Native Cut Flowers Extending Postharvest Life Using 1-MCP Treatment

A report for the Rural Industries Research and Development Corporation by AJ Macnish, DC Joyce, DH Simons and PJ Hofman

October 1999

RIRDC Publication No. 99/155 RIRDC Project No. UQ-63A

ISBN 0 642 57979 2 ISSN 1440-6845

Native Cut Flowers – Extending Postharvest Life Using 1-MCP Treatment Publication no. 99/155 Project no. UQ-63A.

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 Assoc. Prof. David H. Simons School of Land and Food The University of Queensland Gatton College QLD 4345

Phone: 07 5460 1231 Fax: 07 5460 1455 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 October 1999 Printed on environmentally friendly paper by Canprint

ii FOREWORD

Postharvest flower fall from various native Australian cut flowers is induced by ethylene. Silver thiosulfate (STS) solution is commonly used to reduce ethylene-induced flower fall, but may be withdrawn from commercial use due to possible environmental hazards. Recently, an alternative and novel gaseous anti-ethylene agent, 1-MCP, was developed.

This project aimed to:

• Develop dosing (concentration, duration and temperature) relationships for 1-MCP treatment of native cut flowers. • Extend the postharvest longevity of various ethylene-sensitive native cut flowers through treatment with 1-MCP. • Devise and test practical application systems for 1-MCP treatment.

This report documents a study into the effects of 1-MCP treatment on a variety of native Australian cut flowers. The effects of 1-MCP concentration, treatment duration and treatment temperature on the ethylene sensitivity of Grevillea ‘Sylvia’ inflorescences were evaluated. A screening study in which the response of a number of native Australian cut flowers to 1-MCP and ethylene treatments is also presented. A detailed investigation into the effects of temperature on the efficacy of 1-MCP treatments on native cut flowers is reported. Finally, several practical application systems for 1-MCP treatment are examined.

This report, a new addition to RIRDC’s diverse range of over 400 research publications, forms part of our Wildflowers and Native Plants R&D program, which aims to improve the profitability, productivity and sustainability of the Australian wildflower and native plant industry.

Most of our publications are available for viewing, downloading or purchasing online through our website: • downloads at www.rirdc.gov.au/reports/Index.htm • purchases at www.rirdc.gov.au/pub/cat/contents.html

Peter Core Managing Director Rural Industries Research and Development Corporation

iii ACKNOWLEDGMENTS

The authors thank Dr John Faragher of the Victorian Agriculture, Institute for Horticultural Development, Knoxfield laboratory for his interest and collaboration in this study. Special thanks are also due to Tony Slater and Dr David Beardsell for their advice and assistance with experiments conducted at the Knoxfield laboratory.

The skilled technical assistance of Victor Roberston, Alison Van Ansem and Srivong Rangsi is gratefully acknowledged. We thank Allan Lisle for his advice on the statistical analysis of data. We acknowledge Setyadjit for his assistance with experiments during the early part of this study.

The following people are thanked for provision of cut flowers for experiments: Pamela Barrass, John and Barbara Bradshaw, Edward Bunker, Christensen Flower Wholesaler, Graham and Ester Cook, Jamie Creer, Ben Edwards, Brett Gunderson, David and Olive Hockings, Leo Lynch and Sons (Qld) Pty. Ltd., David Matthews, Dr David Tranter, Philip Watkins and Ken Young.

Financial support from the Rural Industries Research and Development Corporation is gratefully acknowledged. In particular, special thanks are due to Dr David Evans for his support throughout this study.

iv TABLE OF CONTENTS

FOREWORD iii ACKNOWLEDGMENTS iv LIST OF ABBREVIATIONS AND SYMBOLS xii EXECUTIVE SUMMARY xv

1 GENERAL INTRODUCTION 1

1.1 BACKGROUND TO RESEARCH 1 1.2 GENERAL OBJECTIVES 2 1.3 REPORT STRUCTURE 3

2 1-MCP TREATMENT PREVENTS ETHYLENE- INDUCED FLOWER ABSCISSION FROM GREVILLEA ‘SYLVIA’ INFLORESCENCES 5

2.1 INTRODUCTION 5 2.2 MATERIALS AND METHODS 6 2.2.1 Plant Material 6 2.2.2 Chemicals 7 2.2.3 Treatment chambers 8 2.2.4 Treatments 10 2.2.5 Assessments 11 2.2.6 Experiment design and data analysis 13 2.3 RESULTS 15 2.3.1 Effect of 1-MCP concentration on the ethylene sensitivity of G. ‘Sylvia’ inflorescences 15 2.3.2 Effect of 1-MCP pre-treatment duration on ethylene sensitivity of G. ‘Sylvia’ inflorescences 25 2.3.3 Effect of temperature on 1-MCP pre-treatment efficacy 32 2.3.4 Effect of 1-MCP pre-treatment on inflorescence physiology 38 2.4 DISCUSSION 45

3 RESPONSES OF A NUMBER OF NATIVE AUSTRALIAN CUT FLOWERS TO 1-MCP AND ETHYLENE TREATMENTS 51 3.1 INTRODUCTION 51 3.2 MATERIALS AND METHODS 52 3.2.1 Plant material 52 3.2.2 Plant material preparation 56 3.2.3 Chemicals 56 3.2.4 Treatments 57 3.2.5 Assessments 58 3.2.6 Experiment design and data analysis 61 3.3 RESULTS 63 3.3.1 Treatment of a range of native cut flowers with 1-MCP and ethylene 63 3.3.2 Treatment of B. heterophylla with 1-MCP, STS and ethylene 108 3.4 DISCUSSION 116

4 EFFECT OF TEMPERATURE ON THE EFFICACY OF 1-MCP TREATMENT OF CUT FLOWERS 125

v 4.1 INTRODUCTION 125 4.2 MATERIALS AND METHODS 126 4.2.1 Plant material 126 4.2.2 Chemicals 127 4.2.3 Treatments 127 4.2.4 Assessments 128 4.2.5 Experiment design and data analysis 128 4.3 RESULTS 129 4.3.1 Duration of persistence of 1-MCP pre-treatment effects on G. ‘Sylvia’ inflorescences 129 4.3.2 Duration of persistence of 1-MCP and STS pre-treatment effects on flowering C. uncinatum sprigs 135 4.4 DISCUSSION 150

5 COMMERCIAL SCALE 1-MCP TREATMENTS PROTECT GERALDTON WAXFLOWER AGAINST ETHYLENE-INDUCED FLOWER ABSCISSION 153

5.1 INTRODUCTION 153 5.2 MATERIALS AND METHODS 151 5.2.1 Plant material and preparation 151 5.2.2 Chemicals 151 5.2.3 Treatments 151 5.2.4 Quality assessment 158 5.2.5 Experiment design and data analysis 158 5.3 RESULTS 159 5.3.1 Application of 1-MCP inside polyethylene 159 5.3.2 Injection of 1-MCP into cartons 173 5.3.3 Application of 1-MCP into a coolroom 183 5.3.4 Application of 1-MCP in cartons by forced-air cooling 187 5.3.5 Slow release of 1-MCP inside cartons 192 5.4 DISCUSSION 197

6 GENERAL DISCUSSION AND CONCLUSIONS 205

6.1 EFFICACY OF 1-MCP TREATMENTS ON CUT FLOWERS 205 6.2 EFFICACY OF COMMERCIAL SCALE 1-MCP TREATMENTS 206 6.3 EFFECT OF TEMPERATURE ON THE EFFICACY OF 1-MCP TREATMENT 207 6.4 DURATION OF PROTECTION AFFORDED BY 1-MCP TREATMENT 208 6.5 EFFECT OF TEMPERATURE ON THE DURATION OF PROTECTION AFORDED BY 1-MCP TREATMENT 209 6.6 DURATION OF PROTECTION AFFORDED BY STS TREATMENT 209 6.7 GENERAL CONCLUSIONS AND RECOMMENDATIONS 210

APPENDICES 213

APPENDIX A LITERATURE REVIEW 213

vi 1.1 ETHYLENE IN PLANT BIOLOGY 213 1.1.1 General roles 213 1.1.2 Abscission 214 1.1.3 Senescence of vegetative tissue 216 1.1.4 Flower senescence 217 1.1.5 Fruit ripening and senescence 220 1.2 ETHYLENE BIOSYNTHESIS 222 1.3 INHIBITORS OF ETHYLENE BIOSYNTHESIS 224 1.3.1 Aminoethoxyvinylglycine 224 1.3.2 Aminooxyacetic acid 225 1.4 ETHYLENE PERCEPTION 225 1.5 INHIBITORS OF ETHYLENE PERCEPTION 229 1.5.1 Silver ions 229 1.5.2 2,5-Norbornadiene 231 1.5.3 Diazocyclopentadiene 231 1.5.4 Cyclopropenes 233 1.6 INTERACTION BETWEEN ETHYLENE BIOSYNTHESIS AND PERCEPTION 239 1.7 ETHYLENE IN POSTHARVEST HORTICULTURE 241 1.7.1 Gas ripening and degreening 241 1.7.2 Acceleration of deterioration 242 1.7.3 Ethylene removal 242 1.7.4 Biosynthesis inhibition 244 1.7.5 Binding inhibition 244

APPENDIX B SUPPORTING AND STATISTICAL DATA 247

APPENDIX C SUMMARY TABLE OF PROJECT ACHIEVEMENTS 329

BIBLIOGRAPHY 331

vii LIST OF ABBREVIATIONS AND SYMBOLS

a.i. active ingredient ACC 1-aminocyclopropane-1-carboxylic acid ACO ACC oxidase ACS ACC synthase Ado-Met S-adenosylmethionine

Ag(S2O3)2 silver thiosulfate Ag+ silver

AgNO3 silver nitrate ANOVA Analysis of variance AOA aminooxyacetic acid AVG aminoethoxyvinylglycine ca. approximately cf. compare χ2 chi-square cm centimetre Co. company

CO2 carbon dioxide Co2+ cobalt ions CP cyclopropene CRD completely randomised design DACP diazocyclopentadiene oC degrees celsius DI deionised water DICA dichloroisocyanurate 3,3-DMCP 3,3-dimethylcyclopropene E east e.g. for example epi epinastic et al. and others FID flame ionisation detector FW fresh weight ggram > greater than ≥ greater than or equal to HCl Hydrochloric acid hr hour i.e. that is

viii IHD Institute for Horticultural Development Inc. Incorporated kd binding dissociation constant kg kilogram (103 g) km kilometre

KMnO4 potassium permanganate KOH potassium hydroxide < less than L litre LSD least significant difference 1-MCP 1-methylcyclopropene mmetre M molar (moles/L) MACC 1-(malonylamino) cyclopropane-1-carboxylic acid µL microlitre (10-6 L) mg milligram (10-3 g) mL millilitre (10-3 L) mm millimetre mM millimolar (10-3 M) mol mole mRNA messenger RNA MTA methylthioadenosine MTR methylthioribose n number of replicates NaOH sodium hydroxide

Na2S2O3 sodium thiosulfate

NH4SO4 ammonium sulphate nL nanolitre (10-9 L) nmol nanomole (10-9 mole) nor non-ripening Nr never-ripe 2,5-NBD 2,5-norbornadiene ns not significant NSW New South Wales % percent ± plus or minus P probability pers. comm. personal communication Qld Queensland  Registered name

ix RH relative humidity rin ripening inhibitor RNA ribonucleic acid s second Ssouth s.e. standard error SLFE shelf life following ethylene treatment STS silver thiosulfate TM Trade Mark UQG The University of Queensland, Gatton College UV ultra violet vvolume viz. namely vs. versus w weight

x EXECUTIVE SUMMARY

Premature flower fall or abscission and flower senescence (e.g. wilting) are ethylene-related postharvest problems for a number of cut flowers. Unintentional exposure of these flowers to ethylene, a plant hormone, reduces their postharvest longevity and marketability. Treatments that inhibit ethylene biosynthesis or action can be used to protect sensitive horticultural commodities against exposure to ethylene. To date, the most successful commercial treatment for preventing ethylene-induced flower abscission and senescence is pulsing flower stems with silver thiosulfate (STS). Because the active ingredient of STS is silver, a heavy metal, legislators in some countries are considering restricting the commercial use of STS. Recently, researchers in the USA developed an alternative, novel gaseous inhibitor of ethylene action in plants, 1-methylcyclopropene (1-MCP). 1-MCP apparently binds irreversibly to ethylene receptors in plant tissue, thereby preventing ethylene action. In studies conducted overseas, 1-MCP applied at low concentrations has been shown to protect several cut flowers including carnation, Cymbidium orchid and Geraldton waxflower against exposure to ethylene. A commercial   preparation of 1-MCP, EthylBloc , has been recently produced. EthylBloc is being evaluated by the  Environmental Protection Agency in the USA. It is anticipated that EthylBloc will soon be registered for use on ornamentals (J. Daly, pers. comm.). In the present study, it was proposed that 1-MCP would prove to be an effective anti-ethylene treatment for a variety of ethylene sensitive native Australian cut flowers.

The objectives of this study were to:

• Develop dosing (concentration, duration and temperature) relationships for 1-MCP treatment of native cut flowers. • Extend the postharvest longevity or vase life of various ethylene-sensitive native cut flowers through treatment with 1-MCP. • Devise and test practical application systems for 1-MCP treatment.

Experiments were conducted to determine an effective 1-MCP pre-treatment protocol (viz. concentration, duration and temperature) for protecting native Australian cut flowers against ethylene. Pre-treatment of Grevillea ‘Sylvia’ inflorescences on day 0 of the experiment with 10 nL 1-MCP/L (10 parts per billion) for 12 hours at 20oC delayed the onset of flower abscission induced by exposure on day 1 to 10 µL ethylene/L (10 parts per million) for 12 hours at 20oC. Pre-treatments using lower 1-MCP concentrations (e.g. 5 nL 1-MCP/L for 12 hours at 20oC) and shorter treatment duration (e.g. 10 nL 1-MCP/L for 3 hours at 20oC) also reduced the sensitivity of G. ‘Sylvia’ inflorescences to exogenous ethylene. These results are consistent with research conducted overseas where similar 1-MCP pre-treatments protected a range of traditional cut flowers, including carnation, snapdragon, Penstemon and phlox against ethylene.

Pre-treatment on day 0 with 10 nL 1-MCP/L for 12 hours at 20oC protected nine other ethylene-sensitive native Australian cut flowers (viz. Alloxylon pinnatum, Ceratopetalum gummiferum, Chamelaucium uncinatum ‘Paddy’s Late’, G. ‘Kay Williams’, G. ‘Sandra Gordon’, Leptospermum petersonii, Telopea speciosissima and Verticordia

xi nitens) against exposure on day 1 to 10 µL ethylene/L for 12 hours at 20oC. In addition, 1-MCP pre-treatment afforded flowering Boronia heterophylla stems with protection against exposure to 10 µL ethylene/L for the longer duration of 72 hours at 20oC. 1-MCP pre-treatment reduced ethylene-induced floral organ abscission and the associated loss in vase life. Ethylene-induced floral organ wilting on B. heterophylla and C. gummiferum was also reduced by 1-MCP pre-treatment. The efficacy of 1-MCP pre-treatment appeared to be similar to reports where STS treatment prevented ethylene-induced floral organ abscission and senescence from various native cut flowers. With the exception of C. gummiferum, 1-MCP pre-treatment did not extend the vase lives of flowers not exposed to ethylene. Thus, 1-MCP appears to be useful as a precautionary treatment for native cut flowers which may be exposed to exogenous ethylene during the postharvest handling phase.

G. ‘Sylvia’ inflorescences pre-treated with 10 nL 1-MCP/L for 12 hours at 2oC were not protected against ethylene. Similarly, C. uncinatum sprigs were only protected against exogenous ethylene treatment for ca. 2 days after pre- treatment with 10 nL 1-MCP/L for 12 hours at 2oC. These results confirm work conducted in the USA, where the efficacy of 1-MCP was shown to be reduced for cut Penstemon and Kalanchoe flowers pre-treated at 2oC. The reason why the efficacy of 1-MCP is reduced at low temperature is unclear. Presumably, 1-MCP binding or diffusion to ethylene receptors is reduced at low temperature.

G. ‘Sylvia’ inflorescences and C. uncinatum sprigs pre-treated on day 0 with 10 nL 1-MCP/L for 12 hours at 20oC remained insensitive to exogenous ethylene for 2 and ca. 4 days, respectively, after 1-MCP pre-treatment. Thereafter, exposure of sprigs to ethylene induced flower abscission and reduced vase life. The synthesis of new ethylene receptors is presumably responsible for the recovery of sensitivity, which appears to be rapid in the abscission zones of both G. ‘Sylvia’ and C. uncinatum flowers. In contrast to 1-MCP, pre-treatment of C. uncinatum sprigs with STS (0.5 mM Ag+) for 12 hours at 2 or 20oC afforded longer term protection against ethylene. Pulsing sprigs with STS provided complete protection against ethylene for the duration of the experiment (viz. 10 days). Silver in the STS complex is thought to bind to ethylene receptors and thereby block ethylene action. Based on the results of this study it seems that silver remains as a ‘pool’ in and around flower abscission zones and binds to new receptors as they are formed.

A number of 1-MCP treatment systems that are potentially suitable for industry were evaluated. Pre-treatment of C. uncinatum bunches standing in buckets of water inside sealed polyethylene tents with 200 nL 1-MCP/L for either 6 hours at 20oC or 14 hours at 2 or 20oC effectively reduced ethylene-induced flower abscission. Similarly, bunches in buckets of water pre-treated with 150 nL 1-MCP/L for 15 hours at 2oC inside a sealed coolroom were afforded protection against ethylene. Thus, 1-MCP pre-treatment at low temperature is effective when high 1-MCP concentrations are used. Similarly, the efficacy of 1-MCP pre-treatment of Kalanchoe flowers was reported to be improved by increasing the pre-treatment temperature from 2 to 24oC and/or increasing the 1-MCP concentration from 10 to 128 nL/L. Thus, from a practical perspective, effective protection against ethylene can be achieved by using high 1-MCP concentrations (e.g. 150-200 nL/L) at low temperature. Moreover, high 1-MCP concentrations take the possibility of small leaks existing from the enclosed structures into account.

1-MCP pre-treatment (200 nL/L) applied at 2oC by forced air movement through cartons or via circulating

xii coolroom air through bunches standing in buckets of water also effectively reduced ethylene-induced flower abscission and the associated loss in vase life. Similar levels of flower abscission and vase lives were found for sprigs at various sampling position within cartons treated by forced air. This similarity indicates that the movement of air containing 1-MCP through cartons was uniform.

Injection of 0.2 or 2 µL 1-MCP/L by syringe into cartons containing C. uncinatum bunches that were then held for 24 hours at 2oC did not consistently reduce ethylene-induced flower abscission. One explanation for reduced efficacy of this treatment is that 1-MCP may have diffused out through the carton wall. To provide sustained release of 1-MCP, glass tubes with rubber seals containing 1-MCP gas were placed at three positions amongst bunches inside cartons. The 1-MCP concentration in tubes decreased from 6928 ± 177 µL/L on day 0 to 1793 ± 48 µL/L on day 6 indicating that 1-MCP diffused through the rubber plugs that sealed the tubes. 1-MCP treatment via this slow release system was fully effective at protecting flowers against ethylene when three tubes were placed into each carton. When one or two tubes were placed into cartons, only flowers adjacent to tubes were protected against ethylene. Sustained release of 1-MCP gas from inside cartons promises to be an effective alternative treatment which, after refinement, may provided extended protection against ethylene.

In summary, 1-MCP treatments developed in this RIRDC sponsored work have potential as a postharvest anti- ethylene treatments for sensitive native Australian cut flowers. Application of 150-200 nL 1-MCP/L for 3-15 hours duration at 2 or 20oC to native cut flowers inside enclosed coolrooms or tents was completely effective in reducing ethylene-induced flower abscission. However, compared to STS pulsing, 1-MCP pre-treatment provides native cut flowers with relatively short term protection against ethylene. Sustained release of 1-MCP from inside cartons has potential, following refinement, to provide cut flowers with longer term protection against ethylene by remaining available to bind to newly formed ethylene receptors. This treatment would be easy to use and allow rapid dispatch of cut flowers to markets by eliminating the need to pre-treat flowers. Additionally, it may appeal to growers without suitable enclosed treatment structures and those concerned about handling chemicals such as 1- MCP and STS. Nevertheless, 1-MCP treatments developed in this study are comparatively easy to apply and can provide protection to sensitive cut flowers where the period of postharvest handling is brief.

xiii CHAPTER 1

GENERAL INTRODUCTION

1.1 BACKGROUND TO RESEARCH

There is increasing demand in the international floral trade for native Australian cut flowers as an exotic alternative to traditional cut flowers such as carnations, roses and chrysanthemums (Joyce et al. 1993). As a result, the native cut flower industry in Australia has grown considerably over the past 15 years. Native cut flower exports have increased from $3 million in 1983 to $27 million in 1995/96 (FECA 1996). However, there has been limited research into the postharvest physiology and horticulture of these flowers particularly in relation to ethylene (Faragher 1989). Ethylene is a gaseous plant hormone that regulates a number of growth, development and senescence processes (Abeles et al. 1992). Exposure of several native cut flowers to ethylene is known to induce floral organ abscission and senescence, thereby reducing vase life and marketability (Joyce et al. 1993). The deleterious effects of ethylene on ornamentals during postharvest can, however, be regulated by chemical inhibitors of ethylene biosynthesis or perception (Sherman 1985).

Ethylene is thought to bind to plants through a membrane-located receptor (Burg and Burg 1967; Sisler et al. 1980; Bleecker et al. 1988). Inhibitors of ethylene perception are presumed to counteract ethylene by binding to these receptors and thereby blocking subsequent signal transduction and translation (Sisler 1979, 1991). A number of compounds have been developed over the past 25 years to inhibit ethylene perception by plants. 2,5-norbornadiene (2,5-NBD) inhibits ethylene perception in a range of ethylene sensitive plants (Sisler and Pian 1973; Sisler et al. 1983; Sisler and Yang 1984b). However, the reversible nature of 2,5-NBD binding to ethylene receptors and offensive odour limit its practical use (Sisler et al. 1986). Another inhibitor of ethylene perception is silver (Ag+) (Beyer 1976). Ag+ in the silver thiosulfate (STS) complex is used successfully as a postharvest treatment to prevent ethylene- induced floral organ abscission and senescence from ethylene sensitive cut flowers and potted plants (Veen 1983; Nowak and Rudnicki 1990). STS solution applied as a pulse moves readily in the transpiration stream of cut flowers and accumulates in their receptacles (Veen and van de Geijn 1978). However, Ag+ is a heavy metal and environmental pollutant and as a result, some countries are starting to restrict its use (Serek et al. 1994a). As a possible alternative to STS, diazocyclopentadiene (DACP) gas was developed and shown to delay ethylene-induced flower abscission and senescence and fruit ripening (Sisler and Blankenship 1993a, b; Sisler et al. 1993). However, a major problem with DACP is that it is only fully effective when irradiated with fluorescent light (Serek et al. 1995b).

Recently, an alternative and novel inhibitor of ethylene perception, 1-methylcyclopropene (1-MCP) gas, was synthesised (Serek et al. 1994b). 1-MCP inhibits ethylene binding by apparently competing with

1 ethylene and binding to the ethylene receptors in an irreversible manner (Sisler and Serek 1997). It has been shown to prevent ethylene perception in a range of cut flowers (Serek et al. 1995a, c; Porat et al. 1995b; Sisler et al. 1996a), potted flowering plants (Serek et al. 1994b), climacteric fruit (Sisler et al. 1996b; Golding et al. 1998) and non-climacteric fruit (Ku et al. 1999; Porat et al. 1999). 1-MCP is reportedly as effective as STS in preventing cut flower abscission and senescence, even when applied at very low concentrations (Serek et al. 1995a). The efficacy of 1-MCP treatment has been shown to be a relationship between 1-MCP concentration, treatment duration and treatment temperature (Serek et al. 1995a; Reid et al. 1996). 1-MCP is generally considered to be non-toxic at active concentrations and thus has considerable potential as a commercial treatment for the regulation of ethylene responses in ornamentals (Sisler and Serek 1997).

1.2 GENERAL OBJECTIVES

The general objectives of this research were to:

1. Develop dosing (concentration, duration and temperature) relationships for 1-MCP treatment of native cut flowers, 2. Extend the postharvest longevity of a number of ethylene-sensitive native cut flowers through treatment with 1-MCP, 3. Devise and test practical application systems for 1-MCP treatment. 1.3 REPORT STRUCTURE

This report is divided into six chapters. Chapter one (General Introduction) identifies the problem and outlines the direction of the investigation. Chapters two, three, four and five report on experiments conducted. An evaluation of the effects of 1-MCP concentration, treatment duration and treatment temperature on the ethylene sensitivity of Grevillea ‘Sylvia’ inflorescences is presented in Chapter two. Chapter three reports on a screening study in which the response of a number of native Australian cut flowers to 1-MCP and ethylene treatments were examined. Chapter four presents a more detailed investigation into the effects of temperature on the efficacy of 1-MCP treatments on native cut flowers. The development and testing of several practical application systems for 1-MCP treatment are presented in Chapter five. Chapter six is the general discussion, which reviews the findings of this report in relation to existing literature and outlines the opportunities for future research. A survey of available literature concerning the content of this report is presented in Appendix A. All supporting and statistical data for the experimental chapters are shown in appendices 2.1 to 5.42 of Appendix B. A summary table of achievements of project milestones is presented in Appendix C.

2 3 CHAPTER 2

1-MCP TREATMENT PREVENTS ETHYLENE-INDUCED FLOWER ABSCISSION FROM GREVILLEA ‘SYLVIA’ INFLORESCENCES

2.1 INTRODUCTION

Grevillea is the largest genus in the family Proteaceae, containing over 340 species that are mostly native to Australia (Olde and Marriott 1994). Many Grevillea hybrids have attractive foliage and colourful inflorescences. Most hybrids have appeal as landscape plants, while some have potential as cut flowers (Costin and Costin 1988). Presently, use of Grevillea hybrids for cut flowers is limited by a typically short vase life of less than 1 week (Joyce et al. 1996). The short vase life of Grevillea hybrids is associated with the fragile nature of inflorescences and rapid perianth abscission (Faragher 1989). Moreover, exposure of Grevillea inflorescences to exogenous ethylene elicits rapid flower and perianth abscission and reduces vase life (Joyce and Haynes 1989). Sensitivity of Grevillea hybrids to ethylene may be reduced by pulse treating inflorescences with STS solution immediately after harvest (Joyce and Haynes 1989; Vuthapanich et al. 1993).

A novel gaseous inhibitor of ethylene perception, 1-MCP was developed recently (Serek et al. 1994b). Treatment with 1-MCP at nanomolar concentrations prevented ethylene-induced senescence of cut carnation flowers (Serek et al. 1995a; Sisler et al. 1996a, b) and abscission from cut Geraldton waxflower (Serek et al. 1995c), Penstemon (Serek et al. 1995a) and phlox (Porat et al. 1995b) flowers. The 1-MCP treatment concentration required to prevent ethylene-induced flower senescence and abscission from cut flowers is inversely related to the treatment time (Serek et al. 1995a; Sisler et al. 1996a) and treatment temperature (Serek et al. 1995a; Reid et al. 1996). Accordingly, 1-MCP efficacy appears to be mediated by treatment conditions with respect to its binding to the ethylene receptor.

The fragile nature of Grevillea inflorescences and their sensitivity to exogenous ethylene suggests that they may benefit, in terms of postharvest longevity, from treatment with 1-MCP. In the present study, 1- MCP was evaluated as a potential postharvest anti-ethylene treatment for G. ‘Sylvia’ (G. banksii x G. whiteana hybrid) inflorescences. The 1-MCP treatment variables of concentration, treatment duration and temperature, were examined in three successive experiments. In addition to basic assessment of vase life parameters, respiration, ACC concentrations in flowers and ethylene production by inflorescences were measured.

2.2 MATERIALS AND METHODS

2.2.1 Plant material

4 G. ‘Sylvia’ cut flowers comprised of 1 terminal inflorescence and the supporting stem were harvested from 5 year old in-ground plants at a commercial nursery near Redland Bay in S.E. Qld (27o 37’S, 153o 18’E). Inflorescences with newly opened flowers at maturity stage 4 (Beal et al. 1995) were cut with secateurs in the morning (0900-1100 hours) to a length of approximately 30 cm. Leaves were trimmed from stems. Inflorescences were then placed into styrofoam boxes between layers of newsprint moistened with deionised (DI) water to minimise moisture loss. Inflorescences were arranged carefully so that they were separated from each other to avoid mechanical injury. A layer of ice was placed in the bottom of each box to minimise heating of inflorescences. They were then taken to The University of Qld, Gatton College (UQG) postharvest laboratory in an air conditioned car within 2 hours of harvest.

At the laboratory, stem ends were recut under DI water to avoid air embolisms by removal of at least 2 cm from the stem base. Inflorescences were then assigned at random to treatment lots. They were each placed into individual vases (375 mL capacity) containing a solution of 10 mg available chlorine/L as the sodium salt of dichloroisocyanurate (DICA), an anti-microbial agent. Smaller vases (100 mL capacity) were used in experiments examining effects of 1-MCP on inflorescence physiology. Vases were closed with a piece of low density polyethylene film secured over the opening with a rubber band. This plastic film minimised evaporation and prevented falling flowers from contaminating the vase solution. Inflorescences were inserted through slits in the plastic film into the vase solution.

2.2.2 Chemicals

2.2.2.1 1-Methylcyclopropene

1-MCP was synthesised using a modified method of Sisler and Serek (1997), whereby lithium diisopropylamide was substituted for phenyllithium (E. Sisler, pers. comm.). 1-MCP stock gas was held in a glass bottle sealed with a rubber port. The volume of gas removed from the bottle with a syringe was simultaneously replaced with saturated ammonium sulphate (NH4SO4) solution to maintain a constant concentration by prevention of pressure imbalances. The stock bottle was kept at 4oC in an inverted position so that the NH4SO4 solution provided an additional seal on the inside of the rubber port. The 1- MCP concentration in the bottle was quantified by injecting 1 mL samples into a Shimadzu GC-8AIT gas chromatograph equipped with a flame ionisation detector (FID). The sample was separated in a 1.22 m long by 3.2 mm internal diameter stainless steel column packed with Chromosorb P-AW with a mesh range of 80/100 (Appendix 2.1). The gas chromatograph was operated at an oven temperature of 40oC and an injector/detector temperature of 50oC. High purity nitrogen gas (2.4 kg/cm2) was the carrier gas. A 97.3 µL iso-butylene/L standard (BOC Gases, β-grade special gas mixture) was used to calibrate the gas chromatograph (L. Dodge, pers. comm.). The detection limit for 1-MCP was 0.1 µL/L in a 1 mL sample. The location of 1-MCP on the gas chromatogram was identified by reacting the 1-MCP sample with 0.1 g elemental iodine in 10 mL absolute ethanol for 1 hour (E. Sisler, pers. comm.). This reaction ‘scrubbed’ or removed 1-MCP from the sample (Appendix 2.2).

5 2.2.2.2 Ethylene

Pure ethylene (BOC Gases) was used in the treatment of inflorescences. A working stock was created by diluting pure ethylene gas in air inside a glass bottle sealed with a rubber port. Saturated NH4SO4 solution was used to replace the volume of gas withdrawn. The ethylene stock was held at 20oC in an inverted position. Ethylene was quantified by injecting 1 mL gas samples into a Shimadzu GC-8AIT FID gas chromatograph. This gas chromatograph was fitted with a 0.9 m long by 3.5 mm internal diameter glass column packed with activated alumina with a mesh range of 80/100. The gas chromatograph was operated at an oven temperature of 90oC and an injector/detector temperature of 120oC. The carrier gas used was high purity nitrogen (1.2 kg/cm2). A 103 µL ethylene/L standard (BOC Gases, β-grade special gas mixture) was used for calibration.

2.2.2.3 Propylene

Pure propylene gas (Matheson Gas Products Inc.) was diluted in air in a glass bottle stoppered with a rubber port. The propylene stock was handled in the same manner described for the ethylene stock (section 2.2.2.2). Propylene gas was quantified by injecting 1 mL samples into the same gas chromatograph used to quantify ethylene (section 2.2.2.2). A laboratory made 1000 µL propylene/L standard was used to calibrate the gas chromatograph. This standard was prepared by diluting pure propylene gas inside a glass bottle containing high purity nitrogen.

2.2.3 Treatment chambers

Glass 60.5 L volume (length, breadth and height each 39.25 cm) chambers, with removable lids were used for adminstering 1-MCP, ethylene and propylene treatments. The lids each had a 10 mm diameter hole at the centre for attachment of a stirring fan and a 75 mm diameter hole to one side for a gas injection and an ambient air admission port. The latter two apertures were through a rubber plug (Plate 2.1). These holes  were sealed around the stirring fan and rubber plug with Vaseline (petroleum jelly). Stirring fans were used to mix air in the chambers and eliminate gas concentration gradients. Beakers each containing 10 mL 1M potassium hydroxide (KOH) solution were placed into chambers to reduce the excessive accumulation of CO2 from respiring inflorescences (Plate 2.1). A filter paper was stood vertically into each beaker to increase the surface area of KOH. To avoid a reduction in chamber air pressure due to sorption of CO2 by KOH, air was admitted through a saturated NH4SO4 solution trap (Plate 2.1).

6 Plate 2.1. Treatment chamber showing a glass structure (A), a stirring fan (B), a gas injection port and an ambient air admission port (C), beakers containing 1M KOH (D) and an air admission trap (E).

7 2.2.4 Treatments

2.2.4.1 Effects of 1-MCP concentration, treatment duration and treatment temperature on cut G. ‘Sylvia’ inflorescences

In a series of three experiments, inflorescences in vases were evenly allocated to treatment chambers each containing 6 to 8 beakers of 1M KOH solution. Once all inflorescences were inside chambers, lids were sealed in place with polyethylene tape. Inflorescences were then treated with 1-MCP on day 0 of each experiment at different concentrations, treatment durations and treatment temperatures. Aliquots of 1- MCP gas were injected through the gas injection port. Control inflorescences were enclosed in matching chambers in air with KOH, but without 1-MCP. The fan in each chamber circulated air for 10 minutes after injection. Inflorescences in chambers were exposed to cool white fluorescent lights providing 6 µmol/m2/s at inflorescence height.

Following 1-MCP treatment, chambers were ventilated by removing their lids either outside the laboratory or in a fume cupboard. After approximately 10-15 minutes, chambers were returned to the laboratory. All inflorescences in their vases were removed from chambers and re-randomised within each treatment lot. Half of the inflorescences from each treatment were then placed back into chambers which were again sealed. They were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. The ethylene treatment protocol was based on similar treatments reported by Joyce (1989). Ethylene gas was injected and the ethylene concentration inside the chambers was checked by gas chromatography. Control inflorescences were enclosed in matching chambers in air and were not treated with ethylene. Following ethylene treatment, inflorescences and their vases were removed from chambers and transferred to a vase life room operating at 20 ± 2oC and 50-70% RH. The room was fitted with overhead cool white fluorescent lights providing 13 µmol/m2/s at inflorescence height on a 12 hour on/off cycle.

Concentrations of 1-MCP required to protect G. ‘Sylvia’ inflorescences against ethylene were investigated by treating inflorescences with 5, 10 or 20 nL 1-MCP/L for 12 hours at 20oC. These 1-MCP concentrations were similar to those used by Serek et al. (1995a) with a range of cut flowers. The effect of different 1-MCP treatment durations was then investigated by treating inflorescences with 10 nL 1- MCP/L for 3, 6, 9 or 12 hours at 20oC. Finally, the efficacy of 1-MCP treatment at a range of temperatures was tested. Inflorescences were placed into vase solutions held at 0, 5, 10 or 20oC. The stem temperature of an additional inflorescence in a vase placed at each temperature was monitored using thermocouples. Once the stem had reached the desired temperature, inflorescences were treated with 10 nL 1-MCP/L for 12 hours at each temperature. Control inflorescences enclosed in chambers held at each temperature remained in air without 1-MCP. Following 1-MCP treatment, inflorescences were transferred to vases kept at 20oC. When the temperature of inflorescences reached 20oC, they were exposed to ethylene.

2.2.4.2 Effect of 1-MCP treatment on the physiology of cut G. ‘Sylvia’

8 inflorescences

In two identical experiments, inflorescences were treated on day 0 with 10 nL 1-MCP/L for 12 hours at 20oC as described in section 2.2.4.1. Control inflorescences were enclosed in chambers in air without 1- MCP. In these experiments, propylene was used to mimic ethylene treatment (Burg and Burg 1967) and thereby facilitate measurement of ethylene production by inflorescences (McMurchie et al. 1972). Following 1-MCP treatment, half of the inflorescences from each treatment were exposed on day 1 to 100 µL propylene/L for 12 hours at 20oC. The remaining half of the inflorescences stayed in air without propylene. At the completion of propylene treatment, inflorescences were placed into a controlled environment room operating at 20oC and 50% RH under cool fluorescent lights giving 6 µmol/m2/s at inflorescence height and a 12 hour light period per day.

