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PubMed1997

Burial and seed survival in Brassica napus subsp. oleifera and Sinapis arvensis including a comparison of transgenic and non-transgenic lines of the crop.

The creation of transgenic plants through genetic engineering has focused interest on how the fitness of a plant species may be altered by small changes in its genome. This study concentrates on a key component of fitness: persistence of seeds overwinter. Seeds of three lines of oilseed rape (Brassica napus subsp. oleifera DC Metzger) and of charlock (Sinapis arvensis L.) were buried in nylon mesh bags at two depths in four habitats in each of three geographically separated sites: Cornwall, Berkshire and Sutherland. Seeds were recovered after 12 and 24 months. Charlock exhibited much greater seed survival (average 60% surviving the first year and 32.5% surviving the second year) than oilseed rape (1.5% surviving the first year and 0.2% surviving the second) at all sites. Charlock showed higher survival at 15 cm burial than 2 cm burial at certain sites, but oilseed rape showed no depth effect. Different genetic lines of oilseed rape displayed different rates of seed survival; non-transgenic rape showed greater survival (2%) than the two transgenic lines, one developed for tolerance to the antibiotic kanamycin (0.3%) and one for tolerance to both kanamycin and the herbicide glufosinate (0.25%). The absolute and relative performances of the different genetic lines of oilseed rape were context specific, illustrating the need to test hypotheses in a wide range of ecological settings.

Brassica↗
PubMed1997

Non-cultivable phytopathogenic mycoplasmas: characterization, detection and perspectives for control.

Phytoplasmas (ex MLOs) and spiroplasmas are important groups of plant pathogenic mollicutes, discovered in 1967 and 1970 respectively. Spiroplasmas, like other mollicutes, can be cultured in artificial media and are thus well characterized. On the contrary, phytoplasmas have resisted in vitro cultivation and their study was difficult until the recent development of molecular techniques. From the sequence of their 16S rDNA, phytoplasmas have been shown to be true mollicutes. Fourteen phytoplasma subclasses have been defined, but only two phytoplasmas have so far been named at the genus and species level. Monoclonal antibodies, DNA probes and PCR primers for the specific detection of various phytoplasmas have been obtained. These showed that a given phytoplasma can infect a broad range of plants, while others are restricted to a single plant species. Specific reagents are also used for identification of insect vectors and reservoir plants of the various phytoplasmas. Plant pathogenic mollicutes cannot be controlled chemically today, since the use of antibiotic treatment is forbidden in agriculture. However, the growth and metabolism of mollicutes are known to be inhibited by antibodies and this provides a hopeful approach for future control of these agents in plants. Indeed, it has been shown recently that plants can be engineered to express and assemble functional immunoglobulin chains. Transgenic tobacco plants expressing an antibody against the stolbur phytoplasmas have been developed. They have now to be challenged with the phytoplasma to determine if they have acquired resistance to this mollicute.

Mycoplasma↗
PubMed1999

Potential side effects of insect-resistant transgenic plants on arthropod natural enemies.

Engineering genes encoding insecticidal proteins into crop plants offers numerous benefits to agriculture. However, like many conventional insecticides, this new technology has the potential to disrupt natural biological control through both direct and indirect side effects of the plants on the fitness or behaviour of arthropod predators and parasitoids. Interactions between transgenic plants and these beneficial insects are being assessed to avoid incompatibility.

Animals↗
PubMed2001

ALKALOID BIOSYNTHESIS IN PLANTS: Biochemistry, Cell Biology, Molecular Regulation, and Metabolic Engineering Applications.

Recent advances in the cell, developmental, and molecular biology of alkaloid biosynthesis have heightened our appreciation for the complexity and importance of plant secondary pathways. Several biosynthetic genes involved in the formation of tropane, benzylisoquinoline, and terpenoid indole alkaloids have now been isolated. The early events of signal perception, the pathways of signal transduction, and the function of gene promoters have been studied in relation to the regulation of alkaloid metabolism. Enzymes involved in alkaloid biosynthesis are associated with diverse subcellular compartments including the cytosol, vacuole, tonoplast membrane, endoplasmic reticulum, chloroplast stroma, thylakoid membranes, and perhaps unique "biosynthetic" or transport vesicles. Localization studies have shown that sequential alkaloid biosynthetic enzymes can also occur in distinct cell types, suggesting the intercellular transport of pathway intermediates. Isolated genes have also been used to genetically alter the accumulation of specific alkaloids and other plant secondary metabolites. Metabolic modifications include increased indole alkaloid levels, altered tropane alkaloid accumulation, elevated serotonin synthesis, reduced indole glucosinolate production, redirected shikimate metabolism, and increased cell wall-bound tyramine formation. This review discusses the biochemistry, cell biology, molecular regulation, and metabolic engineering of alkaloid biosynthesis in plants.

Journal Article↗
PubMed2005

Engineering the genome of Grapevine virus A into a vector for expression of proteins in herbaceous plants.

