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.     

Genotype-specific responses to the effects of commercial Trichoderma formulations in lentil (Lens culinaris ssp. culinaris) in the presence and absence of the oomycete pathogen Aphanomyces euteiches

Members of the endophytic fungal genus Trichoderma have been established as plant-beneficial microbes and are most successful commercial biologicals in the form of bio-fertilisers, biocontrol agents, and growth stimulators. We report the variable interactions among different lentil genotypes and Trichoderma strains in both the presence and absence of biotic stress (root-rot pathogen Aphanomyces euteiches). Two commercial Trichoderma formulations, namely RootShield^® (RS) and RootShield^® Plus (RSP) based on T. harzianum T22 and T. virens G41, respectively, were evaluated for control of Aphanomyces root rot and plant growth promotion in 23 wild and cultivated lentil genotypes. No significant disease control was recorded with either formulation in any lentil genotype. Significant genotype-specific plant growth promotion was observed in terms of root and shoot development and leaf parameters in a genotype-specific manner. Genotypes of Lens culinaris and Lens tomentosus, both in the primary lentil gene pool, demonstrated the maximum response. The overall effect of Trichoderma treatment was markedly higher under biotically stressed conditions in comparison to unstressed conditions. In many cases, negative responses were recorded, particularly in the absence of root-rot disease. L. tomentosus PI 572390 exhibited positive responses for most of the tested parameters. Our findings clearly indicate that, in the case of lentil, plant genotype plays a major role in interactions among the tested Trichoderma strains and the plant. Moreover, the influence of Trichoderma was greater and more favourable under conditions of biotic stress vs. the absence of stress.     

Detecting DNA polymorphism and genetic diversity in Lentil (Lens culinaris Medik.) germplasm: comparison of ISSR and DAMD marker

Genetic diversity and interrelationships among 31 lentil genotypes were evaluated using 10 Inter-Simple Sequence Repeat (ISSR) and 10 directed amplification of minisatellite DNA region (DAMD) primers. A total of 43 and 48 polymorphic bands were amplified by ISSR and DAMD markers, respectively. Average polymorphism information content (PIC) for ISSR and DAMD markers were 0.37 and 0.41, respectively. All 31 lentil genotypes could be distinguished by ISSR markers into three groups and by DAMD markers into two groups. Various molecular markers show a different efficiency for evaluating DNA polymorphism in lentil and indicate that the patterns of variation are clearly influenced by the genetic marker used. Comparatively, the genetic diversity of examined lentil genotypes by two different marker techniques (ISSR and DAMD) was high and indicated that ISSR and DAMD are effective and promising marker systems for fingerprinting in lentil and give useful information on its genetic relationships.     

Production of fertile transgenic lentil (Lens culinaris Medik) plants using particle bombardment

Summary A reproducible system for gene transfer in lentil through particle bombardment is presented. Lentil cotyledonary nodes excised from germinated seedlings were bombarded with a plasmid containing a mutant acetolactate synthase gene (ALS) from tobacco conferring resistance to sulfonylurea herbicides. Putative transgenic shoots regenerated on Murashige and Skoog medium supplemented with 6-benzylaminopurine (BA) and chlorsulfuron (5 nM for first 4 wk followed by 2.5 nM for the remainder of the culture period) were micrografted and successfully transferred to soil. T₀ and selfed progeny plants were screened using metsulfuron herbicide leaflet painting. The non-transformed escapes died and transformed plants survived the test. The surviving plants were phenotypically normal and produced viable seeds. The presence and stable transmission of the transgene into genomic DNA of screened T₁ transformants was confirmed by PCR and Southern hybridization. This method for producing transformed plants will allow new opportunities for lentil breeding to produce improved cultivars.     

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.     

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.     

Early activation of lipoxygenase in lentil (Lens culinaris) root protoplasts by oxidative stress induces programmed cell death.

