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Efficient production of male and female sterile plants by expression of a chimeric repressor in Arabidopsis and rice.

Male and female sterile plants are particularly useful for the effective production of commercial hybrid plants and for preventing the diffusion of seeds or pollen grains of genetically modified plants in the open field. In an attempt to create several types of sterile plant by genetic manipulation, we applied our Chimeric REpressor Gene-Silencing Technology (CRES-T) to four transcription factors, namely APETALA3, AGAMOUS, LEAFY and AtMYB26, involved in the regulation of petal and stamen identity, stamen and carpel identity, floral meristem identity and anther dehiscence, respectively, in Arabidopsis. Transgenic plants expressing each chimeric repressor exhibited, at high frequency, a sterile phenotype that resembled the loss-of-function phenotype of each corresponding gene. Furthermore, in the monocotyledonous crop plant 'rice', expression of the chimeric repressor derived from SUPERWOMAN1, the rice orthologue of APETALA3, resulted in the male sterile phenotype with high efficiency. Our results indicate that CRES-T provides a powerful tool for controlling the fertility of both monocots and dicots by exploiting transcription factors that are strongly conserved amongst plants.

AGAMOUS Protein, Arabidopsis↗

GMOs: building the future on the basis of past experience.

Biosafety of genetically modified organisms (GMOs) and their derivatives is still a major topic in the agenda of government and societies worldwide. The aim of this review is to bring into light that data that supported the decision taken back in 1998 as an exercise to stimulate criticism from the scientific community for upcoming discussions and to avoid emotional and senseless arguments that could jeopardize future development in the field. It must be emphasized that Roundup Ready soybean is just one example of how biotechnology can bring in significant advances for society, not only through increased productivity, but also with beneficial environmental impact, thereby allowing more rational use of agricultural pesticides for improvement of the soil conditions. The adoption of agricultural practices with higher yield will also allow better distribution of income among small farmers. New species of genetically modified plants will soon be available and society should be capable of making decisions in an objective and well-informed manner, through collegiate bodies that are qualified in all aspects of biosafety and environmental impact.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗

The moral difference between intragenic and transgenic modification of plants.

Public policy on the development and use of genetically modified organisms (GMOs) has mainly been concerned with defining proper strategies of risk management. However, surveys and focus group interviews show that although lay people are concerned with risks, they also emphasize that genetic modification is ethically questionable in itself. Many people feel that this technology "tampers with nature" in an unacceptable manner. This is often identified as an objection to the crossing of species borders in producing transgenic organisms. Most scientists reject these opinions as based on insufficient knowledge about biotechnology, the concept of species, and nature in general. Some recent projects of genetic modification aim to accommodate the above mentioned concerns by altering the expression of endogenous genes rather than introducing genes from other species. There can be good scientific reasons for this approach, in addition to strategic reasons related to greater public acceptability. But are there also moral reasons for choosing intragenic rather than transgenic modification? I suggest three interrelated moral reasons for giving priority to intragenic modification. First, we should respect the opinions of lay people even when their view is contrary to scientific consensus; they express an alternative world-view, not scientific ignorance. Second, staying within species borders by strengthening endogenous traits reduces the risks and scientific uncertainty. Third, we should show respect for nature as a complex system of laws and interconnections that we cannot fully control. The main moral reason for intragenic modification, in our view, is the need to respect the "otherness" of nature.

Community Participation↗

Environmental implications of gene flow from sugar beet to wild beet--current status and future research needs.

