Water permeability of seed coats determines differences in dormancy between two orchid species

Seeds of mycoheterotrophic plants including orchids are extremely small and structurally reduced, with two layers of seed coats: large testa and a thin inner seed coat known as the carapace. Germination in many species is limited and often requires chemical pretreatment, suggesting that seed coat permeability could play an important role. However, the relative contributions of the testa and carapace to permeability and germination remain unclear, and it is unknown whether these structures may contribute to a physical dormancy. This study aimed to determine the structural basis of seed coat permeability and its role in regulating germination in two terrestrial orchid species with contrasting germination ability, Dactylorhiza majalis and Epipactis helleborine.

Histochemical and ultrastructural analyses showed that the testa is composed of lignified cell walls and lacks detectable cutin or suberin. In contrast, the carapace consists of several structurally distinct layers, some of them likely containing lipidic material. The overall organization of the carapace was similar in both species, but it was significantly thicker in E. helleborine, particularly in the inner autofluorescent layer, which remained largely resistant to chemical pretreatment. Permeability assays developed in this study demonstrated that the carapace represents the main barrier to water entry, and the proportion of permeable seeds closely corresponded to germination percentages in both species. In contrast, differences in testa permeability were relatively minor and could not explain the large interspecific differences in germination.

Both seed coat layers, the outer testa and the inner carapace, act as barriers to water permeability in orchid seeds. However, the carapace constitutes the dominant barrier controlling water access to the embryo. The permeability of the carapace strongly influences germination ability and explains interspecific differences in germination response. Our findings provide direct evidence that a physical or combinational dormancy may occur even in structurally reduced dust seeds and significantly advance understanding of dormancy mechanisms in mycoheterotrophic plants.