Nodulation ofMedicago sativa in solution culture

Summary The time course of increase in nodule number was observed in solution cultures of controlled composition, over periods of 5 to 7 weeks following the inoculation of 8-day-old seedlings. The plants produced successive crops of nodules two to three weeks apart. Maintaining nitrate at concentrations of 0.2 or 0.5mM greatly reduced the number of nodules in the first crop but only slightly or not at all in the larger following crops, relative to the numbers in nil-nitrate control cultures. This was not due to any decrease in the sensitivity of nodulation to inhibition by nitrate. Nodulation in the nil-nitrate controlplants was restrained following their abundant first-crop nodulation. The experiments ceased to be simple tests of effects of nitrate on rate of nodule production as soon as the first crop of nodules appeared. Subsequent nodule production was then inhibited approximately equally, by prior nodulation in the abundantly nodulated control-plants, and by nitrate in the sparsely nodulated plants in the nitrate treatment.When the effect of nitrate was tested on plants comparable in size and in number of previously established nodules, it inhibited nodule production by 70–80%, whether the plants were seedlings or month-old plants, previously nodulated or not.     

Nodulation ofMedicago sativa in solution culture

Summary Calcium and hydrogen ions interacted on nodulation. Increasing acidity from pH 5.6 to pH 4.8 increased the calcium concentration required to nodulate 50% of the plants, from 0.1 mM to 6 mM. Calcium concentration below 0.2 mM or pH below 4.8 inhibited nodulation at all tested levels of the other variable. Root extension and root-hair production were insufficiently affected by calcium or pH to explain reductions in nodule numbers. Initiation of infection, the most acid-sensitive stage of the nodulation process, was also the most calcium-demanding stage at pH 5.2. Once infections were initiated, infection threads still developed and nodules still grew despite transfer of the plants to solutions too low in calcium to have permitted infection to begin. Pretreatments at 0.5 mM and 8 mM calcium at pH 5.2 before inoculation had no significantly different effects on nodulation. Observations on root-hair distribution suggest that developing nodules can suppress further infection by suppressing the emergence of root hairs on newly developing roots.     

Nodulation and growth ofPhaseolus vulgaris in solution culture

Summary Conditions and techniques for achieving good nodulation ofPhaseolus vulgaris L. in continuously aerated solution were developed from greenhouse experiments.If nodules had been established, their growth and activity and the growth of the plant were at least as good in solution culture as in gravel culture. Nodule formation was observed within 10 days of inoculation in small volumes of solution culture (1 liter). In large volumes (19 liters), similarly prompt nodulation occurred only if the plants were inoculated before or immediately after the seedlings were transferred to the solution from gravel or vermiculite; and the nodules were restricted to the roots that had been present at the time of transfer. Delayed inoculation, 2 days after transfer to large volume solutions, led to sparse nodulation observed only after 3 weeks. Delay or inhibition of nodulation in large volumes of solution could not be explained by failute of bacteria to colonize roots or by sparsity of root hairs.Nodule initiation in solution culture was severely inhibited at pH below 5.4. An additional problem in growing N₂-dependent bean in solution culture was the buildup of Cl^– to toxic levels in the plant in nitrate-free media, even at solution concentrations as low as 0.4 mM Cl^–. Daily addition of 0.5 to 1.0 mg N per plant delayed nodule growth and activity slightly, but increased plant growth and alleviated the severe N-deficiency that otherwise developed before the onset of N₂-fixation.     

Effect of inoculation on the elemental composition ofMedicago sativa

Inoculation with rhizobia ofM. sativa plants grown either in the laboratory on nitrogen-free agar and soil-agar slopes or in field experiments, decreases in all cases the concentration of Mo in plant shoots and increases that of Rb, while differences in the concentration of several elements with atomic numbers between 17 and 42 could also be identified by X-ray fluorescence. These elemental alterations depend on the fixation induced either by inoculation or by the effective indigenous rhizobial population of each growth medium and are not caused by rhizosphere pH alterations but seem to be directly related to the induced biological nitrogen fixation.     

The symmetry of the lateral leaflets ofMedicago sativa L.

The given data shows that between the halves of lateral leaflets ofMedicago sativa there exists an anatomical symmetry of the epidermis as well as in the case when both lateral leaflets are compared one with another. In spite of several differences, which may be caused by the environmental conditions, the analysis of variance shows that these differences have no importance in the total variability of obtained data. Similarly, the symmetry was shown by the study of water loss of the pairs' lateral leaflets. In this case the total course of the water loss curve must be appreciated, which is the same in the curves of a leaflets' pair of the same leaf. Differences in the absolute water loss are caused probably by the manipulation with the material before the experiment.     

