Background

Expression of economically relevant proteins in alternative expression platforms, especially plant expression platforms, has gained significant interest in recent years. A special interest in working with plants as bioreactors for the production of pharmaceutical proteins is related to low production costs, product safety and quality. Among the different properties that plants can also offer for the production of recombinant proteins, protein glycosylation is crucial since it may have an impact on pharmaceutical functionality and/or stability.

Results

The pharmaceutical glycoprotein human Granulocyte-Colony Stimulating Factor was transiently expressed in Nicotiana benthamiana plants and subjected to mammalian-specific mucin-type O-glycosylation by co-expressing the pharmaceutical protein together with the glycosylation machinery responsible for such post-translational modification.

Conclusions

The pharmaceutical glycoprotein human Granulocyte-Colony Stimulating Factor can be expressed in N. benthamiana plants via agroinfiltration with its native mammalian-specific mucin-type O-glycosylation.

Background

Characterization of plant terpene synthases is typically done by production of recombinant enzymes in Escherichia coli. This is often difficult due to solubility and codon usage issues. Furthermore, plant terpene synthases which are targeted to the plastids, such as diterpene synthases, have to be shortened in a more or less empirical approach to improve expression. We report here an optimized Agrobacterium-mediated transient expression assay in Nicotiana benthamiana for plant diterpene synthase expression and product analysis.

Results

Agrobacterium-mediated transient expression of plant diterpene synthases in N. benthamiana led to the accumulation of diterpenes within 3 days of infiltration and with a maximum at 5 days. Over 50% of the products were exported onto the leaf surface, thus considerably facilitating the analysis by reducing the complexity of the extracts. The robustness of the method was tested by expressing three different plant enzymes, cembratrien-ol synthase from Nicotiana sylvestris, casbene synthase from Ricinus communis and levopimaradiene synthase from Gingko biloba. Furthermore, co-expression of a 1-deoxy-D-xylulose-5-phosphate synthase from tomato and a geranylgeranyl diphosphate synthase from tobacco led to a 3.5-fold increase in the amount of cembratrien-ol produced, with maximum yields reaching 2500 ng/cm².

Conclusion

With this optimized method for diterpene synthase expression and product analysis, a single infiltrated leaf of N. benthamiana would be sufficient to produce quantities required for the structure elucidation of unknown diterpenes. The method will also be of general use for gene function discovery, pathway reconstitution and metabolic engineering of diterpenoid biosynthesis in plants.

Background

Safflower (Carthamus tinctorius L.) is a difficult crop to genetically transform being susceptible to hyperhydration and poor in vitro root formation. In addition to traditional uses safflower has recently emerged as a broadacre platform for the production of transgenic products including modified oils and pharmaceutically active proteins. Despite commercial activities based on the genetic modification of safflower, there is no method available in the public domain describing the transformation of safflower that generates transformed T₁ progeny.

Results

An efficient and reproducible protocol has been developed with a transformation efficiency of 4.8% and 3.1% for S-317 (high oleic acid content) and WT (high linoleic acid content) genotypes respectively. An improved safflower transformation T-DNA vector was developed, including a secreted GFP to allow non-destructive assessment of transgenic shoots. Hyperhydration and necrosis of Agrobacterium-infected cotyledons was effectively controlled by using iota-carrageenan, L-cysteine and ascorbic acid. To overcome poor in vitro root formation for the first time a grafting method was developed for safflower in which ~50% of transgenic shoots develop into mature plants bearing viable transgenic T₁ seed. The integration and expression of secreted GFP and hygromycin genes were confirmed by PCR, Southern and Western blot analysis. Southern blot analysis in nine independent lines indicated that 1-7 transgenes were inserted per line and T₁ progeny displayed Mendelian inheritance.

Conclusions

This protocol demonstrates significant improvements in both the efficiency and ease of use over existing safflower transformation protocols. This is the first complete method of genetic transformation of safflower that generates stably-transformed plants and progeny, allowing this crop to benefit from modern molecular applications.

Context

Abundance and diversity of bumblebees have been declining over the past decades. To successfully conserve bumblebee populations, we need to understand how landscape characteristics affect the quantity and quality of floral resources collected by colonies and subsequently colony performance.

