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Leucine-rich repeat-receptor-like kinase (LRR-RLK) is a large subfamily of plant RLKs; however, its role in cold tolerance is still unknown. A novel cold tolerance LRR-RLK gene (MtCTLK1) in Medicago truncatula was identified using the transgenic lines overexpressing MtCTLK1 (MtCTLK1-OE) and mtctlk1 lines with Tnt1 retrotransposon insertion. Compared with the wild-type, MtCTLK1-OE lines had increased cold tolerance and mtctlk1 showed decreased cold tolerance. The impaired cold tolerance in mtctlk1 could be complemented by the transgenic expression of MtCTLK1 or its homolog MfCTLK1 from Medicago falcata. Antioxidant enzyme activities and proline accumulation as well as transcript levels of the associated genes were increased in response to cold, with higher levels in MtCTLK1-OE or lower levels in mtctlk1 lines as compared with wild type. C-Repeat-Binding Factors (CBFs) and CBF-dependent cold-responsive genes were also induced in response to cold, and higher transcript levels of CBFs and CBF-dependent cold-responsive genes were observed in MtCTLK1-OE lines whereas lower levels in mtctlk1 mutants. The results validate the role of MtCTLK1 or MfCTLK1 in the regulation of cold tolerance through the CBF pathway, antioxidant defense system and proline accumulation. It also provides a valuable gene for the molecular breeding program to improve cold tolerance in crops.

The flora of the Altai Mountains is characterized by a rich variety of representatives of the Fabaceae family, which are found in a wide range of habitats and under various anthropogenic loads. The genera Astragalus L. and Oxytropis DC. are the most numerous in terms of species diversity. Many representatives of the genera Vicia L., Trifolium L., Hedysarum L., Latirus L., Melilotus Mill., Medicago L., Pisum L. are good forage plants. The vegetation in the steppe grasslands of the Altai Mountains indicates the presence of predominantly intensive grazing farm animals, in which there is the formation of transient communities by reducing the participation of the prey species and the role of not eaten, eaten bad, harmful and poisonous plants. At the same time, the share of the legume component in the living ground phytomass in meadow steppes is quite high (25–50 %), in real steppes it is about 5–28%, and in desolate steppes – 11–17 %, which is largely due to the species diversity of legumes and their adaptability to natural, climatic and anthropogenic factors. Species Onobrychis arenaria, Medicago falcata are found in the meadow steppe, and Astragalus austrosibiricus, A. tibetanus, A. brevifoliu, and Oxytropis argentata are found in the steppe pastures, which develop a good green mass and have relatively high morphometric indicators. Astragalus testiculatus, A. laguroides, A. dilutes, Oxytropis pumila, Hedysarum gmelinii, Gueldenstaedtia monophylla, and harmful pasture plants – Thermopsis mongolica, Caragana pygmaea – are adapted to drier stony habitats. Of the wild relatives of cultivated plants from familia Fabaceae in the flora of the Altai Mountains there are more than 10 species. According to the results of environmental testing of 13 varieties of annual legumes (Vicia L., Pisum L., Glycine L.), it was found that they are characterized by higher rates of green mass yield (by 12–16%) than traditional varieties of these crops in the mid-mountain zone of the Altai Republic.

Six graded levels of dietary alfalfa Medicago falcata L. powder (0, 5, 10, 15, 20 and 25 g/kg diet, respectively) were fed to Lateolabrax japonicus (initial body weight: 11.41 ± 0.06 g; initial body length: 10.33 ± 0.12 cm) in triplicate groups for 4 weeks in a flow-through system to investigate the effects of alfalfa powder on the growth performance, nonspecific immunity and digestive enzyme activity. The results showed that alfalfa powder reduced the feed conversion ratio (FCR), with the lowest value obtained at 20 g/kg diet among all groups (p

Auxin induced in root culture (AIR12) is a single gene in Arabidopsis and codes for a mono-heme cytochrome b, but it is unknown whether plant AIR12 is involved in abiotic stress responses. MfAIR12 was identified from Medicago falcata that is legume germplasm with great cold tolerance. Transcript levels of MfAIR12 and its homolog MtAIR12 from Medicago truncatula was induced under low temperature. Overexpression of MfAIR12 led to the accumulation of H2 O2 in apoplast and enhanced cold tolerance, which was blocked by H2 O2 scavengers, indicating that the increased cold tolerance was dependent upon the accumulated H2 O2 . In addition, declined cold tolerance was observed in Arabidopsis mutant air12, which could be restored by expressing MfAIR12. Compared to the wild type, higher levels of ascorbic acid and ascorbate redox state, as well as transcripts of the C repeat/dehydration responsive element-binding factor (CBF) transcription factors and their downstream cold-responsive genes, were observed in MfAIR12 transgenic lines, but lower levels of those in air12 mutant. It is suggested AIR12 confers cold tolerance as a result of the altered H2 O2 in the apoplast that is signaling in the regulation of CBF cold response pathway and ascorbate homeostasis.

