In Vitro Propagation of Endangered Vanda coerulea Griff. ex Lindl.: Asymbiotic Seed Germination, Genetic Homogeneity Assessment, and Micro-Morpho-Anatomical Analysis for Effective Conservation

Abstract

In nature, orchid seed germination is extremely low, making in vitro asymbiotic seed germination essential for the propagation and conservation of endangered Vanda coerulea. This study optimized a micropropagation protocol and evaluated the genetic homogeneity of regenerated orchids. The synergistic effect of kinetin (KN) with auxins in the Mitra (M) medium best supported protocorm formation and seedling development. The highest shoot multiplication (5.62 ± 0.09) was achieved with 1.2 mg L−1 KN and 0.6 mg L−1 IBA (indole-3-butyric acid) in the medium. Enhanced leaf production (4.81 ± 0.37) was observed when 3.2 mg L−1 KN was combined with 1.8 mg L−1 IAA (indole-3-acetic acid), while root development was superior when 3.2 mg L−1 KN together with 2.4 mg L−1 IAA was incorporated in the medium. Anatomical sections confirmed well-developed leaf and root structures. Genetic fidelity assessment using random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), inter-primer binding site (iPBS), and start codon targeted (SCoT) markers revealed 97.17% monomorphism (240/247 bands) and low Nei’s genetic distances (0.000–0.039), indicating high similarity among the regenerants. Dendrogram clustering was supported by a high cophenetic correlation coefficient (CCC = 0.806) and strong resolution in Principal Coordinate Analysis (PCoA) (44.03% and 67.36% variation on the first two axes). The Mantel test revealed a significant correlation between both ISSR and SCoT markers with the pooled marker data. Flow cytometry confirmed the genome stability among the in vitro-propagated orchids, with consistently low CV (FL2-A) values (4.37–4.94%). This study demonstrated the establishment of a reliable in vitro protocol for rapidly propagating genetically identical V. coerulea via asymbiotic seed germination.