Inhibiting the Postharvest Wounding Response in Wildflowers

A report for the Rural Industries Research and Development Corporation

by Dr Virginia G. Williamson, Dr John Faragher, Sarah Parsons and Peter Franz

August 2002

RIRDC Publication No 02/114 RIRDC Project No DAV-161A

ISBN 0642 58513 X ISSN 1440-6845

Inhibiting the Postharvest Wounding Response in Wildflowers Publication No. 02/114 Project No. DAV-161A

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

Dr Virginia Williamson Institute for Horticultural Development Department of Natural Resources and Environment Private Bag 15 Ferntree Gully Delivery Centre VIC 3156

Phone: (03) 9210 9222 Fax: (03) 9800 3521 Email: [email protected]

RIRDC Contact Details

Rural Industries Research and Development Corporation Level 1, AMA House 42 Macquarie Street BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604

Phone: 02 6272 4539 Fax: 02 6272 5877 Email: [email protected]. Website: http://www.rirdc.gov.au

Published in August 2002 Printed on environmentally friendly paper by Canprint

Foreword

Cut flowers are dramatically wounded plant tissue. A general response to any mechanical damage to plant tissue is suberin formation. Suberin is an hydrophobic substance that is deposited in plants as a defence response to wounding. It was hypothesised in this project that a suberin barrier caused premature wilting in cut flowers because stems were no longer able to take up water from the vase solution. Although suberin might only coat the xylem walls very thinly in the early stages of its formation, a small amount could still impede water flow.

Several chemicals are known to inhibit suberin formation, and these were tested on a number of Australian wildflowers. If the borderline vase lives (i.e. < 7 days) of many Australian flowers could be improved, the range of species available for export might increase.

This publication examines the effects of numerous wound-inhibiting chemicals and treatments on the vase lives of 13 genera/species of Australian cut flowers and foliage. It provides practical advice on improving the vase lives of several of these flowers, and recommends some easy ways to increase water uptake and vase life. As a by-product of this research, the ethylene sensitivity of some popular Australian native flowers was discovered. The report also shows evidence of the early stage of the wounding response in cut flowers under transmission electron microscopy for the first time.

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 800 research publications, forms part of our Wildflowers and Native Plants R&D program, which aims to understand, strengthen and develop markets; improve existing products and develop new ones; provide profitable and sustainable production systems; and enhance the human capital of the 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/eshop

Simon Hearn Managing Director Rural Industries Research and Development Corporation

iii

Acknowledgements

This work was funded by the Rural Industries Research and Development Corporation (RIRDC) and the Department of Natural Resources and Environment, and sponsored by Longford Flowers.

The project was the equivalent of one year’s full time work (2 years at 50% FTE). In order to attain so many results, assistance was obtained from several people: Lynette Bangay, Trish Grant and Sarah Parsons. Trish and Sarah also provided extremely useful discussions, advice and fun – thanks guys! Sarah Parsons also expertly performed the majority of the data analyses.

Professor Michael Reid (UCLA, Davis) provided useful and stimulating discussions and suggested the “boiling” experiment. Dr John Faragher suggested the “blasting”, deep and shallow water, and recutting experiments,. and the use of TCA. Sarah Parsons took all the photos in this report, except for the TEM photos. Peter Franz provided statistical advice. Bret Henderson supplied horticultural advice. Janyce Truett helped devise the washing experiment apparatus. Joel Saywell (State Chemistry Laboratory) made the soluble form of SHAM. Dr Simon Crawford (University of Melbourne, Transmission Electron Microscope Unit) performed the TEM preparation and operation, and Megan Klemm provided the initial TEM preparation discussions. Professor Dr Walter Liese and Dr Uwe Schmitt (Germany) interpreted the TEM photos for me. Denis Tricks (Longford Flowers) and Bill Frew (Farmgate Flowers) provided floral material. Tony Slater provided Baeckea and Leptospermum material from the IHD plots.

Several people also assisted in particular ways during this project: Dr Wendy Morgan, Denise Millar, Angelika Ziehrl, Bronwyn Clarke and Soehir Salib.

I am grateful to Dr John Faragher for supervising this project, and for the useful discussions, advice and assistance.

Thanks also to Dr Brendan Rodoni, my other 50% FTE supervisor, for all the give and take on his project during this project.

As ever, my long-suffering husband, Dr Patrick Laplagne, put up with my regular experiment monitoring on weekends, and provided invaluable formatting assistance for this final report.

Dedicated to Wendie and Greg Williamson

Table of Contents

Foreword...... iii Acknowledgements...... iv Table of Contents ...... v List of Tables and Figures ...... vii List of Photographs ...... viii List of Abbreviations and Symbols...... ix Executive Summary ...... x Background...... x Objectives ...... x Research outcomes...... x Recommendations...... xi 1. Introduction ...... 1 1.1 Australian flower exports — genera and $...... 1 1.2 The unfulfilled “potential” of Australian native flowers...... 2 1.3 Could untapped genera be used if vase life were increased?...... 2 1.4 Common factors involved in cut flower senescence ...... 3 1.5 The suberin/wounding hypothesis compared with the cavitation hypothesis...... 6 1.6 Suberin biochemistry...... 7 1.7 Suberin synthesis ...... 8 1.8 Suberin detection ...... 9 1.9 Possible suberin inhibitors...... 10 2. Objectives ...... 11 3. Methodology...... 12 3.1 Plant Material ...... 12 3.2 Transport and Preparation of Plant Material ...... 12 3.3 Vase Life Conditions...... 13 3.4 Determination of pH...... 13 3.5 Measurement of Dissolved Oxygen...... 13 3.6 Bacterial Counts ...... 13 3.7 Ethylene Experiments...... 14 3.8 Washing Experiments...... 14 3.9 Measurement of Water Uptake and Transpiration...... 15 3.10 Transmission Electron Microscopy (TEM) ...... 15 3.11 Statistical Design and Analysis of Results ...... 16

4. Results...... 17 4.1 Acacia baileyana ...... 17 4.2 Acacia baileyana, second experiment ...... 19 4.3 Baeckea virgata, vase life experiment...... 21 4.4 B. virgata, ethylene experiment...... 22 4.5 B. virgata, quantification of petal drop experiment...... 24 4.6 B. behrii, a comparison of cutting in air to cutting under water ...... 27 4.7 Crowea exalata...... 28 4.8 C. exalata, testing the effects of STS and SHAM ...... 30 4.9 Grevillea longistyla ...... 31 4.10 Lophomyrtus × ralphii ‘Krinkly’...... 33 4.11 Lophomyrtus × ralphii ‘Krinkly’, determining ethylene sensitivity...... 34 4.12 Washing experiments ...... 36 4.13 Bacterial monitoring of washing experiments...... 40 4.14 Shaker experiment ...... 42 4.15 High water versus low water experiment ...... 44 4.16 Bacterial monitoring of high versus low water experiment...... 46 4.17 Recutting experiment...... 47 4.18 “Blasting” experiment ...... 49 4.19 Second “Blasting” experiment...... 51 4.20 Boiling experiment ...... 52 4.21 Inhibiting suberin chemically ...... 54 4.22 Searching for evidence of a wounding response via transmission electron microscopy... 55 5. Discussion ...... 59 Transmission Electron Microscopy...... 59 Recutting stems...... 59 High water versus low water...... 60 Ethylene 60 6. Recommendations...... 62 References ...... 64 Appendix A ...... 71 Ingredients for Plate Count Agar ...... 71 Appendix B...... 72 Transmission Electron Microscopy Preparation Instructions ...... 72 Appendix C ...... 73 Summary of Vase Life Results ...... 73 Appendix D ...... 77 Chemical Concentration Conversions...... 77 Appendix E...... 82 Silver Thiosulphate Preparation and Disposal ...... 82

List of Tables and Figures

Table 4.1 Mean cumulative percent petal drop in B. virgata ...... 26

Fig. 1.1. Flower exports from Australia by value and weight...... 1 Fig. 4.1. Vase life of A. baileyana foliage...... 18 Fig. 4.2. Simple linear regression showing the relationship between vase life and pH in A. baileyana...... 18 Fig. 4.3. Vase life of A. baileyana foliage...... 20 Fig. 4.4. Vase life of B. virgata...... 22 Fig. 4.5. Vase life of B. virgata after exposure to ethylene...... 24 Fig. 4.6. Cumulative total petal drop (expressed as a % of initial total petal count) in B. virgata. 26 Fig. 4.7. Vase life of B. behrii in which cutting stems in air was compared with cutting stems under water...... 28 Fig. 4.8. Vase life of Crowea exalata...... 29 Fig. 4.9. Vase life of Crowea exalata...... 31 Fig. 4.10. Vase life of Grevillea longistyla foliage...... 32 Fig. 4.11. Vase life of Lophomyrtus × ralphii ‘Krinkly’ foliage...... 34 Fig. 4.12. Vase life of Lophomyrtus × ralphii ‘Krinkly’ foliage...... 35 Fig. 4.13. Vase life of Acacia floribunda flowering stems...... 36 Fig. 4.14. Vase life of flowering Ceratopetalum gummiferum stems...... 37 Fig. 4.16. Vase life of flowering Crowea exalata stems...... 38 Fig. 4.17. Vase life of flowering Hakea teretifolia stems...... 39 Fig. 4.18. Vase life of flowering Sphaerolobium vimineum stems...... 40

Fig. 4.19. The mean number of bacteria (log 10 cfu/mL) in Ceratopetalum gummiferum washing experiment...... 41 Fig. 4.20. Vase life of Ceratopetalum gummiferum...... 42 Fig. 4.21. The amount of dissolved oxygen (expressed as % oxygen saturation) in vases containing Ceratopetalum gummiferum...... 43

Fig. 4.22. The mean number of bacteria (log10 cfu/mL) in Ceratopetalum gummiferum washing experiment...... 43 Fig. 4.23. Vase life of Leptospermum obovatum in high or low water...... 45

Fig. 4.24. The number of bacteria (log10 cfu/mL) in L. obovatum stems kept in either high or low water for 5 d...... 47 Fig. 4.25. Vase life of Leptospermum polygalifolium foliage...... 48 Fig. 4.26. Mean daily water uptake in L. polygalifolium foliage...... 48 Fig. 4.27. Total life (i.e. including 24 h dry period) of L. obovatum when stems were “blasted” with strong chemicals...... 50 Fig. 4.28. Vase life of L. obovatum after a 2 or 5 minute wash with the above chemicals...... 52 Fig. 4.29. Vase life of Rosa hybrida ‘Lambada’...... 53 Fig. 4.30. Vase life of Hakea francisiana...... 55

vii

List of Photographs

Photograph 3.1. Perspex tub used for challenging stems with ethylene...... 14 Photograph 3.2. Experimental apparatus used in washing experiments...... 15 Photograph 4.1. A: Ethylene + STS; B. Ethylene alone...... 23 Photograph 4.2. Experimental set up for quantifying the amount of petal drop in B. virgata...... 25 Photograph 4.3. TEM: Control stem (Hakea francisiana) showing an electron dense, dark pit membrane ...... 56 Photograph 4.4. TEM: After 48 h, stems treated with S-carvone show dark, electron dense staining (arrows) similar to that shown in the control stem...... 57 Photograph 4.5. TEM: Deionised water (48 h): the centrally located part of the pit membrane is less electron dense (arrows) than in the control or S-carvone (48 h) treated stems...... 57

viii

List of Abbreviations and Symbols

ABA abscisic acid a.i. active ingredient AIP 2-aminoindan-2-phosphonic acid ANOVA analysis of variance °C (temperature in) degrees Celsius cfu colony forming units CHI cycloheximide cm centimetre d day/s EPTC S-ethyl-N,N-dipropylthiocarbamate h hour/s IHD Institute for Horticultural Development lsd least significant difference m milli M molar 1-MCP 1-methylcyclopropene mL millilitre mM millimolar msds material safety data sheet P probability PAL phenylalanine ammonia lyase PAR photosynthetically active radiation PCA plate count agar ppm parts per million RH relative humidity rpm revolutions per minute s second/s SHAM salicylhydroxamic acid TCA trichloroacetate TEM transmission electron microscopy µ micro v/v volume for volume w/v weight for volume

Executive Summary

Background Current exports of Australian cut flowers concentrate on only a few of numerous genera. If the borderline vase lives (i.e. < 7 days) of many Australian flowers could be improved, the range of species available for export might increase. This project hypothesised that when flowers are cut, their response to that mechanical damage was to deposit an hydrophobic substance (suberin). A general response to any mechanical damage to plant tissue is suberin formation. A suberin barrier is formed as a generic response to wounding, e.g. to seal off an area invaded by a pathogen. If this occurred in cut flowers, the suberin barrier could cause premature wilting and a shortened vase life because stems were no longer able to take up water from the vase solution. Several chemicals are known to inhibit suberin formation, and these were tested on a number of Australian wildflowers.

Objectives This project was aimed at enhancing the export reputation of Australian native cut flowers and increasing the number of exportable species, with the following objectives:

• to counteract the poor water uptake that characterises most Australian native cut flowers with borderline vase lives (i.e. < 7 days) by inhibiting the initial postharvest wounding response; • to determine whether suberin inhibiting treatments could be used to enhance water uptake and increase the vase life of selected Australian native flowers; • to ascertain by Transmission Electron Microscopy whether suberin is deposited in cut stems; and • to ascertain whether any other factors, such as ethylene sensitivity, are involved in a particular flower’s senescence.

Research outcomes The major outcomes of this research were:

• The first recorded evidence showing the wounding response in cut flowers under the transmission electron microscope (TEM). The observed alteration of pit membranes was indicated by changes in electron density. Such a response is the first part of a two stage suberisation process known to occur in trees as a response to injury or pathogen invasion. • The wounding response observed under TEM was inhibited by the chemical, S-carvone. This chemical is known to delay the appearance of suberin in potato tubers. S-carvone significantly increased the vase life of Hakea francisiana, the only species tested with it. • Vase life was significantly increased by keeping cut stems in deep water rather than in shallow water. This occurred in all three species tested: Acacia baileyana, Leptospermum obovatum and

L. polygalifolium. The beneficial effect of high water is likely to be the result of increased hydrostatic pressure on the stem, but whether that pressure dissolved air emboli or pushed through a suberin barrier is not known. • Daily removal of the basal 1 cm from stems significantly increased the vase life and water uptake of the three species listed above. When these results are considered with the TEM results, it appears likely that recutting removes the early stages of a wounding response. This wounding response can eventually lead to suberin deposition, as mentioned above. • The vase life results of some species indicated the possible involvement of ethylene in their senescence. As a result, ethylene sensitivity was determined in Baeckea virgata, Crowea exalata and Lophomyrtus × ralphii ‘Krinkly’.

Recommendations Several recommendations can be made, both to industry and for future research, as a result of this project: • More TEM work needs to be done, e.g. a daily time course of wounding responses to see whether suberin does eventually form, and the examination of other species, especially short-lived and long-lived cut flowers. • S-carvone needs to be tested on other species to see whether it increases their vase life and also whether a short-term pulse will translate to long-term inhibition of the wounding response; • Daily recutting of stems will inhibit the full effects of a wounding response. It could possibly delay the effects of suberin formation and the impaired water conduction that may result. • A practical, easy way to improve vase life is by standing stems in deep water. This can be done at the packing shed. • Several Australian flowers of commercial and export importance are sensitive to ethylene and therefore should be treated and/or protected against its effects in order to attain optimum vase life: Baeckea virgata, Crowea exalata and Lophomyrtus × ralphii ‘Krinkly’. • Several species used in this project were not tested for ethylene but indications are that they may be sensitive to it. Furthermore, there are dozens more commercially important cut flower species that need to be tested because if they are ethylene sensitive, treatment can provide an easy way to significantly increase vase life.

1. Introduction

1.1 Australian flower exports — genera and $ In 1999-00, Australian flower exports were projected to reach approximately 5,000 tonnes, for a total value of $31.3m (RIRDC 2000). The vast majority of these exports were in the form of fresh cut flowers. Amongst these, Australian native flowers — waxflower in particular — dominated. The largest market for Australian flower exports is Japan, followed by the United States and the Netherlands. With approximately 1,200 species of commercial value (Considine 1993), Western Australia is traditionally the largest exporting State, although Victoria recorded strong growth over the 1990s to become a close second (Brooks 2000). Currently, exports of Australian native cut flowers concentrate on three main genera, Chamelaucium, Anigozanthos and Banksia, which represent approximately 2% of the known flora (Considine 1993). Put in the perspective of world production, Australia’s contribution amounts to only 0.8% (McGeoch 1994).

As Figure 1 shows, the value of Australian flower exports grew rapidly from the mid-1980s to the mid-1990s. Since that time, it has remained fairly stagnant, reflecting flat or falling export volumes. In 1999-00, however, volumes were projected to grow again strongly. This growth was forecast to exceed that of export revenue, indicating a fall in the average price received per kilogram exported.1

35 6000

30 5000

tonnes 25 $m FOB $/kg 4000

$ 3000 t

2000 10

1000 5

0 0 1980-81 1981-82 1982-83 1983-84 1984-85 1985-86 1986-87 1987-88 1988-89 1989-90 1990-91 1991-92 1992-93 1993-94 1994-95 1995-96 1996-97 1997-98 1998-99 1999-00 Year

Fig. 1.1. Flower exports from Australia by value and weight. Sources: ABS, Brooks 2000, Faragher et al. 2000, RIRDC 2000.

1 Indeed, the unit price has been declining steadily since 1993-94. Average price figures must be interpreted with caution, however. FECA indicates that prices received for flowers sold at auction are not known at the time of export, so that token values only are recorded, which may underestimate the actual price received (Brooks 2000).

1.2 The unfulfilled “potential” of Australian native flowers Australia has more than 2,400 genera and an estimated 25,000 species of native plants (Burbidge 1963; George 1981). Yet, despite an account being published in 1953 (Lothian 1953) of 231 Australian native species suitable as cut flowers, up to 40 genera only have been developed commercially to date. These include kangaroo paw (Anigozanthos spp.), Banksia spp., Dryandra spp. Geraldton wax (Chamelaucium uncinatum) and feather flower (Verticordia spp.) (Pegrum 1988).

At the 1999 5th Australian Wildflower Conference in Melbourne, the word “potential” was used so frequently in relation to the Australian flora as cut flowers, it became hackneyed, hence its use in inverted commas here! The word has been associated with Australian native flowers for at least 20 years - a rather long time to wait for “potential” to be realised.

Perhaps the environmentally ‘clean’ angle is one that could be emphasised in the export of Australian cut flowers and foliage, as Australian produce is generally considered by overseas consumers to possess that quality. An interview with German cut flower importers revealed a trend towards ecologically responsible products and production (Laws 1994). In addition, German customers were prepared to pay for “novelty that shows sensitivity to the eco-balance” (Laws 1994). Surely this is an angle that is well suited to Australian cut flowers. Our mild, sunny climate means less heated greenhouses during winter, unlike our northern hemisphere competitors; and ecologically sensitive areas of land are not being cleared for cut flower production, as happens in some other southern hemisphere countries.

1.3 Could untapped genera be used if vase life were increased? Most certainly, the answer to this question is yes. Lamont (1984) discussed the criteria necessary for suitable cut flowers. One of these was a good vase life of at least seven days. At present, numerous Australian native flowers could be used, but they fall into the category of having a “borderline” vase life.

The Australian flora has been described as “particularly tempting and rich” by European scientists collecting plants world wide (von Hentig 1995). In 1986, Dutch scientists, in collaboration with The Research Station for Floriculture at Aalsmeer, visited Australia and collected plant material from 211 Australian native species. They noted that “at the moment a relatively small amount of research and breeding on the Australian native flora is done by the Australians themselves” (Koster and van Raamsdonk 1989).

When the “Kiwi Rose” (Telopea speciosissima) was marketed by New Zealand under Plant Breeder’s Rights, there were numerous indignant Australian protestations. However, there is a case for cultivar exchange: if a plant is popularised overseas, the increased sales as overseas popularity trends infiltrate can benefit the local market (Parvin 1995). Nevertheless, Considine (in Cribb 1988) believed that “the real issue now is not that we give away our genetic resources …, but that we are failing to provide leadership in their commercialisation”.