Background. The collection of wild yellow alfalfas from the Medicago falcata s.l. complex maintained at the N.I. Vavilov All-Russian Institute of Plant Genetic Resources (VIR) is a unique genetic stock collected while exploring the ex-USSR territories from 1924 to 2018. It includes Medicago falcata L. proper, M. borealis Grossh., M. romanica Prod., M. quasifalcata Sinsk., M. difalcata Sinsk., and M. glandulosa David. Gap analysis makes it possible to assess the sufficiency of VIR’s collecting efforts in a certain locality or administrative unit and plan further collecting activities to ensure comprehensive conservation of sickle alfalfa genetic diversity.Material and methods. Databases of the Perennial Forage Crop Genetic Resources Department at VIR, and digital maps of administrative subdivisions of Russia and neighboring countries served as the research material. The ex-USSR administrative subdivisions were accepted as operational units. For each of them, the number of ser. Falcatae Vass. species and the number of accessions per administrative subdivision were taken into account. Statistical data processing included construction of frequency tables and differentiated mapping.Results. The coordinates of collection sites were identified for 446 accessions. Collections of six wild species within the studied complex were analyzed across the ex-USSR space, and in more detail in Krasnodar Territory.Conclusion. The target areas were identified for further collecting activities. For Russia, the prioritized regions where the collection could be replenished are the northern frontier of the area of sickle alfalfa distribution in the European part and its eastern borderline in Siberia, both characterized by extreme environmental conditions. To increase the geographic diversity of the collection, additional collecting efforts are required, along with mobilization of accessions from the neighboring countries.

Topiciality. Flora of Azerbaijan has a diverse vegetation cover and is rich in choice. However, today these sources of raw materials are not fully used to meet the needs of medicine and national economy. We conducted a resource reconnaissance survey of species of the genus Medicago in Azerbaijan and identified regions of mass growth. New natural habitats are located in different environmental conditions and it is possible to change the diagnostic features of the morphological and anatomical structure of plant materials, as well as the chemical composition. The combination of additional morphological and anatomical features allows a more accurate determination of the authenticity of the raw material. Aim. To study the diagnostic signs of morphological and anatomical structure of Medicago falcata L. raw materials from the flora of Azerbaijan. Materials and methods. Plant samples were fixed in a solution made in 0.1 M phosphate buffer (pH = 7.4), containing 2.5 % glutar-aldehyde, 2 % paraformal-aldehyde, 4 % sucrose, and 0.1 % picric acid. The fixed materials have been submitted to the Electron Microscopy Laboratory of the Azerbaijan Medical University for study by electron microscopy. Samples have been postfixed in 1 %-osmium tetraoxide solution prepared in phosphate buffer (pH 7.4) within two hours after being left in the same fixer for one day. Araldite-Epon blocks made from materials using general methods adopted in electron microscopy. The semi-thin (1-2 µm) sections from the blocks taken on a EM UC7 (Leica, Germany) ultramicrotome, stained with methylene blue, azure II, and basic fuchsin were observed under microscope Primo Star (Zeiss, Germany) and images of required parts were shot with EOS D650 (Canon, Chine) digital camera. Results and discussion. Multicellular trichome covered on both leaf sides. Adaxial epidermis a sheet plate have sinuous cells, and abaxial epidermis have differed slightly sinuous cells with clear-shaped walls. In leaflet anatomy of plant vascular bundles are of the collateral type. Stomatas are located from both sides of a leaf. Stomata belong to anisocytic structure. The epidermis of the stem consists of elongated thick-walled cells with anisocytic stomata. The calyx is densely covered by trichomes. The outer epidermis of cross-section of the keel petals cells is the wing horns cells form. And inner epidermis consists of oval cells. Mesophyll cells inside the leaf consist of loose spongy cells on the underside with several conducting bundles (dorsoventral). Conclusions . Morphological and anatomical studying of raw material Medicago falcata L. has shown, that there are prominent features of a structure:four large conductive bundles are located on the cross section of the stem at the corners of the ribs on two sides. Between angular conductive bundles, there are three conductive bundles, and the other two sides are located along one conductive bundle. Therefore, M. Falcata L. differs from M. Sativa L. The epidermis of the stem consists of elongated thickened-walls cells with anisocytic stomata. The indicated diagnostic morphological and anatomical characters could be used in the compilation of a monograph and in identifying plant materials on the raw material of Medicago falcata L.

