Identifying dryland-resilient chickpea genotypes for autumn sowing, with a focus on multi-trait stability parameters and biochemical enzyme activity.

BACKGROUND: Chickpea is a key pulse crop grown in the spring in dryland regions. The cold resistance potential of chickpeas allows for the development of genotypes with varying sowing dates to take advantage of autumn and winter rainfall, particularly in dryland regions. In this study, we assessed grain yield, plant height, 100-seed weight, days to maturity, and days to flowering of 17 chickpea genotypes in five autumn-sown dryland regions from 2019 to 2021. Additionally, the response of selected chickpea genotypes to cold stress was examined at temperatures of -4 °C, 4 °C, and 22 °C by analyzing biochemical enzymes. RESULTS: Mixed linear model of ANOVA revealed a significant genotype × environment interaction for all traits measured, indicating varying reactions of genotypes across test environments. This study reported low estimates of broad-sense heritability for days to flowering (0.34), days to maturity (0.13), and grain yield (0.08). Plant height and seed weight exhibited the highest heritability, with genotypic selection accuracies of 0.73 and 0.92, respectively. Moreover, partial least square regression highlighted the impactful role of rainfall during all months except of October, November, and February on grain yield and its interaction with environments in autumn-planted chickpeas. Among the genotypes studied, G9, G10, and G17 emerged as superior based on stability parameters and grain yield. In particular, genotype G9 stood out as a promising genotype for dryland regions, considering both MTSI and genotype by yield*trait aproaches. The cold assay indicated that - 4 °C is crucial for distinguishing between susceptible and resistant genotypes. The results showed the important role of the enzymes CAT and GPX in contributing to the cold tolerance of genotype G9 in autumn-sown chickpeas. CONCLUSIONS: Significant G×E for agro-morphological traits of chickpea shows prerequisite for multi-trial analysis. Chickpea`s direct root system cause that monthly rainfall during plant establishment has no critical role in its yield interaction with dryland environment. Considering the importance of agro-morphological traits and their direct and indirect effects on grain yield, the utilization of multiple-trait stability approches is propose. Evaluation of chickpea germplasm reaction against cold stress is necessary for autumn-sowing. Finally, autumn sowing of genotype FLIP 10-128 C in dryland conditions can led to significant crop performance.

Using RNA sequencing to unravel molecular changes underlying the defense response in chickpea induced by Phytophthora medicaginis.

Phytophthora root rot (PRR), caused by Phytophthora medicaginis, is a major soil-borne disease of chickpea in Australia. Breeding for PRR resistance is an effective approach to avoid significant yield loss. Genetic resistance has been identified in cultivated chickpea (Cicer arietinum) and in the wild relative C. echinospermum, with previous studies identifying independent genetic loci associated with each of these sources. However, the molecular mechanisms associated with PRR resistance are not known. RNA sequencing analysis employed in this study identified changes in gene expression in roots of three chickpea genotypes grown hydroponically, early post-infection with P. medicaginis zoospores. Analyses of differentially expressed genes (DEG) identified the activation of a higher number of non-specific R-genes in a PRR-susceptible variety than in the resistant genotypes, suggesting a whole plant resistance response occurring in chickpea against the pathogen. Contrasting molecular changes in signaling profiles, proteolysis and transcription factor pathways were observed in the cultivated and wild Cicer-derived resistant genotypes. DEG patterns supported a hypothesis that increased root elongation and reduced adventitious root formation limit the pathogen entry points in the genotype containing the wild Cicer source of PRR resistance. Candidate resistance genes, including an aquaporin and a maltose transporter in the wild Cicer source and GDSL esterases/lipases in the cultivated source of resistance, were oppositely regulated. Increased knowledge of these genes and pathways will improve our understanding of molecular mechanisms controlling PRR resistance in chickpea, and support the development of elite chickpea varieties through molecular breeding approaches.

Phenotypic and genetic characterization of a near-isogenic line pair: insights into flowering time in chickpea.

BACKGROUND: Cicer arietinum is a significant legume crop cultivated mainly in short-season environments, where early-flowering is a desirable trait to overcome terminal constraints. Despite its agricultural significance, the genetic control of flowering time in chickpea is not fully understood. In this study, we developed, phenotyped, re-sequenced and genetically characterized a pair of near-isogenic lines (NILs) with contrasting days to flowering to identify candidate gene variants potentially associated with flowering time. RESULTS: In addition to days to flowering, noticeable differences in multiple shoot architecture traits were observed between the NILs. The resequencing data confirms that the NILs developed in this study serve as appropriate plant materials, effectively constraining genetic variation to specific regions and thereby establishing a valuable resource for future genetic and functional investigations in chickpea research. Leveraging bioinformatics tools and public genomic datasets, we identified homologs of flowering-related genes from Arabidopsis thaliana, including ELF3 and, for the first time in chickpea, MED16 and STO/BBX24, with variants among the NILs. Analysis of the allelic distribution of these genes revealed their preservation within chickpea diversity and their potential association with flowering time. Variants were also identified in members of the ERF and ARF gene families. Furthermore, in silico expression analysis was conducted elucidating their putative roles in flowering. CONCLUSIONS: While the gene CaELF3a is identified as a prominent candidate, this study also exposes new targets in chickpea, such as CaMED16b and LOC101499101 (BBX24-like), homologs of flowering-related genes in Arabidopsis, as well as ERF12 and ARF2. The in silico expression characterization and genetic variability analysis performed could contribute to their use as specific markers for chickpea breeding programs. This study lays the groundwork for future investigations utilizing this plant material, promising further insights into the complex mechanisms governing flowering time in chickpea.