Perhaps the following quote from nearly 150 years’ ago is still uncannily pertinent. Although the comments were in relation to the essential oil potential of Victorian native plants, they are relevant to today’s Australian native cut flowers:

“The native vegetation of Australia is strongly distinguished from that of other countries. Whilst travelling through the bush, a cursory examination has often suggested to me the thought of the immense amount of valuable materials absolutely wasted for want of being represented to the public mind, so as to bring them into competition with the productions of other countries.” (Bosisto 1865)

According to Tjia (1988), Australia should not grow established crops for export such as carnations and chrysanthemums because the market has already been cornered by the world’s leading flower producing nations. Rather, it should concentrate on becoming a world specialist and leader in the growth and marketing of its native flora, which can be identified by the world market as a uniquely Australian specialty (Tjia 1988).

Surveys of cut flower consumers in Australia (Aldous 1983) and overseas (the Netherlands, Koelemeijer 1991) revealed that the most important factor to consumers was flower quality, especially a long vase life. In the United States, potentially new cut flower crops are continuously being evaluated. If any aspect of a crop is found to be weak, e.g. short postharvest life or production problems, it is discarded in favour of less marginal species (Armitage 1993). Some of the common postharvest problems which can lead to a shortened vase life are discussed below.

1.4 Common factors involved in cut flower senescence Numerous factors have been implicated in the senescence of cut flowers and have been discussed in several excellent comprehensive reviews (Halevy and Mayak 1979; Halevy and Mayak 1981; Goszczynska and Rudnicki 1988; Borochov and Woodson 1989; Salunkhe et al. 1990). The most relevant, common factors are discussed briefly below.

1.4.1 Ethylene Cut flowers can be classified broadly into two groups: those which are sensitive to ethylene and those which are not. Although only a small percentage of cut flowers are ethylene-sensitive (Reid 1989), they include many economically profitable genera, including some of the world’s top sellers: roses, chrysanthemums and carnations. Thus, a considerable amount of research has been performed assessing the effects of ethylene on exotic cut flowers. However, little is known of the ethylene sensitivity of many Australian native cut flowers. Ethylene is an important consideration in the vase life of cut flowers because its influence can override other postharvest factors. Hence there is a need to determine a plant’s ethylene response, where indicated.

Treating with silver thiosulphate (STS) is still the most common method of protecting cut stems against ethylene action. Although the use of STS will eventually be phased out because of environmental concerns regarding the heavy metal disposal, it is still regarded as the ethylene action inhibitor, par excellence. Its eventual replacement, 1-methylcyclopropene (1-MCP), is not as effective as STS because its ethylene inhibitory effects may be transient in flowers, particularly at low temperatures and concentrations (Macnish et al. 2000). Furthermore, because our limited knowledge of the effects of ethylene on Australian native flowers has been gathered using STS, experimentally it is beneficial to maintain this point of comparison. Therefore, STS and applied ethylene will be used in this project to determine ethylene sensitivity, where necessary.

1.4.2 Vascular blockage – physiological factors Cavitation and embolisation Unrestricted water uptake through the xylem conduits2 is of paramount importance in providing an adequate supply of water to transpiring cut stems. Zimmermann (1983) noted that if xylem conduits were blocked, either by air (e.g. cavitation and subsequent embolisation), physiological (e.g. tylose formation and gum or pectin deposition) or pathological (e.g. bacteria and fungi) means, water had to come out of storage to meet the transpirational demands of cut stems. This may lead to premature wilting and abbreviated vase life (Williamson 1996).

Plant systems are particularly vulnerable to cavitation and subsequent embolisation of their conduits because they are under negative pressure (or tension). Xylem sap can remain under tension only if the liquid phase exists within the conduit, so if a bubble forms within a conduit (i.e. cavitation occurs), that conduit becomes embolised (gas filled) and is therefore non-conducting as far as its pit

2 The word ‘conduit’ is used throughout this report as a collective term for the conducting xylem units of vessels, tracheids and fibres. ‘Conduit’ was used originally by Arndt (1929) and reintroduced by Milburn and Covey- Crump (1971), who recommended it replace the word ‘vessel’ as a general term, because ‘vessel’ describes only one ontogenetically distinct type of unit.

membranes. When a water column in a plant breaks, water is released to other parts of the plant and the flow path is therefore constricted (Zimmermann 1983). The result of cavitation is that fewer xylem conduits remain which are available for sap transport. In cut flowers, severe loss of xylem function due to cavitation may be irreversible, although postharvest cold storage and capillarity can redissolve gas emboli and rehydrate the smaller xylem elements (Dixon et al. 1988). When a stem is cut in air, air is immediately drawn into the severed, opened xylem conduits because water in the xylem is under negative pressure. Air then fills the xylem conduits that were opened by the cut. The fact that any cut flowers survive this initial treatment indicates that sufficient conduits remained unopened next to the cut to allow water uptake to proceed (Milburn 1979). Furthermore, entrapped air bubbles are dissolved by the surface tension of water (Milburn 1979).

Vascular occlusions Vascular blockages can be divided into two categories – those occurring from (1) physiological and (2) microbiological causes. Microbiological vascular blockage is discussed below. Physiological vascular occlusions include the formation of tyloses, and the deposition of ‘gums’ (e.g. pectins), lignins, tannins, and callose in xylem conduits. Tyloses are balloon-like outgrowths which project from ray or axial parenchyma cells into the lumen of an adjacent cell through a pit cavity (Esau 1977). However, tyloses were found to develop when conduits became gas-filled and were therefore the result of the cessation of water conduction and not the cause (von Reichenbach 1845 in Zimmermann 1979).

The deposition and identity of vascular plugs, including lignins, tannins, pectins (‘gums’) and callose have been studied in an attempt to link decreased hydraulic conduction rates with the appearance of such materials. However, there are two problems associated with such plugs: (1) the time at which they appear is not correlated with observed decreases in flow rates; and (2) only a small percentage (e.g. 2 to 4%, Rasmussen and Carpenter 1974) of xylem vessels exhibit plugging. It has been suggested that the reduced ability of cut stems to take up water over time is a physiological phenomenon, rather than a physical blockage (Rasmussen and Carpenter 1974).

1.4.3 Vascular blockage – microbiological factors Occlusion of xylem conduits by micro-organisms is commonly believed to be the cause of decreased hydraulic conduction in cut stems. Micro-organisms are thought to block xylem conduits and pit membranes physically (Put and Jansen 1989), and also to secrete pectic enzymes (Burdett 1970; Put and Rombouts 1989) and toxic compounds (Put and Klop 1990), which could accelerate senescence. Furthermore, some phytopathogenic bacteria, e.g. Ralstonia solanacearum, produce ethylene (Freebairn and Buddenhagen 1964; Bonn et al. 1975), which may further hasten senescence. Thus, it is commonly recommended that microbial contamination be avoided, and hygienic practices be

employed at each stage of the postharvest chain. Nevertheless, Put and van der Meyden (1988) believed that the extent of decreased conduction after 24 h of infiltration with Pseudomonas putida (104 to 108 cfu mL-1) was too great to be explained by colonisation of infiltrated bacterial cells. They noted that “the vascular water transport system is very sensitive, complicated, and may involve several as yet unknown factors”. Put (1991) observed that vascular blockage, water stress and decreased hydraulic conduction still occurred, despite the absence of micro-organisms.

1.5 The suberin/wounding hypothesis compared with the cavitation hypothesis Kolattukudy (1980) believed that the general response to any mechanical damage to plant tissue, including injury and pathogen invasion, was suberisation. Suberin is an hydrophobic polymeric material that attaches to cell walls (Kolattukudy 1981). It is thought to assist in the effectiveness of compartmentalisation after wounding (Smith and Smart 1955; Schmitt and Liese 1993), and thus inhibit pathogen invasion and water loss. In trees, the CODIT model has been proposed to describe how trees isolate xylem wounds from healthy tissue: Compartmentalisation of Decay In Trees (Shigo and Marx 1977; Shigo 1984). In this model, suberin deposition is thought to assist in the effectiveness of compartmentalisation after wounding. Increased disease resistance is frequently associated with suberisation. Plants that do not form suberin, or only form it in small amounts, are more susceptible to microbial invasion (Gold and Robb 1995). Water loss is prevented through exposed and injured tissues, and a physical barrier is formed to pathogens (Kolattukudy 1980).

In cut stems (which are, after all, dramatically wounded plant tissues), suberin deposition would act as a barrier to water uptake. This would cause premature wilting as water is lost from cells, because stems are no longer able to take up water from the vase solution. There is no published research in the direct area of this project, i.e. inhibition of the initial suberin postharvest wounding response in cut flowers. However, an article relating to suberin deposition and wound healing in geranium cuttings showed that suberin was the first wound response observed within 24 h (Cline and Neely 1983). It was from reading this article and plant pathology literature about pathogenicity and development of a wounding response that the author developed the suberin hypothesis in relation to cut flowers. It is proposed that the formation of an impermeable suberin layer is the early (< 12 h) response to wounding which then precipitates the subsequent documented increase in cavitation (Williamson and Milburn 1995) as hydrophobicity and water stress increase.

Williamson and Milburn (1995) believed that the peak rate of cavitation production occurred as a result of positive feedback, whereby cavitating conduits placed increasing strain on the remaining functional xylem, thereby inducing further cavitation. Hargrave et al. (1994) observed groups of

embolised or non-embolised conduits and suggested that when one conduit embolised, it increased the chances of embolisation in adjacent conduits. Such a phenomenon supports the air seeding hypothesis (Zimmermann 1983) of embolism induction, whereby air is drawn through from the embolised conduit into an adjacent wet pit membrane pore.

However, the initial cause of water stress, and hence cavitation production, remains uncertain. The early rates of cavitation in cut stems seemed too low to explain the familiar decreases in hydraulic conductance that occur over time (Williamson and Milburn 1995). Thus, if cavitation does not cause a decrease in conductance, there must be an earlier cause. Nevertheless, it is possible that early cavitation events decrease conduction markedly. The larger xylem conduits, with flow rates proportional to the fourth power of the capillary radius (Zimmermann 1983), may well be affected first because they are more vulnerable and hence cavitate more easily (the “safety versus efficiency” hypothesis: Zimmermann 1983). Plant tissues may also respond to injury by initiating gas bubbles within their conduits, but it is also likely that there is an earlier response to injury, e.g. prosuberin lamellae visible within 4 h post wounding (Thomson et al. 1995). Thus, I wanted to determine whether cavitation is the first event that precludes water uptake in cut stems, or if it is the universal wounding response of suberisation that inhibits water movement. Van Meeteren and van Gelder (1999) proposed that “an undefined ‘rehydration-inhibiting’ process starts after harvest, which is not due to dehydration of xylem walls or other stem tissue, or to the growth of micro-organisms”.

Since this suberin hypothesis for inhibiting water uptake in cut flowers has been proposed (Williamson and Milburn 1997), there has been some interest in pursuing this area of research, e.g. van Doorn and Cruz (2000) concluded that “preliminary data indicated that material deposited on pit membranes was rich in compounds related to cinnamic acid”. In the biosynthesis of the aromatic domain of suberin there is a channelling of hydroxycinnamic acids into the formation of suberin (Bernards and Lewis 1998).

1.6 Suberin biochemistry Suberin has been eloquently described as an “enigma” because there is no clear definition of what suberin is, despite its obvious importance in plant tissue (Bernards and Lewis 1998). The reason for the lack of definition derives from the inability to obtain pure suberin which is free from other components. Thus, there is uncertainty about both its definition and constitution (Bernards and Lewis 1998). However, it has long been known that suberised tissues contain both polyaromatic (phenolic) and polyaliphatic (fatty acid) domains (Kolattukudy 1981). The aromatic matrix is attached to the cell wall, and the aliphatic components are attached to this matrix (Kolattukudy and Köller 1983). Under Transmission Electron Microscopy (TEM), suberised tissues have a distinctive appearance of

alternating light and dark bands or lamellae (Thomson et al. 1995). These bands are thought to arise from the alternation of the aromatic and aliphatic domains. The presence of the bands under TEM is considered diagnostic of suberised tissue (Bernards and Lewis 1998).

There appears to be a spatial separation of the aliphatic and aromatic domains of suberin. The polyphenols are predominantly within the primary cell wall, while the aliphatic component is in the space between the plasmalemma and the primary wall. Thus, the deposition of the aromatics and aliphatics is not coincident spatially, and neither is it coincident temporally (Bernards and Lewis 1998). The phenolics are detectable from 1 to 2 d in advance of the aliphatics (Rittinger et al. 1987; Thomson et al. 1995). At present, there is no chemical marker to identify the polyaromatic domain of suberised tissues, whereas the α,ω-dioic acids are markers for the aliphatic domain (Bernards and Lewis 1998). The aliphatics are deposited as lamellae under TEM, however, if aliphatics are deposited later than the aromatics, why are they visible so early under TEM as lamellar structures? Also, if the aromatic and aliphatic domains are clearly delineated, it is unlikely that alternating layers of aromatics and aliphatics could explain the lamellar structures (Bernards and Lewis 1998). One hypothesis is that the aliphatic components arrange themselves in a similar way to membrane bilayers (Schmidt and Schönherr 1982).

1.7 Suberin synthesis The formation of the phenolic compounds of suberin begins with the synthesis of trans-cinnamic acid from phenylalanine, catalysed by phenylalanine ammonia lyase (PAL) (Stafford 1974). This is a deamination in which ammonia is split out of phenylalanine by PAL (Salisbury and Ross 1985). In turn, the aromatic amino acids, phenylalanine and tyrosine, are biosynthesised from respiratory intermediates in the shikimic acid pathway. Other relevant phenols to arise from the shikimic acid pathway are cinnamic and ferulic acids, which are derived from phenylalanine and tyrosine. These are converted into, inter alia, lignin and suberin (Salisbury and Ross 1985). Thus, inhibitors of PAL also inhibit suberin synthesis (Kolattukudy 1981). PAL is the first committed enzyme in the phenylpropanoid pathway (Jones 1984), and it can be induced by wounding (Cottle and Kolattukudy 1982). PAL can be inhibited by, e.g., 2-aminoindan-2-phosphonic acid (AIP) (see 1.9).

The ability of suberin to block water flow in the xylem has been questioned because of the common perception that the water conducting parts of xylem are dead (M. Reid, pers. comm.). However, whilst the generality of this statement is true, the detailed reality is different. Upon maturity, the xylem conducting conduits (e.g. vessels, tracheids) are dead as they lack protoplasts, but the xylem fibres and ray parenchyma cells are living. The xylem parenchyma cells are thought to be the most likely site of synthesis of the lipid (or aliphatic) precursors to suberin formation. The lipid components of suberin

must be synthesised in living cells and then moved to coating or suberisation sites (Newcombe and Robb 1989). The ray parenchyma cells are involved in radial water movement in xylem, and thus, would affect water conduction if they were suberised. This is particularly the case if runaway embolisation were to occur, as described above (see The suberin/wounding hypothesis).

Schmitt and Liese (1991) found that suberin was deposited by the cytoplasm of parenchyma cells and that suberised ray and axial parenchyma cells formed a continuous band around the wound. Robb et al. (1987) and Gold and Robb (1995) noted that the coating material first appeared in intercellular spaces between xylem parenchyma cells immediately adjacent to wounded xylem vessels and subsequently moved into pit membranes and spread onto vessel secondary walls.

1.8 Suberin detection Several methods exist to identify suberin. In most of the early work on suberin detection, it is only found 24 h (Cline and Neely 1983), 2 d (Moon et al. 1984) and 3-6 d (Rittinger et al. 1987) after wounding. However, many of these earlier methods used histochemical techniques (e.g. staining with Sudan IV), and it is believed that they are not sensitive enough to detect suberin in the early stages of its formation (Thomson et al. 1995). Suberin would coat the xylem lumen only thinly in the early stages of its formation. Nevertheless, only a small amount would impede water flow.

The aromatic part of suberin can be stained using, e.g. phloroglucinol-HCl, or berberine/aniline blue, which selectively stain for phenolics. The problem with such stains is that it is difficult to distinguish the type of phenolic substance, and therefore between lignin and suberin (Bernards and Lewis 1998). The aliphatic components have been visualised by using the Sudan family of dyes. However, Sudan dyes only stain the aliphatic domain weakly and cannot distinguish between the suberin aliphatic components and other cell lipids (Vaughn and Lulai 1991). While other stains such as neutral red/toluidine blue O suppress cell wall (lignin) autofluorescence (Bernards and Lewis 1998), their ability to detect suberin formation in the early stages is unknown.

Newer techniques such as 13C Nuclear Magnetic Resonance (NMR) have been used to detect suberin, but this method is only able to detect suberin 4 d after wounding (Stark et al. 1994). Therefore, a more sensitive technique such as Transmission Electron Microscopy (TEM) is preferred. Under TEM, prosuberin lamellae have been observed as soon as 4 h post wounding in potato, compared with 24 h using histochemical methods (Sudan IV) and light microscopy (Thomson et al. 1995). Suberin lamellae were then visible 8 h after wounding under TEM (Thomson et al. 1995). Thus, TEM is the preferred method of suberin detection, especially when attempting to determine the early postharvest wounding response.

1.9 Possible suberin inhibitors Several chemicals exist which are known to inhibit suberin production. The most well-known of these is cycloheximide (CHI). CHI is known to inhibit suberin synthesis, however, it is perhaps more widely known as a general inhibitor of protein synthesis (translation) (ap Rees and Bryant 1971).

Salicylhydroxamic acid (SHAM) inhibits suberin formation either by (a) direct inhibition of peroxidase; (b) inhibition of H2O2 production as a substrate for peroxidase-initiated polymerisation of suberin; or a combination of these two (Johnson-Flannagan and Owens 1985).

A chemical which inhibits the activity of phenylalanine ammonia lyase (PAL) is 2-aminoindan-2- phosphonic acid (AIP) (Peiser et al. 1998). It is believed to directly affect PAL enzyme activity and the initial steps in phenylpropanoid (phenolic) metabolism (Peiser et al. 1998).

S-ethyl-N,N-dipropylthiocarbamate (EPTC) is a thiocarbamate herbicide which reduces the chain length of fatty acids in suberin (Schmutz et al. 1996). Cotton fibres treated with EPTC (100 µM) had greatly reduced suberin deposition, formed discontinuous suberin layers, and had a reduced number of lamellae per suberin layer (Schmutz et al. 1996). There was also a decrease in hydroxycinnamic acids (ferulate and caffeate), ω-hydroxyalkanoic acids, and α,ω–hydroxyalkanedioic acids (Schmutz et al. 1996).

Trichloroacetate (TCA) inhibits fatty acid chain elongation (Soliday et al. 1979). Longer chain alkanes (C25) were more severely inhibited than shorter chain hydrocarbons (C19) (Soliday et al. 1979), and the former are characteristic of suberin (Bernards and Lewis 1998). TCA treatment also resulted in disorganisation of suberin ultrastructure (Soliday et al. 1979).

Monoterpenes are one of the main components of essential oils, and the monoterpene, S-carvone, is the major constituent of caraway essential oil. S-carvone is used to suppress sprouting in potatoes (Beveridge et al. 1981), although the mechanism is not known (Oosterhaven et al. 1993). S-carvone has also been found to delay the appearance of suberin in potato tubers, and this was related to the activity of PAL. Potato tissue treated with S-carvone showed delayed suberin formation and also a delay in the increase of PAL activity (Oosterhaven et al. 1995).

Unfortunately, AIP was not available in Australia. Therefore, experiments were conducted with the available suberin inhibitors, CHI, SHAM, EPTC, TCA and S-carvone.

2. Objectives

The objectives of the project were to:

• counteract the poor water uptake that characterises most Australian native cut flowers with borderline vase lives (< 7 days) by inhibiting the initial postharvest wounding response;

• determine whether suberin inhibiting treatments could be used to enhance water uptake and increase the vase life of selected Australian native flowers. This would further enhance the export reputation of Australian native cut flowers and increase the number of exportable species.

