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Genetic engineering of crops as potential source of genetic hazard in human diet
Abstract
The benefits of genetic engineering of crop plants to improve the reliability and quality of the world food supply have been contrasted with public concerns raised about the food safety of the resulting products. Debates have concentrated on the possible unforeseen risks associated with the accumulation of new metabolites in crop plants that may contribute to toxins, allergens and genetic hazards in the human diet. This review examines the various molecular and biochemical mechanisms by which new hazards may appear in foods as a direct consequence of genetic engineering in crop plants. Such hazards may arise from the expression products of the inserted genes, secondary or pleiotropic effects of transgene expression, and random insertional mutagenic effects resulting from transgene integration into plant genomes. However, when traditional plant breeding is evaluated in the same context, these mechanisms are no different from those that have been widely accepted from the past use of new cultivars in agriculture. The risks associated with the introduction of new genes via genetic engineering must be considered alongside the common breeding practice of introgressing large fragments of chromatin from related wild species into crop cultivars. The large proportion of such introgressed DNA involves genes of unknown function linked to the trait of interest such as pest or disease resistance. In this context, the potential risks of introducing new food hazards from the applications of genetic engineering are no different from the risks that might be anticipated from genetic manipulation of crops via traditional breeding. In many respects, the precise manner in which genetic engineering can control the nature and expression of the transferred DNA offers greater confidence for producing the desired outcome compared with traditional breeding.
... Despite the attractive benefits of GM crops, yet there are concerns about their safety [7,8]. Concerns associated with GM food consumption include immunotoxicity development, change in gene expression, reproductive toxicity and other unintended effects [9][10][11]. Consequently, it should be the prime focus of competent authorities to guarantee the biosafety of new biotechnological events before their commercialization [12]. ...
... Transgenic Bt crops have been widely grown for almost four decades for insect control without reporting any harm to human health [50]. Despite benefits of GM and Bt crops, still there are some concerns about their safe use and safety of GM food consumption [9][10][11]. Rigorous risk assessment analysis of GM crops/diets on model animals coupled with performance and toxicological evaluation for substantial equivalence is recommended to address these concerns [51,52]. ...
Genetically modified (GM) crops expressing insecticidal crystal proteins are widely accepted worldwide, but their commercial utilization demands comprehensive risk assessment studies. A 90-day risk assessment study was conducted on Wistar rats fed with GM maize (CEMB-413) expressing binary insect-resistant genes (cry1Ac and cry2Ab) at low (30%) and high (50%) dose along with a control diet group. The study used fifty Wistar rats randomly distributed in five treatment groups. Our study revealed that compared to controls, GM diet had no adverse effects on animal’s health, including body weight, food consumption, clinical pathological parameters, serum hormone levels and histological parameters of testes and ovaries of rats. Differences were observed in transcripts levels of fertility related genes, but these were independent of treatment with GM diet.
... For instance, bean plants that were genetically altered to contain more cysteine and methionine were renounced when it was discovered that the transgene produced extremely allergenic proteins [125]. Similarly, altered metabolic pathways may result in the creation of toxins and other unidentified substances [125,126]. Table 2 shows a summary of the current major ingredients and techniques used in plantbased meat analog production, their positive contributions, and limitations in safety and nutrition of the final product. Table 2. Main approaches used in plant-based meat analog production to improve safety and nutrition of the final products, their positive contribution, and current limitations. ...
Plant-based meat analogs have been shown to cause less harm for both human health and the environment compared to real meat, especially processed meat. However, the intense pressure to enhance the sensory qualities of plant-based meat alternatives has caused their nutritional and safety aspects to be overlooked. This paper reviews our current understanding of the nutrition and safety behind plant-based meat alternatives, proposing fermentation as a potential way of overcoming limitations in these aspects. Plant protein blends, fortification, and preservatives have been the main methods for enhancing the nutritional content and stability of plant-based meat alternatives, but concerns that include safety, nutrient deficiencies, low digestibility, high allergenicity, and high costs have been raised in their use. Fermentation with microorganisms such as Bacillus subtilis, Lactiplantibacillus plantarum, Neurospora intermedia, and Rhizopus oryzae improves digestibility and reduces allergenicity and antinutritive factors more effectively. At the same time, microbial metabolites can boost the final product’s safety, nutrition, and sensory quality, although some concerns regarding their toxicity remain. Designing a single starter culture or microbial consortium for plant-based meat alternatives can be a novel solution for advancing the health benefits of the final product while still fulfilling the demands of an expanding and sustainable economy.
... GE food safety concerns include the belief that new metabolites produced in GE crops, either directly through specific transgene expression or via unanticipated pleiotropic consequences of alterations to the genome, result in potentially toxic or allergenic products. Genetic transformation could in theory result in endogenous gene silencing, aberrant gene activation or epigenetic modifications (Conner and Jacobs, 1999). Moreover, every person is different and may have a harmful reaction to a harmless substance found in food products (Pawankar et al., 2012). ...
Human history is inextricably linked to the introduction of desirable heritable traits in plants and animals. Selective breeding (SB) predates our historical period and has been practiced since the advent of agriculture and farming more than ten thousand years ago. Since the 1970s, methods of direct plant and animal genome manipulation are constantly being developed. These are collectively described as "genetic engineering" (GE). Plant GE aims to improve nutritional value, insect resistance and weed control. Animal GE has focused on livestock improvement and disease control. GE applications also involve medical improvements intended to treat human disease. The scientific consensus built around marketed products of GE organisms (GEOs) is usually well established, noting significant benefits and low risks. GEOs are exhaustively scrutinized in the EU and many non-EU countries for their effects on human health and the environment, but scrutiny should be equally applied to all previously untested organisms derived directly from nature or through selective breeding. In fact, there is no evidence to suggest that natural or selectively bred plants and animals are in principle safer to humans than GEOs. Natural and selectively bred strains evolve over time via genetic mutations that can be as risky to humans and the environment as the mutations found in GEOs. Thus, previously untested plant and animal strains aimed for marketing should be proven useful or harmful to humans only upon comparative testing, regardless of their origin. Highlighting the scientific consensus declaring significant benefits and rather manageable risks provided by equitably accessed GEOs, can mitigate negative predispositions by policy makers and the public. Accordingly, we provide an overview of the underlying technologies and the scientific consensus to help resolve popular myths about the safety and usefulness of GEOs.
... The need for backcrossing to introduce the transgene into the most desirable plant genotypes, which are typically not amenable to transformation, has been suggested [48]. However, with advances in transformation technologies, even recalcitrant genotypes such as maize inbred lines [49], sorghum [50], and cassava [51] are transformed with relative ease. ...
Plant viruses cause yield losses to crops of agronomic and economic significance and are a challenge to the achievement of global food security. Although conventional plant breeding has played an important role in managing plant viral diseases, it will unlikely meet the challenges posed by the frequent emergence of novel and more virulent viral species or viral strains. Hence there is an urgent need to seek alternative strategies of virus control that can be more readily deployed to contain viral diseases. The discovery in the late 1980s that viral genes can be introduced into plants to engineer resistance to the cognate virus provided a new avenue for virus disease control. Subsequent advances in genomics and biotechnology have led to the refinement and expansion of genetic engineering (GE) strategies in crop improvement. Importantly, many of the drawbacks of conventional breeding, such as long lead times, inability or difficulty to cross fertilize, loss of desirable plant traits, are overcome by GE. Unfortunately, public skepticism towards genetically modified (GM) crops and other factors have dampened the early promise of GE efforts. These concerns are principally about the possible negative effects of transgenes to humans and animals, as well as to the environment. However, with regards to engineering for virus resistance, these risks are overstated given that most virus resistance engineering strategies involve transfer of viral genes or genomic segments to plants. These viral genomes are found in infected plant cells and have not been associated with any adverse effects in humans or animals. Thus, integrating antiviral genes of virus origin into plant genomes is hardly unnatural as suggested by GM crop skeptics. Moreover, advances in deep sequencing have resulted in the sequencing of large numbers of plant genomes and the revelation of widespread endogenization of viral genomes into plant genomes. This has raised the possibility that viral genome endogenization is part of an antiviral defense mechanism deployed by the plant during its evolutionary past. Thus, GM crops engineered for viral resistance would likely be acceptable to the public if regulatory policies were product-based (the North America regulatory model), as opposed to process-based. This review discusses some of the benefits to be gained from adopting GE for virus resistance, as well as the challenges that must be overcome to leverage this technology. Furthermore, regulatory policies impacting virus-resistant GM crops and some success cases of virus-resistant GM crops approved so far for cultivation are discussed.
... GM crop offers an unintended effect in the plant genome, resulting in the disruption and rearrangement of the genome and expression of new proteins. Thus, the evaluation of transgenic events and proteome response with induced conditions is essential (Conner and Jacobs, 1999;Gong and Wang, 2013). ...
Drought stress impacts cotton plant growth and productivity across countries. Plants can initiate morphological, cellular, and proteomic changes to adapt to unfavorable conditions. However, our knowledge of how cotton plants respond to drought stress at the proteome level is limited. Herein, we elucidated the molecular coordination underlining the drought tolerance of two inbred cotton varieties, Bacillus thuringiensis-cotton [Bt-cotton + Cry1 Ac gene and Cry 2 Ab gene; NCS BG II BT (BTCS/BTDS)] and Hybrid cotton variety [Non-Bt-cotton; (HCS/HDS)]. Our morphological observations and biochemical experiments showed a different tolerance level between two inbred lines to drought stress. Our proteomic analysis using 2D-DIGE revealed that the changes among them were not obviously in respect to their controls apart from under drought stress, illustrating the differential expression of 509 and 337 proteins in BTDS and HDS compared to their controls. Among these, we identified eight sets of differentially expressed proteins (DEPs) and characterized them using MALDI-TOF/TOF mass spectrometry. Furthermore, the quantitative real-time PCR analysis was carried out with the identified drought-related proteins and confirmed differential expressions. In silico analysis of DEPs using Cytoscape network finds ATPB, NAT9, ERD, LEA, and EMB2001 to be functionally correlative to various drought-responsive genes LEA, AP2/ERF, WRKY, and NAC. These proteins play a vital role in transcriptomic regulation under stress conditions. The higher drought response in Bt cotton (BTCS/BTDS) attributed to the overexpression of photosynthetic proteins enhanced lipid metabolism, increased cellular detoxification and activation chaperones, and reduced synthesis of unwanted proteins. Thus, the Bt variety had enhanced photosynthesis, elevated water retention potential, balanced leaf stomata ultrastructure, and substantially increased antioxidant activity than the Non-Bt cotton. Our results may aid breeders and provide further insights into developing new drought-tolerant and high-yielding cotton hybrid varieties.
... In fact, the transgene insertion site cannot be predetermined and for this reason transgenes may be inserted in functional genomic regions thus disrupting the structure and/or altering the regulation patterns of genes from the plant host genome as previously observed for some commercialized GM crops (Holck et al., 2002;Hernández et al., 2003;Rosati et al., 2008;Morisset et al., 2009;La Paz et al., 2010). Other secondary unintended effects of genetic modification can also arise during conventional breeding as the result of hybridization or spontaneous mutations, processes that are integral to breeding programs (van Gelder and Scheffer, 1991;Conner and Jacobs, 1999;FAO/WHO, 2002). Another documented effect is related to the application of supporting technologies used in the GMO agroecosystem, such as the use of combined herbicides (Bøhn and Millstone, 2019). ...
Profiling technologies, such as proteomics, allow the simultaneous measurement and comparison of thousands of plant components without prior knowledge of their identity. The combination of these non-targeted methods facilitates a more comprehensive approach than targeted methods and thus provides additional opportunities to identify genotypic changes resulting from genetic modification, including new allergens or toxins. The purpose of this study was to investigate unintended changes in GM Bt maize grown in South Africa. In the present study, we used bi-dimensional gel electrophoresis based on fluorescence staining, coupled with mass spectrometry in order to compare the proteome of the field-grown transgenic hybrid (MON810) and its near-isogenic counterpart. Proteomic data showed that energy metabolism and redox homeostasis were unequally modulated in GM Bt and non-GM maize variety samples. In addition, a potential allergenic protein—pathogenesis related protein −1 has been identified in our sample set. Our data shows that the GM variety is not substantially equivalent to its non-transgenic near-isogenic variety and further studies should be conducted in order to address the biological relevance and the potential risks of such changes. These finding highlight the suitability of unbiased profiling approaches to complement current GMO risk assessment practices worldwide.
... The planting area of stacked GM crops accounts for approximately 40% of the global GM crop production area. 2 Although the commercialization of GM crops has many advantages, such as considerable economic benefits and reduced chemical pesticide pollution, more attention is being given to their food and environmental safety. 3,4 Genetic modification might lead to not only the insertion of exogenous DNA fragments but also the rearrangement or deletion of some endogenous genes, 5 therefore interfering with certain biochemical pathways and possibly producing new allergens or toxins. Thus, safety assessments of GM crops must be comprehensively carried out. ...
Unintended effects of genetically modified (GM) crops may pose safety issues. Omics techniques provide researchers with useful tools to assess such unintended effects. Proteomics and metabolomics analyses were performed for three GM maize varieties, 2A-7, CC-2, and 2A-7×CC-2 stacked transgenic maize, and the corresponding non-GM parent Zheng58.
