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Potato (Solanum tuberosum L.) cv. Sante was grown over 2 years under both conventional and organic fertiliser and crop protection regimes. The tuber metabolome was analysed using mass-spectrometry (MS) based approaches, principally liquid chromatography (LC)–MS and gas chromatography (GC)–MS. Data were analysed using Principal Components Analysis (PCA) and Analysis of Variance (ANOVA) to assess any differences between production practices. GC–MS analysis of non-polar metabolites did not detect any statistically significant differences, but GC–MS analysis of polar compounds identified 83 metabolites showing significant differences in the metabolome between the fertiliser treatments. Of the 62 metabolites that were less abundant in tuber samples from organic compared with conventionally fertilised crops, consistent year on year differences were dominated by free amino acids. The effect on free amino acids is associated with the lower nitrogen (N) content of the organically grown potatoes in this instance (50 % lower than for conventional production). LC–MS provided indications that levels of certain glycoalkaloids may be lower under the organic fertiliser regime in one growing season. Differences associated with the crop protection measures used were much less consistent, and relatively small, compared with the fertiliser effects found.

This manuscript provides insights into ongoing debates on the regulatory issues surrounding groups of biotechnology-driven 'New Plant Breeding Techniques' (NPBTs). It presents the outcomes of preliminary discussions and in some cases the initial decisions taken by regulators in the following countries: Argentina, Australia, Canada, EU, Japan, South Africa and USA. In the light of these discussions we suggest in this manuscript a structured approach to make the evaluation more consistent and efficient. The issue appears to be complex as these groups of new technologies vary widely in both the technologies deployed and their impact on heritable changes in the plant genome. An added complication is that the legislation, definitions and regulatory approaches for biotechnology-derived crops differ significantly between these countries. There are therefore concerns that this situation will lead to non-harmonised regulatory approaches and asynchronous development and marketing of such crops resulting in trade disruptions.

This commentary focuses on the potential added value of and need for (sub)-chronic testing of whole genetically modified (GM) foods in rodents to assess their safety. Such routine testing should not be required since, due to apparent weaknesses in the approach, it does not add to current risk assessment of GM foods. Moreover, the demand for routine testing using animals is in conflict with the European Union (EU) Commission's efforts to reduce animal experimentation. Regulating agencies in the EU are invited to respect the sound scientific principles applied to the risk assessment of foods derived from GM plants and not to interfere in the risk assessment by introducing extra requirements based on pseudo-scientific or political considerations.

New plant breeding techniques challenge the current regulation of genetically modified crops. Scientists and regulators must find new ways to guarantee safety whilst supporting innovation and agriculture.

Potato plants (Solanum tuberosum L. cvs Desiree and Record) transformed with sense and antisense constructs of a cDNA encoding the potato fructokinase StFK1 exhibited altered transcription of this gene, altered amount of protein and altered enzyme activities. Measurement of the maximal catalytic activity of fructokinase revealed a 2-fold variation in leaf (from 90 to 180% of wild type activity) and either a 10- or 30-fold variation in tuber (from 10 or 30% to 300% in Record and Desiree, respectively) activity. The comparative effect of the antisense construct in leaf and tuber tissue suggests that this isoform is only a minor contributor to the total fructokinase activity in the leaf but the predominant isoform in the tuber. Antisense inhibition of the fructokinase resulted in a reduced tuber yield; however, its overexpression had no impact on this parameter. The modulation of fructokinase activity had few, consistent effects on carbohydrate levels, with the exception of a general increase in glucose content in the antisense lines, suggesting that this enzyme is not important for the control of starch synthesis. However, when metabolic fluxes were estimated, it became apparent that the transgenic lines display a marked shift in metabolism, with the rate of redistribution of radiolabel to sucrose markedly affected by the activity of fructokinase. These data suggest an important role for fructokinase, acting in concert with sucrose synthase, in maintaining a balance between sucrose synthesis and degradation by a mechanism independent of that controlled by the hexose phosphate-mediated activation of sucrose phosphate synthase.

Germplasm of Solanum tuberosum and Solanum phureja exhibit a wide (over 20-fold) variation in tuber carotenoid content. The levels of carotenoids during tuber development and storage were compared in a high carotenoid-accumulating S. phureja accession (DB375\1) with two S. tuberosum cultivars (Pentland Javelin and Desiree) that accumulate lower levels of tuber carotenoid. On a dry weight basis, total carotenoid levels were at a maximum early in tuber development. However, in the S. phureja accession, carotenoid levels remained at a high level throughout tuber development, whereas in the S. tuberosum accessions, carotenoid content decreased as dry weight increased. The carotenoid profiles of tissues during tuber development were analysed in greater detail by reverse phase HPLC. In S. phureja tubers at maturity the major carotenoids were zeaxanthin, antheraxanthin, and violaxanthin. Following 9 months storage at 4 degrees C the levels of zeaxanthin and antheraxanthin decreased, whereas the level of lutein increased; overall, however, there was only a small decrease in total carotenoid content. In order to explore the reasons for the wide variation in tuber carotenoid content, the expression patterns of the major genes encoding the enzymes of the carotenoid biosynthetic pathway were compared. Significant differences in the profiles were detected, suggesting that transcriptional control or mRNA stability gives rise to the large differences in tuber carotenoid content. In particular, there was an inverse trend between the level of zeaxanthin epoxidase transcript level and tuber carotenoid content in a range of potato germplasm, giving rise to an hypothesis for the regulation of carotenogenesis in potato tubers.

Limit dextrinase (EC 3.2.1.41) from germinating barley (Hordeum vulgare L) can be activated by millimolar concentrations of linear maltodextrins with a degree of polymerisationor = 2. The activation was assay-dependent; it was detected using assays based on the solubilisation of cross-linked dyed pullulan but not in assays that directly measured cleavage events such as the formation of new reducing termini. This strongly suggested that maltodextrins did not increase the catalytic rate of limit dextrinase i.e. this is not a true activation. On the other hand, considerable activation was noted in assays that measured pullulan degradation by reduction in viscosity. Taken together, this suggested that maltodextrins altered the mode of action of limit dextrinase, causing more rapid decreases in viscosity or greater solubilisation of dye-linked pullulan fragments per cleavage event. The proposed mechanism of activation by alteration in action pattern was reminiscent of initial work in the discovery of xyloglucan endotransglycosylase. Therefore, the ability of limit dextrinase to catalyse transglycosylation reactions into pullulan was tested and confirmed by an assay based on the incorporation of a fluorescently labelled maltotriose derivative into higher-molecular-weight products. The transglycosylation reaction was dependent on limit dextrinase activity and was enhanced in more highly purified preparations of limit dextrinase. Transglycosylation was inhibited by unlabelled maltotriose. How transglycosylation accounts for the apparent activation of limit dextrinase by maltodextrins and the physiological relevance of this novel reaction are discussed.

The gene coding for agglutinin from Galanthus nivalis (GNA) was expressed in, and secreted by, the methylotrophic yeast, Pichia pastoris. Transformants of P. pastoris were selected and a process to produce and purify gram quantities of recombinant GNA was developed. GNA was secreted at approximately 80 mg l−1 at the 200 l scale and was purified to 95% homogeneity using hydrophobic interaction chromatography. The recombinant protein was similar to the protein synthesised in plant with respect to structure and biological activity.

