Cotton MON 15985 was developed by biolistic transformation of cotton MON 531 to express Cry2Ab2 a... more Cotton MON 15985 was developed by biolistic transformation of cotton MON 531 to express Cry2Ab2 and GUS in addition to the Cry1Ac and NPTII proteins. Cry proteins in MON 15985 confer resistance to major lepidopteran cotton pests, whereas the GUS and NPTII proteins were used as markers during product development. Molecular characterisation of MON 15985 did not give rise to safety issues. The EFSA GMO Panel could not conclude on the potential occurrence of unintended effects for agronomic and phenotypic characteristics owing to data limitations. Compositional data gave no indication of unintended effects for which further assessment was needed. The Panel concludes that cotton MON 15985, as described in these applications, is as safe and nutritious as its conventional counterpart and other non-genetically modified varieties, and considers it unlikely that the overall allergenicity of the whole plant is changed. Environmental risk assessment was restricted to the exposure through faecal...
Efficient and sustainable agriculture depends on a high degree of predictability. Both in the sho... more Efficient and sustainable agriculture depends on a high degree of predictability. Both in the short-term, for growers and agronomists to make informed management decisions for the immediate upcoming seasons, but also in the long term to establish future agricultural policy and trade agreements, to define crop and livestock breeding goals, and to stimulate innovation in new products with transparent regulatory frameworks for pesticides and biotechnology; all of which require decade-long timeframes or longer. However, there are many factors with implications for world agriculture that are becoming increasingly unpredictable and which pose significant challenges for sustainable future food production. I will highlight two major areas of uncertainty, one which is environmental and beyond the control of humankind in the medium-term and the other, involving regulatory policy that is absolutely with in our short-term grasp. I will argue that providing certainty and transparency in the latter will make a significant contribution to global food security by ameliorating the effects of the former.
Assessment of genetically modified maize 4114 for food and feed uses, under Regulation (EC) No 18... more Assessment of genetically modified maize 4114 for food and feed uses, under Regulation (EC) No 1829/2003
cell permeating peptides (cpps) are attracting great interest for use as molecular delivery vehic... more cell permeating peptides (cpps) are attracting great interest for use as molecular delivery vehicles for the transport of biologically active cargo across the cell membrane. the sequence of a novel cpp sequence, termed 'Cupid', was identified from the genome of Dictyostelium discoideum. A cupid-Green fluorescent protein (cupid-Gfp) fusion protein was tested on mammalian, whole plant cells, plant leaf protoplast and fungal cell cultures and observed using confocal microscopy. GFP fluorescence builds up within the cell cytosol in 60 min, demonstrating Cupid-GFP has permeated them and folded correctly into its fluorescent form. Our combined data suggest Cupid can act as a molecular vehicle capable of delivering proteins, such as Gfp, into the cytosol of a variety of cells.
This document provides guidance for the risk assessment under Regulation (EC) No 1829/2003 of the... more This document provides guidance for the risk assessment under Regulation (EC) No 1829/2003 of the unintended, adventitious or technically unavoidable presence in food and feed of low level of genetically modified plant material intended for markets other than in the European Union. In this context, the presence at low level is defined to be maximum 0.9% of genetically modified plant material per ingredient. This guidance is intended to assist applicants by indicating which scientific requirements of Annex II of Regulation (EU) No 503/2013 are considered necessary for the risk assessment of the presence at low levels of genetically modified plant material in food and feed.
The application of high throughput 1H-NMR of unpurified extracts to determine to determine geneti... more The application of high throughput 1H-NMR of unpurified extracts to determine to determine genetic diversity and the contents of polar components in grain of wheat. milled whole wheat grain was extracted with 80:20 D2O:CD3OD containing 0.05% d4- trimethylsilylpropionate (TSP). 1H-NMR spectra were acquired under automation at 300 °K using an Avance Spectrometer operating at 600.0528 MHz. Regions for individual metabolites were identified by comparison to a library of known standards run under identical conditions. The individual 1H-NMR peaks or levels of known metabolites were then compared by Principal Component Analysis using SIMCA-P software. high throughput 1H-NMR is an excellent tool to compare the extent of genetic diversity within and between wheat species, and to quantify specific components (including glycine betaine, choline and asparagine) in individual genotypes. It can also be used to monitor changes in composition related environmental factors and to support comparisons...
