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NRC publication: Yes

NRC publication: Yes

CRISPR/Cas9 genome editing is a transformative technology that will facilitate the development of crops to meet future demands. However, application of gene editing is hindered by the long life cycle of many crop species and because desired genotypes generally require multiple generations to achieve. Single-celled microspores are haploid cells that can develop into double haploid plants and have been widely used as a breeding tool to generate homozygous plants within a generation. In this study, we combined the CRISPR/Cas9 system with microspore technology and developed an optimized haploid mutagenesis system to induce genetic modifications in the wheat genome. We investigated a number of factors that may affect the delivery of CRISPR/Cas9 reagents into microspores and found that electroporation of a minimum of 75,000 cells using 10–20 µg DNA and a pulsing voltage of 500 V is optimal for microspore transfection using the Neon transfection system. Using multiple Cas9 and sgRNA constructs, we present evidence for the seamless introduction of targeted modifications in an exogenous DsRed gene and two endogenous wheat genes, including TaLox2 and TaUbiL1. This study demonstrates the value and feasibility of combining microspore technology and CRISPR/Cas9-based gene editing for trait discovery and improvement in plants.

Chloroplast transformation technology has become a powerful platform for generating plants that express foreign proteins of pharmaceutical and agricultural importance at high levels. Chloroplasts are often chosen as attractive targets for the introduction of new agronomic traits because they have their own genome and protein synthesis machinery. Certain valuable traits have been genetically engineered into plastid genomes to improve crop yield, nutritional quality, resistance to abiotic and biotic stresses, and the production of industrial enzymes and therapeutic proteins. Synthetic biology approaches aim to play an important role in expressing multiple genes through plastid engineering, without the risk of pleiotropic effects in transplastomic plants. Despite many promising laboratory-level successes, no transplastomic crop has been commercialized to date. This technology is mostly confined to model species in academic laboratories and needs to be expanded to other agronomically important crop species to capitalize on its significant commercial potential. However, in recent years, some transplastomic lines are progressing in field trials, offering hope that they will pass regulatory approval and enter the marketplace. This review provides a comprehensive summary of new and emerging technologies employed for plastid transformation and discusses key synthetic biology elements that are necessary for the construction of modern transformation vectors. It also focuses on various novel insights and challenges to overcome in chloroplast transformation.

Background: Fusarium head blight (FHB) resistance in the durum wheat breeding gene pool is rarely reported. Triticum turgidum ssp. carthlicum line Blackbird is a tetraploid relative of durum wheat that offers partial FHB resistance. Resistance QTL were identified for the durum wheat cv. Strongfield x Blackbird population on chromosomes 1A, 2A, 2B, 3A, 6A, 6B and 7B in a previous study. The objective of this study was to identify the defense mechanisms underlying the resistance of Blackbird and report candidate regulator defense genes and single nucleotide polymorphism (SNP) markers within these genes for high-resolution mapping of resistance QTL reported for the durum wheat cv. Strongfield/Blackbird population. Results: Gene network analysis identified five networks significantly (P < 0.05) associated with the resistance to FHB spread (Type II FHB resistance) one of which showed significant correlation with both plant height and relative maturity traits. Two gene networks showed subtle differences between Fusarium graminearum-inoculated and mock-inoculated plants, supporting their involvement in constitutive defense. The candidate regulator genes have been implicated in various layers of plant defense including pathogen recognition (mainly Nucleotide-binding Leucine-rich Repeat proteins), signaling pathways including the abscisic acid and mitogen activated protein (MAP) kinase, and downstream defense genes activation including transcription factors (mostly with dual roles in defense and development), and cell death regulator and cell wall reinforcement genes. The expression of five candidate genes measured by quantitative real-time PCR was correlated with that of RNA-seq, corroborating the technical and analytical accuracy of RNA-sequencing. Conclusions: Gene network analysis allowed identification of candidate regulator genes and genes associated with constitutive resistance, those that will not be detected using traditional differential expression analysis. This study also shed light on the association of developmental traits with FHB resistance and partially explained the co-localization of FHB resistance with plant height and maturity QTL reported in several previous studies. It also allowed the identification of candidate hub genes within the interval of three previously reported FHB resistance QTL for the Strongfield/Blackbird population and associated SNPs for future high resolution mapping studies.