Oxidative stress caused by hydrogen peroxide (H2O2) triggers the hypersensitive response of plants to pathogens. Here, short pulses of H2O2 are shown to cause death of lentil (Lens culinaris) root protoplasts. Dead cells showed DNA fragmentation and ladder formation, typical hallmarks of apoptosis (programmed cell death). DNA damage was evident 12 h after the H2O2 pulse and reached a maximum 12 h later. The commitment of cells to apoptosis caused by H2O2 was characterized by an early increase of lipoxygenase activity, of ultraweak luminescence and of membrane lipid peroxidation, which reached 720, 350 and 300% of controls, respectively, at 6 h after H2O2 treatment. Increased lipoxygenase activity was paralleled by an increase of its protein and mRNA level. Lipoxygenase inhibitors nordihydroguaiaretic acid, eicosatetraynoic acid and plamitoyl ascorbate prevented H2O2-induced DNA fragmentation and ultraweak luminescence, only when added together with H2O2, but not when added 8 h afterwards. Inhibitory anti-lipoxygenase monoclonal antibodies, introduced into the protoplasts by electroporation, protected cells against H2O2-induced apoptosis. On the other hand, lentil lipoxygenase products 9- and 13-hydroperoxy-octadecadienoic acids and their reduced alcohol derivatives were able to force the protoplasts into apoptosis. Altogether, these findings suggest that early activation of lipoxygenase is a key element in the execution of apoptosis induced by oxidative stress in plant cells, in a way surprisingly similar to that observed in animal cells.     

Morpho-Biochemical characterization of Kalazeera (Bunium persicum Boiss. Fedts) germplasm grown in Global temperate ecologies

The present investigation explores the variability of Bunium persicum populations belonging to different regions. Variability among 74 genotypes for thirty-seven traits (29 quantitative and 8 qualitative) were studied to ascertain the population structure of the Bunium persicum. Among the agro-morphological traits, wide range of variability was recorded in tuber shape, tuber colour, seed shape, seed colour, growth habit, leaf shape, leaf colour, umbel shape, umbel colour, plant height (22.90–96.52 cm), primary branches plant⁻¹ (1–6), umbel diameter of primary umbel (6.17 – 13.67 cm), number of primary umbels plant⁻¹ (1–12), umbels plant⁻¹ (8–40), seed yield per plant (0.55–13.10 g), essential oil content (3.2–9.3 %) etc. Significant and positive association was observed between number of seeds primary^-1 umbel (r = 0.91), plant height (r = 0.65), number of seeds primary^-1 umbel (0.52), number of seeds primary^-1 umbel (0.43), number of seeds secondary^-1 umbel (0.38) with number of umblets secondary^-1 umbel. Cluster analysis classified the genotypes with different geographical origin into two major clusters and sub-clusters. Cluster-I comprises of 50 genotypes and cluster - II of 24 genotypes while the genotype SRS-KZ-189 from Kargil population was separated as an individual sub-group. Principal component (PC1) and (PC2) harbors accounted 20.2% and 14% of total variation. Variability of Kalazeera genotypes would facilitate the plant breeders to implement and design various crop improvement programme in future.     

GABA metabolism and ROS induction in lentil (Lens culinaris Medik) plants by synthetic 1,2,3-Thiadiazole compounds