Background Low temperature is one of the main environmental factors that limits crop growth, development, and production. Medicago falcata is an important leguminous herb that is widely distributed worldwide. M. falcata is related to alfalfa but is more tolerant to low temperature than alfalfa. Understanding the low temperature tolerance mechanism of M. falcata is important for the genetic improvement of alfalfa. Results In this study, we explored the transcriptomic changes in the roots of low-temperature-treated M. falcata plants by combining SMRT sequencing and NGS technologies. A total of 115,153 nonredundant sequences were obtained, and 8849 AS events, 73,149 SSRs, and 4189 lncRNAs were predicted. A total of 111,587 genes from SMRT sequencing were annotated, and 11,369 DEGs involved in plant hormone signal transduction, protein processing in endoplasmic reticulum, carbon metabolism, glycolysis/gluconeogenesis, starch and sucrose metabolism, and endocytosis pathways were identified. We characterized 1538 TF genes into 45 TF gene families, and the most abundant TF family was the WRKY family, followed by the ERF, MYB, bHLH and NAC families. A total of 134 genes, including 101 whose expression was upregulated and 33 whose expression was downregulated, were differentially coexpressed at all five temperature points. PB40804, PB75011, PB110405 and PB108808 were found to play crucial roles in the tolerance of M. falcata to low temperature. WGCNA revealed that the MEbrown module was significantly correlated with low-temperature stress in M. falcata. Electrolyte leakage was correlated with most genetic modules and verified that electrolyte leakage can be used as a direct stress marker in physiological assays to indicate cell membrane damage from low-temperature stress. The consistency between the qRT-PCR results and RNA-seq analyses confirmed the validity of the RNA-seq data and the analysis of the regulatory mechanism of low-temperature stress on the basis of the transcriptome. Conclusions The full-length transcripts generated in this study provide a full characterization of the transcriptome of M. falcata and may be useful for mining new low-temperature stress-related genes specific to M. falcata. These new findings could facilitate the understanding of the low-temperature-tolerance mechanism of M. falcata.

Alfalfa (Medicago sativa L., Medicago varia Mart., Medicago falcata L.) is a perennial leguminous plant well-known as the queen of forages cultivated all over the world. The general biology and morphology of the plant has been described in detail. The typical inflorescence of the plant is raceme. Due to the multistep inbreeding process in this cross-pollinated species, different mutant forms have been found in inbred progenies. They include long racemes, panicle-like racemes (with fertile and sterile flowers), complicated branched racemes, and fasciated inflorescences. The fasciation trait was discovered first in long racemes and then it was introduced into every mutant inflorescence type by hand pollination. By means of pair hybridization, transitional forms of some mutants were isolated and the new mutant forms combined two or three mutant genes. New gene names are proposed for new duplex and triplex mutant types: lpfas, pi1lpfas, brilpfas. Medicago truncatula is a conventional model species for legume genome research. M. truncatula and alfalfa share highly conserved nucleotide sequences and exhibit nearly perfect synteny between the two genomes. The knowledge about inflorescence development in model M. truncatula plants adds to understanding the genetic nature of mutant inflorescence development in alfalfa; therefore, we compiled the information on the genetic regulation of inflorescence development in M. truncatula. The M. truncatula mutant mtpim has a complicated inflorescence structure resembling panicle-like inflorescence in alfalfa. Presently, it is known that the inflorescence architecture in M. truncatula is controlled by spatiotemporal expression of MtTFL1, MtFULc, MtAP1, and SGL1 through reciprocal repression. Some mutants isolated in M. truncatula resemble alfalfa mutants in phenotype. The mutant generated by retrotransposon insertion mutagenesis and named sgl1-1 has a cauliflower-like phenotype looking just like the cauliflower mutant in alfalfa. New data concerning genes regulating inflorescence development in model legumes approach us to understanding the phenomenon of inflorescence mutations in alfalfa. The information of inflorescence mutants in nonmodel crops may augment our knowledge of plant development and help crop improvement.

Background An eukaryotic translation elongation factor-2 (eEF-2) plays an important role in protein synthesis, however, investigation on its role in abiotic stress responses is limited. A cold responsive eEF2 named as MfEF2 was isolated from yellow-flowered alfalfa [Medicago sativa subsp. falcata (L.) Arcang, thereafter M. falcata], a forage legume with great cold tolerance, and transgenic tobacco (Nicotiana tabacum L.) plants overexpressing MfEF2 were analyzed in cold tolerance and proteomic profiling was conducted under low temperature in this study. Results MfEF2 transcript was induced and peaked at 24 h and remained at the high level during cold treatment up to 96 h. Overexpression of MfEF2 in trasngenic tobacco plants resulted in enhanced cold tolerance. Compared to the wild type, transgenic plants showed higher survival rate after freezing treatment, higher levels of net photosynthetic rate (A), maximum photochemical efciency of photosystem (PS) II (Fv/Fm) and nonphotochemical quenching (NPQ) and lower levels of ion leakage and reactive oxygen species (ROS) production after chilling treatment. iTRAQ-based quantitative proteomic analysis identified 336 differentially expressed proteins (DEPs) from leaves of one transgenic line versus the wild type after chilling treatment for 48 h. GO and KEGG enrichment were conducted for analysis of the major biological process, cellular component, molecular function, and pathways of the DEPs involving in. It is interesting that many down-regulated DEPs were grouped into “photosynthesis” and “photosynthesis-antenna”, such as subunits of PSI and PSII as well as light harvesting chlorophyll protein complex (LHC), while many up-regulated DEPs were grouped into “spliceosome”. Conclusions The results suggest that MfEF2 confers cold tolerance through regulating hundreds of proteins synthesis under low temperature conditions. The elevated cold tolerance in MfEF2 transgenic plants was associated with downregulation of the subunits of PSI and PSII as well as LHC, which leads to reduced capacity for capturing sunlight and ROS production for protection of plants, and upregulation of proteins involving in splicesome, which promotes alternative splicing of pre-mRNA under low temperature. Electronic supplementary material The online version of this article (10.1186/s12870-019-1826-7) contains supplementary material, which is available to authorized users.

