In vitro mass production of banana bunchy top virus-free clonal Champa colla (Musa AAB) plantlets

Banana bunchy top virus (BBTV) poses a major threat to global banana production, often leading to complete yield loss in severely infected plants, thereby affecting the global economy. This study aimed to develop a reliable in vitro regeneration protocol for Musa AAB cv. Champa (Champa colla) using immature male flowers (IMFs) as explants, while ensuring the production of genetically stable and BBTV-free plantlets. Immature male flowers (IMFs) were collected from BBTV-free mother plants, confirmed through enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) using DNA-R-specific primers. Explants were cultured on Murashige and Skoog (MS) media supplemented with different dose combinations of plant growth regulators (PGR). Initial development white body-like structures (WBLs) was observed within 15 days on PBT-62 medium (0.5 mg/L α-naphthaleneacetic acid (NAA), 2 mg/L 6-benzylaminopurine (BAP), 2 mg/L thidiazuron (TDZ)), with a maximum mean of 9.20 WBLs. Subsequently elongation was achieved on PBT-70 (0.2 mg/L NAA, 1 mg/L BAP) and PBT-71 (0.2 mg/L NAA, 2 mg/L BAP) media, yielding a mean of 15.40 WBLs. The maximum shoot regeneration and rooting occurred on PBT-81 medium (0.1 mg/L NAA, 2 mg/L BAP), producing an average of 24.20 shoots and 12.60 roots and on PBT-80 (0.1 mg/L NAA, 1 mg/L BAP), producing an average 23.00 shoots and 13.40 roots. Genetic fidelity and ploidy stability of regenerated plantlets were validated using inter simple sequence repeats (ISSR) markers and flow cytometry. The BBTV-free status of the regenerated plantlets were further confirmed via PCR amplification using DNA-R-specific primers. The study successfully demonstrates a protocol for the regeneration of genetically stable and virus-free Musa AAB cv. Champa colla plantlets, offering a viable strategy for banana cultivation in BBTV-endemic regions and contributing to global food security.