Horizontal transfer of antibiotic resistance genes to animals and vertical transfer of herbicide ... more Horizontal transfer of antibiotic resistance genes to animals and vertical transfer of herbicide resistance genes to the weedy relatives are perceived as major biosafety concerns in genetically modified (GM) crops. In this study, five novel vectors which used gusA and bar as a reporter gene and a selection marker gene, respectively, were constructed based on the pCLEAN dual binary vector system. Among these vectors, 1G7B and 5G7B carried two T-DNAs located on two respective plasmids with 5G7B possessing an additional virGwt gene. 5LBTG154 and 5TGTB154 carried two T-DNAs in the target plasmid with either one or double right borders, and 5BTG154 carried the selectable marker gene on the backbone outside of the T-DNA left border in the target plasmid. In addition, 5BTG154, 5LBTG154, and 5TGTB154 used pAL154 as a helper plasmid which contains Komari fragment to facilitate transformation. These five dual binary vector combinations were transformed into Agrobacterium strain AGL1 and used ...
At Rothamsted Research UK, we have developed a robust protocol for the Agrobacterium-mediated tra... more At Rothamsted Research UK, we have developed a robust protocol for the Agrobacterium-mediated transformation of elite wheat and during the last year, made over one hundred independent transgenic lines in one durum and three bread wheat varieties. We are now able to provide training or generate transgenic wheat lines as a not-for-profit service (see www.bract.org). Approximately 40% of our lines
Wheat was among the last of the major crops to be transformed (in 1992), and transformation is st... more Wheat was among the last of the major crops to be transformed (in 1992), and transformation is still difficult, with a lower efficiency than that for maize and rice. However, the recent development of Agrobacterium-based systems is set to improve the precision of the process, while new methods of selection, removal of unnecessary DNA sequences, gene targeting and in vivo mutagenesis will make the process cleaner and more acceptable to regulatory authorities and consumers. Our current work is focussed on using transformation to understand and manipulate aspects of grain processing quality, notably dough strength and texture for milling. However, it is clear that a major priority for future work will be to improve nutritional quality, including vitamin and mineral contents for the developing world and starch digestibility and dietary fibre content and composition for developed countries.
Oat (Avena sativa L.), a worldwide temperate cereal crop, is deficient in tolerance to osmotic st... more Oat (Avena sativa L.), a worldwide temperate cereal crop, is deficient in tolerance to osmotic stress due to drought and/or salinity. To genetically transform the available commercial oat cultivars, a genotype-independent and efficient regeneration system from shoot apical meristems was developed using four oat cultivars: Prairie, Porter, Ogle, and Pacer. All these oat cultivars generated a genotype-independent in vitro differentiated multiple shoots from shoot apical meristems at a high frequency. Using this system, three oat cultivars were genetically co-transformed with pBY520 (containing hva1 and bar) and pAct1-D (containing gus) using biolistic trade mark bombardment. Transgenic plants were selected and regenerated using herbicide resistance and GUS as a marker. Molecular and biochemical analyses of putative transgenic plants confirmed the co-integration of hva1 and bar genes with a frequency of 100%, and 61.6% of the transgenic plants carried all three genes (hva1, bar and gus). Further analyses of R0, R1, and R2 progenies confirmed stable integration, expression, and Mendalian inheritance for all transgenes. Histochemical analysis of GUS protein in transgenic plants showed a high level of GUS expression in vascular tissues and in the pollen grains of mature flowers. Immunochemical analysis of transgenic plants indicated a constitutive expression of hva1 at all developmental stages. However, the level of HVA1 was higher during the early seedling stages. The characteristic of HVA1 expression for osmotic tolerance in transgenic oat progeny was analyzed in vitro as well as in vivo. Transgenic plants exhibited significantly (P<0.05) increased tolerance to stress conditions than non-transgenic control plants. The symptoms of wilting or death of leaves as observed in 80% of non-transgenic plants due to osmotic stress was delayed and detected only in less than 10% of trans-genic plants. These observations confirmed the characteristic of HVA1 protein as providing or enhancing the osmotic tolerance in transgenic plants against salinity and possible water-deficiency stress conditions.