Seeding legumes into degraded grasslands may effectively replace nitrogen fertilization and restore ecosystem stability by retaining or increasing native plant species diversity while increasing forage production. However, previous research indicated limited legume persistence and potential plant community diversity loss following seeding of legumes into grasslands, constraining successful restoration of degraded grasslands. Our research defined optimal management thresholds for successful legume establishment, reconciling both environmental conservation and production priorities. Large areas of degraded steppe in Inner Mongolia, northeast China, were seeded to either native alfalfa (yellow‐flowered alfalfa: Medicago falcata L.), cultivated alfalfa (purple‐flowered alfalfa: Medicago sativa L.), or left nonseeded (control). Within each seeded area, management treatments (P fertilization or mowing) were assigned to plots in the second year of alfalfa establishment. Our results indicated P amendments and mowing improve native alfalfa establishment, increasing plant productivity while maintaining diverse plant communities, thereby successfully rehabilitating degraded meadow steppe grasslands. However, cultivated alfalfa was not persistent under any management treatment. Alfalfa shoot N and P concentration was linearly related to plant‐available P, indicating P fertilization differentially benefits alfalfa compared with the dominate C₃ grass, Leymus chinensis (Trin.) Tzvel. Mowing successfully increased plant species diversity and plant species asynchrony; however, mowing did not alter the temporal stability of plant community productivity, potentially maintaining or enhancing ecosystem stability. Our research showed promise that native alfalfa seeding combined with P fertilization and mowing can restore degraded grasslands to support both forage production and diverse plant communities.

According to climate change scenarios, central Europe may expect extending drought periods during summer. Lower water availability may influence the ruminal digestion of individual forage legume species differently. To test this hypothesis, Lotus corniculatus L. (var. Bull), Medicago lupulina L. (var. Ekola), Medicago falcata L. (wild seeds) and Trifolium repens L. (var. Rivendel) were each grown in parallel lots of control and drought-stressed monocultures. Rainout shelters (installed in May 2011 on a regrowth after first cut until harvest in mid of June) withheld rainfall of 40 mm in the drought stress treatment. Samples of dried (60°C) and milled (5 mm screen) forage legumes were incubated in a simulation experiment using Rusitec to assess drought effects on parameters for microbial metabolism. Degradability of dry matter and organic matter as well as methane production decreased in incubations with drought-stressed compared to control variants of legume species. Degradability of crude protein, neutral detergent fibre, acid detergent fibre and residual organic matter including non-fibre carbohydrates and lipids were affected by interactions between drought stress and species. Significant interactions were also found for ammonia concentrations, molar SCFA proportions and the microbial communities. It is concluded that drought stress for growing forage legumes influences their ruminal degradation and fermentation as well as the ruminal microbial communities of Bacteria and Archaea differently in a legume species-dependent manner.

Alfalfa (Medicago sativa) is an important forage crop belonging to the Fabaceae family. It is cultivated across the world for fodder and originated in Asia. Alfalfa cultivar Regen-SY was used in this study which is a hybrid of first-generation self-parents from Regen-S (M. sativa) and Regen-Y (Medicago falcata) research cultivars. The main objective of the study was to optimize conditions for the isolation and liquid culture of alfalfa Regen-SY protoplasts. Several factors like enzyme combination, incubation time, plant age, centrifugation speed and shaker speed affecting protoplast isolation and culture were optimized in the study. The yield and viability of the protoplasts was determined by using hemocytometer and Fluorescein diacetate (FDA) staining respectively. Results showed that factors like enzyme combination, incubation time, plant age, centrifugation speed and Mannitol concentration significantly (p ≤ 0.05) affect protoplast yield and viability whereas shaker speed didn’t result in any significant difference in the yield and viability of protoplasts. Using optimum conditions protoplasts were cultured in the liquid medium and microcalli formation was achieved after five weeks of the culture. The protocol established in this study will assist researchers in the isolation and culture of protoplasts in alfalfa and will accelerate the research processes like protoplast fusion and genetic engineering.

