Optimization of Sterilization Strategies and Hormonal Regulation for In Vitro Establishment and Early Regeneration of Musa haekkinenii

Musa haekkinenii is an underutilized ornamental banana with recognized horticultural value but limited conventional propagation due to low vegetative multiplication, high endogenous contamination, and pronounced phenolic oxidation during culture initiation. These constraints necessitate plant tissue culture approaches focused on reliable in vitro establishment and early regeneration. This study evaluated six surface-sterilization treatments and six plant growth regulator (PGR) combinations to assess their effects on contamination control and early morphogenic responses. Contamination speed was monitored over a 30-day period. The control treatment (T0) exhibited the highest contamination (14.21%), followed by ethanol- and sodium hypochlorite–based treatments T1 (7.69%) and T2 (6.44%). Multistep disinfection protocols further reduced contamination to 5.08% (T3) and 2.73% (T4), whereas the ultrasonic-assisted treatment (T5) achieved complete asepsis (0.00%). PGR composition significantly influenced all morphological and physiological traits evaluated ( P ≤ 0.05). Treatments T0, T1, and T5 resulted in nonorganogenic callus formation, T4 supported limited morphogenesis, and T2 induced moderate regeneration. In contrast, the combined application of 5 mg/L 6-benzylaminopurine (BAP), 0.2 mg/L thidiazuron (TDZ), and 0.2 mg/L indole-3-acetic acid (IAA) (T3) produced the early morphogenic response, characterized by increased shoot and root formation, leaf production, plant height, and chlorophyll indices. Principal component analysis (PCA) summarized treatment responses along a dominant axis explaining 99.52% of the total variance, clearly separating T3 from the remaining treatments. This study provides a quantitative framework for optimizing sterilization strategies and hormonal regulation during in vitro establishment and early regeneration of M. haekkinenii .