Virus-induced gene silencing (VIGS) is a rapid reverse genetics tool that has been developed in a wide variety of plant species for assessing gene functions. However, while VIGS has been utilized successfully in the diploid model leguminous species Medicago truncatula (Gaertn.) (barrel medic), such a platform has yet to be established in forage legume crop species. Therefore, we evaluated the effectiveness of this method in forage legumes using a previously developed PEBV (pea early browning virus) system whereby a fragment of the pea (Pisum sativum L.) PHYTOENE DESATURASE (PDS) gene was transferred into a range of alfalfa (Medicago sativa L.), sainfoin (Onobrychis viciifolia Scop.), and fenugreek (Trigonella foenum-graecum L.) cultivars using leaf infiltration and apical meristem injection. Barrel medic was used as a positive control. Gene silencing was observed after 10–15 d through the presence of a leaf bleaching phenotype, and was confirmed using quantitative real-time RT-PCR. Silencing of PDS was achieved in a selection of cultivars in all species assessed, with the highest silencing efficiency apparent in fenugreek. The introduction of a highly homologous gene fragment from a heterologous plant species to target endogenous genes for transient VIGS-based silencing in a range of species of interest represents a potentially useful strategy for the rapid functional characterization of candidate genes in forages.

A continuous rise in demand for vegetable oils, which comprise mainly the storage lipid triacylglycerol, is fueling a surge in research efforts to increase seed oil content and improve fatty acid composition in oilseed crops. Progress in this area has been achieved using both conventional breeding and transgenic approaches to date. However, further advancements using traditional breeding methods will be complicated by the polyploid nature of many oilseed crops and associated time constraints, while public perception and the prohibitive cost of regulatory processes hinders the commercialization of transgenic oilseed crops. As such, genome editing using CRISPR/Cas is emerging as a breakthrough breeding tool that could provide a platform to keep pace with escalating demand while potentially minimizing regulatory burden. In this review, we discuss the technology itself and progress that has been made thus far with respect to its use in oilseed crops to improve seed oil content and quality. Furthermore, we examine a number of genes that may provide ideal targets for genome editing in this context, as well as new CRISPR-related tools that have the potential to be applied to oilseed plants and may allow additional gains to be made in the future.

Alfalfa (Medicago sativa L.) is an extensively grown perennial forage legume, and although it is relatively drought tolerant, it consumes high amounts of water and depends upon irrigation in many regions. Given the progressive decline in water available for irrigation, as well as an escalation in climate change-related droughts, there is a critical need to develop alfalfa cultivars with improved drought resilience. M. sativa subsp. falcata is a close relative of the predominantly cultivated M. sativa subsp. sativa, and certain accessions have been demonstrated to exhibit superior performance under drought. As such, we endeavoured to carry out comparative physiological, biochemical, and transcriptomic evaluations of an as of yet unstudied drought-tolerant M. sativa subsp. falcata accession (PI 641381) and a relatively drought-susceptible M. sativa subsp. sativa cultivar (Beaver) to increase our understanding of the molecular mechanisms behind the enhanced ability of falcata to withstand water deficiency. Our findings indicate that unlike the small number of falcata genotypes assessed previously, falcata PI 641381 may exploit smaller, thicker leaves, as well as an increase in the baseline transcriptional levels of genes encoding particular transcription factors, protective proteins, and enzymes involved in the biosynthesis of stress-related compounds. These findings imply that different falcata accessions/genotypes may employ distinct drought response mechanisms, and the study provides a suite of candidate genes to facilitate the breeding of alfalfa with enhanced drought resilience in the future.

