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 potential of legumes as a nitrogen source for the reclamation of derelicht land

Summary White clover (Trifolium repens) growing in association with grasses on colliery spoil amended with lime and complete fertilizer proved to be an effective nitrogen source for the developing ecosystem. Nitrogen transfer from the clover to an associated grass became aparent within 22 months of sowing.     

The effect of source,concentration and time of application of nitrogen on the growth,nodulation and nitrogen fixation ofLotus pedunculatus andTrifolium repens

Summary Studies under growth cabinet conditions investigated the effect of source and concentration of nitrogen and timing of nitrogen application on the growth and nitrogen fixation byLotus pedunculatus cv. Maku andTrifolium repens cv. S184. KNO₃, NaNO₃ and NH₄NO₃ were added at transplanting at the following rates: 3.33, 7.78 and 13.33 mg N/plant. KNO₃ was added at 3.33 and 7.78 mg N/plant at 0, 6, 12, 18, 24 or 30 days after transplanting.Lotus shoot weight increased with all increasing nitrogen sources but clover only responded to KNO₃ and NaNO₃. The root weight of both species increased with increasing KNO₃ and NH₄NO₃. The percentage increase in lotus and clover shoot growth was greater than that of root growth when KNO₃ was added within a week of transplanting. Increases in growth by both species resulted from added nitrogen except with lotus when NaNO₃ was applied where increased nitrogen fixation also contributed to increased growth.Weight and number of effective nodules on both species were increased with 3.33 mg N per plant as KNO₃ but nitrogen fixation was not affected. Addition of 13.33 mg N as NaNO₃ reduced weight and number of effective nodules in both species and also nitrogen fixation by lotus.KNO₃ increased growth and nodulation of both species when applied within one week after transplanting. Nodulated lotus plants responded to KNO₃ by increasing growth but not nodulation.KNO₃ appeared to affect infection and development of nodules on lotus and may affect the growth of existing nodules on clover.     

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.     

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 effects of low concentrations of aluminium on the growth and uptake of nitrate-N by white clover

Summary The effects of aluminium (Al³⁺) at concentrations of 0, 25, 50 and 100 μM on the growth of white clover, dependent upon N supplied as NO ₃ ⁻ , were examined in flowing solution culture. Plants were established with a normal nutrient supply for 7 weeks and then grown with carefully controlled pH (at 4.5) and P concentrations, and with 0, 25, 50 or 100 μM Al³⁺ for a further three weeks. There were rapid visual effects (i.e. symptoms of P deficiency and reduction in root extension) and the dry weights of shoots and roots were reduced at 50 and 100 μM. Less than 10% of Al absorbed from solution was transported to the shoots. The uptake of P, and its transport between roots and shoots, were reduced in plants grown with Al. The uptake of NO ₃ ⁻ stopped immediately after the introduction of 50 or 100 μM Al, and was significantly reduced at 25 μM after three weeks. During a second phase of the experiment, plants previously grown at 0, 25, 50 and 100 μM Al, were grown for a further 2 weeks either with NO ₃ ⁻ (with and without 50 μM Al³⁺) or without NO ₃ ⁻ but with inoculation by Rhizobia (and with or without 50 μM Al³⁺). The effects of the previous treatments with Al on N uptake were small during the second phase, but uptake by all plants was restricted when Al was present. Inoculation did not result in nodulation in the second phase when Al³⁺ was present in the solution, but Al already in the plant from the first phase did not prevent nodulation in the absence of Al during the second phase.     

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 in growth and ion accumulation between two selected populations of Trifolium repens L. differing in salt tolerance

Two divergent populations of T. repens cv. Haifa developed from two generations of recurrent selection for shoot chloride concentration, were grown in the greenhouse at 0 and 40 mol m^–3 NaCl. Over two harvest cycles at 40 mol m^–3 NaCl, the population selected for a low concentration of chloride in the shoot maintained a significantly lower chloride and sodium concentration compared with those plants selected for a high shoot chloride concentration. The distribution of chloride in the shoots was further examined in a subsample of plants from both populations. In all plants, concentrations of chloride were lower in the expanding and fully expanded leaves than in the older leaf tissue or petioles.While there were no significant differences in the photosynthetic rates between lines, shoot yields and relative leaf expansion rates were higher in the low chloride population. Plant death was greater in plants selected for high shoot chloride. These results suggest that selections based on measurements of low shoot chloride concentrations may be successful in developing a cultivar of T. repens with improved salt tolerance.     

Effect of light quality (red: far-red ratio) and defoliation treatments applied at a single phytomer on axillary bud outgrowth in Trifolium repens L.

