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Knowledge of the number of DNA sequences targeted by the taxon-specific reference assays is essential for correct GM quantification and is key to the harmonisation of measurement results. In the present study droplet digital PCR (ddPCR) was used to determine the number of DNA target copies of taxon-specific assays validated for real-time PCR for the four main genetically modified (GM) crops. The transferability of experimental conditions from real-time PCR to ddPCR was also explored, as well as the effect of DNA digestion. The results of this study indicate that for each crop at least one taxon-specific assay can be identified as having a single DNA target. A short list of taxon-specific reference assays is proposed as best candidates for the relative quantification of GM events for soybean, maize, cotton and oilseed rape. The investigated assays could be in most cases transferred to ddPCR without further optimisation. The use of DNA digestion did not improve ddPCR characteristics such as rain and resolution at the conditions tested., Highlights • DdPCR was used to test number of targets of validated reference assays for 4 GM crops. • At least 1 taxon-specific assay for a single DNA target was identified per crop. • Most qPCR based assays tested can be transferred to ddPCR without optimisation. • DNA digestion does not improve ddPCR characteristics at the conditions tested.

Within the MADS-box gene family, the AGAMOUS-subfamily genes are particularly important for plant reproduction, because they control stamen and carpel identity. A number of studies in the last three decades have demonstrated that the AGAMOUS (AG) function has been conserved during land plant evolution. However, gene duplication events have led to subfunctionalization and neofunctionalization of AG-like genes in many species. Here we show that alternative splicing in Oryza sativa produces two variants of the AG ortholog OsMADS3 which differ in just one serine residue, S109. Interestingly, this alternative splicing variant is conserved and specific to the grass family. Since in eudicots the S109 residue is absent in AG proteins, stamen and carpel identity determination activity of the two rice isoforms was tested in Arabidopsis thaliana. These experiments revealed that only the eudicot-like OsMADS3 isoform, lacking the serine residue, had ability to specify stamens and carpels in ag mutant flowers, suggesting an important functional role for the serine residue at position 109 in AG proteins of grasses.

The recent ruling of the European Court of Justice has confirmed that organisms obtained by mutagenesis techniques are genetically modified organisms (GMOs). However, in contrast to organisms originating from conventional mutagenesis techniques, those obtained by new mutagenesis techniques are not exempted from the obligations of the GMO EU regulatory framework. This ruling raises questions about the detectability of the corresponding GM food and feed products. A case study is used in this document to explain and discuss possibilities and limitations for the detection and quantification of (known and unknown) genetic modifications in plant products derived from new mutagenesis techniques. Many of the mutations induced by new mutagenesis techniques cannot be unequivocally distinguished from natural mutations because such genome editing technologies are able to create very precise and limited genome changes that mimic the result of potential naturally occurring mutations. Moreover, mutations obtained by genome editing technologies could also not be differentiated from those introduced by conventional mutagenesis techniques which have been incorporated in traditional breeding programs and are often not thoroughly documented. Products of genome editing could only be detected and identified in imports of commodity products by enforcement laboratories when prior knowledge on the altered genome sequence, a validated detection method with appropriate selectivity and certified reference materials are available, similarly as required for the authorisation of current transgenic GMOs. However, when the modification involves only a SNP or few nucleotide changes, it would not be possible to identify whether the mutation originated spontaneously or was induced by conventional or new (genome editing) mutagenesis techniques. Moreover, it is unlikely that methods for the quantification of GMO products with small genome modifications in complex food or feed materials provide the level of selectivity needed for the enforcement of legislation, such as the one on labelling. In the absence of prior knowledge on the genome-edited changes, it is likely that non-authorised genetically modified food and feed products obtained by genome editing would enter the EU market undetected. The EU control system for GMOs and corresponding food and feed products may not function as efficiently for unauthorised genome-edited products compared to transgenic GMOs. In particular, the principle of zero tolerance for unauthorised GMO on the EU market is more difficult to maintain., JRC.F.5-Food and Feed Compliance

