In vitro shoot regeneration and chloroplast maturase K (matK) gene-based phylogenetic assessment in Phalaenopsis orchid

Phalaenopsis orchids are globally significant ornamental plants, yet traditional propagation is inefficient for commercial demands. While micropropagation is used, standardised protocols for direct organogenesis using floral stalk nodes remain limited, and the influence of genetic lineage on these protocols is poorly understood. This study aimed to optimise a direct regeneration protocol using 6-Benzylaminopurine (BAP) and Naphthalene Acetic Acid (NAA) and to evaluate if phylogenetic relationships based on the chloroplast maturase K (matK) gene correlate with in vitro regeneration potential. Floral stalk nodes and in vitro subcultures were treated with MS medium supplemented with banana homogenate (100 mg/L), coconut water (100 mL/L), and varying PGR concentrations (T0–T6). Phylogenetic analysis of 14 species was conducted using Maximum Likelihood trees. Treatment T5 (2.0 mg/L BAP + 0.6 mg/L NAA) was optimal, yielding shoot initiation in 29.66 days and 18 shootlets per culture. Phylogenetic analysis grouped species into three clades; Clade I species showed superior regeneration compared to the more recalcitrant Clade III. Root development was minimal across all initial treatments, indicating that the current hormonal balance is insufficient for complete plantlet development without specialised rooting induction stages. The study provides a high-throughput protocol for specific Phalaenopsis clades while highlighting the need for genotype-specific modifications for evolutionarily distant species. This integrated approach offers a foundation for scalable propagation and informs future genetic improvement strategies for Phalaenopsis orchids.