Post-market monitoring: legal framework in Brazil and first results

Brazil is presently (2010) the second largest producer of Genetically Modified (GM) crops. The legal framework to support both research activities and the commercial release of Genetically Modified Organisms (GMOs) is, however, very recent, as the Biosafety Law dates back from 2005. After the indispensable risk analysis, done by National Technical Committee for Biosafety (CTNBio), a GM plant can be approved for commercial release. Nevertheless, as stated in the Normative Resolution nr. 5 (NR-5), 2008, the post-market monitoring is mandatory. However, the first GM crop in Brazil, the herbicide tolerant soybean, was harvested in 2005. The CTNBio asked, then, for a monitoring plan and the applicant designed a complex study focused on case-specific monitoring. After four years the detailed field studies proved that no harm derived from this crop. The costs, nevertheless, were enormous. In May 2010, 21 GM plants were already approved for commercial release in Brazil, corresponding to different events in soybean (6), cotton (8) and maize (9). Single and stacked events tolerant to herbicides or insect-resistant are presently been planted for all three crops. If, for each single event and for the stacks, a laborious post market monitoring plan were to be executed, the total costs would be unbearably high. The applicant, i.e. the seed company in the case of the three common crops, is legally responsible for the monitoring in Brazil. It is also responsible to produce an adequate monitoring plan. Since the rules in the Normative Resolution nr. 5 are far from being clear, the applicants either propose an elaborated plan for case-specific monitoring or, conversely, submit a very simple plan, targeting some potential adverse effect that can be more easily evaluated in commercial fields. All data generated must be reported to federal agencies belonging to at least three different ministries. Monitoring is also a responsibility of some federal agencies, as stated in the Biosafety Law, like registration and inspection agencies and other entities, which are responsible for, among other duties, keep updated information in the Biosafety Information Systems (SIB) of institutions and technical responsible individuals that carry out activities and projects related to GMOs and their by products. It is not clearly stated either in the Biosafety Law or in the NR-5 that monitoring should be split in case-specific monitoring and general surveillance, as adopted in Europe. If case-specific monitoring plans are difficult to design, a sensible suggestion for the general surveillance was not yet achieved. Nevertheless, the info-concentrating SIB may be a valuable source for the general surveillance, once it starts to operate. In conclusion, due to the lack of clear information about how to produce an adequate monitoring plan, stakeholders either tend to unnecessarily enlarge the monitoring scope or shorten it to a few objectives. The general surveillance is either not contemplated in the monitoring plans or minimally treated. It is important to keep in mind that the applicants are responsible to produce most of the data, even for the general surveillance, and this can be extremely difficult in a scenario where 20 or more different events are cultivated, frequently in neighboring fields. The EuropaBio approach to general surveillance for cultivation of GM crops (available from Nature Precedings "doi:10.1038/npre.2010.4451.1":http://dx.doi.org/10.1038/npre.2010.4451.1) is difficult to be largely adopted: farmers may be unable to meaningfully answer the questionnaire and the network has a very restricted number of potential participants. CTNBio is aware of these challenges and has already started to adjust the NR-5 to better guide the applicants in their writing of monitoring plans

Post Market Monitoring: Legal Framework in Brazil and First Results(Contribution to the 4th International Workshop on PMEM of Genetically Modified Plants, Quedlinburg)

