Prolific shoot regeneration from immature embryo explants of sainfoin (Onobrychis viciifolia Scop.)

Summary Immature cotyledons and embryo axes of sainfoin were cultured on Murashige and Skoog (MS) media supplemented with various concentrations of 6-benzylaminopurine (BAP) and a-naphthaleneacetic acid (NAA) to induce adventitious shoot regeneration. The highest frequency of shoot regeneration occurred following an initial callus growth on a MS medium containing 0.5 mg/l BAP and 2 mg/l NAA. Immature embryo axes showed higher regeneration capacity than immature cotyledons, however, shoot elongation was best achieved on immature cotyledons. Regenerated shoots were excised and rooted in half strength MS medium with 1 mg/l indole-butyric acid (IBA) or 1 mg/l NAA. The rooted plantlets were finally transferred to compost.     

Condensed tannins in the tissue culture of sainfoin (Onobrychis viciifolia Scop.) and birdsfoot trefoil (Lotus corniculatus L.)

Two forage legumes, birdsfoot trefoil (Lotus corniculatus L.) and sainfoin (Onobrychis viciifolia Scop.), containing condensed tannins in their leaves and stems were used as source material to study condensed tannins in tissue culture. More protoplasts were isolated from mesophyll tissue of a low tannin-containing strain of birdsfoot trefoil than from a high tannin-containing strain, but more tannin-filled protoplasts were observed in the latter. Growth rates of leaf explant-derived callus tissue were greater for the high-tannin than for the low-tannin strain. In sainfoin, callus cultures from leaf explants produced numerous tannin-filled cells by 21 days. Explants from sainfoin cotyledons and roots, tissues which normally do not contain tannins, also formed callus with tannin-filled cells in 21 days but in almost every case, a cytokinin was required for tannin formation to occur. The occurrence of tannin-filled cells in callus from root and cotyledon explants was variable and genotype specific. These results show that endogenous tannins can affect protoplast isolation and possibly callus growth in birds-foot trefoil, and that the formation of condensed tannins in sainfoin callus culture can be influenced by a growth regulator.Abbreviations BAP benzylaminopurine - KIN kinetin - NAA naphthaleneacetic acid - PAR photosynthetically active radiation Contribution no. 920 of Agriculture Canada Research Station, 107 Science Cres., Saskatoon, Saskatchewan, Canada S7N OX2     

Protoplast-derived streptomycin resistant plants of the forage legume Onobrychis viciifolia Scop. (sainfoin)

Approximately 10⁶ protoplast-derived cell colonies of sainfoin were stressed with streptomycin and two resistant colonies were recovered. Plants regenerated from these colonies could be recallused on streptomycin-containing medium three years after growth in the absence of the antibiotic.Ultrastructural studies showed cells of resistant callus grown in the presence of streptomycin to contain chloroplasts with internal thykaloids and grana. Such mutant plants should be useful in designing biochemical selection schemes to recover somatic hybrids and cybrids.Abbreviations BAP 6 - benzylamino purine - NAA - napthaleneacetic acid     

Acylated flavonol glycosides from the forage legume, Onobrychis viciifolia (sainfoin)

Ten acylated flavonol glycosides were isolated from aqueous acetone extracts of the aerial parts of the forage legume, Onobrychis viciifolia, and their structures determined using spectroscopic methods. Among these were eight previously unreported examples which comprised either feruloylated or sinapoylated derivatives of 3-O-di- and 3-O-triglycosides of kaempferol (3,5,7,4'-tetrahydroxyflavone) or quercetin (3,5,7,3',4'-pentahydroxyflavone). The diglycosides were acylated at the primary Glc residue of O-α-Rhap(1→6)-β-Glcp (rutinose), whereas the triglycosides were acylated at the terminal Rha residues of the branched trisaccharides, O-α-Rhap(1→2)[α-Rhap(1→6)]-β-Galp or O-α-Rhap(1→2)[α-Rhap(1→6)]-β-Glcp. Identification of the primary 3-O-linked hexose residues as either Gal or Glc was carried out by negative ion electrospray and serial MS, and cryoprobe NMR spectroscopy. Analysis of UV and MS spectra of the acylated flavonol glycosides provided additional diagnostic features relevant to direct characterisation of these compounds in hyphenated analyses. Quantitative analysis of the acylated flavonol glycosides present in different aerial parts of sainfoin revealed that the highest concentrations were in mature leaflets.     