In this study, we developed, optimized, and in-house validated a real-time PCR method for the event-specific detection and quantification of Golden Rice 2, a genetically modified rice with provitamin A in the grain. We optimized and evaluated the performance of the taxon (targeting rice Phospholipase D α2 gene)- and event (targeting the 3' insert-to-plant DNA junction)-specific assays that compose the method as independent modules, using haploid genome equivalents as unit of measurement. We verified the specificity of the two real-time PCR assays and determined their dynamic range, limit of quantification, limit of detection, and robustness. We also confirmed that the taxon-specific DNA sequence is present in single copy in the rice genome and verified its stability of amplification across 132 rice varieties. A relative quantification experiment evidenced the correct performance of the two assays when used in combination.

Experiments with strong localized electron cyclotron heating (ECH) in the RTP tokamak show that electron heat transport is governed by alternating layers of good and bad thermal conduction. For central deposition hot T-e filaments are observed inside the q = 1 radius. Moving the ECH resonance from the centre to the edge of the plasma results in discrete steps of the central electron temperature. The transitions occur when the minimum q value crosses q = 1, 2, 5/2 or 3, and correspond to the loss of a transport barrier situated close to the rational q value. Close to the transitions a new type of sawtooth activity is observed, characterized by the formation of sharp off-axis maxima on the T, profile, which collapse abruptly. The formation of the off-axis maxima is attributed to heat deposition precisely 'on top of' a transport barrier.

Genes that control ovule identity were first identified in Petunia. Co-suppression of both FLORAL BINDING PROTEIN 7 (FBP7) and FBP11, two D-lineage genes, resulted in the homeotic transformation of ovules into carpelloid structures. Later in Arabidopsis it was shown that three genes, SHATTERPROOF1 (SHP1), SHP2, and SEEDSTICK (STK), redundantly control ovule identity, because in the stk shp1 shp2 triple mutant ovules lose identity and are transformed into carpel and leaf-like structures. Of these three Arabidopsis genes STK is the only D-lineage gene, and its expression, like FBP7 and FBP11, is restricted to ovules. OsMADS13 is the rice ortholog of STK, FBP7, and FBP11. Its amino acid sequence is similar to the Arabidopsis and Petunia proteins, and its expression is also restricted to ovules. We show that the osmads13 mutant is female sterile and that ovules are converted into carpelloid structures. Furthermore, making carpels inside carpels, the osmads13 flower is indeterminate, showing that OsMADS13 also has a function in floral meristem determinacy. OsMADS21 is most likely to be a paralog of OsMADS13, although its expression is not restricted to ovules. Interestingly, the osmads21 mutant did not show any obvious phenotype. Furthermore, combining the osmads13 and the osmads21 mutants did not result in any additive ovule defect, indicating that osmads21 does not control ovule identity. These results suggest that during evolution the D-lineage gene OsMADS21 has lost its ability to determine ovule identity.

This article describes the international validation of the quantitative real-time polymerase chain reaction (PCR) detection method for Golden Rice 2. The method consists of a taxon-specific assay amplifying a fragment of rice Phospholipase D α2 gene, and an event-specific assay designed on the 3' junction between transgenic insert and plant DNA. We validated the two assays independently, with absolute quantification, and in combination, with relative quantification, on DNA samples prepared in haploid genome equivalents. We assessed trueness, precision, efficiency, and linearity of the two assays, and the results demonstrate that both the assays independently assessed and the entire method fulfill European and international requirements for methods for genetically modified organism (GMO) testing, within the dynamic range tested. The homogeneity of the results of the collaborative trial between Europe and Asia is a good indicator of the robustness of the method.

