Micro-propagation of the Gymea Lily A report for the Rural Industries Research and Development Corporation by Jeremy Smith March 2000 RIRDC Publication No 00/36 RIRDC Project No SMA-1A © 2000 Rural Industries Research and Development Corporation. All rights reserved. ISBN 0 642 58066 9 ISSN 1440-6845 Micropropagation of the Gymea Lily Publication No. 00/36 Project No. SMI-1A The views expressed and the conclusions reached in this publication are those of the author and not necessarily those of persons consulted. RIRDC shall not be responsible in any way whatsoever to any person who relies in whole or in part on the contents of this report. This publication is copyright. However, RIRDC encourages wide dissemination of its research, providing the Corporation is clearly acknowledged. For any other enquiries concerning reproduction, contact the Publications Manager on phone 02 6272 3186. Researcher Contact Details Jeremy Smith The Wildflower Farm 20 Grants Road Somersby,2250 Phone: 02 4372 1393 Fax: 02 43 721774 Email: [email protected] RIRDC Contact Details Rural Industries Research and Development Corporation Level 1, AMA House 42 Macquarie Street BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: 02 6272 4539 Fax: 02 6272 5877 Email: [email protected]. Website: http://www.rirdc.gov.au Published in March 2000 Printed on environmentally friendly paper by Canprint ii Foreword Doryanthes excelsa (Doryanthaceae) is one of the most outstanding monocots found in the Australian bushland. It carries massive flower spikes that reach up to 8 metres, which are highly sought after for cutflowers and foliage in the floriculture industry. The commercial value of cutflower and foliage of Doryanthes, presently all cut from wild stands, is steadily increasing, as is the export demand for these unique flowers. The single most constraining factor on the commercial development and horticultural use of Doryanthes is the 10 years or longer lead-time from seed to first flowering. The effective horticultural development of Doryanthes therefore depends upon the derivation of a propagation method, which will enable the rapid multiplication, and early maturation of Doryanthes. This publication outlines some preliminary results achieved for the foundation of a micropropagation protocol that may potentially lead to early flowering. This aspect of early maturation has significant consequences as it will allow for the development of a sound production base and obviate the need to continue harvesting this unique Australian plant from the wild. This will have a major impact on native populations, as this is an example of a species which is well recognized, yet limited in its distribution. This publication provides a background to the micropropagation of a recalcitrant plant species. Information relating to in vitro systems to overcome contaminants, browning of plant tissue and media testing are outlined in detail. Further experimentation relating to ideal concentrations and types of plant growth regulators needs to be addressed before a strict protocol can be recommended for the micropropagation of this plant. It is expected that this project will continue so that a sound protocol can be devised to provide opportunities for commercial row production to proceed. The outcomes of this experimentation are expected to expand the native cutflower industry while conserving a unique Australian monocot. This project was funded from RIRDC Core Funds which are provided by the Federal Government. This report, a new addition to RIRDC’s diverse range of over 450 research publications, forms part of our Wildflowers and Native Plants R&D program, which aims to improve the profitability, productivity and sustainability of the Australian wildflower and native plant industry. Most of our publications are available for viewing, downloading or purchasing online through our website: • downloads at www.rirdc.gov.au/reports/Index.htm • purchases at www.rirdc.gov.au/pub/cat/contents.html Peter Core Managing Director Rural Industries Research and Development Corporation iii Anthony, Stephen Easton and Julia Wilson who Acknowledgements offered technical advise and assistance. Thanks I would like to offer my sincere thanks and also to my family for their support and gratitude to the following people whose support, encouragement especially to my father Alan and time and assistance were invaluable to the my brother Martin of The Wildflower Farm completion of this project: Somersby, for their understanding and endless patience, without which this project would not have Dr Kingsley Dixon, Director of Plant Science at been possible. Kings Park & Botanic Garden for allowing me the opportunity to undertake my project within the Finally I would like to thank RIRDC and the Plant Science division. Many thanks for the University of Technology (Department of suggestions, technical input and continued support. Environmental Science) for funding this work Also to Mr. Eric Bunn and Dr Tissa Senaratna during 1999. (Kings Park & Botanic Garden Research Laboratory) for providing guidance, helpful discussions, advice, encouragement and friendship About the Author throughout the year. Jeremy Smith worked on this project as part of his Honours degree undertaken at the University of Dr Krystyna Johnson (University of Technology Technology, Sydney. He has completed a science Sydney) who, as my university supervisor, offered degree in Environmental and Urban Horticulture assistance and practical advice in all matters and has over 15 years of industry experience in the relating to this project as well as considerable nursery and cutflower industry. Jeremy speaks encouragement. frequently at industry meetings and is a recognized authority on Doryanthes excelsa and the cultivation Thank you to the staff and students at Kings Park & of eastern Australian wildflowers for use as Botanic Garden Research Laboratory who provided cutflowers. For over 15 years Jeremy and his a friendly working environment and help brother Martin have been managing The throughout the year. Special thanks to my Wildflower Farm in Somersby where they grow colleagues in the tissue culture and and promote fresh Australian wildflowers for cryopreservation division:- Dave Merritt, Shane domestic and export markets. Turner, Maggie Panaia, Anthony Bishop, Janet Abbreviations NaOCl sodium hypochlorite tween-80 surfactant or detergent SDW sterile distilled water BA or BAP N6-benzylaminopurine 2iP N6-isopentenyladenine KIN or Kinetin 6-furfurylaminopurine Z zeatin IBA indole-3-butyric acid NAA a-naphthaleneacetic acid NOA 2-naphthoxyacetic acid IAA indoleacetic acid 2,4-D 2,4-dichlorophenoxyacetic acid TDZ thidiazuron (N-phenyl-N-1,2,3,-thidiazol-5-ylurea) standard culture conditions light intensity of 30-40µMolm-2s-1 at culture level, 16/8 hr light/dark at mean temperature 23 ± 2oC PPFD photosynthetic photon flux density MES buffer n- morpholinoethanesulfonic acid MS Murashige and Skoog (1962) basal salts B5 Gamborg et al. (1968) basal media EtOH ethanol NaOH sodium hydroxide HCL hydrochloric acid µM micromole min minute AC activated charcoal K-C:C potassium citrate and citrate NSW New South Wales WA Western Australia iv Contents Foreword ...............................................................................................................................iii About the Author....................................................................................................................iv Abbreviations.........................................................................................................................iv Executive Summary...............................................................................................................ix 1. Micropropagation of Monocotyledons with Special Reference to Australian Plants ............ 1 1.1 Introduction .................................................................................................................. 1 1.2 Species Description (Doryanthaceae) .......................................................................... 1 1.2.1 Aims and Rationale of this Study ........................................................................... 4 1.3 Plant Propagation......................................................................................................... 4 1.3.1 Plant Tissue Culture............................................................................................... 5 1.3.2 In Vitro Propagation of Monocotyledons................................................................. 5 1.4 Explant Sources ........................................................................................................... 5 1.4.1 Shoot Tip Culture................................................................................................... 5 1.4.2 Node and Axillary Bud Culture ............................................................................... 5 1.4.3 Inflorescences as Explant Sources ........................................................................ 6 1.5 Initiation of Tissue Cultures .......................................................................................... 6 1.5.1 Sterilization ............................................................................................................ 6 1.5.2 Multiplication .......................................................................................................... 7 1.5.3 Media Composition ...............................................................................................