Manipulating the pathway of ammonia assimilation through genetic engineering and breeding: consequences to plant physiology and plant development

In this article we discuss the ways in which our understanding of the nature of the molecular controls of nitrogen assimilation have been increased by the use of leguminous and non-leguminous plants with modified capacities for ammonium assimilation. These modifications have been achieved through genetic engineering and breeding. An improved understanding of nitrogen assimilation will be vital if improvements in crop nitrogen use efficiency are to be made to reduce the need for excessive input of fertilisers. In this review we present an overall view of past work and more recent studies on this topic. In our work, using tobacco and Lotus as model plants, glutamine synthetase and glutamate synthase activites have been altered by stimulating or inhibiting in an organ- or tissue-specific manner the expression of the corresponding genes. The physiological impact of these genetic manipulations has been studied on plants grown under different nitrogen regimes. This revised version was published online in June 2006 with corrections to the Cover Date.     

A method for accelerated trait conversion in plant breeding

Backcrossing is often used in cultivar development to transfer one or a few genes to desired genetic backgrounds. The duration necessary to complete such ‘trait conversions’ is largely dependent upon generation times. Constitutive overexpression of the Arabidopsis thaliana gene FT (FLOWERING LOCUS T) induces early-flowering in many plants. Here, we used tobacco (Nicotiana tabacum L.) as a model system to propose and examine aspects of a modified backcross procedure where transgenic FT overexpression is used to reduce generation time and accelerate gene transfer. In this method, the breeder would select for an FT transgene insertion and the trait(s) of interest at each backcross generation except the last. In the final generation, selection would be conducted for the trait(s) of interest, but against FT, to generate the backcross-derived trait conversion. We demonstrate here that constitutive FT overexpression functions to dramatically reduce days-to-flower similarly in diverse tobacco genetic backgrounds. FT-containing plants flowered in an average of 39 days, in comparison with 87–138 days for non-FT plants. Two FT transgene insertions were found to segregate independently of several disease resistance genes often the focus of backcrossing in tobacco. In addition, no undesirable epigenetic effects on flowering time were observed once FT was segregated away. The proposed system would reduce the time required to complete a trait conversion in tobacco by nearly one-half. These features suggest the possible value of this modified backcrossing system for tobacco or other crop species where long generation times or photoperiod sensitivity may impede timely trait conversion.     

The evolution of cultivated plant species: classical plant breeding versus genetic engineering

Agriculture is the most intensive form of environmental exploitation performed by mankind. It involves replacing the natural ecosystem with an artificial plant community comprising one or more crop species, and weeds can invade the cleared land. Initially, the adoption of agriculture did not necessarily imply an improvement in standard of living (there is, in fact, evidence to the contrary), but as agricultural efficiency improved, surpluses were generated on top of mere subsistence levels. It may take many years of labor in order to obtain a crop that has all of the desired traits. It is not possible to control which genes are transferred from the parents to the offspring, and the results are often uncertain. In comparison, the utilization of genetic engineering to improve crops can be a faster and more precise approach. Unlike traditional breeding, genetic engineering makes it possible to select the specific traits desired and insert the genes that code for them into the plant.     

Genome editing: intellectual property and product development in plant biotechnology

Genome editing is a revolutionary technology in molecular biology. While scientists are fascinated with the unlimited possibilities provided by directed and controlled changes in DNA in eukaryotes and have eagerly adopted such tools for their own experiments, an understanding of the intellectual property (IP) implications involved in bringing genome editing-derived products to market is often lacking. Due to the ingenuity of genome editing, the time between new product conception and its actual existence can be relatively short; therefore knowledge about IP of the various genome editing methods is relevant. This point must be regarded in a national framework as patents are instituted nationally. Therefore, when designing scientific work that could lead to a product, it is worthwhile to consider the different methods used for genome editing not only for their scientific merits but also for their compatibility with a speedy and reliable launch into the desired market.     

Assessing the impact of predictive biosimulation on drug discovery and development

Systems biology is creating a context for interpreting the vast amounts of genomic and proteomic data being produced by pharmaceutical companies in support of drug development. While major data collection efforts capitalize on technical advances in miniaturization and automation and represent an industrialization of existing laboratory research, the transition from mental models to predictive computer simulations is setting the pace for advances in this field. This article addresses current approaches to the mathematical modeling of biological systems and assesses the potential impact of predictive biosimulation on drug discovery and development.     