Experiments in H-mode plasmas have shown that both heat and particle transport are sensitive to the ratio between electron and ion temperature (Te/Ti). While decreasing Te/Ti is beneficial for confinement, an increased electron heating in these so called 'hot ion plasmas' deteriorates the confinement. H-mode plasmas with low Te/Ti are often accompanied by high toroidal rotation velocity (v). Its gradient (∇v) can destabilize the ion temperature gradient mode (ITG) through its parallel component in the parallel velocity shear, but it has also stabilizing effects since it produces an E × B shearing rate (ωE × B). In this paper, the effects of electron heating on the ion heat transport is investigated in H-mode plasmas heated by neutral beam injection (NBI) and electron cyclotron heating (ECH). In particular, the correlation on Te/Ti and ∇v is studied and compared with calculations made with GLF23 and GS2. Experimentally it is shown that the normalized gradient length of the ions ( ) is correlated with both Te/Ti and ∇v: peaked ion temperature profiles are only obtained with low Te/Ti and high ∇v, and vice-versa. When ECH is added, both ion heat and momentum transport are enhanced, leading to a drop of both the Ti and v profiles. The effective growth rate γeff = γ−ωE × B is calculated, with the mode growth rate γ determined with GS2 and ωE × B with GLF23. The ion transport is enhanced due to the decrease of the ITG threshold with increasing Te/Ti. Comparison of the dependence of on Te/Ti and ∇v between experiments and modelling indicates that the deterioration of confinement cannot be explained by the changes in only Te/Ti or ∇v, but by the combined effects of both parameters. The changes in Te/Ti act directly on the ITG threshold, while the ones in ∇v modify the ωE × B shearing rate leading to changes in the effective threshold.

Significant progress has been made on ASDEX Upgrade during the last two years in the basic understanding of transport, in the extension of the improved H-mode in parameter space and towards an integrated operating scenario and in the development of control methods for major performance limiting instabilities. The important features were the understanding of particle transport and the control of impurity accumulation based on it, the satisfactory operation with predominantly tungsten-clad walls, the improved H-mode operation over density ranges and for temperature ratios covering (non-simultaneously) the ITER requirements on ν*, n/nGW and Te/Ti, the ELM frequency control by pellet injection and the optimization of NTM suppression by DC-ECCD through variation of the launching angle. From these experiments an integrated scenario has emerged which extrapolates to a 50% improvement in n T τ or a 30% reduction of the required current when compared with the ITER base-line assumptions, with moderately peaked electron and controllable high-Z density profiles.

Electron heat transport properties have been investigated by means of localized steady-state and modulated electron cyclotron heating in the ASDEX Upgrade tokamak. The experimental findings have been successfully compared with the electron temperature with the model assumption of a threshold gradient length, L-Tc, (1/L-T = -delT(e0)/T-e0), T-e0 below which the electron thermal conductivity measured under steady-state conditions, chi(ePB), shows a sudden increase and the heat conduction 'seen' by a temperature perturbation, chi(eHP), switches from low to high values. In this work electron cyclotron heating (ECH) power is used both to modify the steady-state gradient length and to induce a nonlinear transition from low to high electron heat transport at the position where the temperature gradient crosses its critical (or threshold) value. Steady-state ECH has been injected at different radial positions and the changes induced in electron heat transport have been studied by means of cold pulses launched from the plasma boundary. Cold pulses, obtained by injecting radiating impurities by means of laser blow off of silicon, have proved to be a flexible tool for investigating both the radial position of the step in chi(eHp) and the dependence on local plasma parameters of the underlying physical mechanism.