2.2.5 Assessments

2.2.5.1 Vase life

Inflorescences and their vases were separately weighed daily to allow calculations of relative fresh mass [% of initial day 0 fresh weight (FW)] and vase solution uptake (mL/g initial FW), respectively. Flower abscission from inflorescences was determined daily after gently brushing them three times by hand. Abscission was rated using the following scale: 1 = < 10%, 2 = 10-30%, 3 = 30-50%, 4 = 50-80%, 5 = > 80% abscission relative to the initial number of flowers on a inflorescences (Joyce and Poole 1993). Flower wilting and discolouration (fading) were assessed daily using the following rating scale: 1 = none/slight, 2 = moderate, 3 = advanced. Opening of flowers was recorded daily using the following scale: 1 = < 5%, 2 = 5-25%, 3 = > 25% open flowers on an inflorescence. Vase life of inflorescences was judged as the time in days to loss of visual appeal (viz. > 10% flower abscission and/or moderate flower wilt and/or moderate discolouration).

2.2.5.2 Measurement of respiration and ethylene production rates

Inflorescences were placed individually with their vases into 2.2 L glass jars. These jars contained either  10 g Purafil (aluminium oxide pellets coated with potassium permanganate) (ethylene scrubber) or 10 mL 1M KOH (CO2 scrubber) solution. All jars were sealed daily with a plastic screw-on lid and held at o 20 C for 4 and 8 hours to allow accumulation of CO2 and ethylene, respectively. Three replicates were used for each treatment.

Headspace gas samples were taken through a sampling port in the lid of each jar using a 1 mL syringe. Syringes were pumped 5-6 times with air within the jars to stir the air therein before the sample was taken for analysis. Syringes were likewise flushed several times with ambient air between taking subsequent samples from different jars. CO2 was quantified with a Shimadzu GC-8AIF gas chromatograph fitted with a thermal conductivity detector (TCD) and using a 1.5 m long by 1.8 mm internal diameter copper column

9 packed with Porapak R with a mesh size of 80/100. High purity helium gas (2.5 kg/cm2) was the carrier o o gas. Column temperature was 25 C and injector/detector temperature was 30 C. CO2 samples were quantified against a 0.573% CO2 standard (BOC Gases β-grade special gas mixture). Ethylene samples were quantified as detailed previously (section 2.2.2.2) and using a 0.09 µL ethylene/L standard (BOC Gases β-grade special gas mixture). The rates of respiration and ethylene production were calculated as mL CO2/kg FW/hr and µL/kg FW/hr, respectively. 2.2.5.3 Measurement of 1-aminocyclopropane-1-carboxylic acid

ACC concentration in flowers was measured using a modification of the method described by Jobling et al. (1991). At each sampling time, approximately 2 g of flowers from individual inflorescences were stripped by hand. These flowers were sealed into plastic bags and immediately frozen in liquid nitrogen (- 196oC). They were then held in a -20oC freezer pending ACC measurement.

A weighed sample (ca. 1 g) of floral tissue was cut up finely with a scalpel. Five mL of acidified methanol (0.1M HCl in methanol) was added to the tissue in a 110 mL test tube and left to extract for 4 hours at 20oC. Following thorough vortex mixing, a 500 µL aliquot of the sample extract was pipetted into a 15 mL VenojectTM plain blood serum collection tube, and made up to 800 µL with distilled water. Each sample extract was prepared in duplicate. One of the duplicates from each sample was spiked with 100 nmol ACC (Sigma Chemical Co.). The extract was neutralised to the phenolphthalein endpoint (pH 8.5-9, pink colour) by dropwise addition of 10% (w/v) KOH. A 200 µL aliquot of 0.1 M mercuric (II) chloride was added, and the tube was sealed with a rubber septum. Next, a 0.1 mL aliquot of a mixture of liquid chlorine (White KingTM; 40 g available chlorine/L as sodium hypochlorite) and saturated sodium hydroxide (NaOH) in a 2:1 ratio (v/v) were injected into the tube. Following vortex mixing, the mixture was left for 1 hour at 30oC. The concentration of ethylene in a 1 mL sample of headspace gas was determined by gas chromatography. Initial ACC concentration (nmol/g FW) was calculated, with correction for the percent recovery of ACC in the spiked samples.

2.2.6 Experiment design and data analysis

In all experiments, inflorescences were arranged in completely randomised designs (CRD). Three to ten replicate inflorescences were used for each treatment, depending upon the particular experiment. The effects of 1-MCP concentration and treatment duration were examined as 2 (ethylene) x 4 (1-MCP) or 2 (ethylene) x 5 (1-MCP) factorial experiments, respectively. The influence of temperature on the efficacy of 1-MCP treatment was determined using a 2 (ethylene) x 2 (1-MCP) x 4 (temperature) factorial experiment. Inflorescence physiology was examined as 2 (ethylene) x 2 (1-MCP) factorial experiments.  Treatment means ± standard errors were calculated using Microsoft Excel (Version 5.0, Microsoft Inc.)  and are presented for all data. Figures were created using Sigmaplot (Version 2.0, Jandel Corporation) scientific graphing software. Data were analysed by ANOVA unless otherwise stated using the balanced  ANOVA function of Minitab (Release 11.12, Minitab Inc.) biometrics package. All data from the first experiment are presented in the main body of this chapter. Thereafter, data for which there were non-

10 significant differences between treatments are presented in appendices.

Flower abscission, wilting and discolouration data from experiments examining the effects of different 1- MCP concentrations and treatment durations were recorded as the time in days to reach a score of 2 (viz. > 10% flower abscission, moderate flower wilting and moderate flower discolouration) for ANOVA. The time in days to reach moderate flower discolouration on inflorescences used in the physiology experiment was also recorded for ANOVA. Flower abscission data were then analysed as factorial ANOVAs. When flower abscission from inflorescences was 100%, flower wilting, discolouration and opening measurements for these inflorescences were by necessity discontinued. Thus, as unbalanced data sets existed, flower wilting and discolouration data were analysed for the remaining treatments as one-way ANOVAs. Vase life data from all experiments were analysed as factorial ANOVAs.

In subsequent experiments, all replicates did not reach flower abscission, wilting or discolouration scores of 2. In the case of flower abscission data, scores were converted to the corresponding percentage and arcsine transformed to obtain approximately normally distributed data sets for ANOVA (Steel and Torrie 1987). Flower wilting and discolouration data were assigned a binary score, where the absence or presence of moderate to advanced wilting or discolouration on each inflorescence was recorded as a 0 or 1 score for ANOVA (Narula and Levy 1977). Flower abscission, wilting and discolouration data were then anlaysed as split plot for time (i.e. sequential days of measurement) ANOVAs. Relative fresh weight, vase solution uptake, ACC concentration, respiration and ethylene production data were also analysed as split plot for time ANOVAs. The days of measurement on which no variation between data existed were excluded from split plot for time ANOVAs. Flower opening was analysed by testing if an association existed between treatments and opening scores using chi-square (χ2) tests (Conover 1980). Where chi- square tests were invalid, data were analysed using Fisher’s exact test (Conover 1980) by SAS (Release 6.12, SAS Institute 1996).

Following ANOVA, the least significant difference (LSD) test at P = 0.05 was used to separate treatment means. LSDs calculated from split plot for time ANOVAs are for comparisons between treatments, rather than for a particular time within a treatment. LSDs were calculated and presented only when ANOVA showed significant (P < 0.05) differences between treatments. Differences between treatment means referred to in the results are significant at P < 0.05 level. LSDs which relate to ANOVAs performed on data not directly shown in figures are presented in appendices. LSDs from ANOVA on transformed and binary data sets are not presented as they do not correspond to the base data shown in figures.

2.3 RESULTS

2.3.1 Effect of 1-MCP concentration on the ethylene sensitivity of G. ‘Sylvia’ inflorescences

1-MCP pre-treatment protected G. ‘Sylvia’ inflorescences against ethylene (Plate 2.2). Pre-treatment with

11 5 nL 1-MCP/L for 12 hours at 20oC significantly delayed the onset to flower abscission induced by exogenous ethylene treatment (Figure 2.1 and Appendix 2.3). Flower abscission from inflorescences not exposed to exogenous ethylene was significantly reduced by pre-treatment with 5 nL 1-MCP/L. Increasing the 1-MCP concentration to 10 or 20 nL/L was slightly more effective than pre-treatment with 5 nL/L in protecting inflorescences against exogenous and endogenous ethylene-induced flower abscission. The decline of relative fresh weight of inflorescences pre-treated with 5 nL 1-MCP/L was significantly reduced compared to inflorescences not pre-treated with 1-MCP (Figure 2.2). Increasing the 1-MCP concentration to 10 or 20 nL/L did not further reduce the loss of inflorescence relative fresh weight. The loss of inflorescence relative fresh weight was mainly due to the abscission of flowers. 1- MCP pre-treatment was equally effective at 5, 10 or 20 nL 1-MCP/L in significantly delaying the onset to flower wilting from inflorescences exposed to 0 or 10 µL ethylene/L (Figure 2.3 and Appendices 2.3 and 2.4).

Because vase lives of inflorescences were partly based on flower abscission data, pre-treatment with 5 nL 1-MCP/L prevented the exogenous ethylene-induced reduction in vase life (Figure 2.4). Increasing the 1- MCP concentration to 10 or 20 nL/L was not more effective than 5 nL/L in preventing the loss in vase life. Vase lives of inflorescences not exposed to ethylene were only significantly extended by pre- treatment with 10 and 20 nL 1-MCP/L compared to inflorescences not pre-treated with 1-MCP (Figure 2.4). The pronounced counteraction of exogenous ethylene by 1-MCP was reflected in a significant interaction between 1-MCP pre-treatment and ethylene treatment for flower abscission (Appendix 2.5). It followed that a significant interaction between 1-MCP pre-treatment, ethylene treatment and time of measurement for relative fresh weight was also recorded (Appendix 2.6). Likewise, a significant interaction between 1-MCP pre-treatment and ethylene treatment for vase life data was evident (Appendix 2.7).

There were no consistent effects of 1-MCP or ethylene treatments on flower discolouration (Figure 2.5 and Appendices 2.3 and 2.8). 1-MCP or ethylene treatments did not affect flower opening (Figure 2.6 and Appendix 2.9). Nevertheless, there were significant differences between treatments on days 2, 3 and 5, although treatment effects were not consistent. Vase solution uptake by inflorescences tended to increase initially, then declined over time (Figure 2.7). Inflorescences pre-treated with 1-MCP used vase solution at higher rates than inflorescences not pre-treated with 1-MCP, in association with the delay of flower abscission and wilting. These different responses are reflected in a significant interaction between 1-MCP pre-treatment, ethylene and time of measurement for vase solution uptake data (Appendix 2.10).

12 Plate 2.2. G. ‘Sylvia’ inflorescences on day 2 after pre-treatment on day 0 with 0 (LHS) or 5 nL 1- MCP/L (RHS) for 12 hours at 20oC followed by exposure on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Note: extensive flower abscission is evident in the control inflorescence (LHS).

13 5 + Ethylene

5 - Ethylene

Abscission score 4

01234567 Time (days)

Figure 2.1. Flower abscission (scores: 1 = < 10% to 5 = > 80%) from G. ‘Sylvia’ inflorescences treated on day 0 with 0 (z), 5 (), 10 (▲) or 20 (▼) nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Remaining inflorescences were held in air without exogenous ethylene. Vertical bars represent standard errors of means (n = 10). Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD is presented in Appendix 2.3.

14 + Ethylene 100 80 60 40 20 0 - Ethylene 100 80 60 40 Relative fresh weight (% initial FW) 20 0 01234567 Time (days)

Figure 2.2. Relative fresh weight of G. ‘Sylvia’ inflorescences treated on day 0 with 0 (z), 5 (), 10 (▲) or 20 (▼) nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Remaining inflorescences were held in air without exogenous ethylene. Vertical bars represent standard errors of means (n = 10). Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD = 5.3%.

15 + Ethylene 3

- Ethylene

Wilt score 3

01234567 Time (days)

Figure 2.3. Wilting (scores: 1 = none/slight to 3 = advanced) of flowers on G. ‘Sylvia’ inflorescences treated on day 0 with 0 (z), 5 (), 10 (▲) or 20 (▼) nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Remaining inflorescences were held in air without exogenous ethylene. Vertical bars represent standard errors of means (n = 10). Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD is presented in Appendix 2.3.

16 5

2 Vase life (days) Vase

0 0 5 10 15 20 1-MCP concentration (nL/L)

Figure 2.4. Vase life of G. ‘Sylvia’ inflorescences treated on day 0 with 0, 5, 10 or 20 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC (●). The other half of the inflorescences were not exposed to exogenous ethylene (■). Vertical bars represent standard errors of means (n = 10). LSD = 0.3.

17 + Ethylene 3

- Ethylene 3 Discolouration score 2

01234567 Time (days)

Figure 2.5. Discolouration (scores: 1 = none/slight to 3 = advanced) of flowers on G. ‘Sylvia’ inflorescences treated on day 0 with 0 (z), 5 (), 10 (▲) or 20 (▼) nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Remaining inflorescences were held in air without exogenous ethylene. Vertical bars represent standard errors of means (n = 10). Where no vertical bars appear, the standard error was smaller than the size of the symbol.

18 + Ethylene 3

- Ethylene 3 Opening score

01234567 Time (days)

Figure 2.6. Opening (scores: 1 = < 5% to 3 = > 25%) of flowers on G. ‘Sylvia’ inflorescences treated on day 0 with 0 (z), 5 (), 10 (▲) or 20 (▼) nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Remaining inflorescences were held in air without exogenous ethylene. Vertical bars represent standard errors of means (n = 10). Significant differences (P < 0.05) between treatments existed on days 2, 3 and 5 (Appendix 2.9).

19 + Ethylene 0.3

0.2

0.1

0.0 - Ethylene 0.3

0.2

0.1 Solution uptake (mL/g initial FW/day) initial (mL/g uptake Solution

0.0 01234567 Time (days)

Figure 2.7. Vase solution uptake by G. ‘Sylvia’ inflorescences treated on day 0 with 0 (z), 5 (), 10 (▲) or 20 (▼) nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Remaining inflorescences were held in air without exogenous ethylene. Vertical bars represent standard errors of means (n = 10). LSD = 0.022 mL/g initial FW/day.

20 2.3.2 Effect of 1-MCP pre-treatment duration on ethylene sensitivity of G. ‘Sylvia’ inflorescences

Pre-treatment with 10 nL 1-MCP/L for 3 hours at 20oC significantly delayed the onset of flower abscission from inflorescences exposed to exogenous ethylene (Figure 2.8 and Appendix 2.11). 1-MCP pre- treatment for 3 hours also reduced flower abscission from inflorescences not exposed to ethylene. Increasing the 1-MCP pre-treatment duration to 6, 9 or 12 hours was seemingly more effective than the 3 hour pre-treatment in delaying the onset of flower abscission from inflorescencees exposed to exogenous ethylene. However, no significant differences between the 3 and 12 hour treatments existed (Appendix 2.11). Endogenous ethylene-induced flower abscission was reduced on days 6 and 7 by increasing the 1- MCP pre-treatment duration to 12 hours compared to the 3 hour pre-treatment (Figure 2.8).

The decline in relative fresh weight of inflorescences associated with exogenous ethylene-induced flower abscission was significantly reduced by pre-treatment with 1-MCP for 3 hours (Figure 2.9). However, there was no further reduction in the decline of inflorescence relative fresh weight by increasing the 1- MCP pre-treatment to 6, 9 or 12 hours. The loss of inflorescence relative fresh weight from inflorescences not exposed to exogenous ethylene was reduced most effectively by pre-treatment with 1- MCP for 12 hours. Pre-treatment with 1-MCP for 3 hours significantly delayed the onset of flower wilting induced by exposure to exogenous ethylene (Figure 2.10 and Appendix 2.11). The onset of flower wilting was not further delayed by increasing the 1-MCP pre-treatment duration to 6, 9 or 12 hours. 1-MCP pre- treatment did not delay the onset of flower wilting from inflorescences not exposed to ethylene.

Pre-treatment of inflorescences with 1-MCP for 3 hours prevented the reduction in vase life associated with exposure to exogenous ethylene (Figure 2.11). Increasing the 1-MCP pre-treatment duration to 6, 9 or 12 hours was not significantly more effective in preventing the loss in vase life than pre-treatment for 3 hours. However, there was a trend toward extended vase life for inflorescences pre-treated with 1-MCP for 9 or 12 hours compared to inflorescences pre-treated for 3 hours. 1-MCP pre-treatment did not significantly extend the vase lives of inflorescences not exposed to ethylene (Figure 2.11). The delay in the onset of flower abscission primarily for ethylene-treated inflorescences by 1-MCP pre-treatment was reflected in a significant interaction between 1-MCP pre-treatment and ethylene treatment for flower abscission (Appendix 2.12). Likewise, a significant interaction between 1-MCP pre-treatment, ethylene treatment and time of measurement was evident for flower wilting (Appendix 2.11) and relative fresh weight (Appendix 2.14). As a consequence of the strong influence of flower abscission, a significant interaction between 1-MCP pre-treatment and ethylene treatment for vase life was recorded (Appendix 2.15).

There was no consistent effect of 1-MCP pre-treatment on flower discolouration (Appendices 2.11, 2.16 and 2.17). 1-MCP pre-treatment did not affect flower opening (Appendices 2.18 and 2.19). There was more flower opening on days 1 and 2 from inflorescences exposed only to ethylene compared to inflorescences not exposed to ethylene (Appendix 2.18). However, inflorescences exposed only to

21 ethylene had more open flowers than other treatments on day 0. Inflorescences pre-treated with 1-MCP maintained higher rates of vase solution uptake throughout the experiment compared to inflorescences exposed only to ethylene (Figure 2.12). This differential response accounted for the significant interaction between 1-MCP pre-treatment and time of measurement (Appendix 2.20).

22 5 + Ethylene

5 - Ethylene

Abscission score 4

01234567 Time (days)

Figure 2.8. Flower abscission (scores: 1 = < 10% to 5 = > 80%) from G. ‘Sylvia’ inflorescences treated on day 0 with 10 nL 1-MCP/L for 0 (z), 3 (), 6 (▲), 9 (▼) or 12 (◆) hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Remaining inflorescences were held in air without exogenous ethylene. Vertical bars represent standard errors of means (n = 10). Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD is presented in Appendix 2.11.

23 120 + Ethylene 100 80 60 40 20 0 120 - Ethylene 100 80 60

Relative fresh weight (% initial FW) 40 20 0 01234567 Time (days)

Figure 2.9. Relative fresh weight of G. ‘Sylvia’ inflorescences treated on day 0 with 10 nL 1-MCP/L for 0 (z), 3 (), 6 (▲), 9 (▼) or 12 (◆) hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Remaining inflorescences were held in air without exogenous ethylene. Vertical bars represent standard errors of means (n = 10). Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD = 5.7%.

24 + Ethylene 3

- Ethylene

Wilt score 3

01234567 Time (days)

Figure 2.10. Wilting (scores: 1 = none/slight to 3 = advanced) of flowers on G. ‘Sylvia’ inflorescences treated on day 0 with 10 nL 1-MCP/L for 0 (z), 3 (), 6 (▲), 9 (▼) or 12 (◆) hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Remaining inflorescences were held in air without exogenous ethylene. Vertical bars represent standard errors of means (n = 10). Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD is presented in Appendix 2.11.

25 5

2 Vase life (days)Vase life

0 036912 1-MCP pre-treatment duration (hr)

Figure 2.11. Vase life of G. ‘Sylvia’ inflorescences treated on day 0 with 10 nL 1-MCP/L for 0, 3, 6, 9 or 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC (●). The other half of the inflorescences were not exposed to exogenous ethylene (■). Vertical bars represent standard errors of means (n = 10). Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD = 0.8.

26 0.5 + Ethylene 0.4 0.3 0.2 0.1 0.0 0.5 - Ethylene 0.4 0.3 0.2 Solution uptake (mL/g initial FW/day) 0.1 0.0 01234567 Time (days)

Figure 2.12. Vase solution uptake by G. ‘Sylvia’ inflorescences treated on day 0 with 10 nL 1- MCP/L for 0 (z), 3 (), 6 (▲), 9 (▼) and 12 (◆) hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. The other half of the inflorescences were held in air without exogenous ethylene. Vertical bars represent standard errors of means (n = 10). Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD = 0.003 mL/g initial FW/day.

27 2.3.3 Effect of temperature on 1-MCP pre-treatment efficacy

1-MCP pre-treatment (10 nL/L for 12 hours) was equally effective when applied at 0, 5, 10 or 20oC in significantly reducing exogenous ethylene-induced flower abscission (Figure 2.13 and Appendix 2.21). 1- MCP pre-treatment at 0, 5 or 10oC did not significantly reduce flower abscission from inflorescences not exposed to ethylene. Flower abscission from inflorescences pre-treated with 1-MCP at 20oC and that were not exposed to exogenous ethylene was reduced compared to similar inflorescences not pre-treated with 1- MCP (Figure 2.13). The decline in inflorescence relative fresh weight associated with exogenous ethylene-induced flower abscission was significantly reduced by 1-MCP pre-treatment at each temperature (Figure 2.14). However, as for flower abscission data, 1-MCP pre-treatment did not reduce the loss of relative fresh weight of inflorescences not exposed to ethylene, except for those pre-treated at 20oC.

Pre-treatment of inflorescences with 1-MCP at each temperature prevented the loss in vase life associated with exposure to exogenous ethylene (Figure 2.15). 1-MCP pre-treatment at 0, 5 or 10oC did not extend the vase lives of inflorescences not exposed to ethylene. In contrast, pre-treatment with 1-MCP at 20oC significantly extended the vase lives of inflorescences not exposed to ethylene (Figure 2.15), presumably as flower abscission was reduced. The vase lives of inflorescences exposed only to ethylene at 20oC were consistently short due to rapid flower abscission, and were not affected by the pre-treatment temperature. Despite the presence of non-significant pre-treatment temperature effects, there were significant interactions between 1-MCP pre-treatment, ethylene treatment and time of measurement for flower abscission (Appendix 2.21) and relative fresh weight (Appendix 2.22). As a consequence, a significant difference between 1-MCP pre-treatment and ethylene treatment for vase life was recorded (Appendix 2.23).

Flower discolouration was not affected by 1-MCP pre-treatment (Appendix 2.24). However, flower discolouration developed earlier for inflorescences held at 20oC during pre-treatment compared to those held at 0, 5 or 10oC and reflects the significant interaction between treatment (1-MCP and ethylene) and pre-treatment temperature (Appendix 2.25). In contrast to earlier experiments, the onset to flower wilting was not delayed by 1-MCP pre-treatment (Appendix 2.26). Nevertheless, flower wilting was most advanced on inflorescences pre-treated at 0oC and probably gives rise to the significant interaction between treatment (1-MCP and ethylene) and pre-treatment temperature (Appendix 2.27). 1-MCP and ethylene treatments did not affect flower opening (Appendices 2.28 and 2.29). Vase solution uptake by inflorescences exposed only to ethylene was relatively stable over time, due to flower abscission, compared to inflorescences not exposed to ethylene (Figure 2.16). This difference in response is reflected in the presence of a significant pre-treatment temperature, ethylene treatment and time of measurement interaction (Appendix 2.30).

28 + 1-MCP + Ethylene + 1-MCP - Ethylene 5

1 - 1-MCP + Ethylene - 1-MCP - Ethylene 5

Abscission score Abscission 4

0123456701234567 Time (days)

Figure 2.13. Flower abscission (scores: 1 = < 10% to 5 = > 80%) from G. ‘Sylvia’ inflorescences pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 0 (z), 5 (), 10 (▲) and 20oC (▼). Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. The other half of the inflorescences were not exposed to exogenous ethylene. Vertical bars represent standard errors of means (n = 5). Where no vertical bars appear, the standard error was smaller than the size of the symbol.

29 + 1-MCP + Ethylene + 1-MCP - Ethylene 120 100 80 60 40 20 - 1-MCP + Ethylene - 1-MCP - Ethylene 120 100 80 60 Relative fresh weight initial (% FW) 40 20 0123456701234567 Time (days)

Figure 2.14. Relative fresh weight of G. ‘Sylvia’ inflorescences pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 0 (z), 5 (), 10 (▲) and 20oC (▼). Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. The other half of the inflorescences were not exposed to exogenous ethylene. Vertical bars represent standard errors of means (n = 5). Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD = 14.7%.

30 7 + Ethylene 6 5 4 3 2 1 0 7 - Ethylene 6 Vase life life (days) Vase 5 4 3 2 1 0 0 5 10 15 20 Pre-treatment temperature (oC)

Figure 2.15. Vase life of G. ‘Sylvia’ inflorescences treated on day 0 with 0 (●) or 10 nL 1-MCP/L (■) for 12 hours at 0, 5, 10 or 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. The other half of the inflorescences were not exposed to exogenous ethylene. Vertical bars represent standard errors of means (n = 5). Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD = 1.9 days.

31 0.6 + 1-MCP + Ethylene + 1-MCP - Ethylene

0.4

0.2

0.0

0.6 - 1-MCP + Ethylene - 1-MCP - Ethylene

0.4

0.2 Solutionuptake (mL/g initial FW/day) 0.0

0123456701234567 Time (days)

Figure 2.16. Vase solution uptake by G. ‘Sylvia’ inflorescences pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 0 (z), 5 (), 10 (▲) and 20oC (▼). Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. The other half of the inflorescences were not exposed to exogenous ethylene. Vertical bars represent standard errors of means (n = 5). Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD = 0.043 mL/g initial FW/day.

32 2.3.4 Effect of 1-MCP pre-treatment on inflorescence physiology

Pre-treatment with 10 nL 1-MCP/L for 12 hours at 20oC significantly delayed the onset to flower abscission and reduced the associated decline of inflorescence relative fresh weight induced by exposure to 0 or 100 µL propylene/L (Figure 2.17). As a result these responses are manifested as significant interactions between 1-MCP pre-treatment, propylene treatment and time of measurement for flower abscission (Appendix 2.31) and relative fresh weight (Appendix 2.32). Vase solution uptake between days 0 and 1 was lowest for inflorescences pre-treated with 1-MCP (Figure 2.18). In addition, vase solution uptake between days 1 and 2 was lowest for inflorescences exposed to propylene. These responses give rise to the significant interactions between 1-MCP pre-treatment and time of measurement and propylene treatment and time of measurement for vase solution uptake data (Appendix 2.33).

Consequently, the reduction in vase life associated with exposure to propylene treatment was prevented by pre-treatment with 1-MCP (Table 2.1). However, 1-MCP pre-treatment did not significantly extend the vase lives of inflorescences not exposed to propylene. Accordingly, a significant interaction between 1- MCP pre-treatment and propylene treatment for vase life was recorded (Appendix 2.34). 1-MCP pre- treatment did not affect flower discolouration, opening and wilting compared to inflorescences not pre- treated with 1-MCP (Appendices 2.35, 2.36, 2.37, 2.38 and 2.39).

33 5

2 Abscission score Abscission 1 120 100 80 60 40 (% initial FW) (% 20 Relative fresh weight 0 01234567 Time (days)

Figure 2.17. Flower abscission (scores:1 = < 10% to 5 = > 80%) and relative fresh weight for G. ‘Sylvia’ inflorescences treated with 0 nL 1-MCP/L and 0 µL propylene/L (●), 0 nL 1-MCP/L and 100 µL propylene/L (■), 10 nL 1-MCP/L and 0 µL propylene/L (▲) or 10 nL 1-MCP/L and 100 µL propylene/L (▼). 1-MCP and propylene treatments were each conducted for 12 hours at 20oC on day 0 and 1, respectively. Vertical bars represent standard errors of means (n = 6). Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD for relative fresh weight data = 4.5%.

34 0.4

0.2

Solution uptake (mL/g initial FW/day) initial (mL/g uptake Solution 0.0 01234567 Time (days)

Figure 2.18. Vase solution uptake by G. ‘Sylvia’ inflorescences treated with 0 nL 1-MCP/L and 0 µL propylene/L (●), 0 nL 1-MCP/L and 100 µL propylene/L (■), 10 nL 1-MCP/L and 0 µL propylene/L (▲) or 10 nL 1-MCP/L and 100 µL propylene/L (▼). 1-MCP and propylene treatments were each conducted for 12 hours at 20oC on day 0 and 1, respectively. Vertical bars represent standard errors of means (n = 6). Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD = 0.028 mL/g initial FW/day.

35 Table 2.1. Vase life (mean ± s.e.) of G. ‘Sylvia’ inflorescences treated on day 0 with 0 or 10 nL 1- MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 100 µL propylene/L for 12 hours at 20oC. Vase life data followed by the same letter are not significantly different (LSD = 0.7) at P = 0.05 (n = 6).

Treatment Vase life (days)

No propylene (0 µL/L) 0 nL 1-MCP/L 6.0 ± 0.2 b 10 nL 1-MCP/L 6.3 ± 0.3 b Plus propylene (100 µL/L) 0 nL 1-MCP/L 2.0 ± 0.0 a 100 nL 1-MCP/L 6.3 ± 0.2 b

Pre-treatment of inflorescences with 1-MCP reduced ethylene production rates compared to inflorescences exposed only to propylene, which were peduncles without flowers (Figure 2.19). Ethylene production rates by the peduncles of inflorescences exposed only to propylene remained low until day 4, thereafter increased until day 6, and then began to decline. Rates of ethylene production by inflorescences pre- treated with 1-MCP and those not treated with 1-MCP or propylene, slowly increased until day 7 in association with flower abscission (Figures 2.17 and 2.19). However, there was no significant effect of 1- MCP pre-treatment on the rate of ethylene production by inflorescences not exposed to propylene (Appendix 2.40). The respiration rate of all inflorescences declined following initial treatments (Figure 2.19). However, the rate of respiration by inflorescences pre-treated with 1-MCP was maintained at higher rates than inflorescences exposed only to propylene. The rate of respiration started to increase after day 5 or 6 for inflorescences pre-treated with 1-MCP and for those not treated with 1-MCP or propylene, respectively, (Figure 2.19) in association with flower abscission (Figure 2.17). Consequently, a significant interaction between 1-MCP pre-treatment, propylene treatment and time of measurement was evident for respiration (Appendix 2.41).

36 8

2 Rate of ethylene 0 production (µL/kg FW/hr) (µL/kg production 150

100 /kg FW/hr) 2 50 Respiration rate Respiration (mg CO 0 234567 Time (days)

Figure 2.19. Rates of ethylene production and respiration by G. ‘Sylvia’ inflorescences treated with 0 nL 1-MCP/L and 0 µL propylene/L (●), 0 nL 1-MCP/L and 100 µL propylene/L (■), 10 nL 1- MCP/L and 0 µL propylene/L (▲) or 10 nL 1-MCP/L and 100 µL propylene/L (▼). 1-MCP and propylene treatments were each conducted for 12 hours at 20oC on day 0 and 1, respectively. Vertical bars represent standard errors of means (n = 3). Where no vertical bars appear, the standard error was smaller than the size of the symbol. Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD for ethylene production = 1.3 µL/kg FW/hr and respiration = 16.6 mg CO2/kg FW/hr.

37 In the second experiment examining inflorescence physiology, 1-MCP pre-treatment significantly delayed the onset of flower abscission from inflorescences exposed to 0 or 100 µL propylene/L (Figure 2.20). This response accounts for the significant interaction between 1-MCP pre-treatment, propylene treatment and time of measurement (Appendix 2.42). The ACC contents of 1-MCP pre-treated flowers or those not treated with 1-MCP or propylene increased to day 5 (Figure 2.20). Flower ACC content increased rapidly after day 3 and apparently preceded flower abscission (Figure 2.20). However, the ACC content of flowers was not significantly affected by 1-MCP pre-treatment (Appendix 2.43). 1-MCP pre-treatment prevented the loss in vase life for inflorescences exposed to propylene (Table 2.2). The vase lives of inflorescences not exposed to propylene was not extended by 1-MCP pre-treatment. As a result, a significant interaction for 1-MCP pre-treatment and propylene treatment for vase life was recorded (Appendix 2.44). In this experiment, flower discolouration and wilting on inflorescences pre-treated with 1-MCP were significantly more advanced than inflorescences not pre-treated with 1-MCP on days 4 and 5 (Appendices 2.45, 2.46 and 2.47). Flower opening was not affected by 1-MCP or propylene treatments (Appendices 2.45 and 2.48).

38 5

2 Abscission score 1

1 ACC content (n molesACC/g FW) 0 012345 Time (days)

Figure 2.20. Flower abscission (scores: 1 = < 10% to 5 = > 80%) and ACC content from G. ‘Sylvia’ inflorescences treated with 0 nL 1-MCP/L followed by 0 µL propylene/L (z), 0 nL 1-MCP/L followed by 100 µL propylene/L (), 10 nL 1-MCP/L followed by 0 µL propylene/L (▲) or 10 nL 1- MCP/L followed by 100 µL propylene/L (▼). 1-MCP and propylene treatments were each conducted for 12 hours at 20oC on day 0 and 1, respectively. Vertical bars represent the standard errors of means. Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD for ACC is 0.42 n mol/g FW.

39 Table 2.2. Vase life (mean ± s.e.) of G. ‘Sylvia’ inflorescences used in the determination of flower ACC content, which were treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each treatment were then exposed on day 1 to 100 µL propylene/L for 12 hours at 20oC. Vase life data followed by the same letter are not significantly different (LSD = 0.8) at P = 0.05 (n = 3).

Treatment Vase life (days)

No propylene (0 µL/L) 0 nL 1-MCP/L 4.3 ± 0.2 b 10 nL 1-MCP/L 4.3 ± 0.2 b Plus propylene (100 µL/L) 0 nL 1-MCP/L 2.0 ± 0.0 a 10 nL 1-MCP/L 4.0 ± 0.0 b

2.4 DISCUSSION

1-MCP pre-treatment was effective at low concentration (5 nL 1-MCP/L for 12 hours at 20oC) and for short treatment duration (10 nL 1-MCP/L for 3 hours at 20oC) in delaying the onset of flower abscission from G. ‘Sylvia’ inflorescences subsequently exposed to 10 µL ethylene/L (Figures 2.1 and 2.8). Endogenous ethylene-induced flower abscission was also reduced by 1-MCP pre-treatment. The loss of inflorescence relative fresh weight (Figures 2.2 and 2.9) and vase life (Figures 2.4 and 2.11) associated with exogenous ethylene-induced flower abscission were reduced by 1-MCP pre-treatment. These results are in agreement with those of other workers who used similar 1-MCP treatments to prevent ethylene- induced floral organ abscission and senescence from a range of cut flowers (Serek et al. 1995a, b, c; Porat et al. 1995a, b; Sisler et al. 1996a). In particular, 1-MCP pre-treatment protocols used in the present study and their efficacy match those used by Serek et al. (1995a). They found that pre-treatment with 10- 20 nL 1-MCP/L for 6 hours protected various cut flowers including carnation, snapdragon and Penstemon against ethylene.

It is possible that lower 1-MCP concentrations or shorter treatment durations than those used in the present study could also reduce the ethylene sensitivity of G. ‘Sylvia’ inflorescences, since carnation flowers can be protected against ethylene by pre-treatment with 0.5 nL 1-MCP/L for 24 hours at 24oC or 250 nL 1-MCP/L for just 5 minutes at 24oC (Sisler et al. 1996a). Nevertheless, the sensitivity of different plant materials to 1-MCP varies (Sisler and Serek 1997). Protection of G. ‘Sylvia’ inflorescences against ethylene may require a higher 1-MCP concentration and/or longer treatment duration than for carnations. Efficacy of 1-MCP pre-treatment was proposed by Serek et al. (1995a) to be an inverse relationship between 1-MCP concentration and treatment duration. They showed that pre-treatment of Penstemon flowers with 10 nL 1-MCP/L for 0.5 hours or 5 nL 1-MCP/L for 3 hours were equally effective in preventing ethylene-induced flower abscission. Accordingly, from a commercial perspective, a range of effective 1-MCP treatment protocols could be applied depending on the sensitivity of plant tissue to 1-

40 MCP and ethylene and changes in market supply and demand patterns. For example, pre-treatment of cut flowers with high 1-MCP concentrations for short durations could be used to meet increasing market demand.

