Isolation and characterization of a lectin gene from seeds of chickpea (Cicer arietinum L.).

A cDNA library was constructed in lambda TriplEx2 vector using poly (A(+)) RNA from immature seeds of Cicer arietinum. The lectin gene was isolated from seeds of chickpea through library screening and RACE-PCR. The full-length cDNA of Chichpea seed lectin(CpGL)is 972 bp and contains a 807 bp open reading frame encoding a 268 amino acid protein. Analysis shows that CpSL gene has strong homology with other legume lectin genes. Phylogenetic analysis showed the existence of two main clusters and clearly indicated that CpSL belonged to mannose-specific family of lectins. RT-PCR revealed that CAA gene expressed constitutively in various plant tissues including flower, leaf, root and stem. When chickpea lectin mRNA level was checked in developing seeds, it was higher in 10 DAF seeds and decreased throughout seed development.     

A gene for leaf necrosis in chickpea (Cicer arietinum L.)

Necrosis of leaves was observed in the glabrous mutant (ICC 15566) of desi chickpea (Cicer arietinum L.). It was characterized by drying of leaflet margins to drying of complete leaflets of older leaves. The oldest leaves were the most affected and the intensity of necrosis decreased toward the apical meristem. A single recessive gene, designated nec, was found to govern the necrotic characteristic. The nec locus was linked to gl (glabrous shoots) with a map distance of 16 +/- 3 cM. The loci slv (simple leaves), mlv (multipinnate leaves), nlv (narrow leaflets), hg (prostrate growth habit), P (pink corolla), and shp (round seed shape) segregated independently of nec.     

Sequence-tagged microsatellite site markers for chickpea (Cicer arietinum L.).

Two small-insert genomic libraries of chickpea (Cicer arietinum L.) were screened with a set of microsatellite-specific oligonucleotide probes. A total of 121 positive clones were identified among 13,000 plated colonies. Thirty-nine clones were recognized by (TAA)5, 26 by (GA)8, 18 by (GT)8, 27 by a pool of AT-rich trinucleotide repeats [(CAA)5, (CAT)5, and (GAA)5], and 11 by a pool of GC-rich trinucleotides [(TCC)5, (CAC)5, (CAG)5, and (CGA)5]. Of 53 clones selected for sequencing, 43 carried a microsatellite. Flanking primer pairs were designed for 28 loci, and used on a small test-set comprising one C. reticulatum and four C. arietinum accessions. Separation of the PCR products on agarose or polyacrylamide gels revealed single bands of the expected size with 22 of the primer pairs. Sixteen of these "Cicer arietinum sequence-tagged microsatellite site" (CaSTMS) markers were polymorphic at an intraspecific level, detecting 2-4 alleles within the four accessions examined. Primer pairs CaSTMS10 and CaSTMS15 revealed 25 and 16 alleles among 63 C. arietinum accessions from different geographic locations, reflecting gene diversity values of 0.937 and 0.922, respectively. Mendelian inheritance of CaSTMS markers was demonstrated using a set of recombinant inbred lines and their parents.     

Genetic characterization of the acetohydroxyacid synthase (AHAS) gene responsible for resistance to imidazolinone in chickpea (Cicer arietinum L.)

Key message

A point mutation in the AHAS1 gene leading to resistance to imidazolinone in chickpea was identified. The resistance is inherited as a single gene. A KASP marker targeting the mutation was developed.

