Nitrogen Fixation in Hybrids of White Clover (Trifolium repens L.) and Caucasian Clover (Trifolium ambiguum M. Bieb)

Hybrids between white clover (Trifolium repens L.) and Caucasian clover (or Kura clover, Trifolium ambiguum M. Bieb) are a potential route for the improvement of drought tolerance and persistence in white clover. However, to be agronomically viable they must show no significant reduction in their potential for nitrogen fixation relative to white clover. A comparative study of growth rate and nitrogen fixation was carried out in flowing solution culture without a supply of mineral nitrogen to the plants. The two parental species and two generations of backcross hybrids, with white clover as the recurrent parent, were assessed. The growth rate and N content of T. ambiguum were significantly lower than those of the other lines. However, dry matter production, nodule biomass per plant and rates of fixation were similar in second‐generation backcross plants and white clover. The results suggest that the agronomic potential of this novel germplasm is not compromised by limitations with respect to nitrogen fixation.     

Contribution of White Clover Varieties in High-Productivity Systems under Grazing and Cutting

Two medium leaf size varieties of white clover (Trifolium repens L.) were grown in mixture with a range of grass species and varieties in a field experiment conducted over 4 years. The clovers were grown singly or as a blend in swards with a tetraploid perennial ryegrass (Lolium perenne L.), a diploid Italian ryegrass (Lolium multiflorum L.) or tetraploid hybrids between these two species (Lolium x boucheanum Kunth). These grasses were also grown in monoculture. Swards were managed in a regime involving two periods of sheep grazing, two periods of cattle grazing and a silage cut in the same year for 4 years. The objectives of this experiment were 3‐fold: (1) to examine the yield and persistency of white clover in highly productive systems with modern perennial, hybrid and Italian ryegrasses; (2) to compare the performance of white clover varieties grown singly in mixture with grasses with their performance together as a blend, and (3) to examine the persistency of new Italian ryegrass varieties, exemplified by AberComo. The clover content of the sward remained high throughout the experiment, with a mean of 31 % overall. Differences in clover yield between plots were largely attributable to the effects of companion grasses. Total yields were very similar in plots containing different clover varieties and in plots where these varieties had been grown singly or as a blend. Grass yields in mixture were greater than those in monoculture but showed similar relative rankings. The diploid Italian ryegrass variety AberComo showed an unexpectedly high yield in the 4th year of the experiment. The implications of these results for strategies for productive, sustainable agriculture using modern white clover and ryegrass varieties are discussed.     

Introgression as a Route to Improved Drought Tolerance in White Clover (Trifolium repens L.)

Backcross hybrids between the important forage legume white clover Trifolium repens , which is stoloniferous, and the related rhizomatous species T. ambiguum have been produced using T. repens as the recurrent parent. The drought tolerance of parents and two generations of backcross plants, possessing both stolons and rhizomes, was studied in deep soil bins in a glasshouse both as monocultures and in mixtures with perennial ryegrass. Soil moisture content, leaf relative water content, and leaf water potential were measured on plants subjected to drought cycles of 4 weeks duration and those watered normally and maintained at field capacity. Six cycles of drought, carried out over 2 years, showed that T. ambiguum and the first and second generation backcross hybrids maintained a greater leaf relative water content and higher leaf water potential than T. repens at comparable levels of soil moisture. The dry matter yield of T. repens was significantly reduced over each drought cycle compared to the watered treatments, while the yield of T. ambiguum increased over the 4-week cycle in both treatments. The yield of the backcross generations was intermediate between those of the two parental species. The implications of these results for white clover germplasm improvement programmes are discussed.     

