Microheterogeneity of lentil seedling (Lens culinaris) amine oxidase

Purified lentil seedling amino oxidase (LSAO) is homogeneous in the analytical ultracentrifuge, but shows heterogeneity in gel-filtration HPLC and in PAGE. Two components were obtained from HPLC and PAGE, but at least 6-7 subforms were seen by electrofocusing techniques. The chromatographic and electrophoretic forms are not interconvertible, indicating the presence of covalent differences. The electrophoretic pattern, but not the chromatographic pattern, is modified by treating the enzyme with a pool of glycohydrolases. The copper-free enzyme shows the same type of heterogeneity as the native enzyme, this ruling out the possibility that some subforms were due to the presence of apoenzyme.     

Genetic diversity of rhizobia nodulating lentil (Lens culinaris) in Bangladesh

In order to determine the bacterial diversity and the identity of rhizobia nodulating lentil in Bangladesh, we performed a phylogenetic analysis of housekeeping genes (16S rRNA, recA, atpD and glnII) and nodulation genes (nodC, nodD and nodA) of 36 bacterial isolates from 25 localities across the country. Maximum likelihood (ML) and Bayesian analyses based on 16S rRNA sequences showed that most of the isolates (30 out of 36) were related to Rhizobium etli and Rhizobium leguminosarum. Only these thirty isolates were able to re-nodulate lentil under laboratory conditions. The protein-coding housekeeping genes of the lentil nodulating isolates showed 89.1-94.8% genetic similarity to the corresponding genes of R. etli and R. leguminosarum. The same analyses showed that they split into three distinct phylogenetic clades. The distinctness of these clades from closely related species was also supported by high resolution ERIC-PCR fingerprinting and phenotypic characteristics such as temperature tolerance, growth on acid-alkaline media (pH 5.5-10.0) and antibiotic sensitivity. Our phylogenetic analyses based on three nodulation genes (nodA, nodC and nodD) and cross-inoculation assays confirmed that the nodulation genes are related to those of R. leguminosarum biovar viciae, but clustered in a distinct group supported by high bootstrap values. Thus, our multi-locus phylogenetic analysis, DNA fingerprinting and phenotypic characterizations suggest that at least three different clades are responsible for lentil nodulation in Bangladesh. These clades differ from the R. etli-R. leguminosarum group and may correspond to novel species in the genus Rhizobium.     

Ribosomal and mitochondrial peroxidase isoenzymes of the lentil (Lens culinaris) root

Summary Ribosome- and mitochondria-rich preparations were obtained from lentil roots and their peroxidase isoenzymes examined by starch gel electrophoresis. Seven isoenzymes were shown to be associated with both the ribosomal and mitochondrial fractions.The apparent similarity between ribosomal and mitochondrial isoenzyme patterns, together with the observation that a considerable amount of peroxidase activity dissociated from the ribosomes during sedimentation of the ribosomal preparation into a sucrose gradient, suggested that at least some of these isoperoxidases were attached to membranes.     

Differences in responses to iron deficiency between two legumes: lentil (Lens culinaris) and chickpea (Cicer arietinum)

Two legumes, lentil and chickpea, were cultivated in nutrient solutions: Fe lacking or containing 30 microM Fe. After 12 days of Fe starvation, lentil showed a severe yellowing of young leaves, a large decrease in chlorophyll concentration, and a significant decline of plant biomass. Chickpea showed a better response than did lentil, primarily due to a stronger acidification capacity. In addition, no chlorosis symptoms were observed in chickpea until the end of treatment. There was no significant difference in potassium uptake between the two species, but an enrichment of the young leaves at the expense of the old ones was noted in chickpea, and at a lesser extent, in lentil, when they were exposed to Fe deficiency. Moreover, this constraint led to a significant decrease of iron content in the two legumes. However, chickpea displayed higher accumulation levels of HCl-extractible iron in young and old leaves than did lentil. This protection of young leaves against K(+) and Fe(2+) impoverishment confers to these organs the capacity to preserve their chlorophyll status and their photosynthetic integrity. Furthermore, the better performance of chickpea under conditions of low Fe availability could be partially related to its seed iron reserves, higher than those of lentil.     

Plant regeneration of the legume Lens culinaris Medik. (lentil) in vitro

Until recently, grain legumes in general have proven recalcitrant at de novo regeneration in vitro. By culturing portions of lentil (Lens culinaris) shoot meristems and epicotyls on a medium containing kinetin and gibberellic acid, callus tissue was produced which could be induced to regenerate shoots in relatively large numbers, even after several subcultures. The shoots could be rooted in a mist chamber to yield whole, fertile plants.     

Root exudates from lentil (Lens culinaris medic) seedlings in relation to wilt disease

Summary Root exudates from healthy and diseased lentil plants (Lens culinaris) have been investigated in relation to the wilt disease caused byFusarium oxysporum f.lentis. In all ten amino acids and five sugars have been detected. The spore germination of the pathogen in root exudates indicated that 21-days root exudate was inhibitory. Glycine and phenylalanine were detected in 21-days exudate and were found to have an inhibitory effect upon the germination of the spores of the pathogen which may partly explain the lesser disease incidence when plants of more than 3 weeks are inoculated with the pathogen.     

