A macronutrient optimization platform for micropropagation and acclimatization: using turmeric (<Emphasis Type=Italic>Curcuma longa</Emphasis> L.) as a model plant

Tissue culture medium is often overlooked as a factor in plant biotechnology. Most work uses Murashige and Skoog (MS; Physiol Plant in 15:473–497, 1962) inorganic medium formulation, which is not likely optimal for many of the plant systems where it is used. This current study of macronutrient factors simultaneously altered media volume and amount of tissue (plants per vessel), sucrose, nitrogen (as NO₃⁻ and NH₄⁺ ions), and K⁺ in a d-optimal design space with only 55 experimental units (including five true replicates). Meso- and micro-nutrient concentrations were lowered (5% of MS) to determine which elements were most critical to plantlet quality. Plantlet quality was quantified by multiplication in the laboratory and survival and growth in the greenhouse. Plantlets grown at the lowest plant density, the lowest macronutrient concentration (20 mM), and equi-molar proportions of NH₄⁺/K⁺ resulted in the best multiplication ratio and 100% greenhouse survival. Multiplication ratio in vitro and survival in the greenhouse were well correlated with one another. Laboratory dry mass, media use, sucrose use, and the uptake of the macronutrients NO₃⁻, NH₄⁺, and K⁺ were not well correlated with plantlet quality. Plantlets with the greatest uptake of P, Ca, Mg, and Mn had the best multiplication in the laboratory and on subsequent transfer, acclimatized and grew fastest in the greenhouse. Phosphorus was shown to be most depleted in media. This work demonstrates a platform to simultaneously optimize several nutritive components of tissue culture media to produce plantlets that perform well in both laboratory and greenhouse environments. Plant quality was related with factors outside the macronutrient design, and this platform indicated where to expand the experimental space. Fixed, flat-screen presentations revealed less of the response surface than interactive profiles driven by the reader.     

Optimization of expression,purification, and NMR measurement for structural studies of transmembrane region of amyloid β protein

The amyloid precursor protein (APP) is an integral transmembrane protein which has been suggested to play a central role in the pathogenesis of Alzheimer’s disease. Despite the enormous amount of research conducted on amyloid protein, the precise mechanism of its toxic effect is not yet fully understood. To better understand the mechanism and function of amyloid protein, it is critical to elucidate the three-dimensional structure of the single transmembrane spanning region of human APP (hAPP-TM). Unfortunately, it is difficult to prepare the peptide sample because hAPP-TM is a membrane-bound protein that transverses the lipid bilayer of the cell membrane. Generally, the preparation of a transmembrane peptide is very difficult and time-consuming. In fact, high yield production of transmembrane peptides has been limited by experimental difficulties related to insufficient yields and the low solubility of such peptides. In this study, we describe experimental processes developed to optimize the expression, purification, and NMR measurement conditions for hAPP-TM transmembrane peptide.     

Electrocoagulation as a clarifiation and pre-purification step in plant based bioprocesses

Electrocoagulation is a technique that has been applied to water and wastewater treatment. We propose here extension of this technique to the field of plant downstream processing (DSP), where plants are used as a bioreactors for recombinant protein production. The main problem in plant-based bioreactors is the presence of chlorophyll and phenolic compounds in plant extracts, which tend to precipitate and denature proteins. Their removal from the extracts is essential, since they have a large impact on the DSP performance. In the present work we studied the application of an electrocoagulation based technique to clarify tobacco leaf extracts by removing chlorophyll and phenolic compounds. By manipulating the pH of electrocoagulation, 90% of chlorophyll, 65% of phenolic compounds, and only 23% of total protein were removed from the extract. The process preferentially removes proteins with more acidic pI (below 6.03) and the pH of the process differentially effects removal of acidic and basic proteins.     

Conversion of AFLP markers to high-throughput markers in a complex polyploid,sugarcane

The application of DNA markers linked to traits of commercial value in sugarcane may increase the efficiency of sugarcane breeding. The majority of markers generated for quantitative trait locus mapping in sugarcane have been single sequence repeats or AFLPs (amplified fragment length polymorphisms). Since AFLP markers are not adapted for large-scale implementation in plant breeding, our objective was to assess the feasibility of converting AFLP markers to fast, cheap and reliable PCR-based assays in a complex polyploid, sugarcane. Three AFLP markers were selected on the basis of an association to resistance to the fungal pathogen Ustilago scitaminea, the causal agent of smut in sugarcane. We developed an approach which enabled the identification of polymorphisms in these AFLP markers. Towards this goal, we employed GenomeWalking and 454 sequencing to isolate sequences adjacent to the linked AFLP markers and identify SNP (single nucleotide polymorphisms) haplotypes present in the homo(eo)logous chromosomes of sugarcane. One AFLP marker was converted to a cleavage amplified polymorphic sequence marker, another to a SCAR (sequence characteristered amplified region) marker and the final AFLP marker to a SNP PCR-based assay. However, validation of each of the markers in 240 genotypes resulted in 99, 90 and 60% correspondence with the original AFLP marker. These experiments indicate that even in a complex polyploid such as sugarcane, polymorphisms identified by AFLP can be converted to high-throughput marker systems, but due to the complexity this would only be carried out for high-value markers. In some cases, the polymorphisms identified are not transferable to more sequence-specific PCR applications.     