We are witnessing the timely convergence of several technologies that together will have signific... more We are witnessing the timely convergence of several technologies that together will have significant impact on research, human health and in animal and plant breeding. The exponential increase in genome and expressed sequence data, the ability to compile, analyze and mine these data via sophisticated bioinformatics procedures on high-powered computers, and developments in various molecular and in-vitro cellular techniques combine to underpin novel developments in research and commercial biotechnology. Arguably the most important of these is genome editing which encompasses a suite of site directed nucleases (SDN) that can be designed to cut, or otherwise modify predetermined DNA sequences in the genome and result in targeted insertions, deletions, or other changes for genetic improvement. It is a powerful and adaptive technology for animal and plant science, with huge relevance for plant and animal breeding. But this promise will be realized only if the regulatory oversite is propor...
There are many quality targets in cereals that could generate step-change improvements in nutriti... more There are many quality targets in cereals that could generate step-change improvements in nutritional or food-processing characteristics. For instance, levels of acrylamide, soluble and insoluble fibre, antioxidants, allergens and intolerance factors in food are, to a large extent, determined by the genetics of the raw materials used. However, improvements to these traits pose significant challenges to plant breeders. For some traits, this is because the underlying genetic and biochemical basis of the traits is not fully understood but for others, there is simply a lack of natural genetic variation in commercially useful germplasm. One strategy to overcome the latter hindrance is to use wide crosses with more exotic germplasm; however, this can bring other difficulties such as yield loss and linkage drag of deleterious alleles. As DNA sequencing becomes cheaper and faster, it drives the research fields of reverse genetics and functional genomics which in turn will enable the incorpo...
The development of a robust Agrobacterium-mediated transformation protocol for a recalcitrant spe... more The development of a robust Agrobacterium-mediated transformation protocol for a recalcitrant species like bread wheat requires the identification and optimisation of the factors affecting T-DNA delivery and plant regeneration. We have used immature embryos from range of wheat varieties and the Agrobacterium strain AGL1 harbouring the pGreen-based plasmid pAL156, which contains a T-DNA incorporating the bar gene and a modified uidA (beta-glucuronidase) gene, to investigate and optimise major T-DNA delivery and tissue culture variables. Factors that produced significant differences in T-DNA delivery and regeneration included embryo size, duration of pre-culture, inoculation and co-cultivation, and the presence of acetosyringone and Silwet-L77 in the media. We fully describe a protocol that allowed efficient T-DNA delivery and gave rise to 44 morphologically normal, and fully fertile, stable transgenic plants in two wheat varieties. The transformation frequency ranged from 0.3% to 3.3...
Wheat is a major world crop and as such is a primary target for improvement of agronomic characte... more Wheat is a major world crop and as such is a primary target for improvement of agronomic characteristics via genetic engineering. Optimization of transformation is essential in order to overcome the relatively low transformation frequencies encountered with wheat. Transformation of elite wheat varieties is not always successful due to variability in regeneration and transformation frequencies between varieties. In this work, two elite wheat varieties with a relatively high embryogenic capacity were transformed by particle bombardment. A strong correlation between transformation frequency and the age of wheat donor plants was observed in both varieties. The mean transformation frequency rose from 0.7% to 5% when using immature embryos from old and young donor plants, respectively. This was observed in both varieties, the best bombardments achieving up to 7.3% frequency. Using explants at an optimal developmental stage from donor plants grown under environmentally-controlled condition...
