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Oral immune tolerance is a physiological process to achieve tolerance against autoimmunity by oral ingestion of self-antigen(s) or other therapeutics. At the cellular level, oral tolerance suppresses autoimmune diseases by activating FoxP-positive and -negative regulatory T cells (Tregs) and/or causing clonal anergy or deletion of autoreactive T cells, affecting B cell tolerance. However, oral delivery of antigens/biologics is challenging due to their instability in the harsh environment of the gastrointestinal (GI) tract. Several antigen/drug delivery tools and approaches, including micro/nanoparticles and transgenic plant-based delivery systems, have been explored to demonstrate oral immune tolerance for different autoimmune diseases successfully. However, despite the effectiveness, variation in results, dose optimization, and undesirable immune system activation are the limitations of the oral approach to further advancement. From this perspective, the current review discusses the oral tolerance phenomenon, cellular mechanisms, antigen delivery tools and strategies, and its challenges. © 2023 by the authors.

Xylella fastidiosa is a xylem-limited, Gram-negative, gammaproteobacterium, and the causal agent of Pierce’s disease (PD) of grapevine and several other diseases in many economically important crops. In the PD patho-system in California, it is natively vectored by the blue green sharpshooter (Graphocephala atropunctata) and vectored by the invasive glassy-winged sharpshooter (Homalodisca vitripennis). When transmitted into a healthy vine, X. fastidiosa colonizes the xylem leading to blockages both directly through biofilm formation and EPS build up and indirectly through the induction of tyloses and pectin gels. These blockages are believed to contribute to symptom development which includes marginal leaf necrosis, abscission of the leaf blade from the petiole, green islands in the periderm, desiccation of the berries, and eventually vine death. While there is no current cure, common management practices include severe pruning of diseased tissue and insecticide sprays to control vector populations. However, new management strategies are needed to create cost effective and sustainable solutions to protect these culturally valuable crops.As of late, biological control agents, the enhancement of existing treatments, and the development of transgenic disease-resistant or disease-tolerant plants have been implemented in an attempt to cope with phytopathogenic microbes. First, the concept of a biological control agent or biocontrol can be defined as the use of a third organism to act antagonistically against a pathogen and lessening disease symptoms in the host. This antagonism can occur via antibiotic production, direct predation, or nutrient competition and exclusion. We have identified two endophytic bacterial isolates that impart varying levels of protection to the grapevine from X. fastidiosa and have speculated on the mechanisms by which they do so. Second, the transgenesis of plants has been proven to be effective at controlling pathogens. Transcriptomic data can b

‘Isabel’ grape (Vitis labrusca x V. vinifera L. hybrid) is one of the main grape cultivars in Russia and some other countries for processing, due to its vigor, tolerance to the main fungal diseases, high yield and potential for sugar accumulation. The stilbene synthase gene VlvSTS was isolated from the hybrid grape cv. Isabel and cloned into a pSS plant transformation vector under the control of a constitutive 35S RNA double promoter of the cauliflower mosaic virus, CaMV 35SS. VlvSTS-gene containing transgenic tobacco lines were obtained and analyzed. For the first time plants expressing the VlvSTS gene were shown to have an enhanced resistance to the bacterial pathogen Erwinia carotovora subsp. carotovora B15. Transgenic plants were tested for resistance to a number of fungal pathogens. The plants were resistant to the grey mould fungus Botrytis cinerea, but not to the fungi Fusarium oxysporum, F. sporotrichioides, or F. culmorum. According to the results of a high performance liquid chromatography-mass spectrometry analysis, the amount of trans-resveratrol in leaves of transgenic plants with the highest expression of the VlvSTS gene was in a range from 150 to 170 µg/g of raw biomass. Change in the color and a decreased anthocyanin content in the flower corollas of transgenic plants were observed in transgenic lines with the highest expression of VlvSTS. A decrease in total flavonoid content was found in the flower petals but not the leaves of these tobacco lines. High expression of the VlvSTS gene influenced pollen development and seed productivity in transgenic plants. The size of pollen grains increased, while their total number per anther decreased. A decrease in the number of fertile pollen grains resulted in a decreased average weight of a seed boll in transgenic plants. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.

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[ES] Además de por su valor ornamental, el color de las hojas es importante por la influencia que puede tener en la eficacia fotosintética de las plantas. La deteccción de mutantes afectados en el color de las hojas es la mejor estrategia para identificar los genes que controla este carácter. Para lograr esto, la mutagénesis insercional es una de las herramientasla más adecuada entre todas las alternativas metodológicas disponibles. Con esta estrategia, tras la obtención de un gran número de plantas transgénicas, se evalúa su fenotipo para poder detectar alteraciones causadas por la integración del T-DNA. Si se produce la disrupción de un gen endógeno se suele obtener un mutante de anulación de función, mientras que la inserción del T-DNA en una región de control o en la vecindad del mismo puede provocar una alteración de su nivel de expresión. En cualquier caso, como el gen endógeno queda etiquetado por el T-DNA, se puede llegar a la clonación del gen mediante métodos poco exigentes en tiempo y recursos. El desarrollo de un protocolo eficaz de transformación genética en tomate y especies silvestres relacionadas (Solanum pimpinellifolium, S. pennellii, S. galapagense) ha permitido la obtención de más de siete mil líneas T-DNA en el contexto de varios proyectos. Se ha realizado el escrutinio de estas colecciones de líneas T-DNA para identificar mutantes alterados en caracteres del desarrollo vegetativo y reproductivo, así como en el nivel de tolerancia a estreses abióticos, principalmente salinidad y sequía. La frecuencia de mutantes (dominantes o recesivos) para los caracteres evaluados está en torno al 10% y los análisis Southern revelan que el número medio de insertos por línea es moderado (1.7), lo que facilita la identificación del inserto de T-DNA que genera el fenotipo mutante. En el marco de estos proyectos, se ha conseguido la identificación de varios genes a partir de mutantes seleccionados para diversos caracteres. En el presente TFM lo que planteamos es l, [EN] In addition to its ornamental value, the color of the leaves is important due to the influence it can have on the photosynthetic efficiency of plants. The detection of mutants affected in the color of the leaves is the best strategy to identify the genes that control this trait. To achieve this, insertional mutagenesis is one of the most suitable tools among all the methodological alternatives available. With this strategy, after obtaining a large number of transgenic plants, their phenotype is evaluated in order to detect alterations caused by the integration of T-DNA. If an endogenous gene is disrupted, a knock-out mutant is usually obtained, while the insertion of T-DNA in a control region or in the vicinity of it can cause an alteration in its expression level. In any case, as the endogenous gene is tagged by the T-DNA, the cloning of the gene can be achieved by methods that are not very demanding in time and resources. The development of an efficient protocol for genetic transformation in tomato and related wild species (Solanum pimpinellifolium, S. pennellii, S. galapagense) has allowed the obtaining of more than seven thousand T-DNA lines in the context of various projects. These collections of T-DNA lines have been screened to identify mutants altered in characters of vegetative and reproductive development, as well as in the level of tolerance to abiotic stresses, mainly salinity and drought. The frequency of mutants (dominant or recessive) for the evaluated characters is around 10% and Southern analyzes reveal that the average number of inserts per line is moderate (1.7), which facilitates the identification of the T-DNA insert that generates the mutant phenotype. In the framework of these projects, the identification of several genes from mutants selected for various characters has been achieved. In the present TFM what we propose is the identification of the genes responsible for the alterations present in several mutants with differences in the col

