Techniques for Tasmanian native orchid germination for the Department of Primary Industries and Water Jasmine Janes Nature Conservation Report 09/1 Depar tment of Primary Industries and Water Techniques for Tasmanian native orchid germination Jasmine K Janes Resource Management and Conservation Division Department of Primary Industries and Water Nature Conservation Report 09/1 ISSN 1441-0680 Copyright is assigned to the Crown. Apart from fair dealing for the purposes of private study, research, criticism or review, as permitted under the copyright Act, no part may be reproduced by any means without written permission. Published by: Threatened Species Section Biodiversity Conservation Branch Resource Management and Conservation Division Department of Primary Industries and Water GPO Box 44 Hobart, Tasmania, 7001 Australia February 2009 Cover design & book layout: ILS Design Unit, Geodata Services Branch, DPIW Cover images: Jasmine Janes Suggested citation: Janes, JK (2009). Techniques for Tasmanian native orchid germination. Nature Conservation Report 09/1. Department of Primary Industries and Water, Tasmania SUMMARY More than a third of Tasmania’s native Tasmanian Seed Conservation Centre orchid flora and more than half of (Royal Tasmanian Botanical Gardens). its endemic orchids are considered The use of skilled volunteers in threatened. Tasmania has 68 orchid orchid conservation is becoming species listed as threatened under the increasingly important because of the Threatened Species Protection Act 1995. sheer scope of the effort needed, Over half of these species are endemic and a strong community interest in to Tasmania and nationally listed as orchid conservation. In particular, threatened under the Environment The Australian Native Orchid Society Protection and Biodiversity Act 1999. (ANOS) has been extremely active in However, in comparison to other assisting mangers and botanic gardens Australian states with large numbers in establishing and maintaining “ex situ” of threatened terrestrial orchids, orchid collections. Presently there is no conservation targeted research ANOS chapter in Tasmania, however programs have been rare in Tasmania the newly formed Threatened Plants with funding for active research limited. Tasmania (TPT) (Wildcare Inc.), For example between 2003 and established in conjunction with the 2006 there was no specific orchid Threatened Species Section, has conservation program operating in made Tasmanian orchid conservation Tasmania. their priority and recently secured In 2006 the Threatened Species funding (through a Threatened Section (Department of Primary Species Network grant) to make it Industries and Water) secured two their flagship project. This manual years NRM funding to implement was developed as a practical training the Threatened Tasmanian Orchid aid for educational purposes and Flora Recovery Plan 2006-2010. The interested TPT volunteers, consultants aim of the orchid recovery plan is and scientific researchers. It is hoped to improve the conservation status that the practical outcomes from this of native Tasmanian orchids and its manual will strengthen the ongoing implementation involves wide ranging relationship between the numerous in situ recovery actions. An additional collaborators that will assist in the priority of the orchid recovery plan growth and maintenance of the is to establish a threatened orchid collection after the completion of ex situ seed bank and investigate the the Threatened Tasmanian Orchid flora mycorrhizal associations of these Recovery Plan 2006-2010. species in collaboration with the i ACKNOWLEDEMENTS This manual was funded by the Australian Orchid Foundation. This manual was adapted from techniques developed by Andrew Batty (Kings Park and Botanic Gardens) and Richard Thomson (Royal Botanic Gardens Melbourne). I would like to thank Wendy Potts, Stephen Harris, Phil Bell, Michael Pemberton and Penny Wells for comments and suggestions on draft versions of this manual. Laboratory facilities kindly supplied by the Tasmanian Seed Conservation Centre, Royal Tasmanian Botanical Gardens. This project received funding assistance from the Australian Government’s Natural Heritage trust, developed through the cross regional NRM Regional Competitive Grant ‘implement Threatened Species Recovery Plans’ and was supported by NRM South, NRM North and NRM Cradle Coast. ii CONTENTS SUMMARY i ACKNOWLEDGEMENTS ii 1 Background 1 2 Legal requirements and field hygiene 3 3 Hand pollination and collection of mycorrhiza from the field 5 4 Collection of seed from the field 9 5 Isolation of fungal pelotons from orchid tissue 11 6 Subculturing fungi to SSE – first subculture 15 7 Subculturing fungi to PDA – second subculture 17 8 Subculturing of contaminated fungi 19 9 