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.     

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.     

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     

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.     

Somatic embryogenesis and plant regeneration from callus cultures of chickpea (Cicer arietinum L.)

Somatic embryogenesis and plant regeneration were obtained from immature leaflet callus of chickpea. Numerous globular embryos developed on the surface of callus on Murashige and Skoog's (1962) medium containing 25 μM 2,4-dichlorophenoxyacetic acid. These globular embryos differentiated into mature somatic embryos upon removal of 2,4-dichlorophenoxyacetic acid. The maturation of embryos was significantly affected by pH, photoperiod, abscisic acid and genotype. Callus continued to produce somatic embryos for over 8 subcultures at 4 week intervals. Two per cent of the embryos formed plants on medium containing 15 μM gibberellic acid and 1 μM indole-3-butyric acid. Desiccation of embryos for a period of 3 d increased their rate of conversion into plants from 0.9 to 2.8%. All regenerated plants showed normal morphological characteristics.     

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     

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.     

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.     

Isolation,characterization, and effect of phosphate-zinc-solubilizing bacterial strains on chickpea (Cicer arietinum L.) growth

ObjectivePhosphate (P) and zinc (Zn) are essential plant nutrients required for nodulation, nitrogen-fixation, plant growth and yield. Mostly applied P and Zn nutrients in the soil are converted into unavailable form. A small number of soil microbes have the ability to transform unsolvable forms of P and Zn to an available form. P-Zn-solubilizing rhizobacteria are potential alternates for P and Zn supplement. In the present study, the effect of two P-Zn-solubilizing bacterial strains (Bacillus sp. strain AZ17 and Pseudomonas sp. strain AZ5) was evaluated on the growth of chickpea plant.MethodologyBoth strains were purified from the rhizospheric soil of chickpea plant grown-up in sandy soil and rain-fed area (Thal desert). In vitro, both strains solubilize P and Zn as well both strain produce IAA and organic acids. In the field experiments, conducted in the rain-fed area, the positive influence of inoculation with both bacterial isolates AZ5 and AZ17 on chickpea growth was observed.ResultsThe application of inoculum (strains AZ5 and AZ17) resulted in up to 17.47% and 17.34% increase in grain yield of both types of chickpea grown in fertilized and non-fertilized soil, respectively over non-inoculated control. Strain AZ5 was the most effective inoculum, increasing up to 17.47%, 16.04%, 26.32%, 22.53%, 26.12% and 22.59% in grain yield, straw weight, nodules number, dry weight of nodules, Zn uptake and P uptake respectively, over control.ConclusionThese results indicated that Pseudomonas sp. strain AZ5 and Bacillus sp. strain AZ17 can serve as effective microbial inocula for chickpea, particularly in the rain-fed area.     

Isolation and characterization of sequence‐tagged microsatellite sites markers in chickpea (Cicer arietinum L.)

In this study we report the isolation of microsatellite sequences and their conversion to sequence‐tagged microsatellite sites (STMS) markers in chickpea (Cicer arietinum L.). Thirteen putative recombinants isolated from a chickpea genomic library were sequenced, and used to design 10 STMS primer pairs. These were utilized to analyse the genetic polymorphism in 15 C. arietinum varieties and two wild varieties, C. echinospermum and C. reticulatum. All the primer pairs amplified polymorphic loci ranging from four to seven alleles per locus. The observed heterozygosity ranged from 0 to 0.6667. Most of the STMS markers also amplified corresponding loci in the wild relatives suggesting conservation of these markers in the genus. Hence, these polymorphic markers will be useful for the evaluation of genetic diversity and molecular mapping in 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.     

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.     

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.     

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.     

Isolation and characterization of cDNA clones encoding ADP-glucose pyrophosphorylase (AGPase) large and small subunits from chickpea (Cicer arietinum L.)

Four cDNA clones encoding two large subunits and two small subunits of the starch regulatory enzyme ADP-glucose pyrophosphorylase (AGPase) were isolated from a chickpea (Cicer arietinum L.) stem cDNA library. DNA sequence and Southern blot analyses of these clones, designated CagpL1, CagpL2 (large subunits) and CagpS1 and CagpS2 (small subunits), revealed that these isoforms represented different AGPase large and small subunits. RNA expression analysis indicated that CagpL1 was expressed strongly in leaves with reduced expression in the stem. No detectable expression was observed in seeds and roots. CagpL2 was expressed moderately in seeds followed by weak expression in leaves, stems and roots. Similar analysis showed that CagpS1 and CagpS2 displayed a spatial expression pattern similar to that observed for CagpL2 with the exception that CagpS1 showed a much higher expression in seeds than CagpS2. The spatial expression patterns of these different AGPase subunit sequences indicate that different AGPase isoforms are used to control starch biosynthesis in different organs during chickpea development.