Introduction and scientific justification of data transportability for confined field testing for the ERA of GM plants.

The concept of Data Transportability (DT) of Confined Field Testing (CFT) to support the Environmental Risk Assessment (ERA) of Genetically Modified (GM) plants was first introduced in the literature by Garcia-Alonso et al., in 2014. Since then, DT has been discussed in many countries and regions as a concept to prevent duplication of regulatory studies without compromising quality of the ERA. However, despite its usefulness and scientific justification, DT is not well adopted at this time and many regulatory agencies around the world require additional in-country CFT be conducted before approving GM plants. Based on the current circumstances, the authors organized a parallel session entitled "Introduction and Scientific Justification of DT for CFT for the ERA of GM plants" at 16th ISBR (the International Society for Biosafety Research). This session mainly consisted of the following three parts. The first two speakers, Andrew Roberts and Abigail Simmons provided an overview of DT and examples of conditions for the transportability of field data/conclusions advocated in the peer-reviewed scientific journals. Next, the current status of DT adoption in some countries/regions such as Japan and Africa, and a theoretical case study for Argentina were introduced by Kazuyuki Hiratsuka, Douglas Miano, and Facundo Vesprini, respectively. Lastly, a risk hypothesis-based approach for DT which was developed in advance by the five speakers of this parallel session, was introduced. During the discussion, there was a common understanding that transition to the risk hypothesis-based approach for DT was scientifically appropriate, considering the accumulated evidences that several countries have conducted confirmatory local CFT for more than 20 years but they have not detected any differences related to the ERA assessment endpoints in GM crops. The risk hypothesis-based approach for DT introduced here is expected to play an important role in discussions on the implementation of DT in various parts of the world in the future.

RNAi silencing CHS1 gene shortens the mortality time of Plutella xylostella feeding Bt-transgenic Brassica napus.

BACKGROUND: Insect-resistance genetically modified (GM) plants derived from Bacillus thuringiensis (Bt) have been cultivated to control pests, but continuous cultivation of Bt-transgenic plants at large-scale regions leads to the resistance evolution of target insects to transgenic plants. RNA interference (RNAi) technology is considered an effective strategy in delaying the resistance evolution of target insects. RESULTS: We here developed a single transgenic oilseed rape (Brassica napus) line with hairpin RNA of the chitin-synthase 1 gene (CHS1) of Plutella xylostella (hpPxCHS1) and a pyramid transgenic B. napus line harboring hpPxCHS1 and Bt gene (Cry1Ac). Escherichia coli HT115 delivered hpPxCHS1 showed negative effects on the growth of P. xylostella. The single transgenic and pyramid transgenic B. napus significantly reduced the larval weight and length of P. xylostella and increased its lethality rate, with down-regulation expression of the PxCHS1 gene in insects. CONCLUSION: Compared to Bt-transgenic B. napus, pyramid-transgenic B. napus shorted the mortality time of P. xylostella, indicating that RNAi technology synergistic with Bt protein improves the effectiveness of controlling target insects. Our results proved that RNAi can delay the resistance evolution of target insects to Bt-transgenic plants. © 2024 Society of Chemical Industry.

EU regulation of gene-edited plants-A reform proposal.

This article presents a proposal on how the European Union's regulatory framework on genetically modified (GM) plants should be reformed in light of recent developments in genomic plant breeding techniques. The reform involves a three-tier system reflecting the genetic changes and resulting traits of GM plants. The article is intended to contribute to the ongoing debate over how best to regulate plant gene editing techniques in the EU.

Evaluation of existing guidelines for their adequacy for the food and feed risk assessment of genetically modified plants obtained through synthetic biology.

