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.

RNA sequencing of leaf tissues from two contrasting chickpea genotypes reveals mechanisms for drought tolerance.

Chickpea (Cicer arietinum L.) is the second most important winter crop which is consumed globally due to its high nutritional value. Chickpea as one of the leguminous crop is important in crop rotation with cereal crops like wheat and barley. The main constraints for chickpea production are abiotic stresses such as drought, salinity, and heat. Among these, drought is a major cause of the decline in chickpea production in worldwide. Studies conducted so far have provided a limited insight into different genetic pathways associated with drought tolerance/response. In this study, the leaf tissue from shoots apical meristem stage of drought tolerant (ICC8261) and drought sensitive (ICC283) genotypes were analysed using RNA sequencing to identify genes/pathways associated with drought tolerance/sensitivity in both genotypes. It was observed that genes related to ethylene response, MYB-related protein, xyloglucan endotransglycosylase, alkane hydroxylase MAH-like, BON-1 associated, peroxidase 3, cysteine-rich and transmembrane domain, vignain and mitochondrial uncoupling were specifically up-regulated in the tolerant genotype whereas, same genes were down-regulated in sensitive genotype. The crosstalk between the different hormones and transcriptional factors involved in drought tolerance and sensitivity in both genotypes make them great candidates for future research.