Gene flow via seed or pollen is a basic biological process in plant evolution. The ecological and genetic consequences of gene flow depend on the amount and direction of gene flow as well as on the fitness of hybrids. The assessment of potential risks of transgenic plants should take into account the fact that conventional crops can often cross with wild plants. The precautionary approach in risk management of genetically modified plants (GMPs) may make it necessary to monitor significant wild and weed populations that might be affected by transgene escape. Gene flow is hard to control in wind-pollinated plants like beet (Beta vulgaris). In addition, wild beet populations potentially can undergo evolutionary changes which might expand their geographical distribution. Unintended products of cultivated beets pollinated by wild beets are weed beets that bolt and flower during their first year of planting. Weed beets cause yield losses and can delay harvest. Wild beets are important plant genetic resources and the preservation of wild beet diversity in Europe has been considered in biosafety research. We present here the methodology and research approaches that can be used for monitoring the geographical distribution and diversity of Beta populations. It has recently been shown that a century of gene flow from Beta vulgaris ssp. vulgaris has not altered the genetic diversity of wild Beta vulgaris L. ssp. maritima (L.) Arcang. in the Italian sugar beet seed production area. Future research should focus on the potential evolution of transgenic wild beet populations in comparison to these baseline data. Two monitoring models are presented describing how endpoints can be measured: (1) "Pre-post" crop commercialization against today's baseline and (2) "Parallel" to crop commercialization against GMP free reference areas/ populations. Model 2 has the advantage of taking ongoing changes in genetic diversity and population dynamics into account. Model 1 is more applicable if gene flow is so strong that most areas/populations contain GMPs. Important traits that may change the ecology of populations are genes that confer tolerance to biotic and abiotic stress. An assessment of environmental effects can realistically only be based on endpoints and consequences of gene introgression, which may include economic values of biodiversity in littoral and other ecosystems containing wild beet. In general, there is still a great need to harmonize worldwide monitoring systems by the development of appropriate methods to evaluate the environmental impact of introgressed transgenes.

Beta vulgaris↗

Cloning and characterization of red clover polyphenol oxidase cDNAs and expression of active protein in Escherichia coli and transgenic alfalfa.

Red clover (Trifolium pratense) leaves contain high levels of polyphenol oxidase (PPO) activity and o-diphenol substrates. Wounding of leaves during harvest and ensiling results in browning of leaf tissues from activity of PPO on the o-diphenols. In association with browning, leaf proteins remain undegraded during ensiling, presumably due to PPO-generated o-quinone inhibition of leaf proteases. We cloned three red clover PPO cDNAs, PPO1, PPO2, and PPO3, from a leaf cDNA library. Sequence comparisons among the three red clover PPO clones indicated they are 87% to 90% identical at the nucleotide level (80%-83% amino acid identity). All three encode proteins predicted to localize to the chloroplast thylakoid lumen. RNA-blotting and immunoblotting experiments indicated PPO1 is expressed primarily in young leaves, PPO2 in flowers and petioles, and PPO3 in leaves and possibly flowers. We expressed mature PPO1 in Escherichia coli. A portion of the expressed protein was soluble and functional in an assay for PPO activity. We also expressed the red clover PPO cDNAs under the control of a constitutive promoter in alfalfa (Medicago sativa). The expressed red clover PPO proteins were active in alfalfa extracts as evidenced by o-diphenol-dependant extract browning and quantitative assays of PPO activity. Proteolysis in leaf extracts of alfalfa expressing red clover PPO1 was dramatically reduced in the presence of an o-diphenol compared to controls. Transgenic alfalfa expressing red clover PPO should prove an excellent model system to further characterize the red clover PPO enzymes and PPO-mediated inhibition of postharvest proteolysis in forage plants.

Amino Acid Sequence↗

Genetically modified soybeans: false-positive detection in fermented natural soybean (tempe).

Tempe was prepared using mixtures of natural soybean and genetically modified Roundup Ready (RUR) soybean fermented with natural Rhizopus sp. The amount of RUR soybean was quantified using an ELISA plate test. The RUR signal decreased during fermentation. In the control experiments on fermentation of non-RUR soybean, the tempe gave a false-positive RUR signal. The cross-reacting substance was generated only in non-RUR soybean during fermentation by Rhizopus sp., Rhizopus oligosporus, R. oryzae, Mucor rouxii and Aspergillus awamori.

Antigens↗

Widespread horizontal transfer of mitochondrial genes in flowering plants.

