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

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© 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: wffsomersby@ozemail.com.au

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: rirdc@rirdc.gov.au. Website: http://www.rirdc.gov.au Published in March 2000 Printed on environmentally friendly paper by Canprint

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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

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Acknowledgements I would like to offer my sincere thanks and gratitude to the following people whose support, time and assistance were invaluable to the completion of this project: Dr Kingsley Dixon, Director of Plant Science at Kings Park & Botanic Garden for allowing me the opportunity to undertake my project within the Plant Science division. Many thanks for the suggestions, technical input and continued support. Also to Mr. Eric Bunn and Dr Tissa Senaratna (Kings Park & Botanic Garden Research Laboratory) for providing guidance, helpful discussions, advice, encouragement and friendship throughout the year. Dr Krystyna Johnson (University of Technology Sydney) who, as my university supervisor, offered assistance and practical advice in all matters relating to this project as well as considerable encouragement. Thank you to the staff and students at Kings Park & Botanic Garden Research Laboratory who provided a friendly working environment and help throughout the year. Special thanks to my colleagues in the tissue culture and cryopreservation division:- Dave Merritt, Shane Turner, Maggie Panaia, Anthony Bishop, Janet

Anthony, Stephen Easton and Julia Wilson who offered technical advise and assistance. Thanks also to my family for their support and encouragement especially to my father Alan and my brother Martin of The Wildflower Farm Somersby, for their understanding and endless patience, without which this project would not have been possible. Finally I would like to thank RIRDC and the University of Technology (Department of Environmental Science) for funding this work during 1999.

About the Author Jeremy Smith worked on this project as part of his Honours degree undertaken at the University of Technology, Sydney. He has completed a science degree in Environmental and Urban Horticulture and has over 15 years of industry experience in the nursery and cutflower industry. Jeremy speaks frequently at industry meetings and is a recognized authority on Doryanthes excelsa and the cultivation of eastern Australian wildflowers for use as cutflowers. For over 15 years Jeremy and his brother Martin have been managing The Wildflower Farm in Somersby where they grow and promote fresh Australian wildflowers for domestic and export markets.

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

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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 ................................................................................................ 7 1.5.4 Acclimatization....................................................................................................... 8

1.6 Common Problems in Tissue Culture ........................................................................... 8 1.6.1 Contamination........................................................................................................ 8 1.6.2 Hyperhydricity ........................................................................................................ 8 1.6.3 Phenolic Induced Injury.......................................................................................... 9

1.7 Stages of Rooting and In Vitro Root Establishment ...................................................... 9 1.8 Plant Hormones used In Vitro....................................................................................... 9

1.8.1 Auxins.................................................................................................................. 10 1.8.2 Cytokinins ............................................................................................................ 10

1.9 The Need for Commercialization of Doryanthes excelsa ............................................ 11

2. Materials and Methods for Tissue Culture of Doryanthes excelsa .................................... 12 2.1 Introduction ................................................................................................................ 12 2.2 Explant Source and Selection of Doryanthes Cultures................................................ 12 2.3 Disinfestation Procedures for Explant Material ........................................................... 12 2.4 Manipulation Procedures for Explants ........................................................................ 12

2.4.1 Antioxidant Treatment.......................................................................................... 13 2.5 Incubation Conditions for Tissue Cultures .................................................................. 13 2.6 Culture Media............................................................................................................. 13

2.6.1 Basal Media ......................................................................................................... 13 2.6.2 Media Components and Preparation.................................................................... 13

2.7 Liquid Media Pretreatment ......................................................................................... 13 2.8 Plant Growth Regulators ............................................................................................ 14

3. Initiation and In Vitro Survival of Doryanthes excelsa Explants ........................................ 15 3.1 Introduction ................................................................................................................ 15 3.2 Materials and Methods ............................................................................................... 15

3.2.1 Initiation of Explants............................................................................................. 15 3.3 Results ....................................................................................................................... 16 3.4 Discussion.................................................................................................................. 17

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4. Control of Tissue Browning during Explant Preparation ................................................... 19 4.1 Introduction ................................................................................................................ 19 4.2 Materials and Methods ............................................................................................... 20

4.2.1 Detection of Phenolic Compounds using Sodium Hydroxide (NaOH)................... 20 4.2.2 Antioxidant Experiment ........................................................................................ 20 4.2.3 Potassium Citrate and Citrate (citric acid) as Antioxidant Treatments for Excised Tissue........................................................................................................................... 21

4.3 Results ....................................................................................................................... 21 4.3.1 Detection of Phenolic Compounds using Sodium Hydroxide (NaOH)................... 21 4.3.2 Antioxidant Experiment ........................................................................................ 21 4.3.3 Potassium Citrate-Citrate combinations as Antioxidant Treatments for Excised Tissue........................................................................................................................... 22

4.4 Discussion.................................................................................................................. 24

5.Interaction of Inductive Growth Regulator Signals on Explant Growth............................... 26 5.1 Introduction ................................................................................................................ 26 5.2 Materials and Methods ............................................................................................... 26

5.2.1 Response of Various Explants to Media Treatments............................................ 27 5.2.2 Culture Establishment of Mature Flower Tissue ................................................... 27 5.2.3 Induction of Immature Flower Buds to Different Pulse Treatments....................... 29 5.2.4 Liquid Media Pretreatment ................................................................................... 29

5.3 Results ....................................................................................................................... 29 5.3.1 In Vitro Growth of Plant Material in Response to Media Treatments..................... 30 5.3.2 Response of Mature Flower Tissues to Culture Treatments................................. 32 5.3.3 Response of Explant Tissues to Hormonal Pulse Treatments.............................. 35 5.3.4 Response to Liquid Media Pretreatment .............................................................. 37

5.4 Discussion.................................................................................................................. 37 5.4.1 Basal Media Composition .................................................................................... 37 5.4.2 Plant Growth Regulators used In Vitro ................................................................. 38 5.4.3 Floral Organs as Explants.................................................................................... 39 5.4.4 Liquid Media Pretreatment ................................................................................... 39

6.Conclusions and Future Work........................................................................................... 41

7.References ....................................................................................................................... 43

8.Appendices....................................................................................................................... 48 Appendix 1 ....................................................................................................................... 48 Appendix 2 ....................................................................................................................... 49

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List of Tables

Table 3.1. Tissue types and quantity initiated from D. excelsa each scape. ..................................... 17

Table 4.1 Antioxidant treatments ...................................................................................................... 20

Table 4.2 Degree of tissue discolouration after incubation in NaOH solutions for D. excelsa and C. ficifolia (Myrtaceae)............................................................................. 21

Table 4.3. Relative browning of disc sections of D. excelsa treated with antioxidants over a two hour period....................................................................................................... 21

Table 5.1 Media composition and plant growth regulator concentrations used on in vitro grown tissue.. .................................................................................................................... 27

Table 5.2. Plant growth regulators (PGR) used with or without activated charcoal (AC) f or 22 weeks prior to induction. .......................................................................................... 27

Table 5.3. Kinetin and NAA combinations used on explants derived from inflorescence tissues. ..... 28

Table 5.4. Media composition used for culture initiation from mature flower parts............................ 29

Table 5.5. Plant growth regulators used in conjunction with MS basal medium for culture induction and pulse treatments of immature flower buds with or without activated charcoal (AC). ................................................................................................... 29

Table 5.6. Response and in vitro survival of D. excelsa explants to different concentrations of plant growth regulators after 16 weeks of culture. ........................................................ 31

Table 5.7. Response and in vitro survival of different explant material of D. excelsa to various plant growth regulator (PGR) treatments and MS basal media after 9 weeks culture................................................................................................................. 32

Table 5.8. Response and in vitro survival of immature flower buds of D. excelsa to kinetin and NAA treatment after 9 weeks of culture..................................................................... 32

Table 5.9. Response and in vitro survival to different concentrations of plant growth regulators on dissected anther filaments, style bases and ovary sections from mature flowers of D. excelsa. .................................................................................................................... 33

Table 5.10. Response and in vitro survival of immature flower buds of D. excelsa to various plant growth regulators and MS basal medium for varying incubation periods................ 36

Table 5.11. Response and survival of immature flower buds to liquid media pretreatments prior to culture on 20 µM kinetin.. ..................................................................................... 37

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List of Figures Figure 1.1. Known locations of Doryanthes excelsa ............................................................................ 2

Figure 1.2. Doryanthes excelsa flowering in bushland near Somersby, NSW..................................... 3

Figure 1.3. Flowering stem and developing scape of D. excelsa. ........................................................ 3

Figure 1.4. Flowering stems of Doryanthes excelsa featuring in an arrangement in the foyer of The Hotel Intercontinental, Sydney. ............................................................. 11

Figure 3.1. Location of explant source material in Doryanthes excelsa............................................ 16

Figure 3.2. (A) infloresecence bud with sheathing bracts (bar = 10cm), (B) dissected floral bud enclosed in bract tissue (bar = 2cm), (C) immature bud cluster .................... 16

Figure 3.3. Percentage survival* of explants after 15 weeks............................................................. 17

Figure 4.1. NaOH mediated detection of phenolics in pedicel and peduncle sections of D. excelsa.. .................................................................................................................. 22

Figure 4.2. NaOH mediated detection of phenolics in bract tissue of D. excelsa. ........................... 23

Figure 4.3. Results of antioxidant experiment after 120 minutes. ...................................................... 23

Figure 4.4. Excised inflorescence material prepared for culture. ...................................................... 24

Figure 5.1 . Location of explants (anther filament sections) from mature flower of Doryanthes. ................................................................................................................. 28

Figure 5.2. Development of de novo flower buds (arrows) emerging from in vitro grown immature flower buds after 3 weeks culture. ........................................................ 30

Figure 5.3. Globoid outgrowths (arrow) emerging from the upper cut surface of a peduncle disc section after 24 weeks of in vitro culture. ................................................. 30

Figure 5.4 a. Pedicel disc section from mature flower of D. excelsa.................................................... 33

Figure 5.4 b. Enlargement of pedicel disc section from mature flower of D. excelsa. ....................................................................................................................... 34

Figure 5.5. Callus growth developing in vitro from the base of an immature single flower bud. ....................................................................................................................... 34

Figure 5.6. Pink elongating structures developing from the top of a flower bud cultured for a four day pulse on growth regulator TDZ.................................................... 35

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Executive Summary Doryanthes excelsa is one of the most outstanding monocots found in the Australian bushland. It carries massive flower spikes (scapes) that may attain 8 metres in height. These impressive flowering stems are highly sought after to provide floral designers with dramatic feature flowers for large imposing hotel foyer arrangements. At present the commercial appeal for this unique Australian plant is escalating with an increase in demand both on local and overseas markets. At present very few stems come from commercial row production with the vast majority of supply coming from bushland to the north of Sydney. The high returns for the cut flowers has created a situation where stems are being removed illegally from the roadside, private properties and national parks. The purpose of this study was to investigate the use of micropropagation protocols to assist in the early maturation and potential precocious flowering of Doryanthes excelsa. This has implications for commercial cutflower production as it is reported to take 10 years or longer for this giant monocot to develop its first flower. The effective horticultural development of Doryanthes is therefore dependent upon the derivation of a propagation method which facilitates rapid multiplication and potential early maturation. Key factors investigated in this study include the use of immature inflorescence tissues as explant source material, sterilization and culture conditions, control of phenolic exudates in explant preparation and the selection of appropriate growth regulator levels to achieve in vitro regeneration. Future directions involving the in vitro culture of this giant monocotyledon are discussed. This study has resulted in the preliminary development of micropropagation protocols for culture induction of D. excelsa. Tissue culture of Australian arborescent monocotyledons is not widely reported in the literature and this is the first recorded study outlining micropropagation procedures for Doryanthes excelsa which is an important horticultural subject. The study successfully controlled deleterious phenolic reactions in explant tissues, derived an appropriate sterilization procedure and determined explant sources most likely to result in successful regeneration. Successful establishment of cultures using immature floral explants is described, as is the removal of contaminating organisms from explants using sequential sterilizing techniques. Thorough cleaning of explants and the use of the chemical sterilant, sodium hypochlorite (NaOCl) at 1% v/v) followed by several washes in sterile water obviated the need to develop extensive and complicated surface sterilization protocols. The dissection of immature floral buds from inflorescence tissues of D. excelsa revealed high levels of phenolic compounds. This phenomenon is widespread in many monocotyledenous and woody genera and has profound physiological effects on in vitro cultured tissues (Taji and Williams, 1996; Zaid, 1987; Panaia, 1998). The browning of explants became evident immediately upon dissection from the scape. The oxidation of Doryanthes excelsa tissues was severe and proved deleterious to all tissues in the initial stages of explant preparation. Cessation of tissue browning in explants was achieved using an antioxidant solution as a preculture wash. This experimentation demonstrates that the use of an antioxidant treatment, namely the combination of K:citrate and citrate, provides successful protection allowing the establishment of viable cultures of D. excelsa. Following successful explant establishment, a range of cytokinin and auxin combinations were investigated. A selection of different explant tissue types were cultured for 32 weeks on 1/2 MS basal media supplemented with 10 µM BAP and 0.5 µM IBA. Other experimental work undertaken concurrently tested cytokinins and auxin both singularly or in combination at high to very high levels on a variety of explant sources. Culture exposure to growth regulators in these trials ran for four, nine, twelve and sixteen weeks.

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Further work required Due to time constraints the project did not achieve direct organogenesis from inflorescence tissue and further media testing is therefore required before a protocol for the micropropagation of this recalcitrant plant species can be recommended. Throughout this study on D. excelsa a wide range of plant growth hormones were tested. Given the range and concentrations of growth regulators applied to the various inflorescence tissues, longer incubation time is required to achieve complete organogenesis. Furthermore, it is postulated that the lack of response in explants exposed to medium and high concentrations of hormones, reported to give organogenesis in related species, may be an effect of endogenous gibberellins contained in immature floral tissues. This action may be influencing the in vitro activity of cytokinins and auxins on the various explant tissues. Addressing these points in further experimental work may improve the likelihood of in vitro success in Doryanthes excelsa given the results obtained in previous research in related monocotyledonous genera. However, recent elucidation of the phylogenetic status of D. excelsa out of the Agavaceae and creation of its own endemic family, Doryanthaceae, may point to equally unique attributes of the species under in vitro conditions. This is further reinforced by the lack of commonality in in vitro responses in D. excelsa when compared to in vitro growth in taxa of the closely related families Iridaceae, Amaryllidaceae and Liliaceae. The interest in D. excelsa as a cutflower and foliage and for amenity horticulture, both for domestic and export sales has been steadily increasing in recent years. This species has a restricted distribution, which has serious ecological implications as all cutflower material is harvested from wild populations. It is essential that an appropriate method to expedite introduction to cultivation be determined. The success of in vitro propagation methods reported for other giant monocots suggest that D. excelsa can be tissue cultured with further plant growth regulator experimentation. Successful micropropagation will be of major benefit to the floriculture industry making bush harvesting unnecessary thus greatly assisting in the conservation of this unique Australian plant.