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Este producto forma parte de una serie de textos de divulgación científica que buscan reseñar algunas de las investigaciones más importantes en las que ha tenido participación la Universidad EAFIT, publicadas en las revistas especializadas más prestigiosas del mundo

Tecia solanivora is one of the most damaging pest of potato crops in Central and South America. Eight genetically modified (GM) potato lines with Bacillus thuringiensis cry1Ac gene (Bt potato lines) were characterized at molecular, immunological and biological level under biosafety confined, greenhouse and experimental field conditions. Additionally, preliminary assays regarding substantial equivalence were determined. Cry1Ac protein and gene were detected and quantified on leaf and tubers. DC 40.5, DC 40.7A, DC 40.7B, PP 28, PP 28.1 and PP 40.A, Bt potato lines were resistant to Tecia solanivora when they were exposed. Results obtained indicated that two GM potato lines are promising for further assessments and selection tests as well as future possible commercialization.

Tecia solanivora is one of the most damaging pest of potato crops in Central and South America. Eight genetically modified (GM) potato lines with Bacillus thuringiensis cry1Ac gene (Bt potato lines) were characterized at molecular, immunological and biological level under biosafety confined, greenhouse and experimental field conditions. Additionally, preliminary assays regarding substantial equivalence were determined. Cry1Ac protein and gene were detected and quantified on leaf and tubers. DC 40.5, DC 40.7A, DC 40.7B, PP 28, PP 28.1 and PP 40.A, Bt potato lines were resistant to Tecia solanivora when they were exposed. Results obtained indicated that two GM potato lines are promising for further assessments and selection tests as well as future possible commercialization. © 2014 Sociedad Colombiana de Entomologia. All rights reserved.

The C6 aldehydes, alcohols, and the corresponding esters are the most important compounds of virgin olive oil aroma. These C6 volatile compounds are synthesized via the 13-hydroperoxide lyase (13-HPL) branch of the lipoxygenase pathway. In this investigation, a functional analysis of the olive (Olea europaea L.) 13-HPL gene by its overexpression and silencing in olive transgenic lines was carried out. With this aim, sense and RNAi constructs of the olive 13-HPL gene were generated and used for the transformation of embryogenic olive cultures. Leaves from overexpressing lines showed a slight increase in 13-HPL gene expression, whereas RNAi lines exhibited a strong decrease in their transcript levels. Quantification of 13-HPL activity in two overexpressing and two RNAi lines showed a positive correlation with levels of transcripts. Interestingly, RNAi lines showed a high decrease in the content of C6 volatiles linked to a strong increase of C5 volatile compounds, altering the volatile profile in the leaves. In addition, the silencing of the 13-HPL gene severely affected plant growth and development. This investigation demonstrates the role of the 13-HPL gene in the biogenesis of olive volatile compounds and constitutes a functional genomics study in olive related to virgin olive oil quality.

[EN] Background: Secondary metabolites play an important role in the plant defensive response. They are produced as a defence mechanism against biotic stress by providing plants with antimicrobial and antioxidant weapons. In higher plants, the majority of secondary metabolites accumulate as glycoconjugates. Glycosylation is one of the commonest modifications of secondary metabolites, and is carried out by enzymes called glycosyltransferases. Results: Here we provide evidence that the previously described tomato wound and pathogen-induced glycosyltransferase Twi1 displays in vitro activity toward the coumarins scopoletin, umbelliferone and esculetin, and the flavonoids quercetin and kaempferol, by uncovering a new role of this gene in plant glycosylation. To test its activity in vivo, Twi1-silenced transgenic tomato plants were generated and infected with Tomato spotted wilt virus. The Twi1- silenced plants showed a differential accumulation of Twi1 substrates and enhanced susceptibility to the virus. Conclusions: Biochemical in vitro assays and transgenic plants generation proved to be useful strategies to assign a role of tomato Twi1 in the plant defence response. Twi1 glycosyltransferase showed to regulate quercetin and kaempferol levels in tomato plants, affecting plant resistance to viral infection.

This thesis describes how the "In Plant Activation" (INPACT) expression platform was adapted into a molecular resistance strategy against circular single-stranded DNA viruses. The system was first optimized using a model geminivirus in the dicot host tobacco then applied to a commercially relevant pathosystem. When used in conjunction with RNAi, the platform provided immunity to the devastating bunchy top virus in the elite banana cultivar "Cavendish". This work shows that a multi-faceted approach to engineered resistance may be a simple means of generating immunity to this important family of viruses.

