This study developed an efficient, variety-specific in vitro regeneration protocol for the commercially important yet recalcitrant banana cultivar Ney poovan (AB). The investigation aimed to optimise a direct organogenesis system using immature male floral hands as explants and to validate the genetic fidelity and field performance of regenerated plants. Immature male flower buds proved to be a highly effective explant source, offering reduced contamination risk compared to conventional shoot-tip cultures commonly employed for large-scale production of disease-free planting material. Among the growth regulator treatments evaluated, BAP (8 mg L-1) combined with Thidiazuron (TDZ) (0.4 mg L-1) induced the earliest greening response in cv. Ney poovan, with explants exhibiting greening within 23.40 days. The TDZ (0.6 mg L-1) alone was found to be optimal for meristematic clump formation. Maximum shoot proliferation (10.80 shoots per explant) was achieved on medium supplemented with 6-Benzylaminopurine (BAP) (5 mg L-1) and indole-3-acetic acid (IAA) (1 mg L-1). Root induction was significantly enhanced by the combined application of indole-3-butyric acid (IBA) and α-naphthaleneacetic acid (NAA) (1 mg L-1 each), resulting in rapid rooting within 5.20 days. Field evaluation of regenerated plants demonstrated normal vegetative growth, flowering and yield performance. The regenerated plants produced bunch weights comparable to conventionally propagated suckers, recording an average bunch weight of 14.52 kg. Genetic fidelity assessment using inter simple sequence repeats (ISSR) markers revealed very low polymorphism (6.64 %), confirming the high genetic stability and true-to-type nature of the regenerants. The protocol developed in this study provides a reliable, scalable and commercially viable approach for mass propagation of banana cv. Ney poovan banana, facilitating the production of genetically uniform, high-quality planting material for sustainable banana cultivation.
