<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of safflower and the efficient recovery of transgenic plants via grafting

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.

     

Determinants of spread in an urban landscape by an introduced lizard

Context

Urban landscapes are a mixture of built structures, human-altered vegetation, and remnant semi-natural areas. The spatial arrangement of abiotic and biotic conditions resulting from urbanization doubtless influences the establishment and spread of non-native species in a city.

Objectives

We investigated the effects of habitat structure, thermal microclimates, and species coexistence on the spread of a non-native lizard (Anolis cristatellus) in the Miami metropolitan area of South Florida (USA).

Methods

We used transect surveys to estimate lizard occurrence and abundance on trees and to measure vegetation characteristics, and we assessed forest cover and impervious surface using GIS. We sampled lizard body temperatures, habitat use, and relative abundance at multiple sites.

Results

At least one of five Anolis species occupied 79 % of the 1035 trees surveyed in primarily residential areas, and non-native A. cristatellus occupied 25 % of trees. Presence and abundance of A. cristatellus were strongly associated with forest patches, dense vegetation, and high canopy cover, which produced cooler microclimates suitable for this species. Presence of A. cristatellus was negatively associated with the ecologically similar non-native A. sagrei, resulting in reduced abundance and a shift in perch use of A. cristatellus.

Conclusions

The limited spread of A. cristatellus in Miami over 35 years is due to the patchy, low-density distribution of wooded habitat, which limits dispersal by diffusion. The presence of congeners may also limit spread. Open habitats—some parks, yards and roadsides—contain few if any A. cristatellus, and colonization of isolated forest habitat appears to depend on human-mediated dispersal.

     

Disentangling the relative effect of light pollution,impervious surfaces and intensive agriculture on bat activity with a national-scale monitoring program

Context

Light pollution is a global change affecting a major proportion of global land surface. Although the impacts of Artificial Light At Night (ALAN) have been documented locally for many taxa, the extent of effect of ALAN at a landscape scale on biodiversity is unknown.

Objectives

We characterized the landscape-scale impacts of ALAN on 4 insectivorous bat species Pipistrellus pipistrellus, Pipistrellus kuhlii, Eptesicus serotinus, Nyctalus leisleri, and compared the extent of their effects to other major land-use pressures.

Methods

We used a French national-scale monitoring program recording bat activity among 2-km car transect surveys, and extracted landscape characteristics around transects with satellite and land cover layers. For each species, we performed multi-model averaging at 4 landscape scales (from 200 to 1000 m buffers around transects) to compare the relative effects of the average radiance, the proportion of impervious surface and the proportion of intensive agriculture.

Results

For all species, ALAN had a stronger negative effect than impervious surface at the 4 landscape scales tested. This effect was weaker than the effect of intensive agriculture. The negative effect of ALAN was significant for P. pipistrellus, P. kuhlii and E. serotinus, but not for N. leisleri. The effect of impervious surface varied among species while intensive agriculture had a significant negative effect on the 4 species.

Conclusion

Our results highlight the need to consider the impacts of ALAN on biodiversity in land-use planning and suggest that using only impervious surface as a proxy for urbanization may lead to underestimated impacts on biodiversity.

     

Connectivity analysis as a tool for assessing restoration success

Context

Methods for measuring restoration success that include functional connectivity between species’ populations are rare in landscape ecology and restoration practices. We developed an approach that analyzes connectivity between populations of target species and their dispersal probabilities to assess restoration success based on easily accessible input data. Applying this method to landscape development scenarios can help optimize restoration planning.

Objectives

We developed an assessment for restoration success and restoration planning based on functional connectivity between species’ populations and spatially explicit scenarios. The method was used in a case study to test its applicability.

Methods

Based on data on available habitat, species’ occurrence and dispersal ranges, connectivity metrics and dispersal probabilities for target species are calculated using the software Conefor Sensinode. The metrics are calculated for scenarios that reflect possible changes in the landscape to provide a basis for future restoration planning. We applied this approach to floodplain meadows along the Upper Rhine for four plant species and three future scenarios.

