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[Modern biotechnology: a new box for Pandora?].

In a bird's-eye view attention has been paid to a number of items of biotechnology. Amongst others to the beginning of the recombinant DNA technology or genetic modification. Mentioned are the bull Herman, the American tomato 'Flavr Savr' and the sheep Dolly. And further something about the supporters and the opponents of genetic modification and the safety of it. The why of transgenic plants and animals. The legislation and the Netherlands as possible transgene-production country.

Animals↗

A 90-day safety study in Wistar rats fed genetically modified rice expressing snowdrop lectin Galanthus nivalis (GNA).

Genetically modified plants expressing insecticidal traits offer a new strategy for crop protection, but at the same time present a challenge in terms of food safety assessment. The present 90-day feeding study was designed to assess the safety of a rice variety expressing the snowdrop Galanthus nivalis lectin (GNA lectin), and forms part of a EU-funded project where the objective has been to develop and validate sensitive and specific methods to assess the safety of genetically modified foods. Male and female Wistar rats were given a purified diet containing either 60% genetically modified or parental rice for 90 days. This corresponds to a mean daily GNA lectin intake of approximately 58 and 67mg/kg body weight for males and females, respectively. Prior to the animal study comprehensive analytical characterization of both rice materials was performed. The chemical analyses showed a number of statistically significant differences, with the majority being within the ranges reported in the literature. In the animal study a range of clinical, biological, immunological, microbiological and pathological parameters were examined. A number of significant differences were seen between groups fed the two diets, but none of them were considered to be adverse. In conclusion, the design of the present animal study did not enable us to conclude on the safety of the GM food. Additional group(s) where the expressed gene products have been spiked to the diet should be included in order to be able to distinguish whether the observed effects were due to the GNA lectin per se or to secondary changes in the GM rice.

Animals↗

Assuring the safety of genetically modified (GM) foods: the importance of an holistic, integrative approach.

Genes change continuously by natural mutation and recombination enabling man to select and breed crops having the most desirable traits such as yield or flavour. Genetic modification (GM) is a recent development which allows specific genes to be identified, isolated, copied and inserted into other plants with a high level of specificity. The food safety considerations for GM crops are basically the same as those arising from conventionally bred crops, very few of which have been subject to any testing yet are generally regarded as being safe to eat. In contrast a rigorous safety testing paradigm has been developed for GM crops, which utilises a systematic, stepwise and holistic approach. The resultant science based process, focuses on a classical evaluation of the toxic potential of the introduced novel trait and the wholesomeness of the transformed crop. In addition, detailed consideration is given to the history and safe use of the parent crop as well as that of the gene donor. The overall safety evaluation is conducted under the concept known as substantial equivalence which is enshrined in all international crop biotechnology guidelines. This provides the framework for a comparative approach to identify the similarities and differences between the GM product and its comparator which has a known history of safe use. By building a detailed profile on each step in the transformation process, from parent to new crop, and by thoroughly evaluating the significance from a safety perspective, of any differences that may be detected, a very comprehensive matrix of information is constructed which enables the conclusion as to whether the GM crop, derived food or feed is as safe as its traditional counterpart. Using this approach in the evaluation of more than 50 GM crops which have been approved worldwide, the conclusion has been that foods and feeds derived from genetically modified crops are as safe and nutritious as those derived from traditional crops. The lack of any adverse effects resulting from the production and consumption of GM crops grown on more than 300 million cumulative acres over the last 5 years supports these safety conclusions.

Algorithms↗

Development of visible markers for transgenic plants and their availability for environmental risk assessment.

Monitoring of transgenic plants in the field is important, but risk assessment has entailed laborious use of invisible marker genes. Here, we assessed three easily visible marker transgenes--green fluorescent protein (GFP), R, and Nicotiana tabacum homeobox (NTH) 15 genes--for their potential use as marker genes for monitoring genetically modified plants. Transgenic Arabidopsis thaliana plants for each of these genes were visibly distinguished from wild-type plants. We determined the germination rate, 3-week fresh weight, time to first flowering, and seed weight of the transgenic plants to evaluate whether the expression of these marker genes affected the growth of the host. Introduction of GFP gene had no effect on the evaluated parameters, and we then used the GFP gene as a marker to assess the outcrossing frequency between transgenic and two Arabidopsis species. Our results showed that the hybridization frequency between transgenic plants and Arabidopsis thaliana was 0.24%, and between transformants and Arabidopsis lyrata it was 2.6% under experimental condition. Out-crossing frequency was decreased by extending the distance between two kinds of plants. Thus, the GFP gene is a useful marker for assessing the whereabouts of transgenes/transformants in the field. We also demonstrated that the GFP gene is possibly applicable as a selection marker in the process of generation of transgenic plants.

