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     

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.     

Optimization of a protocol for direct organogenesis of red clover (Trifolium pratense L.) meristems for breeding purposes

A series of experiments were carried out in order to optimize a protocol for the direct organogenesis of Chilean red clover germplasm. A range of cultivars were used to analyze the effect of explant source (crown or stem meristems of vegetative plants), culture media and plant growth regulators. Our findings showed that stem meristems were easier to obtain, presented lower levels of contamination and a better development than crown meristems. The L2 medium showed better results than B5 and MS media for the cultivars and experimental lines studied. L2 medium supplemented with 0.003 mg/l of 4-amino-3,5,6-trichloropicolinic acid and 1.0 mg/l of 6-benzylaminopurine gave consistently better results and will be applied in our breeding program to propagate, maintain and eliminate viruses from elite red clover clones.     

The origin of Russian cultivars of red clover ( Trifolium pratense L.) and their genetic relationships to wild populations in the Urals

Propagation and breeding of red clover ( Trifolium pratense L.) in Russia was initiated about 200 years ago but the origin of present-day cultivars is disputed. Some authors argue that most Russian cultivars were derived from western European ones, whereas others support a Russian origin of the cultivars from local wild populations. In the present study we assessed the genetic variation at 17 allozyme loci in seven Russian cultivars, bearing the names of localities of the Urals, two American ones that have been used in Russia for scientific experiments and seven wild populations of the Urals and Western Siberia. Variation at the 17 protein loci supports the western European origin of the cultivars and also indicates that gene flow between cultivars and wild populations was limited or has not acted sufficiently long to affect the genetic composition of the red clover wild populations of the Urals.     

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.     

Improving nitrogen fixation in legumes by plant breeding; the relevance of host selection experiments in red clover (Trifolium pratense L.) and subterranean clover (T. subterraneum L.)

Summary This paper reviews (i) basic studies on the genetics of symbiosis in red clover (a self-sterile species) and subterranean clover (cleistogamous) and (ii) work on selection and plant breeding to increase nitrogen fixation in these hosts.Symbiotic effectiveness in red clover is influenced by many major and minor genes. The highly effective phenotype is inherited in a complex manner associated with early nodulation and the formation of large amounts of persistent bacteroid-containing tissue. Lines bred to fix more nitrogen with one strain ofRhizobium trifolii do so with most but not all other strains examined. They also show slightly increased vigour when grown on nitrate. The highly effective response is correlated with abundant nodulation and an early flowering habit, the evidence from breeding studies indicating that this correlation is not absolute. Normally effective and highly effective nodules have the same specific nitrogenase activities. The expression of the highly effective response is relatively little affected by environmental factors (temperature, light intensity, day length, supplementary carbon-di-oxide). Inbreeding substantially degrades the symbiotic response.Heterosis is shown in crosses between cultivars of subterranean clover but otherwise selection to increase effectiveness in this host was unsuccessful.The relevance of these results (and their physiological aspects) for the improvement of grain legumes is discussed.     

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.     

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.     

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.