Regeneration of foreign genes co-transformed plants ofMedicago sativa L byAgrobacterium rhizogenes

Gene encoding sulphur amino acid-rich protein (HNP) and rol genes were transferred into Medicago sativa L (alfalfa) mediated by Agrobacterium tumafeciens. Regeneration of trans-genie plants was induced successfully from hairy root tissue of cotyledon in alfalfa. Cotyledon tissues were an ideally transformed recipient. There was a negative correlation between age of hairy roots and embryogenesis frequency in alfalfa. Production of co-transformed plants with greater yield and super quality was important for development of new alfalfa varieties.     

Effect of inoculation and nitrogen application on the elemental composition ofMedicago sativa

Medicato sativa plants were grown with and without inoculation with an effectiveR. meliloti strain on nitrogenfree agar slopes. Half of the uninoculated replications were, furthermore, provided with a sufficient amount of the NH₄NO₃ fertilizer, the fertilizer-treated plants (+N plants) achieved dry matter yield equal to that of inoculated variants. The concentration and uptake of Cl, Mn, Cu, Zn, Br, Sr and Mo were found to be significantly lower in inoculated plants relative to the +N plants indicating that these composition alterations are constitutive to the induced biological nitrogen fixation.     

In vitro propagation ofMedicago andLotus species by node culture

Summary Nodes ofMedicago sativa, Lotus corniculatus, Lotus tenuis, andLotus pedunculatus were cultured on MS basal media with different growth regulators. InM. sativa each node produced one shoot and the apical dominance was unaffected by high levels of cytokinins, and subsequent cycles of culture. Shoot development was stimulated by the presence ofN ⁶-isopentenyl-adenine in the culture medium and was dependent on the genotype of the explant. Shoot development was not affected by the original position of the node on the plant nor by the plant age. Shoots rooted in MS medium gelled with starch and containing 2 mg·liter⁻¹ indol-3-acetic acid. In the threeLotus species, node culture was a more effective technique than inM. sativa. The number of shoots per node increased with the time of culture and with the presence of 0.05 mg·liter⁻¹ of 6-benzylaminopurine. The highest number of shoots derived from one node was achieved inL. pedunculatus and inL. tenuis by culturing single nodes, whereas inL. corniculatus stem segments had to be totally covered by the medium for success. Rooting was easily achieved in MS medium with or without auxins.     

Root Surface Association in Relation to Nodulation of Medicago sativa

Nine strains of Rhizobium meliloti, ranging in competitive ability on Medicago sativa from excellent to poor in autoclaved soils, were paired in 29 combinations and used to inoculate M. sativa in a liquid rooting medium. A positive correlation (r = 0.545) between strain ratios in nodules after 28 days and root surface cell ratios after 7 days was determined. Two cell fractions from the root surface, representing loosely and firmly adhering cells, were investigated. Infectivity was linked to the more firmly adhering cells. A significant relationship was established between the cell ratios of competing strains in the two fractions. In another experiment, adherence of cells of both infective and noninfective Rhizobium strains to roots of M. sativa and Trifolium repens was demonstrated; the ratios of loosely to firmly adhering cells on the root surface were significantly narrower with the infective combinations than with noninfective strain-legume associations.     

Morphogenesis in callus tissue ofMedicago sativa: The role of ammonium ion in somatic embryogenesis

Exogenously supplied ammonium ion is critical to alfalfa morphogenesis in vitro. In alfalfa, the ability to induce the formation of either roots or somatic embryos provided an opportunity to examine the effects of ammonium ion on each pattern of morphogenesis. Somatic embryo formation required a minimum of 12.5 mM NH ₄ ⁺ in regeneration medium for optimal expression. Root formation was inhibited by NH ₄ ⁺ levels of 50 mM and above, and occurred in the absence of exogenous NH₄ ⁺. At high levels of NH ₄ ⁺ somatic embryos were formed from cells exposed to the root-inducing combination of hormones. This observation suggests that the growth regulators and exogenously supplied ammonium ion comprise an interactive system controlling the in vitro pattern of alfalfa morphogenesis.     

Elemental composition ofMedicago sativa under saline conditions. Relation to biological nitrogen fixation

The salinity tolerance of aRhizobium meliloti strain, used as inoculum, was established by growing the strain for seven consecutive generations in a broth containing 0–1.2% NaCl. Identical generation times and viable cell numbers were observed. Furthermore, the nodulation, plant yield and elemental composition ofM. sativa grown on agar slopes responded identically to all inocula, irrespective of the levels of NaCl with which they were grown. The effect of salinity on the ability ofM. sativa to grow and fix nitrogen was tested on agar slopes containing 0–1.2 % NaCl. At 0.0, 0.2 and 0.4 % NaCl the induced fixation was identical as indicated by the constant values of nodulation and plant yield. However, a significant reduction at 0.8 and almost a total suppression at 1.2 % NaCl occurred. Commensurate was the effect of inoculation on the elemental composition ofM. sativa as a function of salinity at the agar medium. The concentration of Mo, Mn, Sr, Cu and Zn, is clearly affected by fixation while salinity has no effect. Their concentration in the inoculated plants is significantly lower compared to the uninoculated at 0–0.4 % NaCl levels, when significant fixation occurred. In contrast, at 0.8 and 1.2 % NaCl their concentration in inoculated and uninoculated plants tends to overlap. On the other hand, the concentration of K, Rb, Br and Cl is affected mainly by salinity. Finally, Ni is affected by neither salinity nor nitrogen fixation.     