In this study, we investigated the physiological effects of three synthetic 1,2,3-thiadiazole compounds (compound I: 4-[1,2,3]-Thiadiazole-4-yl-phenol, compound II: 1,3-Bis[4-(1,2,3-thiadiazol-4-yl)phenoxy]propane, compound III: 4-(4-{2,3,4,5,6-Penta[4-(1,2,3-thiadiazole-4-yl)phenoxymethyl]benzyloxy}phenyl)-1,2,3-thiadiazole) treatments on plants via the characterization of γ-aminobutyric acid metabolite level, the accumulation of reactive oxygen species (ROS), total protein contents, total carbohydrate contents, fresh weight and dry mass in two lentil (Lens culinaris Medik) cultivars (Jordan 1 and Jordan 2). In response to the three synthetic 1,2,3-thiadiazole compounds (I, II and III) treatments. A significant increase in γ- aminobutyric acid (GABA) and malondialdehyde (MDA) levels and a significant reduction in carbohydrates and protein levels and fresh weight and dry mass were obtained in both lentil cultivars. Jordan 2 cultivar showed the highest GABA and MDA accumulation and significant reduction in the carbohydrate and protein levels under the various thiadiazole compounds treatments. This suggests that GABA molecule may act as a defense mechanism and signaling molecule in carbohydrate metabolism and other physiological systems in lentil. In conclusion, our results revealed that the synthetic 1,2,3-thiadiazole compound ?? (1,3-Bis[4-(1,2,3-thiadiazol-4-yl)phenoxy]propane) had the strongest physiological inhibitory effect on lentil.     

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.     

Influence of drought and heat stress,applied independently or in combination during seed development,on qualitative and quantitative aspects of seeds of lentil (Lens culinaris Medikus) genotypes,differing in drought sensitivity

Terminal droughts, along with high temperatures, are becoming more frequent to strongly influence the seed development in cool‐season pulses like lentil. In the present study, the lentil plants growing outdoors under natural environment were subjected to following treatments at the time of seed filling till maturity: (a) 28/23 °C day/night temperature as controls; (b) drought stressed, plants maintained at 50% field capacity, under the same growth conditions as in a; (c) heat stressed, 33/28 °C day/night temperature, under the same growth conditions as in a; and (d) drought + heat stressed, plants at 50% field capacity, 33/28 °C day/night temperature, under the same growth conditions as in (a). Both heat and drought resulted in marked reduction in the rate and duration of seed filling to decrease the final seed size; drought resulted in more damage than heat stress; combined stresses accentuated the damage to seed starch, storage proteins and their fractions, minerals, and several amino acids. Comparison of a drought‐tolerant and a drought‐sensitive genotype indicated the former type showed significantly less damage to various components of seeds, under drought as well as heat stress suggesting a cross tolerance, which was linked to its (drought tolerant) better capacity to retain more water in leaves and hence more photo‐assimilation ability, compared with drought‐sensitive genotype.     

Treatment of lentil (Lens culinaris. L) seeds with various concentrations of salts to check their efficiency against seed-borne mycoflora during storage

Seed sample of lentil collected from the Swabi district were treated with NaCl and KCl at 0.01, 0.1 and 1.0% (w/w). Seed-borne mycoflora was observed at 0, 20, 40, 60 and 80 day intervals. Seed treatment with both the salts was found to be effective against storage fungi; however, KCl was more efficient against storage mycoflora such as Aspergillus species when compared with NaCl. With the passage of time, the incidence of deep-seated fungi was observed in salt-treated seed samples while untreated seed sample showed heavy infestation by the species of Aspergillus. Seed viability also remained unaffected in storage, except in seeds heavily infested with seed-borne mycoflora. Aspergillus spp. were the main cause of seed rot. Surface sterilisation of seeds with 1% Na(OCl)₂ reduced the fungal infestation of seeds. Among various concentrations of salts, 0.1% (w/w) of both salts were better in controlling seed-borne mycoflora.     

The potential of lentil (Lens culinaris) as a grain legume crop in the UK: an assessment based on a crop growth model