We studied the effects of light quality and defoliation on the rate of phytomer appearance and axillary bud outgrowth in white clover. The treatments were applied to one phytomer, a phytomer being defined as the structural unit comprising a node, internode, axillary bud, subtending leaf and two nodal root primordia. Light of a low red:far-red (R:FR) ratio (0.27) was applied to a target phytomer either (i) within the apical bud and then to the axillary bud after emergence of the phytomer from the apical bud, or (ii) to the axillary bud only after emergence. The light conditions were directed to these specific parts of the plant by collimating light from small FR light-emitting diodes; with this technique we were able to change the light quality without any change in the level of photosynthetically active radiation. The subtending leaf of the target phytomer was retained or defoliated when it had emerged from the apical bud. FR light applied from the time the phytomer was within the apical bud caused a delay in branch appearance at the target phytomer. In contrast, direct treatment of the axillary bud with FR light after it had emerged from the apical bud did not result in any delay in branch appearance. As the light treatment of the apical bud may have changed the light environment of any of the organs contained in the bud we were unable to ascribe the delay in branch appearance to light perception by any particular organ. However, indirect evidence leads to the conclusion that the likely site of light perception was the developing leaf subtending the axillary bud while it was the outermost phytomer within the apical bud. These results do not support the hypothesis that the R:FR ratio of light incident at an axillary bud site is the environmental factor that controls bud development. Defoliation of the unfolding leaf reduced the rate of phytomer appearance on the main stolon but had no immediate effect on branch appearance. As a consequence there was a reduction in the number of phytomers between the stolon apical meristem and the first phytomer with a branch. This is frequently taken to indicate a relaxation of apical dominance, but in this case was found not to involve a direct effect on bud activity. A current model of white clover growth suggests that there is integration of activity between apical meristems but independence of activity and response to the local micro-environment by axillary buds. In contrast, we found that (i) defoliation reduced phytomer appearance only at the main stolon apical meristem and not at all the meristems in the plant and (ii) that a change in the local light environment of an axillary bud had no discernible effect on bud activity once the bud had emerged from the apical bud but could delay branching if applied before emergence. These results are at variance with the predictions of the model.     

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.     

Developing a breeding strategy to exploit quantitative variation in symbiotic nitrogen fixation

Summary This paper examines evidence which quantifies the relative importance of legume and Rhizobium genotypes as determinants of phenotypic variation in symbiotic nitrogen fixation. It demonstrates potentially large and unpredictable effects of the Rhizobium genotype. The likely importance of such effects on crop yield is considered. The information is then used to assess ways in which legume breeding programmes may be altered to encompass the effects of genetic variation in Rhizobium.     

Soil acidity factors and nodulation ofTrifolium repens

Summary Effects of factors associated with soil acidity (low pH, low calcium, high aluminium and high manganese) on theTrifolium repens-Rhizobium trifolii symbiosis were investigted under laboratory conditions using an axenic solution-culture technique. 200 μM manganese increased root elongation in the range pH 4.3–5.5, but had no effect on root hair formation, the number of Rhizobium in the rhizosphere, or nodule formation. Root elongation and root hair formation were unaffected at pH 4.3 when 500 or 1000μM calcium was supplied, whereas multiplication of Rhizobium in the rhizosphere and nodulation were inhibited at pH 4.3 and 4.7.50–1000μM calcium had no effect either on the multiplication of Rhizobium in the range pH 4.3–5.5, or on nodule formation in the absence of aluminium. 50 μM aluminium inhibited, root elongation and root hair formation at pH 4.3 and 4.7; the effect on root elongation was reduced by increasing the calcium concentration from 50 to 1000μM. 50μM aluminium also inhibited Rhizobium multiplication in the rhizosphere and reduced nodule formation at pH 5.5 (at which aluminium precipitated out of solution), but root elongation and root hair formation were unaffected. These, effects of aluminium at pH 5.5 may explain the poor response to inoculation by white clover in acid mineral soils after liming.     

Inhibition of N2 fixation by white clover (Trifolium repens L.) at low concentrations of NO inf3 sup− in flowing solution culture