• ascertain by Transmission Electron Microscopy whether suberin is deposited in cut stems; and

• ascertain whether any other factors, such as ethylene sensitivity, are involved in a particular flower’s senescence.

3. Methodology

3.1 Plant Material Plant material came from the following local sources: IHD plots (Knoxfield) – Acacia baileyana F. Muell., Baeckea behrii (Schltdl.) F. Muell., B. virgata (Forst. & Forst. f.) Andr., Hakea francisiana F. Muell., (previously H. coriacea Maconochie) grafted onto H. salicifolia (Vent.) B. L. Burtt rootstock; Leptospermum obovatum Sweet, L. polygalifolium Salisb.;

Bill Frew (Farmgate Flowers, Drouin) – Grevillea longistyla Hook., Lophomyrtus × ralphii (J.D. Hook.) Burret ‘Krinkly’;

Berenice & Ted Shaw, Old Emerald Road, Monbulk) – Ceratopetalum gummiferum J. Smith;

Denis Tricks (Longford Flowers, Longford) – Crowea exalata F. Muell. (Longford selection);

Various roadsides – Acacia floribunda (Vent.) Willd.: ~ 201 Burwood Highway, Wantirna, opposite The Knox School; Sphaerolobium vimineum Sm.: ~ 1165 Wellington Road, Belgrave South, just past roundabout at intersection of Belgrave-Hallam Road; Hakea teretifolia (Salisb.) Britten: Jolley Road, Bunyip State Park.

3.2 Transport and Preparation of Plant Material

Plant material was transported to the laboratory in clean buckets containing deionised water. The basal 20 cm were removed from stems under deionised water, and lower leaves were stripped before placing stems into flower tubes. Stems were placed into the vase life room within 5 h of being cut.

3.3 Vase Life Conditions

Experiments were conducted in a specially designed vase life room at IHD, Knoxfield. Standard vase life conditions were used: 20±2°C and RH was between 58-65% (Sytsema 1975; Reid and Kofranek 1980). The lighting used met with the internationally recognised standard (Reid and Kofranek 1980) of 12 h darkness and 12 h Cool White fluorescent light of 20 µmol m-2 s-1 photosynthetically active radiation (PAR) at flower height. The room was vented regularly and automatically. The level of ethylene in the room was tested and was not detectable.

Each glass vase contained a single stem, thus avoiding microbial cross-contamination between stems (Reid and Kofranek 1980). Additionally, the single vase-single stem design meant that the stems were true replicates for statistical analyses. Unless otherwise stated, 10 replicates were used for each treatment. Treatments were arranged in a randomised-block design.

3.4 Determination of pH The pH of solutions was measured with a pH meter (Model 901PH, TPS Pty. Ltd., Brisbane).

3.5 Measurement of Dissolved Oxygen The amount of oxygen present in the vase solution was measured using a dissolved oxygen meter and probe (Model sension6, Hach Company, Colorado USA). This was done in an experiment with Ceratopetalum gummiferum (see 4.14) in which a platform mixer (Model OM6, Ratek Instruments Pty. Ltd., Boronia, Vic.) was used.

3.6 Bacterial Counts Bacterial levels were determined in several experiments (see 4.13, 4.14, 4.16). The number of bacteria, i.e. colony forming units (cfu) per mL of vase water, was determined by removing a 1 mL aliquot from each vase and diluting to an appropriate dilution (up to 10-5). Spread plates were then made by aseptically spreading the diluent onto Plate Count Agar (see Appendix A for recipe). The plates were incubated at 30°C for 48 h, and counted with the aid of magnification from a colony counter (Suntex 560, Model CC-560, Taiwan). Plates were made from two dilutions per vase solution.

Ideally, at least one agar plate in a dilution series will grow between 30 and 300 bacterial colonies. This facilitates manual counting of colonies (Tortora et al. 1986) and eliminates overcrowding and microbial antagonism, which may reduce the count (Harrigan and McCance 1976).

3.7 Ethylene Experiments In two experiments with Baeckea virgata (see 4.4 and 4.5), stems were challenged with 10 ppm ethylene for 24 h. Pure ethylene (1.5 mL) was injected through a gas port into perspex tubs (tub volume = 150 L) sealed by water (see Photograph 3.1). CO2 is a competitive inhibitor of ethylene action (Burg and Burg 1965), therefore the tubs contained potassium hydroxide (20% w/v) (Keys et al. 1975) to absorb respiratory carbon dioxide build up (Kang et al. 1967). The tubs were kept at 20°C, 12 h light and RH in the tubs was ~ 100%. After the ethylene exposure, the stems were well vented before they went into the vase life room with the control flowers.

Photograph 3.1. Perspex tub used for challenging stems with ethylene.

3.8 Washing Experiments An experimental apparatus was designed to test the hypothesis that suberin formation could be prevented or removed by washing (Soliday et al. 1978). Plastic tubing was fitted to a laboratory tap in a sink. This tubing led to a manifold, from which 10 tubes radiated, to feed individually into 10 small glass vials (see Photograph 3.2). The flow rate of the 10 tubes was adjusted with a needle valve. The rate was set to19 mL tube-1 min-1, which resulted in a constant dripping of water from the tubes. The tube was fed to the base of the vial, so it was in close proximity to the base of the flower stem. Thus, there was a constant overflow from these vials down the drain. Another 10 glass vials were randomly arranged amongst the 10 containing tubing to provide a control.

Photograph 3.2. Experimental apparatus used in washing experiments.

3.9 Measurement of Water Uptake and Transpiration In some experiments (see 4.17), the amount of water uptake and transpiration of each stem was quantified. Every 24 h, the amount of water uptake (weight of the vase solution - weight of stem) and transpiration (weight of vase solution + weight of stem) was recorded on a two-place electric balance. Water uptake is the difference in weight of the vase solution - weight of stem between time 1 and time 2, between time 2 and time 3, etc. Transpiration is calculated the same way, using the difference in weight of the vase solution + weight of stem. When stems were deemed to have reached the end of their vase life, no more measurements were made.

3.10 Transmission Electron Microscopy (TEM) Cut stems of Hakea francisiana were kept in either distilled water or S-carvone (0.005% v/v) for 48 h. Control stems were kept in distilled water for 1 h. The basal 1 cm of the stems was cut longitudinally and fixed in Karnovsky’s fixative (2% paraformaldehyde, 2.5% glutaraldehyde in 0.1 M NaPO4 buffer), washed in cacodylate buffer, postfixed in OsO4, and again washed in cacodylate buffer. Material was dehydrated in a graded series of acetone and embedded in Spurr’s epoxy resin. Plant material was examined under TEM (Philips CM120 BioTWIN) at an accelerating voltage of 120 kV. The detailed TEM procedures are set out in Appendix B.

3.11 Statistical Design and Analysis of Results All experiments had a randomised-block design. Data were analysed by ANOVA and F-test at P < 0.05 using Genstat [Genstat 5, Release 4.1, (PC/Windows NT), Copyright 1998, Lawes Agricultural Trust, Rothamsted Experimental Station]. Column graphs were used to present data for visual ease of differentiating between treatments. A bar is shown on each treatment column, indicating the lsd (P < 0.05).

4. Results*

4.1 Acacia baileyana 4.1.1 Introduction and Objectives Acacia baileyana foliage was used because there was a dearth of native flowering material at the time of year this experiment was conducted. Nevertheless, it was believed that the foliage would be a sensitive indicator of vase life as it, like the flowers, dries out quickly in the vase.

The objective was to screen a range of chemicals and concentrations to determine their effect on vase life and to test the hypothesis that suberin inhibition or stimulation would affect vase life. Ascorbic acid was chosen for its antioxidant properties, and its previous beneficial effect on Acacia vase life (Williamson 1989). Citric acid was used for similar reasons (Williamson 1996). Abscisic acid (ABA) is known to promote stomatal closure (Jones and Mansfield 1970), to induce suberisation (Soliday et al. 1978) and inhibit protein synthesis (Thimann 1980). Cycloheximide (CHI) is known to inhibit suberin synthesis, and it is also a general inhibitor of protein synthesis (translation) (ap Rees and Bryant 1971). SHAM inhibits suberin formation by either direct inhibition of peroxidase, inhibition of

H2O2 production as a substrate for peroxidase-initiated polymerisation of suberin, or a combination of these two (Johnson-Flannagan and Owens 1985).

4.1.2 Methodology Abscisic and ascorbic acids are photosensitive, so these chemicals were made and stored in the dark. Test tubes containing these chemicals were covered with foil. SHAM was found to be insoluble in water (Urbañski 1950), therefore after discussions with a Sigma-Aldrich Pty. Ltd. chemist (Dr J. Ribeiro), it was decided to dissolve 1 mg SHAM per 1 mL ethanol (100% v/v).

The pH of all solutions was determined and the influence of pH on vase life was examined using simple linear regression (Genstat).

4.1.3 Senescence Symptoms and Results The symptoms of vase life senescence in A. baileyana foliage are a curling up and separation of the pinnules (≈ leaflets); with a concomitant loss of sheen and suppleness. The end of vase life occurred when any of the above symptoms became evident.

* Note that all vase life results are summarised in Appendix C, and all chemical conversions appear in Appendix D.

The treatment that resulted in the longest vase life was citric acid (5 mM, 12.9 d), which was significantly greater than the deionised water control (7.6 d, Fig. 4.1). Other treatments which were statistically higher than water were citric acid (1.56 mM, 11.4 d) and ascorbic acid (1.7 mM, 10.9 d). The SHAM treatments were not significantly greater than water, nor were the abscisic acid or CHI treatments. All the ABA treatments resulted in a shorter vase life than the deionised water control.

6 Vase life (d)

0 1 uM 5 mM 5 mM 1 mM 2 mM 60 uM 10 uM 3.8 uM 10 mM 20 mM 10 mM 20 mM 100 uM 110 uM 1.8 mM 3.8 mM 1.7 mM 0.5 mM 0.38 mM 1.56 mM ascorbic acid abscisic acid citric acid cycloheximide sham water

Treatment

Fig. 4.1. Vase life of A. baileyana foliage. Data are the means of 10 replicates per treatment. Bars represent the lsd (2.525, P < 0.05).

When the influence of pH on vase life was examined using simple linear regression analysis (Fig. 4.2),

25 y = -0.36x + 10.49 R2 = 0.064 20 (-3.91) (18.47)

10 Vase life (d) 5

0 2345678910 pH

Fig. 4.2. Simple linear regression showing the relationship between vase life and pH in A. baileyana. Although the coefficient for pH is significant (t = -3.91), the variation in pH only explains 6.4% of the variation in vase life (R2 = 0.064).

it was revealed that the pH value had little influence on vase life, as only 6.4% of the variation in vase life was explained by the variation in pH ( R2 = 0.064).

4.1.4 Discussion From the above experiment, it can be seen that the antioxidants, citric and ascorbic acids, were beneficial to vase life. Antioxidants are thought to promote floral longevity by delaying the increase in fatty acid saturation of membrane phospholipids (Paulin 1986). Increased saturation would make membrane lipids less fluid, which is an ubiquitous occurrence among ageing living systems (Borochov and Woodson 1989). Antioxidants might inhibit suberin by inhibiting oxidation of phenolics during suberin synthesis. Bernards and Razem (2001) have recently found that hydrogen peroxide (H2O2) is necessary for the formation of the polyaromatic (phenolic) domain of suberin. The optimum concentration for citric acid (5 mM) was obtained in this experiment because all other concentrations were not as effective at prolonging vase life. A concentration of less than 1.7 mM of ascorbic acid might improve vase life, however, because of its photosensitivity, it is perhaps limited to laboratory use.

The injurious effect of ABA on vase life supports the suberin wounding hypothesis because ABA is known to stimulate suberisation (Soliday et al. 1978). However, the result is complicated by the other roles of ABA in promoting stomatal closure and inhibiting protein synthesis. Thus, it was decided not to include ABA in any future experiments. The suberin hypothesis was not discounted by the present results: it is possible that the correct concentration of CHI (an inhibitor of suberin synthesis) was less than the 1 µM used, and the SHAM results were likely to be confounded by the ethanol used to dissolve SHAM. Thus, a second A. baileyana experiment was performed, testing the effects of SHAM made without ethanol.

The pH of solutions did not greatly influence vase life (Fig. 4.2). Williamson (1996) previously found that different solutions with the same pH had significantly different vase lives, therefore the effect of pH on vase life was not tested in further experiments in the current project.

4.2 Acacia baileyana, second experiment 4.2.1 Introduction and Objectives A second experiment with A. baileyana was designed to test the effects of SHAM dissolved without ethanol. It was realised that SHAM could be readily dissolved in water by preparing a monosodium salt of the acid. Reacting 1 mol of sodium bicarbonate with 1 mol of SHAM produces a sodium salt of SHAM which is then freely soluble in water (Urbañski 1950). The same concentrations of SHAM that were used in the above experiment were then repeated; and a 25% (v/v) ethanol treatment was included to determine the effect of ethanol on vase life. CHI was not included in this second

experiment because its general protein synthesis inhibition (ap Rees and Bryant 1971) was considered too general to attribute any beneficial effects to suberin inhibition.

4.2.2 Results The solution which resulted in the longest vase life was SHAM (10 µM, 16.4 d, Fig.4.3), however, this was not significantly different from water (15.1 d). The effect of increasing SHAM concentration was almost negatively linear, indicating that the optimal concentration of SHAM may not have been used. The use of ethanol had a markedly injurious effect on vase life. In this second A. baileyana experiment, the water control lived much longer (15.1 d) than in the first experiment (7.6 d).

8 Vase life (d)

0 10uM 110uM 1mM 2mM 25% (v/v)

sham water ethanol Treatment

Fig. 4.3. Vase life of A. baileyana foliage. Data are the means of 10 replicates per treatment. Bars represent the lsd (1.615, P < 0.05).

4.2.3 Discussion The improved effects of SHAM were evident in this second experiment, when the results were not masked by ethanol damage. It is likely that an even lower concentration of SHAM than the 10 µM used here may have further increased vase life over that of water. Thus, the effect of SHAM in inhibiting suberin formation, is therefore not discounted. The concentrations of SHAM used in this experiment came from various journal articles, although, obviously, none related to cut flowers as this work has not been done previously.

The effect of ethanol was similar in both experiments: 7.2 d (second experiment) cf. 7.8 d (average of SHAM results in first experiment). Thus, ethanol injury is evident from both experiments. Although ethanol has been used as an inhibitor of ethylene production (Heins 1980) and action (Wu et al. 1992),

the most beneficial concentration of 8% in both those cases, was far below the injurious 25% used here.

The deionised water results were markedly different in both experiments. Climatic conditions may have influenced vase life: the first experiment was performed in late spring, whereas the second experiment was conducted in mid-autumn.

4.3 Baeckea virgata, vase life experiment 4.3.1 Introduction and Objectives Baeckea (Myrtaceae) is useful as an out-of-season native filler flower. It flowers from October to January - a time when most other native species are not in flower. Its small, white flowers are a similar, but reduced form of the popular Geraldton wax (Chamelaucium uncinatum). Despite its short individual flowering period, it can fill a niche market and successive cropping can be manipulated by either pruning regimes or planting different species. The objective of this experiment was to further test the effect of various antioxidants, ascorbic and citric acids, and a suberin-inhibiting chemical, CHI, on Baeckea vase life.

4.3.2 Methodology Vase solutions were prepared as described above (Section 4.1.2). The stage of floral development at which flowers were harvested was the bud stage, just prior to anthesis. B. virgata was used for this experiment.

4.3.3 Senescence Symptoms and Results, B. virgata Symptoms of senescence in B. virgata were a change in petal colour from white to cream, together with a shrivelling of the petals. The petals did not drop. The nectary changed colour from pale green to brown/black, and the leaves fell off when touched.

In this experiment, deionised water produced the longest vase life (11.8 d), although it was not significantly different from CHI (0.01 µM, 11.3 d) (Fig. 4.4). Again, there was a negative linear relationship between concentration and vase life with increasing CHI concentration. In the higher concentrations of citric acid, e.g. 5 mM and 10 mM, there was a marked tendency for the leaves to fall. In 1.56 mM citric acid, the flowers developed well, although this did not result in a longer vase life.

6 Vase life (d)

0 1.7mM 5mM 10mM 1.56mM 5mM 10mM 0.01uM 0.1uM 1uM

ascorbic acid citric acid cycloheximide water

Treatment

Fig. 4.4. Vase life of B. virgata. Data are the means of 10 replicates per treatment. Bars represent the lsd (0.955, P < 0.05).

4.3.4 Discussion The most evident feature from this experiment was that none of the chemicals improved vase life over that of deionised water. Nevertheless, the negative linear relationship between increasing CHI concentration and vase life indicated that a CHI concentration of less than 0.01 µM may have increased longevity. This could indicate that suberin inhibition with CHI need not be discounted and a lower CHI concentration might increase vase life over that of water. The patterns formed by the concentrations of citric acid revealed that the optimum concentrations were likely to have been reached. It is unlikely that a lower concentration of ascorbic acid would dramatically improve vase life more than 100%, to approach that of water.

The result that water produced the longest vase life, together with the ineffectiveness of most other treatments, and the marked leaf drop, indicated to the author that B. virgata might be ethylene sensitive. Thus, a second experiment was designed with B. virgata, in which the effects of exposure to ethylene and protection with an STS pulse were determined.

4.4 B. virgata, ethylene experiment 4.4.1 Introduction and Objectives The objective of this experiment was to test whether B. virgata was sensitive to ethylene (after exposure to ethylene) and whether protection against ethylene action could be achieved by pulsing

with STS. Different concentrations of the STS pulse were used to determine toxicity effects and the optimal treatment. (See Appendix E for STS preparation and disposal methods.)

4.4.2 Methodology The experimental design for the ethylene experiment is described in section 3.7.

4.4.3 Senescence Symptoms and Results, B. virgata The senescence symptoms were similar to those already described for B. virgata above. However, there was a marked amount of petal drop in those stems exposed to ethylene compared with controls, or stems protected by STS (Photograph 4.1).

A B

Photograph 4.1. A: Ethylene + STS: very little petal drop occurred when stems were protected against ethylene action with an STS pulse. B. Ethylene alone: petal drop was markedly greater when stems were exposed to ethylene without the protection of an STS pulse.

Vase life was deemed to have ended when >50% of petals had either fallen (in the case of ethylene exposure), wilted, or changed to an unacceptable colour. Again, the longest vase life occurred in the deionised water control (Fig. 4.5, 12.9 d). Ethylene exposure did not significantly reduce vase life compared with deionised water, although a vast amount of petal drop occurred. Protection against ethylene action with an STS pulse reduced petal drop (Photograph 4.1), but did not result in a longer vase life. When the STS pulse was used without exposure to ethylene, 0.2 mM was the optimal

concentration, however, when flowers were exposed to ethylene, pulsing with 0.4 mM STS increased vase life over that of 0.2 mM, although not significantly.

Vase life (d) 6

0 0.2mM 0.4mM 0.2mM 0.4mM

STS water STS +ethylene ethylene

Treatment

Fig. 4.5. Vase life of B. virgata after exposure to ethylene. Data are the means of 10 replicates per treatment. Bars represent the lsd (1.754, P < 0.05).

4.4.4 Discussion This experiment has shown that B. virgata is sensitive to ethylene, as evidenced by the large amount of petal drop after ethylene exposure. An STS pulse (0.2 or 0.4 mM) markedly inhibited the amount of petal drop, even when plants were challenged with ethylene, however, pulsing did not result in a longer vase life. Symptoms of Ag+ toxicity were evident in the STS pulsed petals, which changed colour from white to cream in both STS concentrations. Such toxicity symptoms might be avoided if stems were pulsed for a shorter period, or a lower concentration of STS was used.

4.5 B. virgata, quantification of petal drop experiment 4.5.1 Introduction and Objectives Quantification of the amount of petal drop was not possible in the above experiment with B. virgata because of the large flowering stems used, therefore an experiment was designed using small sprigs to allow petal drop to be quantified. Quantification of petal drop would enable the efficacy of the treatments in response to ethylene exposure to be determined.

