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 .     

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.     

Growth and reproductive characteristics in backcross hybrids derived from Trifolium repens L. × T. nigrescens Viv. interspecific crosses

Interspecific hybridisation with the close relative, Trifolium nigrescens (Ball clover) is a possible strategy to achieve increased reproductive potential of white clover (Trifolium repens). Fertile F₁ plants have been used as the basis for two generations of backcrossing to T. repens as the recurrent parent. F₁ and backcrossed plants were assessed in both glasshouse and field for a range of morphological traits, including inflorescence production, and the level of water soluble carbohydrates in the stolons. Plants resulting from two generations of backcrossing had an increased allocation of dry matter to inflorescence production in comparison with T. repens. Variation within these plants for agronomic traits (e.g. stolon length, dry weights etc.) suggests that selection for these traits is feasible and in combination with increased inflorescence production offers a potentially valuable approach to germplasm improvement in white clover. This revised version was published online in August 2006 with corrections to the Cover Date.     

Quality characteristics of backcross hybrids between Trifolium repens and Trifolium ambiguum

Two generations of novel backcross hybrids between white clover (Trifolium repens L.) and the related species Trifolium ambiguum,with white clover as the recurrent parent, were analysed for forage quality characteristics. Water soluble carbohydrate (WSC) levels, nitrogen concentrations (%N), and dry matter digestibility (DMD) were recorded for the hybrids and parental species grown in deep soil bins with and without a companion grass. Half the experimental material was subjected to moisture stress by being left unwatered through the four-week period when measurements were made. Four such four -week cycles of drought were imposed. The first and second generation backcrosses had the highest values for WSC, but were lowest for N%. There was no difference between the different legume lines (parents and backcrosses) for DMD. No effects of either moisture stress or presence of a grass companion on quality traits were observed. The WSC and %N of the grass component differed significantly depending on which legume line it was grown with. The importance of these results for the potential use of these hybrids in agriculture is discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

Introgression of reproductive traits from Trifolium nigrescens increases the seed yield of white clover (T. repens)

Interspecific hybrids between white clover and the annual, profuse flowering ball clover have been developed as a strategy to improve the potential seed yield of white clover. The seed yield and seed yield components of advanced white clover × ball clover hybrids were compared with white clover in replicated field experiments over two harvest years. The backcross 3 hybrid had more reproductive nodes per stolon than the control variety ‘AberHerald’ which was reflected in more inflorescences per unit area but inflorescences with fewer florets and seeds. The hybrid had a greater potential seed yield than the control variety in 2002 but not 2001. Backcross 3 hybrids have also been produced across the range of leaf size categories. Medium and large leaved hybrids produced significantly more inflorescences and a higher potential seed yield than control varieties of comparable leaf size in both harvest years. The small leaved hybrid was not significantly greater than the control variety ‘AberDale’ in either inflorescence production or seed yield.     

Modulation of Brassica napus source–sink physiology through film antitranspirant induced drought tolerance amelioration that is dependent on the stress magnitude

Increase in drought conditions during the oilseed rape (OSR) reproductive phase is predicted to occur more often in the temperate zone, leading to significant yield losses. Crop management solutions such as film antitranspirant (AT) applied at key drought‐sensitive growth stages on both wheat and oilseed rape have recently been shown to alleviate drought‐induced yield losses. However, there is a lack of information regarding potential AT effectiveness to reduce drought damage on OSR plants at different soil moisture regimes. Therefore, two similar experiments were performed in a computer‐controlled glasshouse/phenotyping centre to investigate the physiological responses of OSR to well‐watered (WW), moderate water stress (MWS), water stress (WS) and severe water stress (SWS) conditions. Stress treatments were imposed at the initiation of flowering and treated with an AT or water onto the leaf canopy. Stress limited the gas‐exchange and increased leaf temperature, leaf‐to‐air temperature, bud‐to‐air temperature and ABA concentrations which increased with stress intensity in all tissues analysed. Yield components were significantly reduced by WS and SWS treatments when compared to the WW plants. Application of AT counteracted the detrimental effect of WS and SWS by decreasing water use over the first few days of stress application thus improving relative water content and leaf water‐use efficiency, decreasing ABA accumulation in leaf and all the reproductive organs analysed (buds, flowers and pods) and avoiding bud‐to‐air temperature increases. AT application sustained pod formation and seed production under WS but only seed production under SWS conditions. These data suggest that leaf‐canopy application of AT at key phenological stages under particular magnitudes of soil moisture deficit may sustain OSR reproduction and reduce yield losses.     