The influence of urease activity on N distribution and losses after foliar urea application was investigated using wild-type and transgenic potato (Solanum tuberosum cv Désirée) plants in which urease activity was down-regulated. A good correlation between urease activity and 15N urea metabolism (NH3accumulation) was found. The general accumulation of ammonium in leaves treated with urea indicated that urease activity is not rate limiting, at least initially, for the assimilation of urea N by the plant. It is surprising that there was no effect of urease activity on either N losses or 15N distribution in the plants after foliar urea application. Experiments with wild-type plants in the field using foliar-applied 15N urea demonstrated an initial rapid export of N from urea-treated leaves to the tubers within 48 h, followed by a more gradual redistribution during the subsequent days. Only 10% to 18% of urea N applied was lost (presumably because of NH3 volatilization) in contrast to far greater losses reported in several other studies. The pattern of urease activity in the canopy was investigated during plant development. The activity per unit protein increased up to 10-fold with leaf and plant age, suggesting a correlation with increased N recycling in senescing tissues. Whereas several reports have claimed that plant urease is inducible by urea, no evidence for urease induction could be found in potato.

In order to elucidate more fully the function of a potato gene (MAL1) encoding alpha-glucosidase activity, transgenic plants in which MAL1 expression was down-regulated were generated using antisense technology. In transgenic lines severely down-regulated in the expression of MAL1, total alpha-glucosidase activity was not decreased in leaves and tubers, and the contents of starch, glucose, fructose and sucrose remained unchanged in tubers. Phylogenetic analysis indicated that the MAL1 gene product was more similar to the glycoprotein-processing alpha-glucosidase II of mammalian and yeast origin than to other plant alpha-glucosidases. Using [14C-Glc]-labelled Glc2Man9GlcNAc2 as a substrate, it was demonstrated that glucosidase II activity was markedly down-regulated in microsomes isolated from tubers of four independent antisense lines studied in detail, strongly suggesting that MAL1 encodes glucosidase II activity. In field trials (but not in the glasshouse), MAL1 down-regulation produced an extremely stunted phenotype - the leaves were curled and tuber yield was decreased by 90% compared to control values. Microscopic analysis of leaves revealed significant differences between the antisense and control samples. Plants with down-regulated glucosidase II activity showed a greater degree of plasmolysis, and an increase in the size of mesophyll intracellular spaces. Analysis of cell walls also indicated changes in structure as a result of MAL1 down-regulation. In leaves from four antisense lines, the steady-state transcript level corresponding to the endoplasmic reticulum chaperone, BiP, was enhanced. This is diagnostic of stress in the endoplasmic reticulum.

Processes that contribute to the overall phenomenon of fruit ripening are not well understood for many soft fruit species, including raspberry (Rubus idaeus L.) Recent biochemical data implicate ethylene and a range of cell wall hydrolases in ripening processes. However, the genes encoding these activities, and others related to ripening, remain to be characterised. With the advent of high-throughput RNA-fingerprinting techniques it is possible to characterise rapidly the changes in gene expression during developmental processes. This paper describes the application of two RNA-fingerprinting techniques (cDNA amplified fragment length polymorphism and differential display reverse transcription-polymerase chain reaction) to the ripening fruit of Rubus idaeus. Copy-DNA tags were isolated representing 34 genes, up-regulated during fruit ripening. The expression profiles of these genes were determined by RNA-blot analysis and their sequences were compared with those in public databases. Potential roles for some of these gene products are considered, providing valuable insights into the processes that underpin fruit ripening. Many of the cDNAs isolated in this study provide tools for the biotechnological improvement of fruit quality.

Red algae (Rhodophyceae) are photosynthetic eukaryotes that accumulate starch granules in the cytosol. Starch synthase activity in crude extracts of Gracilaria tenuistipitata Chang et Xia was almost 9-fold higher with UDP[U-14C]glucose than with ADP[U-14C]glucose. The activity with UDP[U-14C]glucose was sensitive to proteolytic and oxidative inhibition during extraction whilst the activity with ADP[U-14C]glucose appeared unaffected. This indicates the presence of separate starch synthases with different substrate specificities in G. tenuistipitata. The UDPglucose: starch synthase was purified and characterised. The enzyme appears to be a homotetramer with a native M(r) of 580 kDa and displays kinetic properties similar to other alpha-glucan synthases such as stimulation by citrate, product (UDP) inhibition and broad primer specificity. We propose that this enzyme is involved in cytosolic starch synthesis in red algae and thus is the first starch synthase described that utilises UDPglucose in vivo. The biochemical implications of the different compartmentalisation of starch synthesis in red algae and green algae/plants are also discussed.

S-Adenosylmethionine decarboxylase (SAMDC; EC 4.1.1.50) is a key enzyme in the biosynthesis of the polyamines spermidine and spermine from putrescine and its activity is rate limiting in this pathway. Transgenic potato (Solanum tuberosum L. cv. Desiree) plants containing both sense and antisense SAMDC constructs driven by the tuber-specific patatin promoter have been generated and analysed. In sense transformants, developing tubers expressed higher steady-state levels of the SAMDC-specific transcript, had higher levels of SAMDC activity and contained significantly higher levels of spermidine than vector-transformed controls. Additionally, there was a significant shift in tuber size distribution with larger numbers of smaller tubers but no overall change in tuber yield. In developing tubers from the antisense transformed lines, there was a decrease in SAMDC transcript level, SAMDC activity and total polyamine levels. However, no obvious phenotypic effect was detected in the tuberisation physiology of the antisense lines.

Risk assessment of genetically modified organisms (GMOs) remains a contentious area and a major factor influencing the adoption of agricultural biotech. Methodologically, in many countries, risk assessment is conducted by expert committees with little or no recourse to databases and expert systems that can facilitate the risk assessment process. In this paper we describe DTREEv2, a computer-based decision support system for the identification of hazards related to the introduction of GM-crops into the environment. DTREEv2 structures hazard identification and evaluation by means of an Event-Tree type of analysis. The system produces an output flagging identified hazards and potential risks. It is intended to be used for the preparation and evaluation of biosafety dossiers and, as such, its usefulness extends to researchers, risk assessors and regulators in government and industry.

Conventional plant breeding exploits existing genetic variability and introduces new variability by mutagenesis. This has proven highly successful in securing food supplies for an ever-growing human population. The use of genetically modified plants is a complementary approach but all plant breeding techniques have limitations. Here, we discuss how the recent evolution of targeted mutagenesis and DNA insertion techniques based on tailor-made site-directed nucleases (SDNs) provides opportunities to overcome such limitations. Plant breeding companies are exploiting SDNs to develop a new generation of crops with new and improved traits. Nevertheless, some technical limitations as well as significant uncertainties on the regulatory status of SDNs may challenge their use for commercial plant breeding.

A molecular approach has been used to study the role of polyamines in plant development by manipulating the expression of the S-adenosylmethionine decarboxylase (SAMDC) gene. SAMDC is a key enzyme involved in the biosynthesis of the polyamines spermidine and spermine and is also known to influence the rate of biosynthesis of ethylene. Previously, a cDNA clone of the SAMDC gene of potato has been isolated and characterized. This cDNA clone has been used to make antisense and sense SAMDC constructs under the control of the 35S CaMV or tetracycline-inducible promoters. Agrobacterium-mediated transformation has been used to produce transgenic potato plants with the engineered antisense and sense SAMDC genes in order to downregulate or overexpress the SAMDC transcript, respectively. Decreases or increases in the level of SAMDC transcript in the antisense and sense transgenic plants were observed, respectively. Antisense transgenic plants which expressed the engineered SAMDC gene constitutively under the control of the 35S CaMV promoter with a duplicated enhancer region showed a range of stunted phenotypes with highly branched stems, short internodes, small leaves and inhibited root growth. The abnormal characteristics of the antisense plants correlated with the altered levels of SAMDC transcript, SAMDC activity, polyamine content and rate of ethylene evolution. Attempts to produce sense transgenic plants with the 35S SAMDC sense construct were unsuccessful indicating that a constitutive overexpression of the engineered SAMDC is lethal to the plants. When antisense and sense SAMDC transgenes were expressed under the control of a tetracycline-inducible promoter a number of transgenic plants were produced. In these antisense and sense plants, significant changes in the level of SAMDC transcript, SAMDC activity and free polyamine content were observed on tetracycline-induction of detached leaves.