Increasing the 1-MCP concentration to 10 or 20 nL/L or extending the treatment duration to 12 hours were only slightly more effective in delaying exogenous ethylene-induced flower abscission than a concentration of 5 nL/L or a treatment duration of 3 hours, respectively (Figures 2.1 and 2.8). This is consistent with the results of Serek et al. (1995a), who showed that pre-treatment of Penstemon flowers with 5 or 10 nL 1-MCP/L for 6 hours was slightly more effective in extending flower longevity than a 3 hour pre-treatment. Higher 1-MCP concentrations and/or longer term exposure to 1-MCP may block additional ethylene receptors, although this does not appear to be critical in providing significantly longer protection against ethylene. It is assumed that pre-treatment of inflorescences with 5 nL 1-MCP/L for 12 hours at 20oC or 10 nL 1-MCP/L for 3 hours at 20oC blocked almost all available ethylene receptors (Figures 2.4 and 2.11).

1-MCP pre-treatment (10 nL 1-MCP/L for 12 hours) was equally effective when applied at 0, 5, 10 or 20oC in protecting G. ‘Sylvia’ inflorescences against ethylene (Figure 2.14). These results contrast with those of Serek et al. (1995a) who reported that pre-treatment of Penstemon flowers with 5 or 20 nL 1- MCP/L for 6 hours at 2oC did not prevent ethylene-induced flower abscission. Likewise, Reid et al. (1996) found that 1-MCP treatment of Kalanchoe flowers was not effective when applied at 2oC, possibly because 1-MCP binding was reduced. However, increasing the 1-MCP concentration and/or the treatment duration at 2oC was reported by Reid et al. (1996) to improve the efficacy of treatment. It is possible that the low efficacy of 1-MCP treatment reported by Serek et al. (1995a) and Reid et al. (1996) is related to an inherent low temperature response of the plant material. Exposure of chilling sensitive plants to low temperature is proposed to induce membrane phase changes which, in turn, may alter the conformation of membrane-bound proteins (Lyons 1973). Thus, some degree of conformational change to the membrane- located protein believed to act as the ethylene receptor may reduce its ability to bind 1-MCP molecules. However, whilst Kalanchoe is generally regarded as being chilling sensitive, Penstemon are frost tolerant (Page and Olds 1997). Further, Penstemon flowers were not reported to suffer injury at 2oC (Serek et al. 1995a).

Tropical and sub-tropical flowers are sensitive to chilling at temperatures of 10-15oC (Halevy and Mayak 1981). Because of their sub-tropical origin (Costin and Costin 1988), G. ‘Sylvia’ inflorescences might be considered chilling sensitive. However, no injury was observed for inflorescences kept at 5oC for 5 days (Ligawa et al. 1997). Accordingly, 1-MCP pre-treatment may have protected inflorescences against ethylene as the ethylene receptors were not disturbed by exposure to chilling temperatures. Alternatively, it is possible that the binding of 1-MCP molecules may have taken place during ventilation of chambers at the end of 1-MCP treatment. Chambers were ventilated inside a fume cupboard at 20oC for 10-15 minutes and thus the temperature of inflorescences may have risen while 1-MCP gas was still present. 1-MCP binding is known to be rapid as carnation flowers pre-treated with 250 nL 1-MCP/L for just 5 minutes

41 were protected against ethylene (Sisler et al. 1996a).

Pre-treatment with 10 nL 1-MCP/L for 12 hours at 20oC protected G. ‘Sylvia’ inflorescences against propylene treatment (100 µL/L for 12 hours at 20oC). Propylene treatment mimicked ethylene by inducing flower abscission (Figure 2.17) and reducing vase life (Table 2.1). Consequently, ethylene production by inflorescences pre-treated with 1-MCP was reduced (Figure 2.18). However, increasing ethylene production by 1-MCP pre-treated inflorescences was associated with flower abscission. This result is similar to those of Sisler et al. (1996a), where ethylene production by carnation flowers was reduced and delayed by 1-MCP pre-treatment compared to flowers exposed only to ethylene. In the present study, 1-MCP pre-treatment did not reduce the rate of ethylene production by inflorescences not exposed to propylene. This is in contrast to the findings of Sisler et al. (1996a), where the rate of ethylene production by carnation flowers not exposed to ethylene was reduced by 1-MCP pre-treatment. The retention of flowers on inflorescences pre-treated with 1-MCP was presumably responsible for maintaining higher rates of respiration compared to inflorescences exposed only to ethylene (Figure 2.18). ACC content of flowers not exposed to propylene increased prior to flower abscission, but was not affected by 1-MCP pre-treatment (Figure 2.20). Nonetheless, the association between rates of ethylene production, respiration and natural postharvest flower abscission were similar to results presented by Joyce et al. (1995). The ACC data provide additional evidence that endogenous ethylene production by G. ‘Sylvia’ inflorescences may mediate natural postharvest flower abscission.

The vase solution uptake by inflorescences decreased over time presumably as the transpirational surface area decreased in association with flower abscission and senescence. However, the decrease in vase solution uptake was delayed for inflorescences pre-treated with 1-MCP because flower abscission and the associated decrease in transpirational surface area were also delayed (Figures 2.7, 2.12, 2.16 and 2.18). 1- MCP pre-treatment helped to discriminate between ethylene dependent and ethylene independent senescence processes in G. ‘Sylvia’ inflorescences. Despite 1-MCP pre-treatment delaying flower wilting in the first two experiments, there was no consistent effect of 1-MCP pre-treatment on flower wilting in subsequent experiments. Thus, any potential role of ethylene in flower wilting on G. ‘Sylvia’ inflorescences is unclear. Flower discolouration and opening were not consistently affected by 1-MCP pre-treatment in any way and, therefore, do not appear to be regulated by ethylene.

Overall, 1-MCP pre-treatment was judged only to be effective in protecting inflorescences against exogenous ethylene. It did not consistently extend the vase lives of inflorescences not exposed to ethylene or propylene (Figures 2.4, 2.11, 2.15, Tables 2.1 and 2.2). Nonetheless, 1-MCP pre-treatment was shown to reduce endogenous ethylene-induced flower abscission in most experiments. Consequently, based on its demonstrated capacity to block the sensitivity of G. ‘Sylvia’ inflorescences to exogenous ethylene, 1- MCP pre-treatment may have potential as a postharvest anti-ethylene treatment for sensitive native Australian cut flowers.

42 CHAPTER 3

RESPONSES OF A NUMBER OF NATIVE AUSTRALIAN CUT FLOWERS TO 1-MCP AND ETHYLENE TREATMENTS

3.1 INTRODUCTION

Native Australian cut flowers are gaining acceptance in the international ornamentals trade based on a 10- fold increase in the value of exports between 1980/81 and 1995/96 (FECA 1996). In contrast to traditional flower crops, there has been limited postharvest research on native cut flowers (Faragher 1989). For example, relationships between exogenous and endogenous ethylene-induced flower abscission and senescence have not been thoroughly investigated. Nonetheless, ethylene has been implicated in flower abscission from Chamelaucium uncinatum (Geraldton waxflower) (Joyce 1988, 1989, 1993), Grevillea spp. (Joyce and Haynes 1989), Leptospermum scoparium (tea tree) (Zieslin and Gottesman 1983), Telopea speciosissima (NSW waratah) (Joyce et al. 1993) and Verticordia nitens (yellow Morrison) (Joyce and Haynes 1989; Joyce and Poole 1993). Accelerated senescence of Boronia heterophylla (red boronia) flowers has also been associated with exposure to exogenous ethylene (Joyce and Haynes 1989).

STS treatment has been shown to inhibit ethylene effects on cut B. heterophylla (Joyce and Haynes 1989), C. uncinatum (Joyce 1988, 1989, 1993), Grevillea spp. (Joyce and Haynes 1989; Vuthapanich et al. 1993), L. scoparium (Zieslin and Gottesman 1983), T. speciosissima (Joyce et al. 1993) and V. nitens (Joyce and Haynes 1989; Joyce and Poole 1993). Likewise, the recently developed gaseous inhibitor of ethylene perception, 1-MCP, might also protect native Australian flowers as it does traditional cut flowers against ethylene (Serek et al. 1995a, b, c; Porat et al. 1995a, b; Sisler et al. 1996a).

It is proposed that 1-MCP is likely to prove an effective anti-ethylene treatment for ethylene-sensitive native Australian flowers. The purpose of this screening study was to examine the response in a selection of native Australian cut flowers to 1-MCP and ethylene treatments. In an associated experiment, relationships between B. heterophylla flower senescence, exogenous and endogenous ethylene, and 1- MCP and STS treatments were examined.

3.2 MATERIALS AND METHODS

Experiments were conducted at The University of Qld, Gatton College (UQG), postharvest laboratory or at the Victorian Agriculture, Institute for Horticultural Development (IHD), Knoxfield postharvest laboratory during a visit there by the author. Descriptions and pictures of floral genera used in these experiments can be found in Wrigley and Fagg (1997).

43 3.2.1 Plant material

3.2.1.1 Alloxylon pinnatum

Alloxylon pinnatum (Dorrigo waratah; Proteaceae) inflorescences are comprised of a terminal inflorescence on a stem consisting of approximately 100 flowers. Inflorescences, with 10-20% of flowers open, were harvested from plants growing near Moss Vale, NSW (34o 33’ S, 150o 23’ E). Hydrated water absorbent crystals were placed around cut stem ends and the inflorescences were wrapped in plastic sleeves to minimise water loss. They were placed into a cardboard box and freighted by road to the UQG laboratory within 36 hours of harvest. In a second experiment, A. pinnatum inflorescences at the same stage of development as those used in the first experiment were harvested from plants growing near Maleny in S.E. Qld (26o 46’ S, 152o 51’ E). Inflorescences were packed dry into a cardboard box and transported to the UQG laboratory by road within 3 hours of harvest.

3.2.1.2 Boronia heterophylla

Flowering Boronia heterophylla (red boronia; Rutaceae) stems were harvested from two cut flower farms in Western Australia. Stems were packed into commercial flower cartons and air freighted dry either directly or via a flower exporter in Sydney to the Brisbane airport within 24 or 48 hours of harvest, respectively. Stems were then taken to the UQG laboratory in an air conditioned car within 2 hours. In the first experiment, a form of B. heterophylla with small flowers was used. Flowers used in both experiments arrived at the laboratory with slight flower discolouration (i.e. trace of white on flowers).

3.2.1.3 Cassinia adunca

Flowering Cassinia adunca (Asteraceae) stems with approximately 10-50% of flowers open were harvested from plants growing near Maleny. Cut stem ends were wrapped in moist newsprint and transported to the UQG laboratory by road within 3 hours of harvest.

3.2.1.4 Ceratopetalum gummiferum

Flowering Ceratopetalum gummiferum (NSW Christmas bush; Cunoniaceae) stems were harvested from a farm near Childers in S.E. Qld (25o 14’S, 152o 17’ E). Stems were placed into a cardboard box and transported dry by road to the wholesale flower market in Brisbane. Stems were then collected and taken to the UQG laboratory in an air conditioned car. Stems arrived in the laboratory within 48 hours of harvest.

3.2.1.5 Chamelaucium uncinatum

44 Flowering branches of Chamelaucium uncinatum ‘Paddy’s Late’ (Geraldton waxflower; Myrtaceae) with > 90% of flowers open were harvested from a farm near Esk in S.E. Qld (27o 14’ S, 152o 25’ E). Cut stem ends of branches were stood into buckets containing DI water and taken to the UQG laboratory by road within 1 hour of harvest.

3.2.1.6 Eriostemon scaber

Flowering Eriostemon scaber (Rutaceae) stems were obtained from a farm near Sale in eastern Victoria (38o 07 S, 147o 04 E). Stems were packed dry into a commercial flower carton and delivered by road to the IHD laboratory within 24 hours of harvest.

45 3.2.1.7 Grevillea hybrids

Grevillea ‘Kay Williams’ [(G. sessilis x G. pteridifolia) x G. banksii] and G. ‘Misty Pink’ (G. banksii x G. sessilis) inflorescences (Proteaceae) were harvested from a farm near Gatton in S.E. Qld (27o 34’S 152o 17’E). Inflorescences were selected, harvested and prepared as described in section 2.2.1 and were taken to the UQG laboratory in an air conditioned car within 30 minutes of harvest. G. ‘Sandra Gordon’ (G. sessilis x G. pteridifolia) inflorescences were harvested from a nursery near Redland Bay. Inflorescences were harvested and transported to the laboratory as described in section 2.2.1.

3.2.1.8 Leptospermum spp.

Stems of Leptospermum petersonii (lemon-scented tea tree; Myrtaceae) with > 50% of flowers open were harvested from a residential garden in Brisbane in S.E. Qld (27o 28’S 153o 01’E). Cut stem ends were stood into DI water and transported to the UQG laboratory in an air conditioned car within 1 hour of harvest. Flowering L. scoparium ‘Winter Cheer’ (tea tree) stems were harvested from plants growing near the IHD laboratory. Cut stem ends were immediately stood into DI water and taken inside the laboratory.

3.2.1.9 Ozothamnus diosmifolius

Flowering Ozothamnus diosmifolius ‘Cooks Tall Pink’ (rice flower; Asteraceae) stems were harvested from a farm near Helidon in S.E. Qld (27o 33’ S, 152o 08’ E). This cultivar was a fine leaf form of rice flower. Flowers were harvested at the first sign of flower break or opening. Cut stem ends were stood into DI water and transported in an air conditioned car to the UQG laboratory within 30 minutes of harvest.

3.2.1.10 Platysace lanceolata

Flowering Platysace lanceolata (Apiaceae) stems were harvested from plants near Maleny. Cut stem ends were wrapped in moist newsprint and transported to the UQG laboratory by road within 3 hours of harvest.

3.2.1.11 Telopea speciosissima

Telopea speciosissima (NSW waratah; Proteaceae) inflorescences were obtained from a farm near Monbulk in southern Victoria (37o 53’ S, 145o 25’ E). Inflorescences were harvested from clonally propagated T. speciosissima ‘Shady Lady’ or seed-grown T. speciosissima plants when approximately

46 50% of flowers had opened. Cut stem ends of inflorescences were stood into water and held in a coolroom at 4oC for approximately 48 hours for inflorescences from clonally propagated plants or overnight for inflorescences from seed-grown plants. Cut stem ends of all inflorescences were then stood into DI water and taken to the IHD laboratory in an air conditioned car within 24 (seed-grown) or 48 hours (clonally propagated plants) of harvest.

3.2.1.12 Thryptomene calycina

Flowering Thtyptomene calycina (Grampian’s thryptomene; Myrtaceae) branches were harvested from a farm near Horsham in western Victoria (36o 43’ S, 142o 12’ E). Branches were packed dry into a commercial flower carton and delivered to the IHD laboratory by road within 24 hours of harvest. Branches arrived in the laboratory with approximately 20% of flowers closed.

3.2.1.13 Verticordia nitens

Flowering Verticordia nitens (yellow Morrison; Myrtaceae) stems were bush-picked in Western Australia and air freighted dry to the Brisbane wholesale cut flower market within 30 hours of harvest. On arrival, cut stem ends of flowers were stood into water. Stems were then transferred to buckets of DI water and taken in an air conditioned car to the UQG laboratory within 1 hour. 3.2.1.14 Zieria cytisoides

Flowering Zieria cytisoides (Rutaceae) stems were harvested from a farm near Caboolture in S.E. Qld (27o 05’ S, 152o 57’ E). Cut stem ends were wrapped in moist newsprint and taken to the UQG laboratory by road within 2 hours of harvest.

3.2.2 Plant material preparation

On arrival at the laboratory, stem ends of flowers which were transported dry (viz. A. pinnatum, B. heterophylla, C. gummiferum, E. scaber, T. calycina) were recut under DI water (section 2.2.1) and stood into DI water for 3-6 hours to rehydrate. The stems of all cut flowers were again recut under DI water to a length of 20-30 cm, removing at least 2 cm of the stem base. Leaves were removed from stem portions which would otherwise be submerged in vase solution. In the case of O. diosmifolius, all stem ends were recut under DI water to a length of 30 cm and leaves were removed within 15 cm of the stem base. All cut flower stems were then randomly assigned to individual vases (300-375 mL volume). Vases contained a solution of 10 mg available chlorine provided as DICA/L DI water (section 2.2.1). Vase openings were  covered with a piece of plastic film (section 2.2.1) or Parafilm . Stems were inserted through slits in the film into the vase solution. In a separate study where ethylene production by B. heterophylla stems was measured, 100 mL capacity vases were used.

3.2.3 Chemicals

47 1-MCP gas was synthesised and quantified according to section 2.2.2.1. Similarly, an ethylene gas stock was prepared by diluting gas from a pressurised cylinder as described in section 2.2.2.2. The concentration of the ethylene working stock was determined by gas chromatography (section 2.2.2.2). In experiments conducted at the IHD laboratory, the concentration of the ethylene working stock was quantified by injecting 1 mL samples into a Shimadzu GC-8AIT FID gas chromatograph fitted with a 1.83 m long by 2.2 mm internal diameter stainless steel column packed with activated alumina with a mesh range of 80/100. The column temperature was 140oC and the injector/detector was 180oC. Ethylene samples were quantified against a 1.1 µL ethylene/L standard (BOC Gases β-grade special gas mixture). High purity nitrogen was the carrier gas.

3.2.3.1 Silver thiosulfate

A solution of STS was made according to the method of Joyce (1992). STS solutions used to treat flowers were prepared by pipetting an aliquot of the stock solution into vases containing DI water.

3.2.4 Treatments

3.2.4.1 Treatment of a range of native cut flowers with 1-MCP and ethylene

Flowers in vases were enclosed into 60.5 L glass chambers (section 2.2.3). In experiments conducted at the IHD laboratory, flowers in vases were placed into 120 L perspex chambers or individually into 30 L plastic buckets. Potentially excessive accumulation of CO2 concentrations inside glass chambers or buckets were reduced with KOH solution as previously described (section 2.2.3). In the case of perspex chambers, 50 mL of 1M KOH was poured onto the chamber base. Perspex chambers consisted of a five sided perspex tub and a stainless steel base. The tub was sealed by lowering its walls into matching grooves filled with water on the base. Two inlets in one side of the stainless steel base were connected and sealed by latex tube for gas injection or sampling. Plastic buckets were sealed with press-on lids. A rubber septum for gas injection or sampling was positioned through each lid.

In all experiments, once flowers were inside chambers or buckets, the lids were sealed in place. Based on the results of previous experiments with G. ‘Sylvia’ (Chapter 2) and other cut flowers (Serek et al. 1995a), flowers were then pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC (section 2.2.4.1). Chambers and buckets were ventilated outside each laboratory following 1-MCP pre-treatment. Half of the flowers from each of these treatments were then exposed to 10 µL ethylene/L for 12 hours at 20oC inside the same chambers or buckets containing 1M KOH solution (section 2.2.4.1). Ethylene concentrations inside the chambers or buckets was monitored by gas chromatography (sections 2.2.2.2 and 3.2.3). The other half of the flowers were enclosed in matching chambers or buckets in air with KOH, but without exogenous ethylene. Following ethylene treatment, flowers and their vases were removed from these containers and transferred to vase life rooms. The vase life room at UQG was operated at the

48 conditions decribed in section 2.2.4.1. The IHD vase life room was maintained at 21 ± 0.5oC and 60% RH, and was illuminated with overhead cool white fluorescent lights to 10 µmol/m2/s at flower height for a 12 hour light period each day.

3.2.4.2 Treatment of B. heterophylla with 1-MCP, STS and ethylene

Flowering B. heterophylla stems in vases were pre-treated on day 0 with 0 or 10 nL 1-MCP/L in 60.5 L glass chambers (section 2.2.3) or pulsed with STS (0.5 mM Ag+) for 12 hours at 20oC. Stems were then removed from chambers or STS solutions. The portion of stems that had been submerged in STS solution were rinsed in DI water and placed into standard vase solutions. Half of the stems from each of these anti- ethylene treatments were then exposed on day 1 to 10 µL ethylene/L for the longer period (cf. section 3.2.4.1) of 72 hours at 20oC. The other half of the stems were held in matching chambers in air with KOH, but without exogenous ethylene.

3.2.5 Assessments

3.2.5.1 Vase life

All flowers and their vases were weighed separately daily or every second day (section 2.2.5.1). Floral organ abscission and opening were assessed daily or every second day after gently brushing or tapping stems three times by hand. Depending on the particular genus, floral organ abscission and opening were recorded using a rating scale (1 = < 10%, 2 = 10-30%, 3 = 30-50%, 4 = 50-80%, 5 = > 80%) or as a percentage of the initial number of floral organs present (Table 3.1). Flower opening on Grevillea spp. inflorescences was rated daily using an alternative scale: 1 = < 5%, 2 = 5-25%, 3 = > 25% open flowers on an inflorescence. The number of flowers on T. calycina stems which closed during vase life was expressed as a percentage of the initial number of open flowers.

Table 3.1. Method and frequency of assessing floral organ abscission and opening from native cut flowers.

Flower Abscission Opening

Method Frequency Method Frequency A. pinnatum percentage daily percentage daily B. heterophylla aa aa C. gummiferum score daily aa C. uncinatum score daily aa C. adunca aa score daily E. scaber percentage every second day aa Grevillea spp. score daily score daily L. petersonii percentage daily aa

49 L. scoparium percentage b daily aa O. diosmifolius aa score daily P. lanceolata score daily aa T. calycina percentage every second day aa T. speciosissima percentage daily score every second day V. nitens score daily aa Z. cytisoides score daily aa a Assessment was not made. b Percentage of abscised and senescent flowers.

Wilting at the pedicels and peduncles of C. adunca and O. diosmifolius, flower discolouration (fading) from Grevillea spp. and flower wilting of other flowers were subjectively determined daily using the following rating scale: 1 = none/slight, 2 = moderate, 3 = advanced. Discolouration of B. heterophylla flowers, observed as fading from bright pink to white, was rated daily using the scale of 1 = 0-25%, 2 = 26-50%, 3 = 51-75%, 4 = 76-100% discolouration over the whole stem. Leaf senescence, evident as chlorosis or necrosis, on flowering stems of C. adunca and O. diosmifolius was measured as a percentage leaf area affected. In addition, leaf abscission from O. diosmifolius stems was determined daily and expressed as the length (cm) of stem without leaves, from the stem base to the lowest leaf. Vase life of all cut flowers examined was judged as the time in days to loss of visual appeal (Table 3.2).

Table 3.2. Criteria used to determine the end of vase life for native cut flowers.

Flower Criteria

A. pinnatum ≥ 20% perianth abscission and/or ≥ 10% perianth wilting and/or moderate perianth discolouration B. heterophylla ≥ 50% of flowers with ≥ 50% discolouration and/or moderate wilting C. gummiferum > 10% flower abscission and/or moderate sepal wilting C. uncinatum > 10% flower abscission and/or ≥ 50% of flowers having lost turgor as evidenced by a decreased angle between petals and the style C. adunca ≥ 20% leaf senescence and/or moderate pedicel wilting E. scaber ≥ 10% petal abscission Grevillea spp. > 10% flower abscission and/or moderate flower wilting and/or moderate flower discolouration L. petersonii ≥ 20% flower abscission and/or ≥ 50% of flowers having lost turgor as evidenced by a decreased angle between petals and the style L. scoparium ≥ 20% flower abscission and senescence O. diosmifolius ≥ 20% leaf senescence and/or moderate pedicel wilting

50 P. lanceolata. ≥ 10% flower wilting and/or discolouration T. calycina ≥ 10% flower closing T. speciosissima ≥ 20% perianth abscission V. nitens > 10% flower abscission and/or closing of flowers and/or fading of bracts and/or pedicel wilting Z. cytisoides > 10% flower abscission and/or moderate flower wilting

51 3.2.5.2 Ethylene production rates

Parallel sets of 15 cm long flowering and non-flowering B. heterophylla stems in vases were enclosed in

2.2 L glass jars each containing CO2 scrubber (section 2.2.5.2). Additionally, individual flowers were randomly sampled from flowering stems standing in DI water at 20oC. These detached flowers were placed singly into 15 mL glass test tubes. Five replicate flowering and non-flowering stems and sets of detached flowers were used. All jars and tubes were sealed once daily with plastic screw-on lids and rubber plugs, respectively, and held at 20oC for 10-12 hours. Gas samples were then taken from the headspace of jars and tubes with a 1 mL syringe. The ethylene concentration was measured by gas chromatography (section 2.2.5.2).

3.2.6 Experiment design and data analysis

Following 1-MCP, STS and ethylene treatments, flowers were arranged in a vase life room in a CRD. There were five to ten replicate stems for each treatment, depending upon the particular experiment. The responses of a number of cut flowers to 1-MCP and ethylene treatments were examined as 2 (1-MCP) x 2 (ethylene) factorial experiments. A 3 (anti-ethylene agent) x 2 (ethylene) factorial experiment was used to study the effect of 1-MCP, STS and ethylene treatments on B. heterophylla. Treatment means and  standard errors were calculated using Microsoft Excel (Version 5.0, Microsoft Inc.). Figures were  plotted using Sigmaplot (Version 2.0, Jandel Corporation). Most data were analysed by ANOVA using  the balanced ANOVA function of Minitab (Release 11.12, Minitab Inc.). Data sets from Grevillea spp. and T. speciosissima experiments showing non-significant differences similar to those reported for G. ‘Sylvia’ in Chapter 2 are presented in appendices. Flower opening from these experiments was analysed using χ2 tests or Fisher’s exact test by SAS (release 6.12, SAS Institute 1996) (section 2.2.6).

The time in days to reach 10% flower abscission from G. ‘Kay Williams’, G. ‘Misty Pink’ and Z. cytisoides, or 20% floral organ abscission from L. petersonii, L. scoparium and T. speciosissima was recorded for ANOVA. Likewise, the time in days to reach moderate wilting of sepals on C. gummiferum, pedicels on C. adunca and O. diosmifolius and peduncles on O. diosmifolius was calculated. The time in days to moderate flower discolouration from G. ‘Sandra Gordon’, > 50% flower discolouration from B. heterophylla (second experiment; section 3.2.4.2) and 10% flower opening from O. diosmifolius were also recorded for ANOVA. These data were then analysed as 2 x 2 or 3 x 2 factorial ANOVAs depending upon the particular experiment. In experiments with Grevillea spp., when flower abscission from inflorescences was 100%, flower wilting, discolouration and opening measurements for these inflorescences were discontinued (section 2.2.6). Thus, in the case of G. ‘Sandra Gordon’, flower discolouration of the remaining treatments was analysed as a one-way ANOVA. Ethylene production data from B. heterophylla was also analysed as one-way ANOVAs.

In experiments where all replicates did not reach the abscission and senescence stages described above, data were analysed as split plot for time (i.e. sequential days of measurement) ANOVAs as described in

52 section 2.2.6. For this method of ANOVA, score data were converted to a corresponding percentage. Derived and direct percentage data were then arcsine transformed for ANOVA (Steel and Torrie 1987). Flower discolouration data from G. ‘Kay Williams’ and G. ‘Misty Pink’ and all remaining wilting data were recorded for ANOVA as a binary score, where a 0 or 1 score was assigned to each stem to indicate the absence or presence of moderate to advanced discolouration or wilting (Narula and Levy 1977). Relative fresh weight and vase solution uptake data from all experiments and leaf abscission from O. diosmifolius were also analysed as split plot for time ANOVAs.

Treatment means were separated by the LSD test at P = 0.05. For data analysed as split plots for time, LSDs presented are for between treatments (section 2.2.6). LSDs are presented only when significant differences (P < 0.05) between treatments existed. Differences at the P < 0.05 level between treatment means are referred to in the results as significant. LSDs from ANOVAs on derived data not directly shown in figures are presented in the appendices. LSDs from ANOVA of transformed and binary data sets are not presented (section 2.2.6).

53 3.3 RESULTS

3.3.1 Treatment of a range of native cut flowers with 1-MCP and ethylene

3.3.1.1 A. pinnatum

Treatment of A. pinnatum inflorescences with 1-MCP or ethylene did not significantly affect perianth abscission (Figure 3.1 and Appendix 3.1). The change in relative fresh weight of inflorescences was not affected by 1-MCP pre-treatment (Figure 3.1 and Appendix 3.2). However, inflorescences exposed only to ethylene lost significantly more relative fresh weight compared to inflorescences not exposed to ethylene (Figure 3.1 and Appendix 3.2). Vase solution uptake by inflorescences tended to increase with time (Figure 3.1). Solution uptake by inflorescences exposed to ethylene declined between days 1 and 2. Thereafter, solution uptake by inflorescences exposed to ethylene was higher than by inflorescences not exposed to ethylene and is reflected in a significant interaction between ethylene treatment and time of measurement (Appendix 3.3). There was no significant effect of 1-MCP pre-treatment on solution uptake by inflorescences (Appendix 3.3). Vase lives of inflorescences were not significantly affected by 1-MCP or ethylene treatments (Table 3.3 and Appendix 3.4).

In the second experiment with A. pinnatum, perianth abscission was not significantly affected by 1-MCP or ethylene treatment, although there was a trend toward reduced perianth abscission from inflorescences that were pre-treated with 1-MCP and subsequently exposed to ethylene (Figure 3.2 and Appendix 3.5). Perianth abscission of unopened flowers was also reduced. The loss of relative fresh weight from inflorescences exposed to 0 or 100 µL ethylene/L was significantly reduced by 1-MCP pre-treatment (Figure 3.2 and Appendix 3.6). Inflorescences exposed only to exogenous ethylene lost most relative fresh weight.

Vase solution uptake by inflorescences increased to day 3, and thereafter uptake was low by inflorescences from all treatements (Figure 3.2). Pre-treatment of inflorescences with 1-MCP did not significantly affect solution uptake (Figure 3.2). Inflorescences exposed to ethylene used vase solution at lower rates between days 1 and 2 and at higher rates between days 2 and 3 than inflorescences not exposed to ethylene. These responses are evident as a significant interaction between ethylene treatment and time of measurement for vase solution uptake (Appendix 3.7). Pre-treatment of inflorescences with 1- MCP prevented the exogenous ethylene-induced reduction in vase life (Table 3.4 and Appendix 3.8). Vase lives of inflorescences not exposed to ethylene were marginally extended by pre-treatment with 1- MCP.

54 30

10 Perianth abscission Perianth

(% of initial number) initial of (% 0 110

100

90 (% initial FW) initial (%

Relative fresh weight fresh Relative 80

0.4

0.3

0.2 Solution uptake uptake Solution

(mL/ g initial FW/ day) FW/ initial g (mL/ 0.1 0123456 Time (days)

Figure 3.1. Perianth abscission, relative fresh weight and vase solution uptake for A. pinnatum inflorescences treated with 0 nL 1-MCP/L and 0 µL ethylene/L (●), 0 nL 1-MCP/L and 10 µL ethylene/L (■), 10 nL 1-MCP/L and 0 µL ethylene/L (▲) or 10 nL 1-MCP/L and 10 µL ethylene/L (▼). 1-MCP and ethylene treatments were each conducted for 12 hours at 20oC on days 0 and 1, respectively. Vertical bars represent standard errors of means (n = 10). Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD for relative fresh weight data = 1.7%. LSD for vase solution uptake data = 0.027 mL/g initial FW/day. (Experiment 1).

55 Table 3.3. Vase lives (mean ± s.e. in days) of native cut flowers which did not respond to 1-MCP or ethylene treatments. Flowers were pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the flowers from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Data within each row were not significantly different at P = 0.05.

Flower No ethylene (0 µL/L) Plus ethylene (10 µL/L)

0 nL 1-MCP/L 10 nL 1-MCP/L 0 nL 1-MCP/L 10 nL 1-MCP/L

A. pinnatum 3.7 ± 0.3 3.3 ± 0.4 2.9 ± 0.4 3.6 ± 0.4 B. heterophylla 5.9 ± 0.8 5.5 ± 0.2 5.0 ± 0.3 4.8 ± 0.3 C. adunca 6.7 ± 1.3 4.8 ± 0.8 5.5 ± 1.1 7.0 ± 1.1 E. scaber 2.9 ± 0.3 3.4 ± 0.3 3.0 ± 0.3 3.3 ± 0.3 L. scoparium ‘Winter Cheer’ 2.8 ± 0.2 3.4 ± 0.2 2.6 ± 0.4 3.0 ± 0.3 O. diosmifolius 7.1 ± 1.0 5.5 ± 0.7 5.3 ± 0.9 7.0 ± 0.8 P. lanceolata 20.4 ± 0.9 19.3 ± 0.6 18.5 ± 0.9 20.1 ± 0.8 T. calycina 3.6 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 2.6 ± 0.3 Z. cytisoides 3.6 ± 0.7 4.8 ± 0.4 2.6 ± 0.6 4.4 ± 1.3

56 30

10 (% of initial number) initial of (% Perianth abscission 0

100

90 (% initial FW) initial (% 80 Relative fresh weight fresh Relative 0.4

0.3

0.2

Solution uptake 0.1 (mL/g initial FW/day) initial (mL/g 01234567 Time (days)

Figure 3.2. Perianth abscission, relative fresh weight and vase solution uptake for A. pinnatum inflorescences treated with 0 nL 1-MCP/L and 0 µL ethylene/L (●), 0 nL 1-MCP/L and 10 µL ethylene/L (■), 10 nL 1-MCP/L and 0 µL ethylene/L (▲) or 10 nL 1-MCP/L and 10 µL ethylene/L (▼). 1-MCP and ethylene treatments were each conducted for 12 hours at 20oC on days 0 and 1, respectively. Vertical bars represent standard errors of means (n = 10). Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD for relative fresh weight data = 1.9%. LSD for vase solution uptake data = 0.023 mL/g initial FW/day. (Experiment 2).

57 Table 3.4. Vase lives (mean ± s.e. in days) of native cut flowers which responded to 1-MCP and ethylene treatments. Flowers were pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the flowers from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Data within each row followed by the same letter are not significantly different at P = 0.05.

Flower No ethylene (0 µL/L) Plus ethylene (10 µL/L) LSD

0 nL 1-MCP/L 10 nL 1-MCP/L 0 nL 1-MCP/L 10 nL 1-MCP/L

A. pinnatum 5.0 ± 0.3 ab 5.8 ± 0.4 b 4.3 ± 0.5 a 5.8 ± 0.1 b 0.9 (n= 10) C. gummiferum 11.2 ± 1.1 b 15.6 ± 0.9 c 4.9 ± 1.9 a 14.8 ± 1.1 b 3.8 (n = 10) C. uncinatum ‘Paddy’s Late’ 11.1 ± 0.7 b 11.5 ± 0.2 b 1.0 ± 0.0 a 10.7 ± 0.4 b 1.2 (n = 10) G. ‘Kay Williams’ 4.1 ± 0.1 b 4.4 ± 0.2 b 1.0 ± 0.0 a 4.3 ± 0.2 b 0.5 (n = 7) G. ‘Misty Pink’ 4.1 ± 0.2 b 4.1 ± 0.2 b 1.0 ± 0.0 a 3.9 ± 0.2 b 0.5 (n = 10) G. ‘Sandra Gordon’ 6.0 ± 0.2 b 6.4 ± 0.2 c 2.0 ± 0.0 a 5.8 ± 0.1 b 0.4 (n = 10) L. petersonii 2.5 ± 0.4 b 3.0 ± 0.6 b 1.4 ± 0.2 a 3.4 ± 0.2 b 1.1 (n = 10) T. speciosissima 5.1 ± 0.3 ab 5.5 ± 0.2 b 4.3 ± 0.4 a 5.9 ± 0.3 b 0.8 (n = 10) T. speciosissima ‘Shady Lady’ 2.3 ± 0.4 b 2.5 ± 0.3 b 1.3 ± 0.2 a 2.2 ± 0.4 ab 0.9 (n = 10) V. nitens 11.0 ± 0.9 b 9.8 ± 0.9 b 1.0 ± 0.0 a 11.3 ± 0.9 b 2.2 (n = 10)

58 3.3.1.2 B. heterophylla

Flower discolouration on flowering B. heterophylla stems was not significantly affected by 1-MCP or ethylene treatment (Figure 3.3 and Appendix 3.9). However, there was a trend toward more flower discolouration from stems treated with ethylene as opposed to stems not treated with ethylene (Figure 3.3). This response is reflected in a significant interaction between ethylene treatment and time of measurement (Appendix 3.9). Flower wilting was not significantly affected by 1-MCP pre-treatment (Figure 3.3 and Appendix 3.10). The onset of flower wilting was significantly delayed on stems that received ethylene treatment. However, wilting of flowers not treated with ethylene or pre-treated with 1-MCP was minor. Nonetheless, a significant interaction between 1-MCP pre-treatment and ethylene treatment was recorded (Appendix 3.10). The change in relative fresh weight of stems were not significantly affected by 1-MCP or ethylene treatments when applied either alone or in combination (Figure 3.3 and Appendix 3.11).