Synthetic biology (SynBio) is an interdisciplinary field at the interface of molecular engineering and biology aiming to develop new biological systems and impart new functions to living cells, tissues and organisms. EFSA has been asked by the European Commission to evaluate SynBio developments in agri-food with the aim of identifying the adequacy and sufficiency of existing guidelines for risk assessment and determine if updated guidance is needed. In this context, the GMO Panel has previously adopted an Opinion evaluating the SynBio developments in agri-food/feed and the adequacy and sufficiency of existing guidelines for the molecular characterisation and environmental risk assessment of genetically modified plants (GMPs) obtained through SynBio and reaching the market in the next decade. Complementing the above, in this Opinion, the GMO Panel evaluated the adequacy and sufficiency of existing guidelines for the food and feed risk assessment of GMPs obtained through SynBio. Using selected hypothetical case studies, the GMO Panel did not identify novel potential hazards and risks that could be posed by food and feed from GMPs obtained through current and near future SynBio approaches; considers that the existing guidelines are adequate and sufficient in some Synbio applications; in other cases, existing guidelines may be just adequate and hence need updating; areas needing updating include those related to the safety assessment of new proteins and the comparative analysis. The GMO Panel recommends that future guidance documents provide indications on how to integrate the knowledge available from the SynBio design and modelling in the food and feed risk assessment and encourages due consideration to be given to food and feed safety aspects throughout the SynBio design process as a way to facilitate the risk assessment of SynBio GMPs and reduce the amount of data required.

Non-target Effects of Hyperthermostable α-Amylase Transgenic Nicotiana tabacum in the Laboratory and the Field.

Thermostable α-amylases are important enzymes used in many industrial processes. The expression of recombinant Pyrococcus furiosus α-amylase (PFA) in Nicotiana tabacum has led to the accumulation of high levels of recombinant protein in transgenic plants. The initial steps to registering the transgenic tobacco at a commercial production scale and growing it in the field requires a risk assessment of potential non-target effects. The objective of this study was to assess the effect of feeding on transgenic tobacco with 2 indigenous insect species commonly associated with wild and commercial tobacco involving plants grown and evaluated under laboratory and field conditions. The highest levels of PFA ranged from 1.3 to 2.7 g/kg leaf fresh weight produced in the field-grown cultivars Con Havana and Little Crittenden, respectively. These two cultivars also had the highest nicotine (ranging from 4.6 to 10.9 mg/g), but there was little to no negative effect for either tobacco hornworm Manduca sexta L. or aphid Myzus nicotianae (Blackman). Both laboratory and field trials determined no short term (5 days) decrease in the survival or fecundity of the tobacco aphid after feeding on PFA transgenic tobacco compared to non-transgenic plants. In the field, tobacco hornworm larvae showed no differences in survival, final larval weights or development time to adult stage between transgenic lines of four cultivars and their corresponding wild type controls. Laboratory studies confirmed the field trial results indicating the low risk association of PFA expressed in tobacco leaves with tobacco hornworms and aphids that would feed on the transgenic plants.

Setting an import tolerance for 2,4-D in soyabeans.

In accordance with Article 6 of Regulation (EC) No 396/2005, the applicant Dow AgroSciences submitted a request to the competent national authority in Greece to set an import tolerance for the active substance 2,4-D in genetically modified (GM) soyabeans imported from Canada and the USA. The genetic modification confers tolerance to the herbicide 2,4-D. The data submitted in support of the request provided sufficient evidence to conclude that residues of parent 2,4-D and of the metabolite 2,4-dichlorophenol (2,4-DCP), which was found in the GM soybeans treated with 2,4-D, are unlikely to present a risk for consumers. Sufficiently validated analytical methods are available to enforce the proposed maximum residue level (MRL) in soybeans.

Setting of an import tolerance for 2,4-D in maize.

In accordance with Article 6 of Regulation (EC) No 396/2005, the evaluating Member State (EMS) Greece received an application from Dow AgroSciences to set an import tolerance for the active substance 2,4-D in genetically modified (GM) maize imported from Canada and the USA. The modification confers tolerance to the herbicide 2,4-D. Greece drafted an evaluation report in accordance with Article 8 of Regulation (EC) No 396/2005 which was submitted to the European Commission and forwarded to EFSA. According to EFSA, the data submitted are sufficient to conclude that a change of the existing maximum residue level (MRL) set at the limit of quantification (LOQ) of 0.05 mg/kg in maize is not required. Analytical enforcement methods are available to control the residues of 2,4-D in cereals and no risk for consumers was identified for the notified use on GM maize expressing the aryloxyalkanoate dioxygenase 1 (AAD-1) protein imported from Canada and the USA.

A Rice Gene Homologous to Arabidopsis AGD2-LIKE DEFENSE1 Participates in Disease Resistance Response against Infection with Magnaporthe oryzae.