基因组解析与新药开发

由于ＤＮＡ微阵列技术 ,基因表达的解析已成为可能 ,个体基因差异也正在被发现 ,并产生了一个新的领域———药物基因组学 ,药物开发的模式发生了根本性的改变。基因组解析将为许多新药开发提供目标 ,新的药物筛选系统正在形成 ,基于新的作用功能的先导化合物正在被发现 ,利用ＤＮＡ微阵列技术而实施药理学与安全性评价 ,从基因序列开始对药物标靶的立体构造进行预测 ,从而选择最优秀的化合物。对于临床试验 ,诊断患者的基因多态性 ,筛选最合适的试验人群 ,提高新药的通过率 ,根据个体的基因差异使给药个体化 ,减少副作用 ,加速新药的开发。…     

The impact of systems biology and biosimulation on drug discovery and development

Drug discovery and development involves a series of difficult, systematic decision-making exercises, each of which is based on data acquired from bioassays and clinical trials. Since assays and trials are designed to elucidate the underlying pathophysiology of a disease, it is not sufficient to merely acquire data, but one must also interpret those findings in the context of the physiology they are meant to represent. Recently, these efforts have been enhanced by the use of biosimulation as a means of integrating and interpreting the vast new data sets generated by classically designed systems biology studies. Only when data describing gene expression, cell function, and whole-body physiology are interpreted in the context of integrated system function, will current error rates experienced during drug discovery and development be minimized.     

The impact of recombination on short-term selection gain in plant breeding experiments

Recombination is a requirement for response to selection, but researchers still debate whether increasing recombination beyond normal levels will result in significant gains in short-term selection. We tested this hypothesis, in the context of plant breeding, through a series of simulation experiments comparing short-term selection response (≤20 cycles) between populations with normal levels of recombination and similar populations with unconstrained recombination (i.e., free recombination). We considered additive and epistatic models and examined a wide range of values for key design variables: selection cycles, QTL number, heritability, linkage phase, selection intensity and population size. With few exceptions, going from normal to unconstrained levels of recombination produced only modest gains in response to selection (≈11 % on average). We then asked how breeders might capture some of this theoretical gain by increasing recombination through either (1) extra rounds of mating or (2) selection of highly recombinant individuals via use of molecular markers/maps. All methods tested captured less than half of the potential gain, but our analysis indicates that the most effective method is to select for increased recombination and the trait simultaneously. This recommendation is based on evidence of a favorable interaction between trait selection and the impact of recombination on selection gains. Finally, we examined the relative contributions of the two components of meiotic recombination, chromosome assortment and crossing over, to short-term selection gain. Depending primarily on the presence of trait selection pressure, chromosome assortment alone accounted for 40–75 % of gain in response to short-term selection.     

Genome structural variation discovery and genotyping

Comparisons of human genomes show that more base pairs are altered as a result of structural variation - including copy number variation - than as a result of point mutations. Here we review advances and challenges in the discovery and genotyping of structural variation. The recent application of massively parallel sequencing methods has complemented microarray-based methods and has led to an exponential increase in the discovery of smaller structural-variation events. Some global discovery biases remain, but the integration of experimental and computational approaches is proving fruitful for accurate characterization of the copy, content and structure of variable regions. We argue that the long-term goal should be routine, cost-effective and high quality de novo assembly of human genomes to comprehensively assess all classes of structural variation.     

Metabolic engineering of chloroplasts for artemisinic acid biosynthesis and impact on plant growth

Chloroplasts offer high-level transgene expression and transgene containment due to maternal inheritance, and are ideal hosts for biopharmaceutical biosynthesis via multigene engineering. To exploit these advantages, we have expressed 12 enzymes in chloroplasts for the biosynthesis of artemisinic acid (precursor of artemisinin, antimalarial drug) in an alternative plant system. Integration of transgenes into the tobacco chloroplast genome via homologous recombination was confirmed by molecular analysis, and biosynthesis of artemisinic acid in plant leaf tissues was detected with the help of ¹³C NMR and ESI-mass spectrometry. The excess metabolic flux of isopentenyl pyrophosphate generated by an engineered mevalonate pathway was diverted for the biosynthesis of artemisinic acid. However, expression of megatransgenes impacted the growth of the transplastomic plantlets. By combining two exogenous pathways, artemisinic acid was produced in transplastomic plants, which can be improved further using better metabolic engineering strategies for commercially viable yield of desirable isoprenoid products.     