Proteomics revealed 120, 271 and 135 maize differentially expressed proteins (DEPs) in the 2A-7/Zheng58, CC-2/Zheng58 and 2A-7×CC-2/Zheng58 comparisons, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that most DEPs participated in metabolic pathways and the biosynthesis of secondary metabolite. Metabolomics revealed 179, 135 and 131 differentially accumulated metabolites (DAMs) in the 2A-7/Zheng58, CC-2/Zheng58 and 2A-7×CC-2/Zheng58 comparisons, respectively. Based on KEGG enrichment analysis, most DAMs are involved in the biosynthesis of secondary metabolite and metabolic pathways. According to integrated proteomics and metabolomics analysis, the introduction of exogenous EPSPS did not affect the expression levels of six other enzymes or the abundance of seven metabolites involved in the shikimic acid pathway in CC-2 and 2A-7×CC-2 seeds. Six co-DEPs annotated by integrated proteomics and metabolomics pathway analysis were further analyzed by qRT-PCR.
This study successfully employed integrated proteomic and metabolomic technology to assess unintended changes in maize varieties. The results suggest that GM and gene stacking do not cause significantly unintended effects.
... By contrast, the option to introduce foreign genes by transformation or genetic modification (GM), allows to speed up breeding in plant and add "extra-traits" that are not accessible by other traditional techniques (Limera et al., 2017;Rommens et al., 2007). Although these approaches are faster than the conventional ones, they suffer from serious handicaps since are not widely socially acceptable due to health and environmental concerns (Conner and Jacobs, 1999). ...
Improving fruit quality and resistance to pathogens in strawberry by friendly biotechnological approaches represents a major issue to reduce the use of chemicals and pesticides and an important goal for breeding. We have designed new RNAi intragenic silencing constructions by combining specific strawberry promoters and pathogen defence-related candidate genes to silence endogenous target genes in a fruit ripening related manner thus avoiding any unwanted effect on plant growth and development. For this, a ∼3000-bp DNA fragment upstream FvDOF2 and FvFAAT2 coding regions was fused to RNAi designed cassettes for FaWRKY1 and FaNPR3.1 genes. Four different binary plasmid constructions were generated using pMinMYB as a recipient plasmid frame carrying the MdMYB10 gene from a red-fleshed apple as natural selectable marker. The fruit-specific transcription factor FvDOF2 regulates the production of volatile eugenol in ripe fruit receptacles whereas gene FvAAT2 encodes a fruit-related acyltransferase involved in strawberry aroma biogenesis. Also, FaWRKY1 transcription factor negatively regulates resistance to Colletotrichum acutatum in fruit upon infection and FaNPR3.1 is the strawberry orthologue of the Arabidopsis AtNPR3/AtNPR4 salicylic acid (SA) receptors acting as transcriptional co-repressors in plant immunity. Using these plasmid constructs, stable red transformant intragenic strawberry lines silencing target genes are being produced and will be tested for increase resistance to different pathogens. © 2021 International Society for Horticultural Science. All rights reserved.
... The safety of plant-based GM foods for consumption has remained a hot topic and the rarity of research data in this regard may be a contributory factor. Health risks associated with GM foods fall into three main categories: toxins, allergens, and genetic hazards (Conner and Jacobs, 1999). The Royal Society (2002) without considering gene expression products, suggested that there is no evidence of intact gene transfer to humans (and animals) either from vector organisms used in GM plants in the gut or from foodstuffs, despite daily consumption of GM food. ...
Genetically modified (GM) crops are cultivated in over 30 countries with their products and by-products imported by over 60 countries. This chapter seeks to highlight general concerns and potential lifelong effects of consuming GM plant-based food. The consumption of GM plant-based food is as risky as consuming conventional plant-based food. However, the alien genes in these products may be unstable leading to antinutritional and unintended short-term consequences. Due to the paucity of research, no long-term effects have been attributed to the lifelong consumption of these products. Nonetheless, possible lifelong health and socioeconomic effects may result from outcrossing of genes, increasing antibiotic resistance, development of new diseases, as well as potential effects on the environment and biodiversity. Biotechnology companies need to invest more in interdisciplinary research addressing the potential lifelong effects of these products. Although GM foods are safe for consumption, clarification of current risks and lifelong effects are required.
... No doubt transgenic crops are the best option to cope with the future food scarcity and water shortage probably we have to face due to the exponentially increasing population in the coming years (Bradford et al. 2005). Furthermore, having information about the whole biochemical pathway associated with desired traits we will be able to transfer the complete pathways from one specie to the other (Conner and Jacobs 1999;Pandey et al. 2010). Many transgenic plants has been produced using modern technologies (Omics based) for different required characteristics. ...
World’s population is increasing exponentially and it is expected to be doubled by the year 2050. In many developing countries and rural areas, malnourishment is also making the condition worse. Conventional crop improvemnt strategies e.g. breeding have been used by farmers since ages, but these approaches are not well efficient to get our targets of having more yield, high quality and nutritional food to feed the rapidly growing population. In this chapter, we have summarized the importance and use of Omics-based strategies e.g. genomics, transcriptomics, proteomics, metabolomics and interactomics, for crop improvements. Omics based approaches have opened the doors to improve the varieties with high yield and enhanced nutritional value, together with herbicide and other stresses resistance ability By overcoming few challenges related to the application of Omics in agriculture, this could be the best option to confront with the current needs and future food demands of the exceeding population.
... The safety of plant-based GM foods for consumption has remained a hot topic and the rarity of research data in this regard may be a contributory factor. Health risks associated with GM foods fall into three main categories: toxins, allergens, and genetic hazards (Conner and Jacobs, 1999). The Royal Society (2002) without considering gene expression products, suggested that there is no evidence of intact gene transfer to humans (and animals) either from vector organisms used in GM plants in the gut or from foodstuffs, despite daily consumption of GM food. ...
Genetically modified (GM) crops are cultivated in over 30 countries with their products and by-products imported by over 60 countries. This chapter seeks to highlight general concerns and potential lifelong effects of consuming GM plant-based food. The consumption of GM plant-based food is as risky as consuming conventional plant-based food. However, the alien genes in these products may be unstable leading to antinutritional and unintended short-term consequences. Due to the paucity of research, no long-term effects have been attributed to the lifelong consumption of these products. Nonetheless, possible lifelong health and socioeconomic effects may result from outcrossing of genes, increasing antibiotic resistance, development of new diseases, as well as potential effects on the environment and biodiversity. Biotechnology companies need to invest more in interdisciplinary research addressing the potential lifelong effects of these products. Although GM foods are safe for consumption, clarification of current risks and lifelong effects are required.
... Globally substantial research evaluating the safety of transgenic rice has been done. These studies have been mainly in the discipline of toxicology (animal experiments) involving analysis of metabolites (Anldanl et al., 2002;Zhao et al., 2013;Jiang et al., 2014), toxicokinetics (Kuiper et al., 2004;Delaney et al., 2008), chronic toxicity and sub-chronic toxicity (Kuiper et al., 1999;Hug, 2008), and reproductive and teratogenicity test methods (Conner and Jacobs, 1999;König et al., 2004;Feng et al., 2013;Wang et al., 2014). Safety evaluations were made, mainly by feeding the animal model, to detect the potential effect of transgenic rice on animal organs Gu, 2005;Kok et al., 2008;Fu et al., 2012). ...
With the expanding demand for genetically modified (GM) rice, its safety evaluation is of great significance. Therefore, this study was carried out to assess the acute cytotoxicity of the whole protein extract from GM rice Bar68-1 in Mus musculus lymphocytes in vitro. Cell viability was determined by Cell Counting Kit-8 (CCK-8) and Neutral Red Uptake (NRU) tests. CCK-8 tests was carried out according to the manufacturer’s instructions. Cell dehydrogenase (catalytic redox enzymes) activity was spectrophotometrically determined at 450 nm. The tests result were recorded immediately. NRU tests were completed under yellow light in a dark room according to an improved protocol. Lysosomal uptake of neutral red was spectrophotometrically determined at 540 nm and the results were recorded immediately. The results showed that the survival rate of M. musculus lymphocytes in the positive control group was significantly less than in the blank control group (P < 0.05). Moreover, an exposure- time-effect relationship was observed in the positive control group with CCK-8 and NRU tests. There was no significant difference in survival rate between GM rice Bar68-1group and non-GM rice D68 group (P > 0.05). The GM rice Bar68-1 group also did not show a higher survival rate than non-GM rice D68 group (P > 0.05). These results suggested that the whole protein extract from Bar68-1 and D68 were equivalent in their cytotoxicity, and GM rice Bar68-1 had no acute cytotoxic effect on M. musculus lymphocytes in vitro.
... expression level, or can turn on the existing silent genes (Conner and Jacobs 1999). Alternatively, expression of the Bt proteins may potentially alter the metabolism and biochemical pathways of the plants. ...
... Transformation with promoter-less reporter genes has resulted in the recovery of transgenic plants with reporter gene expression suggesting integration into transcriptionally active gene sequences (Lindsey et al., 1993). Similarly, transcriptional interference by highly expressed promoters on transgene constructs can potentially influence the expression of endogenous genes resulting in unanticipated side effects (Conner and Jacobs, 1999). These side effects compound the problem of lack of predictability in the performance of transgenically derived materials and further compromise the probability of successful product development. ...
The demand for crops requiring increasingly complex combinations of transgenes poses unique challenges for transgenic trait deployment. Future value-adding traits such as those associated with crop performance are expected to involve multiple transgenes. Random integration of transgenes not only results in unpredictable expression and potential unwanted side effects but stacking multiple, randomly integrated, independently segregating transgenes creates breeding challenges during introgression and product development. Designed nucleases enable the creation of targeted DNA double-strand breaks at specified genomic locations whereby repair can result in targeted transgene integration leading to precise alterations in DNA sequences for plant genome editing, including the targeting of a transgene to a genomic locus that supports high-level and stable transgene expression without interfering with resident gene function. In addition, targeted DNA integration via designed nucleases allows for the addition of transgenes into previously integrated transgenic loci to create stacked products. The currently reported frequencies of independently generated transgenic events obtained with site-specific transgene integration without the aid of selection for targeting are very low. A modular, positive selection-based gene targeting strategy has been developed involving cassette exchange of selectable marker genes which allows for targeted events to be preferentially selected, over multiple cycles of sequential transformation. This, combined with the demonstration of intragenomic recombination following crossing of transgenic events that contain stably integrated donor and target DNA constructs with nuclease-expressing plants, points towards the future of trait stacking that is less dependent on high-efficiency transformation.
© 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.
... Also, a jellyfish green fluorescent protein (GFP) gene, another marker gene, was utilized but did not find any risk of toxicity and allergenicity (Richards et al. 2003). There are concerns about the aforementioned potential impacts of genes that can cause gene silencing, changes in Environ Chem Lett (2015) 13:239–249 243 123 Author's personal copy expression level, or can turn on the existing silent genes (Conner and Jacobs 1999). Alternatively, expression of the Bt proteins may potentially alter the metabolism and biochemical pathways of the plants. ...
Genomic technologies have been used to improve cultivated crop species. For example, Bt genes such as Cry1Ac, Cry2Ab, Cry1F and Cry3Bb1 are derived from Bacillus thuringiensis, a soil bacterium. Such genes provide protection against lepidopteran insect pests. Bt genes have been introduced in corn, cotton, soybean, rice, potato and canola. Genetically modified (GM)-cotton, containing the Cry1Ac gene, was released for cultivation in the mid-1990s in the USA and later in 28 countries including China and India. Potential harmful effects of the Bt-crops on non-targets were assessed before release into the environment. Most commonly, cultivation of the Bt-crops was found safe. Safety was tested using various experiments including: the insertional impact of transgene and its regulatory elements on plant phenotype and agronomic performance; effect on non-target organisms; and nutritional impacts on multiple experimental models, albeit the studies were conducted for limited durations. However, skeptics always claim for conducting extensive clinical as well as field trials and also cast doubt on methods and procedures of calculating the ecological risks. This debate got further momentum especially after the publication of reports on substantial reduction in monarch butterfly caterpillars when exposed to Bt-maize pollen—though later nullified—and detection of traces of transgene in various tissues of experimental animals. It is generally accepted that procedures, methods and protocols for evaluating the potential risks of GM-crops and foods should be standardized for building confidence of all stakeholders. Efforts should be exerted in deploying genes of interest, marker genes and regulatory sequences invoking no or little issues of potential risks to the ecosystem.
... Despite the many benefits of GM crops, the biggest problem is controversial on the safety of food that derived from GM crops. An important issue is whether the existence of unintended effects which are caused by random insertion of exogenous specific genes into plant genomes that may result in disruption, modification or rearrangement of the genome [4,5]. These unintended processes may further result in the formation of new biochemical processes or new proteins (especially new allergens or toxins), which have been an important matter of concerns [6,7] . ...
Background
As the rapid growth of the commercialized acreage in genetically modified (GM) crops, the unintended effects of GM crops’ biosafety assessment have been given much attention. To investigate whether transgenic events cause unintended effects, comparative proteomics of cotton leaves between the commercial transgenic Bt + CpTI cotton SGK321 (BT) clone and its non-transgenic parental counterpart SY321 wild type (WT) was performed.
