Quantitative effects of soil nitrate,growth potential and phenology on symbiotic nitrogen fixation of pea (Pisum sativum L.)

The influence of soil nitrate availability, crop growth rate and phenology on the activity of symbiotic nitrogen fixation (SNF) during the growth cycle of pea (Pisum sativum cv. Baccara) was investigated in the field under adequate water availability, applying various levels of fertiliser N at the time of sowing. Nitrate availability in the ploughed layer of the soil was shown to inhibit both SNF initiation and activity. Contribution of SNF to total nitrogen uptake (%Ndfa) over the growth cycle could be predicted as a linear function of mineral N content of the ploughed layer at sowing. Nitrate inhibition of SNF was absolute when mineral N at sowing was over 380 kg N ha^–1. Symbiotic nitrogen fixation was not initiated unless nitrate availability in the soil dropped below 56 kg N ha^–1. However, SNF could no longer be initiated after the beginning of seed filling (BSF). Other linear relationships were established between instantaneous %Ndfa and instantaneous nitrate availability in the ploughed layer of the soil until BSF. Instantaneous %Ndfa decreased linearly with soil nitrate availability and was nil above 48 and 34 kg N ha^–1 for the vegetative and reproductive stages, respectively, levels after which no SNF occurred. Moreover, SNF rate was shown to be closely related to the crop growth rate until BSF. The ratio of SNF rate over crop growth rate decreased linearly with thermal time. Maximum SNF rate was about 40 mg N m^–2 degree-day^–1, equivalent to 7 kg N ha^–1, regardless of the N treatment. From BSF to the end of the growth cycle, the high N requirements of the crop were supported by both SNF and nitrate root absorption but, of the two sources, nitrate root absorption seemed to be less affected by the presence of reproductive organs. However, since soil nitrate availability was low at the end of the growth cycle, SNF was the main source of nitrogen acquisition. The onset of SNF decrease at the end of the growth cycle seemed to be first due to nodule age and then associated to the slowing of the crop growth rate.     

Effect of mineral nitrogen on nitrogen nutrition and biomass partitioning between the shoot and roots of pea (Pisum sativum L.).

The effect of mineral N availability on nitrogen nutrition and biomass partitioning between shoot and roots of pea (Pisum sativum L., cv Baccara) was investigated under adequately watered conditions in the field, using five levels of fertiliser N application at sowing (0, 50, 100, 200 and 400 kg N ha^–1). Although the presence of mineral N in the soil stimulated vegetative growth, resulting in a higher biomass accumulation in shoots in the fertilised treatments, neither seed yield nor seed nitrogen concentration was affected by soil mineral N availability. Symbiotic nitrogen fixation was inhibited by mineral N in the soil but it was replaced by root mineral N absorption, which resulted in optimum nitrogen nutrition for all treatments. However, the excessive nitrogen and biomass accumulation in the shoot of the 400 kg N ha^–1 treatment caused crop lodging and slightly depressed seed yield and seed nitrogen content. Thus, the presumed higher carbon costs of symbiotic nitrogen fixation, as compared to root mineral N absorption, affected neither seed yield nor the nitrogen nutrition level. However, biomass partitioning within the nodulated roots was changed. The more symbiotic nitrogen fixation was inhibited, the more root growth was enhanced. Root biomass was greater when soil mineral N availability was increased: root growth was greater and began earlier for plants that received mineral N at sowing. Rooting density was also promoted by increased mineral N availability, leading to more numerous but finer roots for the fertilised treatments. However, the maximum rooting depth and the distribution of roots with depth were unchanged. This suggested an additional direct promoting effect of mineral N on root proliferation.     

