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.     

Flowering and gibberellins in a mutant red clover (Trifolium pratense L.)

Relationships between gibberellins and floral initiation were investigated in a conditional non-flowering mutant of red clover, Trifolium pratense. Untreated mutant plants will not flower under long-days, but will do so when certain GAs are applied. Gibberellins, A₃, A₁, A₇, and A₅ all resulted in both stem elongation and flowering whilst GA₄ produced the elongation only. Applications of GA₂₀, GA₈ and GA₁₃ under long-days had no detectable effect. Thus, by combining the use of the mutant with the application of different GAs, the correlation between the processes of stem elongation and floral initiation, which is normally strongly expressed in this species, was broken. Endogenous gibberellins shown to be present in normal plants were also found in the mutant genotype. Gibberellins alone were not sufficient to initiate floral development in the mutant, there being an essential element of interaction with long-days. These results are discussed in relation to the nature of the lesion in the mutant and the signal provided by the applied gibberellin.     

Genotype-dependent whole plant regeneration from protoplasts of red clover (Trifolium pratense L.)

Protoplasts are useful for subcellular studies, in vitro selection, somatic hybridization and transformation. Whole plant regeneration from protoplasts is a prerequisite to producing altered crop plants using these methods. Whole plant regeneration was achieved from leaf- and suspension culture-derived protoplasts of T. pratense. Regeneration was most dependent upon identifying genotypes with genetic capacity to regenerate. Additional factors that were used to select genotypes, but which proved to be less important, were a high rate of cell growth in culture and a high plating efficiency of protoplasts. One genotype was identified which had a regeneration response equivalent to that of T. rubens and which regenerated from both leaf- and suspension culture-derived protoplasts.Research supported by USDA/CRGO Grant No. 81 CRCR-1-0613     

Effects of sulphur nutrition on growth and nitrogen fixation of pea (Pisum sativum L.)

A S-deficient soil was used in pot experiments to investigate the effects of S addition on growth and N₂-fixation in pea (Pisum sativum L.). Addition of 100 mg S pot⁻¹ increased seed yield by more than 2-fold. Numbers of pods formed were the most sensitive yield component affected by S deficiency. Sulphur addition also increased the concentration of N in leaves and stems, and the total content of N in the shoots. The amounts of N fixed by pea were determined at four growth stages from stem elongation to maturity, using the ¹⁵N dilution technique. Sulphur addition doubled the amount of N fixed at all growth stages. In contrast, leaf chlorophyll content and shoot dry weight were increased significantly by S addition only after the flowering and pod fill stage, respectively. Pea roots were found to have high concentrations of S, reaching approximately 10 mg g⁻¹ dry weight and being 2.6–4.4 times the S concentration in the shoots under S-sufficient conditions. These results suggest that roots/nodules of pea have a high demand for S, and that N₂-fixation is very sensitive to S deficiency. The effects of S deficiency on pea growth were likely to be caused by the shortage of N, due to decreased N₂-fixation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Improving nitrogen fixation in legumes by plant breeding; the relevance of host selection experiments in red clover (Trifolium pratense L.) and subterranean clover (T. subterraneum L.)

Summary This paper reviews (i) basic studies on the genetics of symbiosis in red clover (a self-sterile species) and subterranean clover (cleistogamous) and (ii) work on selection and plant breeding to increase nitrogen fixation in these hosts.Symbiotic effectiveness in red clover is influenced by many major and minor genes. The highly effective phenotype is inherited in a complex manner associated with early nodulation and the formation of large amounts of persistent bacteroid-containing tissue. Lines bred to fix more nitrogen with one strain ofRhizobium trifolii do so with most but not all other strains examined. They also show slightly increased vigour when grown on nitrate. The highly effective response is correlated with abundant nodulation and an early flowering habit, the evidence from breeding studies indicating that this correlation is not absolute. Normally effective and highly effective nodules have the same specific nitrogenase activities. The expression of the highly effective response is relatively little affected by environmental factors (temperature, light intensity, day length, supplementary carbon-di-oxide). Inbreeding substantially degrades the symbiotic response.Heterosis is shown in crosses between cultivars of subterranean clover but otherwise selection to increase effectiveness in this host was unsuccessful.The relevance of these results (and their physiological aspects) for the improvement of grain legumes is discussed.     

