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"Plant Biotechnology"
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Corporate crops : biotechnology, agriculture, and the struggle for control
Frontmatter -- Contents -- List of Acronyms -- Introduction -- 1 Agricultural Biotechnologies on the Farm and around the World -- 2 The Coming of the Third Regime? Agricultural Biotechnology Regulation in Canada and the United States -- 3 Biotechnology on the Prairies: The Rise of Canola . . . -- 4 . . . And the Fall of Wheat -- 5 Legal Offense and Defense on the Canadian Prairies -- 6 From When Cotton Was King to King Monsanto -- 7 Starting a New Regime: Training the Locals -- 8 Conclusion -- Appendix: Log of Interviews -- Notes -- Bibliography -- Index
eBook
GMOs
An overview of genetic modification and how this biotechnology affects food, humans, animals, and medicine. Includes a discussion of the ethical concerns of genetic biotechnology.
Book
Validation of the ITS2 Region as a Novel DNA Barcode for Identifying Medicinal Plant Species
Background: The plant working group of the Consortium for the Barcode of Life recommended the two-locus combination of rbcL + matK as the plant barcode, yet the combination was shown to successfully discriminate among 907 samples from 550 species at the species level with a probability of 72%. The group admits that the two-locus barcode is far from perfect due to the low identification rate, and the search is not over. Methodology/Principal Findings: Here, we compared seven candidate DNA barcodes (psbA-trnH, matK, rbcL, rpoC1, ycf5, ITS2, and ITS) from medicinal plant species. Our ranking criteria included PCR amplification efficiency, differential intra- and inter-specific divergences, and the DNA barcoding gap. Our data suggest that the second internal transcribed spacer (ITS2) of nuclear ribosomal DNA represents the most suitable region for DNA barcoding applications. Furthermore, we tested the discrimination ability of ITS2 in more than 6600 plant samples belonging to 4800 species from 753 distinct genera and found that the rate of successful identification with the ITS2 was 92.7% at the species level. Conclusions: The ITS2 region can be potentially used as a standard DNA barcode to identify medicinal plants and their closely related species. We also propose that ITS2 can serve as a novel universal barcode for the identification of a broader range of plant taxa.
Journal Article
Fruit-Surface Flavonoid Accumulation in Tomato Is Controlled by a SlMYB12-Regulated Transcriptional Network
The cuticle covering plants' aerial surfaces is a unique structure that plays a key role in organ development and protection against diverse stress conditions. A detailed analysis of the tomato colorless-peel y mutant was carried out in the framework of studying the outer surface of reproductive organs. The y mutant peel lacks the yellow flavonoid pigment naringenin chalcone, which has been suggested to influence the characteristics and function of the cuticular layer. Large-scale metabolic and transcript profiling revealed broad effects on both primary and secondary metabolism, related mostly to the biosynthesis of phenylpropanoids, particularly flavonoids. These were not restricted to the fruit or to a specific stage of its development and indicated that the y mutant phenotype is due to a mutation in a regulatory gene. Indeed, expression analyses specified three R2R3-MYB-type transcription factors that were significantly down-regulated in the y mutant fruit peel. One of these, SlMYB12, was mapped to the genomic region on tomato chromosome 1 previously shown to harbor the y mutation. Identification of an additional mutant allele that co-segregates with the colorless-peel trait, specific down-regulation of SlMYB12 and rescue of the y phenotype by overexpression of SlMYB12 on the mutant background, confirmed that a lesion in this regulator underlies the y phenotype. Hence, this work provides novel insight to the study of fleshy fruit cuticular structure and paves the way for the elucidation of the regulatory network that controls flavonoid accumulation in tomato fruit cuticle.
Journal Article
A One Pot, One Step, Precision Cloning Method with High Throughput Capability
Current cloning technologies based on site-specific recombination are efficient, simple to use, and flexible, but have the drawback of leaving recombination site sequences in the final construct, adding an extra 8 to 13 amino acids to the expressed protein. We have devised a simple and rapid subcloning strategy to transfer any DNA fragment of interest from an entry clone into an expression vector, without this shortcoming. The strategy is based on the use of type IIs restriction enzymes, which cut outside of their recognition sequence. With proper design of the cleavage sites, two fragments cut by type IIs restriction enzymes can be ligated into a product lacking the original restriction site. Based on this property, a cloning strategy called 'Golden Gate' cloning was devised that allows to obtain in one tube and one step close to one hundred percent correct recombinant plasmids after just a 5 minute restriction-ligation. This method is therefore as efficient as currently used recombination-based cloning technologies but yields recombinant plasmids that do not contain unwanted sequences in the final construct, thus providing precision for this fundamental process of genetic manipulation.