Brasilien war 2010 der zweitgrößte Anbauer von gentechnisch veränderten Pflanzen. Die rechtliche Basis für die Forschung und kommerzielle Freisetzung von gentechnisch veränderten Organismen geht zurück auf das Jahr 2005. Nach der notwendigen Risikobewertung durch die nationale Technische Kommission für Biologische Sicherheit (CTNBio), kann eine Genehmigung für die kommerzielle Verwertung einer gentechnisch veränderten Pflanze erteilt werden. Allerdings erfordert die Normregel Nr. 5 (NR-5) aus dem Jahre 2008 ein begleitendes Monitoring. Allerdings wurde bereits im Jahre 2005 die erste transgene Pflanze, eine Herbizid-tolerante Sojabohne, in Brasilien angebaut. CTNBio forderte die Ausarbeitung eines Monitoringplanes, dem die Antragsteller mit einem komplexen, fallspezifisch fokussierten Monitoringdesign nachkamen. Nach vier Jahren detaillierter Feldstudien konnten keine Schäden durch den Anbau der transgenen Pflanzen nachgewiesen werden. Der Kostenaufwand war jedoch enorm. Im Juni 2010 waren bereits 23 gentechnisch veränderte Pflanzen mit verschiedenen transgenen Merkmalen für die kommer­zielle Nutzung in Brasilien zugelassen: Soja (6 Zulassungen), Baumwolle (8) und Mais (9). Einzelne oder gekoppelte Merkmale für Herbizidtoleranz und Insektenresistenz werden derzeit genutzt. Müsste für jedes einzelne Merkmal oder gekoppelte Merkmale ein eigenständiges Monitoringprogramm durchgeführt werden, wären die Kosten praktisch untragbar. Die Antragsteller sind nach brasilianischem Recht für die Planung und Durchführung des Monitoring verantwortlich. Da die Normregel Nr. 5 keine klaren Vorgaben macht, reichen die Antragsteller entweder sehr detaillierte, fallspezifisch orientierte oder sehr einfache, allgemeine Monitoringpläne ein, die sich auf einfach nachzuweisende, mögliche Effekte im kommerziellen Anbau beziehen. Im Gegensatz zum euro­päischen Recht unterscheidet weder das brasilianische Biosicherheitsgesetz noch Normregel Nr. 5 ein fallspezi­fisches Monitoring und eine Allgemeine Beobachtung. Sollte ein fallspezifischer Monitoringplan schwer herzuleiten sein, gibt es keine verbindlichen Vorgaben für eine Allgemeine Beobachtung. Hier kann ein zukünftiges brasilianisches Informationssystem für Biosicherheit wertvolle Unterstützung bieten. Derzeit jedoch ist eine Allgemeine Beobachtung gar nicht oder nur sehr begrenzt in den Monitoringplänen vorgesehen. Es ist zu beachten, dass die Antragsteller in jedem Fall für das Monitoring verantwortlich bleiben. Dies kann jedoch bei zunehmendem Anbau transgener Pflanzen mit verschiedenen (ähnlichen), einzelnen oder gekoppelten Merkmalen auf benachbarten Feldern zu großen Schwierigkeiten bei der Interpretation der einzelnen Monitoringdaten führen. Deshalb ist es notwendig, eine breit angelegte und mit verschiedenen Interessengruppen kommunizierte Monitoringstrategie zu entwickeln. CTNBio ist sich dieser Herausforderungen bewusst und hat in einem ersten Schritt die Normregel Nr. 5 überarbeitet, um den Antragstellern klarere Vorgaben für den Entwurf von Monitoringplänen zu bieten.    Brazil is presently (2010) the second largest producer of genetically modified (GM) crops. The legal framework to support both research activities and the commercial release of Genetically Modified Organisms (GMOs) is, however, very recent, as the Biosafety Law dates back from 2005. After the indispensable risk analysis, done by National Technical Commission for Biosafety (CTNBio), a GM plant can be approved for commercial release. Nevertheless, as stated in the Normative Resolution Nr. 5 (NR-5), of 2008, the post-market monitoring is mandatory. However, the first GM crop in Brazil, the herbicide tolerant soybean, was harvested in 2005. The CTNBio then asked for a monitoring plan and the applicant designed a complex study focused on case-specific monitoring. After four years the detailed field studies proved that no harm derived from this crop. The costs, nevertheless, were enormous. In June 2010, 21 GM plants were already approved for commercial release in Brazil, corresponding to different events in soybean (6), cotton (8) and maize (9). Single and stacked events tolerant to herbicides or insect-resistant are presently been planted. If, for each single event and for the stacks, a laborious post market monitoring plan were to be executed, the total costs would be unbearably high. The applicant is legally and financially responsible for the monitoring in Brazil. It is also responsible to produce an adequate monitoring plan. Since the NR-5 rules are far from being clear, the applicants either proposed an elabo­rated plan for case-specific monitoring or, conversely, submitted a very simple plan, targeting some potential adverse effect that can be more easily evaluated in commercial fields. It is not clearly stated either in the Biosafety Law or in the NR-5 that monitoring should be split in case-specific monitoring and general surveillance, as adopted in Europe. If case-specific monitoring plans are difficult to design, a sensible suggestion for the general surveillance was not yet achieved. Never­theless, the info-concentrating Brazilian Biosafety Infor­mation System may be a valuable source for the general surveillance, once it starts to operate. The general surveillance is presently either not contemplated in the monito­ring plans or only minimally treated. It is important to keep in mind that the applicants are responsible to produce most of the data, even for the general surveillance, and this can be extremely difficult in a scenario where many different events are cultivated, frequently in neighbouring fields. A conceptual framework for post market monitoring must therefore be established following a dia­log with all stakeholders. CTNBio is aware of these challenges and has already started to adjust the NR-5 to better guide the applicants in their writing of monitoring plans.   &nbsp