Results

In the case study, habitats of the target species were poorly connected. Peucedanum officinale and Sanguisorba officinalis were more successful in recolonizing new habitats than Iris spuria and Serratula tinctoria. The scenarios showed that restoration of species-rich grassland was beneficial for dispersal of the target species. As expected in the agriculturally dominated study area, restoration of former arable land significantly increased dispersal probabilities.

Conclusions

In the case study, the developed approach was easily applicable and provided reasonable results. Its implementation will be helpful in decision-making for future restoration planning.

     

Estimating landscape resistance from habitat suitability: effects of data source and nonlinearities

Context

Conservation corridors must facilitate long-distance dispersal movements to promote gene flow, prevent inbreeding, and allow animals to shift ranges with climate change. Least-cost models are used to identify areas that support long-distance movement. These models rely on estimates of landscape resistance, which are typically derived from habitat suitability.

Objectives

We examine two key steps in estimating resistance from habitat suitability: choosing a procedure to estimate habitat suitability, and choosing a transformation function to translate habitat suitability into resistance.

Methods

We used linear and nonlinear functions to convert three types of habitat suitability estimates (from expert opinion, resource selection functions, and step selection functions) into resistances for elk (Cervus canadensis) and desert bighorn sheep (Ovis canadensis nelsoni). We evaluated the resulting resistance maps on an independent set of observed long-distance, prospecting movements.

Results

A negative exponential function best described the relationship between resistance values and habitat suitability for desert bighorn sheep indicating long-distance movers readily travel through moderately-suitable areas and avoid only the least suitable habitat. For desert bighorn sheep, all three suitability estimates performed better than chance, and resource and step selection functions outperformed expert opinion. For elk, all three suitability estimates performed the same as chance.

Conclusions

When designing corridors to facilitate long-distance movements of mobile animals, we recommend transforming habitat suitability into resistance with a negative exponential function. Use of an exponential transformation means that larger fractions of the landscape offer low resistance, allowing greater flexibility in where a corridor is located.

     

Spatiotemporal dynamics of the agricultural landscape mosaic drives distribution and abundance of dominant carabid beetles

Context

Agroecosystems are dynamic, with yearly changing proportions of crops. Explicit consideration of this temporal heterogeneity is required to decipher population and community patterns but remains poorly studied.

Objectives

We evaluated the impact on the activity-density of two dominant carabid species (Poecilus cupreus and Anchomenus dorsalis) of (1) local crop, current year landscape composition, and their interaction, and (2) inter-annual changes in landscape composition due to crop rotations.

Methods

Carabids were sampled using pitfall-traps in 188 fields of winter cereals and oilseed rape in three agricultural areas of western France contrasting in their spatial heterogeneity. We summarized landscape composition in the current and previous years in a multi-scale perspective, using buffers of increasing size around sampling locations.

Results

Both species were more abundant in oilseed rape, and in landscapes with a higher proportion of oilseed rape in the previous year. P. cupreus abundance was negatively influenced by oilseed rape proportion in the current year landscape in winter cereals and positively by winter cereal proportion in oilseed rape. A. dorsalis was globally impacted at finer scales than P. cupreus.

Conclusions

Resource concentration and dilution-concentration processes jointly appear to cause transient dynamics of population abundance and distribution among habitat patches. Inter-patch movements across years appear to be key drivers of carabids’ survival and distribution, in response to crop rotation. Therefore, the explicit consideration of the spatiotemporal dynamics of landscape composition can allow future studies to better evidence ecological processes behind observed species patterns and help developing new management strategies.

     

Surface metrics: scaling relationships and downscaling behavior

Context

Considerable research has examined scale effects for patch-based metrics with the ultimate goal of predicting values at finer resolutions (i.e., downscaling), but results have been inconsistent. Surface metrics have been suggested as an alternative to patch-based metrics, although far less is known about their scaling relationships and downscaling potential. If successful, downscaling would enable integration of disparate datasets and comparison of landscapes using different resolution datasets.

Objectives

(1) Determine how surface metrics scale as resolution changes and how consistent those scaling relationships are across landscapes. (2) Test whether these scaling relationships can be accurately downscaled to predict metric values for finer resolutions.