Vase solution uptake by stems fluctuated over time regardless of 1-MCP or ethylene treatment (Figure 3.3). However, solution uptake by stems pre-treated with 1-MCP was less variable compared to stems not pre-treated with 1-MCP. In addition, stems exposed to ethylene used vase solution at a higher rate between days 0 and 1 than stems not exposed to ethylene. Collectively, these variable responses resulted in a significant interaction between 1-MCP pre-treatment, ethylene treatment and time of measurement for vase solution upatke (Appendix 3.12). 1-MCP and ethylene treatments did not significantly affect vase lives of stems (Table 3.3 and Appendix 3.13).

59 4

1 Discolouration score

2 Wilt score Wilt 1

100

60 (% of initial (% of FW)

Relative fresh weight 40 2

Solution uptake 0 (mL/g initial FW/day) 024681012 Time (days)

Figure 3.3 Flower discolouration (scores: 1 = 0-25% to 4 = 76-100%), flower wilting (scores: 1 = none/slight to 3 = advanced), relative fresh weight and vase solution uptake of flowering B. heterophylla stems treated with 0 nL 1-MCP/L and 0 µL ethylene/L (●), 0 nL 1-MCP/L and 10 µL ethylene/L (■), 10 nL 1-MCP/L and 0 µL ethylene/L (▲) or 10 nL 1-MCP/L and 10 µL ethylene/L (▼). 1-MCP and ethylene treatments were each conducted for 12 hours at 20oC on days 0 and 1, respectively. Vertical bars represent the standard errors of means (n = 8). Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD for vase solution uptake data = 0.106 mL/g initial FW/day.

60 3.3.1.3 C. adunca

Leaf senescence and flower opening on C. adunca stems were not significantly affected by 1-MCP or ethylene treatments (Figure 3.4, Appendices 3.14 and 3.15). However, there was trend toward reduced flower opening from stems pre-treated with 1-MCP (Figure 3.4). Pedicel wilting from stems pre-treated with 1-MCP and exposed to ethylene was significantly delayed compared to stems pre-treated with 1- MCP and not exposed to ethylene (Figure 3.4 and Appendix 3.16). This difference is reflected by a significant interaction between 1-MCP pre-treatment and ethylene treatment (Appendix 3.17). 1-MCP pre-treatment apparently slightly reduced peduncle wilting on stems and consequently a significant interaction between 1-MCP pre-treatment and ethylene treatment was evident (Figure 3.4 and Appendix 3.18).

There was no significant effect of 1-MCP and ethylene treatments on the loss of relative fresh weight of C. adunca stems (Figure 3.4 and Appendix 3.19). Vase solution uptake by stems increased between days 2 and 4, but thereafter decreased over time (Figure 3.4). The increase in solution uptake by stems between days 2 and 4 varied according to each of the four treatments tested. Consequently, a significant interaction between 1-MCP pre-treatment, ethylene treatment and time of measurement was recorded (Appendix 3.20). Vase lives of stems were not significantly affected by either 1-MCP or ethylene treatment (Table 3.3 and Appendix 3.21).

61 80 60 40 20 (% of leaf area) of leaf (%

Leaf senescence senescence Leaf 0 5 4 3 Flower 2

opening score opening 1 3

2 score

Pedicel wilt wilt Pedicel 1

2 score 1 Peduncle wilt wilt Peduncle

100 80 60 Relative fresh weight weight fresh (% initial FW) 40 3 2 1 FW/2 days) (mL/g initial

Solution uptake uptake Solution 0 0 2 4 6 8 10 12 Time (days)

Figure 3.4. Leaf senescence, flower opening (scores: 1 = < 10% to 5 = > 80%), pedicel wilting (scores: 1 = none/slight to 3 = advanced), peduncle wilting (scores: 1 = none/slight to 3 = advanced), relative fresh weight and vase solution uptake for flowering C. adunca stems treated with 0 nL 1- MCP/L and 0 µL ethylene/L (●), 0 nL 1-MCP/L and 10 µL ethylene/L (■), 10 nL 1-MCP/L and 0 µL ethylene/L (▲) or 10 nL 1-MCP/L and 10 µL ethylene/L (▼). 1-MCP and ethylene treatments were conducted for 12 hours at 20oC on days 0 and 1, respectively. Vertical bars represent standard errors of the means (n = 10). LSD for pedicel wilting data is presented in Appendix 3.16. LSD for solution uptake data = 0.127 mL/g initial FW/2 days. 3.3.1.4 C. gummiferum

Pre-treatment of flowering C. gummiferum stems with 1-MCP prevented exogenous ethylene-induced 62 flower abscission (Figure 3.5). Exposure of stems to ethylene caused moderate levels of flower abscission, whereas no flower abscission at all was observed from stems not exposed to ethylene (Figure 3.5). This response is reflected in a significant interaction between 1-MCP pre-treatment, ethylene treatment and time of measurement (Appendix 3.22). Pre-treatment of stems with 1-MCP also significantly delayed the onset of sepal wilting associated with exposure of stems to 0 or 10 µL ethylene/L (Figure 3.5, Appendices 3.23 and 3.24). The loss of stem relative fresh weight associated with exogenous ethylene-induced flower abscission and sepal wilting was significantly reduced by 1-MCP pre-treatment (Figure 3.5 and Appendix 3.25). Likewise, the decline of relative fresh weight from stems not exposed to ethylene was significantly greater than from stems pre-treated with 1-MCP, presumably as sepal wilting was delayed. These responses are evident as significant interactions between 1-MCP pre-treatment and ethylene treatment and between 1-MCP pre-treatment and time of measurement for relative fresh weight (Appendix 3.25).

Vase solution uptake by stems fluctuated during the first 7 days of the experiment (Figure 3.5). Thereafter, solution uptake tended to decrease with time. After day 8, stems pre-treated with 1-MCP used vase solution at higher rates than stems not pre-treated with 1-MCP. This variation resulted in a significant interaction for 1-MCP pre-treatment and time of measurement (Figure 3.5 and Appendix 3.26). 1-MCP pre-treatment prevented the exogenous ethylene-induced reduction in vase life (Table 3.4). Vase lives of stems not exposed to ethylene were significantly extended by 1-MCP pre-treatment. Accordingly, a significant interaction between 1-MCP pre-treatment and ethylene treatment was evident (Appendix 3.27).

63 5 4 3 2

Abscission score 1

2 Wilt score Wilt 1

100 80 60

(% of initial FW) initial of (% 40 Relativefresh weight 1.0 0.8 0.6 0.4 0.2 Solution uptake

(mL/g initial FW/day) 0.0 0 2 4 6 8 10 12 14 16 18 Time (days)

Figure 3.5. Flower abscission (scores: 1 = < 10% to 5 = > 80%), sepal wilting (scores: 1 = none/slight to 3 = advanced), relative fresh weight and vase solution uptake for flowering C. gummiferum stems treated with 0 nL 1-MCP/L and 0 µL ethylene/L (●), 0 nL 1-MCP/L and 10 µL ethylene/L (■), 10 nL 1-MCP/L and 0 µL ethylene/L (▲) or 10 nL 1-MCP/L and 10 µL ethylene/L (▼). 1-MCP and ethylene treatments were each conducted for 12 hours at 20oC on days 0 and 1, respectively. Vertical bars represent the standard errors of means (n = 10). Where no vertical bar appears, standard errors were smaller than the size of the symbol. LSD for sepal wilting data is presented in Appendix 3.23. LSD for relative fresh weight data = 5.7%. LSD for vase solution uptake = 0.046 mL/g initial FW/day. 3.3.1.5 C. uncinatum ‘Paddy’s Late’

64 1-MCP pre-treatment protected C. uncinatum ‘Paddy’s Late’ sprigs against exogenous ethylene (Plate 3.1). Pre-treatment with 1-MCP significantly delayed the onset of flower abscission from sprigs that were exposed to exogenous ethylene (Figure 3.6). Exposure of sprigs only to ethylene caused rapid and extensive flower abscission. By the end of the experiment, flower abscission from sprigs not exposed to ethylene was significantly greater than that from sprigs pre-treated with 1-MCP (Figure 3.6). Thus, a significant interaction for 1-MCP pre-treatment, ethylene treatment and time of measurement was recorded (Appendix 3.28). As a result of 1-MCP pre-treatment having delayed exogenous ethylene- induced flower abscission, the associated decline of sprig relative fresh weight was significantly reduced (Figure 3.6). However, the decline of relative fresh weight from sprigs pre-treated with 1-MCP was not significantly different from that of sprigs not treated with 1-MCP or ethylene. This response was reflected in a significant interaction between 1-MCP pre-treatment, ethylene treatment and time of measurement (Appendix 3.29).

Vase solution uptake by sprigs fluctuated over time (Figure 3.6). Sprigs exposed only to ethylene had the lowest uptake rate during vase life. However, convergence of the uptake patterns by sprigs from all treatments towards the end of the experiment probably accounts for the significant interaction between 1- MCP pre-treatment, ethylene treatment and time of measurement (Figure 3.6 and Appendix 3.30). Vase life was based partly on flower abscission data (Table 3.2). Thus, the exogenous ethylene-induced loss in vase life was prevented by pre-treatment with 1-MCP (Table 3.4). 1-MCP pre-treatment did not, however, significantly extend the vase lives of sprigs which were not exposed to ethylene. This differential response is manifested in a significant interaction between 1-MCP pre-treatment and ethylene treatment (Appendix 3.31).

65 Plate 3.1. Flowering C. uncinatum ‘Paddy’s Late’ sprigs on day 2 after treatment on day 0 with 0 (control treatment) (LHS) or 10 nL 1-MCP/L (RHS) followed by exposure on day 1 to 10 µL ethylene/L. Note: extensive flower abscission is evident in the control sprig (LHS).

66 5

2 Abscission score 1 120 100 80 60

(% of initial FW) initial of (% 40

Relative freshweight 20 1.5

1.0

0.5 Solution uptake

(mL/g initial FW/day) initial (mL/g 0.0 02468101214 Time (days)

Figure 3.6. Flower abscission (scores: 1 = < 10% to 5 = > 80%), relative fresh weight and vase solution uptake for flowering sprigs of C. uncinatum ‘Paddy’s Late’ treated with 0 nL 1-MCP/L and 0 µL ethylene/L (z), 0 nL 1-MCP/L and 10 µL ethylene/L (), 10 nL 1-MCP/L and 0 µL ethylene/L (▲) or 10 nL 1-MCP/L and 10 µL ethylene/L (▼). 1-MCP and ethylene treatments were conducted for 12 hours at 20oC on days 0 and 1, respectively. Vertical bars represent the standard errors of means (n = 10). Where no vertical bars appear, standard errors are smaller than the size of the symbol. LSD for relative fresh weight = 3.5%. LSD for vase solution uptake = 0.052 mL/g initial FW/day.

67 3.3.1.6 E. scaber

Petal abscission from flowers on E. scaber stems was slightly reduced for stems pre-treated with 1-MCP compared to stems exposed only to ethylene (Figure 3.7). Exposure of stems to exogenous ethylene induced petal abscission (Appendix 3.32). Changes in the relative fresh weights of stems were not significantly altered by 1-MCP or ethylene treatments applied either alone or in combination (Figure 3.7 and Appendix 3.33). Vase solution uptake by stems increased over the short duration of this experiment (Figure 3.7). Solution uptake by stems pre-treated with 1-MCP was significantly higher between days 0 and 2, but not between days 2 and 4, relative to stems not pre-treated with 1-MCP. As a result, a significant interaction between 1-MCP pre-treatment and time of measurement for vase solution uptake was evident (Appendix 3.34). 1-MCP or ethylene treatments applied either alone or in combination did not significantly affect stem vase life relative to stems not treated with 1-MCP or ethylene (Table 3.3 and Appendix 3.35).

68 100

40 Petal abscission petals and buds) 20 (% initial number of of number initial (% 0

100

90 (% initial FW) initial (% 80 Relative fresh weight fresh Relative

1.2

1.0

0.8 Solution uptake Solution uptake

(mL/ g initial FW/ 2 days) 2 FW/ initial g (mL/ 0.6 024 Time (days)

Figure 3.7. Petal abscission, relative fresh weight and vase solution uptake for flowering E. scaber stems of treated with 0 nL 1-MCP/L and 0 µL ethylene/L (●), 0 nL 1-MCP/L and 10 µL ethylene/L (■), 10 nL 1-MCP/L and 0 µL ethylene/L (▲) or 10 nL 1-MCP/L and 10 µL ethylene/L (▼). 1- MCP and ethylene treatments were each conducted for 12 hours at 20oC on days 0 and 1, respectively. Vertical bars represent standard errors of means (n = 10). LSD for vase solution uptake data = 0.069 mL/g initial FW/2 days.

69 3.3.1.7 Grevillea hybrids 3.3.1.7.1 G. ‘Kay Williams’

Pre-treatment of G. ‘Kay Williams’ inflorescences with 1-MCP significantly delayed the onset of flower abscission and reduced the associated loss of relative fresh weight stimulated by exposure to exogenous ethylene (Figure 3.8 and Appendix 3.36). 1-MCP pre-treatment did not significantly reduce flower abscission or the loss of relative fresh weight from inflorescences not exposed to ethylene. Accordingly, a significant interaction between 1-MCP pre-treatment and ethylene treatment for flower abscission was evident (Appendix 3.37). Likewise, a significant interaction between 1-MCP pre-treatment, ethylene treatment and time of measurement for relative fresh weight was recorded (Appendix 3.38). Vase solution uptake by inflorescences pre-treated with 1-MCP was consistently higher during the experiment compared to inflorescences exposed only to ethylene (Figure 3.8). Despite apparent similarities in the uptake patterns, significant interactions between 1-MCP and time of measurement and between ethylene treatment and time of measurement were recorded (Appendix 3.39).

Each of flower discolouration, opening and wilting for inflorescences were not significantly affected by 1- MCP or ethylene treatment applied either alone or in combination (Appendices 3.40, 3.41, 3.42 and 3.43). As vase life was partly based on flower abscission (Table 3.2), the exogenous ethylene-induced loss in vase life was prevented by 1-MCP pre-treatment (Table 3.4). However, 1-MCP pre-treatment did not significantly extend vase life of inflorescences not exposed to ethylene. Thus, a significant interaction between 1-MCP pre-treatment, ethylene treatment and vase life was evident (Appendix 3.44).

70 5

2 Abscission score Abscission 1 120 100 80 60

(% initial FW) 40 Relative fresh weight 20 0.4

0.3

0.2 Solution uptake

(mL/ g initial FW/day) 0.1 012345 Time (days)

Figure 3.8. Flower abscission (scores: 1 = < 10% to 5 = > 80%), relative fresh weight and vase solution uptake for G. ‘Kay Williams’ inflorescences treated with 0 nL 1-MCP/L and 0 µL ethylene/L (z), 0 nL 1-MCP/L and 10 µL ethylene/L (), 10 nL 1-MCP/L and 0 µL ethylene/L (▲) or 10 nL 1-MCP/L and 10 µL ethylene/L (▼). 1-MCP and ethylene treatments were conducted for 12 hours at 20oC on days 0 and 1, respectively. Vertical bars represent the standard errors of means (n = 7). Where no vertical bars appear, standard errors are smaller than the size of the symbol. LSD for flower abscission is presented in Appendix 3.36. LSD for relative fresh weight data = 4.0%. LSD for vase solution uptake data = 0.087 mL/g initial FW/day.

71 3.3.1.7.2 G. ‘Misty Pink’

1-MCP pre-treatment protected G. ‘Misty Pink’ inflorescences against exogenous ethylene (Plate 3.2). Pre-treatment with 1-MCP significantly delayed the onset of flower abscission from inflorescences exposed to exogenous ethylene (Figure 3.9, Appendix 3.45). 1-MCP pre-treatment did not significantly reduce flower abscission from inflorescences not exposed to ethylene. Consequently, a significant interaction between 1-MCP pre-treatment and ethylene treatment was evident (Appendix 3.46). 1-MCP pre-treatment significantly reduced the exogenous ethylene-induced loss of inflorescence relative fresh weight (Figure 3.9). However, the decline in relative fresh weight of inflorescences not treated with ethylene was not reduced by 1-MCP pre-treatment. Thus, this differential response was reflected in a significant interaction between 1-MCP pre-treatment, ethylene treatment and time of measurement (Appendix 3.47). Inflorescences exposed only to ethylene used vase solution at lower rates than inflorescences pre-treated with 1-MCP (Figure 3.9). This difference in solution use pattern was reflected in significant interactions between 1-MCP pre-treatment and time of measurement and, between ethylene treatment and time of measurement (Appendix 3.48).

Flower discolouration and wilting were not significantly affected by 1-MCP or ethylene treatments (Appendices 3.49, 3.50 and 3.51). There were no consistent effects of treatments on flower opening (Appendices 3.49 and 3.52). Nonetheless, on day 1, flower opening from inflorescences pre-treated with 1-MCP was slightly but significantly reduced compared to inflorescences not pre-treated with 1-MCP (Appendices 3.49 and 3.52).

Vase lives of inflorescences were partly based on flower abscission (Table 3.2). Accordingly, 1-MCP pre-treatment prevented the exogenous ethylene-induced reduction in vase life (Table 3.4). 1-MCP pre- treatment did not significantly extend the vase lives of inflorescences not exposed to ethylene. As a result, there was a significant interaction between 1-MCP pre-treatment and ethylene treatment (Appendix 3.53).

72 Plate 3.2. G. ‘Misty Pink’ inflorescences on day 2 after treatment on day 0 with 0 (LHS) or 10 nL 1-MCP/L (RHS) followed by exposure on day 1 to 10 µL ethylene/L. Note: extensive flower abscission is evident in the control inflorescence (LHS).

73 5

2 Abscission score Abscission 1 120 100 80 60 40 (% of initial FW) (%initial of

Relative fresh weight weight fresh Relative 20 0.4

0.3

0.2 Solution uptake Solution

(mL/g initial FW/day) initial (mL/g 0.1 012345 Time (days)

Figure 3.9. Flower abscission (scores: 1 = < 10% to 5 = > 80%), relative fresh weight and vase solution uptake for G. ‘Misty Pink’ inflorescences treated with 0 nL 1-MCP/L and 0 µL ethylene/L (z), 0 nL 1-MCP/L and 10 µL ethylene/L (), 10 nL 1-MCP/L and 0 µL ethylene/L (▲) or 10 nL 1- MCP/L and 10 µL ethylene/L (▼). 1-MCP and ethylene treatments were conducted for 12 hours at 20oC on days 0 and 1, respectively. Vertical bars represent the standard errors of means (n = 10). Where no vertical bars appear, standard errors are smaller than the size of the symbol. LSD for flower abscission data is presented in Appendix 3.45. LSD for weight loss data = 5.0%. LSD for vase solution uptake data = 0.022 mL/g initial FW/day.

74 3.3.1.7.3 G. ‘Sandra Gordon’

The onset of flower abscission and associated loss of relative fresh weight from G. ‘Sandra Gordon’ inflorescences exposed to exogenous ethylene were significantly delayed by 1-MCP pre-treatment (Plate 3.3 and Figure 3.10). However, 1-MCP did not significantly reduce flower abscission or the decline of relative fresh weight from inflorescences not exposed to ethylene (Figure 3.10). Accordingly, there were significant interactions between 1-MCP pre-treatment, ethylene treatment and time of measurement for flower abscission (Appendix 3.54) and relative fresh weight (Appendix 3.55). Vase solution uptake by inflorescences pre-treated with 1-MCP was more highly variable over time compared to inflorescences not pre-treated with 1-MCP (Figure 3.10). This differential pattern of response resulted in a significant interaction between 1-MCP pre-treatment and time of measurement for vase solution uptake (Appendix 3.56).

There was slightly, but significantly more flower discolouration on day 7 for inflorescences not treated with 1-MCP or ethylene compared to inflorescences pre-treated with 1-MCP (Appendices 3.57, 3.58 and 3.59). However, effects of 1-MCP pre-treatment on flower discolouration were not consistent (Appendices 3.57 and 3.58). Similarly, there were no consistent treatment effects on flower opening (Appendices 3.57 and 3.60). Flower wilting was not significantly affected by 1-MCP pre-treatment compared to inflorescences not treated with 1-MCP or ethylene (Appendices 3.57 and 3.61). Pre- treatment of G. ‘Sandra Gordon’ inflorescences with 1-MCP treatment prevented the exogenous ethylene- induced loss in vase life (Table 3.4). 1-MCP pre-treatment did not significantly extend vase lives of inflorescences not exposed to ethylene. As a result, there was a significant interaction between 1-MCP pre-treatment and ethylene treatment (Appendix 3.62).

75 Plate 3.3. G. ‘Sandra Gordon’ inflorescences on day 5 after treatment on day 0 with 0 (control treatment) (LHS) or 10 nL 1-MCP/L (RHS) followed by exposure on day 1 to 10 µL ethylene/L. Note: extensive flower abscission is evident in the control inflorescence (LHS).

76 5

2 Abscission score 1 120 100 80 60

(% initial FW) 40 Relative fresh weight 20 0.4

0.3

0.2

Solution uptake 0.1 (mL/ g initial FW/ day)

01234567 Time (days)

Figure 3.10. Flower abscission (scores: 1 = < 10% to 5 = > 80%), relative fresh weight and vase solution uptake for G. ‘Sandra Gordon’ inflorescences treated with 0 nL 1-MCP/L and 0 µL ethylene/L (z), 0 nL 1-MCP/L and 10 µL ethylene/L (), 10 nL 1-MCP/L and 0 µL ethylene/L (▲) or 10 nL 1-MCP/L and 10 µL ethylene/L (▼). 1-MCP and ethylene treatments were conducted for 12 hours at 20oC on days 0 and 1, respectively. Vertical bars represent the standard errors of means (n = 10). Where no vertical bars appear, standard errors are smaller than the size of the symbol. LSD for weight loss = 3.6%. LSD for vase solution uptake data = 0.051 mL/g initial FW/day.

77 3.3.1.8 Leptospermum spp. 3.3.1.8.1 L. petersonii

Pre-treatment of flowering L. petersonii stems with 1-MCP significantly reduced exogenous ethylene- induced petal abscission (Plate 3.4, Figure 3.11 and Appendix 3.63). However, 1-MCP pre-treatment did not significantly reduce petal abscission from stems not exposed to ethylene. 1-MCP pre-treatment significantly reduced the decline of stem relative fresh weight associated with petal abscission from stems exposed to exogenous ethylene (Figure 3.11 and Appendix 3.64). Vase solution uptake by stems increased over time to day 3 and then declined (Figure 3.11). Stems exposed only to ethylene used vase solution at a lower rate throughout the experiment compared to stems pre-treated with 1-MCP. As a result, there were significant interactions between 1-MCP pre-treatment and time of measurement and between ethylene treatment and time of measurement (Appendix 3.65). Because vase life was based on petal abscission data (Table 3.2), 1-MCP pre-treatment also prevented the exogenous ethylene-induced loss in vase life (Table 3.4). 1-MCP pre-treatment did not significantly extend vase lives of stems not exposed to ethylene (Table 3.4 and Appendix 3.63).

3.3.1.8.2 L. scoparium ‘Winter Cheer’

Flower abscission and senescence (flower wilting and discolouration) for L. scoparium ‘Winter Cheer’ stems were not consistently affected by 1-MCP or ethylene treatments, although there was a trend toward reduced flower abscission and senescence from stems pre-treated with 1-MCP (Figure 3.12 and Appendix 3.66). Exposure of stems to ethylene increased the number of abscised and senescent flowers and, thereby, significantly reduced stem relative fresh weight (Figure 3.12 and Appendix 3.67). However, 1- MCP pre-treatment did not significantly reduce the loss of stem relative fresh weight. Vase solution uptake by stems exposed to ethylene was lower between days 2 and 3 than stems not exposed to ethylene (Figure 3.12). This variable pattern resulted in a significant interaction for ethylene treatment and time of measurement for vase solution uptake (Appendix 3.68). Vase life was not significantly affected by 1- MCP or ethylene treatments (Table 3.3 and Appendix 3.66). Nonetheless, there was a trend toward reduced vase lives of stems exposed to exogenous ethylene.

78 Plate 3.4. Flowering L. petersonii stems on day 2 after treatment on day 0 with 0 (LHS) or 10 nL 1- MCP/L (RHS) followed by exposure on day 1 to 10 µL ethylene/L. Note: petal abscission is evident in the control stem (LHS).

79 60

20 petals and buds) Petal abscission (% initial number of of number initial (% 0 120

100

80 (% initial FW) initial (%

Relative fresh weight 60 2

1 Solution uptake

(mL/ g initial FW/ day) FW/ initial g (mL/ 0 0123456 Time (days)

Figure 3.11. Petal abscission, relative fresh weight and vase solution uptake for flowering L. petersonii stems treated with 0 nL 1-MCP/L and 0 µL ethylene/L (z), 0 nL 1-MCP/L and 10 µL ethylene/L (), 10 nL 1-MCP/L and 0 µL ethylene/L (▲) or 10 nL 1-MCP/L and 10 µL ethylene/L (▼). 1-MCP and ethylene treatments were conducted for 12 hours at 20oC on days 0 and 1, respectively. Vertical bars represent the standard errors of means (n = 10). Where no vertical bars appear, standard errors are smaller than the size of the symbol. LSD for petal abscission data is presented in Table 3.4. LSD for relative fresh weight data = 5.1%. LSD for vase solution uptake data = 0.111 mL/g initial FW/day.

80 60

40 (%) 20 Abscised and senescent flowers 0 100 90 80 70

(% initial FW) initial (% 60

Relative fresh weight weight fresh Relative 50 0.8

0.6

0.4

0.2 Solution uptake

(mL/g initial FW/day) initial (mL/g 0.0 01234 Time (days)

Figure 3.12. Abscised and senescent flowers (% of initial number of open flowers and buds), relative fresh weight and vase solution uptake for flowering stems of L. scoparium ‘Winter Cheer’ pre-treated with 0 nL 1-MCP/L and 0 µL ethylene/L (z), 0 nL 1-MCP/L and 10 µL ethylene/L (), 10 nL 1-MCP/L and 0 µL ethylene/L (▲) or 10 nL 1-MCP/L and 10 µL ethylene/L (▼). 1-MCP and ethylene treatments were conducted for 12 hours at 20oC on days 0 and 1, respectively. Vertical bars represent the standard errors of means (n = 7). Where no vertical bars appear, standard errors are smaller than the size of the symbol. LSD for relative fresh weight data = 3.2%. LSD for solution uptake data = 0.051 mL/g initial FW/day.

81 3.3.1.9 O. diosmifolius

Pre-treatment of flowering O. diosmifolius stems with 1-MCP or ethylene did not significantly affect pedicel wilting and flower opening (Figure 3.13, Appendices 3.69, 3.70 and 3.71). There was more leaf senescence on stems treated with 1-MCP or ethylene alone until day 10 compared to stems not treated with 1-MCP or ethylene or those treated with 1-MCP and ethylene in combination (Figure 3.13 and Appendix 3.72). Leaf abscission, as indicated by the length of stems with no leaves, was greatest from stems pre-treated with 1-MCP or from stems not treated with 1-MCP or ethylene (Figure 3.13 and Appendix 3.73). These responses probably account for the significant interaction between 1-MCP pre- treatment and ethylene treatment evident for leaf senescence (Appendix 3.72) and leaf abscission (Appendix 3.73).

The loss of relative fresh weight was reduced from stems treated with 1-MCP or ethylene alone compared to stems not treated with 1-MCP and ethylene (Figure 3.14). However, the variable pattern of stem relative fresh weight loss on the last day of the experiment probably accounts for a significant interaction for 1-MCP pre-treatment, ethylene treatment and time of measurement (Appendix 3.74). Pre-treatment of stems with 1-MCP did not prevent peduncle wilting (Figure 3.14). Peduncle wilting increased following exposure to exogenous ethylene compared to stems not exposed to exogenous ethylene (Appendices 3.69 and 3.75). Vase solution uptake by stems declined after day 4 of the experiment (Figure 3.14). Solution uptake by stems exposed to ethylene was lower between days 0 and 2 relative to stems not exposed to ethylene. Stems pre-treated with 1-MCP used vase solution at relatively constant rates after day 4 of the experiment compared to stems not pre-treated with 1-MCP. These initially variable data led to significant interactions between 1-MCP pre-treatment and time of measurement and between ethylene treatment and time of measurement (Figure 3.14 and Appendix 3.76). Vase lives of stems were not significantly affected by 1-MCP or ethylene treatments applied either alone or in combination (Table 3.3 and Appendix 3.77).

82 3

2 Pedicel wilt score 1

5 4 3 2 Opening score Opening 1 60 50 40 30 20 10 (% of leaf area) Leaf senescence 0 12 10 8 6 4 2 Length of stem 0 with no leaves (cm) 0 2 4 6 8 10 12 14 Time (days)

Figure 3.13. Pedicel wilting (scores: 1 = none/slight to 3 = advanced), flower opening (scores: 1 = < 10% to 5 = > 80%), leaf senescence and the length of stems without leaves for flowering O. diosmifolius stems treated with 0 nL 1-MCP/L and 0 µL ethylene/L (z), 0 nL 1-MCP/L and 10 µL ethylene/L (), 10 nL 1-MCP/L and 0 µL ethylene/L (▲) or 10 nL 1-MCP/L and 10 µL ethylene/L (▼). 1-MCP and ethylene treatments were each conducted for 12 hours at 20oC on days 0 and 1, respectively. Vertical bars represent the standard errors of means (n = 10). Where no vertical bars appear, standard errors are smaller than the size of the symbol. LSD for leaf abscission data = 1.8 cm.

83 110 100 90 80 (% initial FW) initial (%

Relative fresh weight weight fresh Relative 70

2 wilt score wilt Peduncle 1

1.5

1.0

0.5 Solution uptake

(mL/g initial FW/2 days) FW/2 initial (mL/g 0.0 02468101214 Time (days)

Figure 3.14. Relative fresh weight, peduncle wilting (scores: 1= none/slight to 3 = advanced) and vase solution uptake for flowering O. diosmifolius stems pre-treated with 0 nL 1-MCP/L and 0 µL ethylene/L (●), 0 nL 1-MCP/L and 10 µL ethylene/L (■), 10 nL 1-MCP/L and 0 µL ethylene/L (▲) or 10 nL 1-MCP/L and 10 µL ethylene/L (▼). 1-MCP and ethylene treatment were each conducted for 12 hours at 20oC on days 0 and 1, respectively. Vertical bars represent standard errors of means (n = 10). LSD for peduncle wilting data is presented in Appendix 3.69. LSD for relative fresh weight data = 3.2%. LSD for solution uptake data = 0.070 mL/g initial FW/2 days.

84 3.3.1.10 P. lanceolata

1-MCP pre-treatment did not significantly affect the loss of relative fresh weight of flowering P. lanceolata stems during vase life evaluation (Figure 3.15). The loss of stem relative fresh weight was slightly, but significantly greater from stems treated with ethylene compared to stems not treated with ethylene (Figure 3.15 and Appendix 3.78). The separation of the pattern of stem relative fresh weight loss from all treatments towards the end of the experiment probably accounts for the significant interaction between 1-MCP pre-treatment, ethylene treatment and time of measurement (Appendix 3.78). Vase solution uptake by stems fluctuated during the experiment, but was highest between days 2 and 4 for all treatments, particularly for stems pre-treated with 1-MCP (Figure 3.15). A small initial variation in response (Figure 3.15) was reflected in a significant interaction between 1-MCP pre-treatment and time of measurement for vase solution uptake (Appendix 3.79). Treatment of stems with 1-MCP or ethylene either alone or in combination did not significantly affect vase life (Table 3.3 and Appendix 3.80).

85 110

100

(% of initial(% of FW) 80 Relative fresh weight 3

1 Solution uptake

(mL/g initial FW/2 days) 0 0 4 8 121620 Time (days)

Figure 3.15. Relative fresh weight and vase solution uptake for flowering P. lanceolata stems treated with 0 nL 1-MCP/L and 0 µL ethylene/L (●), 0 nL 1-MCP/L and 10 µL ethylene/L (■), 10 nL 1-MCP/L and 0 µL ethylene/L (▲) or 10 nL 1-MCP/L and 10 µL ethylene/L (▼). 1-MCP and ethylene treatments were each conducted for 12 hours at 20oC on days 0 and 1, respectively. Vertical bars represent standard errors of means (n = 10). Where no vertical bars appear, standard errors are smaller than the size of the symbol. LSD for relative fresh weight data = 2.1%. LSD for vase solution uptake data = 0.156 mL/g initial FW/2 days.

86 3.3.1.11 T. speciosissima

Perianth abscission from clonally propagated T. speciosissima inflorescences exposed to exogenous ethylene was reduced by 1-MCP pre-treatment (Figure 3.16 and Appendix 3.81). However, when no ethylene was applied, 1-MCP had no significant effect on perianth abscission (Appendix 3.81). 1-MCP pre-treatment significantly reduced the loss of relative fresh weight from inflorescences treated with ethylene (Figure 3.16). 1-MCP pre-treatment did not significantly reduce the loss of relative fresh weight from inflorescences not treated with ethylene. Accordingly, a significant interaction between 1-MCP pre- treatment, ethylene treatment and time of measurement was evident for relative fresh weight (Appendix 3.82). Flower opening and vase solution uptake by inflorescences were not significantly affected by 1- MCP or ethylene treatments (Appendices 3.83, 3.84 and 3.85). Pre-treatment of inflorescences with 1- MCP did not prevent the loss in vase life (time to 20% perianth abscission) for inflorescences exposed to ethylene (Figure 3.16, Table 3.4 and Appendix 3.81).

1-MCP pre-treatment protected seed-grown T. speciosissima inflorescences against exogenous ethylene by significantly delaying the onset of perianth abscission (Plate 3.5 and Figure 3.17). However, 1-MCP pre- treatment did not significantly reduce perianth abscission from inflorescences not exposed to ethylene. As a result, a significant interaction between 1-MCP pre-treatment and ethylene treatment was recorded (Appendix 3.86). Pre-treatment of inflorescences with 1-MCP significantly reduced the loss of relative fresh weight from inflorescences exposed to 0 or 10 µL ethylene/L (Figure 3.17). These responses reflected significant interactions between 1-MCP pre-treatment and time of measurement and between ethylene treatment and time of measurement for relative fresh weight (Appendix 3.87).

1-MCP and ethylene treatments did not consistently affect flower opening and vase solution uptake by inflorescences (Appendices 3.88, 3.89 and 3.90). The exogenous ethylene-induced loss in vase life associated with perianth abscission was prevented by 1-MCP pre-treatment (Table 3.4). 1-MCP pre- treatment did not significantly extend vase lives of inflorescences not exposed to ethylene. Thus, there was a significant interaction between 1-MCP pre-treatment and ethylene treatment (Appendix 3.86).

87 100

(% of initial) of (% 20 Perianth abscission Perianth 0

100

(% initial FW) 70

Relativeweight fresh 60 0123456 Time (days)

Figure 3.16. Perianth abscission and relative fresh weight for clonally propagated T. speciosissima ‘Shady Lady’ inflorescences treated with 0 nL 1-MCP/L and 0 µL ethylene/L (z), 0 nL 1-MCP/L and 10 µL ethylene/L (), 10 nL 1-MCP/L and 0 µL ethylene/L (▲) or 10 nL 1-MCP/L and 10 µL ethylene/L (▼). 1-MCP and ethylene were conducted for 12 hours at 20oC on days 0 and 1, respectively. Vertical bars represent standard errors of means (n = 10). Where no vertical bars appear, standard errors are smaller than the size of symbols. LSD for relative fresh weight data = 2.1%.

88 Plate 3.5. Seed-grown T. speciosissima inflorescences on day 2 after treatment on day 0 with 0 (control treatment) (LHS) or 10 nL 1-MCP/L (RHS) followed by exposure on day 1 to 10 µL ethylene/L. Note: perianth abscission is evident in the control inflorescence (LHS) as the white/yellow separation zones at the base of individual styles.

89 40

(% of initial) of (% 10

Perianth abscission abscission Perianth 0 110

100

90 (% initial FW) initial (% 80 Relativeweight fresh

01234567 Time (days)

Figure 3.17. Perianth abscission and relative fresh weight for seed-grown T. speciosissima inflorescences treated with 0 nL 1-MCP/L and 0 µL ethylene/L (z), 0 nL 1-MCP/L and 10 µL ethylene/L (), 10 nL 1-MCP/L and 0 µL ethylene/L (▲) or 10 nL 1-MCP/L and 10 µL ethylene/L (▼). 1-MCP and ethylene were conducted for 12 hours at 20oC on days 0 and 1, respectively. Vertical bars represent standard errors of means (n = 10). Where no vertical bars appear, standard errors are smaller than the size of symbols. LSD for perianth abscission data is presented in Table 3.4. LSD for relative fresh weight data = 1.1%.