4.5.2 Methodology Stems were challenged with ethylene in sealed perspex tubs, as described previously (section 3.7). Small glass vials containing a sprig of B. virgata were placed inside larger plastic “capture” vials to

ensure that petal drop could be quantified for each sprig (Photograph 4.2). The number of petals on each sprig was counted at the beginning of the experiment. Every 24 h, each sprig was tapped gently 20 times onto black cardboard to determine petal drop. The amount of fallen petals for each sprig was then counted. Cumulative petal drop data were expressed as a percentage of the initial number of petals on each sprig. Data were analysed by two-way ANOVA, examining the differences in petal drop between treatments at days 2, 5 and 10.

Photograph 4.2. Experimental set up for quantifying the amount of petal drop in B. virgata.

4.5.3 Senescence Symptoms and Results, B. virgata quantification experiment Senescence symptoms were as described above (section 4.4.3) for B. virgata. Petal drop was determined for each sprig until the end of that sprig’s vase life. However, no vase life results are shown because the small sprigs were not deemed representative of a true vase life experiment. Cumulative petal drop, expressed as a percentage of the initial number of petals, is shown in Fig. 4.6.

At day 2, the only significant difference in cumulative % petal drop occurred in the petals not treated with STS (Table 4.1). A significant difference in petal drop also occurred at day 5 in the petals not treated with STS. By day 10, however, no significant differences were found in any of the treatments.

30 0.2 STS 0.2 STS + eth 0.4 STS 20 0.4 STS + eth Cumulative total petal drop (%) ethylene 10 water

0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Time (d)

Fig. 4.6. Cumulative total petal drop (expressed as a % of initial total petal count) in B. virgata. Data are the means of 10 replicates per treatment.

Table 4.1 Mean cumulative percent petal drop in B. virgata Day 2 Day 5 Day 10 STS concentration Air Ethylene Air Ethylene Air Ethylene 0 11.7 56.9 31.3 65.9 62.1 73.0 0.2 mM 16.0 27.4 37.2 41.1 68.6 75.7 0.4 mM 8.8 9.3 23.3 15.6 57.4 46.6 lsd 13.42 16.54 16.53

4.5.4 Discussion From this experiment, it can be seen that exposing flowering stems of B. virgata to ethylene results in a significant initial amount of petal drop. Petal drop can be inhibited if stems are either not exposed to ethylene, or if stems are protected against ethylene action with STS (both at 0.2 and 0.4 mM concentrations). After 5 days, that difference was still evident, however, by day 10, no significant differences in cumulative petal drop occurred. The effects of STS pulsing are not temporary, therefore, this raises the question of whether B. virgata begins to produce its own ethylene after several days, akin to a climacteric in some fruits. The logical continuation of this experiment would thus be to test ethylene production in B. virgata over time: do the flowers produce ethylene, and is an increase in ethylene production evident?

The 0.2 mM concentration of STS was not as effective as 0.4 mM in retarding petal drop. By day 10, 76% petal drop had occurred in the 0.2 mM STS pulse treatment, compared with 47% in the 0.4 mM STS pulse treatment, however, these were not significantly different. It is tempting to think that the higher 0.4 mM STS concentration was the better one, however, it should be recalled from section 4.4.4 that symptoms of Ag+ toxicity were evident in both concentrations of STS. Perhaps pulsing with 1- MCP might improve results with Baeckea. STS protected against ethylene-induced petal drop, but STS alone did not stop natural petal drop in B. virgata.

4.6 B. behrii, a comparison of cutting in air to cutting under water 4.6.1 Introduction and Objectives Previous work with Leptospermum (J. Faragher, unpublished) indicated that cutting stems in air and then storing them for 24 h at high humidity was more beneficial to vase life than cutting stems under water and placing them into water immediately. Theoretically, cutting stems under water would delay the onset of cavitation. Cavitation results in embolisation of xylem conduits, which results in an inability of stems to take up water, and therefore in decreased longevity (Williamson and Milburn 1995). The aim of this experiment was to determine whether cutting stems under water was more beneficial than cutting in air. It was also wondered whether the suberin inhibiting chemicals would be effective when stems were cut in air.

4.6.2 Methodology Flowering B. behrii was used in this experiment. The experiment compared two organic acid antioxidants, ascorbic and citric acids, with the suberin (lipoxygenase) inhibitor and antioxidant, n- propyl gallate, and the suberin inhibitor, SHAM. Stems were either cut under water, or in air. The stems that had been cut in air were transferred into a plastic bag, containing cotton wool moistened with sterile distilled water. The stems that were cut under water were placed into water immediately. There were 10 stems in each of these bags, which were sealed and placed in the dark for 24 h at 20°C. The concentrations of the chemicals were as follows: citric acid (5 mM), ascorbic acid (100 ppm), CHI (0.01 µM), SHAM (1 µM), n-propyl gallate (4 mM). These concentrations were either the most beneficial from other experiments done in this project, or were taken from the literature (n-propyl gallate, van Doorn and Cruz 2000).

4.6.3 Senescence Symptoms and Results, B. behrii Floral senescence followed a similar pattern to that observed in B. virgata, i.e. the nectary colour changed from green to red with age. The petal colour also changed from white to cream and the petals either inrolled and desiccated, or abscised, depending on treatment. The n-propyl gallate solution was

obviously too strong because the leaves and petals were clearly desiccated by day 5. In that solution the petals desiccated and adhered rather than abscised.

Stems that had been kept in humid air for 24 h prior to placement in SHAM produced the significantly longest vase life (= total vase life) (Fig. 4.7). When the stems were cut under water and placed directly into SHAM, the vase life was not significantly improved over that of several other treatments.

Vase life (d) Vase life 6

0 air water air water air water air water air water air water ascorbic acid citric acid cycloheximide n-propyl gallate sham water

Treatment Fig. 4.7. Vase life of B. behrii in which cutting stems in air was compared with cutting stems under water. Data are the means of 10 replicates per treatment. Bars represent the lsd (1.734, P < 0.05).

4.6.4 Discussion The results of this experiment were surprising and confounded previous literature (van Meeteren 1992, van Doorn and Cruz 2000). Previous research found that cutting in air resulted in stem occlusion and a shorter vase life compared with cutting in water (van Meeteren 1992). Nevertheless, the result with SHAM is encouraging because it significantly increased vase life compared with all other treatments. This may indicate a role of suberin formation in decreasing vase life. In terms of transport, the results are promising because they show that if stems are transported in air under high humidity (for one day), vase life is not reduced.

4.7 Crowea exalata 4.7.1 Introduction and Objectives Crowea exalata (Rutaceae) is one of three species in this Australian genus. It has attractive pink flowers, which appear along the stems from summer to late autumn. It is a popular flower among Japanese consumers (D. Tricks, pers. comm.) and already has a long vase life (> 7 d), however, little is

known of its postharvest behaviour. The aim of this experiment was to test a range of chemicals and concentrations on the vase life of C. exalata.

4.7.2 Methodology A form of C. exalata growing at Longford was used (D. Tricks, pers. comm.). This form has very attractive pale pink flowers and the foliage releases a pleasant aroma when touched. The following chemicals were used: ascorbic acid and citric acid (organic acid antioxidants), CHI and SHAM (suberin inhibitors) and STS (ethylene action inhibitor). STS was prepared as described in Appendix E, and the flowers were pulsed at 4 °C for 16 h.

4.7.3 Senescence Symptoms and Results Symptoms of senescence in C. exalata are a fading of the flowers from pale pink to white. The petals desiccate and roll back. Flower abscission at the pedicel also occurs. The end of vase life occurred when >50% of the flowers exhibited signs of senescence.

In the stems which were pulsed with 0.5 mM STS, there was a blackening of the leaves 24 h after the pulse had finished. Flowers in the 0.5 mM STS concentration also lived a significantly shorter time than flowers pulsed with 0.2 mM STS (Fig. 4.8). The highest concentrations of both citric and ascorbic acids were also too strong because the leaves looked acceptable, but dropped off when touched. The lowest concentrations of SHAM, ascorbic and citric acids were the most beneficial, as indicated by the almost linear relationships in vase life.

Vase life (d) 6

0 1 mM 1 uM 10um 5 mM 5 mM 2 mM 0.1 uM 1.7 mM 10 mM 15 mM 10 mM 15 mM 0.2 mM 0.5 mM 0.01 uM 110 uM 1.56 mM 0.001 uM ascorbic acid citric acid cycloheximide sham STS water Treatment

Fig. 4.8. Vase life of Crowea exalata. Data are the means of 10 replicates per treatment. Bars represent the lsd (1.731, P < 0.05).

The treatment which produced the longest vase life was STS at 0.2 mM (15.6 days), then SHAM at 10 µM (15 days), however, these two treatments were not significantly different from each other (Fig. 4.8). STS reduced the amount of petal drop which occurred, however, this was not quantified.

4.7.4 Discussion The leaf blackening that occurred in the 0.5 mM STS pulse indicated a toxicity level had been reached and therefore the 0.5 mM pulse was either too strong, or too long. It was noted that a large number of petals abscised in this experiment. This raised the question of the possibility of ethylene involvement in the senescence of C. exalata. Therefore, a second experiment was devised to test the effects of treatment with STS, SHAM and a combination of STS and SHAM.

4.8 C. exalata, testing the effects of STS and SHAM 4.8.1 Introduction and Objectives The results of the above C. exalata experiment (section 4.7) indicated that ethylene may be involved in its senescence. The two treatments which produced the significantly longest vase life in that experiment were STS and SHAM. Thus, it was decided to test the effects of a combination of STS and SHAM in this next experiment.

4.8.2 Methodology The methodology was the same as described above (section 4.7.2), i.e. the flowers were pulsed at 4 °C for 16 h, but only in a 0.2 mM STS concentration.

4.8.3 Results The treatment that resulted in the longest vase life was the STS + SHAM combination (14.2 d), however, this was not significantly different from the STS treatment on its own (13.6), or water (12.4 d) (Fig. 4.9). SHAM by itself resulted in a significantly lower vase life (11 d).

4.8.4 Discussion Although the combination of STS + SHAM resulted in the longest vase life of 14.2 d, as this was not significantly different from STS on its own, it does not appear necessary to include SHAM in the vase solution. Whilst this might also be statistically true of the water control (12.4 d) as well, one sometimes has to weigh up statistics versus practical results. It is up to the grower to decide whether the extra effort of STS pulsing is worth up to two more (non-significant) days of vase life; or whether the decreased amount of petal drop after STS treatment is perceived well enough along the marketing chain to be worthwhile. It should be noted that the amount of petal drop was not determined in this

6 Vase life (d)

0 10uM 0.2mM 0.2mM+10uM

sham sts sts+sham water Treatment

Fig. 4.9. Vase life of Crowea exalata. Data are the means of 10 replicates per treatment. Bars represent the lsd (2.006, P < 0.05). experiment, thus, vase life may well have been shorter in the water treatment if petal drop had been carefully enumerated. It should also be pointed out that this second C. exalata experiment was performed with plant material that was one month later in the flowering season, and may well have been past “its prime”. Further evidence for this comes from the fact that the water control in the first C. exalata experiment lived 13.5 d compared with 15.6 d for the 0.2 mM STS treatment, and these results were significantly different from each other.

4.9 Grevillea longistyla 4.9.1 Introduction and Objectives Grevillea longistyla has attractive red flowers and long, narrow dark green foliage up to 30 cm long. The linear revolute foliage is so attractive that it is also used alone, and therefore can be harvested year round. However, little is known of its postharvest requirements, so this experiment was designed to test the effect of two organic acid antioxidants, citric and ascorbic acids, together with the suberin inhibitor, SHAM.

4.9.2 Methodology This experiment tested four concentrations of citric acid, three of ascorbic acid and three concentrations of SHAM on G. longistyla foliage. Plant material was prepared as described previously (section 3.2).

4.9.3 Senescence Symptoms and Results Senescence symptoms in G. longistyla foliage are desiccation and a curling inwards of the ends of the leaves. The leaves sometimes exhibit a speckled blackening from the tips of the leaves downwards, which then results in the entire leaf becoming black. Vase life was deemed to have ended when >50% of the foliage exhibited blackening, curling, or desiccation.

The treatment that resulted in the longest vase life was SHAM 1 (0.1 µM, Fig. 4.10), although none of the treatments was significantly different from the other as the lsd was quite large (5.392). One of the SHAM 1 treatments lived 54 d, however, redoing the analysis without that result did not change significance levels.

10 Vase life (d)

0 0.01mM 0.1mM 1mM 0.5mM 1mM 2mM 5mM 0.1uM 1uM 10uM ascorbic acid citric acid sham water

Treatment

Fig. 4.10. Vase life of Grevillea longistyla foliage. Data are the means of 10 replicates per treatment. Bars represent the lsd (5.392, P < 0.05).

4.9.4 Discussion The effect of SHAM concentration was linear (Fig. 4.10), so it would be interesting to try a 0.01 µM concentration of SHAM with G. longistyla to see if any further increase in vase life occurred. G. longistyla foliage is long-lived, as indicated by the distilled water control (18.1 d), and does not appear to be ethylene sensitive, therefore, like C. exalata, it is really up to the end user whether they consider it worth including any vase life additives. It would be used as a greenery filler, so it is unlikely the vase life needs to be extended more than 18 d, as that would probably outlive the other flowers in the arrangement. The effect of SHAM was not significantly greater than that of all other treatments (except SHAM 3), so inhibiting the wounding response does not appear to be necessary in G.

longistyla. This may be because (1) the foliage naturally lives a long time and so any beneficial effect of SHAM is masked by longevity; (2) suberin may not be involved in G. longistyla senescence; (3) SHAM may not inhibit suberin formation in G. longistyla; or (4) a lower concentration of SHAM might significantly increase vase life. Given that suberin is a universal wounding response (Kolattukudy 1980), the answers are more likely to be (1), (3) or (4) above.

4.10 Lophomyrtus × ralphii ‘Krinkly’ 4.10.1 Introduction and Objectives Lophomyrtus (Myrtaceae) is native to New Zealand, however, no Australian native flora were available at the time of this experiment. Lophomyrtus is popular among some Australian cut flower growers and florists because of its attractive year-round crimson foliage, however, nothing is known of its postharvest response to chemical treatment. Therefore, the objective of this experiment was to test several chemicals and concentrations in order to obtain vase life information about this relatively new crop.

4.10.2 Methodology Lophomyrtus × ralphii ‘Krinkly’ foliage was used in this experiment. Three concentrations of the organic acid antioxidants, citric and ascorbic acids, were tested, together with the suberin inhibitor, CHI.

4.10.3 Senescence Symptoms and Results The first visible symptom of senescence was a bending over of the tips of branches. A few days later, other leaves appeared desiccated and curled under on the margins. At this time they also lost their shine and appeared dull. In addition, both the shiny and dull leaves dropped off when they were touched. Leaf drying and/or drop determined the end of vase life.

The longest vase life occurred in the deionised water control (14.5 d, Fig.4.11). This result was not significantly different from citric acid (5 mM, 14 d), CHI (0.01 µM, 13.3 d), CHI (0.1 µM, 12.91 d) or ascorbic acid (1.77 mM, 12.7 d). There was virtually a negative linear relationship between vase life and increasing ascorbic acid concentration; to a lesser extent this pattern was repeated in the CHI treatment.

4.10.4 Discussion The pattern created by vase life and increasing concentration for both ascorbic acid and CHI revealed that the lowest concentrations used were the most beneficial to vase life. Extrapolating from this

8 Vase life (d) 6

0 1.7mM 5mM 10mM 1.56mM 5mM 10mM 0.01uM 0.1uM 1uM

ascorbic acid citric acid cycloheximide water Treatment

Fig. 4.11. Vase life of Lophomyrtus × ralphii ‘Krinkly’ foliage. Data are the means of 10 replicates per treatment. Bars represent the lsd (2.325, P < 0.05). pattern, it is possible that lower concentrations of those chemicals may have further increased vase life. However, because none of the other treatments improved vase life over that of water, together with the observed leaf fall, indicated a possible sensitivity to ethylene in this cultivar. Therefore, a further experiment was done in which the effect of STS on vase life was determined.

4.11 Lophomyrtus × ralphii ‘Krinkly’, determining ethylene sensitivity 4.11.1 Introduction and Objectives In the experiment with L. × ralphii ‘Krinkly’ described above, no chemical known to improve water uptake had a more beneficial effect on vase life than water. This result, together with the observation that senescent and non-senescent leaves fell in all treatments, indicated a possible role of ethylene in the postharvest senescence of this cultivar. Thus, the objective of this experiment was to determine the effects of ethylene inhibition with an STS pulse on the vase life of L. × ralphii ‘Krinkly’.

4.11.2 Methodology Three concentrations of an STS pulse were used, 0.1 mM, 0.2 mM and 0.5 mM. These pulses occurred at 4°C for 16 h. In addition, three concentrations of the suberin-inhibiting chemical, SHAM, were used, as well as three concentrations of ascorbic acid.

4.11.3 Senescence Symptoms and Results Senescence symptoms were similar to those described above, except that in the STS treatments, leaf drop did not occur. In those treatments, senescence was characterised by a gradual dulling of the leaves’ shiny appearance. The end of vase life was determined by either leaf dullness or leaf drop.

The treatments which produced the significantly longest vase lives were the three STS pulse treatments: 0.5 mM STS (30.4 d), 0.2 mM STS (29.8 d) and 0.1 mM STS 1 (29 d) (Fig. 4.12). These treatments were not significantly different from each other. Treatment with SHAM (1 µM) produced a vase life of 23 d whereas water was 20.7 d, however, these results were not significantly different (lsd = 6.680). All of the ascorbic acid treatments resulted in a shorter vase life than in water.

Vase life (d) 15

0 0.01mM 0.1mM 1mM 0.1uM 1uM 10uM 0.1mM 0.2mM 0.5mM ascorbic acid sham sts water Treatment

Fig. 4.12. Vase life of Lophomyrtus × ralphii ‘Krinkly’ foliage. Data are the means of 10 replicates per treatment. Bars represent the lsd (6.680, P < 0.05).

4.11.4 Discussion The above results reveal the importance of ethylene in the vase life of those flowers which are sensitive to it. Treatment with any chemical known to prolong vase life has no effect on longevity if ethylene is involved in that flower’s senescence unless it addresses ethylene sensitivity. In such cases, it is imperative that protection against ethylene production or action occur if optimum vase life is to be attained. Such a finding could explain the ineffectiveness of SHAM in prolonging vase life in this case.

4.12 Washing experiments 4.12.1 Introduction and Objectives Previous research has shown that washing can severely inhibit suberisation of potato disks (Soliday et al. 1978). Therefore, an experimental apparatus was designed to test this hypothesis (see section 3.8 and Photograph 3.2). This section details experiments with several genera: Acacia, Ceratopetalum, Crowea, Hakea and Sphaerolobium.

4.12.2 Methodology The methodology is described in section 3.8. Stems with a tube that constantly dripped water into the vial were considered to be “washed”, while those without tubes were referred to as “not washed”.

4.12.3 Senescence Symptoms and Results Acacia floribunda In Acacia floribunda, there was no significant difference between the vase life of stems that were washed (5.8 d) compared with stems that were not washed (5.2 d, Fig. 4.13).

3 Vase life (d)

0 Washed Not washed Treatment

Fig. 4.13. Vase life of Acacia floribunda flowering stems. Stems which were washed had a constant change over of water, whereas stems which were not washed did not have the water changed. Data are the means of 10 replicates per treatment. Bars represent the lsd (0.672, P < 0.05).

Ceratopetalum gummiferum This experiment was conducted when there was a lack of suitable Australian native flowering material. C. gummiferum material was used which was past the optimum harvest time, therefore, the criterion for end of vase life was when 100% of the red bracts (= ‘flowers’) had reached the end of

vase life. There was a significant difference between the vase life of stems that were washed (16.56 d) compared with stems that were not washed (11.6 d, Fig. 4.14).

8 Vase life (d)

0 Washed Not washed Treatment

Fig. 4.14. Vase life of flowering Ceratopetalum gummiferum stems. Stems which were washed had a constant change over of water, whereas stems which were not washed did not have the water changed. Data are the means of 10 replicates per treatment. Bars represent the lsd (3.898, P < 0.05).