The importance of standardizing on sampling procedures was shown by examining diurnal variation of petiole sap nitrate-N concentration, [NO3--N], and its variation within the plant. There was important variability in petiole sap [NO3--N] about the general decline in values through the season, in time, between cultivars, between nitrogen fertilizer treatments, and between replicates. We have shown the lack of a consistent relation between petiole sap [NO3--N] and rates of uptake of nitrogen either in the period around the time of sampling, or integrated over the whole growing season. Finally, we have examined the literature and have been unable to find reports of critical, independent tests of the use of petiole sap [NO3-] to guide the application of fertilizer supplements. We conclude that a new, critical attitude should be taken to testing petiole sap [NO3-] if the technique is to be used properly and to its seeming potential. Comparisons were made between measurements using a hand-held system and an auto-analyser.

EFSA’s remit in the risk assessment of GMOs is very broad encompassing genetically modified plants, microorganisms and animals and assessing their safety for humans, animals and the environment. The legal frame for GMOs is set by Directive 2001/18/EC on their release into the environment, and Regulation (EC) No 1829/2003 on GM food and feed. The main focus of EFSA’s GMO Panel and GMO Unit lies in the evaluation of the scientific risk assessment of new applications for market authorisation of GMOs, and in the development of corresponding guidelines for the applicants. The EFSA GMO Panel has elaborated comprehensive guidance documents on GM plants, GM microorganisms and GM animals, as well as on specific aspects of risk assessment such as the selection of comparators. EFSA also provides special scientific advice upon request of the European Commission; examples are post-market environmental monitoring of GMOs, and consideration of potential risks of new plant breeding techniques. The GMO Panel regularly reviews its guidance documents in the light of experience gained with the evaluation of applications, technological progress in breeding technologies and scientific developments in the diverse areas of risk assessment.

This scientific opinion reports an evaluation of a risk assessment for placing on the market the genetically modified (GM) insect-resistant maize MIR162 for food and feed uses, import and processing. Maize MIR162 contains a single insert consisting of the vip3Aa20 and pmi expression cassettes. The Vip3Aa20 protein confers resistance against certain lepidopteran pests and phosphomannose isomerase (PMI) serves as a selection marker. Bioinformatic analyses of the inserted DNA and flanking regions do not raise safety concerns. The levels of Vip3Aa20 and PMI proteins in maize MIR162 have been sufficiently analysed. The stability of the genetic modification has been demonstrated. Comparative analyses established that there are no biologically relevant differences in the compositional, agronomic or phenotypic characteristics of maize MIR162 compared with its conventional counterpart, and its composition falls within the range of non-GM commercial varieties, except for the expression of the Vip3Aa20 and PMI proteins. The safety assessment identified no concerns regarding potential toxicity and allergenicity of Vip3Aa20 and PMI proteins or of maize MIR162. A feeding study on broiler chickens confirmed that grain produced by maize MIR162 is as nutritious as that produced by its conventional counterpart and a non-GM commercial variety. There are no indications of an increased likelihood of establishment and spread of feral maize plants. Considering its intended use as food and feed, interactions with the biotic and abiotic environment were not considered to be an issue. Risks associated with an unlikely but theoretically possible horizontal gene transfer from maize MIR162 to bacteria have not been identified. The monitoring plan and reporting intervals are in line with the intended uses of maize MIR162. In conclusion, the EFSA GMO Panel considers that information available for maize MIR162 addresses scientific comments raised by Member States and that maize MIR162, as described in this application, is as safe as its conventional counterpart and non-GM commercial varieties with respect to potential effects on human and animal health and the environment in the context of its intended uses.

This Scientific Opinion reports on an evaluation of a risk assessment for the placing on the market for cultivation of genetically modified soybean 40-3-2, and updates the previous EFSA GMO Panel Scientific Opinion on the renewal applications for the continued marketing of soybean 40-3-2. The EFSA GMO Panel considered that soybean 40-3-2 is unlikely to raise additional environmental safety concerns compared with conventional soybean, but that the management of its cultivation could result in environmental harm under certain conditions. The Panel therefore recommended managing the use of glyphosate on soybean 40-3-2 in ways that result in similar or reduced environmental impacts compared with conventional soybean cultivation. There is no evidence of adverse effects on non-target organisms (including pollinators) due to the expression of the CP4 EPSPS protein, and there are no indications of the occurrence of adverse effects on non-target predators, herbivores and decomposers due to potential unintended changes in soybean 40-3-2. Owing to the lack of event-specific data on plant-pollinator interactions, scientific uncertainty on the occurrence of adverse effects on pollinators, due to potential unintended changes in soybean 40-3-2, remains, and strategies for resolving this uncertainty are discussed. The Panel recommended the deployment of case-specific monitoring to address: (1) changes in weed community diversity; and (2) the evolution of resistance to glyphosate in weeds due to changes in herbicide and cutivation regimes. The Panel agreed with the general surveillance plan of the applicant, but requested that the Panel‟s proposals to strengthen general surveillance are implemented. The Panel concluded that the information available for soybean 40-3-2 addresses the scientific comments raised by Member States and that soybean 40-3-2, as described in this application, is as safe as its conventional counterpart and commercial non-GM soybean varieties with respect to potential adverse effects on human and animal health. If subjected to appropriate management measures, the cultivation of soybean 40-3-2 is unlikely to have environmental effects any more adverse than those associated with conventional soybean cultivation.

Austria notified to the European Commission its ordinance implementing a national safeguard measure prohibiting the placing on the market of GM potato EH92‐527‐1 for cultivation purposes in Austria, after which the European Commission asked the European Food Safety Authority to assess the scientific elements supporting the prohibition. Having considered the information package provided by Austria and all relevant scientific publications, the GMO and BIOHAZ Panels concluded that: i) no new data specific to the safety of the nptII gene have been provided; ii) the risk posed by the formation of mosaic structures of aminoglycoside phosphotransferase genes could not be assessed without data documenting the existence of such structures among the existing gene variants and such data were not provided; iii) the therapeutic relevance of kanamycin and neomycin was already addressed in the EFSA's opinion on ARM genes and as of yet there is no evidence to indicate that resistance to these antibiotics in clinically‐relevant bacteria has developed as a result of acquisition of the nptII gene; iv) the knowledge gaps and uncertainties highlighted in the Austrian document have already been considered in the EFSA's opinion on ARM genes. Austria did not provide any new or additional information on the molecular characterisation or PMEM of potato EH92‐527‐1 after the date of consent for this GM event that would require the reassessment of existing information. The EFSA GMO Panel reiterates its scientific opinion on the 2010 monitoring report of GM potato EH92‐527‐1 in which it provides specific recommendations to improve the methodology of the PMEM of the GM potato. Further, the EFSA GMO Panel concludes that no grounds exist to date that would lead to reconsideration of its opinion on GM potato EH92‐527‐1.