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1 Micropropagation of Monocotyledons with Special Reference to Australian Plants

1.1 Introduction

Throughout history the introduction of plants into cultivation has arisen as an essential need for supply of food, medicines or material for industry. Another aspect that has influenced the introduction of wild plants to gardens has been by the dedicated enthusiast seeing the beautifying elements that a plant may exhibit. The first European explorers, navigators and botanists arriving in Australia were astounded at the vast range of plant taxa never seen before. Realizing the uniqueness of the flora, ardent botanists and horticulturists of the day fostered the introduction of new and novel species into England and Europe throughout the nineteenth century (Wrigley and Fagg, 1979). The main source of propagated material used to supply the horticulture industry then and up to the present has been via traditional methods of seed production, cuttings and grafting. However, developments in plant science over the past 40 years have seen in vitro propagation technology provide horticulturists with an alternative to conventional propagation methods, which has gained worldwide acceptance. Plant tissue culture involves selection of plant tissues (source material), disinfestation of tissues and introducing them into a sterile environment on a defined growth medium (Vasil and Thorpe, 1994). In vitro propagation allows for the mass production of plants where minimal propagation material is available or alternative methods of propagation are not achievable. In vitro culture is of particular importance in ornamental horticulture where rare colour variants or forms may exist which have high commercial value.

1.2 Species Description (Doryanthaceae)

Doryanthes excelsa Correa, which has been assigned to the monogeneric Australian family Doryanthaceae, was first described by Joseph Correa de Serra in 1802 (Newman, 1928). An early study by Newman (1928) placed D. excelsa in the Agavoideae section of the Amaryllidaceae until later classification positioned it in the family Agavaceae. This placement has been the cause of much taxonomic debate (Nash, 1996), however, the most recent molecular genetics information confirms placement in Doryanthaceae with distant relationships in the Iridaceae (M. Fay pers. com., Royal Botanic Gardens, Kew). The genus Doryanthes occurs only on the east coast of Australia (Newman, 1928; Fairley & Moore, 1989; Nash, 1996) and consists of two species D. excelsa and D. palmeri. Doryanthes excelsa occurs with a limited distribution on the central and mid north coast of NSW (Fairley & Moore, 1989; Nash, 1996), (Figure 1.1) with D. palmeri occurring in northern NSW and southern Queensland on the coastal ranges (Wilson, 1993; Nash, 1996).

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Figure 1.1. Known locations of Doryanthes excelsa (adapted from Nash, 1996). Doryanthes excelsa is one of the most outstanding monocots found in the Australian bushland. It carries massive flower spikes (scapes) that may attain 8 metres in height (Figure 1.2). This species has a shortened rhizome and consists of many closely packed evergreen bulbs that arise from the short, thick fleshy rootstock. The leaves are linear, 10-15 cm wide, up to 3 metres long and are spirally arranged, ascending from a short basal stem (Pedley, 1986; Rymer, 1983). The inflorescence of Doryanthes excelsa is held on a long leafy scape and consists of many individual florets arranged in a compound raceme (Newman 1928; Nash 1996) (Figure 1.3). A flowering head can contain over 100 individual flowers, which begin to mature as they emerge from protective sheathing bracts. The dark, red leathery bracts are forced apart as the inflorescence expands. In the Sydney region Doryanthes excelsa is well conserved in National Parks and other reserved land occurring in Heathcote and Royal National Park to the south, Brisbane Water, Girrakool and Dharug National Parks in the north. The populations not protected in National parks and reserves have continued to decline in recent years, particularly on the Somersby plateau north of Sydney. Industrial and agricultural land use has contributed to the demise of the species through clearing and competition from weeds. Similarly, the increased interest in the cut flowers has seen the illegal removal of plant material from roadsides and national parks. Propagation methods, rapid multiplication and a technique that may lead to early maturation of D. excelsa are of utmost importance to alleviate the pressure placed on the wild populations.

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Figure 1.2. Doryanthes excelsa flowering in bushland near Somersby, NSW.

Figure 1.3. Flowering stem and developing scape of D. excelsa.

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1.2.1 Aims and Rationale of this Study

This study will aim to investigate micropropagation methods that will lead to early maturation (time to flowering) of Doryanthes excelsa. Key study areas will include detailed investigation of source tissues including all components of the inflorescence likely to lead to establishment of explants and maintenance of the adult (non-juvenile) state. Explants such as nodal segments, pedicel sections, immature flower buds of different age, vegetative apices in the flower stem and ground and procambial tissue from the pith regions of the extended flowering stem will be examined. Establishment of explant sources will then be used to induce direct shooting and organogenesis. Throughout the program, retention of the adult rather than juvenile state will be an imperative. It is expected that this project will yield important information that will be of major benefit to the floriculture industry as the demand for this species for both export and domestic sales continues to escalate. The specific hypotheses to be tested are:- • that flower meristems of D. excelsa can be induced to revert to vegetative meristems in vitro. • that regeneration can be achieved from inflorescence sections in such a way that tissue maturity (non-juvenile) is likely to be retained. The aims of the experimental design will address the following aspects of in vitro development:- • that sterilization procedures can be developed which will optimize tissue survival in vitro. • that antioxidant treatments minimize phenolic exudation. • that cytokinin and auxin ratios will facilitate tissue regeneration. • that certain tissue types will respond to successful regeneration. From the literature presented relating to other arborescent monocotyledons in this review, appropriate protocols will be devised to address all aspects of micropropagation of Doryanthes excelsa in vitro and ex vitro.

1.3 Plant Propagation

The single most constraining factor affecting the commercial development and horticultural potential of D. excelsa is the 10 years or longer lead time from seed to first flowering. The effective horticultural development of Doryanthes is therefore dependent upon the derivation of a propagation method, which facilitates rapid multiplication and early maturation. In vitro propagation using sections of immature inflorescences has been shown to reduce the time to first flowering as demonstrated by Collins and Dixon (1992) for early maturity in the Australian terrestrial orchid Diuris longifolia. Similar outcomes using micropropagation protocols have been established in large monocots with similar growth form to Doryanthes such as the genera Yucca (Attaalla and Vanstaden, 1997), Euterpe (Guerra and Handro, 1998) and Elaeis (Teixeira et al., 1994). Tissue culture technologies have been successfully developed for many Australian ornamental species (Hutchinson et al., 1985; Johnson, 1996). There are detailed methodologies and principles for commercially important Australian monocotyledons; however, the main focus has been restricted to genera in Haemodoraceae, Orchidaceae, Restionaceae, Xanthorrhoeaceae and Liliaceae (Gorst, 1996). In vitro micropropagation has been documented for many arborescent monocotyledenous genera including Bambusa (Lin and Chang, 1998), Cocos (Verdeil et al., 1994), Cordyline (Kunisaki, 1977; Mee, 1978; Evaldsson and Welander, 1985), Elaeis (Teixeira et al., 1994), Euterpe (Guerra and Handro, 1998), Musa (Smith and Drew, 1990; Khatri et al., 1997), Phoenix (Zaid, 1987), Yucca (Attaalla and Vanstaden, 1997) and the monotypic Australian genus Dasypogon (Bunn and Dixon, 1992).

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1.3.1 Plant Tissue Culture

The use of in vitro propagation is an important tool in horticulture. The applications include rapid multiplication of economically important plants where suitable vegetative material is in short supply, rendering conventional propagation techniques difficult to employ (Johnson, 1996; Bunn and Dixon, 1996). It is also especially advantageous for the rapid multiplication and ex situ conservation of rare and endangered plants as well as recalcitrant species. Another interesting application of micropropagation is that of initiating adventitious shoots from immature inflorescences which reduces the time to flowering in some species (Collins and Dixon, 1992). 1.3.2 In Vitro Propagation of Monocotyledons

Gorst (1996) outlines tissue culture protocols for Australian monocotyledonous genera from 11 families. Other works dealing with exotic monocots are well represented with well over 500 published papers dealing with important flower bulb crops. Procedures for in vitro production of important agricultural monocots such as Musa sp., Zingiber sp., Ananas sp. are well researched (Smith and Drew, 1990). Research involving tissue culture of Australian monocotyledons has identified that many protocols for exotic monocotyledenous species can be implemented, particularly in the field of media composition and explant sources.

1.4 Explant Sources

Plant tissue culture is a technology that involves initiation and growth of organized and unorganized tissues under aseptic conditions (Nehra and Kartha, 1994). Manipulation of the media components and the physical environment in vitro allows for a wide range of protocols to be implemented. Common source material used as explants include meristematic tissue, shoot tips, axillary buds or nodes, inflorescences and seeds (Hutchinson et al., 1985; Johnson, 1996). 1.4.1 Shoot Tip Culture

Shoot tips are the predominant source of explant material for initiation of many Australian species (McComb, 1983). The growing apex of main shoot tips or lateral branches are excised and induced to multiply in vitro on a cytokinin enriched basal medium (Hutchinson et al., 1985; Johnson, 1996). The mode of action of cytokinins is to release the axillary meristems from apical dominance and encourage shoot proliferation (Taji and Williams, 1996). The subsequent shoot sections are sub-cultured onto media containing auxins leading to the formation of adventitious roots. The newly developed plantlets are transferred from in vitro to in vivo conditions and acclimatized in a glasshouse environment. 1.4.2 Node and Axillary Bud Culture

This method of micropropagation utilizes unbranched stems having single or multiple dormant axillary buds. The source material is obtained from above ground parts such as suckers or slips for Pineapple, Ananas comosus (Smith and Drew, 1990) or below ground organs such as rhizomes, bulbs, tubers and corms. Hussey (1976) and Van der Linde (1992) list many commercial bulbous crops that are propagated in vitro using axillary buds including genera from the Liliaceae, Iridaceae and Amaryllidaceae. The bud material is place horizontally on the culture medium with added hormones which forces the outgrowth of lateral unbranched shoots (George, 1996). The procedure for initiating further subcultures and adventitious roots on new shoot material is outlined later in this review.

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1.4.3 Inflorescences as Explant Sources

Utilization of whole flowers, buds, ovary sections and inflorescence sections as primary explant source material has been reported (Slabbert et al., 1995; Wildi et al., 1998; Amomarco and Ibanez, 1998; Richwine et al., 1995; Holme and Petersen, 1996). Bunn and Dixon (1997) demonstrated that adventitious shoot formation arises directly from the perianth or external ovary tissues in Blandfordia grandiflora, with this technique proving advantageous for rapid shoot regeneration with minimal explant material. This protocol is also useful for conservation of endangered species as it is a non-destructive method utilizing seasonal structures of the plant therefore allowing for the preservation of the mother plant (Amomarco and Ibanez, 1998). This method of in vitro propagation demonstrates that many hundreds of clones can be obtained successfully from a single inflorescence of date palms (Loutfi and Chlyah, 1998) and regeneration of recalcitrant species in vitro can be overcome (Verdeil et al., 1994). The utilization of immature material also favours minimal contamination rates compared to other tissues.

1.5 Initiation of Tissue Cultures

When initiating a new species in vitro the selection of appropriate explant material is a key requirement for successful micropropagation (Hartmann et al., 1990). Assessment of the tissue types used will determine the media and hormone components. The successful initiation of many Australian species has resulted from meristem-tip culture (Johnson, 1996). However, leaf sections and segments of immature inflorescences of flower scapes are alternative sources (Wildi et al., 1998; Richwine et al., 1995). The establishment of geophytic species from meristematic material poses greater difficulties due to high levels of contaminants (George, 1996). The culture incubation conditions vary greatly depending on species type and selected source material. Complete darkness when establishing explants has been shown to reduce oxidation and the deleterious effects of phenolic exudation (George, 1996). Stimart (1986) states that darkness is essential for callus and shoot proliferation in Hosta sp. using scape material as the explant source. For many Australian

species suitable incubation conditions include 25ºC, 16 hours of light (40 µmol m-2s-1) supplied by cool white fluorescent tubes with an 8 hour dark period. Polycarbonate tubes (30 ml volume) are selected for the initiation of cultures and once the initial establishment phase is complete, the stable cultures are transferred to 250ml glass jars or equivalent. Basal media consisting of 1/2 MS (Murashige and Skoog, 1962) basal salts is able to sustain a wide range of Australian species in vitro (Bunn, 1994). Full strength MS has been shown to have detrimental effects on the in vitro development of some Australian species (Meney and Dixon, 1995). This may be related to the fact that many Australian species have evolved to cope with highly nutrient depleted soils. Successful in vitro establishment has been achieved for many monocot species using 1/2 MS media including Lilium longiflorum (Nhut, 1998), Ranunculus lyallii (Bicknell et al., 1996), Yucca aloifolia (Attaalla and Vanstaden, 1997) and the rare Australian species, Sowerbaea multicaulis (Rossetto et al., 1992a). 1.5.1 Sterilization

Microbial contamination caused by bacteria, fungi and insect contamination are serious problems in plant tissue culture in any form because of competition for nutrients, release of toxins and predation resulting in death of plant tissue. The loss of plant material can be very costly, especially if contamination rates go undetected at early stages of culture and appear in later stages. It is therefore necessary to remove external micro organisms with physical or chemical disinfectants or sterilants before culture initiation. Culture media are rich in organic compounds such as sugar, amino acids and vitamins and thus provide favourable conditions for the development of bacteria, fungi and other micro organisms (George, 1996).