Medicago and its relatives, Trigonella and Melilotus comprise the most important forage resources globally. The alfalfa selected from the wild relatives has been cultivated worldwide as the forage queen. In the Flora of China, 15 Medicago, eight Trigonella, and four Melilotus species are recorded, of which six Medicago and two Trigonella species are introduced. Although several studies have been conducted to investigate the phylogenetic relationship within the three genera, many Chinese naturally distributed or endemic species are not included in those studies. Therefore, the taxonomic identity and phylogenetic relationship of these species remains unclear. In this study, we collected samples representing 18 out of 19 Chinese naturally distributed species of these three genera and three introduced Medicago species, and applied an integrative approach by combining evidences from population-based morphological clusters and molecular data to investigate species boundaries. A total of 186 individuals selected from 156 populations and 454 individuals from 124 populations were collected for genetic and morphological analyses, respectively. We sequenced three commonly used DNA barcodes (trnH-psbA, trnK-matK, and ITS) and one nuclear marker (GA3ox1) for phylogenetic analyses. We found that 16 out of 21 species could be well delimited based on phylogenetic analyses and morphological clusters. Two Trigonella species may be merged as one species or treated as two subspecies, and Medicago falcata should be treated as a subspecies of the M. sativa complex. We further found that major incongruences between the chloroplast and nuclear trees mainly occurred among the deep diverging lineages, which may be resulted from hybridization, incomplete lineage sorting and/or sampling errors. Further studies involving a finer sampling of species associated with large scale genomic data should be employed to better understand the species delimitation of these three genera.

Industrial waste sites, although extremely difficult to revegetate, may be suitable for rare plants such as Orobanche lutea that are condemned to extinction due to their low ability to compete in their natural habitats. The presence of potentially toxic metals seems to facilitate the expansion of O. lutea (parasitizing Medicago falcata) and was found in hundreds of exemplars per m2 in south Poland and potentially could spread to other localities, causing yield loss in agricultural plants. The main aim of this research was to characterize the interaction between the host, the parasitic plant and symbiotic microbes under different metal concentration in the substratum. The parasite was more common on more polluted soil and when the parasite was connected to the host, potentially toxic metals (Zn, Cd and Pb) were shared by both plants; thus, the content and concentration of these potentially toxic metals in the host were lower than those in plants without parasites. While the performance index (PIABS) of photosynthesis was lower in parasitized plants on control soil, on metal-rich industrial waste soil, PIABS was higher in the parasitized plants than in cases where M. falcata grew alone. This result suggests a role of this parasite in toxicity attenuation, although the biomass of parasitized plants and those growing on polluted sites was lower than that in control sites. In the described case, mycorrhizal colonization and arbuscular richness in M. falcata were even more highly developed on polluted sites than in control ones. The data presented support the hypothesis that the expansion of O. lutea is most likely supported by the increased concentrations of Zn and Cd in areas connected with industrial waste. Although, on industrial wastes the host yield was decreased in the parasite presence, its photosynthetic capacity was even increased.

When identifying biological materials popular is the use of universal DNA-barcode. At present, the study of the genetic diversity of the flora of Northern Kazakhstan at different taxonomic levels (generic, species and subspecies) was not carried out using DNA markers. The purpose of this work was the identification and phylogenetic analysis for flora of national parks «Bayanaul» and «Burabay» by using ITS. As a source material, plant material was used, which was collected from May to July months 2015-2017 in national parks «Bayanaul» (90 species ) and «Burabay» (69 species). As a result of the research, 64 endemic and rare plant species of two national parks were visually identified. The level of genetic diversity of plants of the family Fabaceae is estimated. In phylogenetic analysis, the Lathyrus pisiformis, Trifolium repens, Medicago falcata, Oxytropis floribunda, Glycyrrhiza uralensis Fisch., the family is divided into 2 clusters of five plant genera. The intraspecific level of the genetic diversity of the Juniperus sabina L. formed two subclusters, depending on the type of population, where the Tajima index was 1.183269. The obtained data on the basis of classical approaches of taxonomy and molecular genetic methods will allow to create in the future an electronic database on endemic, rare and economically valuable plant species. Key words: Bayanaul SNNP, Burabay SNNP, flora, DNA genotyping, ITS (internal transcribed spacer)

The genetic resources of wild populations of Medicago falcate L. of the Cretaceous South of the Srednerussky Upland in ecotopic conditions of chalky slopes and floodplain meadows in the basins of small rivers Tikhaya Sosna and Manjokha were studied to find the source material for breeding work to create varieties for different cultivation conditions. Evaluation of phytocenotic features, density of cenopopulations, forage and seed species was carried out. The species M. falcata forms, both on meadows in floodplains and on chalk slopes, full-membered normal cenopopulations, which have a continual (continuous) distribution of individuals by age groups, stable in time and in space. The centralized ontogenetic spectrum indicates the stable status of M. falcata cenopopulations in plant communities in various ecotopes of small river basins. The density of individuals of M. falcata was on average 18.9 % higher in cenopopulations of floodplains than in chalk slopes. The density of generative individuals in all studied cenopopulations was on average close and varied within 81.2-83.7 %, which indicates their stability in time and high adaptive potential. The weight of one fruiting model plant of floodplain meadows was 2.28 times higher than on chalky slopes. Seed yield on floodplain meadows was 2.02 times higher than on chalky slopes. Valuable source material for breeding M. falcatum varieties adapted to different ecotopic conditions was obtained.