Genome mining and prospects for antibiotic discovery

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Lignin: genetic engineering and impact on pulping

Lignin is a major component of wood, the most widely used raw material for the production of pulp and paper. Although the biochemistry and molecular biology underpinning lignin production are better understood than they are for the other wood components, recent work has prompted a number of re-evaluations of the lignin biosynthetic pathway. Some of the work on which these revisions have been based involved the investigation of transgenic plants with modified lignin biosynthesis. In addition to their value in elucidating the lignin biosynthetic pathway, such transgenic plants are also being produced with the aim of improving plant raw materials for pulp and paper production. This review describes how genetic engineering has yielded new insights into how the lignin biosynthetic pathway operates and demonstrates that lignin can be improved to facilitate pulping. The current technologies used to produce paper are presented in this review, followed by a discussion of the impact of lignin modification on pulp production. Fine-tuned modification of lignin content, composition, or both is now achievable and could have important economic and environmental benefits.     

Bradykinin antagonists: discovery and development

Practical bradykinin antagonists were discovered in 1984 by Vavrek and Stewart and reported in "Peptides." At that time there was already much evidence for involvement of bradykinin in inflammation and pain, so the specific, competitive antagonists were widely accepted and applied. The key to conversion of bradykinin into an antagonist was replacement of the proline residue at position 7 with a D-aromatic amino acid. Other modifications converted the initial weak antagonists into modern peptides which are totally resistant to all degrading enzymes, are orally available, and have been used in clinical trials. Non-peptide bradykinin antagonists have also been developed.     

Manipulation of the mouse genome: a multiple impact resource for drug discovery and development

Few would deny that the pharmaceutical industry's investment in genomics throughout the 1990s has yet to deliver in terms of drugs on the market. The reasons are complex and beyond the scope of this review. The unique ability to manipulate the mouse genome, however, has already had a positive impact on all stages of the drug discovery process and, increasingly, on the drug development process too. We give an overview of some recent applications of so-called 'transgenic' mouse technology in pharmaceutical research and development. We show how genetic manipulation in the mouse can be employed at multiple points in the drug discovery and development process, providing new solutions to old problems.     

Monoclonal antibodies: technologies for early discovery and engineering

Antibodies are essential in modern life sciences biotechnology. Their architecture and diversity allow for high specificity and affinity to a wide array of biochemicals. Combining monoclonal antibody (mAb) technology with recombinant DNA and protein expression links antibody genotype with phenotype. Yet, the ability to select and screen for high affinity binders from recombinantly-displayed, combinatorial libraries unleashes the true power of mAbs and a flood of clinical applications. The identification of novel antibodies can be accomplished by a myriad of in vitro display technologies from the proven (e.g. phage) to the emerging (e.g. mammalian cell and cell-free) based on affinity binding as well as function. Lead candidates can be further engineered for increased affinity and half-life, reduced immunogenicity and/or enhanced manufacturing, and storage capabilities. This review begins with antibody biology and how the structure and genetic machinery relate to function, diversity, and in vivo affinity maturation and follows with the general requirements of (therapeutic) antibody discovery and engineering with an emphasis on in vitro display technologies. Throughout, we highlight where antibody biology inspires technology development and where high-throughput, “big data” and in silico strategies are playing an increasing role. Antibodies dominate the growing class of targeted therapeutics, alone or as bioconjugates. However, their versatility extends to research, diagnostics, and beyond.     

A functional trait perspective on plant invasion

Background and Aims

Global environmental change will affect non-native plant invasions, with profound potential impacts on native plant populations, communities and ecosystems. In this context, we review plant functional traits, particularly those that drive invader abundance (invasiveness) and impacts, as well as the integration of these traits across multiple ecological scales, and as a basis for restoration and management.

Scope

We review the concepts and terminology surrounding functional traits and how functional traits influence processes at the individual level. We explore how phenotypic plasticity may lead to rapid evolution of novel traits facilitating invasiveness in changing environments and then ‘scale up’ to evaluate the relative importance of demographic traits and their links to invasion rates. We then suggest a functional trait framework for assessing per capita effects and, ultimately, impacts of invasive plants on plant communities and ecosystems. Lastly, we focus on the role of functional trait-based approaches in invasive species management and restoration in the context of rapid, global environmental change.

