Drought poses a significant challenge to cowpea growth and productivity, necessitating the development of drought-tolerant cultivars through detailed morpho-physiological and molecular analyses. This study evaluated drought stress responses in cowpea cultivars using polypropylene plastic boxes under greenhouse conditions. RT-qPCR was conducted to assess the relative expression of five photosynthetic and abiotic stress-related genes in a subset of seven contrasting cultivars at 7-, 14-, and 28-days post-treatment initiation (DPTI) and 24 h post-rewatering. Drought-stressed plants showed progressive wilting and a declining chlorophyll content, with plant greenness scores ranging from 2.2 (TVu11987) to 4.7 (TVu2428). California Blackeye (72.2%) and TVu11987 (69.4%) had the highest recovery rates, indicating greater drought tolerance, while TVu2428 had the lowest (2.8%). Gene expression analyses revealed significant drought-induced variation across cultivars and time points. Transcript levels were notably higher in drought-tolerant cultivars, particularly at 14 DPTI and 24 h post-rewatering, aligning with the morpho-physiological screening results. However, gene expression declined as the drought severity increased. These results suggest that California Blackeye, TVu11987, Lobia-I-Sefade, K929, and Aloomba were more drought tolerant compared to Mississippi Silver and TVu2428. Future research using transcriptomic profiling could unravel the complex molecular mechanisms of drought responses in cowpeas, providing valuable insights for breeding genotypes with improved resiliency to drought.

CRISPR-Cas9 technology has opened new perspectives in genome editing of clonally, asexually propagated and polyploid plants by enabling multiple allelic gene edits. Traditional Agrobacterium- and particle bombardment-mediated transformations, which rely on integration of gene-editing transgene cassettes, have been efficiently applied to several plants; however, concerns about the acceptability of resultant edited transgenic genotypes make these methods less attractive for vegetatively propagated crops. We leveraged and optimized the CRISPR-Cas9/sgRNA-RNPs system for delivery into protoplasts of the hexaploid sweetpotato cultivar PI-318846, targeting eukaryotic translation initiation factor isoform 4E genes to enhance resistance to SPFMV potyviruses. To evaluate the efficiency of pre-assembled Cas9/sgRNA-RNP in sweetpotato transfection, single guide RNAs were designed to target putative host susceptibility genes: IbeIF4E, IbeIF(iso)4E, and IbCBP. Freshly isolated leaf protoplasts were subjected to CRISPR-CAS9-RNP PEG-mediated transfection under different parameters. Sweetpotato regenerants screened using PCR-RE-T7 assay, sequencing, and Inference CRISPR Edit analyses of target-site amplicons revealed the most efficient editing conditions utilizing 25% PEG with a 3:1 (15 µg:45 µg) ratio of Cas9/sgRNA-RNP for 25 min and 48 h incubation period. Different allelic InDels were obtained with editing efficiencies of 10–20% in regenerated plantlets, demonstrating that PEG-mediated CRISPR-RNP transfection system is key for advancing DNA-free editing tools in polyploid and vegetatively propagated crops.