Results
Using enzyme linked immunosorbent assay (ELISA), Cry1Ac toxin protein was detected in the BT leaves, while its content was only 0.31 pg/g. By 2-DE, 58 differentially expressed proteins (DEPs) were detected. Among them 35 were identified by MS. These identified DEPs were mainly involved in carbohydrate transport and metabolism, chaperones related to post-translational modification and energy production. Pathway analysis revealed that most of the DEPs were implicated in carbon fixation and photosynthesis, glyoxylate and dicarboxylate metabolism, and oxidative pentose phosphate pathway. Thirteen identified proteins were involved in protein-protein interaction. The protein interactions were mainly involved in photosynthesis and energy metabolite pathway.
Conclusions
Our study demonstrated that exogenous DNA in a host cotton genome can affect the plant growth and photosynthesis. Although some unintended variations of proteins were found between BT and WT cotton, no toxic proteins or allergens were detected. This study verified genetically modified operation did not sharply alter cotton leaf proteome, and the target proteins were hardly checked by traditional proteomic analysis.
Electronic supplementary material
The online version of this article (doi:10.1186/s12953-015-0071-8) contains supplementary material, which is available to authorized users.
Agricultural soil salinity poses a significant threat to global crop productivity, contributing to ongoing land degradation. Conventional food crops (glycophytes) display limited tolerance to high-salinity levels, while halophytes, naturally adapted to high saline environments, harbor a wealth of genes responsible for salinity tolerance. Leveraging these genes through techniques such as plant breeding, genetic engineering, and genome editing holds promise for transforming conventional crop plants. However, traditional plant breeding is labor-intensive and inefficient. Introduction of genes from other salt-tolerant species (halophytes) through genetic engineering has gained prominence due to its reliability, long-term efficacy, and potential cost savings while maintaining environmental sustainability, but it also faces commercial acceptance challenges in many crops due to concerns about transgenics and GMOs. The emergence of genome editing, facilitating tools such as zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeat (CRISPR), offers a more optimistic avenue for enhancing the salt tolerance of glycophytes. This cutting-edge biotechnological approach opens up new prospects for further improving the salinity tolerance capacity of conventional crops which has been widely discussed in this chapter.
Genetically modified organisms (GMOs) are an important nexus of biotechnology, agriculture, and research. GMOs have gained popularity because of their potential to address global food insecurity. However, the widespread adoption of GMOs has sparked debates and controversies. This study collected data on the threats and effects of the GMO ban and the status of implementation of Biosafety Act and Cartagena Protocol on biosafety in Kenya. A cross-sectional survey was used with 216 participants sampled purposively and 16 key informants interviewed in a qualitative survey with triangulation of data from a scoping literature review. Fifty-seven percent of the respondents indicated GMOs and genetically modified (GM) foods are a solution to food security in Africa. A majority believe that the introduction of GMO technology and the use of GM food is beneficial (52.3%). Fifty-five percent of the respondents indicated that GM foods are safe. Most respondents (>90%) indicated that there is a low threat of GMO technology on the environment, human health, and animal health. The respondents (54.3%) agreed there is good adherence to the Cartagena Protocol. The correlation between awareness of the Cartagena Protocol on biosafety and views on whether there are adequate legal and regulatory frameworks was r = 0.4, indicating a weak but positive relationship between the two. Respondents who reported that the legal and regulatory frameworks were adequate were, therefore, likely (r = 0.4) to be aware of the Cartagena Protocol. There was a moderate positive correlation (r = 0.67) on views that GM food is a solution to food insecurity. Understandably, the relationship between GM food health concerns and GM food as safe was moderately negative (r = −0.4591). The findings are important in addressing gaps in the current GMO regulations and implementation aimed at increasing awareness of GMO technology as well as informing policy on biotechnology.
To evaluate the reproductive toxicity of gene modified rice generated by introducing phytoene synthase (Psy) and bacterial phytoene desaturase (CrtI) from maize and Erwinia uredovora, Wistar rats were allocated into 3 groups and fed with Psy and CrtI gene modified rice mixture diet (GM group), non-gene modified rice mixture diet (non-GM group), and AIN-93 diet (Blank control group) from parental generation (F0) to the offsprings (F1). GM rice, Heijinmi (HJM) and Non-GM rice, Heishuai (HS), were both formulated into diets at ratios of 73.5% and 75.5% according to the AIN93 diet for rodent animals, respectively. Relative to the non-GM group, no biologically relevant differences were observed in GM group rats concerning reproductive performance such as fertility rate, gestation rate, mean duration, hormone level, and reproductive organ pathology. The developmental parameters results were not significantly different from the non-GM group such as body weight, food consumption, developmental neurotoxicity, behavior, hematology, and serum chemistry. In terms of immunotoxicity, the IgG indicators of offspring from the GM group improved in contrast with the non-GM group. Additional gut flora analysis of F0 generation rats resulted as that the treatment elicited an increased gut microflora diversity of F0 rats. And no horizontal gene transfer of Psy and CrtI genes in rats fed a GM rice HJM diet. In conclusion, we found no adverse effects related to GM rice in the extended one-generation reproductive toxicity study, indicating that GM rice is a safe alternative for its counterpart rice regarding reproductive toxicity.
Label-free quantitative proteomic (LFQ) and widely targeted metabolomic analyses were applied in the safety evaluation of three genetically modified (GM) maize varieties, BBL, BFL-1, and BFL-2, in addition to their corresponding non-GM parent maize. A total of 76, 40, and 25 differentially expressed proteins (DEPs) were screened out in BBL, BFL-1, and BFL-2, respectively, and their abundance compared was with that in their non-GM parents. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that most of the DEPs participate in biosynthesis of secondary metabolites, biosynthesis of amino acids, and metabolic pathways. Metabolomic analyses revealed 145, 178, and 88 differentially accumulated metabolites (DAMs) in the BBL/ZH58, BFL-1/ZH58, and BFL-2/ZH58×CH72 comparisons, respectively. KEGG pathway enrichment analysis showed that most of the DAMs are involved in biosynthesis of amino acids, and in arginine and proline metabolism. Three co-DEPs and 11 co-DAMs were identified in the seeds of these GM maize lines. The proteomic profiling of seeds showed that the GM maize varieties were not dramatically different from their non-GM control. Similarly, the metabolomic profiling of seeds showed no dramatic changes in the GM/non-GM maize varieties compared with the GM/GM and non-GM/non-GM maize varieties. The genetic background of the transgenic maize was found to have some influence on its proteomic and metabolomic profiles.
Plant breeding is a continuous process in which genetic variation and heritability of a trait are essential. Cisgenesis and intragenesis are biotechnology techniques employed to generate additional genetic diversity in the current germplasm to improve their productivity. The development in sequencing approaches and the genome information accessibility simplifies isolation of integral Cis-genes, in conjunction with related promoter/terminator from the same species, and are introduced into the identical or a closely related genome, sexually compatible species, whereas in intragenesis, diverse coding and controlling sequences are accumulated either in a sense or in antisense alignment. This chapter illustrates the present status, applications, constraints, and prospects of cisgenesis and intragenesis to improve crop disease resistance. Further comparisons of cisgenesis, intragenesis, and transgenesis in an era of genome editing to develop cisgenic crops were described. This technique has been utilized to develop disease-resistant cultivars, such as the late blight-resistant potato, by transferring gene Rpi-sto1, Rpi-vnt1.1; similarly, scab resistance gene HcrVF2 for the development of apple cultivars and Vvtl-1, NtpII genes for grapes resistant to fungal diseases. Therefore, we can infer cisgenesis as an influential substitute to transfer the desired gene without linkage drag. Hence, it can be concluded that both cisgenic and intragenic derived genetically modified plants (GMP) are as sustainable and eco-friendly as traditionally bred plants and, therefore, excused from GMP regulation.KeywordsCisgenesisDisease resistanceGenome editingIntragenesisPlant breeding
An intragene involves the rearrangement of gene configurations based on switching coding sequences and regulatory regions within a species. The overexpression of specific GSL (Gibberellin Stimulated-Like) genes is known to confer enhanced disease resistance. A potato intragenic expression cassette, with the potato GSL1 gene placed under the regulatory control of the light-inducible Lhca3 gene from potato, was designed and constructed. The resulting intragene was transformed using Agrobacterium-mediated gene transfer into potato cultivar Iwa to investigate whether overexpression of the potato GSL1 intragene can confer disease resistance. The transgenic status of the resulting plants was confirmed by PCR. The majority of the transgenic potato lines were determined to overexpress the GSL1 gene at the mRNA level using quantitative RT–PCR analysis. From these results, seven lines were selected for further characterisation and thoroughly evaluated in bioassays for resistance to Pectobacterium atrosepticum which causes blackleg disease in potato. These pathogenicity bioassays demonstrated that high transcriptional overexpression of GSL1 confers high resistance to blackleg disease. This confirms a functional role of the GSL1 gene in plant defence against pathogens and provides an intragenic approach for disease resistance in potatoes.
Under climate change, the spread of pests and pathogens into new environments has a dramatic effect on crop protection control. Strawberry (Fragaria spp.) is one the most profitable crops of the Rosaceae family worldwide, but more than 50 different genera of pathogens affect this species. Therefore, accelerating the improvement of fruit quality and pathogen resistance in strawberry represents an important objective for breeding and reducing the usage of pesticides. New genome sequencing data and bioinformatics tools has provided important resources to expand the use of synthetic biology-assisted intragenesis strategies as a powerful tool to accelerate genetic gains in strawberry. In this paper, we took advantage of these innovative approaches to create four RNAi intragenic silencing cassettes by combining specific strawberry new promoters and pathogen defense-related candidate DNA sequences to increase strawberry fruit quality and resistance by silencing their corresponding endogenous genes, mainly during fruit ripening stages, thus avoiding any unwanted effect on plant growth and development. Using a fruit transient assay, GUS expression was detected by the two synthetic FvAAT2 and FvDOF2 promoters, both by histochemical assay and qPCR analysis of GUS transcript levels, thus ensuring the ability of the same to drive the expression of the silencing cassettes in this strawberry tissue. The approaches described here represent valuable new tools for the rapid development of improved strawberry lines.
Background
Widely targeted metabolomics was applied to estimate the differences in the metabolite profiles of maize seeds from 3 natural genotypic varieties and 4 genetically modified (GM) lines.
Results
Pairwise comparison with their isogenic controls revealed 71, 121, 43 and 95 differentially accumulated metabolites (DAMs) in GM maize seeds of C0030.2.4, C0030.3.5, C0010.1.1 and C0010.3.1, respectively. KEGG pathway enrichment analysis showed that most of these DAMs participated in the biosynthesis of secondary metabolites and purine metabolism in GM maize C0030.2.4 and C0030.3.5, but participated in tryptophan metabolism and 2-oxocarboxylic acid metabolism in C0010.3.1 seeds and in metabolic pathways and the biosynthesis of secondary metabolites in C0010.1.1 seeds. The data also showed that the differences in metabolite accumulation, both total DAMs and co-DAMs, among the different natural genotypic varieties (418 DAMs and 39 co-DAMs) were greater than those caused by genetic modification (330 DAMs and 3 co-DAMs).
Conclusions
None of the DAMs were identified as new or unintended, showing only changes in abundance in the studied maize seeds. The metabolite profile differences among the 3 non-GM lines were more notable than those among GM lines. Different genetic backgrounds affect metabolite profiling more than gene modification itself.
Graphic abstract
The term ‘genetically modified organisms (GMO)’ is still an under-discussion topic since it was introduced decades ago. Beside public concerns against GM products, some of the scientists claim it is a vital tool to improve plant breeding in the future, while others emphasize potential harmful effects of the technology. In this paper, advantages, risks, and some technological developments of GMOs technology were discussed with old and recent studies to understand where we are at GMO technology in 2020s. GMO technology also has a great impact on global economy with a total of US$ 224.9 billion profit between 1996 and 2018. Its economic impact makes the technology inevitable in the future. Thus, this paper will also explain legal perspectives of the countries against GM crop breeding and marketing.
To study reproductive toxicity of gene modified wheat generated by introducing DREB3 (drought response element binding protein 3) gene, Wistar rats of were allocated into 3 groups and fed with DREB3 gene modified wheat mixture diet (GM group), non-gene modified wheat mixture diet (Non-GM group) and AIN-93 diet (Control group) from parental generation (F0) to the second offspring (F2). GM wheat and Non-GM wheat, Jimai22, were both formulated into diets at a ratio of 69.55% according to AIN93 diet for rodent animals. Compared with non-GM group, no biologically related differences were observed in GM group rats with respect to reproductive performance such as fertility rate, gestation rate, mean duration, hormone level, reproductive organ pathology and developmental parameters such as body weight, body length, food consumption, neuropathy, behavior, immunotoxicity, hematology and serum chemistry. In conclusion, no adverse effect were found relevant to GM wheat in the two generation reproduction toxicity study, indicating the GM wheat is a safe alternative for its counterpart wheat regarding to reproduction toxicity.