Grapevine virus A (GVA), a species of the genus Vitivirus, consists of a approximately 7.4 kb single-stranded RNA genome of positive polarity, organized into five open reading frames (ORFs). In addition to grape varieties, GVA infects Nicotiana benthamiana plants and protoplasts. We engineered the genome of GVA as a vector that includes duplication of homologous sequences that contain the promoter of the movement protein (MP) sgRNA, supplemented by enzymatic restriction sites to be used as a convenient tool for transient expression of foreign genes from an individual sgRNA. The resulting vector was able to infect and to move in N. benthamiana plants in a manner similar to the wild-type GVA, but it was not stable and the inserted sequence was lost from the genome. Replacing the duplicated promoter with a GVA-MP promoter derived from a distantly related isolate of GVA improved the stability of the inserted sequence. The resulting vector was successfully used to express the reporter gene beta-glucuronidase (GUS) and the coat protein gene of Citrus tristeza virus in inoculated N. benthamiana plants. Development of a useful GVA vector is expected to find a use as a biotechnological tool for improvement of grapevines and it may enable vine breeders to bypass obstacles involved in genetic manipulation of perennial and fruiting plants.

Binding Sites↗
PubMed2002

Biosynthesis and metabolic engineering of glucosinolates.

Glucosinolates are amino acid-derived natural plant products found throughout the Capparales order. Glucosinolates and their degradation products have a wide range of biological activities, e.g. in plant defense as deterrents against insect and fungi. The conversion of amino acids to aldoximes is a key step in glucosinolate biosynthesis. This step is catalyzed by cytochromes P450 from the CYP79 family. The post-aldoxime enzymes in the glucosinolate pathway have high substrate-specificity for the functional group and low substrate-specificity for the side chain. Therefore, we have been able to metabolically engineer new glucosinolate profiles into Arabidopsis by altering the levels of endogenous CYP79s and by introducing exogenous CYP79s. The approach has great potential for design of metabolically engineered plants with improved pest resistance and increased nutritional value.

Arabidopsis↗
PubMed2006

Recent developments and future prospects of Vitreoscilla hemoglobin application in metabolic engineering.

In hypoxic conditions, bacteria express a kind of hemoglobin, which is proposed to enhance respiration and energy metabolism by promoting oxygen delivery. Bacteria hemoglobin from Vitreoscilla stercoraria - Vitreoscilla hemoglobin (VHb), when expressed in various hosts in oxygen-limited conditions, has been shown to improve growth, protein secretion, metabolite productivity and stress resistance of hosts, thus rendering the protein promising in metabolic engineering, especially in plant metabolism optimization. In this review, many well-studies areas are presented to illustrate the potential of VHb application in biotechnology industry, to discuss the cellular mechanisms of VHb function and to show the wide variety of approaches taken within the field.

Bacteria↗
PubMed2004

Expression of a feedback-resistant anthranilate synthase in Catharanthus roseus hairy roots provides evidence for tight regulation of terpenoid indole alkaloid levels.

Different plant species produce a variety of terpenoid indole alkaloids, which are of interest as plant defensive secondary metabolites and as valuable pharmaceuticals. Although significant progress has been made, the mechanisms regulating the levels of this important class of compounds require continued elucidation. Previous precursor feeding studies have indicated that alkaloid accumulation can be improved during the exponential growth phase of hairy root cultures through enhanced tryptophan availability. To test this relationship, transgenic hairy root cultures of Catharanthus roseus were established with a glucocorticoid-inducible promoter controlling the expression of an Arabidopsis feedback-resistant anthranilate synthase alpha subunit. Enzyme assays demonstrated that the Arabidopsis alpha subunit is compatible with the native beta subunit and that anthranilate synthase activity is more resistant to tryptophan inhibition in induced than in uninduced extracts. The metabolic effects of expressing the feedback-resistant anthranilate synthase alpha subunit were also dramatic. Over a 6-day induction period during the late exponential growth phase, tryptophan and tryptamine specific yields increased from almost undetectable levels to 2.5 mg/g dry weight and from 25 microg/g to 267 microg/g dry weight, respectively. The greater than 300-fold increase in tryptophan levels observed in these studies under certain induction conditions compares favorably with the fold increases obtained in previous constitutive expression studies. Despite the large increases in tryptophan and tryptamine, the levels of most terpenoid indole alkaloids were not significantly altered, with the exception of lochnericine, which increased 81% after a 3-day induction period. These results suggest that terpenoid indole alkaloid levels are tightly controlled.

Anthranilate Synthase↗
PubMed2004

Use of the tobacco feedback-insensitive anthranilate synthase gene (ASA2) as a selectable marker for legume hairy root transformation.