Objectives

We therefore investigated how amount and composition of pollen collected by buff-tailed bumblebee Bombus terrestris colonies was affected by the surrounding landscape (i.e. the proportion of forest, urban, semi-natural habitats) and how they were related to colony growth.

Methods

Thirty B. terrestris colonies were placed at grassland sites differing in surrounding landscape. Colonies were established in spring when availability of flowering plants was highest, and their weight gain was monitored for 1 month. We additionally recorded the quantity and compared plant taxonomic composition and nutritional quality (i.e. amino acid composition) of pollen stored.

Results

Bumblebee colonies varied little in the pollen spectra collected despite differences in surrounding landscape composition. They collected on average 80 % of pollen from woody plants, with 34 % belonging to the genus Acer. Early colony growth positively correlated with total amount of woody pollen and protein collected and decreased with increasing proportions of semi-natural habitats and total amino acid concentrations.

Conclusions

Our results suggest that woody plant species represent highly important pollen sources for the generalist forager B. terrestris early in the season. We further show that colony growth of B. terrestris is predominantly affected by the quantity, not quality, of forage, indicating that several abundant plant species flowering throughout the bumblebees’ foraging season may cover the colonies’ nutritional needs.

Context

Submersed aquatic vegetation (SAV) performs water quality enhancing functions that are critical to the overall health of estuaries such as the Chesapeake Bay. However, eutrophication and sedimentation have decimated the Bay’s SAV population to a fraction of its historical coverage. Understanding the spatial distribution of and connectedness among patches is important for assessing the dynamics and health of the remaining SAV population.

Objectives

We seek to explore the distribution of SAV patches and patterns of potential connectivity in the Chesapeake Bay through time.

Methods

We assess critical distances, from complete patch isolation to connection of all patches, in a merged composite coverage map that represents the sum of all probable Vallisneria americana containing patches between 1984 and 2010 and in coverage maps for individual years within that timeframe for which complete survey data are available.

Results

We have three key findings: First, the amount of SAV coverage in any given year is much smaller than the total recently occupied acreage. Second, the vast majority of patches of SAV that are within the tolerances of V. americana are ephemeral, being observed in only 1 or 2 years out of 26 years. Third, this high patch turnover results in highly variable connectivity from year to year, dependent on dispersal distance and patch arrangement.

Conclusions

Most of the connectivity thresholds are beyond reasonable dispersal distances for V. americana. If the high turnover in habitat occupancy is due to marginal water quality, relatively small improvements could greatly increase V. americana growth and persistence.

Context

Common species important for ecosystem restoration stand to lose as much genetic diversity from anthropogenic habitat fragmentation and climate change as rare species, but are rarely studied. Salt marshes, valuable ecosystems in widespread decline due to human development, are dominated by the foundational plant species black needlerush (Juncus roemerianus Scheele) in the northeastern Gulf of Mexico.

Objectives

We assessed genetic patterns in J. roemerianus by measuring genetic and genotypic diversity, and characterizing population structure. We examined population connectivity by delineating possible dispersal corridors, and identified landscape factors influencing population connectivity.

Methods

A panel of 19 microsatellite markers was used to genotype 576 samples from ten sites across the northeastern Gulf of Mexico from the Grand Bay National Estuarine Research Reserve (NERR) to the Apalachicola NERR. Genetic distances (F_ST and D_ch) were used in a least cost transect analysis (LCTA) within a hierarchical model selection framework.

Results

Genetic and genotypic diversity results were higher than expected based on life history literature, and samples structured into two large, admixed genetic clusters across the study area, indicating sexual reproduction may not be as rare as predicted in this clonal macrophyte. Digitized coastal transects buffered by 500 m may represent possible dispersal corridors, and developed land may significantly impede population connectivity in J. roemerianus.

Conclusions

Results have important implications for coastal restoration and management that seek to preserve adaptive potential by sustaining natural levels of genetic diversity and conserving population connectivity. Our methodology could be applied to other common, widespread and understudied species.

