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    Callus induction and efficient in vitro plant regeneration protocol for Chickpea
    (Springer Nature Publishing AG, 2024) Jangid, Vinod Kumar; Senthil-Kumar, Muthappa; Chandran, Divya; Sinharoy, Senjuti
    The development of an efficient and consistent callus-mediated in vitro regeneration protocol is crucial for biotechnological approaches aimed at improving chickpea, an economically important crop legume. In this study, we assess the effectiveness of callus-mediated regeneration in different chickpea genotypes. Through in vitro screening of explants, we identified the Indian cultivar Pusa 240 as a favourable genotype with higher efficiency of somatic embryogenesis and in vitro plant regeneration. Building upon this finding, we have successfully established two distinct protocols for chickpea callus-mediated somatic embryogenesis, utilizing leaf and hypocotyl explants obtained from the Pusa 240 genotype. These protocols achieved plant regeneration efficiencies of 27% using leaf explants and 46.6 − 66% using hypocotyl explants. Extensive literature review and comparative analysis underscored the superiority of our current protocol. Subsequently, the regenerated plants were successfully acclimatized and transferred to the greenhouse, exhibiting normal phenotypic growth. This detailed regeneration method will provide a valuable resource for chickpea genetic transformations and the generation of large mutant populations where embryogenesis via callus formation is required. The protocol presented here establishes a powerful tool for studying the functional genomics of chickpea plants and lays the foundation for future advancements in this field.
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    Genome-wide discovery and differential regulation of conserved and novel microRNAs in chickpea via deep sequencing
    (Oxford University Press, 2014) Jain, Mukesh; Chevala, VVS Narayana; Garg, Rohini
    MicroRNAs (miRNAs) are essential components of complex gene regulatory networks that orchestrate plant development. Although several genomic resources have been developed for the legume crop chickpea, miRNAs have not been discovered until now. For genome-wide discovery of miRNAs in chickpea (Cicer arietinum), we sequenced the small RNA content from seven major tissues/organs employing Illumina technology. About 154 million reads were generated, which represented more than 20 million distinct small RNA sequences. We identified a total of 440 conserved miRNAs in chickpea based on sequence similarity with known miRNAs in other plants. In addition, 178 novel miRNAs were identified using a miRDeep pipeline with plant-specific scoring. Some of the conserved and novel miRNAs with significant sequence similarity were grouped into families. The chickpea miRNAs targeted a wide range of mRNAs involved in diverse cellular processes, including transcriptional regulation (transcription factors), protein modification and turnover, signal transduction, and metabolism. Our analysis revealed several miRNAs with differential spatial expression. Many of the chickpea miRNAs were expressed in a tissue-specific manner. The conserved and differential expression of members of the same miRNA family in different tissues was also observed. Some of the same family members were predicted to target different chickpea mRNAs, which suggested the specificity and complexity of miRNA-mediated developmental regulation. This study, for the first time, reveals a comprehensive set of conserved and novel miRNAs along with their expression patterns and putative targets in chickpea, and provides a framework for understanding regulation of developmental processes in legumes.
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    Validation of internal control genes for quantitative gene expression studies in chickpea (Cicer arietinum L.)
    (Elsevier B.V., 2010) Garg, Rohini; Sahoo, Annapurna; Tyagi, Akhilesh K.; Jain, Mukesh
    The real-time polymerase chain reaction (PCR) data requires normalization with an internal control gene expressed at constant levels under all the experimental conditions being analyzed for accurate and reliable gene expression results. In this study, the expression of 12 candidate internal control genes, including ACT1, EF1alpha, GAPDH, IF4a, TUB6, UBC, UBQ5, UBQ10, 18SrRNA, 25SrRNA, GRX and HSP90, in a diverse set of 18 tissue samples representing different organs/developmental stages and stress conditions in chickpea (Cicer arietinum L.) has been validated. Their expression levels vary considerably in various tissue samples analyzed. The expression levels of EF1alpha and HSP90 are most constant across various organs/developmental stages analyzed. Similarly, the expression levels of IF4a and GAPDH are most constant across various stress conditions. A set of two most stable genes is found sufficient for accurate and reliable normalization of real-time PCR data in the given set of tissue samples of chickpea. The genes with most constant expression identified in this study should be useful for normalization of gene expression data in a wide variety of tissue samples in chickpea.