An optimized fine root sampling methodology balancing accuracy and time investment

Aims

Tree roots are spatially highly heterogeneous and it thus requires large numbers of samples to detect statistically significant changes in root biomass. The objectives of this study were to understand and quantify the sources of error in the assessment of fine root biomass (<2 mm) during the second year of a high-density Populus plantation.

Methods

Soil cores were collected in winter (n?=?35) and in summer (n?=?20), and fine roots were picked by hand for varying lengths of time: 1, 2, 5, 20, 40, and 60 min. The root biomass data were used to identify the best combination of the time spent for root picking and the number of samples collected, that minimizes the overall uncertainty (i.e. the combination of the spatial error due to the incomplete sampling and the temporal error due to the incomplete core processing).

Results

On average, 25 min was enough time to pick 90 % of the fine root biomass in winter, while in summer only 10 min were needed. In winter fewer samples were needed, but more time for picking was necessary as compared to summer when root biomass was higher.

Conclusions

Fine root sampling can be optimized by minimizing the uncertainty of the biomass estimates and simultaneously decreasing root sampling time investment.     

Discovery of induced point mutations in maize genes by TILLING

Background

Going from a gene sequence to its function in the context of a whole organism requires a strategy for targeting mutations, referred to as reverse genetics. Reverse genetics is highly desirable in the modern genomics era; however, the most powerful methods are generally restricted to a few model organisms. Previously, we introduced a reverse-genetic strategy with the potential for general applicability to organisms that lack well-developed genetic tools. Our TILLING (Targeting Induced Local Lesions IN Genomes) method uses chemical mutagenesis followed by screening for single-base changes to discover induced mutations that alter protein function. TILLING was shown to be an effective reverse genetic strategy by the establishment of a high-throughput TILLING facility and the delivery of thousands of point mutations in hundreds of Arabidopsis genes to members of the plant biology community.

Results

We demonstrate that high-throughput TILLING is applicable to maize, an important crop plant with a large genome but with limited reverse-genetic resources currently available. We screened pools of DNA samples for mutations in 1-kb segments from 11 different genes, obtaining 17 independent induced mutations from a population of 750 pollen-mutagenized maize plants. One of the genes targeted was the DMT102 chromomethylase gene, for which we obtained an allelic series of three missense mutations that are predicted to be strongly deleterious.

Conclusions

Our findings indicate that TILLING is a broadly applicable and efficient reverse-genetic strategy. We are establishing a public TILLING service for maize modeled on the existing Arabidopsis TILLING Project.     

A randomization method for efficiently and accurately processing fine roots, and separating them from debris, in the laboratory

Background and Aims

We developed a method for processing roots from soil cores and monoliths in the laboratory to reduce the time and cost devoted to separating roots from debris and improve the accuracy of root variable estimates. The method was tested on soil cores from a California oak savanna, with roots from trees, Quercus douglasii, and annual grasses.

Methods

In the randomized sampling method, one isolates the sample fraction consisting of roots and organic debris?<?= 1 cm in length, and randomizes it through immersion in water and vigorous mixing. Sub-samples from the mixture are then used to estimate the percentage of roots in this fraction, thereby enabling an estimate of total sample biomass.

Results

We found that root biomass estimates, determined through the randomization method, differed from total root biomass established by meticulously picking every root from a sample with an error of 3.0 % +/? 0.6 %?s.e.

Conclusions

This method greatly reduces the time and resources required for root processing from soil cores and monoliths, and improves the accuracy of root variable estimates compared to standard methods. This gives researchers the ability to increase sample frequency and reduce the error associated with studying roots at the landscape and plant scales.     

Effects of disturbance scale on soil microbial communities in the Western Cascades of Oregon

Aims

To gain a better understanding of how rapidly microbial communities respond to different magnitudes of perturbation that mimic minor or catastrophic disturbances.

