Optimization of CRISPR/Cas9-mediated CtPDS knockout in guar protoplasts

Guar (Cyamopsis tetragonoloba L. Taub.) is a climate-resilient legume with industrial and agricultural applications. Recently, gene editing has emerged as a key genetic tool for crop improvement. Despite its recent increasing value as a commodity for various uses, there is no documented report of gene editing work in guar to date. In this study, we present the first optimized protocol for protoplast-based clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) genome editing in guar. The most intact and viable protoplasts were observed in the cotyledons of 6-day-old seedlings that were isolated using the tape-sandwich method. Enzymatic digestion with 1.5% cellulase RS and 0.3% pectinase in 600 mM mannitol, followed by 10 min of vacuum infiltration, increased protoplast release and viability. Polyethylene glycol (PEG)-mediated transformation had been tailored using 40% PEG for 5 min with green fluorescent protein plasmid, and the cestrum yellow leaf curling virus promoter at room temperature showed the highest transient expression efficiency (∼57%). A multiplexed CRISPR/Cas9 construct was designed to target the Cyamopsis tetragonoloba phytoene desaturase (CtPDS) gene in guar. Polymerase chain reaction amplification and Sanger sequencing of transfected protoplasts confirmed highly efficient editing, with fragment deletions ranging from 714 to 1061 bp in CtPDS. Overall, we achieved 100% editing efficiency, as all successfully transformed samples showed CRISPR/Cas9-induced mutations. These findings establish a reliable, transient protoplast system for functional genomics and targeted trait improvement in guar, providing a key foundation for future crop improvement.