Horizontal gene transfer--the exchange of genes across mating barriers--is recognized as a major force in bacterial evolution. However, in eukaryotes it is prevalent only in certain phagotrophic protists and limited largely to the ancient acquisition of bacterial genes. Although the human genome was initially reported to contain over 100 genes acquired during vertebrate evolution from bacteria, this claim was immediately and repeatedly rebutted. Moreover, horizontal transfer is unknown within the evolution of animals, plants and fungi except in the special context of mobile genetic elements. Here we show, however, that standard mitochondrial genes, encoding ribosomal and respiratory proteins, are subject to evolutionarily frequent horizontal transfer between distantly related flowering plants. These transfers have created a variety of genomic outcomes, including gene duplication, recapture of genes lost through transfer to the nucleus, and chimaeric, half-monocot, half-dicot genes. These results imply the existence of mechanisms for the delivery of DNA between unrelated plants, indicate that horizontal transfer is also a force in plant nuclear genomes, and are discussed in the contexts of plant molecular phylogeny and genetically modified plants.

Base Sequence↗

Pathogenesis-related (PR)-proteins identified as allergens.

Type 1 allergies are recognized as an important disease affecting around 25% of the population of industrialized countries of the Northern hemisphere. Allergic patients produce specific IgE antibodies after frequent exposure to either inhaled or nutritive allergens. Of the plant allergens listed in the Official Allergen Database of the International Union of Immunological Societies, approx. 25% belong to the group of pathogenesis-related proteins (PR-proteins). PR-proteins are defined as proteins that are induced upon stress, pathogen attack and abiotic stimuli. This inhomogeneous group of proteins has been classified into 14 PR-protein families. So far, plant-derived allergens have been identified with sequence similarities to PR-protein families 2, 3, 4, 5, 8, 10 and 14. In general, both protein groups, i.e. PR-proteins and allergens, comprise rather small proteins, which are stable at low pH and resistant to proteolysis. These features, and their level of expression, make PR-proteins good candidates for evoking an immune response in predisposed humans, when coming into contact with mucosal surfaces. The identification of PR-proteins with allergenic potential and their homologues is of importance for the allergic patient and the management of this disease. Firstly, plant foods derived from genetically modified plants could represent new allergen sources, and therefore should be evaluated carefully for their potential allergenicity. Secondly, complex plant-derived foods should be analysed for hidden allergens and labelled accordingly.

Allergens↗

Protein immunoassay methods for detection of biotech crops: applications, limitations, and practical considerations.

Immunoassay methods are available for detection and quantitation of proteins expressed by most biotechnology-derived crops in commercial production. The 2 most common test formats are enzyme-linked immunosorbent assay (ELISA) and immunochromatographic (lateral flow) strip tests. Two ELISA methods, one for Roundup Ready soybeans and one for MON810 CrylAb corn, were the subject of large international collaborative studies and were demonstrated to quantitatively determine the concentrations of biotech crops in samples of ground grain. Quantitative ELISA methods are also useful for analysis of processed fractions of agricultural commodities such as soybean toasted meal or corn flour. Both strip tests and ELISAs for biotech crops are currently being used on a large scale in the United States to manage the sale and distribution of grain. In these applications, tests are used to determine if the concentration of biotech grain is above or below specified threshold limits. Using existing U.S. Department of Agriculture sampling techniques, the reliability of the threshold determination is expressed in terms of statistical confidence rather than analytical precision. Combining the use of protein immunoassays with Identity Preservation systems provides an effective means of characterizing the raw and processed agricultural inputs to the food production system in a way that allows food producers to comply with labeling laws.

Crops, Agricultural↗

Novel and transgenic food crops: overview of scientific versus public perception.

Recombinant DNA technology offers opportunities to develop new products in many different fields, including agriculture and the agro-food area. Transgenic plants with improved agronomic traits currently grow in field trials and a few varieties have already reached the European market. By and large, new technologies raise both concerns and expectations and modern biotechnology is no exception. Indeed, a voluntary moratorium on experiments involving recombinant DNA molecules was called for in 1974. At the present time, although a majority of academic and industrial scientists agree that transgenic food crops pose no risk for the environment or human health, some others believe that certain applications of modern plant biotechnology are hazardous. In particular, deliberate releases of genetically modified plants are regarded as risky. There is also a disparity between expert and lay perception of r-DNA technology applications to food crops, which makes public information a difficult task. This paper aims at exposing these conflicting points of view on the agricultural applications of modern biotechnology. We also propose some recommendations pertaining to public information in Europe. It appears that consensus conferences might be a good approach to stimulate public information and public debate in Europe, although this approach has to be adapted to the cultural context of each country.