Current and futuristic pattern of global climate change are a major concern and is a major threat for the entire ecosystem. Man-made centric climate change is majorly through emissions of greenhouse gases, that is, carbon dioxide, methane, and nitrous oxide, etc. These variables and their implications remain a challenge for crop growth and productivity. Many attempts in past were made to address this challenge and green revolution is one such breakthrough but the extent of climate change effects on plants has varied over time. The mainstreamed agriculture involving high yielding crop varieties, chemical fertilizer input, and chemical pesticides usage are of limited significance under changing climatic conditions. So, intensifying crop production and addressing climate change must be done in an integrated sustainable way and development of genetically modified crops can act as an effective adaptive strategy against climate change. There shall always be challenge though in dealing with the unexplored dimension of climate change and also the extent to which a particular genetically modified crop would be able to deal with the climate change. This chapter is therefore emphasized on genetically modified crop development to resist different variables of climate change, singly and in combination and the related challenges.
Peroxisome proliferator‐activated receptor gamma (PPARγ) is a master regulator of adipogenesis and lipogenesis. To understand its roles in fiber formation and fat deposition in skeletal muscle, we successfully generated muscle‐specific overexpression of PPARγ in two pig models by random insertion and CRISPR/Cas9 transgenic cloning procedures. The content of intramuscular fat was significantly increased in PPARγ pigs while had no changes on lean meat ratio. PPARγ could promote adipocyte differentiation by activating adipocyte differentiating regulators such as FABP4 and CCAAT/enhancer‐binding protein (C/EBP), along with enhanced expression of LPL, FABP4, and PLIN1 to proceed fat deposition. Proteomics analyses demonstrated that oxidative metabolism of fatty acids and respiratory chain were activated in PPARγ pigs, thus, gathered more Ca²⁺ in PPARγ pigs. Raising of Ca²⁺ could result in increased phosphorylation of CAMKII and p38 MAPK in PPARγ pigs, which can stimulate MEF2 and PGC1α to affect fiber type and oxidative capacity. These results support that skeletal muscle‐specific overexpression of PPARγ can promote oxidative fiber formation and intramuscular fat deposition in pigs.
The recent advances in ‘omics’ technologies have impacted biological science research in many ways. This includes the realm diversity studies, physiological, biochemical and molecular level researches to the development of breakthrough products and approaches. They are strengthening our attempts to fight with emerging diseases, global food crisis, environmental degradation, production of quality value-added products values, and in better understanding of the underlying mechanism(s) of host-pathogen interactions and stress (both biotic and abiotic) tolerance in plants and microbes. However, many of these techniques are still under refinement and resulting data need careful integration for attending meaningful conclusions. Although not a big concern, but we must be attentive to some of the biosafety measures and ethical concerns associated with these techniques, which we have tried to discuss in this chapter
Proteomics provides a powerful approach to evaluate the unintended effects of transgenic crops. In this study, iTRAQ (isobaric tags for relative and absolute quantitation)-based quantitative proteomics was applied to estimate the differences in the proteomic profiles of maize seeds from 3 natural genotypic varieties and 4 genetically modified (GM) lines. Compared with their isogenic controls, there were 108, 69, 180 and 204 differentially expressed proteins (DEPs) in GM maize C0030.2.4, C0030.3.5, C0010.1.1 and C0010.3.1 seeds. Molecular functional classification showed that these DEPs were mainly involved in catalytic activity and binding. KEGG pathway annotation showed that most of these DEPs participated in metabolic pathways, the biosynthesis of secondary metabolites and microbial metabolism in diverse environments. In addition to the foreign protein EPSPS, 4 maize proteins were simultaneously identified as being differentially expressed among the 4 GM maize lines. Nevertheless, the regulation trends of these DEPs were not exactly consistent among the different GM maize lines. The data also showed that the differences in protein expression among the different natural genotypic varieties were greater than those caused by genetic modification. These results provide new information regarding the unintended effects of GM maize seeds through iTRAQ-based quantitative proteomic analysis.
Plant genetic transformation is an important technological advancement in modern science, which has not only facilitated gaining fundamental insights into plant biology but also started a new era in crop improvement and commercial farming. However, for many crop plants, efficient transformation and regeneration still remain a challenge even after more than 30 years of technical developments in this field. Recently, FokI endonuclease-based genome editing applications in plants offered an exciting avenue for augmenting crop productivity but it is mainly dependent on efficient genetic transformation and regeneration, which is a major roadblock for implementing genome editing technology in plants. In this chapter, we have outlined the major historical developments in plant genetic transformation for developing biotech crops. Overall, this field needs innovations in plant tissue culture methods for simplification of operational steps for enhancing the transformation efficiency. Similarly, discovering genes controlling developmental reprogramming and homologous recombination need considerable attention, followed by understanding their role in enhancing genetic transformation efficiency in plants. Further, there is an urgent need for exploring new and low-cost universal delivery systems for DNA/RNA and protein into plants. The advancements in synthetic biology, novel vector systems for precision genome editing and gene integration could potentially bring revolution in crop-genetic potential enhancement for a sustainable future. Therefore, efficient plant transformation system standardization across species holds the key for translating advances in plant molecular biology to crop improvement.
Background:
Food safety in the food market is one of the key areas of focus in public health, because it affects people of every age, race, gender, and income level around the world. The local and international food marketing continues to have significant impacts on food safety and health of the public. Food supply chains now cross multiple national borders which increase the internationalization of health risks. This systematic review of literature was, therefore, conducted to identify common public health risks related to food safety issues in the food market.
Methods:
All published and unpublished quantitative, qualitative, and mixed method studies were searched from electronic databases using a three step searching. Analytical framework was developed using the PICo (population, phenomena of interest, and context) method. The methodological quality of the included studies was assessed using mixed methods appraisal tool (MMAT) version 2018. The included full-text articles were qualitatively analyzed using emergent thematic analysis approach to identify key concepts and coded them into related non-mutually exclusive themes. We then synthesized each theme by comparing the discussion and conclusion of the included articles. Emergent themes were identified based on meticulous and systematic reading. Coding and interpreting the data were refined during analysis.
Results:
The analysis of 81 full-text articles resulted in seven common public health risks related with food safety in the food market. Microbial contamination of foods, chemical contamination of foods, food adulteration, misuse of food additives, mislabeling, genetically modified foods (GM foods), and outdated foods or foods past their use-by dates were the identified food safety-related public health risks in the food market.
Conclusion:
This systematic literature review identified common food safety-related public health risks in the food market. The results imply that the local and international food marketing continues to have significant impacts on health of the public. The food market increases internationalization of health risks as the food supply chains cross multiple national borders. Therefore, effective national risk-based food control systems are essential to protect the health and safety of the public. Countries need also assure the safety and quality of their foods entering international trade and ensure that imported foods conform to national requirements.
Stable transgenic rice line (named KRSV-1) with strong resistance against rice stripe virus was generated using the gene sequence of disease-specific protein by RNA interference. Comprehensive safety assessment of transgenic plants has turned into a significant field of genetic modification food safety. In this study, a safety assessment of KRSV-1 was carried out in a stepwise approach. The molecular analysis exhibited that KRSV-1 harbored one copy number of transgene, which was integrated into the intergenic non-coding region of chromosome 2 associated with inter-chromosomal translocations of 1.6-kb segments of chromosome 8. Then, transcriptomics and proteomics analyses were carried out to detect the unintended effects as a result of the integration of the transgene. Although 650 dramatically differentially expressed genes (DDEGs) and 357 differentially expressed proteins were detected between KRSV-1 and wild-type (WT) by transcriptomics and proteomics analyses, no harmful members in the form of toxic proteins and allergens were observed. Encouragingly, the nutritional compositions of seeds from KRSV-1 were comparable with WT seeds. The results of this entire study of molecular analysis, transcriptome and proteome profile of KRSV-1 revealed that no detrimental changes in the form of toxic proteins and allergens were detected in the transgenic rice line due to the integration of the transgene.
The unintended effects of transgenesis have increased food safety concerns, meriting comprehensive evaluation. Proteomic profiling provides an approach to directly assess the unintended effects. Herein, the isobaric tags for relative and absolute quantitation (iTRAQ) comparative proteomic approach was employed to evaluate proteomic profile differences in seed cotyledons from 4 genetically modified (GM) and 3 natural genotypic soybean lines. Compared with their non-GM parents, there were 67, 61, 13 and 22 differentially expressed proteins (DEPs) in MON87705, MON87701 × MON89788, MON87708, and FG72. Overall, 170 DEPs were identified in the 3 GM soybean lines with the same parents, but 232 DEPs were identified in the 3 natural soybean lines. Thus, the differences in protein expression among the genotypic varieties were greater than those caused by GM. When considering ≥2 replicates, 4 common DEPs (cDEPs) were identified in the 3 different GM soybean lines with the same parents and 6 cDEPs were identified in the 3 natural varieties. However, when considering 3 replicates, no cDEPs were identified. Regardless of whether ≥2 or 3 replicates were considered, no cDEPs were identified among the 4 GM soybean lines. Therefore, no feedback due to GM was observed at the common protein level in this study.
Food products from genetically modified (GM) crops for human consumption, referred to as ‘GM foods’ in this chapter, have been available on the market since 1994. A few years after their introduction, controversy and negative reactions arose because of their potentially harmful environmental effects and the health risks associated with their intake. In spite of ongoing concerns, the growing of GM crops has increased more than 100-fold during the last 20 years. Nowadays, one of the most controversial aspects of GM foods consumption is still the risk to human health. The first complaint is that genes are inserted randomly into the crop genome that in turn may modify non-target gene sequences or cause genetic mutations during the transformation process. Therefore, the insertion site and copies of the new DNA sequence should be analysed before the approval of the product for human consumption. This raises the possibility of new conjugated proteins or peptides with unknown biological effects on human health. Another critical point to consider is the presence of proteins without a natural history of consumption. Recently, the potential carcinogenicity of herbicides used in GM crop land has been used to argue for the lack of safety of GM foods. The majority of results show that GM foods have no adverse effects on model animals. Only a few studies have detected serious risks attributable to some GM foods, principally at the microscopic and molecular levels. In this respect, assessment of the health risk associated with GM foods has not been standardized and, as a result, a wide variety of protocols are being used. In the near future, it will be possible to study the effects of specific nutrients, foods or whole diets on the expression of thousands of genes at the same time, as well as changes in metabolic pathways or the use of specific metabolites as biomarkers for human intake or animal models. This ‘omics’ approach will be an alternative for characterizing the health risks of any GM food. This chapter reviews the current published information on nutrition and health concerns associated with the consumption of GM foods, presents the pros and cons for GM food consumption, and discusses some future perspectives on the risk assessment based on current science and technology developments.
The U.S. Food and Drug Administration (FDA), which has statutory authority for protecting the safety and quality of the food supply, provides the oversight for genetically modified foods cultivated and marketed within the American agricultural system. This article examines the FDA's policies on genetically modified foods including its voluntary consultation program and its proposed rule on premarket notification and data submission. The FDA's consultations of foods modified for delayed ripening and herbicide tolerance are reviewed. The article also discusses the FDA's science-based approach for evaluating whether there are any potential adverse health effects of genetically modified food products. The agency has chosen an approach to risk assessment that takes account of both product characteristics and the process through which the food is developed.
From the increasing number of patent registrations, the European Commission (EC) concludes that nanotechnology ranks within the six most promising key technologies. Most successful at inventing nanomaterials—measuring one billionth of a meter—is nature, which provides blueprints of self-organizing physical-chemical nanoparticles (NPs) properties and opens new dimensions for researchers in exploiting nature’s NPs and developing new products for increasing the agricultural and industrial productivity.
After summarizing product processing effects, advantages and risks for agriculture, food, nutrition, and medicine, this book chapter discusses reasons why NPs use should occur balanced and carefully controlled by health and landscape policy. Only then a successful and profitable use for the overall benefit at lowest environmental pollution is achieved.
The European Commission requested that the EFSA Panel on Genetically Modified Organisms deliver a scientific opinion related to risk assessment of cisgenic and intragenic plants. The EFSA GMO Panel considers that the Guidance for risk assessment of food and feed from genetically modified plants and the Guidance on the environmental risk assessment of genetically modified plants are applicable for the evaluation of food and feed products derived from cisgenic and intragenic plants and for performing an environmental risk assessment and do not need to be developed further. It can be envisaged that on a case-by-case basis lesser amounts of event- specific data are needed for the risk assessment. The EFSA GMO Panel compared the hazards associated with plants produced by cisgenesis and intragenesis with those obtained either by conventional plant breeding techniques or by transgenesis. The Panel concludes that similar hazards can be associated with cisgenic and conventionally bred plants, while novel hazards can be associated with intragenic and transgenic plants. The Panel is of the opinion that all of these breeding methods can produce variable frequencies and severities of unintended effects. The frequency of unintended changes may differ between breeding techniques and their occurrence cannot be predicted and needs to be assessed case by case. Independent of the breeding method, undesirable phenotypes are generally removed during selection and testing programmes by breeders. The risks to human and animal health and the environment will depend on exposure factors such as the extent to which the plant is cultivated and consumed.
