Cytokinin group of phytohormones regulate root elongation and branching during postembryonic development. Cytokinin-degrading enzymes cytokinin oxidases/dehydrogenases (CKXs) have been deployed to investigate biological activities of cytokinin and to engineer root growth. We expressed chickpea cytokinin oxidase 6 (CaCKX6) under the control of a chickpea root-specific promoter of CaWRKY31 in Arabidopsis thaliana and chickpea having determinate and indeterminate growth patterns, respectively, to study the effect of cytokinin depletion on root growth and drought tolerance. Root-specific expression of CaCKX6 led to a significant increase in lateral root number and root biomass in Arabidopsis and chickpea without any penalty to vegetative and reproductive growth of shoot. Transgenic chickpea lines showed increased CKX activity in root. Soil-grown advanced chickpea transgenic lines exhibited higher root-to-shoot biomass ratio and enhanced long-term drought tolerance. These chickpea lines were not compromised in root nodulation and nitrogen fixation. The seed yield in some lines was up to 25% higher with no penalty in protein content. Transgenic chickpea seeds possessed higher levels of zinc, iron, potassium and copper. Our results demonstrated the potential of cytokinin level manipulation in increasing lateral root number and root biomass for agronomic trait improvement in an edible legume crop with indeterminate growth habit.

In our recent publication, we have shown that a member of the Laccase family, LACCASE2 (LAC2) acted as a negative regulator of lignin deposition in the root xylem tissue of Arabidopsis thaliana. LAC2 messenger RNA (mRNA) level was post-transcriptionally regulated by microRNA 397b, which showed increased expression under water and phosphate deficiency, resulting in the downregulation of LAC2 expression In this report, we have investigated root growth and lignin deposition in an economically important legume crop chickpea (Cicer arietinum L.) in response to natural drought in soil-grown condition. In contrast to the growth retardation of Arabidopsis root in mannitol-supplemented medium, chickpea root showed an increase in length in low soil moisture condition. Lignin estimation in the primary root showed an increase in lignin content, which was substantiated by staining of root xylem. Drought treatment enhanced the expression of four out of six LAC genes tested, while the expression of two was downregulated. Our preliminary study indicateed a molecular mechanism of lignin deposition in chickpea root xylem during drought.

Chickpea is considered recalcitrant to in vitro tissue culture. The Clustered, Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) based genome editing in chickpea can remove the bottleneck of limited genetic variation in this cash crop rich in nutrients and protein. However, the generation of stable mutant lines using CRISPR/Cas9 requires efficient and highly reproducible transformation approaches. We modified a binary vector pPZP200 by introducing a codon-optimized Cas9 gene for chickpea and the promoters of Medicago truncatula U6 snRNA for expressing guide RNA targeted to the Phytoene Desaturase (PDS) gene. The dissected single cotyledons with half embryo of chickpea were used as explants for genetic transformation. A single gRNA was found sufficient to achieve high efficiency (42%) editing with the generation of PDS mutants with albino phenotypes. A simple, rapid, highly reproducible, stable transformation and CRISPR/Cas9-based genome editing system for chickpea was established. For the first time, this study aimed to demonstrate this system's applicability by performing a gene knockout of the chickpea phytoene desaturase gene (CaPDS) in stable shoots using an improved chickpea transformation protocol.

Chickpea is considered recalcitrant to in vitro tissue culture. The Clustered, Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) based genome editing in chickpea can remove the bottleneck of limited genetic variation in this cash crop rich in nutrients and protein. However, the generation of stable mutant lines using CRISPR/Cas9 requires efficient and highly reproducible transformation approaches. We modified a binary vector pPZP200 by introducing a codon-optimized Cas9 gene for chickpea and the promoters of Medicago truncatula U6 snRNA for expressing guide RNA targeted to the Phytoene Desaturase (PDS) gene. The dissected single cotyledons with half embryo of chickpea were used as explants for genetic transformation. A single gRNA was found sufficient to achieve high efficiency (42%) editing with the generation of PDS mutants with albino phenotypes. A simple, rapid, highly reproducible, stable transformation and CRISPR/Cas9-based genome editing system for chickpea was established. For the first time, this study aimed to demonstrate this system's applicability by performing a gene knockout of the chickpea phytoene desaturase gene (CaPDS) in stable shoots using an improved chickpea transformation protocol. Keywords: Chickpea transformation, CRISPR/Cas9, PDS, Codon optimization, Inference of CRISPR Edits (ICE)

Plants deposit lignin in the secondary cell wall as a common response to drought and pathogen attacks. Cell wall localised multicopper oxidase family enzymes LACCASES (LACs) catalyse the formation of monolignol radicals and facilitate lignin formation. We show an upregulation of the expression of several LAC genes and a downregulation of microRNA397 (CamiR397) in response to natural drought in chickpea roots. CamiR397 was found to target LAC4 and LAC17L out of twenty annotated LACs in chickpea. CamiR397 and its target genes are expressed in the root. Overexpression of CamiR397 reduced expression of LAC4 and LAC17L and lignin deposition in chickpea root xylem causing reduction in xylem wall thickness. Downregulation of CamiR397 activity by expressing a short tandem target mimic (STTM397) construct increased root lignin deposition in chickpea. CamiR397‐overexpressing and STTM397 chickpea lines showed sensitivity and tolerance, respectively, towards natural drought. Infection with a fungal pathogen Macrophomina phaseolina, responsible for dry root rot (DRR) disease in chickpea, induced local lignin deposition and LAC gene expression. CamiR397‐overexpressing and STTM397 chickpea lines showed more sensitivity and tolerance, respectively, to DRR. Our results demonstrated the regulatory role of CamiR397 in root lignification during drought and DRR in an agriculturally important crop chickpea.