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PubMed2001

The production of foreign proteins from genetically modified plant cells.

While traditionally used to produce natural products, plant suspension cultures can also be utilized for the production of foreign proteins. Production of these high-value products in plant cells is an economically viable alternative to other systems, particularly in cases where the protein must be biologically active. There are several advantages to using plant cells for the large-scale production of secreted proteins. Plant cell media are composed of simple sugars and salts and are therefore less expensive and complex than mammalian media. Consequently, purification of secreted protein is simpler and more economical. Additionally, plant cell derived proteins are likely to be safer than those derived from other systems, since plant cell pathogens are not harmful to humans. In this chapter, we will review foreign protein production from plant cells. To begin, we will discuss the behavior of plant cell cultures, products produced by plant cells, protein secretion and its relationship to purification, and the performance of plant cells as compared to whole plants and other alternative hosts. After a brief discussion of gene transfer techniques, we will present strategies to overcome the limitations of protein production, including protein stabilization, novel production schemes, modeling, and scale-up considerations. To conclude, we will discuss implications for future development of this technology.

Animals↗

PubMed2001

Genetically modified plants: the stakes.

Generically modified plants (GMP) are massively used on the American continent in Australia and in China, since they represent an unquestionable potential for progress. New attributes are therefore devoted to the human and animal diet, to the facilitating of culture management, to the reducing of the chemical fertilizer and pesticide usage, and to the conquest of new cultural spaces. Considering itself to be flawed by a too hasty plunge into the market, concomitant with sagging evaluations of other innovations, Europe is confronted by a strong societal debate which blocks GMP cultures and orientates the research towards an evaluation of the environmental and public health risks and an evaluation of their economical and sociological impacts. The authors encourage this societal debate in order to arbitrate the presence of transgenes in conventional productions and products, to define the accepted rules of responsibility, to decide what is not acceptable, and to involve the more upstream actors and operators of the innovation process, all that keeping in mind the agronomical, ecological and economical repercussions of their decisions.

Animals↗

PubMed2002

Monitoring the persistence of genes deriving from genetically modified plants in the soil environment.

To study the gene persistence in the soil environment, soil samples were collected from sugar beet (Beta vulgaris) and chicory (Cichorium intybus) experimental fields just before harvest. They were homogenized, mixed and stored at constant humidity in a non-heated room. Sub-samples of soils were subsequently collected at regular intervals, dried and sieved through a 1.8-mm mesh before DNA was extracted. Specific primers were then used for the detection of plant DNA by hot start PCR. Results reveal that, under laboratory conditions, transgenic and non-transgenic sugar beet DNA was still detected after 25 days incubation in the soil taken from a sugar beet experimental plot while detection of chicory DNA was still possible after 50 days incubation in soil taken from the chicory experimental plot. This might be in correlation with the stronger resistance of chicory radicles to decomposition as compared to radicles from sugar beets.

Beta vulgaris↗

PubMed2003

[HRCA and application in detection of genetically modified plant].

In this article primary studies of the application of hyperbranched rolling cycle amplification (HRCA) in exogenous genes detection of transgenic plants were done. Four padlock probes were designed according to the sequences of four genes/DNA fragments that are used widely in transgenic plants; part of the sequence of pKK233 was chosen as the linking part of padlock probes and a pair of HRCA primers was designed according to the sequence of linking part. Study of the specificity of ligation in HRCA with isotope labeled padlock probes indicated padlock probes could be ringed effectively only when corresponding target DNA exited in the same reaction system and could not be ringed when there was no corresponding target DNA exited. Ligation time is very different according to the characteristic of target DNA being used. 5 min to 10 min is enough if the target DNA is plasmid; 30 min to 60 min is needed if the target is genome DNA of plant because it's sequence is more complex than that of plasmid's. HRCA time was analyzed which indicated longer reaction time can obviously increase the amount of products. Quantity of enzyme in HRCA was also analyzed. Different amount of enzyme (from 0.5 unit to 4 units) can give similar result when other conditions are not changed. On the basis of the research, transgenic tobacco was detected with these four padlock probes and the results were just as prospective. In order to increase the efficiency of detection, multiplex HRCA (MHRCA)was used. In MHRCA more than one padlock probes are used at the same time in the same reaction system to detect more than one targets. Because the amplification products of MHRCA will be complex and it is almost impossible to analyze with electrophoresis, so reverse-blot is used. Detection results of transgenic tobacco with this method are the same with anticipation. Compare to MPCR method we established before MHRCA is more convenient to operate and more effective in detecting exogenous genes in transgenic plants.

