Differential effects of low concentrations of aluminium on the growth of four genotypes of white clover

Summary The effects of aluminium (Al³⁺) on the growth of four cultivars of white clover dependent upon NO₃ ⁻−N were examined. Plants were grown in flowing solution culture with carefully maintained low concentrations (0, 12.5, 25 and 50 mmolm⁻³) of Al, and with P and pH (4.5) also held constant and appropriately low. A three-week treatment period resulted in major effects on the growth and elemental composition of shoots and roots at all concentrations of added Al. There were inherent differences between the cultivars in growth but the relative effects of Al were similar in each case. Examination by S.E.M. and x-ray microanalysis of one cultivar grown at 50 mmolm⁻³ Al, indicated that Al in the roots was associated with P, especially in old, outer epidermal cells. Aluminium reduced NO₃ ⁻ uptake and there were significant effects of Al on nitrate reductase activity (NRA). In contrast to the other characteristics, there were differential effects between the cultivars in NRA, both in the presence and absence of Al.     

Plant factors influencing phosphorus uptake by white clover from solution culture

Summary The phosphorus (P) uptake rate of several white clover populations was determined in two solution culture experiments. Populations and cultivars differed in P uptake per plant and per unit root length in both experiments. Correlation and multiple regression analysis showed that differences between populations for P uptake per plant were largely related (r²>80%) to differences in leaf area and absolute growth rate, when plants had been grown at high-P levels, and by differences in root size and absolute growth rate when plants had been grown at low-P levels. Differences between populations for P uptake per unit root length were related (r²≈50%) to leaf area and relative growth rate in experiment 1 and to transpiration rate and water influx in experiment 2, when plants were pretreated at high-P levels. Differences between populations for P uptake per unit root length were negatively related to root size when plants had been grown at low-P levels. On the basis of these and other results it is suggested that P uptake per plant is determined largely by shoot factors. However, P uptake per unit root length is negatively related to root size, because demand for P is largely determined by shoot factors, and so differences in root size lead to an apparent difference in uptake per unit of root size.     

Variation for aluminium tolerance in white clover

Aluminium (Al) tolerance of fourteen white clover (Trifolium repens L.) cultivars from eleven countries was compared in the greenhouse in the Wainui silt loam (Typic Dystrochrept) to which Al had been added at nine levels (0, 2.5, 5, 20, 50, 150, 250, 500 and 750 mg kg⁻¹ of soil) as Al₂ (SO₄)₃ and incubated for 30 days. None of the white clover cultivars, including those either referred to as Al-tolerant, Dusi and Pathfinder, or from countries that have large areas of acid soils, El Lucero M.A.G., Bayucua, Bage and Zapican, showed greater Al-tolerance than ‘Grasslands Huia’ white clover. Subsequent screening for Al-tolerance can therefore be restricted to germplasm with wide agronomic adaptation.     

Auxin-stimulated somatic embryogenesis from immature cotyledons of white clover

Cotyledons from immature embryos of white clover (Trifolium repens L.) cv. Osceola were exposed to 2,4-D or NAA to induce somatic embryogenesis. NAA at 10 or 20 mg 1^–1 was very inefficient at stimulating embryogenesis, while concentrations of 30 or 40 mg 1^–1 resulted in death of the explant tissue. Continuous exposure of cotyledons to 40 mg 1^–1 2,4-D resulted in somatic embryos which were arrested at the globular stage, or which underwent cycles of secondary embryogenesis, never proceeding beyond the globular stage. A 10 day exposure time to 2,4-D at the same concentration led to formation of somatic embryos, most of which had poorly developed cotyledons. Almost 10% of the somatic embryos converted into plants following transfer to medium devoid of growth regulators. Attempts to improve morphology of somatic embryos by using shorter exposure times to 2,4-D at 40 mg 1^–1, or by maintaining the 10 day exposure time while varying the concentration of 2,4-D, were not successful. Plants were obtained from all parents evaluated, although at different frequencies.     

The growth and asymmetry of neighbouring plants of white clover (Trifolium repens L.)

