Biochemical and transcriptomic analyses of the symbiotic interaction between Cremastra appendiculata and the mycorrhizal fungus Coprinellus disseminatus

Background Cremastra appendiculata is a rare terrestrial orchid with a high market value as an ornamental and medicinal plant. However, the species depends entirely on fungi for seed germination under natural conditions. In a previous study, we have successfully isolated and identified the mycorrhizal fungus Coprinellus disseminatus which was able to induce the germination of C. appendiculata seeds. We then speculated that C. disseminatus may do so by breaking the testa imposed dormancy of the seeds. In this study, biochemical and transcriptomic analyses were used to characterize the germination of C. appendiculata seeds, collected at different stages of germination, as affected by C. disseminatus. Results The lignocellulose in the seeds coat of C. appendiculata was degraded by the mycorrhizal fungus resulting in facilitated absorption of water. The rate of decline in lignin content was 67 and 73% at 6 and 12 days after sowing, respectively. The water content increased from 13 to 90% during symbiosis. A total of 15,382 genes showing significantly different levels of expression (log2 FPKM≥2.0, Qvalue≤0.05) were successfully identified among all libraries, where the highest number of DEGs was shared between 6 days versus 0 day after symbiotic germination. Gene annotation results suggested that 15 key genes related water-status, such as DHN gene family and Xero 1 were down-regulated. The genes zeaxanthin epoxidase ZEP, 9-cis-epoxycarotenoid dioxygenase NCED3 and β-carotene hydroxylase involved in the biosynthesis of abscisic acid (ABA) were significantly down-regulated in 6 days as compared to 0 day after symbiotic germination. Conclusions This work demonstrates that mycorrhizal fungus C. disseminatus can stimulate C. appendiculata seeds germination through a mechanism of breaking the testa imposed dormancy and inducing water absorption of the embryo.

Influence of Culture Conditions on In Vitro Asymbiotic Germination of Anacamptis longicornu and Ophrys panormitana (Orchidaceae)

This study is the first approach to in vitro asymbiotic germination of two species of Sicilian threatened terrestrial orchids, Anacamptis longicornu and Ophrys panormitana. Seeds were collected in the wild and cultured in two different media—Orchimax medium (OM) and Murashige and Skoog (MS)—and exposed to different photoperiods and temperatures to evaluate the best conditions for the specific stages of development. The germination of A. longicornu was very high on OM (95.5%) and lower on MS medium (21.4%), whereas O. panormitana germinated only on OM medium, with significantly lower percentages (12.0%), compared with A. longicornu. This difference is caused by variation in quality and quantity of nutrients used, primarily by nitrogen source. The results show that temperature and photoperiod widely affect seed germination and development. Although further investigations on asymbiotic and symbiotic germination are needed for the improvement of conservation of Mediterranean terrestrial orchids, our results contribute to the conservation of this group of plants.

Tulasnella calospora (UAMH 9824) retains its effectiveness at facilitating orchid symbiotic germination in vitro after two decades of subculturing

Background The technique of symbiotic germination—using mycorrhizal fungi to propagate orchids from seed in vitro—has been used as one method to cultivate orchids in North America and abroad for > 30 years. A long-held assumption is that mycorrhizal fungi used for this purpose lose their effectiveness at germinating seeds over time with repeated subculturing. Results We provide evidence for the lingering efficacy of one particular strain of Tulasnella calospora (266; UAMH 9824) to stimulate seed germination exemplified by the North American terrestrial orchid, Spiranthes cernua, as a case study. This fungus was originally acquired from roots from Spiranthes brevilabris in 1999 and sub-cultured during the two decades since. Seeds inoculated with the fungus in vitro developed to an advanced protocorm stage after 16 days, and leaf elongation was pronounced after 42 days. In a pilot study, seedlings co-cultured with Tulasnella calospora 266 were deflasked after 331 days and later transferred to soil under greenhouse conditions where they eventually initiated anthesis. During the course of two decades, seeds of 39 orchid species, cultivars and hybrids spanning 21 genera, germinated in vitro co-cultured with Tulasnella calospora 266. These orchids included temperate terrestrials and tropical epiphytes alike. Conclusions The sustained effectiveness of this fungus is noteworthy because it argues against the concept of mycorrhizal fungi losing their symbiotic capability through prolonged subculturing. This study serves as an example of why in situ habitat preservation is essential for the conservation of orchids as a source of potentially useful mycorrhizal fungi.

