Inheritance of somatic embryogenesis in red clover (Trifolium pratense L.)

 Red clover genotypes capable of regenerating plantlets in vitro from non-meristematic tissue-derived callus are rare. Selection for genotypes capable of somatic embryogenesis identified a clone comprised of a group of plantlets regenerated from a hypocotyl-derived callus culture on L2-based media and another group of plantlets originating from crown divisions of the epicotyl-derived plant. The callus-derived plants of this clone were highly regenerative when reintroduced to callus culture, but the epicotyl-derived plants produced nonregenerative callus cultures. F₁, F₂ and BC₁ populations were evaluated to determine the mode of inheritance of the regeneration trait. Reciprocal crosses did not differ, indicating a lack of maternal effects. Results were compatible with genetic control of regeneration by two complementary genes. We propose the genotype Rn1-Rn2- for regenerative plants. Three petiole segment explants were sufficient to evaluate regenerative ability in seedlings. Regenerative ability was often associated with abnormal leaf morphology in a few to several leaves. Received: 17 February 1998 / Accepted: 7 April 1998     

Flowering and gibberellins in a mutant red clover (Trifolium pratense L.)

Relationships between gibberellins and floral initiation were investigated in a conditional non-flowering mutant of red clover, Trifolium pratense. Untreated mutant plants will not flower under long-days, but will do so when certain GAs are applied. Gibberellins, A₃, A₁, A₇, and A₅ all resulted in both stem elongation and flowering whilst GA₄ produced the elongation only. Applications of GA₂₀, GA₈ and GA₁₃ under long-days had no detectable effect. Thus, by combining the use of the mutant with the application of different GAs, the correlation between the processes of stem elongation and floral initiation, which is normally strongly expressed in this species, was broken. Endogenous gibberellins shown to be present in normal plants were also found in the mutant genotype. Gibberellins alone were not sufficient to initiate floral development in the mutant, there being an essential element of interaction with long-days. These results are discussed in relation to the nature of the lesion in the mutant and the signal provided by the applied gibberellin.     

Mechanisms of microbially enhanced Fe acquisition in red clover (Trifolium pratense L.)

Soil microorganisms may play an important role in plant Fe uptake from soils with low Fe bioavailability, but there is little direct experimental evidence to date. We grew red clover, an Fe-efficient leguminous plant, in a calcareous soil to investigate the role of soil microbial activity in plant Fe uptake. Compared with plants grown in non-sterlie (NS) grown plants, growth and Fe content of the sterile(s) grown plants was significantly inhibited, but was improved by foliar application of Fe EDTA, indicating that soil microbial activity should play an important role in plant Fe acquisition. When soil solution was incubated with phenolic root exudates from Fe-deficient red clover, a few microbial species thrived while growth of the rest was inhibited, suggesting that the Fe-deficient (-Fe) root exudates selectively influenced the rhizosphere's microbial community. Eighty six per cent of the phenolic-tolerant microbes could produce siderophore [the Fe(III) chelator] under -Fe conditions, and 71% could secrete auxin-like compounds. Interestingly, the synthetic and microbial auxins (MAs) significantly enhanced the Ferric reduction system, suggesting that MAs, in addition to siderophores, are important to plant Fe uptake. Finally, plant growth and Fe uptake in sterilized soil were significantly increased by rhizobia inoculation. Root Fe-EDTA reductase activity in the -Fe plant was significantly enhanced by rhizobia infection, and the rhizobia could produce auxin but not siderophore under Fe-limiting conditions, suggesting that the contribution of nodulating rhizobia to plant Fe uptake can be at least partially attributed to stimulation of turbo reductase activity through nodule formation and auxin production in the rhizosphere. Based on these observations, we propose as a model that root exudates from -Fe plants selectively influence the rhizosphere microbial community, and the microbes in turn favour plant Fe acquisition by producing siderophores and auxins.     

QTL analysis of seed yield components in red clover (Trifolium pratense L.)

