Development and optimisation of a hybrid surface sterilisation protocol for in-vitro culture initiation from leaf explants of Capsicum frutescens and Solanum lycopersicum

As global agriculture grapples with the dual crises of climate change and food insecurity, the demand for resilient, disease-free crops has never been greater. Amid shrinking resources and rising population pressures, Plant Tissue Culture (PTC) stands at the forefront of agricultural innovation, offering a precise, sterile method to massproduce healthy plants for food, medicine, and conservation. Yet, despite its transformative potential, standard protocols are often repetitive, labour-intensive, and heavily reliant on chemical sterilants and contamination control agents, which risk promoting antimicrobial resistance (AMR) and limit scalability. Therefore, this study introduces a Hybrid surface-sterilisation protocol for In-Vitro Culture (HP-IVC) as an innovative and scalable solution to reduce contamination, physical labour, and callus yield inconsistency and address these limitations. Using two Solanaceae model species; Capsicum frutescens with waxy adaxial foliar (WAF) and Solanum lycopersicum with trichomes adaxial foliar (TAF), the research investigated four objectives: developing an IVC leaf sampling kit using hydrogen peroxide and vacuum-sealed bags; optimising semi-manual sonozonation through ozonation, ultrasonic cleaning, and surfactant washing; enhancing sterilisation through cutting phase order, silica beads, and highfrequency electromagnetic treatment (HFET); and comparing callus induction performance using minimal Plant Preservative Mixture (PPM) and hormone treatments. Thus, Stage 1 results showed that 15% hydrogen peroxide with vacuum-sealed bags reduced Gram-positive bacteria by 12% and preserved sample freshness for 3–5 days. Stage 2's sonozonation protocol increased explant survival by 33.33% with container "C" and power sweep settings, while Treatment 4 further reduced mechanical stress, prolonging survival to one month. Pre-cutting delayed contamination by ~27 days (C. frutescens) and ~25 days (S. lycopersicum), with silica beads improving survival by over 60%, and HFET lowering mortality to 20% across leaf types. In Stage 3, HP-IVC showed 98.33% (C. frutescens) and 98.07% (S. lycopersicum) callus induction using 1.0 g/L PPM, compared to <15% under standard conditions; both maintained ~90% callus growth, validating callus formation as a key indicator of protocol success. Operational time also improved: HP-IVC needed only 27.5 minutes for 60 explants and 82.5 minutes for 180, versus 25–30 and 90 minutes using standard IVC. Overall, HPIVC sterilisation protocol demonstrated a practical and biologically efficient alternative to standard protocols by reducing potential AMR risks, minimising chemical usage, decreasing procedural repetition and labour fatigue, while consistently supporting high product yield, making it a sustainable tool for various plant research.