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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

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.

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.

In this study, we aimed to understand effects and mechanisms of legume species and their relative abundance on soil N, species recruitment, and productivity in mixed grassland. We also assessed the utilization prospect of several legumes in natural grassland. In 2006, grass-legume combinations (GLCs) with different ratios (GLR) were established using Leymus chinensis and four legume species (Medicago ruthenica, Lespedeza daurica, Medicago falcata, and Medicago sativa). In 2009, plant and soil samples were taken to examine the differences in soil nitrogen (N) properties, biological N fixation (BNF), recruitment characteristics, and aboveground biomass of the community under different mixture patterns. Soil total N and available N concentration increased when legume component increased from GLR 1:0 (grass monoculture) to GLR 1:1 (grass:legume 1:1) but decreased as legume component increased further from GLR 1:3 (grass:legume 1:3) to GLR 0:1 (legume monoculture). GLR 1:1 had the highest BNF in most GLCs except for L. chinensis-M. falcata. For any GLR with legume, L. chinensis-M. sativa combination (L-MS) had higher soil total N, available N, water content, and BNF. The legume combinations with a GLR lower than 1:1 resulted in more species recruitments compared with the grass monoculture, and L-MS induced relatively more species recruitments than other GLCs, which were correlated with improved soil water and NO3 −-N status. The grassland productivity increased as the GLR decreased; in any GLRs with legume, higher productivity was found under L-MS combination, and current results showed that grassland productivity was positively linked to soil NO3 −-N availability influenced by grass-legume mixture. We concluded that the species and relative abundance of legumes had great impact on soil N status, species recruitment, and productivity in this temperate grassland ecosystem. In this study site, legume introduction can be considered as an alternative to N fertilization for increasing grassland productivity. M. sativa has the greatest economic and ecological potential species to be mixed into natural temperate steppe grassland, and grass-legume ratio 1:1 is the best combination in mixed communities.

Background Plasma membrane intrinsic proteins (PIPs), which belong to aquaporins (AQPs) superfamily, are subdivided into two groups, PIP1 and PIP2, based on sequence similarity. Several PIP2s function as water channels, while PIP1s have low or no water channel activity, but have a role in water permeability through interacting with PIP2. A cold responsive PIP2 named as MfPIP2-7 was isolated from Medicago falcata (hereafter falcata), a forage legume with great cold tolerance, and transgenic tobacco plants overexpressing MfPIP2-7 were analyzed in tolerance to multiple stresses including freezing, chilling, and nitrate reduction in this study. Results MfPIP2-7 transcript was induced by 4 to 12 h of cold treatment and 2 h of abscisic acid (ABA) treatment. Pretreatment with inhibitor of ABA synthesis blocked the cold induced MfPIP2-7 transcript, indicating that ABA was involved in cold induced transcription of MfPIP2-7 in falcata. Overexpression of MfPIP2-7 resulted in enhanced tolerance to freezing, chilling and NO3− deficiency in transgenic tobacco (Nicotiana tabacum L.) plants as compared with the wild type. Moreover, MfPIP2-7 was demonstrated to facilitate H2O2 diffusion in yeast. Higher transcript levels of several stress responsive genes, such as NtERD10B, NtERD10C, NtDREB1, and 2, and nitrate reductase (NR) encoding genes (NtNIA1, and NtNIA2) were observed in transgenic plants as compared with the wild type with dependence upon H2O2. In addition, NR activity was increased in transgenic plants, which led to alterations in free amino acid components and concentrations. Conclusions The results suggest that MfPIP2-7 plays an important role in plant tolerance to freezing, chilling, and NO3− deficiency by promoted H2O2 diffusion that in turn up-regulates expression of NIAs and multiple stress responsive genes. Electronic supplementary material The online version of this article (doi:10.1186/s12870-016-0814-4) contains supplementary material, which is available to authorized users.

Sexual hybridization is an important generator of biodiversity and a powerful breeding tool. Hybridization can also overcome ploidy barriers when it involves 2n gametes, as in the case of unilateral sexual polyploidization (USP) that has been utilized in several crops, among which alfalfa. This research was aimed at gaining insights into the effects of USP on genome methylation and on phenotypic traits in alfalfa, an important forage species. The Methylation-Sensi- tive Amplified Polymorphism technique was used to estimate the cytosine methylation changes occurring in a tetraploid (2n = 4x = 32) USP progeny from crosses between a diploid Medicago sativa subsp. falcata genotype that produces 2n eggs and a cultivated tetraploid Medicago sativa subsp. sativa variety. De novo methylation or demethylation in the USP progeny were observed for 13% of the detected genomic sites, indicating that methylation changes can be relevant. USP plants showed larger surface area of the leaf epidermis cells than both parents, but this did not result in larger leaf size or higher plant biomass. They displayed significant higher ovule sterility than the tetraploid parent, but normal fertility was observed in crosses with unrelated male testers. We conclude that hybridization and sexual polyploidization resulted in novel variation in terms of remodeling of the methylation landscape as well as changes in phenotypic traits in alfalfa.