Transient kinetics of copper-containing lentil (Lens culinaris) seedling amine oxidase.

The reaction between lentil (Lens culinaris) seedling amine oxidase and its chromogenic substrate, p-dimethylaminomethylbenzylamine, has been studied by the stopped-flow technique. Upon being mixed with substrate in the absence of oxygen, the enzyme is bleached in a complex kinetic process. A yellow intermediate absorbing at 464 nm and the first product (aldehyde) are formed in subsequent steps. When oxygenated buffer is mixed with substrate-reduced amine oxidase, the 496 nm absorption of the oxidized enzyme is very rapidly restored in a second-order process (k = 2.5 X 10(7) M-1 X S-1). This reaction is appreciable even at very low oxygen concentration, in keeping with the fairly low Km for O2 measured by steady-state kinetics.     

Inheritance of mitochondrial DNA in lentil (Lens culinaris Medik.)

Restriction fragment analysis was used to examine the inheritance of lentil mitochondrial DNA (mtDNA) in F₁ and F₅ progeny from intrasubspecific (Lens culinaris ssp. culinaris) crosses and in F₁ progeny from intersubspecific (Lens culinaris ssp. orientalis x L. culinaris ssp. culinaris) crosses. Southern blots of digested parental and progeny DNA were hybridized to heterologous maize mtDNA probes specific to coxI and atp6 genes. Two restriction fragment polymorphisms separated L.c. ssp. culinaris Laird and Eston from L.c. ssp. culinaris ILL5588, and one restriction fragment polymorphism distinguished L.c. ssp. culinaris Laird and Eston from L.c. ssp. orientalis LO4. Twelve of 13 f₁ progeny and all F₅ progeny from the intrasubspecific crosses, and all F₁ progeny from intersubspecific crosses had only maternal mtDNA restriction fragments. One f₁ plant from an Eston x ILL5588 cross inherited mtDNA fragments from both parents. Nuclear DNA inheritance was biparental in all F₁ progeny.NRCC No. 38451     

Effect of acetylcholine on growth and isoperoxidases of the lentil(Lens culinaris) root

The effects of acetylcholine (Ach) on growth, the total peroxidase activity and the isoperoxidase spectrum of the roots ofLens culinaris were studied and compared with actual and earlier results obtained with an auxin (IAA) treatment. The general growth and peroxidase activity patterns of Ach treated roots and IAA treated ones showed many important similarities.     

Aspartate aminotransferase allozyme variation in a germplasm collection of the domesticated lentil (Lens culinaris)

Summary Variation at a polymorphic Aspartate aminotransferase locus was assayed in a sample of 298 accessions from the ICARDA germplasm collection of the domesticated lentil (Lens culinaris). Two alleles Aat-1 ^F and Aat-1 ^S were detected with global frequencies of 0.51 and 0.49, respectively. Fifty-nine percent of accessions were polymorphic for both alleles. The frequency of outcrossing was estimated from the observed heterozygosity to be about 1%. This is higher than direct estimates of outcrossing and implicates selection in favour of heterozygous gene combinations. Significant variation in allele frequency and in the occurrence of polymorphic accessions was observed between countries or geographic areas. Significant associations were observed between the allozymes and agronomic characters. In particular high frequency of Aat-1 ^F appeared to be associated with late flowering and maturity and low yield.     

Agrobacterium tumefaciens-mediated beta-glucuronidase (GUS) gene expression in lentil (Lens culinaris Medik.) tissues

Summary Lentil (Lens culinaris Medik.) shoot apex, epicotyl, and root expiants were capable of expressing an intron-containing beta-glucuronidase (GUS) gene after inoculation with the disarmed Agrobacterium strain GV2260:p35SGUSINT. Expression occurred at all wound sites on these expiants except at the end of the root expiants proximal to the cotyledonary node. GUS expression was detected using both histochemical and fluorescence assays and was stable for at least nine days after inoculation for epicotyl and root expiants, and for at least seventeen days for shoot apices. Non-inoculated controls, or controls inoculated with an Agrobacterium strain lacking the GUS gene, did not produce any background blue staining in the histochemical assay. Expression levels for all lentil expiants were substantially lower than for comparable flax (Linum usitatissimum L.) expiants which served as a positive control.     

Comparative study of different cytokinins in the induction of morphogenesis in lentil (Lens culinaris Medik)

Summary This study presents the first comparative analysis of the effect of four different cytokinins, applied to different lentil (Lens culinaris Medik.) explants in four different concentrations. with regard to the regeneration of shoots and roots of the pulse crop. The variables explant, phytohormone and concentration were all found to be highly significant. Mature seed explants showed the highest shoot regeneration over all the phytohormones and concentrations tested. Thidiazuron (TDZ) and benzyladenine (BA) showed a higher number of regenerated shoots than kinetin (KIN) and zeatin (ZEA); an increase from 1.25 μM to 10 μM of any cytokinin in general doubled the number of shoots regenerated. The average length of regenerated shoots was found to be inversely proportional to the number of shoots regenerated. TDZ and BA were found to inhibit root development more than KIN and ZEA. The highest root regeneration frequency was obtained from shoots regenerated on media containing 1.25 μM ZEA. The study concludes that in order to obtain whole plants it is best to regenerate shoots on media containing the cytokinins KIN or ZEA at low concentrations, in order to be able subsequently to regenerate roots.     