Abstract

Weed control in chickpea (Cicer arietinum L.) is challenging due to poor crop competition ability and limited herbicide options. A chickpea genotype with resistance to imidazolinone (IMI) herbicides has been identified, but the genetic inheritance and the mechanism were unknown. In many plant species, resistance to IMI is caused by point mutation(s) in the acetohydroxyacid synthase (AHAS) gene resulting in an amino acid substitution preventing herbicide attachment to the molecule. The main objective of this research was to characterize the resistance to IMI herbicides in chickpea. Two homologous AHAS genes namely AHAS1 and AHAS2 sharing 80 % amino acid sequence similarity were identified in the chickpea genome. Cluster analysis indicated independent grouping of AHAS1 and AHAS2 across legume species. A point mutation in the AHAS1 gene at C675 to T675 resulting in an amino acid substitution from Ala205 to Val205 confers the resistance to IMI in chickpea. A KASP marker targeting the point mutation was developed and effectively predicted the response to IMI herbicides in a recombinant inbred (RI) population of chickpea. The RI population was used in molecular mapping where the major locus for the reaction to IMI herbicide was mapped to chromosome 5. Segregation analysis across an F₂ population and RI population demonstrated that the resistance is inherited as a single gene in a semi-dominant fashion. The simple genetic inheritance and the availability of KASP marker generated in this study would speed up development of chickpea varieties with resistance to IMI herbicides.     

Relationship between P and Mn in chickpea (Cicer arietinum L.)

Summary Phosphorus and Mn relationship was studied in chickpea at two stages of growth in pot culture using 0, 7.5, 15 and 30 ppm P and 0, 5, 10 and 15 ppm Mn. The dry matter yield increased with P at both stages of growth. Manganese improved the yield only in the first stage. Initial levels of Mn enhanced while higher levels had a depressing effect on tissue P. Addition of 7.5 ppm P enhanced Mn concentration at first stage and at higher levels a marked reduction in Mn content was observed at both the stages.     

Plant regeneration via somatic embryogenesis in chickpea (Cicer arietinum L.)

Efficient plant regeneration via somatic embryogenesis has been developed in chickpea cultivar C235. Leaf explants, on MS medium supplemented with 1.25 mg/l 2,4-D and 0.25 mg/l kinetin, yielded somatic embryos with high efficiency during dark incubation. MS medium supplemented with B₅ vitamins, 0.125 mg/l IBA and 2 mg/l BAP was found suitable for embryo maturation. The well formed embryos germinated into plantlets on basal B₅ medium supplemented with 0.25 mg/l BAP. Further development into healthy plantlets was obtained on basal B₅ medium. Hardened plantlets produced normal, fertile plants upon transfer to soil.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-Benzyl-aminopurine - IAA IndoIe-3-acetic acid - IBA Indole-3-butyric acid - NAA 1-Naphthalene acetic acid - Kinetin 6-furfuryl aminopurine - Zeatin 6-(4-hydroxy-3-methylbut-2-enylamino)-purine     

Purification and characterization of an N-acetyl-D-galactosamine-specific lectin from seeds of chickpea (Cicer arietinum L.)

A novel lectin (CAA-II) was isolated and purified from the seeds of Cicer arietinum by ammonium sulphate fractionation and affinity chromatography on an N-acetyl-D-galactosamine-linked agarose column. The lectin is composed of four identical subunits of 30 kDa and the molecular mass of the native lectin was estimated to be 120 kDa by gel filtration chromatography and confirmed by mass spectrometry. The lectin showed agglutination activity against rabbit erythrocytes (trypsin-treated and untreated) as well as against human erythrocytes. Haemagglutination inhibition assays showed that the lectin is a galactose-specific protein having a high affinity for N-acetyl-D-galactosamine. The molecular weight, haemagglutination pattern, carbohydrate specificity and N-terminal amino acid sequence indicated that the lectin is clearly distinct from the previously reported chickpea lectin CAA-I.     

Agrobacterium mediated transformation of chickpea (Cicer arietinum L.) embryo axes

 Embryo axes of four accessions of chickpea (Cicer arietinum L.) were treated with Agrobacterium tumefaciens strains C58C1/GV2260 carrying the plasmid p35SGUSINT and EHA101 harbouring the plasmid pIBGUS. In both vectors the GUS gene is interrupted by an intron. After inoculation shoot formation was promoted on MS medium containing 0.5 mg/l BAP under a selection pressure of 100 mg/l kanamycin or 10 mg/l phosphinothricin, depending on the construct used for transformation. Expression of the chimeric GUS gene was confirmed by histochemical localization of GUS activity in regenerated shoots. Resistant shoots were grafted onto 5-day-old dark-grown seedlings, and mature plants could be recovered. T-DNA integration was confirmed by Southern analysis by random selection of putative transformants. The analysis of 4 plantlets of the T1 progeny revealed that none of them was GUS-positive, whereas the presence of the nptII gene could be detected by polymerase chain reaction. Received: 30 May 1997 / Revision received: 18 September 1997 / Accepted: 22 March 1999     

Purification and characterization of trypsin inhibitor from seeds of faba bean (Vicia faba L.)