Synergistic effects of melatonin and 24-epibrassinolide on chickpea water deficit tolerance.

BACKGROUND: Water deficiency stress reduces yield in grain legumes, primarily due to a decrease in the pods number. Melatonin (ML) and 24-epibrassinolide (EBL) are recognized for their hormone-like properties that improve plant tolerance to abiotic stresses. This study aimed to assess the impact of different concentrations of ML (0, 100, and 200 µM) and EBL (0, 3, and 6 µM) on the growth, biochemical, and physiological characteristics of chickpea plants under water-stressed conditions. RESULTS: The study's findings indicated that under water-stressed conditions, a decrease in seed (30%) and pod numbers (31%), 100-seed weight (17%), total chlorophyll content (46%), stomatal conductance (33%), as well as an increase in H2O2 (62%), malondialdehyde content (40%), and electrolyte leakage index (40%), resulted in a 40% reduction in chickpea plants grain yield. Our findings confirmed that under water-stressed conditions, seed oil, seed oil yield, and seed protein yield dropped by 20%, 55%, and 36%, respectively. The concurrent exogenous application of ML and EBL significantly reduces oxidative stress, plasma membrane damage, and reactive oxygen species (ROS) content. This treatment also leads to increased yield and its components, higher pigment content, enhanced oil and protein yield, and improved enzymatic and non-enzymatic antioxidant activities such as catalase, superoxide dismutase, polyphenol oxidase, ascorbate peroxidase, guaiacol peroxidase, flavonoid, and carotenoid. Furthermore, it promotes the accumulation of osmoprotectants such as proline, total soluble protein, and sugars. CONCLUSIONS: Our study found that ML and EBL act synergistically to regulate plant growth, photosynthesis, osmoprotectants accumulation, antioxidant defense systems, and maintain ROS homeostasis, thereby mitigating the adverse effects of water deficit conditions. ML and EBL are key regulatory network components in stressful conditions, with significant potential for future research and practical applications. The regulation metabolic pathways of ML and EBL in water-stressed remains unknown. As a result, future research should aim to elucidate the molecular mechanisms by employing genome editing, RNA sequencing, microarray, transcriptomic, proteomic, and metabolomic analyses to identify the mechanisms involved in plant responses to exogenous ML and EBL under water deficit conditions. Furthermore, the economical applications of synthetic ML and EBL could be an interesting strategy for improving plant tolerance.

Implementation of theoretical non-photochemical quenching (NPQ(T)) to investigate NPQ of chickpea under drought stress with High-throughput Phenotyping.

Non-photochemical quenching (NPQ) is a protective mechanism for dissipating excess energy generated during photosynthesis in the form of heat. The accelerated relaxation of the NPQ in fluctuating light can lead to an increase in the yield and dry matter productivity of crops. Since the measurement of NPQ is time-consuming and requires specific light conditions, theoretical NPQ (NPQ(T)) was introduced for rapid estimation, which could be suitable for High-throughput Phenotyping. We investigated the potential of NPQ(T) to be used for testing plant genetic resources of chickpea under drought stress with non-invasive High-throughput Phenotyping complemented with yield traits. Besides a high correlation between the hundred-seed-weight and the Estimated Biovolume, significant differences were observed between the two types of chickpea desi and kabuli for Estimated Biovolume and NPQ(T). Desi was able to maintain the Estimated Biovolume significantly better under drought stress. One reason could be the effective dissipation of excess excitation energy in photosystem II, which can be efficiently measured as NPQ(T). Screening of plant genetic resources for photosynthetic performance could take pre-breeding to a higher level and can be implemented in a variety of studies, such as here with drought stress or under fluctuating light in a High-throughput Phenotyping manner using NPQ(T).

Chasmophyte associated stress tolerant bacteria confer drought resilience to chickpea through efficient nutrient mining and modulation of stress response.