The feedback-insensitive anthranilate synthase ( ASA2) cDNA--isolated from a 5-methyltryptophan (5MT)-resistant tobacco cell line--driven by the CaMV 35S promoter or 606 bp of the native ASA2 promoter, was introduced into the forage legume plant Astragalus sinicus or soybean ( Glycine max), using Agrobacterium rhizogenes strains DC-AR2 or K599, respectively. Hairy roots of A. sinicus transformed with 35S-ASA2 but not 606- ASA2 could be directly selected using 20-75 micro M 5MT. ASA2 mRNA was expressed in all A. sinicus lines selected with 5MT, but nptII mRNA was expressed only in some lines even though the gene was present. Free tryptophan was increased 8- to 26-fold in A. sinicus and 3- to 6-fold in soybean (selected with kanamycin). An HPLC method was used to measure anthranilate synthase (AS) activity since there was a fluorescent compound or compounds present in the soybean hairy root extracts. The transformed soybean hairy roots contained more feedback-resistant AS activity, showing that there is interaction of the tobacco ASA2 alpha-subunit with the soybean beta-subunit to form an active enzyme. Soybean hairy roots that express ASA2 also exhibit 5MT resistance. These results demonstrate that the tobacco feedback-insensitive ASA2 gene can be used as a selectable marker for transformation of the legume A. sinicus.

Anthranilate Synthase↗
PubMed2005

Agrobacterium-mediated transformation of Solanum phureja.

A population of transgenic plants was produced by the transformation of internodal explants of Solanum phureja, DB337/37 (the cultivar Mayan Gold) using an Agrobacterium tumefaciens LBA4404-based vector containing a phytoene synthase gene (crtB). The regeneration strategy utilised a two-step protocol, with a 12-day callus induction stage mediated by 1.07 microM alpha-napthaleneacetic acid (NAA), 7.10 microM zeatin riboside and 0.06 microM gibberellic acid (GA3), followed by a prolonged (up to 90 day) shoot induction stage on medium containing 0.11 microM NAA, 7.10 microM zeatin riboside and 0.06 microM GA3 supplemented with kanamycin at 50 mg l(-1) as the selection agent. Southern analysis of the transgenic population revealed that the transgene copy number varied between one and five in the lines tested. Northern blot analysis showed significant expression of the introduced crtB gene in some lines during tuber development. Cytological analysis of the material showed a high incidence of chromosome doubling in the transgenic population with over 80% of all lines tested having doubled their chromosome complement during the transformation process.

Agrobacterium tumefaciens↗
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↗
PubMed1999

Tandem constructs: preventing the rise of superweeds.

Transgenic crops may interbreed with nearby weeds, increasing their competitiveness, and may themselves become a 'volunteer' weed in the following crop. The desired transgene can be coupled in tandem with genes that would render hybrid offspring or volunteer weeds less able to compete with crops, weeds and wild species. Genes that prevent seed shatter or secondary dormancy, or that dwarf the recipient could all be useful for mitigation and may have value to the crop. Many such genes have been isolated in the past few years.

Crops, Agricultural↗
PubMed1999

Application of remedy studies to the development of a soil washing pilot plant that uses mineral processing technology: a practical experience.

Soil washing employing mineral processing technology to treat radionuclide-contaminated soils has been examined as a remedy alternative to the exclusive excavation, transportation, and disposal of the soil. Successful application depends on a thorough remedy study, employing a systematic tiered approach that is efficient, self-limiting, and cost effective. The study includes: (1) site and soil characterization to determine the basic mineral and physical properties of both the soil and contaminants and to identify their relative associations; (2) treatment studies to evaluate the performance of process units for contaminant separation; (3) conceptual process design to develop a treatment pilot plant; and (4) engineering design to construct, test, and optimize the actual full-scale plant. A pilot plant using soil washing technology for the treatment of radium-contaminated soil was developed, tested, and demonstrated. The plant used particle-size separation to produced a remediated product that represented approximately 50% of the contaminated soil. Subsequently, it was modified for more effective performance and application to soil with alternate characteristics; it awaits further testing. The economic analysis of soil washing using the pilot plant as a model indicates that a remedy plan based on mineral processing technology is very competitive with the traditional alternative employing excavation, transportation, and disposal exclusively, even when disposal costs are modest or when recovery of remediated soil during treatment is low. This paper reviews the tiered approach as it applies to mineral processing technology to treat radionuclide-contaminated soils and a pilot plant developed to test the soil washing process.

Cost-Benefit Analysis↗
PubMed2000

Genomic approaches to plant disease resistance.

Genomic approaches are beginning to revolutionize our understanding of plant disease resistance. Large-scale sequencing will reveal the detailed organization of resistance-gene clusters and the genetic mechanisms involved in generating new resistance specificities. Global functional analyses will elucidate the complex regulatory networks and the diversity of proteins involved in resistance and susceptibility.

Genes, Plant↗
PubMed1997

A dominant mutant receptor from Arabidopsis confers ethylene insensitivity in heterologous plants.

Ethylene (C2H4) is a gaseous hormone that affects many aspects of plant growth and development. Ethylene perception requires specific receptors and a signal transduction pathway to coordinate downstream responses. The etr1-1 gene of Arabidopsis encodes a mutated receptor that confers dominant ethylene insensitivity. Evidence is presented here that etr1-1 also causes significant delays in fruit ripening, flower sensecence; and flower abscission when expressed in tomato and petunia plants. The ability of etr1-1 to function in heterologous plants suggests that this pathway of hormone recognition and response is highly conserved and can be manipulated.

Amino Acid Sequence↗