A trypsin inhibitor from seeds of faba bean (Vicia faba L.) was purified to near homogeneity as judged by native-PAGE with about 11 % recovery using ammonium sulphate fractionation, ion-exchange chromatography on DEAE-cellulose and gel filtration through Sephadex G-100. The inhibitor had a molecular weight of 18 kD as determined by SDS-PAGE and Sephadex G-100. The inhibitor inhibited trypsin and chymotrypsin to the extent of 48 and 12 %, respectively. The inhibtion was of non-competitive type with dissociation constant for the enzyme inhibitor complex in the region of 0.07 mg·ml⁻¹. The inhibtor was stable between pH 4 and 5. It completely lost its activity when heated at 125 °C for 1 h or at 100 °C for 2 h. The inhibitor also lost its activity on exposure to 2-mercaptoethanol. Based on these properties, it could be concluded that Vicia faba trypsin inhibitor belongs to Bowman-Birk type of inhibitors, as it has molecular weight lower than generally observed for Kunitz type inhibitors.     

Pathotype-specific genetic factors in chickpea (Cicer arietinum L.) for quantitative resistance to ascochyta blight

Ascochyta blight in chickpea (Cicer arietinum L.) is a devastating fungal disease caused by the necrotrophic pathogen, Ascochyta rabiei (Pass.) Lab. To elucidate the genetic mechanism of pathotype-dependent blight resistance in chickpea, F₇-derived recombinant inbred lines (RILs) from the intraspecific cross of PI 359075(1) (blight susceptible) × FLIP84-92C(2) (blight resistant) were inoculated with pathotypes I and II of A. rabiei. The pattern of blight resistance in the RIL population varied depending on the pathotype of A. rabiei. Using the same RIL population, an intraspecific genetic linkage map comprising 53 sequence-tagged microsatellite site markers was constructed. A quantitative trait locus (QTL) for resistance to pathotype II of A. rabiei and two QTLs for resistance to pathotype I were identified on linkage group (LG)4A and LG2+6, respectively. A putative single gene designated as Ar19 (or Ar21d) could explain the majority of quantitative resistance to pathotype I. Ar19 (or Ar21d) appeared to be required for resistance to both pathotypes of A. rabiei, and the additional QTL on LG4A conferred resistance to pathotype II of A. rabiei. Further molecular genetic approach is needed to identify individual qualitative blight resistance genes and their interaction for pathotype-dependent blight resistance in chickpea.     

Zeatin-induced shoot regeneration from immature chickpea (Cicer arietinum L.) cotyledons

For the purpose of developing an in vitro regeneration system for chickpea (Cicer arietinum L.), an important food legume, immature cotyledons approximately 5 mm long were excised from developing embryos and cultured on B5 basal medium supplemented with 1.5% sucrose and various growth regulator combinations. Only non-morphogenic callus was formed in response to concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), naphthaleneacetic acid (NAA) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) previously reported to induce somatic embryogenesis on immature soybean cotyledons. However, 4.6, 13.7, and 45.6 M zeatin induced formation of white, cotyledon-like structures (CLS) at the proximal end of immature cotyledons placed with adaxial surface facing the agar medium. No morphogenesis, or occasional formation of fused, deformed CLS, was observed when zeatin was replaced with kinetin or 6-benzyladenine, respectively. The highest response frequency, 64% of explants forming CLS, was induced by 13.7 M zeatin plus 0.2 M indole-acetic acid (IAA). Within 20–40 days culture on zeatin, shoots formed at the base of CLS on approximately 50% of CLS-bearing explants, and proliferated upon subsequent transfer to basal medium with 4.4 M BA or 4.6 M kinetin. This regeneration system may be useful for genetic transformation of chickpea.     

