Transgenic plant factories for the production of biopolymers and plateform chemicals

In the mid-to long-term, resource constraints will force society to cover a significant share of the future demand for fuels, materials and chemicals by renewable resources. This trend is already visible in the increasing conversion of carbohydrates and plant oils to fuels, chemicals, and polymers. In this perspective, we discuss current efforts and ideas to produce platform chemicals and polymers directly in transgenic plants.

Immunogenicity of transgenic plant-derived hepatitis B surface antigen.

The focus of the Children's Vaccine Initiative is to encourage the discovery of technology that will make vaccines more readily available to developing countries. Our strategy has been to genetically engineer plants so that they can be used as inexpensive alternatives to fermentation systems for production of subunit antigens. In this paper we report on the immunological response elicited in vivo by using recombinant hepatitis B surface antigen (rHBsAg) purified from transgenic tobacco leaves. The anti-hepatitis B response to the tobacco-derived rHBsAg was qualitatively similar to that obtained by immunizing mice with yeast-derived rHBsAg (commercial vaccine). Additionally, T cells obtained from mice primed with the tobacco-derived rHBsAg could be stimulated in vitro by the tobacco-derived rHBsAg, yeast-derived rHBsAg, and by a synthetic peptide that represents part of the a determinant located in the S region (139-147) of HBsAg. Further support for the integrity of the T-cell epitope of the tobacco-derived rHBsAg was obtained by testing the ability of the primed T cells to proliferate in vitro after stimulation with a monoclonal anti-idiotype and an anti-idiotype-derived peptide, both of which mimic the group-specific a determinant of HBsAg. In total, we have conclusively demonstrated that both B- and T-cell epitopes of HBsAg are preserved when the antigen is expressed in a transgenic plant.

Culture-Independent Assessment of Rhizobiales-Related Alphaproteobacteria and the Diversity of Methylobacterium in the Rhizosphere and Rhizoplane of Transgenic Eucalyptus

The rhizosphere is an ecosystem exploited by a variety of organisms involved in plant health and environmental sustainability. Abiotic factors influence microorganism-plant interactions, but the microbial community is also affected by expression of heterologous genes from host plants. In the present work, we assessed the community shifts of Alphaproteobacteria phylogenetically related to the Rhizobiales order (Rhizobiales-like community) in rhizoplane and rhizosphere soils of wild-type and transgenic eucalyptus. A greenhouse experiment was performed and the bacterial communities associated with two wild-type (WT17 and WT18) and four transgenic (TR-9, TR-15, TR-22, and TR-23) eucalyptus plant lines were evaluated. The culture-independent approach consisted of the quantification, by real-time polymerase chain reaction (PCR), of a targeted subset of Alphaproteobacteria and the assessment of its diversity using PCR-denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone libraries. Real-time quantification revealed a lesser density of the targeted community in TR-9 and TR-15 plants and diversity analysis by principal components analysis, based on PCR-DGGE, revealed differences between bacterial communities, not only between transgenic and nontransgenic plants, but also among wild-type plants. The comparison between clone libraries obtained from the transgenic plant TR-15 and wild-type WT17 revealed distinct bacterial communities associated with these plants. In addition, a culturable approach was used to quantify the Methylobacterium spp. in the samples where the identification of isolates, based on 16S rRNA gene sequences, showed similarities to the species Methylobacterium nodulans, Methylobacterium isbiliense, Methylobacterium variable, Methylobacterium fujisawaense, and Methylobacterium radiotolerans. Colonies classified into this genus were not isolated from the rhizosphere but brought in culture from rhizoplane samples, except for one line of the transgenic plants (TR-15). In general, the data suggested that, in most cases, shifts in bacterial communities due to cultivation of transgenic plants are similar to those observed when different wild-type cultivars are compared, although shifts directly correlated to transgenic plant cultivation may be found.