Arabidopsis↗

Different expression of an S-adenosylmethionine synthetase gene in transgenic tobacco callus modifies alkaloid biosynthesis.

Transformed callus cultures of Nicotiana tabacum were generated in which the SAM-1 gene from Arabidopsis thaliana encoding S-adenosylmethionine synthetase (SAM-S), under the control of the 35S promoter, had been integrated. The presence of the SAM-1 gene was detected in all tested transformants and the SAM-S activity correlated with the accumulation of SAM in the tobacco callus cultures. Three distinct phenotypic classes were identified among the transgenic cell lines in relation to growth of the cells, structure of the calli, and level of SAM. Transgene silencing was observed in several cultivated transgenic calli and this phenomenon was correlated directly with a low level of SAM-1 mRNA accompanied by a decrease of the SAM-S activity. The transgenic calli overexpressing the SAM-1 gene accumulated a high SAM level. The modifications in SAM-S activity were reflected in the pattern of secondary products present in the different cell lines, thereby demonstrating that the flux through the biosynthetic pathway of a plant secondary product can be modified by means of genetic engineering.

Alkaloids↗

Effects of seed-specific expression of a cytokinin biosynthetic gene on canola and tobacco phenotypes.

The Agrobacterium tumefaciens isopentenyl transferase gene (ipt), a cytokinin biosynthetic gene, was placed under the control of 1.9 kb of promoter sequence from the 2S albumin AT2S1 gene isolated from an Arabidopsis thaliana library. The construct was introduced into canola (Brassica napus) and tobacco (Nicotiana tabacum). ipt transcripts were followed during embryo development of transgenic plants by northern hybridizations. The phenotype of transformed plants from the T1 generation was analysed and we observed an increased branching of inflorescences in tobacco and canola plants expressing the ipt gene. Comparing with controls, the average number of capsules and siliques in AT2S1-ipt plants was 82.6 and 24.8% higher, respectively. This result was correlated with an increase in cytokinin levels in transgenic plants, as revealed by RIA. Indeed, cytokinin contents of T1 AT2S1-ipt B. napus seeds were found 2.2-fold higher than cytokinin contents of control seeds, and T1 AT2S1-ipt N. tabacum capsules contained 2.6-fold more cytokinins than control capsules. In tobacco, the average seed weight per capsule was lower in AT2S1-ipt plants while the seed number per silique and the average seed weight were not modified in canola carrying this construct. The average seed yield per plant was not significantly increased in AT2S1-ipt tobacco or canola plants.

Agrobacterium tumefaciens↗

Cytosine methylation at CG and CNG sites is not a prerequisite for the initiation of transcriptional gene silencing in plants, but it is required for its maintenance.

Transgenes integrated into plant chromosomes, and/or endogenous plant genes, may be subjected to epigenetic silencing at the transcriptional or post-transcriptional level. Transcriptional inactivation is correlated with hypermethylation of CG/CNG sites at the silent loci. It is not known whether local hypermethylation is part of the inactivation process, or just an outcome of the silent state. To address this issue, we generated transgenic tobacco lines containing a selectable marker gene controlled by a derivative of the 35S promoter of the cauliflower mosaic virus (CaMV) devoid of CG and CNG methylation acceptor sites. Silencing was triggered by crossing to the silencer locus of tobacco line 271. This line contains inactive and methylated copies of the 35S promoter and is able to silence homologous promoter copies at ectopic chromosomal positions. The mutated promoter lacking CG/CNG methylation acceptor sites was as susceptible to Trans-silencing as the unmodified 35S promoter control. Thus, methylation at CG and CNG sites is not a prerequisite for the initiation of epigenetic gene inactivation. Interestingly, while methylation of the remaining cytosines is usually only slightly affected by silencing, it was significantly increased in the absence of CG/CNG sequences. Since this sequence preference is the same as that of known methyltransferases, this may imply that silencing is accompanied or directly followed by recruitment of methyltransferase, which, in the absence of cytosines in the optimal sequence context, modifies other C residues in the affected area. However, silencing without CG/CNG methylation was immediately relieved in the absence of the silencer. Thus, CG/CNG methylation is probably essential for the maintenance of previously established epigenetic states.

Aminobutyrates↗

The development of a plant-based vaccine for measles.

Plant-based vaccination strategies have the potential to overcome the limitations of the current measles vaccine. The measles virus hemagglutinin (MV-H) protein has been expressed in tobacco. Oral immunisation of mice with plant-derived MV-H protein resulted in MV-specific antibodies and secretory IgA, indicative of humoral and mucosal immune responses. In addition, boosting with oral plant-derived MV-H protein following a MV-H DNA prime, resulted in a greater response than could be induced with either vaccine alone. Collectively, this research represents a significant step towards an effective oral measles vaccine that would be temperature-stable, easy to administer and amenable to inexpensive manufacture.