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.     

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.     

白三叶种质资源主要数量性状的变异与相关性研究

为了探讨白三叶(Trifolium repens L.)种质资源农艺性状变异和相关性,揭示各种质材料的特征特性和种质资源群间的遗传关系,本研究对78份白三叶种质的12个主要数量性状的变异和相关性进行了分析。结果表明：这些种质材料存在着丰富的遗传多样性,各个数量性状间均呈正相关,植株高度与小叶长度、小叶宽度、小叶大小、叶柄长度呈显著正相关;小叶长度与小叶宽度、小叶大小呈极显著正相关,与叶柄长度、叶柄直径呈显著正相关;小叶宽度与小叶大小呈极显著正相关,与叶柄长度、花柄直径呈显著正相关;小叶大小与叶柄长度、叶柄直径、花柄直径呈显著正相关。该批种质材料能为各类白三叶育种和遗传研究提供较为丰富的亲本材料。     

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.     

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.     

干旱胁迫对白三叶光合参数日变化的影响

通过白三叶(Trifolium repens cv.Haifa)的盆栽试验,研究了连续干旱对白三叶叶片相对含水量、叶绿素含量和光合参数的影响。结果表明,当植株遭受较长时间干旱胁迫时,白三叶叶片相对含水量显著下降;净光合速率、蒸腾速率下降明显,日变化由双峰曲线转变为单峰曲线;水分利用效率仅在早晨有小幅升高,但仍以下降为主。气孔导度显著降低,而胞间CO2浓度显著升高,说明干旱胁迫下白三叶的光合作用降低的原因主要是非气孔限制的结果。     

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.     

Morphological characteristics of hybrids between white clover, Trifolium repens L., and Caucasian clover, Trifolium ambiguum M. Bieb

Hybrids between the stoloniferous Trifolium repens (2n = 4x = 32) and the rhizomatous Trifolium ambiguum (2n = 4x = 32) have been produced using T. repens as the recurrent parent. Morphological characteristics of the parent species and the F₁, BC1, BC2 and BC3 hybrids were examined in a glasshouse experiment. Leaflet ratios and general plant shape indicated that the BC3 hybrid was similar to T. repens. Separation of plants into components of above- and below-ground growth showed that T. repens had a greater total plant dry weight than T. ambiguum but 24% of its dry weight was in roots compared with 22% in roots and 44% in rhizomes in T. ambiguum. The BC3 generation contained plants that were predominantly T. repens-like with stolons, but also had a small proportion of their total dry weight (3%) as rhizome, confirming the potential to produce plants that combine stoloniferous and rhizomatous growth. The proportions of rhizome and stolon varied within the BC3 generation, enabling farther selection and plant breeding. The BC3 hybrids produced similar numbers of inflorescences to T. repens however, fertility was lower, although there was some variation within the BC3 for both characters.     

Role of the maternal effect phenomena in improving water stress tolerance in narrow‐leafed lupine (Lupinus angustifolius)

This paper describes experiments concerning improvement of drought tolerance in narrow‐leafed lupine (Lupinus angustifolius L.) by maternal effects. The first step involved harvesting seeds from plants exposed and not exposed to drought during flowering and seed ripening. The next‐generation plants grown from these seeds were exposed to drought and the effects of this stress on their physiological processes were examined. To find out whether drought applied to parent plants may affect tolerance to this stress in progeny plants such features as plant growth, tissue water content, abscisic acid concentration and yield‐related parameters were assessed. The study revealed that the progeny plants grown from the seeds of drought‐treated plants were more tolerant to this stress than the plants grown from the seeds harvested from optimally watered maternal plants. Drought tolerance was manifested by a reduced concentration of abscisic acid, increased plant height and maintaining high leaf water content. Most importantly, these plants produced significantly higher yield when exposed to drought than the plants grown from the seeds harvested from optimally watered plants.     