In the present study, ten (10) selected bacteria isolated from chasmophytic wild Chenopodium were evaluated for alleviation of drought stress in chickpea. All the bacterial cultures were potential P, K and Zn solubilizer. About 50% of the bacteria could produce Indole-3-acetic acid (IAA) and 1-aminocyclopropane-1-carboxylate (ACC) deaminase. The bacteria showed wide range of tolerance towards pH, salinity, temperature and osmotic stress. Bacillus paralicheniformis L38, Pseudomonas sp. LN75, Enterobacter hormachei subsp. xiangfengensis LJ89, B. paramycoides L17 and Micrococcus luteus LA9 significantly improved growth and nutrient (N, P, K, Fe and Zn) content in chickpea under water stress during a green house experiment conducted following a completely randomized design (CRD). Application of Microbacterium imperiale LJ10, B. stercoris LN74, Pseudomonas sp. LN75, B. paralicheniformis L38 and E. hormachei subsp. xiangfengensis LJ89 reduced the antioxidant enzymes under water stress. During field experiments conducted following randomized block design (RBD), all the bacterial inoculations improved chickpea yield under water stress. Highest yield (1363 kg ha-1) was obtained in plants inoculated with Pseudomonas sp. LN75. Pseudomonas sp. LN75, B. paralicheniformis L38 and E. hormachei subsp. xiangfengensis LJ89 have potential as microbial stimulants to alleviate the water stress in chickpea. To the best of our knowledge this is the first report of using chasmophyte associated bacteria for alleviation of water stress in a crop plant.

Comprehensive Transcriptome Profiling Uncovers Molecular Mechanisms and Potential Candidate Genes Associated with Heat Stress Response in Chickpea.

Chickpea (Cicer arietinum L.) production is highly susceptible to heat stress (day/night temperatures above 32/20 °C). Identifying the molecular mechanisms and potential candidate genes underlying heat stress response is important for increasing chickpea productivity. Here, we used an RNA-seq approach to investigate the transcriptome dynamics of 48 samples which include the leaf and root tissues of six contrasting heat stress responsive chickpea genotypes at the vegetative and reproductive stages of plant development. A total of 14,544 unique, differentially expressed genes (DEGs) were identified across different combinations studied. These DEGs were mainly involved in metabolic processes, cell wall remodeling, calcium signaling, and photosynthesis. Pathway analysis revealed the enrichment of metabolic pathways, biosynthesis of secondary metabolites, and plant hormone signal transduction, under heat stress conditions. Furthermore, heat-responsive genes encoding bHLH, ERF, WRKY, and MYB transcription factors were differentially regulated in response to heat stress, and candidate genes underlying the quantitative trait loci (QTLs) for heat tolerance component traits, which showed differential gene expression across tolerant and sensitive genotypes, were identified. Our study provides an important resource for dissecting the role of candidate genes associated with heat stress response and also paves the way for developing climate-resilient chickpea varieties for the future.

A Comprehensive Review on Chickpea (Cicer arietinum L.) Breeding for Abiotic Stress Tolerance and Climate Change Resilience.

Chickpea is one of the most important pulse crops worldwide, being an excellent source of protein. It is grown under rain-fed conditions averaging yields of 1 t/ha, far from its potential of 6 t/ha under optimum conditions. The combined effects of heat, cold, drought, and salinity affect species productivity. In this regard, several physiological, biochemical, and molecular mechanisms are reviewed to confer tolerance to abiotic stress. A large collection of nearly 100,000 chickpea accessions is the basis of breeding programs, and important advances have been achieved through conventional breeding, such as germplasm introduction, gene/allele introgression, and mutagenesis. In parallel, advances in molecular biology and high-throughput sequencing have allowed the development of specific molecular markers for the genus Cicer, facilitating marker-assisted selection for yield components and abiotic tolerance. Further, transcriptomics, proteomics, and metabolomics have permitted the identification of specific genes, proteins, and metabolites associated with tolerance to abiotic stress of chickpea. Furthermore, some promising results have been obtained in studies with transgenic plants and with the use of gene editing to obtain drought-tolerant chickpea. Finally, we propose some future lines of research that may be useful to obtain chickpea genotypes tolerant to abiotic stress in a scenario of climate change.