DNA damage is one of the most harmful stress inducers in living organisms. Studies have shown that exposure to high doses of various types of radiation cause DNA sequence changes (mutation) and disturb protein synthesis, hormone balance, leaf gas exchange and enzyme activity. Recent discovery of a protein called Damage Suppressor Protein (Dsup), found in the tardigrade species Ramazzotius varieornatus, has shown to reduce the effects of radiation damage in human cell lines. We have generated multiple lines of tobacco plants expressing the Dsup gene and preformed numerous tests to show viability and response of these transgenic plants when exposed to mutagenic chemicals, UV radiation and ionizing radiation. We have also investigated Dsup function in association to DNA damage and repair in plants by analyzing the expression of related genes using RT-qPCR. We have also analyzed DNA damage from X-ray and UV treatments using an Alkaline Comet Assay. This project has the potential to help generate plants that are tolerant to more extreme stress environments, particularly DNA damage and mutation, unshielded by our atmosphere. The possibility of growing plants accompanying human space travel and extraterrestrial colonization inspires our imagination. Extremotolerant tardigrade genes such as Dsup may be a valuable avenue in helping to cultivate crops in these future endeavors., 2019, Includes bibliography., Degree granted: Thesis (M.S.)--Florida Atlantic University, 2019., Collection: FAU Electronic Theses and Dissertations Collection

Сьогодні ГМО заборонені в Україні. Проте йде масове та безконтрольне використання модифікованого ріпаку, сої, кукурудзи тощо. Трансгени (генетично модифіковані організми ‒ ГМО) ‒ це організми, в ДНК яких (тварини, рослини чи мікроорганізми) втілено сторонні гени. Першим ГМО організмом була кишкова паличка., Сегодня ГМО запрещены в Украине. Однако идет массовое и бесконтрольное использование модифицированного рапса, сои, кукурузы и др. Трансгены (генетически модифицированные организмы ‒ ГМО) ‒ это организмы, в ДНК которых (животные, растения или микроорганизмы) внедрены посторонние гены. Первым ГМО организмом была кишечная палочка., Today, GMOs are banned in Ukraine. However, there is mass and uncontrolled use of modified rapeseed, soybeans, corn and so on. Transgenes (Genetically Modified Organisms ‒ GMOs) ‒ are organisms in which DNA (animals, plants or microorganisms) are embodied in foreign genes. The first GMO organism was E. coli.

Huanglongbing (HLB), also known as citrus greening, is one of the most destructive diseases of citrus worldwide. It is caused by unculturable phloem-limited bacteria that belong to the Candidatus Liberibacter genus including Ca. L. asiaticus (CLas), Ca. L. africanus, and Ca. L. americanus. Currently, there is no effective control strategy for HLB and no known cure for the disease. We have previously generated transgenic ‘Duncan’ grapefruit and ‘Hamlin’ sweet orange expressing the Arabidopsis NPR1 (AtNPR1) gene, which encodes a master regulator of systemic acquired resistance. Characterization of the transgenic lines indicated that overexpression of AtNPR1 confers resistance to citrus canker, another serious bacterial disease in citrus. In this study, we intensively screened these transgenic lines for resistance or tolerance to HLB under greenhouse conditions. Three independent transgenic lines (one line in the ‘Duncan’ grapefruit background and two lines in the ‘Hamlin’ sweet orange background) only occasionally displayed mild HLB symptoms in the presence of C Las and have kept growing normally over a period of nine years. Significantly, all vegetatively propagated progeny plants of these lines have retained the same levels of HLB tolerance. Furthermore, immunoblot analysis revealed that the three transgenic lines accumulate high levels of AtNPR1 protein. These results, together with the previous finding that the same three transgenic lines are resistant to citrus canker, demonstrate that overexpression of the AtNPR1 protein in citrus is able to provide robust tolerance to HLB.

Colombia es actualmente uno de los países más prometedores en cuanto a su potencial para el desarrollo agrícola y para generar alimentos para las generaciones humanas actuales y futuras. Esto se debe a factores como la disponibilidad de tierra, agua, diversidad topográfica, así como a factores políticos. Sin embargo, Colombia alcanzará su pleno potencial de desarrollo agrícola sostenible si adopta las tecnologías disponibles que puedan hacer frente a los desafíos globales actuales que enfrenta la agricultura en el siglo XXI: entre otros, el crecimiento de la población mundial, el incremento en el promedio de la esperanza de vida, alto grado de desnutrición, cambio climático, uso de practicas agrícolas equivocadas. Aquí es presentado cómo la biotecnología moderna es un aliado importante ya que se puede aplicar una amplia gama de tecnologías y sistemas innovadores donde más se necesitan: aumentar la productividad y sostenibilidad del cultivo, resistir los factores de estrés bióticos y abióticos y garantizar la seguridad alimentaria. En este estudio se muestra evidencia sobre los beneficios significativos de la adopción de cultivos biotecnológicos para contribuir a la inocuidad de los alimentos y como ellos ya se están implementando en los países desarrollados y en vía de desarrollo. Usando tecnologías modernas, hay oportunidades abiertas para el país en la búsqueda de una economía circular de base biológica, que fortalezca su soberanía alimentaria y para servir como despensa agrícola para América Latina y el mundo., Colombia is currently one of the most promising countries with regard to its potential for agricultural development and for generating food supply for current and future human generations. This is owing to factors such as availability of land, water, topographical diversity, as well as political factors. Nevertheless, Colombia will reach this full potential if it adopts available technologies that can meet the current global challenges faced by the agriculture in the 21st century: among others, world population growth, increase in average life expectancy, high degree of malnutrition, climate change, wrong agricultural practices. Here is presented how modern biotechnology is an important ally as a wide range of technologies and innovative systems can be applied where they are most needed: for increasing cultivation productivity, resisting both biotic and abiotic factors, and ensuring food safety. In this study is showed evidence with regard to significant benefits of adopting biotechnological crops to contribute to food safety and how they are already being implemented in both developed and developing countries. Using modern technology, there are open opportunities for the country in search of circular bio-based economy, strengthen its food sovereignty and to serve as an agricultural breadbasket to Latin America and the World

