Protoplasts as tools for plant genome modifications

We present here the application of protoplast technology in the selection and recovery of rare, spontaneous plant genome alterations. Using protoplasts as a cell cloning system allowed the detection and molecular characterization of intrachromosomal recombination events between genomic repeats. The mechanism, frequencies and the induction of intrachromosomal recombination are discussed as well as its application for genome mutagenesis.     

Restriction fragment polymorphisms as probes for plant diversity and their development as tools for applied plant breeding

Summary Maize and tomato cDNA clones have been hybridized in Southern blotting experiments to plant genomic DNA prepared from different lines to detect restriction fragment polymorphisms (RFPs). In maize we have found that a high degree of genetic variability is present, even among domestic inbred lines. Most randomly chosen maize cDNA clones can be used to detect elements of this variability. Similar levels of polymorphism are observed when genomic DNA is digested with any of a number of different restriction enzymes and probed with individual clones. When a clone is hybridized to genomic DNAs prepared from several different maize lines, a number of different alleles are often detected at a single locus. At the same time one clone can often detect more than one independently segregating locus by cross hybridization to related sequences at other loci. As expected these markers are inherited as simple codominant Mendelian alleles from one generation to the next and colinkage of these markers can be demonstrated in the progeny from a heterozygous parent. In similar studies with tomato, remarkably different results were found. Few RFPs were demonstrable among domestic Lycopersicon esculentum lines although a higher level of variability could be detected when comparing esculentum with its wild Lycopersicon relatives. These results are discussed in relation to the applied uses of RFPs in plant breeding as well as the inherent variability of different plant genomes.This work was supported in part by funds from Sandoz Ltd. (Basel, Switzerland) and its subsidiary company, Northrup King Co. (Minneapolis, Minn., U.S.A.) as well as by NSF SBIR grant #BSR-8360870.     

Hyaluronan-chondroitin hybrid oligosaccharides as new life science research tools

Mitochondrial trifunctional protein (MTP), which consists of the MTPα and MTPβ subunits, catalyzes long-chain fatty acid β-oxidation. MTP deficiency in humans results in Reye-like syndrome. Here, we generated Drosophila models of MTP deficiency by targeting two genes encoding Drosophila homologs of human MTPα and MTPβ, respectively. Both Mtpα(KO) and Mtpβ(KO) flies were viable, but demonstrated reduced lifespan, defective locomotor activity, and reduced fecundity represented by the number of eggs laid by the females. The phenotypes of Mtpα(KO) flies were generally more striking than those of Mtpβ(KO) flies. Mtpα(KO) flies were hypersensitive to fasting, and retained lipid droplets in their fat body cells as in non-fasting conditions. The amount of triglyceride was also unchanged upon fasting in Mtpα(KO) flies, suggesting that lipid mobilization was disrupted. Finally, we showed that both Mtpα(KO) and Mtpβ(KO) flies accumulated acylcarnitine and hydroxyacylcarnitine, diagnostic markers of MTP deficiencies in humans. Our results indicated that both Mtpα(KO) and Mtpβ(KO) flies were impaired in long-chain fatty acid β-oxidation. These flies should be useful as a model system to investigate the molecular pathogenesis of MTP deficiency.     

Genetic elements of plant viruses as tools for genetic engineering.

Viruses have developed successful strategies for propagation at the expense of their host cells. Efficient gene expression, genome multiplication, and invasion of the host are enabled by virus-encoded genetic elements, many of which are well characterized. Sequences derived from plant DNA and RNA viruses can be used to control expression of other genes in vivo. The main groups of plant virus genetic elements useful in genetic engineering are reviewed, including the signals for DNA-dependent and RNA-dependent RNA synthesis, sequences on the virus mRNAs that enable translational control, and sequences that control processing and intracellular sorting of virus proteins. Use of plant viruses as extrachromosomal expression vectors is also discussed, along with the issue of their stability.     