Methods

Various scaling functions were fit to 16 surface metrics computed for multiple resolutions for a set of landscapes. Best-fitting functions were then extrapolated to test downscaling behavior (i.e., predict metric value for a finer resolution) for an independent set of validation landscapes. Relative error was assessed between the predicted and true values to determine downscaling robustness.

Results

Seven surface metrics (Sa, Sq, S10z, Sdq, Sds, Sdr, Srwi) fit consistently well (R² > 0.99) with a 3rd order polynomial or power law. Of those, the scaling functions for Sa, Sq, and S10z were able to predict metric values at a finer resolution within 5 %. Three metrics, (Ssk, Sku, Sfd) were also notable in terms of fit and downscaling.

Conclusions

Many metrics exhibit consistent scaling relations across resolution, and several are able to accurately predict values at finer resolutions. However, prediction accuracy is likely related to the amount of information lost during aggregation.

     

Landscape genetics of the foundational salt marsh plant species black needlerush (<Emphasis Type="Italic">Juncus roemerianus</Emphasis> Scheele) across the northeastern Gulf of Mexico

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.

     

A landscape index of ecological integrity to inform landscape conservation

Context

Conservation planning is increasingly using “coarse filters” based on the idea of conserving “nature’s stage”. One such approach is based on ecosystems and the concept of ecological integrity, although myriad ways exist to measure ecological integrity.

Objectives

To describe our ecosystem-based index of ecological integrity (IEI) and its derivative index of ecological impact (ecoImpact), and illustrate their applications for conservation assessment and planning in the northeastern United States.

Methods

We characterized the biophysical setting of the landscape at the 30 m cell resolution using a parsimonious suite of settings variables. Based on these settings variables and mapped ecosystems, we computed a suite of anthropogenic stressor metrics reflecting intactness (i.e., freedom from anthropogenic stressors) and resiliency metrics (i.e., connectivity to similar neighboring ecological settings), quantile-rescaled them by ecosystem and geographic extent, and combined them in a weighted linear model to create IEI. We used the change in IEI over time under a land use scenario to compute ecoImpact.

Results

We illustrated the calculation of IEI and ecoImpact to compare the ecological integrity consequences of a 70-year projection of urban growth to an alternative scenario involving securing a network of conservation core areas (reserves) from future development.

Conclusions

IEI and ecoImpact offer an effective way to assess ecological integrity across the landscape and examine the potential ecological consequences of alternative land use and land cover scenarios to inform conservation decision making.

     

Transient co-expression with three <Emphasis Type="Italic">O</Emphasis>-glycosylation enzymes allows production of GalNAc-<Emphasis Type="Italic">O</Emphasis>-glycosylated Granulocyte-Colony Stimulating Factor in <Emphasis Type="Italic">N. benthamiana</Emphasis>

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.

     

Landscape capability models as a tool to predict fine-scale forest bird occupancy and abundance

Context

Species-specific models of landscape capability (LC) can inform landscape conservation design. Landscape capability is “the ability of the landscape to provide the environment […] and the local resources […] needed for survival and reproduction […] in sufficient quantity, quality and accessibility to meet the life history requirements of individuals and local populations.” Landscape capability incorporates species’ life histories, ecologies, and distributions to model habitat for current and future landscapes and climates as a proactive strategy for conservation planning.

Objectives

We tested the ability of a set of LC models to explain variation in point occupancy and abundance for seven bird species representative of spruce-fir, mixed conifer-hardwood, and riparian and wooded wetland macrohabitats.

Methods

We compiled point count data sets used for biological inventory, species monitoring, and field studies across the northeastern United States to create an independent validation data set. Our validation explicitly accounted for underestimation in validation data using joint distance and time removal sampling.

Results

Blackpoll warbler (Setophaga striata), wood thrush (Hylocichla mustelina), and Louisiana (Parkesia motacilla) and northern waterthrush (P. noveboracensis) models were validated as predicting variation in abundance, although this varied from not biologically meaningful (1%) to strongly meaningful (59%). We verified all seven species models [including ovenbird (Seiurus aurocapilla), blackburnian (Setophaga fusca) and cerulean warbler (Setophaga cerulea)], as all were positively related to occupancy data.