Transport studies are presented in this chapter. Global scaling studies have been performed using several transport codes. Ohmic plasmas are found to follow the ITER97 L-mode scaling. Transport coefficients are discussed for improved confinement scenarios achieved in the Frascati Tokamak Upgrade (FTU): the repetitive pellet enhanced plasma mode, showing neoclassical confinement with H-factors up to 1.6, and the electron internal transport barriers (ITBs) with large transport barriers and H-factors up to 1.3. Heat transport models have been tested using electron cyclotron resonance heating (ECRH), steady or modulated, as a probe. The electron temperature stiffness observed in the main bulk of steady FTU plasmas can be interpreted both with a critical gradient transport model and with a model based on the existence of canonical profiles. ECRH has also been used to benefit from the improved confinement generally associated with low or negative magnetic shear, and large electron temperatures have been achieved in these conditions. Profile resiliency is observed so that heat transport is not consistent with a constant thermal diffusivity. Experimental optimization is discussed together with the analysis of transport coefficients. Thorough discussions of impurity transport are given, both for intrinsic and injected (from laser blow-off) impurities. Code simulation and experimentalmore » data are compared for a series of FTU experiments focusing on the improved confinement modes (pellets and ITBs). A moderate inward pinch velocity is generally required to reproduce the data.« less

Perturbative and steady-state heat transport of FTU tokamak in current ramp-up discharges are investigated by means of modulated electron cyclotron heating (MECH). Perturbative and steady-state transport experiments are coherent with an electron heat transport which switches from low to high values when electron temperature gradient length reaches a threshold value 1/L_Tc. The threshold value 1/L_Tc is shown to be proportional to the ratio s/q. The experimental findings are compared to predictions of an empirical model based on the assumption of a threshold gradient length, L_Tc (1/L_T = |grad Te/Te|), in the electron temperature Te below which electron thermal diffusivity, chi_e, switches from low to high values. Plasma responses to steady state and MECH are modelled assuming the electron diffusivity as chi_PB = chi_0 + alpha Te^(3/2) (1/L_T - 1/L_Tc)^(1/2); here Te^(3/2) reflects the gyro-Bohm assumption, chi_0 represents the heat transport for 1/L_T < 1/LT_c and the term (1/L_T - 1/L_Tc)^(1/2), which sets in for 1/L_T > 1/L_Tc , mimics an extra transport possibly due to electron temperature gradient (ETG) modes. In agreement with ETG threshold 1/L_Tc is shown to be correlated with the magnetic shear s.

The EU-RL GMFF has previously validated individually, and declared fit for purpose, the detection methods for the single line soybean events MON 87705 and MON 89788 and has published the corresponding reports (see http://gmo-crl.jrc.ec.europa.eu/StatusOfDossiers.aspx). In line with the approach defined by the ENGL (http://gmo-crl.jrc.ec.europa.eu/doc/Min_Perf_Requirements_Analytical_methods.pdf) the EU-RL GMFF therefore has carried out only an in-house verification of the performance of each validated method when applied to DNA extracted from the GM stack MON 87705 x MON 89788 soybean. The hereby reported in-house verification study led to the conclusion that the individual methods meet the ENGL requirements also when applied to DNA extracted from the GM stack MON 87705 x MON 89788 soybean., JRC.I.3-Molecular Biology and Genomics

An application was submitted by Monsanto Company to request the authorisation of genetically modified stack (GM stack) MON 87708 × MON 89788 soybean (tolerant to dicamba and to glyphosate) and all sub-combinations of the individual events as present in the segregating progeny, for food and feed uses, and import and processing, in accordance with articles 5 and 17 of Regulation (EC) N° 1829/2003 GM Food and GM Feed. The unique identifier assigned to GM stack MON 87708 × MON 89788 soybean is MON-877Ø8-9 x MON-89788-1. The GM stack MON 87708 × MON 89788 soybean has been obtained by conventional crossing between two genetically modified soybean events: MON 87708 and MON 89788, without any new genetic modification. The EU-RL GMFF has previously validated individually, and declared fit for purpose, the detection methods for the single events MON 87708 and MON 89788 (see http://gmo-crl.jrc.ec.europa.eu/StatusOfDossiers.aspx). In line with the approach defined by the ENGL (http://gmo-crl.jrc.ec.europa.eu/doc/Min_Perf_Requirements_Analytical_methods.pdf) the EU-RL GMFF has carried out only an in-house verification of the performance of each validated method when applied to genomic DNA extracted from GM stack MON 87708 × MON 89788 soybean. The results of the in-house verification led to the conclusion that the individual methods meet the ENGL performance criteria also when applied to genomic DNA extracted from the GM stack MON 87708 × MON 89788 soybean., JRC.I.3-Molecular Biology and Genomics