The impact of sustained low external concentrations of NO ₃ ^– (0, 10, 100 and 1000 mmol m^–3) on plant growth and the relative acquisition of N through N₂ fixation and NO ₃ ^– uptake by established, nodulated white clover (Trifolium repens L. cv. Blanca) was studied over 28 days in flowing solution culture. Nitrogen fixation was measured by N difference and ¹⁵N dilution methods. Plants supplied with NO ₃ ^– achieved higher relative growth rates (% MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x% fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaGabmiEayaara% aaaa!3702!\[\bar x\]=0.091 d^–1) compared with control plants dependent on N₂ fixation (0.073 d^–1). Nitrate plants showed progressive increases in shoot: root d.w. ratios from 4 to 6.5–7.6 between days 0–28, compared with 5.1 on day 28 for control plants. Increases in both nodule d.w. and numbers per plant were inhibited after day seven at all concentrations of NO ₃ ^– . The severity of inhibition of N₂ fixation increased with increasing NO ₃ ^– concentration and with time. The total amounts of N₂ fixed per plant between days 0–7 after supplying 10, 100 and 1000 mmol m^–3 NO ₃ ^– , respectively, were 37–39, 28–30 and 0–13%, of the total N acquired. Between days 7–28 the proportional contributions of N₂ fixation to total N acquisition declined to 3, 0.5 and 0%, respectively, in these treatments. The corresponding mean specific rates of N₂ fixation between days 0–7 were, respectively, 5.4, 3.2, and 2.0 mmol N d^–1 g^–1 nodule d.w., compared with 7.9 mmol N d^–1 g^–1 nodule d.w. for zero NO ₃ ^– plants. There was no evidence of a transitory increase in N₂ fixation following the addition of NO ₃ ^– , even at the lowest supply concentration.     

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 mM¹⁵N-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.     

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.     

Aluminium toxicity and nodulation ofTrifolium repens

Summary Effects of aluminium on theTrifolium repens var Huia-Rhizobium trifolii strain HP3 symbiosis were studied using an axenic solution-culture system. With, 10 μM phosphate, 50 μM aluminium reduced or inhibited root elongation at pH<5.0, root hair formation at pH< 5.0–5.5, and Rhizobium multiplication in the rhizosphere and nodule formation at pH<6.0. In the absence of aluminium, root elongation and root hair formation were reduced at pH<4.3, and Rhizobium multiplication and nodule formation were inhibited at pH<5.0. Root hair formation was more sensitive to aluminium at pH<5 than was root elongation. No effect of aluminium on Rhizobium multiplication and nodule formation at pH<5 was detected because both were sensitive to pH alone. At pH 5.5 most of the aluminium changed immediately to a form which was susceptible to low-speed centrifugation, but which was detected by the aluminon method of analysis, and after 24 h a precipitate formed. the concentration of phosphate was reduced also, to approximately 1μM. Toxicity was overcome by either increasing the phosphate concentration from 10 to 50 μM, or by increasing the pH to 6.0 and the calcium, concentration to 1000μM.     

Heritability of,and relationships between phosphorus and nitrogen concentration in shoot,stolon and root of white clover (Trifolium repens L.)

Ninety eight white clover genotypes were cloned and grown in pots at two levels of phosphorus (P) supply in soil. After harvest the nitrogen (N) and P content of shoot (leaf, petiole and unrooted stolon), stolon and root tissue was determined. Broad sense heritabilities for %N, %P, and proportion of total N or P in each tissue type were calculated. Heritabilities ranged from 0.22 to 0.68. They were generally higher for %P than %N; and higher in shoot and stolon tissue than root tissue for %P, %N, and proportion of N or P. Level of P in which plants were grown had little effect on heritability values. Genotypes from bred cultivars differed from those collected from hill country pastures for plant size, and partitioning of N and P to shoot, stolon and root. Relationships between plant characters were examined to determine the consequences of selection.     

Transposon-induced symbiotic mutants of Bradyrhizobium japonicum: isolation of two gene regions essential for nodulation

Summary Two strains of the soybean endosymbiont Bradyrhizobium japonicum, USDA 110 and 61 A101 C, were mutagenized with transposon Tn5. After plant infection tests of a total of 6,926 kanamycin and streptomycin resistant transconjugants, 25 mutants were identified that are defective in nodule formation (Nod^-) or nitrogen fixation (Fix^-). Seven Nod^- mutants were isolated from strain USDA 110 and from strain 61 A101 C, 4 Nod^- mutants and 14 Fix^- mutants were identified. Subsequent auxotrophic tests on these symbiotically defective mutants identified 4 His^- Nod^- mutants of USDA 110. Genomic Southern analysis of the 25 mutants revealed that each of them carried a single copy of Tn5 integrated in the genome. Three 61 A101 C Fix^- mutants were found to have vector DNA co-integrated along with Tn5 in the genome. Two independent DNA regions flanking Tn5 were cloned from the three nonauxotrophic Nod^- mutants and one His^-Nod^- mutant of USDA 110. Homogenotization of the cloned fragments into wild-type strain USDA 110 and subsequent nodulation assay of the resulting homogenotes confirmed that the Tn5 insertion was responsible for the Nod^- phenotype. Partial EcoR1 restriction enzyme maps around the Tn5 insertion sites were generated. Hybridization of these cloned regions to the previously cloned nod regions of R. meliloti and nif and nod regions of B. japonicum USDA 110 showed no homology, suggesting that these regions represent new symbiotic clusters of B. japonicum.