Intersubspecific hybrids between Medicago sativa subsp. sativa and subsp. falcata show biomass yield heterosis in space planted plots. Relative biomass yield of alfalfa is known to differ in space planted versus sward plots, although the magnitude of the difference is variable. The objective of this study was to determine if and how much of the biomass yield heterosis observed in space planted alfalfa hybrids was retained in sward planted plots. Two falcata genotypes (WISFAL-6 and PI502453-1) and three elite sativa genotypes were crossed in a diallel mating design. Progeny were transplanted into space-planted plots and harvested three times per year or drilled into sward plots harvested four times per year. Hybrids of WISFAL-6 with the sativa genotypes produced as much first harvest biomass yield as intra-sativa hybrids in space-planted plots and more in sward plots. Yield in subsequent harvests was lower than intra-sativa hybrids in sward plots only. Hybrids of PI502453-1 with sativa were generally lower yielding than intra-sativa crosses. A moderate correlation was observed between biomass yield in space planted and sward plots. Heterosis expression in swards was lower than that in spaced planted nurseries for progeny of both falcata genotypes. The increased plant-to-plant competition in swards is the likely cause of the loss of heterosis.

Summary Semi-hybrid alfalfa cultivars offer the possibility of capturing non-additive genetic variation. Medicago sativa subsp. falcata and subsp. sativa have been shown to form a heterotic pattern for biomass yield. Objectives of this study were to examine morphological variation in a broad range of falcata germplasm and to determine how falcata morphological variation per se is related to the performance of falcata germplasm in hybrid crosses with subsp. sativa. Falcata genotypes from 40 populations spanning the subspecies native range were selected and biomass yield, plant width, plant height, growth angle, biomass density, plant maturity, and regrowth after cutting were measured on the genotypes and their hybrid progeny three times throughout the growing season. In addition weekly plant heights were measured and growth rates were determined with a Gompertz function. Falcata parental genotypes exhibited a full range of phenotypes for plant width, plant height, growth angle, density, and maturity. Heterosis was not only observed for biomass yield but also for plant width, plant height, and more erect growth habit. The top yielding sativa-falcata hybrids had increased plant width, plant height, and plant density. European germplasm was taller and had faster regrowth than Asian material. Sativa-falcata hybrids produced biomass yield superior to the mid-subspecies mean only after two to three weeks of growth prior to first and third harvests. Prior to second harvest, biomass production was inferior to the mid-subspecies mean for 30 days. Hybrids using falcata as one parent are not currently adapted to intensive harvest management due to their slower regrowth. Abbreviations: FC – Falcata clones; SC – Sativa clones; SFC – Sativa by falcata crosses; SSC – Sativa by sativa crosses

Three somatic hybrid plants produced by protoplast fusion between Medicago sativa and each of the three species Medicago coerulea, Medicago falcata and Medicago arborea have been analysed for the composition of their mitochondrial DNA. Restriction fragment length polymorphism (RFLP) analysis of mitochondrial genes in somatic hybrids and their parental lines showed various degrees of rearrangement. The M. sativa+M. coerulea hybrid retained all of the M. coerulea-specific bands but lost all the major M. sativa- specific bands. The M. sativa+M. falcata hybrid showed only M. sativa-specific bands together with non-parental bands, and the M. sativa+M. arborea hybrid showed a partial incorporation of bands from both parents together with non-parental bands. The three different outcomes were attributed mainly to differences in the genetic distance between the parents of each hybrid. Analysis of the sexual progeny of the M. sativa+M. coerulea hybrid showed that a residual mitochondrial DNA subunit of M. sativa was retained in the hybrid cytoplasm. This subunit was amplified and inherited in a mutually exclusive, allelic-like fashion with its M. coerulea homologous counterpart in the sexual progeny of the hybrid. Possible mechanisms for the partitioning of mitochondrial DNA in the generative lineage of the somatic hybrids are discussed in relation to the creation of new nucleus-cytoplasm assortments otherwise impossible to obtain by a sexual cross in Medicago.