Summary Transplants of white clover (Trifolium repens L.) were grown isolated from each other and in pairs placed at different distances apart. The paired plants developed asymmetrically and at the interface between paired clones both the density of nodes and of stolons appeared to reach ceiling values that were of the same order as those achieved in isolated clones. It is argued that the growth of plants of T. repens is controlled by the local conditions experienced by the plant parts and not by integrated growth of the whole. Transplants of three different genotypes of T. repens, which differed in growth form, were grown as neighbouring pairs and the calculated asymmetry of the plants was used to compare their mutual aggressivenes. The more compact (phalangeal) genotypes induced greater asymmetry in their neighbours than the more diffuse forms.     

The potential for breeding white clover (Trifolium repens L.) with improved nodulation and nitrogen fixation when grown with combined nitrogen

Summary Sodium nitrate applications ranging from 0.36 to 22.84 mM N were shown to depress rates of nodule formation and reduce total nitrogen fixation (acetylene reduction) in white clover plants grown in aseptic test tube culture.Low nitrate levels gave an initial depression in symbiotic activity but the reduction was of short duration and these treatments were subsequently associated with enhanced rates of nodule formation and nitrogen fixation. As a result, phenotypic variation appeared to be strongly differentially affected by the amount of nitrate present. A subsequent experiment suggested that much of the variation was a consequence of early enhancement of plant growth rates by low levels of nitrate followed by rapid depletion thus giving a transitory inhibitory effect. This was confirmed in a third experiment in which the range of nitrate concentration was held constant. Differential effects on variability in nodule formation and nitrogen fixation were then greatly reduced but there was still a residual level of plant-to-plant variation. The results have clear implications for selecting genetic variants capable of fixing di-nitrogen in the presence of combined N. The provision of a single limiting dose of combined nitrogen to a population containing individuals with inherently different growth rates can bring about variations in the phenotypic expression of symbiotic characters. These variations are unlikely to be based on genetic factors which have a direct and stable effect on nodule development and nitrogenase activity. The implications of the results for plant breeding are discussed.     

Genetic differences in phosphorus absorption among white clover populations

Summary Eight semi-natural white clover populations and two cultivars were grown in culture solutions containing 10 ppm and 0.01 ppm phosphorus (P). The rate of P uptake by the intact plants was then measured in solutions containing 10 ppm P.Phosphorus uptake per unit root length was twice as great by plants previously grown at 0.01 ppm P than those grown at 10 ppm P. Large differences in total P uptake were found among populations regardless of the pretreatment; most of this variation was accounted for by differences in root length. Only small differences were found between populations for P uptake per unit root length, and then only after pretreatment with 10 ppm P; this variation was largely accounted for by relative growth rate and shoot %P.     

Effects of aluminium and pH on growth and potassium uptake by three ectomycorrhizal fungi in liquid culture

Clonal plants of white clover (Trifolium repens L.) were grown in a controlled environment with either low or high rates of applied nitrate-N (providing, notionally, insufficient or sufficient N for unrestricted growth), or in the absence of applied N. Plants receiving no nitrate-N were inoculated with Rhizobia and fixed their own N₂. All plants were maintained with a maximum of three fully unfolded leaves per apex (lenient defoliation) until day 68 when half of the plants were severely defoliated. The export and translocation of carbohydrates from the first fully unfolded main stolon leaf was measured three days later using ¹⁴C.Reduced carbon translocation to stolon tissue and roots, and increased translocation to young branches, occurred following severe defoliation in all three nitrogen treatments. However, N-deficient plants showed large reductions in total export of carbohydrates (44 vs. 17% of ¹⁴C assimilated for lenient vs. severe defoliation) whereas N-sufficient plants (either receiving nitrate-N or fixing their own N₂) showed small increases in total export (means of 54% vs. 62% in the respective defoliation treatments). Furthermore, carbohydrate translocation to old branches ceased altogether in severely defoliated, N-deficient plants, but increased in severely defoliated, N-sufficient plants, illustrating that plant responses to multiple-factor stresses may differ greatly from those seen as the result of single-factor stresses. Interactions between nitrogen nutrition and defoliation in total carbohydrate export, and in carbohydrate supply to old branches, could have serious negative effects on the short-term C economy and physiological integration, and hence on the adaptability, of clonal plants growing with a mineral deficiency in the presence of grazing animals.     