C. gummiferum, second experiment A second experiment was performed with C. gummiferum, in which the flowers were harvested at the optimal time. Vase life was deemed to have ended when > 50% of the flowers exhibited signs of senescence. There was also a significant difference between the vase life of stems that were washed (19.1 d) compared with stems that were not washed (14 d, Fig. 4.15).

10 Vase life (d)

0 Washed Not washed Treatment Fig. 4.15. Vase life of flowering C. gummiferum stems, second experiment. Washed stems had a constant change over of water, whereas stems which were not washed did not have the water changed. Data are the means of 10 replicates per treatment. Bars represent the lsd (1.861, P < 0.05).

Crowea exalata There was a significant difference between the vase life of stems that were washed (13.7 d) compared with stems that were not washed (12.1 d, Fig. 4.16).

8 Vase life (d) Vase life 6

0 Washed Not washed Treatment

Fig. 4.16. Vase life of flowering Crowea exalata stems. Stems which were washed had a constant change over of water, whereas stems which were not washed did not have the water changed. Data are the means of 10 replicates per treatment. Bars represent the lsd (1.315, P < 0.05).

Hakea teretifolia There was no significant difference between the vase life of stems that were washed (8.7 d) compared with stems that were not washed (8.2 d, Fig. 4.17) (lsd 2.026).

6 Vase life (d)

0 Washed Not washed Treatment

Fig. 4.17. Vase life of flowering Hakea teretifolia stems. Stems which were washed had a constant change over of water, whereas stems which were not washed did not have the water changed. Data are the means of 10 replicates per treatment. Bars represent the lsd (2.026, P < 0.05).

Sphaerolobium vimineum There was no significant difference between the vase life of stems that were washed (7 d) compared with stems that were not washed (7.6 d, Fig. 4.18).

4.12.4 Discussion In all of the above experiments, vase life was longer when stems were washed compared with the stems that were not washed, however, this was only significantly different in three of the six experiments. The flowers with delicate floral structures, e.g. staminiferous Acacias and Hakeas, and Sphaerolobium, were the ones in which there was no significant improvement in vase life by washing. Crowea and Ceratopetalum have much more robust obvious floral parts, and these were the genera in which vase life was improved. However, if washing removes suberin, then why wasn’t the experiment successful in all cases? There are several possibilities: (1) washing may not remove suberin; (2) suberin may not be involved in cut flower senescence; (3) ethylene may be involved in the senescence of those flowers; (4) the vase life of those flowers may have been too short, irrespective of treatment, to show any improvement, or (5) another factor may be involved. We know that washing does remove suberin (Soliday et al. 1978) and that suberin is a general response to cutting or wounding. Ethylene is

Vase life (d) Vase life 4

0 Washed Not washed Treatment

Fig. 4.18. Vase life of flowering Sphaerolobium vimineum stems. Stems which were washed had a constant change over of water, whereas stems which were not washed did not have the water changed. Data are the means of 10 replicates per treatment. Bars represent the lsd (2.216, P < 0.05). involved in the senescence of the two species that responded to washing, Ceratopetalum and Crowea – its involvement in the other genera used is not known. Therefore, it may be either that the vase life of the flowers that did not respond to washing was too short, or that another factor was involved. In the washed stem treatment, the vase solution was constantly removed and renewed, which would not allow a build up of a microbial population. This hypothesis was tested by monitoring the number of bacteria in a washing experiment.

4.13 Bacterial monitoring of washing experiments 4.13.1 Introduction and Objectives The aim of this experiment was to monitor the bacterial levels in a washing experiment to determine whether there was a relationship between vase life and bacterial numbers.

4.13.2 Methodology C. gummiferum was used in this experiment. Every 2 d, the number of bacteria in the vase solutions was monitored as described previously (section 3.6).

4.13.3 Results The mean number of bacteria throughout the experiment is shown below (Fig. 4.19). Differences in bacterial numbers were analysed after 8 d in both treatments. The mean number of bacteria for the no tube (i.e. not washed) treatment was 5.17 (log 10 cfu/mL), whereas for the tube (washed) treatment it was 0.25 (log 10 cfu/mL). These results were significantly different when analysed by ANOVA (lsd 0.959), however, there was too much variation for ANOVA analysis. In the tube treatment, eight of the ten tubes had no bacterial counts, and the other two had very low numbers: too many “zeroes” for ANOVA analysis. All that can be presented are the treatment means of 5.17 and 0.25 and the standard error of the mean (0.35).

4 cfu/mL) 10

2 NT T No. of bacteria (log

0 Day 0 Day 2 Day 4 Day 6 Day 8 Day 10 Day 12 Day 14 Day 16 Day 18 Day 20 Day 22 Time (d)

Fig. 4.19. The mean number of bacteria (log 10 cfu/mL) in Ceratopetalum gummiferum washing experiment. Data are the means of 10 replicates per treatment. NT = no tube (i.e. not washed); T = tube (i.e. washed).

4.13.4 Discussion The tubes in the washed treatment contained significantly lower numbers of bacteria than tubes which were not washed. The vase life results were already presented in Fig. 4.15, and showed that the washed tubes had a significantly longer vase life than the not washed tubes. Thus, it is likely that the beneficial effect of the washing treatment was due to a reduction in bacterial numbers. These results led to the experiment described below (section 4.14).

4.14 Shaker experiment 4.14.1 Introduction and Objectives This experiment was the sequel to the washing experiment. A platform mixer was used to “wash” the stems, without reducing the number of bacteria in the vase water. “Washing” potato disks in the same liquid on a platform mixer was previously shown to inhibit suberin formation (Soliday et al. 1978). The number of bacteria was also determined in this experiment.

4.14.2 Methodology Cut flowering stems of Ceratopetalum gummiferum were used and prepared as described previously (section 3.2). Stems were placed in individual 250 mL conical flasks containing 150 mL deionised water. Ten flasks were placed on a platform mixer, set to a speed of 110 rpm. This speed ensured a constant movement of water around the stem, without exposing the cut end to air.

The amount of dissolved oxygen in all flasks was determined as described in section 3.5. The number of bacteria in the flasks was monitored every 2 d as described previously (section 3.6).

4.14.3 Results Stems that were shaken lived a significantly shorter time (10.5 d) compared with stems that were not shaken (16.2 d) (Fig. 4.20). The flowering bracts of the shaken stems exhibited symptoms of dehydration far earlier than the non-shaken stems.

8 Vase life (d) Vase life

0 Shaken Not Shaken Treatment

Fig. 4.20. Vase life of Ceratopetalum gummiferum. Shaken vases were kept on a platform mixer at 110 rpm. Data are the means of 10 replicates per treatment. Bars represent the lsd (2.871, P < 0.05).

There was a significant difference in the amount of dissolved oxygen in the vase solutions that were shaken (96.89%) compared with those that were not shaken (89.67%, Fig. 4.21).

100

88 % oxygen saturation

82 Shaken Not shaken Treatment

Fig. 4.21. The amount of dissolved oxygen (expressed as % oxygen saturation) in vases containing Ceratopetalum gummiferum. Shaken vases were kept on a platform mixer at 110 rpm. Data are the means of 10 replicates per treatment. Bars represent the lsd (2.453, P < 0.05).

The number of bacteria in the shaken and not shaken treatments is shown below (Fig. 4.22). Bacterial numbers were analysed after 8 d and were not found to be significantly different in either the shaken

(5.31 log10 cfu/mL) or not shaken (5.34 log10 cfu/mL) treatments (lsd 0.785, data not shown).

6 cfu/mL) 10 5

No. of bacteria (log 2

1 Shaker No Shaker

0 Day 0 Day 2 Day 4 Day 6 Day 8 Day 10 Day 12 Day 14 Day 16 Day 18 Day 20 Day 22 Time (d)

Fig. 4.22. The mean number of bacteria (log10 cfu/mL) in Ceratopetalum gummiferum washing experiment. Data are the means of 10 replicates per treatment.

4.14.4 Discussion The results from the shaker experiment are inconclusive because they are complicated by both the differences in dissolved oxygen and the increased amount of transpiration that would have occurred in the stems that were shaken. Removal of air from water has been known for some time to improve conduction and, therefore, vase life (Dickson and Blackman 1938; Hamner et al. 1945; Stocking 1948; Durkin 1979a, b). Xylem conduits will remain functional if they do not contain entrapped air, resulting in the maintenance of hydraulic conduction and, therefore, increased cut flower longevity. Williamson and Milburn (1995) found that the vase life of stems was significantly greater in degassed water than in distilled water, but no statistical relationship was found between the loss of hydraulic conductance and higher levels of dissolved oxygen. Conrado et al. (1980) found that oxygen concentration had no significant effect on solution uptake after 72 h. However, their results should be interpreted with caution because deoxygenated solutions were prepared by replacing oxygen with nitrogen, which is equally capable of initiating cavitation.

It is unfortunate that this experiment was complicated by the above factors, because the hypothesis of suberin inhibition by washing was unable to be tested satisfactorily in cut stems. The shaker experiment would have worked well for potato disks, as they were not exposed to air, but the increased transpirational demands placed on cut stems were too great. Nevertheless, the monitoring of bacterial numbers indicated that the significant difference in vase life was not caused by bacterial levels since they were the same in both treatments.

4.15 High water versus low water experiment 4.15.1 Introduction and Objectives It is frequently recommended that cut stems be placed in deep water rather than shallow water. Deep water is believed to be beneficial because the increase in water head pressure at the cut end helps “push” water into stems and dissolve trapped air inside xylem conduits (Durkin 1986; Staby 1994). This results in improved water uptake. Stems are not usually able to take up water directly through the stem, apart from at the cut surface, however, some hollow stemmed flowers such as gerberas take up water through the inside of stems.

Initial experiments performed with vegetative stems of Acacia baileyana, Leptospermum obovatum and L. polygalifolium showed that all stems in the high water treatment lived significantly longer: up to 5 d longer (data not shown). Therefore, a further experiment was designed to test the effect on vase life of both high and low water levels, together with a vaseline coating on stems. Two questions were asked in this experiment: (1) if the increased head of pressure can dissolve air emboli, could it also

“push” past a suberin layer?; and (2) is water taken up through the outside of woody stems, especially through the areas opened up by leaf removal?

4.15.2 Methodology Vegetative stems of Leptospermum obovatum were used for this experiment. The foliage was removed from the lower 35 cm of the stem. Half the stems were coated with vaseline around the circumference. Stems were then assigned to either a high water (23 cm) treatment or a low water (5 cm) treatment. Data were analysed using a two-way ANOVA.

4.15.3 Senescence Symptoms and Results In L. obovatum foliage, senescence symptoms are a greying of the foliage, together with a concomitant loss of sheen. The venation also becomes prominent. The results revealed a marked difference in longevity between the water level treatments. High water levels resulted in a significantly longer vase life (22.6 and 25.6 d) than low water levels (8.7 and 8.5 d, Fig. 4.23). The effect of coating was not significant in either the high or low water treatments, or within treatments.

15 Vase life (d)

0 Coated Not coated Coated Not coated High Low Treatment

Fig. 4.23. Vase life of Leptospermum obovatum in high or low water. Coated = stems were coated with vaseline. Data are the means of 10 replicates per treatment. Bars represent the lsd (13.84, P < 0.05).

4.15.4 Discussion The results have shown that high water levels do improve vase life in woody stemmed plants such as the Acacia and Leptospermum foliage tested in these experiments. It was shown that water is not taken up through the outside of L. obovatum, because coating with vaseline had no significant effect on vase life. However, the question of whether suberin is involved was not answered by this

experiment. The beneficial effect of high water is likely the result of increased pressure, but whether this pressure dissolved air emboli or pushed through a suberin barrier is not known. Valle et al. (2000) found that water uptake was greatest when stems were kept at higher water levels. They used Magnetic Resonance Imaging to examine water emboli and found that 20 and 25 cm water levels (hydrostatic pressures) accelerated water uptake and emboli resorbtion.

4.16 Bacterial monitoring of high versus low water experiment 4.16.1 Introduction and Objectives The high and low water levels of the above experiment (section 4.15) may have led to differences in the amount of bacteria in the vase solutions. It was thought that the low water level might have concentrated the number of bacteria in the vase solution, but conversely, the high water level could have put more phylloplane bacteria from the stem surface in contact with the vase solution. Thus, bacterial levels in the vase solutions were monitored to determine that the beneficial effects of high water level were not attributable to differences in the number of bacteria.

4.16.2 Methodology The number of bacteria in the vase solutions was monitored at day 0 and day 5 as described previously (section 3.6). Data were analysed using a two-way ANOVA.

4.16.3 Results As expected, the number of bacteria in all treatments at day 0 was not significantly different from each other (data not shown). By day 5, however, there were significant differences in the number of bacteria in the coated and not coated treatments at both high and low water levels (Fig. 4.24). Significant differences in bacterial numbers also occurred in the coated treatments at both water levels. However, no significant differences occurred in the not coated treatment at both high and low water levels.

4.16.4 Discussion These results revealed that coating significantly reduced the bacterial numbers within each water level treatment. This was not unexpected, as it was thought that coating the stems with vaseline would form a barrier, thereby preventing the phylloplane bacteria from being washed off into the vase water. However, the most interesting result is that there was no significant difference in bacterial numbers between the uncoated stems in both water level treatments (Fig. 4.24), yet there was a significant difference in vase life between the water level treatments (Fig. 4.23). This indicates that differences in

3 cfu/mL 10 log 2

0 coated not coated coated not coated High Low Treatment

Fig. 4.24. The number of bacteria (log10 cfu/mL) in L. obovatum stems kept in either high or low water for 5 d. Coated = stems were coated with vaseline. Data are the means of 10 replicates per treatment. Bars represent the lsd (0.2623, P < 0.05). bacterial numbers were not the reason for the vastly different vase lives between the high and low water level treatments. Therefore, the hypothesis discussed in 4.15 above is still valid: an increased hydrostatic pressure improved vase life, rather than differences in bacterial numbers.

4.17 Recutting experiment 4.17.1 Introduction and Objectives A blockage at the cut end of stems has long been implicated as the cause of impaired water uptake (see sections 1.4.2 and 1.4.3). If suberin formation occurs as a response to wounding (i.e. cutting at harvest), would daily recutting remove the damaged – and possibly suberised – cut end? Recutting the stem end has previously been shown to increase hydraulic conduction in Acacia (Williamson 1989). The aim of this experiment was to test the hypothesis that daily recutting of the stem end might improve vase life and remove a suberised layer. Initial experiments were done with foliage of Acacia baileyana, Leptospermum obovatum and L. polygalifolium. In each case, there was a significantly longer vase life when stems were recut daily.

4.17.2 Methodology L. polygalifolium foliage was used for this experiment. Stems that were in the cut treatment had the basal 1 cm of stem removed under water daily. Water uptake was monitored daily in this experiment (see section 3.9).

4.17.3 Senescence Symptoms and Results The end of vase life in L. polygalifolium foliage was deemed to have occurred when the stem tips wilted as far as the horizontal plane. In L. polygalifolium, there was a significant difference between treatments: the recut stems lived significantly longer (10.1 d) than the uncut stems (7.6 d) (Fig. 4.25). Water uptake was consistently higher in the recut treatment (Fig. 4.26), however, this was not a

6 Vase life (d)

0 cut uncut Treatment

Fig. 4.25. Vase life of Leptospermum polygalifolium foliage. Stems which were in the cut treatment had the basal 1 cm of stem removed under water daily. Data are the means of 10 replicates per treatment. Bars represent the lsd (2.113).

cut mean 20 Water uptake (mL) uncut mean

0 0 2 4 6 8 10 12 14 16 Time (d)

Fig. 4.26. Mean daily water uptake in L. polygalifolium foliage. Stems which were in the cut treatment had the basal 1 cm of stem removed under water daily.

significantly higher value than in the uncut treatment, even when initial fresh weight was used as a covariate in the ANOVA.

4.17.4 Discussion Recutting stems daily resulted in a significantly longer vase life in L. polygalifolium foliage (Fig. 4.25). This could indicate that a blockage at the cut end was removed, allowing unobstructed water uptake to occur. Whilst water uptake was greater in the stems which were recut daily, this result was not significantly different, even when analysed using the initial fresh weight of stems as a covariate. Nevertheless, recutting obviously had a beneficial effect on vase life in L. polygalifolium. This is not surprising because recutting improves hydraulic conduction and such increased water uptake results in longer vase life. Williamson (1989) found that removal of a 2 mm segment from the base of cut stems every 30 min restored hydraulic conductivity in Acacia subulata, however, the result was only temporary. The time could be extended if stems were perfused with citric acid (10 mM), rather than distilled water.

However, the exact mechanism by which recutting works is unclear: does it remove bacteria, particulate matter and cell debris from the cut end; does it open up new xylem conduits, or does it remove suberin from the cut end?

4.18 “Blasting” experiment 4.18.1 Introduction and Objectives A recent article investigated the use of strong chemicals to remove a deposit on the pit membranes of xylem conduits (van Doorn and Cruz 2000). These authors found that a 15 min pulse with either KOH, acetone or hypochlorite delayed leaf wilting in chrysanthemum (Dendranthema grandiflora cv. Viking) stems. Thus, the following experiment was based on the above work with chrysanthemum (van Doorn and Cruz 2000), and is termed a “blasting” experiment because it was thought the strong chemicals used would “blast” any deposits from pit membranes because of their solvent, oxidising or hydrolysing action.

4.18.2 Methodology Leptospermum obovatum was used in this experiment because it has a short vase life of < 6 d, and the flowers desiccate rather than abscise. This indicates a water stress, rather than ethylene involvement, senescence process. The basal 3 cm of stems was recut in air (except for the under water treatment). Stems were either kept in air for 24 h under high humidity (sealed plastic bags containing moistened cotton wool) and darkness; or in distilled water and light for 24 h. After 24 h, stems were transferred to one of the following chemicals: acetone (33%), germicide (4.7 ppm available Cl-), NaOH (0.8 M),

NaClO (0.2 M), or water and shaken for 15 min on a platform mixer (Model OM6, Ratek Instruments Pty. Ltd., Boronia, Vic.) at 110 rpm. Stems were then rinsed in distilled water and all stems were transferred to distilled water.

4.18.3 Senescence Symptoms and Results The end of vase life was deemed to have occurred when >50% of the open flowers had closed. All flowers lived a very short time (< 7 d). The stems that lived the longest were in the air/water treatment (5.1 d), however, this was not significantly different from several other treatments: cut under water (4.8 d), air germicide treatment (4.8 d), air acetone (4.2 d) and air NaClO (4.1 d) (Fig. 4.27). The stems that took up the most water (visually only – not measured) were the stems that were cut under water, however, this did not translate into a significantly longer vase life. It is evident from Fig. 4.27 that all of the 24 h air treatments were more beneficial to total life than keeping stems in water for that time, although not all these differences were significant.

3 Vase life (d)

0 A acet V acet A germ V germ A NaOH V NaOH A V A water Contin Under NaClO NaClO water water Treatment

Fig. 4.27. Total life (i.e. including 24 h dry period) of L. obovatum when stems were “blasted” with strong chemicals. Stems were either kept in air (A) or water (V) for the initial 24 h. After 24 h, stems were washed for 15 min in the indicated solutions. Stems were then rinsed and transferred to distilled water. Acet = acetone; germ = germicide, contin water = continuous water; under water = cut under water. Data are the means of 10 replicates per treatment. Bars represent the lsd (1.042).

4.18.4 Discussion None of the treatments prolonged vase life over the 6 d previously recorded for this species (Faragher, unpublished). Perhaps the naturally short vase life of this species negated any possible advantage conferred by the treatments. It is evident, however, that the air treatments resulted in a longer vase life – albeit not always significantly – than that of the equivalent stems which were kept in water for the preceding 24 h. This may have been due to the reduced transpiration that would have occurred in the

dark, humid conditions, compared with stems kept in light at 60% RH. The observation that the stems which were cut under water took up a large amount of water, but did not have the longest vase life is surprising, especially if the Discussion of 4.17.4 is considered. It was thought that 15 min was too long a time to expose the cut stems to such strong chemicals, so a second experiment was designed in which a shorter exposure time was planned.