This scientific opinion evaluates the risk assessment for the authorisation for (continued) marketing of genetically modified insect‐resistant and herbicide‐tolerant cotton MON 531 × MON 1445 for food and feed produced from it. Cotton MON 531 × MON 1445 was produced by conventional crossing methods, and the stack is homozygous for the newly introduced traits. The integrity and the stable co‐inheritance of the parental events were demonstrated in the stack. Molecular characterisation did not reveal safety issues. No biologically relevant differences were identified in the compositional, phenotypic and agronomic characteristics of cotton MON 531 × MON 1445 in comparison with its conventional counterpart and its composition fell within the range of non‐GM cotton varieties, except for the Cry1Ac, CP4 EPSPS and NPTII proteins. No safety issues were identified with regard to toxicity and allergenicity of food and feed produced from cotton MON 531 × MON 1445. A feeding study in catfish confirmed that toasted cottonseed meal from MON 531 × MON 1445 is as nutritious as toasted cottonseed meal from its conventional counterpart and four commercial non‐GM cotton varieties. Products from cotton MON 531 × MON 1445 do not contain viable plant parts. The insert structure in cotton MON 531 × MON 1445 may facilitate the stabilisation of the nptII gene in plasmids of environmental bacteria through double homologous recombination. However, considering the overall limited occurrence of horizontal transfer of DNA in plant material to bacteria, and the very low exposure to DNA from cotton MON 531 × MON 1445, the EFSA GMO Panel concludes that it is highly unlikely that cotton MON 531 × MON 1445 will contribute to the environmental prevalence of nptII genes. Potential interactions of cotton MON 531 × MON 1445 with non‐target organisms and the abiotic environment were not considered to be an issue because of low exposure levels. A post‐market environmental monitoring plan is not required.

The cloning of a 1332 bp cDNA from a potato (Solanum tuberosumL.) cv. Cara leaf cDNA expression library, using an antibody raised against a purified tuber protein preparation with sucrolytic activity, is described. The corresponding gene in potato is of low copy number, is expressed in a variety of tissues, and encodes a protein which includes several domains with similarity to database sequences, including ferredoxin from Clostridium pasteurianum. Expression of the cDNA in E. coli yields a fusion protein with sucrolytic activity.

Amino acid release from roots of sterile and non-sterile, solution-grown, 7-, 21- and 60-days-old forage rape plants (Brassica napus L.), was measured over periods of up to 6 hours. With sterile plants, release of amino acids into a fixed volume of collection medium (6, 12, 70 mL) was concentration-limited, giving rise to similar convex accumulation profiles for individual acids. In contrast, amino acid accumulation in continuously circulating collection medium was not concentration limited, giving a linear accumulation pattern. The compositions of accumulating amino acids, which were similar to those measured in root extracts, did not change significantly. However, the proportions of ALA, GABA, GLU and ILE in both root extracts and root-derived amino acids increased as plants aged. Older plants released more amino acids per plant, while younger plants released more amino acids g-1 root DW. Using non-sterile plants, the patterns of change in amino acid concentration and composition in the collection medium were completely different from those determined with sterile plants. In general, with 7-days-old plants, and 60-days-old plants that had recently become non-sterile, an initial rise in the concentration of all acids was followed by a fall to low levels. The loss of amino acids was apparently due to microbial consumption. Individual amino acids attained maximum concentration at different times during the collection process. This is attributed mainly to concentration-dependent differential assimilation of amino acids, since those with the highest initial concentrations, the major components of the mixtures released from roots, declined the earliest. When calculated rates of amino acid release from roots (Rr) and microbial consumption of amino acids (Rc) were compared (for 7-days-old plants), the highest ratios of Rc/Rr were found for ASN, ARG, GLU, GLN, and LYS. This suggests a degree of selectivity for glutamate and nitrogen-rich acids on the part of the consuming micro-organisms. With 21-days old plants and 60-days old plants grown entirely under non-sterile conditions, fluctuations in amino acid concentration were similar for all acids.

Amino acid loss from the roots of 25-day-old, sterile and non-sterile sand-grown forage rape plants, was determined over periods of up to 3.5 hours. Amino acid accumulation in the root-zone of sterile plants was concentration-dependent giving a convex accumulation profile. Amino acid levels in the root zone of non-sterile plants rapidly attained steady state values. Microbial assimilation of amino acids within the root zone appeared to lower amino acid concentrations, resulting in an underestimation of rates of amino acid loss from roots. The concentrations of most amino acids were higher after selected amino acids were supplied to the root zone. The response to exogenous acids was dependent on the concentration and composition of the acids added. Addition of a mixture containing ASN, GLN and GABA, each at 0.25 mM resulted in a greater increase in individual and total acid levels compared with a mixture containing ALA, SER, GLY and THR at the same concentration. Apparently, amino acids supplied exogenously competed with acids lost from the plant, by providing an alternative nutrient source for root zone micro-organisms. Addition of glucose and citric acid had a similar effect to addition of ALA, SER, GLY and THR, but were less effective than ASN, GLN and GABA at all concentrations tested. The nitrogen-rich amino acids ASN and GLN, and the γ-amino acid, GABA, appeared to compete more effectively with plant-derived acids than did ALA, SER, GLY and THR, the most abundent constituents of the plant-derived acids, which had the highest calculated rates of microbial consumption. Therefore, although bacterial consumption showed a dependence on amino acid concentration, a degree of selectivity for nitrogen-rich acids and gaba was also apparent.

In this Scientific Opinion, the EFSA GMO Panel supplements its previous evaluations of the potential impact of maize 1507 cultivation on a range of non‐target lepidopteran species using existing data on species susceptibility and considering various scenarios of exposure which may occur across Europe. The mathematical model, developed for maize MON 810, was recalibrated and extended to estimate the efficacy of certain mitigation measures. In situations where highly sensitive non‐target Lepidoptera populations might be at risk, the EFSA GMO Panel recommends that mitigation measures are adopted to reduce exposure. Risk managers are provided with tools to estimate global and, where needed local, mortality of exposed non‐target Lepidoptera, both before and after different mitigation measures are put in place, and for different host‐plant densities. Mitigation measures are only needed when the proportion of maize and uptake of maize 1507 are sufficiently high, regardless of the other parameters. If maize 1507 cultivation remains below 5% of the Utilized Agricultural Area, then risk mitigation measures are not required. In addition, the EFSA GMO Panel recommends case‐specific monitoring to assess the efficacy of risk mitigation measures put in place to reduce levels of risk and scientific uncertainty for (1) the possible resistance evolution to the Cry1F protein in target pests, and (2) the risk to sensitive non‐target Lepidoptera from maize 1507 pollen. The EFSA GMO Panel also considers that the plan for general surveillance, and in particular the methodology, needs further details according to the requirements of its 2011 Guidance Document on post‐market environmental monitoring of generically modified plants, as well as its Scientific Opinion on the annual 2009 monitoring report on maize MON 810. The EFSA GMO Panel concludes that, subject to appropriate management measures, maize 1507 cultivation is unlikely to raise safety concerns for the environment.