Adventitious shoot production from immature embryos of white clover

Cotyledonary-stage embryos of Haifa white clover, collected 13 days after cross-pollination, were induced to form adventitious shoots primarily from the hypocotyl region. The culture medium used for the production of adventitious shoots contained 5 M thidiazuron and 0.5 M -naphthaleneacetic acid. Numerous shoot meristems were produced within the first week, discrete shoots developed by week three, small plantlets by week eight, and whole plants in soil by week ten. 95–100% of all embryos, regardless of genotype, produced adventitious shoots within four weeks with an average production of 17.5 shoots per embryo. The majority of shoots (on average 77%) were easily converted to whole plants in soil. The white clover regeneration system described is prolific, rapid and effective on a large number of genotypes.Abbreviations BA N⁶-benzylaminopurine - MS medium Murashige & Skoog medium (1962) - NAA -naphthaleneacetic acid - thidiazuron N-phenyl-N-1,2,3-thiadiazol-5-ylurea     

Repetitive somatic embryogenesis and plant recovery in white clover

Summary Breeding and selection was used to generate a population of white clover (Trifolium repens L.) from cultivar Osceola with a high embryogenic capacity. Somatic embryos were obtained from immature cotyledons of white clover placed onto EC6 basal medium containing 40 mg L^–1 of 2,4-D and 6% sucrose. The effects of 2,4-D at 20 and 40 mg L^–1 and of the carbohydrates, sucrose and maltose, were evaluated for their influence in the establishment of repetitive somatic embryogenesis. To determine the optimal protocol for plant recovery from somatic embryos, the effects of MS vs. EC6 basal salts, sucrose vs. maltose, B5 vitamins vs. yeast extract, and inclusion or exclusion of activated charcoal were evaluated. Repeated subculture of white clover somatic embryos on EC6 basal medium containing 6% sucrose with 2,4-D at 20 or 40 mg L^–1 effectively maintains repetitive embryogenesis. Medium containing MS salts with 6% maltose as the carbohydrate source was the most efficient for plant recovery.     

Interactions between nitrate uptake and N2 fixation in white clover

Summary White clover (Trifolium repens L.) plants grown in pots and supplied with the same concentration x days of¹⁵N labelled nitrate, but in contrasting patterns and doses had similar N concentrations but differed in the proportions devived from N₂ fixation and nitrate. N₂-fixation and nodule dry weight responded rapidly (2–3 days) to changes in nitrate availability. Plants exposed frequently to small doses of nitrate took up more nitrate (and hence relied less on N₂-fixation) and had greater dry weights and shoot: root ratios than those exposed to larger doses less often. In mixed ryegrass (Lolium perenne L.)/clover communities clover's ability to either successfully compete for nitrate or fix N₂ gave it consistently higher N concentrations than grass whether they were given high or low nitrate nutrient. This higher N concentration was accompanied by greater dry weights than grass in the low nitrate swards but not where high levels of nitrate were applied.     

The effect of canopy filtered light on the growth of white clover Trifolium repens

Summary Plants of white clover (Trifolium repens) were grown under canopies of clover leaves floating on shallow glass tanks of water and their growth was compared with that of plants under canopies of black polythene leaves. The experimental design allowed the growth of the clover plants in canopy filtered light to be compared with that in unfiltered light at different intensities of Photosynthetically Active Radiation (P.A.R.). The effect of canopy filtered light was to exaggerate the effects of reduced P.A.R. especially in promoting petiole extension and inhibiting stolon branching. Two clones of white clover differed in their responses to P.A.R. and to light quality and there were significant interactions between the effects of the intensity of P.A.R. and type of shade. It is argued that it may be important for both ecologists and plant breeders to recognise the role of radiation quality in regulating the dynamics of pasture.     

Response to soil aluminium of two white clover (Trifolium repens L.) genotypes

An aluminium (Al) tolerant genotype of white clover was compared with an Al susceptible genotype in artificial soil profiles in which exchangeable Al increased with depth. The tolerant genotype had a greater proportion of its root mass deeper in the soil than the susceptible genotype. Nitrogenase activity showed a similar pattern. Shoot Al concentration did not vary between the genotypes but root Al in the susceptible line was twice that in the tolerant genotype. Plant potassium content in the susceptible line was relatively less, probably in response to higher aluminium content.     