Methods

Two montane sites in the western Cascade Mountains of Oregon with adjacent areas of forest and meadow vegetation were studied. A reciprocal transplant experiment evaluated both minor (soil cores remaining in the same vegetation type) or more severe disturbance (soil cores transferred to a different vegetation type). The biomass and composition of the bacterial and fungal communities were measured for 2 years following the establishment of the experiment.

Results

Minor disturbance (coring) had little impact on microbial biomass but transferring between vegetation type showed greater fungal biomass in soil incubated in the forest environment. The composition of bacterial communities was not influenced by coring but responded strongly to transfers between vegetation sites, changing to reflect their new environment after 2 years. Fungal community composition responded somewhat to coring, probably from disrupting mycorrhizal fungal hyphae, but more strongly to being transferred to a new environment.

Conclusions

The response of the microbial community to major disturbance was rapid, showing shifts reflective of their new environment within 2 years, suggesting that microbial communities have the capacity to quickly adjust to catastrophic disturbances.     

Investigating the utility of combining Φ29 whole genome amplification and highly multiplexed single nucleotide polymorphism BeadArray™ genotyping

Background

Sustainable DNA resources and reliable high-throughput genotyping methods are required for large-scale, long-term genetic association studies. In the genetic dissection of common disease it is now recognised that thousands of samples and hundreds of thousands of markers, mostly single nucleotide polymorphisms (SNPs), will have to be analysed. In order to achieve these aims, both an ability to boost quantities of archived DNA and to genotype at low costs are highly desirable. We have investigated Φ29 polymerase Multiple Displacement Amplification (MDA)-generated DNA product (MDA product), in combination with highly multiplexed BeadArray? genotyping technology. As part of a large-scale BeadArray genotyping experiment we made a direct comparison of genotyping data generated from MDA product with that from genomic DNA (gDNA) templates.

Results

Eighty-six MDA product and the corresponding 86 gDNA samples were genotyped at 345 SNPs and a concordance rate of 98.8% was achieved. The BeadArray sample exclusion rate, blind to sample type, was 10.5% for MDA product compared to 5.8% for gDNA.

Conclusions

We conclude that the BeadArray technology successfully produces high quality genotyping data from MDA product. The combination of these technologies improves the feasibility and efficiency of mapping common disease susceptibility genes despite limited stocks of gDNA samples.     

A holistic high-throughput screening framework for biofuel feedstock assessment that characterises variations in soluble sugars and cell wall composition in <Emphasis Type="Italic">Sorghum bicolor</Emphasis>

Background

A major hindrance to the development of high yielding biofuel feedstocks is the ability to rapidly assess large populations for fermentable sugar yields. Whilst recent advances have outlined methods for the rapid assessment of biomass saccharification efficiency, none take into account the total biomass, or the soluble sugar fraction of the plant. Here we present a holistic high-throughput methodology for assessing sweet Sorghum bicolor feedstocks at 10 days post-anthesis for total fermentable sugar yields including stalk biomass, soluble sugar concentrations, and cell wall saccharification efficiency.

Results

A mathematical method for assessing whole S. bicolor stalks using the fourth internode from the base of the plant proved to be an effective high-throughput strategy for assessing stalk biomass, soluble sugar concentrations, and cell wall composition and allowed calculation of total stalk fermentable sugars. A high-throughput method for measuring soluble sucrose, glucose, and fructose using partial least squares (PLS) modelling of juice Fourier transform infrared (FTIR) spectra was developed. The PLS prediction was shown to be highly accurate with each sugar attaining a coefficient of determination (R ² ) of 0.99 with a root mean squared error of prediction (RMSEP) of 11.93, 5.52, and 3.23 mM for sucrose, glucose, and fructose, respectively, which constitutes an error of <4% in each case. The sugar PLS model correlated well with gas chromatography–mass spectrometry (GC-MS) and brix measures. Similarly, a high-throughput method for predicting enzymatic cell wall digestibility using PLS modelling of FTIR spectra obtained from S. bicolor bagasse was developed. The PLS prediction was shown to be accurate with an R ² of 0.94 and RMSEP of 0.64 μg.mgDW^-1.h^-1.