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.     

Effects of water deficit and plant interaction on morphological growth parameters and yield of white clover (Trifolium repens L.) and ryegrass (Lolium perenne L.) mixtures

The effects of soil water deficit and interspecific plant interaction were studied on the dry matter (DM) yield of white clover and ryegrass and on the morphogenesis of white clover. Plants were grown either: (1) individually (no interaction); or in a mixture of equal plant numbers with either (2) just shoot interaction, or (3) both shoot+root interaction. Plants were subjected to soil water deficits corresponding to no (0 MPa), moderate (−0.5 MPa) or severe (−1.0 MPa) water deficit. Ryegrass had higher above- and below-ground DM yields than white clover. The above- and below-ground DM yield for ryegrass, and the above-ground DM yield, stolon growth and relative growth rate (RGR), and leaf appearance rate (LAR) for white clover decreased as soil water deficit increased. In shoot+root interaction at no and moderate soil water deficit levels, white clover had the highest proportion of above-ground growth in the leaf form (69%) and had, respectively, 11% and 32% more above-ground DM yield than when grown in just shoot interaction; ryegrass had, respectively, 20% and 25% more above-ground DM yield than ryegrass grown in just shoot interaction. In shoot+root interaction at severe soil water deficit, ryegrass had twice as much above-ground DM yield as white clover (3.50 g per plant versus 1.59 g per plant, respectively) and white clover had 60% less above-ground DM yield than when grown in just shoot interaction. In a soil column of restricted depth (30 cm) at no and moderate soil water deficit levels, remarkable increases in shoot biomass yield were observed for both white clover and ryegrass grown in shoot+root interaction. The increased shoot biomass yield of ryegrass can be attributed to benefits from white clover's N fixing ability, whereas the causes for increased white clover biomass yield need to be studied further. However, at severe soil water deficit, ryegrass had a competitive advantage over white clover when grown in shoot+root interaction. This was due to the larger root system of ryegrass and its ability to control transpirational losses more efficiently, thus prolonging its growth period compared with white clover.     

Influence of Seed Quality on Field Establishment and Forage Yield of White Clover (Trifolium repens L.)

Two seed lots of white clover ( Trifolium repens L.) cultivar 'Makibashiro' classified as high and poor quality seed lots, based on the speed of germination (T50), were used to examine the influence of seed quality on field establishment and forage yield. Field emergence rate was estimated approximately three weeks after sowing and found that plots sown with high quality seeds (Lot 1) had 452 seedlings m^-2 compared with 392 seedlings in the plots sown with low quality seed lot (Lot 2), but the difference was not significant. However, plant density in autumn (November, 1993), 3 months after sowing, was significantly (P < 0.05) higher in the plots of the Lot 1 seed than that from the Lot 2 seed. The percentage of clover plants surviving over the winter from Lot 1 plots was 55% compared with 39% from the Lot 2 plots, but the difference was not significant. Total forage yield in Lot 1 plots harvested in July and monthly intervals to October, was significantly increased by 44% (P < 0.05) over that of Lot 2 plots, but this greater forage yield in high seed quality seed lot plots was produced early in the season. The results strongly imply that the primary advantage to be gained from high quality seed is an improved field establishment, which led to increased yield of clover. In Lot 1 plots, the percentage of weed dry matter in all the four harvests remained below 1% of die seasonal dry matter production, whereas in Lot 2 plots die percentage of weed dry matter ranged from 3.3–16.9%.     

Effects of divergent selection on correlations between morphological traits in white clover (Trifolium repens L.)

The effects of two generations of divergent selection for stolon diameter and stolon amount on the pattern of phenotypic correlations between important agronomic traits in white clover (Trifolium repens L.) were studied. The effects of selection were: the proportions of the variances of traits under direct selection were considerably lower within selection lines than overall and this was associated with a loss of correlation between traits. Unselected traits showed higher proportions of variances remaining within the selection lines and shifts from non-significant to significant correlations from the plants overall to selection lines. The significance of these results and their relevance to strategies of germplasm improvement is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.