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     

Development of sequence‐tagged microsatellite site markers for chickpea (Cicer arietinum L.)

Microsatellite loci were identified from chickpea (Cicer arietinum L.), the third most important grain legume crop in the world. A total of 13 sequence‐tagged microsatellite markers were developed using two different approaches: (i) amplification using degenerate primers and (ii) cloning of intersimple sequence repeat (ISSR)‐amplified fragments. Thirty‐five chickpea accessions were analysed, which resulted in a total of 30 alleles at the 13 loci. The observed heterozygosity ranged from 0.1143 to 0.4571 with an average of 0.2284. The cross‐species transferability of the sequence‐tagged microsatellite site (STMS) markers was checked in Cicer reticulatum, the wild annual progenitor of chickpea. These microsatellite markers will be useful for assessing the genetic diversity patterns within chickpea as well as aid in construction of intra‐ and interspecific genetic linkage maps.     

Embryo rescue and plant regeneration in vitro of selfed chickpea (Cicer arietinum L.) and its wild annual relatives

The main constraint to the transfer of desired traits into cultivated chickpea from wild Cicer relatives is the presence of post-zygotic barriers which result in abortion of the immature embryo following interspecific hybridisation. Rescue of hybrid embryos in vitro and regeneration of hybrid plantlets could allow chickpea breeders to transfer desirable traits from wild relatives of chickpea. The development of embryo rescue techniques using selfed chickpea and selfed wild relatives is being used as a first step to protocols for wide hybrids. Optical microscopy studies of embryogenesis, in both selfs and hybrids, identified deleterious changes in the fertilised hybrid seed as early as 2–4 days after pollination in some crosses. These observations suggest that the appropriate time to rescue chickpea × C. bijugum hybrids is at the early globular stage of embryogenesis (2–7 days old), which requires the development of a complex tissue culture medium. In contrast hybrids between chickpea × C. pinnatifidum abort later (up to 15–20 days old) at the heart-shaped or torpedo stages, and are easier to rescue in vitro. Genotype also plays a significant role in the ability of immature selfed ovules to germinate in vitro. In this paper we report on the optimisation of␣protocols for rescueing immature embryos using selfed chickpea and its wild relatives in ovule, and subsequently to regenerate plantlets.     

UPLC‐HRMS‐based untargeted metabolic profiling reveals changes in chickpea (Cicer arietinum) metabolome following long‐term drought stress

Genetic improvement for drought tolerance in chickpea requires a solid understanding of biochemical processes involved with different physiological mechanisms. The objective of this study is to demonstrate genetic variations in altered metabolic levels in chickpea varieties (tolerant and sensitive) grown under contrasting water regimes through ultrahigh‐performance liquid chromatography/high‐resolution mass spectrometry‐based untargeted metabolomic profiling. Chickpea plants were exposed to drought stress at the 3‐leaf stage for 25 days, and the leaves were harvested at 14 and 25 days after the imposition of drought stress. Stress produced significant reduction in chlorophyll content, F_v/F_m, relative water content, and shoot and root dry weight. Twenty known metabolites were identified as most important by 2 different methods including significant analysis of metabolites and partial least squares discriminant analysis. The most pronounced increase in accumulation due to drought stress was demonstrated for allantoin, l ‐proline, l ‐arginine, l ‐histidine, l ‐isoleucine, and tryptophan. Metabolites that showed a decreased level of accumulation under drought conditions were choline, phenylalanine, gamma‐aminobutyric acid, alanine, phenylalanine, tyrosine, glucosamine, guanine, and aspartic acid. Aminoacyl‐tRNA and plant secondary metabolite biosynthesis and amino acid metabolism or synthesis pathways were involved in producing genetic variation under drought conditions. Metabolic changes in light of drought conditions highlighted pools of metabolites that affect the metabolic and physiological adjustment in chickpea that reduced drought impacts.     