白三叶基因工程改良研究进展

白三叶（Trifolium repens L.）作为优良牧草和地被植物具有重要的应用价值。但其耐盐碱、干旱能力差等原因致使应用受到广泛限制,转基因技术的快速发展为白三叶种质创新提供了有效技术手段,为开展白三叶分子育种奠定了基础。本文就近30年来有关白三叶组织培养、转化方法、转基因遗传改良方面的研究进展进行了综述,并对其应用进行了展望。     

Comparing mating designs to restore seed production of interspecific hybrids between Trifolium repens (white clover) and Trifolium uniflorum

Interspecific hybrids between Trifolium uniflorum and cultivated white clover (Trifolium repens) have highly useful characteristics for temperate pastoral systems derived from both parent species. However, the early hybrids (F₁ and BC₁) also have unacceptably poor seed production for commerce. This study analysed the basis for the poor seed production and investigated breeding strategies for overcoming the problem. The BC₁F₁ generation produced lower‐than‐expected numbers of heads per plant and seeds per floret. Backcrossing of selected hybrids to white clover corrected these deficiencies and created new variation. Seed numbers were also returned to near target levels by recurrent selection within the BC₁ generation. Thus, it was possible to retain a theoretical average of 25% of T. uniflorum genome and still achieve high seed production per plant. The BC₁F₂ and BC₂F₁ generations produced high seed numbers per plant, along with reasonable variation. Both of these second‐generation hybrid forms have high reproductive potential and should be the focus for the selection of the desired combinations of agronomic and seed production traits.     

Performance of Timothy-based Grass/Legume Mixtures in Cold Winter Region

This study sought to identify grass/legume mixtures that increase the yield and persistence of forage stands with improved nutritive quality in cold‐winter regions, compared with the standard mixture of timothy (Phleum pratense L.)/red clover (Trifolium pratense L.)/alsike clover (Trifolium hybridum L.). Timothy was mixed with either perennial ryegrass (Lolium perenne L.), meadow fescue (Festuca pratensis L.) or Kentucky bluegrass (Poa pratensis L.). The legumes in mixtures were red clover, alfalfa (Medicago sativa L.) or white clover (Trifolium repens L.). Averaged over three production years, the majority of mixtures had greater dry matter (DM) yields than the standard (8.35 t ha^?1). Timothy, grown alone and in three mixtures, outyielded the standard by 19–30 %. Yield reductions in mixtures over the 3‐year period were greatest with red clover, and least with bluegrass. Mixtures with alfalfa were highest in nitrogen (28.4 g kg^?1), while grasses grown alone (24.6 g kg^?1) and the standard mixture (25.1 g kg^?1) were the lowest in N. Mixtures with red clover or alfalfa had the least neutral detergent fibre (NDF), averaging 418 and 429 g kg^?1 respectively. Mixtures including white clover were initially low in NDF at 347 g kg^?1 in year 1 but increased to 550 g kg^?1 in year 3 as white clover composition declined in the sward.     

Variation for reproductive and agronomic traits among T. repens × T. nigrescens third generation backcross hybrids in the field

Interspecific hybridisation with Trifolium nigrescens Viv. is a possible strategy to improve the reproductive potential of white clover (Trifolium repens L.). Following the development of a fertile F1 hybrid, three generations of backcrossing have been carried out usingT. repens as the recurrent parent. Vegetative characteristics, stolon growth and seed yield components of the backcross (BC) 2 and 3 generations, as well as the parental species were measured on spaced plants grown in the field. Leaf size and plant spread of the BC2 and BC3 generations were less than T. repens but there was no difference in plant fresh weight. Numbers of inflorescences per plant and florets per inflorescence of the backcrosses were greater than T. repens however this was not reflected indifferences in seed yield per plant asT. repens had more seeds per floret and per plant than the backcrosses. Differences in stolon length, the proportion of flowering nodes and the pattern of axillary bud development were observed between T. repens and the backcrosses. Significant variation among the BC 3 generation for vegetative and reproductive traits was observed. Individual plants among the BC 3 generation were identified that combine high forage yield, substantial inflorescence production and good fertility, and these will form the basis of further selection. This revised version was published online in August 2006 with corrections to the Cover Date.     