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Surface sterilization of plant material is essential prior to initiation to eliminate any micro-organisms that may be present. Various sterilants that are commonly used include alcohol (ethyl-, methyl- and isopropylalcohol at 80% v/v), calcium hypochlorite, sodium hypochlorite and mercuric chloride (Vassil and Thorpe, 1994; Hartmann et al., 1990). The effectiveness of chemical sterilization is dependent on the type and age of explant material, concentration of the sterilant and exposure time. Over exposure of plant tissue to sterilants is a common cause of damage and subsequent death of plant cells. It is therefore necessary to competently investigate disinfestation procedures for different tissue types before attempting initiation of valuable explant material. Addition of surfactant to the disinfectant helps to improve the surface wetting of tissues and enhance effectiveness of the chemical sterilant. The variations in responses by different tissue types and species to chemical disinfestation has been documented by Johnson (1996) and others for Australian taxa. For example, Leucopogon obtectus (Epacridaceae) can only survive a mild sterilization procedure (0.05% tween-80 for 30 sec, 80% ethanol for 2-5 sec, washed and stored in sterilized distilled water (SDW), then each shoot treated with 0.5% NaOCl v/v for 15 sec, while outer leaves removed and washed in SDW) due to its sensitivity to hypochlorite (Bunn et al., 1989). Jusaitis (1991) outlines procedures for sterilization of less sensitive species such as Phebalium spp. using 10% v/v sodium hypochlorite (washing in 70% v/v ethanol for 5 sec, then 10% NaOCl v/v for 15 minutes). The majority of sterilization procedures documented utilize sodium or calcium hypochlorite (NaOCl or CaOCl) at 0.5 to 1% v/v for between 5 and 15 minutes exposure followed by several washes in sterile distilled water. Once a desirable disinfectant is selected the time of exposure usually varies between 5 and 15 minutes followed by 3-6 washes in sterilized distilled water (SDW). Aseptic conditions are maintained throughout the entire process of micropropagation until plants are transferred ex vitro, that is axenic culture being the usual micropropagation regime. However, in terrestrial orchid culture (Collins and Dixon, 1992; Ramsay et al., 1986), cultures are inoculated with mycorrhizal fungi or the appropriate fungus is introduced at a later stage of the culture cycle. 1.5.2 Multiplication

Many Australian species have different responses to plant growth regulators. Essentially for in vitro

shoot multiplication cytokinins including N6-benzylaminopurine (BAP), N6-isopentenyladenine (2iP)

and kinetin are selected (Johnson, 1996; Gorst, 1996; Bunn and Dixon, 1996b). The type of regeneration and the capacity to rapidly multiply depends on the tissue type from which the explant is derived (George, 1996). Similarly, explant source and species type dictates the hormone combinations and concentration for desirable in vitro development. Development of callus and root induction is influenced by auxin levels in the culture media. Indole-3-butyric acid (IBA), indoleacetic acid (IAA) and naphthaleneacetic acid (NAA) have been shown to successfully promote rooting in many monocotyledons (Gorst, 1996; Bunn and Dixon, 1996; Rossetto et al., 1992b). The determination of optimal ranges and combinations of hormones requires detailed experimentation. Bicknell et al. (1996), demonstrated that very low concentrations of BAP (0.1 mg/L) completely inhibits root development in Ranunculus lyallii. Similar low levels of BAP as a result of carry over have been shown to limit the amount of root development in Caustis dioica (Rossetto et al., 1992b). This demonstrates the sensitivity of different species and tissue types to hormone type, combinations and concentrations. 1.5.3 Media Composition

Different media compositions used for establishment of explants have been tested across a wide range of Australian species (Williams et al., 1985). Development of specific media constituents for desirable establishment of cultures is dependent on each species particular requirements. The first tissue culture of Australian plants relied on basal media composed of Murashige and Skoog (1962) basal salts. In addition to this, vitamins, amino acids, sucrose and plant growth regulators were added to promote various responses from the explant. Recent work in micropropagation research indicates a

8

high level of media sophistication where hormone adjustments are modified for particular development of end products e.g. shoots, root induction, somatic embryos and organogenesis. With advancement of many in vitro procedures media constituents have also developed extensively. Traditional protocols relied on gelling agents to solidify the media. Agar is the most commonly used gelling agent for explant establishment (Hartmann et al., 1990; George, 1996). The gel provides support along with water and nutrient essential for plant growth (Taji and Williams, 1996). However, more recent experimentation has identified the efficacy of liquid media for explant growth. When dealing with very small organs such as anthers and excised embryos liquid media provides a desirable substrate for suspension cultures (Guerra and Handro, 1998). The small tissues are floated on top of the liquid media or suspended on filter paper bridges with gentle and continuous agitation (George, 1996; Doran, 1996). 1.5.4 Acclimatization

For many species micropropagated under axenic conditions a significant problem is the protection of explants from microbial contamination when transferring from axenic culture to ex vitro situations. “Hardening” or acclimation procedures can help to minimize the impact of losses due to microbial infection in the soil transfer phase. The areas of acclimation of cultures are the subject of major reviews (Johnson, 1996; Gorst, 1996; Bunn and Dixon; 1996).

1.6 Common Problems in Tissue Culture

There are several problems that need to be overcome in establishment of new explant material in vitro. They are contamination, hyperhydricity and phenolic injury. Plant material suffering from hyperhydricity is described as being “glassy” and having a translucent appearance. It is a condition of metabolic and morphological derangement that leads to a high degree of difficulty in establishing the plantlets ex vitro (Barlass and Hutchinson, 1996; Johnson, 1996; Ziv, 1991). 1.6.1 Contamination

Achieving and maintaining asepsis throughout the entire culture process is essential if viable explants are to be established. The decontamination and preparation of explants is a vital step in the culture process and therefore good hygiene and laboratory procedures must be observed (Krikorian, 1994). The induction of cultures from young actively growing tissue raised in greenhouses, laboratory or growth rooms is preferable to source material collected from the wild (George, 1996). It is not always possible to obtain such material and subsequently there is greater degree of microbial contamination and therefore the development of suitable disinfestation protocols is needed (Barlass and Hutchinson, 1996). 1.6.2 Hyperhydricity

Hyperhydricity or vitrification is a widespread problem in tissue culture. Johnson (1996) outlines this obstacle associated within in vitro culture systems which results in poor structural wax development, non-functioning stomata and lack of mesophyll organisation preventing photosynthesis and impeding growth of cultures. There are a number of factors that contribute to this phenomenon such as low potassium concentrations, high cytokinin levels and low concentrations of solidifying agents or sugar in the culture media. This is a common problem with woody plant cultures and can be overcome by modification of culture conditions such as improving aeration (Rosssetto et al., 1992) cooling and adjusting the gelling agent used in the media (Johnson, 1996).

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1.6.3 Phenolic Induced Injury

High levels of phenolic oxidation appears to be widespread across many monocot species e.g. oil palm (Teixeira et al., 1994), Ensete (Zeweldu et al., 1998) and banana clones (Khatri et al., 1997; Zaid, 1987). Phenolic compounds are contained in plant tissues and consist of at least one hydroxy group on a benzene ring. These are strong reducing agents and when phenolic compounds react with enzymes, usually separated by cell vacuoles, the hydroxy group is oxidized resulting in the formation of quinones and water (Taji and Williams, 1996). This irreversible process of hydrogen bonding to proteins inhibits enzyme activity and leads to cell death. Oxidation and browning of tissue occurs instantaneously when the cut plant surfaces are exposed to air with damage appearing on and around the cut surface. Other reported factors influencing tissue oxidation includes age of parent material and time of collection. This poses major problems in the establishment of explants particularly with stem sections where a large surface area is exposed. Removal of phenolic compounds through rigorous washing can reduce the damage to some plant tissues and repeated washing with antioxidants such as citrate or ascorbate have also been shown to minimize damage (George, 1996). Other methods that are known to reduce the incidence of browning in other species include activated charcoal in the media for Ensete (Zeweldu, 1998), oil palm (Teixeira et al., 1994) and apple and pear cultivars (Wang et al., 1994). The oxidation of phenolic compounds was shown to be controlled in banana clones using L-cysteine HCl at 40 mg/L incorporated in the media (Khatri et al., 1997).

1.7 Stages of Rooting and In Vitro Root Establishment

It is generally accepted that there are four distinct phases in the in vitro rooting process (Hartmann et al., 1990). These are the induction, initiation, organisation and growth or elongation phases. The difficulty in establishing roots in vitro with many woody Australian plants has been well reviewed (Williams et al., 1985; Gorst, 1981; Johnson, 1996). Generally, herbaceous and juvenile plants are easier to root (Hutchinson et al., 1985), with juvenility a key factor in ease of micropropagation of many taxa (George, 1996). Many studies report complete omission of cytokinins and increased auxin concentration for promoting in vitro root development. Johnson (1996) comments on the enhancement of rooting in Eremophila lanii taking place in the presence of cytokinins (1µM K and 1µM BAP) rather than an increase in auxin levels. Other research identifies inhibition of rooting, an increase in callus and deterioration of the shoot when explant material is exposed to auxins over extended periods (George, 1996). Rooting responses vary greatly between species with many species forming roots rapidly while others are much slower. Hartmann et al. (1990) reports that some plants root best if stored in darkness during the auxin treatment phase. Following this exposure to light is thought to degrade the auxin and enhance root initiation and growth (Fabijan et al., 1981). Some studies have suggested that a cytokinin carry over from the shoot multiplication phase is enough to suppress root development. Bunn et al. (1989) found that shoots of a difficult to root Australian species, Leucopogon obtectus, formed roots on a minimal medium containing only agar and water. Later studies on the Australian orchid Diuris by Collins and Dixon (1992) demonstrated that high levels of sucrose and the addition of activated charcoal (AC) to the media gave rise to root induction. For many Australian species the controls which direct adventitious rooting in vitro remains obscure and one of the most difficult areas of micropropagation research (Bunn and Dixon, 1996).

1.8 Plant Hormones used In Vitro

Plant growth regulators are a class of compounds, which are used in the canalization of plant development and growth in vitro. For a compound to be classed as a plant hormone it should have clear effects on plant growth at very low (micromolar) concentrations i.e. not at nutrient concentrations. The important growth regulators commonly used are auxins, cytokinins and gibberellins. The combination and concentrations of these in the tissue culture media have been

10

widely researched (de Fossard, 1976; Johnson, 1996). For development of new species in vitro, detailed assessment of the optimal ratios and concentrations of plant hormones are often required. 1.8.1 Auxins

Auxins are responsible for cell elongation and root induction. The most commonly used auxins applied in tissue culture include indole-3-butyric acid (IBA), indoleacetic acid (IAA), naphthaleneacetic acid (NAA), 2-naphthoxyacetic acid (NOA) and 2,4-dichlorophenoxyacetic acid (2,4-D). The major influences exogenous auxins play in plant development can be described in two ways. These are incorporation into the basal media or a pulse treatment applied at higher concentrations. Johnson and Burchett (1991) have shown that root induction in the Australian monocotyledon, Blandfordia grandiflora varies according to auxin type and concentration. The greatest number of roots for this species was produced on media containing 8µM IBA and 32µM IAA but the greatest number of plants to survive were produced on media with 2µM IBA and 0.5µM IAA. Short pulse treatments of auxins are used to overcome shoot deterioration and problems of excess callus formation. The pulse application can be done in two ways. Firstly shoots can have their cut ends placed in a solution of auxins before being transferred on to a weak medium with no added growth regulators. Alternatively, shoot material is placed on a medium high in auxins for a short period (usually several days until root induction takes place) and then transferred to an auxin free medium to allow roots to develop and grow (George, 1996). Incorporation of activated charcoal (AC) is often used to neutralize any excess auxins that may be carried over after the initial pulse phase. 1.8.2 Cytokinins

Cytokinins are responsible for cell division and stem growth. The mode of action is to release axillary meristems from apical dominance and cytokinins are therefore widely used in plant tissue culture for shoot multiplication (Taji and Williams, 1996; Hutchinson et al., 1985). The most commonly used

cytokinins are N6-benzylaminopurine (BAP), N6-isopentenyladenine (2iP) and kinetin. These are all potent growth regulators and work within a narrow concentration range (0.25 - 10µM) (Taji and Williams, 1996). The rates of multiplication obtained on shoots of Blandfordia grandiflora are described by Johnson and Burchett (1991). These show 2µM BAP, 8µM 2iP and 8µM kinetin all giving rise to rapid shoot proliferation on solid basal media. The use of 0.5µM BAP in liquid media was also shown to improve elongation and multiplication of shoots, giving rise to 30 shoots per explant in Blandfordia (Johnson, 1992).

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1.9 The Need for Commercialization of Doryanthes excelsa

For many years D. excelsa has been harvested from the wild to provide floral designers with dramatic feature flowers for large imposing hotel foyer arrangements (Figure 1.4). At present very few stems come from commercial row production with the vast majority of supply coming from bushland to the north of Sydney on the Mangrove Mountain, Peats Ridge and Somersby plateaux. The high returns for the cut flowers has created a situation where stems are being removed illegally from the roadside, private properties and national parks. This study will aim to investigate micropropagation methods that will lead to early maturation (time to flowering) of Doryanthes excelsa. Key study areas will include detailed investigation of source tissues including all components of the inflorescence likely to lead to establishment of explants and maintenance of the adult (non-juvenile) state. From the literature presented relating to other arborescent monocotyledons in this review, appropriate protocols will be devised to address all aspects of micropropagation of Doryanthes excelsa in vitro.

Figure 1.4. Flowering stems of Doryanthes excelsa featuring in an arrangement in the foyer of The Hotel Intercontinental, Sydney.

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2. Materials and Methods for Tissue Culture of Doryanthes excelsa 2.1 Introduction

This chapter details the general methodology of this study on Doryanthes excelsa (Corr.). The chapter is divided into sections dealing with collection of plant material, surface sterilization, antioxidant treatments to prevent tissue browning, tissue culture media and culture conditions. In vitro methods detail the initiation of cultures selected from immature inflorescence sections (from developing flower scapes) including axillary buds, single flower buds, flower bud clusters, ovary sections, anther filaments and pedicel and peduncle sections. Sterilization methods used include chemical treatment with sodium hypochlorite, followed by washes in sterile distilled water. Post sterilization treatments of explant tissues to control phenolic leakage are described. All new cultures are incubated in the dark for the first three weeks. Media are solidified with agar, unless otherwise stated, with media formula consisting of MS (Murashige and Skoog, 1962) used at full and 1/2 strength and B5 basal media (Gamborg et al., 1968). Liquid media (MS) were also used for some culture initiation. Cultures are incubated in plastic 30 ml tubes containing medium in a culture room with artificial illumination from fluorescent bulbs with average light intensity of 30-40 µMolm-2s-1. Tissue survival and morphogenic reactions to various media treatments were monitored recorded and analysed.