Lectin from sainfoin (Onobrychis viciifolia scop.). Complete amino acid sequence

The complete amino acid sequence of a lectin from sainfoin ( Onobrychis viciifolia Scop . var. Eski ) has been determined by sequential Edman analyses of the intact protein and peptides derived from digests with trypsin and thermolysin. Peptides were purified by pH fractionation, by gel filtration, and by cation-exchange and reverse-phase high-performance liquid chromatography. Seven segments of continuous sequence, accounting for the entire protein, were aligned through sequence comparison with several homologous leguminous lectins to give the final structure. Sainfoin lectin monomer, a glycoprotein which contains a single polypeptide chain of 236 amino acid residues with a molecular weight of 26 509, has amino- and carboxyl-terminal residues of alanine and threonine, respectively. A single residue of cysteine, located at position 33, is the only sulfur-containing amino acid present. Asparagine-118 is the single oligosaccharide attachment site. At least two apparent allelomorphic forms of the protein, having valine or isoleucine at position 49 in equal amounts, were detected. The amino acid sequence of sainfoin lectin exhibits circular permutation relative to that of the homologous protein concanavalin A.     

Asymmetric somatic hybrid plants between Medicago sativa L. (alfalfa,lucerne) and Onobrychis viciifolia Scop. (sainfoin)

This paper reports on the production of intergeneric somatic hybrid plants between two sexually incompatible legume species. Medicago sativa (alfalfa, lucerne) leaf protoplasts were inactivated by lethal doses of iodoacetamide. Onobrychis viciifolia (sainfoin) suspension-cell protoplasts were gamma-irradiated at lethal doses. Following electrofusion under optimized conditions about 50,000 viable heterokaryons were produced in each test. The fusion products were cultured with the help of alfalfa nurse protoplasts. Functional complementation permitted only the heterokaryons to survive. A total of 706 putative heterokaryon-derived plantlets were regenerated and 570 survived transplantation to soil. Experimentation was aimed at the introduction of proanthocyanidins (condensed tannins) from sainfoin, a bloat-safe plant, to alfalfa, a bloat-causing forage crop; however, no tannin-positive regenerant plants were detected. Most regenerant plants have shown morphological differences from the fusion parents, although, as expected, all resembled the recipient parent, alfalfa. Southern analysis using an improved total-genomic probing technique has shown low levels of sainfoin-specific DNA in 43 out of 158 tested regenerants. Cytogenetic analysis of these asymmetric hybrids has confirmed the existence of euploid (2n=32; 17%) as well as aneuploid (2n=30, 33–78; 83%) plants. Pollen germination tests have indicated that the majority of the hybrids were fertile, while 35% had either reduced fertility or were completely sterile.     

Influence of benzyladenine on condensed tannin formation and callus growth in cultures from sainfoin (Onobrychis viccifolia Scop.) cotyledons