The engineering of plants with enhanced tolerance to abiotic stresses typically involves complex multigene networks and may therefore have a greater potential to introduce unintended effects than simple monogenic traits. Therefore it is essential to study the unintended effects in transgenic plants engineered for stress tolerance. Drought- and salt-tolerant transgenic wheat overexpressing the transcription factor, GmDREB1, was selected to investigate unintended pleiotropic effects using RNA-sequence analysis. As a control, we compared the transcriptome alteration of transgenic plants with wild-type plants subjected to salt stress. We found that GmDREB1 overexpression had a minimal impact on gene expression under normal condition. GmDREB1 overexpression resulted in transcriptional reprogramming of the salt response, but many of the genes with differential expression are known to mitigate salt stress and contribute incrementally to the enhanced stress tolerance of transgenic wheat. GmDREB1 overexpression did not activate unintended gene networks with respect to gene expression in the roots of transgenic wheat. This work is important for establishing a method of detecting unintended effects of genetic engineering and the safety of such traits with the development of marketable transgenic crops in the near future.
Genomic technologies started in the early 1980s to improve the genomes of cultivated crop species. For example the term “Bt” comes from the soil bacterium Bacillus thuringiensis containing genes, e.g. Cry1Ac, Cry2Ab, Cry1F, Cry3Bb1, that provides protection against lepidopteran insect pests. Those genes have been inserted in crops such as corn, cotton, soybean, rice, potato and canola released for cultivation in mid 1990s in USA, and later in many other countries like China and India. About 29 countries commercialized genetically-modified (GM) or ‘transgenic’ crops while 30 countries granted regulatory approvals for planting GM-crops; together making 75 % of the world population. Potential harmful effects of the Bt-crops on non-targets were quantified before releasing such non-conventional crops into the environment. The cultivation of Bt-crops were most commonly found safe, based on various studies including the insertional impact of transgene and its regulatory elements on plant phenotype and agronomic performance, effect on non-target organisms (NTOs) and nutritional impacts on multiple experimental models. Albeit the studies were conducted for limited durations. However, the skeptics always claim for conducting extensive clinical as well as field trials, and also doubt on methods and procedures of calculating the ecological risks. This debate is still on-going, especially after reports on substantial reduction of monarch butterfly caterpillars exposed to Bt-maize pollen, though later nullified; and detection of traces of transgene in various tissues of experimental animals. Procedures, methods and protocols for evaluating potential risks of GM-crops and foods should be standardized as the first step to build trust of researchers and end-users. Many efforts should be exerted in deploying genes of interest, marker genes and regulatory sequences invoking no or little issues of potential risks to the ecosystem.
To assess the safety of Bar-transgenic rice, its allergenicity and mutagenicity were tested on 100 Kunming mice (Mus musculus). The test mice were randomly divided into five groups of twenty mice each and were given diets containing varying doses of genetically modified (GM) Bar68-1 rice, D68 (non-GM) rice, and routine feed. On the 180 th day, five mice from each group were randomly sampled, and the IgE and DAO levels in their serum and the sIgA in their small intestinal mucus were quantified. The quantification of these chemicals was done for three generations of mice. Moreover, the 12S rDNA and 16S rDNA conserved region of the small intestinal mitochondrial DNA (mtDNA) were sequenced using the double sequencing method. The results indicate no significant difference in the serum sIgA, DAO and IgE between the Bar68-1 GM rice group and the non-GM D68 rice groups (P>0.05). The Bar-transgenic rice did not have allergenic effects on the mice and no mutation was found in the 12S rDNA and 16S rDNA conserved region of the intestinal mtDNA.
Environmental Health Ethics illuminates the conflicts between protecting the environment and promoting human health. In this study, David B. Resnik develops a method for making ethical decisions on environmental health issues. He applies this method to various issues, including pesticide use, antibiotic resistance, nutrition policy, vegetarianism, urban development, occupational safety, disaster preparedness, and global climate change. Resnik provides readers with the scientific and technical background necessary to understand these issues. He explains that environmental health controversies cannot simply be reduced to humanity versus environment and explores the ways in which human values and concerns – health, economic development, rights, and justice – interact with environmental protection. © Cambridge University Press 2012 except in the United States of America.
This study is an attempt to identify the status of genetically modified food consumption in the developing countries like Malaysia. From preceding literature, a theoretical framework was developed to show the effects of perceived risks, perceived quality and social norms on the consumer purchase intention for genetically modified food. The sample of 392 respondents was randomly selected from two leading hypermarkets in Johor Bahru. Results from Multiple Regression revealed that among the three predicting variables, perceived quality and social norms had significant and positive relationship with the consumer purchase intentions while perceived risks did not have any significant relationship with it. A comparative analysis of Muslim and non-Muslim consumers revealed significant difference of the purchase intentions for GMF between the two groups. Implications and future research suggestions are also discussed.
Through commercial application, new technologies may improve health, agriculture, farming practices and the quality of foods. However, along with the array of potential benefits are potential risks and uncertainties surrounding the commercial applications of these technologies. In this study, a survey was prepared to receive opinion of Turkish People living in the most populated area of Turkey about irradiated foods, food additives and organic foods and genetically modified foods. Survey questions were asked to 1226 people (626 women and 600 men) in 4 different age groups (15-30, 31-45, 46-60, 61-90 years old). Questions were generally prepared to expose their knowledge and awareness about food additives, organic foods, irradiation, genetically modified foods or hormone residues in foods. Most of them think that consuming food containing some of the food additives can lead to some health problems. The half of the participants did not have knowledge about food irradiation. The results of the current study also indicated that 9% of respondents did not concern for the safety of irradiated foods; however, 69% of them concerned very much. In addition, the present study indicated that only 8% of respondents thought that GM foods were safe, 69% thought that they were little risky, 23% of them thought they were extremely risky for our health. All participants with different age, gender, income level or education level had negative thought about hormone residues in food products. Interestingly, 29 and 18% of them did not have knowledge about organic foods and believed that they can be unhealthy, respectively. In conclusion, the most of the participants, especially 31-45 years old and high income people, were cautious about what they purchase.
Biological systems are highly complex, and for this reason there is a considerable degree of uncertainty as to the consequences of making significant interventions into their workings. Since a number of new technologies are already impinging on living systems, including our bodies, many of us have become participants in large-scale "social experiments". I will discuss biological complexity and its relevance to the technologies that brought us BSE/vCJD and the controversy over GM foods. Then I will consider some of the complexities of our social dynamics, and argue for making a shift from using the precautionary principle to employing the approach of evaluating the introduction of new technologies by conceiving of them as social experiments.
The progress made in plant biotechnology has provided an opportunity to new food crops being developed having desirable traits for improving crop yield, reducing the use of agrochemicals and adding nutritional properties to staple crops. However, genetically modified (GM) crops have become a subject of intense debate in which opponents argue that GM crops represent a threat to individual freedom, the environment, public health and traditional economies. Despite the advances in food crop agriculture, the current world situation is still characterised by massive hunger and chronic malnutrition, representing a major public health problem. Biofortified GM crops have been considered an important and complementary strategy for delivering naturally-fortified staple foods to malnourished populations. Expert advice and public concern have led to designing strategies for assessing the potential risks involved in cultivating and consuming GM crops. The present critical review was aimed at expressing some conflicting points of view about the potential risks of GM crops for public health. It was concluded that GM food crops are no more risky than those genetically modified by conventional methods and that these GM crops might contribute towards reducing the amount of malnourished people around the world. However, all this needs to be complemented by effective political action aimed at increasing the income of people living below the poverty-line.
In the 1940's the root-knot nematode resistance gene (Mi) was introgressed into the cultivated tomato from the wild species, L. peruvianum, and today it provides the only form of genetic resistance against this pathogen. We report here the construction of a high resolution RFLP map around the Mi gene that may aid in the future cloning of this gene via chromosome walking. The map covers the most distal nine map units of chromosome 6 and contains the Mi gene, nine RFLP markers, and one isozyme marker (Aps-1). Based on the analysis of more than 1,000 F2 plants from four crosses, we were able to pinpoint the Mi gene to the interval between two of these markers — GP79 and Aps-1. In crosses containing the Mi gene, this interval is suppressed in recombination and is estimated to be 0.4 cM in length. In contrast, for a cross not containing Mi, the estimated map distance is approximately 5 times greater (ca. 2 cM).
Using RFLP markers around Mi as probes, it was possible to classify nematode resistant tomato varieties into three types based on the amount of linked peruvianum DNA still present. Two of these types (representing the majority of the varieties tested) were found to still contain more than 5 cM of peruvianum chromosome — a result that may explain some of the negative effects (e.g. fruit cracking) associated with nematode resistance. The third type (represented by a single variety) is predicted to carry a very small segment of peruvianum DNA (<2 cM) and may be useful in the identification of additional markers close to Mi and in the orientation of clones during a chromosome walk to clone the gene.
A genetic map of potato (Solanum tuberosum L.) integrating molecular markers with morphological and isozyme markers was constructed using a backcross population of 67 diploid potato plants. A general method for map construction is described that differs from previous methods employed in potato and other outbreeding plants. First, separate maps for the female and male parents were constructed. The female map contained 132 markers, whereas the male map contained 138 markers. Second, on the basis of the markers in common the two integrated parental maps were combined into one with the computer programme JoinMap. This combined map consisted of 175 molecular markers, 10 morphological markers and 8 isozyme markers. Ninety-two of the molecular markers were derived from DNA sequences flanking either T-DNA inserts in potato or reintegrated maize transposable elements originating from these T-DNA constructs. Clusters of distorted segregation were found on chromosomes 1,2,8 and 11 for the male parent and chromosome 5 for both parents. The total length of the combined map is 1120 cM.
Using genomic in situ hybridization to chromosomes, we identified introgressed segments in a tetrageneric hybrid of Triticum, Avena, Thinopyrum, and Secale, which conferred high resistance to leaf rust, stem rust, stripe rust, powdery mildew, and root rot to wheat. The disease-resistance traits of the hybrid originated from three wild related genera of Triticum, namely Avena, Thinopyrum, and Secale. The new breeding system that combined traditional wide hybridization with anther culture was efficient and rapid in creating wheat germplasms resistant to major diseases.Key words: Triticum aestivum, Avena fatua, Thinopyrum intermedium, Secale cereale, wide hybridization, anther culture, genomic in situ hybridization, GISH.
Transformed root cultures of Nicotiana rustica have been generated in which the gene from the yeast Saccharomyces cerevisiae coding for ornithine decarboxylase has been integrated. The gene, driven by the powerful CaMV35S promoter with an upstream duplicated enhancer sequence, shows constitutive expression throughout the growth cycle of some lines, as demonstrated by the analysis of mRNA and enzyme activity. The presence of the yeast gene and enhanced ornithine decarboxylase activity is associated with an enhanced capacity of cultures to accumulate both putrescine and the putrescine-derived alkaloid, nicotine. Even, however, with the very powerful promoter used in this work the magnitude of the changes seen is typically only in the order of 2-fold, suggesting that regulatory factors exist which limit the potential increase in metabolic flux caused by these manipulations. Nevertheless, it is demonstrated that flux through a pathway to a plant secondary product can be elevated by means of genetic manipulation.
Nuclear DNA contents of more than 100 important plant species were measured by flow cytometry of isolated nuclei stained with
propidium iodide.Arabidopsis exhibits developmentally regulated multiploidy and has a 2C nuclear DNA content of 0.30 pg (145 Mbp/1C), twice the value
usually cited. The 2C value for rice is only about three times that ofArabidopsis. Tomato has a 2C value of about 2.0 pg, larger than commonly cited. This survey identified several horticultural crops in
a variety of families with genomes only two or three times as large asArabidopsis; these include several fruit trees (a pricot, cherry, mango, orange, papaya, and peach). The small genome sizes of rice and
the horticultural plants should facilitate molecular studies of these crops.
The rice Xa21 gene, which confers resistance to Xanthomonas oryzae pv. oryzae race 6, was isolated by positional cloning. Fifty transgenic rice plants carrying the cloned Xa21 gene display high levels of resistance to the pathogen. The sequence of the predicted protein, which carries both a leucine-rich
repeat motif and a serine-threonine kinase-like domain, suggests a role in cell surface recognition of a pathogen ligand and
subsequent activation of an intracellular defense response. Characterization of Xa21 should facilitate understanding of plant disease resistance and lead to engineered resistance in rice.
Intact roots of 109 radish ( Raphanus sativus L.) cultivars were analyzed for glucosinolates (GS’s) and found to contain primarily 4-methylthio-3-butenyl-GS with small amounts of 4-methylsulfinylbutyl-, 4-methylsulfinyl-3-butenyl-, and 3-indolylmethyl-GS’s. Cultivars included oil radishes (ssp. oleifera) and food radishes (ssp. radicola ) available in European, European-American, Japanese, and Korean markets. Regarding total GS’s, 80% or more of the red European-American radishes had 100-199 pmole/100 g, the Korean 100-299, and the Japanese 200-399. No correlation was found between root size and 4-methylthio-3-butenyl-, 3-indolylmethyl-, or total GS’s. Japanese radish peelings contained significantly greater concentrations of these 3 constituents than did the peeled root.
Six transgenic tobacco lines, each homozygous for the β-glucuronidase (GUS) gene at a different locus, and wild type were selfed and intercrossed to evaluate GUS activity in all possible hemizygous, homozygous and dihybrid combinations of GUS alleles. The transgenic lines are characterized by their GUS activity (two low, three intermediate, one high), T-DNA complexity (four single-copy, two more complex single-locus) and the presence of the chicken lysozyme matrix-associated region (MAR) around the full T-DNA (two lines). Gene action and interaction was analyzed by weighted linear regression with parameters for additivity, dominance and epistasis. The analysis showed that each of the four single-copy lines acted fully additively. In contrast, the two complex single-locus lines showed classical single-locus overdominance and were epistatic dominant over all other GUS alleles. The latter is manifested in severe suppression of GUS activity in dihybrid lines, irrespective of the presence of MAR elements around the GUS gene. Such elements apparently do not protect against epistatic dominance. The quantitative data suggested that the epistatic dominance and overdominance are based on the same molecular mechanism. Our approach of a genetic analysis of quantitative variation in well-characterized transgenic lines provides a powerful tool to gain insight into complex plant traits.