90 3.3.1.12 T. calycina

Flower abscission from flowering T. calycina stems was slightly greater from stems pre-treated with 1- MCP than from stems treated only with ethylene (Figure 3.18). These unexpected responses were reflected in a significant interaction between 1-MCP pre-treatment and ethylene treatment (Appendix 3.91). Flower closing on stems treated with 1-MCP and ethylene in combination or ethylene alone was higher on day 4 than on stems pre-treated with 1-MCP alone (Figure 3.18). Accordingly, a significant interaction between 1-MCP pre-treatment and ethylene treatment for flower closing was evident (Appendix 3.92). The loss of stem relative fresh weight was greatest from stems not treated with 1-MCP or ethylene (Figure 3.18). 1-MCP pre-treatment did not significantly reduce the decline of relative fresh weight of stems treated with ethylene (Figure 3.18 and Appendix 3.93). This variable response was reflected in a significant interaction between 1-MCP pre-treatment and ethylene treatment (Appendix 3.93).

Vase solution uptake by stems exposed only to ethylene or pre-treated with 1-MCP were consistently higher than that by stems not treated with 1-MCP or ethylene (Figure 3.18). Accordingly, there was a significant interaction between 1-MCP pre-treatment, ethylene treatment and time of measurement for vase solution uptake (Appendix 3.94). Vase lives of stems were not significantly affected by 1-MCP or ethylene treatment applied either alone or in combination (Table 3.3 and Appendix 3.95).

91 6 5 4 3 2 (% initial

open flowers) open 1 Flower abscission Flower 0 40 30 20 (% initial

open flowers) open 10 Closed flowers 0 100

80 (% initial FW) 70 Relative fresh weight fresh Relative 2.0

1.5

1.0 FW/2 days) (mL/g initial initial (mL/g Solution uptake 0.5 0246 Time (days)

Figure 3.18. Flower abscission, flower closure, relative fresh weight and vase solution uptake for flowering T. calycina stems treated with 0 nL 1-MCP/L and 0 µL ethylene/L (●), 0 nL 1-MCP/L and 10 µL ethylene/L (■), 10 nL 1-MCP/L and 0 µL ethylene/L (▲) or 10 nL 1-MCP/L and 10 µL ethylene/L (▼). 1-MCP and ethylene treatments were each conducted for 12 hours at 20oC on days 0 and 1, respectively. Vertical bars represent standard errors of means (n = 10). LSD for relative fresh weight data = 3.3%. LSD for vase solution uptake data = 0.201 mL/g initial FW/2 days.

92 3.3.1.13 V. nitens

Pre-treatment of V. nitens stems with 1-MCP prevented exogenous ethylene-induced flower abscission (Plate 3.6 and Figure 3.19). There was no flower abscission at all from stems pre-treated with 1-MCP or from stems not exposed to ethylene. Consequently, there was a significant interaction between 1-MCP pre-treatment, ethylene treatment and time of measurement (Appendix 3.96). 1-MCP pre-treatment significantly reduced the decline in relative fresh weight of stems associated with exogenous ethylene- induced flower abscission (Figure 3.19). However, 1-MCP pre-treatment of stems did not significantly reduce the decline of relative fresh weight of stems not exposed to ethylene. As a result of the pervading effect of flower abscission, there was a significant interaction between 1-MCP pre-treatment and ethylene treatment for relative fresh weight (Appendix 3.97).

Vase solution uptake by stems pre-treated with 1-MCP was consistently higher during the experiment than that by stems not pre-treated with 1-MCP (Figure 3.19). Accordingly, a significant interaction between 1- MCP pre-treatment and time of measurement was evident (Appendix 3.98). The exogenous ethylene- induced reduction in vase life was prevented by pre-treating inflorescences with 1-MCP (Table 3.4). Pre- treatment of stems with 1-MCP did not extend the vase lives of flowering stems not exposed to ethylene. Thus, there was a significant interaction between 1-MCP pre-treatment and ethylene treatment for vase life (Appendix 3.99).

93 Plate 3.6. Flowering V. nitens stems on day 2 after treatment on day 0 with 0 (control) (LHS) or 10 nL 1-MCP/L (RHS) followed by exposure on day 1 to 10 µL ethylene/L. Note: flower abscission is evident in the control stem (LHS).

94 5

2 Abscission score Abscission 1 120

100

60 (% of initial FW) initial of (%

Relative fresh weight weight fresh Relative 40 0.4

0.3

0.2

0.1 Solution uptake uptake Solution

(mL/g initial FW/day) 0.0

02468101214 Time (days)

Figure 3.19. Flower abscission (scores: 1 = < 10% to 5 = > 80%), relative fresh weight and vase solution uptake for flowering V. nitens stems treated with 0 nL 1-MCP/L and 0 µL ethylene/L (●), 0 nL 1-MCP/L and 10 µL ethylene/L (■), 10 nL 1-MCP/L and 0 µL ethylene/L (▲) or 10 nL 1- MCP/L and 10 µL ethylene/L (▼). 1-MCP and ethylene treatments were each conducted for 12 hours at 20oC on days 0 and 1, respectively. Vertical bars represent standard errors of means (n = 10). Where no vertical bars appear, standard errors are smaller than the size of the symbol. LSD for relative fresh weight data = 3.6%. LSD for vase solution uptake data = 0.027 mL/g initial FW/day.

95 3.3.1.14 Z. cytisoides

Flower abscission from flowering Z. cytisoides stems was not significantly affected by 1-MCP or ethylene treatments, when applied alone or in combination (Figure 3.20 and Appendix 3.100). However, there was a trend toward reduced flower abscission from stems pre-treated with 1-MCP. Stems pre-treated with 1- MCP and not exposed to ethylene lost significantly less relative fresh weight than stems not pre-treated with 1-MCP (Figure 3.20). However, the loss of relative fresh weight from stems exposed to ethylene was not significantly different from stems not exposed to ethylene. This variable response resulted in a significant interaction between 1-MCP pre-treatment and ethylene treatment (Appendix 3.101).

Vase solution uptake by stems pre-treated with 1-MCP and exposed to ethylene was higher, but erratic between days 2 and 9 of the experiment relative to stems not pre-treated with 1-MCP or stems not exposed to ethylene (Figure 3.20). This response probably accounts for the significant interaction between 1-MCP pre-treatment, ethylene treatment and time of measurement (Appendix 3.102). Flower abscission from stems was the sole factor in limiting vase life in this experiment (Table 3.2). The vase lives of stems were not significantly affected by 1-MCP or ethylene treatments (Table 3.3 and Appendix 3.100).

96 5 4 3 2

Abscission score 1 110 100 90 80 (% initial FW) initial (% 70 Relativefresh weight

1 Solution uptake Solution

(mL/g initialFW/day) 0 0246810121416 Time (days)

Figure 3.20. Flower abscission (scores: 1 = < 10% to 5 = > 80%), relative fresh weight and vase solution uptake for flowering Z. cytisoides stems treated with 0 nL 1-MCP/L and 0 µL ethylene/L (●), 0 nL 1-MCP/L and 10 µL ethylene/L (■), 10 nL 1-MCP/L and 0 µL ethylene/L (▲) or 10 nL 1- MCP/L and 10 µL ethylene/L (▼). 1-MCP and ethylene treatments were each conducted for 12 hours at 20oC on days 0 and 1, respectively. Vertical bars represent standard errors of means (n = 5). Where no vertical bars appear, the standard errors were smaller than the size of the symbol. LSD for relative fresh weight data = 4.9%. LSD for solution uptake data = 0.075 mL/g initial FW/day.

97 3.3.2 Treatment of B. heterophylla with 1-MCP, STS and ethylene

Pre-treatment of B. heterophylla stems with 1-MCP was more effective than STS pre-treatment in delaying the onset of flower wilting stimulated by exposure to exogenous ethylene (Figure 3.21). Exposure of stems to 10 µL ethylene/L for the longer period of 72 hours, compared to the 12 hour exposure duration used previously (section 3.2.4.1), stimulated flower wilting (Figure 3.21) and induced flower and leaf abscission (> 10% of total number of flowers and leaves) (Plate 3.7). Pre-treatment with 1-MCP did not significantly delay the onset of flower wilting from stems not exposed to ethylene. Conversely, pre-treatment with STS stimulated flower wilting from stems not exposed to ethylene (Figure 3.21). As a result, there was a significant interaction between the anti-ethylene treatment (1-MCP or STS), ethylene treatment and time of measurement for flower wilting (Appendix 3.103). Accumulation of Ag+ from STS solution by stems was 0.25 ± 0.01 µmol Ag+/g initial stem FW (n = 20).

Flower wilting and abscission induced by exposure to ethylene were also reflected by accelerated loss of stem relative fresh weight (Figure 3.21). 1-MCP and STS pre-treatments significantly reduced the loss of relative fresh weight from stems exposed to ethylene (Figure 3.21). Loss of stem relative fresh weight was not significantly reduced by either 1-MCP or STS treatment for stems not exposed to ethylene. Thus, a significant interaction between the anti-ethylene pre-treatment, ethylene treatment and time of measurement was recorded (Appendix 3.104).

The exogenous ethylene-induced loss in vase life was prevented by 1-MCP pre-treatment (Table 3.5). STS pre-treatment did not significantly reduce the loss in vase life, although there was a trend toward extended vase life for STS-treated stems (Table 3.5). 1-MCP pre-treatment did not significantly extend the vase lives of stems not exposed to ethylene. In contrast, STS pre-treatment significantly reduced vase lives of stems not exposed to ethylene. Due to these responses, a significant interaction between the anti- ethylene treatment and ethylene treatment was recorded (Appendix 3.105).

1 - Ethylene + Ethylene 3

2 Wilt score

- Ethylene + Ethylene

100

60 (% of initial FW) initial of (% Relative freshweight 40 04812160481216 Time (days)

Figure 3.21. Flower wilting (scores: 1 = none/slight to 3 = advanced) and relative fresh weight for flowering B. heterophylla stems pre-treated on day 0 with 10 nL 1-MCP/L (■), STS (0.5 mM Ag+) (▲) or not pre-treated with 1-MCP or STS (control treatment) (●) for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 72 hours at 20oC. Vertical bars represent the standard errors of means (n = 10). LSD for relative fresh weight data = 4.2%.

2 Plate 3.7. Flowering B. heterophylla stems on day 7 after treatment on day 1 with 0 nL 1-MCP/L and 0 mM Ag+ (control) (LHS), 10 nL 1-MCP/L (centre) or STS (0.5 mM Ag+) (RHS) followed by exposure on day 1 to 10 µL ethylene/L for 72 hours. Note: flower and leaf abscission are evident in the control stem (LHS).

3 Table 3.5. Vase life (mean ± s.e.) of flowering B. heterophylla stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L or with STS (0.5 mM Ag+) for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 72 hours at 20oC. Data followed by the same letters are not significantly different at P = 0.05. LSD = 1.6 days.

Treatment Vase life (days)

No ethylene (0 µL/L) Control (0 nL 1-MCP/L or 0 mM Ag+) 10.8 ± 0.9 b 10 nL 1-MCP/L 10.0 ± 0.7 b 0.5 mM Ag+ 9.1 ± 0.2 a Plus ethylene (10 µL/L) Control (0 nL 1-MCP/L or 0 mM Ag+) 8.2 ± 0.4 a 10 nL 1-MCP/L 10.4 ± 0.5 b 0.5 mM Ag+ 9.7 ± 0.5 a

Pre-treatment of stems with 1-MCP did not significantly affect flower discolouration from stems exposed to 0 or 10 µL ethylene/L (Figure 3.22 and Appendix 3.106). However, pre-treatment of stems with STS significantly delayed the onset of flower discolouration from stems not exposed to ethylene (Figure 3.22, Appendices 3.106 and 3.107). Vase solution uptake by stems pre-treated with STS was lower than stems pre-treated with 1-MCP when they were not exposed to ethylene (Figure 3.22). In contrast, when stems were exposed to ethylene, vase solution uptake by stems pre-treated with STS was higher compared to stems pre-treated with 1-MCP until day 11 of the experiment (Figure 3.22). These different responses resulted in a significant interaction between the anti-ethylene treatments, ethylene treatments and time of measurement for vase solution uptake (Appendix 3.108).

4 - Ethylene + Ethylene 4

Discolouration score 1

1.0 - Ethylene + Ethylene

0.8

0.6

0.4

Solution uptake Solution 0.2

(mL/g initial FW/day) initial (mL/g 0.0

0 4 8 12 16 0481216 Time (days)

Figure 3.22. Flower discolouration (scores: 1 = 0-25% to 4 = 76-100%) and vase solution uptake for flowering B. heterophylla stems pre-treated on day 0 with 10 nL 1-MCP/L (■), STS (0.5 mM Ag+) (▲) or not pre-treated with 1-MCP or STS (control treatment) (●) for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 72 hours at 20oC. Vertical bars represent the standard errors of means (n = 10). Where no vertical bars appear, standard errors are smaller than the size of the symbol. LSD for flower discolouration data is presented in Appendix 3.106. LSD for vase solution uptake data = 0.035 mL/g initial FW/day.

5 Increased rates of ethylene production by detached B. heterophylla flowers were associated with the onset of flower wilting and advanced stages of flower discolouration (Figure 3.23, Appendices 3.109, 3.110, 3.111 and 3.112). Ethylene production by intact flowering stems fluctuated during vase life (Figure 3.24). Nonetheless, increased rates of ethylene production were associated with flower wilting (Appendices 3.109 and 3.113). However, the association between changes in flower discolouration and the rate of ethylene production by inatct flowering stems was less pronounced (Figure 3.24, Appendices 3.110 and 3.114) than for detached flowers. Ethylene production by non-flowering stems of B. heterophylla was generally low (Figure 3.24). Increased rates of ethylene production by these stems on day 12 were associated with leaf wilting and disease development.

6 8 4 7

6 L/kg FW/hr)

3 µ 5

2 3

2 Wilt/discolouration score Wilt/discolouration

1 1 Ethylene production ( 0 02468101214 Time (days)

Figure 3.23. Flower wilting (scores: 1 = none/slight to 3 = advanced) (■), discolouration (scores: 1 = 0-25% to 4 = 76-100%) (▲) and ethylene production (bars) by detached B. heterophylla flowers held at 20oC. Vertical lines represent standard errors of means (n = 5).

7 8 4 7

6 L/kg FW/hr)

3 µ 5

2 3

2 Wilt/discolouration score

1 1 Ethylene production ( 0 024681012 Time (days)

Figure 3.24. Flower wilting (scores: 1 = none/slight to 3 = advanced) (■), discolouration (scores: 1 = 0-25% to 4 = 76-100%) (▲) and ethylene production by flowering (open bars) or non-flowering (shaded bars) B. heterophylla stems held at 20oC. Vertical lines represent standard errors of means (n = 5).

8 3.4 DISCUSSION

Pre-treatment of nine ethylene-sensitive native Australian cut flowers (viz. A. pinnatum, C. gummiferum, C. uncinatum, G. ‘Kay Williams, G. ‘Misty Pink’, G. ‘Sandra Gordon’, L. petersonii, T. speciosissima and V. nitens) with 10 nL 1-MCP/L for 12 hours at 20oC provided protection against subsequent treatment with ethylene (10 µL/L for 12 hours at 20oC). Exposure of these flowers to this ethylene treatment protocol induced floral organ abscission and, in turn, reduced vase life (Table 3.4). Wilting of sepals on cut C. gummiferum stems was also associated with exposure to exogenous ethylene (Figure 3.5). The efficacy of the 1-MCP treatment protocol used in this study in counteracting ethylene supports the findings of Serek et al. (1995a). They found that treatment of a range of cut flowers with 10-20 nL 1- MCP/L for 6 hours prevented ethylene treatment effects.

In general, pre-treatment with 1-MCP did not significantly extend the base level vase lives of flowers not exposed to exogenous ethylene (Table 3.4). By way of an exception, 1-MCP pre-treatment extended the base vase lives of C. gummiferum flowers by delaying the onset of sepal wilting. There is limited published research into the postharvest characteristics of C. gummiferum (Wade and Satyan 1997; Worrall et al. 1999). The results of the present study indicate that the vase lives of flowers can be limited, at least in part, by flower abscission and sepal wilting induced by exposure to exogenous ethylene. Moreover, sepal wilting is likely to be regulated by endogenous ethylene. Presumably as a result of sepal wilting, C. gummiferum stems that were not exposed to ethylene lost relative fresh weight at a significantly greater rate than similar stems pre-treated with 1-MCP (Figure 3.5). 1-MCP pre-treatment was therefore apparently effective in preventing both endogenous and exogenous ethylene effects.

Loss of relative fresh weight associated with flower abscission and senescence for G. ‘Sandra Gordon’ and seed-grown T. speciosissima inflorescences (Figures 3.10 and 3.17) and flower abscission from C. uncinatum ‘Paddy’s Late’ sprigs (Figure 3.6) not exposed to ethylene were also significantly reduced by pre-treatment with 1-MCP. These results support earlier assertions by Faragher (1986), Joyce (1993) and Joyce et al. (1995) that endogenous ethylene may mediate flower senescence and abscission processes in T. speciosissima, C. uncinatum and G. ‘Sylvia’.

The delay or prevention by 1-MCP pre-treatment of the onset of flower abscission from cut C. uncinatum sprigs (Figure 3.6) and V. nitens flowers (Figure 3.19) exposed to exogenous ethylene correlates with reports that STS treatment prevented ethylene-induced flower abscission from cut C. uncinatum (Joyce 1988, 1989, 1993) and V. nitens (Joyce and Poole 1993). Likewise, the prevention of extensive ethylene- induced flower abscission from inflorescences of Grevillea hybrids by 1-MCP pre-treatment (Figures 3.8, 3.9 and 3.10, respectively) is similar to the results of earlier experiments with G. ‘Sylvia’ (Chapter 2). Thus, the efficacy of 1-MCP pre-treatment in preventing ethylene-induced flower abscission is common for a number of Grevillea hybrids. This finding complements those of Joyce and Haynes (1989) and Vuthapanich et al. (1993) who reported that pre-treatment of hybrid Grevillea spp. with STS prevented ethylene-induced flower abscission.

9 Exogenous ethylene has been reported to induce flower and petal abscission from L. scoparium stems (Zieslin and Gottesman 1983) and is suspected to cause similar problems in several other Leptospermum species (T. Slater, pers. comm.). 1-MCP pre-treatment reduced exogenous ethylene-induced petal abscission from L. petersonii stems (Figure 3.11). This observation supports the findings of Zieslin and Gottesman (1983), who reported that STS prevented ethylene-induced flower abscission from L. scoparium stems. L. petersonii stems not exposed to ethylene were observed to be susceptible to dessication and wilting of petals. Wilting of L. petersonii petals in this study occurred independently of 1- MCP treatment. Thus, petal wilting does not appear to be mediated by ethylene. In contrast, wilting of L. scoparium flowers is thought to be an ethylene-mediated process, at least in drier atmospheres (70% RH) (Zieslin and Gottesman 1983).

Vase life of L. scoparium was not significantly affected by 1-MCP or ethylene treatments in the present study (Table 3.3). However, exposure of stems to ethylene tended to reduce vase life as a result of increased flower abscission. Whilst 1-MCP pre-treatment of stems tended to reduce the number of abscised and senescent flowers and associated loss of relative fresh weight induced by exposure to ethylene, no significant differences between treatments existed (Figure 3.12). Exposure of L. scoparium stems to 10 µL ethylene/L for 48 hours at 21oC is known to induce flower abscission and senescence (Zieslin and Gottesman 1983). Thus, it is possible that the ethylene treatment protocol chosen for this study (viz. 10 µL ethylene/L for 12 hours at 20oC) was not optimal for inducing ethylene-induced effects on cut L. scoparium stems. Accordingly, the protective effect of 1-MCP was minor.

Ethylene production by T. speciosissima flowers increases prior to perianth abscission and is, thus, likely to regulate perianth abscission from inflorescences (Faragher 1986). Furthermore, treatment of inflorescences with ethylene or ACC can stimulate perianth abscission (J. Faragher, unpublished data). Moreover, pre-treatment of inflorescences with STS and AOA can prevent this abscission (J. Faragher, unpublished data). The results of the present study support these observations in that exposure of inflorescences to exogenous ethylene caused perianth abscission. 1-MCP pre-treatment of T. speciosissima inflorescences was shown to reduce ethylene-induced flower abscission (Figure 3.16 and 3.17). 1-MCP pre-treatment was more effective in reducing exogenous ethylene-induced perianth abscission from seed-grown inflorescences than from clonally propagated inflorescences. It is possible that the efficacy of 1-MCP pre-treatment of clonally-propagated inflorescences was poorer because of the 48 hour delay between harvest and 1-MCP pre-treatment. Endogenous ethylene production by flowers may have commenced before 1-MCP pre-treatment, thereby reducing treatment efficacy. 1-MCP treatment efficacy has been found by Reid et al. (1996) to be reduced when endogenous ethylene production by plant tissue is high.

1-MCP pre-treatment significantly reduced exogenous ethylene-induced loss of fresh weight and vase life of A. pinnatum inflorescences as a consequence of reduced perianth abscission, wilting and discolouration (Figure 3.2 and Table 3.4). However, 1-MCP pre-treatment was only significantly effective in reducing

10 the ethylene-induced loss in vase life for inflorescences used in the second of two similar experiments in this study. The reasons for the differing responses of A. pinnatum inflorescences to 1-MCP between the first and second experiments are unclear but may be related to the growing region. Perianth abscission was found to occur only from opened flowers, while perianth wilting and discolouration was generally associated with unopened flowers. However, as inflorescences used in both experiments were selected with similar numbers of open and unopened flowers, it is unlikely that different degrees of flower opening were responsible for this differential response.

Cut flowers that did not respond to pre-treatment with 10 nL 1-MCP/L for 12 hours at 20oC or exposure to 10 µL ethylene/L for 12 hours at 20oC were B. heterophylla, C. adunca, E. scaber, O. diosmifolius, P. lanceolata, T. calycina and Z. cytisoides (Table 3.3). Flower wilting and discolouration and stem relative fresh weight loss from cut B. heterophylla stems were not affected by exposure to 10 µL ethylene/L for 12 hours at 20oC (Figure 3.3). Further, 1-MCP pre-treatment did not extend the vase lives of stems not exposed to ethylene. This observation is apparently contrary to the findings of Williamson (1996), where STS treatment extended vase lives of stems not treated with ethylene.

Peduncle and pedicel wilting on C. adunca stems were significantly reduced for stems pre-treated with 1- MCP and then exposed to ethylene (Figure 3.4). However, the absence of a similar response for stems pre-treated with 1-MCP and not exposed to ethylene suggests it is unlikely that pedicel wilting is controlled by ethylene. Likewise, peduncle wilting from flowering O. diosmifolius stems exposed only to ethylene increased on day 4 of the experiment (Figure 3.14). Pre-treatment with 1-MCP did not prevent this response. Vase life was not significantly affected by peduncle wilting (Table 3.3). Nonetheless, it is possible that ethylene is involved in regulation of peduncle wilting. Leaf abscission from a broad leaf form of O. diosmifolius was prevented by treatment with STS (Johnston 1992). As a result, vase life was extended. However, STS treatment did not prevent leaf abscission from a fine leaf form of O. diosmifolius. Thus, despite the variable response of different plant forms to STS treatment, it is conceivable, in view of apparent ethylene responsiveness, that exogenous ethylene did mediate peduncle wilting of O. diosmifolius in the present study.

Exposure of E. scaber stems to 1-MCP and ethylene treatments did not affect their vase lives. However, petal abscission from stems pre-treated with 1-MCP and exposed to ethylene was significantly less than that from stems exposed only to ethylene (Figure 3.7). Nevertheless, the involvement of ethylene in this process is unclear since there was an absence of significant differences between other treatments and the change in level of flower abscission during vase life was only small. P. lanceolata stems were not sensitive to the ethylene treatment protocol used in this study. This assertion is evidenced by no significant flower quality changes (e.g. wilting or abscission) or loss of relative fresh weight during vase life (Figure 3.15). Flower abscission and closure for T. calycina stems were not consistently affected by exposure to ethylene (Figure 3.18). Therefore, ethylene does not appear to be involved in flower abscission and senescence of T. calycina. These data tend to support the opinion of Joyce et al. (1993), who reported that in which applied ethylene gas and STS did not affect flower abscission in this species.

11 Treatment of Z. cytisoides with 1-MCP and ethylene did not significantly affect flower abscission (Figure 3.20) or vase life (Table 3.3). However, stems pre-treated with 1-MCP had somewhat reduced levels of flower abscission and, in turn, longer vase lives than stems exposed only to ethylene. Thus, it is possible that flower abscission from Z. cytisoides is an ethylene-mediated process.

Higher vase solution uptake by several flowers (i.e. C. gummiferum, C. uncinatum, E. scaber, G. ‘Kay Williams’, G. ‘Misty Pink’, L. petersonii, L. scoparium, P. lanceolata and V. nitens) was associated with 1-MCP pre-treatment (Figures 3.5, 3.6, 3.7, 3.8, 3.9, 3.11, 3.12, 3.15 and 3.19, respectively). With the exception of E. scaber and P. lanceolata, exposure of these flowers to exogenous ethylene induced floral organ abscission and/or wilting which presumably account for reduced transpiration and solution uptake. In contrast, 1-MCP pre-treatment delayed floral organ abscission and/or wilting and, thereby, maintained transpiration and solution uptake. It is unclear why E. scaber and P. lanceolata stems pre-treated with 1- MCP used vase solution at higher rates compared to stems exposed only to ethylene, as no major changes in postharvest flower quality were evident. In contrast to these species, solution uptake by A. pinnatum inflorescences exposed to ethylene was consistently higher than that by similar inflorescences not exposed to ethylene (Figures 3.1 and 3.2). Unexpectedly, perianth abscission, wilting and discolouration induced by exposure to ethylene did not reduce solution uptake.

In other flowers, relationships between vase solution uptake and 1-MCP or ethylene treatments over the duration of the experiments were not clear. Solution uptake by B. heterophylla stems and G. ‘Sandra Gordon’ inflorescences fluctuated during the experiments (Figures 3.3 and 3.10). This fluctuation sometimes probably reflected variations in the vase life room temperature and RH (i.e. vapour pressure deficit). Transpiration and vase solution uptake by cut flowers is known to vary in association with changes to vapour pressure deficit (Halevy and Mayak 1981). Low solution uptake by C. adunca, O. diosmifolius and T. calycina stems not treated with 1-MCP or ethylene or by stems exposed only to ethylene was found to correspond to reduced relative fresh weight, possibly indicating adverse water relations (Figures 3.4, 3.14 and 3.18). Adverse water relations have been reported to limit vase life of T. calycina (Jones et al. 1993). It is unclear why Z. cytisoides stems pre-treated with 1-MCP and exposed to ethylene used solution at highly variable rates compared to stems from other treatments (Figure 3.20). This different response presumably reflects inherent differences in randomly selected stems.

Exposure of B. heterophylla flowers to 10 µL ethylene/L for the longer period of 72 hours at 20oC induced flower wilting and abscission and, thereby, reduced stem fresh weight and vase life (Figure 3.21 and Table 3.5). Joyce and Haynes (1989) also found that treatment of B. heterophylla with 10 µL ethylene/L for 72 hours at 22oC induced rapid loss of stem fresh weight and flower wilting. However, flower abscission was not reported in that study. In the present study, 1-MCP pre-treatment was more effective than STS in reducing exogenous ethylene effects. Silver toxicity from STS treatment may have limited vase life as evidenced by premature flower wilting and delayed flower discolouration (Figures 3.21 and 3.22). Vase solution uptake by stems pre-treated with 1-MCP and STS was consistently higher

12 than that by stems exposed only to ethylene. This relative difference was presumably reflecting delayed flower wilting and abscission and the associated loss in functional transpirational area (Figure 3.22). Conversely, premature flower wilting induced by STS pre-treatment when stems were not exposed to ethylene was reflected in lower vase solution uptake (Figure 3.21 and 3.22).

Pre-treating B. heterophylla stems with STS was reported by Joyce and Haynes (1989) to prevent exogenous ethylene-induced flower wilting and loss of stem fresh weight. Further, Williamson (1996) found that treatment of flowers with STS (0.5 mM Ag+ for 10.5 hours at 20oC) extended the vase lives of stems not exposed to exogenous ethylene by delaying flower wilting. In both these studies, STS treatment was not reported to be toxic. In the present study, stems were calculated to accumulate 0.25 ± 0.01 µmol Ag+/g initial FW following STS treatment. Joyce (1988) reported that a safe, effective range for Ag+ uptake by C. uncinatum was 0.1-0.6 µmol Ag+/g stem FW. However, the safe range could vary according to genotype and/or phenotype. Thus, it is possible that the effective range for B. heterophylla is lower than that for C. uncinatum. This potential problem highlights a difficulty with using STS treatments. The effective concentration can be close to those which can cause phytotoxicity (Cameron and Reid 1981). In contrast, 1-MCP is thought to be completely non-phytotoxic (Serek et al. 1994b).

The finding by Williamson (1996) that treating B. heterophylla with STS extended vase lives of stems not exposed to exogenous ethylene provides circumstantial evidence that endogenous ethylene is involved in flower senescence. In the present study, this assertion was supported since increased ethylene production rates were found to correspond to moderate flower wilting and advanced stages of flower discolouration (Figure 3.23). The early stages of flower discolouration do not appear to be associated with increased ethylene production. Thus, flower discolouration of B. heterophylla, as suggested by Williamson (1996), may not be associated with ethylene production. It is possible that flower discolouration is regulated by earlier changes in tissue pH and in the carbohydrate content of flowers. Discolouration was delayed when sucrose was included in vase solutions for B. heterophylla (Williamson 1996). Moreover, treatment of cut flowers, such as red rose, with solutions containing sucrose is known to delay proteolysis, thereby delaying increases in tissue pH and pigment changes (Mayak and Halevy 1980). The association between flower discolouration and ethylene production by intact flowering stems was, however, less pronounced than that of detached flowers (Figure 3.24). Ethylene production by non-flowering stems of B. heterophylla was generally low compared to flowering stems, suggesting that flowers are the principal site of ethylene production.

This study has shown that 1-MCP pre-treatment at a nanomolar concentration level can protect 10 different ethylene sensitive native Australian cut flowers against exposure to exogenous ethylene; viz. A. pinnatum, B. heterophylla, C. gummiferum, C. uncinatum, G. ‘Kay Williams, G. ‘Misty Pink’, G. ‘Sandra Gordon’, L. petersonii, T. speciosissima and V. nitens. With the exception of C. gummiferum, 1-MCP did not afford flowers with protection against endogenous ethylene. Nonetheless, 1-MCP gas, which is comparatively easy to apply, has potential as a postharvest anti-ethylene treatment for ethylene-sensitive native Australian cut flowers. In an associated study, increased rates of ethylene production by B.

13 heterophylla flowers were found to be associated with their natural senesecence.

14 15 CHAPTER 4

EFFECT OF TEMPERATURE ON THE EFFICACY OF 1-MCP TREATMENT OF CUT FLOWERS

4.1 INTRODUCTION

Ethylene is responsible for regulating a number of processes in plants which have commercial significance in postharvest horticulture (Abeles et al. 1992). Unintentional exposure to ethylene can reduce the postharvest life of cut flowers by eliciting abscission and/or accelerating senescence (Reid 1985b). Such effects of ethylene can, however, be prevented by pre-treating sensitive cut flowers with chemical inhibitors of ethylene biosynthesis or perception (Sherman 1985).

STS can be used as a treatment to protect sensitive cut flowers and potted flowering plants against ethylene-induced floral organ abscission and senescence (Veen 1983). Ag+ in the STS complex is thought to bind and block ethylene receptors thereby preventing ethylene perception (Sisler 1982). However, the use of STS is being reconsidered in some countries due to possible environmental hazards (Serek et al. 1994a). The recently synthesised novel inhibitor of ethylene perception, 1-MCP gas may have potential as an alternative treatment for a range of ethylene-sensitive crops (Serek et al. 1994b, 1995a, b; Sisler et al. 1996a, b).

In contrast to STS, 1-MCP efficacy has been reported to be poor when applied at low temperature. For example, 1-MCP pre-treatment at 20oC was highly effective in protecting cut Penstemon flowers against ethylene, but no protection was afforded when applied at 2oC (Serek et al. 1995a). Increasing the concentration of 1-MCP applied during low temperature treatment improved treatment efficacy to levels similar to treatment at 20oC (Reid et al. 1996). Reasons for this variable temperature response are unclear.

Sisler et al. (1996a) reported that 1-MCP molecules bind permanently to ethylene receptors and thus prevent ethylene action irreversibly. Nonetheless, 1-MCP pre-treated cut carnation flowers, banana and tomato fruit regain sensitivity to ethylene between 10 and 15 days after 1-MCP treatment (Sisler et al. 1996b; Sisler and Serek 1997). Recovery of competence to respond to ethylene is thought to be due to the synthesis of ethylene receptors (Sisler and Serek 1997).

Exposure of several native Australian cut flowers including Grevillea hybrids and C. uncinatum to ethylene elicits rapid flower abscission and, thereby, reduces vase life (Joyce 1988, 1989; Joyce et al. 1993). Therefore, the postharvest longevity of native Australian cut flowers may be extended by treatment with 1-MCP. However, it is proposed that low 1-MCP concentrations will not protect native cut

16 flowers against ethylene when applied at low temperature. The purpose of this study was to test these hypotheses by examining the influence of temperature on 1-MCP pre-treatment efficacy in preventing ethylene-induced flower abscission from two cut flowers, G. ‘Sylvia’ and C. uncinatum. In addition, the duration of 1-MCP effects for each flower was examined. In the case of C. uncinatum, protection was compared with the duration of ethylene insensitivity afforded by STS treatment.

4.2 MATERIALS AND METHODS

4.2.1 Plant material

4.2.1.1 G. ‘Sylvia’ inflorescences

G. ‘Sylvia’ inflorescences were harvested from plants grown at a nursery near Redland Bay. Inflorescences were harvested and transported to the laboratory within 2 hours of harvest. They were prepared for treatment as described in section 2.2.1.

4.2.1.2 C. uncinatum sprigs

Flowering branches of C. uncinatum cultivars ‘Lollypop’, ‘Alba’ and ‘Mid Pink’ were harvested from a farm near Gatton. Stems were immediately stood into buckets containing DI water and taken to the laboratory in an air conditioned car within 30 minutes of harvest. Flowering sprigs were cut from stems with secateurs to 20 cm in length. Thereafter, they were prepared as described for G. ‘Sylvia’ inflorescences (section 2.2.1). Leaves were removed from the lower section of sprigs which were submerged into vase solution.

4.2.2 Chemicals

The preparation of 1-MCP, ethylene and STS stocks was by procedures outlined in sections 2.2.2.1, 2.2.2.2 and 3.2.3.1, respectively.

4.2.3 Treatments

4.2.3.1 Treatment of G. ‘Sylvia’ inflorescences with 1-MCP

G. ‘Sylvia’ inflorescences standing in individual vases were enclosed in 60.5 L glass chambers, each containing 6 beakers of KOH solution (section 2.2.3). Inflorescences were pre-treated on day 0 with 10 nL 1-MCP/L for 12 hours at 2 or 20oC. Control inflorescences were kept in other chambers in air with KOH solution, but without 1-MCP. Different sub-samples of 1-MCP treated inflorescences were then exposed to 10 µL ethylene/L for 12 hours at 20oC (section 2.2.4.1) daily from day 1 until the end of vase life (day 5). When not receiving 1-MCP or ethylene treatments, inflorescences were held in a vase life

17 room operating at the same conditions as described in section 2.2.4.1.

4.2.3.3 Treatment of C. uncinatum sprigs with 1-MCP or STS

In three separate experiments, C. uncinatum ‘Lollypop’, ‘Alba’ and ‘Mid Pink’ sprigs were pre-treated on day 0 with 10 nL 1-MCP/L in chambers with KOH solution or pulsed with STS (0.5 mM Ag+) for 12 hours. Both chemicals were applied to sprigs at 2 or 20oC. Untreated sprigs (i.e. 0 nL 1-MCP/L or 0 mM Ag+) were also maintained in air at 2 or 20oC. Following these treatments, different sub-samples of sprigs treated with 1-MCP or STS were exposed to 10 µL ethylene/L for 12 hours at 20oC daily from day 1 until the end of vase life (ca. day 10). When not receiving 1-MCP, STS or ethylene treatments, sprigs were kept in the same vase life room used for G. ‘Sylvia’ inflorescences.

4.2.4 Assessments

Flower abscission, wilting, discolouration and opening from G. ‘Sylvia’ inflorescences were assessed daily using the subjective scales described in section 2.2.5.1. Flower abscission was determined after gently brushing inflorescences three times by hand. Vase life of inflorescences was based on the same criteria used in section 2.2.5.1. G. ‘Sylvia’ inflorescences and C. uncinatum sprigs and their vases were weighed separately daily (section 2.2.5.1). Flower abscission from C. uncinatum sprigs was assessed daily after gently brushing them three times by hand. Flower abscission was expressed as the percentage of flowers abscised out of the initial number (Day 0) on a sprig. Vase life of sprigs was judged using the criteria in section 3.2.5.1.