As a perennial forage crop, alfalfa (Medicago L.) can be cultivated in marginal lands and has a high yield and good quality of high-protein content. Alfalfa has deep vertical roots; this species is able to absorb even depth waters about 5 m depth and more. This advantage saves plant’s life in long term drought. The objective of this study was to compare the performance of drought stress during germination and growth stage of 2 species (Medicago sativa, Medicago falcata) and 2 varieties (Mongolian Medicago varia Marthz var.Burgaltai and Inner Mongolian Medicago varia Martyn var.Nutag Belcheer-2) in laboratory condition. In order to expose the drought stresses in plant by polyethylene glycol (PEG 4000) with 0 (control), -0.3, -0.5 and -1.0 MPa for 2 weeks respectively. The results represented that Medicago sativa had higher seed germination percentage than other species and varieties. Water uptake, dry matter index are observed in M.sativa higher than others.The highest chlorophyll content was in Medicago varia Marthz var.Burgaltai. The highest result of root, shoot length and weight were observed in Medicago sativa. In contrast, the significantly lowest result of morphological parameters was in Mediago varia Martyn var.Nutag Belcheer-2. M.sativa is highly tolerant to drought stress among species. M.varia Marth var.Burgaltai was moderately tolerant to drought stress among varieties.

MfLEA3 is involved in protection of catalase activity and confers multiple abiotic stress tolerance. Late embryogenesis abundant (LEA) proteins are involved in plant growth, development and abiotic stress tolerance. A member of group 3 LEA proteins from Medicago sativa subsp. falcata (L.) Arcang, MfLEA3, was investigated in the study. MfLEA3 transcript was induced in response to cold, dehydration, and abscisic acid (ABA), while the cold-induced transcript of MfLEA3 was blocked by pretreatment with inhibitor of ABA synthesis. Constitutive expression of MfLEA3 led to enhanced tolerance to cold, drought, and high-light stress in transgenic tobacco plants. Compared to accumulated reactive oxygen species (ROS) in the wild-type in response to treatments with low temperature, drought, and high light, ROS were not accumulated in transgenic plants. Superoxide dismutase, catalase (CAT), and ascorbate-peroxidase activities were increased in all plants after treatments with the above stresses, while higher CAT activity was maintained in transgenic plants compared with wild-type. However, transcript level of CAT-encoding genes including CAT1, CAT2, and CAT3 showed no significant difference between transgenic plants and wild-type, indicating that the higher CAT activity was not associated with its gene expression. ABA sensitivity and transcripts of several ABA and stress-responsive genes showed no difference between transgenic plant and wild-type, indicating that ABA signaling was not affected by constitutive expression of MfLEA3. The results suggest that MfLEA3 may be involved in the protection of CAT activity and confers multiple abiotic stress tolerance.

The role of nitric oxide (NO) signaling in the cold acclimation of forage legumes was investigated in this study. Medicago sativa subsp. falcata (L.) Arcang. (hereafter M. falcata) is a forage legume with a higher cold tolerance than Medicago truncatula, a model legume. Cold acclimation treatment resulted in increased cold tolerance in both M. falcata and M. truncatula, which was suppressed by pretreatment with tungstate, an inhibitor of nitrate reductase (NR), and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), a scavenger of NO. Likely, NITRATE REDUCTASE 1 (NIA1), but not NIA2 transcript, NR activity, and NO production were increased after cold treatment. Treatments with exogenous NO donors resulted in increased cold tolerance in both species. Superoxide dismutase (SOD), catalase (CAT), and ascorbate-peroxidase (APX) activities and Cu,Zn-SOD2, Cu,Zn-SOD3, cytosolic APX1 (cAPX1), cAPX3 and chloroplastic APX1 (cpAPX1) transcript levels were induced in both species after cold treatment, which was suppressed by tungstate and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO). Treatment with exogenous NO resulted in enhanced activities of SOD, CAT, and APX. Moreover, higher levels of NIA1 transcript, NR activity, NO production, and antioxidant enzyme activities and transcripts were observed in M. falcata as compared with M. truncatula after cold treatment. The results suggest that NR-derived NO production and upregulated antioxidant defense are involved in cold acclimation in both species, while the higher levels of NO production and its derived antioxidant enzymes are associated with the higher cold tolerance in M. falcata as compared with M. truncatula.

A 3-year study (2016, 2017, and 2018) was conducted to compare the effects of alfalfa (ALF; cv. Yellowhead) (Medicago falcata L.) or sainfoin (SF; cv. AAC Mountainview) (Onobrychis viciifolia Scop.) in mixtures with either Russian wildrye (RWR; cv. Tom) (Psathyrostachys junceus [Fisch.]) or hybrid bromegrass (HBG; cv. Success) (Bromus riparius Rehm × B. inermis Leyss) on herbage dry matter yield (DMY) and nutritive value, and beef yearlings performance at two sites: Swift Current (Brown soil zone) and Lanigan (Thin Black soil zone), Saskatchewan, Canada. In 2015, in each site, sixteen 0.7-ha (in Lanigan) or 0.8-ha (in Swift Current) paddocks were established with replicated (n = 4) ALF-HBG, ALF-RWR, SF-HBG, or SF-RWR binary mixtures. Each year in August, yearling steers (16 ± 1 month and BW = 391 ± 1.7 kg and 350 ± 0.04 kg for Swift Current and Lanigan sites, respectively) were allocated to paddocks for an average 30-d grazing trial to utilize 40–60% of available herbage. The DMY (3629 ± 154 kg/ha) did not differ (P > 0.05) among mixtures at Swift Current. The HBG included (ALF-HBG and SF-HBG) mixtures produced 54% more (P