Cultivar differences in assimilate partitioning and capacity to maintain N2 fixation rate in pea during pod-filling

Effects of plant development on the rate of N₂ fixation and assimilate partitioning in pea were investigated. In growth cabinets, N₂ fixation declined with the onset of pod-filling in a small, determinant cultivar of field pea (cv. Express). In contrast, in a larger, indeterminant variety (cv. Century) N₂ fixation rate did not peak until several weeks into the pod-filling period. The smaller cultivar, Express, fixed 66% less nitrogen than the cultivar Century. Dry matter and nitrogen content increased during pod-filling in nodules but declined, or held steady in leaves, stems, and roots for Century. This indicates that nodules could compete successfully with pods for assimilates during pod-filling. In contrast, dry matter and nitrogen content did not increase in all non-reproductive plant parts (including nodules) for the smaller cultivar, Express. Under field conditions, rates of N₂ fixation declined severely for cv. Century with the onset of pod-filling. It is proposed that maintenance of the rate of N₂ fixation with the onset of pod-filling is dependent on genetic and environmental factors which influence the source-to-sink ratio of carbon in the plant at the start of pod-filling. This hypothesis is incorporated into a proposed scheme of how to maximize nitrogen accumulation by a legume in a growing season.     

The contribution of associative and symbiotic nitrogen fixation to the nitrogen nutrition of non-legumes

During the past 10 years estimates of N₂ fixation associated with sugar cane, forage grasses, cereals and actinorhizal plants grown in soil with and without addition of inoculum have been obtained using the ¹⁵N isotope dilution technique. These experiments are reviewed in this paper with the aim of determining the proportional and absolute contribution of N₂ fixation to the N nutrition of non-legumes, and its role as a source of N in agriculture. The review also identifies deficiencies in both the totality of data which are currently available and the experimental approaches used to quantify N₂ fixation associated with non-legumes.Field data indicate that associative N₂ fixation can potentially contribute agronomically-significant amounts of N (>30–40 kg N ha^-1 y^-1) to the N nutrition of plants of importance in tropical agriculture, including sugar cane (Saccharum sp.) and forage grasses (Panicum maximum, Brachiaria sp. and Leptochloa fusca) when grown in uninoculated, N-deficient soils. Marked variations in proportions of plant N derived from the atmosphere have been measured between species or cultivars within species.Limited pot-culture data indicate that rice can benefit naturally from associative N₂ fixation, and that inoculation responses due to N₂ fixation can occur. Wheat can also respond to inoculation but responses do not appear to be due to associative N₂ fixation. ¹⁵N dilution studies confirm that substantial amounts of N₂ can be fixed by actinorhizal plants.     

Yield and biological nitrogen fixation of common bean (Phaseolus vulgaris L.) in Peru

Two field experiments were performed to evaluate the nitrogen fixation potential of twenty common bean cultivars and breeding lines during summer and winter seasons of 1986 and 1988, respectively. The ¹⁵N isotope dilution method was used to quantify N₂ fixation. The cultivars and breeding lines were variable in terms of their N₂ fixation. The cv. Caballero was very efficient, with more than 50% N derived from the atmosphere and 60–80 kg N ha^–1 fixed in both seasons. Other cultivars were less efficient, since the poorest ones derived less than 30% of their nitrogen from the atmosphere and fixed less than 20 kg N ha^–1. After additional testing the best cultivars may be used directly by the farmers for cultivation. The experiments have provided information about which genotypes may be used to breed for enhanced fixation in common bean.     

Optimising biological N2 fixation by legumes in farming systems

Whether grown as pulses for grain, as green manure, as pastures or as the tree components of agro-forestry systems, the value of leguminous crops lies in their ability to fix atmospheric N₂, so reducing the use of expensive fertiliser-N and enhancing soil fertility. N₂ fixing legumes provide the basis for developing sustainable farming systems that incorporate integrated nutrient management. By exploiting the stable nitrogen isotope ¹⁵N, it has been possible to reliably measure rates of N₂ fixation in a wide range of agro-ecological field situations involving many leguminous species. The accumulated data demonstrate that there is a wealth of genetic diversity among legumes and their Rhizobium symbionts which can be used to enhance N₂ fixation. Practical agronomic and microbiological means to maximise N inputs by legumes have also been identified.     