2.2 Explant Source and Selection of Doryanthes Cultures

Floral scape material (containing mainly immature inflorescences) from plants in the field are the main explant source type in this study. Six developing scapes were chosen and harvested from the wild at three locations on The Wildflower Farm in Somersby, NSW. Material was kept cool and moist after collection and the length of the scape trimmed down to 1.2 metre lengths. Explant material was selected which was juvenile, healthy and free of visible pests and disease. Due to quarantine regulations in Western Australia, material was treated with a post harvest insecticidal dip (deltamethrin), wrapped in ‘Long-life Film’ and packaged for transport from NSW to Kings Park & Botanic Garden, West Perth, WA. The immature inflorescences were enclosed within several bracts and were processed in the laboratory 48 hours after collection.

2.3 Disinfestation Procedures for Explant Material

The surface sterilization procedure began with dissection of explant material into manageable units. Stem sections (250 mm - 300 mm) containing axillary buds and immature inflorescences were treated by initially removing the small leaflets and cleaning away surface detritus under running tap water for 1 to 2 minutes. A plastic vessel (130 mm x 320 mm x 120 mm) was used for treatments with surfactant and sterilant solutions. Sterilization was undertaken for 15 minutes using a 1% v/v sodium hypochlorite (NaOCl) treatment with sufactant added (Tween-80 at 0.05%). Explants were transferred to a separate vessel for the washing phase in three changes of sterile distilled water.

2.4 Manipulation Procedures for Explants

Aseptic manipulation of explants involved trimming away damaged tissue after surface sterilization. Axillary buds, single flower buds, flower bud clusters, ovary sections and pedicel and peduncle sections were excised under aseptic conditions using sterilized scalpel and forceps. Instruments were flamed regularly and appropriate sterile techniques were employed throughout all manipulation procedures.

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2.4.1 Antioxidant Treatment

The extracted buds were placed in petri dishes containing an antioxidant wash of 0.125% potassium-citrate:citrate (K-C:C in a ratio of 4:1 w/w) solution. A concentrated stock of the antioxidant wash was filter sterilized and frozen in 10ml units until required. The concentrate was later thawed and further diluted with SDW to give the final 0.125% concentration. Petri dishes (90 mm x 14 mm) were filled with sufficient antioxidant solution to fully cover the explants. Peduncle sections were cut into discs under the antioxidant solution to minimise browning during initial preparation. Each explant was placed in 30 cc polycarbonate culture tube containing 20 ml of media after five minutes in the antioxidant treatment.

2.5 Incubation Conditions for Tissue Cultures

Cultures were maintained at mean temperatures of 23 ± 2ºC and photoperiod of 16 hours per day using 36 W cool white fluorescent tubes providing 30-40 µMolm-2s-1 PPFD (photosynthetic photon flux density) at culture level in a culture room. During the initial 3 weeks all cultures were wrapped with aluminium foil to provide darkness.

2.6 Culture Media

2.6.1 Basal Media

Half strength MS (Murashige and Skoog, 1962) basal salts were used for most culture initiation in this study. Other basal media used include full strength MS and B5 (Gamborg et al., 1968) basal salts (see Appendix 1). 2.6.2 Media Components and Preparation

All basal salt formulations were prepared in small volumes in concentrated form and frozen. Growth factors and plant hormones were prepared as stock solutions and added during preparation of media treatments. All components of the media, including sugar and agar, were dissolved in the desirable amount of tissue culture grade distilled water and the pH adjusted accordingly with NaOH or HCl prior to autoclaving. Heat stable hormones were added prior to autoclaving whilst heat sensitive hormones (IAA, 2iP and Zeatin) were filter sterilized using a 0.2 µM Millex ® millipore syringe-driven filter and added post-autoclaving. Basal salt formulation for all media are listed in Appendix 1. The following additions were made to the MS basal media: 3µM Nicotinic acid, 3µM Thiamine-HCl, 3µM Pyridoxine-HCl, 500µM meso-Inositol, 60mM sucrose and 100mg/L MES buffer (n-morpholinoethanesulfonic acid). The pH of all media was adjusted to 6.0 and gelled with 0.8% agar unless otherwise stated. The media were then autoclaved according to usual guidelines for autoclaving media (107 kPa and 121ºC) for 20 minutes. Filter sterilized components were added to the media after cooling to 50ºC and aseptically dispensed into pre-sterilized culture vessels (10 ml of medium per 30 ml polycarbonate tubes).

2.7 Liquid Media Pretreatment

Excised floral buds were incubated in liquid basal salts using full strength MS formulation. Three different treatment supplements were used (i) activated charcoal (AC at 0.5% w/v), (ii) ancymidol (20 µM) and (iii) paclobutrazol (PAC) (3 mg/L) respectively. Media were prepared according to the procedures previously outlined without gelling agent. Tissues were placed in glass screw-top jars of 250ml capacity containing 25 ml of liquid media and stirred (in darkness) on an orbital shaker at 100 rpm. Tissues were maintained under these conditions for 14 days with media replenished daily.

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Treated tissue were then subcultured to a solid media (MS) after 3, 7 and 14 days incubation in liquid pretreatments. Morphogenic responses to the various treatments were monitored and recorded.

2.8 Plant Growth Regulators

Growth regulators were added to all basal media (except where considered unnecessary and otherwise stated). For initiation, most explants are placed on 1/2 MS media supplemented with 10 µM BA (N6-benzylaminopurine) and 0.5 µM IBA (indole-3-butyric acid) with these heat stable auxins and cytokinins added before autoclaving. In other experimentation to assess various growth responses,

cytokinins are used singularly or together with auxins. Cytokinins used in this study included N6-

benzylaminopurine (BAP), N6-isopentenyladenine (2iP), 6-furfurylaminopurine (kinetin) and zeatin (Z). Auxins applied included indole-3-butyric acid (IBA), indoleacetic acid (IAA), naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D). The plant hormone Thidiazuron (N-phenyl-N-1,2,3,-thidiazol-5-ylurea) or TDZ was also used in this study. Plant growth regulators were prepared as stock solutions (see appendix 2) and stored at 5ºC until required. Concentrations for various experimental work are outlined in Chapter 5.

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3. Initiation and In Vitro Survival of Doryanthes excelsa Explants 3.1 Introduction

The physiological condition and developmental stage of the inflorescence is critical for obtaining a desirable in vitro response (Vasil and Thorpe, 1994). Selection of the ideal type of source material and the size of explant are some of the most important factors determining successful growth and development in vitro. The larger the explant the more determinative are its endogenic signals (Augé, 1995). Consequently the composition of the media, including exogenous growth regulators, may have only a limited influence on the tissue type or require prolonged exposure and incubation. Similarly, higher than average concentrations may be implemented to achieve organogenesis. Sterilization and establishment of aseptic cultures from tissues taken from whole plants grown in non-sterile conditions requires a great deal of vigilance. Stock plants from ex vitro conditions are invariably infested externally with fungi, bacteria and other contaminants. Most microbial organisms, particularly fungi and bacteria, compete adversely with plant tissue growing in vitro (George, 1996). It is therefore essential that superficial contamination be controlled or eliminated before the explants are introduced to the culture medium. A variety of sterilization methods have been successfully implemented for many Australian species (Johnson, 1996). The degree of surface infection of different tissue types and the response of various tissues to sterilants are highly variable. Investigation of disinfestation procedures to facilitate optimal in vitro tissue growth of Doryanthes excelsa was recognized as essential to successful culture. The process of identifying suitable explant source material and sterilization methods is the focus of this chapter. The specific aims of this component of the study are: • achievement of successful disinfestation of the full range of explant tissue types. • identifying which tissue types provide the best explant source material for establishment of cultures likely to lead to plant regeneration.

3.2 Materials and Methods

Collection of explant material and general methods are according to the procedures described in Chapter 2. The steps for tissue sterilization are detailed in Section 2.3. Cultures were examined and observations were made immediately after the first 3 days of incubation. Visual assessments of the tissue and media for signs of fungal and bacterial contamination were made at weekly intervals for 15 weeks. 3.2.1 Initiation of Explants

Six scapes comprising of mostly immature or unopened flower buds and axillary buds between stem and leaf sheaths were used to provide explants. Axillary buds were removed from directly below the developing flower bracts with the position of the bud on the stem noted. Outer leaf sheaths covering the buds were removed taking care not to damage the developing buds. Bract tissue was carefully peeled away to reveal developing flower buds of various ages. The apices were initiated as complete clusters while larger buds were initiated as single buds. A careful cut across individual buds exposed ovary tissues that were then cultured on basal media with added growth regulators. Figure 3.1, 3.2 and 3.3 details location of explants sources from inflorescence sections. The medium utilized for initiation was 1/2 MS and supplemented with 10 µM BA (N6-benzylaminopurine) and 0.5 µM IBA (indole-3-butyric acid) as is described in Section 2.6. Explant survival was determined as green viable tissues free of visible contamination and phenolic injury suitable for continued culturing.

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3.3 Results

The response of the different explant sources to the sterilization treatment of NaOCl (1%v/v) varied between tissue types. The highest survival was for the dissected single flower buds with tissues almost entirely free from observable contamination (97%). Ovary sections produced the next highest survival followed by flower bud clusters and peduncle sections. The excised axillary buds were the most contaminated with only 35% tissue survival after 15 weeks (Figure 3.3).

Figure 3.1. Location of explant source material in Doryanthes excelsa.

Figure 3.2. (A) infloresecence bud with sheathing bracts (bar = 10cm), (B) dissected floral bud enclosed in bract tissue (bar = 2cm), (C) immature bud cluster (bar = 1cm).

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Table 3.1. Tissue types and quantity initiated from D. excelsa each scape. Explant source No. of explants

initiated Mean No. of explants/stem

Axillary buds 110 18.3 ± 1.8 Peduncle sections 24 3.0 ± 0.6 Bud clusters 14 2.3 ± 0.4 Single buds 32 5.3 ± 0.3 Ovary sections 91 15.2 ± 1.5

0

20

40

60

80

100

axillary buds peduncle sections bud clusters single buds ovary sections

Explant sourc e

Figure 3.3. Percentage survival* of explants after 15 weeks. Note: * Survival was determined as green viable tissues free of visible contamination and phenolic injury suitable for continued culturing. Survival was based on a count of total number of surviving explants at the conclusion of experimentation in all treatments. The excised axillary buds found in the lower part of the inflorescence (region II) (Figure 3.1) showed a greater degree of contamination compared to explants selected from the upper region, where tissues were protected within the sheathing floral bracts (Figure 3.2). All tissue from region I gave survival values in excess of 71% indicating that the disinfestation process employed was less successful for explants derived from material found on the elongating scape (region II).

3.4 Discussion

Principal sources of contamination experienced when initiating new material in vitro result from residual microorganisms on explants or from inadequate laboratory hygiene or operator error (Barlass and Hutchinson, 1996). The sterilization procedure employed in this study is commonly used with many plant species and tissues to initiate explants to the culture medium. It proved to be successful for most inflorescence tissue of D. excelsa except axillary buds. This observation is similar to the results reported by Hussey (1975) with the induction of inflorescence explants in monocot families such as Liliaceae, Iridaceae and Amaryllidaceae. The objective of this initiation phase was to obtain a high percentage of viable explants in aseptic culture. The origin of tissue and developmental stage also determine the success of a culture (Guerra and Handro, 1998). Delicate explant tissue such as small shoot tips or immature flower parts within

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protective outer coverings are known to be free of superficial microbial contaminants and are considered ideal source tissue for culture induction (George, 1996). Amomarco and Ibanez (1998) demonstrated that inflorescence sections (as explant material) are easy to establish as aseptic cultures. In vitro establishment of bulbous plants using floral stems give less contamination of cultures than that of subterranean organs, i.e. bulb scales (Slabbert et al., 1995). The technique also offers convenience in micropropagation development as once the structure containing the explant source material (i.e. bract tissue) is surface sterilized the inflorescence tissues within can be dissected out under sterile conditions and placed directly into culture. From this study, sodium hypochlorite used at 1% v/v concentration was shown to be an effective sterilant on immature inflorescence tissue of D. excelsa. An advantage of using NaOCl is that it is cost effective, easy to prepare and can disinfest the surface of tissues effectively (Johnson, 1996). It is also considered safer than sterilants such as mercuric chloride and is generally effective against a broad range of microorganisms (Barlass and Hutchinson, 1996). Other sterilants such as calcium hypochlorite or benzalkonium chloride are reportedly less effective for highly contaminated tissue (George,1996). Excised axillary bud material from D. excelsa also proved to be non-sensitive to NaOCl but the degree of contamination in this tissue type (65%) would indicate that longer exposure time and more rigorous washing before sterilization may have resulted in a better survival rate. The efficacy of this treatment was similar to the successful initiation of similar monocotyledonous species where inflorescence stem sections were used e.g. Crinum macowanii, (Slabbert et al., 1995), Aloe sp., Gasteria sp. and Haworthia sp. (Richwine, 1995), Iris sp. (Meyer et al., 1975) and other members of the Liliaceae, Iridaceae and Amaryllidaceae (Hussey, 1975). The sterilization procedures outlined in this chapter were employed throughout all experimental work in this project. The use of sodium hypochlorite proved to be a cost effective method of sterilization and is readily obtainable. It was also proven to be effective on floral stem tissue contained within the protective bract tissue. The methods outlined for treating plant material with NaOCl, along with the selection of source material for D. excelsa is beneficial for culture induction as it is relatively easy to obtain isolated tissue types with minimal contamination

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4. Control of Tissue Browning during Explant Preparation 4.1 Introduction