Conclusions

This methodology has been demonstrated as an efficient and effective way to screen large biofuel feedstock populations for biomass, soluble sugar concentrations, and cell wall digestibility simultaneously allowing a total fermentable yield calculation. It unifies and simplifies previous screening methodologies to produce a holistic assessment of biofuel feedstock potential.

     

Methods to account for tree-scale variability in soil- and plant-related parameters in oil palm plantations

Background and aims

Lateral tree-scale variability in plantations should be taken into account when scaling up from point samples, but appropriate methods for sampling and calculation have not been defined. Our aim was to define and evaluate such methods.

Methods

We evaluated several existing and new methods, using data for throughfall, root biomass and soil respiration in mature oil palm plantations with equilateral triangular spacing.

Results

Three ways of accounting for spatial variation within the repeating tree unit (a hexagon) were deduced. For visible patch patterns, patches can be delineated and sampled separately. For radial patterns, measurements can be made in radial transects or a triangular portion of the tree unit. For any type of pattern, including unknown patterns, a triangular sampling grid is appropriate. In the case studies examined, throughfall was 79 % of rainfall, with 95 % confidence limits being 62 and 96 % of rainfall. Root biomass and soil respiration, measured on a 35-point grid, varied by an order of magnitude. In zones with steep gradients in parameters, sampling density has a large influence on calculated mean values.

Conclusions

The methods defined here provide a basis for representative sampling and calculation procedures in studies requiring scaling up from point sampling, but more efficient methods are needed.     

Effect of nitrogen form on the rhizosphere dynamics and uptake of cadmium and zinc by the hyperaccumulator Thlaspi caerulescens

Information about root distribution is important for characterization and modeling of water and nutrient uptake, biomass, and yield. Due to the heterogeneous distribution of roots in row crops, the reliability and representativeness of estimates of RL and root morphology using core sampling depend on the number of samples and their location. The objectives of this study were to evaluate errors when assessing RLD and root morphology from auger core sampling schemes, to estimate 2D distributions of RLD by auger core sampling, and to assess the number of samples necessary for representative estimates of RLD and RD classes. The reference dataset utilized in this study is based on completely sampled 3D soil monoliths under maize, taken at three different dates (55, 78, and 104 days after planting) and two different row spacings (75 and 37.5 cm). A hypothetical auger core sampling scheme with one core within the row and another midway between rows mostly overpredicted total RLD. Bias was lower when using an 1:3 weighting scheme. Estimation of 2D vertical RLD distribution by calculating the ratio of RLD in the plant row to RLD midway between rows yielded reasonable estimates only when the average of eight cores was used. An assessment of the proportions of RD classes yielded high bias values, even when using the average of eight cores. An analysis of sampling errors using successively more cores revealed that for total RLD, to attain a bias <20%, more than ten samples would be necessary. This suggests that the number of core samples taken in many root studies could be too low. This bias was even higher when taking core samples for estimation of proportions of RD classes, where a reasonably low bias between estimated and “true” values could not be attained in this study even with ten core samples. Consequently, when taking samples for measurement of root morphological parameters, more detailed and site-adapted sampling schemes have to be devised. For estimating total averaged RLD, two core samples (within-row and midway between rows) could be sufficient when using a 1:3 weighting scheme for calculating the average RLD. When samples are taken in a more random manner and no weighting is applied, the necessary number of samples would be at least ten.     

Fine root biomass and turnover of two fast-growing poplar genotypes in a short-rotation coppice culture

Background and aims

The quantification of root dynamics remains a major challenge in ecological research because root sampling is laborious and prone to error due to unavoidable disturbance of the delicate soil-root interface. The objective of the present study was to quantify the distribution of the biomass and turnover of roots of poplars (Populus) and associated understory vegetation during the second growing season of a high-density short rotation coppice culture.

