A highly mutable GST is essential for bract colouration in Euphorbia pulcherrima Willd. Ex Klotsch.

BACKGROUND: Mutation breeding is an extraordinary tool in plant breeding to increase the genetic variability, where mutations in anthocyanin biosynthesis are targets to generate distinctive phenotypes in ornamental species. In poinsettia, ionizing radiation is routinely applied in breeding programs to obtaining a range of colours, with nearly all pink and white varieties being obtained after γ- or X-ray mutagenesis of red varieties. In the present study we performed a thorough characterization of a potential mutagenesis target gene as the main responsible for the 'white paradox' in poinsettia. RESULTS: We identified a GST gene in poinsettia (Bract1) as an essential factor for the expression of anthocyanin-based red colouration of bracts, which presents a high phylogenetic similarity to known anthocyanin-related GSTs. Red poinsettia varieties and white mutants generated from these varieties by X-ray were analysed for polymorphisms related to the 'white paradox' in the species. A 4 bp mutation in a short repeat within the coding region of Bract1 is most likely responsible for the appearance of white phenotypes upon irradiation treatment. The polymorphism between wild-type and mutant alleles co-segregates with the phenotype in progeny from heterozygous red and white parents. Moreover, overexpression of Bract1 wild-type allele in Arabidopsis tt19 mutants restored the anthocyanin phenotype, while the Bract1 mutated allele showed to be non-functional. CONCLUSIONS: The identified repeat seems to be highly unstable, since mutated plants can be easily detected among fewer than 200 shoots derived from 10 mutated plants. Our data indicate that particular short repeat sequences, similar to microsatellite sequences or so-called dynamic mutations, might be hot spots for genetic variability. Moreover, the identification of the Bract1 mutation fills a gap on the understanding on the molecular mechanism of colour formation in poinsettia.

Hybrid de novo transcriptome assembly of poinsettia (Euphorbia pulcherrima Willd. Ex Klotsch) bracts.

BACKGROUND: Poinsettia is a popular and important ornamental crop, mostly during the Christmas season. Its bract coloration ranges from pink/red to creamy/white shades. Despite its ornamental value, there is a lack of knowledge about the genetics and molecular biology of poinsettia, especially on the mechanisms of color formation. We performed an RNA-Seq analysis in order to shed light on the transcriptome of poinsettia bracts. Moreover, we analyzed the transcriptome differences of red- and white-bracted poinsettia varieties during bract development and coloration. For the assembly of a bract transcriptome, two paired-end cDNA libraries from a red and white poinsettia pair were sequenced with the Illumina technology, and one library from a red-bracted variety was used for PacBio sequencing. Both short and long reads were assembled using a hybrid de novo strategy. Samples of red- and white-bracted poinsettias were sequenced and comparatively analyzed in three color developmental stages in order to understand the mechanisms of color formation and accumulation in the species. RESULTS: The final transcriptome contains 288,524 contigs, with 33% showing confident protein annotation against the TAIR10 database. The BUSCO pipeline, which is based on near-universal orthologous gene groups, was applied to assess the transcriptome completeness. From a total of 1440 BUSCO groups searched, 77% were categorized as complete (41% as single-copy and 36% as duplicated), 10% as fragmented and 13% as missing BUSCOs. The gene expression comparison between red and white varieties of poinsettia showed a differential regulation of the flavonoid biosynthesis pathway only at particular stages of bract development. An initial impairment of the flavonoid pathway early in the color accumulation process for the white poinsettia variety was observed, but these differences were no longer present in the subsequent stages of bract development. Nonetheless, GSTF11 and UGT79B10 showed a lower expression in the last stage of bract development for the white variety and, therefore, are potential candidates for further studies on poinsettia coloration. CONCLUSIONS: In summary, this transcriptome analysis provides a valuable foundation for further studies on poinsettia, such as plant breeding and genetics, and highlights crucial information on the molecular mechanism of color formation.

First genome edited poinsettias: targeted mutagenesis of flavonoid 3'-hydroxylase using CRISPR/Cas9 results in a colour shift.