Modification of symbiotic interaction of pea (Pisum sativum L.) andRhizobium leguminosarum by induced mutations

Summary In pea (Pisum sativum L.), mutants could be induced, modified in the symbiotic interaction withRhizobium leguminosarum. Among 250 M₂-families, two nodulation resistant mutants (K₅ and K₉) were obtained. In mutant K₅ the nodulation resistance was monogenic recessive and not Rhizobium strain specific. Out of 220 M₂-families one mutant nod₃ was found which could form nodules at high nitrate concentrations (15 mM KNO₃). This mutant nodulated abundantly with severalRhizobium strains, both in the absence and presence of nitrate. Probably as the result of a pleiotropic effect, its root morphology was also changed. Among 1800 M₂-families, five nitrate reductase deficient mutants were obtained and one of them (mutant E₁) was used to study the inhibitory effect of nitrate on nodulation and nitrogen fixation.The results of the present investigation show that pea mutants which are modified in their symbiosis withRhizobium leguminosarum, can readily be obtained. The significance of such mutants for fundamental studies of the legume-Rhizobium symbiosis and for applications in plant breeding is discussed.     

Inheritance and mapping of isoenzymes in pea (Pisum sativum L.)

Summary Three isoenzyme systems (amylase, esterase and glutamate oxaloacetate transaminase) were examined in seeds of pea (Pisum sativum L.) and shown to give clear variation in their band patterns on gel electrophoresis between different lines. The inheritance of these isoenzyme systems, and the location of their genes on the pea genome was investigated. Reciprocal crosses were made between lines, F2 seeds were analysed for segregation in the band patterns of the isoenzymes, and F2 plants were investigated to find linkage between the genes for these isoenzymes and genes for selected morphological markers. The results obtained showed that each of the investigated isoenzyme systems is genetically controlled by co-dominant alleles at a single locus. The gene for amylase was found to be on chromosome 2, linked to the loci k and wb (wb ... 9 ... k ... 25 ... Amy). The gene for esterase was found to be linked with the gene Br (chromosome 4) but the exact location is uncertain because of the lack of the morphological markers involved in the cross. The gene for glutamate oxaloacetate transaminase was found to be on chromosome 1 and linked with the loci a and d (a... 24... Got... 41 ... d).     

15N estimates of nitrogen fixation by white clover (Trifolium repens L.) growing in a mixture with ryegrass (Lolium perenne L.)

The ¹⁵N isotope dilution technique and the N difference method were used to estimate N₂ fixation by clover growing in a mixture with ryegrass, in a field experiment and a controlled environment experiment. Values obtained using N difference were approximately 25% lower than those estimated using ¹⁵N isotope dilution. In the field experiment there was a measured N benefit to grass growing with clover, equivalent to 42.7 kgN ha^-1. The grass in the mixture had a lower atom %¹⁵N content and a higher N content than grass in a monoculture; therefore values for N₂ fixation were different depending on choice of control plant i.e. monoculture or mixture grass. In the controlled environment experiment there were no significant differences between either the atom %¹⁵N contents or the N contents of monoculture grass and grass growing in a mixture with clover. It is concluded that there is a long term indirect transfer of N from clover to associated grass which can lead to errors in estimates of N₂ fixation.     

