From recalcitrance to precision: a robust regeneration, transformation and targeted gene editing framework in Cajanus...

Pigeonpea (Cajanus cajan (L.) Millsp.; 2n = 2× = 22) is a drought-tolerant perennial grain legume commonly cultivated in India's rain-fed and dry land zones, and it is a remarkable natural source of minerals and protein worldwide. Despite decades of research, the constraints associated with tissue culture continue to hinder the genetic improvement of the crop, including the explant's inability to produce embryogenic calli, direct shoot formation, limited regeneration potential, and a lack of an effective transformation system. Moreover, traditional or molecular breeding approaches for crop improvement is time, resource, and labour-intensive. CRISPR/Cas9-mediated approach for targeted trait improvement has emerged as a robust technology for introducing desired genetic modifications in several crop plants. We sought to report an improved protocol for calli production, in vitro regeneration, and a CRISPR-mediated genome editing of the phytoene desaturase (PDS) gene via a biolistic-mediated transformation system in pigeonpea. The indigenously developed construct (CcPDS_NICTK-2_pCRISPR-Cas9) harboring pigeonpea codon-optimized Cas9 and target-specific sgRNA was used for transformation in pigeonpea explants (embryonic axis and cotyledonary nodes). The addition of tailored growth regulators and silver nitrate to shoot-induction media boosted plant regeneration to about 86% (±0.04) and transformation efficiency to 46% (±0.04). Sequencing analysis revealed the incurred mutations in the native CcPDS gene, with an editing efficiency of approximately 10%. Moreover, this optimized approach can be utilized in the future to generate marker-free genome-edited plants, addressing biosafety concerns and facilitating the acceptance and commercialization of genetically improved crops.