Cotton MON 15985 was developed by biolistic transformation of cotton MON 531 to express Cry2Ab2 a... more Cotton MON 15985 was developed by biolistic transformation of cotton MON 531 to express Cry2Ab2 and GUS in addition to the Cry1Ac and NPTII proteins. Cry proteins in MON 15985 confer resistance to major lepidopteran cotton pests, whereas the GUS and NPTII proteins were used as markers during product development. Molecular characterisation of MON 15985 did not give rise to safety issues. The EFSA GMO Panel could not conclude on the potential occurrence of unintended effects for agronomic and phenotypic characteristics owing to data limitations. Compositional data gave no indication of unintended effects for which further assessment was needed. The Panel concludes that cotton MON 15985, as described in these applications, is as safe and nutritious as its conventional counterpart and other non-genetically modified varieties, and considers it unlikely that the overall allergenicity of the whole plant is changed. Environmental risk assessment was restricted to the exposure through faecal...
Efficient and sustainable agriculture depends on a high degree of predictability. Both in the sho... more Efficient and sustainable agriculture depends on a high degree of predictability. Both in the short-term, for growers and agronomists to make informed management decisions for the immediate upcoming seasons, but also in the long term to establish future agricultural policy and trade agreements, to define crop and livestock breeding goals, and to stimulate innovation in new products with transparent regulatory frameworks for pesticides and biotechnology; all of which require decade-long timeframes or longer. However, there are many factors with implications for world agriculture that are becoming increasingly unpredictable and which pose significant challenges for sustainable future food production. I will highlight two major areas of uncertainty, one which is environmental and beyond the control of humankind in the medium-term and the other, involving regulatory policy that is absolutely with in our short-term grasp. I will argue that providing certainty and transparency in the latter will make a significant contribution to global food security by ameliorating the effects of the former.
Assessment of genetically modified maize 4114 for food and feed uses, under Regulation (EC) No 18... more Assessment of genetically modified maize 4114 for food and feed uses, under Regulation (EC) No 1829/2003
cell permeating peptides (cpps) are attracting great interest for use as molecular delivery vehic... more cell permeating peptides (cpps) are attracting great interest for use as molecular delivery vehicles for the transport of biologically active cargo across the cell membrane. the sequence of a novel cpp sequence, termed 'Cupid', was identified from the genome of Dictyostelium discoideum. A cupid-Green fluorescent protein (cupid-Gfp) fusion protein was tested on mammalian, whole plant cells, plant leaf protoplast and fungal cell cultures and observed using confocal microscopy. GFP fluorescence builds up within the cell cytosol in 60 min, demonstrating Cupid-GFP has permeated them and folded correctly into its fluorescent form. Our combined data suggest Cupid can act as a molecular vehicle capable of delivering proteins, such as Gfp, into the cytosol of a variety of cells.
This document provides guidance for the risk assessment under Regulation (EC) No 1829/2003 of the... more This document provides guidance for the risk assessment under Regulation (EC) No 1829/2003 of the unintended, adventitious or technically unavoidable presence in food and feed of low level of genetically modified plant material intended for markets other than in the European Union. In this context, the presence at low level is defined to be maximum 0.9% of genetically modified plant material per ingredient. This guidance is intended to assist applicants by indicating which scientific requirements of Annex II of Regulation (EU) No 503/2013 are considered necessary for the risk assessment of the presence at low levels of genetically modified plant material in food and feed.
The application of high throughput 1H-NMR of unpurified extracts to determine to determine geneti... more The application of high throughput 1H-NMR of unpurified extracts to determine to determine genetic diversity and the contents of polar components in grain of wheat. milled whole wheat grain was extracted with 80:20 D2O:CD3OD containing 0.05% d4- trimethylsilylpropionate (TSP). 1H-NMR spectra were acquired under automation at 300 °K using an Avance Spectrometer operating at 600.0528 MHz. Regions for individual metabolites were identified by comparison to a library of known standards run under identical conditions. The individual 1H-NMR peaks or levels of known metabolites were then compared by Principal Component Analysis using SIMCA-P software. high throughput 1H-NMR is an excellent tool to compare the extent of genetic diversity within and between wheat species, and to quantify specific components (including glycine betaine, choline and asparagine) in individual genotypes. It can also be used to monitor changes in composition related environmental factors and to support comparisons...