Methods for enhancing symbiotic nitrogen fixation

Biological nitrogen fixation of leguminous crops is becoming increasingly important in attempts to develop sustainable agricultural production. However, these crops are quite variable in their effectiveness in fixing nitrogen. By the use of the ¹⁵N isotope dilution method some species have been found to fix large proportions of their nitrogen, while others like common bean have been considered rather inefficient. Methods for increasing N₂ fixation are therefore of great importance in any legume work. Attempts to enhance nitrogen fixation of grain legumes has been mainly the domain of microbiologists who have selected rhizobial strains with superior effectiveness or competitive ability. Few projects have focused on the plant symbiont with the objective of improving N₂ fixation as done in the FAO/IAEA Co-ordinated Research Programme which is being reported in this volume. The objective of the present paper is to discuss some possibilities available for scientists interested in enhancing symbiotic nitrogen fixation in grain legumes. Examples will be presented on work performed using agronomic methods, as well as work on the plant and microbial symbionts. There are several methods available to scientists working on enhancement of N₂ fixation. No one approach is better than the others; rather work on the legume/Rhizobium symbiosis combining experience from various disciplines in inter-disciplinary research programmes should be pursued.     

Compared cycling in a soil-plant system of pea and barley residue nitrogen

Field experiments were carried out on a temperate soil to determine the decline rate, the stabilization in soil organic matter and the plant uptake of N from ¹⁵N-labelled crop residues. The fate of N from field pea (Pisum sativum L.) and spring barley (Hordeum vulgare L.) residues was followed in unplanted and planted plots and related to their chemical composition. In the top 10 cm of unplanted plots, inorganic N was immobilized after barley residue incorporation, whereas the inorganic N pool was increased during the initial 30 days after incorporation (DAI) of pea residues. Initial net mineralization of N was highly correlated to the concentrations of soluble C and N and the lignin: N ratio of residues. The contribution of residue-derived N to the inorganic N pool was at its maximum 30 DAI (10–55%) and declined to on average 5% after 3 years of decomposition.Residual organic labelled N in the top 10 cm soil declined rapidly during the initial 86 DAI for all residue types. Leaching of soluble organic materials may have contributed to this decline. At 216 DAI 72, 59 and 45% of the barley, mature pea and green pea residue N, respectively, were present in organic N-forms in the topsoil. During the 1–3 year period, residual organic labelled N from different residues declined at similar rates, mean decay constant: 0.18 yr^-1. After 3 years, 45% of the barley and on average 32% of the pea residue N were present as soil organic N. The proportion of residue N remaining in the soil after 3 years of decomposition was most strongly correlated with the total and soluble N concentrations in the residue. The ratio (% inorganic N derived from residues): (% organic N derived from residues) was used as a measure of the rate residue N stabilization. From initial values of 3–7 the ratios declined to on average 1.9 and 1.6 after 2 and 3 yrs, respectively, indicating that a major part of the residue N was stabilized after 2 years of decomposition. Even though the largest proportion of residue N stabilized after 3 years was found for barley, the largest amount of residue N stabilized was found with incorporation of pea residues, since much more N was incorporated with these residues.In planted plots and after one year of decomposition, 7% of the pea and 5% of the barley residue N were recovered in perennial ryegrass (Lolium perenne L.) shoots. After 2 years the cumulative recovery of residue N in ryegrass shoots and roots was 14% for pea and 15% for barley residue N. The total uptake of non-labelled soil N after 2 years of growth was similar in the two residue treatments, but the amount of soil N taken up in each growth period varied between the treatments, apparently because the soil N immobilized during initial decomposition of residues was remineralized later in the barley than in the pea residue treatment. Balances were established for the amounts of barley and mature pea residue N remaining in the 0–10 cm soil layer and taken up in ryegrass after 2 years of decomposition. About 24% of the barley and 35% of the pea residue N were unaccounted for. Since these apparent losses are comparable to almost twice the amounts of pea and barley residue N taken up by the perennial ryegrass crop, there seems to be a potential for improved crop residue management in order to conserve nutrients in the soil-plant system.     