Colombia is currently one of the most promising countries with regard to its potential for agricultural development and for generating food supply for current and future human generations. This is owing to factors such as availability of land, water, topographical diversity, as well as political factors. Nevertheless, Colombia will reach this full potential if it adopts available technologies that can meet the current global challenges faced by the agriculture in the 21st century: among others, world population growth, increase in average life expectancy, high degree of malnutrition, climate change, wrong agricultural practices. Here is presented how modern biotechnology is an important ally as a wide range of technologies and innovative systems can be applied where they are most needed: for increasing cultivation productivity, resisting both biotic and abiotic factors, and ensuring food safety. In this study is showed evidence with regard to significant benefits of adopting biotechnological crops to contribute to food safety and how they are already being implemented in both developed and developing countries. Using modern technology, there are open opportunities for the country in search of circular bio-based economy, strengthen its food sovereignty and to serve as an agricultural breadbasket to Latin America and the World., Colombia es actualmente uno de los países más prometedores en cuanto a su potencial para el desarrollo agrícola y para generar alimentos para las generaciones humanas actuales y futuras. Esto se debe a factores como la disponibilidad de tierra, agua, diversidad topográfica, así como a factores políticos. Sin embargo, Colombia alcanzará su pleno potencial de desarrollo agrícola sostenible si adopta las tecnologías disponibles que puedan hacer frente a los desafíos globales actuales que enfrenta la agricultura en el siglo XXI: entre otros, el crecimiento de la población mundial, el incremento en el promedio de la esperanza de vida, alto grado de desnutrición, cambio climático, uso de practicas agrícolas equivocadas. Aquí es presentado cómo la biotecnología moderna es un aliado importante ya que se puede aplicar una amplia gama de tecnologías y sistemas innovadores donde más se necesitan: aumentar la productividad y sostenibilidad del cultivo, resistir los factores de estrés bióticos y abióticos y garantizar la seguridad alimentaria. En este estudio se muestra evidencia sobre los beneficios significativos de la adopción de cultivos biotecnológicos para contribuir a la inocuidad de los alimentos y como ellos ya se están implementando en los países desarrollados y en vía de desarrollo. Usando tecnologías modernas, hay oportunidades abiertas para el país en la búsqueda de una economía circular de base biológica, que fortalezca su soberanía alimentaria y para servir como despensa agrícola para América Latina y el mundo.

We describe the computational design of proteins that bind the potent analgesic fentanyl. Our approach employs a fast docking algorithm to find shape complementary ligand placement in protein scaffolds, followed by design of the surrounding residues to optimize binding affinity. Co-crystal structures of the highest affinity binder reveal a highly preorganized binding site, and an overall architecture and ligand placement in close agreement with the design model. We use the designs to generate plant sensors for fentanyl by coupling ligand binding to design stability. The method should be generally useful for detecting toxic hydrophobic compounds in the environment.

[EN] The use of genetically modified organisms (GMOs) as biofactories for the production of recombinant proteins of commercial interest is at present one of the major applications of `molecular¿ biotechnology, and the business basis of many modern biotech companies. Since marketing in the early 1980s of recombinant human insulin, synthesised in Escherichia coli, hundreds of proteins with pharmacological activity, used for the diagnosis, treatment of prevention of human (and animal) diseases, have been produced in different platforms. The intrinsic limitations of bacterial cell cultures ¿ especially the lack of the machinery for post-translanslational modifications of proteins, which are required for the synthesis of pharmacologically active proteins ¿ have made mammalian cell cultures the system of choice for the industrial production of biopharmaceuticals. These are robust, reliable and highly controlled production systems, optimised over the years and for which GMP (`good manufacturing practice¿) procedures have been established and approved by the competent authorities. Mammalian cell cultures, however, have also important limitations and drawbacks, mostly regarding their high costs, relatively low productivity and lack of flexibility to scale-up or -down the production, in response to market demands. Many of these limitations could be overcome with the use of plant biofactories, the so-called `3rd generation¿ of genetically modified plants used for the commercial production of recombinant proteins including, more specifically, pharmaceutical proteins: `molecular pharming¿ or `pharma crops. However, despite the important advantages ¿ at least theoretically ¿ of GM plants, development of this kind of production platforms has been slow and the first plant-made biopharmaceuticals have been approved for human use only recently. This has been due mostly to regulatory issues rather than to scientific or technical problems, but recent developments indicate a rapid growt

Full Text, Thesis (PhD Doctorate), Doctor of Philosophy (PhD), Griffith School of Environment, Science, Environment, Engineering and Technology, Biotechnological development of transgenic crops and widespread international adoption has stimulated comprehensive research into their effects on the soil environment. Insects (both target and non-target), weeds, aquatic environments, and soils have all been extensively researched. Since the first commercial GM crop in 1995, the debate surrounding controversial aspects of their biology has polarised issues concerning insects, weeds, and the concentration of technology within a few multinational biotechnology corporations. The effect of transgenic crops on the biology of soils seemed to attract less attention. This thesis examines the interactions between transgenic canola plants through their root exudates and residues with the surrounding soil, its microbes, and their biology. The discovery of the commensal relationship of root exudates and their microbial partners has revolutionised the research into soil microbiology. From this knowledge of soil science, scientists now understand the critical importance of a healthy and abundant soil biome. This thesis focuses on the microbiology of soil. The effects of genotype on soil bacterial and fungal physiology of genetically modified (GM) canola are compared statistically with other cultivars which are genetically modified through conventional plant breeding rather than through genomic engineering, and with the canola cultivar from which the GM variety was developed. The research differentiates the effects of four canola genotypes on microbial DNA and enzymology in a greenhouse trial format. Specifically, the objective of the research is to focus on changes in microbial enzymatic activity of key functional enzymes in protein metabolism, and carbon (C), phosphorus (P), and sulphur (S) mineralisation. The effects on DNA concentration are also studied. The main results of this research generally suggest the effects of cultivar genetic differences with respect to their interactions through root exudates and residues on micro

Plant-pathogenic microbes secrete effector molecules to establish disease on their hosts, whereas plants in turn employ immune receptors to try and intercept such effectors in order to prevent pathogen colonization. Based on structure and subcellular location, immune receptors fall into two major classes; cell surface-localized receptors that comprise receptor kinases (RKs) and receptor-like proteins (RLPs) that monitor the extracellular space, and cytoplasm-localized nucleotide-binding domain leucine-rich repeat receptors (NLRs) that survey the intracellular environment. Race-specific resistance to Verticillium wilt in tomato (Solanum lycopersicum) is governed by the tomato extracellular leucine-rich repeat (eLRR)-containing RLP-type cell surface receptor Ve1 upon recognition of the effector protein Ave1 that is secreted by race 1 strains of the soil-borne vascular wilt Verticillium dahliae. Homologues of V. dahliae Ave1 (VdAve1) are found in plants and in a number of plant pathogenic microbes, and some of these VdAve1 homologues are recognized by tomato Ve1. The research presented in this thesis aims to characterize the role of the tomato cell surface-localized immune receptor Ve1, and its homologues in other diverse plant species, in Verticillium wilt resistance.