The CRISPR/Cas9 system is a remarkably promising tool for targeted gene mutagenesis, and becoming ever more popular for modification of ornamental plants. In this study we performed the knockout of flavonoid 3'-hydroxylase (F3'H) with application of CRISPR/Cas9 in the red flowering poinsettia (Euphorbia pulcherrima) cultivar 'Christmas Eve', in order to obtain plants with orange bract colour, which accumulate prevalently pelargonidin. F3'H is an enzyme that is necessary for formation of cyanidin type anthocyanins, which are responsible for the red colour of poinsettia bracts. Even though F3'H was not completely inactivated, the bract colour of transgenic plants changed from vivid red (RHS 45B) to vivid reddish orange (RHS 33A), and cyanidin levels decreased significantly compared with the wild type. In the genetically modified plants, an increased ratio of pelargonidin to cyanidin was observed. By cloning and expression of mutated proteins, the lack of F3'H activity was confirmed. This confirms that a loss of function mutation in the poinsettia F3'H gene is sufficient for obtaining poinsettia with orange bract colour. This is the first report of successful use of CRISPR/Cas9 for genome editing in poinsettia. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11240-021-02103-5.

Bioscaffold Stiffness Mediates Aerosolized Nanoparticle Uptake in Lung Epithelial Cells.

In this study, highly porous, ultrasoft polymeric mats mimicking human tissues were formed from novel polyurethane soft dendritic colloids (PU SDCs). PU SDCs have a unique fibrillar morphology controlled by antisolvent precipitation. When filtered from suspension, PU SDCs form mechanically robust nonwoven mats. The stiffness of the SDC mats can be tuned for physiological relevance. The unique physiochemical characteristics of the PU SDC particles dictate the mechanical properties resulting in tunable elastic moduli ranging from 200 to 800 kPa. The human lung A549 cells cultured on both stiff and soft PU SDC membranes were found to be viable, capable of supporting the air-liquid interface (ALI) cell culture, and maintained barrier integrity. Furthermore, A549 cellular viability and uptake efficiency of aerosolized tannic acid-coated gold nanoparticles (Ta-Au) was found to depend on elastic modulus and culture conditions. Ta-Au nanoparticle uptake was twofold and fourfold greater on soft PU SDCs, when cultured at submerged and ALI conditions, respectively. The significant increase in endocytosed Ta-Au resulted in a 20% decrease in viability, and a 4-fold increase in IL-8 cytokine secretion when cultured on soft PU SDCs at ALI. Common tissue culture materials exhibit super-physiological elastic moduli, a factor found to be critical in analyzing nanomaterial cellular interactions and biological responses.

IgG stability in fresh and conditioned medium of tobacco (Nicotiana tabacum) and larch (Larix decidua) embryogenic suspension cultures.

To explore the feasibility of larch (Larix decidua Mill.) embryogenic cell culture as alternative plant expression system, protein stability in fresh and conditioned medium was characterized in this study and compared to tobacco BY2 suspension culture. Fresh and conditioned media were spiked with 1 microg human IgG and IgG content was determined by ELISA after 24 h incubation. In fresh media, IgG recovery rate decreased to 12-23%. Adsorption on vessel walls probably is the best explanation for this IgG loss and EDTA in the medium strongly influenced wall adsorption. A high IgG recovery rate occurred in all conditioned cell culture media (7 or 14 days after inoculation). Changes in the low molecular weight-constitution of conditioned medium, rather than co-secreted polymers, are responsible for IgG stability in the cell suspension cultures.

Phenotypic Selection in Ornamental Breeding: It's Better to Have the BLUPs Than to Have the BLUEs.

Plant breeders always face the challenge to select the best individuals. Selection methods are required that maximize selection gain based on available data. When several crosses have been made, the BLUP procedure achieves this by combining phenotypic data with information on pedigree relationships via an index, known as family-index selection. The index, estimated based on the intra-class correlation coefficient, exploits the relationship among individuals within a family relative to other families in the population. An intra-class correlation coefficient of one indicates that the individual performance can be fully explained based on the family background, whereas an intra-class correlation coefficient of zero indicates the performance of individuals is independent of the family background. In the case the intra-class correlation coefficient is one, family-index selection is considered. In the case the intra-class correlation coefficient is zero, individual selection is considered. The main difference between individual and family-index selection lies in the adjustment in estimating the individual's effect depending on the intra-class correlation coefficient afforded by the latter. Two examples serve to illustrate the application of the BLUP method. The efficiency of individual and family-index selection was evaluated in terms of the heritability obtained from linear mixed models implementing the selection methods by suitably defining the treatment factor as the sum of individual and family effect. Family-index selection was found to be at least as efficient as individual selection in Dianthus caryophyllus L., except for flower size in standard carnation and vase life in mini carnation for which traits family-index selection outperformed individual selection. Family-index selection was superior to individual selection in Pelargonium zonale in cases when the heritability was low. Hence, the pedigree-based BLUP procedure can enhance selection efficiency in production-related traits in P. zonale or shelf-life related in D. caryophyllus L.