Exogenously applied nutrients can improve the chickpea productivity under water stress conditions by modulating the antioxidant enzyme system / Nutrientes aplicados exogenamente melhoram a produtividade do grão-de bico em condições de estresse hídrico, modulando o sistema de enzimas antioxidantes

Water stress is one of the major factor restricting the growth and development of chickpea plants by inducing various morphological and physiological changes. Therefore, the present research activity was designed to improve the chickpea productivity under water stress conditions by modulating antioxidant enzyme system. Experimental treatments comprised of two chickpea genotypes i.e. Bhakhar 2011 (drought tolerant) and DUSHT (drought sensitive), two water stress levels i.e. water stress at flowering stage and water stress at flowering + pod formation + grain filling stage including well watered (control) and three exogenous application of nutrients i.e. KCl 200 ppm, MgCl2, 50 ppm and CaCl2, 10 mM including distilled water (control). Results indicated that water stress at various growth stages adversely affects the growth, yield and quality attributes of both chickpea cultivars. Exogenous application of nutrients improved the growth, yield and antioxidant enzyme activities of both chickpea genotypes even under water stress conditions. However, superior results were obtained with foliar spray of potassium chloride on Bhakhar 2011 under well-watered conditions. Similarly, foliar spray of potassium chloride on chickpea cultivar Bhakhar 2011 cultivated under stress at flowering + pod formation + grain filling stage produced significantly higher contents of superoxide dismutase, peroxidase and catalase. These results suggests that the application of potassium chloride mitigates the adverse effects of water stress and enhanced tolerance in chickpea mainly due to higher antioxidant enzymes activity, demonstrating the protective measures of plant cells in stress conditions.

O estresse hídrico é um dos principais fatores que restringem o crescimento e o desenvolvimento das plantas de grão-de-bico, induzindo várias alterações morfológicas e fisiológicas. Portanto, a presente atividade de pesquisa foi projetada para melhorar a produtividade do grão-de-bico em condições de estresse hídrico, por meio da modulação do sistema de enzimas antioxidantes. Tratamentos experimentais compostos de dois genótipos de grão-de-bico, ou seja, Bhakhar 2011 (tolerante à seca) e DUSHT (sensível à seca), dois níveis de estresse hídrico, ou seja, estresse hídrico na fase de floração e estresse hídrico na floração + formação de vagens + estágio de enchimento de grãos incluindo bem irrigado (controle) e três aplicações exógenas de nutrientes, ou seja, KCl 200 ppm, MgCl2 50 ppm e CaCl2 10 mM, incluindo água destilada (controle). Os resultados indicaram que o estresse hídrico em vários estágios de crescimento afeta negativamente os atributos de crescimento, rendimento e qualidade de ambas as cultivares de grão-de-bico. A aplicação exógena de nutrientes melhorou o crescimento, o rendimento e as atividades das enzimas antioxidantes de ambos os genótipos de grão-de-bico, mesmo em condições de estresse hídrico. No entanto, resultados superiores foram obtidos com pulverização foliar de cloreto de potássio em Bhakhar 2011, em condições bem irrigadas. Da mesma forma, a pulverização foliar de cloreto de potássio na cultivar de grão-de-bico Bhakhar 2011 cultivada sob estresse na fase de floração + formação de vagens + enchimento de grãos produziu teores significativamente maiores de superóxido dismutase, peroxidase e catalase. Esses resultados sugerem que a aplicação de cloreto de potássio atenua os efeitos adversos do estresse hídrico e aumenta a tolerância no grão-de-bico, principalmente em razão de mais atividade de enzimas antioxidantes, demonstrando as medidas protetoras das células vegetais em condições de estresse.

Characterization and differential expression of sucrose and starch metabolism genes in contrasting chickpea (Cicer arietinum L.) genotypes under low temperature.

Low temperature (LT) causes significant yield losses in chickpea (Cicer arietinum L.). The sucrose starch metabolism is associated with abiotic-stress tolerance or sensitivity in plants. The changes in sugars and starch contents under LT in chickpea have already been studied, however, no information is available on LT-induced alterations in transcription of carbohydrate metabolic pathway genes in chickpea. To understand the differences in the regulation of sucrose and starch metabolism under LT, the expression of sucrose and starch metabolism genes was studied in leaves of cold-sensitive (GPF2) and cold-tolerant (ICC 16349) chickpea genotypes. The mRNA sequences of chickpea genes were retrieved from the public databases followed by confirmation of identity and characterization. All the genes were functional in chickpea. Between the two paralogues of cell wall invertase, cell wall invertase 3×2 (CWINx2) was the truncated version of cell wall invertase 3×1 (CWINx1) with the loss of 241 bases in the mRNA and 67 amino acids at N terminal of the protein. Comparison of expression of the genes between control (22°C day / 16°C night) and LT treated (4°C; 72 h) plants revealed that granule bound starch synthase 2 (GBSS2) and ß-amylase 3 (BAM3) were upregulated in ICC 16349 whereas sucrose phosphate synthase 2 (SPS2), CWINx1, CWINx2 and ß-amylase 1 (BAM1) were downregulated. In contrast to this, SPS2, CWINx1, CWINx2 and BAM1 were upregulated and GBSS2 downregulated in GPF2 under LT. The gene expression data suggested that UGPase, CWINs, GBSS2 and BAM3 are important components of cold-tolerance machinery of chickpea.

Validation of the markers linked with drought tolerance related traits for use in MAS programme in chickpea.