ALD1 (ABERRANT GROWTH AND DEATH2 [AGD2]-LIKE DEFENSE1) is one of the key defense regulators in Arabidopsis thaliana and Nicotiana benthamiana. In these model plants, ALD1 is responsible for triggering basal defense response and systemic resistance against bacterial infection. As well ALD1 is involved in the production of pipecolic acid and an unidentified compound(s) for systemic resistance and priming syndrome, respectively. These previous studies proposed that ALD1 is a potential candidate for developing genetically modified (GM) plants that may be resistant to pathogen infection. Here we introduce a role of ALD1-LIKE gene of Oryza sativa, named as OsALD1, during plant immunity. OsALD1 mRNA was strongly transcribed in the infected leaves of rice plants by Magnaporthe oryzae, the rice blast fungus. OsALD1 proteins predominantly localized at the chloroplast in the plant cells. GM rice plants over-expressing OsALD1 were resistant to the fungal infection. The stable expression of OsALD1 also triggered strong mRNA expression of PATHOGENESIS-RELATED PROTEIN1 genes in the leaves of rice plants during infection. Taken together, we conclude that OsALD1 plays a role in disease resistance response of rice against the infection with rice blast fungus.

High CO2 concentration as an inductor agent to drive production of recombinant phytotoxic antimicrobial peptides in plant biofactories.

Cationic α-helical antimicrobial peptides such as BP100 are of increasing interest for developing novel phytosanitary or therapeutic agents and products with industrial applications. Biotechnological production of these peptides in plants can be severely impaired due to the toxicity exerted on the host by high-level expression. This can be overcome by using inducible promoters with extremely low activity throughout plant development, although the yields are limited. We examined the use of modified atmospheres using the increased levels of [CO2], commonly used in the food industry, as the inductor agent to biotechnologically produce phytotoxic compounds with higher yields. Here we show that 30% [CO2] triggered a profound transcriptional response in rice leaves, including a change in the energy provision from photosynthesis to glycolysis, and the activation of stress defense mechanisms. Five genes with central roles in up-regulated pathways were initially selected and their promoters successfully used to drive the expression of phytotoxic BP100 in genetically modified (GM) rice. GM plants had a normal phenotype on development and seed production in non-induction conditions. Treatment with 30 % [CO2] led to recombinant peptide accumulation of up to 1 % total soluble protein when the Os.hb2 promoter was used. This is within the range of biotechnological production of other peptides in plants. Using BP100 as a proof-of-concept we demonstrate that very high [CO2] can be considered an economically viable strategy to drive production of recombinant phytotoxic antimicrobial peptides in plant biofactories.

Transgenic rice seed expressing flavonoid biosynthetic genes accumulate glycosylated and/or acylated flavonoids in protein bodies.

Plant-specialized (or secondary) metabolites represent an important source of high-value chemicals. In order to generate a new production platform for these metabolites, an attempt was made to produce flavonoids in rice seeds. Metabolome analysis of these transgenic rice seeds using liquid chromatography-photodiode array-quadrupole time-of-flight mass spectrometry was performed. A total of 4392 peaks were detected in both transgenic and non-transgenic rice, 20-40% of which were only detected in transgenic rice. Among these, 82 flavonoids, including 37 flavonols, 11 isoflavones, and 34 flavones, were chemically assigned. Most of the flavonols and isoflavones were O-glycosylated, while many flavones were O-glycosylated and/or C-glycosylated. Several flavonoids were acylated with malonyl, feruloyl, acetyl, and coumaroyl groups. These glycosylated/acylated flavonoids are thought to have been biosynthesized by endogenous rice enzymes using newly synthesized flavonoids whose biosynthesis was catalysed by exogenous enzymes. The subcellular localization of the flavonoids differed depending on the class of aglycone and the glycosylation/acylation pattern. Therefore, flavonoids with the intended aglycones were efficiently produced in rice seeds via the exogenous enzymes introduced, while the flavonoids were variously glycosylated/acylated by endogenous enzymes. The results suggest that rice seeds are useful not only as a production platform for plant-specialized metabolites such as flavonoids but also as a tool for expanding the diversity of flavonoid structures, providing novel, physiologically active substances.