A crop growth model developed in Canterbury, New Zealand was used to assess the potential of lentil (Lens culinaris) as a grain legume crop in the UK. The model was validated using five sowing dates at Durham (54.77°N, 1.58°W) in 1999. Predicted time to flowering was within 7 days of actual time to flowering and predicted seed yields were within 9% of actual yields. Actual yields ranged from 1.40 to 1.65 t ha‐¹. Seed was of high quality. The model was used to predict rate of development and yields of spring and autumn sown lentils at eight sites along a transect from NW Scotland (Stornoway, 58.22°N, 6.32°W) to SE England (East Mailing, 51.28°N, 0.45°E) chosen to encompass important environmental gradients in the UK. In general, for a 1 May sowing with 150 or 250 mm plant available water (PAW) and a 1 October sowing with 150 mm PAW, predicted mean values over the period 1987–95 for maximum crop growth rate, maximum leaf area index, radiation intercepted, total dry matter produced and seed yield were closely positively related to monthly mean values for mean daily air temperature and increased along the transect from NW to SE UK. Time to flowering generally decreased along the transect from NW to SE UK ranging from 28 June to 9 July and from 20 May to 14 June with the May and October sowings respectively. For the 1 May sowing with 250 mm PAW, predicted mean seed yield ranged from 1.00 to 1.90 t ha‐1. For all sites, yield was very stable over the 9 yr period. For the 1 May sowing with 150 mm PAW, predicted mean seed yield ranged from 0.97–1.23 t ha‐1. Yields for the four more southerly sites were more variable at the lower PAW and, in exceptionally dry years, were substantially lower than average. For these sites, autumn sowing increased seed yields in exceptionally dry years and gave similar or greater mean seed yields to spring sowing with 250 mm PAW. For East Mailing, predicted yields for autumn sowing were on average 2.78 t ha‐1. Also, for Stornoway, because of its relatively high overwinter temperatures, the model predicted substantial increases in yield with autumn sowing. It is concluded that lentil has considerable potential as a grain legume crop in the UK.     

用发光酶基因(luxAB)标记法研究慢生花生根瘤菌的竞争结瘤能力

luxAB基因标记是一种新型基因标记技术,在很多研究领域都有着良好的应用前景。研究通过三亲本杂交将luxAB基因成功地向慢生型花生根瘤菌进行了转移,并获得了一株带LuxAB基因标记的菌株Cspr7-1。对Cspr7-1进行性状、标记基因的遗传稳定性检测,结果表明,LuxAB基因不仅能有效表达,而且性状稳定。在无氮水培条件下进行标记菌株与土著根瘤菌的竞争结瘤试验。结果证实,Cspr7-1在植物根系上的占瘤率平均达到61.3％,比土著根瘤菌的竞争结瘤能力强,而且Cspr7-1在主根上的侵染能力远较侧根上的强,平均高出22.3％-39.6％。     

Studies on interaction of Meloidogyne incognita (Kofoid and White) Chitwood and Fusarium solani (Mart.) Sacc forming a disease complex in lentil (Lens culinaris Medik.)

Abstract

An experiment was conducted to study the effects of interaction between Meloidogyne incognita and Fusarium solani on plant length, fresh and dry weights, number of pods, chlorophyll, carotenoid, nitrogen and phosphorus contents and nitrate reductase activity in lentil plants. The results reveal a maximum damage occurring in all the plant growth, biochemical and nutrient parameters, in plants inoculated with M. incognita 10 days prior to F. solani (Mi?→?Fs). This was followed by simultaneous (Mi?+?Fs) inoculations, fungus inoculation 10 days prior to nematode (Fs?→?Mi), M. incognita alone and F. solani alone treatments. Nematode reproduction factor and root galling were highest in individual inoculation of M. incognita, while root rotting percentage was highest when nematode was inoculated 10 days prior to fungus followed by simultaneous inoculation with both nematode and fungus.     