Background

Switchgrass (Panicum virgatum), a robust perennial C4-type grass, has been evaluated and designated as a model bioenergy crop by the U.S. DOE and USDA. Conventional breeding of switchgrass biomass is difficult because it displays self-incompatible hindrance. Therefore, direct genetic modifications of switchgrass have been considered the more effective approach to tailor switchgrass with traits of interest. Successful transformations have demonstrated increased biomass yields, reduction in the recalcitrance of cell walls and enhanced saccharification efficiency. Several tissue culture protocols have been previously described to produce transgenic switchgrass lines using different nutrient-based media, co-cultivation approaches, and antibiotic strengths for selection.

Results

After evaluating the published protocols, we consolidated these approaches and optimized the process to develop a more efficient protocol for producing transgenic switchgrass. First, seed sterilization was optimized, which led to a 20% increase in yield of induced calluses. Second, we have selected a N₆ macronutrient/B₅ micronutrient (NB)-based medium for callus induction from mature seeds of the Alamo cultivar, and chose a Murashige and Skoog-based medium to regenerate both Type I and Type II calluses. Third, Agrobacterium-mediated transformation was adopted that resulted in 50–100% positive regenerated transformants after three rounds (2 weeks/round) of selection with antibiotic. Genomic DNA PCR, RT-PCR, Southern blot, visualization of the red fluorescent protein and histochemical β-glucuronidase (GUS) staining were conducted to confirm the positive switchgrass transformants. The optimized methods developed here provide an improved strategy to promote the production and selection of callus and generation of transgenic switchgrass lines.

Conclusion

The process for switchgrass transformation has been evaluated and consolidated to devise an improved approach for transgenic switchgrass production. With the optimization of seed sterilization, callus induction, and regeneration steps, a reliable and effective protocol is established to facilitate switchgrass engineering.

Background

Genome editing of monocot plants can be accomplished by using the components of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR associated Cas9) technology specifically optimized for these types of plants. Here, we present the development of RNA-guided Cas9 system for simplex and multiplex genome editing in barley.

Results

We developed a set of customizable RNA-guided Cas9 binary vectors and sgRNA modules for simplex and multiplex editing in barley. To facilitate the design of RNA-guided Cas9 constructs, the pBract derived binary vectors were adapted to Gateway cloning and only one restriction enzyme was required for construction of the sgRNA. We designed a synthetic, codon optimized Cas9 gene containing the N terminal SV40 nuclear localization signal and the UBQ10 Arabidopsis 1st intron. Two different sgRNAs were constructed for simplex editing and one polycistronic tRNA-gRNA construct (PTG) for multiplex editing using an endogenous tRNA processing system. The RNA-guided Cas9 constructs were validated in transgenic barley plants produced by Agrobacterium-mediated transformation. The highest mutation rate was observed in simplex editing of the cytokinin oxidase/dehydrogenase HvCKX1 gene, where mutations at the hvckx1 locus were detected in 88% of the screened T₀ plants. We also proved the efficacy of the PTG construct in the multiplex editing of two CKX genes by obtaining 9 plants (21% of all edited plants) with mutations induced in both HvCKX1 and HvCKX3. Analysis of the T₁ lines revealed that mutations in the HvCKX1 gene were transmitted to the next generation of plants. Among 220 screened T₁ plants we identified 85 heterozygous and 28 homozygous mutants, most of them bearing frameshift mutations in the HvCKX1 gene. We also observed independent segregation of mutations and the Cas9-sgRNA T-DNA insert in several T₁ plants. Moreover, the knockout mutations of the Nud gene generated phenotype mutants with naked grains, and the phenotypic changes were identifiable in T₀ plants.

Conclusions

We demonstrated the effectiveness of an optimized RNA-guided Cas9 system that can be used for generating homozygous knockout mutants in the progeny of transgenic barely plants. This is also the first report of successful multiplex editing in barley using a tRNA processing system.

Background

CRISPR-Cas is a recent and powerful addition to the molecular toolbox which allows programmable genome editing. It has been used to modify genes in a wide variety of organisms, but only two alga to date. Here we present a methodology to edit the genome of Thalassiosira pseudonana, a model centric diatom with both ecological significance and high biotechnological potential, using CRISPR-Cas.