Influence of silicon on antioxidant mechanisms and lipid peroxidation in chickpea (Cicer arietinum L.) cultivars under drought stress

Abstract

The effects of exogenous silicon (Si) on leaf relative water content (RWC), and the growth, Si concentrations, lipid peroxidation (MDA), lipoxygenase (LOX) activity, proline and H₂O₂ accumulation, non-enzymatic antioxidant activity (AA) and the activity of some antioxidant enzymes (superoxide dismutase, SOD; catalase, CAT; ascorbate peroxidase, APX) in shoots of ten chickpea cultivars grown under drought were investigated. Drought stress decreased the growth of all the cultivars while applied Si improved the growth at least five of the 10 chickpea cultivars. Silicon applied to the soil at 100 mg kg^?1 significantly increased Si concentrations of the cultivars and counteracted the deleterious effects of drought in 5 of the ten chickpea cultivars by increasing their RWC. In most cultivars tested H₂O₂, proline and MDA content and LOX activity were increased by drought whereas application of Si decreased their levels. APX activity was increased by drought but it was depressed by Si. In general, SOD and CAT activities of the cultivars were decreased by drought. Depending on cultivars, the CAT activity was decreased, and increased or unchanged in response to applied Si, while the SOD activity of the cultivars increased or unchanged by Si. The non-enzymatic antioxidant activity of the cultivars was also increased by Si. These observations implied an essential role for Si in minimizing drought stress-induced limitation of the growth and oxidative membrane damage in chickpea plants.     

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.     

Synergistic effect of Pseudomonas putida and Bacillus amyloliquefaciens ameliorates drought stress in chickpea (Cicer arietinum L.)

Two plant growth promoting rhizobacteria (PGPR) Pseudomonas putida NBRIRA and Bacillus amyloliquefaciens NBRISN13 with ability to tolerate abiotic stress along with multiple PGP traits like ACC deaminase activity, minerals solubilisation, hormones production, biofilm formation, siderophore activity were evaluated for their synergistic effect to ameliorate drought stress in chickpea. Earlier we have reported both the strains individually for their PGP attributes and stress amelioration in host plants. The present study explains in detail the possibilities and benefits of utilizing these 2 PGPR in consortium for improving the chickpea growth under control and drought stressed condition. In vitro results clearly demonstrate that both the PGPR strains are compatible to each other and their synergistic growth enhances the PGP attributes. Greenhouse experiments were conducted to evaluate the effect of inoculation of both strains individually and consortia in drought tolerant and sensitive cultivars (BG362 and P1003). The growth parameters were observed significantly higher in consortium as compared to individual PGPR. Colonization of both PGPR in chickpea rhizosphere has been visualized by using gfp labeling. Apart from growth parameters, defense enzymes, soil enzymes and microbial diversity were significantly modulated in individually PGPR and in consortia inoculated plants. Negative effects of drought stress has been ameliorated and apparently seen by higher biomass and reversal of stress indicators in chickpea cultivars treated with PGPR individually or in consortia. Findings from the present study demonstrate that synergistic application has better potential to improve plant growth promotion under drought stress conditions.     

Pseudomonas putida modulates the expression of miRNAs and their target genes in response to drought and salt stresses in chickpea (Cicer arietinum L.)

MicroRNAs are small non-coding regulatory RNA molecules that play an important role in the modulation of gene expression during various environmental stresses. Pseudomonas putida RA, a plant growth promoting rhizobacteria (PGPR) colonizes the root surface of plants improving their growth and development during abiotic stresses modulating the expression of stress-responsive genes; however, the impact of RA on stress responsive-miRNA remains elusive. The present study was an attempt to delineate the role of PGPR in modulating stress responsive-miRNAs in a tolerant desi chickpea genotype exposed to drought and salt stresses. The existence of variable expression patterns of individual miRNAs and their target genes under these stresses at different time points indicate a distinct miRNA-mediated perception and response mechanisms operating under these stresses in the presence or absence of RA in chickpea.     