Chickpea is an important cool season legume crop. The breeding efforts in chickpea are often hampered due to the narrow genetic base. Availability of diverse germplasm is an essential requirement for any crop improvement programme. This can facilitate development of desirable gene combinations and subsequently the improved cultivars. In any marker-assisted selection (MAS) programme, study of parental polymorphism using QTL linked markers is a pre-requisite for screening of desired genotypes. Any such study involving use of markers chosen randomly can only tell the diversity of the parents, but does not guarantee success of the MAS. The present study was undertaken to study the suitability of the SSR markers from the QTL-hotspot region linked with drought tolerance related traits in different genetic background. The study of polymorphism of the QTL-hotspot linked SSR markers NCPGR127, NCPGR21, TAA170, ICCM0249, STMS11, TR11 and GA24 between drought tolerant genotype ICC-4958 and remaining 32 chickpea genotypes revealed that most of the genotypes had monomorphic alleles as that of ICC-4958, while only a few genotypes showed polymorphic alleles. The markers that are found polymorphic between ICC-4958 and other chickpea genotypes can be used directly for foreground selection in MAS as they are mapped in the QTL-hotspot region. However, in cases where these are monomorphic, additional markers from QTL-hotspot region need to be screened. Besides validating the suitability of these markers, we also validated SSR markers that can be used for the background selection. Of the 21 SSR markers, 15 were found polymorphic between ICC-4958 and other genotypes suggesting their usefulness in the background selection.

Discerning molecular diversity and association mapping for phenological, physiological and yield traits under high temperature stress in chickpea (Cicer arietinum L.).

High temperature (HT) stress is assuming serious production constraint for chickpea production worldwide. A collection of 182 diverse chickpea genotypes was assessed for genetic variation in 15 traits including phenological, physiological and yield-related traits under both normal sown (NS) and late sown (LS) conditions for two years 2017-2018 and 2018-2019, which revealed significant variation for all the traits. Association mapping of chickpea genotypes was also conducted with 120 simple sequence repeat markers distributed across all the chickpea chromosomes to discern the molecular diversity and to capture the significant marker-trait association (MTA). MTA analysis based on mixed linear model (MLM) revealed a total of 24 and 14 significant associations for various traits evaluated under NS conditions in 2017 and 2018, respectively. Similarly, a total of 17 and 34 significant associations for various traits were also recorded under LS conditions in 2018 and 2019, respectively. Notably, ICCM0297, NCPGR150, TAA160 and NCPGR156 markers showed significant MTA under both NS and LS conditions and GA11 exhibited significant MTA for filled pod% under late sown condition for both years. Thus, these markers could be useful for genomics-assisted breeding for developing heat-tolerant chickpea genotype.

Effect of cold stress on polyamine metabolism and antioxidant responses in chickpea.

Metabolic and genomic characteristics of polyamines (PAs) may be associated with the induction of cold tolerance (CT) responses in plants. Characteristics of PAs encoding genes in chickpea (Cicer arietinum L.) and their function under cold stress (CS) are currently unknown. In this study, the potential role of PAs along with the antioxidative defense systems were assessed in two chickpea genotypes (Sel96th11439, cold-tolerant and ILC533, cold-sensitive) under CS conditions. Six days after exposure to CS, the leaf H2O2 content and electrolyte leakage index increased in the sensitive genotype by 47.7 and 59 %, respectively, while these values decreased or remained unchanged, respectively, in the tolerant genotype. In tolerant genotype, the enhanced activity of superoxide dismutase (SOD) (by 50 %) was accompanied by unchanged activities of ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and catalase (CAT) as well as the accumulation of glutathione (GSH) (by 43 %) on the sixth day of CS. Higher levels of putrescine (Put) (322 %), spermidine (Spd) (45 %), spermine (Spm) (69 %) and the highest ratio of Put/(Spd + Spm) were observed in tolerant genotype compared to the sensitive one on the sixth day of CS. Gamma-aminobutyric acid (GABA) accumulation was 74 % higher in tolerant genotype compared to the sensitive one on the sixth day of CS. During CS, the activity of diamine oxidase (DAO) and polyamine oxidase (PAO) increased in tolerant (by 3.02- and 2.46-fold) and sensitive (by 2.51- and 2.8-fold) genotypes, respectively, in comparison with the respective non-stressed plants (normal conditions). The highest activity of DAO and PAO in the tolerant genotype was accompanied by PAs decomposition and a peak in GABA content on the sixth day of CS. The analysis of chickpea genome revealed the presence of five PAs biosynthetic genes, their chromosomal locations, and cis-regulatory elements. A significant increase in transcript levels of arginine decarboxylase (ADC) (24.26- and 7.96-fold), spermidine synthase 1 (SPDS1) (3.03- and 1.53-fold), SPDS2 (5.5- and 1.62-fold) and spermine synthase (SPMS) (3.92- and 1.65-fold) genes was detected in tolerant and sensitive genotypes, respectively, whereas the expression of ornithine decarboxylase (ODC) genes decreased significantly under CS conditions in both genotypes. Leaf chlorophyll and carotenoid contents exhibited declining trends in the sensitive genotype, while these photosynthetic pigments were stable in the tolerant genotype due to the superior performance of defensive processes under CS conditions. Overall, these results suggested the specific roles of putative PAs genes and PAs metabolism in development of effective CT responses in chickpea.

