory nontarget bug Orius majusculus(Reuter)

Genetically engineered(GE)cotton,MON 88702,is protected against cer-tain sucking pests,such as plant bugs and thrips,by producing mCry51Aa2,a mod-ified protein from Bacillus thuringiensis(Bt).Predatory pirate bugs(Orius spp.),natural enemies contributing to biological pest control,are also sensitive to the insecti-cidal protein when exposed continuously to high concentrations.We evaluated effects of MON 88702 on Orius majusculus when fed prey types with different mCry51Aa2 concen-trations.When neonates were provided exclusively Tetranychus urticae spider mites reared on MON 88702(high mCry51Aa2 content),adverse effects on predator survival and de-velopment were confirmed,compared with specimens fed prey from near-isogenic non-Bt cotton.When fed a mixture of T.urticae and Ephestia kuehniella eggs(mCry51Aa2-free),predator life table parameters were similar to the treatment where eggs were fed exclu-sively.When mCry51Aa2-containing spider mites were provided for a limited time at the beginning or the end of juvenile development,effects were less pronounced.While pirate bug nymphs showed similar consumption rates for prey from Bt and non-Bt cotton,choice experiments revealed a preference for E.kuehniella eggs over spider mites.Lepidopteran larvae(Spodoptera littoralis,high mCry51Aa2 content)or cotton aphids(Aphis gossypii,mCry51Aa2-free)reared on MON 88702 as alternative prey did not result in adverse ef-fects on O.majusculus.Our study suggests limited risk of mCry51Aa2-producing cotton for O.majusculus,because its sensitivity for the Bt protein is relatively low and its natural food consists of diverse prey species with varying concentrations of Bt protein.

Brazilian biosafety law and the new breeding technologies

Globally,the area of land cultivated with genetically modified(GM)crops has increased a thousand-fold over the last two decades.Although this technology has become important for food production,the regulatory frameworks that underpin these outcomes are based on a list of requirements for a risk assessment that differ from country to country.In recent years,policymakers have had the opportunity to learn from the controversies over transgenics to create effective regulatory milestones for emerging technologies,allowing them to reach their potential for a more sustainable agriculture,ensuring food security.In Brazil,Law No.11.105 of 24 March 2005 established a framework with four main organizations responsible for risk assessment and management.However,most of new breeding technologies did not exist at that time and were not considered in this law.In2016,Normative Resolution No.16 of the National Biosafety Technical Commission(CTNBio)was established to address this gap based on the evaluation of the products obtained through these techniques(termed Innovative Precision Improvement Techniques in the resolution),in a case-by-case consultation system.Briefly,if the product is designated to be a GM,the developer will have to go through the biosafety requirements and will be approved only after CTNBio risk assessment.If the product is designated not to be GM(for the purposes of the legislation),then it can be registered using the existing procedures.Currently,152 GM products are commercially approved in Brazil.In 2018,CTNBio assessed the first consultation on commercial release of plants generated using the new breeding technologies and has subsequently approved six products.It is expected that many institutions would be able to participate in Brazilian and world markets,developing and introducing new biotechnological solutions and products through a more sustainable approach and without facing public disapproval,a common issue for GM crops.

Evaluation and application of an efficient plant DNA extraction protocol for laboratory and field testing

Nucleic acids in plant tissue lysates can be captured quickly by a cellulose filter paper and prepared for amplification after a quick purification.In this study,a published filter paper strip method was modified by sticking the filter paper on a polyvinyl chloride resin(PVC)sheet.This modified method is named EZ-D,for EASY DNA extraction.Compared with the original cetyl trimethylammonium bromide(CTAB)method,DNA extracted by EZ-D is more efficient in polymerase chain reaction(PCR)amplification due to the more stable performance of the EZ-D stick.The EZ-D method is also faster,easier,and cheaper.PCR analyses showed that DNA extracted from several types of plant tissues by EZ-D was appropriate for specific identification of biological samples.A regular PCR reaction can detect the EZ-D-extracted DNA template at concentration as low as 0.1 ng/μL.Evaluation of the EZ-D showed that DNA extracts could be successfully amplified by PCR reaction for DNA fragments up to 3000 bp in length and up to 80%in GC content.EZ-D was successfully used for DNA extraction from a variety of plant species and plant tissues.Moreover,when EZ-D was combined with the loop-mediated isothermal amplification(LAMP)method,DNA identification of biological samples could be achieved without the need for specialized equipment.As an optimized DNA purification method,EZ-D shows great advantages in application and can be used widely in laboratories where equipment is limited and rapid results are required.