This Scientific Opinion reports on an evaluation of a risk assessment for placing on the market of genetically modified maize MON 88017 for cultivation. The EFSA GMO Panel considers that maize MON 88017 is unlikely to have any adverse effect on the environment, except for the possible resistance evolution to the Cry3Bb1 protein in coleopteran target pests; resistance evolution may lead to altered pest control practices that may cause adverse environmental effects. The cultivation management of maize MON 88017 could result in environmental harm. The EFSA GMO Panel therefore recommends managing the use of glyphosate on maize MON 88017 within diversified cropping regimes that have similar or reduced environmental impacts compared with conventional maize cultivation. The EFSA GMO Panel recommends the deployment of insect resistance management strategies and case‐specific monitoring to address (1) the possible resistance evolution to the Cry3Bb1 protein in coleopteran target pests, (2) changes in botanical diversity within fields due to novel herbicide regimes, and (3) resistance evolution to glyphosate in weeds due to novel herbicide regimes. The EFSA GMO Panel agrees with the general surveillance plan of the applicant, but requests that the proposals made to strengthen general surveillance are implemented. Whilst the scope of this application only covers the cultivation of maize MON 88017, this Scientific Opinion also updates the previous EFSA GMO Panel safety evaluation of the food and feed uses, import and processing of maize MON 88017 and derived products. The EFSA GMO Panel concludes that the information available for maize MON 88017 addresses the scientific comments raised by Member States and that maize MON 88017, as described in this application, is as safe as its conventional counterpart and commercial maize varieties with respect to potential adverse effects on human and animal health. If subjected to appropriate management measures, the cultivation management of maize MON 88017 is unlikely to raise safety concerns for the environment.

In this statement the EFSA GMO Panel responds to a request from the European Commission on whether data provided within application RX‐MON810 on maize MON810 are sufficient to conclude on the safety of MON810 pollen as or in food and, if it is the case, to confirm MON810 pollen safety. Data on molecular characterisation did not identify features of maize MON810 pollen with a potential to raise any safety concerns. The EFSA GMO Panel has previously assessed the safety of the Cry1Ab protein in MON810 and the assessment and conclusions of the GMO Panel on safety of the protein Cry1Ab (including toxicity and allergenicity) reached for food/feed aspects also apply to pollen. While the EFSA GMO Panel is not in a position to conclude on the safety of maize pollen in or as food in general, it concludes that the genetic modification in MON810 maize does not constitute an additional health risk if MON810 maize pollen were to replace maize pollen from non‐GM maize in or as food.

modified insect resistant cotton MON531 for food and feed produced from it. MON531 was transformed via Agrobacterium. It contains single copies of functional Cry1Ac and NPTII expression cassettes; two fragments of the Cry1Ac cassette and aadA as non-functional elements. Stability of the inserted DNA was confirmed over several generations. Bioinformatic analyses and the levels of recombinant proteins did not reveal safety concerns. Analysis of compositional, phenotypic and agronomic characteristics indicated that MON531 is not different from its conventional counterpart and is compositionally within the range observed among conventional cotton varieties, except for Cry1Ac and NPTII. Safety assessment of Cry1Ac and NPTII proteins and cotton MON531 identified no concerns regarding potential toxicity and allergenicity. Products from MON531 do not contain viable plant parts. The aadA and oriV sequences in MON531 may facilitate the stabilisation of nptII through double homologous recombination in plasmid sequences in the environment. However, considering the expected low frequency of gene transfer from MON531 to bacteria compared to that between bacteria, and the very low exposure to MON531 DNA, the GMO Panel concludes that gene transfer from MON531 to bacteria does not indicate a risk to human or animal health or to the environment in the context of its intended uses. The exposure of potentially sensitive non-target organisms to Cry1Ac protein is likely to be low and of no biological relevance. A PMEM plan is not required. The EFSA GMO Panel considers that information available for cotton MON531 addresses the questions raised by the Member States and that cotton MON531, as described in this application, is as safe as its conventional counterpart and is unlikely to have adverse effects on human and animal health and the environment in the context of its intended uses.

This scientific opinion is an evaluation of a risk assessment of the genetically modified, insect resistant, soybean MON 87701 for food and feed uses, import and processing. Soybean MON 87701 was developed through Agrobacterium‐mediated transformation. It contains a single insert consisting of a cry1Ac expression cassette, encoding the Cry1Ac protein that confers resistance against specific lepidopteran insects. The stability of the insert was confirmed over multiple generations. Bioinformatic analyses of the insert and its flanking regions, and levels of newly expressed protein did not raise safety concerns. Comparative analyses of compositional, phenotypic and agronomic characteristics indicated that soybean MON 87701 is not different from its conventional counterpart (A5547) and equivalent to commercial soybean varieties, except for having an increased vitamin E content (still within normal ranges) and expressing the Cry1Ac protein. The safety assessment of the Cry1Ac protein and soybean MON 87701 identified no concerns regarding potential toxicity and allergenicity. A feeding study on broiler chickens confirmed that defatted soybean MON 87701 meal is as nutritious as conventional defatted soybean meal. There are no indications of an increased likelihood of establishment and spread of feral soybean plants. Considering its intended use as food and feed, environmental risks associated of an unlikely but theoretically possible horizontal gene transfer from soybean MON 87701 to bacteria have not been identified. Potential interactions of soybean MON 87701 with the biotic and abiotic environment were not considered due to the low level of exposure. The monitoring plan and reporting intervals are in line with the intended uses of soybean MON 87701. The EFSA GMO Panel considers that the soybean MON 87701, as described in this application, is as safe as its conventional counterpart with respect to potential effects on human and animal health and the environment in the context of its intended uses.

This scientific opinion is an evaluation of a risk assessment for the genetically modified herbicide tolerant soybean 356043 for food and feed uses, import and processing. Soybean 356043 contains a single copy of intact gat4601 and Glycine max‐hra cassettes at a single insertion locus. The results of the bioinformatic analyses of the insert and the flanking regions, and the levels of newly expressed proteins did not raise a safety concern. The comparative analysis of phenotypic and agronomic characteristics indicated that soybean 356043 is not different from its conventional counterpart. In the composition, differences were identified between 356043 soybean and its conventional counterpart in the newly expressed proteins Glycine max‐HRA and GAT4601, and the levels of the fatty acids heptadecanoic, heptadecenoic and heptadecadienoic acid and the acetylated amino acids N‐acetylaspartate (NAA) and N‐acetylglutamate (NAG). The safety assessment of the newly expressed proteins Glycine max‐HRA and GAT4601 identified no concerns regarding potential toxicity and allergenicity. Heptadecanoic, heptadecenoic and heptadecadienoic acid are present in the diet and the intake of small amounts of these fatty acids via food or feed is not expected to produce adverse effects. NAA and NAG are normal constituents in the mammalian metabolism and the estimated increases in their intake are considered low when related to the normal intake of L‐aspartic acid and L‐glutamic acid. Further toxicological, allergenicity and nutritional analysis provided no indications of adverse effects. There are no indications of an increased likelihood of establishment and spread of feral soybean plants, except in the presence of the glyphosate and ALS‐inhibiting herbicides neither a risk caused by a possible transfer of the recombinant gene from soybean 356043 to environmental micro‐organisms. The EFSA GMO Panel considers that the information available for soybean 356043 addresses the scientific comments raised by the Member States and states that the soybean 356043, as described in this application, is as safe as its conventional counterpart with respect to potential effects on human and animal health and the environment in the context of its intended uses.