Currently, fungicides are necessary to protect potato crops against late blight, Phytophthora infestans, one of the world’s most damaging crop pathogens. The introgression of plant resistance genes from wild potato species targeted specifically to the late blight pathogen into susceptible potato varieties may alleviate the environmental impact of chemical control. Genetically modified plants are subject to an environmental risk assessment, and this includes testing for risks to the non-target arthropod community associated with the crop. The thesis begins with a review about the main plant defense responses and their role in influencing sequential interactions between herbivores and plant pathogens. The experimental chapters each focus on different aspects of the interaction between potato plants (both resistant and susceptible), the target pathogen (P. infestans) and several non-target insects. With each chapter, the scope widens: from the molecular gene expression in potato leaves in response to sequential attacks, to field scale biodiversity analyses. At the molecular level, one of the main findings was that the genomic position of the Rpi-vnt1 insertion conferring resistance to P. infestans influenced potato gene expression measured in leaves, when interacting with the non-target insect pests Myzus persicae (Green peach aphid) and Leptinotarsa decemlineata (Colorado potato beetle). Insect performance differed between the resistant GM and susceptible non-GM comparator. In the following chapter, the differences in insect performance were tested across a range of conventionally bred cultivars varying in resistance to P. infestans. Differences in M. persicae performance between several cultivars greatly outweighed the differences previously detected between the GM and non-GM comparator. These results are crucial in shaping the way risk is assessed in the context of GM crops, and these results are supported in our experiments assessing effects on biodiversity with pi

[ES] La morfogénesis in vitro es la base de todas las aplicaciones que se derivan del cultivo de tejidos y células vegetales. En efecto, para emplear alternativas biotecnológicas encaminadas a la producción de plantas, como la micropropagación y el saneamiento del material vegetal, o para emprender programas de mejora genética basados en la obtención de haploides y doblehaploides, el aprovechamiento de la variación somaclonal, la hibridación sexual o somática entre especies sexualmente incompatibles, así como la generación de híbridos gametosomáticos, asimétricos y cíbridos, se necesitan métodos que permitan la regeneración de plantas a partir de explantes o de protoplastos. Del mismo modo, salvo en alguna especie modelo como Arabidopsis thaliana, donde es factible la transformación in planta, la obtención de plantas transgénicas precisa de métodos adecuados para la regeneración en cultivo in vitro. En realidad, en este caso, se requieren métodos de regeneración mucho más eficaces porque ciertos tratamientos (e.g. infección con Agrobacterium tumefaciens) conducen a un descenso de la respuesta morfogenética del material vegetal. La regeneración in vitro se puede conseguir a través de dos vías, la organogénesis y la embriogénesis somática. En este proyecto pretendemos poner a punto un método de regeneración mediante cualquiera de estas alternativas. Conviene resaltar que, si bien se han publicado algunos artículos en los que se describen métodos de regeneración en esta especie, las eficacias no son muy elevadas y, en cualquier caso, son dependientes del material de partida. Esto hace necesario que, para cada nueva variedad, cultivar o línea con la que se quiera trabajar, sea necesario ajustar las condiciones idóneas para la regeneración. Teniendo en cuenta la problemática existente, el objetivo de este trabajo es desarrollar una metodología eficaz para regenerar plantas a partir de explantes primarios de colza para abrir una vía que permita explotar las posibilidades, [EN] : In vitro morphogenesis is the basis of all applications derived from vegetal cell and tissue culture. Indeed, to use biotechnology alternatives aimed at plant production, as micropropagation and vegetal material sanitation, or to undertake breeding programs based on obtaining haploid and double-haploid plants, taking profit of somaclonal variation, sexual or somatic (between species sexually uncompatible) hybridization, as well as for generation of gameto-somatic and asymmetric hybrids or cybrids, methods that allow regeneration of plants from explants or protoplasts are required. Similarly, except in some model species such as Arabidopsis thaliana, where in planta transformation is feasible, the production of transgenic plants requires regeneration methods suitable for in vitro culture. Actually, in this case, much more effective regeneration methods are required because certain treatments (e.g. Agrobacterium tumefaciens infection) lead to a decrease in plant material morphogenetic response. The in vitro regeneration can be achieved through two ways, organogenesis and somatic embryogenesis. In this project, we intend to develop a regeneration method using any of these alternatives. It should be noted that although some articles have been published in which regeneration methods in this species are described, the efficiencies are not very high and, in any case, are dependent on the starting material. This requires that, for each new variety, cultivar or line is needed to adjust the conditions for regeneration. Given the existing problems, the objective of this work is to develop an effective methodology to regenerate plants from primary explants of rape in order to open a path that allows exploiting the possibilities offered by the in vitro culture on improving this species

Wild-type and two genetically engineered hybrid poplar lines were pyrolyzed in a micro-pyrolysis (Py-GC/MS) and a bench scale setup for fast and intermediate pyrolysis studies. Principal component analysis showed that the pyrolysis vapors obtained by micro-pyrolysis from wood of caffeic acid O-methyltransferase (COMT) and caffeoyl-CoA O-methyltransferase (CCoAOMT) down-regulated poplar trees differed significantly from the pyrolysis vapors obtained from non-transgenic control trees. Both fast micro-pyrolysis and intermediate pyrolysis of transgenic hybrid poplars showed that downregulation of COMT can enhance the relative yield of guaiacyl lignin-derived products, while the relative yield of syringyl lignin-derived products was up to a factor 3 lower. This study indicates that lignin engineering via genetic modifications of genes involved in the phenylpropanoid and monolignol biosynthetic pathways can help to steer the pyrolytic production of guaiacyl and syringyl lignin-derived phenolic compounds such as guaiacol, 4-methylguaiacol, 4-ethylguaiacol, 4-vinylguaiacol, syringol, 4-vinylsyringol, and syringaldehyde present in the bio-oil. (C) 2016 Elsevier Ltd. All rights reserved., Bio4Energy

