Optimization of In Vitro Propagation, Contamination Control, and Acclimatization of Wild Enset (Ensete ventricosum (Welw.) Cheesman, Musaceae)

Enset (Ensete ventricosum (Welw.) Cheesman) is an important perennial food crop that contributes to food security and has potential for climate-resilient agriculture. Its diverse germplasm represents an important genetic resource for conservation and future crop improvement. However, the development of efficient in vitro propagation systems for wild Enset remains constrained by microbial contamination, phenolic browning, accession-dependent responses, and suboptimal plant regeneration. This study evaluated and optimized key factors affecting indirect organogenesis and greenhouse establishment in four wild Kenyan Enset accessions: Chepseoni, Limuru, Kanjuiri, and Kinangop. Sterilization treatment ST11 (70% ethanol for 3 min followed by 70% NaOCl for 30 min) produced the highest sterilization efficiency, with highly significant differences detected among treatments and accessions (p < 0.0001). Activated charcoal treatment ACT3 (2 g L−1) was the most effective in suppressing phenolic browning, with a highly significant treatment effect (p < 0.0001) but no significant accession effect (p = 0.808). Ascorbic acid treatment AAT3 (10 mg L−1) produced the greatest reduction in phenolic browning, with a highly significant treatment effect (p < 0.0001), whereas accession and treatment × accession interaction effects were not statistically significant (p > 0.05). Cefuroxime treatment CT4 (500 mg L−1) effectively reduced bacterial contamination, with neither accession nor treatment × accession interaction effects being statistically significant. The highest callus induction response was obtained using CIT7 (4.5 mg L−1 BAP + 1.5 mg L−1 NAA), with pronounced accession-dependent responses (p < 0.0001). Root elongation was generally promoted by RIT7 (0.5 mg L−1 NAA), while the treatment producing the highest root number varied among accessions (p < 0.001). FCT4 (irrigation when soil moisture declined below 70% field capacity) was identified as the most favorable irrigation treatment, with a highly significant effect on growth performance (p < 0.0001). Temperature treatments did not produce statistically significant differences, and soil substrate composition had no significant effect on growth performance. Overall, the optimized protocol improved contamination control, phenolic-browning suppression, shoot regeneration, rooting, and greenhouse establishment across the four tested wild Enset accessions. The protocol provides a practical foundation for the conservation and propagation of wild Enset germplasm and may support future breeding efforts aimed at utilizing valuable traits from wild populations.