Nitrogen Fixation by Hybrids of White Clover (Trifolium repens L.) and Trifolium nigrescens

Development of hybrids between white clover ( Trifolium repens L.) and Trifolium nigrescens provides a novel route for genetically improving the reproductive capacity of white clover, provided the hybrids are agronomically viable, particularly with respect to N₂ fixation. A comparative study of growth and rates of N₂ fixation over 21 days was conducted with the parental species, F ₁ hybrids and backcross hybrids, in flowing solution culture, without a supply of mineral N to the plants. T. nigrescens was unable to fix N₂ in association with the strains of Rhizobium leguminosarum biovar. trifolii selected for inoculation. Rates of N₂ fixation per plant increased in the order T. nigrescens < F ₁ hybrid < T. repens < backcross 1. Specific rates of N₂ fixation (days 0–21) increased in the order T. nigrescens < F ₁ hybrid < backcross 1 <  T. repens . Dry matter production and nodule biomass per plant increased at a higher rate in backcross 1 hybrids than in T. repens. The results suggest that the potential for N₂ fixation by backcross 1 hybrids is at least as great as that by T. repens .     

Effects of Soil Moisture Deficit on Forage Quality, Digestibility, and Protein Fractionation of Kura Clover

Kura clover (Trifolium ambiguum M.B.) is a persistent rhizomatous forage legume with great potential for permanent pastures. The effects of a soil moisture deficit on forage quality, digestibility and protein fractionation of two cultivars of Kura clover (Endura and Rhizo) were investigated in this study for 1 year. The responses of alfalfa (Medicago sativa L.) and red clover (Trifolium pratense L.) were also characterized. Stands of each species were field‐grown and submitted to two soil water regimes promoting soil moisture deficits and well‐watered (i.e. control) conditions. There were no interactions between species and soil water regimes. Soil moisture deficit increased acid detergent fibre (ADF) but reduced acid detergent lignin (ADL) content and consequently increased forage digestibility. It had only minor effects on protein content and fractionation. Species varied for most parameters measured. Kura clover generally had the lowest neutral detergent fibre (NDF), ADF and ADL contents, and consequently the greatest digestibility (83.9 %). Species also differed in their crude protein fractionation. Kura clover and red clover had a lower proportion of non‐protein nitrogen (NPN; A fraction) and a greater proportion of true protein (B fraction) (30.9 and 64.5 %, respectively) than alfalfa (36.4 and 57.4 %, respectively). Kura clover also had the lowest proportion (i.e. 4.7 %) of acid detergent insoluble protein (ADIP; C fraction) of all species tested. Endura Kura clover often had a higher forage quality than Rhizo. This study confirms that Kura clover produces high‐quality forage and provides the first estimates of protein fractionation in this species.     

Bulked segregant AFLP analysis to identify markers for the introduction of the rhizomatous habit from Trifolium ambiguum into T. repens (white clover)

Bulked segregant analysis (BSA) using the technique of amplified fragment length polymorphism (AFLP^TM) was carried out on backcross hybrids between the important pasture species white clover and its relative Trifolium ambiguum. The introgression of the rhizomatous trait fromTrifolium ambiguum, previously shown to be associated with enhanced drought tolerance but which takes eighteen months to manifest itself, was analysed in a series of third generation backcross families with white clover as the recurrent parent. Eleven families differed in the proportions of plants classified according to rhizome content. Three of these families were selected for BSA-AFLP based on the creation of separate DNA pools from plants with large amounts of rhizomes or no rhizomes. Following an initial screen with119 primer combinations, 53 combinations were selected on the basis of the number of clear, reproducible polymorphisms they produced. Polymorphic bands were found that distinguished between rhizomatous and non-rhizomatous bulks in the three families. However, only one polymorphic band was found that unambiguously reflected the presence of the rhizomatous trait in individual plants in these families. This polymorphic band was also shown to be present in the BC2 parent, the BC1 and the original T. ambiguum parent. Given the aneuploid nature of the BC2 plants this band would appear to be a chromosome-specific marker tightly linked to the locus controlling rhizome development. The identification of a consistent marker for the rhizomatous trait opens up the possibility of applying marker assisted introgression and thereby facilitating the rapid incorporation of this trait into diverse germplasm. This revised version was published online in July 2006 with corrections to the Cover Date.     