A protocol for the fluorometric quantification of mGFP5-ER and sGFP(S65T) in transgenic plants

The Green Fluorescent Protein (GFP) from Aequorea victor-in has begun to be used as a reporter protein in plants. It is particularly useful as GFP fluorescence can be detected in a non-destructive manner, whereas detection of enzyme-based reporters often requires destruction of the plant tissue. The use of GFP as a reporter enables transgenic plant tissues to be screened in vivo at any growth stage. Quantification of GFP in transgenic plant extracts will increase the utility of GFP as a reporter protein. We report herein the quantification of a mGFP5-ER Variant in tobacco leaf extracts by UV excitation and a sGFP(S65T) variant in sugarcane leaf and callus extracts by blue light excitation using the BioRad VersaFluor(TM) Fluorometer System or the Labsystems Fluoroskan Ascent FL equipped with a narrow band emission filter (510 +/- 5 nm). The GFP concentration in transgenic plant extracts was determined from a GFP-standard series prepared in untransformed plant extract with concentrations ranging from 0.1 to 4 mu g/ml of purified rGFP. Levels of sgfp(S65T) expression, driven by the maize ubiquitin promoter, in sugarcane calli and leaves ranged up to 0.525 mu g and 2.11 mu g sGFP(S65T) per mg of extractable protein respectively. In tobacco leaves the expression of mgfPS-ER, driven by the cauliflower mosaic virus (CaMV) 35S promoter, ranged up to 7.05 mu g mGFP5-ER per mg extractable protein.

A simple and reliable method for the screening of transgenic tobacco plants

Even though much improvement has been made in plant transformation methods, the screening of transgenic plants is often a laborious work. Most approaches for detecting the transgene in transformed plants are still timeconsuming, and can be quite expensive. The objective of this study was to search for a simpler method to screen for transgenic plants. The infiltration of kanamycin (100 mg/mL) into tobacco leaves resulted in conspicuous chlorotic spots on the non-transgenic plant leaves, while no spots were seen on the leaves of transformed plants. This reaction occurred regardless of age of the tested plants, and the method has proven to be simple, fast, non-destructive, relatively cheap, and reliable. These results were comparable to those obtained by the polymerase chain reaction (PCR) amplification of the transgene using specific primers.

Transgenic passionfruit expressing RNA derived from Cowpea aphid-borne mosaic virus is resistant to passionfruit woodiness disease

Sixteen transgenic yellow passionfruit (Passiflora spp.) plants (R0) were obtained which express a non-translatable transgenic RNA corresponding to the 3' region of the NIb gene and the 5' region of the CP gene, derived from the genome of a Brazilian isolate of Cowpea aphid-borne mosaic virus (CABMV). The transgenic plants were propagated by stem cuttings and challenged by sap inoculation with isolates CABMV-MG1 and CABMV-PE1. One transgenic plant (TE5-10) was resistant to the isolate CABMV-MG1, but susceptible to CABMV-PE1. The remaining transgenic plants developed systemic symptoms, equal to non-transformed plants, when inoculated with either isolate. The absence of virus in TE5-10 plants was confirmed by indirect ELISA. Transcription analysis of the transgene demonstrated that the TE5-10 plant did not accumulate transgenic mRNA, even before inoculation. After inoculation, viral RNA was only detected in plants inoculated with CABMV-PE1. These results confirm that the transgenic plant TE5-10 is resistant to isolate CABMV-MG1, and suggest that the resistance mechanism is post-transcriptional gene silencing, which is already activated in the transgenic plants before virus inoculation.

Gene flow, risk assessment and the environmental release of transgenic plants

The release of genetically modified plants is governed by regulations that aim to provide an assessment of potential impact on the environment. One of the most important components of this risk assessment is an evaluation of the probability of gene flow. In this review, we provide an overview of the current literature on gene flow from transgenic plants, providing a framework of issues for those considering the release of a transgenic plant into the environment. For some plants gene flow from transgenic crops is well documented, and this information is discussed in detail in this review. Mechanisms of gene flow vary from plant species to plant species and range from the possibility of asexual propagation, short- or long-distance pollen dispersal mediated by insects or wind and seed dispersal. Volunteer populations of transgenic plants may occur where seed is inadvertently spread during harvest or commercial distribution. If there are wild populations related to the transgenic crop then hybridization and eventually introgression in the wild may occur, as it has for herbicide resistant transgenic oilseed rape (Brassica napus). Tools to measure the amount of gene flow, experimental data measuring the distance of pollen dispersal, and experiments measuring hybridization and seed survivability are discussed in this review. The various methods that have been proposed to prevent gene flow from genetically modified plants are also described. The current "transgenic traits'! in the major crops confer resistance to herbicides and certain insects. Such traits could confer a selective advantage (an increase in fitness) in wild plant populations in some circumstances, were gene flow to occur. However, there is ample evidence that gene flow from crops to related wild species occurred before the development of transgenic crops and this should be taken into account in the risk assessment process.