Methods

Roots were manually picked from soil samples collected with a soil core from narrow (75 cm apart) and wide rows (150 cm apart) of the double-row planting system from two genetically contrasting poplar genotypes. Several methods of estimating root production and turnover were compared.

Results

Poplar fine root biomass was higher in the narrow rows than in the wide rows. In spite of genetic differences in above-ground biomass, annual fine root productivity was similar for both genotypes (ca. 44 g DM m^?2 year^?1). Weed root biomass was equally distributed over the ground surface, and root productivity was more than two times higher compared to poplar fine roots (ca. 109 g DM m^?2 year^?1).

Conclusions

Early in SRC plantation development, weeds result in significant root competition to the crop tree poplars, but may confer certain ecosystem services such as carbon input to soil and retention of available soil N until the trees fully occupy the site.     

A soil-free root observation system for the study of root-microorganism interactions in maize

Background and aims

The root surface of a plant usually exceeds the leaf area and is constantly exposed to a variety of soil-borne microorganisms. Root pathogens and pests, as well as belowground interactions with beneficial microbes, can significantly influence a plants' performance. Unfortunately, the analysis of these interactions is often limited because of the arduous task of accessing roots growing in soil. Here, we present a soil-free root observation system (SF-ROBS) designed to grow maize (Zea mays) plants and to study root interactions with either beneficial or pathogenic microbes.

Methods

The SF-ROBS consists of pouches lined with wet filter paper supplying nutrient solution.

Results

The aspect of maize grown in the SF-ROBS was similar to soil-grown maize; the plant growth was similar for the shoot but different for the roots (biomass and length increased in the SF-ROBS). SF-ROBS-grown roots were successfully inoculated with the hemi-biotrophic maize fungal pathogen Colletotrichum graminicola and the beneficial rhizobacteria Pseudomonas putida KT2440. Thus, the SF-ROBS is a system suitable to study two major belowground phenomena, namely root fungal defense reactions and interactions of roots with beneficial soil-borne bacteria.

Conclusions

This system contributes to a better understanding of belowground plant microbe interactions in maize and most likely also in other crops.     

Spatial distribution and phenotypic variation in root cortical aerenchyma of maize (Zea mays L.)

Background and aims

Previous research has suggested that root cortical aerenchyma (RCA) can enhance soil exploration and crop performance by decreasing root respiration. This trait is a potential breeding target for adaptation to abiotic stresses such as drought and low nutrient availability. However, little is known of phenotypic variation in aerenchyma or its distribution among root classes.

Methods

The spatial distribution of RCA was evaluated in freehand sections from 13 sites in the root systems of 11 recombinant inbred and commercial lines of maize (Zea mays). RCA variation was evaluated in 583 recombinant inbred lines of maize at one sampling position.

Results

RCA varied significantly among root classes and axial positions. Genotypic differences were observed for the amount of RCA at corresponding sampling locations and for the mean amount of RCA across all sampling locations, but genotypes did not differ in the proportional distribution of RCA within the whole root system. The amount of RCA in a cross-section was independent of several other anatomical traits.

Conclusions

There is substantial genetic variation for RCA, and this variation is independent of other anatomical traits. RCA can be phenotyped in greenhouse-grown plants by sampling the middle parts of second- or third-whorl crown roots.     

