Seed germination evaluation of Phalaenopsis amabilis in various media for long-term conservation

Puspitaningtyas DM, Handini E. 2021. Seed germination evaluation of Phalaenopsis amabilis in various media for long-term conservation. Biodiversitas 22: 5231-5238. Phalaenopsis amabilis (L.) Blume (moon orchid) is one of Indonesia's national flowers, which is mentioned in Presidential Decree No. 4/1993. Study on the orchid seed of P. amabilis was conducted to observe the longevity of seeds storage. The purpose of this study was to determine the viability of P. amabilis seeds after storage for years, then to predict the life span of the seeds, and to find out the best media germination for P. amabilis seeds. After harvesting, P. amabilis seeds were stored at –20 °C and the germination tests were carried out periodically in different periods until the seeds lose their viability to determine the optimum conditions for long-term seed storage. Four trial media cultures have been used to test seed germination by means of in vitro culture. The result showed that seeds of P. amabilis were able to germinate in 4 media i.e. Knudson C, modified Knudson C, modified Vacin & Went and modified leaf fertilizer. The best germination was on leaf fertilizer medium. Based on the research showed that the viability seeds of P. amabilis decreased sharply after 6 years stored. By using regression analysis, the life span of the seeds could be predicted can reach 9 years of storage which is sowing on the best media (leaf fertilizer).

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.

Fungal diversity driven by bark features affects phorophyte preference in epiphytic orchids from southern China

Epiphytic orchids exhibit varying degrees of phorophyte tree specificity. We performed a pilot study to investigate why epiphytic orchids prefer or avoid certain trees. We selected two orchid species, Panisea uniflora and Bulbophyllum odoratissimum co-occurring in a forest habitat in southern China, where they showed a specific association with Quercus yiwuensis and Pistacia weinmannifolia trees, respectively. We analysed a number of environmental factors potentially influencing the relationship between orchids and trees. Difference in bark features, such as water holding capacity and pH were recorded between Q. yiwuensis and P. weinmannifolia, which could influence both orchid seed germination and fungal diversity on the two phorophytes. Morphological and molecular culture-based methods, combined with metabarcoding analyses, were used to assess fungal communities associated with studied orchids and trees. A total of 162 fungal species in 74 genera were isolated from bark samples. Only two genera, Acremonium and Verticillium, were shared by the two phorophyte species. Metabarcoding analysis confirmed the presence of significantly different fungal communities on the investigated tree and orchid species, with considerable similarity between each orchid species and its host tree, suggesting that the orchid-host tree association is influenced by the fungal communities of the host tree bark.

The Diverse Assemblage of Fungal Endophytes from Orchids in Madagascar Linked to Abiotic Factors and Seasonality

The inselbergs of the Central Highlands of Madagascar are one of many ‘micro-hotspots’ of biodiversity on the island, particularly for Orchidaceae. In this region are several genera that have a large number of endemic species that are in serious decline or edging towards extinction. Studies relating to diversity of orchids and their fungal partners (both mycorrhizal and non-mycorrhizal root associates) deserve more attention, as climate change and human induced decline in resilience of species in the wild is at an all-time high. Identification of mycorrhizal fungi (MF) via conventional seed baited-protocorms has limitations for large scale studies and its application for time-bound conservation projects. The paper describes the value of understanding fungal diversity in the roots of orchids at different stages of maturity. The first part of the study was a preliminary investigation mainly to identify culturable Rhizoctonia endophytes, and the second part looked at all life forms of available taxa together with associated soil characteristics. We isolated and identified 19 putative MF from 18 of the 50 taxa spread over an area of 250 sq. km, covering three life forms, growth phases of the orchid taxa, and habitat types. In the rest of the taxa, we were unable to detect any putative MF, but had varying numbers of non-mycorrhizal endophytes. We also found that diversity of putative MF was higher in plants from soils with the lowest P levels recorded. Putative mycorrhizal OTUs were predominantly from the Tulasnella lineage, followed by Ceratobasidium and Serendipita. Within a small subset of samples, a difference in colonised endophytes depending on the collection season was observed. In vitro germination studies using 10 OTUs of mycorrhizal fungi in 14 orchid species showed mostly generalist associations. When orchid seed and fungal sources were studied irrespective of habitat, life form, and distance from each other (orchid seed and fungal source), compatibility for symbiotic seed germination was observed in most cases. Issues with the identification of compatible MF and symbiotic system of seed germination are discussed.