This opinion provides guidance in the area of comparators taking into account the requirements for the molecular characterisation, the food and feed and the environmental risk assessments. A key step in the risk assessment of genetically modified (GM) plants and derived food and feed is the identification of intended and unintended differences and equivalences between the GM plant and its comparator(s), taking into account the range of natural variation. In line with Regulation (EC) No 1829/2003 and Directive 2001/18/EC, the EFSA GMO Panel has, to date, required the use of non-GM lines with comparable genetic background as comparators. In the case of vegetatively propagated crops, these are the isogenic lines. In the case of sexually propagated crops these are non-GM lines as close as possible genetically to the GM plant under assessment. The identification and production of such comparators is becoming increasingly challenging due to the increasing complexity of GM plants, e.g. those developed by combining (stacking) events through conventional crosses, or those in which extensive compositional changes are targeted. Consequently, the EFSA GMO Panel has developed this guidance on the selection of comparators for the risk assessment of GM plants and derived food and feed. Whilst considering the requirements of Directive 2001/18/EC and Regulation (EC) No 1829/2003, the EFSA GMO Panel provides options which introduce flexibility in the selection of comparators based on sound scientific principles. This document addresses the selection of comparators for GM plants containing single or multiple events stacked by either conventional breeding, or by other approaches such as re-transformation, co-transformation and the use of multiple gene cassettes. The EFSA GMO Panel also considers situations where additional comparators may be required on a case-by-case basis and scenarios where appropriate comparators are not available (e.g. where extensive compositional changes are targeted). The EFSA GMO Panel recognises the different requirements for comparators for the molecular characterisation, food and feed and environmental components of the risk assessment.

This document provides updated guidance for the risk assessment of food and feed containing, consisting or produced from genetically modified (GM) plants, submitted within the framework of Regulation (EC) No 1829/2003 on GM food and feed. The risk assessment strategy for GM plants and derived food and feed proposed seeks to deploy appropriate approaches to compare GM plants and derived food and feed with their respective comparators. The underlying assumption of this comparative approach is that traditionally cultivated crops have gained a history of safe use for consumers and/or domesticated animals. The document provides guidance on how to perform the comparative analysis of the relevant characteristics of the GM plant. The document addresses the details of the different components of the risk assessment: the molecular characterisation, which provides information on the structure and expression of the insert(s) and on the stability of the intended trait(s); the toxicological assessment, which addresses the impact of biologically relevant change(s) in the GM plant and/or derived food and feed resulting from the genetic modification; the assessment of potential allergenicity, of the novel protein(s) as well as of the whole food derived from the GM plant; the nutritional assessment to evaluate whether food and feed derived from a GM plant is not nutritionally disadvantageous to humans and/or animals. In addition every section of the document addresses specifically the requirements for GM plants containing a combination of transformation events, providing guidance on how to establish that the combination is stable and that no interactions occurs between the events that may raise safety concerns. The document does not cover the environmental risk assessment of GM plants which is addressed in a stand-alone environmental risk assessment (ERA) guidance document developed by the EFSA GMO Panel.

This scientific opinion reports an evaluation of a risk assessment for placing on the market of insect resistant genetically modified (GM) cotton (Gossypium hirsutum L.) 281‐24‐236 × 3006‐210‐23 (Unique Identifier DAS‐24236‐5 × DAS‐21Ø23‐5) for food and feed uses, import and processing. The GM cotton 281‐24‐236 × 3006‐210‐23 has been produced by conventional crossing between lines of the single cotton events 281‐24‐236 and 3006‐210‐23. The cotton 281‐24‐236 × 3006‐210‐23 expresses combined traits encoded by cry1F in event 281‐24‐236 and by cry1Ac in event 3006‐210‐23, both genes conferring resistance to certain lepidopteran pests. Both single events contain pat genes used as a selectable marker. The structure of the inserts in the single cotton events was retained in the stack 281‐24‐236 × 3006‐210‐23. Expression levels of Cry1Ac, Cry1F and PAT proteins were demonstrated to be comparable to those of the respective single events. The results of comparative analyses indicated that cotton 281‐24‐236 × 3006‐210‐23 and its respective single events are compositionally, agronomically and phenotypically equivalent to its conventional counterpart, except for the expression of Cry1Ac, Cry1F and PAT proteins. The safety assessment identified no concerns regarding toxicity and allergenicity of cotton 281‐24‐236 × 3006‐210‐23. A feeding study on broiler chickens confirmed the nutritional equivalence of this GM cotton to its conventional counterpart and two commercial non‐GM cotton varieties. The intended uses of cotton 281‐24‐236 × 3006‐210‐23 exclude cultivation within the European Union. The environmental risk assessment is therefore restricted to the indirect exposure through manure and faeces mainly from animals fed with cotton products of 281‐24‐236 × 3006‐210‐23 and with the accidental release into the environment of cotton 281‐24‐236 × 3006‐210‐23 grains during transportation and processing. There are no indications of increased likelihood of establishment or survival of feral cotton plants. If accidental spillage and subsequent release into the environment of cotton 281‐24‐236 × 3006‐210‐23 seeds occur, cotton 281‐24‐236 × 3006‐210‐23 plants would have a selective advantage only under infestation of target pest species or in presence of glufosinate‐ammonium herbicides, which are not commonly used on cultivated cotton or in most areas where the GM cotton might be spilled. In conclusion, the EFSA GMO Panel considers that information available for cotton 281‐24‐236 × 3006‐210‐23 addresses the scientific comments raised by Member States and that the cotton 281‐24‐236 × 3006‐210‐23 as described in this application is as safe as its conventional counterpart and other appropriate comparators with respect to potential effects on human and animal health and the environment in the context of its intended uses. The EFSA GMO Panel concludes that cotton 281‐24‐236 × 3006‐210‐23 is unlikely to have any adverse effect on human and animal health and the environment, in the context of its intended uses.

This scientific opinion reports on an evaluation of an application (reference EFSA-GMO-RX-MON863[8.1.b/20.1.b]) for renewal of the authorisation for continued marketing of existing feed materials, feed additives and food additives produced from genetically modified (GM) insect resistant maize MON863. Maize MON863 has been modified with a gene encoding the Cry3Bb1 protein which confers protection against coleopteran pests, principally the corn rootworm (Diabrotica spp.). In addition, a selectable marker gene nptII encoding neomycin phosphotransferase II has been introduced. In 2004 the EFSA GMO Panel issued scientific opinions on a Notification for the placing on the market of GM maize MON863 for import and processing and on a request from the European Commission for the placing on the market of foods and food ingredients derived from GM maize MON863. In delivering the present opinion, the EFSA GMO Panel considered the information provided in the application EFSA-GMO-RX-MON863[8.1.b/20.1.b] as well as additional information provided by the applicant and information published in the scientific literature. The new data in the application included bioinformatic analyses using updated databases which confirmed that no relevant similarities exist between the newly expressed proteins and known allergens or proteins toxic to mammals. The EFSA GMO Panel has evaluated the new information provided by the applicant and the scientific literature and concluded that there was no new information that would require changes of its previous scientific opinions on maize MON863 (EFSA, 2004a,b). Therefore, the EFSA GMO Panel reiterates its previous conclusions that GM maize MON863 and its products which are the subject of this application are unlikely to have an adverse effect on human and animal health or the environment in the context of its intended uses (EFSA, 2004a,b).

According to Article 95(5) of the EC Treaty, Portugal notified to the European Commission its intention to declare the Autonomous Region of Madeira as a region free from the cultivation of GM plants. Portugal therefore proposed to introduce a regional decree to prohibit the cultivation of GM seeds and propagating plant material in the Autonomous Region of Madeira. Two reports were provided in support of the national measure. Following the notification of Portugal, the European Commission requested a scientific opinion from the Scientific Panel on Genetically Modified Organisms (GMO Panel) of the European Food Safety Authority (EFSA) to investigate whether the information mentioned in the supporting documents contains any new scientific evidence in terms of protection of the environment in the Autonomous Region of Madeira, that would justify such a prohibition of the use of GMOs, including those that have already been authorised under Directive 90/220/EEC or Directive 2001/18/EC. In its evaluation, the EFSA GMO Panel only investigated and commented on aspects that relate to the protection of human and animal health and the environment. Socio-economic aspects related to the cultivation of GM plants and the coexistence between cropping systems fall outside the remit of EFSA, and are not addressed in this scientific opinion. Following investigation of the evidence presented in the Portuguese submission, the EFSA GMO Panel did not identify new scientific information on the environmental or human and animal health impacts of EU approved GM plants. The EFSA GMO Panel therefore concludes that, based on the supporting documents submitted by Portugal, no new scientific evidence, in terms of risk to human and animal health and the environment, was provided that would justify a prohibition of the cultivation of GM plants in the Autonomous Region of Madeira.