Biodetoxification of toxins generated from lignocellulose pretreatment using a newly isolated fungus, Amorphotheca resinae ZN1, and the consequent ethanol fermentation

Background

Enzymes for plant cell wall deconstruction are a major cost in the production of ethanol from lignocellulosic biomass. The goal of this research was to develop optimized synthetic mixtures of enzymes for multiple pretreatment/substrate combinations using our high-throughput biomass digestion platform, GENPLAT, which combines robotic liquid handling, statistical experimental design and automated Glc and Xyl assays. Proportions of six core fungal enzymes (CBH1, CBH2, EG1, β-glucosidase, a GH10 endo-β1,4-xylanase, and β-xylosidase) were optimized at a fixed enzyme loading of 15 mg/g glucan for release of Glc and Xyl from all combinations of five biomass feedstocks (corn stover, switchgrass, Miscanthus, dried distillers' grains plus solubles [DDGS] and poplar) subjected to three alkaline pretreatments (AFEX, dilute base [0.25% NaOH] and alkaline peroxide [AP]). A 16-component mixture comprising the core set plus 10 accessory enzymes was optimized for three pretreatment/substrate combinations. Results were compared to the performance of two commercial enzymes (Accellerase 1000 and Spezyme CP) at the same protein loadings.

Results

When analyzed with GENPLAT, corn stover gave the highest yields of Glc with commercial enzymes and with the core set with all pretreatments, whereas corn stover, switchgrass and Miscanthus gave comparable Xyl yields. With commercial enzymes and with the core set, yields of Glc and Xyl were highest for grass stovers pretreated by AP compared to AFEX or dilute base. Corn stover, switchgrass and DDGS pretreated with AFEX and digested with the core set required a higher proportion of endo-β1,4-xylanase (EX3) and a lower proportion of endo-β1,4-glucanase (EG1) compared to the same materials pretreated with dilute base or AP. An optimized enzyme mixture containing 16 components (by addition of α-glucuronidase, a GH11 endoxylanase [EX2], Cel5A, Cel61A, Cip1, Cip2, β-mannanase, amyloglucosidase, α-arabinosidase, and Cel12A to the core set) was determined for AFEX-pretreated corn stover, DDGS, and AP-pretreated corn stover. The optimized mixture for AP-corn stover contained more exo-β1,4-glucanase (i.e., the sum of CBH1 + CBH2) and less endo-β1,4-glucanase (EG1 + Cel5A) than the optimal mixture for AFEX-corn stover. Amyloglucosidase and β-mannanase were the two most important enzymes for release of Glc from DDGS but were not required (i.e., 0% optimum) for corn stover subjected to AP or AFEX. As a function of enzyme loading over the range 0 to 30 mg/g glucan, Glc release from AP-corn stover reached a plateau of 60-70% Glc yield at a lower enzyme loading (5-10 mg/g glucan) than AFEX-corn stover. Accellerase 1000 was superior to Spezyme CP, the core set or the 16-component mixture for Glc yield at 12 h, but the 16-component set was as effective as the commercial enzyme mixtures at 48 h.

Conclusion

The results in this paper demonstrate that GENPLAT can be used to rapidly produce enzyme cocktails for specific pretreatment/biomass combinations. Pretreatment conditions and feedstock source both influence the Glc and Xyl yields as well as optimal enzyme proportions. It is predicted that it will be possible to improve synthetic enzyme mixtures further by the addition of additional accessory enzymes.     

A TMA de-arraying method for high throughput biomarker discovery in tissue research

Background

Tissue MicroArrays (TMAs) represent a potential high-throughput platform for the analysis and discovery of tissue biomarkers. As TMA slides are produced manually and subject to processing and sectioning artefacts, the layout of TMA cores on the final slide and subsequent digital scan (TMA digital slide) is often disturbed making it difficult to associate cores with their original position in the planned TMA map. Additionally, the individual cores can be greatly altered and contain numerous irregularities such as missing cores, grid rotation and stretching. These factors demand the development of a robust method for de-arraying TMAs which identifies each TMA core, and assigns them to their appropriate coordinates on the constructed TMA slide.

Methodology

This study presents a robust TMA de-arraying method consisting of three functional phases: TMA core segmentation, gridding and mapping. The segmentation of TMA cores uses a set of morphological operations to identify each TMA core. Gridding then utilises a Delaunay Triangulation based method to find the row and column indices of each TMA core. Finally, mapping correlates each TMA core from a high resolution TMA whole slide image with its name within a TMAMap.