The abrupt demise of in vitro cultured explants, accompanied by darkening of culture medium has been attributed to phenolic compounds exuded from tissues and accumulating in the culture medium. This process is initiated by browning of the surface of plant tissues due to the oxidation of phenolic compounds resulting in the formation of quinones which are highly reactive and toxic to plant tissue (Zaid, 1987; Salisbury and Ross, 1992; Taji and Williams, 1996). Understanding the processes contributing to the oxidation of phenols and how these can be minimized when initiating explants is critical for successful in vitro culture particularly with woody species. Removal of the phenols as they are formed is essential for growth and establishment of explants as the presence of phenolic substances often causes explant material to create more exudates (i.e. autocatalytic reactions). Various methods for control and minimization of phenolic injury have been reported in the literature. These include removal of phenolic compounds through rigorous washing, repeated washing with antioxidants, inclusion of activated charcoal in the media and incorporation of cysteine HCl and other chemicals into the culture medium as well as frequent transfer of cultures to fresh media (Zaid, 1987; George, 1996; Taji and Williams, 1996). Phenolic compounds consist of a benzene ring with various substitute groups attached. At least one hydroxy group is present (Zaid, 1987; Salisbury and Ross, 1992; Taji and Williams, 1996). These are strong reducing agents and when phenolic compounds react with certain enzymes, the hydroxy group is oxidized resulting in the formation of quinones and water (Taji and Williams, 1996). The process of oxidation is initiated when a compound loses an electron while the process of reduction is the reverse i.e.. the gaining of electrons. Redox reactions occur simultaneously in a variety of chemical reactions under well controlled conditions in plant tissues. Phenols and phenolic oxidases in healthy plant tissue are usually compartmentalised. Tissue blackening results when decompartmentalisation occurs through wounding or during senescence and the oxidation process is initiated and continues as a chain reaction (Zaid, 1987). During this process irreversible hydrogen bonding of phenols to proteins may occur inhibiting enzyme activity and ultimately leading to cell death. It has been suggested that thorough rinsing after sterilization is essential to wash away biocidal chemicals that may have antagonistic effects on plant tissue. Many chemicals used on delicate material should be only applied for a short duration as damage by sterilants may provoke the synthesis of phenolics in some species. Furthermore some of the sterilants being oxidants may enhance the phenol oxidation. Prolonged rinsing of sterilized explants also removes any oxidized phenols that may have begun to accumulate. High levels of phenolic oxidation appear to be widespread across many arborescent monocot species e.g. oil palm (Teixeira et al., 1994), Ensete (Zeweldu et al., 1998) and banana clones (Khatri et al., 1997; Zaid, 1987). The determination of an appropriate protocol to combat phenolic oxidation is essential before successful in vitro induction can proceed. A study by Panaia (1998) on Symonanthus bancroftii demonstrated that control of phenolic exudates with a 0.1% solution of antioxidant potassium-citrate:citrate was pivotal to the successful culture of this critically rare Australian species. D. excelsa stems are susceptible to tissue browning and elimination or minimization of this process is an essential prerequisite to successful culture establishment. Therefore identification of a suitable treatment to minimize tissue browning in the various explant source material of D. excelsa will be the focus of this chapter. The specific aim of this component of the study was to: • research methods for reducing phenolic induced injury in D. excelsa during explant preparation, with particular emphasis on the use of appropriate antioxidant treatments.

20

4.2 Materials and Methods

Collection of explant material and general methods were according to the procedures described in Chapter 2. The following materials and methods outlined are for the detection of phenolic compounds in plant tissue and antioxidant experimentation. 4.2.1 Detection of Phenolic Compounds using Sodium Hydroxide (NaOH)

Detection of phenolic leakage was tested in the study species Doryanthes excelsa and an Australian woody species Corymbia ficifolia (Myrtaceae). C. ficifolia was selected because it is known to contain high levels of phenolic compounds and would provide evidence that a phenolic reaction will occur under experimental condition (Panaia, 1998). Flowering stems of D. excelsa were collected in Somersby NSW and flown to Kings Park and Botanic Gardens Plant Science laboratory for assessment while material of C. ficifolia was collected within the confines of Kings Park and Botanic Gardens. Concentrations of sodium hydroxide (NaOH) were made at rates 1M, 0.1M, 0.01M and 0.001M to treat the various plant tissues. NaOH oxidizes phenols which causes darkening of affected or damaged tissue (Panaia, 1998). Peduncle sections and pedicel slices of D. excelsa were cut in 2 mm thick discs and submerged in petri dishes containing the concentrations of NaOH for 5 min. Similarly, bract tissue was cut into squares approximately 15 mm x 15 mm and also placed in the NaOH solutions while whole leaves of C. ficifolia were submerged in the various treatments. Observations were made of any browning on the surface or cut edges of the various plant tissue to assess if phenolic leakage had occurred. 4.2.2 Antioxidant Experiment

Pedicel and peduncle disc sections from D. excelsa were collected from a mature flower stem and treated with various antioxidant solutions (Table 4.1). Disc sections were selected as they have a large surface area and have been shown to be prone to oxidation. Table 4.1 Antioxidant treatments Treatment Number

Treatment

Volume of antioxidant treatment (ml)

1 Exposed to air, cut on wet filter paper 0 2 Cut in petri dish plus H2O (SDW) 100 3 Cut in petri dish plus H2O (reverse osmosis) 100 4 Cut in K-C:C 0.125% w/v 100 5 Cut in K-C:C 0.125% w/v, + L-cysteine 100 6 Cut in K-C:C 0.125% w/v + L-cysteine + ascorbic acid 100

see abbreviations page A stock solution of potassium citrate and citrate (K-C:C) was made up using 1g K-C and 0.25g citrate and dissolved in 10 ml of SDW. The concentrate was then diluted and used at a final concentration of 0.125%. For treatments 5 and 6, 0.02g /L L-cysteine HCl was added to the 0.125% solution of K-C:C and 0.25g/L ascorbic acid was added to treatment 6. One hundred millilitres of the various solutions were used to fully cover the disc sections with the control treatment cut on filter paper and exposed to air. All other material was cut under the various treatments to avoid exposure to the air. The prepared disc sections were placed onto water agar petri dishes and results were recorded at time at intervals of 0, 7, 30, 60 and 120 minutes. Observations of the extent of browning were recorded.

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4.2.3 Potassium Citrate and Citrate (citric acid) as Antioxidant Treatments for Excised Tissue.

Methods are according to the procedures described in Section 2.4.1. Cultures were examined and observations were made immediately after the first day in vitro and then daily. Tissues were examined and the degree of darkening of tissues and media was recorded.

4.3 Results

4.3.1 Detection of Phenolic Compounds using Sodium Hydroxide (NaOH)

Leaves of C. ficifolia showed signs of phenolic leakage in the higher concentrations of NaOH. Pedicel and peduncle sections produced a large degree of discolouration after having been sliced into discs and placed in the NaOH (Table 4.2 and Figure 4.1). The bract tissue of D. excelsa developed a distinct green/brown line, approximately 2 mm wide around the cut edges (Figure. 4.2). Table 4.2 Degree of tissue discolouration after incubation in NaOH solutions for D. excelsa and C. ficifolia (Myrtaceae).

Species Control (SDW)

0.001M NaOH

0.01M NaOH

0.1M NaOH

1M NaOH

D. excelsa Peduncle sections

Ο

Ο

+

++

+++

Pedicel sections Ο Ο + ++ +++ Bract tissue Ο Ο + ++ +++ C. ficifolia Leaves

Ο

Ο

Ο

++

+++

Key: Ο no discolouration + low discolouration ++ medium discolouration +++ high (darkly stained)

4.3.2 Antioxidant Experiment

All cut surfaces in the control appeared to oxidize rapidly once exposed to air as evidenced by tissue browning. Subsequently all other tissues were prepared under each of the antioxidant treatments. Pedicel sections showed similar rates of browning to excised flower bud material when exposed to air without antioxidant treatment (Figure 4.3). Treatments 4, 5 and 6 initially reduced browning of the disc sections and after 2 hours, treatment 4 (K-C:C) was visually better than the other 2 antioxidant treatments (Table 4.3 and Figure 4.3). Table 4.3. Relative browning of disc sections of D. excelsa treated with antioxidants over a two hour period.

Treatment Number Time (min) 1 2 3 4 5 6 0 +++ ++ + Ο Ο Ο 7 +++ +++ ++ Ο + Ο 30 +++ +++ +++ Ο + + 60 +++ +++ +++ Ο ++ ++ 120 +++ +++ +++ Ο +++ +++

Key: Ο no oxidation; + low oxidation; ++ medium oxidation; +++ high oxidation.

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Treatment numbers: 1 Exposed to air, cut on wet filter paper. 2 Cut in petri dish plus H2O (SDW). 3 Cut in petri dish plus H2O 4 Cut in K-C:C 0.125% w/v. 5 Cut in K-C:C 0.125% w/v, + L-cysteine. 6 Cut in K-C:C 0.125% w/v + L-cysteine + ascorbic acid.

4.3.3 Potassium Citrate-Citrate combinations as Antioxidant Treatments for Excised Tissue

All tissues initiated into culture were treated with the K-C:C treatment as it proved to be the best treatment type from experimental results (Table 4.3). The antioxidant treatment reduced browning in all tissue types after 24 hours in culture. The cut surfaces and any damaged areas of untreated tissue (particularly peduncle sections) turned brown within 15 minutes after the excision from the developing scape. These explants continued to oxidize under culture conditions and were completely brown after 1 hour and were subsequently discarded. After 4 weeks of culture the phenolic leakage in treated tissues was minimized and in most cases controlled. Many of the explants had remained pale while others had started showing signs of greening. Some minor staining of the media was evident in some explant tissues predominantly in peduncle sections. Treatment of tissue with antioxidant K-C:C greatly reduced the incidence of browning in all explant sources.

Figure 4.1. NaOH mediated detection of phenolics in pedicel and peduncle sections of D. excelsa. Note: Treatments exposed to high concentrations of NaOH exhibited a greater degree of browning than the sterile water (control).

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Figure 4.2. NaOH mediated detection of phenolics in bract tissue of D. excelsa. Note: Treatments at 1.0M, 0.1M NaOH showed a distinct green/brown margin on the cut surfaces.

Figure 4.3. Results of antioxidant experiment after 120 minutes. See Table 4.1 for treatment details. The K-citrate and citrate (K-C:C) solution minimized browning while other treatments were not as effective in reducing phenolic oxidation. (bar = 2 cm)

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Figure 4.4. Excised inflorescence material prepared for culture. Explants on the right are treated with K-citrate and citrate while untreated tissues are shown on the left. (bar = 1 cm)

4.4 Discussion

One of the most common problems associated with the in vitro establishment of many monocotyledonous and woody species is the deleterious effects of oxidized phenols (Vasil and Thorpe, 1994; Teixeira et al., 1994; Khatri et al., 1997; Zweldu and Ludders, 1998). The results of the detection of phenolic compounds experiment (Table 4.2; Figure 4.1, 4.2 and 4.4) clearly indicate that D. excelsa has phenolic compounds present in inflorescence tissue. Similarly, C. ficifolia exhibited signs of phenolic leakage (Table 4.2) indicated by the discolouration on the leaf’s surface and leakage of a yellow halo in the NaOH solution consistent with presence of phenols in a basic solution. This experiment provides a simple technique for detecting the presence of phenolic compounds in D. excelsa inflorescence tissue. This procedure assists in early detection of phenols (Panaia, 1998) and assists in preparation of explant source material to reduce injury associated with phenolic oxidation. George (1996) describes an antioxidant as an electron donor (reducing agent) which inhibits the oxidation of labile substrates. It is suggested that prevention of oxidation of phenols through the use of antioxidants is attributed to the modification of the redox potential and therefore prevents blackening of isolated plant tissue. Blackening of the tissue is thought to be facilitated by the activity of oxidase enzymes (polyphenol oxidases). Arresting the rate of oxidation in plant tissues susceptible to browning has been successfully treated by washing the excised tissues in antioxidant compounds. The antioxidant compounds utilized in the experimental work in this chapter were selected because they have been used successfully in the past to delay browning in other arborescent monocotyledonous species (Drew, 1986; Zaid, 1987; Khatri et al., 1997). George (1996) details the use of citric acid and ascorbic acid combinations to delay browning. The successful prevention of browning in explants of

25

Musa textilis by using a mixture of ascorbic acid, citric acid and cysteine are reported by Mante and Tepper (1983). The behaviour of the citrate in citric acid works as a chelating agent bonding to ions responsible for activating polyphenol oxidative enzymes (George, 1996). Ascorbate behaves as a reducing agent and is converted to dehydro-ascorbic acid (Panaia, 1998). Ascorbate is able to scavenge oxygen radicals produced when tissue is damaged and therefore cells are protected from oxidative injury. Oxygen radicals are attributed to exacerbating oxidative injury. Antioxidants containing citrate and ascorbate reduce browning of tissue by detoxifying these free radicals. The results provide evidence that K-C:C combination is a useful antioxidant for explant preparation for Doryanthes excelsa (Figure 4.4). Source tissue must be prepared and immersed in the treatment solution during excision to avoid exposure to the atmosphere thus avoiding the initiation of oxidation reactions. Oxidation rates are influenced by the amount of available oxygen and minimizing exposure to air (oxygen) will reduce the rate of oxidation. The rate of adsorption in liquid is slow and subsequently preparation of explants immersed in liquid will also delay the oxidation process. Efficacy of the K-C:C treatment may be further enhanced if tissue is treated for several hours in the solution. Also, future experimentation with antioxidants incorporated in the culture media may further minimize explant demise through polyphenolic oxidation. Similarly, in further culture initiation, peduncle sections should be embedded slightly under the agar to minimize exposure to the air and therefore reduce the incidence of browning.. The K-C:C treatment investigated in this study showed that the pH of the antioxidant was acidic (pH 5) which would assist in preventing oxidative enzymes becoming active, and that reducing agents in solution considerably minimized the degree of browning of sectioned tissues. Although the nature of the compounds, which exuded from D. excelsa inflorescence tissue, was not tested in this study they are assumed to be a mixture of phenolic compounds. From this research using K-C:C as an antioxidant, many of the explants became green after four weeks of culture indicating that there was a reduction or cessation in the phenol oxidation activity. The potassium component in the treatment acts by competing with oxidative enzymes responsible for tissue browning and buffering the solution (Panaia, 1998). Many proteins show an affinity for potassium and by binding to them become active enzymes due to a change in their configuration (George, 1996). It is this action, competition for proteins, that is believed to be responsible for slowing or minimizing the process of oxidation. The incubation of cultures in the dark for the first three weeks also contributed to the suppression of tissue browning. Drew (1986) reports that maintaining Musa sp. callus in the dark is an effective method to suppress browning as enzymatic activity is greatly reduced in the dark as does Stimart (1986) for callus and shoot proliferation in Hosta sp. The results from this study indicate the browning phenomenon in D. excelsa tissue can be greatly reduced by pre-soaking of explants in antioxidant solution of 0.125% w/v potassium citrate and citrate prior to culture. Also, incubation in the dark for the first 3 weeks may arrest the rate of tissue browning by slowing the enzymatic activity responsible for tissue oxidation. Frequent subculturing to fresh medium may also assist so that toxic phenolic compounds do not hinder the activity of plant growth regulators on tissues. The combined effects of the treatments outlined above proved beneficial to explant survival in vitro of Doryanthes excelsa inflorescence tissue.

