Eliciting Targeted Mutations in Medicago sativa Using CRISPR/Cas9-Mediated Genome Editing: A Potential Tool for the Improvement of Disease Resistance.

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) has become a breeding tool of choice for eliciting targeted genetic alterations in crop species as a means of improving a wide range of agronomic traits, including disease resistance, in recent years. With the recent development of CRISPR/Cas9 technology in Medicago sativa (alfalfa), which is an important perennial forage legume grown worldwide, its use for the enhancement of pathogen resistance is almost certainly on the horizon. In this chapter, we present detailed procedures for the generation of a single nonhomologous end-joining-derived indel at a precise genomic locus of alfalfa via CRISPR/Cas9. This method encompasses crucial steps in this process, including guide RNA design, binary CRISPR vector construction, Agrobacterium-mediated transformation of alfalfa explants, and molecular assessments of transformed genotypes for transgene and edit identification.

Elucidation of Physiological, Transcriptomic and Metabolomic Salinity Response Mechanisms in Medicago sativa.

Alfalfa (Medicago sativa L.) is a widely grown perennial leguminous forage crop with a number of positive attributes. However, despite its moderate ability to tolerate saline soils, which are increasing in prevalence worldwide, it suffers considerable yield declines under these growth conditions. While a general framework of the cascade of events involved in plant salinity response has been unraveled in recent years, many gaps remain in our understanding of the precise molecular mechanisms involved in this process, particularly in non-model yet economically important species such as alfalfa. Therefore, as a means of further elucidating salinity response mechanisms in this species, we carried out in-depth physiological assessments of M. sativa cv. Beaver, as well as transcriptomic and untargeted metabolomic evaluations of leaf tissues, following extended exposure to salinity (grown for 3-4 weeks under saline treatment) and control conditions. In addition to the substantial growth and photosynthetic reductions observed under salinity treatment, we identified 1233 significant differentially expressed genes between growth conditions, as well as 60 annotated differentially accumulated metabolites. Taken together, our results suggest that changes to cell membranes and walls, cuticular and/or epicuticular waxes, osmoprotectant levels, antioxidant-related metabolic pathways, and the expression of genes encoding ion transporters, protective proteins, and transcription factors are likely involved in alfalfa's salinity response process. Although some of these alterations may contribute to alfalfa's modest salinity resilience, it is feasible that several may be disadvantageous in this context and could therefore provide valuable targets for the further improvement of tolerance to this stress in the future.

The CRISPR/Cas9-Mediated Modulation of SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 8 in Alfalfa Leads to Distinct Phenotypic Outcomes.

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.

Identification of Differential Drought Response Mechanisms in Medicago sativa subsp. sativa and falcata through Comparative Assessments at the Physiological, Biochemical, and Transcriptional Levels.

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.

The Potential of Genome Editing for Improving Seed Oil Content and Fatty Acid Composition in Oilseed Crops.

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.

Potential effects of a high CO2 future on leguminous species.

Legumes provide an important source of food and feed due to their high protein levels and many health benefits, and also impart environmental and agronomic advantages as a consequence of their ability to fix nitrogen through their symbiotic relationship with rhizobia. As a result of our growing population, the demand for products derived from legumes will likely expand considerably in coming years. Since there is little scope for increasing production area, improving the productivity of such crops in the face of climate change will be essential. While a growing number of studies have assessed the effects of climate change on legume yield, there is a paucity of information regarding the direct impact of elevated CO2 concentration (e[CO2]) itself, which is a main driver of climate change and has a substantial physiological effect on plants. In this review, we discuss current knowledge regarding the influence of e[CO2] on the photosynthetic process, as well as biomass production, seed yield, quality, and stress tolerance in legumes, and examine how these responses differ from those observed in non-nodulating plants. Although these relationships are proving to be extremely complex, mounting evidence suggests that under limiting conditions, overall declines in many of these parameters could ensue. While further research will be required to unravel precise mechanisms underlying e[CO2] responses of legumes, it is clear that integrating such knowledge into legume breeding programs will be indispensable for achieving yield gains by harnessing the potential positive effects, and minimizing the detrimental impacts, of CO2 in the future.