4.2.5 Experiment design and data analysis

When flowers were not receiving 1-MCP, ethylene or STS treatments, they were arranged in vase life rooms in CRDs. There were five replicate inflorescences or sprigs per treatment. The effect of temperature on the efficacy of 1-MCP treatment of G. ‘Sylvia’ inflorescences was examined as a 2 (temperature) x 8 (treatment) factorial experiment. Treatments consisted of 5 ethylene application times and 3 controls. In similar studies with C. uncinatum, 2 (temperature) x 11 (treatment), 2 (temperature) x 12 (treatment) or 2 (temperature) x 10 (treatment) factorial experiments were used for ‘Lollypop’, ‘Alba’ and ‘Mid Pink’ sprigs, respectively. In each case, treatments included 2 controls. Remaining treatments were the different ethylene application times. In all experiments, control treatments were excluded from ANOVA.

 Treatment means ± standard errors were calculated using Microsoft Excel (Version 5.0, Microsoft Inc.).  Figures were prepared using Sigmaplot (Version 2.0, Jandel Corporation). Most data were analysed as factorial (depending upon the particular experiment) ANOVAs by the balanced ANOVA function of  Minitab (Release 11.12, Minitab Inc.). Flower abscission score data from G. ‘Sylvia’ were converted to a corresponding percentage. The percent

18 change in flower abscission from G. ‘Sylvia’ and C. uncinatum immediately following ethylene treatment was recorded. A logistic transformation of this data was then performed for ANOVA (McCullagh and Nelder 1989). The change in relative fresh weight of G. ‘Sylvia’ inflorescences and C. uncinatum sprigs immediately after ethylene treatment was calculated for ANOVA. Vase solution uptake data were analysed as split plot for time ANOVAs. When flower abscission from G. ‘Sylvia’ inflorescences reached 100%, measurement of flower wilting, discolouration and opening was discontinued. Based on the assumption that no meaningful conclusions could be drawn from statistical analysis of these unbalanced data sets, ANOVAs were not performed.

The LSD test at P = 0.05 was used to separate treatment means. For vase solution uptake data, LSDs presented are for comparisons between treatments (rather than for a particular time within a treatment). LSDs are shown only when significant (P < 0.05) differences between treatments existed. Differences between treatment means referred to in the results are significant at the P < 0.05 level. LSDs from ANOVA of derived data not presented in figures are shown in appendices. Data sets which show non- significant differences are also presented in appendices.

4.3 RESULTS

4.3.1 Duration of persistence of 1-MCP pre-treatment effects on G. ‘Sylvia’ inflorescences

G. ‘Sylvia’ inflorescences pre-treated with 10 nL 1-MCP/L at 2oC were not protected from subsequent exposure to ethylene as evidenced by rapid flower abscission (Plate 4.1, Figure 4.1 and Appendix 4.1). Pre-treatment of inflorescences with 1-MCP at 2oC did not reduce the ethylene-induced loss of fresh weight associated with flower abscission (Figure 4.2 and Appendix 4.2).

In contrast, inflorescences pre-treated with 1-MCP at 20oC were afforded protection against ethylene- induced flower abscission for 2 days after 1-MCP pre-treatment (Plate 4.1, Figure 4.1 and Appendix 4.1). Thereafter, ethylene treatment stimulated flower abscission. However, flower abscission from inflorescences pre-treated with 1-MCP at 20oC was not as rapid following ethylene treatment. Furthermore, flower abscission was not as complete compared to inflorescences pre-treated with 1-MCP at 2oC (Figure 4.1 and Appendix 4.1). This differential response reflected a significant interaction between 1-MCP pre-treatment and pre-treatment temperature (Appendix 4.3). Changes in flower abscission were also reflected in similar changes in the loss of relative fresh weight from inflorescences pre-treated with 1-MCP at 20oC (Figure 4.2 and Appendix 4.2). However, the loss of relative fresh weight from inflorescences pre-treated with 1-MCP at 20oC was significantly less after exposure to ethylene than from inflorescences pre-treated with 1-MCP at 2oC. Consequently, a significant interaction between 1- MCP pre-treatment and pre-treatment temperature existed (Appendices 4.2 and 4.4).

Vase solution uptake by inflorescences pre-treated with 1-MCP at 2 or 20oC tended to decrease in

19 association with flower abscission induced by ethylene treatment, presumably as the transpirational area was reduced (Figure 4.3). A significant interaction between 1-MCP pre-treatment, pre-treatment temperature and time of measurement for vase solution uptake reflected the differential response of inflorescences to 1-MCP at 2 and 20oC (Appendix 4.5).

As vase life was partly based on flower abscission, 1-MCP pre-treatment at 2oC did not prevent the exogenous ethylene-induced loss in vase life (Table 4.1). However, 1-MCP pre-treatment at 20oC significantly reduced the ethylene-induced loss in vase life for up to 2 days after 1-MCP pre-treatment (Table 4.1). As a result, there was a significant interaction between 1-MCP pre-treatment and pre- treatment temperature for vase life (Appendix 4.6). Flower wilting, opening and discolouration were not affected by 1-MCP pre-treatment at 2 or 20oC (Appendices 4.7, 4.8 and 4.9).

20 Plate 4.1. G. ‘Sylvia’ inflorescences on day 5 after pre-treatment on day 0 with 10 nL 1-MCP/L at 20 (LHS) or 2oC (RHS) followed by exposure on day 1 to 10 µL ethylene/L at 20oC. Note: extensive flower abscission is evident in the inflorescence pre-treated with 1-MCP at 2oC (RHS).

21 Control treatments Sequential treatments 5 4 3

1 2oC 2oC Control treatments Sequential treatments 5

Abscission score 4 3 2 o o 1 20 C 20 C

01234560123456 Time (days)

Figure 4.1. Flower abscission (scores: 1 = < 10% to 5 = > 80%) from G. ‘Sylvia’ inflorescences pre- treated with 10 nL 1-MCP/L at 2 or 20oC. Different sub-samples of inflorescences were then sequentially exposed to 10 µL ethylene/L at 20oC days 1 (●), 2 (■), 3 (▲), 4 (▼) or 5 (◆). Control inflorescences were treated with 0 nL 1-MCP/L at 2 or 20oC and 0 µL ethylene/L at 20oC (●), 0 nL 1-MCP/L at 2 or 20oC and 10 µL ethylene/L at 20oC (■) or 10 nL 1-MCP/L at 2 or 20oC and 0 µL ethylene/L at 20oC (▲). 1-MCP and ethylene treatments were each conducted for 12 hours on days 0 or 1, respectively, unless otherwise stated. Vertical bars represent standard errors of means (n = 5). LSD is presented in Appendix 4.1.

22 120 Control treatments Sequential treatments 100 80 60 40 o o 20 2 C2C 120 Control treatments Sequential treatments 100 80 60

Relative fresh weight (% of initial FW) (% Relative fresh weight 40 o o 20 20 C 20 C 01234560123456 Time (days)

Figure 4.2. Relative fresh weight of G. ‘Sylvia’ inflorescences pre-treated with 10 nL 1-MCP/L at 2 or 20oC. Different sub-samples of inflorescences were then sequentially exposed to 10 µL ethylene/L at 20oC on days 1 (●), 2 (■), 3 (▲), 4 (▼) or 5 (◆). Control inflorescences were treated with 0 nL 1-MCP/L at 2 or 20oC and 0 µL ethylene/L at 20oC (●), 0 nL 1-MCP/L at 2 or 20oC and 10 µL ethylene/L at 20oC (■) or 10 nL 1-MCP/L at 2 or 20oC and 0 µL ethylene/L at 20oC (▲). 1- MCP and ethylene treatments were each conducted for 12 hours on days 0 or 1, respectively, unless otherwise stated. Vertical bars represent standard errors of means (n = 5). LSD is presented in Appendix 4.2.

23 Control treatments Sequential treatments

0.4

0.2

o o 0.0 2 C 2 C Control treatments Sequential treatments

0.4

0.2 Solution uptake (mL/g initial FW/day)

20oC 20oC 0.0 01234560123456 Time (days)

Figure 4.3. Vase solution usage by G. ‘Sylvia’ inflorescences pre-treated with 10 nL 1-MCP/L at 2 or 20oC. Different sub-samples of these inflorescences were then sequentially exposed to 10 µL ethylene/L at 20oC days 1 (●), 2 (■), 3 (▲), 4 (▼) or 5 (◆). Control inflorescences were treated with 0 nL 1-MCP/L at 2 or 20oC and 0 µL ethylene/L at 20oC (●), 0 nL 1-MCP/L at 2 or 20oC and 10 µL ethylene/L at 20oC (■) or 10 nL 1-MCP/L at 2 or 20oC and 0 µL ethylene/L at 20oC (▲). 1-MCP and ethylene treatments were each conducted for 12 hours on days 0 or 1, respectively, unless otherwise stated. Vertical bars represent standard errors of means (n = 5). LSD = 0.037 mL/g initial FW/day.

24 Table 4.1. Vase life (mean ± s.e.) of G. ‘Sylvia’ inflorescences pre-treated at 2 or 20oC with 0 or 10 nL 1-MCP/L for 12 hours. Different sub-samples of inflorescences pre-treated with 10 nL 1- MCP/L were then treated at 20oC daily until day 5 with 10 µL ethylene/L for 12 hours. Values in parentheses show vase life relative to the longest recorded (%). Data followed by the same letters are not significantly different (LSD = 1.0) at P = 0.05 (n = 5).

1-MCP treatment Days between 1-MCP Vase life (days) pre-treatment and exposure to ethylene

Part A: Control 1-MCP treatment at 1-MCP treatment at treatments 2oC 20oC

0 nL 1-MCP/L - 4.0 ± 0.4 a (87) 3.6 ± 0.4 a (78) 1 2.0 ± 0.0 a (43) 2.0 ± 0.0 a (43) 10 nL 1-MCP/L - 4.6 ± 0.4 a (100) 4.6 ± 0.2 a (100)

Part B: Sequential treatments

10 nL 1-MCP/L 1 2.0 ± 0.0 a (43) 4.6 ± 0.4 c (100) 2 3.0 ± 0.0 bc (65) 4.2 ± 0.5 c (91) 3 3.8 ± 0.2 c (83) 3.8 ± 0.4 c (83) 4 4.2 ± 0.4 c (91) 4.4 ± 0.4 c (96) 5 4.4 ± 0.4 c (96) 4.6 ± 0.5 c (100) a Control inflorescences were excluded from the statistical analysis of vase life.

4.3.2 Duration of persistence of 1-MCP and STS pre-treatment effects on flowering C. uncinatum sprigs

Pre-treatment of C. uncinatum ‘Lollypop’, ‘Alba’ and ‘Mid Pink’ sprigs with 10 nL 1-MCP/L at 2oC provided protection against exogenous ethylene-induced flower abscission for 1, 2 and 2 days, respectively (Figures 4.4, 4.5, 4.6 and Appendices 4.10, 4.11, 4.12). Thereafter, flower abscission in response to ethylene treatment was rapid. Ethylene-induced flower abscission was accompanied by the loss in sprig relative fresh weight (Figures 4.7, 4.8, 4.9 and Appendices 4.13, 4.14, 4.15). Vase life of ‘Lollypop’, ‘Alba’ and ‘Mid Pink’ sprigs pre-treated with 1-MCP at 2oC was reduced when exposed to ethylene treatment applied 2, 3 and 3 days after 1-MCP pre-treatment, respectively (Tables 4.2, 4.3, 4.4). In contrast, flower abscission and associated loss of relative fresh weight from ‘Lollypop’, ‘Alba’ and ‘Mid Pink’ sprigs were low throughout these experiments (10, 11 and 9 days, respectively) from parallel sets of sprigs pre-treated with STS at 2oC (Figures 4.4, 4.5, 4.6, 4.7, 4.8 and 4.9). Thus, vase life of sprigs pre-treated with STS was effectively extended relative to sprigs pre-treated with 1-MCP (Tables 4.2, 4.3 and 4.4).

The onset of ethylene-induced flower abscission from ‘Lollypop’, ‘Alba’ and ‘Mid Pink’ sprigs pre- treated with 1-MCP at 20oC was delayed for 6, 4 and 3 days, respectively (Figures 4.4, 4.5, 4.6 and Appendices 4.10, 4.11, 4.12). Consequently, the loss of relative fresh weight from sprigs pre-treated with 1-MCP at 20oC was reduced during this period compared to sprigs pre-treated with 1-MCP at 2oC (Figures 4.7, 4.8, 4.9 and Appendices 4.13, 4.14, 4.15). Thereafter flower abscission and associated loss 25 of relative fresh weight were rapid. Vase life of ‘Lollypop’, ‘Alba’ and ‘Mid Pink’ sprigs pre-treated with 1-MCP at 20oC was not reduced by exposure to ethylene for up to 6, 4 and 3 days after 1-MCP pre- treatment, respectively (Tables 4.2, 4.3 and 4.4). As found for parallel sets of sprigs pre-treated with STS at 2oC, those treated with STS at 20oC remained insensitive to ethylene for the duration of the experiments. There was virtually no flower abscission at all from sprigs pre-treated with STS (Figures 4.4, 4.5 and 4.6) except for ‘Lollypop’, which may have been due to STS phytotoxicity. Furthermore, the loss of relative fresh weight from sprigs pre-treated with STS was significantly reduced compared to sprigs pre-treated with 1-MCP (Figures 4.7, 4.8 and 4.9). Accordingly, vase life of sprigs pre-treated with STS at 20oC was longer than sprigs pre-treated with 1-MCP at 20oC except for ‘Lollypop’ (Tables 4.2, 4.3 and 4.4).

The differential response of ‘Lollypop’, ‘Alba’ and ‘Mid Pink’ sprigs to 1-MCP and STS pre-treatments at 2 or 20oC was reflected in a significant interaction between 1-MCP or STS pre-treatment, time of ethylene treatment and pre-treatment temperature for flower abscission (Appendices 4.16, 4.17 and 4.18) and loss of relative fresh weight (Appendices 4.19, 4.20 and 4.21). As a result, there was also a significant interaction between 1-MCP or STS pre-treatment, time of ethylene treatment and pre-treatment temperature for vase life of ‘Lollypop’ and ‘Mid Pink’ sprigs (Appendices 4.22 and 4.23). For ‘Alba’ sprigs, a significant interaction between 1-MCP or STS pre-treatment and pre-treatment temperature for vase life was evident (Appendix 4.24).

26 Control (2oC) Control (20oC) 100 80 60 40 20 0 + 1-MCP (2oC) + 1-MCP (20oC) 100 80 60 40 20 0 + 1-MCP (2oC) + 1-MCP (20oC) 100 80 60 40 20 0 o + STS (20oC) 100 + STS (2 C) Flower abscission (%) abscission Flower 80 60 40 20 0 o + STS (20oC) 100 + STS (2 C) 80 60 40 20 0 02468100246810 Time (days)

Figure 4.4. Flower abscission from C. uncinatum ‘Lollypop’ sprigs pre-treated on day 0 with 10 nL 1-MCP/L or STS (0.5 mM Ag+) at 2 or 20oC. Different sub-samples of sprigs were then sequentially exposed to 10 µL ethylene/L for 12 hours at 20oC on days 1 (●), 2 (■), 3 (▲), 4 (▼), 5 (◆), 6 (), 7 (d), 8 (U) or 9 (V). Control sprigs were treated with 0 nL 1-MCP/L at 2 or 20oC and 0 µL ethylene/L at 20oC (p) or 0 nL 1-MCP/L at 2 or 20oC and 10 µL ethylene/L at 20oC (●). 1- MCP/STS and ethylene treatments were each conducted for 12 hours on days 0 or 1, respectively, unless otherwise stated. Vertical bars represent standard errors of means (n = 5). Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD is presented in Appendix 4.10.

27 o 100 Control (2oC) Control (20 C) 80 60 40 20 0 100 + 1-MCP (2oC) + 1-MCP (20oC) 80 60 40 20 0 100 + 1-MCP (2oC) + 1-MCP (20oC) 80 60 40 20 0 100 + STS (2oC) + STS (20oC)

Flower abscission (%) 80 60 40 20 0 100 + STS (2oC) + STS (20oC) 80 60 40 20 0 02468100246810 Time (days)

Figure 4.5. Flower abscission from C. uncinatum ‘Alba’ sprigs pre-treated on day 0 with 10 nL 1- MCP/L or STS (0.5 mM Ag+) at 2 or 20oC. Different sub-samples of sprigs were then sequentially exposed to 10 µL ethylene/L for 12 hours at 20oC on days 1 (●), 2 (■), 3 (▲), 4 (▼), 5 (◆), 6 (), 7 (d), 8 (U), 9 (V) or 10 (p). Control sprigs were treated with 0 nL 1-MCP/L at 2 or 20oC and 0 µL ethylene/L at 20oC ( ) or 0 nL 1-MCP/L at 2 or 20oC and 10 µL ethylene/L at 20oC (●). 1- MCP/STS and ethylene treatments were each conducted for 12 hours on days 0 or 1, respectively, unless otherwise stated. Vertical bars represent standard errors of means (n = 5). Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD is presented in Appendix 4.11.

28 Control (2oC) o 100 Control (2 C) 80 60 40 20 0 o o 100 + 1-MCP (2 C) + 1-MCP (20 C) 80 60 40 20 0 o o 100 + 1-MCP (2 C) + 1-MCP (20 C) 80 60 40 20 0 o o 100 + STS (2 C) + STS (20 C) Flower abscission (%) abscission Flower 80 60 40 20 0 o o 100 + STS (2 C) + STS (20 C) 80 60 40 20 0 01234567890123456789 Time (days)

Figure 4.6. Flower abscission from C. uncinatum ‘Mid Pink’ sprigs pre-treated on day 0 with 10 nL 1-MCP/L or STS (0.5 mM Ag+) at 2 or 20oC. Different sub-samples of sprigs were then sequentially exposed to 10 µL ethylene/L for 12 hours at 20oC on days 1 (●), 2 (■), 3 (▲), 4 (▼), 5 (), 6 (d), 7 (U) or 8 (V). Control sprigs were treated with 0 nL 1-MCP/L at 2 or 20oC and 0 µL ethylene/L at 20oC (p) or 0 nL 1-MCP/L at 2 or 20oC and 10 µL ethylene/L at 20oC (●). 1-MCP/STS and ethylene treatments were each conducted for 12 hours on days 0 or 1, respectively, unless otherwise stated. Vertical bars represent standard errors of means (n = 5). Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD is presented in Appendix 4.12.

29 120 Control (2oC) Control (20oC) 100 80 60 40 120 + 1-MCP (2oC) + 1-MCP (20oC) 100 80 60 40 120 + 1-MCP (2oC) + 1-MCP (20oC) 100 80 60 40 120 + STS (2oC) + STS (20oC) 100 80 60 Relative fresh weight (% of initial FW) initial of (% weight fresh Relative 40 120 + STS (2oC) + STS (20oC) 100 80 60 40 02468100246810 Time (days)

Figure 4.7. Relative fresh weight of C. uncinatum ‘Lollypop’ sprigs pre-treated on day 0 with 10 nL 1-MCP/L or STS (0.5 mM Ag+) at 2 or 20oC. Different sub-samples of sprigs were then sequentially exposed to 10 µL ethylene/L for 12 hours at 20oC on days 1 (●), 2 (■), 3 (▲), 4 (▼), 5 (◆), 6 (), 7 (d), 8 (U) or 9 (V). Control sprigs were treated with 0 nL 1-MCP/L at 2 or 20oC and 0 µL ethylene/L at 20oC (p) or 0 nL 1-MCP/L at 2 or 20oC and 10 µL ethylene/L at 20oC (●). 1- MCP/STS and ethylene treatments were each conducted for 12 hours on days 0 or 1, respectively, unless otherwise stated. Vertical bars represent standard errors of means (n = 5). Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD is presented in Appendix 4.13.

30 120 Control (2oC) Control (20oC) 100 80 60 40 120 + 1-MCP (2oC) + 1-MCP (20oC) 100 80 60 40 120 + 1-MCP (2oC) + 1-MCP (20oC) 100 80 60 40 120 + STS (2oC) + STS (20oC) 100 80 60 Relative fresh weight of (% initial FW) 40 120 + STS (2oC) + STS (20oC) 100 80 60 40 02468100246810 Time (days)

Figure 4.8. Relative fresh weight of C. uncinatum ‘Alba’ sprigs pre-treated on day 0 with 10 nL 1- MCP/L or STS (0.5 mM Ag+) at 2 or 20oC. Different sub-samples of sprigs were then treated with 10 µL ethylene/L for 12 hours at 20oC on days 1 (●), 2 (■), 3 (▲), 4 (▼), 5 (◆), 6 (), 7 (d), 8 (U), 9 (V) or 10 (p). Control sprigs were treated with 0 nL 1-MCP/L at 2 or 20oC and 0 µL ethylene/L at 20oC ( ) or 0 nL 1-MCP/L at 2 or 20oC and 10 µL ethylene/L at 20oC (●). 1-MCP/STS and ethylene treatments were each conducted for 12 hours on days 0 or 1, respectively, unless otherwise stated. Vertical bars represent standard errors of means (n = 5). Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD is presented in Appendix 4.14.

31 120 Control (2oC) Control (20oC) 100 80 60 40 120 + 1-MCP (2oC) + 1-MCP (20oC) 100 80 60 40 120 + 1-MCP (2oC) + 1-MCP (20oC) 100 80 60 40 120 + STS (2oC) + STS (20oC) 100 80 60 Relative fresh weight (% of initial FW) initial of (% weight fresh Relative 40 120 + STS (2oC) + STS (20oC) 100 80 60 40 02468 02468 Time (days)

Figure 4.9. Relative fresh weight of C. uncinatum ‘Mid Pink’ sprigs pre-treated on day 0 with 10 nL 1-MCP/L or STS (0.5 mM Ag+) at 2 or 20oC. Different sub-samples of sprigs were then treated with 10 µL ethylene/L for 12 hours at 20oC on days 1 (●), 2 (■), 3 (▲), 4 (▼), 5 (), 6 (d), 7 (U) or 8 (V). Control sprigs were treated with 0 nL 1-MCP/L at 2 or 20oC and 0 µL ethylene/L at 20oC (p) or 0 nL 1-MCP/L at 2 or 20oC and 10 µL ethylene/L at 20oC (●). 1-MCP/STS and ethylene treatments were each conducted for 12 hours on days 0 or 1, respectively, unless otherwise stated. Vertical bars represent standard errors of means (n = 5). Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD is presented in Appendix 4.15.

32 Table 4.2. Vase life (mean ± s.e.) of C. uncinatum ‘Lollypop’ sprigs pre-treated at 2 or 20oC with 0 or 10 nL 1-MCP/L or pulsed with STS (0.5 mM Ag+) for 12 hours. Different sub-samples of sprigs pre-treated with 10 nL 1-MCP/L or 0.5 mM Ag+ were then treated at 20oC daily until day 9 with 10 µL ethylene/L for 12 hours. Values in parentheses show vase life relative to the longest recorded (%). Data followed by the same letters are not significantly different (LSD = 1.9) at P = 0.05 (n = 5).

1-MCP treatment Days between 1-MCP or Vase life (days) STS pre-treatment and exposure to ethylene

Part A: Control treatments 1-MCP or Ag+ treatment 1-MCP or Ag+ at 2oC treatment at 20oC 0 nL 1-MCP/L or - 8.6 ± 0.9 z (88) 6.4 ± 0.9 z (65) 0 mM Ag+ 1 2.0 ± 0.0 z (20) 2.0 ± 0.0 z (20) Part B: Sequential 10 nL 1-MCP/L at 2oC 0.5 mM Ag+ at 2oC 10 nL 1-MCP/L at 0.5 mM Ag+ at 20oC treatments 20oC 1-MCP or Ag+ 1 7.4 ± 1.0 cd (76) 7.4 ± 0.6 cd (76) 7.2 ± 0.7 cd (73) 3.2 ± 0.5 ab (33) 2 3.0 ± 0.0 ab (31) 6.6 ± 0.7 bc (67) 7.6 ± 1.0 cd (78) 2.6 ± 0.6 a (27) 3 4.4 ± 0.2 ab (45) 7.6 ± 0.6 cd (78) 6.8 ± 0.5 c (69) 3.8 ± 0.5 ab (39) 4 5.0 ± 0.0 bc (51) 9.0 ± 0.4 d (92) 7.8 ± 0.4 cd (80) 3.8 ± 0.5 ab (39) 5 5.6 ± 0.4 bc (57) 6.0 ± 0.8 bc (61) 8.0 ± 1.3 cd (82) 5.2 ± 1.2 bc (53) 6 5.4 ± 1.0 bc (55) 7.4 ± 0.9 cd (76) 8.8 ± 0.8 d (90) 4.2 ± 0.2 ab (43) 7 7.2 ± 0.8 cd (73) 4.8 ± 0.6 b (49) 5.4 ± 0.9 bc (55) 3.2 ± 0.5 ab (33) 8 8.0 ± 1.0 cd (82) 4.8 ± 0.6 b (49) 7.6 ± 0.4 cd (78) 3.6 ± 0.4 ab (37) 9 9.8 ± 0.2 d (100) 8.2 ± 0.6 cd (84) 7.0 ± 0.5 cd (71) 3.6 ± 0.4 ab (37) z Control sprigs were excluded from the statistical analysis of vase life. Table 4.3. Vase life (mean ± s.e.) of C. uncinatum ‘Alba’ sprigs pre-treated at 2 or 20oC with 0 or 10 nL 1-MCP/L or pulsed with STS (0.5 mM Ag+) for 12 hours. Different sub-samples of sprigs pre-treated with 10 nL 1-MCP/L or 0.5 mM Ag+ were then treated at 20oC daily until day 10 with 10 µL ethylene/L for 12 hours. Values in parentheses show vase life relative to the longest recorded (%). Data followed by the same letters are not significantly different (LSD = 2.6) at P = 0.05 (n = 5).

1-MCP treatment Days between 1-MCP or STS Vase life (days) pre-treatment and exposure to

33 ethylene

Part A: Control treatments 1-MCP or Ag+ 1-MCP or Ag+ treatment at 2oC treatment at 20oC 0 nL 1-MCP/L or - 6.2 ± 1.4 z (62) 4.4 ± 0.4 z (44) 0 mM Ag+ 1 2.0 ± 0.0 z (20) 2.0 ± 0.0 z (20) Part B: Sequential 10 nL 1-MCP/L at 2oC 0.5 mM Ag+ at 2oC 10 nL 1-MCP/L at 0.5 mM Ag+ at 20oC treatments 20oC 1-MCP or Ag+ 1 8.8 ± 1.3 bc (88) 8.6 ± 1.0 bc (86) 8.4 ± 0.5 bc (84) 7.0 ± 0.7 b (70) 2 4.4 ± 0.7 a (44) 8.0 ± 1.6 bc (80) 7.2 ± 1.2 bc (72) 6.6 ± 1.3 a (66) 3 4.6 ± 0.6 a (46) 10.0 ± 0.8 c (100) 6.2 ± 0.6 a (62) 5.8 ± 0.7 a (58) 4 5.4 ± 0.4 a (54) 8.6 ± 1.5 bc (86) 8.0 ± 0.9 bc (80) 8.0 ± 0.4 bc (80) 5 5.6 ± 0.4 a (56) 9.0 ± 1.2 bc (90) 6.0 ± 0.0 a (60) 9.6 ± 1.0 c (96) 6 7.0 ± 0.0 b (70) 9.8 ± 1.0 c (98) 7.2 ± 0.2 bc (72) 8.2 ± 1.0 bc (82) 7 7.8 ± 0.4 bc (78) 9.2 ± 0.7 bc (92) 8.6 ± 0.2 bc (86) 7.0 ± 1.3 b (70) 8 7.2 ± 0.8 bc (72) 8.8 ± 1.2 bc (88) 7.4 ± 0.7 bc (74) 7.2 ± 1.0 bc (72) 9 6.6 ± 1.2 a (66) 10.0 ± 0.4 c (100) 8.4 ± 0.5 bc (84) 7.2 ± 1.2 bc (72) 10 7.6 ± 1.2 bc (76) 9.6 ± 0.5 c (96) 8.2 ± 1.7 bc (82) 9.8 ± 0.6 c (98) z Control sprigs were excluded from the statistical analysis of vase life. Table 4.4. Vase life (mean ± s.e.) of C. uncinatum ‘Mid Pink’ sprigs pre-treated at 2 or 20oC with 0 or 10 nL 1-MCP/L or pulsed with STS (0.5 mM Ag+) for 12 hours. Different sub-samples of sprigs pre-treated with 10 nL 1-MCP/L or 0.5 mM Ag+ were then treated at 20oC daily until day 8 with 10 µL ethylene/L for 12 hours. Values in parentheses show vase life relative to the longest recorded (%). Data followed by the same letters are not significantly different (LSD = 1.6) at P = 0.05 (n = 5).

1-MCP treatment Days between 1-MCP or STS Vase life (days) pre-treatment and exposure to ethylene

Part A: Control 1-MCP or Ag+ 1-MCP or Ag+ treatments treatment at 2oC treatment at 20oC 0 nL 1-MCP/L or - 5.8 ± 0.6 z (78) 4.4 ± 0.2 z (59)

34 0 mM Ag+ 1 2.0 ± 0.0 z (27) 2.0 ± 0.0 z (27)

Part B: Sequential 10 nL 1-MCP/L at 2oC 0.5 mM Ag+ at 2oC 10 nL 1-MCP/L at 20oC 0.5 mM Ag+ at 20oC treatments 1-MCP or Ag+ 1 5.4 ± 1.5 ab (73) 5.8 ± 0.5 bc (78) 5.6 ± 0.6 b (77) 4.4 ± 0.2 ab (59) 2 3.8 ± 0.5 a (51) 6.4 ± 0.9 bc (86) 4.8 ± 0.4 ab (65) 6.4 ± 0.9 bc (86) 3 4.0 ± 0.0 ab (54) 6.8 ± 0.5 bc (92) 5.0 ± 0.0 ab (68) 5.8 ± 0.5 bc (78) 4 4.6 ± 0.2 ab (62) 6.6 ± 0.5 bc (89) 4.4 ± 0.2 ab (59) 5.6 ± 0.7 b (76) 5 5.4 ± 0.2 ab (73) 5.0 ± 0.5 ab (68) 4.4 ± 0.2 ab (59) 5.0 ± 0.5 ab (68) 6 4.8 ± 0.2 ab (65) 4.6 ± 0.4 ab (62) 5.4 ± 0.5 ab (73) 4.6 ± 0.4 ab (62) 7 5.0 ± 0.5 ab (68) 6.8 ± 1.0 bc (92) 6.6 ± 0.4 bc (89) 4.4 ± 0.4 ab (59) 8 7.4 ± 0.5 c (100) 5.4 ± 0.7 ab (73) 6.4 ± 0.7 bc (86) 6.2 ± 0.8 bc (84) z Control sprigs were excluded from the statistical analysis of vase life.

35 ‘Lollypop’ sprigs pre-treated with STS at 20oC used vase solution at a higher rate between days 0 and 1 than parallel sets of sprigs pre-treated at 2oC and sprigs pre-treated with 1-MCP at 2 or 20oC (Figure 4.10). This response was reflected in a significant interaction between the anti-ethylene agent, pre- treatment temperature and time of measurement (Appendix 4.25). After day 2, vase solution uptake by sprigs declined with time and was not affected by 1-MCP or STS pre-treatment. Vase solution uptake by ‘Alba’ sprigs pre-treated with STS was consistently lower and more stable throughout the experiment than sprigs pre-treated with 1-MCP (Figure 4.11). Furthermore, sprigs pre-treated with 1-MCP and STS at 20oC used less vase solution between days 1 and 2 than parallel sets of sprigs pre-treated at 2oC. As a result there was a significant interaction between the anti-ethylene agent, the timing of ethylene treatment, pre-treatment temperature and time of measurement (Appendix 4.26). Vase solution uptake by ‘Mid Pink’ sprigs pre-treated with STS at 2 or 20oC was lower and more consistent throughout the experiment than sprigs pre-treated with 1-MCP (Figure 4.12). These responses resulted in a significant interaction between the anti-ethylene agent, the timing of ethylene treatment and time of measurement (Appendix 4.27).

Overall, based on solution volume uptake measured for STS pulsing at 2oC, ‘Lollypop’, ‘Alba’ and ‘Mid Pink’ sprigs accumulated 0.067 ± 0.003, 0.031 ± 0.005 and 0.054 ± 0.002 µmol Ag+/g sprig FW, respectively. Accumulation of Ag+ by sprigs of ‘Lollypop’, ‘Alba’ and ‘Mid Pink’ pulsed with STS at 20oC was 0.353 ± 0.010, 0.213 ± 0.006 and 0.160 ± 0.005 µmol Ag+/g sprig FW, respectively.

1 Control treatments o o 1.2 2 C Control treatments 20 C 0.8 0.4 0.0 1-MCP treatment o 1-MCP treatment o 1.2 2 C 20 C 0.8 0.4 0.0 1-MCP treatment o 1-MCP treatment o 1.2 2 C 20 C 0.8 0.4 0.0 STS treatment o STS treatment o 1.2 2 C 20 C 0.8 0.4 Solution uptake (mL/g initial FW/day) initial (mL/g uptake Solution 0.0 STS treatment o STS treatment o 1.2 2 C 20 C 0.8 0.4 0.0 02468100246810 Time (days)

Figure 4.10. Vase solution uptake by C. uncinatum ‘Lollypop’ sprigs pre-treated on day 0 with 10 nL 1-MCP/L or STS (0.5 mM Ag+) at 2 or 20oC. Different sub-samples of sprigs from each of these treatments were then sequentially exposed to 10 µL ethylene/L for 12 hours at 20oC on days 1 (●), 2 (■), 3 (▲), 4 (▼), 5 (◆), 6 (), 7 (d), 8 (U) or 9 (V). Control sprigs were treated with 0 nL 1-MCP/L at 2 or 20oC and 0 µL ethylene/L at 20oC (p) or 0 nL 1-MCP/L at 2 or 20oC and 10 µL ethylene/L at 20oC (●). 1-MCP/STS and ethylene treatments were each conducted for 12 hours on days 0 or 1, respectively, unless otherwise stated. Vertical bars represent standard errors of means (n = 5). Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD = 0.142 mL/g initial FW/day.

2 o 1.5 Control treatments 2 C Control treatments 20oC 1.0 0.5 0.0 1.5 1-MCP treatment 2oC 1-MCP treatment 20oC 1.0 0.5 0.0 1.5 1-MCP treatment 2oC 1-MCP treatment 20oC 1.0 0.5 0.0 1.5 STS treatment 2oC STS treatment 20oC 1.0 0.5 Solution uptake (mL/g initial FW/day) (mL/g uptake Solution 0.0 1.5 STS treatment 2oC STS treatment 20oC 1.0 0.5 0.0 02468100246810 Time (days)

Figure 4.11. Vase solution uptake by C. uncinatum ‘Alba’ sprigs pre-treated on day 0 with 10 nL 1- MCP/L or STS (0.5 mM Ag+) at 2 or 20oC. Different sub-samples of sprigs from each of these treatments were then sequentially exposed to 10 µL ethylene/L for 12 hours at 20oC on days 1 (●), 2 (■), 3 (▲), 4 (▼), 5 (◆), 6 (), 7 (d), 8 (U), 9 (V) or 10 (p). Control sprigs were treated with 0 nL 1- MCP/L at 2 or 20oC and 0 µL ethylene/L at 20oC ( ) or 0 nL 1-MCP/L at 2 or 20oC and 10 µL ethylene/L at 20oC (●). 1-MCP/STS and ethylene treatments were each conducted for 12 hours on days 0 or 1, respectively, unless otherwise stated. Vertical bars represent standard errors of means (n = 5). Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD = 0.184 mL/g initial FW/day.

3 Control treatments o Control treatments o 1.2 2 C 20 C 0.8 0.4 0.0 1-MCP treatment o 1-MCP treatment o 1.2 2 C 20 C 0.8 0.4 0.0 1-MCP treatment o 1-MCP treatment o 1.2 2 C 20 C 0.8 0.4 0.0 STS treatment o o 1.2 2 C STS treatment 20 C 0.8 0.4 Solution uptake (mL/g initial FW/day) 0.0 STS treatment o STS treatment 20oC 1.2 2 C 0.8 0.4 0.0 02468 02468 Time (days)

Figure 4.12. Vase solution uptake by C. uncinatum ‘Mid Pink’ sprigs pre-treated on day 0 with 10 nL 1-MCP/L or STS (0.5 mM Ag+) at 2 or 20oC. Different sub-samples of sprigs from each of these treatments were then sequentially exposed to 10 µL ethylene/L for 12 hours at 20oC on days 1 (●), 2 (■), 3 (▲), 4 (▼), 5 (), 6 (d), 7 (U) or 8 (V). Control sprigs were treated with 0 nL 1-MCP/L at 2 or 20oC and 0 µL ethylene/L at 20oC (p) or 0 nL 1-MCP/L at 2 or 20oC and 10 µL ethylene/L at 20oC (●). 1-MCP/STS and ethylene treatments were each conducted for 12 hours on days 0 or 1, respectively, unless otherwise stated. Vertical bars represent standard errors of means (n = 5). Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD = 0.143 mL/g initial FW/day.