Influence of lead,cadmium, and nickel on the growth ofMedicago sativa (L.)

Summary Alfalfa (Medicago sativa L.), cv. Iroquois, was grown in the greenhouse in soils amended with additions of either lead, cadmium, or nickel. Metals, at rates varying from 0–250 ppm, were not uniformly mixed but were placed close to the soil surface so as to simulate surface deposition. In one series of experiments the sulphate salt of each metal and two soils were used. In a second series of experiments the nitrate salts and one soil were used. Neither salt of lead significantly depressed alfalfa yields. Both salts of either cadmium or nickel significantly depressed yields. Additions of all metals to the soil resulted in both increased metal uptake and concentrations in alfalfa tissue, particularly for cadmium and nickel. The highest tissue concentrations of cadmium and nickel were associated with plant stunting and necrosis. However, at rates of 125 ppm and less, substantial increases in cadmium and nickel concentrations were obtained frequently without serious yield reductions. Generally, metal concentrations were greatest in the first harvest following metal application. Concentration and uptake of lead and cadmium were greater when the metal was applied to the soil as nitrate than when applied as the sulphate salt.     

Root Hair Deformation Activity of Nodulation Factors and Their Fate on Vicia sativa

We used a semiquantitative root hair deformation assay for Vicia sativa (vetch) to study the activity of Rhizobium leguminosarum bv viciae nodulation (Nod) factors. Five to 10 min of Nod factor-root interaction appears to be sufficient to induce root hair deformation. The first deformation is visible within 1 h, and after 3 h about 80% of the root hairs in a small susceptible zone of the root are deformed. This zone encompasses root hairs that have almost reached their maximal size. The Nod factor accumulates preferentially to epidermal cells of the young part of the root, but is not restricted to the susceptible zone. In the interaction with roots, the glucosamine backbone of Nod factors is shortened, presumably by chitinases. NodRlv-IV(C18:4,Ac) is more stable than NodRlv-V(C18:4,Ac). No correlation was found between Nod factor degradation and susceptibility. Degradation occurs both in the susceptible zone and in the mature zone. Moreover, degradation is not affected by NH4NO3 and is similar in vetch and in the nonhost alfalfa (Medicago sativa).     

Phytotoxicity of aluminium-fluoride complexes and their uptake from solution culture by Avena sativa and Lycopersicon esculentum

Avena sativa (oats) and Lycopersiconesculentum (tomatoes) were grown in dilute nutrientsolutions supplemented with Al only, F only or acombination of both. In solutions containing Al andF, shoot growth was limited when predicted Al_r(Al³⁺, AlOH²⁺ and Al(OH) )activities were < 0.1 M, activities three orders ofmagnitude lower than the critical value determinedwith Al only. The data suggest that at the activitiesused in these experiments, Al_r is most toxic,AlF²⁺ and AlF are toxic to a lesserextent, and AlF₃, AlF and F^-are least toxic. Fluoride concentrations in shootsgrown in solutions correlated best with positivelycharged AlF species (i.e.AlF²⁺, AlF ) and the molar ratio ofF:Al in most plants shoots was about 3:2. However,when activities of positively chargedAlF species were low (< 50 M)and theactivity of AlF₃ species high (500 M) the molarratio of F:Al in plant shoots suggested AlF₃ wastaken up. These findings are discussed in relation toplant uptake and toxicity. Measured concentrations of Al_r and F^- insolutions containing Al and F were compared withconcentrations predicted by a computer model(GEOCHEM-PC). The method for measuring F^-concentrations, using a non-complexing buffer and Fion-selective electrode, gave good agreement withpredicted F^- concentrations. The8-hydroxyquinoline method for measuring Al_rconcentrations did not agree with predictedconcentrations, highlighting the limitations of thismethod when measuring Al_r in the presence of F ina multi-ligand system with high concentrations of Fand Al.     