Isolation and partial characterization of clathrin-coated vesicles from pea (Pisum sativum L.) cotyledons

Summary Clathrin-coated vesicles have been isolated from cotyledons of both developing and germinating pea seeds using differential centrifugation, ribonuclease treatment, discontinuous sucrose gradients, and isopycnic centrifugation on a linear D₂O-Ficoll gradient. The yield of coated vesicles from developing pea cotyledons was exceptional, being 1.6 × higher than the yield from hog and bovine brain, 5.3 × higher than the yield from carrot suspension cultures, and 13 × the yield from cotyledons of germinating pea seeds. The pea coated vesicles are similar to other plant coated vesicles in size (approximately 80 nm in diameter) and in having a clathrin heavy chain of 190,000 M_r. The lipid phosphorus to protein ratio, 190–250 nmol P per mg protein, of the coated vesicles from plants is comparable to that reported for highly purified coated vesicles from animals. The nondenatured pea clathrin reacted weakly with an antiserum to bovine brain clathrin, but pea clathrin denatured by sodium dodecyl sulfate did not.Abbreviations CLC Clathrin light chain - CHC clathrin heavy chain - CV coated vesicle - DTT dithiothreitol - EGTA ethyleneglycol-bis-(-aminoethyl ether) N,N-tetraacetic acid - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TBS Tris buffered saline     

Sulphate uptake and xylem loading of young pea (Pisum sativum L.) seedlings

Sulphate uptake and xylem loading was analysed in young pea (Pisum sativum) seedlings. The rate of sulphate uptake into intact 8-days-old pea seedlings (determined by a 1 h exposure to radiolabelled sulphate in the nutrient solution) was 585 nmol sulphate g^–1 root fresh weight h^–1. When the cotyledons were removed on day 6 the 8-days-old seedlings took up only 7% of the controls. Interruption of the phloem transport by steam girdling of the stem or the root (1 h before incubation with radiolabelled sulphate) diminished sulphate uptake by approximately 50%. The addition of sucrose to the nutrient solution during incubation did not restore sulphate uptake rates indicating that the decrease was not due to a lack of energy. Apparently, a signal from the shoot and/or the cotyledons is necessary to stimulate sulphate uptake into the roots of pea seedlings. Glutathione fed to the roots for 3 h prior to incubation with radiolabelled sulphate diminished sulphate uptake by approximately 50%. The relative proportion of the sulphate taken up that was loaded into the xylem remained unchanged (between 7 and 9% of total uptake), even when the stem was girdled above the cotyledons or when the seedlings were pre-exposed to glutathione. Only removal of the cotyledons or girdling of the root below the cotyledons increased the proportion of sulphate loaded into the xylem to 13–15% of total uptake upon exposure to glutathione. Apparently, a signal from the cotyledons represses xylem loading to some extent.     

Study of the factors influencing Agrobacterium-mediated transformation of pea (Pisum sativum L.)

Factors influencing the efficiency of Agrobacterium-mediated transformation of pea were tested using highly efficient, direct regeneration system. The virulence of three Agrobacterium strains (octopine LBA 4404, nopaline C58C1 and succinamopine, hypervirulent EHA 105) clearly varied giving 1 transgenic plant per 100 explants for LBA 4404, 2.2 for C58C1 and 8.2 for EHA 105. To test the efficacy of selection agents we used the hypervirulent EHA 105 strain carrying pGPTV binary vector with one of four different selection genes: nptII, hpt, dhfr or bar. The mean number of transgenic, kanamycin-resistant plants for two cultivars tested was 4.2 per 100 explants and was slightly higher than the number of phosphinothricin-resistant plants (3.6 plants per 100 explants). The proportion of transgenics among kanamycin-selected plants was also higher than among phosphinothricin-resistant plants (35% and 28% respectively). There was no regeneration on hygromycin or methotrexate media (transformation with hpt and dhfr genes). Acetosyringone had no apparent influence on efficiency of transformation with hypervirulent EHA 105 strain, however it did affect the rate of transformation when moderately virulent C58C1 was used. Recovery of transgenic plants was enhanced after application of 5-azacytidine. The presence of integrated T-DNA was checked by PCR and confirmed by Southern hybridization. T-DNA was stably transmitted to the next generation.     