Two simple, rapid and efficient protocols for theregeneration of transformed tetraploid lines ofalfalfa (Medicago falcata L.) have beendeveloped and compared. Leaf explants fromembryogenic lines 47/1-150 and 47/1-5 were inoculatedwith Agrobacterium tumefaciens containingconstructs carrying the nptII selectable markergene and promoter:gusA gene fusions under thecontrol of the CaMV 35S or Arabidopsis cdc2a,CycB1 and CycA2 promoters. In the firstregeneration system (the MSH system), inoculated leafexplants were incubated on MS medium supplemented with2,4-D and kinetin and then subcultured onto plantgrowth regulator-free MS medium in order to inducedirect somatic embryogenesis. In the secondregeneration system (the B5h system), the inoculatedexplants were incubated on B5h medium to induceindirect production of somatic embryos viaembryogenic callus. In both systems, an effectivekanamycin selection regime was employed and wasmaintained when the embryos were subcultured onto arecovery medium (Boi2Y) to promote further embryodevelopment. The use of Boi2Y medium was particularlyimportant for shortening the regeneration time andpromoting a higher frequency of healthy plantletproduction from the somatic embryos. The maturesomatic embryos were finally transferred to plantgrowth regulator-free MS medium for plantletformation. Transgenic plantlets were produced within10–14 weeks in the MSH system and 12–16 weeks in theB5h system. The MSH system appears to be the fastesttransformation system reported for leguminous speciesto date. Confirmation of transformation was obtainedusing a re-callusing assay on kanamycin and subsequentSouthern blot hybridisation and PCR analysis. Theability to induce expression of GUS activity in leafexplants containing the cell division cycle genepromoter:gusA constructs by 2,4-D treatment alsoproved to be a reliable indicator of transformation.

The aim of this study was to produce somatic hybrids between tetraploid (2n=4x=32) M. sativa and diploid (2n=2x=16) M. falcata and analyse their genomic structure. Protoplasts from genotypes selected for regeneration ability from the cultivar Rangelander of M. sativa and Wisfal-1 of M. falcata were electrofused. Seven somatic hybrid calli were produced and one of them regenerated plants. The hybrid nature of these plants and their genetic composition were assessed with morphological, cytological, and molecular analyses. The resulting plants were hyper-aneuploid (2n=33) and contained one extra long chromosome, indicating that a translocation had taken place. The presence of both types of parental sequences in the RAPDs analysis confirmed the true hybrid nature of the plants. Rearrangements within the parental genomes and the presence of somaclonal variation among hybrid plants were observed through an RFLP analysis of the nucleolar organizing region (NOR). The possible causes for the gross genomic alterations, and the suitability of this method for transferring useful agronomic traits from wild species to cultivated alfalfa, are discussed.

Drought is an important environmental factor that can affect rhizobial competition and N2 fixation. Three alfalfa (Medicago sativa L. and M. falcata L.) accessions were grown in pots containing soil from an irrigated (Soil 1) and a dryland (Soil 2) alfalfa field in northern Utah, USA. Mutants of three strains of Rhizobium meliloti Dang. from Pakistan (UL 136, UL 210, and UL 222) and a commercial rhizobial strain 102F51a were developed with various levels of resistance to streptomycin. Seeds inoculated with these individual streptomycin-resistant mutants were sown in the two soils containing naturalized rhizobial populations. Soils in the pots were maintained at −0.03, −0.5, and −1.0 MPa. After 10 weeks, plants were harvested and nodule isolates were cultured on agar medium with and without streptomycin to determine nodule occupancy (proportion of the nodules occupied by introduced rhizobial strains). Number of nodules, nodule occupancy, total plant dry weight, and shoot N were higher for Soil 1 than Soil 2. Number of nodules, plant dry weight, and shoot N decreased as drought increased from −0.03 to −1.0 MPa in the three alfalfa accessions. Rhizobial strains UL 136 and UL 222 were competitive with naturalized alfalfa rhizobia and were effective at symbiotic N2 fixation under drought. These results suggest that nodulation, growth, and N2 fixation in alfalfa can be improved by inoculation with competitive and drought-tolerant rhizobia and may be one economically feasible way to increase alfalfa production in water-limited environments.