Conclusions

LC models represent a useful tool for conservation planning owing to their predictive ability over a regional extent. As improved remote-sensed data become available, LC layers are updated, which will improve predictions.

     

How do field margins contribute to the functional connectivity of insect-pollinated plants?

Context

Habitat fragmentation generates a loss of functional connectivity detrimental to the persistence of biodiversity. The French agricultural intensification initiated in the 1950s has caused a decline in field margins.

Objectives

As field margins may facilitate species dispersal while providing socio-economic benefits, it is of interest to assess their contribution to the functional connectivity of insect-pollinated plants in agro-ecosystems. This will help develop appropriate management strategies mitigating fragmentation.

Methods

We addressed this issue by studying the links between landscape structure and the patterns of abundance and pollen dispersal (using fluorescent dye particles) for two contrasted insect-pollinated plants occurring in field margins (Crepis sancta and Euphorbia serrata). We investigated the influence of field margins quality and of the surrounding matrix on pollen dispersal and compared the relevance of the least-cost algorithm with a straight-line approach to depict pollinators’ movements.

Results

The influence of landscape structure on plant abundance is species and scale-specific. Pollen dispersal decreases with distance from the source. For E. serrata, it was preferentially dispersed via field margins, confirming the relevance of the least-cost algorithm, while C. sancta dispersal followed a straight-line.

Conclusions

Euphorbia serrata, which grows strictly on field margins with a greater dispersal ability and a more diversified pollinator guild than C. sancta, is less affected by land-use changes. Our study demonstrates the contrasting contributions of field margins to pollen dispersal as they may act as functional corridors favouring pollinators’ movement depending on the species of interest.

     

Occupancy pattern of a long-horned beetle in a variegated forest landscape: linkages between tree quality and forest cover across spatial scales

Context

Interactions between landscape-scale processes and fine-grained habitat heterogeneity are usually invoked to explain species occupancy in fragmented landscapes. In variegated landscapes, however, organisms face continuous variation in micro-habitat features, which makes necessary to consider ecologically meaningful estimates of habitat quality at different spatial scales.

Objectives

We evaluated the spatial scales at which forest cover and tree quality make the greatest contribution to the occupancy of the long-horned beetle Microplophorus magellanicus (Coleoptera: Cerambycidae) in a variegated forest landscape.

Methods

We used averaged data of tree quality (as derived from remote sensing estimates of the decay stage of single trees) and spatially independent pheromone-baited traps to model the occurrence probability as a function of multiple cross-scale combinations between forest cover and tree quality (with scales ranging between 50 and 400 m).

Results

Model support and performance increased monotonically with the increasing scale at which tree quality was measured. Forest cover was not significant, and did not exhibit scale-specific effects on the occurrence probability of M. magellanicus. The interactive effect between tree quality and forest cover was stronger than the independent (additive) effects of tree quality and particularly forest cover. Significant interactions included tree quality measured at spatial scales ≥200 m, but cross-scale interactions occurred only in four of the seven best-supported models.

Conclusions

M. magellanicus respond to the high-quality trees available in the landscape rather than to the amount of forest per se. Conservation of viable metapopulations of M. magellanicus should consider the quality of trees at spatial scales >200 m.

     

Towards an energy–landscape integrated analysis? Exploring the links between socio-metabolic disturbance and landscape ecology performance (Mallorca,Spain, 1956–2011)

Context

The role of agricultural landscapes in biodiversity conservation is an emerging topic in a world experiencing a worrying decrease of species richness. Farm systems may either decrease or increase biological diversity, depending on land-use intensities and management.

Objectives

We present an intermediate disturbance-complexity model (IDC) of cultural landscapes aimed at assessing how different levels of anthropogenic disturbance on ecosystems affect the capacity to host biodiversity depending on the land matrix heterogeneity. It is applied to the Mallorca Island, amidst the Mediterranean biodiversity hotspot.