Thirteen-year monitoring of the vegetation growing in the industrial and adjacent areas of an oil refinery showed the prevalence of yellow medick (Medicago falcata L.) over other plant species, including alfalfa (Medicago sativa L.). A comparative field study of the two Medicago species established that yellow medick and alfalfa exhibited similar resistance to soil petroleum hydrocarbons and that the pollutant concentration in their rhizosphere was 30% lower than that in the surrounding bulk soil. In laboratory pot experiments, yellow medick reduced the contaminant content by 18% owing to the degradation of the major heavy oil fractions, such as paraffins, naphthenes, and alcohol and benzene tars; and it was more successful than alfalfa. Both species were equally effective in stimulating the total number of soil microorganisms, but the number of hydrocarbon-oxidizing microorganisms, including polycyclic aromatic hydrocarbon degraders, was larger in the root zone of alfalfa. In turn, yellow medick provided a favorable balance of available nitrogen. Both Medicago species equally stimulated the dehydrogenase and peroxidase activities of the soil, and yellow medick increased the activity of soil polyphenol oxidase but reduced the activity of catalase. The root tissue activity of catalase, ascorbate oxidase, and tyrosinase was grater in alfalfa than in yellow medick. The peroxidase activity of plant roots was similar in both species, but nondenaturing polyacrylamide gel electrophoresis showed some differences in the peroxidase profiles of the root extracts of alfalfa and yellow medick. Overall, this study suggests that the phytoremediation potentials of yellow medick and alfalfa are similar, with some differences.

Along with the wide distribution and use of alfalfa grass there is no comprehensive scientific pharmacognosy studies about representatives of the plants of this kind. Aim. To study the content of the group of true coumarins and 4-oxyderivatives which presence must be characteristic for the yellow alfalfa. Materials and methods. The determination of diagnostic anatomical features of the plant Medicago falcata L. subsp. romanica (Prodan) O. Schwarz & Klink has been carried out. The leaf petiole has such diagnostic characteristics: elongated epidermal cells, parenchymal or prosenchymal, 4-, or (rarely) 5-6 gonal; pubescence is of simple single-celled hairs with warty cuticle, which can be found on the upper and lower epidermis of the leaf. The stomatas are rare. The rosette is formed from basal epithelial cells near the base of the hairs. Results. 66 Components have been revealed by chromatography of native raw, 38 of components have been identified, namely - dihydrocoumarin (1.60%) and coumarin (13.13%). After the acid hydrolysis 52 components have been revealed, 41 of which have been identified. These substances are the complex of biologically active compounds: primary alcohols, aldehydes, ketones. 4-Oxycumarin derivatives have been identified: coumarin - 79.38% dihydrocoumarin - 4.95%, and 6-methylcoumarin - 0.39%. While studying the amino acid composition of the above-ground parts of alfalfa yellow we got interested in a significant amount of aspartic acid, proline and phenylalanine.

Iron deficiency is the most common nutritional disorder, affecting over 30% of the world’s human population. The primary method used to alleviate this problem is nutrient biofortification of crops so as to improve the iron content and its availability in food sources. The over-expression of ferritin is an effective method to increase iron concentration in transgenic crops. For the research reported herein, sickle alfalfa (Medicago falcata L.) ferritin was transformed into wheat driven by the seed-storage protein glutelin GluB-1 gene promoter. The integration of ferritin into the wheat was assessed by PCR, RT-PCR and Western blotting. The concentration of certain minerals in the transgenic wheat grain was determined by inductively coupled plasma-atomic emission spectrometry, the results showed that grain Fe and Zn concentration of transgenic wheat increased by 73% and 44% compared to non-transformed wheat, respectively. However, grain Cu and Cd concentration of transgenic wheat grain decreased significantly in comparison with non-transformed wheat. The results suggest that the over-expression of sickle alfalfa ferritin, controlled by the seed-storage protein glutelin GluB-1 gene promoter, increases the grain Fe and Zn concentration, but also affects the homeostasis of other minerals in transgenic wheat grain.