International audience; Twenty-five lucerne populations of the Medicago sativa complex, which were either diploid or tetraploid and wild or cultivated, were analysed for their resistance to four different fungal diseases and to stem nematode. Forage quality, including stem digestibility and saponin content, was also tested. Populations varied in susceptibility to the diseases caused by Colletotrichum trifolii, Verticillium albo-atrum, Sclerotinia trifoliorum and Pseudopezizza medicaginis, and to the nematode Ditylenchus dipsaci. Except for Sclerotinia rot, sativa and falcata subspecies differed in susceptibility, but this grouping of populations did not account for the full range of variation among them. However, the resistance to P. medicaginis was much lower in the sativa than in the falcata populations. Populations also varied significantly in stem fiber content and digestibility. Stem digestibility was negatively correlated to forage yield. Wild sativa and falcata populations had lower fiber content and higher digestibility than cultivated sativa populations. The medicagenic acid was the sapogenin responsible for the anti-nutritional effect of the lucerne measured by the yellow mealworm larvae Tenebrio molitor. The medicagenic acid content was lowest for the pure sativa populations, highest for the pure falcata populations, and intermediate for the French sativa varieties that have some traits originating from falcata germplasm. Some populations could be used in breeding pro rams to improve disease and nematode resistance, and forage quality.

A procedure for the development of alfalfa (Medicago falcata L.) somatic embryos to the torpedo stage in air-lift vessels is described. Embryos were initiated from chopped leaf explants and were formed by direct somatic embryogensis. The system produced a high number of torpedo stage embryos. The effect of various inoculation densities on embryo development was studied. A procedure for the development and maturation of embryos in aerated liquid media was established. The rate of conversion of the torpedo stage embryos formed in the vessels was 83%.

Gametes formed without meiosis in alfalfa would be useful in basic and applied research. Therefore, the cytological analysis of macrosporogenesis of a diploid plant of Medicago sativa subsp. falcata (L.) Arcangeli (named PG-F9), previously selected as a good 2n egg producer, was conducted. A stain-clearing technique was applied which also allowed the analysis of microsporogenesis to be performed. Two mechanisms of 2n egg formation were determined: absence of the second meiotic division (mechanism of the SDR type) and apomeiosis. In particular, it is noteworthy that 2n eggs produced via apomeiosis should retain the same parental genotype, because apomeiotic cells give rise directly to the female gametophyte without undergoing meiosis. The presence of binucleate female gametophytes in the ovules at an early stage of development confirmed the presence of apomeiosis in PG-F9. These abnormalities concerned only macrosporogenesis; in fact, the analysis of microsporogenesis showed the production of normal tetrads of microspores after normal meiosis. Possible application of the discovered mechanisms of 2n egg production in breeding research are discussed.

A method for direct somatic embryogenesis in alfalfa (Medicago falcata) is described. The time course in the development phase has been followed for fresh weight, cell density, pH, sugar uptake and embryo number and type. The method of disrupting the explant material has also been shown to influence subsequent embryo formation.

Lucerne (alfalfa —Medicago sativa) pollen, cultured at the late unicellular stage, followed one of two developmental pathways: (1) A pathway involving symmetric mitosis which produces pollen containing two vegetative (2 V) or two generative (2 G) pollen. This morphology was only observed in culture, and pollen which followed this developmental path is defined as non-physiological. Occasionally the formation of multi-nucleate pollen grains containing from 4–9 cells were observed. Sustained divisions were not observed. (2) The production of bicellular (V+G) pollen followed by tricellular (V+2G) pollen. Since these types of grains are encountered during development in vivo, pollen following this developmental pathway is defined as physiological. The proportion of pollen that divided was enhanced by a cold treatment at 4°C for one week, prior to culture. The ratio of non-physiological (i.e., 2V or 2G) to physiological pollen (G+V or 2G+V) was found to be affected by the nature of the osmoticum in the medium. Media containing maltose or melibiose gave higher proportions of non-physiological pollen than media containing glucose or sucrose.

A highly efficient system for direct somatic embryogenesis is described. Leaf sections originating from young trifoliate leaves of Medicago falcata line 47/1-5 and Medicago sativa line No2/9R, directly produced embryos after cultivation in liquid B5IV induction medium. In comparison with indirect somatic embryogenesis the system omits the callus stage and thus allows shortening of the process of somatic embryogenesis in alfalfa by 35-40 days. It permits the avoidance of secondary changes occurring during the process of dedifferentiation. A modified B5/3H medium containing Polyethylene Glycol 6000 promoted embryo development from globular up to torpedo stage. It was clearly shown that 2.5% Polyethylene Glycol stimulated this process for both H. falcata 47/1-5 and M. sativa No 2/9R. Maturation of torpedo stage embryos was carried out on solidified or liquid abscisic acidcontaining medium. A 30μM abscisic acid concentration was optimal in allowing one embryo to yield one plant. Somatic embryo conversion to plants and plant regeneration was performed on Murashige and Skoog medium. Regenerated plants showed a normal morphology.