DNA variations in regenerated plants of pea (Pisum sativum L.)

Summary The aim of this study was to determine whether DNA variations could be detected in regenerated pea plants. Two different genotypes were analyzed by cytogenetic and molecular techniques: the Dolce Provenza cultivar and the 5075 experimental line. Dolce Provenza regenerated plants showed a reduction in DNA content, particularly at the level of unique sequences and ribosomal genes. Moreover, regeneration was associated with an increase in DNA methylation of both internal and external cytosines of the CCG sequence. On the other hand, the DNA content of the 5075 line remained stable after regeneration. DNA reduction was found only in 5075 plants regenerated from callus cultures maintained for long incubation periods (about a year). The DNA variations observed are discussed both in relation to the genotype source and the role of tissue-culture stress.     

Nuclear cytology of callus and plantlets regenerated from pea (Pisum sativum L.) meristems

Summary The chromosomal status of calli and plantlets regenerated fromPisum sativum shoot apical meristems was studied. Chromosome mosaicism (aneusomaty) occurs during callus induction and proliferation, mostly owing to nuclear fragmentation prior to mitosis in the first days of culture. Plantlets regenerated from calli are diploid or aneusomatic, but a selective advantage of diploid cells (diplontic selection) takes place with plantlet growth. The results are discussed in relation to the possibility of inducing chromosomal and/or genetic variability by using meristematic tissues as expiants.     

Mechanism of uptake and translocation of zinc by pea plants (Pisum sativum L.)

Summary Radiotracer studies revealed that the mechanism of zinc uptake was not purely non-metabolic. The over all uptake of Zn by intact plant ofPisum sativum L. (Var. T-163) occurred through an initial nonmetabolic phase followed by a metabolically mediated absorption. The translocation of zinc from plant roots to shoot was more sensitive to metabolic inhibitors and thus was a metabolic function.Research Publication No. 1541/116/0 through Experiment Station, G. B. Pant University, Pantnagar-263145, India.     

The effect of surface soil consolidation on the early root development of pea (Pisum sativum L.)

Cores of repacked soil were consolidated with a compressive strength testing machine, after peas had been planted in the centre of the core. The number that emerged were counted and root and shoot lengths and diameters were measured. Consolidation had no effect on emergence, root length or root diameter of the peas grown in a loamy sand, whereas emergence, root length and root diameter were affected by a small increase in load in a clay loam.     

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.     

Interspecific hybridization of red clover (Trifolium pratense L.) with T. sarosiense Hazsl. Using in vitro embryo rescue

Summary Interspecific hybrid clover plants from the cross Trifolium sarosiense Hazsl. X T. pratense L. were obtained in the present investigation. Immature hybrid embryos were excised aseptically from the pistillate parent, T. sarosiense (2 n = 48), and cultured in vitro prior to in situ abortion. Agar-solidified nutrient media modified from that developed previously for tissue and cell cultures of red clover (2 n = 14) were used for embryo rescue.The heart shaped embryos obtained were cultured for 8 to 14 days on a medium containing a high level of sucrose, a moderate level of auxin, and low cytokinin activity. Viable embryos were then transferred to a standard medium with low auxin and moderate cytokinin levels for the direct germination of shoots. Some embryos produced only callus. Plants were regenerated from callus using an alternate culture scheme. Hybrid shoot numbers were increased on a low auxin, high cytokinin medium and subsequently rooted before transfer to soil in the greenhouse.About 10% of the hybrid embryos were rescued using the optimal culture sequence. Five full-sib families of the F₁ hybrid were successfully grown to maturity. Root-tip cells of hybrid plants possessed the expected somatic chromosome number of 31. The genetically determined leaf-mark trait carried by the staminate parent and the rhizomatous root habit of the pistillate parent were expressed in hybrid plants.The investigations reported in this paper (No. 81-3-151) were performed in connection with projects of the Kentucky Agricultural Experiment Station and the paper is published with the approval of the Director     

Evaluation of a cotyledonary node regeneration system forAgrobacterium-mediated transformation of pea (Pisum sativum L.)