Cultivars of red clover (Trifolium pratense L.), an important forage crop in temperate regions, are often characterised by an unsatisfactory level of seed yield, leading to high production costs. This complex trait is influenced by many components and negatively correlated with other important traits, such as forage yield or persistence. Therefore, seed yield has proven to be difficult to improve. Thus, the objectives of this study were to assess association among seed yield components and to provide the basis for identifying molecular markers linked to QTLs for seed yield components to assist breeding for improved red clover cultivars. A total of 42 SSR and 216 AFLP loci were used to construct a molecular linkage map with a total map length of 444.2 cM and an average distance between loci of 1.7 cM. A total of 38 QTLs were identified for eight seed yield components. The traits seed number per plant, seed yield per head, seed number per head, head number per plant and percent seed set were highly correlated with seed yield per plant, and QTLs for several of these traits were often detected in the same genome region. Head number per plant may present a particularly useful character for the improvement of seed yield since it can easily be determined before seed maturity. In addition, two genome regions containing four or five QTLs for different seed yield components, respectively, were identified representing candidate regions for further characterisation of QTLs. This study revealed several key components which may facilitate further improvement of seed yield. The QTLs identified represent an important first step towards marker-assisted breeding in red clover.     

Genotype-dependent whole plant regeneration from protoplasts of red clover (Trifolium pratense L.)

Protoplasts are useful for subcellular studies, in vitro selection, somatic hybridization and transformation. Whole plant regeneration from protoplasts is a prerequisite to producing altered crop plants using these methods. Whole plant regeneration was achieved from leaf- and suspension culture-derived protoplasts of T. pratense. Regeneration was most dependent upon identifying genotypes with genetic capacity to regenerate. Additional factors that were used to select genotypes, but which proved to be less important, were a high rate of cell growth in culture and a high plating efficiency of protoplasts. One genotype was identified which had a regeneration response equivalent to that of T. rubens and which regenerated from both leaf- and suspension culture-derived protoplasts.Research supported by USDA/CRGO Grant No. 81 CRCR-1-0613     

Correlative effects of leaf age on reproductive growth in red clover (Trifolium pratense L.)

Summary The contribution of individual leaves towards the flowering response in red clover (Trifolium pratense L.) seedlings at the three-leaf stage is described. Removal of the first (oldest) or first and second leaves resulted in large increases (up to 300%) in both the rate of stem extension and the degree of apical differentiation.Removal of the youngest leaves depressed both processes. Application of cycloheximide to specific leaves produced effects similar to defoliation, but chloramphenicol was generally inhibitory and kinetin substantially ineffective. Translocation patterns between leaves and the shoot apex were studied using [¹⁴C] sucrose and [³H] gibberellin A₁. There was appreciable movement between leaves, but exposure to long-days depressed the transport of labelled assimilate. Label arriving at the apex was increasingly derived from the younger leaves as floral induction proceeded.Abbreviations CAP D-threo-chloramphenicol - CH cycloheximide - K Kinetin - Tris Tris(hydroxymethyl) amino methane - G.S.H. Glutathione (reduced) - E.D.T.A. ethylene diamino tetra acetic acid disodium salt     

接种不同VA菌根真菌对红三叶草利用不同磷源的影响

以红三叶草为材料,利用三室隔网培养方法,研究了施用不同磷源条件下,接种VA菌根真菌(Glomusmosseae和Glomusversiforme)对外加有机磷源及土壤有机磷的利用效率.植物生长10周后,测定植株干物重、含磷量、菌根侵染率及根系长度.结果表明接种菌根真菌能明显增加植株干物重和含磷量.接种条件下无机磷(KH2PO4)对植株生长的促进作用大于有机磷(Na-phytate)处理,接种Glomusversoiforme的作用明显大于接种Glomusmosseae.接种Glomusmosseae,植株对磷酸二氢钾中磷的吸收量明显大于植酸钠,而接种Glomusversiforme时则植株对植酸钠的磷吸收量明显高于磷酸二氢钾.上述结果说明接种两菌种对不同磷源的作用不同,接种Glomusmosseae可提高磷酸二氢钾中磷的利用率,接种Glomusversiforme则可提高对植酸钠的磷利用率.     

Changes in gene products associated with flowering in red clover (Trifolium pratense L.)