Horizontal transfer of antibiotic resistance genes to animals and vertical transfer of herbicide ... more Horizontal transfer of antibiotic resistance genes to animals and vertical transfer of herbicide resistance genes to the weedy relatives are perceived as major biosafety concerns in genetically modified (GM) crops. In this study, five novel vectors which used gusA and bar as a reporter gene and a selection marker gene, respectively, were constructed based on the pCLEAN dual binary vector system. Among these vectors, 1G7B and 5G7B carried two T-DNAs located on two respective plasmids with 5G7B possessing an additional virGwt gene. 5LBTG154 and 5TGTB154 carried two T-DNAs in the target plasmid with either one or double right borders, and 5BTG154 carried the selectable marker gene on the backbone outside of the T-DNA left border in the target plasmid. In addition, 5BTG154, 5LBTG154, and 5TGTB154 used pAL154 as a helper plasmid which contains Komari fragment to facilitate transformation. These five dual binary vector combinations were transformed into Agrobacterium strain AGL1 and used ...
At Rothamsted Research UK, we have developed a robust protocol for the Agrobacterium-mediated tra... more At Rothamsted Research UK, we have developed a robust protocol for the Agrobacterium-mediated transformation of elite wheat and during the last year, made over one hundred independent transgenic lines in one durum and three bread wheat varieties. We are now able to provide training or generate transgenic wheat lines as a not-for-profit service (see www.bract.org). Approximately 40% of our lines
Wheat was among the last of the major crops to be transformed (in 1992), and transformation is st... more Wheat was among the last of the major crops to be transformed (in 1992), and transformation is still difficult, with a lower efficiency than that for maize and rice. However, the recent development of Agrobacterium-based systems is set to improve the precision of the process, while new methods of selection, removal of unnecessary DNA sequences, gene targeting and in vivo mutagenesis will make the process cleaner and more acceptable to regulatory authorities and consumers. Our current work is focussed on using transformation to understand and manipulate aspects of grain processing quality, notably dough strength and texture for milling. However, it is clear that a major priority for future work will be to improve nutritional quality, including vitamin and mineral contents for the developing world and starch digestibility and dietary fibre content and composition for developed countries.
Oat (Avena sativa L.), a worldwide temperate cereal crop, is deficient in tolerance to osmotic st... more Oat (Avena sativa L.), a worldwide temperate cereal crop, is deficient in tolerance to osmotic stress due to drought and/or salinity. To genetically transform the available commercial oat cultivars, a genotype-independent and efficient regeneration system from shoot apical meristems was developed using four oat cultivars: Prairie, Porter, Ogle, and Pacer. All these oat cultivars generated a genotype-independent in vitro differentiated multiple shoots from shoot apical meristems at a high frequency. Using this system, three oat cultivars were genetically co-transformed with pBY520 (containing hva1 and bar) and pAct1-D (containing gus) using biolistic trade mark bombardment. Transgenic plants were selected and regenerated using herbicide resistance and GUS as a marker. Molecular and biochemical analyses of putative transgenic plants confirmed the co-integration of hva1 and bar genes with a frequency of 100%, and 61.6% of the transgenic plants carried all three genes (hva1, bar and gus). Further analyses of R0, R1, and R2 progenies confirmed stable integration, expression, and Mendalian inheritance for all transgenes. Histochemical analysis of GUS protein in transgenic plants showed a high level of GUS expression in vascular tissues and in the pollen grains of mature flowers. Immunochemical analysis of transgenic plants indicated a constitutive expression of hva1 at all developmental stages. However, the level of HVA1 was higher during the early seedling stages. The characteristic of HVA1 expression for osmotic tolerance in transgenic oat progeny was analyzed in vitro as well as in vivo. Transgenic plants exhibited significantly (P<0.05) increased tolerance to stress conditions than non-transgenic control plants. The symptoms of wilting or death of leaves as observed in 80% of non-transgenic plants due to osmotic stress was delayed and detected only in less than 10% of trans-genic plants. These observations confirmed the characteristic of HVA1 protein as providing or enhancing the osmotic tolerance in transgenic plants against salinity and possible water-deficiency stress conditions.