First Report of CRISPR/Cas9 Mediated DNA-Free Editing of 4CL and RVE7 Genes in Chickpea Protoplasts.

The current genome editing system Clustered Regularly Interspaced Short Palindromic Repeats Cas9 (CRISPR/Cas9) has already confirmed its proficiency, adaptability, and simplicity in several plant-based applications. Together with the availability of a vast amount of genome data and transcriptome data, CRISPR/Cas9 presents a massive opportunity for plant breeders and researchers. The successful delivery of ribonucleoproteins (RNPs), which are composed of Cas9 enzyme and a synthetically designed single guide RNA (sgRNA) and are used in combination with various transformation methods or lately available novel nanoparticle-based delivery approaches, allows targeted mutagenesis in plants species. Even though this editing technique is limitless, it has still not been employed in many plant species to date. Chickpea is the second most crucial winter grain crop cultivated worldwide; there are currently no reports on CRISPR/Cas9 gene editing in chickpea. Here, we selected the 4-coumarate ligase (4CL) and Reveille 7 (RVE7) genes, both associated with drought tolerance for CRISPR/Cas9 editing in chickpea protoplast. The 4CL represents a key enzyme involved in phenylpropanoid metabolism in the lignin biosynthesis pathway. It regulates the accumulation of lignin under stress conditions in several plants. The RVE7 is a MYB transcription factor which is part of regulating circadian rhythm in plants. The knockout of these selected genes in the chickpea protoplast using DNA-free CRISPR/Cas9 editing represents a novel approach for achieving targeted mutagenesis in chickpea. Results showed high-efficiency editing was achieved for RVE7 gene in vivo compared to the 4CL gene. This study will help unravel the role of these genes under drought stress and understand the complex drought stress mechanism pathways. This is the first study in chickpea protoplast utilizing CRISPR/Cas9 DNA free gene editing of drought tolerance associated genes.

Transgenic chickpea (Cicer arietinum L.) harbouring AtDREB1a are physiologically better adapted to water deficit.

BACKGROUND: Chickpea (Cicer arietinum L.) is the second most widely grown pulse and drought (limiting water) is one of the major constraints leading to about 40-50% yield losses annually. Dehydration responsive element binding proteins (DREBs) are important plant transcription factors that regulate the expression of many stress-inducible genes and play a critical role in improving the abiotic stress tolerance. Transgenic chickpea lines harbouring transcription factor, Dehydration Responsive Element-Binding protein 1A from Arabidopsis thaliana (AtDREB1a gene) driven by stress inducible promoter rd29a were developed, with the intent of enhancing drought tolerance in chickpea. Performance of the progenies of one transgenic event and control were assessed based on key physiological traits imparting drought tolerance such as plant water relation characteristics, chlorophyll retention, photosynthesis, membrane stability and water use efficiency under water stressed conditions. RESULTS: Four transgenic chickpea lines harbouring stress inducible AtDREB1a were generated with transformation efficiency of 0.1%. The integration, transmission and regulated expression were confirmed by Polymerase Chain Reaction (PCR), Southern Blot hybridization and Reverse Transcriptase polymerase chain reaction (RT-PCR), respectively. Transgenic chickpea lines exhibited higher relative water content, longer chlorophyll retention capacity and higher osmotic adjustment under severe drought stress (stress level 4), as compared to control. The enhanced drought tolerance in transgenic chickpea lines were also manifested by undeterred photosynthesis involving enhanced quantum yield of PSII, electron transport rate at saturated irradiance levels and maintaining higher relative water content in leaves under relatively severe soil water deficit. Further, lower values of carbon isotope discrimination in some transgenic chickpea lines indicated higher water use efficiency. Transgenic chickpea lines exhibiting better OA resulted in higher seed yield, with progressive increase in water stress, as compared to control. CONCLUSIONS: Based on precise phenotyping, involving non-invasive chlorophyll fluorescence imaging, carbon isotope discrimination, osmotic adjustment, higher chlorophyll retention and membrane stability index, it can be concluded that AtDREB1a transgenic chickpea lines were better adapted to water deficit by modifying important physiological traits. The selected transgenic chickpea event would be a valuable resource that can be used in pre-breeding or directly in varietal development programs for enhanced drought tolerance under parched conditions.