Nucleic Acid Amplification Techniques↗

PubMed1995

Applying the Good Laboratory Practice regulations to studies involving genetically modified plants.

How can the Environmental Protection Agency's Good Laboratory Practice (GLP) regulations, originally written primarily for mammalian toxicology studies, be applied to regulatory studies conducted for genetically modified plants? Do they fit? Can they be applied and still make sense? How is a Quality Assurance Unit (QAU) to interpret the requirements in this new area of biotechnology? The answers to these questions are discussed in this brief presentation of how one team within the Monsanto QAU, along with the researchers, developed am effective and comprehensive compliance program by applying the "traditional approach" to the GLP regulations to a new and important scientific field in regulatory compliance. Topics discussed will address the differences in the approach between traditional toxicity testing and the newer technology and how the differences were resolved, new and innovative definitions of particular phases and other aspects of regulatory studies, and how the draft regulations for pesticidal plants will help this area of technology in the future.

Animals↗

PubMed2000

Analysis of genetically modified plant gene expression using GUS fluorimetry.

A fluorimetric assay method for the analysis of beta-glucuronidase (GUS) reporter gene expression in genetically modified plants is described. Optimization of this method for woody plants and a statistical approach suitable for comparisons of gene expression in different transformants or tissues of the same plant is described. Example data from elm (Ulmus procera) SR4 regenerant plants, shown to be genetically modified by PCR and DNA-DNA hybridizations, in which higher GUS expression levels are found in stems than in leaves demonstrates the utility of this approach.

DNA, Plant↗

PubMed2003

Overview of the current status of genetically modified plants in Europe as compared to the USA.

Genetically modified crops have been tested in 1,726 experimental releases in the EU member states and in 7,815 experimental releases in the USA. The global commercial cultivation area of genetically modified crops is likely to reach 50 million hectares in 2001, however, the commercial production of genetically modified crops in the EU amounts to only a few thousand hectares and accounts for only some 0.03% of the world production. A significant gap exists between the more than fifty genetically modified crop species already permitted to be cultivated and to be placed on the market in the USA, Canada and other countries and the five genetically modified crop species permitted for the same use in the EU member states, which are still pending inclusion in the Common Catalogue of agricultural plant species. The further development of the "green gene technology" in the EU will be a matter of public acceptance and administrative legislation.

Agriculture↗

PubMed2004

An assessment of the risks associated with the use of antibiotic resistance genes in genetically modified plants: report of the Working Party of the British Society for Antimicrobial Chemotherapy.

Development of genetically modified (GM) plants is contentious, in part because bacterial antibiotic resistance (AR) genes are used in their construction and often become part of the plant genome. This arouses concern that cultivation of GM plants might provide a reservoir of AR genes that could power the evolution of new drug-resistant bacteria. We have considered bacterial DNA transfer systems (conjugation, transduction and transformation) and mechanisms of recombination (homologous recombination, transposition, site-specific recombination and DNA repair) that together might productively transfer AR genes from GM plants to bacterial cells, but are unable to identify a credible scenario whereby new drug-resistant bacteria would be created. However, we cannot entirely rule out the possibility of rare transfer events that involve novel mechanisms. Hence, we also considered if occasional transfers of AR genes (bla(TEM), aph(3'), aadA) from GM plants into bacteria would pose a threat to public health. These AR genes are common in many bacteria and each is found on mobile genetic elements that have moved extensively between DNA molecules and bacterial cells. This gene mobility has already severely compromised clinical use of antibiotics to which resistance is conferred. Accordingly, the argument that occasional transfer of these particular resistance genes from GM plants to bacteria would pose an unacceptable risk to human or animal health has little substance. We conclude that the risk of transfer of AR genes from GM plants to bacteria is remote, and that the hazard arising from any such gene transfer is, at worst, slight.