Cotyledons from aseptically grown seedlings of sainfoin (Onobrychis viciifolia Scop.) were used as explant material to grow callus tissue for periods of 21 and 31 days. The formation of cells containing condensed tannins was induced by adding a range of BAP concentrations to an mB5 culture medium containing 2,4-D. After 21 days the fresh weight of calli treated with BAP was much greater than the control and appeared highest at the 0.6 mg/L level. Fresh weight was reduced at high BAP levels (2 to 8 mg/L) but still remained well above the control. The formation of tannin-filled cells was genotype-specific but occurred in all treatments with BAP. After 21 days in culture, fresh weight and tannin formation increased with the BAP level at the lower BAP concentrations. After 31 days, the growth rate slowed in the control and the lower BAP concentration, but continued at a high rate in the remaining treatments; the number of tannin-filled cells appeared to decline. The results show that in sainfoin callus culture, BAP induces the formation of condensed tannin, a secondary metabolite, and concomitantly produces a high growth rate.Abbreviations BAP 6-benzylaminopurine - KIN kinetin - PAR photosynthetically active radiation Contribution No. 974 of Agriculture Canada, Research Station, 107 Science Cres., Saskatoon, Saskatchewan, S7N 0X2 Canada     

An assessment of the cultural capabilities of Trifolium repens L. (white clover) and Onobrychis viciifolia Scop. (sainfoin) mesophyll protoplasts

Mesophyll protoplasts isolated from white clover and sainfoin divided to form callus under similar cultural conditions. White clover protoplasts showed varietal differences in their plating efficiency. Sainfoin tissues regenerated readily by forming shoots, but induction of morphogenesis in white clover was only achieved after testing several media and culture sequences. Many of the white clover shoots were abnormal in being fused together to form green plate-like structures, but the latter still developed into plantlets while attached to the parent callus. The ability to isolate, culture, and regenerate mesophyll protoplasts of these two forage legumes is discussed in relation to future attempts to produce somatic hybrids between high tannin containing bloat-safe sainfoin and other major forage legumes such as alfalfa, white clover, and red clover.     

Polyphenol metabolism provides a screening tool for beneficial effects of Onobrychis viciifolia (sainfoin)

Onobrychis viciifolia (sainfoin) is a traditional fodder legume showing multiple benefits for the environment, animal health and productivity but weaker agronomic performance in comparison to other legumes. Benefits can be mainly ascribed to the presence of polyphenols. The polyphenol metabolism in O. viciifolia was studied at the level of gene expression, enzyme activity, polyphenol accumulation and antioxidant activity. A screening of 37 accessions regarding each of these characters showed a huge variability between individual samples. Principal component analysis revealed that flavonols and flavan 3-ols are the most relevant variables for discrimination of the accessions. The determination of the activities of dihydroflavonol 4-reductase and flavonol synthase provides a suitable screening tool for the estimation of the ratio of flavonols to flavan 3-ols and can be used for the selection of samples from those varieties that have a specific optimal ratio of these compounds for further breeding.     

Effects of Sainfoin (Onobrychis viciifolia Scop.) Condensed Tannins on Growth and Proteolysis by Four Strains of Ruminal Bacteria

Sainfoin leaf condensed tannins inhibited growth and protease activity in Butyrivibrio fibrisolvens A38 and Streptococcus bovis 45S1 but had little effect on Prevotella ruminicola B(1)4 or Ruminobacter amylophilus WP225. Tannins bound to cell coat polymers in all strains. Morphological changes in B. fibrisolvens and S. bovis implicated the cell wall as a target of tannin toxicity.     

Photosynthesis, Water Relations, Temperature and Canopy Structure as Factors Influencing the Growth of Sainfoin (Onobrychis viciifolia Scop.) and Lucerne (Medicago sativa L.)