We are witnessing the timely convergence of several technologies that together will have signific... more We are witnessing the timely convergence of several technologies that together will have significant impact on research, human health and in animal and plant breeding. The exponential increase in genome and expressed sequence data, the ability to compile, analyze and mine these data via sophisticated bioinformatics procedures on high-powered computers, and developments in various molecular and in-vitro cellular techniques combine to underpin novel developments in research and commercial biotechnology. Arguably the most important of these is genome editing which encompasses a suite of site directed nucleases (SDN) that can be designed to cut, or otherwise modify predetermined DNA sequences in the genome and result in targeted insertions, deletions, or other changes for genetic improvement. It is a powerful and adaptive technology for animal and plant science, with huge relevance for plant and animal breeding. But this promise will be realized only if the regulatory oversite is propor...
There are many quality targets in cereals that could generate step-change improvements in nutriti... more There are many quality targets in cereals that could generate step-change improvements in nutritional or food-processing characteristics. For instance, levels of acrylamide, soluble and insoluble fibre, antioxidants, allergens and intolerance factors in food are, to a large extent, determined by the genetics of the raw materials used. However, improvements to these traits pose significant challenges to plant breeders. For some traits, this is because the underlying genetic and biochemical basis of the traits is not fully understood but for others, there is simply a lack of natural genetic variation in commercially useful germplasm. One strategy to overcome the latter hindrance is to use wide crosses with more exotic germplasm; however, this can bring other difficulties such as yield loss and linkage drag of deleterious alleles. As DNA sequencing becomes cheaper and faster, it drives the research fields of reverse genetics and functional genomics which in turn will enable the incorpo...
The development of a robust Agrobacterium-mediated transformation protocol for a recalcitrant spe... more The development of a robust Agrobacterium-mediated transformation protocol for a recalcitrant species like bread wheat requires the identification and optimisation of the factors affecting T-DNA delivery and plant regeneration. We have used immature embryos from range of wheat varieties and the Agrobacterium strain AGL1 harbouring the pGreen-based plasmid pAL156, which contains a T-DNA incorporating the bar gene and a modified uidA (beta-glucuronidase) gene, to investigate and optimise major T-DNA delivery and tissue culture variables. Factors that produced significant differences in T-DNA delivery and regeneration included embryo size, duration of pre-culture, inoculation and co-cultivation, and the presence of acetosyringone and Silwet-L77 in the media. We fully describe a protocol that allowed efficient T-DNA delivery and gave rise to 44 morphologically normal, and fully fertile, stable transgenic plants in two wheat varieties. The transformation frequency ranged from 0.3% to 3.3...
Wheat is a major world crop and as such is a primary target for improvement of agronomic characte... more Wheat is a major world crop and as such is a primary target for improvement of agronomic characteristics via genetic engineering. Optimization of transformation is essential in order to overcome the relatively low transformation frequencies encountered with wheat. Transformation of elite wheat varieties is not always successful due to variability in regeneration and transformation frequencies between varieties. In this work, two elite wheat varieties with a relatively high embryogenic capacity were transformed by particle bombardment. A strong correlation between transformation frequency and the age of wheat donor plants was observed in both varieties. The mean transformation frequency rose from 0.7% to 5% when using immature embryos from old and young donor plants, respectively. This was observed in both varieties, the best bombardments achieving up to 7.3% frequency. Using explants at an optimal developmental stage from donor plants grown under environmentally-controlled condition...
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