This conference report summarizes the contributions to the EU-US workshop on Transport in Fusion Plasmas, Transport Barrier Physics held in Varenna, Italy, 4-7 September 2000. The workshop, whose main focus was on transport barriers, was organized into four sections: transport barrier physics, MHD effects concerning barriers, turbulence and core transport. This report is organized in a like manner.

Experiments on electron heat transport were performed in the tokamak ASDEX Upgrade, mainly in ohmically heated plasmas, applying either edge cooling with laser blow-off impurity injection or edge heat pulses with ECRH. Repetitive pulses within one plasma discharge were made allowing Fourier transformation of the temperature perturbation, yielding a good signal to noise ratio up to high harmonics and a detailed investigation of the pulse propagation. For densities lower than 1.8 x 10(19) m(-3), an increase of central electron temperature was found as the response to edge cooling via impurity injection similar to observations made in the TEXT, TFTR, RTP and Tore Supra tokamaks. The inversion does not appear instantaneously, but with a time delay roughly compatible with diffusion. Modelling of the propagation of the cold pulses in the framework of the IFS-PPPL model yields qualitative agreement. However, the predicted increase of ion temperature is not observed experimentally on a fast timescale. The response to ECRH heat pulses is not perfectly symmetrical to cold pulse experiments, but the similarities suggest a common underlying physical mechanism. No inversion of the heat pulse is found, instead the initial pulse from the edge is associated with a second, much slower, heat pulse in the centre. This central reaction is in fact quite similar (and not symmetrical) to that of the cold pulses. By varying the position of the ECRH deposition it could be shown that the central increase is related to the arrival of the pulse close to the inversion radius and not to the initial pulse. Modelling was performed to investigate the possible physics mechanisms and for comparison with the cold pulses.

An application was submitted by Pioneer Overseas Corporation to request the authorization of the genetically modified maize stack 1507 x 59122 x MON 810 x NK603, resistant against certain lepidopteran pests, protected against corn rootworm larvae, and glufosinate-ammonium and glyphosate tolerant, and all sub-combinations of the individual events as present in the segregating progeny, for food and feed uses, and import and processing, in accordance with articles 5 and 17 of Regulation (EC) N° 1829/2003 GM Food and GM Feed. The unique identifier assigned to 1507 x 59122 x MON 810 x NK603 maize is DAS-Ø15Ø7-1xDAS-59122-7xMON-ØØ81Ø-6xMON-ØØ6Ø3-6. The genetically modified maize line 1507 x 59122 x MON 810 x NK603 has been obtained by conventional crossing of four genetically modified single maize events: 1507, 59122, MON 810 and NK603 without any new genetic modification. The EU-RL GMFF has previously validated, and declared fit for purpose, the detection methods for the single events 1507, 59122, MON 810 and NK603 (see: http://gmo-crl.jrc.ec.europa.eu/statusofdoss.htm). In line with the approach defined by the ENGL (http://gmo-crl.jrc.ec.europa.eu/doc/Min_Perf_Requirements_Analytical_methods.pdf) the EU-RL GMFF therefore has carried out only an in-house verification of the performance of each validated method when applied to DNA extracted from 1507 x 59122 x MON 810 x NK603. The herewith reported in-house verification study lead to the conclusion that the individual methods meet the ENGL performance criteria also when applied to DNA extracted from the GM maize stack 1507 x 59122 x MON 810 x NK603., JRC.I.3-Molecular Biology and Genomics