Universal Stress Proteins (USPs) are ubiquitously responsive to environmental stresses, but function of most of them remains largely unknown. A novel USP from Medicago falcata (MfUSP1) was identified in the present study. MfUSP1 transcript is induced by low temperature, salinity, osmotic stress, abscisic acid (ABA) and H2O2. Overexpression of MfUSP1 resulted in increased tolerance to multiple abiotic stresses including freezing, salinity, osmotic stress, and methyl viologen (MV) induced oxidative stress. Higher activities of superoxide dismutase (SOD), catalase (CAT) and ascorbate dependent peroxidase (APX) were observed in transgenic plants than in the wild type (WT) under the stressed conditions with lower reactive oxygen species (ROS) accumulation. Compared to WT, higher transcript levels of the genes encoding Cu, Zn-SOD, CAT, and APX were observed in transgenic plants under salinity and osmotic stress, and higher transcript levels of CAT and APX encoding genes under low temperature condition. Moreover, more proline was accumulated in transgenic plants than in WT under the stressed conditions, with upregulation of pyrroline-5-carboxylate synthetase 2 (P5CS2) and downregulation of proline oxidase 1 and 2 (PROX1 and PROX2). It is suggested that MfUSP1 confers tolerance to multiple abiotic stresses by regulating antioxidant defense and proline accumulation under stressed conditions.

The purpose of this study was to evaluate the effects of Chinese herbal medicines mixture (CHMM: consisted of asafoetida Ferula sinkiangensis K. M. Shen, Medicago falcata L.and Allium sativum) on feeding attraction activity (FAA), growth performance, nonspecific immunity and digestive enzyme activity of juvenile Japanese seabass (Lateolabrax japonicus). Six diets containing 0, 5, 10, 15, 20 and 25 g/kg CHMM were designed for the luring and feeding experiment. A clear attractive trend of the CHMM on Japanese seabass was found after a 5-day luring trial. The 28-day feeding experimental results indicated that dietary CHMM significantly influenced weight gain (WG) and specific growth rate (SGR) of fish (P < 0.05), with the highest WG and SGR observed in fish fed 20 g/kg CHMM diet. The feed conversion ratio (FCR) of each treatment group was significantly reduced by the CHMM. The body crude lipid content was significantly increased except for the 15 g/kg group (P < 0.05). The supplementation of CHMM could significantly increase the activity of lysozyme (LZM) and total superoxide dismutase (T-SOD) in serum with the highest values observed in the 20 mg/kg group while the content of total protein (TP) was increased significantly in the fish fed the diet with 10−15 g/kg CHMM (P < 0.05). Digestive enzymes including lipase (LPS) and trypsin activities were obviously affected by the CHMM and reached the highest among all groups at 20 g/kg diet. Regression analyse of the FAA, FCR, WG, SGR, TP, LZM, T-SOD, LPS and trypsin were performed, showing the optimal additional amount was 12.75, 15.09, 20.14, 20.00, 16.13, 18.31, 17.33, 21.38 and 21.17 g/kg, respectively. Comprehensive consideration of the current experimental results and the regression analyses, we suggested the additive suitable dosage of the CHMM was 20 g/kg diet.

The No apical meristem–Arabidopsis transcription activation factor–Cup-shaped cotyledon (NAC) proteins play vital roles in plant development processes and responses to abiotic stress. In this study, 146 unigenes were identified as NAC genes from wild Medicago falcata L. by RNA sequencing. Among these were 30 full-length NACs, which, except for MfNAC63, MfNAC64 and MfNAC91, contained a complete DNA-binding domain and a variable transcriptional activation region. Sequence analyses of MfNACs along with their Arabidopsis thaliana (L.) Heynh. counterparts allowed these proteins to be phylogenetically classified into nine groups. MfNAC35, MfNAC88, MfNAC79, MfNAC26 and MfNAC95 were found to be stress-responsive genes. The eight MfNAC genes that were chosen for further analysis had different expression abilities in the leaves, stems and roots of M. falcata. Additionally, their expression levels were regulated by salinity, drought and cold stress, and ABA. This study will be useful for understanding the roles of MfNACs in wild M. falcata and could provide important information for the selection of candidate genes associated with stress tolerance.

Ethylene responsive factor (ERF) subfamily transcription factors play an important role in plant abiotic and biotic stress tolerance. A cold responsive ERF, MfERF1, was isolated from Medicago falcata, an important forage legume that has great cold tolerance. Overexpression of MfERF1 resulted in an increased tolerance to freezing and chilling in transgenic tobacco plants, whereas down-regulation of the ortholog of MfERF1 in Medicago truncatula resulted in reduced freezing tolerance in RNAi plants. Higher transcript levels of some stress responsive genes (CHN50, OSM, ERD10C, and SAMS) and those involved in spermidine (Spd) and spermine (Spm) synthesis (SAMDC1, SAMDC2, SPDS1, SPDS2, and SPMS) and catabolism (PAO) were observed in transgenic plants than in wild type. However, neither Spd nor Spm level was accumulated in transgenic plants as a result of promoted polyamine oxidase activity. Transgenic plants had higher activities of antioxidants associated with the induced encoding genes including Cu, Zn-SOD, CAT1, CAT2, CAT3, and cpAPX and accumulated more proline associated with induced P5CS and reduced PROX2 transcription as compared with wild type. The results suggest that MfERF1 confers cold tolerance through promoted polyamine turnover, antioxidant protection, and proline accumulation.