Agriculture↗

Techniques for the removal of marker genes from transgenic plants.

The presence of marker genes encoding antibiotic or herbicide resistances in genetically modified plants poses a number of problems. Various techniques are under development for the removal of unwanted marker genes, while leaving required transgenes in place. The aim of this brief review is to describe the principal methods used for marker gene removal, concentrating on the most recent and promising innovations in this technology.

Cell Nucleus↗

Horizontal gene transfer from transgenic plants to terrestrial bacteria--a rare event?

Today, 12 years after the first field release of a genetically modified plant (GMP), over 15,000 field trials at different locations have been performed. As new and unique characteristics are frequently introduced into GMPs, risk assessment has to be performed to assess their ecological impact. The possibilities of horizontal gene transfer (HGT; no parent-to-offspring transfer of genes) from plants to microorganisms are frequently evaluated in such risk assessments of GMPs before release into the field. In this review we indicate why putative HGT from plants to terrestrial (soil and plant associated) bacteria has raised concern in biosafety evaluations. Further, we discuss possible pathways of HGT from plants to bacteria, outline the barriers to HGT in bacteria, describe the strategies used to investigate HGT from plants to bacteria and summarize the results obtained. Only a few cases of HGT from eukaryotes such as plants to bacteria have been reported to date. These cases have been ascertained after comparison of DNA sequences between plants and bacteria. Although experimental approaches in both field and laboratory studies have not been able to confirm the occurrence of such HGT to naturally occurring bacteria, recently two studies have shown transfer of marker genes from plants to bacteria based on homologous recombination. The few examples of HGT indicated by DNA sequence comparisons suggest that the frequencies of evolutionarily successful HGT from plants to bacteria may be extremely low. However, this inference is based on a small number of experimental studies and indications found in the literature. Transfer frequencies should not be confounded with the likelihood of environmental implications, since the frequency of HGT is probably only marginally important compared with the selective force acting on the outcome. Attention should therefore be focused on enhancing the understanding of selection processes in natural environments. Only an accurate understanding of these selective events will allow the prediction of possible consequences of novel genes following their introduction into open environments.

Bacteria↗

Prevention of flower development in birch and other plants using a BpFULL1::BARNASE construct.

The prevention of flower formation is important for avoiding the spread of transgenes from genetically modified plants into wild populations. Moreover, the resources not expended for the generation of flowers and fruits might be allocated to increased vegetative growth. We have been developing methods for preventing flower formation in silver birch (Betula pendula), a tree species of considerable economical importance in the boreal region. Here we study the suitability of the promoter of BpFRUITFULL-LIKE1 (BpFULL1, formerly BpMADS5) for tissue-specific ablation of inflorescences in Arabidopsis, tobacco and birch. With all these species, the development of inflorescences was successfully prevented. The results show that the BpFULL1::BARNASE construct has potential biotechnological applications in different plant species.

Arabidopsis↗

Intra-chromosomal rearrangements generated by Cre-lox site-specific recombination.

Chromosomal rearrangements are useful genetic and breeding tools but are often difficult to detect and characterize. To more easily identify and define chromosome deletions and inversions, we have used the bacteriophage P1 Cre-lox site-specific recombination system to generate these events in plants. This involves three steps: (i) the introduction of two lox sites into one locus in a plant genome, including one site within a modified Ds transposon; (ii) Ac transposase-mediated transposition of the Ds-lox element to a new locus on the same chromosome; (iii) Cre-mediated site-specific recombination between the two lox sites that bracket a chromosome segment. We report the production of a deletion and three inversion events in tobacco. The utility of chromosomal segments bracketed by lox sites for targeted manipulation and cloning is discussed.

Base Sequence↗

Study of phytochelatins and other related thiols as complexing biomolecules of As and Cd in wild type and genetically modified Brassica juncea plants.