Biological nitrogen fixation in trees in agro-ecosystems

The integration of trees, especially nitrogen fixing trees (NFTs), into agroforestry and silvo-pastoral systems can make a major contribution to sustainable agriculture by restoring and maintaining soil fertility, and in combating erosion and desertification as well as providing fuelwood. The particular advantage of NFTs is their biological nitrogen fixation (BNF), their ability to establish in nitrogen-deficient soils and the benefits of the nitrogen fixed (and extra organic matter) to succeeding or associated crops.The importance of NFTs leads to the question of how we can maximise or optimize their effects and how we can manage BNF and the transfer of nitrogen to associated or succeeding plantings. To be able to achieve these goals, suitable methods of measuring BNF in trees are necessary. The total nitrogen difference (TND) method is simple, but is better suited for low than high soil N conditions. The acetylene reduction assay (ARA), although sensitive and simple, has many technical limitations especially for NFTs, and the estimates of BNF have generally been very low, compared to other methods. For NFTs, the ¹⁵N techniques are still under development, but have already given some promising results (e.g., has been used to measure large genetic variability in BNF within different NFTs).Various factors affect BNF in trees. They include the age of trees, the microbial component, soil moisture, temperature, salinity, pH, soil N level and plant nutrient deficiencies. Some of the factors, e.g. temperature, affect the symbiosis more than plant growth, and differences in the effects of these factors on BNF in different NFT genotypes have been reported. These factors and research needs for improving BNF in trees are discussed.     

Biological nitrogen fixation in four Gliricidia sepium genotypes

A field experiment was conducted at the Coconut Research Institute in Sri Lanka to examine the biological nitrogen fixation potential of three Gliricidia sepium provenances (OFI 14/84, 17/84, 12/86) and a local landrace (designated LL), using the ¹⁵N isotope dilution method. There was marked variation in dry matter, total N, nodulation and ¹⁵N enrichment among the Gliricidia genotypes (=0.001), and the dry matter yield of Cassia siamea (syn. Senna siamea), the non-N₂ fixing reference plant was higher than for G. sepium. In all cases, highest biomass and total N were aboveground, with roots on average accounting for < 20 % of total dry matter or the total N in plants. Atom % ¹⁵N excess was highest in C. siamea, and lowest in OFI 14/84. Although atom % ¹⁵N excess was lower in Gliricidia leaves than in the other organs (all of which had similar ¹⁵N enrichments), values of % N derived from atmospheric N₂ fixation (% Ndfa) calculated for any individual organ or for the whole plant were similar. This was because the relative distribution of ¹⁵N in the different parts of the fixing plant followed the same trend as in the reference plant. There were significant differences (p=0.01) in N₂ fixation between the Gliricidia genotypes. The values ranged from 17.8 g N tree^-1 (equivalent to 86 kg N ha^-1 at 5000 trees ha^-1) in OFI 12/86 to 61.7g N tree^-1 (equivalent to 309 kg N ha^-1) in OFI 14/84. Although most of this variability was due to differences in both % Ndfa and total N in plant, amount of N fixed was more correlated with total N in plant (r=0.935) than with % Ndfa (r=0.707). On average, % Ndfa in all three G. sepium provenances and LL was about 55 % or 34.6 g N tree^-1 (equivalent to some 166 kg N ha^-1) in the 9 months within which N₂ fixation was measured. This represents a substantial contribution of N into the soil-plant system.     

Methods for measuring biological nitrogen fixation in grain legumes

To assure proper management and fully realize the benefits of the legume-Rhizobium symbiosis it is necessary to be able to quantify the amount of nitrogen fixed. Having measured the effectiveness of atmospheric N₂ fixation the macro- or micro-symbionts as well as agronomic factors can be manipulated with the objective to maximize biological nitrogen fixation. A suitable method to quantify nitrogen fixation is therefore necessary in any programme aiming at increasing N₂ fixation, like the one being reported in this volume. There are several methods available to quantify nitrogen fixation and most of the commonly used ones are described in the present paper listing their advantages and disadvantages.     