Sterilisation and symbiotic germination of orchid seed 21 10 Sterilisation and asymbiotic germination of orchid seed 25 11 Potting out protocorms to OMA and sand 27 12 Transfer of seedlings to soil 29 Appendix I. Preparation of stock solutions for Soil Solution Equivalent (SSE) 31 Appendix II. Making Soil Solution Equivalent media with Streptomycin sulphate 33 Appendix III. Making Fungal Isolation Media with Streptomycin sulphate 35 Appendix IV. Making Potato Dextrose Agar (PDA) 37 Appendix V. Making Oatmeal Agar (OMA) 39 Glossary 41 References 43 iii 1 Background Orchids belong to one of the most the fungus forms hyphal coils that feed species rich and floristically diverse the plant, allowing for a heterotrophic families in the world. Orchids are form of growth known as believed to have spread from “mycotrophy” or “myco-heterotrophy” the Malaysian tropics during the (Selosse et al. 2002). Initial contact Cretaceous (65-144 mya), the between the fungus and the seed geographical range now extending appears to be haphazard with no from Alaska through to subantarctic attractant released by the orchid, Macquarie Island (Dressler 1981, although seeds do appear to be able Rittershausen 2002). The vast to resist entry from incompatible fungi colonising capacity of orchids is (Clements 1988). Thus, initial infection attributed to their dust-like seed. A can have one of three results: the single orchid capsule may contain fungus and embryo form a functional between 1,000–100,000 seed (Selosse mycorrhiza, the fungus colonizes all et al. 2002), although fruit set in some plant tissues and parasitizes them, or orchid species is often low due to infection does not occur. Subsequently, a unique pollination system, which some seeds germinate and form a relies on sexually deceiving male protocorm, some senesce and others insects (Cozzolino and Widmer 2005). fail to germinate, presumably because Further complicating the issue, orchid of a lack of recognition or specificity seeds are almost devoid of reserves between the orchid seed and fungus. (Selosse et al. 2002). Consequently, It is not clear how long the orchid- the undifferentiated embryo relies fungus relationship is maintained during on a symbiotic fungal infection for seedling development. Typically orchids its carbon, water and nutrient supply become chlorophyllous and thereby (Batty et al. 2001). capable of photosynthesizing, although Similar to other plant species, orchid some species are saprophytic (Bayman germination begins with the seed et al. 2002). Regardless of habit, the imbibing water however, at this point, vast majority of orchid species retain a fungus penetrates the testa of the the mycorrhizal fungus or fungi for life, seed and enters either through the restricting them to the velamen of the epidermal hairs or the suspensor root in epiphytic species or the collar of the undifferentiated embryo cells of the tuber in terrestrial species (Masuhara and Katsuya 1994). After (Smith and Read 1997). It is thought invagination of the plasma membrane that this arrangement is particularly 1 useful for Australian terrestrial species 1995. Very little orchid research has that undergo prolonged periods been conducted within Tasmania, of dormancy because the orchid but research on other terrestrial can continue to acquire inorganic orchids in the Southern Hemisphere nutrition whilst supplying the fungal suggests that orchid species have partner with carbon in return (Irwin highly variable population sizes and et al. 2007). Furthermore, orchid display irregular patterns in flowering, mycorrhizal associations typically have pollination, germination and dormancy a degree of specificity. For example, (Feuerherdt et al. 2005, Coates et al. some relationships show absolute 2006, Gregg and Kéry 2006). Such specificity or a “one orchid-one fungal studies also indicate that habitat species” arrangement (i.e. Glossodia loss, human induced changes in fire major) (Warcup 1971), others exhibit frequency, introduced and invasive taxon-level specificity, where one plant species (Jones et al. 1999), orchid species is infected by more casual picking of flowers grazing than one member of the same fungal and digging from animals and the genus/family (i.e. Microtis parviflora) use of fungicides are all recognised (Warcup 1981). Alternatively, non- threats to the current and future specificity, or the presence of a range management of orchids (Dockrill 1992, of unrelated fungal taxa in a single Jones et al. 1999, Feuerherdt et al. orchid species, has also been reported 2005, Flora Recovery Plan: Tasmanian (i.e. Caladenia reticulata) (Warcup Threatened Orchids 2006). With 1971, 1981). Absolute and taxon- such a high proportion of endemic level specificity are suspected to be and endangered