28-Homobrassinolide ameliorates the saline stress in chickpea (Cicer arietinum L.)

The response of chickpea (Cicer arietinum L.) cv. KPG-59 to pre-sowing seed treatment with 28-homobrassinolide (HBR) and/or sodium chloride (NaCl) was investigated. The seeds imbibed in aqueous solution of 10⁻¹⁰ or 10⁻⁸ M of HBR for 8 h, resulted in an increase in the values for most of the characteristics of shoot and root at 90-day stage and seed yield, at harvest. The plants resulting from the seeds soaked in HBR (10⁻⁸ M) possessed 23% and 31% higher leaf nitrate reductase (E.C. 1.6.6.1) and carbonic anhydrase (E.C. 4.2.2.1) activities, 34% more dry mass, 30% higher nodule number, 31% and 18% more nodule fresh and dry mass, compared with water soaked, control. Leghaemoglobin content and nitrogenase activity (E.C. 1.7.99.2) were 28% and 30% higher while nodule nitrogen and carbohydrate contents decreased by 5% and 6%, compared with the control. Moreover, seed yield increased by 26% over the control, at harvest. The values for all the above characteristics declined significantly, in the plants raised from the seeds soaked in NaCl. However, this ill effect was overcome, if NaCl treatment was given before or after HBR treatment.     

Genomic regions associated with chocolate spot (Botrytis fabae Sard.) resistance in faba bean (Vicia faba L.)

Molecular Breeding - Maize (Zea mays L.) is the most important food crop in the world, with significant acreage and production across the globe. However, it is affected by low temperatures...     

Effects of selection criteria on yield stability in chickpea (Cicer arietinum L.)

Summary Selection in the F₃ generation for seed yield, fruiting branches/plant, effective pods/plant, and seed index (100-seed weight) was carried out in two chickpea crosses. Sixty F₅ lines (15 lines/selection criterion) along with check variety were evaluated for seed yield in three distinct environments. The effects of selection criteria on yield stability was examined using linear regression approach and genotype-grouping technique. There were no differences between selection criteria for linear yield responses of F₅ lines to different environments. Within all four selection criteria the lines showed similar linear responses. The non-linear component was relatively higher for lines selected for effective pods and seed index than lines selected for yield and fruiting branches. On the basis of mean yield and coefficient of variation across environments, the seed index was the least effective selection criterion for developing high yielding and stable lines. When the results of stability parameters and genotype-grouping technique were considered together, selection for yield and fruiting branches was highly effective for isolating stable and high yielding lines.     

Integrated physical,genetic and genome map of chickpea (Cicer arietinum L.)

Physical map of chickpea was developed for the reference chickpea genotype (ICC 4958) using bacterial artificial chromosome (BAC) libraries targeting 71,094 clones (~12× coverage). High information content fingerprinting (HICF) of these clones gave high-quality fingerprinting data for 67,483 clones, and 1,174 contigs comprising 46,112 clones and 3,256 singletons were defined. In brief, 574 Mb genome size was assembled in 1,174 contigs with an average of 0.49 Mb per contig and 3,256 singletons represent 407 Mb genome. The physical map was linked with two genetic maps with the help of 245 BAC-end sequence (BES)-derived simple sequence repeat (SSR) markers. This allowed locating some of the BACs in the vicinity of some important quantitative trait loci (QTLs) for drought tolerance and reistance to Fusarium wilt and Ascochyta blight. In addition, fingerprinted contig (FPC) assembly was also integrated with the draft genome sequence of chickpea. As a result, ~965 BACs including 163 minimum tilling path (MTP) clones could be mapped on eight pseudo-molecules of chickpea forming 491 hypothetical contigs representing 54,013,992 bp (~54 Mb) of the draft genome. Comprehensive analysis of markers in abiotic and biotic stress tolerance QTL regions led to identification of 654, 306 and 23 genes in drought tolerance “QTL-hotspot” region, Ascochyta blight resistance QTL region and Fusarium wilt resistance QTL region, respectively. Integrated physical, genetic and genome map should provide a foundation for cloning and isolation of QTLs/genes for molecular dissection of traits as well as markers for molecular breeding for chickpea improvement.     