Results

A single construct was assembled using Golden Gate cloning. Two sgRNAs were used to introduce a precise 37 nt deletion early in the coding region of the urease gene. A high percentage of bi-allelic mutations (≤61.5%) were observed in clones with the CRISPR-Cas construct. Growth of bi-allelic mutants in urea led to a significant reduction in growth rate and cell size compared to growth in nitrate.

Conclusions

CRISPR-Cas can precisely and efficiently edit the genome of T. pseudonana. The use of Golden Gate cloning to assemble CRISPR-Cas constructs gives additional flexibility to the CRISPR-Cas method and facilitates modifications to target alternative genes or species.

Context

Multispecies and multiscale habitat suitability models (HSM) are important to identify the environmental variables and scales influencing habitat selection and facilitate the comparison of closely related species with different ecological requirements.

Objectives

This study explores the multiscale relationships of habitat suitability for the pine (Martes martes) and stone marten (M. foina) in northern Spain to evaluate differences in habitat selection and scaling, and to determine if there is habitat niche displacement when both species coexist.

Methods

We combined bivariate scaling and maximum entropy modeling to compare the multiscale habitat selection of the two martens. To optimize the HSM, the performance of three sampling bias correction methods at four spatial scales was explored. HSMs were compared to explore niche differentiation between species through a niche identity test.

Results

The comparison among HSMs resulted in the detection of a significant niche divergence between species. The pine marten was positively associated with cooler mountainous areas, low levels of human disturbance, high proportion of natural forests and well-connected forestry plantations, and medium-extent agroforestry mosaics. The stone marten was positively related to the density of urban areas, the proportion and extensiveness of croplands, the existence of some scrub cover and semi-continuous grasslands.

Conclusions

This study outlines the influence of the spatial scale and the importance of the sampling bias corrections in HSM, and to our knowledge, it is the first comparing multiscale habitat selection and niche divergence of two related marten species. This study provides a useful methodological framework for multispecies and multiscale comparatives.

Context

Although small isolated habitat patches may not be able to maintain a minimum viable population, small patches that are structurally isolated may be functionally connected if individuals can cross the gaps between them, in which case, their areas could be added to form a larger habitat patch, eventually surpassing the size threshold for holding a viable population.

Objectives

We studied whether models based on the size and isolation of habitat patches could be used to predict the distribution of the Chestnut-throated Huet-Huet (Pteroptochos castaneus) in fragmented landscapes of the coastal range of the Maule region, central Chile.

Methods

We selected seven 10,000-ha landscapes (8.4–70.7% forest cover). For each habitat patch we made 18 predictions of the presence of the species based on the combination of two thresholds: three critical patch sizes for maintaining a viable population (62.5, 125 and 250 ha) and six critical isolation distances between patches (0, 10, 50, 100, 150 and 200 m). We used playbacks in 59 sampling points to estimate the species’ presence/absence. We used logistic regressions to test whether the output of the patch-matrix models could explain part of the variation in the presence of Pteroptochos castaneus.

Results

The best predictions for the presence of P. castaneus were obtained with the most conservative scenarios (125–250 ha to 0–10 m), including a positive effect of the understory cover and a lack of effect of the forest type (native or exotic).

Conclusions

Our findings suggest that the long term persistence of P. castaneus may depend on the existence of large and/or very connected forest tracts.

Background

Genetic studies and breeding of agricultural crops frequently involve phenotypic characterization of large collections of genotypes grown in field conditions. These evaluations are typically based on visual observations and manual (destructive) measurements. Robust image capture and analysis procedures that allow phenotyping large collections of genotypes in time series during developmental phases represent a clear advantage as they allow non-destructive monitoring of plant growth and performance. A L. perenne germplasm panel including wild accessions, breeding material and commercial varieties has been used to develop a low-cost, high-throughput phenotyping tool for determining plant growth based on images of individual plants during two consecutive growing seasons. Further we have determined the correlation between image analysis-based estimates of the plant’s base area and the capacity to regrow after cutting, with manual counts of tiller number and measurements of leaf growth 2 weeks after cutting, respectively. When working with field-grown plants, image acquisition and image segmentation are particularly challenging as outdoor light conditions vary throughout the day and the season, and variable soil colours hamper the delineation of the object of interest in the image. Therefore we have used several segmentation methods including colour-, texture- and edge-based approaches, and factors derived after a fast Fourier transformation. The performance of the procedure developed has been analysed in terms of effectiveness across different environmental conditions and time points in the season.

