First integrated in vitro regeneration protocol for the endangered aquatic plant Hydrocera triflora (Balsaminaceae) via optimized cytokinin–auxin balance

Hydrocera triflora, a rare and endangered aquatic species of phylogenetic importance within the family Balsaminaceae, lacks any reported in vitro regeneration system. This study establishes the first complete in vitro propagation protocol for this taxon, integrating optimized sterilization, shoot induction, proliferation, and rooting. Stem explants (~ 3 cm) were disinfected using ethanol, 0.1% mercuric chloride with Tween, and 20% sodium hypochlorite, with the most effective method combining ethanol pre-wash and 0.1% mercuric chloride for 15 min, resulting in 35% contamination and 13% browning. Shoot induction reached 96% on medium supplemented with 1.0 mg L⁻¹ 6-benzylaminopurine and 0.2 mg L⁻¹ indole-3-butyric acid, producing 3-12 healthy shoots per explant with minimal callus. Moderate cytokinin concentrations optimized shoot proliferation, maximizing multiplication coefficient and vigor while avoiding excessive callusing. Rooting was highest on Murashige and Skoog medium with 0.2 mg L⁻¹ indole-3-butyric acid, where roots appeared by day 5, averaged 6.3 cm in length with 16.1 roots per plantlet, and achieved a 100% rooting rate. These results indicate that precise cytokinin-auxin balance governs organized morphogenesis in H. triflora, providing insights into the developmental plasticity and tissue-specific hormonal responses of this species. The established protocol offers a reliable platform not only for large-scale micropropagation and ex situ conservation but also for future biotechnological applications, including genetic improvement, secondary metabolite production, and translational studies in related recalcitrant aquatic species.