4.19 Second “Blasting” experiment 4.19.1 Introduction and Objectives Based on the results of the above experiment (4.18), a second experiment was done in which the stems were exposed to strong chemicals for a much shorter time. It was thought that 15 min was too long for the cut stems to be in strong chemicals, so a shorter exposure time was designed. An extra chemical treatment was included (NaOH in methanol) because it had been found to yield suberin fragments from cork oak (Quercus suber) (Cordeiro et al. 1998).

4.19.2 Methodology Flowering stems of L. obovatum were kept in darkness for 24 h in sealed plastic bags containing moistened cotton wool. Stems were then “washed” with chemicals for either 2 or 5 min using a platform mixer (Model OM6, Ratek Instruments Pty. Ltd., Boronia, Vic.) at 110 rpm. Stems were washed in the chemicals to ensure that any wounded areas at the cut surface were given good contact with the chemicals. The same chemicals were used in this experiment as were used in 4.18 above, with the addition of a 0.1 M NaOH in methanol (100% v/v) treatment. Stem ends were then rinsed with distilled water and placed into tubes containing distilled water.

4.19.3 Results The results of this second “blasting” experiment are also disappointing because vase life was not increased and in fact was shorter when stems were exposed to strong chemicals for either 2 or 5 min (Fig. 4.28) than the original 15 min (Fig. 4.27). In this latter experiment, all but two of the treatments (NaClO 5 min, NaOH 2 min) were not significantly different from each other.

4.19.4 Discussion All that can really be said about this experiment was that washing stems of L. obovatum with strong chemicals for either 2 or 5 min did not increase vase life. Conversely, using such strong chemicals did not have a negative effect on vase life because all but two of the treatments were not significantly different from water. Thus, the beneficial results of strong chemicals with chrysanthemum in delaying the time to leaf wilting (van Doorn and Cruz 2000) were not transferable to L. obovatum. It is unclear what causes such rapid senescence in L. obovatum.

3 Vase life (d) 2

0 2 min 5 min 2 min 5 min 2 min 5 min 2 min 5 min 2 min 5 min 2 min 5 min acetone germicide NaOH/Methanol NaClO NaOH water

Washing treatment

Fig. 4.28. Vase life of L. obovatum after a 2 or 5 minute wash with the above chemicals. All stems were kept in humidified air in the dark for the initial 24 h. Data are the means of 10 replicates per treatment. Bars represent the lsd (1.278).

4.20 Boiling experiment 4.20.1 Introduction and Objectives As discussed in section 1.7, suberin formation begins with the synthesis of trans-cinnamic acid from phenylalanine, which is catalysed by the enzyme, phenylalanine ammonia lyase (PAL) (Stafford 1974). Enzyme activity is influenced by such factors as temperature, pH, substrate concentration and inhibitors (Tortora et al. 1986). Enzymes are proteins and, as such, are subject to denaturation beyond a certain temperature. Boiling is one of the common methods of irreversibly denaturing an enzyme.

The debate about whether microorganisms reduce vase life began nearly 100 years’ ago (Fourton and Ducomet 1906). The debate shall not be entered into here: the subject was discussed in Williamson (1996). Suffice it to say that there are two schools of thought about whether bacteria reduce vase life. Nevertheless, millipore filters were fitted onto stems in this experiment in order to determine whether “bacterially-filtered” water improved vase life.

4.20.2 Methodology Flowering stems of Rosa hybrida ‘Lambada’ were obtained from a local grower. Roses subjected to the boiling treatment were placed with cut stems submerged to approximately 7 cm in boiling water for 3 min. The flowers were protected from steam. Filters (0.22 µM, Millipore) were fitted onto the cut ends in other treatments.

4.20.3 Results The control stems lived longest (13 d), followed by the stems with filters (12.1 d), although there was no significant difference between these treatments (Fig. 4.29). Stems in the boiling treatments lived 6.6 and 7 days, which were significantly shorter than in the non-boiled treatments.

Vase life (d) Vase life 6

0 control filters boiled filters+boiled Treatment

Fig. 4.29. Vase life of Rosa hybrida ‘Lambada’. Filters = 0.22 µM Millipore filters were fitted to cut ends; boiled = stems were kept in boiling water for 3 min. Data are the means of 8 replicates per treatment. Bars represent the lsd (1.544, P < 0.05).

4.20.4 Discussion It is evident from the above experiment that boiling was injurious to vase life. The stems that were subjected to boiling exhibited damage at the edge of their petals. It is uncertain whether this was caused directly by some steam escaping to injure petals, or whether the effects of the boiling water were able to travel directly to the flower and cause damage. Certainly, in florist’s Icelandic poppies (Papaver nudicaule), they are placed into recently boiled water to “sear” the stem, thus preventing latex from blocking the xylem. This technique is also used by florists to ensure that the flowers open, which indicates that the heated water must rapidly travel up the stem to the flower. If this occurred in the above rose experiment, damage is likely to have occurred. In retrospect, a momentary boiling of the stem ends would have been sufficient to denature the PAL enzyme involved in suberin synthesis.

Fitting millipore filters onto the cut ends of stems did not confer any special advantage on vase life, as the results were not significantly different from water. However, this result and experiment raises the following questions: (1) are bacteria unimportant in R. hybrida ‘Lambada’ vase life; (2) were bacteria involved in this flower’s senescence processes; (3) was there a “microclimate” of water between the cut stem end and the plastic tubing; and (4) what pressures were required to draw the water through the millipore filter to the cut stem end? Durkin (1979a) found that millipore filtration of water

increased flow rates through stems, but they filtered the water prior to use, rather than fitting the ends of stems with filters. Furthermore, their beneficial effects were noticed with well waters (Durkin 1979a) and re-used flower water (Durkin 1979b), rather than distilled or deionised water.

It is possible that this experiment would be worth repeating, exposing the cut stem ends to boiling water only momentarily. However, it is unclear whether other factors such as cell debris, or damage to other enzymes would complicate the results. The use of a specific phenylalanine ammonia lyase (PAL) inhibitor in the vase water, e.g. S-carvone (Oosterhaven et al. 1995), is a preferable way of examining its effect on cut flower longevity.

4.21 Inhibiting suberin chemically 4.21.1 Introduction and Objectives S-carvone, the major constituent of caraway essential oil, has been found to delay the appearance of suberin in potato tubers, which was related to the activity of PAL. Potato tissue treated with S- carvone showed delayed suberin formation and also a delay in the increase of PAL (Oosterhaven et al. 1995). Two other chemicals which affect suberin are S-ethyl-N,N-dipropylthiocarbamate (EPTC) and trichloroacetate (TCA). EPTC is a thiocarbamate herbicide which reduces suberin deposition (Schmutz et al. 1996). TCA inhibits fatty acid chain elongation and results in a disorganisation of suberin ultrastructure (Soliday et al. 1979). (See 1.9 for a more detailed discussion of these two chemicals.) “Special” water was also included in this experiment as there have recently been claims that “clustered” water can increase vase life, inter alia. Thus, the objectives of this experiment were to test the effects on vase life of the above chemicals.

4.21.2 Methodology Flowering Hakea francisiana was used for this experiment. Stems were harvested when no more than half of the flowering spike was at the “loopy” stage of style exsertion.

4.21.3 Senescence Symptoms and Results Symptoms of senescence in H. francisiana were a loss of style turgor and colour (from pinky-red to pale pink), with an angling downward of the style by about 20°. The tip of the flowering spike also exhibited browning.

The stems in S-carvone (0.005% and 0.0005%) lived significantly longer than the other treatments (8.4 and 8 d respectively). There was no significant difference in vase life between the “special” water and deionised water (6.4 d and 5.8 d respectively). At the concentrations used, neither TCA nor EPTC were significantly different from either water.

4 Vase life (d) Vase life

0 1 uM 1 mM 10 uM 0.05% 100 uM 0.1 mM 0.005% 0.01 mM 0.0005% Deionised Special S-carvone EPTC TCA Water Water Treatment

Fig. 4.30. Vase life of Hakea francisiana. Data are the means of 10 replicates per treatment. Bars represent the lsd (1.266, P < 0.05).

4.21.4 Discussion The results of this experiment were exciting as they revealed that a chemical known to delay suberin formation, S-carvone, significantly increased the vase life of H. francisiana. The optimal concentration appeared to have been reached (0.005%), as neither concentration on either side of this significantly improved vase life. Compared with all the other suberin-inhibiting chemicals used in this project, S-carvone is probably the safest and easiest to make up. After this experiment was completed, Dr John Faragher performed a preliminary study which indicated that H. francisiana may be sensitive to ethylene. It awaits further testing to see whether inhibiting ethylene action would improve the vase life above that of S-carvone. There would appear to be no special benefit to be gained by using “special” water for extending the vase life of H. francisiana.

4.22 Searching for evidence of a wounding response via transmission electron microscopy 4.22.1 Introduction and Objectives It was mentioned previously (section 1.8) that Transmission Electron Microscopy (TEM) was the most sensitive method available to detect suberin, particularly in the early stages of its formation and deposition. The results of experiment 4.21 revealed that stems kept in the suberin inhibitor, S- carvone, produced a significantly longer vase life than other treatments and the deionised water control. Therefore, the optimum concentration of S-carvone (0.005% v/v) was compared with a

deionised water control to determine whether there was TEM evidence of a wounding response in Hakea francisiana stems kept in deionised water compared with those in S-carvone.

4.22.2 Methodology The methods are described briefly in 3.10 and details of TEM fixation and infiltration are set out in Appendix B.

4.22.3 Results Fine structural analyses revealed evidence of an early wounding response after 48 h in the deionised water stems compared with the stems in S-carvone and the deionised water control. In the freshly cut (control) stems (Photograph 4.3), the pit membrane showed a dark, uniform electron dense staining. This is also the case in the stems kept in S-carvone for 48 h (Photograph 4.4).

Photograph 4.3. TEM: Control stem (Hakea francisiana) showing an electron dense, dark pit membrane (arrow). Bar = 1 µm.

In contrast, the pit membranes of stems kept in deionised water for 48 h reveal a lower electron density than the control or 48 h S-carvone treated stems (Photograph 4.5). The centrally-located portion of the pit membrane has been modified. Preliminary observations by W. Liese and U. Schmitt (pers. comm.; see 4.22.4 below) indicate that the structures resemble wound reactions by the parenchyma, however, suberin is not formed. These structures were observed in all stem preparations.

Photograph 4.4. TEM: After 48 h, stems treated with S-carvone show dark, electron dense staining (arrows) similar to that shown in the control stem. Bar = 2 µm.

Photograph 4.5. TEM: Deionised water (48 h): the centrally located part of the pit membrane is less electron dense (arrows) than in the control or S-carvone (48 h) treated stems. Bar = 2 µm.

4.22.4 Discussion Because of the difficulty in interpreting whether suberin was in fact formed, and the nature of the particular wounding response, the TEM micrographs were sent to Germany for interpretation by Professor Dr Walter Liese and Dr Uwe Schmitt, the world experts in this field. They believed that the material shown in Photograph 4.5 (arrow) resembles that seen by them in the early stages of wound reactions. They observed similar structures after wounding in birch (Betula pendula) (Liese et al.

1995), linden (Tilia americana) (Schmitt and Liese 1992) and ash (Fraxinus excelsior) (Schmitt et al. 1997). In such cases, there was evidence of initial structural alterations to the pit membrane: the centrally located portion of the pit membrane appeared to be less electron dense than in control stems. In the present study, this was most evident when comparing the 48 h distilled water and 48 h S- carvone stems. Schmitt et al. (1997) believe that the lower electron density of the pit membrane reflected a modification in its structure, which was the preliminary stage of increased pit membrane permeability. Such permeability then leads to secretions from a parenchyma cell into the pit cavity and lumen of an adjoining xylem vessel or fibre. It is likely that the material is transported from the synthesis site within the cytoplasm to the pit areas, where it is extruded into the adjacent cell (Schmitt et al. 1995). Schmitt and Liese (1992) believe that the pit membrane becomes increasingly permeable to the material synthesised by the parenchyma cell. The material that is then deposited is darkly stained, indicating the presence of phenolics (Liese et al. 1995), of which suberin is partly composed (see section 1.6).

Although suberin was not observed in H. francisiana after 48 h, it is possible that the initial stages of pit membrane modification seen in Photograph 4.5 may be followed by a deposition of granular/fibrillar material and then suberin. This occurred in Acacia mangium, in which a two-stage process occurred: the first was an alteration of pit membranes, indicated by changes in electron density (as seen here in H. francisiana), and the second process was a wound-associated secretion followed by the suberisation of parenchyma cells, with secreted material occurring in adjacent xylem vessels and fibres (Schmitt et al. 1995) (see also section 1.7). In A. mangium, the second stage of the process was evident one week post-wounding. Thus, the formation of similar suberised structures in H. francisiana after one week is not inconceivable.

Schmitt et al. (1995) noted that the suberised parenchyma in A. mangium was observed 1 cm from the wounded surface. A practical outcome of their finding could come from recutting stem ends so that the suberised layer does not form during the life of the cut flower. However, there may well already be a detrimental effect on water conduction from the changes in pit membrane structure that are already seen here after 48 h. Daily recutting of stems ends may well assume a new, more meaningful importance.

This is the first time that cut flower stems have been examined under TEM for a wounding response and it provides tangible evidence about the hypothesised anatomical nature of the physiological change leading to occlusion in cut flowers. These results will be discussed more fully in a forthcoming joint paper (Williamson, V.G., Liese, W. and Schmitt, U. Evidence of a Physiological Blockage in Cut Flower Stems: A Transmission Electron Microscopy Study, in prep.).

5. Discussion

Numerous and differing experiments were performed during this project in order to study the hypothesised wounding response from several aspects. Thus, for clarity, this general discussion will be grouped under broad headings. A more detailed discussion appears at the end of each individual experiment.

Transmission Electron Microscopy TEM was used as the most sensitive way to detect the hypothesised wounding response in cut flowers. Ultrastructural analyses revealed evidence of an early wounding response in stems kept in deionised water for 48 h, compared with those in the suberin inhibitor, S-carvone. Stems in deionised water exhibited an alteration to pit membranes, indicated by changes in electron density. Conversely, in the S-carvone treated stems, the pit membrane remained intact. Although suberin was not observed 48 h post-cutting, it is possible that the initial stages of pit membrane modification seen here (Photograph 4.5) may be followed by a deposition of granular/fibrillar material and then suberin. Such a two-stage process is known to occur: the first is an alteration of pit membranes, indicated by changes in electron density (as seen here), and the second process is a wound-associated secretion followed by the suberisation of parenchyma cells, with secreted material occurring in adjacent xylem vessels and fibres. The second stage of the process is evident one week post-wounding. Thus, the formation of suberised structrures is still possible. However, are the early changes in pit membrane structure that we have seen under TEM enough to inhibit water uptake and reduce vase life? Certainly, the stems kept in S-carvone lived significantly longer than the other treatments.

This was the first time that cut flowers have been examined under TEM for a wounding response and it provides tangible evidence about the often hypothesised anatomical nature of the physiological change leading to occlusion in cut flowers.

Recutting stems These experiments tested the hypothesis that the daily recutting of stems might remove a suberised layer. Daily removal of the basal 1 cm from stems significantly improved the vase lives of all the species tested: Acacia baileyana, Leptospermum obovatum and L. polygalifolium. Recutting might remove bacteria, particulate matter and cell debris from the cut end, or perhaps it opens up new xylem conduits, or it might remove suberin from the cut end. However, when these results are considered with the TEM results (section 4.22), it is tempting to hypothesise that recutting removes the early stages of a wound response. This wounding response can eventually lead to suberin deposition, as discussed in section 4.22.4. The suberised parenchyma that was observed 1 cm from the wounded

surface in Acacia mangium (Schmitt et al. 1995) would have been removed with recutting. The old recommendation to recut stem ends daily may well now assume a new, more meaningful importance, however, each cut would initiate another wound reaction. The need for and benefit of frequent recutting supports the suberin/wounding hypothesis, although, of course, it doesn’t completely discount the air/bacteria/particulate matter hypothesis. Clearly, this is an area for further research.

High water versus low water This research has shown that high levels of vase water significantly increased vase life compared with low water levels in the two genera tested: Acacia and Leptospermum foliage. Although it has been known for some time that high (= deep) water improves vase life and water uptake, the mechanism/s involved are not known. Proposed mechanisms have included an increased amount of water pressure on the stem, which could force water up the stem and past a blockage, or perhaps water was taken up through the outside of the stem that was under water. The latter hypothesis was tested by coating the outside of Leptospermum obovatum stems with vaseline. The results indicated that water was not taken up through the outside of the stems because coating with vaseline had no significant effect on vase life.

Some researchers have proposed that the improved vase life in high water levels occurs because the bacterial levels are more diluted in the larger volume of water. Conversely, however, a high water level may have put more phylloplane bacteria in contact with the vase solution. We tested this hypothesis by monitoring the bacterial levels and found that stems coated with vaseline significantly reduced bacterial numbers within each water level. More interestingly, we found no significant differences in bacterial numbers between the uncoated stems in both water level treatments, but there was a significant difference in vase life between the water level treatments. This indicated that differences in bacterial numbers were not the reason for the vastly different vase lives between the high and low water level treatments. Therefore, it was more likely that an increased hydrostatic pressure improved vase life, rather than differences in bacterial numbers. This work will be pursued further with Professor Michael Reid, UC, Davis.

Ethylene As a result of this project, we now know of more Australian flowers that are sensitive to ethylene. This research, rather than being a direct testing of the suberin hypothesis, was more a by-product of those experiments. The species that were found to be sensitive to ethylene are: Baeckea virgata, Crowea exalata and Lophomyrtus × ralphii ‘Krinkly’ foliage. (Lophomyrtus is native to New Zealand and belongs to the family Myrtaceae. It was used in the absence of any Australian native plant experimental material at the time.) It is possible that several other species may be sensitive to ethylene

and these will require further testing: Grevillea longistyla foliage (leaf abscission occurred during the vase life experiment), and Baeckea behrii flowers (petal inroll, desiccation and/or abscission during the experiment). In addition, mixed reports already exist as to the ethylene sensitivity of New South Wales Christmas Bush/Festival Bush (Ceratopetalum gummiferum). The large amount of bract abscission that occurred in the C. gummiferum washing experiment fuels the ethylene sensitivity hypothesis, but this obviously requires refined testing. Hakea francisiana may also be sensitive to exogenous ethylene as abscission occurred during a separate preliminary trial performed by Dr John Faragher for another project.

6. Recommendations

Several recommendations can be made, both to industry and for future research, as a result of this project:

• Daily recutting of stems will inhibit the full effects of a wounding response. It will nevertheless trigger a new wounding response, but it could possibly delay the effects of suberin formation (the two-stage process described in 4.22.4) and the impaired water conduction that may result. Beneficial results were evident from the daily recutting experiments performed in this project.

• A practical, easy way to improve vase life is by standing stems in deep water. This can be done at the packing shed.

• The effects and optimal concentrations of S-carvone need to be tested further on other genera. Can benefit be obtained from a short-term ‘pulse’ of S-carvone? This is a new, safe and easy-to- use suberin inhibitor for cut flower growers/exporters/consumers to use.

• As a by-product of this research, it is now known that several Australian flowers of commercial and export importance are sensitive to ethylene and therefore should be treated and/or protected against its effects in order to attain optimum vase life: Baeckea virgata, Crowea exalata, Lophomyrtus × ralphii ‘Krinkly’. Acacia floribunda was previously shown to be ethylene insensitive (Williamson 1996).

• The ethylene sensitivity of the following flowers and/or foliage was either not tested (NT) or remains unclear (U): NT & U: Acacia baileyana NT & U: Baeckea behrii NT & U: NSW Christmas Bush/Festival Bush (Ceratopetalum gummiferum) NT: Grevillea longistyla (but probably not sensitive) NT: Hakea teretifolia NT: Hakea francisiana NT: Leptospermum obovatum NT: Leptospermum polygalifolium NT: Rosa hybrida ‘Lambada’ NT: Sphaerolobium vimineum

• There are still dozens more commercially important cut flower species for which we know nothing about their ethylene sensitivity.