Overall picture of expressed Heat Shock Factors in Glycine max, Lotus japonicus and Medicago truncatula

Heat shock (HS) leads to the activation of molecular mechanisms, known as HS-response, that prevent damage and enhance survival under stress. Plants have a flexible and specialized network of Heat Shock Factors (HSFs), which are transcription factors that induce the expression of heat shock proteins. The present work aimed to identify and characterize the Glycine max HSF repertory in the Soybean Genome Project (GENOSOJA platform), comparing them with other legumes (Medicago truncatula and Lotus japonicus) in view of current knowledge of Arabidopsis thaliana. The HSF characterization in leguminous plants led to the identification of 25, 19 and 21 candidate ESTs in soybean, Lotus and Medicago, respectively. A search in the SuperSAGE libraries revealed 68 tags distributed in seven HSF gene types. From the total number of obtained tags, more than 70% were related to root tissues (water deficit stress libraries vs. controls), indicating their role in abiotic stress responses, since the root is the first tissue to sense and respond to abiotic stress. Moreover, as heat stress is related to the pressure of dryness, a higher HSF expression was expected at the water deficit libraries. On the other hand, expressive HSF candidates were obtained from the library inoculated with Asian Soybean Rust, inferring crosstalk among genes associated with abiotic and biotic stresses. Evolutionary relationships among sequences were consistent with different HSF classes and subclasses. Expression profiling indicated that regulation of specific genes is associated with the stage of plant development and also with stimuli from other abiotic stresses pointing to the maintenance of HSF expression at a basal level in soybean, favoring its activation under heat-stress conditions.35124725

Overall picture of expressed Heat Shock Factors in Glycine max, Lotus japonicus and Medicago truncatula

Heat shock (HS) leads to the activation of molecular mechanisms, known as HS-response, that prevent damage and enhance survival under stress. Plants have a flexible and specialized network of Heat Shock Factors (HSFs), which are transcription factors that induce the expression of heat shock proteins. The present work aimed to identify and characterize the Glycine max HSF repertory in the Soybean Genome Project (GENOSOJA platform), comparing them with other legumes (Medicago truncatula and Lotus japonicus) in view of current knowledge of Arabidopsis thaliana. The HSF characterization in leguminous plants led to the identification of 25, 19 and 21 candidate ESTs in soybean, Lotus and Medicago, respectively. A search in the SuperSAGE libraries revealed 68 tags distributed in seven HSF gene types. From the total number of obtained tags, more than 70% were related to root tissues (water deficit stress libraries vs. controls), indicating their role in abiotic stress responses, since the root is the first tissue to sense and respond to abiotic stress. Moreover, as heat stress is related to the pressure of dryness, a higher HSF expression was expected at the water deficit libraries. On the other hand, expressive HSF candidates were obtained from the library inoculated with Asian Soybean Rust, inferring crosstalk among genes associated with abiotic and biotic stresses. Evolutionary relationships among sequences were consistent with different HSF classes and subclasses. Expression profiling indicated that regulation of specific genes is associated with the stage of plant development and also with stimuli from other abiotic stresses pointing to the maintenance of HSF expression at a basal level in soybean, favoring its activation under heat-stress conditions

Inter-genus gene expression analysis in livestock fibroblasts using reference gene validation based upon a multi-species primer set.