Methods

The model uses the disturbance exerted when farmers alter the Net Primary Production through land-use change as well as when they remove a share of it (HANPP), together with Shannon–Wiener index (H′) of land-cover diversity. The model is tested with a twofold-scalar experimental design (1:50,000 and 1:5000) of a set of landscape units along three time points (1956, 1989, 2011). Species richness of breeding and wintering birds, taken as a biodiversity proxy, is used in an exploratory factor analysis.

Results

The results clearly show that when intermediate levels of HANPP are performed within intermediate levels of complexity (H′) in landscape patterns, like agro-forest mosaics, great bird species richness and high socio-ecological resilience can be maintained. Yet, these complex-heterogeneous landscapes are currently vanishing due to industrial farm intensification, rural abandonment and urban sprawl.

Conclusions

The results make apparent the usefulness of transferring the concept of intermediate disturbance-complexity interplay to cultural landscapes. Our spatial-explicit IDC model can be used as a tool for strategic environmental assessment of land-use planning.

     

Predicting effects of future development on a territorial forest songbird: methodology matters

Context

Projected increases in human population size are expected to increase forest loss and fragmentation in the next century at the expense of forest-dwelling species.

Objectives

We estimated landscape carrying capacity (N _k) for Ovenbirds in urban, suburban, exurban, and rural areas for the years 2000 and 2050, and compared changes in N _k with changes in occupancy probability.

Methods

Maximum clique analysis, a branch of mathematical graph theory, was used to estimate landscape carrying capacity, the maximum potential number of territories a given landscape is capable of supporting (N _k). We used occupancy probability maps as inputs for calculating Ovenbird N _k in the northeastern USA and a spatially explicit growth model to forecast future development patterns in 2050. We compared occupancy probability with estimates of N _k for urban, suburban, exurban, and rural areas for the years 2000 and 2050.

Results

In response to human population growth and development, Ovenbird N _k was predicted to decrease 23% in urban landscapes, 28% in suburban landscapes, 43% in exurban landscapes, and 20% in rural landscapes. These decreases far exceeded decreases in mean occupancy probabilities that ranged between 2 and 5% across the same development categories. Thus, small decreases in occupancy probability between 2000 and 2050 translated to much larger decreases in N _k.

Conclusions

For the first time, our study compares occupancy probability with a species population metric, N _k, to assess the impact of future development. Maximum clique analysis is a tool that can be used to estimate N _k and inform landscape management and communication with stakeholders.

     

Exploring dispersal barriers using landscape genetic resistance modelling in scarlet macaws of the Peruvian Amazon

Context

Dispersal is essential for species persistence and landscape genetic studies are valuable tools for identifying potential barriers to dispersal. Macaws have been studied for decades in their natural habitat, but we still have no knowledge of how natural landscape features influence their dispersal.

Objectives

We tested for correlations between landscape resistance models and the current population genetic structure of macaws in continuous rainforest to explore natural barriers to their dispersal.

Methods

We studied scarlet macaws (Ara macao) over a 13,000 km² area of continuous primary Amazon rainforest in south-eastern Peru. Using remote sensing imagery from the Carnegie Airborne Observatory, we constructed landscape resistance surfaces in CIRCUITSCAPE based on elevation, canopy height and above-ground carbon distribution. We then used individual- and population-level genetic analyses to examine which landscape features influenced gene flow (genetic distance between individuals and populations).

Results

Across the lowland rainforest we found limited population genetic differentiation. However, a population from an intermountain valley of the Andes (Candamo) showed detectable genetic differentiation from two other populations (Tambopata) located 20–60 km away (F _ST = 0.008, P = 0.001–0.003). Landscape resistance models revealed that genetic distance between individuals was significantly positively related to elevation.

Conclusions

Our landscape resistance analysis suggests that mountain ridges between Candamo and Tambopata may limit gene flow in scarlet macaws. These results serve as baseline data for continued landscape studies of parrots, and will be useful for understanding the impacts of anthropogenic dispersal barriers in the future.

     

A simple and efficient CRISPR/Cas9 platform for induction of single and multiple,heritable mutations in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

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.

     

Editing of the urease gene by CRISPR-Cas in the diatom <Emphasis Type="Italic">Thalassiosira pseudonana</Emphasis>

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.