Ontologies for behavior

SUMMARY: Although dozens of biological ontologies have been created and deployed, relatively little attention has been given to using ontologies to represent behavior. Ontologies for two different behavior systems are described here. One ontology was a translation of a published ethogram, and the second was coded from video clips in a comparative study of jumping spider courtship. AVAILABILITY: http://mesquiteproject.org/ontology/.     

The utility of transposable elements as tools for the isolation of plant genes

Transposable elements are segments of DNA which have the unique capability of being able to excise from one site in the genome and reintegrate into new, different sites elsewhere in the genome. When transposition takes place and integration occurs within a gene locus, mutations are frequently generated producing variegated or recessive phenotypes. This ability of transposable elements to act as mutagenic agents through their association with particular gene sequences has lead to the development of the procedure of transposon tagging or gene tagging in higher plants. Through this technique, transposable elements can be used to clone and isolate genes of interest for which little or nothing is known about the final product (i.e., polypeptide). This offers tremendous potential for the isolation of a variety of agronomically important genes, which are virtually impossible to recover by other currently available gene cloning methodologies. To date, the technique has been used successfully to isolate genes from corn and snapdragon. Using gene transfer technologies, the potential now exists to extend this approach to clone genes from other plant species. Advantages and limitations of transposon tagging for isolating plant genes will be discussed.     

Astrobiology,the transcendent science: the promise of astrobiology as an integrative approach for science and engineering education and research

Astrobiology is rapidly gaining the worldwide attention of scientists, engineers and the public. Astrobiology's captivation is due to its inherently interesting focus on life, its origins and distribution in the Universe. Because of its remarkable breadth as a scientific field, astrobiology touches on virtually all disciplines in the physical, biological and social sciences as well as engineering. The multidisciplinary nature and the appeal of its subject matter make astrobiology ideal for integrating the teaching of science at all levels in educational curricula. The rationale for implementing novel educational programs in astrobiology is presented along with specific research and educational policy recommendations.     

Bioreactors for plant cells: hardware configuration and internal environment optimization as tools for wider commercialization

Mass production of value-added molecules (including native and heterologous therapeutic proteins and enzymes) by plant cell culture has been demonstrated as an efficient alternative to classical technologies [i.e. natural harvest and chemical (semi)synthesis]. Numerous proof-of-concept studies have demonstrated the feasibility of scaling up plant cell culture-based processes (most notably to produce paclitaxel) and several commercial processes have been established so far. The choice of a suitable bioreactor design (or modification of an existing commercially available reactor) and the optimization of its internal environment have been proven as powerful tools toward successful mass production of desired molecules. This review highlights recent progress (mostly in the last 5 years) in hardware configuration and optimization of bioreactor culture conditions for suspended plant cells.     

Cre-lox recombination: Cre-ative tools for plant biotechnology

Targeted insertion and the precise deletion of DNA from transgenic plant chromosomes increase the potential of plant biotechnology for commercial applications and basic research. The Cre–lox recombination system is one of the best characterized and most widely used systems for these purposes. Cre–lox has many applications, but it is primarily used for the controlled excision of DNA fragments, in particular selectable marker genes, from the nuclear and chloroplast genomes, and for the targeted insertion of DNA into specific sites in the nuclear genome. Recent developments, including regulated expression of cre and the creative use of wild-type and modified lox sites, have improved the potential of these applications. After almost 15 years of research and development in plants, the Cre–lox system continues to provide an efficient and precise tool for plant biotechnologists.     

Chromosome engineering: power tools for plant genetics

The term "chromosome engineering" describes technologies in which chromosomes are manipulated to change their mode of genetic inheritance. This review examines recent innovations in chromosome engineering that promise to greatly increase the efficiency of plant breeding. Haploid Arabidopsis thaliana have been produced by altering the kinetochore protein CENH3, yielding instant homozygous lines. Haploid production will facilitate reverse breeding, a method that downregulates recombination to ensure progeny contain intact parental chromosomes. Another chromosome engineering success is the conversion of meiosis into mitosis, which produces diploid gametes that are clones of the parent plant. This is a key step in apomixis (asexual reproduction through seeds) and could help to preserve hybrid vigor in the future. New homologous recombination methods in plants will potentiate many chromosome engineering applications.     