High density molecular linkage maps, comprised of more than 1000 markers with an average spacing between markers of approximately 1.2 cM (ca. 900 kb), have been constructed for the tomato and potato genomes. As the two maps are based on a common set of probes, it was possible to determine, with a high degree of precision, the breakpoints corresponding to 5 chromosomal inversions that differentiate the tomato and potato genomes. All of the inversions appear to have resulted from single breakpoints at or near the centromeres of the affected chromosomes, the result being the inversion of entire chromosome arms. While the crossing over rate among chromosomes appears to be uniformly distributed with respect to chromosome size, there is tremendous heterogeneity of crossing over within chromosomes. Regions of the map corresponding to centromeres and centromeric heterochromatin, and in some instances telomeres, experience up to 10-fold less recombination than other areas of the genome. Overall, 28% of the mapped loci reside in areas of putatively suppressed recombination. This includes loci corresponding to both random, single copy genomic clones and transcribed genes (detected with cDNA probes). The extreme heterogeneity of crossing over within chromosomes has both practical and evolutionary implications. Currently tomato and potato are among the most thoroughly mapped eukaryotic species and the availability of high density molecular linkage maps should facilitate chromosome walking, quantitative trait mapping, marker-assisted breeding and evolutionary studies in these two important and well studied crop species.
Vectors were constructed for the isolation of random transcriptional and translational beta-glucuronidase gene fusions in plants. This system is based on the random integration of the transferred DNA (T-DNA) into the plant nuclear genome. The Escherichia coli beta-glucuronidase coding sequence without promoter, and also devoid of its ATG initiation site in the translational gene fusion vector, was inserted in the T-DNA with its 5' end at a distance of 4 base pairs from the right T-DNA border sequence. Transgenic plants can be selected by using a chimeric (P35S-nptII-3' ocs) kanamycin-resistance gene present in the same T-DNA. Subsequent screening of these for beta-glucuronidase expression allows the identification of clones harboring a fusion of the beta-glucuronidase coding sequence with plant 5' regulatory sequences. After transformation of Arabidopsis thaliana C24 root explants, beta-glucuronidase expression was detected in 54% and 1.6% of the plants transformed with the transcriptional and translational fusion vectors, respectively. Several different patterns of tissue-specific beta-glucuronidase expression were identified. The plant upstream sequence of a beta-glucuronidase fusion that is specifically expressed in the phloem of all organs was cloned and sequenced. After introduction in A. thaliana C24 and Nicotiana tabacum SR1, this sequence mediates the same highly phloem-specific beta-glucuronidase expression pattern as in the original transgenic plant from which it was isolated. These data demonstrate that this system facilitates the isolation and analysis of plant DNA sequences mediating regulated gene expression.
Twenty-nine cultivars of turnip [Brassica campestris L. ssp. rapifera (Metzg.) Sinsk.] and twelve of rutabaga [Brassica napus L. ssp. rapifera (Metzg.) Sinsk.] were analyzed for fourteen glucosinolates. Major glucosinolates in turnip roots are 2-hydroxy-3-butenyl, 2-phenylethyl, and 3-indolylmethyl glucosinolates; in rutabaga roots there are these three plus 4-(methylthio)butyl glucosinolate. Only a few small turnips show a negative correlation between glucosinolate concentration and root weight. Whereas the amounts of individual glucosinolates within a cultivar fluctuated from year to year, there was little change in the relative proportions of these glucosinolates. Both 4-(methylthio)butyl and 5-(methylthio)pentyl glucosinolates are lower in peelings and tops (above-ground parts) of turnips than in peeled roots, whereas 3-butenyl and total glucosinolates were higher. However, 2-phenylethyl and 3-indolylmethyl glucosinolates are concentrated in turnip peelings. Other glucosinolate distributions within the plant are also discussed.
Genes introduced into cultivated plants by backcross breeding programs are flanked by introgressed segments of DNA derived from the donor parent. This phenomenon is known as linkage drag and is frequently thought to affect traits other than the one originally targeted. The Tm-2 gene of Lycopersicon peruvianum, which confers resistance to tobacco mosaic virus, was introduced into several different tomato cultivars (L. esculentum) by repeated backcrossing. We have measured the sizes of the introgressed segments flanking the Tm-2 locus in several of these cultivars using a high density map of restriction fragment length polymorphic (RFLP) markers. The smallest introgressed segment is estimated to be 4 cM in length, while the longest is over 51 cM in length and contains the entire short arm of chromosome 9. Additionally, RFLP analysis was performed on remnant seed from different intermediate generations corresponding to two different backcross breeding programs for TMV resistance. The results reveal that plants containing desirable recombination near the resistance gene were rarely selected during backcrossing and, as a result, the backcross breeding method was largely ineffective in reducing the size of linked DNA around the resistance gene. We propose that, by monitoring recombination around genes of interest with linked RFLP markers, one can quickly and efficiently reduce the amount of linkage drag associated with introgression. Using such a procedure, it is estimated that an introgressed segment can be obtained in two generations that is as small as that which would otherwise require 100 backcross generations without RFLP selection.
Genomic in situ hybridization was used to identify alien chromatin in chromosome spreads of wheat, Triticum aestivum L., lines incorporating chromosomes from Leymus multicaulis (Kar. and Kir.) Tzvelev and Thinopyrum bessarabicum (Savul. and Rayss) Löve, and chromosome arms from Hordeum chilense Roem. and Schult, H. vulgare L. and Secale cereale L. Total genomic DNA from the introgressed alien species was used as a probe, together with excess amounts of unlabelled blocking DNA from wheat, for DNA:DNA in-situ hybridization. The method labelled the alien chromatin yellow-green, while the wheat chromosomes showed only the orange-red fluorescence of the DNA counterstain. Nuclei were screened from seedling root-tips (including those from half-grains) and anther wall tissue. The genomic probing method identified alien chromosomes and chromosome arms and allowed counting in nuclei at all stages of the cell cycle, so complete metaphases were not needed. At prophase or interphase, two labelled domains were visible in most nuclei from disomic lines, while only one labelled domain was visible in monosomic lines. At metaphase, direct visualization of the morphology of the alien chromosome or chromosome segment was possible and allowed identification of the relationship of the alien chromatin to the wheat chromosomes. The genomic in-situ hybridization method is fast, sensitive, accurate and informative. Hence it is likely to be of great value for both cytogenetic analysis and in plant breeding programmes.
The Agropyron intermedium chromosome 7Ai #2 is the source of the leaf rust resistance gene Lr38 which was transferred to wheat by irradiation. The chromosomal constitutions of eight radiation-induced rust-resistant wheat-Agropyron intermedium derivatives were analyzed by C-banding and genomic in-situ hybridization (GISH). Five lines were identified as wheat Ag. intermedium chromosome translocation lines with the translocation chromosomes T2AS·2AL-7Ai#2L, T5AL · 5AS-7Ai # 2L, T1DS · 1DL-7Ai # 2L, T3DL · 3DS-7Ai#2L, and T6DS · 6DL-7Ai#2L. The sizes of the 7Ai#2L segments in mitotic metaphases of these translocations are 2.42 μm, 4.20 μm, 2.55 μm, 2.78 μm, and 4.19 μm, respectively. One line was identified as a wheat-Ag. intermedium chromosome addition line. The added Ag. intermedium chromosome in this line is different from 7Ai # 2. This line has resistance to leaf rust and stem rust. Based on the rust reactions, and the C-banding and GISH results, the remaining two lines do not contain any Ag. intermedium-derived chromatin.
Asymmetric hybrid plants were obtained from fused protoplasts of a monocotyledon (Oryza sativa L.) and a dicotyledon (Daucus carota L.). X-ray-irradiated protoplasts isolated from a cytoplasmic malesterile (cms) carrot suspension culture were fused with iodoacetoamide-treated protoplasts isolated from a 5-methyltryptophan (5MT)-resistant rice suspension culture by electrofusion. The complementary recovered cells divided and formed colonies, which were then cultivated on regeneration medium supplemented with 25mg/l 5MT to eliminate any escaped carrot cells. Somatic hybrids were regenerated from 5 of the 5MT-resistant colonies. The morphologies of most of the regenerated plants closely resembled that of the parental carrot plants. A cytological analysis of callus cultures induced from these plants indicated that most of the cells possessed 20-22 chromosomes and were resistant to 5MT. An isozyme analysis revealed that several regenerated plants had the peroxidase isozyme patterns of both parents. A Southern hybridization analysis with non-radioactively labelled DNA fragments of the rgp1 gene showed that regenerated plants had hybridizing bands from both rice and carrot. Chloroplast (cp) and mitochondrial (mt) DNAs were also analyzed by Southern hybridization by using several probes. CpDNA patterns of the regenerated plants were indistinguishable from those of the carrot parent. However 1 of the regenerated plants had a novel band pattern of mtDNA that was not detected in either of the parents, indicating a possible recombination of mitochondrial genomes.
In the course of a heterologous transposon tagging experiment in Petunia hybrida (n=7), 135 independent T-DNA loci were tested for linkage to the target genes Hf1 and Fl, which are located on the two largest chromosomes. Approximately one-third (47) of these T-DNA loci were linked to one of these two markers. Of these 47 linkedloci, 19 mapped within 1 cM of its marker, indicating a highly non-random genetic distribution of introduced loci. However, rather than non-random integration within both of the marked chromosomes, this probably reflects a suppression of recombination around these marker loci in the particular wide hybrids used for mapping. This hypothesis was tested by measuring recombination between linked T-DNAs in an inbred background. Inbred recombination levels were found to be at least 3-fold higher around the Hf1 locus and 12-fold higher around Fl compared to the wide hybrids. These findings may reflect the origin of P. hybrida by hybridization of wild species, and while relevant to genetic mapping in petunia in particular they may also have more general significance for any mapping strategies involving the use of wide hybrids in other species.
Genetic engineering offers considerable potential for the development of insect‐resistant crop plants through the transfer and expression of Bacillus thuringiensis genes encoding insecticidal proteins. A potential problem with the use of such crops is the possibility of insects adapting to overcome the resistance mechanism. Strategies to minimize this risk have included concepts of seed mixtures and crop rotations. This may involve genotypes transformed with a series of genes encoding different resistance mechanisms and/or manipulating the manner in which these genes are expressed. The aim of this paper is to put these strategies into perspective with respect to the limitations imposed by the nature of plant transformation, the traditional approaches to plant breeding for cultivar development and seed marketing options for commercial release of transgenic crops. The optimum strategy for any specific crop varies depending on whether the cultivar targeted for release is a clone, an inbred line, a hybrid or an open pollinated population.
Transgenic hairy root lines were developed for six Brassica cultivars: B. napus cv. Giant (rape), B. oleracea cvs. Kapeti, Midas, Rawara and Medium Stem (all kales) and B. campstris cv. Red Globe (turnip). Leaf explants or petioles of intact cotyledons were cocultivated with Agrobacterium rhizogenes strain A4T harbouring the binary vector pKIWI110, the T-DNA of which contains genes encoding β-glucuronidase (GUS), neomycin phosphotransferase II (NPTII) and chlorsulfuron resistance. Hairy root lines grew prolifically on hormone-free medium containing selective levels of kanamycin (50 or 100 mg/l) or chlorsulfuron (10 μg/l). Transgenic shoots were regenerated from hairy roots of all cultivars and plants have been successfully transferred to soil. Molecular analyses using polymerase chain reaction (PCR) and Southern blotting confirmed the trnasgenic nature of these plants.
To induce post-transcriptional silencing of flower pigmentation genes by homologous sense transgenes in transgenic petunias, it is not necessary for the transgenes to be highly transcribed. Even promoterless transgenes can induce silencing. Here it is shown that in these cases silencing is mediated by multimeric transgene/T-DNA loci in which the T-DNAs are arranged as inverted repeats (IRs). With the transgene constructs used, monomeric T-DNA loci are unable to confer silencing even though they modulate IR-induced silencing. IRs with the silencing sequences proximal to the centre (IR(c)) induce a more severe silencing than IRs with these sequences distal to the centre (IR(n)). Somatic reversion of silencing, as observed in a side branch of one of the chalcone synthase (Chs) transformants, was associated with a deletion of the IR locus from L1 cells, the meristematic cell layer that expresses the endogenous Chs genes in the flower corolla. Taken together, these data indicate that the post-transcriptional silencing mechanism can be activated by inverted transgene repeats. It is also shown that a silent IR UidA-ChsA locus silences the expression of a monomeric 35S promoter-driven UidA-ChsA transgone only in corollas where the endogenous Chs genes are highly transcribed. These results are consistent with a model in which an IR, by virtue of its palindromic sequence organization, is able to promote the production of aberrant RNAs from the endogenous homologs as a result of ectopic pairing.