Interspecific hybridization in the genus Trifolium

The genus Trifolium (Leguminosae) or clovers includes a number of agriculturally important forage species. In particular Trifolium repens (white clover) and Trifolium pratense (red clover) are widely used in temperate livestock agriculture. Interspecific crossing programmes within this genus have been carried out for more than 50 years, in many cases involving embryo rescue or ovule culture techniques. In general, the aims of these have been twofold: to aid understanding of evolutionary relationships within the genus and to introgress useful traits, predominantly into white or red clover. In the case of white clover, the greatest efforts have been made with respect to crossing with Trifolium nigrescens and Trifolium ambiguum . Novel germplasm has been developed and useful traits transferred, although no material has yet been commercialized. The main emphasis with respect to red clover has been increasing longevity through crosses with more persistent species, particularly Trifolium medium . Again, this work has not yet resulted in new varieties. Wider crossing programmes, allied in recent years to comparisons at the DNA level, have shed considerable light on the systematics and evolution of the genus. The future of interspecific hybridization for improvement of the agriculturally important clovers and the role of molecular approaches in this are discussed.     

Effects of inbreeding on nodal root system morphology and architecture of white clover (<Emphasis Type="Italic">Trifolium repens</Emphasis> L.)

Plants of white clover (Trifolium repens L.) cultivar Crau, a self-fertile Crau genotype, and nine generations of inbred progeny were raised in sand culture in a glasshouse experiment. Digital images of the root systems were made and root morphological characteristics were determined on all the plants. Root architectural parameters were measured on the Crau parent and the S₁, S₄, S₆, and S₉ inbred lines. The clover roots became shorter and thicker with inbreeding but the number of root tips per plant was unchanged. Root architecture (branching pattern) was largely unaffected by inbreeding. It is concluded that inbreeding white clover will lead to shorter, thicker roots, and reduced nutrient uptake efficiency compared with the parent clover. The degree to which these deleterious traits are overcome during the development of F₁ hybrids needs to be determined.     

Development of breeding populations from interspecific hybrids between Trifolium repens L. and T. occidentale Coombe

Trifolium occidentale is a diploid wild relative of T. repens (white clover) with adaptation to dry, saline coastal habitats. Transfer of drought and salt‐tolerant adaptive traits to white clover could be potentially valuable if interspecific hybridization can be achieved efficiently and leads to fertile hybrid populations. To achieve hybridization, 4x plants of T. occidentale were generated. Efficient techniques for generation of 4x plants and their identification using dry pollen shape are described. Interspecific 4x F₁ plants were achieved without embryo rescue. F₂ populations and first backcross hybrids to white clover were also efficiently achieved. Although male and female fertility were lower than in white clover, they were adequate to produce large amounts of seed from small numbers of inflorescences. Thus, early generation interspecific hybrid prebreeding populations can be readily developed, opening the way for transfer of traits from T. occidentale to white clover.     

Effect of Legume Management on Forage Production and Residual Effects on Upland Rice

An experiment was conducted over the period 1995–96 in a warm-temperate environment in Nepal to investigate the effect of cutting frequency on forage yields of three temperate legume species, grown during the winter season, and the residual treatment effects on a subsequent upland rice crop. The three species, Persian clover ( Trifolium resupinatum ), white clover ( Trifolium repens ) and vetch ( Vicia benghalensis ), proved to be well adapted to the winter growing conditions and produced cumulative forage yields between 6.8 t DM ha⁻¹ (vetch) and 9.2 t DM ha⁻¹ (Persian clover). Vetch grew vigorously throughout the winter months and provided reasonable forage yields between December and February, whereas the clovers provided green fodder up to July. These species may therefore make a substantial contribution to alleviating the severe shortage of quality feed during the winter months. Grain yields of the subsequent rice crop ranged from 3.6 to 7.3 t ha⁻¹. Rice yields were greatly affected by the previous legume species and forage management practices. In general, the removal of legume forage greatly reduced the residual effect, and farmers will have to seek a compromise between maximizing green fodder production and the immediate beneficial residual effect of the legume crop on rice yield.     