The accumulation of As and Cd in Brassica juncea plants and the formation of complexes of these elements with bioligands such as glutathione and/or phytochelatins (PCs) is studied. The genetic manipulation of these plants to induce higher As and Cd accumulation has been achieved by overexpressing the genes encoding for gamma-glutamyl cysteine synthetase (gamma-ECS) and glutathione synthetase (GS). These two enzymes are responsible for glutathione (GSH) formation in plants, which is the first step in the production of PCs. The biomass produced in both the wild type and the genetically modified plants, has been evaluated. Additionally, the total Cd and As concentration accumulated in the plant tissues was measured by inductively coupled plasma mass spectrometry (ICP-MS) after extraction. Speciation studies on the extracts were conducted using size exclusion liquid chromatography (SEC) coupled online with ICP-MS to monitor As, Cd and S. For further purification of the As fractions, reversed phase high performance liquid chromatography (RP-HPLC) was used. Structural elucidation of the PCs and other thiols, as well as their complexes with As and Cd, was performed by electrospray-quadrupole-time-of-flight (ESI-Q-TOF). In both the Cd and As exposed plants it was possible to observe the presence of oxidized PC2 ([M + H]+, m/z 538), GS-PC2(-Glu) ([M + H]+, m/z 716) as well as reduced GSH ([M + H]+, m/z 308) and oxidized glutathione (GSSG) ([M + H]+, m/z 613). However, only the GS plants exhibited the presence of As(GS)3 complex ([M + H]+, m/z 994) that was further confirmed by MS/MS. This species is reported for the first time in B. juncea plant tissues.

Arsenic↗

Experience with environmental issues in GM crop production and the likely future scenarios.

In the Cartagena Protocol on Biosafety, standards for risk assessment of genetically modified organisms (GMOs) have been set. The criteria and information basis for the risk assessment of GMOs have been modified by the EU Directive 2001/18/EC. Various approaches to further improve the criteria for environmental risk assessment of GMOs are described in this study. Reports on the ecological impacts of the cultivation of certain non-transgenic crop plants with novel or improved traits as analogy models to transgenic plants showed that the effects of agricultural practice can be at least equally important as the effects of gene transfer and invasiveness, although the latter currently play a major role in risk assessment of transgenic crops. Based on these results the applicability of the methodology of 'Life Cycle Analysis (LCA)' for genetically modified plants in comparison with conventionally bred and organically grown crop plants was evaluated. The methodology was regarded as applicable with some necessary future improvements. In current projects, the assessment of toxicology and allergenicity of GM crops are analysed, and suggestions for standardization are developed. Based on results and recommendations from these efforts there are still the challenges of how to operationalize the precautionary principle and how to take into account ecologically sensitive ecosystems, including centres of origin and centres of genetic diversity.

Agriculture↗

An amino-terminal deletion of rice phytochrome A results in a dominant negative suppression of tobacco phytochrome A activity in transgenic tobacco seedlings.

Overexpression of phytochrome A results in an increased inhibition of hypocotyl elongation under red and far-red light. We used this approach to assay for the function of N-terminal mutations of rice (Oryza sativa L.) phytochrome A. Transgenic tobacco seedlings that express the wild-type rice phytochrome A (RW), a rice phytochrome A lacking the first 80 amino acids (NTD) or a rice phytochrome A with a conversion of the first 10 serines into alanine residues (S/A) were compared with untransformed wild-type tobacco (Nicotiana tabacum L. cv. Xanthi) seedlings. Experiments under different fluence rates showed that RW and, even more strongly, S/A increased the response under both red and far-red light, whereas NTD decreased the response under far-red light but hardly altered the response under red light. These results indicate that NTD not only lacks residues essential for an increased response under red light but also distorts the wild-type response under far-red light. Wild-type rice phytochrome A and, even more so, S/A mediate an enhanced phytochrome A as well as phytochrome B function, whereas NTD interferes with the function of endogenous tobacco phytochrome A as well as that of rice phytochrome A when co-expressed in a single host. Experiments with seedlings of different ages and various times of irradiation under far-red light demonstrated that the effect of NTD is dependent on the stage of development. Our results suggest that the lack of the first 80 amino acids still allows a rice phytochrome A to interact with the phytochrome transduction pathway, albeit non-productively in tobacco seedlings.

Base Sequence↗