Results

The procedure developed was able to analyse correctly 77.2 % of the 24,048 top view images processed. High correlations were found between plant’s base area (image analysis-based) and tiller number (manual measurement) and between regrowth after cutting (image analysis-based) and leaf growth 2 weeks after cutting (manual measurement), with r values up to 0.792 and 0.824, respectively. Nevertheless, these relations depend on the origin of the plant material (forage breeding lines, current forage varieties, current turf varieties, and wild accessions) and the period in the season.

Conclusions

The image-derived parameters presented here deliver reliable, objective data, complementary to the breeders’ scores, and are useful for genetic studies. Furthermore, large variation was shown among genotypes for the parameters investigated.

Background

Plant biomass consists primarily of carbohydrates derived from photosynthesis. Monitoring the assimilation of carbon via the Calvin-Benson cycle and its subsequent utilisation is fundamental to understanding plant growth. The use of stable and radioactive carbon isotopes, supplied to plants as CO₂, allows the measurement of fluxes through the intermediates of primary photosynthetic metabolism, long-distance transport of sugars in the vasculature, and the synthesis of structural and storage components.

Results

Here we describe the design of a system for supplying isotopically labelled CO₂ to single leaves of Arabidopsis thaliana. We demonstrate that the system works well using short pulses of ¹⁴CO₂ and that it can be used to produce robust qualitative and quantitative data about carbon export from source leaves to the sink tissues, such as the developing leaves and the roots. Time course experiments show the dynamics of carbon partitioning between storage as starch, local production of biomass, and export of carbon to sink tissues.

Conclusion

This isotope labelling method is relatively simple to establish and inexpensive to perform. Our use of ¹⁴CO₂ helps establish the temporal and spatial allocation of assimilated carbon during plant growth, delivering data complementary to those obtained in recent studies using ¹³CO₂ and MS-based metabolomics techniques. However, we emphasise that this labelling device could also be used effectively in combination with ¹³CO₂ and MS-based techniques.

Background

Sorghum (Sorghum bicolor L.) is one of the world’s most important cereal crops grown for multiple applications and has been identified as a potential biofuel crop. Despite several decades of study, sorghum has been widely considered as a recalcitrant major crop for transformation due to accumulation of phenolic compounds, lack of model genotypes, low regeneration frequency and loss of regeneration potential through sub-cultures. Among different explants used for genetic transformation of sorghum, immature embryos are ideal over other explants. However, the continuous supply of quality immature embryos for transformation is labour intensive and expensive. In addition, transformation efficiencies are also influenced by environmental conditions (light and temperature). Despite these challenges, immature embryos remain the predominant choice because of their success rate and also due to non-availability of other dependable explants without compromising the transformation efficiency.

Results

We report here a robust genetic transformation method for sorghum (Tx430) using differentiating embryogenic calli (DEC) with nodular structures induced from immature embryos and maintained for more than a year without losing regeneration potential on modified MS media. The addition of lipoic acid (LA) to callus induction media along with optimized growth regulators increased callus induction frequency from 61.3 ± 3.2 to 79 ± 6.5% from immature embryos (1.5–2.0 mm in length) isolated 12–15 days after pollination. Similarly, the regeneration efficiency and the number of shoots from DEC tissue was enhanced by LA. The optimized regeneration system in combination with particle bombardment resulted in an average transformation efficiency (TE) of 27.2 or 46.6% based on the selection strategy, 25% to twofold higher TE than published reports in Tx430. Up to 100% putative transgenic shoots were positive for npt-II by PCR and 48% of events had < 3 copies of transgenes as determined by digital droplet PCR. Reproducibility of this method was demonstrated by generating ~ 800 transgenic plants using 10 different gene constructs.

Conclusions

This protocol demonstrates significant improvements in both efficiency and ease of use over existing sorghum transformation methods using PDS, also enables quick hypothesis testing in the production of various high value products in sorghum.