A conditional non-flowering mutant of red clover was used toconfirm the coincidence of increases in two apical proteinswith the switch from vegetative to floral development. Apparentlyidentical proteins in leaves did not respond to florally inductiveconditions in the same way. Key words: Flowering, gene prodgcts, red clover     

Genetic map-based location of the red clover (Trifolium pratense L.) gametophytic self-incompatibility locus

Red clover is a hermaphroditic allogamous diploid (2n = 2x = 14) with a homomorphic gametophytic self-incompatibility (GSI) system (Trifolium pratense L.). Red clover GSI has long been studied, and it is thought that the genetic control of GSI constitutes a single locus. Although GSI genes have been identified in other species, the genomic location of the red clover GSI-locus remains unknown. The objective of this study was to use a mapping-based approach to identify simple sequence repeats (SSR) that were closely linked to the GSI-locus. Previously published SSR markers were used in this effort (Sato et al. in DNA Res 12:301–364, 2005). A bi-parental cross was initiated in which the parents were known to have one self-incompatibility allele (S-allele) in common. S-allele genotypes of 100 progeny were determined through test crosses and pollen compatibility. Pseudo F₁ linkage analysis isolated the GSI-locus on red clover linkage-group one within 2.5 cM of markers RCS5615, RCS0810, and RCS3161. A second 256 progeny mapping testcross population of a heterozygous self-compatible mutant revealed that this specific self-compatible mutant mapped to the same location as the GSI-locus. Finally, 82 genotypes were identified whose parents putatively shared one S-allele in common from maternal halfsib families derived from two random mating populations in which paternal identity was determined using molecular markers. Unique S-allele identity in the two random mating populations was tentatively inferred based on haplotypes of two highly allelic linkage-group one SSR (RCS0810 and RCS4956), which were closely linked to each other and the GSI-locus. Paternally derived pollen haplotype linkage analysis of RCS0810 and RCS4956 SSR and the GSI-locus again revealed tight linkage at 2.5 and 4.7 cM between the GSI-locus and RCS0810 and RCS4956, respectively. The map-based location of the GSI-locus in red clover has many immediate applications to red clover plant breeding and could be useful in helping to sequence the GSI-locus.     

Inhibition of angiogenesis and inflammation by an extract of red clover (Trifolium pratense L.)

Antiangiogenic compounds are gaining more and more interest as a new approach in the prevention and treatment of cancer and inflammatory diseases. The objective of this study was the evaluation of the antiangiogenic effect of a red clover extract (RCE) used in food supplements for menopausal complaints as well as of its main isoflavones in an in vivo system, the chorioallantoic membrane assay of fertilized hen's eggs. At a dosage of 250 μg/pellet the red clover extract showed excellent inhibition of angiogenesis. The antiangiogenic activity of the non-methylated isoflavones daidzein and genistein was higher than that of the methylated compounds formononentin and biochanin A. The results demonstrate that RCE is not only suitable for menopausal complaints, but might also be a powerful chemopreventive agent against chronic diseases e.g. which have a high incidence especially in elderly female.     

Inhibition of angiogenesis and inflammation by an extract of red clover (Trifolium pratense L.)

Antiangiogenic compounds are gaining more and more interest as a new approach in the prevention and treatment of cancer and inflammatory diseases. The objective of this study was the evaluation of the antiangiogenic effect of a red clover extract (RCE) used in food supplements for menopausal complaints as well as of its main isoflavones in an in vivo system, the chorioallantoic membrane assay of fertilized hen's eggs. At a dosage of 250 μg/pellet the red clover extract showed excellent inhibition of angiogenesis. The antiangiogenic activity of the non-methylated isoflavones daidzein and genistein was higher than that of the methylated compounds formononentin and biochanin A. The results demonstrate that RCE is not only suitable for menopausal complaints, but might also be a powerful chemopreventive agent against chronic diseases e.g. which have a high incidence especially in elderly female.     

Leaf area index, canopy structure and photosynthesis of red clover (Trifolium pratense L.)

Abstract. The influence of leaf age, total leaf area and its dispersion in space on canopy photosynthesis were studied using microswards of red clover ( Trifolium pratense L.) which were established in the greenhouse. Two varieties, Renova (flowering) and Molstad (non-flowering), were sown in separate plastic boxes at densities of 225, 400 and 625 plants per m².
Vertical distribution of photosynthetically active radiation (PAR), leaf area, leaf age and ¹⁴CO₂-fixation were determined periodically. Net photosynthesis and dark respiration of canopies were measured. Maximum photosynthetic capacity of individual leaves was measured on plants taken from the intact canopy or from plants where shading of the growing leaves had been prevented.
Net photosynthetic rate of canopies increased linearly with leaf area index (LAI) up to an LAI of 3.5 and then declined at higher LAI, independent of variety and sowing density. Below the optimum LAI, net photosynthesis depended mainly on interception of PAR. Decrease in canopy photosynthesis above the optimum LAI was due to a higher proportion of old leaves with decreased photosynthetic capacity, and not to an increase in respiring plant parts. It is concluded that LAI and position of leaf age categories in the canopy are more important than vertical distribution of leaf area in determining canopy photosynthesis of red clover.     

