Cryopreservation without vitrification suitable for large scale cryopreservation of orchid seeds

Abstract

BackgroundOrchids are under threat from human activities and climate change, with populations limited to small geographic hotspots. This makes them ideal candidates for ex situ conservation. Orchid seeds are desiccation tolerant, but often have poor longevity in seed banks and cryopreservation of orchid protocorms is complex and expensive. Therefore, simple methods for large-scale storage programs are essential to store orchid seeds of different life forms. Seeds of five species representing epiphytic, lithophytic and terrestrial orchids from the Central Highlands of Madagascar were studied to find a simple and effective system of cryopreservation. The use of a vitrification solution prior to cryopreservation to improve survival was investigated, as well as the use of symbiotic and asymbiotic germination media to maximise germination after cryopreservation. Using the filter paper packet method, dried seeds were stored in vapour phase above liquid nitrogen and recovered after thawing with both symbiotic and asymbiotic media.ResultsThe study revealed that cryoprotection is not essential for the species in this study, which represented a range of lifeforms. Vitrification generally led to a decrease in germination post cryopreservation. The use of a symbiotic germination medium post cryopreservation was found to be successful in the species in which it was tested. However, the use of an asymbiotic medium was successful for all the species in this study.ConclusionsVitrification was not essential for the species in this study as the orchid seeds were already ultralow temperature and desiccation tolerant. However, further studies using more species are required to validate this approach. This may be an ecophysiological or genetic trait of these species. Therefore, this form of dry seed cryopreservation could form part of ex situ orchid seed conservation using a standard method. The methods developed here will store greater genetic diversity compared to what can be achieved with protocorms and are suitable for both asymbiotic and symbiotic recovery after cryopreservation. This will help reduce the time and cost of ex situ conservation, and help develop universal protocols for large genera, compared to custom protocols required for protocorm cryopreservation.Electronic supplementary materialThe online version of this article (10.1186/s40529-018-0229-7) contains supplementary material, which is available to authorized users.

Orchid Mycorrhizal Interactions on the Pacific Side of the Andes from Chile. A Review

“…Mycorrhizal fungi and seed conservation are considered an effective measure to improve the population numbers of several endangered species. Orchid seeds for banking need to be dried to low moisture (3 to 7%) and stored at different temperatures, whereas the fungal associates are commonly grown on agar-based culture media and stored at room temperature or 4°C (Popova et al 2016;Schofield et al 2018). All scientific studies analyzing seed germination of endemic orchids must consider applying ex situ conservations methods.…”

Seed viability testing for research and conservation of epiphytic and terrestrial orchids

“…A seed viability test is defined as any technique used to determine whether individual seeds appear to be dead or alive within a sample, which enables the proportion of live seeds in a population to be estimated (Gosling 2003 ). Although conservation in their natural habitat, in situ, is the best option for safeguarding diminishing species numbers, owing to increased habitat loss, fragmentation and habitat degradation, ex situ conservation efforts, such as seed banking (Pant 2013 ; Schofield et al 2018 ). Seed banking provides a long-term security back up for the species and their genetic diversity.…”

Fungal Diversity of Selected Habitat Specific Cynorkis Species (Orchidaceae) in the Central Highlands of Madagascar

“…Those that displayed morphological characteristics of Rhizoctonia-complex (e.g., Tulasnellaceae, Ceratobasidiaceae), such as monolioid cells, were retained for further identification by ribosomal DNA internal transcribed spacer (rDNA ITS) amplification and sequencing. To safeguard the mycorrhizal OTUs (Operational Taxonomic Units) for the purposes of future work (e.g., symbiotic seed germination) and long-term conservation, they were stored in cryopreservation in vapor phase as described by Schofield et al [26].…”