Interactive effects of exogenous combined nitrogen and phosphorus on growth and nitrogen fixation by azolla

Effects of N source and media-N and P levels were examined on growth, N uptake, and N₂ fixation ofAzolla pinnata withAnabaena azollae association (azolla) at two inoculum-P concentrations. Each expeiment was conducted for 7 days in a growth chamber using azolla at a predetermined inoculum-P concentration and the growth media containing a combination of four levels of P (0, 15, 75, and 200 M) and three levels (0, 1, and 5 mM) of either¹⁵N-enriched NH ₄ ⁺ as ammonium sulfate or¹⁵N-enriched NO ₃ ^– as potassium nitrate. Nitrogen uptake and N₂ fixation were measured by¹⁵N isotopic dilution method. Tissue P and N, N uptake, and N₂-fixation increased with increasing P concentration in the media regardless of the inoculum-P level of azolla. Increasing P concentration in the media increased growth of azolla at low inoculum P, but the effect on high inoculum-P azolla was either small or absen. High inoculum-P concentration resulted in increased growth, tissue-N and P concentrations, N uptake, and N₂ fixation by azolla. Ammonium in the growth media caused larger increase in tissue-N and greater repression of N₂ fixation than equimolar concentration of NO ₃ ^– . In the presence of NH ₄ ⁺ or NO ₃ ^– , in the growth media, N uptake by azolla exceeded the corresponding decrease in N₂ fixation, resulting in an overall increase in tissue-N concentration. Phosphorus in the media tended to negate the inhibitory effect of NH ₄ ⁺ or NO ₃ ^– on N₂ fixation. A multiple regression model showed that the effect of tissue-N on N₂ fixation was negative while that of tissue-P was positive. Therefore, a relative change in tissue-N and P appeared to regulate N₂ fixation. Tissue-N and P had similar effects on relative growth rate of azolla also. Inoculum-P level of azolla was important in determining the response to media-P.This research was supported by a grant from USAID under Indo-US Science and Technology Initiative.     

Variability in nitrogen fixation of common bean (Phaseolus vulgaris L.) intercropped with maize

Thirty one selected bean lines were evaluated in the field for ability to support N₂ fixation when intercropped with maize which received 0, 30 and 60 kg N ha^–1 as ammonium sulphate. The amount of fixed N₂ was estimated using the natural variation of ¹⁵N and wheat as the standard non-fixing crop. Nitrogen as low as 15 kg N ha^–1 at sowing suppressed nodule weight and activity (acetylene reduction activity) but not nodule number, suggesting that the main effect of mineral N was on nodule development and function. ¹⁵N data revealed a high potential of the bean genotypes to fix N₂, with the most promising ones averaging between 50–60% of seed N coming from fixation. Bean lines CNF-480, Puebla-152, Mexico-309, Negro Argel, CNF-178, Venezuela-350 and WBR22-3, WBR22-50 and WBR22-55 were ranked as good fixers.     

Genotypic variation in biological nitrogen fixation by common bean

Field experiments were performed in Austria, Brazil, Chile, Colombia, Guatemala, Mexico and Peru as part of an FAO/IAEA Co-ordinated Research Programme to investigate the nitrogen fixing potential of cultivars and breeding lines of common bean (Phaseolus vulgaris L.). Each experiment included approximately 20 bean genotypes which were compared using the ¹⁵N isotope dilution method. Great differences in nitrogen fixation were observed between and within experiments, with average values of 35% N derived from atmosphere (% Ndfa) and highest values of 70% Ndfa being observed. These values which were larger than had been reported previously for common bean, were observed only when environmental factors were favorable. Therefore, common bean lines are available, which can support high biological nitrogen fixation. These can be used either directly as cultivars for production or in breeding programmes to enhance nitrogen fixation in other cultivars.     