Selection of Rhizobium leguminosarum strains for lentil (Lens culinaris) under growth room and field conditions

Most of the production of lentil (Lens culinaris) on the Great Plains occurs on soils that are free of indigenous Rhizobium leguminosarum. Inoculation is required to increase yields through N₂ fixation. A screening program to evaluate the effectiveness of R. leguminosarum strains for lentil was initially carried out under controlled environments followed by an evaluation under field conditions. In two separate growth room experiments, the effectiveness of 185 and 24 different strains of R. leguminosarum were tested for Laird and Eston lentil. Significant differences between strains in number of nodules, shoot weight and nitrogenase activity (acetylene reduction activity, ARA) were found for lentil grown for 5 weeks. When lentil were grown for 7 weeks, significant differences between strains in number of nodules, total plant weight, total N, and % N were observed.Fourteen strains plus Nitragin C inoculant were selected for further field testing on Eston and Laird lentil at two locations in 1986 and one site in 1987. Inoculation increased yield up to 135%. Percent Ndfa and total N₂ fixed ranged from 0 to 76 and 0 to 105 kg ha^-1, respectively. N₂-fixing activity was site specific and higher spring soil NO₃-levels resulted in lower N₂-fixing activity. Depending on site and growing conditions, strains 99A1 and I-ICAR-SYR-Le20 appeared to be superior to the other strains tested. A good agreement was found between the estimates for N₂ fixation based upon the ¹⁵N-isotope dilution and the classical N difference methods. Number of nodules, dry weight of nodules and ARA of Eston and Laird lentil grown under growth room conditions failed to show positive correlations with total dry matter production, total N or total N₂ fixed of field grown lentil. However, total plant weight and total N of lentil grown under growth room conditions were highly correlated with field parameters, and were the most reliable screening parameters for the selection of superior rhizobial strains.     

Screening of local/exotic accessions of lentil (Lens culinaris Medic.) for salt tolerance at two growth stages

Screening of available local/exotic germplasm of a crop for salinity tolerance is of considerable value for the economic utilization of salt-affected soils of arid and semi-arid regions.The response of 133 lentil (Lens culinaris Medic.) accessions, to NaCl at the germination and seedling stage, was examined. A great amount of variation of NaCl tolerance in lentil was observed at both the growth stages but, in general, there was no consistent relationship between tolerance assessed at germination or at the seedling stage. In the NaCl treatment five accessions, ILL 5845, ILL 6451, ILL 6788, ILL 6793, and ILL 6796 produced significantly greater fresh and dry plant biomass in both absolute and relative terms than the others, but these accessions performed as well as other intermediate or low biomass producing accessions in total germination percentage and rate of germination. In view of the existence of the great amount of variability of tolerance to NaCl among lentil varieties improvement in NaCl tolerance in this species is possible.     

Effect of potassium deficiency on growth and some biochemical characteristics in two lines of lentil (Lens culinaris Medic.)

The effect of low potassium regimes on two lines, ILL 6796 (salt-tolcrant) and ILL 6439 (salt-sensitive), of lentil (Lens culinaris) was studied in a pot experiment under glasshouse conditions. Plants of the two lines were treated with 78, 39, 19.5, and 9.75 mg K·L⁻¹ in Rorison’s nutrient solution. ILL 6796 excelled ILL 6439 in biomass production under varying K regimes. Of the various organic solutes measured in the present study only total soluble sugars proved to be helpful in discriminating the lines, i.e., total sugars increased consistently in ILL 6796 with a decrease in K level of the growth medium, whereas in ILL 6439 they decreased significantly with a decrease in K levels. ILL 6796 showing high efficiency for K utilisation (high shoot growth) could be of great economic value in terms of its use in soils deficient in K.     

Transformation of lentil (Lens culinaris M.) cotyledonary nodes by vacuum infiltration ofAgrobacterium tumefaciens

Lentil cotyledonary nodes are some of the most regenerative tissues in legumes. Attempts to transform them by vacuum filtration have been limitedly successful. This report describes a rapid and convenient transient expression protocol based on vacuum infiltration ofAgrobacterium cells into lentil cotyledonary nodes. Vacuum-infiltrated tissues had significantly (P<.05) higher transient GUS expression than did noninfiltrated tissues. Under optimal conditions (infiltration at 200 mmHg for 20 min), 95% of theAgrobacterium-infiltrated explants exhibited an average of 16 blue foci. We believe this to be the first report of this technique for transient gene expression in lentil cotyledonary nodes.