Microalgae are attracting the interest of agrochemical industries and farmers, due to their biostimulant and biofertiliser properties. Microalgal biostimulants (MBS) and biofertilisers (MBF) might be used in crop production to increase agricultural sustainability. Biostimulants are products derived from organic material that, applied in small quantities, are able to stimulate the growth and development of several crops under both optimal and stressful conditions. Biofertilisers are products containing living microorganisms or natural substances that are able to improve chemical and biological soil properties, stimulating plant growth, and restoring soil fertility. This review is aimed at reporting developments in the processing of MBS and MBF, summarising the biologically-active compounds, and examining the researches supporting the use of MBS and MBF for managing productivity and abiotic stresses in crop productions. Microalgae are used in agriculture in different applications, such as amendment, foliar application, and seed priming. MBS and MBF might be applied as an alternative technique, or used in conjunction with synthetic fertilisers, crop protection products and plant growth regulators, generating multiple benefits, such as enhanced rooting, higher crop yields and quality and tolerance to drought and salt. Worldwide, MBS and MBF remain largely unexploited, such that this study highlights some of the current researches and future development priorities.

Saponins, a group of glycosidic compounds present in several plant species, have aglycone moieties that are formed using triterpenoid or steroidal skeletons. In spite of their importance as antimicrobial compounds and their possible benefits for human health, knowledge of the genetic control of saponin biosynthesis is still poorly understood. In the Medicago genus, the hemolytic activity of saponins is related to the nature of their aglycone moieties. We have identified a cytochrome P450 gene (CYP716A12) involved in saponin synthesis in Medicago truncatula using a combined genetic and biochemical approach. Genetic loss-of-function analysis and complementation studies showed that CYP716A12 is responsible for an early step in the saponin biosynthetic pathway. Mutants in CYP716A12 were unable to produce hemolytic saponins and only synthetized soyasaponins, and were thus named lacking hemolytic activity (lha). In vitro enzymatic activity assays indicate that CYP716A12 catalyzes the oxidation of β-amyrin and erythrodiol at the C-28 position, yielding oleanolic acid. Transcriptome changes in the lha mutant showed a modulation in the main steps of triterpenic saponin biosynthetic pathway: squalene cyclization, β-amyrin oxidation, and glycosylation. The analysis of CYP716A12 expression in planta is reported together with the sapogenin content in different tissues and stages. This article provides evidence for CYP716A12 being a key gene in hemolytic saponin biosynthesis.

Genetic progress for forage quality has been poor in alfalfa (Medicago sativa L.), the most-grown forage legume worldwide. This study aimed at exploring opportunities for marker-assisted selection (MAS) and genomic selection of forage quality traits based on breeding values of parent plants. Some 154 genotypes from a broadly-based reference population were genotyped by genotyping-by-sequencing (GBS), and phenotyped for leaf-to-stem ratio, leaf and stem contents of protein, neutral detergent fiber (NDF) and acid detergent lignin (ADL), and leaf and stem NDF digestibility after 24 hours (NDFD), of their dense-planted half-sib progenies in three growing conditions (summer harvest, full irrigation; summer harvest, suspended irrigation; autumn harvest). Trait-marker analyses were performed on progeny values averaged over conditions, owing to modest germplasm × condition interaction. Genomic selection exploited 11,450 polymorphic SNP markers, whereas a subset of 8,494 M. truncatula-aligned markers were used for a genome-wide association study (GWAS). GWAS confirmed the polygenic control of quality traits and, in agreement with phenotypic correlations, indicated substantially different genetic control of a given trait in stems and leaves. It detected several SNPs in different annotated genes that were highly linked to stem protein content. Also, it identified a small genomic region on chromosome 8 with high concentration of annotated genes associated with leaf ADL, including one gene probably involved in the lignin pathway. Three genomic selection models, i.e., Ridge-regression BLUP, Bayes B and Bayesian Lasso, displayed similar prediction accuracy, whereas SVR-lin was less accurate. Accuracy values were moderate (0.3–0.4) for stem NDFD and leaf protein content, modest for leaf ADL and NDFD, and low to very low for the other traits. Along with previous results for the same germplasm set, this study indicates that GBS data can be exploited to improve both quality traits (by genomic selection or MAS) and forage yield.

In the Medicago genus, triterpene saponins are a group of bioactive compounds extensively studied for their different biological and pharmaceutical properties. In this work, the CRISPR/Cas9-based approach with two single-site guide RNAs was used in Medicago truncatula (barrel medic) to knock-out the CYP93E2 and CYP72A61 genes, which are responsible for the biosynthesis of soyasapogenol B, the most abundant soyasapogenol in Medicago spp. No transgenic plants carrying mutations in the target CYP72A61 gene were recovered while fifty-two putative CYP93E2 mutant plant lines were obtained following Agrobacterium tumefaciens-mediated transformation. Among these, the fifty-one sequenced plant lines give an editing efficiency of 84%. Sequencing revealed that these lines had various mutation patterns at the target sites. Four T0 mutant plant lines were further selected and examined for their sapogenin content and plant growth performance under greenhouse conditions. The results showed that all tested CYP93E2 knock-out mutants did not produce soyasapogenols in the leaves, stems and roots, and diverted the metabolic flux toward the production of valuable hemolytic sapogenins. No adverse influence was observed on the plant morphological features of CYP93E2 mutants under greenhouse conditions. In addition, differential expression of saponin pathway genes was observed in CYP93E2 mutants in comparison to the control. Our results provide new and interesting insights into the application of CRISPR/Cas9 for metabolic engineering of high-value compounds of plant origin and will be useful to investigate the physiological functions of saponins in planta.