Partial purification (approximately 270-fold) of sucrose synthase (EC 2.4.1.13) from developing cotyledons of Vicia faba L. cv Maris Bead was achieved by ammonium sulfate fractionation and hydrophobic, affinity, anion-exchange, and gel filtration chromatography. Further purification to homogeneity resulted from gel elution of single bands from native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was identified as a homotetramer with a total molecular mass of 360 kD and subunits of 92 to 93 kD. Antibodies were raised to both native and denatured protein. The identity of the polypeptide was confirmed in western blots using antibodies raised against soybean nodule sucrose synthase. The enzyme has a pH optimum of 6.4 (cleavage direction) and an isoelectric point of 5.5. The affinity of the enzyme for sucrose (K(m)) was estimated at 169 mm, and for UDP at 0.2 mm. With uridine diphosphate as the nucleoside diphosphate, the V(max) is 4-fold higher than with adenosine diphosphate. Fructose acts as a competitive inhibitor with an inhibitor constant (K(i)) of 2.48 mm.

Following a request from the European Commission, the Panel on Genetically Modified Organisms was asked to review the published scientific paper of Lu & Yang (2009) and to indicate whether this paper contains new information that would alter the previous EFSA GMO Panel environmental safety conclusions on the genetically modified rice event LLRice62. This scientific paper reviews vertical gene flow between cultivated GM rice and cross-compatible wild/weedy rice relatives and the potential ecological consequences thereof. In 2007, the EFSA GMO Panel issued a scientific opinion on the risk assessment evaluation of application EFSA/GMO/UK/2004/04 for the market authorisation of LLRice62 for food/feed uses, import and processing in the EU. In light of the new scientific paper provided by the European Commission and, having considered relevant scientific publications on vertical gene flow in rice, the EFSA GMO Panel concludes that, in terms of risk to the environment, no new scientific evidence has been provided that invalidates the previous environmental risk assessment evaluation of LLRice62 for its intended uses, which exclude cultivation.

Hybrid oilseed rape Ms8 x Rf3 has been produced by conventional crossing between the genetically modified (GM) parental lines Ms8 and Rf3. The hybrid system is achieved using a pollination control system by insertion and expression of barnase and barstar genes into two separate oilseed rape lines; each gene is linked to the bar gene conferring tolerance to glufosinate-containing herbicides. The scope of this application covers the continued marketing of existing food (processed oil) and feeds produced from oilseed rape Ms8, Rf3 and Ms8 x Rf3 which were lawfully placed on the market in the Community before the date of entry into force of Regulation (EC) No 1829/2003. In 2005 the EFSA GMO Panel had issued a scientific opinion on the safety of glufosinate-tolerant oilseed rape Ms8 x Rf3 derived from parental lines Ms8 and Rf3 for import and processing for feed and industrial uses. In delivering the present opinion, the EFSA GMO Panel considered the information provided in the application EFSA-GMO-RX-MS8-RF3 as well as additional information submitted by the applicant. The new data included molecular and bioinformatic analyses and a 42-day feeding study on chickens. The animal study showed that the tested material of oilseed rape Ms8 x Rf3 was nutritionally equivalent to its non-GM counterpart. In addition, bioinformatic studies using updated databases confirmed that no relevant similarities exist between the newly expressed proteins and known allergens or toxic proteins. Therefore, the EFSA GMO Panel reiterates the conclusions of its previous scientific opinion of 2005 that GM oilseed rape Ms8, Rf3 and Ms8 x Rf3 is unlikely to have an adverse effect on human and animal health or, in the context of its proposed uses, on the environment. This also applies to the products which are the subject of the present application.

This scientific opinion reports on an evaluation of a risk assessment for placing on the market the genetically modified insect resistant and herbicide tolerant maize Bt11 x MIR604 x GA21 for food and feed uses, import and processing. Conventional crossing methods were used in the production of maize Bt11 x MIR604 x GA21 from lines of the respective single maize events. The structure of the inserts in the single maize events as well as the phenotypes were both retained in the stacked maize events. The expression levels of the Cry1Ab, PAT, mCry3A, PMI and mEPSPS proteins in maize Bt11 x MIR604 x GA21 were demonstrated to be comparable with those of the respective single maize events. The comparative analysis of ompositional, phenotypic and agronomic characteristics indicated equivalence of maize Bt11 x MIR604 x GA21 with its conventional counterpart except for the newly expressed proteins which provided resistance to certain lepidopteran and coleopteran target pests and tolerance to glufosinate-ammonium- and/or glyphosate-based herbicides. The safety assessment identified no concerns regarding potential toxicity and allergenicity of maize Bt11 x MIR604 x GA21. A feeding study on broiler chickens confirmed the nutritional equivalence of this GM maize to its conventional counterpart and a commercial non-GM maize variety. Considering the intended uses of maize Bt11 x MIR604 x GA21, which excludes cultivation within the European Union, no scientific assessment of potential environmental effects associated with cultivation of maize Bt11 x MIR604 x GA21 was required. In case of accidental release of viable maize Bt11 x MIR604 x GA21 grains into the environment during transportation and processing, there are no indications of increased likelihood of establishment or survival of feral maize plants except in the presence of the glufosinate-ammonium- and/or glyphosate-based herbicides. It is highly unlikely that the recombinant DNA will transfer and establish in the genome of bacteria in the environment or human and animal digestive tracts. In conclusion, the EFSA GMO Panel considers that the information available for maize Bt11 x MIR604 x GA21 addresses the scientific comments raised by Member States and that the maize Bt11 x MIR604 x GA21, as assessed in this application, is as safe as its conventional counterpart and other appropriate comparators with respect to potential effects on human and animal health and the environment, in the context of its intended uses. The EFSA GMO Panel concludes that maize Bt11 x MIR604 x GA21 is unlikely to have an adverse effect on human and animal health and the environment, in the context of its intended uses.

This scientific opinion reports on an evaluation of a risk assessment for placing on the market the genetically modified insect resistant and herbicide tolerant maize Bt11xGA21 for food and feed uses, import and processing. Conventional breeding methods were used in the production of maize Bt11xGA21 from inbred lines of the respective parental events. The structural integrity of the inserts in the single maize events as well as the phenotypes were retained in the stacked maize event. The expression levels of the Cry1Ab, PAT and mEPSPS proteins in maize Bt11xGA21 were demonstrated to be comparable with those of the single maize events. The comparative analysis of phenotypic, agronomic and compositional characteristics of this GM maize indicated equivalence with its non-GM maize counterpart and conventional maize, except for the expression of the target proteins Cry1Ab, PAT and mEPSPS, providing resistance to certain lepidopteran pests and tolerance to glufosinate-ammonium- and glyphosate-based herbicides. The safety assessment identified no concerns regarding potential toxicity and allergenicity of maize Bt11xGA21. A feeding study on broiler chickens confirmed the nutritional equivalence of this GM maize to its non-GM maize counterpart and conventional maize. Considering the intended uses of maize Bt11xGA21, which excludes cultivation within the European Union, no scientific assessment of potential environmental effects associated with cultivation of maize Bt11xGA21 was required. In case of accidental release of viable maize Bt11xGA21 grains into the environment during transportation and processing, there are no indications of increased likelihood of establishment or survival of feral maize plants, except in the presence of glufosinte-ammonium- and/or glyphosate-based herbicides. The EFSA GMO Panel concludes that maize Bt11xGA21 is as safe as its non-GM maize counterpart with respect to effects on human and animal health and the environment, and is unlikely to have any adverse effect on human and animal health or on the environment in the context of its intended uses.

