ENHANCING IN VITRO MICROPROPAGATION AND EXUDATION REGULATION IN STRAWBERRY (FRAGARIA × ANANASSA)

Abstract

Introduction: Strawberry (Fragaria × ananassa) is extensively cultivated crop; however, enhanced in vitro micropropagation techniques are required to have disease-free planting material throughout the year. However, the success of micropropagation is limited and depend on phenolic exudation and microbial contamination, which can result in explant browning and mortality. Objectives: The main goal of this study are developing an optimized in vitro propagation protocol for strawberry that treats these issues. Specially, the goals included establishing an effective explant sterilization method to reduce contamination, mitigating oxidative browning via antioxidant treatments, and optimizing shoot proliferation and rooting conditions. Methods: Runner tip explants collected from virus free plants were subjected to sequential experiments in a completely randomized design. Two disinfection treatments (5% sodium hypochlorite vs. 0.1% mercuric chloride) were compared for initial explant sterilization. To control phenolic browning, three antioxidants (ascorbic acid, citric acid, and polyvinylpyrrolidone) were tested both as medium supplements and 24-hour presoaks (in a 4×4 factorial including a no-antioxidant control) . For shoot multiplication, explants were cultured on MS media with different cytokinin regimes: (i) 6.0 mg/L BA, (ii) 3.0 mg/L BA, and (iii) 3.0 mg/L BA + 1.0 mg/L thidiazuron (TDZ) . Rooting was evaluated on MS medium with either 0.5 or 1.0 mg/L NAA auxin . Data on contamination, browning, shoot number, and rooting were statistically analyzed. Results: Mercuric chloride sterilization drastically reduced contamination (16% vs 52.5% with bleach, p < 0.001). Adding ascorbic acid (0.03%) to the medium halved the incidence of lethal browning and roughly doubled explant survival (~60% survival vs ~30% without antioxidant) . A combined antioxidant treatment (ascorbic acid presoak + PVP in medium) nearly eliminated browning, yielding ~96% explant survival. In the proliferation stage, a moderate cytokinin level (3 mg/L BA) with or without TDZ produced significantly more shoots per explant (~5–6) than a higher BA dose (~4 shoots) . Both NAA levels yielded high rooting rates (~76% at 0.5 mg/L and ~89% at 1.0 mg/L), with the higher auxin concentration producing longer, more vigorous roots (average ~3.5 cm vs 2.7 cm) . Conclusion: The optimized protocol effectively overcomes contamination and phenolic exudation challenges, resulting in a reliable, high-efficiency micropropagation system for strawberry. Improvements at each stage (disinfection, browning control, multiplication, and rooting) synergistically enabled robust plantlet regeneration. This protocol can be applied to produce healthy, true-to-type strawberry plantlets on a commercial scale, supporting sustainable cultivation programs.