4 4.4. DISCUSSION

The efficacy of 1-MCP pre-treatment applied to cut Penstemon and Kalanchoe flowers at low temperatures (2oC) and at low concentrations (5-20 nL/L) has been reported as being poor (Serek et al. 1995a; Reid et al. 1996). This assertion was confirmed in the present study. Pre-treatment of G. ‘Sylvia’ inflorescences with 10 nL 1-MCP/L for 12 hours at 2oC did not prevent ethylene-induced flower abscission (Figure 4.1). This response differs from results presented in section 2.3.3 where pre-treatment of inflorescences with 10 nL 1-MCP/L for 12 hours at 0, 5, 10 or 20oC afforded similar levels of protection against ethylene. These contrasting results may reflect the different temperatures at which chambers were ventilated (i.e. 2 vs. 20oC).

C. uncinatum sprigs pre-treated with 10 nL 1-MCP/L for 12 hours at 2oC were afforded only short term (ca. 2 days) protection against ethylene (Tables 4.2, 4.3 and 4.4). Pre-treatment of sprigs with 10 nL 1- MCP/L for 12 hours at 20oC provided comparatively longer term (ca. 4 days) protection against ethylene (Tables 4.2, 4.3 and 4.4). 1-MCP evidently blocked ethylene receptors during pre-treatment at 2 or 20oC, since the sprigs were fully protected against ethylene immediately after the pre-treatment.

The reasons why higher 1-MCP concentrations are needed at low temperature are unclear. Poor 1-MCP binding at low temperature may be due to conformational changes in the membrane-located protein(s) that is/are the ethylene receptor(s). Conformational changes to membranes and membrane-bound proteins are proposed to occur at low temperature in chilling sensitive plant tissue (Lyons 1973). In addition, slower diffusion of 1-MCP molecules to the ethylene receptor may occur at low temperature.

According to Sisler and Serek (1997), plants regain sensitivity to ethylene presumably by producing new ethylene receptors. An alternative explanation to that tendered above is that exposure to low temperature treatment may induce rapid receptor synthesis. Mayak and Kofranek (1976) reported that holding cut carnation flowers at 2oC for several days increased their sensitivity to subsequent ethylene treatment. Sisler et al. (1996a) also reported that carnation flowers pre-treated with 5 nL 1-MCP/L at 24oC and then held at 4oC for 4 days had more unbound ethylene receptors than parallel sets of flowers held at 24oC. However, the duration of pre-treatment at 2oC in the present study was only 12 hours. It is possible that this was sufficient to induce a temporary ‘stress’ which may have enhanced ethylene sensitivity. Low temperature stress is also known to enhance ethylene production by chilling-sensitive plant tissue upon return to warmer temperatures (Wang 1989). Further, the number of ethylene receptors has been recently proposed to increase in response to enhanced ethylene biosynthesis (Klee and Tieman 1997). However, G. ‘Sylvia’ and C. uncinatum are not considered chilling sensitive crops (Ligawa et al. 1997; Joyce 1988, respectively).

Pre-treatment of G. ‘Sylvia’ inflorescences with 10 nL 1-MCP/L at 20oC afforded protection against ethylene-induced flower abscission for only 2 days after 1-MCP pre-treatment (Table 4.1). This observation is similar to that discussed above for C. uncinatum, and suggests that synthesis of ethylene

5 receptors in the abscission zones of G. ‘Sylvia’ and C. uncinatum flowers is very rapid. Other horticultural commodities such as carnation flowers, banana and tomato fruit were reported by Sisler et al. (1996b) and Sisler and Serek (1997) to remain insensitive to ethylene for 10-15 days after 1-MCP pre- treatment.

In contrast to 1-MCP pre-treatment , pulsing C. uncinatum sprigs with STS (0.5 mM Ag+) at 2 or 20oC provided complete protection against ethylene for the duration of experiments (ca. 10 days) (Tables 4.2, 4.3 and 4.4). Minor flower abscission from ‘Lollypop’ sprigs pulsed with STS at 20oC was, however, associated with the highest accumulation of Ag+ (0.353 µmol Ag+/g sprig FW) during STS pulsing. This flower abscission associated with STS treatment suggests that sprigs suffered STS phytotoxicity. Uptake of Ag+ by C. uncinatum above 0.6 µmol Ag+/g sprig FW was shown by Joyce (1988) to be toxic and to cause flower abscission. The safe and effective range of Ag+ accumulation by C. uncinatum may vary with genotype. Cameron and Reid (1981) reported that effective STS treatment concentrations are usually close to the phytotoxic level. Relatively low Ag+ accumulation was observed for sprigs pulsed with STS at 2oC, presumably because transpiration was reduced at low temperature.

Higher vase solution uptake rates were observed during the pre-treatment period for ‘Lollypop’, ‘Alba’ and ‘Mid Pink’ sprigs which were not enclosed in chambers (i.e. those pre-treated with STS and those not pre-treated with 1-MCP or STS) (Figures 4.10, 4.11 and 4.12). Possibly, the presence of lower relative humidity and greater air circulation in the controlled environment rooms caused an increase in the transpiration and hence solution uptake by these sprigs. Vase solution uptake by ‘Alba’ and ‘Mid Pink’ sprigs tended to decrease and became more stable in association with ethylene-induced flower abscission, possibly reflecting reduced transpirational surface area.

Earlier workers have shown that effective Ag+ treatments provide long term protection against ethylene.

For example, new growth from pea seedlings following spray pre-treatment with AgNO3 showed no sensitivity to ethylene, indicating that silver acted systemically (Beyer, 1976). Additionally, STS pre- treatment prevented ethylene-induced flower abscission from Zygocactus for at least 4 weeks (Cameron and Reid 1981). Ag+ is presumably retained as a ‘pool’ in or around C. uncinatum flower abscission zones and may remain available to bind to newly formed receptors.

In the present study, 1-MCP pre-treatment was shown to protect G. ‘Sylvia’, C. uncinatum against ethylene, thereby extending their postharvest longevity. However, the efficacy of 1-MCP pre-treatment was reduced at low temperature. STS pre-treatment was shown to provide longer term protection against ethylene than 1-MCP pre-treatment. The duration of persistence of 1-MCP pre-treatment effects varied depending on treatment temperature (e.g. 2 vs. 20oC) and genus (e.g. G. ‘Sylvia’ vs. C. uncinatum). These results highlight the need for research into refining 1-MCP treatments to provide cut flowers with commercially viable longer term protection against ethylene. Compared with STS, 1-MCP is safe and easy to apply. Moreover, the results of this study indicate that for cut flowers, effective protection during the relatively brief but critical period of unrefrigerated export by airplane can be achieved.

6 CHAPTER 5

COMMERCIAL SCALE 1-MCP TREATMENTS PROTECT GERALDTON WAXFLOWER AGAINST ETHYLENE-INDUCED FLOWER ABSCISSION

5.1 INTRODUCTION

Native Australian flowers are traded internationally as exotic alternatives to traditional cut flowers such as roses, carnations and chrysanthemums (Joyce et al. 1993). Geraldton waxflower (Chamelaucium uncinatum, Myrtaceae) is Australia’s most valuable native cut flower export (FECA 1996). C. uncinatum stems have small flowers and leaves and are attractive as a filler in floral arrangements or when displayed alone (Joyce 1993). However, exposure of cut C. uncinatum stems to ethylene elicits flower abscission thereby reducing postharvest quality and marketability (Joyce 1988, 1989, 1993). Consignments of C. uncinatum can often reach export destinations suffering from extensive flower abscission due to unintentional exposure to exogenous sources of ethylene or the accumulation of endogenous ethylene within cartons (Joyce 1993).

Pulse treating C. uncinatum stems with STS solution prevents ethylene-induced flower abscission (Joyce 1988). STS is presumed to bind to ethylene receptors in plant tissue and block ethylene perception (Sisler 1982). Because Ag+ in STS is a heavy metal and possible environmental risk, legislators in some countries are reconsidering its commercial use (Serek et al. 1994a). The alternative gaseous inhibitor of ethylene perception, 1-MCP, is apparently non-toxic and can prevent ethylene-induced flower abscission from cut phlox (Porat et al. 1995b), Penstemon (Serek et al. 1995a), Kalanchoe (Reid et al. 1996) and C. uncinatum (Serek et al. 1995c).

It was hypothesised that effective 1-MCP treatments developed by Serek et al. (1995c) for C. uncinatum under laboratory conditions could be replicated on a commercial scale. The purpose of the present study was to devise several 1-MCP pre-treatment systems suitable for use on a commercial scale and to test the efficacy of each system in preventing ethylene-induced flower abscission from C. uncinatum.

5.2 MATERIALS AND METHODS

5.2.1 Plant material and preparation

Branches of C. uncinatum cultivars ‘CWA Pink’, ‘Fortune Cookie’, ‘Lollypop’ and ‘Purple Pride’ with 50% or more flowers open were harvested from a cut flower farm near Gatton (27o 34’S 152o 17’E) in

7 S.E. Qld. C. uncinatum cultivars ‘Paddy’s Late’ and ‘Alba’ were harvested from farms near Esk (27o 14’S 152o 25’E) in S.E. Qld and Cunnamulla (28o 04’S 145o 40’E) in southern Qld, respectively, and transported dry to the Gatton farm within 24 hours of harvest. Branches were trimmed to 50 cm in length and bunched to approximately 350-400 g. Flowering ends of bunches were then treated in a fungicide dip   [1mL Rovral (a.i. iprodione) and 5 mL Cislin (a.i. deltamethrin)/L of rain water] for 20-30 seconds.

5.2.2 Chemicals

1-MCP and ethylene stocks were prepared and quantified as previously described (sections 2.2.2.1 and 2.2.2.2). STS stock solution (8 mM Ag+) was prepared by the grower using the method of Joyce (1992). STS solutions for the treatment of cut C. uncinatum were diluted to 0.2 mM Ag+.

5.2.3 Treatments

5.2.3.1 Application of 1-MCP inside polyethylene tents

Bunches of ‘CWA Pink’ were stood into buckets of rain water and placed inside a 30m3 polyethylene tent (dimensions: 5 m long, 3 m wide, 2 m high) positioned inside a covered packing shed (Plate 5.1). The tent had an opening at one end which permitted access to the inside. An electric fan was placed inside the tent to stir the air. A bottle containing 1-MCP gas calculated to create a concentration of 200 nL 1- MCP/L inside the tent was placed inside and opened. The tent opening was then immediately closed and sealed with polethylene tape for 6 hours. The stirring fan was operated for 15 minutes. During the 6 hour pre-treatment, temperature around the tent was recorded to be ca. 20oC. Other bunches were either stood into buckets of water or STS (0.2 mM Ag+) and remained outside the tent.

At the completion of 1-MCP pre-treatment, an exhaust fan was fitted to the rear of the tent and used for 10-15 minutes to expel air and 1-MCP to the outside of the packing shed. Six bunches from each treatment (1-MCP, STS or water) were randomly selected and packed into individual fibreboard flower cartons (internal dimensions: 100 cm long, 26.5 cm wide and 8 cm high) lined with blank newsprint. Bunches were packed so that the flower end was alternated with the cut stem end. Lids of cartons were secured in place with polyethylene strapping tape. Ventilation holes in the ends of cartons were left open. Flowering sprigs 20 cm in length were randomly sampled from the remaining bunches from each treatment and placed immediately into buckets containing DI water. Cartons and sprigs were then taken in an air conditioned car to the UQG laboratory within 20 minutes.

At the laboratory, cartons were arranged in a CRD inside a controlled temperature room operating at 20oC and 50% RH. Cartons remained in this room for 6 days under continuous illumination by cool white fluorescent lights (10 µmol/m2/s at carton height). The cut ends of sprigs were recut under DI water, removing at least 2 cm from the stem base and placed into 375 mL capacity vases containing the same solution described in section 2.2.1. All sprigs and their vases were randomly placed into 60.5 L glass

8 chambers each containing 4 jars of 10 mL 1M KOH and a filter paper (section 2.2.3). Chamber lids were then sealed in place with polyethylene tape. Sprigs were exposed to 10 µL ethylene/L for 12 hours at 20oC (section 2.2.4.1). At the completion of ethylene treatment, sprigs and their vases were transferred to a vase life room operating at the same conditions as described in section 2.2.4.1. At the end of the 6 day storage period, cartons were opened and sprigs removed at random from bunches. Stem ends of sprigs were recut under DI water and placed into vase solutions as described above. Sprigs and their vases were then taken to the vase life room.

Plate 5.1. Treatment tent showing polyethylene structure (A), stirring fan (B), open bottle containing 1-MCP gas (C) and bunches of C. uncinatum (D).

9 In a second experiment, bunches of ‘Fortune Cookie’ were stood into buckets of rain water and taken by an air conditioned car to the laboratory. Bunches in water were then evenly allocated to two 470 L volume polyethylene tents. Tents were placed individually inside coolrooms operating at 2 and 20oC. Additional bunches remained in water or were stood into STS (0.2 mM Ag+) and placed next to each tent. Tent openings were closed and sealed with polyethylene tape. Stem temperature was monitored with thermocouples on additional bunches until they reached the desired temperature. Bunches inside tents were treated with 200 nL 1-MCP/L for 14 hours at 2 or 20oC in the dark. At the completion of 1-MCP pre-treatment, tents were opened and ventilated outside the laboratory. All bunches were stood into rain water. Flowering sprigs were pruned from bunches in each treatment at random. The cut ends of sprigs were recut under DI water, placed into vase solutions and treated with ethylene as described for the first experiment. All remaining bunches were taken in an air conditioned car to the Gatton farm.

At the farm, bunches were randomly selected from each treatment and packed into flower cartons as described for the first experiment. Cartons were then returned to the laboratory by car and arranged in a CRD inside the same 20oC room used for the first experiment. Cartons remained in this room for 6 days. Sprigs exposed to ethylene and those that were removed from bunches after storage in cartons for 6 days were placed into vases (section 2.2.1) and kept in the vase life room used in the first experiment.

5.2.3.2 Injection of 1-MCP into cartons

Bunches of ‘Lollypop’ were stood in buckets of rain water to hydrate for 3 hours at 20oC. Bunches were then packed into two flower cartons. The ventilation holes at each end of cartons were closed with fibreboard disks and sealed onto the carton with polyethylene tape. A rubber septum was inserted tightly through one end of the cartons and acted as a gas injection port. Cartons were placed into a coolroom operating at 2oC. An aliquot of 1-MCP gas was injected through the septum into one carton such that an internal concentration of 200 nL 1-MCP/L was created. Bunches inside the other carton remained in air without 1-MCP. After 24 hours, cartons were opened outside the coolroom and sprigs removed from bunches at 5, 25, 75 and 95 cm from the injection point (Plate 5.2). Sprigs were recut under DI water, placed in vase solution and treated with ethylene as described in section 5.2.3.1. Following ethylene treatment, sprigs and their vases were taken to the vase life room. In a second experiment, the same procedure was applied to bunches of ‘Purple Pride’ with the exception that the 1-MCP concentration was increased to 2 µL/L.

5.2.3.3 Application of 1-MCP into a coolroom

Bunches of ‘Paddy’s Late’ that had been held dry for 24 hours after harvest were stood into buckets of rain water and placed at several positions in a coolroom operating at 2oC. Stem temperature of an additional bunch was monitored with a thermocouple until it reached 2oC. A stock bottle of 1-MCP was then placed in the centre of the room and opened. The stock was calculated to create a 1-MCP concentration of 150 nL/L inside the room. Sliding access doors to the room were closed and circulation

10 fans operated for the duration of treatment. Additional bunches were stood into buckets of rain water and transported to the laboratory. These bunches were placed into a controlled temperature room operating at 2oC. After 15 hours, the room fumigated with 1-MCP was opened and ventilated for 10-15 minutes before bunches were removed. Bunches from the controlled temperature room at the laboratory were then removed and taken to the Gatton farm. Bunches from each treatment were then selected at random and packed into flower cartons as described in section 5.2.3.1. Flowering sprigs from the remaining bunches in each treatment were removed at random and stood into DI water. Cartons and sprigs were transported to the laboratory. At the laboratory, cartons were arranged at 20oC (section 5.2.3.1). Cut ends of sprigs were recut under DI water and placed into vases (section 5.2.3.1). Half of the sprigs from each of these treatments were then treated with ethylene as described in section 5.2.3.1. The other half of the sprigs were held in air without exogenous ethylene. Cartons were opened after 6 days and sprigs removed at random from bunches. Sprigs and their vases were kept in the vase life room.

5.2.3.4 Application of 1-MCP by forced-air cooling

Bunches of ‘Purple Pride’ were stood into buckets of rain water and hydrated for 3 hours. Half of the bunches were then packed into flower cartons (section 5.2.3.1). Half of the bunches remaining in water and cartons were then placed into a coolroom operating at 2oC. Bunches in water were placed at several positions within the room while cartons were arranged against a forced-air cooler. Sliding doors to the room were closed and the forced-air cooler was operated. The other half of the bunches in water and cartons remained in air at ca. 20oC. After 3 hours the room was opened and bunches and cartons were removed and placed at ca. 20oC. The other half of the bunches in water and cartons were then placed into the coolroom and against the forced-air cooler, respectively. A stock bottle of 1-MCP was opened and released as described in section 5.2.3.3. The 1-MCP concentration created inside the room was calculated to be 200 nL/L. After 3 hours the room was opened and ventilated for 10-15 minutes and bunches and cartons were removed. Bunches in water and cartons from all treatments were then taken to the laboratory by car. Flowering sprigs were removed at random from bunches in each treatment and prepared for ethylene treatment. Flowering sprigs were removed from bunches in cartons at 5, 25, 75 and 95 cm from the carton end furtherest from the forced-air cooler wall (Plate 5.2). Sprigs were prepared for ethylene treatment as previously described (section 5.2.3.1.). Half of the sprigs from each treatment were then treated with ethylene (section 5.2.3.1). The other half of the sprigs remained in air without exogenous ethylene. At the completion of ethylene treatment, sprigs and their vases were transferred to the vase life room.

5.2.3.5 Slow release of 1-MCP inside cartons

Bunches of ‘Alba’ were stood in buckets of rain water for 7 days at 2oC. Bunches were then packed into cartons (section 5.2.3.1). Glass 33 mL volume tubes containing 6928 ± 177 µL 1-MCP/L that were sealed with a rubber septa were included in cartons either at one end only, both ends or at both ends and the centre of cartons (Plate 5.2). Additional cartons were packed with bunches but without the inclusion of

11 tubes and acted as the control treatment. Cartons were then transported to the laboratory by car and arranged in a CRD inside a 20oC coolroom (section 5.2.3.1). After 6 days, cartons were opened. Flowering sprigs were pruned from bunches at 5, 25, 75 and 95 cm from the position of the first tube (Plate 5.2). Sprigs were then prepared for ethylene treatment as described in section 5.2.3.1. Half of the sprigs from each treatment were then treated with ethylene (section 5.2.3.1). The other half of the sprigs remained in air without exogenous ethylene. Following ethylene treatment, all sprigs and their vases were taken to the vase life room.

Plate 5.2. Commercial flower carton showing fibreboard wall structure (A), flowering C. uncinatum bunches (B), 1-MCP injection point/intake end (C), sprig sampling positions (D1, D2, D3 and D4) and glass tubes containing 1-MCP gas (E1, E2 and E3).

12 5.2.4 Quality assessment

Sprigs and their vases were weighed separately daily during vase life to allow determination of relative fresh weight and vase solution uptake, respectively. Flower abscission from sprigs was assessed daily using the 5 point scale described in section 2.2.5.1. Sprig vase life was judged using the same criteria presented in section 3.2.5.1.

Bunches were weighed individually before being packed into cartons and again at the end of the 6 day storage period to enable calculation of weight loss. The accumulation of abscised flowers and leaves from bunches in each carton after the storage period was weighed and expressed as a percentage of the initial bunch weight. The concentration of 1-MCP gas inside glass tubes used in the final experiment was quantified before and after the 6 day treatment period by gas chromatography (section 2.2.5.2). Postharvest longevity was used to describe the time in days from harvest to the end of vase life (section 3.2.5.1) for sprigs from bunches held inside cartons for 6 days.

5.2.5 Experiment design and data analysis

In all experiments, sprigs were arranged in a CRD in the vase life room. Three to ten replicates were used for each treatment, depending upon the particular experiment. The application of 1-MCP inside polyethylene tents was examined in two experiments. The first experiment was a one factor (treatment) design while the second experiment was a 3 (treatment) x 2 (temperature) factorial design. Injection of 1- MCP into cartons was examined as 2 (1-MCP) x 4 (position) factorial experiments. A 2 (1-MCP) x 2 (ethylene) factorial experiment was used to study the application of 1-MCP into a coolroom. The efficacy of 1-MCP treatment applied by forced-air cooling was determined using a 2 (1-MCP) x 2 (ethylene) x 4 (position) factorial experiment. A 4 (1-MCP) x 2 (ethylene) x 4 (position) factorial experiment was used to study the slow release of 1-MCP inside cartons.

 Treatment means ± standard errors were calculated using Microsoft Excel (Version 5.0, Microsoft Inc.).  Figures were created using Sigmaplot (Version 2.0, Jandel Corporation). Most data were analysed as  split plot for time ANOVAs by the balanced ANOVA function of Minitab (Release 11.12, Minitab Inc.). Bunch weight loss, accumulated abscised flowers and leaves from bunches and sprig longevity data were analysed as one-way ANOVAs. Flower abscission score data were converted to a corresponding percentage and arcsine transformed for ANOVA (Steel and Torrie 1987). Treatment means were separated by the LSD test at P = 0.05. LSDs are presented for between treatments for data analysed as split plots for time (section 2.2.6). LSDs are only presented when significant (P < 0.05) differences between treatment means existed. Differences between treatment means at the P < 0.05 level are referred to in the results as significant. LSDs from ANOVAs of transformed data sets are not presented (section 2.2.6).

5.3 RESULTS

13 5.3.1 Application of 1-MCP inside polyethylene tents

Pre-treatment of C. uncinatum ‘CWA Pink’ bunches with 200 nL 1-MCP/L for 6 hours at 20oC inside a polyethylene tent protected flowering sprigs against exposure to 10 µL ethylene/L for 12 hours at 20oC (Plate 5.3). 1-MCP pre-treatment significantly reduced ethylene-induced flower abscission from sprigs (Figure 5.1). The STS pre-treatment protocol (0.2 mM Ag+ for 6 hours at 20oC) used in this experiment was only partially effective in reducing ethylene-induced flower abscission compared to 1-MCP pre- treatment (Figure 5.1). This differential response accounted for the significant interaction between treatment and time of measurement for flower abscission (Appendix 5.1). The decrease in sprig relative fresh weight was significantly reduced by 1-MCP or STS pre-treatments (Figure 5.1 and Appendix 5.2). Solution uptake by sprigs increased to day 2 or 3, then declined over time for sprigs from all treatments (Figure 5.1). Solution uptake between days 2 and 4 was lowest for sprigs pre-treated with STS and highest for sprigs pre-treated with 1-MCP. This uptake pattern probably resulted in the significant interaction between treatment and time of measurement for vase solution uptake (Appendix 5.3). As vase life was partly based on flower abscission, 1-MCP pre-treatment prevented the ethylene-induced loss in vase life (Table 5.1 and Appendix 5.4). Vase lives of sprigs pre-treated with STS were only marginally, but significantly longer than sprigs exposed only to ethylene. The loss of weight from ‘CWA Pink’ bunches pre-treated with 200 nL 1-MCP/L or 0.2 mM Ag+ for 6 hours at 20oC and held in cartons for 6 days at 20oC was significantly reduced compared to bunches not pre-treated with 1-MCP or STS (Table 5.2 and Appendix 5.5). Bunches not pre-treated with 1-MCP or STS accumulated the most abscised flowers and leaves in cartons during storage (Table 5.2). However, during the subsequent vase life of sprigs taken from these bunches, there was no flower abscission at all. The loss of sprig relative fresh weight during vase life was greatest from sprigs pre-treated with 1-MCP compared sprigs pre-treated with STS or those not pre-treated with 1-MCP or STS (Figure 5.2 and Appendix 5.6). Sprigs pre-treated with 1-MCP used significantly less vase solution between days 6 and 7 (i.e. the first day after storage) than sprigs pre-treated with STS or those not pre-treated with 1-MCP or STS (Figure 5.2 and Appendix 5.7). Longevity of sprigs pre-treated with 1-MCP or STS were significantly extended compared to sprigs not pre-treated with 1-MCP or STS (Table 5.2 and Appendix 5.8).

14 Plate 5.3. C. uncinatum ‘CWA Pink’ sprigs on day 2 after treatment on day 0 with 0 nL 1-MCP/L (control) (LHS), STS (0.2 mM Ag+) (centre) or 200 nL 1-MCP/L (RHS) followed by exposure to 10 µL ethylene/L on day 1. Note: extensive flower abscission is evident in the control sprig (LHS).

15 5

2 Abscission score 1

100

80 (% of initial FW)

Relative fresh weight 60

0.8

0.6

0.4

0.2 Solution uptake

(mL/ g initial FW/ day) 0.0 0246810 Time (days)

Figure 5.1. Flower abscission (scores: 1 = < 10% to 5 = > 80%), relative fresh weight and vase solution uptake for C. uncinatum ‘CWA Pink’ sprigs pre-treated on day 0 with 200 nL 1-MCP/L (■), STS (0.2 mM Ag+) (▲) or 0 nL 1-MCP/L and 0 mM Ag+ (●) for 6 hours at 20oC. Sprigs from each of these treatments were then immediately exposed on day 0 to 10 µL ethylene/L for 12 hours at 20oC. Vertical bars represent standard errors of means (n = 10). Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD for relative fresh weight data = 2.9%. LSD for solution uptake data = 0.051 mL/g initial FW/day.

16 Table 5.1. Vase life (mean ± s.e.) of C. uncinatum ‘CWA Pink’ sprigs pre-treated on day 0 with 200 nL 1-MCP/L, STS (0.2 mM Ag+) or 0 nL 1-MCP/L and 0 mM Ag+ for 6 hours at 20oC. Sprigs were then immediately exposed on day 0 to 10 µL ethylene/L for 12 hours at 20oC. Data followed by the same letter are not significantly different (LSD = 1.6) at P = 0.05 (n = 10).

Treatment Vase life (days)

200 nL 1-MCP/L 8.0 ± 0.5 c 0.2 mM Ag+ 3.0 ± 0.1 b 0 nL 1-MCP/L and 0 mM Ag+ 1.2 ± 0.1 a

Table 5.2. Weight loss (mean ± s.e.) and proportion of abscised flowers and leaves from bunches of C. uncinatum ‘CWA Pink’ pre-treated on day 0 with 200 nL 1-MCP/L, STS (0.2 mM Ag+) or 0 nL 1-MCP/L and 0 mM Ag+ for 6 hours at 20oC. Following treatment, bunches were packed into commercial flower cartons and held for 6 days at 20oC. Longevity (mean ± s.e.) of sprigs from bunches is presented. Data within each column followed by the same letter are not significantly different (LSD = 6.6 and 1.2 for weight loss and longevity, respectively) at P = 0.05 (n = 10).

Treatment Weight loss (% of Abscised flowers and Longevity (days) initial FW) leaves (% of initial FW)

200 nL 1-MCP/L 23.5 ± 1.6 a 0.16 9.8 ± 0.5 b 0.2 mM Ag+ 20.1 ± 1.3 a 0.19 9.7 ± 0.5 b 0 nL 1-MCP/L and 0 mM Ag+ 31.6 ± 3.1 b 0.20 7.3 ± 0.3 a

17 120

100 (% of initial FW) Relative fresh weight 80

0.8

0.6

0.4

Solution uptake 0.2 (mL/ g initial FW/ day) 0.0 67891011 Time (days)

Figure 5.2. Relative fresh weight and vase solution uptake for C. uncinatum ‘CWA Pink’ sprigs from bunches pre-treated on day 0 with 200 nL 1-MCP/L (■), STS (0.2 mM Ag+) (▲) or 0 nL 1- MCP/L and 0 mM Ag+ (●) for 6 hours at 20oC. Bunches were then packed into commercial flower cartons and held for 6 days at 20oC. Vertical bars represent standard errors of means. Where no vertical bars appear, the standard error was smaller than the size of the symbol (n = 10). LSD for relative fresh weight data = 4.0%. LSD for vase solution uptake data = 0.040 mL/g initial FW/day.

18 In the second experiment, pre-treatment of ‘Fortune Cookie’ bunches with 200 nL 1-MCP/L or 0.2 mM Ag+ for 14 hours at 2 or 20oC inside polyethylene tents prevented ethylene-induced flower abscission from flowering sprigs (Figure 5.3). Pre-treatment with 1-MCP or STS at 2 or 20oC significantly reduced the decline in sprig relative fresh weight associated with flower abscission (Figure 5.4). However, the loss in sprig relative fresh weight after day 6 was most effectively reduced from sprigs pre-treated with 1-MCP compared to sprigs pre-treated with STS, particularly for sprigs pre-treated at 2oC. These responses account for the significant interactions between treatment and time of measurement and between pre- treatment temperature and time of measurement (Appendix 5.9). Solution uptake by sprigs from all treatments fluctuated over time (Figure 5.5). Sprigs pre-treated with STS used vase solution at the highest rate until day 5, thereafter, solution uptake was greatest by sprigs pre-treated with 1-MCP. This response reflects the significant interaction between treatment and time of measurement for vase solution uptake (Appendix 5.10). As vase life was partly based on flower abscission, 1-MCP and STS pre-treatments at 2 or 20oC prevented the ethylene-induced loss in vase life (Table 5.3). Vase lives of sprigs pre-treated with STS at 2oC were significantly longer than parallel sets of sprigs pre-treated with 1-MCP (Appendix 5.11).

Bunches pre-treated with 1-MCP at 2oC and then held in cartons for 6 days at 20oC lost significantly more weight than parallel sets of bunches pre-treated with STS and bunches not pre-treated with 1-MCP or STS (Table 5.4). Conversely, bunches pre-treated with 1-MCP at 20oC lost significantly less weight than bunches not pre-treated with 1-MCP or STS. The loss of weight from bunches pre-treated at 20oC was slightly reduced by STS pre-treatment compared to bunches not pre-treated with STS. As a result, a significant interaction between pre-treatment and pre-treatment temperature was evident (Appendix 5.12). The accumulation of abscised flowers and leaves in cartons was greatest from bunches not pre-treated with 1-MCP or STS at 2 and 20oC and least from bunches pre-treated with STS at 2 and 20oC (Table 5.4).

No flower abscission from sprigs was recorded during the subsequent vase life. Sprigs pre-treated with 1- MCP or STS at 2oC lost significantly more relative fresh weight during vase life than parallel sets of sprigs pre-treated at 20oC (Figure 5.6). This reponse probably reflects the significant interactions between pre-treatment temperature and time of measurement and between pre-treatment and pre-treatment temperature for relative fresh weight (Appendix 5.13). Vase solution uptake by sprigs not pre-treated with 1-MCP and STS at 2oC was higher between days 1 and 2 than for parallel sets of sprigs pre-treated at 20oC (Figure 5.7). This reponse probably accounts for the significant interactions between pre-treatment and time of measurement and between pre-treatment temperature and time of measurement for vase solution uptake (Appendix 5.14). 1-MCP pre-treatment at 2 and 20oC extended the longevity of sprigs marginally compared to sprigs not pre-treated with 1-MCP, although no significant difference between these treatments existed (Table 5.3 and Appendix 5.15). STS pre-treatment did not extend the longevity of sprigs.

19 5 2oC 4

1 5 20oC

Abscission score Abscission 4

024681012 Time (days)

Figure 5.3. Flower abscission (scores: 1 = < 10% to 5 = > 80%) from C. uncinatum ‘Fortune Cookie’ sprigs pre-treated on day 0 with 200 nL 1-MCP/L (■), STS (0.2 mM Ag+) (▲) or 0 nL 1- MCP/L and 0 mM Ag+ (control treatment) (●) for 14 hours at 2oC or 20oC. Sprigs from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Vertical bars represent standard errors of means (n = 5). Where no vertical bars appear, the standard error was smaller than the size of the symbol.

20 o 100 2 C

20 o 100 20 C

Relative fresh weight (% of initial FW) initial of (% weight fresh Relative 40

20 024681012 Time (days)

Figure 5.4. Relative fresh weight of C. uncinatum ‘Fortune Cookie’ sprigs pre-treated on day 0 with 200 nL 1-MCP/L (■), STS (0.2 mM Ag+) (▲) or 0 nL 1-MCP/L and 0 mM Ag+ (control treatment) (●) for 14 hours at 2oC or 20oC. Sprigs from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Vertical bars represent standard errors of means (n = 5). Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD = 4.8%.

21 o 1.6 2 C

1.2

0.8

0.4

0.0 o 1.6 20 C

1.2

0.8

Solution uptake (mL/g initial FW/day) (mL/g uptake Solution 0.4

0.0 024681012 Time (days)

Figure 5.5. Vase solution uptake by C. uncinatum ‘Fortune Cookie’ sprigs pre-treated on day 0 with 200 nL 1-MCP/L (■), STS (0.2 mM Ag+) (▲) or 0 nL 1-MCP/L and 0 mM Ag+ (control treatment) (●) for 14 hours at 2oC or 20oC. Sprigs from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Vertical bars represent standard errors of means (n = 5). Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD = 0.149 mL/g initial FW/day.

22 Table 5.3. Vase life (mean ± s.e.) of C. uncinatum ‘Fortune Cookie’ sprigs pre-treated on day 0 with 200 nL 1-MCP/L, STS (0.2 mM Ag+) or 0 nL 1-MCP/L and 0 mM Ag+ for 14 hours at 2 or 20oC. Sprigs were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Data followed by the same letters are not significantly different (LSD = 1.9) at P = 0.05 (n = 5).

Treatment Vase life (days)

2oC 200 nL 1-MCP/L 7.0 ± 0.6 b 0.2 mM Ag+ 9.0 ± 1.0 c 0 nL 1-MCP/L and 0 mM Ag+ 2.0 ± 0.0 a

20oC 200 nL 1-MCP/L 7.6 ± 0.5 bc 0.2 mM Ag+ 8.4 ± 1.0 bc 0 nL 1-MCP/L and 0 mM Ag+ 2.0 ± 0.0 a

Table 5.4. Weight loss (mean ± s.e.) and proportion of abscised flowers and leaves from bunches of C. uncinatum ‘Fortune Cookie’ pre-treated on day 0 with 200 nL 1-MCP/L, STS (0.2 mM Ag+) or 0 nL 1-MCP/L and 0 mM Ag+ for 14 hours at 2 or 20oC. Bunches were then packed into commercial flower cartons and held for 6 days at 20oC. Longevity (mean ± s.e.) of sprigs from bunches is presented. Data within each column followed by the same letters are not significantly different (LSD = 2.8 and 1.2 for weight loss (n = 6) and longevity (n = 5), respectively) at P = 0.05.

Treatment Weight loss (% of Abscised flowers and Longevity initial FW) leaves (% of initial (days) FW)

2oC 200 nL 1-MCP/L 14.4 ± 1.3 c 1.04 11.2 ± 0.4 b 0.2 mM Ag+ 9.4 ± 0.7 ab 0.63 9.8 ± 0.5 a 0 nL 1-MCP/L and 0 mM Ag+ 7.6 ± 0.5 a 1.54 10.4 ± 0.7 ab

20oC 200 nL 1-MCP/L 10.7 ± 0.7 b 1.06 10.4 ± 0.2 ab 0.2 mM Ag+ 12.4 ± 1.3 bc 0.39 9.6 ± 0.2 a 0 nL 1-MCP/L and 0 mM Ag+ 16.3 ± 1.0 c 1.16 10.2 ± 0.4 ab

23 2oC 120

100

20oC 120

100 Relative fresh weight (% of initial FW) initial of (% weight fresh Relative 80

789101112 Time (days)

Figure 5.6. Relative fresh weight of C. uncinatum ‘Fortune Cookie’ sprigs from bunches pre- treated on day 0 with 200 nL 1-MCP/L (■), STS (0.2 mM Ag+) (▲) or 0 nL 1-MCP/L and 0 mM Ag+ (control treatment) (●) for 14 hours at 2oC or 20oC. Bunches were then packed into commercial flower cartons and held for 6 days at 20oC. Vertical bars represent standard errors of means (n = 5). Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD = 8.2%.