Relative roles of Arbuscular Mycorrhizae in establishing a correlation between soil properties, carbohydrate utilization and yield in Cicer arietinum L. under As stress.

Accumulation of As (metalloid) degrades soil by negatively affecting the activities of soil enzymes, which in turn reduce growth and yield of the inhabiting plant. Arbuscular mycorrhizal (AM) symbiosis can impart metalloid tolerance in plants by secreting glomalin-related soil protein (GRSP) which binds with As or inertly adsorb in the extraradical mycelial surface. However, profitable use of AM requires selection of the most efficient combination of host plant and fungal species. The current study, therefore designed to study the efficacy of 3 a.m. fungal species: Rhizoglomus intraradices (Ri), Funneliformis mosseae (Fm) and Claroideoglomus claroideum (Cc) in imparting arsenate As(V) and arsenite As(III) stress tolerance in Cicer arietinum (chickpea) genotypes (G) - relatively metalloid tolerant- HC 3 and sensitive- C 235. Roots were found to be more severly affected as compared to shoots which resulted into a major decline in uptake of nutrients, chlorophyll concentrations and yield with As(III) inducing more toxic effects than As(V). HC 3 established more effective mycorrhizal symbiosis and was able to extract higher nutrients from the soil than C 235. Ri was most beneficial in improving plant biomass, carbohydrate utilization and productivity followed by Fm and Cc which could be due to its capability to initiate highest percent colonization and least metalloid uptake in roots through higher glomalin production in the soil. Moreover, Ri was highly efficient in improving soil enzymes activities-phosphatases (PHAs), ß-glucosidase (BGA) and invertase (INV), thereby, imparting metalloid tolerance in chickpea genotypes. The results suggested use of Ri-chickpea symbiosis as a promising strategy for ameliorating As stress in chickpea.

Physiological disruption, structural deformation and low grain yield induced by neonicotinoid insecticides in chickpea: A long term phytotoxicity investigation.

Arbitrary use of insecticides in agricultural practices cause severe environmental hazard that adversely affects the growth and productivity of edible crops. Considering theses, the aim of the present study was to evaluate the toxicological potential of two neonicotinoid insecticides, imidacloprid (IMID) and thiamethoxam (THIA) using chickpea as a test crop. Application of insecticides at three varying doses revealed a gradual decrease in biological performance of chickpea plants which however, varied noticeably among insecticides and their doses. Significant (P ≤ 0.05) decline in germination efficiency, length of plant organs under in vitro condition was observed in a dose related manner. Among insecticides, 300 µgIMIDkg-1 (3X dose) maximally and significantly (P ≤ 0.05) inhibited germination efficiency, vigor index, length, dry matter accumulation, photosynthetic pigment formation, nodule volume and mass, nutrient uptake, grain yield and protein over untreated control. In contrast, 75 µgTHIAkg-1 (3X dose) considerably declined the leghaemoglobin content, shoot phosphorus and root nitrogen. Enhanced expression of stress biomarkers including proline, malondialdehyde (MDA), and antioxidant defence enzymes was noticed in the presence of insecticides. For instance, at 3X IMID, shoot proline, MDA, ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and peroxidase (POD) were increased significantly (P ≤ 0.05) by 66%, 81%, 36% and 35%, respectively. Additionally, electrolyte leakage was maximally (77%) increased at 3X dose of IMID, whereas, H2O2 in foliage was maximally accumulated (0.0156 µ moles min-1 g-1 fw) at 3X dose of THIA which was 58% greater than untreated foliage. A clear distortion/damage in tip and surface of roots and ultrastructural deformation in xylem and phloem of plant tissues as indication of insecticidal phytotoxicity was observed under scanning electron microscope (SEM). For oxidative stress and cytotoxicity assessment, root tips were stained with a combination of acridine orange and propidium iodide, and Evan blue dyes and examined. Confocal laser scanning microscopic (CLSM) images of roots revealed a 10-fold and 13.5-fold increase in red and blue fluorescence when 3X IMID treated roots were assessed quantitatively. Conclusively, the present investigation recommends that a careful and protected approach should be adopted before the application of insecticides in agricultural ecosystems.

Effect of chromium (VI) toxicity on morpho-physiological characteristics, yield, and yield components of two chickpea (Cicer arietinum L.) varieties.