Nitrogen fixation by white clover when competing with grasses at moderately low temperatures

It was the aim of this study to determine the way in which low temperature modifies the effect of a competing grass on nitrogen fixation of a forage legume. White clover (Trifolium repens L.) was grown in monoculture or in different planting ratios with timothy (Phleum pratense L.) or perennial ryegress (Lolium perenne L.) in growth chambers at either 7.5/5°C (LoT) or 15/10°C (HiT) average day/night temperatures, and with 2.5 or 7.5 mM ¹⁵N-labelled nitrate in the nutrient solution.Competition with grass led to a marked increase in the proportion of clover nitrogen derived from symbiosis (% N_sym). This increase was slower at LoT where % N_sym was reduced considerably; it was closely related to the reduction in the amount of available nitrate as a result of its being utilized by the grass.Nitrogen concentration in white clover herbage and dry matter yield per clover plant were reduced, for the most part, when a competing grass was present. The amount of nitrogen fixed per plant of white clover decreased markedly with temperature. Low temperature consequently accentuated competition for nitrate. The capacity of white clover to compete successfully was limited by its slower growth and nitrogen accumulation.     

Senescence and decomposition of white clover stolons in grazed upland grass/clover swards

The fate of marked sections of stolons of white clover (Trifolium repens) over a 50-week period from May 1987 was followed in grazed grass/clover swards maintained at 5-cm sward surface height with and without N fertiliser. There was little effect of N treatment on the pattern of survival of stolon sections. The proportion of live stolons recovered decreased during the experiment, and in May 1988 on average only 29% of the marked sections remained alive. At all harvests only a small percentage of stolon sections showed signs of senescence; the maximum percentage, on average 20% of those marked, occurred in autumn, 15–20 weeks after marking. Following this period, i.e. in late autumn/winter, the most rapid increase in percentage of decomposed stolons was measured. Over 50% of stolon sections were buried within the 5-week period following marking and nearly all were buried after 20 weeks; generally a much smaller proportion of stolon tips was buried. Nutrient concentrations of N, P and K fell to their lowest levels in autumn, before increasing in the following spring. Results are discussed in relation to the cycling of nutrients via stolon senescence.     

Acquisition of phosphorus and copper by VA-mycorrhizal hyphae and root-to-shoot transport in white clover

White clover (Trifolium repens L.) plants were grown in a calcareous soil in pots with three compartments, a central one for root growth and two outer ones for growth of vesicular-arbuscular (VA) mycorrhizal (Glomus mosseae [Nicol. & Gerd.] Gerdemann & Trappe) hyphae (hyphal compartments). Phosphorus (P) was applied at three levels (0, 20 and 50 mg kg⁻¹ soil) in the outer compartments in mycorrhizal treatments. Root and shoot dry weight were increased in mycorrhizal plants with hyphal access to outer compartments. Growth of the mycorrhizal hyphae in the outer compartments was not significantly affected by variation in P level in these compartments. However, both concentration and amount of P in roots and shoots sharply increased with increasing P supply in the outer (hyphal) compartments. With increasing P levels the calculated delivery of P by the hyphae from the outer compartments increased from 34% to 90% of total P uptake. Hyphal access to the outer compartments also significantly increased both concentration and quantity of Cu in the plants. The calculated delivery of Cu by the hyphae from the outer compartments ranged from 53% to 62% of total Cu uptake, irrespective of the P levels and the amounts of P taken up and transported by the hyphae. However, the distribution of Cu over roots and shoots was largely dependent on P levels. With increase in P level in the outer compartments the calculated hyphal contribution to the total amount of Cu in the shoots increased from 12% to 58%, but decreased in the roots from 75% to 46%. In conclusion, uptake and transport by VA-mycorrhizal hyphae may contribute substantially not only to P nutrition, but also to Cu nutrition of the host.     

The effects of low,regulated supplies of nitrate and ammonium nitrogen on the growth and composition of perennial ryegrass