Administration, Oral↗

A biotechnological approach to improving the nutritive value of alfalfa.

The postruminal supply of the sulfur-containing amino acids, methionine and cysteine, has been reported to be a major limitation to wool growth in sheep. We aim to improve the protein quality of forage for ruminants by introducing into alfalfa chimeric genes encoding a ruminally stable, sulfur amino acid-rich protein from sunflower seeds. Four gene constructs were transferred to Australian commercial cultivars of alfalfa using Agrobacterium tumefaciens-mediated transformation and selection with phosphinothricin (PPT). Modification of the sunflower seed albumin protein-coding region by addition of the coding information for an endoplasmic reticulum (ER) retention signal was found to greatly increase the level to which the sulfur amino acid-rich protein accumulated in the leaves of transgenic alfalfa plants. The Cauliflower Mosaic Virus (CaMV) 35S promoter and two light-regulated plant gene promoter regions were compared for their ability to direct high-level expression of the introduced genes in alfalfa leaves. The highest expression of sunflower seed albumin was found in transformants bearing a gene incorporating the promoter from the Arabidopsis thaliana ats1A gene, which encodes the ribulose bisphosphate carboxylase small subunit. The highest level of sunflower seed albumin found in transgenic alfalfa leaves was estimated to constitute .1% of soluble leaf protein. This level of accumulation of the foreign protein would be predicted to supply an extra 40 mg of sulfur amino acids daily to sheep fed the modified forage. Published studies in which wool growth rates were significantly increased employed supplementation of approximately 1 to 2 g of sulfur amino acids daily.

Agrobacterium tumefaciens↗

Stringent repression and homogeneous de-repression by tetracycline of a modified CaMV 35S promoter in intact transgenic tobacco plants.

A cauliflower mosaic virus (CaMV) 35S promoter derivative, which is tightly repressed by the Tn 10 encoded Tet repressor in a transient expression system as well as in transgenic plants has been constructed. After treatment of transgenic plants with tetracycline (Tc) the activity of the reporter enzyme beta-glucuronidase (GUS) increased up to 500-fold in tissue culture as well as under greenhouse conditions. Efficient de-repression was achieved by Tc uptake through the roots as well as by Tc treatment of leaves of intact plants. As Tc is not very stable in the plants, this system can also be used for a transient expression of a transgene. This system provides a unique tool for regenerating transgenic plants carrying a repressed transgene and for efficiently de-repressing its activity by a specific inducer at any time point of further development.

Base Sequence↗

Isolation and expression analysis of peanut chlorotic streak caulimovirus (PClSV) full-length transcript (FLt) promoter in transgenic plants.

A promoter fragment from peanut chlorotic streak caulimovirus (PClSV) full-length transcript (FLt) was identified and later modified to have duplicated enhancer domain. The FLt promoter with its single or double enhancer domains, fused with the GUS reporter gene to form chimeric gene constructs, showed a high level of expression of these genes in cells and transgenic plants. The FLt promoter with its double enhancer domain gives an average threefold greater expression of genes compared to the FLt promoter with its single enhancer domain in transgenic plants. In young seedlings the expression was in the order root > leaf > stem. The histochemical GUS assay in young seedlings showed more activity in root tips and leaf midribs, veins, and other vascular tissues. The expression from the PClSV FLt promoter was compared with that from the figwort mosaic virus promoter in transgenic plants. These constitutive promoters were comparable in respect to GUS expression level.

Caulimovirus↗

Effect of the M-EcoRII methyltransferase-encoding gene on the phenotype of Nicotiana tabacum transgenic cells.

The EcoRII DNA methyltransferase (M-EcoRII; MTase) modifies a cytosine in the DNA sequence CCWGG which contains a CNG methylation motif characteristic of plant DNA. The gene (ecoRIIM) encoding this MTase has been cloned into the T-DNA of the wild-type Agrobacterium Ti-plasmid pTiC58 downstream from the plant expression nopaline synthase-encoding gene promoter. Nicotiana tabacum cells have been transformed with Agrobacterium tumefaciens harbouring this recombinant Ti-plasmid. The primary transformed tabacco tissue line has given rise to novel stable lines which are morphologically distinctive. Southern hybridization analysis of all transformed tissue lines has shown the presence, in each of them, of ecoRIIM. The tissue studied differed in morphology in callus culture, dependence on phytohormones and the ability to synthesize nopaline.

Agrobacterium tumefaciens↗

Expression of a modified Cry1Ie gene in E. coli and in transgenic tobacco confers resistance to corn borer.