The high gene density in Arabidopsis thaliana leaves only relatively short intergenic regions for potential cis-regulatory sequences. To learn more about the regulation of genes harbouring only very short upstream intergenic regions, this study investigates a recently identified novel microbe-associated molecular pattern (MAMP)-responsive cis-sequence located within the 101 bp long intergenic region upstream of the At1g13990 gene. It is shown that the cis-regulatory sequence is sufficient for MAMP-responsive reporter gene activity in the context of its native promoter. The 3' UTR of the upstream gene has a quantitative effect on gene expression. In context of a synthetic promoter, the cis-sequence is shown to achieve a strong increase in reporter gene activity as a monomer, dimer and tetramer. Mutation analysis of the cis-sequence determined the specific nucleotides required for gene expression activation. In transgenic A. thaliana the synthetic promoter harbouring a tetramer of the cis-sequence not only drives strong pathogen-responsive reporter gene expression but also shows a high background activity. The results of this study contribute to our understanding how genes with very short upstream intergenic regions are regulated and how these regions can serve as a source for MAMP-responsive cis-sequences for synthetic promoter design.

Homozygosity is highly desirable in transgenic plants research to ensure the stable integration and inheritance of transgene(s). Simple, reliable and high-throughput techniques to detect the zygosity of transgenic events in plants are invaluable tools for biotechnology and plant breeding companies. Currently, a number of basic techniques are being used to determine the zygosity of transgenic plants in T1 generation. For successful application of any technique, precision and simplicity of approach combined with the power of resolution are important parameters. On the basis of simplicity, resolution and cost involved, the available techniques have been classified into three major classes which are conventional methods, current methods and next generation methods. Conventional methods include antibiotic marker-based selection and the highly labour intensive Southern blot analysis. In contrast, methods such as real time PCR, TAIL PCR and competitive PCR are not only cost effective but rapid as well. Moreover, methods such as NGS, Digital PCR and Loop-mediated isothermal amplification also provide cost effective, fast and not so labour-intensive provide substitute of current methods. In this review we have attempted to compare and contrast all the available efficient methods to distinguish the homozygous plant in progeny of transgenics. This review also provides plenty of information regarding the various techniques available for determining zygosity in plants so as to permit the scientists to make informed choices of techniques that best suit their analyses. More importantly, detection and subsequent selection of homozygous individuals is central for facilitating the movement of transgenic plant from the laboratory to the field.

Bunchy top, caused by banana bunchy top virus (BBTV), is the most important viral disease affecting banana worldwide, for which control is very difficult. In this thesis, a molecular approach was used to generate genetically modified banana plants, some of which were resistant to BBTV infection. Further, sources of natural resistance to BBTV were identified in a range of different banana cultivars which may be exploited in future control strategies. The results from this study provide a strong platform for the future development of BBTV-resistant banana plants.

Transgenic plants with improved salt and drought stress tolerance have been developed with a large number of abiotic stress-related genes. Among these, the most extensively used genes are the glycine betaine biosynthetic codA, the DREB transcription factors, and vacuolar membrane Na⁺/H⁺ antiporters. The use of codA, DREBs, and Na⁺/H⁺ antiporters in transgenic plants has conferred stress tolerance and improved plant phenotype. However, the future deployment and commercialization of these plants depend on their safety to the environment. Addressing environmental risk assessment is challenging since mechanisms governing abiotic stress tolerance are much more complex than that of insect resistance and herbicide tolerance traits, which have been considered to date. Therefore, questions arise, whether abiotic stress tolerance genes need additional considerations and new measurements in risk assessment and, whether these genes would have effects on weediness and invasiveness potential of transgenic plants? While considering these concerns, the environmental risk assessment of abiotic stress tolerance genes would need to focus on the magnitude of stress tolerance, plant phenotype and characteristics of the potential receiving environment. In the present review, we discuss environmental concerns and likelihood of concerns associated with the use of abiotic stress tolerance genes. Based on our analysis, we conclude that the uses of these genes in domesticated crop plants are safe for the environment. Risk assessment, however, should be carefully conducted on biofeedstocks and perennial plants taking into account plant phenotype and the potential receiving environment.

It was assessed in genetically modified (GM) maize with Agrisure ® Viptera TM 3111, the incidence and severity of ear rot by Fusarium sp., in two locations from Culiacan, Sinaloa. In El Camalote, the hybrid showed incidence and severity of 47.2 and 22.5% respectively; compared to conventional hybrid with pest control showed 54.7 and 27.1% of incidence and severity of rot and conventional hybrid (control), had further damage with an incidence of 59.4 and severity of 35.2%. In assessing Oso Viejo, the hybrid Agrisure ® Viptera TM 3111 showed less damage with 25.7 and 7.6% incidence and severity of the disease respectively; the conventional hybrid with pest control had an incidence of 67.5% and severity of 38.7%, and control had the highest damage 80.8 and 53.5% of incidence and severity. Agrisure ® Viptera TM 3111 corn was not developed for disease management, however; the findings show that the genetically modified maize confers an indirect additional benefit to pest resistance, preventing the formation of entry points for pathogens that develop in susceptible tissue, causing ear rot., Se evaluó en maíz genéticamente modificado (GM) con el evento Agrisure ® Viptera TM 3111, la incidencia y severidad de pudrición de mazorca por Fusarium sp., en dos localidades de Culiacán, Sinaloa. En El Camalote, el híbrido observó una incidencia y severidad de 47.2 y 22.5% respectivamente; comparado con el híbrido convencional con control de plagas que presentó 54.7 y 27.1% de incidencia y severidad de pudrición y el híbrido convencional (testigo absoluto), tuvo un mayor daño con una incidencia de 59.4 y 35.2% de severidad. En la evaluación de Oso Viejo, el hibrido Agrisure ® Viptera TM 3111 presentó menor daño con 25.7 y 7.6% de incidencia y severidad de la enfermedad respectivamente; el híbrido convencional con control de plagas tuvo una incidencia de 67.5% y una severidad de 38.7%, y el testigo absoluto tuvo el mayor daño 80.8 y 53.5% de incidencia y severidad. El maíz Agrisure ® Viptera TM 3111 no fue desarrollado para el manejo de enfermedades, no obstante; el resultado encontrado demuestra que el maíz genéticamente modificado confiere un beneficio adicional indirecto al de resistencia a insectos plaga, previniendo la formación de puntos de entrada para fitopatógenos que se desarrollan en el tejido susceptible, provocando la pudrición de mazorca

