Combating aggressive weeds: Reinforcing herbicide resistance in pigeonpea (Cajanus cajan L.) through genome editing.

Pigeonpea (Cajanus cajan L.) is a drought-tolerant, tropical grain legume, rich in dietary proteins, vitamins, and micronutrients. However, the longstanding problem of weed infestation in the fields is a major constraint that significantly hampers the productivity of pigeonpea. Glyphosate, a widely used post-emergent, broad-spectrum, systemic herbicide, has emerged as an effective weed management strategy at the field level. It inhibits the chloroplastic enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the shikimate pathway by competitively inhibiting its substrate, phosphoenol pyruvate (PEP), thus curtailing the biosynthesis of essential aromatic amino acids (phenylalanine, tyrosine, and tryptophan). This makes glyphosate lethal to weeds and the main crop as well. To address this susceptibility towards glyphosate, we developed glyphosate-resistant pigeonpea plants by modifying the PEP-binding site within the native CcEPSPS enzyme at positions 182G-to-A, 183T-to-I, and 187P-to-S, for reducing glyphosate binding affinity. The targeted base-editing was achieved using the CRISPR-Cas9-based homology-directed repair (HDR) technique. T2-edited plants harbouring mCcEPSPS exhibited stable inheritance of the these GATIPS mutations, reduced glyphosate binding affinity, and maintained optimal photosynthetic and agronomic parameters post-glyphosate application. The mCcEPSPS enzyme efficiently catalysed the transformation of PEP and S3P to EPSP and demonstrated in vitro resistance to glyphosate. In contrast to treated control (TC) plants, the edited plants possessed excellent photosynthetic, agronomic, and physiological metrics following a post-foliar Roundup (6 ml/L) spray. This work offers the first efficient and precise gene-editing report in pigeonpea, offering an effective, sustainable strategy for broad-spectrum weed management to mitigate both quantitative and qualitative crop losses.