A Simple and Highly Efficient Regeneration Protocol for Bougainvillea glabra: Achieving Callus Induction, Shoot Organogenesis, and Elongation on a Single Medium

Bougainvillea is among the most economically and horticulturally important flowering shrubs worldwide, yet its genetic improvement is severely constrained by low natural seed-set and frequent embryo abortion. Existing micropropagation protocols rely predominantly on nodal stem segments and thus bypass a totipotent callus phase, limiting their utility for molecular breeding and genetic manipulation. In this study, we systematically evaluated and compared the in vitro morphogenic competence of young leaf versus seed-derived juvenile hypocotyl explants in B. glabra Choisy ‘Light Violet’. Although young leaf explants readily formed vigorous calli on medium containing 2.0 mg/L 6-benzylaminopurine (6-BA) and 0.2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), they consistently failed to differentiate into adventitious buds across all tested conditions, indicating an intrinsic morphogenic recalcitrance. In sharp contrast, juvenile hypocotyl explants successfully overcame this developmental block, establishing a complete and highly efficient callus-mediated whole-plant regeneration system. Among six tested combinations of 6-BA, 2,4-D, and kinetin on Murashige and Skoog (MS) medium, the combination of 2.0 mg/L 6-BA and 0.2 mg/L 2,4-D uniquely induced compact, actively dividing callus that spontaneously initiated bud primordia within 25 days, achieving the highest adventitious bud differentiation rate of 90.4 ± 2.5%. Regenerated shoots rooted most effectively on half-strength MS (1/2 MS) medium supplemented with 1.25 mg/L 1-naphthaleneacetic acid (NAA) and 0.75 mg/L indole-3-butyric acid (IBA), reaching a rooting rate of 95.2 ± 0.7%. Crucially, the entire developmental sequence from callus induction on hypocotyl segments and de novo bud organogenesis to shoot elongation was coordinated on a unified single medium (2.0 mg/L 6-BA + 0.2 mg/L 2,4-D), substantially streamlining the operational process and enhancing multiplication efficiency. This robust protocol provides a practical pipeline for high-efficiency mass propagation and establishes an indispensable cellular foundation for future genetic transformation and molecular breeding in Bougainvillea.