Efficient In Vitro Regeneration from Cotyledon Nodes and In Planta Genetic Transformation in Elite Peanut Cultivars

Abstract Peanut ( Arachis hypogaea L.), a vital oilseed and food legume, is cultivated across the globe. Genetic improvement via conventional breeding faces limitations from narrow diversity and reproductive barriers, underscoring the need for tissue culture-based regeneration and transformation platforms. This study optimizes an efficient, reproducible in vitro regeneration protocol using cotyledonary node explants from three Indian elite cultivars: GG-20, GJG-9, and TAG-37A. Explants from aseptically germinated seedlings were cultured on Murashige and Skoog (MS) medium with varying cytokinins (e.g., BAP 0–4 mg/L) and auxins (e.g., NAA 0.1-0.9mg/L), yielding direct multiple shoot induction without callus, minimizing somaclonal variation. Optimal shoot proliferation occurred on full-strength MS + 2 mg/L BAP for GG-20/GJG-9 (88.9% efficiency) and 4 mg/L BAP for TAG-37A (∼89–100% efficiency); rooting peaked on half-MS + NAA (up to 88.9% in GG-20). Regenerated plants acclimatized successfully in greenhouse conditions. Additionally, a robust in planta Agrobacterium tumefaciens (EHA105, pGFPGUSPlus) transformation via plumular meristem pricking in GG-20 achieved 7.69% efficiency. Transgene integration was confirmed by GUS assay and PCR (GUS/hptII), with ∼64% soil establishment.