Storage starch is an energy reservoir for plants and the major source of calories in the human diet. Starch is used in a broad range of industrial applications, as a cheap, abundant, renewable and biodegradable biopolymer. However, starch needs to be modified before it can fulfill the required properties for specific industrial applications. Genetic modification of starch, as a green technology with environmental and economic advantages, has attracted increasingly attention. Many achievements obtained from earlier studies have demonstrated the feasibility and potential of using this approach to produce starches with novel properties (Chapter 2). The main objective of this research was to produce novel starches with enhanced functionalities through genetic modification, while gaining a better understanding of storage starch biosynthesis. A focus on potato was warranted as it represents a superior model system for storage starch biosynthesis studies and for the production of starches with novel properties. To this end, a number of enzymes from various sources have been expressed in potato tubers to modify starch phosphate content and polysaccharide structure, since these two characteristics have long been recognized as key features in starch properties. To modify starch phosphate content and explore starch (de)phosphorylation, a human phosphatase enzyme named laforin, and modifications of it, were introduced into potato (Chapter 3). Interestingly, modified starches exhibited a significantly higher phosphate content rather than the expected lower phosphate content. Transcriptome analysis showed that the increase in phosphate content was a result of upregulation of starch phosphorylating genes, which revealed a compensatory response to the loss of phosphate content in potato starch. Furthermore, the increase of phosphate content in potato starch was reached to a threshold level. This was in line with the observations in the modified starches from overexpressed- Glucan w

Genetic modifications for perturbing the lignin pathway in three different species of angiosperm plants, including non-woody (Arabidopsis and alfalfa) and woody (poplar) plants, were readily evaluated by analytical pyrolysis coupled to gas chromatography-mass spectrometry (Py-GC/MS). Pyrolysis showed that the composition of Arabidopsis plants was severely altered when the expression of the gene encoding the enzyme caffeic acid O-methyltransferase (COMT) was downregulated, resulting in a lignin largely enriched in guaiacyl (G) units (88%). Alfalfa plants in which lignin biosynthesis was modified by down-regulation of the p-coumarate 3-hydroxylase (C3H) gene, showed extremely high proportions of p-hydroxyphenyl (H) units (71%) relative to the naturally prevailing guaiacyl (G) and syringyl (S) units. Finally, Py-GC/MS analyses indicated that overexpression in poplar of the gene that encodes the enzyme ferulate 5-hydroxylase (F5H) resulted in a lignin with a higher content of syringyl lignin units (88%) compared to the wild-type control (71%). In conclusion, Py-GC/MS is a useful and convenient tool for the rapid evaluation of compositional changes in lignin from genetically modified plants.

If the Netherlands, alongside Germany, France, Poland, and Hungary, decides to ban genetically modified (GM) soy in animal feed, the use of soy products in animal feed in these five countries will have to decrease by 40 to 50% to ensure that the EU demand for non-GM soy does not exceed the supply on the world market. The extra costs to Dutch livestock farmers over a period of 3 to 5 years as a result of the more expensive non-GM soy and alternative protein sources are estimated at between €60 and €100 million a year, with approximately 80% being borne by poultry farmers. Livestock numbers and productivity will then be maintained. A partial shift in trade flows from animal feed ingredients can be expected from import in the west of the EU - for example, through the port of Rotterdam - to intra-EU flows from production areas within the EU to consumers and via the waterway axis from regions east of the EU, such as Ukraine. Less soy will enter the EU via the Netherlands. This deficit can be offset by the increased demand for alternative protein sources, which will be partly imported from overseas. The effects on Dutch ports, the transport sector, and employment will depend on the nature of the trade flow shifts., Als Nederland - naast Duitsland, Frankrijk, Polen en Hongarije - kiest voor een verbod op het gebruik van genetisch gemodificeerde (gg-)soja in veevoeder, dan zal het verbruik van sojaproducten in veevoeder in deze vijf landen moeten dalen met 40 tot 50%, om ervoor te zorgen dat de vraag naar niet-genetisch gemodificeerde soja vanuit de EU niet groter is dan de beschikbare hoeveelheid op de wereldmarkt. Op een termijn van 3 tot 5 jaar worden de extra kosten voor de Nederlandse veehouderij, vanwege het gebruik van duurdere niet-gg-soja en alternatieve eiwitbronnen, geschat op € 60 tot € 100 miljoen per jaar, waarvan circa 80% voor de pluimveehouderij. Hierbij blijft het aantal dieren en de productiviteit van de dieren gehandhaafd. Handelsstromen van veevoedergrondstoffen worden verwacht deels te verschuiven van invoer in het westen van de EU, bijvoorbeeld via de haven van Rotterdam, naar aanvoer vanuit Oost-Europa, via de weg, het spoor en het water. Er zal minder soja via Nederland de EU binnenkomen. Dit kan worden opgevangen door de toegenomen behoefte aan alternatieve eiwitbronnen. De gevolgen voor de Nederlandse havens en de transportsector en voor de werkgelegenheid hangen af van hoe handelsstromen uiteindelijk verschuiven.