Low-specific Symbiotic Germination of Dendrobium Huoshanense in Vitro, an Endangered Epiphytic Orchid Endemic From South China

Background: Dendrobium huoshanense is an endangered epiphytic orchid and is endemic to Anhui province of China with a narrow distribution. In nature, orchid seed germination depends on specific mycorrhizal fungi and it is generally assumed that there is a high specific symbiotic germination in narrow distribution orchid. To evaluate the mycorrhizal compatibility and germination-promoting activity of mycorrhizal fungi of D. huoshanense in vitro, we isolated and identified cultured mycorrhizal fungi from the roots of D. huoshanense and D. officinale. A total of 20 mycorrhizal fungal strains (D. huoshanense (n = 6), D. officinale (n = 12), D. nobile (n = 1), D. moniliforme (n = 1)) were used to test the germination-promoting activity of D. huoshanense. Results: In symbiotic germination tests, the mycorrhizal fungi displayed different efficiency in stimulating D. huoshanense seed germination, nine strains stimulated seed development to an advanced seedling stage, while the rest promoted only initial seed germination to stage 2 or stage 3, and the most effective in promoting rapid seed germination was strain 12825(seb) isolated from D. moniliforme, besides, the second most effective fungus was strain WX-7(seb) isolated from D. officinale, both of them belong to Sebacina isolated from non-D. huoshanense. In this study, all twenty fungal strains formed pelotons inside seeds of D. huoshanense based on the trypan blue staining results. Conclusions: Despite its narrow specific habitat preference, D. huoshanense was found to be a mycorrhizal generalist during symbiotic seed germination in vitro. The study is the first to demonstrate the different abilities of orchid mycorrhizal fungi to promote seed germination, protocorm formation and seedling development of D. huoshanense in vitro, providing important microbiological resources for its conservation and reintroduction in nature.

In Vitro Symbiotic Germination: A Revitalized Heuristic Approach for Orchid Species Conservation

As one of the largest families of flowering plants, Orchidaceae is well-known for its high diversity and complex life cycles. Interestingly, such exquisite plants originate from minute seeds, going through challenges to germinate and establish in nature. Alternatively, orchid utilization as an economically important plant gradually decreases its natural population, therefore, driving the need for conservation. As with any conservation attempts, broad knowledge is required, including the species’ interaction with other organisms. All orchids establish mycorrhizal symbiosis with certain lineages of fungi to germinate naturally. Since the whole in situ study is considerably complex, in vitro symbiotic germination study is a promising alternative. It serves as a tool for extensive studies at morphophysiological and molecular levels. In addition, it provides insights before reintroduction into its natural habitat. Here we reviewed how mycorrhiza contributes to orchid lifecycles, methods to conduct in vitro study, and how it can be utilized for conservation needs.

‘Dust seeds’ with undifferentiated embryos and their germination in mycoheterotrophic Monotropoideae (Ericaceae)

‘Dust seeds’ with an undifferentiated (organless) embryo are known to be produced by mycoheterotrophic species (MH) in nine families of angiosperms. However, aside from the numerous studies on seed germination of orchids, relatively little is known about germination in MH families. In the Ericaceae, some degree of mycoheterotrophy (full, partial or initial) and dust seeds with an undifferentiated embryo occur in all species in the three tribes of Monotropoideae, the only subfamily of Ericaceae with this combination of characters. In most species, the seed is <0.90 mm in the greatest dimension, the endosperm is absent (Pityopus) or consists of few to many (30–40) cells, and the embryo is minute, consisting of as few as two cells in Monotropa. Germination in Monotropoideae is monopolar, with only the radicular pole of the embryo participating in germination. Thus, germination polarity differs from that of the dust seeds of orchids in which only the plumular pole of the embryo (protocorm) participates in germination. The dust seeds in Monotropoideae require the presence of fungi, either direct contact with a fungus or the presence of a diffusible substance therefrom, to germinate (symbiotic germination). Recently, representatives of the four genera of tribe Pyroleae have been successfully germinated asymbiotically in vitro. We present a broad overview of dust-size seeds in angiosperms and conclude that they should be subdivided into at least two major categories.