Symbiotic N2 fixation in pea and field bean estimated by15N fertilizer dilution in field experiments with barley as a reference crop

Summary The total amount of nitrogen derived from symbiotic nitrogen fixation in two pea and one field bean cultivar, supplied with 50 kg N ha⁻¹ at sowing (‘starter’-N), was estimated to 165, 136, and 186 kg N ha⁻¹, respectively (three-year means). However, estimates varied considerably between the three years. At the full bloom/flat pod growth stage from 30 to 59 per cent of total N₂ fixation had taken place. The proportion of total N derived from N₂ fixation at maturity was higher in seeds than in vegetative plant parts and amounted to 59.5, 51.3 and 66.3 per cent of total above-ground plant N in the two pea cultivars and field bean, respectively (three-year means). The recovery of fertilizer N was 62.2, 70.2, 52.1, and 69.5 per cent in the two pea cultivars, field bean and barley, respectively. Growth analysis indicated that barley did not meet the claims for an ideal reference crop in the¹⁵N fertilizer dilution technique for estimating N₂ fixation in pea and field bean. ‘Starter’-N neither increased the seed yield nor the N content of the grain legumes.     

Estimation of residual N effect of faba bean and pea on two succeeding cereals using15N methodology

A field experiment was conducted using¹⁵N methodology to study the effect of cultivation of faba bean (Vicia faba L.), pea (Pisum sativum L.) and barley (Hordeum vulgare L.) on the N status of soil and their residual N effect on two succeeding cereals (sorghum (Sorghum vulgare) followed by barley). Faba bean, pea and barley took up 29.6, 34.5 and 53.0 kg N ha^–1 from the soil, but returned to soil through roots only 11.3, 10.8 and 5.7 kg N ha^–1, respectively. Hence, removal of faba bean, pea and barley straw resulted in a N-balance of about –18, –24, and –47 kg ha^–1 respectively. A soil nitrogen conserving effect was observed following the cultivation of faba bean and pea compared to barley which was of the order of 23 and 18 kg N ha^–1, respectively. Cultivation of legumes resulted in a significantly higher A_N value of the soil compared to barley. However, the A_N of the soil following fallow was significantly higher than following legumes, implying that the cultivation of the legumes had depleted the soil less than barley but had not added to the soil N compared to the fallow. The beneficial effect of legume cropping also was reflected in the N yield and dry matter production of the succeeding crops. Cultivation of legumes led to a greater exploitation of soil N by the succeeding crops. Hence, appreciable yield increases observed in the succeeding crops following legumes compared to cereal were due to a N-conserving effect, carry-over of N from the legume residue and to greater uptake of soil N by the succeeding crops when previously cropped to legumes.     

Measurement of N2-fixation in field-grown pigeonpea [Cajanus cajan (L.) Millsp.] using15N-labelled fertilizer

Two experiments were carried out from 1981 to 1983 in Vertisol field at ICRISAT Center, Patancheru, India to measure N₂-fixation of pigeonpea [Cajanus cajan (L.) Millsp.] using the¹⁵N isotope dilution technique. One experiment examined the effect of control of a nodule-eating insect on fixation while another in vestigated the effect of intercroping with cereals on fixation and the residual effect of pigeonpea on a succeeding cereal crop. Although both experiments indicated that at least 88% of the N in pigeonpea was fixed from the atmosphere, one result is considered fortuitous in view of the differential rates of growth of the legume and the control, sorghum [Sorghum bicolor (L.) Moench]. The difference method of calculation in dieated negative fixation and the results emphasized the problem of finding a suitable nonfixing control. In a second experiment, when all plants were confined to a known volume of soil to which¹⁵N fertilizer was added in the field, these problems were overcome, and isotope dilution and difference methods gave similar results of N₂-fixation of about 90%. In intercropped pigeonpea 96% of the total N was derived from the atmosphere. This estimate might be an artifact. There was no evidence of benefit from N fixed by pigeonpea to intercropped sorghum plants. Plant tissue¹⁵N enrichments of cereal crops grown after pigeonpea indicated that the cereal derived some N fixed by the previous pigeonpea. Thus residual benefits to cereals are not only an effect of ‘sparing’ of soil N.     

