Comparative seed germination and seedling development of the ghost orchid,Dendrophylax lindenii(Orchidaceae), and molecular identification of its mycorrhizal fungus from South Florida

Abstract

Background and Aims The endangered leafless ghost orchid, Dendrophylax lindenii, one of the most renowned orchids in the world, is difficult to grow under artificial conditions. Published information on asymbiotic and symbiotic (co-culture with a mycobiont) seed germination, seedling anatomy and developmental morphology of this leafless orchid is completely lacking. This information is critical for the development of efficient procedures for ghost orchid production for successful reintroduction.Methods Ghost orchid seedling early development stages were morphologically and anatomically defined to compare germination, embryo and protocorm maturation and seedling development during asymbiotic and symbiotic culture with one of two mycorrhizal strains (Dlin-379 and Dlin-394) isolated from ghost orchid roots in situ.Key Results Seeds symbiotically germinated at higher rates when cultured with fungal strain Dlin-394 than with strain Dlin-379 or asymbiotically on P723 medium during a 10-week culture period. Fungal pelotons were observed in protocorm cells co-cultured with strain Dlin-394 but not Dlin-379. Some 2-year-old seedlings produced multinode inflorescences in vitro. Production of keikis from inflorescence nodes indicated the capacity for clonal production in the ghost orchid.Conclusions Ghost orchid embryo and seedling development were characterized into seven stages. Fungal strain Dlin-394 was confirmed as a possible ghost orchid germination mycobiont, which significantly promoted seed germination and seedling development. Internal transcribed spacer sequencing data confirmed that Dlin-394 belongs within the genus Ceratobasidium. These results offer the opportunity to examine the benefits of using a mycobiont to enhance in vitro germination and possibly ex vitro acclimatization and sustainability following outplanting.

Precocious flowering of plants resulting from in vitro germination of Cycnoches haagii seeds on mycorrhizal fungi presence

“…Plants of the remaining treatments continued in the vegetative stage. Hoang et al (2017) reported a similar flowering phenomenon in some plants from ghost orchid (Dendrophylax lindenii), after two years of in vitro germination on symbiotic conditions. Indeed, we are convinced that this reproductive precocity is a consequence of the benefits of the orchid-mycorrhizal symbiosis, allowing an increase in plant growth and development.…”

Micropropagation of Dendrobium phalaenopsis Orchid Through Overexpression of Embryo Gene AtRKD4

“…For histological preparation, seeds and protocorms (0-8 weeks after planting) were treated using paraffin method following the method of Hoang, Kane, Radcliffe, Zettler, & Richardson (2017). The anatomy slides were observed by a light microscope.…”

Pollinia Removal and Suspected Pollination of the Endangered Ghost Orchid, Dendrophylax lindenii (Orchidaceae) by Various Hawk Moths (Lepidoptera: Sphingidae): Another Mystery Dispelled

“…The mean D. lindenii nectar spur length measured on artificially propagated plants (n = 102) was 136.5 mm (H. Ray, personal communication). These artificially propagated plants were grown from D. lindenii seeds that were collected on the Florida Panther National Wildlife Refuge (Hoang et al 2016). Nectar spur lengths measured on 5 natural Florida Panther National Wildlife Refuge D. lindenii flowers ranged from 90 to 120 mm, and averaged 100 mm (M. LaRusso & J. Lu, personal communication).…”

“…For orchid conservation to be successful, Swarts and Dixon (2009) proposed 3 actions that must be met: (1) management of natural reserves with emphasis on the specialized needs of orchids (e.g., pollinators, mycorrhizal fungi); (2) establishment of ex situ seed and mycorrhiza banks for orchids under immediate threat; and (3) development of orchid restoration techniques. For D. lindenii, all 3 of these actions have been achieved largely due to coordinated research efforts in southern Florida and Cuba during the past decade (see Hoang et al 2016;Mujica et al 2018;Zettler et al 2019;Coopman & Kane 2019). Collectively, all of these efforts have aligned in favor of D. lindenii with 1 important exception -virtually no information existed on the pollination biology of the ghost orchid until now.…”

“…The 2 most critical biotic agents needed for reproduction by orchids are pollinators for fruit set, and mycorrhizal fungi for seedling recruitment (Swarts & Dixon 2009). In recent years, much has been learned about D. lindenii, including its specific habitat requirements (Mujica et al 2018), artificial propagation with and without mycorrhizal fungi (Hoang et al 2016), and greenhouse acclimatization leading to field establishment (Coopman & Kane 2019). Other than anecdotal reports (Tuttle 2007), little information exists with regards to pollination of D. lindenii.…”