In vitro bioproduction and enhancement of moscatilin from a threatened tropical epiphytic orchid, Dendrobium ovatum (Willd.) Kraenzl

Moscatilin, a bibenzyl derivative (stilbenoid), mostly found in one of the largest genera of Orchidaceae; Dendrobium has many therapeutic benefits. Its function as an anticancer agent has been widely demonstrated through many research investigations. However, the compound has not been produced in vitro to date. The present study highlights the development of cultures viz., seedling generation, callus induction and callus regeneration (transformation of callus into plantlets). These cultures were devised to conserve the threatened tropical epiphytic orchid species, Dendrobium ovatum and identify their potential towards moscatilin bioproduction in vitro. Among the three culture platforms, callus-derived plantlets could yield high moscatilin when treated with l-Phenylalanine as a precursor. Tissue differentiation was found to be indispensable for the high production of this polyphenol. These cultures also offer potential commercial benefits as they can serve as appropriate platforms to decode moscatilin biosynthesis and other significant bibenzyl derivatives. Elicitors, such as chitosan, salicylic acid, and methyl jasmonate, were found, causing an enhancement in moscatilin content in the cultures. The seedlings obtained can serve towards ecorestoration and preservation of the studied species. Callogenesis was useful in plantlet regeneration, as callus-derived plantlets could be utilized for the enrichment and commercial scale-up of moscatilin-like chemicals.

Getting a Grip on the Adhesion Mechanism of Epiphytic Orchids – Evidence From Histology and Cryo-Scanning Electron Microscopy

Plants and animals evolve different attachment structures and strategies for reversible or permanent adhesion to different substrate types. For vascular epiphytes, having the ability to permanently attach to their host plants is essential for establishment and survival. Unlike mistletoe roots, roots of vascular epiphytes do not penetrate the host tissues but instead achieve attachment by growing in close contact to the surface of the substrate. However, the fundamental understanding of the attachment functions of epiphytic roots remains scarce, where majority of studies focused on the general root morphology, their functional properties and the descriptions of associated microbial endophytes. To date, research on attachment strategies in plants is almost entirely limited to climbers. Therefore, this study aims to fill the knowledge gap and elucidate the attachment functions of roots of epiphytic orchids. With the use of histology and high-resolution cryo-scanning electron microscopy (cryo-SEM) technique with freeze fracturing, the intimate root-bark substrate interface of epiphytic orchid Epidendrum nocturnum Jacq was investigated. Results showed a flattened underside of the root upon contact with the substrate surface, and the velamen layer appeared to behave like a soft foam, closely following the contours of the substrate. Root hairs emerged from the outermost velamen layer and entered into the crevices in the substrate, whenever possible. A layer of amorphous substance (glue-like substance) was observed on the surface of the root hairs. Combining the observations from this study and knowledge from previous studies, we hypothesised that epiphytic orchid roots produced a layer of glue-like substance to adhere the root to the substrate. Then root hairs are produced and enter into the voids and crevices of the substrate. This further generates a mechanical interlocking mechanism between root and substrate, thus reinforcing the attachment of the root (and hence the whole plant) to its substrate.

Endophytic Fungal Diversity of Aerides crispa, an Epiphytic Orchid

Aerides crispa is an epiphytic plant that belongs to Orchidaceae family. Re-quirement of special habitat makes epiphytic orchids different from other plants. Day by day favourable conditions for these plants are decreasing. Here we have isolated six endophytic fungi from a single orchid. Isolated fungal cultures were molecularly characterized using ITS sequence region comparison with known fungi from NCBI data base and submitted to Gen-Bank. Homology of the fungi were confirmed by similarity matrix generation and phylogenetic tree construction. Thus, the endophytic fungi obtained were Cladosprium cladosporioides, Nigrospora sphaerica, Colletotricum gloe-osporioides, Fusarium circinatum, F.equiseti, Cl.cladosporioides. From the research study, it is revealed that genotypic characterization is a better method for identification of fungi than traditional methods, since same fungi were identified from different parts and also different species from same part of the same plant shows the interesting part of biodiversity. Richness of fungi in A crispaspeaks a lot about the importance of conservation of or-chids, endophytes and thereby biodiversity. The study describes endophytic fungi from an epiphytic orchid Acrispa to its sequence level identification.

Epiphytic Orchid Diversity along an Altitudinal Gradient in Central Nepal

Epiphytic orchids are common in subtropical forests, but little is known about the factors that determine their diversity. We surveyed two sites (north-facing Phulchowki and south-facing Shivapuri hills), in the sub-tropical forest in the Kathmandu valley, central Nepal. Along five transects per site, spanning an altitudinal gradient of 1525–2606 m a.s.l., we recorded all epiphytic orchids and the host species on which they were growing. The data were analyzed using a generalized linear model (GLM) and redundancy analysis (RDA). Species richness significantly decreased with increasing altitude and was higher in larger hosts and in places with high temperature. Species composition was affected by altitude, distance from the forest edge, host type, and precipitation. This study indicates that the most important factors affecting epiphytic orchid diversity was altitude, even if other factors were associated with patterns in composition. The low-altitude habitats with high species diversity are the best places for epiphytic orchids in this region. The altitudinal species richness and patterns in composition revealed by this study provide a baseline for further studies on epiphytic orchids.

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.

Mating system and female reproductive success of the endemic and endangered epiphyte Rhynchostele cervantesii (Orchidaceae) in a cloud forest in Michoacan, Mexico

Background and aims – The Orchidaceae family is vulnerable, because of the destruction of their habitat, as well as the extraction of individuals from natural populations. This is the case of the genus Rhynchostele Rchb.f.; among the actions considered important for appropriate conservation strategies for this genus is the generation of fundamental knowledge, such as on its reproductive biology. The objective of this work is to understand the mating system and reproductive success of Rhynchostele cervantesii, an endangered epiphytic orchid endemic to Mexico.Material and methods – Manual and open-pollination treatments were conducted during 2014 and 2015 in a cloud forest in Michoacan, Mexico. In each period, 30 to 40 randomly selected inflorescences were subjected to the following treatments: a) spontaneous-self-pollination, b) emasculation, c) self-pollination, d) cross-pollination, and e) open-pollination. The developed fruits were counted and harvested, the viability of the seeds was determined, through the observation and evaluation of embryos using microscopy.Key results – Significant differences were recorded between the treatments in both 2014 and 2015, with higher fruit production in cross-pollination than in self-pollination and natural-pollination. There were significant differences in seed viability, with higher values for seeds from open-pollination and cross-pollination and lower values for seeds from self-pollination.Conclusions – Rhynchostele cervantesii is a species that requires pollinators for sexual reproduction because there is no fruit production with spontaneous-self-pollination. Under pollen limitation, the fruit set of natural pollination was a lot lower than in cross-pollination although fruits were the same quality. R. cervantesii had a mixed mating system with a tendency to exogamy, presenting high values of female reproductive success compared to other tropical epiphytic orchid species reported in the literature.