Optimization of sterilization strategies and hormonal regulation for in vitro establishment and early regeneration of Musa haekkinenii

Authors: MUNOZ-SALAS, MILAGROS - Florida International University; Nageswara Rao, Madhugiri; KHODDAMZADEH, AMIR - Florida International University

Submitted to: HortTechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/20/2026
Publication Date: 3/31/2026
Citation: Munoz-Salas, M.N., Nageswara Rao, M., Khoddamzadeh, A. 2026. Optimization of sterilization strategies and hormonal regulation for in vitro establishment and early regeneration of Musa haekkinenii. HortTechnology. https://doi.org/10.21273/HORTTECH05850-26.
DOI: https://doi.org/10.21273/HORTTECH05850-26

Interpretive Summary: This study successfully developed the first reproducible micropropagation protocol for Musa haekkinenii, an endangered ornamental banana with significant horticultural potential but constrained by its limited natural propagation capacity. The research addressed two critical challenges for in vitro establishment: microbial contamination and phenolic oxidation. Regarding disinfection, the novel ultrasonic-assisted treatment (T5) was the only protocol that achieved 100% explant asepsis while maintaining viability, proving superior to conventional chemical-only methods. To mitigate tissue browning, the medium was supplemented with activated charcoal and ascorbic acid, which effectively adsorbed phenolic compounds and improved culture establishment. For stimulating regeneration, the plant growth regulator (PGR) combination T3 significantly outperformed all other treatments. T3 promoted the most vigorous and coordinated development, supporting the highest number of shoots, roots, and leaves, along with superior plant height and chlorophyll content, a finding strongly supported by principal component analysis. This integrated, optimized protocol provides a reliable framework for the commercial-scale propagation of M. haekkinenii, supporting its conservation and wider availability in the ornamental industry.

Technical Abstract: 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.