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5. Interaction of Inductive Growth Regulator Signals on Explant Growth 5.1 Introduction

The development of a suitable nutrient media is considered to be one of the most crucial phases in in vitro growth and differentiation of excised plant tissue (de Fossard et al., 1974; Williams et al., 1985; Johnson, 1996). The factors affecting growth of plant tissues, organs and cells in vitro are similar to those limiting growth of whole plants in vivo (Leifert et al., 1995). The precise nutritive requirements for many Australian species remains largely unknown, which is an added complication when dealing with the establishment of micropropagation protocols and the derivation of appropriate basal media (Bunn and Dixon, 1996). Many basal media are modified to facilitate and improve growth of plant cells and tissues. It is regarded as a common procedure to compare previously published nutrient and hormone compositions for closely related species and then modify the nutrient and growth regulator constituents to maximize in vitro development of the target species. Murashige and Skoog (1962) basal medium, or modified versions, are the most frequently encountered basal media used for micropropagation of Australian species (Williams et al., 1985; Taji and Williams, 1996; Gorst, 1996; Bunn et al., 1989; Bunn and Dixon, 1996). The selection of a basal medium is considered by many as straightforward, however, some deleterious responses to certain media can occur leading to shoot death or browning (Bunn & Dixon; 1996). The influence of plant growth regulators on various tissue types and organs is widely reported in the literature. Plant growth regulators such as cytokinins, auxins and gibberellins and their interactions with plant tissue types determine the response of growth in vitro. Many studies have assessed hormone ratios and concentrations to maximize in vitro development. de Fossard et al (1974) developed a broad spectrum strategy for testing media constituents so that a systematic approach to media formulation for previously untried plant species can progress. A large majority of in vitro research into Australian plant species has concentrated on growth responses to plant hormones at different stages of tissue development (Johnson, 1996). Researchers often extrapolate responses to related genera or species but care is needed when interpreting such data as optimal growth rates may be quite different and require altered levels of cytokinins, auxins and other components to achieve optimal in vitro growth for the target species. The aims of this study were to investigate an appropriate basal medium composition in conjunction with a cytokinin and auxin ratio likely to facilitate successful in vitro regeneration from inflorescence tissue of D. excelsa.

5.2 Materials and Methods

Preparation of media and general methods were according to procedures described in Chapter 2. The following methods outlined are for testing a variety of plant growth regulators on various tissue types from different sources. All treatments were maintained for various incubation periods on the different media types and tissue survival and morphogenic reactions to various treatments were monitored and recorded weekly. Twenty explants per treatment (with 10 explants per replicate) were examined on each medium with a minimum of ten explants per treatment (no replicates) where material was limited. All abbreviations used are stated in the preface. Tissue types cultured from experimentation in Chapter 3 (Section 3.2.2) were held as stock cultures and maintained on 1/2 MS supplemented with 10 µM BAP (N6-benzylaminopurine) and 0.5 µM IBA (indole-3-butyric acid) for the duration of this study (32 weeks). This media was successful in initiating direct adventitious shoots from flower explants of Blandfordia (Bunn and Dixon, 1996). A trial was also established to examine lower concentrations of growth regulators on immature flower bud explants. Basal media (1/2 MS) was supplemented with 5 µM BAP and 0.25 µM IBA for 8 weeks

27

of in vitro culture. The explant material incubated on this medium, along with all other cultures listed were subcultured at three to four weekly intervals. Regular inspections were made and morphogenic reaction to the culture media was measured and recorded. Summaries of results were recorded at the termination of this study and appear in Tables 5.6 to 5.11. Survival was scored as a percentage and defined as green, viable tissue suitable for continued culturing. Explant enlargement was measured based on an increase in volume of explant tissue from initiation after continued culture on the various media types. Results are recorded as the number of explants exhibiting this enlargement (expressed as a percentage). 5.2.1 Response of Various Explants to Media Treatments

Single flower buds, ovary sections, peduncle disc section and axillary buds were sourced from established tissues proliferated on BAP 10µM and IBA 0.5µM for 14 weeks prior to transfer to the media treatments detailed in Table 5.1. Table 5.1 Media composition and plant growth regulator concentrations used on in vitro grown tissue. Explants incubated on BAP 10 µM and IBA 0.5 µM for 14 weeks prior to induction.

Basal media

Cytokinin Conc. µM) Auxin Conc. µM)

1/2 MS kinetin 5 IAA 0.25 1/2 MS 2iP 5 IAA 0.25 1/2 MS kinetin 10 IAA 0.5 1/2 MS Zeatin 10 IAA 0.5 1/2 MS 2iP 10 IAA 0.5 B5 kinetin 5 IAA 0.25 B5 2iP 5 IAA 0.25 B5 kinetin 10 IAA 0.5 B5 Zeatin 10 IAA 0.5

Further investigation of different media was undertaken using single flower buds and peduncle disc sections. These tissues were sourced from in vitro grown explants cultured as above for 22 weeks, enlarged bract tissue was dissected and treated as explant source material. Basal medium and plant growth regulators utilized in this experiment are detailed in Table 5.2 and 5.3. Table 5.2. Plant growth regulators (PGR) used with or without activated charcoal (AC) for 22 weeks prior to induction in conjunction with MS basal media for the culture of explants incubated on BAP 10µM and IBA 0.5µM

PGR Conc. (µM) kinetin 20, 50, 100 2,4-D 0 + AC, 20 + AC, 50 + AC, 100 + AC, 500 + AC 2,4-D 20, 50, 100, 500

5.2.2 Culture Establishment of Mature Flower Tissue

Inflorescence material was collected from the wild in July 1999 (details described in Section 2.2) when flowers were at maturity (Figure 1.4and Figure 5.1). Experimentation was undertaken to examine anther filaments, bases of the style and ovary sections of mature flowers as explant source material. Anther filaments were dissected into 2 mm pieces and cut longitudinally with the cut surface in contact with the medium. Ovary sections were cut in discs of 2 mm thickness. Basal medium and plant growth regulators utilized in this experiment are detailed in Table 5.4. Where indicated, activated charcoal (AC) was incorporated in the media at 0.1% w/v. Observations were made weekly to assess tissue survival (i.e. maintenance of tissue integrity, asepsis and absence of oxidation) and morphogenic changes recorded.

28

Figure 5.1. Location of explants (anther filament sections) from mature flower of Doryanthes. (bar = 10 mm) Table 5.3. Kinetin and NAA combinations used on explants derived from inflorescence tissues incubated on BAP 10 µM and IBA 0.5 µM for 22 weeks prior to induction.

Basal media

Cytokinin Conc. (µM)

Auxin Conc (µM)

MS Kinetin 20 NAA 10 MS Kinetin 50 NAA 20

dissected anther filament

longitudinally cut anther filament

(cut surface placed face down on media)

29

Table 5.4. Media composition used for culture initiation from mature flower parts.

Basal media

PGR Conc. (µM)

Auxin Conc. (µM)

1/2 MS kinetin 10 IAA 0.5 1/2 MS 2 iP 10 - - MS BAP 10 IAA 1.5 MS kinetin 20 - - MS kinetin 100 - - MS - - 2,4-D 20 MS - - 2,4-D 20 + AC

5.2.3 Induction of Immature Flower Buds to Different Pulse Treatments

Freshly excised immature single flower buds were cultured on various plant growth regulators (outlined in Table 5.5). Culture exposure times on the different media were for periods of four, ten and 20 days. Activated charcoal was added to the various concentrations of 2,4-D at 0.1% w/v. Tissues were transferred to MS basal media with no added growth regulators where tissue survival and morphogenic reactions were measured. Table 5.5. Plant growth regulators used in conjunction with MS basal medium for culture induction and pulse treatments of immature flower buds with or without activated charcoal (AC).

PGR Conc. (µM)

Auxin Conc. (µM)

- - 2,4-D 20 + AC - - 2,4-D 50 + AC - - 2,4-D 100 + AC - - 2,4-D 500 + AC kinetin 20 IAA 10 kinetin 50 IAA 25 kinetin 100 IAA 50 kinetin 50 - - kinetin 100 - - TDZ 50 - -

5.2.4 Liquid Media Pretreatment

Methods and liquid pretreatment types are according to procedures described in section 2.7. After the liquid pretreatment, cultures were incubated on MS basal medium supplemented with 20 µM kinetin. Results of treatments were recorded weekly for four weeks

5.3 Results

Explant material cultured on 1/2 MS supplemented with 5 µM BAP and 0.25 µM IBA exhibited only minor enlargement compared to the cultures maintained on 1/2 MS with 10 µM BAP and 0.5 µM IBA. The explants on the higher hormone rates showed swelling and enlargement in all tissue types. Development of small floral structures from inflorescence explants were observed during the culture period, particularly from single flower bud source tissue. These developed as pale globular outgrowths after 3 weeks of in vitro culture (Figure 5.2) and continued to develop into immature flower buds with attached bracts. These buds and bract tissue were dissected and further used as source material for various media testing. One explant from this treatment, a peduncle disc section, developed de novo growth with green globoid bodies emerging from the upper cut surface after 24 weeks of culture (Figure. 5. 3).

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5.3.1 In Vitro Growth of Plant Material in Response to Media Treatments

The growth regulators tested on all tissue types produced a high percentage of tissue survival which was maintained throughout the culture period of 16 weeks (Table 5.6). Peduncle disc sections had the highest response (explant enlargement) on both 1/2 MS and B5 basal media supplemented with 10 µM Zeatin and 0.5 µM IAA. The enlargement in these explants was predominantly from the epidermal region or rind of explants rather than the adjacent pith tissues. Seventy percent of single flower bud explants on B5 media supplemented with 10 µM Zeatin and 0.5 µM IAA showed visible signs of expansion and swelling throughout the culture period. None of the explants cultured for the 16 week period showed signs of de novo development.

Figure 5.2. Development of de novo flower buds (arrows) emerging from in vitro grown immature flower buds after 3 weeks culture. (bar = 10mm)

Figure 5.3. Globoid outgrowths (arrow) emerging from the upper cut surface of a peduncle disc section after 24 weeks of in vitro culture. Tissue incubated on 1/2 MS basal media with 10 µM BAP and 0.5 µM IBA. (bar = 2mm)

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Table 5.6. Response and in vitro survival of D. excelsa explants to different concentrations of plant growth regulators after 16 weeks of culture. Explants incubated on BAP 10 µM and IBA 0.5 µM for 14 weeks prior to induction.

Basal media

Cytokinin Conc. (µM)

Auxin Conc. (µM)

Source tissue

Mean survival (%)

Mean explant enlargement (%)

1/2 MS kinetin 5 IAA 0.25 SB 83 46 1/2 MS 2iP 5 IAA 0.25 SB 70 61 1/2 MS 2iP 5 IAA 0.25 AB 78 34 1/2 MS kinetin 10 IAA 0.5 OS 69 31 1/2 MS Zeatin 10 IAA 0.5 OS 60 25 1/2 MS Zeatin 10 IAA 0.5 PD 100 100 1/2 MS 2iP 10 IAA 0.5 OS 82 47 B5 kinetin 5 IAA 0.25 AB 83 50 B5 2iP 5 IAA 0.25 AB 100 37 B5 kinetin 10 IAA 0.5 OS 71 38 B5 Zeatin 10 IAA 0.5 OS 73 60 B5 Zeatin 10 IAA 0.5 SB 70 70 B5 Zeatin 10 IAA 0.5 PD 100 100

Key: SB = single bud; OS = ovary section; PD = peduncle disc section; AB = axillary bud. Note: No de novo growth was observed in any treatments after 16 weeks of in vitro culture. Table 5.7 summarizes survival and explant enlargement of various tissue types cultured from in vitro grown material. A high percentage of explant survival was evident in all treatments except for single flower buds on high concentrations of 2,4-D. Peduncle disc sections showed higher survival rates and tissue expansion compared to the single buds cultured on the same auxin concentrations. All single flower buds cultured on 500 µM 2,4-D and 500 µM 2,4-D with 0.1% AC died within the first week of exposure to this treatment. A high percentage of single flower buds and bract tissue showed signs of enlargement on the kinetin treatments at 20 and 100 µM concentrations (Table 5.7). After the nine week culture period no de novo development was observed. Response of single flower buds to kinetin and NAA are outlined in Table 5.8. Callus like growth developed in one explant on 20 µM kinetin plus 10 µM NAA after 7 weeks of culture. This callus developed from the basal tissue of the flower bud and continued to expand 50% in size over the following 2 week period of the trial. Elongated, torpedo-like callus tissue was observed after 9 weeks. The appearance of this callus may be the early development of shoot like structures (Figure 5.5). All explants survived the high level kinetin and NAA exposure with 80% of the single buds showing enlargement and visible swelling. Apart from the single explant showing callus like development no other explants exhibited de novo growth in the nine week culture period.

32

Table 5.7. Response and in vitro survival of different explant material of D. excelsa to various plant growth regulator (PGR) treatments and MS basal media after 9 weeks culture. Explants incubated on BAP 10µM and IBA 0.5µM for 22 weeks prior to induction.

PGR Conc. (µM)

Source tissue

Mean survival (%)

Mean explant enlargement (%)

kinetin 20 SB + bracts 89 67 kinetin 50 SB + bracts 56 42 kinetin 100 SB + bracts 82 77 2,4-D 0 + AC SB 58 3 2,4-D 0 + AC PD 62 10 2,4-D 20 + AC SB 56 51 2,4-D 20 + AC PD 70 31 2,4-D 50 + AC SB 75 60 2,4-D 50 + AC PD 71 59 2,4-D 100 + AC SB 79 68 2,4-D 500 + AC PD 71 59 2,4-D 500 + AC SB 0 0 2,4-D 20 SB + bracts 39 30 2,4-D 50 SB 12 0 2,4-D 50 PD 76 29 2,4-D 100 SB 14 0 2,4-D 100 PD 56 56 2,4-D 500 SB + bracts 0 0

Key: SB = single bud + dissected bract tissue; PD = peduncle disc section; AC = activated charcoal incorporated in the media at 0.1 % Note: No de novo growth was observed in any treatments after 9 weeks of in vitro culture. Table 5.8. Response and in vitro survival of immature flower buds of D. excelsa to kinetin and NAA treatment after 9 weeks of culture. Explants incubated on BAP 10 µM and IBA 0.5 µM for 22 weeks prior to induction.