Ribozyme-mediated high resistance against potato spindle tuber viroid in transgenic potatoes

A hammerhead ribozyme [R(-)] targeting the minus strand RNA of potato spindle tuber viroid (PSTVd) and a mutated nonfunctional ribozyme [mR(-)] were designed, cloned, and transcribed. As predicted, both monomer and dimer transcripts of the active R(-) ribozyme gene could cleave the PSTVd minus strand dimer RNA into three fragments of 77, 338, and 359 bases in vitro at 25 and 50°C. The tandem dimer genes of R(-) and mR(-) were subcloned separately into the plant expression vector pROK2. Transgenic potato plants (cultivar Desirée) were generated by Agrobacterium tumefaciens-mediated transformation. Twenty-three of 34 independent transgenic plant lines expressing the active ribozyme R(-) resulted in having high levels of resistance to PSTVd, being free of PSTVd accumulation after challenge inoculation with PSTVd, but the remaining lines showed weaker levels of resistance to PSTVd with low levels of PSTVd accumulation. In contrast, 59 of 60 independent transgenic lines expressing the mutated ribozyme mR(-) were susceptible to PSTVd inoculation and had levels of PSTVd accumulation similar to that of the control plants transformed with the empty vector. The resistance against PSTVd replication was stably inherited to the vegetative progenies.

Pollen selection: a transgenic reconstruction approach.

A transgenic reconstruction experiment has been performed to determine the feasibility of male gametophytic selection to enhance transmission of genes to the next sporophytic generation. For tobacco pollen from a transgenic plant containing a single hygromycin-resistance (hygromycin phosphotransferase, hpt-) gene under control of the dc3 promoter, which is active in both sporophytic and gametophytic tissues, 3 days of in vitro maturation in hygromycin-containing medium was sufficient to result in a 50% reduction of germinating pollen, as expected for meiotic segregation of a single locus insert. Pollination of wild-type plants with the selected pollen yielded 100% transgenic offspring, as determined by the activity of the linked kanamycin-resistance gene--present within the same transferred T-DNA borders--under control of the nos promoter. This is direct proof that selection acting on male gametophytes can be a means to alter the frequency of genes in the progeny.

Plant-based immunocontraceptive control of wildlife - "potentials, limitations, and possums"

Possums (Trichosurus vulpecula), originally introduced from Australia, are spread over 90% of New Zealand and cause major economic and environmental damage. Immunocontraception has been suggested as a humane means to control them. Marsupial-specific reproductive antigens expressed at high levels in edible transgenic plant tissue might provide a safe, effective, and cheap oral delivery bait for immuno-contraceptive control. As proof of concept, female possums vaccinated with immunocontraceptive antigens showed reduced fertility, and possums fed with potato-expressed heat labile toxin-B (LT-B) had mucosal and systemic immune responses to the antigen. This demonstrated that immunocontraception was effective in possums and that oral delivery in edible plant material might be possible. Nuclear transformation with reporter genes showed that transgenic carrot roots accumulate high levels of foreign protein in edible tissues, indicating their potential as a delivery vector. However, prior to attempts at large scale production, more effective immunocontraceptive antigen-adjuvant formulations are probably required before plant-based immunocontraception can become a major tool for immunocontraceptive control of overabundant vertebrate pests. (c) 2004 Elsevier Ltd. All rights reserved.

The Rg1 allele as a valuable tool for genetic transformation of the tomato 'Micro-Tom' model system

Background: The cultivar Micro-Tom (MT) is regarded as a model system for tomato genetics due to its short life cycle and miniature size. However, efforts to improve tomato genetic transformation have led to protocols dependent on the costly hormone zeatin, combined with an excessive number of steps. Results: Here we report the development of a MT near-isogenic genotype harboring the allele Rg1 (MT-Rg1), which greatly improves tomato in vitro regeneration. Regeneration was further improved in MT by including a two-day incubation of cotyledonary explants onto medium containing 0.4 mu M 1-naphthaleneacetic acid (NAA) before cytokinin treatment. Both strategies allowed the use of 5 mu M 6-benzylaminopurine (BAP), a cytokinin 100 times less expensive than zeatin. The use of MT-Rg1 and NAA pre-incubation, followed by BAP regeneration, resulted in high transformation frequencies (near 40%), in a shorter protocol with fewer steps, spanning approximately 40 days from Agrobacterium infection to transgenic plant acclimatization. Conclusions: The genetic resource and the protocol presented here represent invaluable tools for routine gene expression manipulation and high throughput functional genomics by insertional mutagenesis in tomato.