Characterization of novel microsatellite loci for red clover (Trifolium pratense L.) from enriched genomic libraries

Twenty‐seven polymorphic microsatellite markers were isolated from red clover (Trifolium pratense). Allelic variability and cross‐species amplification were assessed on 24 red clover and eight white clover (Trifolium repens) genotypes. The number of alleles detected in red clover ranged from two to 25. Observed and expected heterozygosities were high with average values of 0.71 and 0.88, respectively. Five of the 27 loci were also successfully amplified from white clover, where two to 13 alleles were detected. These highly polymorphic microsatellite loci provide powerful tools for population genetic studies as well as for marker‐assisted selection in this important forage legume species.     

Diversity study on Sclerotinia trifoliorum Erikks., the causal agent of clover rot in red clover crops (Trifolium pratense L.)

Since the 16th century, red clover has been an important crop in Europe. Since the 1940s, the European areal of red clover has been severely reduced, due to the availability of chemical fertilizers and the growing interest in maize. Nowadays there is a growing interest in red clover again, although some setbacks still remain. An important setback is the low persistence of red clover crops. Clover rot, caused by the ascomycete fungus Sclerotinia trifoliorum Erikss., is a major disease in Europe and reduces the persistence of red clover crops severely. The fungus infects clover plants through ascospores in the autumn, the disease develops during the winter and early spring and can kill many plants in this period. In early spring, black sclerotia, serving as surviving bodies, are formed on infected plants. Sclerotia can survive up to 7 years in the soil (Ohberg, 2006). The development of clover rot is highly dependent on the weather conditions: a humid fall, necessary for the germination of the ascospores and an overall warm winter with short periods of frost are favourable for the disease. Cold and dry winters slow the mycelial growth down too much and prevent the disease from spreading. Clover rot is difficult to control and completely resistant red clover varieties have yet to be developed. Because of the great annual variation in disease severity, plant breeders cannot use natural infection as an effective means to screen for resistant material. Breeding for resistant cultivars is being slowed down by the lack of a bio-test usable in breeding programs. When applying artificial infections, it is necessary to have an idea of the diversity of the pathogen. A diverse population will require resistance screening with multiple isolates. The objective of this research is to investigate the genetic diversity among isolates from the pathogen S. trifoliorum from various European countries. We assessed diversity using a species identification test based on the sequence of the beta-tubulin gene, vegetative compatibility grouping and AFLP.     

A copper-deficiency-induced root reductase is different from the iron-deficiency-induced one in red clover (Trifolium pratense L.)

There is increasing evidence that Cu deficiency can induce root reductase activity, but the ecological and physiological significance of this is unknown. This study compared the characteristics of root reductase activity induced by Cu deficiency with those induced by Fe deficiency in red clover (Trifolium pratenseL. cv. Kenland), a Fe-efficient plant. Effects of other nutritional stresses were also investigated for comparison. Compared with the effect of Fe deficiency, Cu deficiency induced only a moderate level of root reductase activity, while other nutrient stresses had no effect, or even inhibited the root reductases activity, especially in the case of Zn deficiency. Compared with Fe deficiency-induced Fe(III)-chelate reductase, Cu deficiency-induced reductase displayed a different pattern of induction. The activity of the Cu deficiency-induced reductase in intact plants increased with time; in decapitated plants it showed a distinct peak at a later stage of the treatment. The Fe concentration in the roots was significantly increased under Cu deficiency. Furthermore, the reductase activity was presented in the entire root system, contrary to what was observed for the Fe-deficiency-induced reductase activity, which was confined to the root apex. Cu deficiency did not increase proton extrusion from the roots, even when growth was significantly affected. The present results suggest that in red clover Cu deficiency induces a root reductase that is different from the reductase induced by Fe deficiency.