Basal media

Cytokinin Conc. (µM)

Auxin Conc. (µM)

Source tissue

Mean survival (%)

Mean explant enlargement (%)

MS kinetin 20 NAA 10

SB 94 50*

MS kinetin 50 NAA 20 SB 100 80 Key: SB = Single buds. Note: * One explant from this treatment gave callus-like growth after 9 weeks of in vitro culture. 5.3.2 Response of Mature Flower Tissues to Culture Treatments

From Table 5.9 seventy three percent of mature ovary sections responded positively to the MS basal media supplemented with 20 µM kinetin and no added auxin. The thickness of the disc sections in these tissues had enlarged to 4 mm indicating a direct reaction to the growth regulator. Figure 5.4.a and 5.4.b shows the enlargement (thickening) of the epidermal tissue and the formation of clusters of opaque globoid cells from around the ovary walls. These pale structures developed after 8 weeks of culture and were similar to the early structural developments observed in the peduncle disc section where green globoid bodies developed from the pith region (Figure 5.3). After 12 weeks of in vitro culture, anther filaments and the bases of the styles had survival rates greater than 50% on the various treatments except for explants cultured on 100 µM kinetin. Only 3% and 5% of anther filaments showed signs of tissue expansion on 20 µM 2,4-D and 20 µM 2,4-D plus AC respectively. No de novo growth was observed on any treatment after 12 weeks of in vitro culture.

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Table 5.9. Response and in vitro survival to different concentrations of plant growth regulators on dissected anther filaments, style bases and ovary sections from mature flowers of D. excelsa.

Basal media

Cytokinin Conc. (µM)

Auxin Conc. (µM)

Source tissue

Mean survival (%)

Mean explant enlargement (%)

1/2 MS kinetin 10 IAA 0.5 AF 82 0 1/2 MS 2 iP 10 - - S 71 18 MS BAP 10 IAA 1.5 AF 87 0 MS MS

kinetin kinetin

20 20

- -

- -

AF OS

53 91

0 73

MS kinetin 100 - - AF 20 0 MS - - 2,4-D 20 AF 52 3 MS - - 2,4-D 20 + AC AF 80 5

Key: AF = anther filament; S = base of style; OS = ovary section; AC = activated charcoal incorporated in the media at 0.1 %.

Figure 5.4a. Pedicel disc section from mature flower of D. excelsa. Ovary tissue exposed with clusters of new cells (arrows) developing externally around the ovary wall. (bar = 3 mm)

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Figure 5.4b. Enlargement of pedicel disc section from mature flower of D. excelsa. Note the clustering of newly formed cells (arrows) around the ovary wall and the enlarged epidermal tissue (bar = 1 mm)

Figure 5.5. Callus growth developing in vitro from the base of an immature single flower bud. (bar = 1 mm)

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Figure 5.6. Pink elongating structures developing from the top of a flower bud cultured for a four day pulse on growth regulator TDZ. (bar = 1 mm) 5.3.3 Response of Explant Tissues to Hormonal Pulse Treatments

All immature flower buds cultured had greater than 50% tissue survival after four days exposure to the different growth regulators except for the explants exposed to 50 µM kinetin and 25 µM IAA (Table 5.10). Visible enlargement was noted in 100% of explants exposed to the four day pulse treatments of 20 µM 2,4-D and 50 µM TDZ after 6 weeks in vitro culture on MS basal medium with no added hormones. One explant from the four day TDZ pulse treatment developed opaque and pink elongated structures around the top section of the immature flower bud (Figure 5.6). These were discovered during the final stages of the 6 week culture period and are thought to be the early signs of somatic embryos. No other explants showed de novo growth from this trial. The results of the 10 day incubation (pulse) on different growth regulators is also detailed in Table 5.10. The prolonged exposure to the TDZ and high concentrations of 2,4-D had deleterious effects on explant survival with only 14% of explants surviving on 100 µM 2,4-D +AC and none surviving on the 500 µM 2,4-D +AC or TDZ treatments. All the explants treated on the 10 day pulse of 20 µM kinetin and 10 µM IAA and 20 µM 2,4-D +AC showed positive reactions to these growth regulators observed as tissue greening and expansion of perianth tissue of the immature flower bud. The 10 day exposure trial gave no de novo growth after 6 weeks of culture. The 20 day incubation period on the various media constituents are outlined at the bottom of Table 5.10. Similar to the results of the 10 days exposure trial, the explant survival on the TDZ and high concentration of 2,4-D were damaging to the immature flower buds with none of these explants surviving the exposure times. All explants surviving induction on the pulse treatments of 20 µM kinetin, 20 µM kinetin plus 10 µM IAA and 50 µM 2,4-D showed a marked increase in size compared to the original explant cultured. No callus growth or other de novo structures were observed after the 6 weeks culture period that followed the pulse exposures.

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Table 5.10. Response and in vitro survival of immature flower buds of D. excelsa to various plant growth regulators and MS basal medium for varying incubation periods. Explants cultured on MS media for 6 weeks after treatment

Cytokinin Conc. (µM)

Auxin Conc. (µM) Mean survival (%)

Mean explant enlargement (%)

4 DAYS - - 2,4-D 20 + AC 100 100 - - 2,4-D 50 + AC 50 50 - - 2,4-D 100 + AC 50 38 - - 2,4-D 500 + AC 100 0 kinetin 20 IAA 10 (100)* - kinetin 50 IAA 25 40 20 kinetin 100 IAA 50 71 57 kinetin 50 - - 71 71 kinetin 100 - - 100 33 TDZ 50 - - 100 100

10 DAYS - - 2,4-D 20 + AC 100 100 - - 2,4-D 50 + AC (100)* - - - 2,4-D 100 + AC 14 14 - - 2,4-D 500 + AC 0 0 kinetin 20 IAA 10 100 100 kinetin 50 IAA 25 40 40 kinetin 100 IAA 50 50 0 kinetin 20 - - 100 67 kinetin 100 - - 66 0 TDZ 50 - - 0 0

20 DAYS - - 2,4-D 20 + AC 100 50 - - 2,4-D 50 + AC 100 100 - - 2,4-D 100 + AC 45 / (55)* 27 - - 2,4-D 500 + AC 0 0 kinetin 20 IAA 10 100 100 kinetin 50 IAA 25 66 66 kinetin 100 IAA 50 100 0 kinetin 20 - 100 100 kinetin 100 - 50 0 TDZ 50 - 0 0

Key. AC = activated charcoal. Note: (55)* and (100)* denotes % lost through contamination. No de novo growth was observed in any treatments after 6 weeks in vitro culture.

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5.3.4 Response to Liquid Media Pretreatment

After a four week culture period tissue survival rates in all treatments were above 53%. The percentage of explants showing tissue expansion ranged from 20 to 46%. No observations of organogenesis were recorded from this experimentation. Table 5.11 Response and survival of immature flower buds to liquid media pretreatments prior to culture on 20 µM kinetin. Explants cultured for a 4 week period.

Basal media

Cytokinin Conc. (µM)

Treatment Mean survival (%)

Mean tissue enlargement (%)

3 DAYS MS kinetin 20 AC 80 40 MS kinetin 20 PAC 60 33 MS kinetin 20 ANC 60 46 7 DAYS MS kinetin 20 AC 40 26 MS kinetin 20 PAC 53 20 MS kinetin 20 ANC 60 26 14 DAYS MS kinetin 20 AC 60 26 MS kinetin 20 PAC 73 26 MS kinetin 20 ANC 53 33

Key: AC = activated charcoal (0.1%); PAC = paclobutrazol at 3 mg/ L; ANC = ancymidol at 20 µM. Note: No de novo growth was observed in any treatments after a 4 week culture period.

5.4 Discussion

This is the first reported in vitro study of D. excelsa and the selection of media and the amounts of growth regulators used on different explant sources were determined based on several factors. These included available literature on related plant families and type and maturity of different inflorescence source material. The general biology of D. excelsa was compared to other arborescent monocotyledons with growth regulator types, concentrations and in vitro culture protocols devised and reported. A variety of basal media (see Appendix 1 and 2), cytokinins and auxins were tested over various in vitro culture periods (Tables 5.6 to 5.11) throughout this 32 week study. 5.4.1 Basal Media Composition

Basal medium consisting of 1/2 MS (Murashige and Skoog, 1962) is reported to be favourable for the culture induction of many Australian monocotyledons (Bunn, 1994), whereas full strength MS can be deleterious to others (Meney and Dixon, 1995). This study demonstrated that D. excelsa tissues incubated on both 1/2 and full strength MS showed high survival rates. Other basal media, B5 medium (Gamborg et al, 1968) gave similar results in terms of tissue survival. In vitro culture of genera in the Liliaceae, Iridaceae and Amaryllidaceae, reported by Hussey (1975), details the successful induction of twelve species where full strength MS was utilized as the basal medium. Establishment of shoots from inflorescence explants in genera from the Agavaceae family also relied on full MS basal salts for culture establishment (Richwine et al, 1995). In vitro micropropagation has

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been documented for many arborescent monocotyledonous genera and the habit of Doryanthes excelsa can be likened to this feature. Australian and exotic genera such as Bambusa (Lin and Chang, 1998), Cordyline (Kunisaki, 1977; Mee, 1978; Evaldsson and Welander, 1985), Elaeis (Teixeira et al., 1994), Euterpe (Guerra and Handro, 1998), Musa (Khatri et al., 1997), and Dasypogon (Bunn and Dixon, 1992) are cultured using full strength MS as the basal media. It is known that seed grown material of D. excelsa in nursery conditions have high demands for fertilizer (pers. obs). The mineral composition of Murashige and Skoog (Appendix 1) is very rich in salts and the successful culture of many Australian plants, which have evolved on nutrient depleted soils, requires the dilution of the MS salts to half strength (Bunn, 1994; Meney and Dixon, 1995; Rossetto et al., 1992). The results of this study would indicate that in vitro cultured explants of D. excelsa can survive continued culturing on full MS basal salts, but it not clear which basal media formulation (i.e. 1/2 MS, MS or B5) is significantly better for in vitro growth of D. excelsa. 5.4.2 Plant Growth Regulators used In Vitro

In this study, explants were initially exposed to concentrations of cytokinin and auxin on 1/2 MS basal medium (5 µM BAP + 0.25 µM IBA and 10 µM BAP + 0.5 µM IBA). The response of tissues to the low level treatment showed minimal enlargement compared to explants cultured on higher levels, therefore further experimentation focussed on the use of higher concentrations of growth regulators. The subsequent experimentation that followed utilized cytokinin and auxin concentrations used singly or in combination at various levels. These were selected as they had been successful in the past in promoting organogenesis in other arborescent and monocotyledonous genera (Khatri et al., 1997; Wildi et al., 1998; Collins and Dixon, 1992; Teixeira et al., 1994). For the duration of this study (32 weeks) immature inflorescence explants were continuously cultured on 10 µM BAP and 0.5 µM IBA. This cytokinin and auxin combination proved successful in the culture of Blandfordia grandiflora (Bunn and Dixon, 1996b) where adventitious shoots arose from floral explants after 8 weeks culture duration. One explant of D. excelsa cultured on this medium developed green globoid structures directly from the upper cut surface of a peduncle disc section after 24 weeks culturing (Figure 5.3) but no further development into shoot structures was noticed. In a study by Bunn (1994) on Villarsia calthifolia, 10 µM BAP plus auxin, mediated direct adventitious shoot production (22 shoots per explant) from petiole pith sections. It would appear likely that these globoid bodies observed in D. excelsa might eventually develop shoots in a similar fashion with further culturing over time. These globoid structures are scattered over the cut surface and appear to develop from cells around the vascular bundles. These locations are known to be highly active in terms of cellular divisions. Unfortunately the results involving the further culturing of this explant and histological analysis will not be able to be completed within this project. The most commonly encountered cytokinin used for in vitro culture of many Australian monocotyledons is BAP (Bunn, 1994). Reports demonstrate it has been effective in stimulating adventitious growth from inflorescence tissues in some genera (Collins and Dixon, 1992; Kawase et al., 1995; Loutfi and Chlyah, 1998; Wildi et al., 1998). This plant growth regulator was initially chosen for testing on D. excelsa explants because the in vitro responses resulting in organogenesis was demonstrated in genera from related plant families. After a 14 week culture period explant material exposed to this medium showed low response, in terms of organogenesis, to the BAP concentrations. Flower buds generated from the subsequent in vitro culture stages (Figure 5.2) were dissected and exposed to a range of different cytokinin and auxin supplements. The levels tested in the following experimentation utilized cytokinin and auxin concentrations considered very high in published reports. However, an investigation involving the in vitro culture of immature inflorescence of Elaeis guineensis indicate that 500 µM 2,4-D supplemented with 0.5% AC was pivotal to the regeneration of this large monocotyledon (Teixeira et al., 1994). Explant material of D. excelsa exposed to this level of auxin died within one week of exposure. The plant growth regulator TDZ was examined as an alternative hormone owing to its high activity compared to BAP (10 times), and reported success in promoting organogenesis with woody plant taxa (George, 1996; Gill et al., 1994).