The distribution of glucosinolates in the pith, cambial-cortex, and leaves were determined for cabbage heads from 13 varieties of white, red, and Savoy types. The cambial-cortex contains about twice the weight concentration of total glucosinolate compared to that in the pith or the leaves. This increased glucosinolate concentration is due primarily to increased amounts of allyl, 3-butenyl, and phenylethyl glucosinolates. The concentrations of 3-methylthiopropyl and 4-methylthiobutyl glucosinolate in the pith were high. In contrast, the amounts of their oxidized counterparts, 3-methylsulfinylpropyl and 4-methylsulfinylbutyl glucosinolates, were high in cambial-cortex and leaves. Of all glucosinolates in the leaves, 3-indolylmethyl glucosinolates comprised 45, 28, and 68% in the white, red, and Savoy types, respectively.
The first food products derived from transgenic plants that are resistant to diseases, insects or viruses are now reaching the market and there is growing public concern about problems of allergenicity and toxicological changes in such transgenic food plants. We review the modifications being carried out or envisaged in molecular resistance breeding and specifically consider the allergenic and toxicological potential of the gene products used. Several protein families that contribute to the defence mechanisms of food plants have members which are allergens or putative allergens and some of these proteins are used in molecular approaches to increase resistance. These include α-amylase and trypsin inhibitors, lectins and pathogenesis-related proteins. An assessment procedure to avoid the transfer of such allergens is described. The source of the transgene is of great importance for the application of immunological assays. In addition to putative changes in the allergenic potential, the toxicological implications of classical and molecular resistance breeding are discussed. Several ‘self defence’ substances made by plants are highly toxic for mammals, including humans. Examples of molecular approaches that could be of toxicological concern are given. The source of the transgene is of no relevance in assessing the toxicological aspects of foods from transgenic plants. Food safety can also be severely influenced by invading pathogens and their metabolic products. This may result in a trade-off situation between ‘nature's pesticides’ produced by transgenic plants or varieties from traditional breeding programmes, synthetic pesticides and mycotoxins or other poisonous products of pests.
In this study the T-DNA composition of four antisense potato transformants showing complete or very strong inhibition of granule-bound starch synthase (GBSS) activity was analysed in detail. By Southern blot hybridizations, it was determined that all four transformants contained T-DNAs on multiple linkage groups and that most linkage groups contained multiple T-DNA copies, often in combination with non-T-DNA vector sequences. Subsequently, fluorescence in situ hybridization was performed on extended DNA fibres (‘fibre-FISH’) of three progeny plants each containing a single linkage group with a complex T-DNA organization. By using two differently labelled probes, one consisting of T-DNA sequences and the other of vector DNA sequences, it was possible to visualize the composition of complex loci. DNA sequences of 5–6 kb were well distinguishable. With this technique it is possible to determine T-DNA copy number, and arrangement of T-DNA and vector DNA sequences in a locus, more accurately than by Southern blot analysis alone. Therefore, fibre-FISH is a valuable supplementary tool to study T-DNA integrations in detail.
We have previously described substantial variation in the level of expression of two linked genes which were introduced into transgenic petunia plants using Agrobacterium tumefaciens. These genes were (i) nopaline synthase (nos) and (ii) a chimeric chlorophyll a/b binding protein/octopine synthase (cab/ocs) gene. In this report we analyze the relationship between the level of expression of the introduced genes and T-DNA structure and copy number in 40 transgenic petunia plants derived from 26 transformed calli. Multiple shoots were regenerated from 8 of these calli and in only 6 cases were multiple regenerated shoots from each callus genotypically identical to each other. Many genotypes showed no nos gene expression (22/28). Most of the plants (16/22) which lacked nos gene expression did contain nos-encoding DNA with the expected restriction enzyme map. Similarly, amongst the genotypes showing no cab/ocs gene expression, the majority (11/28) did not show any alterations in restriction fragments corresponding to the expected cab/ocs coding sequences (10/11). Approximately half of the plants carried multiple copies of T-DNA in inverted repeats about the left or right T-DNA boundaries. No positive correlation was observed between the copy number of the introduced DNA and the level of expression of the introduced genes. However, plants with high copy number complex insertions composed of multiple inverted repeats in linear arrays usually showed low levels of expression of the introduced genes.
In order to obtain plants that were somatic hybrids of barley (Hordeum vulgare L.) and carrot (Daucus carota L.), we fused protoplasts that had been isolated from 6-month-old suspension cultures of carrot cells with protoplasts isolated
from barley mesophyll by electrofusion. After culture for 1 month at 25°C , the cells were cultured for 5 weeks at 4°C , and
were then returned to 25°C for culture on a shoot-inducing medium. Three plants (nos. 1, 2 and 3) were regenerated from the
cells. The morphology of the regenerated plants closely resembled that of the parental carrot plants. A cytological analysis
of callus cultures induced from these plants indicated that most of the cells had about 24 chromosomes, fewer than the sum
of the numbers of parent chromosomes which was 32. Southern hybridization analysis with fragments of the rgp1 gene used as probe showed that the regenerated plants contained both barley and carrot genomic DNA. Chloroplast (ct) and
mitochondrial (mt) DNAs were also analyzed with several probes. The ctDNA of the regenerated plants yielded hybridization
bands specific for both barley and carrot when one fragment of rice ctDNA was used as probe. Furthermore, the regenerated
plants yielded a barley specific band and a novel band with another fragment of rice ct DNA as a probe. One of the regenerated
plants (no. 1) yielded a novel pattern of hybridized bands of mt DNA (with an atp6 probe) that was not detected with either of the parents. These results indicated that the regenerated plants were somatic
hybrids of barley and carrot and that recombination of both the chloroplast genomes and the mitochondrial genomes might have
occurred.
DNA repeats are associated with gene instability and silencing phenomena in plants. Therefore, the presence of a direct repeat of matrix-associated region (MAR) DNA, that considerably reduced position effects between independent transformants, may increase (epi)genetic instability. To investigate the influence of such a repeat on the stability of the expression of embedded transgenes, the meiotic stability of transgene expression was assessed in eighteen homozygous 1-locus transgenic tobacco lines carrying the kanamycin resistance (NPTII) and the -glucuronidase (GUS) gene. Half of the lines carry a 3 kb direct repeat of MAR DNA flanking the transgenes. Large progeny populations, totalling over a million seedlings, were screened for kanamycin resistance with the help of a newly developed high-density seedling screen. Kanamycin-sensitive seedlings were detected in selfed progeny at a frequency of 0.5-5.910-4. The frequency became as high as 210-2 when embryo development occurred under heat and/or drought stress. In backcrossed progeny only, a joint loss of NPTII and GUS gene expression was observed at an average frequency of 2.910-5. In selfed and backcrossed progeny we observed similar frequencies of reversion to kanamycin sensitivity, indicating that epigenetic silencing mechanisms rather than MAR repeat-related homologous recombination underlie the reversal to kanamycin sensitivity. Different lines, hence different areas of the tobacco genome, differed in their genetic stability. No significant differences in reversal frequencies were apparent between lines with or without the MAR elements. The use of the MAR repeat is, therefore, not compromised by any increased (epi)genetic instability.
The detailed structural organization of DNA sequences transferred to the plant genome via Agrobacterium tumefaciens has been determined in 11 transgenic tomato plants that carry the transferred DNA (T-DNA) at a single genetic locus. The majority (seven) of these plants were found to carry multiple copies of T-DNA arranged in inverted repeat structures. Such a high frequency of inverted repeats among transgenotes has not been previously reported and appears to be characteristic of transformation events caused by C58/pGV3850 strains of Agrobacterium. The inverted repeats were found to be centered on either the left or the right T-DNA boundary and both types were observed at similar frequency. In several plants both types of inverted repeat were found to coexist in the same linear array of elements. Direct repeats were observed in two plants, each time at the end of an array of inverted repeat elements, and at a lower frequency than inverted repeats. The junctions between T-DNA elements and plant DNA sequences and the junctions between adjacent T-DNA elements were mapped in the same 11 plants, allowing the determination of the distribution of junction points at each end for both types of junction. Based on a total of 17 distinct junctions at the right end of T-DNA and 19 at the left end, the distribution of junction points was found to be much more homogeneous at the right end than at the left end. Left end junctions were found to be distributed over a 3 kb region of T-DNA with two thirds of the junctions within 217 bp of the left repeat. Two thirds of the right end junctions were found to lie within 11 bp of the right repeat with the rest more than 39 bp from the right repeat. T-DNA::plant DNA junctions and T-DNA::T-DNA inverted repeat junctions showed similar distributions of junction points at both right and left ends. The possibilities that T-DNA inverted repeats are unstable in plants and refractory to cloning in wild type Escherichia coli is discussed. Two distinct types of mechanisms for inverted repeat formation are contrasted, replication and ligation mechanisms.
In several plant systems expression of structurally intact genes may be silenced epigenetically when a transgenic construct increases the copy number of DNA sequences. Here we report epigenetic silencing inArabidopsis lines containing transgenic inserts of defined genetic structure, all at the same genomic locus. These comprise an allelic series that includes a single copy of the primary insert, which carries repeated drug resistance transgenes, and a set of its derivatives, which as a result of recombination within the insert carry different numbers and alleles of resistance genes. Although the drug resistance genes remained intact, both the primary and some recombinant lines nevertheless segregated many progeny that were partly or fully drug-sensitive because of silencing. As in other systems silencing was reversible, and correlated with decreased steady-state mRNA and increased DNA methylation. Each different number and combination of genes, on the same or different (i.e., homologous) chromosomes, conditioned its own idiosyncratic segregation pattern. Strikingly, lines with a single gene segregated only a few slightly drug-sensitive progeny whereas multi-gene lines segregated many highly sensitive progeny, indicating dependence of silencing at this locus on repeated sequences. This argues strongly against explanations based on antisense RNA, but is consistent with explanations based on ectopic DNA pairing. One possibility is that silencing reflects the interaction of paired homologous DNA with flanking heterologous DNA, which induces condensation of chromatin into a non-transcribable state.
Allium fistulosum harbours a number of desirable agronomical traits for the breeding of onions. However exploitation of A. fistulosum for onion breeding via direct sexual hybridization is problematic. Therefore, we examined if a bridge cross, using A. roylei as a bridging species, might provide an alternative. By means of genomic in situ hybridization (GISH) we showed that each
of the three parental genomes can be distinguished from the others in interspecific hybrids, suggesting that these genomes
contain sufficiently different repetitive DNA families. We succeeded in carrying out multi-colour GISH to metaphase spreads
of a first-generation bridge-cross individual [A. cepa× (A. fistulosum×A. roylei], which is composed of three parental genomes. Recombination between the genomes of A. fistulosum and A. roylei took place to a large extent: 7 recombined chromosomes were observed, and it could be shown that the proximal regions of
the recombined A. fistulosum/A. roylei chromosomes belonged to the former, whereas the distal parts belonged to the latter. The high percentage of bound bivalent
arms in metaphase I of pollen mother cells of a fertile bridge-cross individual suggests the introgression of A. fistulosum genes, mediated by A. roylei, into the genome of A. cepa. However, the presence of univalents reflects decreased pairing and recombination between the three genomes. Pollen fertility
and pollen-tube growth of the first-generation bridge-cross individual seem to be sufficient to produce a second generation
bridge-cross (A. cepa×first-generation bridge cross) progeny.
We are developing a system for isolating tomato genes by transposon mutagenesis. In maize and tobacco, the transposon Activator (Ac) transposes preferentially to genetically linked sites. To identify transposons linked to various target genes, we have determined the RFLP map locations of Ac- and Dissociation (Ds)-carrying T-DNAs in a number of transformants. T-DNA flanking sequences were isolated using the inverse polymerase chain reaction (IPCR) and located on the RFLP map of tomato. The authenticity of IPCR reaction products was tested by several criteria including nested primer amplification, DNA sequence analysis and PCR amplification of the corresponding insertion target sequences. We report the RFLP map locations of 37 transposon-carrying T-DNAs. We also report the map locations of nine transposed Ds elements. T-DNAs were identified on all chromosomes except chromosome 6. Our data revealed no apparent chromosomal preference for T-DNA integration events. Lines carrying transposons at known map locations have been established which should prove a useful resource for isolating tomato genes by transposon mutagenesis.
By backcrossing three BC1 genotypes of potato (+) tomato fusion hybrids to different tetraploid potato pollinators, BC2 populations were produced. A combined total of 97 BC2 plants from three BC2 populations were analysed with chromosome-specific probes through restriction fragment length polymorphism (RFLP) for the
presence of alien tomato chromosomes. The number of different alien tomato chromosomes transmitted through the female BC1 parent ranged from 0 to 6, and the average number of different alien chromosomes transmitted per BC2 plant varied between 1.7 and 3.4 in the different populations. This variation corresponded to the chromosome constitution
of the individual BC1 parents: parent 6739, which possessed 11 different alien chromosomes in a single condition, gave rise to progeny with a lower
average number of alien chromosomes per plant than the BC1 parent 2003 that possessed 2 of the 12 alien chromosomes in a disomic condition. In the latter case, the higher transmission
rate was attributed to the more regular distribution of the two alien chromosomes in the disomic condition because of regular
bivalent formation during meiosis as revealed by genomic in situ hybridisation (GISH) and fluorescent in situ hybridisation
(FISH). The transmission frequencies of individual alien chromosomes were subjected to statistical analysis to test whether
the maternal genotypes had an effect on alien-chromosome transmission. Among the BC2 plants, a total of 27 single additions were detected for as many as seven different chromosomes (1, 2, 4, 6, 8, 10 and 12)
out of the 12 possible types.