Summary A rapid regeneration system was used for studies ofAgrobacterium-mediated transformation inPisum sativum L. Cotyledonary node explants were inoculated withAgrobacterium tumefaciens strains containing binary vectors carrying genes for nopaline synthase (NOS),β-glucuronidase (GUS), and neomycin phosphotransferase (NPTII) and placed on selection medium containing either 75 or 150 mg/liter kanamycin. A GUS encoding gene (uidA) containing an intron was used to monitor gene expression from 6 to 21 days postinoculation. GUS activity could be observed 6 days after inoculation in the area of the explant in which regeneration-occurred. Regenerating tissue containing transformed cells was observed in explants on selection medium 21 days postinoculation. Using this system, a single transgenic plant was obtained. Progeny of this plant, which contained two T-DNA inserts, demonstrated segregation for the inserts and for expression of the NOS gene in the selfed R₁ progeny. NPTII activity was observed in the R₂ generation, indicating inheritance and expression of the foreign DNA over at least two generations. Attempts to repeat this procedure were unsuccessful.     

A dehydrin cognate protein from pea (Pisum sativum L.) with an atypical pattern of expression

Dehydrins are a family of proteins characterised by conserved amino acid motifs, and induced in plants by dehydration or treatment with ABA. An antiserum was raised against a synthetic oligopeptide based on the most highly conserved dehydrin amino acid motif, the lysine-rich block (core sequence KIKEK-LPG). This antiserum detected a novel M _r 40 000 polypeptide and enabled isolation of a corresponding cDNA clone, pPsB61 (B61). The deduced amino acid sequence contained two lysine-rich blocks, however the remainder of the sequence differed markedly from other pea dehydrins. Surprisingly, the sequence contained a stretch of serine residues, a characteristic common to dehydrins from many plant species but which is missing in pea dehydrin.The expression patterns of B61 mRNA and polypeptide were distinctively different from those of the pea dehydrins during seed development, germination and in young seedlings exposed to dehydration stress or treated with ABA. In particular, dehydration stress led to slightly reduced levels of B61 RNA, and ABA application to young seedlings had no marked effect on its abundance.The M _r 40 000 polypeptide is thus related to pea dehydrin by the presence of the most highly conserved amino acid sequence motifs, but lacks the characteristic expression pattern of dehydrin. By analogy with heat shock cognate proteins we refer to this protein as a dehydrin cognate.     

Linkage mapping of quantitative trait loci controlling seed weight in pea (Pisum sativum L.)

Quantitative trait loci (QTLs) affecting seed weight in pea (Pisum sativum L.) were mapped using two populations, a field-grown F₂ progeny of a cross between two cultivated types (Primo and OSU442-15) and glasshouse-grown single-seed-descent recombinant inbred lines (RILs) from a wide cross between a P. sativum ssp. sativum line (Slow) and a P. sativum ssp. humile accession (JI1794). Linkage maps for these crosses consisted of 199 and 235 markers, respectively. QTLs for seed weight in the Primo x OSU442-15 cross were identified by interval mapping, bulked segregant analysis, and selective genotyping. Four QTLs were identified in this cross, demonstrating linkage to four intervals on three linkage groups. QTLs for seed weight in the JI1794 x Slow cross were identified by single-marker analyses. Linkage were demonstrated to four intervals on three linkage groups plus three unlinked loci. In the two crosses, only one common genomic region was identified as containing seed-weight QTLs. Seed-weight QTLs mapped to the same region of linkage group III in both crosses. Conserved linkage relationships were demonstrated for pea, mungbean (Vigna radiata L.), and cowpea (V. unguiculata L.) genomic regions containing seed-weight QTLs by mapping RFLP loci from the Vigna maps in the Primo x OSU442-15 and JI1794 x Slow crosses.