Effects of culture media composition on protocorm-like body formation and seedling mortality in Holcoglossum amesianum: Insights from micromorphological observations

Holcoglossum amesianum is an endangered orchid threatened by habitat destruction and overexploitation. Research on its fundamental biology, particularly the nutrient requirements for seed germination, is scarce. This study aimed to enhance the in vitro propagation efficiency of H. amesianum by optimising the culture medium to improve protocorm-like body (PLB) formation and seedling survival. We systematically evaluated the effects of different basal media (½MS, Hyponex, N6), organic additives (apple homogenate, AH; banana homogenate, BH), and plant growth regulators on seed germination, PLB development, and seedling mortality. The results showed that Hyponex medium supplemented with 30 g/L BH and 20 g/L AH, as well as N6 medium with 30 g/L BH, significantly promoted seed germination. However, these media combinations were associated with high subsequent PLB mortality. In contrast, half-strength Murashige and Skoog (½MS) medium supplemented with 20 g/L AH yielded a high seed germination rate and the lowest seedling mortality. These results suggest that H. amesianum benefits from the nutrient-rich formulation of ½MS medium supplemented with AH for successful seedling establishment in vitro. Our findings provide an optimized protocol for the conservation-oriented micropropagation of this endangered species.