• This project has begun a new area of cut flower research: that of examining cut flower stems under Transmission Electron Microscopy. The results presented here show for the first time the often-hypothesised anatomical nature of the physiological occlusion that occurs in cut flowers. Clearly, more work needs to be done in this area, e.g. a daily time-course of the wounding response in H. francisiana to see whether suberin does eventually form and, of course, the examination of numerous other species, especially short-lived and long-lived cut flowers. However, funding constraints will limit this research in Australia, unless a postgraduate student (and university partner) might be inspired to take up the challenge of this latest and most exciting area of cut flower research.

References

Aldous, D.E. 1983. Cut flower purchases in Victoria. Australian Horticulture, July: 29, 31-32. ap Rees, T. and Bryant, J.A. 1971. Effects of cycloheximide on protein synthesis and respiration in disks of carrot storage tissue. Phytochemistry 10: 1183-1190.

Armitage, A.M. 1993. Research in the United States on specialty cut flowers, an overview. Acta Horticulturae 337: 189-199.

Arndt, C.H. 1929. The movement of sap in Coffea arabica L. American Journal of Botany 16: 179- 181.

Australian Bureau of Statistics. Various issues. Exports, Australia Annual Summary Tables, Catalogue No. 5424.0.

Bernards, M.A. and Lewis, N.G. 1998. The macromolecular aromatic domain in suberized tissue: A changing paradigm. Phytochemistry 47: 915-933.

Bernards, M.A. and Razem, F.A. 2001. The poly(phenolic) domain of potato suberin: A non-lignin cell wall bio-polymer. Phytochemistry 57: 1115-1122.

Beveridge, J.L., Dalziel, J. and Duncan, H.J. 1981. The assessment of some volatile organic compounds as sprout suppressants for ware and seed potatoes. Potato Research 24: 61-76.

Bonn, W.G., Sequeira, L. and Upper, C.D. 1975. Technique for the determination of the rate of ethylene production by Pseudomonas solanacearum. Plant Physiology 56: 688-691.

Borochov, A. and Woodson, W.R. 1989. Physiology and biochemistry of flower petal senescence. Horticultural Reviews 11: 15-43.

Bosisto, J. 1865. Abstract of a paper on the yield and uses of volatile oils, from native and imported plants, in the colony of Victoria. Transactions and Proceedings of the Royal Society of Victoria 6: 52-61.

Brooks, P. 2000. Australian Export Market Statistics – The FECA Perspective. In: Proceedings of the First Australian Flower Conference, Gosford, August. pp. 1-7.

Burbidge, N.T. 1963. Dictionary of Australian Plant Genera. Angus & Robertson, Sydney.

Burdett, A.N. 1970. The cause of bent neck in cut roses. Journal of the American Society for Horticultural Science 95: 427-431.

Burg, S.P. and Burg, E.A. 1965. Ethylene action and the ripening of fruits. Science 148: 1190-1196.

Cline, M.N. and Neely, D. 1983. The histology and histochemistry of the wound-healing process in geranium cuttings. Journal of the American Society for Horticultural Science 108: 496-502.

Conrado, L.L., Shanahan, R. and Eisinger, W. 1980. Effects of pH, osmolarity and oxygen on solution uptake by cut rose flowers. Journal of the American Society for Horticultural Science 105: 680- 683.

Considine, J.A. 1993. Progress in selection and cultivation of Australian native plants for floriculture. Acta Horticulturae 337: 11-18.

Cordeiro, N., Belgacem, M.N., Silvestre, A.J.D., Pascoal Neto, C. and Gandini, A. 1998. Cork suberin as a new source of chemicals. 1. Isolation and chemical characterization of its composition. International Journal of Biological Macromolecules 22: 71-80.

Cottle, W. and Kolattukudy, P.E. 1982. Abscisic acid stimulation of suberization. Plant Physiology 70: 775-780.

Cribb, J. 1988. The wilting of our flower power. The Australian, 1 May.

Dickson, H. and Blackman, V.H. 1938. The absorption of gas bubbles present in xylem vessels. Annals of Botany 2: 293-299.

Dixon, M.A., Butt, J.A., Murr, D.P. and Tsujita, M.J. 1988. Water relations of cut greenhouse roses: The relationships between stem water potential, hydraulic conductance and cavitation. Scientia Horticulturae 36: 109-118.

Durkin, D.J. 1979a. Some characteristics of water flow through isolated rose stem segments. Journal of the American Society for Horticultural Science 104: 777-783.

Durkin, D.J. 1979b. Effect of millipore filtration, citric acid, and sucrose on peduncle water potential of cut rose flower. Journal of the American Society for Horticultural Science 104: 860-863.

Durkin, D. 1986. Getting the most out of cut flowers. Minnesota State Florists Bulletin 35(2): 1-4.

Esau, K. 1977. Anatomy of Seed Plants. (second ed.) John Wiley & Sons, Inc., London.

Faragher, J., Slater, T., Beardsell, D. and Cass, A. 2000. Flowers 2000 Final Report. ExpHORT Publication No. 86, 35 pp.

Fourton, L. and Ducomet, V. 1906. Sur la conservation des fleurs coupées. (On the conservation of cut flowers.) Revue Horticole 70: 260-262.

Freebairn, H.T. and Buddenhagen, I.W. 1964. Ethylene production by Pseudomonas solanacearum. Nature 202: 313-314.

George, A.S. 1981. The background to the Flora of Australia. Flora of Australia, Volume 1. Australian Government Publishing Service, Canberra.

Gold, J. and Robb, J. 1995. The role of the coating response in Craigella tomatoes infected with Verticillium dahliae, races 1 and 2. Physiological and Molecular Plant Pathology 47: 141-157.

Goszczynska, D.M. and Rudnicki, R.M. 1988. Storage of cut flowers. Horticultural Reviews 10: 35- 62.

Hahlbrock, K. and Scheel, D. 1989. Physiology and molecular biology of phenylpropanoid metabolism. Annual Review of Plant Physiology and Plant Molecular Biology 40: 347-369.

Halevy, A.H. and Mayak, S. 1979. Senescence and postharvest physiology of cut flowers, part 1. Horticultural Reviews 1: 204-236.

Halevy, A.H. and Mayak, S. 1981. Senescence and postharvest physiology of cut flowers, part 2. Horticultural Reviews 3: 59-143.

Hamner, C.L., Carlson, R.F. and Tukey, H.B. 1945. Improvement in keeping quality of succulent plants and cut flowers by treatment under water in partial vacuum. Science 102: 332-333.

Hargrave, K.R., Kolb, K.J., Ewers, F.W. and Davis, S.D. 1994. Conduit diameter and drought-induced embolism in Salvia mellifera Greene (Labiatae)[sic Lamiaceae]. New Phytologist 126: 695-705.

Harrigan, W.F. and McCance, M.E. 1976. Laboratory Methods in Food and Dairy Microbiology. Academic Press, London.

Heins, R.D. 1980. Inhibition of ethylene synthesis and senescence in carnation by ethanol. Journal of the American Society for Horticultural Science 105: 141-144.

Johnson-Flanagan, A.M. and Owens, J.N. 1985. Peroxidase activity in relation to suberization and respiration in white spruce (Picea glauca [Moench] Voss) seedling roots. Plant Physiology 79: 103-107.

Jones, D.H. 1984. Phenylalanine ammonia-lyase: Regulation of its induction and its role in plant development. Phytochemistry 23: 1349-1351.

Jones, R.J. and Mansfield, T.A. 1970. Suppression of stomatal opening in leaves treated with abscisic acid. Journal of Experimental Botany 21: 714-719.

Joyce, D. and Haynes, Y. 1989. Postharvest treatment and shipping of W.A. Flora. W.A. Flora Summit, September 14-15.

Kang, B.G., Yocum, C.S., Burg, S.P. and Ray, P.M. 1967. Ethylene and carbon dioxide: Mediation of hypocotyl hook-opening response. Science 156: 958-959.

Keys, R.D., Smith, O.E., Kumamoto, J. and Lyon, J.L. 1975. Effect of gibberellic acid, kinetin, and ethylene plus carbon dioxide on the thermodormancy of lettuce seed (Lactuca sativa L. cv. Mesa 659). Plant Physiology 56: 826-829.

Koelemeijer, K. 1991. Florists' physical distribution customer services in the marketing of roses. Acta Horticulturae 295: 149-158.

Kolattukudy, P.E. 1980. Biopolyester membranes of plants: Cutin and suberin. Science 208: 990-1000.

Kolattukudy, P.E. 1981. Structure, biosynthesis, and biodegradation of cutin and suberin. Annual Review of Plant Physiology 32: 539-567.

Kolattukudy, P.E. and Köller, W. 1983. Fungal penetration of the first line defensive barriers of plants. pp. 79-100. In Callow, J.A. (ed.) Biochemical Plant Pathology, John Wiley & Sons Ltd., Chichester.

Koster, J. and van Raamsdonk, L.W.D. 1989. Between kangaroos and kangaroo paws. (A plant exploration trip to Australia.) Acta Horticulturae 252: 67-70.

Lamont, G. 1984. The potential of Australian native plants for cut flowers. Australian Horticulture 1: 8-17.

Laws, N. 1994. In other words. Accelerating the rate of change. FloraCulture International, January/February 4(1), 42.

Liese, W., Schmidt, O. and Schmitt, U. 1995. On the behaviour of hardwood pits towards bacteria during water storage. Holzforschung 49: 389-393.

Lothian, N. 1953. Native flowers as cut flowers. The South Australian Naturalist, December: 20-22.

Macnish, A.J., Joyce, D.C., Hofman, P.J., Simons, D.H. and Reid, M.S. 2000. 1-methylcyclopropene treatment efficacy in preventing ethylene perception in banana fruit and grevillea and waxflower flowers. Australian Journal of Experimental Agriculture 40: 471-481.

McGeoch, J. 1994. Overview, future directions and threats. Third National Workshop for Australian Native Flowers, University of Queensland Gatton College, February, pp. 2.1—2.5.

Milburn, J.A. 1979. Water Flow in Plants. Longman Group Limited, London.

Milburn, J.A. and Covey-Crump, P.A.K. 1971. A simple method for the determination of conduit length and distribution in stems. New Phytologist 70: 427-434.

Moon, G.J., Peterson, C.A. and Peterson, R.L. 1984. Structural, chemical, and permeability changes following wounding in onion roots. Canadian Journal of Botany 62: 2253-2259.

Newcombe, G. and Robb, J. 1989. The chronological development of a lipid-to-suberin response at Verticillium trapping sites in alfalfa. Physiological and Molecular Plant Pathology 34: 55-73.

Oosterhaven, K., Hartmans, K.J. and Huizing, H.J. 1993. Inhibition of potato (Solanum tuberosum) sprout growth by the monoterpene S-carvone: Reduction of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity without effect on its mRNA level. Journal of Plant Physiology 141: 463-469.

Oosterhaven, K., Hartmans, K.J., Scheffer, J.C. and van der Plas, L.H.W. 1995. S-carvone inhibits phenylalanine ammonia lyase (PAL) activity and suberization during wound healing of potato tubers. Journal of Plant Physiology 146: 288-294.

Parvin, P. 1995. in Middelmann, M. and Coetzee, C. The Fynbos Genebank Trust, South Africa. Chronica Horticulturae 35(2): 16.

Paulin, A. 1986. Influence of exogenous sugars on the evolution of some senescence parameters of petals. Acta Horticulturae 181: 183-193.

Peiser, G., López-Gálvez, G., Cantwell, M. and Saltveit, M.E. 1998. Phenylalanine ammonia lyase inhibitors control browning of cut lettuce. Postharvest Biology and Technology 14: 171-177.

Pegrum, J. 1988. The Western Australian wildflower industry - Finally coming of age. Proceedings of Australian Floriculture Conference, Adelaide, South Australia, 30-31 July, pp. 29-47.

Put, H.M.C. 1991. Factors Affecting the Vase Life of Rosa cultivar ‘Sonia’: Microbiological and Scanning Electron Microscopic Investigations. Ph.D. thesis, Landbouwuniverstiteit de Wageningen. Vijfsprong, Deventer, Netherlands.

Put, H.M.C. and Jansen, L. 1989. The effects on the vase life of cut Rosa cultivar ‘Sonia’ of bacteria added to the vase water. Scientia Horticulturae 39: 167-179.

Put, H.M.C. and Klop, W. 1990. The effects of microbial exopolysaccharides (EPS) in vase water on the water relations and the vase life of Rosa cv. Sonia. Journal of Applied Bacteriology 68: 367- 384.

Put, H.M.C. and Rombouts, F.M. 1989. The influence of purified microbial pectic enzymes on the xylem anatomy, water uptake and vase life of Rosa cultivar ‘Sonia’. Scientia Horticulturae 38: 147-160.

Put, H.M.C. and van der Meyden, T. 1988. Infiltration of Pseudomonas putida cells, strain 48, into xylem vessels of cut Rosa cv. ‘Sonia’. Journal of Applied Bacteriology 64: 197-208.

Rasmussen, H.P. and Carpenter, W.J. 1974. Changes in the vascular morphology of cut rose stems: A scanning electron microscope study. Journal of the American Society for Horticultural Science 99: 454-459.

Reid, M.S. 1989. The role of ethylene in flower senescence. Acta Horticulturae 261: 157-169.

Reid, M.S. and Kofranek, A.M. 1980. Recommendations for standardized vase life evaluations. Acta Horticulturae 113: 171-173.

Rural Industries Research & Development Corporation. 2000. R&D Plan for the Wildflowers and Native Plants Program 2000-2005. RIRDC, 25 pp.

Rittinger, P.A., Biggs, A.R. and Peirson, D.R. 1987. Histochemistry of lignin and suberin deposition in boundary layers formed after wounding in various plant species and organs. Canadian Journal of Botany 65: 1886-1892.

Robb, J., Powell, D.A. and Street, P.F.S. 1987. Time course of wall-coating secretion in Verticillium- infected tomatoes. Physiological and Molecular Pathology 31: 217-226.

Salisbury, F.B. and Ross, C.W. 1985. Plant Physiology. (third ed.) Wadsworth Publishing Company, Belmont, California.

Salunkhe, D.K., Bhat, N.R. and Desai, B.B. 1990. Postharvest Biotechnology of Flowers and Ornamental Plants. Springer-Verlag, Berlin.

Schmidt, H.W. and Schönherr, J. 1982. Fine structure of isolated and non-isolated potato tuber periderm. Planta 154: 76-80.

Schmitt, U. and Liese, W. 1991. Suberin in wound reaction parenchyma of birch xylem (Betula pendula Roth): An electron microscopic study. Holzforschung 45: 313-315.

Schmitt, U. and Liese, W. 1992. Seasonal influences on early wound reactions in Betula and Tilia. Wood Science and Technology 26: 405-212.

Schmitt, U. and Liese, W. 1993. Response of xylem parenchyma by suberization in some hardwoods after mechanical injury. Trees 8: 23-30.

Schmitt, U., Liese, W., Thong, H.L. and Killmann, W. 1995. The mechanisms of wound response in Acacia mangium. IAWA Journal 16: 425-432.

Schmitt, U., Richter, H.G. and Muche, C. 1997. TEM study of wound-induced vessel occlusions in European ash (Fraxinus excelsior L.). IAWA Journal 18: 401-404.

Schmutz, A., Buchala, A.J. and Ryser, U. 1996. Changing the dimensions of suberin lamellae of green cotton fibers with a specific inhibitor of the endoplasmic reticulum-associated fatty acid elongases. Plant Physiology 110: 403-411.

Shigo, A.L. 1984. Compartmentalization: A conceptual framework for understanding how trees grow and defend themselves. Annual Review of Phytopathology 22: 189-214.

Shigo, A.L. and Marx, H.G. 1977. Compartmentalization of decay in trees. USDA Forestry Service Information Bulletin 405: 73 pp.

Smith, W.L.Jr. and Smart, H.F. 1955. Relation of soft rot development to protective barriers in Irish potato slices. Phytopathology 45: 649-654.

Soliday, C.L., Dean, B.B. and Kolattukudy, P.E. 1978. Suberization: Inhibition by washing and stimulation by abscisic acid in potato disks and tissue culture. Plant Physiology 61: 170-174.

Soliday, C.L., Kolattukudy, P.E. and Davis, R.W. 1979. Chemical and ultrastructural evidence that waxes associated with the suberin polymer constitute the major diffusion barrier to water vapor in potato tuber (Solanum tuberosum L.). Planta 146: 607-614.

Staby, G. 1994. Flower & Plant Care Manual. A Contemporary Approach. Society of American Florists, Alexandria, Virginia.

Stafford, H.A. 1974. The metabolism of aromatic compounds. Annual Review of Plant Physiology 25: 459-486.

Stark, R.E., Sohn, W., Pacchiano, R.A. Jr., Al-Bashir, M. and Garbow, J.R. 1994. Following 13 suberization in potato wound periderm by histochemical and solid-state C nuclear magnetic resonance methods. Plant Physiology 104: 527-533.

Stocking, C.R. 1948. Recovery of turgor by cut shoots after wilting. Plant Physiology 23: 152-155.

Sytsema, W. 1975. Conditions for measuring vase life of cut flowers. Acta Horticulturae 41: 217-226.

Thimann, K.V.(ed.) 1980. Senescence in Plants. CRC Press, Inc., Florida.

Thomson, N., Evert, R.F. and Kelman, A. 1995. Wound healing in whole potato tubers: A cytochemical, fluorescence, and ultrastructural analysis of cut and bruise wounds. Canadian Journal of Botany 73: 1436-1450.

Tjia, B. 1988. New crops to consider for New Zealand and Australia to enter the world market. International Plant Propagators' Society, Combined Proceedings 37: 166-171.

Tortora, G.J., Funke, B.R. and Case, C.L. 1986. Microbiology: An Introduction. The Benjamin/Cummings Publishing Company, Inc., California.

Urbañski, T. 1950. Salicylhydroxamic acid as an antitubercular agent. Nature 166: 267-268.

Valle, R.S., Evans, R.Y. and Reid, M.S. 2000. Magnetic resonance imaging investigation of the rehydration of cut roses. 4th International Conference on Postharvest Science, Jerusalem, Israel, March 26-31. van Doorn, W.G. and Cruz, P. 2000. Evidence for a wounding-induced xylem occlusion in stems of cut chrysanthemum flowers. Postharvest Biology and Technology 19: 73-83. van Meeteren, U. 1992. Role of air embolism and low water temperature in water balance of cut chrysanthemum flowers. Scientia Horticulturae 51: 275-284.

van Meeteren, U. and van Gelder, H. 1999. Effect of time since harvest and handling conditions on rehydration ability of cut chrysanthemum flowers. Postharvest Biology and Technology 16: 169-177.

Vaughn, S.F. and Lulai, E.C. 1991. Suberin stains comparison of fluorescent stains for the detection of suberin in potato periderm. American Potato Journal 68: 667-674. von Hentig, W.-U. 1995. The development of "new ornamental plants" in Europe. Acta Horticulturae 397: 9-29.

Williamson, V. 1989. Studies in the disruption of water flow through the xylem of excised flowering Acacia stems. B.Sc.(Hons) thesis, University of New England, Armidale, 132 pp.

Williamson, V.G. 1996. Physiological and microbiological processes of cut flower senescence in two Australian native genera, Acacia and Boronia. Ph.D. thesis, University of New England, Armidale, 300 pp.

Williamson, V.G., Liese, W. and Schmitt, U. Evidence of a Physiological Blockage in Cut Flower Stems: A Transmission Electron Microscope Study, in prep.

Williamson, V.G. and Milburn, J.A. 1995. Cavitation events in cut stems kept in water: Implications for cut flower senescence. Scientia Horticulturae 64: 219-232.

Williamson, V.G. and Milburn, J.A. 1997. When cohesion breaks down: Consequences of cavitation in cut flowers (Abstract only). Australian Society of Plant Physiologists conference, Melbourne, 29 September – 2 October.