Toward supportive data collection tools for plant metabolomics

Over recent years, a number of initiatives have proposed standard reporting guidelines for functional genomics experiments. Associated with these are data models that may be used as the basis of the design of software tools that store and transmit experiment data in standard formats. Central to the success of such data handling tools is their usability. Successful data handling tools are expected to yield benefits in time saving and in quality assurance. Here, we describe the collection of datasets that conform to the recently proposed data model for plant metabolomics known as ArMet (architecture for metabolomics) and illustrate a number of approaches to robust data collection that have been developed in collaboration between software engineers and biologists. These examples also serve to validate ArMet from the data collection perspective by demonstrating that a range of software tools, supporting data recording and data upload to central databases, can be built using the data model as the basis of their design.     

Statistical tests as inappropriate tools for data analysis performed on non-random samples of plant communities

Many authors apply statistical tests to sets of relevés obtained using non-random methods to investigate phytosociological and ecological relationships. Frequently applied tests include thet-test, ANOVA, Mann-Whitney test, Kruskal-Wallis test, chi-square test (of independence, goodness-of-fit, and homogeneity), Kolmogorov-Smirnov test, concentration analysis, tests of linear correlation and Spearman rank correlation coefficient, computer intensive methods (such as randomization and re-sampling) and others. I examined the extent of reliability of the results of such tests applied to non-random data by examining the tests requirements according to statistical theory. I conclude that when used for such data, the statistical tests do not provide reliable support for the inferences made because non-randomness of samples violated the demand for observations to be independent, and different parts of the investigated communities did not have equal chance to be represented in the sample. Additional requirements, e.g. of normality and homoscedasticity, were also neglected in several cases. The importance of data satisfying the basic requirements set by statistical tests is stressed.     

Tobraviruses—plant pathogens and tools for biotechnology

The tobraviruses, Tobacco rattle virus (TRV), Pea early‐browning virus (PEBV) and Pepper ringspot virus (PepRSV), are positive‐strand RNA viruses with rod‐shaped virus particles that are transmitted between plants by trichodorid nematodes. As a group, these viruses infect many plant species, with TRV having the widest host range. Recent studies have begun to dissect the interaction of TRV with potato, currently the most commercially important crop disease caused by any of the tobraviruses. As well as being successful plant pathogens, these viruses have become widely used as vectors for expression in plants of nonviral proteins or, more frequently, as initiators of virus‐induced gene silencing (VIGS). Precisely why tobraviruses should be so effective as VIGS vectors is not known; however, molecular studies of the mode of action of the tobravirus silencing suppressor protein are shedding some light on this process.     

Prescribed fire and cutting as tools for reducing woody plant succession in a created salt marsh

This paper reports on efforts to reduce woody successional growth by the native shrub Iva frutescens L. in a created salt marsh by using prescribed fire and cutting. Experimental treatments included a winter burn, cutting plants at ground level, and a combination burn-and-cut treatment, with replicate plots of each. Iva frutescens proved to be extremely hardy, with zero mortality following the cutting, burning, or combination treatment; similar levels of regrowth were observed for all treatments. Individual shrub response, however, was found to be related to initial plant size, ground water level and salinity, and two fire characteristics (total heating >60°C and total heat index >60°C). Fire severity, sediment nutrient concentrations, and other abiotic factors had no observable effects.     

Glycopeptides as versatile tools for glycobiology

This review describes the recent advances in the field of glycopeptide and small glycoprotein synthesis. The strategies covered include chemical and chemoenzymatic synthesis, native chemical ligation (NCL), and expressed chemical ligation. The importance of glycopeptide synthesis is exemplified by giving the reader an overview of how versatile and important these well-defined glycopeptides are as tools in glycobiology.