Bacteria↗

PubMed2003

Genetically modified plants for improved trace element nutrition.

Deficiencies of iron and zinc are common worldwide. Various strategies have been used to combat these deficiencies including supplementation, food fortification and modification of food preparation and processing methods. A new possible strategy is to use biotechnology to improve trace element nutrition. Genetic engineering can be used in several ways; the most obvious is to increase the trace element content of staple foods such as cereals and legumes. This may be achieved by introduction of genes that code for trace element-binding proteins, overexpression of storage proteins already present and/or increased expression of proteins that are responsible for trace element uptake into plants. However, even very high levels of expression may not substantially increase the iron and zinc contents unless many atoms of trace elements are bound per protein molecule. Another possibility is to introduce a protein that specifically enhances trace element absorption even in the presence of naturally occurring inhibitors, thus improving bioavailability. Genetically modifying plants so that their contents of inhibitors of trace element absorption such as phytate are substantially reduced is another approach. Increasing the expression of compounds that enhance trace element absorption such as ascorbic acid is also a possibility, although this has received limited attention so far. Iron absorption may be increased by higher ascorbic or citric acid content but require overexpression of enzymes that are involved in the synthetic pathways. Finally, a combination of all of these approaches perhaps complemented with conventional breeding techniques may prove successful.

Absorption↗

PubMedDate not supplied

[Detection and identification of the transgenes in genetically modified plants on the example of Bt-potato].

We have developed diagnostic system for revealing 35S promoter CaMV and transgene CryIIIA in transgenic potato NL Russet Burbank and NL Superior companies Monsanto. Using the data about nucleotide sequences of 35S promoter (AF234316 GenBank) and gene CryIIIA (X70979 GenBank) oligonucleotides primers were synthesized for polymerase chain reaction (PCR). The technique of genomic DNA isolation from plant material, suitable for PCR has been optimized. Using Touchdown PCR method presence of specific transgene in a Bt-potato was shown. This method can be used in routine laboratory practice for revealing 35S promoter in genetically modified plants and for identification of specific transgene CryIIIA in the transformed Bt-plants.

Bacillus thuringiensis Toxins↗

PubMed2005

Antibiotic resistance markers in genetically modified plants: a risk to human health?

Cotransformation with an antibiotic-resistance marker is often necessary in the process of creating a genetically modified (GM) plant. Concern has been expressed that the release of these markers in GM plants may result in an increase in the rate of antibiotic resistance in human pathogens. For such an event to occur, DNA must not be totally degraded in field conditions, and the antibiotic-resistance marker must encounter potential recipient bacteria and be taken up by them, before being integrated into the bacterial genome, and the genes then expressed. In addition, the new recombinant must overcome the physiological disadvantage of acquisition of a piece of foreign DNA, probably in conditions where the new gene does not provide a selective advantage. We review each of these stages, summarising the investigations that have followed each of these steps. We contrast the potential increase in the antibiotic resistance reservoir created by antibiotic-resistance markers in GM plants with the current situation created by medical antibiotic prescribing. We conclude that, although fragments of DNA large enough to contain an antibiotic-resistance gene may survive in the environment, the barriers to transfer, incorporation, and transmission are so substantial that any contribution to antibiotic resistance made by GM plants must be overwhelmed by the contribution made by antibiotic prescription in clinical practice.

Drug Resistance↗

PubMed2003

Safety of foods derived from genetically modified plants.