Trifolium ambiguum as a source of variation in the breeding of white clover

Summary Hybrids between T. ambiguum and T. repens were successfully established after ovule culture. The hybrids were male sterile but one of the hybrids produced a single seed when backcrossed to T. repens, while the other produced a significantly greater number of seeds. The majority of the backcross hybrids from the latter had 48 chromosomes, being produced from the fertilisation of unreduced gametes of the F1 hybrid by T. repens pollen. Regular meiosis in the BC1 hybrid resulted in the production of balanced gametes containing the full complement of T. repens chromosomes and the polyhaploid set of T. ambiguum chromosomes. There was no difficulty in establishing a substantial population of BC2 hybrids. The meiotic behaviour of the BC1 hybrid indicated that it should be possible to transfer genes from T. ambiguum into white clover T. repens. The significance of the results in terms of the use of alien variation in the improvement of white clover is discussed.     

Forage legumes for future dry climates: Lower relative biomass losses of minor forage legumes compared to Trifolium repens under conditions of periodic drought stress

Dairy livestock production systems rely on high‐quality forage legumes, which are widely present in grassland swards all over Europe. A future climatic scenario with higher average annual temperatures and lower precipitation is expected to affect grassland productivity in general and the productivity of the most important forage legume species Trifolium repens in particular. One way to cope with such constraints is the adoption of currently underutilized minor legume species with a higher tolerance towards drought stress. Therefore, the present study investigated legume species with lower moisture requirements than T. repens, these are Lotus corniculatus, Medicago falcata, Medicago lupulina and Onobrychis viciifolia in comparison with T. repens. Legumes were grown in containers as monocultures in a roofed open‐sided greenhouse under conditions of optimal water supply or periodic drought stress. Generally, drought stress decreased the biomass production, but species differed in their reaction. Particularly, M. lupulina and L. corniculatus had lower relative biomass losses (?26%) compared to T. repens (?43%, as averaged over drought stress periods). However, in overall biomass production T. repens still was at one level with M. lupulina and L. corniculatus under drought stress. This was related to high stomata control as indicated by the intrinsic water‐use efficiency. We conclude that there are promising future options of forage legumes as alternatives to T. repens.     

Genetic variability in herbage and seed yield in selected half-sib families of berseem clover, Trifolium alexandrinum L.

Berseem clover Trifolium alexandrinum L. is an annual forage legume commonly grown in pure stands and in grass mixtures in the Medi-terranean basin. Six populations were naturally cross-pollinated in 1990 and 1991 by a half-sib breeding method. In 1992 and 1993. 54 half-sib maternal plants, six original populations, and six advanced populations were field evaluated for forage (short cycle, harvest made at seven or eight inlernodes: long cycle, harvested at flowering) and seed yield (no forage harvest) in experiments at the Forage Crop Institute at Foggia, Italy (typical Mediterranean location). Genetic variability, narrow-sense herilability, genetic and phenotypic correlations among forage and seed yield component trails were investigated. The genetic variance in dry matter among maternal half-sib populations in short cycle was 51% greater than in long cycle. Narrow-sense heritabilily was 35% higher in short cycle than long cycle for dry matter and 26% higher for seed weight than seed yield. The magnitude of the genetic variance components and genetic correlations suggested that selection among plants of maternal half-sib populations would be more effective for improving dry matter in short than in long cycle harvests. The selection applied in the study was not effective for increasing seed yield per se however, the trait may be increased by selecting indirectly for seed weight.     