Conclusion

This study describes a genuine robust TMA de-arraying algorithm for the rapid identification of TMA cores from digital slides. The result of this de-arraying algorithm allows the easy partition of each TMA core for further processing. Based on a test group of 19 TMA slides (3129 cores), 99.84% of cores were segmented successfully, 99.81% of cores were gridded correctly and 99.96% of cores were mapped with their correct names via TMAMaps. The gridding of TMA cores were also extensively tested using a set of 113 pseudo slide (13,536 cores) with a variety of irregular grid layouts including missing cores, rotation and stretching. 100% of the cores were gridded correctly.     

Dent and Flint maize diversity panels reveal important genetic potential for increasing biomass production

Key message

Genetic and phenotypic analysis of two complementary maize panels revealed an important variation for biomass yield. Flowering and biomass QTL were discovered by association mapping in both panels.

Abstract

The high whole plant biomass productivity of maize makes it a potential source of energy in animal feeding and biofuel production. The variability and the genetic determinism of traits related to biomass are poorly known. We analyzed two highly diverse panels of Dent and Flint lines representing complementary heterotic groups for Northern Europe. They were genotyped with the 50 k SNP-array and phenotyped as hybrids (crossed to a tester of the complementary pool) in a western European field trial network for traits related to flowering time, plant height, and biomass. The molecular information revealed to be a powerful tool for discovering different levels of structure and relatedness in both panels. This study revealed important variation and potential genetic progress for biomass production, even at constant precocity. Association mapping was run by combining genotypes and phenotypes in a mixed model with a random polygenic effect. This permitted the detection of significant associations, confirming height and flowering time quantitative trait loci (QTL) found in literature. Biomass yield QTL were detected in both panels but were unstable across the environments. Alternative kinship estimator only based on markers unlinked to the tested SNP increased the number of significant associations by around 40 % with a satisfying control of the false positive rate. This study gave insights into the variability and the genetic architectures of biomass-related traits in Flint and Dent lines and suggests important potential of these two pools for breeding high biomass yielding hybrid varieties.     

Sea ice meiofauna distribution on local to pan‐Arctic scales

Arctic sea ice provides microhabitats for biota that inhabit the liquid‐filled network of brine channels and the ice–water interface. We used meta‐analysis of 23 published and unpublished datasets comprising 721 ice cores to synthesize the variability in composition and abundance of sea ice meiofauna at spatial scales ranging from within a single ice core to pan‐Arctic and seasonal scales. Two‐thirds of meiofauna individuals occurred in the bottom 10 cm of the ice. Locally, replicate cores taken within meters of each other were broadly similar in meiofauna composition and abundance, while those a few km apart varied more; 75% of variation was explained by station. At the regional scale (Bering Sea first‐year ice), meiofauna abundance varied over two orders of magnitude. At the pan‐Arctic scale, the same phyla were found across the region, with taxa that have resting stages or tolerance to extreme conditions (e.g., nematodes and rotifers) dominating abundances. Meroplankton, however, was restricted to nearshore locations and landfast sea ice. Light availability, ice thickness, and distance from land were significant predictor variables for community composition on different scales. On a seasonal scale, abundances varied broadly for all taxa and in relation to the annual ice algal bloom cycle in both landfast and pack ice. Documentation of ice biota composition, abundance, and natural variability is critical for evaluating responses to decline in Arctic sea ice. Consistent methodology and protocols must be established for comparability of meiofauna monitoring across the Arctic. We recommend to (1) increase taxonomic resolution of sea ice meiofauna, (2) focus sampling on times of peak abundance when seasonal sampling is impossible, (3) include the bottom 30 cm of ice cores rather than only bottom 10 cm, (4) preserve specimens for molecular analysis to improve taxonomic resolution, and (5) formulate a trait‐based framework that relates to ecosystem functioning.     