Conclusions

To understand how the abundance and impacts of invasive plants will respond to rapid environmental changes it is essential to link trait-based responses of invaders to changes in community and ecosystem properties. To do so requires a comprehensive effort that considers dynamic environmental controls and a targeted approach to understand key functional traits driving both invader abundance and impacts. If we are to predict future invasions, manage those at hand and use restoration technology to mitigate invasive species impacts, future research must focus on functional traits that promote invasiveness and invader impacts under changing conditions, and integrate major factors driving invasions from individual to ecosystem levels.     

Molecular markers and plant breeding

The achievements of modern biotechnology allow to modernize significantly the traditional plant breeding. The use of molecular codominant markers reduces considerably the quantity of breeding material and promotes the selection of genotypes, which posses desirable genes in the homozygous state. Molecular marking systems of agronomically important simple and quantitative traits have been developed using mono- and multiloci systems. Markers of the plant type and development rate, alleles of the storage protein genes, Wx-genes, short-stem genes, etc., have been created and tested at the South Biotecnology Center, National Academy of Agricultural Sciences. The technology of the application of DNA-typing for the identification and registration of varieties that has been developed in the South Biotecnology Center is of great importance for the systematization of germplasm sources and the protection of the rights of breeders.     

Optimized breeding strategies for multiple trait integration: II. Process efficiency in event pyramiding and trait fixation

Multiple trait integration (MTI) is a multi-step process of converting an elite variety/hybrid for value-added traits (e.g. transgenic events) through backcross breeding. From a breeding standpoint, MTI involves four steps: single event introgression, event pyramiding, trait fixation, and version testing. This study explores the feasibility of marker-aided backcross conversion of a target maize hybrid for 15 transgenic events in the light of the overall goal of MTI of recovering equivalent performance in the finished hybrid conversion along with reliable expression of the value-added traits. Using the results to optimize single event introgression (Peng et al. Optimized breeding strategies for multiple trait integration: I. Minimizing linkage drag in single event introgression. Mol Breed, 2013) which produced single event conversions of recurrent parents (RPs) with ≤8 cM of residual non-recurrent parent (NRP) germplasm with ~1 cM of NRP germplasm in the 20 cM regions flanking the event, this study focused on optimizing process efficiency in the second and third steps in MTI: event pyramiding and trait fixation. Using computer simulation and probability theory, we aimed to (1) fit an optimal breeding strategy for pyramiding of eight events into the female RP and seven in the male RP, and (2) identify optimal breeding strategies for trait fixation to create a ‘finished’ conversion of each RP homozygous for all events. In addition, next-generation seed needs were taken into account for a practical approach to process efficiency. Building on work by Ishii and Yonezawa (Optimization of the marker-based procedures for pyramiding genes from multiple donor lines: I. Schedule of crossing between the donor lines. Crop Sci 47:537–546, 2007a), a symmetric crossing schedule for event pyramiding was devised for stacking eight (seven) events in a given RP. Options for trait fixation breeding strategies considered selfing and doubled haploid approaches to achieve homozygosity as well as seed chipping and tissue sampling approaches to facilitate genotyping. With selfing approaches, two generations of selfing rather than one for trait fixation (i.e. ‘F2 enrichment’ as per Bonnett et al. in Strategies for efficient implementation of molecular markers in wheat breeding. Mol Breed 15:75–85, 2005) were utilized to eliminate bottlenecking due to extremely low frequencies of desired genotypes in the population. The efficiency indicators such as total number of plants grown across generations, total number of marker data points, total number of generations, number of seeds sampled by seed chipping, number of plants requiring tissue sampling, and number of pollinations (i.e. selfing and crossing) were considered in comparisons of breeding strategies. A breeding strategy involving seed chipping and a two-generation selfing approach (SC + SELF) was determined to be the most efficient breeding strategy in terms of time to market and resource requirements. Doubled haploidy may have limited utility in trait fixation for MTI under the defined breeding scenario. This outcome paves the way for optimizing the last step in the MTI process, version testing, which involves hybridization of female and male RP conversions to create versions of the converted hybrid for performance evaluation and possible commercial release.     

Patenting of plant varieties and plant breeding methods

This article considers the relationship between patenting and plant variety rights protection, through a detailed analysis of the recent determination by the Extended Board of Appeal of the European Patent Office that methods for breeding broccoli and tomatoes were not patentable. It concludes that the right to patent agricultural innovations is increasingly located within a political context.