Keragaman genetik Anggrek Grammatophyllum scriptum asal biji dari hasil Kultur In Vitro berdasarkan penanda RAPD

Propagation through tissue culture by using orchid seed as explants will produce a lot of orchid plants. This study aims to measure the genetic character of orchid plantlets that were regenerated from seeds which have been resulted from in vitro culture. The genetic character of the original orchid plants produced from in vitro culture was determined using Random Amplified Polymorphic DNA (RAPD) molecular markers. The results showed that the primers used in the RAPD analysis showed a polymorphic band pattern of 14 DNA bands, with sizes between 500 bp - 8000 bp. The genetic distance of Grammatophyllum scriptum orchids that was regenerated from seeds is between 0.229 and 0.649. The progenis produced from in vitro culture were clustered into seven groups at a dissimilarity coefficient of 45%.

Seed viability test of orchids native to the Brazilian Savanna1

ABSTRACT An essential factor for the formation of a native orchid seed bank is the identification of the viability of stored seeds. This study aimed to determine a methodology for optimizing the tetrazolium test, in the identification of the viability of stored seeds of two Orchidaceae medicinal species native to the Brazilian Savanna. Seeds of Miltonia flavescens Lindl. and Schomburgkia crispa Lindl. were submitted to three pre-conditioning conditions: no soaking (control), sucrose solution or distilled water. The seeds were then submitted to a tetrazolium solution, with three types of conditioning: oven, water bath or ambient temperature. The M. flavescens seeds showed a germination of 86.91 %, with a higher percentage of viable seeds (62.32 %) when submitted to pre-conditioning with sucrose + oven conditioning. For S. crispa, the germination was 97.78 %, with a higher percentage of viable seeds for the control treatment + ambient temperature (89.49 %). These results suggest that specific protocols should be used to conduct the tetrazolium test in Orchidaceae. Moreover, when performed on a sample basis, the tetrazolium test should only be used to indicate the seed viability.

Ex-situ conservation of Cymbidium finlaysonianum by seed storage

Puspitaningtyas DM, Handini E. 2020. Ex-situ conservation of Cymbidium finlaysonianum by seed storage. Biodiversitas 21: 3519-3524. Cymbidium finlaysonianum Lindl. is an orchid that is native to Indonesia which has a pendulous flowers and relatively attractive colors. As a part of the Orchid Seed Storage for Sustainable Use (OSSSU) project, a preliminary study on seed of Cymbidium finlaysonianum Lindl. was conducted to test its viability at Bogor Botanic Gardens, Indonesian Institute of Sciences. The aim of this research was to evaluate the seed viability and to predict the seed longevity of C. finlaysonianum, after storage for different periods at freezing temperature-20°C. Storing of orchid seeds under cold conditions was the best conventional method in prolonging seed viability. Four different culture media were tested for asymbiotic germination. Seed viability was assessed after the seeds were stored for 0, 1, 2, 3, 6, 9, 12 months. Testing continued every year until there was no seed germination, and the viability was lost. The direct method of counting the number of germinating seeds was used to test seed viability. The result showed that seeds of C. finlaysonianum were able to germinate in 4 media i.e. Knudson C, modified Knudson C, modified Vacin & Went, and modified Hyponex fertilizer. The research showed that the viability seeds of C. finlaysonianum decreased after 2 years of storage, and the seeds were almost completely non-viable after six years of storage.

Endophytic fungi associated with roots of epiphytic orchids in two Andean forests in Southern Ecuador and their role in germination

Orchids are known to establish complex relationships with endophytic fungi throughout their life cycle, and particularly during germination of their reserves-deprived seeds. Characterizing generalist or specialist interactions between orchids and associated fungi is key to supporting orchid conservation efforts. Here, endophytic fungi associated with roots of epiphytic orchids were studied in two montane Andean forests in Southern Ecuador. Orchid root samples were collected from ten ~500 m2 plots distributed between two neighboring forests. Endophytic fungi associated with these roots were then isolated, cultured, and identified by sequencing of rDNA markers. In total, 52 pure isolates were recovered from the roots of 10 orchid species. These isolates were classified into nine taxonomic groups except for one isolate that remained unclassified. Most fungal isolates were found in roots of up to two different orchid species; however, Coprinellus radians was found in the roots of all sampled orchids. The potential of C. radians to promote germination of orchid seeds was tested in a separate assay using seeds from two orchid species different than those found in the experimental forest plots. Of the two C. radians isolates tested, one improved germination in the two orchids evaluated to a level about half of that observed in seeds germinated in nutrient-rich medium (Phytamax) and above the null germination observed in plates without the fungus. Together, these results revealed a generalist relationship between C. radians and all the studied epiphytic orchids and the potential role of this fungus as a promoter of orchid seed germination. Key Words: Agaricales, Epidendrum, symbiotic germination