Fine root and aboveground carbon stocks in riparian forests: the roles of diking and environmental gradients

Aims

We analysed current carbon (C) stocks in fine root and aboveground biomass of riparian forests and influential environmental parameters on either side of a dike in the Donau-Auen National Park, Austria.

Methods

On both sides of the dike, carbon (C) stock of fine roots (CFR) under four dominant tree species and of aboveground biomass (CAB) were assessed by topsoil cores (0–30 cm) and angle count sampling method respectively (n?=?48). C stocks were modeled, performing boosted regression trees (BRT).

Results

Overall CFR was 2.8 t ha^?1, with significantly higher C stocks in diked (DRF) compared to flooded riparian forests (FRF). In contrast to CFR, mean CAB was 123 t ha^?1 and lower in DRF compared to FRF. However, dike construction was consistently ruled out as a predictor variable in BRT. CFR was influenced by the distance to the Danube River and the dominant tree species. CAB was mainly influenced by the magnitude of fluctuations in the groundwater table and the distances to the river and the low groundwater table.

Conclusions

Despite pronounced differences in FRF and DRF, we conclude that there is only weak support that dikes directly influence C allocation in floodplain forests within the time scale considered (110 years).     

Alpine plant functional group responses to fertiliser addition depend on abiotic regime and community composition

Background and aims

We ask how productivity responses of alpine plant communities to increased nutrient availability can be predicted from abiotic regime and initial functional type composition.

Methods

We compared four Caucasian alpine plant communities (lichen heath, Festuca varia grassland, Geranium-Hedysarum meadow, snow bed community) forming a toposequence and contrasting in productivity and dominance structure for biomass responses to experimental fertilization (N, P, NP, Ca) and irrigation for 4–5?years.

Results

The dominant plants in more productive communities monopolized added N and P, at the expense of their neighbors. In three out of four communities, N and P fertilizations gave greater aboveground biomass increase than N or P fertilization alone, indicating overall co-limitation of N and P, with N being most limiting. Relative biomass increase in NP treatment was negatively related to biomass in control plots across the four communities. Grasses often responded more vigorously to P, but sedges to N alone. Finally, we present one of the rare examples of a forb showing a strong N or NP response.

Conclusion

Our findings will help improve our ability to predict community composition and biomass dynamics in cool ecosystems subject to changing nutrient availability as induced by climate or land-use changes.     

Scale-up from microtiter plate to laboratory fermenter: evaluation by online monitoring techniques of growth and protein expression in Escherichia coli and Hansenula polymorpha fermentations

Background

In industry and academic research, there is an increasing demand for flexible automated microfermentation platforms with advanced sensing technology. However, up to now, conventional platforms cannot generate continuous data in high-throughput cultivations, in particular for monitoring biomass and fluorescent proteins. Furthermore, microfermentation platforms are needed that can easily combine cost-effective, disposable microbioreactors with downstream processing and analytical assays.

Results

To meet this demand, a novel automated microfermentation platform consisting of a BioLector and a liquid-handling robot (Robo-Lector) was sucessfully built and tested. The BioLector provides a cultivation system that is able to permanently monitor microbial growth and the fluorescence of reporter proteins under defined conditions in microtiter plates. Three examplary methods were programed on the Robo-Lector platform to study in detail high-throughput cultivation processes and especially recombinant protein expression. The host/vector system E. coli BL21(DE3) pRhotHi-2-EcFbFP, expressing the fluorescence protein EcFbFP, was hereby investigated. With the method 'induction profiling' it was possible to conduct 96 different induction experiments (varying inducer concentrations from 0 to 1.5 mM IPTG at 8 different induction times) simultaneously in an automated way. The method 'biomass-specific induction' allowed to automatically induce cultures with different growth kinetics in a microtiter plate at the same biomass concentration, which resulted in a relative standard deviation of the EcFbFP production of only ± 7%. The third method 'biomass-specific replication' enabled to generate equal initial biomass concentrations in main cultures from precultures with different growth kinetics. This was realized by automatically transferring an appropiate inoculum volume from the different preculture microtiter wells to respective wells of the main culture plate, where subsequently similar growth kinetics could be obtained.