The wild-type Cry1Ie gene from Bacillus thuringiensis was modified for its efficient expression in transgenic plants. Modified Cry1Ie gene (designated as Cry1Iem) was cloned into prokaryotic expression vector pET28b and its expression in E. coli was confirmed by SDS-PAGE analysis. Bioassays using crude expression products in E. coli revealed that Cry1Iem protein had a similar toxicity to corn borer as wild-type Cry1Ie. Cry1Iem gene was then inserted downstream of the maize ubiquitin-1 promoter in plant expression vector p3301. Transgenic tobacco plants carrying Cry1Iem showed insecticidal activity against corn borer.

Animals↗

Novel gene expression system for plant cells based on induction of alpha-amylase promoter by carbohydrate starvation.

The 5' regulatory region and putative signal sequence of a rice alpha-amylase gene, alpha Amy8, was fused to a bacterial gene encoding beta-glucuronidase (GUS) and introduced into rice, tobacco, and potato via Agrobacterium-mediated transformation systems. Expression of this chimeric gene in suspension cells of transgenic plants was suppressed by the presence of sucrose in the medium and induced by its absence. Induction or suppression of GUS expression in transgenic rice could be reversed by the deprivation or replenishment, respectively, of sucrose in the medium. The expressed GUS fusion protein was translocated to the endoplasmic reticulum, modified by glycosylation, and secreted into the culture medium of transgenic cells. In the presence of a glycosylation inhibitor, tunicamycin, the enzymatically active form of GUS was assembled in the endoplasmic reticulum. The yield of GUS secreted by transgenic cells was estimated to be as high as 40% of total secreted proteins. The reversible induction of the alpha-amylase promoter in culture cells by sugar level in the medium provides an excellent inducible expression system for basic research in plant science. Combination of the alpha-amylase promoter and signal sequence also offers a novel approach for large scale production of low cost, easily purified, secreted recombinant proteins.

Amino Acid Sequence↗

The use of a thermostable beta-glucanase gene from Clostridium thermocellum as a reporter gene in plants.

In order to take advantage of the high thermostability of its product, beta-1,3;1,4-glucanase (lichenase), we used a modified version of the licB gene from Clostridium thermocellum as a reporter gene for the analysis of gene expression in transformed plants. The coding region of the licB gene was truncated at both ends. The truncated enzyme retained its activity and thermostability. The modified gene (m-licB), with and without a plant leader peptide-encoding sequence, was expressed in tobacco plants under control of either the Agrobacterium octopine TR-DNA 2' gene promoter or the promoter of the gene for the small subunit of ribulose-1,5-bisphosphate carboxylase. Expression of licB can be measured quantitatively and accurately, the assay is sensitive and simple enough to be used for analysis of various gene fusion systems or for screening of transformants. The enzyme is very stable and remains active in tissue extracts even after storage for 1 year and survives many thawing-freezing cycles. The lichenase-encoding gene was expressed at high levels in transformed tobacco plants without any apparent detrimental effects on vegetative growth or flowering.

Clostridium↗

Premature dissolution of the microsporocyte callose wall causes male sterility in transgenic tobacco.

Male sterility in a petunia cytoplasmic male sterile line has been attributed to the early appearance of active callase, a beta-1,3-glucanase, in the anther locule. This leads to premature dissolution of the callose walls surrounding the microsporogenous cells. We have mimicked this aspect of the petunia line in transgenic tobacco by engineering the secretion of a modified pathogenesis-related vacuolar beta-1,3-glucanase from the tapetum prior to the appearance of callase activity in the locule. Plants expressing the modified glucanase from tapetum-specific promoters exhibited reduced male fertility, ranging from complete to partial male sterility. Callose appearance and distribution are normal in the male sterile transgenic plants up to prophase I, whereupon callose is prematurely degraded. Meiosis and cell division occur normally. The resultant microspores have an abnormally thin cell wall that lacks sculpturing. The tapetum shows hypertrophy. Male sterility is probably caused by bursting of the aberrant microspores at a time corresponding to microspore release. These results demonstrate that premature callose degradation is sufficient to cause male sterility and suggest that callose is essential for the formation of a normal microspore cell wall.

Base Sequence↗

Modification of carbonic anhydrase activity by antisense and over-expression constructs in transgenic tobacco.

The activity and location of carbonic anhydrase has been modified by transformation of tobacco with antisense and over-expression constructs. Antisense expression resulted in the inhibition of up to 99% of carbonic anhydrase activity but had no significant impact on net CO2 assimilation. Stomatal conductance and susceptibility to water stress appeared to increase in response to the decline in carbonic anhydrase activity. An over-expression construct designed to increase cytosolic carbonic anhydrase abundance resulted in a significant increase in net activity, a small increase in stomatal conductance but little impact on CO2 assimilation. Chloroplastic carbonic anhydrase activity was enhanced by the expression of an additional construct which targeted the polypeptide to the organelle. The increase in chloroplastic carbonic anhydrase appeared to be accompanied by a concomitant increase in Rubisco activity.

Antisense Elements (Genetics)↗