Effect of spring fertilization on ecosystem services of organic wheat and clover relay intercrops

Nitrogen (N) deficiency and weed infestation are main factors limiting yield and yield stability in organic wheat. Organic fertilizers may be used to improve crop performance but off-farm input costs tend to limit profitability. Instead, forage legumes may be inserted into the crop rotation to improve the N balance and to control weed infestation. In opposition to simultaneous cropping, relay intercropping of legumes in organic winter wheat limits resource competition for the legume cover crop, without decreasing the performance of the associated wheat.The aim of this study is to evaluate the effect of spring organic fertilization on the performance of intercropped legumes and wheat, and on services provided by the legume cover.Two species of forage legumes (Trifolium pratense L. and Trifolium repens L.) were undersown in winter wheat (Triticum aestivum L. cv Lona) in five organic fields during two consecutive crop seasons. Organic fertilizer was composed of feather meal and applied on wheat at legume sowing. The cover crop was maintained after the wheat harvest and destroyed just before sowing maize.Spring organic nitrogen fertilization increased wheat biomass (+35%), nitrogen (+49%), grain yield (+40%) and protein content (+7%) whatever the intercropping treatment. At wheat harvest, red clover biomass was significantly higher than white clover one (1.4 vs. 0.7 t ha⁻¹). Nitrogen fertilization decreased forage legume above-ground biomass at wheat harvest, at approximately 0.5 t ha⁻¹ whatever the specie. No significant difference in forage legume biomass production was observed at cover killing. Nitrogen accumulation in legume above-ground tissues was significantly higher for white clover than for red clover. Both red and white clover species significantly decreased weed infestation at this date. Nitrogen fertilization significantly increased weed biomass whatever the intercropping treatment and decreased nitrogen accumulation in both clover species (−12%).We demonstrated that nitrogen fertilization increased yield of wheat intercropped with forage legume while the performance of legumes was decreased. Legume growth was modified by spring fertilization whatever the species.     

Self-compatibility and heterosis in white clover (Trifolium repens L.)

Inbred lines of white clover (Trifolium repens L.) have been produced utilising the rare self-fertility (Sf) allele. Twenty-two lines of four distinct groups have been maintained through five generations of selfing by single seed descent. Fourteen lines were used in crosses to produce F¹s. Both parents and hybrids were analysed for a range of morphological characters. Positive heterosis for dry matter production was observed in half the hybrids. No other trait showed significant heterosis. The degree of heterosis appears to be related to the extent of variation in morphological characters between the parental lines, some combinations of lines yielding heterotic hybrids in all cases, others in none. The majority of the F¹ hybrids are superior in terms of dry matter production to eight control varieties. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic variation within and between two cultivars of red clover (Trifolium pratense L.): Comparisons of morphological,isozyme, and RAPD markers

Summary Morphological, isozyme and random amplified polymorphic DNA (RAPD) markers were used to estimate genetic variation within and between cultivars of red clover (Trifolium pratense L.), an important temperate forage legume. Two cultivars of red clover, Essi from Europe and Ottawa from Canada, were evaluated. Six monogenic morphological characters were observed for 80 plants from each of these two cultivars. All six morphological loci were polymorphic in the cultivar Essi whereas only four loci were polymorphic in the cultivar Ottawa. Forty plants from each cultivar were assayed for isozyme markers. A total of 21 enzyme-coding loci with 43 alleles was detected using twelve enzyme systems. Thirteen and nine of these loci were polymorphic in Essi and Ottawa, respectively. The mean number of alleles per locus was 1.81 in Essi and 1.67 in Ottawa. Seventeen random 10-mer primers were screened for RAPD markers. Nine primers which gave clear and consistent amplified products were used to assay 20 individuals from each cultivar. Each primer gave from 7 to 20 amplified bands with an average of 14.8 bands per primer. One hundred and eight of 116 putative loci were polymorphic in Essi and 90 of 98 loci were polymorphic in Ottawa. High within-cultivar variation was observed in both cultivars using both isozyme and RAPD markers. This high polymorphism makes these markers useful for germplasm characterization and genetic studies in red clover.