Development of an Agrobacterium-delivered codon-optimized CRISPR/Cas9 system for chickpea genome editing.

Chickpea is considered recalcitrant to in vitro tissue culture amongst all edible legumes. 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, which is rich in nutrients and protein. However, generating stable mutant lines using CRISPR/Cas9 requires efficient and highly reproducible transformation protocols. As an attempt to solve this problem, we developed a modified and optimized protocol for chickpea transformation. This study transformed the single cotyledon half-embryo explants using CaMV35S promoter to drive two marker genes (β-glucuronidase gene; GUS and green fluorescent protein; GFP) through binary vectors pBI101.2 and modified pGWB2, respectively. These vectors were delivered in the explants through three different strains of Agrobacterium tumefaciens, viz., GV3101, EHA105, and LBA4404. We found better efficiency with the strain GV3101 (17.56%) compared with two other strains, i.e., 8.54 and 5.43%, respectively. We recorded better regeneration frequencies in plant tissue culture for the constructs GUS and GFP, i.e., 20.54% and 18.09%, respectively. The GV3101 was further used for the transformation of the genome editing construct. For the development of genome-edited plants, we used this modified protocol. We also used a modified binary vector pPZP200 by introducing a CaMV35S-driven chickpea codon-optimized SpCas9 gene. The promoter of the Medicago truncatula U6.1 snRNA gene was used to drive the guide RNA cassettes. This cassette targeted and edited the chickpea phytoene desaturase (CaPDS) gene. 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. This study aimed to demonstrate this system's applicability by performing a gene knockout of the chickpea PDS gene using an improved chickpea transformation protocol for the first time.