Alfalfa (Medicago sativa L.) is the most widely grown perennial leguminous forage and is an essential component of the livestock industry. Previously, the RNAi-mediated down-regulation of alfalfa SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 8 (MsSPL8) was found to lead to increased branching, regrowth and biomass, as well as enhanced drought tolerance. In this study, we aimed to further characterize the function of MsSPL8 in alfalfa using CRISPR/Cas9-induced mutations in this gene. We successfully generated alfalfa genotypes with small insertions/deletions (indels) at the target site in up to three of four MsSPL8 alleles in the first generation. The efficiency of editing appeared to be tightly linked to the particular gRNA used. The resulting genotypes displayed consistent morphological alterations, even with the presence of up to two wild-type MsSPL8 alleles, including reduced leaf size and early flowering. Other phenotypic effects appeared to be dependent upon mutational dosage, with those plants with the highest number of mutated MsSPL8 alleles also exhibiting significant decreases in internode length, plant height, shoot and root biomass, and root length. Furthermore, MsSPL8 mutants displayed improvements in their ability to withstand water-deficit compared to empty vector control genotypes. Taken together, our findings suggest that allelic mutational dosage can elicit phenotypic gradients in alfalfa, and discrepancies may exist in terms of MsSPL8 function between alfalfa genotypes, growth conditions, or specific alleles. In addition, our results provide the foundation for further research exploring drought tolerance mechanisms in a forage crop.

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The Leguminosae family comprises approximately 800 genera and 20,000 species, and constitutes the third largest family of flowering plants (Stagnari et al., 2017). In terms of agronomically important crops, grain legumes (pulses), soybean, and certain forage species (e.g., alfalfa [Medicago sativa]) provide value as both food and feed due to their high levels of protein and health-promoting properties (Darmadi-Blackberry et al., 2004; Graham & Vance, 2003; Singh et al., 2017). In addition, their capacity to fix nitrogen through their symbiotic relationship with rhizobia allows approximately 50-70 Mt of nitrogen to be fixed annually in agricultural systems (Herridge, Peoples, & Boddey, 2008). This reduces the need for synthetic nitrogen fertilizers, which currently support 30%-50% of non-leguminous crop yields (Erisman et al., 2008), with more than 50% of applied fertilizer typically lost from cereal crops as runoff or to the

Cercospora leaf spot (CLS) caused by Cercospora traversiana is an important phyto-pathological problem of fenugreek (Trigonella foenum-graecum L.), a multiuse legume crop. Field screenings for resistant plants, although accurate and effective, demand significant time and a sizable workforce to accomplish. Also, weather conditions in the field may not always be favourable for uniform disease spread, which eventually may lead to failure of the overall experiment. Whole-plant assays (WPA) and detached leaf assays (DLA) with artificial inoculation not only help in scaling up the number of plants screened but also reduce the space, time, and amount of inoculum needed for the experiment. The results from our two experiments indicate that both the WPA and DLA methods can be used reliably to differentiate resistant and susceptible genotypes of fenugreek. In addition, the correlation coefficient between WPA and DLA (r = 0.875, P < 0.01), derived from the mean disease score of each genotype, shows that they can be used interchangeably while screening fenugreek for CLS. DLA was found to be temperature-sensitive for the development of CLS symptoms and wounded leaves developed symptoms faster than non-wounded leaves. These indoor methods can be used for the development of CLS-resistant fenugreek cultivars in areas where disease development is difficult under field conditions.

Rising emissions of anthropogenic greenhouse gases such as carbon dioxide (CO2), nitrous oxide (N2O) and methane (CH4) are a key driver of climate change, which is predicted to have myriad detrimental consequences in coming years if not kept in check. Given the potency of CH4 in terms of trapping heat in the atmosphere in the short term, as well as the fact that ruminant production currently contributes approximately 30% of anthropogenic emissions, there is an impetus to substantially decrease the generation of ruminant-derived CH4. While various strategies are being assessed in this context, a multi-faceted approach is likely required to achieve significant reductions. Feed supplementation is one strategy that has shown promise in this field by attenuating methanogenesis in rumen archaea; however, this can be costly and sometimes impractical. In this review, we examine and discuss the prospect of directly modulating forages and/or rumen archaea themselves in a manner that would reduce methanogenesis using CRISPR/Cas-mediated gene editing platforms. Such an approach could provide a valuable alternative to supplementation and has the potential to contribute to the sustainability of agriculture, as well as the mitigation of climate change, in the future.