What role does the seed coat play during symbiotic seed germination in orchids: an experimental approach with Dendrobium officinale

Abstract

Background: Orchids require specific mycorrhizal associations for seed germination. During symbiotic germination, the seed coat is the first point of fungal attachment, and whether the seed coat plays a role in the identification of compatible and incompatible fungi is unclear. Here, we compared the effects of compatible and incompatible fungi on seed germination, protocorm formation, seedling development, and colonization patterns in Dendrobium officinale; additionally, two experimental approaches, seeds pretreated with NaClO to change the permeability of the seed coat and fungi incubated with in vitro-produced protocorms, were used to assess the role of seed coat played during symbiotic seed germination.

Results: The two compatible fungi, Tulasnella sp. TPYD-2 and Serendipita indica PI could quickly promote D. officinale seed germination to the seedling stage. Sixty-two days after incubation, 67.8 ± 5.23% of seeds developed into seedlings with two leaves in the PI treatment, which was significantly higher than that in the TPYD-2 treatment (37.1 ± 3.55%), and massive pelotons formed inside the basal cells of the protocorm or seedlings in both compatible fungi treatments. In contrast, the incompatible fungus Tulasnella sp. FDd1 did not promote seed germination up to seedlings at 62 days after incubation, and only a few pelotons were occasionally observed inside the protocorms. NaClO seed pretreatment improved seed germination under all three fungal treatments but did not improve seed colonization or promote seedling formation by incompatible fungi. Without the seed coat barrier, the colonization of in vitro-produced protocorms by TPYD-2 and PI was slowed, postponing protocorm development and seedling formation compared to those in intact seeds incubated with the same fungi. Moreover, the incompatible fungus FDd1 was still unable to colonize in vitro-produced protocorms and promote seedling formation.

Conclusions: Compatible fungi could quickly promote seed germination up to the seedling stage accompanied by hyphal colonization of seeds and formation of many pelotons inside cells, while incompatible fungi could not continuously colonize seeds and form enough protocorms to support D. officinale seedling development. The improvement of seed germination by seed pretreatment may result from improving the seed coat hydrophilicity and permeability, but seed pretreatment cannot change the compatibility of a fungus with an orchid. Without a seed coat, the incompatible fungus FDd1 still cannot colonize in vitro-produced protocorms or support seedling development. These results suggest that seed coats are not involved in symbiotic germination in D. officinale.

Keywords: Compatible fungi; Dust seeds; Incompatible fungi; Orchid mycorrhizal fungi; Plant-fungus interactions; Symbiotic seed germination.

Fig. 1

The status of seed germination, fungal hyphae on seeds, and pelotons inside protocorms or seedlings at 32 and 62 days after the incubation of seeds with compatible or incompatible fungal strains in Dendrobium officinale. a, Protocorms (Stage 2 or 3) in two compatible fungal treatments at 32 days after incubation; b, Cross-sections of protocorm showing pelotons in the basal cells in two compatible fungal treatments at 32 days after incubation; c, Fungal hyphae clustered on the seed surfaces under the incompatible fungal FDd1 treatment at 32 days after incubation; d, Seedlings in the compatible PI treatment bore two leaves (Stage 5) and some had one or two roots at 62 days after incubation; e, Cross-sections of seedling showing massive pelotons inside basal cells in two compatible fungal treatments at 62 days after incubation; f, Cross-sections of protocorm showing pelotons occasionally observed in the basal cells in the incompatible fungal FDd1 treatment at 62 days after incubation. The red arrows indicate pelotons inside protocorm or seedling cells

Fig. 2

Effects of different treatments (seeds on OMA and MS and seeds on OMA with addition of the fungi TPYD-2, PI or FDd1) on seed germination, protocorm formation and seedling development at 42, 52 and 62 days after incubation in Dendrobium officinale. In each panel, different letters indicate significant differences (p < 0.05) based on one-way ANOVA and the least significant difference (LSD) method where the data are normally distributed and the generalized linear model (GLM) where the data are not normally distributed

Fig. 3

Effects on seedling development from seeds pretreated with 1% (w/v) NaClO for 5, 10, 20, 30 and 40 min under two compatible fungi (TPYD-2 and PI) treatments. The significant differences among seed pretreatment durations in the two fungal treatments are shown by different lowercase letters at p < 0.05

Fig. 4

Effects of in vitro-produced protocorms incubated with different mycorrhizal fungi (TPYD-2, PI or FDd1) on protocorm differentiation and seedling formation at 32, 42, 52 and 62 days after incubation in Dendrobium officinale. The significant differences among fungal treatments are shown by different lowercase letters at p < 0.05. The protocorms were obtained by in vitro-seed germination on MS medium for 22 days to Stage 2

Fig. 5

Protocorm or seedling, status of fungal hyphae colonization, and cross-sections of protocorms and seedlings showing pelotons under the treatments of in vitro-produced protocorms incubated with three different mycorrhizal fungi at 62 days after incubation. (a1-a3) Compatible fungus TPYD-2 treatment; (b1-b3) compatible fungus PI treatment; (c1-c3) incompatible fungus FDd1 treatment. The red arrows indicate pelotons inside the cells of protocorms or seedlings