The European Commission proposed the opportunity for individual EU Member States to restrict or prohibit the use of GMOs in food or feed on their territory (a national ‘opt-out’). The economic impact on individual sectors of the feed and food chain (the vegetable oil and meal industry, trade, animal feed industry, livestock sector) of a possible opt-out policy for soy by individual Member States has been assessed by LEI Wageningen UR.A single scenario was defined in which the four countries France, Germany, Poland and Hungary choose an ‘opt-out’ policy for soy. Consequences of this switch to non-GM soy and substitutes were assessed both quantitatively and qualitatively for feed prices, for production costs for animal production, for crushing industry and for trade, with a focus on the medium term

Beschrijving van een voorstel voor een concreet co-existentiemonitoringprogramma (CMP) voor suikerbiet dat is aangepast aan de specifieke gewaseigenschappen van suikerbiet. De gemaakte keuzen t.b.v. een pragmatische invulling van het voorgestelde CMP worden in de opvolgende hoofdstukken toegelicht op basis van de huidige stand van zaken in het wetenschappelijk onderzoek aan (trans)genverspreiding in suikerbiet. Er is nog geen ervaring met een CMP in suikerbiet, noch is er een (Europese) standaard voor. Voor het bereiken van een zo pragmatisch mogelijke aanpak wordt zoveel mogelijk aangesloten bij al bestaande evaluatiepraktijken in de suikerbietenteelt

In het rapport wordt een voorstel voor een concreet co-existentiemonitoringprogramma (CMP) voor maïs beschreven dat is aangepast aan de specifieke gewaseigenschappen van maïs. De gemaakte keuzen t.b.v. een pragmatische invulling van het voorgestelde CMP worden in de opvolgende hoofdstukken toegelicht op basis van de huidige stand van zaken in het wetenschappelijk onderzoek aan (trans)genverspreiding in maïs. Er is nog beperkte ervaring met een CMP in maïs, bijvoorbeeld in Portugal, Tsjechië en Slowakije waar Bt MON810 maïs op beperkte schaal verbouwd wordt. Er is ook geen (Europese) standaard voor een CMP, maar er is wel voor maïs als eerste een Best Practice Document door het European Co-existence Bureau (ECoB) van het JRC uitgebracht (Rizov & Rodríguez-Cerezo 2014).

Het rapport is als volgt opgezet. Eerst wordt een voorstel voor een concreet coexistentiemonitoringprogramma (CMP) voor aardappel beschreven dat is aangepast aan de specifieke gewaseigenschappen van aardappel. De gemaakte keuzen t.b.v. een pragmatische invulling van het voorgestelde CMP worden in de opvolgende hoofdstukken toegelicht op basis van de huidige stand van zaken in het wetenschappelijk onderzoek aan (trans)genverspreiding in aardappel. Er is nog geen ervaring met een CMP in aardappel, noch is er een (Europese) standaard voor. Voor het bereiken van een zo pragmatisch mogelijke aanpak wordt zoveel mogelijk aangesloten bij al bestaande evaluatiepraktijken in de aardappelteelt. Dat betekent dat zoveel mogelijk aangesloten is bij de bestaande controlepraktijk zoals die uitgevoerd wordt door de NAK. Dat laat onverlet dat co-existentiemonitoring niet tot de staande praktijk van de NAK gerekend kan worden en dat nu ook nog niet vastligt dat de NAK deze monitoring inderdaad gaat doen. Praktische uitvoering hangt af van besluitvorming over de uiteindelijke invulling van de co-existentiemonitoring zodra GG aardappelteelt geïntroduceerd zou gaan worden.

Transgenic tobacco plants expressing ACR2 gene of Arabidopsis thaliana exhibit reduced accumulation of arsenics and increased tolerance to arsenate. Toxic metals such as arsenics, lead, cadmium or chromium are the major environmental pollutants that severely contribute to contamination of the global food chain directly through their accumulation in the edible parts of the cultivated crops or indirectly via meat-milk pathway. Fortunately, plant genetic engineering has the potential for developing new crop cultivars for removal of the toxic substances from the polluted sources or for avoiding accumulation of these contaminants in the edible parts. Previously, we have identified and studied four key genes that are involved in accumulation of arsenics in plants (J Biol. Systems 2010, 18/1:1-19; J Mol. Modeling 2012, DOI 10.1007/s00894-012-1419 and J Mol. Modeling 2014, DOI 10.1007/s00894-014-2104). In this study, we have cloned and transformed the ACR2 gene (arsenic reductase 2) of Arabidopsis thaliana into tobacco plants (Nicotiana tabacum). Our results revealed that the transgenic tobacco plants are more tolerant to arsenic than the wild-type control plants. These plants can grow on medium containing 200 µM arsenate, whereas the non-transgenic plants can hardly survive under this condition. Furthermore, when exposed to 100 µM arsenate for 35 days accumulation of arsenics in shoots of the transgenic plants decreases significantly (28 µg/g d wt.) compared to that observed in the non-transgenic control plants (40 µg/g d wt.). This study shows that A. thaliana ACR2 gene is a potential candidate for genetic engineering of plants to develop new cultivars that can be grown on arsenic contaminated fields and can supply harmless foods containing no or significantly reduced amount of arsenics., Bioremediation of toxic metals

De weigering van de Indiaase regering om rijst toe te laten die langs genetische weg verrijkt is met vitamine A, kostte al 1,4 miljoen levensjaren. Dat stelt economiehoogleraar Justus Wesseler. Zijn berekeningen werpen een nieuw licht op de gmo-discussie.

Overheidsregels rond biotechnologie en genetisch gemodificeerde gewassen leiden vaak tot substantieel hoge investeringskosten. Die hebben weer een lager niveau van productontwikkeling tot gevolg en een concentratie in de industrie, een herschikking van onderzoeksprioriteiten en een verschuiving van onderzoek en ontwikkeling naar landen met minder stringente regelgeving. Die trend leidde zelfs tot schade aan duurzame ontwikkeling uit oogpunt van milieu en volksgezondheid.

We have increased the productivity and yield of potato (Solanum tuberosum) by developing a novel method to enhance photosynthetic carbon fixation based on expression of a polyprotein (DEFp) comprising all three subunits (D, E and F) of Escherichia coli glycolate dehydrogenase (GlcDH). The engineered polyprotein retained the functionality of the native GlcDH complex when expressed in E. coli and was able to complement mutants deficient for the D, E and F subunits. Transgenic plants accumulated DEFp in the plastids, and the recombinant protein was active in planta, reducing photorespiration and improving CO2 uptake with a significant impact on carbon metabolism. Transgenic lines with the highest DEFp levels and GlcDH activity produced significantly higher levels of glucose (5.8-fold), fructose (3.8-fold), sucrose (1.6-fold) and transitory starch (threefold), resulting in a substantial increase in shoot and leaf biomass. The higher carbohydrate levels produced in potato leaves were utilized by the sink capacity of the tubers, increasing the tuber yield by 2.3-fold. This novel approach therefore has the potential to increase the biomass and yield of diverse crops.