Pretreatment of discs excised from developing tubers of potato (Solanum tuberosum L.) with 10 millimolar sodium fluoride induced a transient increase in 3-phosphoglycerate content. This was followed by increases in triose-phosphate, fructose 1,6-bisphosphate and hexose-phosphate (glucose 6-phosphate + fructose 6-phosphate + glucose 1-phosphate). The effect of fluoride is attributed to an inhibition of glycolysis and a stimulation of triose-phosphate recycling (the latter confirmed by the pattern of (13)C-labeling [NMR] in sucrose when tissue was supplied with [2-(13)C]glucose). Fluoride inhibited the incorporation of [U-(14)C] glucose, [U-(14)C]sucrose, [U-(14)C]glucose 1-phosphate, and [U-(14)C] glycerol into starch. The incorporation of [U-(14)C]ADPglucose was unaffected. Inhibition of starch biosynthesis was accompanied by an almost proportional increase in the incorporation of (14)C into sucrose. The inhibition of starch synthesis was accompanied by a 10-fold increase in tissue pyrophosphate (PPi) content. Although the subcellular localization of PPi was not determined, a hypothesis is presented that argues that the PPi accumulates in the amyloplast due to inhibition of alkaline inorganic pyrophosphatase by fluoride ions.

Utilisation de clones de pomme de terre ayant des capacites tres differentes a accumuler des hexoses afin d'etudier le role de l'invertase et de son inhibiteur dans l'accumulation d'hexoses dans les tubercules stockes a 10°C ou transferres ensuite a 3°C

Contains fulltext : 35876pub.pdf (Publisher’s version ) (Closed access) Laser photoacoustic spectroscopy continuously quantified the ethylene (C2H4) produced by strawberry flowers and fruits developing in planta. C2H4 was first detected as flower buds opened and exhibited diurnal oscillations (to approximately 200 pl flower(-1) h(-1)) before petal abscission. Exogenous application of silver thiosulphate (STS) to detached flowers inhibited petal abscission and flower senescence. In fruit, C2H4 production was maintained at a 'low level' (10-60 pl fruit(-1) h(-1)) until fruit expanded when levels increased in a diurnal pattern (to 200 pl fruit(-1) h(-1)). After expansion, C2H4 production declined to a low level until fruit attained the red-ripe stage for at least 24 h. After this time, C2H4 levels increased linearly (no diurnal fluctuation) to approximately 1 nL fruit(-1) h(-1). Twenty-four hours after the re-initiation of C2H4 production by red fruit, CO2 levels increased approximately three-fold, indicative of a respiratory climacteric. STS applied to fruits developing in planta and dissected fruit parts ex situ established that C2H4 production is regulated by negative feedback until fruits had expanded. The C2H4 produced by red-ripe fruit was regulated by positive feedback. Anti-1-amino-cyclopropane-1-carboxylic acid oxidase IgG localization identified immunoreactive antigens of 40 and 30 kDa (M-r) within the fruit achenes of expanding and red-ripe fruit. Analysis of dissected fruit showed that seed C2H4 accounts for 50% the C2H4 that is detectable from ripe fruit.

Crop improvement by genetic modification remains controversial, one of the major issues being the potential for unintended effects. Comparative safety assessment includes targeted analysis of key nutrients and antinutritional factors, but broader scale-profiling or “omics” methods could increase the chances of detecting unintended effects. Comparative assessment should consider the extent of natural variation and not simply compare genetically modified (GM) lines and parental controls. In this study, potato (Solanum tuberosum) proteome diversity has been assessed using a range of diverse non-GM germplasm. In addition, a selection of GM potato lines was compared to assess the potential for unintended differences in protein profiles. Clear qualitative and quantitative differences were found in the protein patterns of the varieties and landraces examined, with 1,077 of 1,111 protein spots analyzed showing statistically significant differences. The diploid species Solanum phureja could be clearly differentiated from tetraploid (Solanum tuberosum) genotypes. Many of the proteins apparently contributing to genotype differentiation are involved in disease and defense responses, the glycolytic pathway, and sugar metabolism or protein targeting/storage. Only nine proteins out of 730 showed significant differences between GM lines and their controls. There was much less variation between GM lines and their non-GM controls compared with that found between different varieties and landraces. A number of proteins were identified by mass spectrometry and added to a potato tuber two-dimensional protein map.

Globally, urea is the most widely used nitrogen fertilizer and is made accessible to plants via the urease reaction. However, sequence information for the plant enzyme is scarce. A cDNA encoding urease from soybean (Glycine max) has been cloned and sequence information has been obtained for two alleles (11 and 19 kbp, respectively) of the potato (Solanum tuberosum, cv. Desiree) urease gene and the corresponding cDNAs. It was found that urease is encoded by a single copy gene in several solanaceous species, and maps to chromosome V in potato. By contrast, the presence of two urease genes was reported for soybean. Comparative analysis of 11 kbp overlapping allelic DNA allowed the quantification of single nucleotide polymorphisms and revealed the presence of a truncated Ty1-copia retrotransposon in one of the alleles. Both alleles contained a copy of a terminal-repeat retrotransposon in miniature (TRIM). 25-50% of urease pre-mRNAs from both alleles were alternatively spliced in a variety of different ways. The retrotransposons had no effect on splicing. While urease is expressed in all tissues tested, its mRNA and protein are of low abundance. The TATA-less urease promoter appears to drive low-level housekeeping transcription. An in silico analysis showed that eukaryotic urease protein sequences are very similar to sequences from prokaryotic enzymes, conserving all residues of known functional importance. It is therefore likely that all known ureases are structurally similar, employing the same catalytic mechanism.

The commercialisation of GM crops in Europe is practically non-existent at the present time. The European Commission has instigated changes to the regulatory process to address the concerns of consumers and member states and to pave the way for removing the current moratorium. With regard to the safety of GM crops and products, the current risk assessment process pays particular attention to potential adverse effects on human and animal health and the environment. This document deals with the concept of unintended effects in GM crops and products, i.e. effects that go beyond that of the original modification and that might impact primarily on health. The document first deals with the potential for unintended effects caused by the processes of transgene insertion (DNA rearrangements) and makes comparisons with genetic recombination events and DNA rearrangements in traditional breeding. The document then focuses on the potential value of evolving "profiling" or "omics" technologies as non-targeted, unbiased approaches, to detect unintended effects. These technologies include metabolomics (parallel analysis of a range of primary and secondary metabolites), proteomics (analysis of polypeptide complement) and transcriptomics (parallel analysis of gene expression). The technologies are described, together with their current limitations. Importantly, the significance of unintended effects on consumer health are discussed and conclusions and recommendations presented on the various approaches outlined.

The gene coding for agglutinin from Galanthus nivalis (GNA) was expressed in, and secreted by, the methylotrophic yeast, Pichia pastoris. Transformants of P. pastoris were selected and a process to produce and purify gram quantities of recombinant GNA was developed. GNA was secreted at approximately 80 mg l(-1) at the 200 1 scale and was purified to 95% homogeneity using hydrophobic interaction chromatography. The recombinant protein was similar to the protein synthesised in plant with respect to structure and biological activity.