Quantitative reverse transcription PCR (RT-qPCR) remains as an accurate approach for gene expression analysis but requires labor-intensive validation of reference genes using species-specific primers. To ease such demand, the aim was to design and test a multi-species primer set to validate reference genes for inter-genus RT-qPCR gene expression analysis. Primers were designed for ten housekeeping genes using transcript sequences of various livestock species. All ten gene transcripts were detected by RT-PCR in Bos taurus (cattle), Bubalus bubalis (buffaloes), Capra hircus (goats), and Ovis aries (sheep) cDNA. Primer efficiency was attained for eight reference genes using B. taurus-O. aries fibroblast cDNA (95.54-98.39%). The RT-qPCR data normalization was carried out for B. taurus vs. O. aries relative gene expression using Bestkeeper, GeNorm, Norm-finder, Delta CT method, and RefFinder algorithms. Validation of inter-genus RT-qPCR showed up-regulation of TLR4 and ZFX gene transcripts in B. taurus fibroblasts, irrespectively of normalization conditions (two, three, or four reference genes). In silico search in mammalian transcriptomes showed that the multi-species primer set is expected to amplify transcripts of at least two distinct loci in 114 species, and 79 species would be covered by six or more primers. Hence, a multi-species primer set allows for inter-genus gene expression analysis between O. aries and B. taurus fibroblasts and further reveals species-specific gene transcript abundance of key transcription factors

An overall evaluation of the resistance (R) and pathogenesis-related (PR) superfamilies in soybean, as compared with Medicago and Arabidopsis

Plants have the ability to recognize and respond to a multitude of pathogens, resulting in a massive reprogramming of the plant to activate defense responses including Resistance (R) and Pathogenesis-Related (PR) genes. Abiotic stresses can also activate PR genes and enhance pathogen resistance, representing valuable genes for breeding purposes. The present work offers an overview of soybean R and PR genes present in the GENOSOJA (Brazilian Soybean Genome Consortium) platform, regarding their structure, abundance, evolution and role in the plantpathogen metabolic pathway, as compared with Medicago and Arabidopsis. Searches revealed 3,065 R candidates (756 in Soybean, 1,142 in Medicago and 1,167 in Arabidopsis), and PR candidates matching to 1,261 sequences (310, 585 and 366 for the three species, respectively). The identified transcripts were also evaluated regarding their expression pattern in 65 libraries, showing prevalence in seeds and developing tissues. Upon consulting the SuperSAGE libraries, 1,072 R and 481 PR tags were identified in association with the different libraries. Multiple alignments were generated for Xa21 and PR-2 genes, allowing inferences about their evolution. The results revealed interesting insights regarding the variability and complexity of defense genes in soybean, as compared with Medicago and Arabidopsis

In silico identification of known osmotic stress responsive genes from Arabidopsis in soybean and Medicago

Plants experience various environmental stresses, but tolerance to these adverse conditions is a very complex phenomenon. The present research aimed to evaluate a set of genes involved in osmotic response, comparing soybean and medicago with the well-described Arabidopsis thaliana model plant. Based on 103 Arabidopsis proteins from 27 categories of osmotic stress response, comparative analyses against Genosoja and Medicago truncatula databases allowed the identification of 1,088 soybean and 1,210 Medicago sequences. The analysis showed a high number of sequences and high diversity, comprising genes from all categories in both organisms. Genes with unknown function were among the most representative, followed by transcription factors, ion transport proteins, water channel, plant defense, protein degradation, cellular structure, organization & biogenesis and senescence. An analysis of sequences with unknown function allowed the annotation of 174 soybean and 217 Medicago sequences, most of them concerning transcription factors. However, for about 30% of the sequences no function could be attributed using in silico procedures. The establishment of a gene set involved in osmotic stress responses in soybean and barrel medic will help to better understand the survival mechanisms for this type of stress condition in legumes