Measurements of the growth of sainfoin and lucerne were madein the field after cutting on 31 May 1977. Sainfoin reacheda total above-ground dry weight of 408 g m^–2 over thegrowing period of 48 days compared with 598 g m^–2 in lucerne.Final leaf area indices (LAIs) were 2.8 in sainfoin and 6.1in lucerne. The specific leaf areas (SLAs) for sainfoin wereapproximately half those of lucerne throughout the regrowthperiod. The maximum rates of leaf appearance were 0.12 leavesper day in sainfoin and 0.85 leaves per day in lucerne. Themaximum mean rate of plant extension growth for lucerne of 2.12mm h^–1 occurred during the night, whereas, in sainfointhe maximum rate of 1.72 mm h^–1 occurred during the day. Measurements of extinction coefficients for PAR ranged from0.45 to 0.89 in sainfoin and from 0 42 to 0.57 in lucerne. Asthe lucerne crop increased in size leaf water potentials andsolute potentials became more negative. In sainfoin leaf waterpotentials remained remarkably high throughout the growth period,solute potentials decreased and turgor potentials increased.The stomatal conductances of the two species were similar. The photosynthetic capacities and rates of dark respirationper unit leaf area in both species were similar. The rate ofcanopy ‘gross’ photosynthesis at 295 W m^–2was always greater in lucerne than in sainfoin. This was largelya matter of differences between the species in LAI, althoughat higher LAIs the more erect structure of lucerne leads toa better utilization of photosynthetically active radiation. Onobrychis vicifolia Scop, sainfoin, Medicago sativa L., lucerne, photosynthesis, water relations, temperature, canopy structure     

Ultrastructural changes in the third-stage, infective larvae of ruminant nematodes treated with sainfoin (Onobrychis viciifolia) extract

Plants rich in condensed tannins are an alternative to chemical anthelmintics to control gastrointestinal nematodes (GINs) in ruminants. Previous functional studies have shown that sainfoin extracts affect the two forms of infective larvae (L3), ensheathed and exsheathed. However, the mechanisms of action remain unknown. The aim of this study was thus to compare ultrastructural changes in ensheathed and exsheathed L3 of two GIN species after in vitro contact with sainfoin extracts using transmission electron microscopy. The main changes identified were an alteration of the hypodermis, the presence of numerous vesicles in the cytoplasm and degeneration and/or death of muscular and intestinal cells. The changes suggested similar and nonspecies-specific lesions in the two nematode species. Comparison of the modifications found in the ensheathed vs. exsheathed L3s revealed different locations of the main cellular changes depending on the larval form. It is hypothesized that these spatial differences in lesions are mainly influenced by the presence of the sheath which favors contact between the active compounds and either the cuticle or the digestive tract. Overall, our observations suggest that the functional changes observed in the biology of GIN L3s after contact with sainfoin extracts are mediated through a direct mode of action, i.e. different interactions between the bioactive plant metabolites and the nematode structure depending on the route of contact.     

Effects of low temperatures on nitrogenase activity in sainfoin (Onobrychis viciifolia) nodulated by arctic rhizobia

The temperate forage legume sainfoin (Onobrychis viciifolia) is readly nodulated by rhizobia isolated from arctic legumes (Astragalus and Oxytropis species). We have investigated the effects of low temperatures on nitrogenase activity in sainfoin nodulated by arctic and temperate (homologous) rhizobia. At low temperatures, nitrogenase activity of arctic rhizobia measured either with detached nodules or with whole plants, was higher than that of temperate rhizobia. At 5°C and 10°C, nitrogenase activity values of arctic rhizobia represented 12% and 33% of those measured at 20°C, while lower values of 3.7% and 22.4% were observed with temperate rhizobia. This cold adaptation was also reflected on bacterial growth where, at 5°C and 10°C, arctic rhizobia showed a shorter doubling time and synthesized more protein than temperate rhizobia.     

Enhanced N2-fixing ability of a deletion mutant of arctic rhizobia with sainfoin (Onobrychis viciifolia)

Summary Mutagenesis provoked by exposure at elevated temperature of the cold-adapted, arctic Rhizobium strain N31 resulted in the generation of five deletion mutants, which exhibited loss of their smaller plasmid (200 kb), whereas the larger plasmid (> 500 kb) was still present in all mutants. Deletion mutants did not show differences from the wild type in the antibiotic resistance pattern, the carbohydrates and organic acids utilization, and the growth rate at low temperature. However, deletion mutants differed from the wild type and among themselves in the ex planta nitrogenase activity, the nodulation index, and the symbiotic effectiveness. The deletion mutant N31.6rif ^r showed higher nodulation index and exhibited higher nitrogenase activity and symbiotic efficiency than the other deletion mutants and the wild type. The process of deletion mutation resulted in the improvement of an arctic Rhizobium strain having an earlier and higher symbiotic nitrogen fixation efficiency than the wild type.     