Summary Perennial ryegrass was grown in flowing solution culture with nitrogen supplied in amounts that increased exponentially,i.e. in parallel with the rate of increase in growth. Nitrogen was supplied as either NO ₃ ⁻ or NH ₄ ⁺ , and the amounts to be added were calculated on the basis of extrapolated values for dry weights obtained from fitted curves. There were two rates of addition for each form of N aimed at providing adequate (5.0 per cent) and less than adequate (2.75 per cent) contents in the plants in each case. Measured plant weights and N concentrations were in close agreement with predicted values over a four week experimental period. There was no effect of N-form at high N, and these plants produced 46 per cent more dry matter than the plants at low N. Only minor differences in overall growth occurred with NO ₃ ⁻ or NH ₄ ⁺ plants at low N, but the NH ₄ ⁺ plants had a greater shoot:root ratio. The absorption rate (m mol Ng⁻ root d⁻¹) for NH ₄ ⁺ -N was therefore greater than for NO ₃ ⁻ -N. The cation/anion composition of the plants was affected in a predicable way, and to a greater or lesser extent at high or low N, respectively, in NO ₃ ⁻ or NH ₄ ⁺ plants. The major changes in cation composition came through effects on potassium absorption. Plants with low NO ₃ ⁻ appeared to be under greater N stress than those with low NH ₄ ⁺ because of the lower shoot:root ratio and the greater C∶N ratio in the shoots.     

Influence of pH,exchangeable aluminium and 0.02M CaCl2-extractable aluminium on the growth and nitrogen-fixing activity of white clover (Trifolium repens) in some New Zealand soils

The effects of soil acidity on the growth and N₂-fixing activity of white clover in seven acid topsoils and subsoils of New Zealand were investigated using a glasshouse experiment.The application of phosphate (Ca(H₂PO₄)₂) to the soils resulted in very large increases in white clover growth on all soils. The application of phosphate, as well as increasing P supply, also decreased 0.02M CaCl₂-extractable Al levels, but had little effect on exchangeable Al levels.Where adequate phosphate was applied, increasing rates of lime (CaCO₃) resulted in increased plant growth on most soils. N₂[C₂H₂]-fixing activity was increased by the first level of lime for one soil, but generally remained approximately constant or declined slightly at higher rates of lime. Up to the point of maximum yield, white clover top weight was more highly correlated with 0.02M CaCl₂-extractable soil Al than with exchangeable Al or pH. At pH values greater than 5.5, plant yield declined on some soils, apparently because of Zn deficiency. The data suggest that white clover is unlikely to be affected by Al toxicity at 0.02M CaCl₂-extractable Al levels of less than about 3.3 g g^–1. However, there were differences between soils in apparent plant tolerance to 0.02M CaCl₂-extractable Al, which appeared to be caused by differing C levels in the 0.02M CaCl₂ extracts.     

Prolific direct plant regeneration from cotyledons of white clover

Summary A facile procedure has been developed to regenerate white clover (Trifolium repens L.) plants, rapidly and directly from cotyledon explants of 3 day old seedlings. Scanning electron microscopy and histological sectioning demonstrated that shoot meristems developed from individual epidermal cells on the adaxial surface of the cotyledonary stalk, proximal to the site of excision. Initial cell divisions occurred after 2 days of culture and regenerated plants were transferred to soil within 6–8 weeks. Regenerated plants were normal, flowered and set seed. The highest shoot regeneration frequency (an average of 20 shoots per cotyledon) was obtained using an MS based medium containing 1.0 mg 1^-1 6-benzylaminopurine and 0.05 mg 1^-1 -napthaleneacetic acid. A similar regeneration frequency was obtained from cotyledon explants taken from eight different white clover cultivars.Abbreviations BAP 6-benzylaminopurine - NAA -napthaleneacetic acid - MS Murashige and Skoog medium     

Earlier flowering between 1962 and 2002 in agricultural varieties of white clover

Date of first flowering was recorded for 40 years in the same agricultural varieties of white clover ( Trifolium repens L.). Standard procedures, experimental conditions and germplasm allowed the unmediated effects of climatic trends to be observed. Flowering dates of the same varieties at the same site have become significantly earlier over this period, with an advance of first flowering of approximately 7.5 days per decade since 1978. Annual maximum, minimum and soil temperatures at the site increased during the 40-year period. First flowering dates (FFDs) were significantly negatively correlated with minimum and maximum temperatures during February and March and soil temperatures between January and April. Maximum, minimum and soil temperatures increased between 1962 and 2002 for these months. No significant correlation was seen between FFD and soil temperatures at other times of the year including the period of vernalisation during winter. The occurrence of ground frost in February was significantly correlated with later flowering. Total annual rainfall and the number of wet days in a year increased between 1962 and 2002. However, only rainfall in February had a significant impact on FFD.