Journal Article
Nicotiana benthamiana as a Production Platform for Artemisinin Precursors
Production of pharmaceuticals in plants provides an alternative for chemical synthesis, fermentation or natural sources. Nicotiana benthamiana is deployed at commercial scale for production of therapeutic proteins. Here the potential of this plant is explored for rapid production of precursors of artemisinin, a sesquiterpenoid compound that is used for malaria treatment.
Biosynthetic genes leading to artemisinic acid, a precursor of artemisinin, were combined and expressed in N. benthamiana by agro-infiltration. The first committed precursor of artemisinin, amorpha-4,11-diene, was produced upon infiltration of a construct containing amorpha-4,11-diene synthase, accompanied by 3-hydroxy-3-methylglutaryl-CoA reductase and farnesyl diphosphate synthase. Amorpha-4,11-diene was detected both in extracts and in the headspace of the N. benthamiana leaves. When the amorphadiene oxidase CYP71AV1 was co-infiltrated with the amorphadiene-synthesizing construct, the amorpha-4,11-diene levels strongly decreased, suggesting it was oxidized. Surprisingly, no anticipated oxidation products, such as artemisinic acid, were detected upon GC-MS analysis. However, analysis of leaf extracts with a non-targeted metabolomics approach, using LC-QTOF-MS, revealed the presence of another compound, which was identified as artemisinic acid-12-β-diglucoside. This compound accumulated to 39.5 mg x kg(-1) fwt. Apparently the product of the heterologous pathway that was introduced, artemisinic acid, is further metabolized efficiently by glycosyl transferases that are endogenous to N. benthamiana.
This work shows that agroinfiltration of N. bentamiana can be used as a model to study the production of sesquiterpenoid pharmaceutical compounds. The interaction between the ectopically introduced pathway and the endogenous metabolism of the plant is discussed.
Journal Article
Effector genomics accelerates discovery and functional profiling of potato disease resistance and Phytophthora infestans avirulence genes
Potato is the world's fourth largest food crop yet it continues to endure late blight, a devastating disease caused by the Irish famine pathogen Phytophthora infestans. Breeding broad-spectrum disease resistance (R) genes into potato (Solanum tuberosum) is the best strategy for genetically managing late blight but current approaches are slow and inefficient. We used a repertoire of effector genes predicted computationally from the P. infestans genome to accelerate the identification, functional characterization, and cloning of potentially broad-spectrum R genes. An initial set of 54 effectors containing a signal peptide and a RXLR motif was profiled for activation of innate immunity (avirulence or Avr activity) on wild Solanum species and tentative Avr candidates were identified. The RXLR effector family IpiO induced hypersensitive responses (HR) in S. stoloniferum, S. papita and the more distantly related S. bulbocastanum, the source of the R gene Rpi-blb1. Genetic studies with S. stoloniferum showed cosegregation of resistance to P. infestans and response to IpiO. Transient co-expression of IpiO with Rpi-blb1 in a heterologous Nicotiana benthamiana system identified IpiO as Avr-blb1. A candidate gene approach led to the rapid cloning of S. stoloniferum Rpi-sto1 and S. papita Rpi-pta1, which are functionally equivalent to Rpi-blb1. Our findings indicate that effector genomics enables discovery and functional profiling of late blight R genes and Avr genes at an unprecedented rate and promises to accelerate the engineering of late blight resistant potato varieties.
Journal Article
A Plant‐Based Platform for the Production of Bark Beetle Pheromones
Bark beetle species of the genera Ips and Dendroctonus represent a threat to forests in both North America and Europe. Under normal circumstances, these beetles recycle dying trees into nutrients, but under certain conditions, growing populations can overcome healthy tree defenses and cause severe economic loss in forestry. The most economically relevant bark beetle species communicate with aggregation pheromones such as ipsdienol, cis ‐verbenol and trans‐ verbenol. These pheromones are currently used in synthetic baits as part of control strategies for bark beetles, although their chemical synthesis makes them expensive to use. Here, we explore the possibility of producing bark beetle pheromones in plant factories, since these compounds can be derived from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) from the mevalonic acid (MVA) and methylerythritol phosphate (MEP) pathways in plants. By the combined expression of enzymes from plants and bark beetles, we show that Arabidopsis thaliana can produce the intermediates β‐myrcene (ipsdienol biosynthesis pathway) and α‐pinene (verbenol biosynthesis pathway). Furthermore, we were able to produce the final products cis‐ verbenol and trans‐ verbenol in stably transformed Arabidopsis, without the addition of external substrates. Finally, we achieved the production of verbenone, an anti‐aggregation pheromone derived from verbenol, which deters bark beetles from a host. These results are an important step towards using plants as biofactories for a cheaper and greener production of pheromones and repellent components for artificial baits.
Journal Article