The effect of dietary sainfoin ( Onobrychis viciifolia) on local cellular responses to Trichostrongylus colubriformis in sheep

The effect of sainfoin (Onobrychis viciifolia) hay consumption on the pathophysiology and local cellular responses of growing lambs during infection with Trichostrongylus colubriformis was investigated. Thirty-two lambs, 16 weeks of age, were allocated to 1 of 4 treatment groups (n=8) that were offered either grass (G) or sainfoin (S) hay while concurrently either infected (+), or not (-) with 12,000 L3 T. colubriformis larvae per week for 6 weeks. Liveweight gains were affected by diet (P=0.002) and reduced by infection (P<0.05). Faecal egg count was reduced in S+compared to G+ from days 35 to 42 (P=0.001); however, total egg output, worm burdens at day 42 and worm fecundity were similar between diets (P>0.05).Feeding sainfoin appeared to enhance immune cell development with tissue eosinophils, mast cells and pan T cells present in greater concentrations in S+ than in G+ animals. However, further studies are required to determine if the enhanced immune cell development is a consequence of a greater nutrient supply or a direct influence of sainfoin metabolites on local inflammatory responses to the gastrointestinal nematode T. colubriformis.     

Molecular phylogeny and ecogeography of Onobrychis viciifolia Scop. (Fabaceae) based on nrDNA ITS sequences and genomic ISSR fingerprinting

In this study 40 specimens from 8 representative Iranian populations of Onobrychis viciifolia SCOP. were collected from their natural habitats. The specimens were biometrically assessed using 43 quantitative and 15 qualitative morphological characters. At each sample site we recorded ecogeographical variables. In order to evaluate the difference between the variation due to phenotypic responses and genetic adaptation, we generated nucleotide sequence data from the internal transcribed spacer of the nuclear rDNA genes (ITS) and carried out genomic fingerprints using inter‐simple sequence repeat PCR (ISSR). Cluster analysis of morphological characters divided the eight populations into three major groups. Leaf length, leaflet length and width, calyx length, plant height, and stem length were introduced as diagnostic characters for three different morphological groups. Furthermore, canonical correspondence analysis (CCA) of ecogeographical data showed correlations between morphological variations and ecogeographical factors. Clay%, saturation percentage (SP), total neutralizing value (TNV%), texture, minimum temperature, and geographic separation were the main environmental variables associated with morphological groups of O. viciifolia. ISSR analysis revealed three main groups which are in correspondence with morphological groups, indicating that the three morphological groups have been shaped by genetic and phenotypic responses to environmental conditions. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Effect of feeding dehydrated and ensiled tanniferous sainfoin (Onobrychis viciifolia) on nitrogen and mineral digestion and metabolism of lambs

The effects of tanniferous sainfoin on digestion and metabolism have been investigated in 12 lambs in an incomplete cross-over design (n = 6). Effects of condensed tannins (CT) were evaluated by comparing dehydrated and ensiled sainfoin treated with and without polyethylene glycol (PEG). Dehydrated and ensiled grass-clover mixtures served as controls. The lambs were fed the treatment diets, including a mineral supplement, for 21 d. During the last 7 d excreta, rumen fluid and blood were sampled. The CT of sainfoin decreased rumen fluid ammonia concentration (p < 0.001) and increased the plasma concentration mainly of essential amino acids (p < 0.001). Body retention of phosphorus, calcium and magnesium was lower with sainfoin compared to PEG-treated sainfoin (p < 0.05). Sainfoin without PEG resulted in lower digestibilities of organic matter and neutral detergent fibre than sainfoin with PEG and the grass-clover mixture (p < 0.001). Ensiling of sainfoin led to the lowest N-retention. In conclusion, the reduction in ruminal ammonia and urine-N losses by sainfoin CT did not improve N-retention.     