Conclusion

The Robo-Lector generates extensive kinetic data in high-throughput cultivations, particularly for biomass and fluorescence protein formation. Based on the non-invasive on-line-monitoring signals, actions of the liquid-handling robot can easily be triggered. This interaction between the robot and the BioLector (Robo-Lector) combines high-content data generation with systematic high-throughput experimentation in an automated fashion, offering new possibilities to study biological production systems. The presented platform uses a standard liquid-handling workstation with widespread automation possibilities. Thus, high-throughput cultivations can now be combined with small-scale downstream processing techniques and analytical assays. Ultimately, this novel versatile platform can accelerate and intensify research and development in the field of systems biology as well as modelling and bioprocess optimization.     

Effects of climate and vegetation on soil nutrients and chemistry in the Great Basin studied along a latitudinal-elevational climate gradient

Background and aims

Roots have morphological plasticity to adapt to heterogeneous nutrient distribution in soil, but little is known about crop differences in root plasticity. The objective of this study was to evaluate root morphological strategies of four crop species in response to soil zones enriched with different nutrients.

Methods

Four crop species that are common in intercropping systems [maize (Zea mays L.), wheat (Triticum aestivum L.), faba bean (Vicia faba L.), and chickpea (Cicer arietinum L.)] and have contrasting root morphological traits were grown for 45 days under uniform or localized nitrogen and phosphorus supply.

Results

For each species tested, the nutrient supply patterns had no effect on shoot biomass and specific root length. However, localized supply of ammonium plus phosphorus induced maize and wheat root proliferation in the nutrient-rich zone. Localized supply of ammonium alone suppressed the whole root growth of chickpea and maize, whereas localized phosphorus plus ammonium reversed (maize and chickpea ) the negative effect of ammonium. The localized root proliferation of chickpea in a nutrient-rich zone did not increase the whole root length and root surface area. Faba bean had no significant response to localized nutrient supply.

Conclusions

The root morphological plasticity is influenced by nutrient-specific and species-specific responses, with the greater plasticity in graminaceous (eg. maize) than leguminous species (eg. faba bean and chickpea).     

SNP high-throughput screening in grapevine using the SNPlex™ genotyping system

Background

Until recently, only a small number of low- and mid-throughput methods have been used for single nucleotide polymorphism (SNP) discovery and genotyping in grapevine (Vitis vinifera L.). However, following completion of the sequence of the highly heterozygous genome of Pinot Noir, it has been possible to identify millions of electronic SNPs (eSNPs) thus providing a valuable source for high-throughput genotyping methods.

Results

Herein we report the first application of the SNPlex? genotyping system in grapevine aiming at the anchoring of an eukaryotic genome. This approach combines robust SNP detection with automated assay readout and data analysis. 813 candidate eSNPs were developed from non-repetitive contigs of the assembled genome of Pinot Noir and tested in 90 progeny of Syrah × Pinot Noir cross. 563 new SNP-based markers were obtained and mapped. The efficiency rate of 69% was enhanced to 80% when multiple displacement amplification (MDA) methods were used for preparation of genomic DNA for the SNPlex assay.

Conclusion

Unlike other SNP genotyping methods used to investigate thousands of SNPs in a few genotypes, or a few SNPs in around a thousand genotypes, the SNPlex genotyping system represents a good compromise to investigate several hundred SNPs in a hundred or more samples simultaneously. Therefore, the use of the SNPlex assay, coupled with whole genome amplification (WGA), is a good solution for future applications in well-equipped laboratories.