Biopharmaceuticals have been available for clinical use for nearly three decades, but foods derived from agribiotechnology have been available for just under a decade. Controversy surrounding foods from genetically modified (GM) plants has focused primarily upon their allergenicity, with lesser concerns about antibiotic resistance genes. Concerns are related to possible environmental impacts on non-human species, including effects on non-target species (e.g., butterflies) and on the development of so-called "super weeds." Food allergies are no more prevalent in foods from GM plants than in conventional foods. Further, the use of antibiotics in the development of GM plants does not pose a significant risk to the human population. Foods from the current GM plant products have been shown not to pose any detrimental effects to humans, and, in fact, nutritionally enhanced products are being developed. GM foods are subjected globally to intense regulatory scrutiny, and extensive data have been provided consistently to regulatory agencies in the United States on a voluntary basis, with mandatory reporting of data soon to be in force. Existing environmental concerns appear to be unjustified on the basis of existing data and experience.

Consumer Product Safety↗

PubMed2006

Review: Genetically modified plants for the promotion of human health.

Plants are attractive biological resources because of their ability to produce a huge variety of chemical compounds, and the familiarity of production in even the most rural settings. Genetic engineering gives plants additional characteristics and value for cultivation and post-harvest. Genetically modified (GM) plants of the "first generation" were conferred with traits beneficial to producers, whereas GM plants in subsequent "generations" are intended to provide beneficial traits for consumers. Golden Rice is a promising example of a GM plant in the second generation, and has overcome a number of obstacles for practical use. Furthermore, consumer-acceptable plants with health-promoting properties that are genetically modified using native genes are being developed. The emerging technology of metabolomics will also support the commercial realization of GM plants by providing comprehensive analyzes of plant biochemical components.

Carotenoids↗

PubMedDate not supplied

Validation of PCR methods for quantitation of genetically modified plants in food.

For enforcement of the recently introduced labeling threshold for genetically modified organisms (GMOs) in food ingredients, quantitative detection methods such as quantitative competitive (QC-PCR) and real-time PCR are applied by official food control laboratories. The experiences of 3 European food control laboratories in validating such methods were compared to describe realistic performance characteristics of quantitative PCR detection methods. The limit of quantitation (LOQ) of GMO-specific, real-time PCR was experimentally determined to reach 30-50 target molecules, which is close to theoretical prediction. Starting PCR with 200 ng genomic plant DNA, the LOQ depends primarily on the genome size of the target plant and ranges from 0.02% for rice to 0.7% for wheat. The precision of quantitative PCR detection methods, expressed as relative standard deviation (RSD), varied from 10 to 30%. Using Bt176 corn containing test samples and applying Bt176 specific QC-PCR, mean values deviated from true values by -7to 18%, with an average of 2+/-10%. Ruggedness of real-time PCR detection methods was assessed in an interlaboratory study analyzing commercial, homogeneous food samples. Roundup Ready soybean DNA contents were determined in the range of 0.3 to 36%, relative to soybean DNA, with RSDs of about 25%. Taking the precision of quantitative PCR detection methods into account, suitable sample plans and sample sizes for GMO analysis are suggested. Because quantitative GMO detection methods measure GMO contents of samples in relation to reference material (calibrants), high priority must be given to international agreements and standardization on certified reference materials.

European Union↗

PubMed2003

A plant genetically modified that accumulates Pb is especially promising for phytoremediation.

From a number of wild plant species growing on soils highly contaminated by heavy metals in Eastern Spain, Nicotiana glauca R. Graham (shrub tobacco) was selected for biotechnological modification, because it showed the most appropriate properties for phytoremediation. This plant has a wide geographic distribution, is fast-growing with a high biomass, and is repulsive to herbivores. Following Agrobacterium mediated transformation, the induction and overexpression of a wheat gene encoding phytochelatin synthase (TaPCS1) in this particular plant greatly increased its tolerance to metals such as Pb and Cd, developing seedling roots 160% longer than wild type plants. In addition, seedlings of transformed plants grown in mining soils containing high levels of Pb (1572 ppm) accumulated double concentration of this heavy metal than wild type. These results indicate that the transformed N. glauca represents a highly promising new tool for use in phytoremediation efforts.