Effects of sainfoin (Onobrychis viciifolia) extract and monomers of condensed tannins on the association of abomasal nematode larvae with fundic explants

Tannin-rich forages offer an alternative to anthelmintic chemicals to control gastrointestinal nematodes. However, the mode of action of such bioactive plants still needs to be assessed. Previous studies have shown that extracts of tannin-rich plants interfere with the first phase of host invasion, i.e., the exsheathment of infective larvae (L3s). In the current study, we examined the hypothesis that exposure to tannins could also affect the second phase of larval establishment, i.e., the tissue association/penetration of the exsheathed L3s into the digestive mucosae. An in vitro direct challenge technique using fundic explants was applied in this study. The main parasite model was Haemonchus contortus. The objectives were to verify: (i) whether a modification of the association/penetration of L3s with the mucosae occurred after contact with sainfoin extract; (ii) whether this is a dose-dependent phenomenon; (iii) whether tannins were responsible for these effects; (iv) whether these effects were dependent on the parasite species; and (v) how the biochemical structure of tannins might influence these effects. Following 3 h contact with sainfoin extract at 1,200 μg/ml, the penetration of exsheathed L3s of H. contortus and Teladorsagia circumcincta into fundic explants was significantly reduced. Moreover, a dose–response relationship was found for H. contortus. For both nematodes, the changes were totally alleviated after addition of polyvinyl polypyrrolidone, an inhibitor of tannins, to the sainfoin extract, suggesting that tannins play a major role in the observed effects. Comparison of results obtained with different monomers of condensed tannins confirms a relationship between structure and activity, the prodelphinidin monomers and galloyl-derivatives being more effective than the procyanidin monomers. Combined with the delay or the inhibition of larval exsheathment previously shown, these effects could explain how tanniniferous plants reduce the establishment of infective larvae in small ruminants.     

Effects of condensed tannins in wrapped silage bales of sainfoin (Onobrychis viciifolia) on in vivo and in situ digestion in sheep

The objective of this study was to characterize the condensed tannins (CTs) in wrapped silage bales of sainfoin (Onobrychis viciifolia) and examine their potential action on in vivo and in situ digestive characteristics in sheep. Silage was made from sainfoin, cut at two phenological stages. The first phenological stage, at which silage was made, was from the first vegetation cycle at the end of flowering and the second stage silage was made from regrowth, 5 weeks after the first cut, but before flowering. The silages made from the two phenological stages were fed to 12 rumen-fistulated sheep in a crossover design. Of the 12 sheep, six received polyethylene glycol (PEG) to bind with and remove the effects of CT, whereas the other six were dosed with water. Organic matter digestibility, total-tract N digestibility and N (N) balance were measured over 6 days. Kinetic studies were performed on total N, ammonia N (NH3-N) and volatile fatty acids (VFAs) in rumen fluid before and 1.5, 3 and 6 h after feeding. The kinetics of degradation of dry matter and N from Dacron bags suspended in the rumen were also determined. Biological activity of CT (protein-binding capacity) and CT concentration were greater for the silage made from sainfoin at the early flowering stage. Total-tract N digestibility was increased by the addition of PEG (P < 0.001) to the sainfoin silage before flowering (P < 0.001). CTs decreased N excretion in urine (P < 0.05) and increased faecal N excretion (P < 0.001), but had no effect on body N retention, which is beneficial for the animal. Ruminal N degradability was smaller in the presence of active CT (P < 0.001) at both phenological stages; however, soluble N (P = 0.2060) and NH3-N (P = 0.5225) concentrations in rumen fluid remained unchanged. The results of this experiment indicate that CT in the sainfoin retain their ability to affect the nutritive value of preserved forage legumes.