Genetics of resistance to anthracnose and identification of AFLP and RAPD markers linked to the resistance gene in PI 320937 germplasm of lentil (<Emphasis Type="Italic">Lens culinaris</Emphasis> Medikus)

Anthracnose, caused by Colletotrichum truncatum, is a major disease problem and production constraint of lentil in North America. The research was conducted to examine the resistance to anthracnose in PI 320937 lentil and to identify molecular markers linked to the resistance gene in a recombinant inbred line (RIL) population developed from a cross of Eston lentil, the susceptible parent, and PI 320937, the resistant parent. A total of 147 F(5:6) RILs were evaluated for resistance to anthracnose in the greenhouse using isolate 95B36 of C. truncatum. Bulked segregant analysis (BSA) strategy was employed and two contrasting DNA bulks were constructed based on greenhouse inoculation of F(5)-derived F(6) RILs. DNA from the parents and bulks were screened with 700 RAPD primers and seven AFLP primer combinations. Analysis of segregation data indicated that a major dominant gene was responsible for resistance to anthracnose while variations in the resistance level among RILs could be the influences of minor genes. We designate the major gene as LCt-2. MapMaker analysis produced two flanking RAPD markers OPEO6(1250) and UBC-704(700) linked to LCt-2 locus in repulsion (6.4 cM) and in coupling (10.5 cM), respectively. Also, three AFLP markers, EMCTTACA(350) and EMCTTAGG(375) in coupling, and EMCTAAAG(175) in repulsion, were linked to the LCt-2 locus. These markers could be used to tag the LCt-2 locus and facilitate marker-assisted selection for resistance to anthracnose in segregating populations of lentil in which PI 320937 was used as the source of resistance. Also, a broader application of the linked RAPD markers was also demonstrated in Indianhead lentil, widely used as a source of resistance to anthracnose in the breeding program at the Crop Development Centre, University of Saskatchewan. Further selection within the few F(5:6) lines should be effective in pyramiding one or several of the minor genes into the working germplasm of lentil, resulting in a more durable and higher level of resistance.     

Assessment of genetic diversity in indigenous turmeric (Curcuma longa) germplasm from India using molecular markers

Curcuma longa L., commonly known as turmeric, is one of the economically and medicinally important plant species. It is predominantly cultivated in the tropical and sub tropical countries. India is the largest producer, and exporter of turmeric in the world, followed by China, Indonesia, Bangladesh and Thailand. In the present study, Directed Amplification of Minisatellite DNA (DAMD) and Inter Simple Sequence Repeats (ISSR), methods were used to estimate the genetic variability in indigenous turmeric germplasm. Cumulative data analysis for DAMD (15) and ISSR (13) markers resulted into 478 fragments, out of which 392 fragments were polymorphic, revealing 82 % polymorphism across the turmeric genotypes. Wide range of pairwise genetic distances (0.03–0.59) across the genotypes revealed that these genotypes are genetically quite diverse. The UPGMA dendrogram generated using cumulative data showed significant relationships amongst the genotypes. All 29 genotypes studied grouped into two clusters irrespective of their geographical affiliations with 100 % bootstrap value except few genotypes, suggesting considerable diversity amongst the genotypes. These results suggested that the current collection of turmeric genotypes preserve the vast majority of natural variations. The results further demonstrate the efficiency and reliability of DAMD and ISSR markers in determining the genetic diversity and relationships among the indigenous turmeric germplasm. DAMD and ISSR profiling have identified diverse turmeric genotypes, which could be further utilized in various genetic improvement programmes including conventional as well as marker assisted breeding towards development of new and desirable turmeric genotypes.     

A genetic linkage map of azuki bean constructed with molecular and morphological markers using an interspecific population (Vigna angularis x V. nakashimae)

A genetic linkage map of azuki bean (Vigna angularis) was constructed with molecular and morphological markers using an F₂ population of an interspecific cross between azuki bean and its wild relative, V. nakashimae. In total, 132 markers (108 RAPD, 19 RFLP and five morphological markers) were mapped in 14 linkage groups covering 1250 cM; ten remained unlinked. The clusters of markers showing distorted segregation were found in linkage groups 2, 8 and 12. By comparing the azuki linkage map with those of mungbean and cowpea, using 20 RFLP common markers, some sets of the markers were found to belong to the same linkage groups of the respective maps, indicating that these linkage blocks are conserved among the three Vigna species. This map provides a tool for markerassisted selection and for studies of genome organization in Vigna species.