Plant and soil resistance to water flow in faba bean (Vicia faba L. major Harz.)

An experiment was conducted to determine soil and plant resistance to water flow in faba bean under field conditions during the growing season. During each sampling period transpiration flux and leaf water potential measured hourly were used with daily measurements of root and soil water potential to calculate total resistance using Ohm's law analogy. Plant growth, root density and soil water content distributions with depth were measured. Leaf area and root length per plant reached their maximum value during flowering and pod setting (0.31 m² and 2200 m, respectively), then decreasing until the end of the growing period. Root distribution decreased with depth ranging, on average, between 34.2% (in the 0–0.25 m soil layer) and 18.1% (in the 0.75–1.0 m soil layer). Mean root diameter was 0.6 mm but most of the roots were less than 0.7 mm in diameter. Changes in plant and soil water potentials reflected plant growth characteristics and climatic patterns. The overall relationship between the difference in water potential between soil and leaf and transpiration was linear, with the slope equal to average plant resistance (0.0165 MPa/(cm³ m^-1 h^-1 10^-3). Different regression parameters were obtained for the various measurement days. The water potential difference was inversely related to transpiration at high leaf stomatal resistance and at high values of VPD. Total resistance decreased with transpiration flux in a linear relationship (r=−0.68). Different slope values were obtained for the different measurement days. Estimated soil resistance was much lower than the observed total resistance to water flow. The change from vegetative growth to pod filling was accompanied by an increase in plant resistance. The experimental results support previous findings that resistance to water flow through plants is not constant but is influenced by plant age, growth stage and environmental conditions. A more complex model than Ohm's law analogy may be necessary for describing the dynamic flow system under field conditions. This revised version was published online in June 2006 with corrections to the Cover Date.     

Paenibacillus lentimorbus enhances growth of chickpea (Cicer arietinum L.) in chromium-amended soil

Chromium (Cr), with its great economic importance in industrial use, is a major metal pollutant of the environment. It affects soil microbial activity and soil fertility, resulting in losses in yield of plants. Paenibacillus lentimorbus B-30488^r (B-30488^r) tolerated 200 μg ml⁻¹ of Cr under in vitro conditions and produced the plant growth promoting substance indole acetic acid in the presence of Cr. Our in vitro study indicates enhancement in B-30488^r biofilm formation by sodium alginate (SA) and calcium chloride (CaCl₂) both in absence and presence of supplemented Cr(VI) as compared to unsupplemented control. The plant growth promoting effects caused by the B-30488^r biofilm in rhizosphere of chickpea under Cr(VI) stress suggests a phytoprotective role of B-30488^r biofilm. Our study reflects the multifarious role of strain B-30488^r and presents it as a potent plant growth promoting and bioremediation agent useful in Cr-contaminated rhizosphere soil, whereby the SA and CaCl₂ induced B-30488^r biofilm on plant root acts as a shield in preventing the direct access of toxic Cr to plant tissues, thus reducing its uptake in plants.     

Early generation yield testing versus visual selection in chickpea (Cicer arietinum L.)

Summary Four F₃ populations of chickpea (Cicer arietinum L.) were simultaneously evaluated for yield in an F₃ yield trial and in single plant progeny rows. Ten high yielding, 10 low yielding and 10 randomly sampled lines, along with 10 lines visually selected for yield from the progeny rows, were retained for further evaluation. The lines from each of the four selection groups in each population were bulked and evaluated in a replicated yield trial at three locations and four environments. The bulk of visually selected lines was not superior in yield to the bulk of randomly sampled lines at all locations. The present results indicate that an early generation yield testing selection procedure is more efficient than visual selection for yield improvements in chickpea.