Biodegradation, Environmental↗

PubMed1990

Production of a foreign protein product with genetically modified plant cells.

Plant cells (Nicotiana tabacum) were genetically engineered to produce a foreign protein, chloramphenicol acetyltransferase (CAT), and the CAT production from suspension cultures was investigated. Suspension cultures were grown in a shake flask, a stirred fermenter, and a bubble-column fermenter. The CAT production was growth related and the maximum activity was reached during the early stationary phase. A 41-day, semicontinuous stirred fermenter run, consisting of five sequential batch runs, demonstrated long-term CAT production. Continuous CAT production was also accomplished in a bubble-column fermenter at a medium flow rate of 3.1 ml h-1, which was equivalent to a dilution rate of 0.25 day-1.

Cells, Cultured↗

PubMed2006

Pyrolysis-field ionization mass spectrometry of rhizodeposits - a new approach to identify potential effects of genetically modified plants on soil organisms.

The objectives of the present study were (1) to investigate the qualitative composition of rhizodeposits leached from soils cropped with non-transgenic and genetically modified (GM) potatoes, and disclose if there were GM-specific modifications in potato rhizodeposition, and (2) to compare these results with conventional bulk parameters of microbial activity in soil. We have raised potatoes from a non-transgenic line (Solanum tuberosum L. cv. Désirée) and three GM lines, which expressed a gene for the resistance to kanamycin (DLH 9000) and a gene for T4 lysozyme (DL10 and DL12). A sandy soil placed in 340 cm3-"CombiSart" containers was used, from which the rhizodeposit was leached after a six-week growth period. The freeze-dried leachates were analyzed by pyrolysis-field ionization mass spectrometry (Py-FIMS). The Py-FI mass spectra gave detailed molecular-chemical information about the composition of leachates, indicating that the potato growth generally altered the composition of the soil solution. Moreover, a principal component analysis of the mass spectra showed differences between the leachates from the non-transgenic parent line and the GM potatoes as well as among the latter group. However, these differences in molecular composition could not be assigned to the release of T4-lysozyme into soil. Dehydrogenase activity and substrate-induced soil respiration as more common bulk parameters of soil microbial activity failed to disclose any significant effects of the various potatoes grown. The limitations of the described rhizodeposit leaching and analysis for risk assessment of GM potato cropping under field conditions are discussed critically. However, it could be concluded that the Py-FI mass spectrometric "fingerprint" can be developed as a fast, comprehensive, highly sensitive and reproducible analytical approach to discern any effects GM-crops may exert on soil ecological parameters.

Animals↗

PubMed2005

Genetically modified plants and food hypersensitivity diseases: usage and implications of experimental models for risk assessment.

The recent advances in biotechnology in the plant industry have led to increasing crop production and yield that in turn has increased the usage of genetically modified (GM) food in the human food chain. The usage of GM foods for human consumption has raised a number of fundamental questions including the ability of GM foods to elicit potentially harmful immunological responses, including allergic hypersensitivity. To assess the safety of foods derived from GM plants including allergenic potential, the US FDA, Food and Agriculture Organization of the United Nations (FAO)/World Health Organization (WHO), and the EU have developed approaches for evaluation assessment. One assessment approach that has been a very active area of research and debate is the development and usage of animal models to assess the potential allergenicity of GM foods. A number of specific animal models employing rodents, pigs, and dogs have been developed for allergenicity assessment. However, validation of these models is needed and consideration of the criteria for an appropriate animal model for the assessment of allergenicity in GM plants is required. We have recently employed a BALB/c mouse model to assess the potential allergenicity of GM plants. We have been able to demonstrate that this model is able to detect differences in antigenicity and identify aspects of protein post-translational modifications that can alter antigenicity. Furthermore, this model has also enabled us to examine the usage of GM plants as a therapeutic approach for the treatment of allergic diseases. This review discusses the current approaches to assess the allergenic potential of GM food and particularly focusing on the usage of animal models to determine the potential allergenicity of GM foods and gives an overview of our recent findings and implications of these studies.

Animals↗