Taxon-specific multiplex-PCR for quick,easy, and accurate identification of encyrtid and aphelinid parasitoid species attacking soft scale insects in California citrus groves

Citricola scale, Coccus pseudomagnoliarum Kuwana (Hemiptera: Coccidae), is a serious pest of citrus in California’s San Joaquin Valley, but not in southern California where a complex of Metaphycus spp. Mercet (Hymenoptera: Encyrtidae) suppress it. This has created interest in using these (and other Metaphycus) species for biological control in the San Joaquin Valley. A critical step in assessing an organism’s potential for biological control is the ability to accurately identify it. For Metaphycus spp., this currently requires slide mounted adult specimens and expert taxonomic knowledge. We present a simple, quick and accurate method to identify any life stage of the ten major parasitoids of soft scales in California citrus, based on amplification of ribosomal DNA, using the polymerase chain reaction (PCR). Three multiplex-PCR protocols amplify products of taxon-specific sizes, allowing direct diagnosis of taxa accommodated by the PCR, and reducing identification time to a fraction of that of existing methods.     

The Harlequin ladybird continues to invade southeastern Europe

The ladybird Harmonia axyridis (Pallas) is considered one of the most serious invasive species around the globe. It has spread all over North America and Western Europe, while data from southeastern Europe, especially in the Mediterranean region, are scarce. In this study we present the first confirmed data of the spread of H. axyridis throughout Croatia. Specimens were sampled and identified during the period 2008–2010. The species was recorded at 18 localities in all three colour forms in various habitat types. Light trapping was found to be a satisfactory method for collecting H. axyridis. Since there is no evidence to suggest the deliberate introduction of H. axyridis in Croatia, it can be assumed that it has spread southwards from Central and Eastern Europe, and that it will probably continue to spread. Further investigations should focus on monitoring and detailed mapping of H. axyridis in Croatia and neighbouring countries, especially in the Mediterranean region, to determine whether stable populations are present.     

Ring-based versus disc-based separation of spatial scales: a case study on the impact of arable land proportions on invertebrates in freshwater streams

The impact of different land-use types on species is traditionally estimated by correlating landscape proportions recorded in buffer areas around focal points with species data observed at these sites. If a high proportion of a specific land-use type exists within a small radius, it will be accumulated in larger buffers and may confound the interpretation at larger scales. We sampled freshwater invertebrates in ten streams using cages with artificial substrate and compared the effects of arable land proportions calculated in disc-shaped buffers of increasing radius versus areas calculated from non-overlapping rings of increasing radius. We hypothesize that (1) the accumulative disc-based approach leads to confounding effects across increasing buffer size and that (2) the use of ring-based methods facilitates the identification of relevant scales for conservation measures. The abundance of crustaceans showed a positive relationship with arable land proportions, but Plecoptera abundance and the taxonomic richness of Ephemeroptera and Plecoptera decreased with increasing arable land proportions in the surrounding landscape. Our results further support the presence of confounding effects in disc-based analyses, as correlations between arable land proportions and Crustacea, or Plecoptera, respectively, were affected by the accumulation of small-scale area proportions. The distance at which arable land proportions significantly affected benthic fauna in freshwater streams was consistently shorter if calculated from rings rather than from discs. Although an a priori definition of ring width introduces new challenges, a combined use of disc- and ring-based techniques for the estimation of land-use effects may substantially improve the realization of conservation and protection measures in terrestrial and aquatic systems.     

Increased abundance of the non-indigenous zooplanktivore, <Emphasis Type=Italic>Bythotrephes longimanus</Emphasis>, is strongly correlated with greater spring prey availability in Canadian Shield lakes

The non-indigenous zooplanktivore, Bythotrephes longimanus, is a large Palaearctic cladoceran that is spreading rapidly in the Great Lakes watershed in North America. As a voracious predator, Bythotrephes can reduce herbivorous cladoceran abundance and diversity; however, the variables that affect its abundance are not well understood. To determine what bottom-up factors are associated with the abundance and seasonal dynamics of established Bythotrephes populations, two Bythotrephes datasets from lakes in south-central Ontario, Canada, were analysed using multiple regression and multivariate analyses: a multi-lake dataset of nine lakes sampled in 2003 and a multi-year dataset of one of these lakes, Harp Lake, sampled from 1994–1998 and 2001–2004. Bottom-up variables tested were Secchi disk depth, epilimnetic temperature, cladoceran (prey) density, total phosphorus, dissolved organic carbon and Chlorophyll a, as well as maximum depth for the multi-lake dataset. In both analyses and datasets, springtime abundance of herbivorous cladocerans was consistently found to be a significant factor associated with Bythotrephes (June–September) abundance; Bythotrephes annual abundance was significantly and positively associated with mean May and June prey abundance, along with mean Secchi disk depth for the multi-lake dataset, and groups of lakes or years with similar Bythotrephes seasonal abundance patterns were predicted by June prey abundance. Additionally, prey availability was the dominant contributor towards changes in weekly Bythotrephes birth rates calculated for two of the study lakes. Our study suggests that prey availability influences Bythotrephes abundance, which provides evidence that Bythotrephes establishment success is affected by the abundance of its prey.