Effects of a diet containing genetically modified rice expressing the Cry1Ab/1Ac protein (Bacillus thuringiensis toxin) on broiler chickens.

The aim of this study was to evaluate the effect of feeding Bacillus thuringiensis (Bt) rice expressing the Cry1Ab/1Ac protein on broiler chicken. The genetically modified (GM) Bt rice was compared with the corresponding non-GM rice regarding performance of feeding groups, their health status, relative organ weights, biochemical serum parameters and occurrence of Cry1Ab/1Ac gene fragments. One hundred and eighty day-old Arbor Acres female broilers with the same health condition were randomly allocated to the two treatments (6 replicate cages with 15 broilers in each cage per treatment). They received diets containing GM rice (GM group) or its parental non-GM rice (non-GM group) at 52-57% of the air-dried diet for 42 days. The results show that the transgenic rice had a similar nutrient composition as the non-GM rice and had no adverse effects on chicken growth, biochemical serum parameters and necropsy during the 42-day feeding period. In birds fed the GM rice, no transgenic gene fragments were detected in the samples of blood, liver, kidneys, spleen, jejunum, ileum, duodenum and muscle tissue. In conclusion, the results suggest that Bt rice expressing Cry1Ab/1Ac protein has no adverse effects on broiler chicken. Therefore, it can be considered as safe and used as feed source for broiler chicken.

Ocorrência do ácaro fitófago Catarhinus tricholaenae Keifer (Acari: Diptilomiopidae) em cultivares de milho Bt / Occurrence of the phytophagous mite Catarhinus tricholaenae Keifer (Acari: Diptilomiopidae) on Bt corn cultivars

O objetivo deste trabalho foi avaliar a ocorrência do microácaro-da-face-inferior-das-folhas-de-milho Catarhinus tricholaenae Keifer (Acari: Diptilomiopidae) em cultivares transgênicas de milho, contendo as proteínas Cry1F e Cry 1 A(b) e milho não Bt. Durante o período de junho de 2010 a janeiro de 2011, foram coletadas, quinzenalmente, cinco amostras aleatórias de quatro folhas em talhões de milho Bt, contendo a proteína Cry 1F e Cry 1 A(b), e de milho não Bt em áreas experimentais da Embrapa Milho e Sorgo, em Sete Lagoas, MG. As amostras de folhas foram vistoriadas por 15 minutos na região da nervura central, em busca de adultos de C. tricholaenae. Foram registrados 2.930 indivíduos de C. tricholaenae, sendo que 1.114 no milho Bt Cry 1F, 753 em Cry 1 A(b) e 1063 indivíduos em folhas das cultivares não Bt. As maiores abundâncias populacionais médias ocorreram nos meses de novembro e dezembro. Os fatores estágio fenológico das plantas e precipitação afetaram positivamente a abundância de C. tricholaenae. A abundância média do período de coleta de C. tricholaenae foi reduzida pela cultivar de milho contendo a proteína Cry 1 A(b). Esse é o primeiro registro de ácaros sobre cultivares de milho transgênico no Brasil.

The objective of this study was to evaluate the occurrence of "microácaro-da-face-inferior-das-folhas-de-milho" Catarhinus tricholaenae Keifer (Acari: Diptilomiopidae) on transgenic cultivars of corn containing proteins Cry1F and Cry 1 A (b) and non-Bt corn. During the period from June 2010 to January 2011 were collected, every two weeks, five random samples of four leaves in plots of Bt corn containing the protein Cry 1F and Cry 1 A (b) and non-Bt corn in the experimental area of Embrapa Corn and Sorghum, Sete Lagoas, MG. The leaf samples were examined for 15 minutes in the central region of leaf in search of adult C. tricholaenae. We recorded 2930 individuals of C. tricholaenae, 1114 on Bt Cry 1F, 752 on Cry 1 A (b) and 1063 individuals on leaves of non-Bt cultivars. The highest average population abundances occurred in the months of November and December. Factors of plant phenological stage and rainfall positively affected the abundance of C. tricholaenae. The average abundance of the collection period of C. tricholaenae was reduced by cultivar corn containing Cry 1 A (b) protein. This is the first report of mites on the cultivars of transgenic corn in Brazil.