39

Exposure to TDZ of more than 4 days was deleterious to D. excelsa tissue explants (Table 5.10). A brief exposure however, of four days to TDZ followed by culturing on MS basal media, produced opaque and pink elongated structures around the top section of immature flower bud (Figure 5.6). These are thought to be the early signs of somatic embryos but determination of this could not be addressed as they were observed at the completion of this project. Kinetin was selected as an alternative cytokinin to promote organogenesis in single flower bud explants of the target species. The use of kinetin promoted the development of callus and formation of shoots in explants derived from inflorescence tissue in genera with similar habit to D. excelsa as reported in the literature (Amomarco and Ibanez, 1998; Slabbert et al., 1995; Kintzios and Michaelakis, 1999; Meyer et al., 1975). Callus like growth developed from a single explant of D. excelsa cultured on 20 µM kinetin with 10 µM NAA (Figure 5.5). It developed from the basal tissue of a flower bud which is the same region which gave regenerative responses with in vitro cultured date palm (Loutfi and Chlyah, 1998) and Japanese iris (Kawase et al., 1995). The appearance of this callus may be the early developmental stages of shoot like structures (Figure 5.5). Kinetin also gave a positive response to ovary sections (pedicle discs) derived from mature flower tissue. The development of opaque globoid cell clusters from around the ovary walls was achieved which is similar to the in vitro response obtained by Kawase et al.(1995). The thickness of the disc sections in these tissues increased two fold as is shown in Figure 5.3 a and 5.3 b. 5.4.3 Floral Organs as Explants

The response of different ages of inflorescence tissue types to various growth regulators and basal media were examined in this chapter. The results of experimental work show that excised single flower buds and ovary sections of immature inflorescences gave rise to further flower bud tissue. This phenomena is useful as it provided additional explant material for further media testing without having to sterilize and begin new cultures from ex vitro material. Meristems that would normally produce flowers or floral parts can be induced to give vegetative shoots in vitro and is reported in the literature. The exact origin of the shoots produced is not always known but histological studies can assist in determination of origin (Guerra and Handro, 1998; Loutfi and Chlyah, 1998). Positive results were gained in only a few explants sourced from single flower buds, peduncle disc section and ovary sections (pedicle discs) from a mature flower. The in vitro experimentation and investigation of inflorescence tissue of D. excelsa undertaken in this study would indicate these sources of explants are responsive and therefore ideal for further media and in vitro experiments. 5.4.4 Liquid Media Pretreatment

This experimentation was undertaken as it was postulated that the lack of regeneration over the duration of the study was due to endogenous gibberellins contained in the young inflorescence tissue influencing the in vitro action of cytokinin and auxin treatments. The scape of D. excelsa begins as a leafy shoot and undergoes continued elongation for a minimum of 12 months until it terminates as a fully developed inflorescence. It is assumed that such a prolonged period of elongation is controlled by a strong gibberellin signal (Salisbury and Ross, 1992). The results of this experimentation demonstrate that liquid pretreatment can be undertaken with no contamination and relatively good tissue survival. The results however were not conclusive as the culture duration (4 weeks) was not considered long enough to detect any clear trends. The different liquid pretreatments were designed to nullify the mode of action of gibberellins in vitro and this aspect of D. excelsa culture is a key area for further investigation. Culture in liquid media is also advantageous in leaching out harmful phenolic compounds in the early stages of culture initiation. Therefore liquid culture addresses two perceived problems with the successful in vitro propagation of floral explants of D. excelsa. From this study, it was clear that the culture incubation period may need to be greatly extended to achieve regeneration from the variety of experimental trials. The response to medium and high concentrations gave very minor results in comparison to the same levels tested on comparable genera

40

to the target species. The basal media testing and plant growth regulator responses outlined do however provide a useful platform for further experimental work. The sheer size and long life cycle of this species may explain the in vitro recalcitrance of Doryanthes. A similar life cycle influence on in vitro response in an arborescent species was found for Elaeis guineensis which required 81 weeks of culture to obtain embryogenic tissue from very young inflorescence tissues (Teixeira et al., 1994). Regeneration from flower disc sections of the terrestrial orchid, Diuris longifolia (Collins and Dixon, 1992), showed that 11 months of continuous culturing was essential for induction of growth (K. Dixon pers. com) whereas shorter in vitro culture periods (to achieve shoot structures from floral tissue) are reported in other monocotyledonous genera (Meyer et al., 1975; Slabbert et al., 1995; Richwine et al., 1995; Bunn and Dixon, 1996b). Reversal of reproductive to vegetative phase can be achieved in cultured flower buds and is the subject of many reviews (Hussey, 1975; Hussey, 1976; Kawase et al., 1995; Gulati et al., 1996; George, 1996). It is expected that with further experimentation involving the use of “anti-gibberellins”, media testing and patience, similar outcomes may be achieved with D. excelsa.

41

6. Conclusions and Future Work This study examined the application of micropropagation protocols to assist in the early maturation and potential precocious flowering of Doryanthes excelsa. This has implications for commercial cutflower production as it is reported to take 10 years or longer for this giant monocot to develop its first flower. The aim was to investigate the use of immature inflorescence tissues as explant source material. Key factors investigated in this study include selection of explant material, sterilization and culture conditions, control of phenolic exudates in explant preparation and the selection of appropriate growth regulator levels to achieve successful in vitro regeneration. Future directions involving the in vitro culture of this giant monocotyledon are discussed. This study has resulted in the preliminary development of a micropropagation protocol for culture induction of D. excelsa. Tissue culture of Australian arborescent monocotyledons is not widely reported in the literature and this is the first recorded study outlining micropropagation procedures for Doryanthes excelsa which is an important horticultural subject. The study successfully controlled deleterious phenolic reactions in explant tissues, derived an appropriate sterilization procedure and determined explant sources most likely to result in successful regeneration. In this study plant growth hormone combinations have been widely tested, but may need further examination to determine the concentrations likely to provide successful organogenesis. One of the most commonly encountered problems in in vitro culture establishment is the containment of microbial contaminants. Many reports suggest mature, field grown explant material as difficult to establish free from contamination in vitro whereas improved establishment is reported using cultivated material. All explant tissues sourced for this study were collected from developing flower scapes growing in natural habitats. Successful establishment of cultures using immature floral explants is described. Removal of contaminating organisms from explants was achieved using sequential sterilizing techniques (as outlined in Chapter 2). Thorough cleaning of explants and the use of the chemical sterilant, sodium hypochlorite (NaOCl at 1% v/v) followed by several washes in sterile water obviated the need to develop extensive and complicated surface sterilization protocols. This study found that inflorescence tissues of D. excelsa were high in phenolic compounds. This phenomenon is widespread in many monocotyledenous and woody genera and has profound physiological effects on in vitro cultured tissues (Taji and Williams, 1996; Zaid, 1987; Panaia, 1998). The browning of explants became evident immediately upon dissection from the scape. The oxidation of Doryanthes excelsa tissues was severe and proved deleterious to all tissues in the initial stages of explant preparation. Cessation of tissue browning in explants was achieved using potassium citrate and citrate (0.125% w/v) as a preculture wash. K:citrate and citrate was effective in minimizing the oxidative process and can be attributed firstly to the low pH (5.4) of the treatment solution which is likely to reduce the activity of enzymes (Panaia, 1998). Secondly, the proteins found in cells have a high affinity for potassium (K+ ions in the potassium citrate) and compete with the oxidative enzymes combining with proteins, resulting in a change in enzymatic activity in the cell thus preventing phenols binding to proteins and causing a loss of enzymatic activity leading to cell death (George, 1996). Thirdly, the citrate component is thought to behave as a chelating agent binding to ions in the cell that are needed by the phenolase to initiate the browning process. Finally, excision of explants under solution reduces exposure of cut surfaces to oxygen in the atmosphere and slows the rate of oxidation of phenols at the wound site, thus delaying polyphenolic production and subsequent explant demise. This study demonstrates that the use of an antioxidant treatment, namely the combination of K:citrate and citrate, provided successful protection allowing the establishment of viable cultures of D. excelsa. Following successful explant establishment, ranges of cytokinin and auxin combinations were investigated. A selection of different explant tissue types were cultured for 32 weeks on 1/2 MS basal medium supplemented with 10 µM BAP and 0.5 µM IBA. Other experimental work undertaken concurrently tested cytokinins and auxin both singularly or in combination at high to very high levels on a variety of explant sources. Culture exposure to growth regulators in these trials ran for four, nine,

42

twelve and sixteen weeks. Callus like growth was observed in one explant developing from the basal region of a single flower bud on MS basal medium supplemented with 20 µM Kinetin and 10 µM NAA after 7 weeks of in vitro culture. A pedicle section from a mature flower (with exposed ovary tissue) developed clusters of opaque globoid cells around the ovary wall. These were observed at the conclusion of this project and were not able to be examined histologically. No other tissue types tested on the various plant growth regulator treatments showed further tissue development beyond initial explant expansion. This in vitro study on D. excelsa ran for 32 weeks and it is assumed, given the range and concentrations of growth regulators tested, longer incubation time is required to achieve the objectives set at the commencement of the project. Furthermore, it is postulated that the lack of response in explants exposed to medium and high concentrations of hormones, reported to give organogenesis in related species, may be an effect of endogenous gibberellins contained in immature floral tissues. This action may be influencing the in vitro activity of cytokinins and auxins on the various explant tissues. Addressing these points in further experimental work may improve the likelihood of in vitro success in Doryanthes excelsa given the results obtained in previous research in related monocotyledonous genera. However, recent elucidation of the phylogenetic status of D. excelsa out of the Agavaceae and creation of its own endemic family, Doryanthaceae, may point to equally unique attributes of the species under in vitro conditions. This is further reinforced by the lack of commonality in in vitro responses in D. excelsa when compared to in vitro growth in taxa of the closely related families Iridaceae, Amaryllidaceae and Liliaceae. The interest in D. excelsa as a cutflower and foliage and for amenity horticulture, both for domestic and export sales has been steadily increasing in recent years. This species has a restricted distribution which has serious ecological implications as all cutflower material is harvested from wild populations. It is essential that an appropriate method to expedite introduction to cultivation be determined. The success of in vitro propagation methods reported for other giant monocots suggest that D. excelsa can be tissue cultured with further plant growth regulator experimentation. Successful micropropagation will be of major benefit to the floriculture industry making bush harvesting unnecessary thus greatly assisting in the conservation of this unique Australian plant.

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7. References Amomarco, J.B. and Ibanez, M.R. (1998). Micropropagation of Limonium cavanillesii Erben, a threatened statice, from inflorescence stems. Plant Growth Regulation 24(1): 49-54 Attaalla, H. and Vanstaden, J. (1997). Micropropagation and establishment of Yucca aloifolia L. Plant Cell Tissue & Organ Culture, 48 (3): 209-212 Augé, R. (1995). Chapter 2 The physiological phenomena related to the realisation of cultures in vitro. pp7-31. In: In vitro Culture and its Applications in Horticulture. Vidalie, H. (Ed) Science Publishers, North Lebanon, NH, USA Barlass, M. and Hutchinson, J. (1996) Chapter 6 Commercial micropropagation of Australian native plants. pp.180-203. In: Tissue Culture of Australian Plants: past, present and future. Taji, A.M. and Williams, R.R. (Eds) University of New England Press, Armidale, Australia Bicknell, R.A. , Braun, R.H. , Evans, A.C. , Morgan, E.R. (1996). Tissue culture of Ranunculus lyllii Hook F. New Zealand Journal of Crop & Horticultural Science. 24(4): 303-306 Bunn, E., Dixon, K.W. and Langley, M.A. (1989). In vitro propagation of Leucopogon obtectus Benth. Epacridaceae. Plant Cell, Tissue and Organ Culture 19, 77-84 Bunn, E. and Dixon, K.W. (1992). Micropropagation of the pineapple lily (Dasypogon hookeri J. Drumm.). HortScience 27(4): 369 Bunn, E. (1994). Micropropagation of recalcitrant Australian plants, with special emphasis on rare and endangered taxa. Thesis for M.Sc. Degree, University of Western Australia. Bunn, E. and Dixon, K.W. (1996a). Chapter 5 Tissue culture of rare and endangered Australian plants. pp.157-179. In: Tissue Culture of Australian Plants: past, present and future. Taji, A.M. and Williams, R.R. (Eds) University of New England Press, Armidale, Australia Bunn, E. and Dixon, K.W. (1996b). In vitro propagation methods for Blandfordia grandiflora, Hibbertia miniata, Newcastelia chrysophylla and Eucalyptus graniticola (ms). pp. 157-163 In: Proceeding of International Association for Plant Tissue Culture-Australian Branch 5th Meeting. Collins, M.T. and Dixon, K.W. (1992). Micropropagation of an Australian terrestrial orchid, Diuris longifolia R.Br. Australian Journal of Experimental Agriculture, 32: 262-262 de Fossard, R.A., Myint, A. and Lee, E.C.M (1974) A broad spectrum tissue culture experiment with tobacco (Nicotiana tabacum) pith tissue callus. Physiology Plantarum 30: 125-130. de Fossard, R.A. (1976). Tissue culture for plant propagators. University of New England Printery. Armidale, Australia Doran, P.M. (1996) Chapter 8 Cell culture technology for secondary metabolite production with reference to Australian plants. pp.240-283. In: Tissue Culture of Australian Plants: past, present and future. Taji, A.M. and Williams, R.R. (Eds) University of New England Press, Armidale, Australia Drew, R.A (1986). The use of tissue culture in the search for Panama disease resistant clones of banana. Combined Proceedings International PlantPropagators Society. 35: 44-53 Evaldsson, I.E. and Welander, N.T. (1985). The effects of media composition on in vitro propagation and in vivo growth of Cordyline terminalis cv Atoom. Journal of Horticultural Science 60: 525-530

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8. Appendices Appendix 1

Mineral salts used for in vitro culture of D. excelsa.(concentrations in mg/L)

Components Murashige and Skoog, (1962) (MS)

1/2 strength MS Basal Salts

Gamborg et al. (1968) B5 Basal Salts

NH4NO3 1601 800.5 - (NH4)2SO4 - - 134 KNO3 2022 1011 2500 CaCl2.2H2O 441 220.5 150 KH2PO4 204 102 - MgSO4.7H2O 370 185 370 MnSO4.4H2O 22 11 10 FeNa.EDTA 38 19 37.25 H3BO3 6.2 3.1 3.0 ZnSO4.7H2O 8.6 4.3 2.0 Na2MoO4.2H2O 0.25 0.125 0.25 NaH2PO4H2O - - 150 KI 0.83 0.415 0.75 CuSO4.5H2O 0.025 0.0125 0.025 CoCl2.6H2O 0.24 0.012 0.025

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Appendix 2

Outline of procedures for preparation of plant hormone stock solutions. Hormone Stock Solution Preparation

Abbreviation g per 100ml for 1 mM stock soln

solvent* sterilization**

Auxin indoleacetic acid IAA 0.01752 EtOH/1 M NaOH F indole-3-butyric acid IBA 0.02032 EtOH/1 M NaOH A a-naphthaleneacetic acid NAA 0.01860 1 M NaOH A 2,4-dichlorophenoxyacetic acid

2,4-D 0.02210 EtOH/1 M NaOH A

Cytokinin

N6-benzylaminopurine BA or BAP 0.02253 1 M NaOH A

N6-isopentenyladenine 2iP 0.02032 1 M NaOH F

thidiazuron TDZ 0.02202 A 6-furfurylaminopurine kinetin 0.02152 1 M NaOH A zeatin Z 0.02192 1 M NaOH F

* Note: Only small volumes (typically 1-2ml) of solvents required for initial solubilizing then water added to make 100 ml. Stock solutions were therefore slightly basic or acidic. ** A= Autoclavable, F= Filter sterilized