Optimisation of surface sterilisation protocols to improve explant survival and reduce microbial contamination in local banana (Musa spp.) germplasm from Malawi

<title>Abstract</title> <p> Microbial contamination during culture initiation severely constrains micropropagation of banana ( <italic>Musa</italic> spp.), limiting the large-scale production of disease-free planting materials. In Malawi, lack of optimised sterilisation protocols for indigenous banana germplasm hinders tissue culture adoption. This study optimised surface sterilisation protocols to maximise explant survival and minimise microbial contamination during the initiation of three widely cultivated local cultivars: 'Kabuthu', 'Sukali', and 'Ndoki'. The experiment was laid out in a Completely Randomised Design (CRD) with a factorial arrangement. Eight sterilisation treatments, comprising varying combinations and exposure times of 70% ethanol, 0.1% mercuric chloride, 3.5% sodium hypochlorite, and Tween 20, were evaluated. Data were analysed using analysis of variance (ANOVA). Sterilisation treatments significantly (p &lt; 0.001), affected explant survival, whereas genotypic effects were not significant (p &gt; 0.05), indicating broad protocol applicability across the evaluated cultivars. The optimal treatment (T8) involved sequential immersion in 70% ethanol for 5 min followed by 3.5% supplemented with Tween 20 for 20 min. This protocol achieved peak explant survival rates of 96.7%, 91.7%, and 73.3% for 'Sukali', 'Kabuthu', and 'Ndoki', respectively. Multi-step treatments consistently outperformed single-agent methods. Fungal contamination was more prevalent than bacterial contamination across all treatments. The study demonstrates that an integrated, multi-step surface sterilisation protocol substantially enhances explant survival and reduces microbial contamination in local banana germplasm. This optimised protocol provides a reliable foundation for mass propagation, germplasm conservation, and strengthening banana seed systems in Malawi and sub-Saharan Africa. </p>