By relatively simple breeding procedures, seven different modifications of fruit and pedicel have been successfully transferred
from wild tomatoes of the Galápagos Islands to large-fruited cultivars. Some of these traits, the majority of which are simply
inherited, may have horticultural value. Such introgression from the wild forms is expedited by the remarkable freedom from
barriers at any stage of the breeding processes.
The length was estimated of the chromosome segment with the desired marker gene introgressed from a donor by backcrossing into a recurrent parent. It was found that, for instance, for a chromosome with a length of 100 cM the length of the segment is 32 cM in BC6.These estimates were compared with those made by Hanson (1959). Two corrections proved to be necessary.The implications for breeders and researchers are discussed. Differences between two near-isogenic lines often ascribed to the difference in genotype at the marker locus, may also be caused by genes linked to the marker gene and dragged into the backcross product.
Southern hybridisation was performed on ninety-six transgenic petunias that had been selected for resistance to kanamycin. Just over half of the plants contained intact copies of the T-DNA. The most common rearrangements (at least 24 plants out of 96) were simple deleted derivatives that had lost one or both ends of the T-DNA. T-DNAs lacking the left border occurred at a frequency of 20%, and estimates of the frequency of T-DNAs lacking the right border were at least this high. Three plants contained grossly rearranged T-DNAs, of which all expressed the kanamycin resistance gene but only one transmitted the gene to progeny. Two plants lacked T-DNA homology altogether and did not express kanamycin resistance in their leaves or their progeny. Circumstantial evidence suggests that plants containing a chimaeric kanamycin resistance gene driven by the ocs promoter do not root efficiently in the presence of kanamycin. There was no correlation between intactness of the T-DNA and Mendelian inheritance of the kanamycin-resistance phenotype. However, a disproportionate number of plants showing non-Mendelian inheritance had a high copy number of their T-DNA.
Solanum vernei is widely used in potato breeding for the introgression of desired genes into the cultivated potato. The composition of the natural toxicants (steroidal glycoalkaloids) of tubers of S. vernei, of S. vernei x S. tuberosum hybrids, and of potato cultivars was studied. Extremely high levels of solanidine glycosides and varying levels of glycosidic-bound solasodine, tomatidenol and minor steroidal alkaloids, amongst which the 22R,25R epimer of solanidine, were present in tubers of S. vernei. The growing conditions did not influence the total content, but did significantly influence the composition of the steroidal alkaloids in S. vernei. Potentially toxic concentrations of solanidine glycosides can be present in tubers of inter-specific hybrids and of new potato cultivars produced by repeated backcrossing. In addition alien steroidal glycoalkaloids can be transmitted from the wild species to the cultivated potato. Itis recommended that the steroidal glycoalkaloid composition be used as a major criterion for assessing new potato cultivars prior to being released for human consumption.
A full diallel analysis is a tool for selection in plant breeding that has been subject to many discussions and controversies regarding its interpretation and merits. The analysis of well-defined transgenes by such an approach permits assessment of the value of diallel analyses. The performance of the Eberhart/Gardner diallel approach is analysed for the β-glucuronidase (GUS) activity of six well-defined, homozygous one-locus tobacco (Nicotiana tabacum L.) transgenic lines, each carrying differently located alleles of the GUS gene, and the nulliplex wild type. Tobacco is an inbreeding plant species, therefore all these lines are fully isogenic apart from the T-DNA insertion. The analysis shows that additivity of GUS gene activity as well as epistatic gene silencing translate well in the diallel parameters of general combining ability (GCA) and specific combining ability (SCA) or more detailed versions thereof, when compared to a parsimonious model based on the precise genetic constitution of the transgenic plants lines used as parents. The tobacco line with the highest GUS activity also has the highest GCA, demonstrating that an evaluation of parental phenotype would be sufficient for determining breeding potential. In case of the epistatic gene silencing, however, there is no positive correlation between GCA and parental performance, the reduction in GUS activity is more severe than is to be expected on the basis of parental performance.
Tuberiferous and nontuberiferous wild Solanum species are increasingly being used in potato breeding as a source of genes for disease and pest resistances and for other valuable characteristics. A disadvantage of Solanum species, from a consumers point of view, is that they contain steroidal glycoalkaloids (SGAs), which are natural toxins occurring in all parts of these plants. The SGAs consist of a C 27 -steroidal alkaloid (SA) and a sugar moiety, often a tri- or tetrasaccharide. The tubers of the cultivated potato usually contain small quantities of one type of SGAs, the solanidine glycosides.Evidence has been presented that utilization of wild Solanum species can result in the introduction of hazardous levels of solanidine glycosides into the cultivated potato. However, little is known of the qualitative and quantitative SGA composition of wild Solanum species used in potato breeding. Such information on hybrid offspring or on cultivars containing germplasm from wild species is entirely lacking.The aim of the studies described in this thesis was to evaluate the possible health hazards of the SGAs from the genus Solanum and to develop and apply methods for analysis of the SGA compositions of Solanum species. The collected information was placed into the perspectives of potato breeding and of food safety, in order to point out possible consequences of introducing undesired levels or types of SGAs into the cultivated potato.In Chapter I, the literature on the distribution and accumulation of solanidine glycosides in the cultivated potato and on the biosynthesis of the SGAs is reviewed. It was shown that many factors during the growth and post-harvest period of potatoes can lead to levels of solanidine glycosides exceeding 200 mg/kg fresh weight. This information is important also with respect to introduction of SGAs from wild Solanum species into the household potato, as alien SGAs will almost certainly accumulate similarly, because the biosynthetic pathways of the various SGAs are closely related.The objective of Chapter II was to evaluate the toxicity of the SGAs, based on the data available in the literature, in order to assess possible consumer hazards and to derive safe levels for household potatoes. The data showed that acute poisoning in man may occur due to consumption of potatoes with a solanidine glycoside content above 200 mg/kg fresh weight. This level is only two to three times higher than levels that are considered normal for tubers of current cultivars. Because of the virtual absence of chronic toxicity data, an adequate 'no-adverse effect level' for potato SGAs could not be assessed. Consequently an acceptable daily intake (ADI) figure for man or an acceptable level for potatoes could not be derived. It was concluded that the SGA content of new household potato cultivars should not be allowed to rise above the average level of the current cultivars; preferably it should be lower. SGAs alien to S. tuberosum must not be introduced into the household potato as acceptable levels have not been established.The methods for SGA analysis described in the literature are not adequate for determining -qualitatively and quantitatively- the SGA composition of breeding material. Therefore, a comprehensive, quantitative and efficient method applicable to diverse plant material, was developed.In Chap ter III, a new hydrolysis technique employing a two-phase system is described. The technique was developed in order to prevent the losses of aglycones which do usually occur during conventional hydrolysis of different SGAs. The two-phase system consists of an aqueous acid phase, in which the glycosides are hydrolysed, and an immiscible nonpolar organic phase, which serves as a protective phase for the unstable nonpolar aglycones. Using the technique quantitative recoveries of various SAs after simultaneous hydrolysis of different SGAs were obtained.In Chapter IV a capillary gas chromatography (GC) method is described, which enabled -for the first time- the separation of the Δ5-and 5α-aglycone pair solanidine and demissidine as well as separation of the other SAs studied. Derivatization of the SAs was not required. The capillary GC method in combination with the bisolvent extraction and two- phase hydrolysis described in Chapter III offered prospects for quantitative analysis of complex SGA compositions of Solanum species in a single run.Chapter V deals with the development of a method that enabled an unambiguous differentiation during capillary GC between peaks of unknown SAs and peaks of closely related nonnitrogen-containing (non-N) compounds such as sterols and steroidal sapogenins. The method involves a splitting device that connects a capillary column to a dual detector system, for flame ionization detection (FID) and N-specific detection (NPD), respectively. This system is connected to a two-channel system for interactive processing of the two sets of data. The application of hydrogen as carrier gas was introduced to achieve a short analysis duration. As tubers of wild Solanum species appeared to vary strongly in SGA contents, identification by retention times was not possible. Therefore a retention index system for the SAs was introduced. The retention indices were sufficiently reproducible, even for identification of the closely eluting Δ5-and 5α-aglycone pair solanidine and demissidine. Application of NPD/FID response ratios together with retention indices proved to be useful in the identification and characterization of SAs. Costly spectrometric techniques will have to be employed for characterization only when new SAs are detected.Chapter VI deals with an evaluation of the developed methods using diverse plant tissues of wild Solanum and Lycopersicon species, cultivars and hybrid progeny. The results showed that the methods were useful for qualitative and quantitative analysis of the SAs in the diverse samples. A potato extract spiked with 20 SAs, including three Δ5-and 5α-aglycone pairs, could be analysed in a single GC run. A comparison with procedures described in the literature showed that the procedure developed for sample preparation followed by capillary GC should be applied, because literature procedures for sample preparation can lead to losses of various SGAs or SAs, and other chromatographic techniques do not offer the efficiency required for separation of the various SAs.In Chapter VII, the SGA compositions of a large number of wild Solanum species used in potato breeding, are presented. The tubers of most of the species contained high concentrations of solanidine glycosides. Some species contained glycosidic-bound demissidine, solasodine and/or tomatidine. Unidentified compounds were also detected, which were most probably SAs as was shown by their NPD/FID response ratios. The total SGA contents of the tubers of the wild species varied from 123 to 7348 mg/kg. In order to place these contents in a realistic perspective, tubers of cultivars, corresponding in small size with and grown under the same conditions as the tubers of the wild species, were analysed. The contents of solanidine glycosides of these tubers were two to three times higher than those of field-grown normal tubers. Thus, even when this factor was taken into consideration, the SGA contents of the tubers of several wild species were extremely high. This study also revealed that samples of some wild species contained high or low levels of one or many other glycosidic-bound SAs, and that the SGA composition can vary within species and between different organs of one plant. The results further suggested that the SGA synthesis can be organ specific or regulated by light and that translocation of SGAs from leaves to tubers and viceversa did not occur. It was concluded that wild crossing parents should be analysed as to their SA composition, before they are used in a breeding programme.A procedure developed for the characterization of unidentified SAs present in plant extracts with complex SA compositions, is described in Chapter VIII. The combined results of GC-mass spectrometry (MS) applying electron impact and chemical ionization, and high-resolution MS, of the application of retention indices and NPD/FID response ratios, and of a comparison of the degree of dehydration of SAs in different twophase hydrolysis systems, were used. This study revealed that the SAs all possessed a solanidane skeleton; they were characterized as substituted, e.g. hydroxylated or methylated, forms; dehydrogenated forms; or substituted saturated forms of solanidine. Most of these SAs had not been reported before. The complete SA composition, including the minor new SAs, of Solanum (sub)species used in potato breeding, is presented in this chapter and the importance of analysis of the minor SAs is indicated.S. vernei is widely being used in potato breeding. Although the SGA composition of its tubers had not been reported, cultivars containing S. vernei germplasm have been released and an increasing number will be released in the future.Chapter IX describes the identification and characterization of the SAs of S. vernei tubers produced under various growth conditions, and deals with a tentative study on the transmission of these SAs to hybrid offspring. High levels of solanidine and solasodine glycosides were found together with tomatidenol and novel glycosidic-bound SAs, amongst which the 22R,25R epimer of solanidine, a structural configuration not reported before for naturally occurring solanidanes. It was revealed that the SA composition can vary significantly in tubers grown under different cultivation conditions.In the tubers of S. vernei offspring, high levels of solanidine glycosides were found but the newly identified and characterized SAs of S. vernei were not detected. Solasodine was also found in the offspring, even in tubers of cultivars obtained after several times backcrossing, but fortunately, the levels were low. However, it can not be totally excluded that S. vernei offspring may synthesize hazardous levels of solasodine glycosides under particular growth or post-harvest conditions, and in this respect the possible teratogenic potency of solasodine should be kept in mind.In conclusion it can be stated that the ingestion by the public of the amount of solanidine glycosides should not be allowed to rise but should preferably be reduced. SGAs alien to S. tuberosum should not be introduced into the household potato. The basis for the production of a potato crop safe for consumption is, to grow current cultivars and to breed new ones which accumulate low levels of only solanidine glycosides under various growth and post-harvest conditions. Utilization of germplasm from wild Solanum species must be approached with caution. Therefore it is recommended to analyse the SGAs of potential wild crossing parents before they are used in a breeding programme, in order to select the genotypes that combine a desired trait with the least unfavourable SCA composition. Depending on the SGA composition of the parents, the offspring should be monitored too.For analysis of SGA compositions, the newly developed procedures for separation, quantification and identification/characterization, described in this thesis, must be applied, as the conventional methods described in the literature do not meet the required standards.The accumulation of SGAs in new cultivars containing wild-species germplasm should be studied under various environmental conditions before such cultivars are registered. The guideline of 60-70 mg solanidine glycosides per kg fresh tuber recommended in the literature from the viewpoint of consumer safety (see Chapter II), should be used in potato breeding until an adequate acceptable level for these compounds has been established.