Efficient transformation and regeneration of diverse cultivars of peanut (Arachis hypogaea L.) by particle bombardment into embryogenic callus produced from mature seeds

Peanut, one of the world's most important oilseed crops, has a narrow germplasm base and lacks sources of resistance to several major diseases. The species is considered recalcitrant to transformation, with few confirmed transgenic plants upon particle bombardment or Agrobacterium treatment. Reported transformation methods are limited by low efficiency, cultivar specificity, chimeric or infertile transformants, or availability of explants. Here we present a method to efficiently transform cultivars in both botanical types of peanut, by (1) particle bombardment into embryogenic callus derived from mature seeds, (2) escape-free (not stepwise) selection for hygromycin B resistance, (3) brief osmotic desiccation followed by sequential incubation on charcoal and cytokinin-containing media; resulting in efficient conversion of transformed somatic embryos into fertile, non-chimeric, transgenic plants. The method produces three to six independent transformants per bombardment of 10 cm(2) embryogenic callus. Potted, transgenic plant lines can be regenerated within 9 months of callus initiation, or 6 months after bombardment. Transgene copy number ranged from one to 20 with multiple integration sites. There was ca. 50% coexpression of hph and luc or uidA genes coprecipitated on separate plasmids. Reporter gene (luc) expression was confirmed in T-1 progeny from each of six tested independent transformants. Insufficient seeds were produced under containment conditions to determine segregation ratios. The practicality of the technique for efficient cotransformation with selected and unselected genes is demonstrated using major commercial peanut varieties in Australia (cv. NC-7, a virginia market type) and Indonesia (cv. Gajah, a spanish market type).

Detection of lateral gene transfer among microbial genomes

An increasingly comprehensive assessment is being developed of the extent and potential significance of lateral gene transfer among microbial genomes. Genomic sequences can be identified as being of putatively lateral origin by their unexpected phyletic distribution, atypical sequence composition, differential presence or absence in closely related genomes, or incongruent phylogenetic trees. These complementary approaches sometimes yield inconsistent results. Not only more data but also quantitative models and simulations are needed urgently.

L’éthique et l’acceptation sociale des innovations technologiques. Le cas des vaccins transgéniques

La technologie est une manière pour la science d’exprimer son côté pratique en plus d’être un moyen de traduire les connaissances lors de vraies applications scientifiques, mais ce processus peut engendrer une variété de défis moraux et éthiques. Le champ des biotechnologies laisse entrevoir de grandes réalisations pour les sociétés, y compris des traitements médicaux révolutionnaires et des aliments modifiés génétiquement, lesquels seraient sécuritaires, accessibles et largement disponibles. Mais peu de produits ont réussi le saut dans le panier du consommateur. Dans un des domaines d'application les plus prometteurs, tel celui des biotechnologies agricoles, certaines technologies n’ont pas encore entièrement émergé des laboratoires et ces produits, qui sont à l’heure actuelle sur le marché, ont été la source de polémiques significatives. L’étude présente se concentre sur le cas des vaccins faits à partir de plantes transgéniques qui, au cours des 15 dernières années, a peine à passer outre l’étape de la preuve de conception. Ces vaccins stagnent là où ils auraient dû accomplir la « promesse d'or » de fournir à bas coût une inoculation efficace pour les populations pauvres des pays en voie de développement. La question examinée dans cet essai est pourquoi, au-delà du processus de la découverte et de la conceptualisation, de telles technologies éprouvent des difficultés à atteindre leur maturité et ainsi retarde l’implantation dans les sociétés contemporaines ? Quels facteurs particuliers, sous l’angle de la bioéthique, auront besoin d’une reconsidération dans le cas échéant d’une mise en application de ces technologies pour être acceptées par les consommateurs, et avoir ainsi un impact positif sur la santé globale et l’accès équitable aux soins de santé ?