Aluminum (Al) toxicity is a serious environmental constraint facing crop production in acidic soils, primarily due to the oxidative damage it causes to plant tissues. Alfalfa (Medicago sativa), a globally important forage crop, is highly susceptible to Al-induced stress, necessitating the development of Al-tolerant cultivars for sustainable forage production. In this study, we investigated the regulatory role of miR156 in Al stress response in alfalfa. Transcript analysis revealed significant downregulation of miR156 in alfalfa roots after 8 h of Al exposure, suggesting a negative role for miR156 in response to Al. To further investigate the role of miR156 in regulating agronomic traits and alfalfa’s Al tolerance, we utilized the short tandem target mimic (STTM) method to silence miR156 in alfalfa (MsSTTM156), which led to an upregulation of SQUAMOSA PROMOTER BINDING-LIKE (SPL) target genes, albeit with variable miR156 dose-dependent effects across different transgenic genotypes. Morphological characterization of MsSTTM156 plants revealed significant negative changes in root architecture, root and shoot biomass, as well as flowering time. Under Al stress, overexpression of miR156 in alfalfa (MsmiR156OE) resulted in stunted growth and reduced biomass, whereas moderate MsmiR156 silencing enhanced root dry weight and increased stem basal diameter. In contrast, MsmiR156OE reduced plant height, stem basal diameter, shoot branching, and overall biomass under Al stress conditions. At the molecular level, silencing miR156 modulated the transcription of cell wall-related genes linked to Al tolerance, such as polygalacturonase 1(MsPG1) and polygalacturonase 4 (MsPG4). Furthermore, miR156 influenced the expression of indole-3-acetic acid (IAA) transport-related genes auxin transporter-like protein (MsAUX1) and auxin efflux carrier components 2 (MsPIN2), with MsSTTM156 and MsmiR156OE plants showing lower and higher transcript levels, respectively, upon Al exposure. These findings reveal the multi-layered role of miR156 in mediating Al tolerance, providing valuable insights into the genetic strategies that regulate response to Al stress in alfalfa.

Sainfoin ( Onobrychis viciifolia ) is a forage legume crop with resurgent popularity due to its anti-bloating and anthelmintic properties attributed to its relatively high content of condensed tannins. Although this property of sainfoin is desirable, sainfoin lags behind other popular forage crops in attributes, such as biomass yield, waterlogging tolerance, and stand persistence. Traditional breeding efforts to improve sainfoin have been constrained by its tetraploid genome and obligatory outcrossing nature. To accelerate the development of competitive cultivars using molecular-based approaches, a method for stable genetic transformation was needed. Here we describe a method for Agrobacterium tumefaciens -mediated transformation of sainfoin ‘AAC Mountainview’ cotyledons using A. tumefaciens strain GV3101 that produces an average transformation efficiency of 10%. We used the FB_35S::RUBY binary vector for transformation which harbors the RUBY reporter gene to enable quick and easy identification of transformation events early in the tissue culturing process. This research establishes the foundation for future advances in sainfoin through molecular-based improvement.