The ever-increasing industrial activities over the decades have generated high toxic metal such as chromium (Cr) that hampers the crop productivity. This study evaluated the effect of Cr on two chickpea (Cicer arietinum L.) varieties, Pusa 2085 and Pusa Green 112, in hydroponic and pot-grown conditions. First, growth parameters (seed germination, seedling growth, and biomass production) and physio-biochemical parameters (oxidative stress and the content of antioxidants and proline) were measured to evaluate the performance of both varieties grown hydroponically for 21 days at concentrations of 0, 30, 60, 90 and 120 µM Cr in the form of potassium dichromate (K2Cr2O7). In both varieties, significantly deleterious effects on germination and seedling growth parameters were observed at 90 and 120 µM, while growth was stimulated at 30 µM Cr. Significant increases in malondialdehyde and hydrogen peroxide content and electrolyte leakage demonstrated enhanced oxidative injury to seedlings caused by higher concentrations of Cr. Further, increasing concentrations of Cr positively correlated with increased proline content, superoxide dismutase activity, and peroxide content in leaves. There was also an increase in peroxisomal ascorbate peroxidase and catalase in the leaves of both varieties at lower Cr concentrations, whereas a steep decline was recorded at higher Cr concentrations. In the pot experiments conducted over two consecutive years, growth, yield, yield attributes, grain protein, and Cr uptake and accumulation were measured at different Cr concentrations. Pusa Green 112 showed a significant reduction in plant growth, chlorophyll content, grain protein, pod number, and grain yield per plant when compared with Pusa 2085. Overall, our results indicate that Pusa 2085 has a higher Cr tolerance than Pusa Green 112. Therefore, Pusa 2085 could be used to further elucidate the mechanisms of Cr tolerance in plants and in breeding programmes to produce Cr-resistant varieties.

Biogenic iron oxide nanoparticles enhance callogenesis and regeneration pattern of recalcitrant Cicer arietinum L.

This study is the first report on the biosynthesized iron oxide nanoparticles (IONPs) which mediate in-vitro callus induction and shoot regeneration in economically important recalcitrant chickpea crop (Cicer arietinum L.). Here, we used leaf extract of Cymbopogon jwarancusa for the synthesis of IONPs in order to achieve a better biocompatibility. The bioactive compounds in C. jwarancusa leaf extract served as both reducing and capping agents in the fabrication process of IONPs. Field emission scanning electron microscopy (FE-SEM) revealed rods like surface morphology of IONPs with an average diameter of 50±0.2 nm. Energy-dispersive X-ray spectroscopy (EDS) depicted formation of pure IONPs with 69.84% Fe and 30.16% O2. X-ray diffractometry (XRD) and attenuated total reflectance-fourier transform infrared (ATR-FTIR) validate the crystalline structure, chemical analysis detect the presence of various biomolecular fingerprints in the as synthesized IONPs. UV-visible absorption spectroscopy depicts activity of IONPs under visible light. Thermo-gravimetric analysis (TGA) displayed thermal loss of organic capping around 500°C and confirmed their stabilization. The biosynthesized IONPs revealed promising results in callus induction, shoot regeneration and root induction of chickpea plants. Both chickpea varieties Punjab-Noor 09 and Bittle-98 explants, Embryo axes (EA) and Embryo axes plus adjacent part of cotyledon (EXC) demonstrated dose-dependent response. Among all explants, EXC of Punjab-Noor variety showed the highest callogenesis (96%) and shoot regeneration frequency (88%), while root induction frequency was also increased to 83%. Iron content was quantified in regenerated chickpea varieties through inductively coupled plasma-optical emission spectrometry. The quantity of iron is significantly increased in Punjab-Noor regenerated plants (4.88 mg/g) as compare to control treated plants (2.42 mg/g). We found that IONPs enhance chickpea growth pattern and keep regenerated plantlets infection free by providing an optimum environment for rapid growth and development. Thus, IONPs synthesized through green process can be utilized in tissue culture studies in other important recalcitrant legumes crops.

Gene co-expression analysis reveals transcriptome divergence between wild and cultivated chickpea under drought stress.

Ancestral adaptations in crop wild relatives can provide a genetic reservoir for crop improvement. Here we document physiological changes to mild and severe drought stress, and the associated transcriptome dynamics in both wild and cultivated chickpea. Over 60% of transcriptional changes were related to metabolism, indicating that metabolic plasticity is a core and conserved drought response. In addition, changes in RNA processing and protein turnover were predominant in the data, suggestive of broad restructuring of the chickpea proteome in response to drought. While 12% of the drought-responsive transcripts have similar dynamics in cultivated and wild accessions, numerous transcripts had expression patterns unique to particular genotypes, or that distinguished wild from cultivated genotypes and whose divergence may be a consequence of domestication. These and other comparisons provide a transcriptional correlate of previously described species' genetic diversity, with wild accessions well differentiated from each other and from cultivars, and cultivars essentially indistinguishable at the broad transcriptome level. We identified metabolic pathways such as phenylpropanoid metabolism, and biological processes such as stomatal development, which are differentially regulated across genotypes with potential consequences on drought tolerance. These data indicate that wild Cicer reticulatum may provide both conserved and divergent mechanisms as a resource in breeding for drought tolerance in cultivated chickpea.