Gibberellin A3 reverses the effect of salt stress in chickpea (Cicer arietinum L.) seedlings by enhancing amylase activity and mobilization of starch in cotyledons

The percentage germination of chickpea seeds (Cicer arietinum L.cv. PBG-1) gradually decreased with increasing concentration of NaCl in the growth medium and was completely inhibited with 200 mM NaCl. In the presence of 75 mM NaCl, only 51% of the seeds germinated. Gibberellic acid (GA3) and kinetin at 6 µM concentration induced the maximum increase in % germination and seedling growth under salt stress. However, IAA further inhibited both the germination and growth of stressed seedlings. The reduction in amylase activity in cotyledons of stressed seedlings was partially reversed with GA3 and kinetin whereas IAA did not show any positive effect. GA3 was more effective than kinetin in enhancing the reduced germination and seedling growth of chickpea seeds along with amylase activity in cotyledons under NaCl induced saline conditions. The reduced uptake of radiolabelled 14C sucrose by cotyledons and its reduced distribution in the shoots and roots of stressed seedlings was increased with addition of GA3 in the medium. Cotyledonary amylase was separated into amylase 1 and amylase 2 by sephadex G 150 column chromatography. The reduced activities of both amylase 1 and amylase 2 in cotyledons under salt stress was returned to near normal levels with GA3 and there was also an increase in starch utilization, resulting in its lower concentration in cotyledons of GA3-supplemented stressed cotyledons.     

Salinity tolerant Trichoderma harzianum reinforces NaCl tolerance and reduces population dynamics of Fusarium oxysporum f.sp. ciceri in chickpea (Cicer arietinum L.) under salt stress conditions

The antagonistic potential of salinity tolerant (ST) Trichoderma (Th) isolates against Fusarium oxysporum f.sp. ciceri (foc) was tested, along with their capability to induce relative salt stress tolerance in chickpea with the aim to exploit their use as biological agents in reducing deleterious effects of salinity and controlling Fusarium wilt of chickpea under saline soil conditions. Under laboratory conditions, salt stress was created by supplementing nutrient medium with different concentrations of NaCl viz. 0, 70, 150 and 240?mM NaCl and a pot experiment was conducted using natural saline soil (EC – 6.6 dS?m^?1). Out of 45 Th isolates studied, only five isolates viz. Th-13, Th-14, Th-19, Th-33 and Th-50 were selected to be ST as these were able to grow and sporulate in growth medium containing up to 240?mM NaCl. In saline medium, ST isolates greatly surpassed salinity sensitive (SS) isolate with respect to growth rate, mycelial dry weight, sporulation and biological proficiency against foc. Out of five ST isolates that retained their tolerance to different salt stress levels, Th-14 and Th-19 showed maximum antagonism against foc. Under greenhouse conditions, chickpea plants obtained from seeds bioprimed with Th-14 and Th-19 performed well both at germination and seedling stage in comparison to control in saline soil. As compared to untreated plants, characterisation of Th treated plants confirmed that they had reinforced contents of proline along with relatively higher levels of total phenols, membrane stability index and superoxide dismutase activity while lower accumulation of hydrogen peroxide and malondealdehyde contents. ST isolates, Th-14 and Th-19 significantly reduced foc-induced wilt disease incidence in chickpea plants. The population density of both the Th isolates in rhizosphere far exceeded that of foc under both saline and non-saline soils. However, Th-14 was found more efficient in increasing relative salt stress tolerance in chickpea and reducing the foc growth in rhizosphere under present materials and conditions. These findings provide a novel paradigm for developing alternative, environmentally safe strategy to alleviate salt stress and manage fungal diseases such as foc that aggravates under saline soils.     

Active extrusion of protons and exudation of carboxylic acids in response to iron deficiency by roots of chickpea (Cicer arietinum L.)

A chickpea cultivar, K-850, acidified the nutrient solution in response to iron deficiency, with subsequent re-greening of chlorotic leaves. No recovery of chlorosis was observed when the nutrient solution was buffered at a pH 6.3. During the period of acidification induced by iron deficiency, the roots of K-850 exuded more carboxylic acids than when supplied with sufficient iron. However, the rate of extrusion of protons was much higher than the rate of exudation of carboxylic acids during the acidification period. The extrusion of protons was inhibited by the addition of vanadate at the beginning of the decrease in pH. It appeared that acidification of the solution in response to iron deficiency was mediated by a proton-pumping ATPase, located at the plasma membrane. The presence of cations in the solution was essential for the extrusion of protons under iron deficiency, but the species of cation made no significant difference to the rate of extrusion of protons from roots. Therefore, we concluded that non-specific H+/cation antiport was involved in the acidification process.     