Misconceptions and practical problems in the use of 15N soil enrichment techniques for estimating N2 fixation

The ¹⁵N methods are potentially accurate for measuring N₂ fixation in plants. The only problem with those methods is, how to ensure that the ¹⁵N/¹⁴N ratio in the plant accurately reflects the integrated ¹⁵N/¹⁴N ratio (R) in soil which is variable in time and with soil depth. However, the consequences of using an inappropriate reference plant vary with the level of N₂ fixation and the conditions under which the study was made. For example, the errors introduced into the values of N₂ fixation are higher at low levels of fixation, and decrease with increasing rates of fixation. At very high N₂ fixation rates, the errors are often insignificant. Also, the magnitude of error is proportional to the rate of decline of the ¹⁵N/¹⁴N ratio with time. Since N₂ fixation in most plants would be expected to below 60%, the question of how to select a good reference plant is still pertinent. In this paper, we have discussed some of the criteria to adopt in selecting reference plants, e.g. how to ensure that the reference plant is not fixing N₂, is absorbing most of its N from the same zone as the fixing plant, and in the same pattern with time, etc. In addition, we have discussed ¹⁵N labelling materials and methods that are likely to minimize any errors even when the fixing and reference plants don't match well in certain important criteria. The use of slow release ¹⁵N fertilizer or ¹⁵N labelled plant materials results in slow changes in the ¹⁵N/¹⁴N ratio of soil, and is strongly recommended. Where ¹⁵N inorganic fertilizers are used, the application of the fertilizer in small splits at various intervals is recommended over a one-time application. The problem with the reference crop, which has sometimes discouraged potential users of the ¹⁵N methods, is surmountable, as discussed in this paper.     

Contributions and limitations to symbiotic nitrogen fixation in common bean (Phaseolus vulgaris L.) in Romania

Common beans usually achieve grain yields less than the genotypic potential of the cultivar under Romanian field conditions. To understand better the contribution of nitrogen fixation to the yield formation, I made a long-term evaluation (1977–1994) of inoculation effects of 19 Rhizobium leguminosarum bv. phaseoli strains on common bean cultivated in several locations. Grain yields were significantly influenced by all selected strains, years and locations in all experimental cycles, and only partially by the interactions between strains and years or strains and locations. The average yield increases induced by these strains during the four cycles ranged from 6 to 20%. Four bacterial strains proved to be more stable in their field performances, taking into account the yield increases greater than 10% over controls observed in all individual trials. Mean yields and variation limits recorded during the long-term evaluation of strain efficacy in locations with different soil pH values showed similar patterns of yield increases from soils with acidic to neutral pH values. Linear regression between mean grain yields and average temperatures demonstrated the limiting effect of temperature on yield. The interaction between bacterial strain and nitrogen fertiliser rate demonstrated the ability of dinitrogen fixation to satisfy the crop requirements for this element. An evaluation of the amounts of nitrogen fixed in three common bean cultivars inoculated with two bacterial strains showed different N2-fixing capacities among plant genotypes.     

Survival and growth of pea protoplasts after transformation by electroporation

Protoplasts from two different pea cultivars, Belman and Filby, were stably transformed by direct gene transfer using electroporation. Transgenic calli could be obtained after selection, when hygromycin resistance was used as the selective trait introduced into the protoplasts, while no transformants were obtained when kanamycin resistance was used as selective marker in either of the two pea cultivars tested. The effect of the field strength on survival and division rates of the protoplasts was studied. Two different culture systems and osmotica were compared for induction of sustained divisions in and regeneration of transgenic callus from the protoplasts. The choice of the culture system had a considerable effect on the initial division frequency of the treated protoplasts, as well as on the later growth of the colonies. Transformation efficiency was monitored by histochemical GUS assay, and the transgenic nature of the calli selected for resistance against antibiotics was confirmed by DNA analysis.