Bioproduction of therapeutic proteins in the 21st century and the role of plants and plant cells as production platforms.

In the last decade, the technique to genetically modify crop plants has gained more and more interest in terms of bioproduction of heterologous proteins. Plants have been discovered as a possible source for large amounts of cost effective recombinant protein. Main application fields are therapeutics for use in animal and human health, diagnostics, and technical enzymes. This review is focused on the recent progress in this field of molecular farming. After a comparison with hitherto established protein production systems, the advantages of plants as an alternative production system are discussed. An overview about the different host plants and possible expression strategies is given and the progress in commercialization of the techniques is highlighted. Finally, the role of plant cell cultures for the production of recombinant proteins is discussed.

Identifying Effective Design Approaches to Allocate Genotypes in Two-Phase Designs: A Case Study in Pelargonium zonale.

Robust phenotypic data allow adequate statistical analysis and are crucial for any breeding purpose. Such data is obtained from experiments laid out to best control local variation. Additionally, experiments frequently involve two phases, each contributing environmental sources of variation. For example, in a former experiment we conducted to evaluate production related traits in Pelargonium zonale, there were two consecutive phases, each performed in a different greenhouse. Phase one involved the propagation of the breeding strains to obtain the stem cutting count, and phase two involved the assessment of root formation. The evaluation of the former study raised questions regarding options for improving the experimental layout: (i) Is there a disadvantage to using exactly the same design in both phases? (ii) Instead of generating a separate layout for each phase, can the design be optimized across both phases, such that the mean variance of a pair-wise treatment difference (MVD) can be decreased? To answer these questions, alternative approaches were explored to generate two-phase designs either in phase-wise order (Option 1) or across phases (Option 2). In Option 1 we considered the scenarios (i) using in both phases the same experimental design and (ii) randomizing each phase separately. In Option 2, we considered the scenarios (iii) generating a single design with eight replicates and splitting these among the two phases, (iv) separating the block structure across phases by dummy coding, and (v) design generation with optimal alignment of block units in the two phases. In both options, we considered the same or different block structures in each phase. The designs were evaluated by the MVD obtained by the intra-block analysis and the joint inter-block-intra-block analysis. The smallest MVD was most frequently obtained for designs generated across phases rather than for each phase separately, in particular when both phases of the design were separated with a single pseudo-level. The joint optimization ensured that treatment concurrences were equally balanced across pairs, one of the prerequisites for an efficient design. The proposed alternative approaches can be implemented with any model-based design packages with facilities to formulate linear models for treatment and block structures.

Selection for production-related traits in Pelargonium zonale: improved design and analysis make all the difference.

Ornamental plant variety improvement is limited by current phenotyping approaches and neglected use of experimental designs. The present study was conducted to show the benefits of using an experimental design and corresponding analysis in ornamental breeding regarding simulated response to selection in Pelargonium zonale for production-related traits. This required establishment of phenotyping protocols for root formation and stem cutting counts, with which 974 genotypes were assessed in a two-phase experimental design. The present paper evaluates this protocol. The possibility of varietal improvement through indirect selection on secondary traits such as branch count and flower count was assessed by genetic correlations. Simulated response to selection varied greatly, depending on the genotypic variances of the breeding population and traits. A varietal improvement of over 20% is possible for stem cutting count, root formation, branch count and flower count. In contrast, indirect selection of stem cutting count by branch count or flower count was found to be ineffective. The established phenotypic protocols and two-phase experimental designs are valuable tools for breeding of P. zonale.

Characterization of photosynthetically active duckweed (Wolffia australiana) in vitro culture by Respiration Activity Monitoring System (RAMOS).

The feasibility of oxygen transfer rate (OTR) measurement to non-destructively monitor plant propagation and vitality of photosynthetically active plant in vitro culture of duckweed (Wolffia australiana, Lemnaceae) was tested using Respiration Activity Monitoring System (RAMOS). As a result, OTR proofed to be a sensitive indicator for plant vitality. The culture characterization under day/night light conditions, however, revealed a complex interaction between oxygen production and consumption, rendering OTR measurement an unsuitable tool to track plant propagation. However, RAMOS was found to be a useful tool in preliminary studies for process development of photosynthetically active plant in vitro cultures.