Genetic engineering of chickpea (Cicer arietinum L.) with the P5CSF129A gene for osmoregulation with implications on drought tolerance

Abiotic stresses including water deficit severely limits crop yields in the semi-arid tropics. In chickpea, annual losses of over 3.7 million tones have been estimated to be due to water deficit conditions alone. Therefore, major efforts are needed to improve its tolerance to water deficit, and genetic engineering approaches provide an increasing hope for this possibility. We have used transgenic technology for the introduction of an osmoregulatory gene P5CSF129A encoding the mutagenized Δ¹-pyrroline-5-carboxylate synthetase (P5CS) for the overproduction of proline. A total of 49 transgenic events of chickpea were produced with the 35S:P5CSF129A gene through Agrobacterium tumefaciens-mediated gene transfer through the use of axillary meristem explants. Eleven transgenic events that accumulated high proline (2–6 folds) were further evaluated in greenhouse experiments based on their transpiration efficiency (TE), photosynthetic activity, stomatal conductance, and root length under water stress. Almost all the transgenic events showed a decline in transpiration at lower values of the fraction of transpirable soil water (dryer soil), and extracted more water than their untransformed parents. The accumulation of proline in the selected events was more pronounced that increased significantly in the leaves when exposed to water stress. However, the overexpression of P5CSF129A gene resulted only in a modest increase in TE, thereby indicating that the enhanced proline had little bearing on the components of yield architecture that are significant in overcoming the negative effects of drought stress in chickpea.     

Trends in integrated pest management strategies for the control of Helicoverpa armigera (Hübner) caterpillars on chickpea (Cicer arietinum L.)

A study was carried out at the University of Arid Agriculture, Rawalpindi, Pakistan to integrate different control measures against Helicoverpa armigera (Hübner) on chickpea, to minimize the use of hazardous insecticides and develop an eco-friendly management program. Cultural (weeding), mechanical (hand picking), biological (release of Trichogramma chilonis ), microbial (spraying of Bacillus thruingiensis ) and chemical (Steward) control practices were applied three times at 10 day intervals individually and also in various combinations. The application of Steward proved most effective when applied alone, with 0.41 larvae/plant, pod infestation of 9.31% and the highest grain yield (1203.66 g/plot); however, the integration of weeding, hand picking and Steward proved to be the most effective in reducing the larval population (0.12 larvae/plant) with minimum pod infestation (5.45%) on variety CM-2000, which resulted in the maximum grain yield (1260.33 g/plot). The cost–benefit ratio in the treatment where Steward was applied alone was 1:2.20, and it was 1:3.53 where weeding and hand picking practices were integrated.     

Chitinase and β-1,3-glucanase activities in chickpea (Cicer arietinum). Induction of different isoenzymes in response to wounding and ethephon

Chitinase and β-1,3-glucanase activities were assayed in roots, stems and leaves of 12-day-old chickpea ( Cicer arietinum L.) plants. While glucanase activity was higher in roots than in the aerial parts of the plant, leaves had higher Chitinase activity. Both glucanase and chitinase activities were induced in roots and stems in response to wounding (excision into 1-cm pieces), with activity increasing 6 h after treatment, reaching a maximum between 24 and 48 h, and thereafter remaining nearly constant up to 72 h. Ethephon treatment also induced β-1,3-glucanase and chitinase activities in stems but not in roots. Both enzymes occurred in root and stem tissues as a complex mixture of isoenzymes. At least four different peaks with glucanase and chitinase activities could be resolved by gel filtration chromatography on Sephacryl S-200 and chromatofocusing on PBE 94 (pH 4–7). Following ammonium sulfate precipitation and ion exchange on CM- and DEAE-Trisacryl, three β-1,3-glucanase and chitinase fractions, referred to as basic, neutral and acidic, were separated on the basis of their chromatographic behaviour. Most of the total protein (75%) of stem extracts was found in the acidic fraction, whereas the major glucanase (53%) and chitinase (62%) activities were in the basic and neutral fractions, respectively. While wounding resulted in an increase in the neutral glucanase and chitinase activities, the activities of the acidic fractions were promoted by ethephon.