In Vitro Proliferation of Black Betel (Piper betle L. var. Nigra) Nodal Explants Using Different Combinations and Concentrations of Plant Growth Regulators

Faculty of Agriculture, Mulawarman University, Jl. Pasir Balengkong, Kampus Gunung Kelua, Samarinda 75123, East Kalimantan, Indonesia

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Abstract

Black betel (Piper betle L. var. Nigra) contains valuable secondary metabolites with significant medicinal potential. The increasing demand for its leaves as raw material in the biopharmaceutical industry underscores the need for efficient propagation methods to ensure a sustainable supply. However, conventional vegetative propagation of this species is challenging, necessitating the development of a reliable in vitro propagation protocol. This study aimed to evaluate the effects of the plant growth regulators IAA, BAP, and kinetin on the in vitro culture of black betel nodal explants and to determine the optimal combination for effective propagation, comprising BAP at 0.5, 1.0, 1.5, and 2.0 mg L⁻¹; kinetin at 0, 0.5, 1.0, 1.5, and 2.0 mg L⁻¹; and IAA at 0 and 1.0 mg L⁻¹. Single-node explants were cultured on MS media containing these growth regulator combinations. Despite rigorous sterilization, contamination remained high with an overall rate of 60.62%, including 25.75% fungal and 36.12% bacterial infections. Explant viability as well as viable callus formation ranged from 0% to 100% depending on hormonal treatments, with the highest survival achieved at moderate BAP (0.5 mg L⁻¹) and kinetin levels (0.5 and 1.0 mg L⁻¹) under IAA supplementation. Elevated BAP concentrations markedly reduced viability, highlighting the importance of hormonal equilibrium in phenolic-rich medicinal plants. Callus induction yielded four color categories, white, green, cream, and pink, with white callus predominating, indicative of undifferentiated proliferative tissue. These findings emphasize that successful in vitro propagation of black betel requires optimal combinations of growth regulators and improved sterilization protocols, providing a foundation for high-quality callus production and secondary metabolite extraction.