Cotton is a globally important fiber crop, but many elite cultivars are recalcitrant to in vitro regeneration. We developed a callus-mediated plant regeneration protocol for the local G. barbadense L. cultivar Surkhan-106. Sterilized seeds were germinated in vitro, and hypocotyl and cotyledon explants were cultured on Murashige and Skoog (MS) medium with various auxin/cytokinin combinations. Robust callus induction was achieved from hypocotyl explants within 2–3 weeks. Somatic embryogenesis was induced from callus after approximately 12–14 weeks on medium supplemented with indole-3-butyric acid (IBA), 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin, and 6-benzylaminopurine (BAP). Embryogenic callus was periodically subcultured to prevent browning, and torpedo-stage somatic embryos were matured on hormone-free medium. These embryos germinated into plantlets within 10–15 days on MS medium. The entire regeneration cycle, from seed to plantlet, was completed in about 4–5 months. Hypocotyl explants showed significantly higher callus formation frequency (85%) and regeneration efficiency than cotyledon explants (60%) (ANOVA, p < 0.05). This study establishes the first efficient callus-based regeneration system for Surkhan-106, an important Uzbek cotton variety. The optimized protocol can facilitate rapid clonal propagation and genetic transformation of Surkhan-106 and other recalcitrant cotton cultivars, accelerating breeding programs for improved stress tolerance and yield.

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