Wu, M.J., Zacarias, L., Saltveit, M.E. and Reid, M.S. 1992. Alcohols and carnation senescence. HortScience 27: 136-138.

Zimmermann, M.H. 1979. The discovery of tylose formation by a Viennese lady in 1845. IAWA Bulletin 2-3: 51-56.

Zimmermann, M.H. 1983. Xylem Structure and the Ascent of Sap. Springer-Verlag, Berlin.

Appendix A

Ingredients for Plate Count Agar 5 g Tryptone (Oxoid) 2.5 g Yeast Extract (Oxoid) 1 g D-glucose (Merck) 15 g Agar (Oxoid Technical, Agar No. 3) 1 L Distilled water

Appendix B

Transmission Electron Microscopy Preparation Instructions • Hakea francisiana stem samples were cut longitudinally using double sided razor blades. • The stem segments were immediately fixed in half strength Karnovsky’s mixture, consisting of 2% formaldehyde, 2.5% glutaraldehyde in 0.1M phosphate buffer, and left under vacuum for 24 h. • Samples were rinsed in three changes of buffer for 1 h each, before being post-fixed in 1% osmium tetroxide in 0.1M buffer for 24 h under vacuum. • The samples were then rinsed in three changes of distilled water for a total of 24 h. • The fixed samples were dehydrated in a graded acetone series, consisting of 10, 30, 50, 70, 90, 100% and 100% anhydrous acetone for 1 h each. • Dehydrated samples were gradually infiltrated with increasing concentrations of Spurr’s resin in acetone, consisting of resin/acetone mixtures of 25:75, 50:50, 75:25 and finally 100% resin without the catalyst (DMAE) for 24 h each. • The samples were then left to infiltrate in 100% resin without the catalyst for 14 d under vacuum. • The samples were then infiltrated in fresh 100% resin containing the catalyst for 48 h under vacuum. • Finally, the infiltrated samples were placed into fresh resin (with catalyst) in gelatin capsules and polymerised in the oven at 60°C for 24 h. • Sections of embedded Hakea stem (in cross-section) were cut 50-100 nm thick, using a Leica Ultracut R microtome with a diamond knife and collected onto 200 mesh copper hexagonal grids. • Grids were stained sequentially with saturated uranyl acetate for 15 min, and triple lead stained for 10 min and viewed in a Phillips Biotwin transmission electron microscope at 120 kV.

Appendix C

Summary of Vase Life Results (Chemical abbreviations appear at end of table)* Species Chemical Conc- Vase Species Chemical Conc- Vase entration life entration life (d) (d) Acacia aa1 1.7 mM 10.9 Baeckea a aa 100 ppm 11.1 baileyana #1 aa2 5 mM 9.6 behrii w aa 100 ppm 9.3 aa3 10 mM 9.9 aa4 20 mM 8.9 a ca 5 mM 10.5 w ca 5 mM 10.3 aba1 3.8 µM 7.5 aba2 0.38 mM 7.3 a chi 0.01 µM 11.7 aba3 1.8 mM 7.5 w chi 0.01 µM 11.2 aba4 3.8 mM 6.7 a pg 4 mM 5.8 ca1 1.56 mM 11.4 w pg 4 mM 5.9 ca2 5 mM 12.9 ca3 10 mM 9.5 a sham 1 µM 13.4 ca4 20 mM 6.5 w sham 1 µM 11.4

chi1 1 µM 9.5 a water 11.1 chi2 60 µM 6.6 w water 11.8 chi3 100 µM 6.4 chi4 0.5 mM 6.1 lsd = 1.734

sham1 10 µM 7.1 B. virgata aa1 300 ppm 5.3 sham2 110 µM 8.3 aa2 5 mM 4.4 sham3 1 mM 8 aa3 10 mM 4.3 sham4 2 mM 7.6 ca1 300 ppm 7.2 water 7.6 ca2 5 mM 7.7 ca3 10 mM 6.3 lsd = 2.525 chi1 0.01 µM 11.3 A. baileyana etoh 25% (v/v) 7.2 chi2 0.1 µM 9.7 #2 sham1 10 µM 16.4 chi3 1 µM 7 sham2 110 µM sham3 1 mM 14.1 water 11.8 sham4 2 mM 14.4 lsd = 0.955 water 15.1

lsd = 1.615

Species Chemical Conc- Vase Species Chemical Conc- Vase entration life entration life (d) (d) B. virgata sts 0.2 mM 11 Grevillea aa1 0.01 mM 19.9 ethylene sts 0.4 mM 10 longistyla aa2 0.1 mM 18.6 expt aa3 1 mM 18.5 sts+eth 0.2 mM 9.2 sts+eth 0.4 mM 9.7 ca1 0.5 mM 20.2 ca2 1 mM 20.9 eth 11.6 ca3 2 mM 17.4 ca4 5 mM 19 water 12.9 sham1 0.1 µM 21 lsd = 1.754 sham2 1 µM 17.3 sham3 10 µM 15.5 Crowea aa1 1.7 mM 12.8 exalata #1 aa2 5 mM 11.6 water 18.1 aa3 10 mM 10.1 aa4 15 mM 10.5 lsd = 5.392

ca1 1.56 mM 11.7 Hakea carv1 6.7 ca2 5 mM 11.2 francisiana carv2 8.4 ca3 10 mM 10.5 carv3 8 ca4 15 mM 9.8 eptc1 6.2 chi1 0.001 µM 12.6 eptc2 6.8 chi2 0.01 µM 13.8 eptc3 5.7 chi3 0.1 µM 12.2 chi4 1 µM 12.7 tca1 6.5 tca2 6.4 sham1 10 µM 15 tca3 5.6 sham2 110 µM 13.6 sham3 1 mM 11.9 de water 5.8 sham4 2 mM 9 sp water 6.4 sts1 0.2 mM 15.6 sts2 0.5 mM 10.8 lsd = 1.266

water 13.5

lsd = 1.731

C. exalata sham 10 µM 11 #2 sts 0.2 mM 13.6

sts+sham 0.2mM,10µM 14.2

water 12.4

lsd = 2.006

Species Chemical Conc- Vase Species Chemical Conc- Vase entration life entration life (d) (d) Lepto- A acet 4.2 Lepto- cut 10.1 spermum V acet 3.5 spermum obovatum polygali- uncut 7.6 “blasting” A germ 4.8 folium expt #1 V germ 3.5 recutting lsd = 2.113 expt A NaOH 4 V NaOH 2.7 Lopho- aa1 300 ppm 12.7 myrtus aa2 5 mM 10.8 A NaCO 4.1 × ralphii aa3 10 mM 9.3 V NaClO 3.3 ‘Krinkly’ #1 ca1 300 ppm 12.1 A water 5.12 ca2 5 mM 14 V water 3.5 ca3 10 mM 11.2

cont water 3.5 chi1 0.01 µM 13.3 und water 4.8 chi2 0.1 µM 12.9 chi3 1 µM 8.5 lsd = 1.042 water 14.5 Lepto- acet 2 4 spermum acet 5 3.8 lsd = 2.325 obovatum “blasting” germ 2 3.4 Lopho- aa1 0.01 mM 12.7 expt #2 germ 5 3.5 myrtus aa2 0.1 mM 13.8 × ralphii aa3 1 mM 16.1 meth 2 3.5 ‘Krinkly’ meth 5 4.2 #2 sham1 0.1 µM 19.7 sham2 1 µM 23 NaClO 2 4.1 sham3 10 µM 16.8 NaClO 5 2.5 sts1 0.1 mM 29. NaOH 2 2 sts2 0.2 mM 29.8 NaOH 5 3.4 sts3 0.5 mM 30.4

water 2 3.3 water 20.7 water 5 4.2 lsd = 6.680 lsd = 1.278 Rosa cv. control 13.1 Lepto- hi water coated 22.6 Lambada spermum hi water uncoated 25.6 boiling boiled 6.6 obovatum expt #3 low water coated 8.7 filters 12.1 high/low low water uncoated 8.5 water filters+ 7 lsd = ?? boiled

lsd = 1.544

*Abbreviations: A air aa ascorbic acid aba abscisic acid acet acetone ca citric acid carv S-carvone chi cycloheximide coated vaseline coating cont water continuous water cut 1 cm cut daily from base de water deionised water eptc S-ethyl-N,N-dipropylthiocarbamate eth ethylene etoh ethanol germ germicide hi high KOH potassium hydroxide meth methanol NaOCl sodium hypochlorite NaOH sodium hydroxide pg n-propyl gallate sham salicylhydroxamic acid sp water special water sts silver thiosulphate tca trichloroacetate uncoated no vaseline coating uncut stem not recut daily und water cut under water V vase 2 2 minute washing @ 110 rpm 5 5 minute washing @ 110 rpm

Appendix D

Chemical Concentration Conversions

Chemical abbreviation Acacia baileyana experiment #1 ascorbic acid 1 300 ppm (0.3 g/L, 1.7 mM) aa1 ascorbic acid 2 5 mM (0.88 g/L) aa2 ascorbic acid 3 10 mM (1.76 g/L) aa3 ascorbic acid 4 20 mM (3.52 g/L) aa4 citric acid 1 300 ppm (0.3 g/L, 1.56 mM) ca1 citric acid 2 5 mM (0.96 g/L) ca2 citric acid 3 10 mM (1.92 g/L) ca3 citric acid 4 15 mM (2.88 g/L) ca4 cycloheximide 1 1 µM (0.0003 g/L) chi1 cycloheximide 2 60 µM (0.0169 g/L) chi2 cycloheximide 3 100 µM (= 0.1 mM, 0.028 g/L) chi3 cycloheximide 4 0.5 mM (0.141 g/L) chi4 salicylhydroxamic acid 1 10 µM (0.0015 g/L) sham1 salicylhydroxamic acid 2 110 µM (0.0168 g/L) sham2 salicylhydroxamic acid 3 1 mM (0.153 g/L) sham3 salicylhydroxamic acid 4 2 mM (0.3063 g/L) sham4 abscisic acid 1 3.8 µM (0.001 g/L) aba1 abscisic acid 2 0.38 mM (0.01 g/L) aba2 abscisic acid 3 1.8 mM (0.05 g/L) aba3 abscisic acid 4 3.8 mM (0.1 g/L) aba4

Acacia baileyana experiment #2 salicylhydroxamic acid 1 10 µM (0.0015 g/L) sham1 salicylhydroxamic acid 2 110 µM (0.0168 g/L) sham2 salicylhydroxamic acid 3 1 mM (0.153 g/L) sham3 salicylhydroxamic acid 4 2 mM (0.3063 g/L) sham4 ethanol 25 mL in 100 mL distilled water ethanol

Baeckea behrii ascorbic acid 100 ppm (0.1 g/L) aa citric acid 5 mM (0.96 g/L) ca cycloheximide 0.01 µM (1: make up stock solution: chi 0.0003 g/L = 1 µM; 2: take 100 mL of 1 and add 900 mL water = 0.1 µM; 3: take 100 mL of 2 and add 900 mL water = 0.01µM) n-propyl gallate 4 mM (0.848 g/L) pg

B. behrii continued salicylhydroxamic acid 1 µM (0.0002 g/L) sham

Baeckea virgata ascorbic acid 1 300 ppm (0.3 g/L, 1.7 mM) aa1 ascorbic acid 2 5 mM (0.88 g/L) aa2 ascorbic acid 3 10 mM (1.76 g/L) aa3 citric acid 1 300 ppm (0.3 g/L, 1.56 mM) ca1 citric acid 2 5 mM (0.96 g/L) ca2 citric acid 3 10 mM (1.92 g/L) ca3 cycloheximide 1 0.01 µM (1: make up stock solution: chi1 0.0003 g/L = 1 µM; 2: take 100 mL of 1 and add 900 mL water = 0.1 µM; 3: take 100 mL of 2 and add 900 mL water = 0.01 µM) cycloheximide 2 0.1 µM (as for 2 immediately above) chi2 cycloheximide 3 1 µM (0.0003 g/L) chi3

B. virgata ethylene experiment silver thiosulphate 1 0.2 mM (as per Appendix E, but take sts1 5 mL of Stock Solution and add 995 mL distilled water) silver thiosulphate 2 0.4 mM (as per Appendix E, but take sts2 10 mL of Stock Solution and add 990 mL distilled water)

Crowea exalata #1 ascorbic acid 1 300 ppm (0.3 g/L, 1.7 mM) aa1 ascorbic acid 2 5 mM (0.88 g/L) aa2 ascorbic acid 3 10 mM (1.76 g/L) aa3 ascorbic acid 4 15 mM (2.64 g/L) aa4 citric acid 1 300 ppm (0.3 g/L, 1.56 mM) ca1 citric acid 2 5 mM (0.96 g/L) ca2 citric acid 3 10 mM (1.92 g/L) ca3 citric acid 4 15 mM (2.88 g/L) ca4 cycloheximide 1 0.001 µM (1: make up stock solution: chi1 0.0003 g/L = 1 µM; 2: take 100 mL of 1 and add 900 mL water = 0.1 µM; 3: take 100 mL of 2 and add 900 mL water = 0.01 µM; 4: take 100 mL of 3 and add 900 mL water = 0.001 µM) cycloheximide 2 0.01 µM (as for 3 immediately chi2 above) cycloheximide 3 0.1 µM (as for 2 immediately above) chi3 cycloheximide 4 1 µM (0.0003 g/L) chi4 salicylhydroxamic acid 1 10 µM (0.0015 g/L) sham1 salicylhydroxamic acid 2 110 µM (0.0168 g/L) sham2 salicylhydroxamic acid 3 1 mM (0.153 g/L) sham3 salicylhydroxamic acid 4 2 mM (0.3063 g/L) sham4

C. exalata #1 continued silver thiosulphate 1 0.2 mM (as per Appendix E, but take sts1 5 mL of Stock Solution and add 995 mL distilled water) silver thiosulphate 2 0.5 mM (as per Appendix E, but take sts2 12.5 mL of Stock Solution and add 987.5 mL distilled water)

C. exalata #2 salicylhydroxamic acid 10 µM (0.0015 g/L) sham silver thiosulphate 0.2 mM (as per Appendix E, but take sts 5 mL of Stock Solution and add 995 mL distilled water)

Grevillea longistyla ascorbic acid 1 0.01 mM (0.0018 g/L) aa1 ascorbic acid 2 0.1 mM (0.0176 g/L) aa2 ascorbic acid 3 1 mM (0.176 g/L) aa3 citric acid 1 0.5 mM (0.096 g/L) ca1 citric acid 2 1 mM (0.192 g/L) ca2 citric acid 3 2 mM (0.384 g/L) ca3 citric acid 4 5 mM (0.96 g/L) ca4 salicylhydroxamic acid 1 0.1 µM (10 mL of 10 µM stock sham1 solution in 1 L) salicylhydroxamic acid 2 1 µM (10 mL of 100 µM stock sham2 solution in 1 L) salicylhydroxamic acid 3 10 µM (0.0015 g/L, or 10 mL of 1 sham3 mM stock solution in 1 L. 1 mM = 0.153 g/L)

Hakea francisiana S-carvone 1 0.0005% (5 µL in 1 L) carv1 S-carvone 2 0.005% (0.05 mL in 1 L) carv2 S-carvone 3 0.05% (0.5 mL in 1 L) carv3

S-ethyl-N,N- 1 µM (Take 1 mL of 1 mM stock eptc1 dipropylthiocarbamate 1 solution [see eptc3 below] and add 999 mL water.) S-ethyl-N,N- 10 µM (Take 10 mL of 1 mM stock eptc2 dipropylthiocarbamate 2 solution and add 990 mL water.) S-ethyl-N,N- 100 µM (0.0189 g/L. Make up 1 mM eptc3 dipropylthiocarbamate 3 stock solution: 189.35 g/L. Take 100 mL of this and add 900 mL water.) trichloroacetate 1 0.01 mM (Take 10 mL of 1 mM tca1 stock solution [see tca3 below] and add 990 mL water.) trichloroacetate 2 0.1 mM (Take 100 mL of 1 mM tca2 stock solution and add 900 mL water.) trichloroacetate 3 1 mM (0.1854 g/L) tca3

special water Add 1.5 mL of Dr Flanagan’s sp water Microcluster liquid to 998.5 mL distilled water.

Leptospermum obovatum “blasting” experiment #1 acetone 33% (Add 33 mL acetone to 67 mL acet distilled water.) germicide (sodium hypochlorite) 10 ppm (= 0.01 g/L = 4.7 ppm germ available Cl) sodium hydroxide 0.8 M (32 g/L) NaOH sodium hypochlorite 0.2 M (14.8 g/L of 12.5% a.i.) NaClO

Leptospermum obovatum “blasting” experiment #2 acetone 33% (Add 33 mL acetone to 67 mL acet distilled water.) germicide (sodium hypochlorite) 10 ppm (= 0.01 mL/L = 4.7 ppm germ available Cl)

NaOH in methanol 0.1 M NaOH in 100% methanol meth sodium hypochlorite 0.2 M (14.8 g/L) NaClO sodium hydroxide 0.2 M (14.8 mL/L) NaOH

Lophomyrtus × ralphii ‘Krinkly’ experiment #1 ascorbic acid 1 300 ppm (0.3 g/L, 1.7 mM) aa1 ascorbic acid 2 5 mM (0.88 g/L) aa2 ascorbic acid 3 10 mM (1.76 g/L) aa3 citric acid 1 300 ppm (0.3 g/L, 1.56 mM) ca1 citric acid 2 5 mM (0.96 g/L) ca2 citric acid 3 10 mM (1.92 g/L) ca3 cycloheximide 1 0.01 µM (1: make up stock solution: chi1 0.0003 g/L = 1 µM; 2: take 100 mL of 1 and add 900 mL water = 0.1 µM; 3: take 100 mL of 2 and add 900 mL water = 0.01µM) cycloheximide 2 0.1 µM (as for 2 immediately above) chi2 cycloheximide 3 1 µM (0.0002814 g/L) chi3

Lophomyrtus × ralphii ‘Krinkly’ experiment #2 ascorbic acid 1 0.01 mM aa1 ascorbic acid 2 0.1 mM aa2 ascorbic acid 3 1 mM aa3 salicylhydroxamic acid 1 0.1 µM (10 mL of 10 µM stock sham1 solution in 1 L)

salicylhydroxamic acid 2 1 µM (10 mL of 100 µM stock sham2 solution in 1 L) salicylhydroxamic acid 3 10 µM (0.0015 g/L, or 10 mL of 1 sham3 mM stock solution in 1 L. 1 mM = 0.153 g/L) silver thiosulphate 1 0.1 mM (as per Appendix E, but take sts1 2.5 mL of Stock Solution and add 997.5 mL distilled water) silver thiosulphate 2 0.2 mM (as per Appendix E, but take sts2 5 mL of Stock Solution and add 995 mL distilled water) silver thiosulphate 3 0.5 mM (as per Appendix E, but take sts3 12.5 mL of Stock Solution and add 987.5 mL distilled water)

Appendix E

Silver Thiosulphate Preparation and Disposal

Preparation of Silver Thiosulphate Complex (from Joyce and Haynes 1989)

A. PREPARATION OF THE STS STOCK SOLUTION (40 mM Ag+)

1. Dissolve 6.8 g of AgNO3 in 500 mL of distilled water.

2. Dissolve 25.3 g of Na2S203 in 500 mL of distilled water.

3. With vigorous stirring (preferably using a magnetic stirrer), slowly pour the AgNO3 solution

into the Na2S203 solution to obtain a final volume of 1 L. 4. Store the 40 mM STS stock solution in a dark bottle (aluminium foil wrap) in a coldroom for up to one week.

B. DILUTING THE STOCK SOLUTION FOR PULSING FLOWERS IN THE COLDROOM (0.5 to 2°C) OVERNIGHT (8 to 12 h) 1. Dilute 12.5 mL of 40 mM STS stock solution to 1,000 mL (by adding 987.5 mL of distilled water) to obtain a 0.5 mM STS pulsing solution.

Disposal of STS STS can be precipitated prior to disposal by pouring used solutions into a bucket containing steel wool. Theoretically, the silver can then be reused, or the entire solution can be disposed of by contacting the Environment Protection Authority (EPA).