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From recalcitrance to precision: a robust regeneration; transformation and targeted gene editing framework in Cajanus cajan
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DOI：10.3389/fgeed.2026.1815812.png)
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Abstract
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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.
Keywords:
CRISPR/Cas9
genome editing
transformation
efficient regeneration
phytoene desaturase
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