Production of somatic hybrid tissues following chemical and electrical fusion of protoplasts from albino cell suspensions ofMedicago sativa andM. borealis

Protoplasts isolated from cell suspensions of albinoMedicago borealis andM. sativa were fused chemically, using two methods, and electrically. Although a small scale method of chemical fusion gave the highest fusion frequency, electrofusion was the superior technique on the basis of throughput of green somatic hybrid cell colonies. Chlorophyll-containing tissues were confirmed as being somatic hybrid by isoenzyme and cytological analyses. This is the first report of the application of albino complementation to produce somatic hybrid cells in forage legumes.Abbreviations AC alternating current - DC direct current - 2,4-D 2,4-dichlorophenoxyacetic acid - f.wt. fresh weight - PEG polyethylene glycol - K8P and K8 Kao (1977) protoplast and cell culture media - MS Murashige and Skoog (1962) medium - UM Uchimiya and Murashige (1974) medium     

Phytotoxicity of the fluoride ion and its uptake from solution culture by Avena sativa and Lycopersicon esculentum

Avena sativa (oats) and Lycopersicon esculentum (tomatoes) were grown in dilute nutrient solutions containing varying concentrations of fluoride (F). Shoot and root growth of tomatoes were limited when calculated F ion activities (F^-) were greater than 1473 M in solution. However, F^- activities up to 5130 m had no effect on the dry weights of oat shoots or roots, suggesting that tomatoes are more sensitive to F toxicity. At low F activities in solution (<1684 M) F concentrations in plant shoots increased almost linearly with activity, but then increased rapidly before reaching an upper asymptote. These findings are discussed in relation to plant uptake and toxicity of F. The complexation of Ca with F has been proposed as the mechanism of selectively altering membrane permeability to F, but further studies are required to confirm the role of Ca. Data recalculated from the literature and from this paper also suggest that growth solution ionic strength is positively related to uptake of F^- by plants.     

Iodate and iodide effects on iodine uptake and partitioning in rice (Oryza sativa L.) grown in solution culture

In the Xinjiang province of western China, conventional methods of iodine (I) supplementation (i.e, goiter pills and iodinated salt) used to mitigate I deficiencies were ineffectual. However, the recent addition of KIO₃ to irrigation waters has proven effective. This study was conducted to determine the effects of I form and concentration on rice (Oryza sativa L.) growth, I partitioning within the plant, and ultimately to assist in establishing guidelines for incorporating I into the human food chain. We compared IO₃ ⁻ vs. I⁻ in order to determine how these chemical species differ in their biological effects. Rice was grown in 48 L aerated tubs containing nutrient solution and IO₃ ⁻ or I⁻ at 0, 1, 10, or 100 μM concentrations (approximately 0, 0.1, 1, and 10 mg kg⁻¹ I). The IO₃ ⁻ at 1 and 10 μM had no effect on biomass yields, and the 100 μM treatment had a small negative effect. The I⁻ at 10 and 100 μM was detrimental to biomass yields. The IO₃ ⁻ treatments had more I partitioning to the roots (56%) on average than did the I⁻ treatments (36%), suggesting differences in uptake or translocation between I forms. The data support the theory that IO₃ ⁻ is electrochemically or biologically reduced to I⁻ prior to plant uptake. None of the treatments provided sufficient I in the seed to meet human dietary requirements. The I concentration found in straw at 100 μM IO₃ ⁻ was several times greater than seed, and could provide an indirect source of dietary I via livestock feeding on the straw. This revised version was published online in June 2006 with corrections to the Cover Date.     

溶液培养及缺素培养的实验改进

介绍了溶液培养及缺素培养的概念及重要性,并重点从幼苗的选择、溶液的配制、培养容器的选择及通气用品等几方面提出了对溶液培养及缺素培养实验改进的建议。     

Phytotoxicity of hydrogen fluoride and fluoroborate and their uptake from solution culture by Lycopersicon esculentum and Avena sativa

The aims of this paper were to determine the phytoavailability and phytotoxicity of hydrogen fluoride (HF) and fluoroborate (BF ₄ ^- ) in solution when exposed to the root of the plant. As fluoroborate undergoes a slow hydrolysis to F and borate ions, the stability of BF ₄ ^- under solution culture conditions was determined. Fluoroborate was found to have a zero order rate constant of 0.0136 and took approximately 72 days to hydrolyse completely.Tomato (Lycopersicon esculentum) and oat (Avena sativa) plants were grown in dilute nutrient solutions which contained a range of activities of HF and BF ₄ ^- . Dry matter production of both tomato and oat plants grown in nutrient solutions were found to be restricted by increased activity of HF and BF ₄ ^- in solution. Tomatoes were more sensitive to HF and BF ₄ ^- than oats. Limitations to dry matter production coincided with increased uptake of F for F concentrations in tissue of both tomatoes and oats. Fluoride uptake of both HF and BF by tomatoes and oats was orders of magnitude higher compared to similar activities of other ionic species of F reported in previous studies. Possible mechanisms of uptake are discussed.