Background Plant RNA virus–mediated guide RNA (gRNA) delivery represents a transformative advance in genome editing technologies. Unlike conventional transformation methods that rely on labor-intensive tissue culture and regeneration for each individual gRNA delivery, viral vectors can rapidly and systemically transmit gRNAs into pre-established Cas-expressing plants, providing an accelerated route for functional genomics and trait discovery directly in planta . However, key design parameters, including subgenomic promoter choice, transcript architecture, and their effects on viral fitness and editing outcomes, remain to be elucidated for most viral platforms. Results We developed five Barley stripe mosaic virus (BSMV) vectors, each with distinct subgenomic promoter elements to drive single gRNA expression. These were initially evaluated in Cas9-expressing transgenic Nicotiana benthamiana plants targeting the Phytoene desaturase ( PDS ) gene to compare their editing efficiencies. Single gRNAs expressed under the duplicated γb subgenomic promoter or when fused directly to the γb genome achieved the highest mutation frequencies (up to 90% at 60 days post-inoculation), whereas β1- and β2-driven sgRNAs produced delayed and reduced editing. Thus, promoter selection critically determines gRNA accumulation and the efficacy of BSMV-mediated genome editing. The top-performing design was then applied to Cas9-expressing barley ( Hordeum vulgare ) targeting HvCMF7 (conferring green-white variegation) and HvGW2.1 (impacts grain width and weight). BSMV spread systemically throughout barley, inducing somatic and heritable mutations at frequencies up to 100%, with virus-free edited progeny. In contrast, despite robust somatic editing in N. benthamiana , no heritable mutations were detected indicating species-dependent limitations in germline transmission. Conclusion Our systematic comparison of subgenomic promoter architectures establishes clear design principles for optimizing viral vector–mediated delivery. Promoter choice and transcript structure critically shape editing efficiency and viral stability. The host-specific boundary for germline editing, defined by efficient heritable editing in barley but not N. benthamiana , highlights where BSMV offers advantages and where alternative vectors or hybrid strategies are required, guiding rational platform selection for diverse crop species and applications. Collectively, these findings establish BSMV as a promising next-generation vector for rapid, tissue culture–free, and transformation-independent genome editing in cereals and other recalcitrant monocots.

NRC publication: Yes

Fusarium head blight (FHB) is a devastating fungal disease of small-grain cereals that results in severe yield and quality losses. FHB resistance is controlled by resistance components including incidence, field severity, visual rating index, Fusarium damaged kernels (FDKs), and the accumulation of the mycotoxin deoxynivalenol (DON). Resistance conferred by each of these components is partial and must be combined to achieve resistance sufficient to protect wheat from yield losses. In this study, two biparental mapping populations were analyzed in Canadian FHB nurseries and quantitative trait loci (QTL) mapped for the traits listed above. Nine genomic loci, on 2AS, 2BS, 3BS, 4AS, 4AL, 4BS, 5AS, 5AL, and 5BL, were enriched for the majority of the QTL controlling FHB resistance. The previously validated FHB resistance QTL on 3BS and 5AS affected resistance to severity, FDK, and DON in these populations. The remaining seven genomic loci colocalize with flowering time and/or plant height QTL. The QTL on 4B was a major contributor to all field resistance traits and plant height in the field. QTL on 4AL showed contrasting effects for FHB resistance between Eastern and Western Canada, indicating a local adapted resistance to FHB. In addition, we also found that the 2AS QTL contributed a major effect for DON, and the 2BS for FDK, while the 5AL conferred mainly effect for both FDK/DON. Results presented here provide insight into the genetic architecture underlying these resistant components and insight into how FHB resistance in wheat is controlled by a complex network of interactions between genes controlling flowering time, plant height, local adaption, and FHB resistance components.

Fusarium head blight (FHB) resistance is quantitatively inherited, controlled by multiple minor effect genes, and highly affected by the interaction of genotype and environment. This makes genomic selection (GS) that uses genome-wide molecular marker data to predict the genetic breeding value as a promising approach to select superior lines with better resistance. However, various factors can affect accuracies of GS and better understanding how these factors affect GS accuracies could ensure the success of applying GS to improve FHB resistance in wheat. In this study, we performed a comprehensive evaluation of factors that affect GS accuracies with a multi-parental population designed for FHB resistance. We found larger sample sizes could get better accuracies. Training population designed by CDmean based optimization algorithms significantly increased accuracies than random sampling approach, while mean of predictor error variance (PEVmean) had the poorest performance. Different genomic selection models performed similarly for accuracies. Including prior known large effect quantitative trait loci (QTL) as fixed effect into the GS model considerably improved the predictability. Multi-traits models had almost no effects, while the multi-environment model outperformed the single environment model for prediction across different environments. By comparing within and across family prediction, better accuracies were obtained with the training population more closely related to the testing population. However, achieving good accuracies for GS prediction across populations is still a challenging issue for GS application.

This research examined the physicochemical, functional, and nutritional aspects of pea (PPI) and faba bean protein isolates (FBPI) modified using trypsin at varying levels of hydrolysis. Enzyme hydrolysis decreased the protein content for both pulse protein isolates and increased the lipid content for pea. The surface charge became more negative, and the surface hydrophobicity was enhanced. The surface and interfacial tension, in general, decreased upon hydrolysis. The oil holding capacity for both pulses increased while the water holding capacity decreased. The foaming capacity of FBPI was significantly lowered by hydrolysis, while in the case of PPI, it increased at pH 4.5 and was unaffected at 7.0. The foaming stability increased for both pulses at pH 4.5 but was reduced at pH 7.0. At pH 4.5, hydrolysis enhanced the emulsion activity index of FBPI, while it decreased the index of PPI. The indices increased for both pulses at pH 7.0. The emulsion stability index was generally reduced for FBPI but improved for PPI after hydrolysis. All samples were limiting in tryptophan and the sulfur-containing amino acids, and in vitro protein digestibility decreased after hydrolysis for both pulses. As the results of these changes, overall protein quality improved for FBPI but not PPI. The degree of protein hydrolysis was an important factor in determining the functional and nutritional attributes of modified pulse proteins along with pH and pulse type.