24 1.0 2oC 0.8 0.6 0.4 0.2 0.0 1.0 20oC 0.8 0.6 0.4

Solution uptake (mL/g initial FW/day) 0.2 0.0 7 8 9 10 11 12 Time (days)

Figure 5.7. Vase solution uptake by C. uncinatum ‘Fortune Cookie’ sprigs from bunches pre- treated on day 0 with 200 nL 1-MCP/L (■), STS (0.2 mM Ag+) (▲) or 0 nL 1-MCP/L and 0 mM Ag+ (control treatment) (●) for 14 hours at 2oC or 20oC. Bunches were then packed into commercial flower cartons and held for 6 days at 20oC. Vertical bars represent standard errors of means (n = 5). Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD = 0.076 mL/g initial FW/day.

25 5.3.2 Injection of 1-MCP into cartons

Injection of 200 nL 1-MCP/L at 2oC into cartons containing ‘Lollypop’ bunches did not prevent ethylene- induced flower abscission from sprigs (Figure 5.8). Unexpectantly, sprigs not pre-treated with 1-MCP and sampled 5 cm from the carton end sustained moderate flower abscission. This response probably accounted for the significant interaction between 1-MCP pre-treatment, sampling position and time of measurement (Appendix 5.16). Consequently, 1-MCP pre-treatment did not reduce the loss of sprig relative fresh weight (Figure 5.9). Sprigs not pre-treated with 1-MCP and sampled 25 cm from the end of the carton lost significantly more relative fresh weight than sprigs sampled 5 cm from the end of the carton. This reponse probably reflects the significant interaction between 1-MCP pre-treatment, sampling position and time of measurement (Appendix 5.17). Vase solution uptake by sprigs from all treatments decreased over time until day 4 (Figure 5.10). Solution uptake by sprigs not pre-treated with 1-MCP and sampled 25 cm from the end of the carton was lower between days 1 and 2 and between days 4 and 5 compared to sprigs pre-treated with 1-MCP. As a result, a significant interaction between 1-MCP pre- treatment and sampling position was evident (Appendix 5.18). Because vase life was partly based on flower abscission, there was no effect of 1-MCP on vase lives of sprigs (Table 5.5).

Injection of 2 µL 1-MCP/L at 2oC into cartons containing ‘Purple Pride’ bunches did not afford flowering sprigs with protection against ethylene-induced flower abscission (Figure 5.11). Unexpectantly, higher levels of flower abscission were recorded for sprigs pre-treated with 1-MCP and sampled 25, 75 and 95 cm from the injection point compared to sprigs not pre-treated with 1-MCP. This response probably accounts for the significant interaction between 1-MCP pre-treatment, sampling position and time of measurement for flower abscission (Appendix 5.19). With the exception of sprigs sampled 5 cm from the end of the carton, the loss of relative fresh weight from sprigs pre-treated with 1-MCP was significantly greater than from sprigs not pre-treated with 1-MCP (Figure 5.12). Accordingly, a significant interaction between 1-MCP pre-treatment, sampling position and time of measurement for relative fresh weight was recorded (Appendix 5.20). With the exception of sprigs not pre-treated with 1-MCP and sampled from each end of the carton (i.e. 5 and 95 cm from the injection end), the use of vase solution by sprigs decreased over time (Figure 5.13). Vase solution uptake by sprigs pre-treated with 1-MCP was less variable than that by sprigs not pre-treated with 1-MCP. These responses probably account for the significant interactions between 1-MCP pre-treatment and time of measurement and between sampling position and time of measurement (Appendix 5.21). Injection of 1-MCP into cartons prevented the ethylene-induced loss in sprig vase life (Table 5.6). However, 1-MCP pre-treatment was only fully effective in protecting sprigs sampled 5 cm from the injection point against ethylene on the basis of extended vase life. Accordingly, a significant interaction between 1-MCP pre-treatment and sampling position was recorded for vase life (Appendix 5.22).

26 5 - 1-MCP

5 + 1-MCP

Abscission score Abscission 4

12345 Time (days)

Figure 5.8. Flower abscission (scores: 1 = < 10% to 5 = > 80%) from C. uncinatum ‘Lollypop’ sprigs on bunches pre-treated on day 0 with 0 or 200 nL 1-MCP/L in sealed flower cartons for 24 hours at 2oC. Flower abscission is presented for sprigs sampled at 5 (●), 25 (■), 75 (▲) and 95 cm (▼) from the 1-MCP injection point. Sprigs were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Vertical bars represent the standard errors of means (n = 5). Where no vertical bars appear, the standard error was smaller than the size of the symbol.

27 - 1-MCP 100

40 + 1-MCP 100

60 Relative fresh weight (% of initial FW) of initial (% weight fresh Relative

40 12345 Time (days)

Figure 5.9. Relative fresh weight of C. uncinatum ‘Lollypop’ sprigs from bunches pre-treated on day 0 with 0 or 200 nL 1-MCP/L in sealed flower cartons for 24 hours at 2oC. Relative fresh weight is presented for sprigs sampled at 5 (●), 25 (■), 75 (▲) and 95 cm (▼) from the 1-MCP injection point. Sprigs were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Vertical bars represent the standard errors of means (n = 5). Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD = 5.1%.

28 3 - 1-MCP

0 3 + 1-MCP

1 Solution uptake (mL/ g initial FW/ day) FW/ g initial uptake (mL/ Solution

0 12345 Time (days)

Figure 5.10. Vase solution uptake by C. uncinatum ‘Lollypop’ sprigs from bunches pre-treated on day 0 with 0 or 200 nL 1-MCP/L in sealed flower cartons for 24 hours at 2oC. Solution uptake is presented for sprigs sampled at 5 (●), 25 (■), 75 (▲) and 95 cm (▼) from the 1-MCP injection point. Sprigs were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Vertical bars represent the standard errors of means (n = 5). Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD = 0.130 mL/g initial FW/day.

29 Table 5.5. Vase life (mean ± s.e.) of C. uncinatum ‘Lollypop’ sprigs from bunches pre-treated on day 0 with 0 or 200 nL 1-MCP/L for 24 hours at 2oC inside a commercial flower carton. Sprigs were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Flowering sprigs were sampled from stems located 5, 25, 75 and 95 cm from the point of 1-MCP injection within a carton (positions 1, 2, 3 and 4, respectively).

Vase life (days)

Treatment Position means (n = 5) Treatment means (n = 20)

0 nL 1-MCP/L 2.0 ± 0.0 Position 1 2.0 ± 0.0 Position 2 2.0 ± 0.0 Position 3 2.0 ± 0.0 Position 4 2.0 ± 0.0 200 nL 1-MCP/L 2.0 ± 0.0 Position 1 2.0 ± 0.0 Position 2 2.0 ± 0.0 Position 3 2.0 ± 0.0 Position 4 2.0 ± 0.0

30 5 - 1-MCP

5 + 1-MCP

Abscission score Abscission 4

12345678 Time (days)

Figure 5.11. Flower abscission (scores: 1 = < 10% to 5 = > 80%) from C. uncinatum ‘Purple Pride’ sprigs on bunches pre-treated on day 0 with 0 or 2 µL 1-MCP/L in sealed flower cartons for 24 hours at 2oC. Flower abscission is presented for sprigs sampled at 5 (●), 25 (■), 75 (▲) and 95 cm (▼) from the 1-MCP injection point. Sprigs were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Vertical bars represent the standard errors of means (n = 5). Where no vertical bars appear, the standard error was smaller than the size of the symbol.

31 120 - 1-MCP

100

60 120 + 1-MCP

100

80 Relative fresh weight of (% initial FW) 60 12345678 Time (days)

Figure 5.12. Relative fresh weight of C. uncinatum ‘Purple Pride’ sprigs from bunches pre-treated on day 0 with 0 or 2 µL 1-MCP/L in sealed flower cartons for 24 hours at 2oC. Relative fresh weight is presented for sprigs sampled at 5 (●), 25 (■), 75 (▲) and 95 cm (▼) from the 1-MCP injection point. Sprigs were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Vertical bars represent the standard errors of means (n = 5). Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD = 5.0%.

32 0.4 - 1-MCP

0.3

0.2

0.1

0.0

0.4 + 1-MCP

0.3

0.2

Solution uptake (mL/g initialSolution uptake (mL/g FW/day) 0.1

0.0

12345678 Time (days)

Figure 5.13. Vase solution uptake by C. uncinatum ‘Purple Pride’ sprigs from bunches pre-treated on day 0 with 0 or 2 µL 1-MCP/L in sealed flower cartons for 24 hours at 2oC. Vase solution uptake is presented for sprigs sampled at 5 (●), 25 (■), 75 (▲) and 95 cm (▼) from the 1-MCP injection point. Sprigs were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Vertical bars represent the standard errors of means (n = 5). Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD = 0.042 mL/g initial FW/day.

33 Table 5.6. Vase life (mean ± s.e.) of C. uncinatum ‘Purple Pride’ sprigs from bunches pre-treated on day 0 with 0 or 2 µL 1-MCP/L for 24 hours at 2oC inside a commercial flower carton. Sprigs were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Flowering sprigs were sampled from stems 5, 25, 75 and 95 cm from the point of 1-MCP injection within a carton (positions 1, 2, 3 and 4, respectively). Data within each column followed by the same letters are not significantly different (LSD = 2.1 and 1.0 for position and treatment means, respectively) at P = 0.05.

Vase life (days)

Treatment Position means (n = 5) Treatment means (n = 20)

0 nL 1-MCP/L 3.6 ± 0.4 a Position 1 2.4 ± 0.4 a Position 2 3.6 ± 0.8 ab Position 3 2.8 ± 0.8 a Position 4 5.4 ± 0.9 bc 200 nL 1-MCP/L 5.3 ± 0.4 b Position 1 7.2 ± 0.4 c Position 2 5.0 ± 0.5 b Position 3 4.4 ± 0.8 ab Position 4 4.4 ± 1.1 ab

34 5.3.3 Application of 1-MCP into a coolroom

Pre-treatment of ‘Paddy’s Late’ bunches standing in buckets of water with 150 nL 1-MCP/L for 15 hours at 2oC inside a coolroom significantly reduced ethylene-induced flower abscission from sprigs and the associated loss of relative fresh weight (Figure 5.14). 1-MCP pre-treatment did not reduce flower abscission or the associated loss in relative fresh weight from sprigs not exposed to ethylene. Accordingly, a significant interaction between 1-MCP pre-treatment, ethylene treatment and time of measurement was recorded for flower abscission (Appendix 5.23) and relative fresh weight (Appendix 5.24). Vase solution uptake by sprigs from all treatments fluctuated over time (Figure 5.14). Sprigs pre- treated with 1-MCP used significantly more vase solution than sprigs exposed only to ethylene. Vase solution uptake by sprigs pre-treated with 1-MCP and exposed to ethylene was consistently lower than sprigs pre-treated only with 1-MCP except between days 5 and 6. These responses probably account for the significant interactions between 1-MCP pre-treatment and ethylene treatment and between 1-MCP pre- treatment and time of measurement (Appendix 5.25). 1-MCP pre-treatment prevented the ethylene- induced loss in vase life (Table 5.7). Vase lives of sprigs not exposed to ethylene were not significantly extended by 1-MCP pre-treatment. Consequently, a significant interaction between 1-MCP pre-treatment and ethylene treatment for vase life was evident (Appendix 5.26).

Pre-treatment of bunches with 1-MCP at 2oC followed by storage in cartons for 6 days at 20oC did not reduce the loss of fresh weight or the accumulation of abscised flowers and leaves in cartons (Table 5.8, Appendices 5.27 and 5.28). Similarly, flower abscission, the associated loss of relative fresh weight and vase solution uptake by sprigs during the subsequent vase life were not significantly affected by 1-MCP pre-treatment (Figure 5.15, Appendices 5.29, 5.30 and 5.31). Longevity of sprigs were not significantly extended by 1-MCP pre-treatment (Table 5.8 and Appendix 5.32).

35 5

2 Abscission score Abscission 1 100 80 60 40

(% of initial FW) initial of (% 20

Relative fresh weight weight fresh Relative 0 0.8

0.6

0.4

0.2 Solution uptake uptake Solution

(mL/g initial FW/day) initial (mL/g 0.0 12345678 Time (days)

Figure 5.14. Flower abscission (scores: 1 = < 10% to 5 = > 80%), relative fresh weight and vase solution uptake for C. uncinatum ‘Paddy’s Late’ sprigs treated with 0 nL 1-MCP/L and 0 µL ethylene/L (●), 0 nL 1-MCP/L and 10 µL ethylene/L (■), 150 nL 1-MCP/L and 0 µL ethylene/L (▲) or 150 nL 1-MCP/L and 10 µL ethylene/L (▼). 1-MCP pre-treatment was conducted on day 0 for 15 hours at 2oC. Ethylene treatment was conducted on day 1 for 12 hours at 20oC. Vertical bars represent standard errors of means (n = 10). Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD for relative fresh weight data = 5.4%. LSD for vase solution uptake data = 0.075 mL/g initial FW/day.

36 Table 5.7. Vase life (mean ± s.e.) of C. uncinatum ‘Paddy’s Late’ sprigs pre-treated on day 0 with 0 or 150 nL 1-MCP/L for 15 hours at 2oC. Half of the sprigs from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Data followed by the same letters are not significantly different (LSD = 1.0) at P = 0.05 (n = 10).

Treatment Vase life (days)

0 nL 1-MCP/L 0 µL ethylene/L 4.2 ± 0.5 b 10 µL ethylene/L 2.0 ± 0.0 a 150 nL 1-MCP/L 0 µL ethylene/L 4.4 ± 0.2 b 10 µL ethylene/L 3.7 ± 0.5 b

Table 5.8. Fresh weight loss and proportion of abscised flowers and leaves (mean ± s.e.) from bunches of C. uncinatum ‘Paddy’s Late’ pre-treated on day 0 with 0 or 150 nL 1-MCP/L for 15 hours at 2oC and held inside commercial flower cartons for 6 days at 20oC. Longevity (mean ± s.e.) of sprigs from bunches is presented. Data within each column were not significantly different at P = 0.05.

Treatment Weight loss (% of Abscised flowers and leaves Longevity (days)b initial FW)a (% of initial FW)a

0 nL 1-MCP/L 43.4 ± 2.3 15.7 ± 0.2 8.1 ± 0.1 150 nL 1-MCP/L 44.8 ± 1.3 16.5 ± 0.5 8.2 ± 0.1 a n = 6. b n = 10.

37 5

2 Abscission score 1 100

60 (% of initial FW) initial of (%

Relative fresh weight 40 0.8

0.6

0.4 Solution uptake

(mL/ g initialFW/ day) 0.2 789 Time (days)

Figure 5.15. Flower abscission (scores: 1 = < 10% to 5 = > 80%), relative fresh weight and vase solution uptake for C. uncinatum ‘Paddy’s Late’ sprigs from bunches pre-treated on day 0 with 0 nL 1-MCP/L (●) or 150 nL 1-MCP/L (■) for 15 hours at 2oC. Bunches were then packed into commercial flower cartons and held for 6 days at 20oC. Vertical bars represent standard errors of means (n = 10). Where no vertical bars appear, the standard error was smaller than the size of the symbol.

38 5.3.4 Application of 1-MCP in cartons by forced-air cooling

Application of 200 nL 1-MCP/L for 3 hours at 2oC into a coolroom containing ‘Purple Pride’ bunches either standing in buckets of water or in cartons against a forced-air cooler reduced ethylene-induced flower abscission (Figure 5.16). 1-MCP pre-treatment did not significantly reduce flower abscission from sprigs not exposed to ethylene. Accordingly, a significant interaction between 1-MCP pre-treatment, ethylene treatment and time of measurement for flower abscission was recorded (Appendix 5.33). Pre- treatment with 1-MCP reduced the loss of relative fresh weight of sprigs associated with flower abscission (Figure 5.17). 1-MCP pre-treatment did not significantly reduce the loss of relative fresh weight from sprigs not exposed to ethylene. However, sprigs pre-treated against the forced-air cooler and sampled 95 cm from the carton end nearest the coolroom air (or those closest to the forced-air cooler wall) had the greatest loss of relative fresh weight irrespective of 1-MCP pre-treatment. Thus, these responses probably account for the significant interaction between 1-MCP pre-treatment, ethylene treatment and sampling position for relative fresh weight (Appendix 5.34).

Vase solution uptake by sprigs not exposed to ethylene decreased over time (Figure 5.18). In contrast, vase solution uptake by sprigs exposed to ethylene increased between days 0 and 1 and then declined over time. This different uptake pattern is reflected in a significant interaction between ethylene treatment and time of measurement (Appendix 5.35). 1-MCP pre-treatment of bunches standing in water or in cartons did not extend the vase lives of sprigs not exposed to ethylene (Table 5.9). Vase lives of sprigs from bunches pre-treated with 0 or 200 nL 1-MCP/L in cartons against a forced-air cooler and not exposed to ethylene were reduced compared to sprigs sampled from similarly treated bunches standing in buckets of water. 1-MCP pre-treatment prevented the loss in vase life for sprigs exposed to ethylene. As a result there were significant interactions between 1-MCP pre-treatment and ethylene treatment and between ethylene treatment and the pre-treatment vessel (i.e. bucket vs. carton) (Appendix 5.36). There was no consistent or significant effect of the sampling position on sprig vase life (Table 5.9 and Appendix 5.37).

39 - 1-MCP (bucket) - Ethylene - 1-MCP (bucket) + Ethylene 5 4 3 2 1 - 1-MCP (forced-air) - Ethylene - 1-MCP (forced-air) + Ethylene 5 4 3 2 1 + 1-MCP (bucket) - Ethylene + 1-MCP (bucket) + Ethylene 5

Abscission score 4 3 2 1 + 1-MCP (forced-air) - Ethylene + 1-MCP (forced-air) + Ethylene 5 4 3 2 1 0123456789 0123456789 Time (days)

Figure 5.16. Flower abscission (scores: 1 = < 10% to 5 = > 80%) from C. uncinatum ‘Purple Pride’ sprigs from bunches pre-treated on day 0 with 0 or 200 nL 1-MCP/L in either buckets of water or cartons against a forced air cooler for 3 hours at 2oC. Half of the sprigs from each of these treatments were then exposed on day 0 to 10 µL ethylene/L for 12 hours at 20oC. Flower abscission from sprigs held inside cartons against the forced-air cooler is presented for sprigs sampled from stems 5 (●), 25 (■), 75 (▲) and 95 cm from the carton end closest to the incoming coolroom air (▼). Vertical bars represent the standard errors of means (n = 3). Where no vertical bars appear, the standard error was smaller than the size of the symbol.

40 120 - 1-MCP (bucket) - Ethylene - 1-MCP (bucket) + Ethylene 100 80 60 40 120 - 1-MCP (forced-air) - Ethylene - 1-MCP (forced-air) + Ethylene 100 80 60 40 120 + 1-MCP (bucket) - Ethylene + 1-MCP (bucket) + Ethylene 100 80 60

Relative fresh weight (% of initial FW) initial of (% weight fresh Relative 40 120 + 1-MCP (forced-air) - Ethylene + 1-MCP (forced-air) + Ethylene 100 80 60 40 0123456789 0123456789 Time (days)

Figure 5.17. Relative fresh weight of C. uncinatum ‘Purple Pride’ sprigs from bunches pre-treated on day 0 with 0 or 200 nL 1-MCP/L in either buckets of water or cartons against a forced air cooler for 3 hours at 2oC. Half of the sprigs from each of these treatments were then exposed on day 0 to 10 µL ethylene/L for 12 hours at 20oC. Relative fresh weight of sprigs held inside cartons against the forced-air cooler is presented for sprigs sampled from stems 5 (●), 25 (■), 75 (▲) and 95 cm from the carton end closest to the incoming coolroom air (▼). Vertical bars represent the standard errors of means (n = 3). Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD = 6.5%.

41 1.0 - 1-MCP (buckets) - Ethylene - 1-MCP (buckets) + Ethylene 0.8 0.6 0.4 0.2 0.0 1.0 - 1-MCP (forced-air) - Ethylene - 1-MCP (forced-air) + Ethylene 0.8 0.6 0.4 0.2 0.0 1.0 + 1-MCP (bucket) - Ethylene + 1-MCP (bucket) + Ethylene 0.8 0.6 0.4 0.2

Solution uptake (mL/g initial FW/day) initial (mL/g uptake Solution 0.0 1.0 + 1-MCP (forced-air) - Ethylene + 1-MCP (forced-air) + Ethylene 0.8 0.6 0.4 0.2 0.0 0123456789 0123456789 Time (days)

Figure 5.18. Vase solution uptake by C. uncinatum ‘Purple Pride’ sprigs from bunches pre-treated on day 0 with 0 or 200 nL 1-MCP/L in either buckets of water or cartons against a forced air cooler for 3 hours at 2oC. Half of the sprigs from each of these treatments were then exposed on day 0 to 10 µL ethylene/L for 12 hours at 20oC. Vase solution uptake by sprigs held inside cartons against the forced-air cooler is presented for sprigs sampled from stems 5 (●), 25 (■), 75 (▲) and 95 cm from the carton end closest to the incoming coolroom air (▼). Vertical bars represent the standard errors of means (n = 3). Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD = 0.068 mL/g initial FW/day.

42 Table 5.9. Vase life (days; mean ± s.e.) of C. uncinatum ‘Purple Pride’ sprigs from bunches pre-treated on day 0 with 0 or 200 nL 1-MCP/L for 3 hours while standing in buckets of water or in cartons against a forced air cooler inside a coolroom operating at 2oC. Flowering sprigs were removed at random from bunches standing in water or from four positions within each carton (5, 25, 75 and 95 cm from the carton end closest to the incoming coolroom air; positions 1, 2, 3 and 4, respectively) and exposed to 10 µL ethylene/L for 12 hours at 20oC. Data followed by the same letters are not significantly different (LSD = 1.9 for position data (n = 3) and 0.9 for treatment data (n = 12)) at P = 0.05.

Treatment Position 1 Position 2 Position 3 Position 4 Treatment means and s.e.

No 1-MCP Bucket 0 µL ethylene/L zzzz7.3 ± 0.4 c 10 µL ethylene/L zzzz1.9 ± 0.1 a Carton 0 µL ethylene/L 6.0 ± 0.6 b 7.3 ± 0.9 c 6.0 ± 1.2 b 5.0 ± 1.0 b 6.1 ± 0.5 bc 10 µL ethylene/L 2.0 ± 0.0 a 2.0 ± 0.0 a 1.7 ± 0.3 a 2.0 ± 0.0 a 1.9 ± 0.1 a

Plus 1-MCP Bucket 0 µL ethylene/L zzzz6.9 ± 0.2 c 10 µL ethylene/L zzzz6.7 ± 0.4 bc Carton 0 µL ethylene/L 6.3 ± 0.7 b 5.7 ± 0.9 b 5.7 ± 0.7 b 6.3 ± 0.7 b 6.0 ± 0.3 b 10 µL ethylene/L 5.7 ± 0.7 b 7.3 ± 0.9 c 6.7 ± 0.3 b 6.3 ± 0.3 b 6.5 ± 0.3 bc z Position means and standard errors are not presented for these treatments as sprigs were randomly sampled from bunches standing in buckets of water.

43 5.3.5 Slow release of 1-MCP inside cartons

Inclusion of a tube containing 1-MCP gas into cartons packed with bunches of ‘Alba’ reduced ethylene- induced flower abscission and the associated loss of relative fresh weight from sprigs sampled adjacent to the tube (Figures 5.20 and 5.21). Unexpectantly, the additional of two tubes of 1-MCP gas into cartons only reduced ethylene-induced flower abscission and the loss of relative fresh weight from sprigs sampled next to one tube only. However, the inclusion of three tubes of 1-MCP gas into cartons significantly reduced ethylene-induced flower abscission and the loss of relative fresh weight from sprigs sampled from all four positions within the carton. The addition of tubes of 1-MCP to cartons did not consistently affect flower abscission and changes in relative fresh weight from bunches not exposed to ethylene. Based on these responses, a significant interaction between 1-MCP pre-treatment, ethylene treatment, sampling position and time of measurement was evident for flower abscission (Appendix 5.38) and relative fresh weight (Appendix 5.39).

Vase solution uptake by sprigs tended to decrease over time (Figure 5.22). However, in cartons that contained only one tube of 1-MCP, the vase solution uptake between days 7 and 11 by sprigs sampled 75 cm from this tube was higher than that by sprigs from all other treatments. This response probably reflects the significant interactions between ethylene treatment, sampling position and time of measurement and between 1-MCP pre-treatment, ethylene treatment and sampling position for vase solution uptake (Appendix 5.40). Sprigs from bunches held in cartons with one tube only were slightly, but not significantly protected against the ethylene-induced loss in longevity (Table 5.10). However, the inclusion of two or three tubes into cartons prevented the ethylene-induced loss in longevity. There was no consistent effect of sampling position on sprig longevity. Nevertheless, for sprigs sampled adjacent to tubes of 1-MCP gas and subsequently exposed to ethylene, there was a trend toward extended longevity compared to sprigs not exposed to 1-MCP. The addition of tubes of 1-MCP gas into cartons did not significantly extend the subsequent longevity of sprigs not exposed to ethylene. As a result, there was a significant interaction between 1-MCP treatment, ethylene treatment and sampling position for longevity (Appendix 5.41).

44 - 1-MCP - Ethylene - 1-MCP + Ethylene 5 4 3 2 1 + 1-MCP (1 tube) - Ethylene + 1-MCP (1 tube) + Ethylene 5 4 3 2 1 + 1-MCP (2 tubes) - Ethylene + 1-MCP (2 tubes) + Ethylene 5 4

Abscission score 3 2 1 + 1-MCP (3 tubes) - Ethylene + 1-MCP (3 tubes) + Ethylene 5 4 3 2 1 67891011126 7 8 9 101112 Time (days)

Figure 5.20. Flower abscission (scores: 1 = < 10% to 5 = > 80%) from C. uncinatum ‘Alba’ sprigs on bunches pre-treated on day 0 in cartons with 0 (control), 1, 2 or 3 tubes of 1-MCP gas for 6 days at 20oC. Half of the sprigs from each of these treatments were then exposed on day 6 to 10 µL ethylene/L for 12 hours at 20oC. Flower abscission is presented for sprigs sampled 5 (●), 25 (■), 75 (▲) and 95 cm from 1-MCP tube 1 (▼). Vertical bars represent the standard errors of means (n = 3). Where no vertical bars appear, the standard error was smaller than the size of the symbol.

45 120 - 1-MCP - Ethylene - 1-MCP + Ethylene 100 80 60 40 20 120 + 1-MCP (1 tube) - Ethylene+ 1-MCP (1 tube) + Ethylene 100 80 60 40 20 120 + 1-MCP (2 tubes) - Ethylene+ 1-MCP (2 tubes) + Ethylene 100 80 60 40

Relative fresh weight (% of initial FW) 20 120 + 1-MCP (3 tubes) - Ethylene+ 1-MCP (3 tubes) + Ethylene 100 80 60 40 20 67891011126 7 8 9 10 11 12 Time (days)

Figure 5.21. Relative fresh weight of C. uncinatum ‘Alba’ sprigs from bunches pre-treated on day 0 in cartons with 0 (control), 1, 2 or 3 tubes of 1-MCP gas for 6 days at 20oC. Half of the sprigs from each of these treatments were then exposed on day 6 to 10 µL ethylene/L for 12 hours at 20oC. Relative fresh weight is presented for sprigs sampled 5 (●), 25 (■), 75 (▲) and 95 cm from 1-MCP tube 1 (▼). Vertical bars represent the standard errors of means (n = 3). Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD = 9.4%.

46 0.8 - 1-MCP - Ethylene - 1-MCP + Ethylene 0.6 0.4 0.2 0.0 0.8 + 1-MCP (1 tube) - Ethylene + 1-MCP (1 tube) + Ethylene 0.6 0.4 0.2 0.0 0.8 + 1-MCP (2 tubes) - Ethylene + 1-MCP (2 tubes) + Ethylene 0.6 0.4 0.2

Solution uptake (mL/g initial FW/day) 0.0 0.8 + 1-MCP (3 tubes) - Ethylene + 1-MCP (3 tubes) + Ethylene 0.6 0.4 0.2 0.0 6 7 8 9 10 11 12 6789101112 Time (days)

Figure 5.22. Vase solution uptake by C. uncinatum ‘Alba’ sprigs from bunches pre-treated on day 0 in cartons with 0 (control), 1, 2 or 3 tubes of 1-MCP gas for 6 days at 20oC. Half of the sprigs from each of these treatments were then exposed on day 6 to 10 µL ethylene/L for 12 hours at 20oC. Vase solution uptake is presented for sprigs sampled 5 (●), 25 (■), 75 (▲) and 95 cm from 1-MCP tube 1 (▼). Vertical bars represent the standard errors of means (n = 3). Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD = 0.059 mL/g initial FW/day.

47 Table 5.10. Longevity (days; mean ± s.e.) of C. uncinatum ‘Alba’ sprigs from bunches pre-treated with 0, 1, 2 or 3 tubes containing 1-MCP for 6 days at 20oC. 1- MCP gas was slowly released from test tubes stoppered with a rubber septa. Immediately following 1-MCP treatment, flowering sprigs were removed from four positions (5, 25, 75 and 95 cm from tube 1; positions 1, 2, 3 and 4, respectively) within each carton and half of the sprigs from each position were treated with 10 µL ethylene/L at 20oC for 12 hours. Data followed by the same letters are not significantly different (LSD = 2.5 for position data (n = 3) and 1.3 for treatment data (n = 12)) at P = 0.05.

Treatment Position 1 Position 2 Position 3 Position 4 Treatment means and s.e.

No 1-MCP (0 tubes) 0 µL ethylene/L 8.7 ± 0.3 ab 6.7 ± 0.7 a 8.3 ± 1.2 ab 8.7 ± 1.2 ab 8.1 ± 0.5 y 10 µL ethylene/L 7.0 ± 0.0 ab 7.0 ± 0.0 ab 6.3 ± 0.3 a 6.7 ± 0.3 a 6.8 ± 0.1 z Plus 1-MCP 1 tube 0 µL ethylene/L 8.0 ± 0.0 ab 8.7 ± 0.9 ab 9.7 ± 1.9 b 10.3 ± 0.7 b 9.2 ± 0.5 y 10 µL ethylene/L 8.7 ± 1.7 ab 8.7 ± 1.7 ab 6.7 ± 0.3 a 7.0 ± 0.0 ab 7.8 ± 0.6 zy 2 tubes 0 µL ethylene/L 8.0 ± 1.2 ab 8.7 ± 0.3 ab 7.7 ± 0.9 ab 7.7 ± 0.3 ab 8.0 ± 0.3 zy 10 µL ethylene/L 7.0 ± 0.0 ab 6.7 ± 0.3 a 8.0 ± 1.0 ab 11.0 ± 0.6 b 8.2 ± 0.6 y 3 tubes 0 µL ethylene/L 8.0 ± 0.0 ab 8.0 ± 1.2 ab 10.0 ± 1.2 b 8.7 ± 1.5 ab 8.7 ± 0.5 y 10 µL ethylene/L 7.7 ± 0.3 ab 8.0 ± 1.0 ab 9.3 ± 0.7 b 9.3 ± 0.3 b 8.6 ± 0.4 y

48 The loss of weight from bunches of ‘Alba’ during the 6 day treatment period was significantly reduced by the addition of tubes containing 1-MCP into cartons (Table 5.11 and Appendix 5.41). Similarly, the accumulation of abscised flowers and leaves in cartons from bunches was significantly reduced by the inclusion of a tube of 1-MCP (Table 5.11 and Appendix 5.42). The inclusion of 2 or 3 tubes in cartons was not as effective in reducing flower and leaf abscission as the use of 1 tube. The diffusion of 1-MCP gas from 33 mL volume tubes was presumably through a 2mm thick rubber septa with a surface area of 56.75 mm2. The concentration in tubes decreased from 6928 ± 177 µL 1-MCP/L on day 0 to 1793 ± 48 µL 1-MCP/L on day 6.

Table 5.11. Weight loss and proportion of abscised flowers and leaves (mean ± s.e.) from C. uncinatum ‘Alba’ bunches pre-treated on day 0 in cartons with 0, 1, 2 or 3 tubes of 1-MCP gas for 6 days at 20oC. Data followed by the same letters are not significantly different (LSD = 3.2 and 1.1 for weight loss and flower and leaf abscission, respectively) at P = 0.05.

Treatment Weight loss (% of initial FW)a Abscised flowers and leaves (% of initial FW)b

No 1-MCP 0 tubes (control) 37.0 ± 1.5 b 9.3 ± 0.7 c Plus 1-MCP 1 tube 27.4 ± 1.1 a 5.3 ± 0.1 a 2 tubes 27.3 ± 1.0 a 6.5 ± 0.1 b 3 tubes 29.2 ± 0.9 a 6.3 ± 0.3 ab a n = 18. b n = 3.

5.4 DISCUSSION

Application of 1-MCP inside sealed polyethylene tents or coolrooms containing C. uncinatum reduced ethylene-induced flower abscission and thus appear to be practical treatments. Pre-treatment of ‘CWA Pink’ bunches standing in buckets of water with 200 nL 1-MCP/L for 6 hours at 20oC inside a sealed polyethylene tent reduced flower abscission (Figure 5.1) and associated loss in vase life (Table 5.1) induced by exposure to 10 µL ethylene/L for 12 hours at 20oC. This result confirms the findings of Serek et al. (1995c) where pre-treatment of C. uncinatum ‘Wendy’ sprigs with 200 nL 1-MCP/L for 6 hours at 21oC inside sealed perspex chambers reduced ethylene-induced flower and bud abscission. The 1-MCP concentration used was higher than those used in laboratory experiments (Chapter 4) to take into account the possibility of small leaks existing from the enclosed tent. 1-MCP pre-treatment was more effective than STS pre-treatment (0.2 mM Ag+ for 6 hours at 20oC) in reducing ethylene-induced flower abscission from ‘CWA Pink’ sprigs (Figure 5.1). Joyce (1988, 1989, 1993) showed that pulsing C. uncinatum stems with STS can reduce ethylene-induced flower abscission. STS treatments were reported to be effective when the accumulation of Ag+ in C. uncinatum tissue was in the range of 0.1 to 0.6 µmol/g (Joyce 1989).

1 As the STS concentration required to protect C. uncinatum is inversely related to treatment duration (Joyce 1988, 1989) it is possible that in the present study, the duration of STS pulsing was too short for the concentration used.

1-MCP and STS pre-treatments were equally effective in reducing the loss of weight and abscission of flowers and leaves from bunches held dry in cartons for 6 days at 20oC after pre-treatment (Table 5.2). The postharvest longevity of sprigs from these bunches was extended by 1-MCP and STS pre-treatments presumably indicating that endogenous ethylene production limited longevity (Table 5.2). These results are similar to those of Serek et al. (1995c) where 1-MCP pre-treatment reduced flower and bud abscission from C. uncinatum ‘Wendy’ bunches held dry for 72 hours at 21oC after 1-MCP pre-treatment.

Pre-treatment of ‘Fortune Cookie’ bunches standing in water with 200 nL 1-MCP/L inside sealed polyethylene tents or 0.2 mM Ag+ for the longer duration of 14 hours were equally effective in preventing ethylene-induced flower abscission from sprigs when applied at 2 or 20oC (Figure 5.3). Presumably, pre- treatment with high 1-MCP concentration at low temperature can afford C. uncinatum with protection against ethylene. Pre-treatment of C. uncinatum with the low 1-MCP concentration of 10 nL/L for 12 hours at 2oC did not provide long term protection against ethylene (Chapter 4). Reid et al. (1996) reported that the 1-MCP treatment concentration required to protect cut Kalanchoe flowers against ethylene was inversely related to treatment temperature. For example, the efficacy of 1-MCP pre- treatment applied to Kalanchoe flowers at 2oC was improved by increasing the 1-MCP concentration from 10 to 128 nL/L.

Pre-treatment of ‘Fortune Cookie’ bunches with STS at 2oC was more effective than 1-MCP in preventing the ethylene-induced loss in sprig vase life (Table 5.3). As both 1-MCP and STS pre-treatments prevented flower abscission, the termination of vase life was based entirely on flower wilting. Accordingly, in this experiment it appears that STS was more effective than 1-MCP in delaying flower wilting. Conversely, 1-MCP pre-treatment was more effective than STS pre-treatment in extending the postharvest longevity of sprigs from bunches held dry in cartons for 6 days at 20oC after pre-treatments (Table 5.4). Abscission of flowers and leaves from bunches during storage was more effectively reduced by STS pre-treatment compared to 1-MCP (Table 5.4). It is possible that rapid synthesis of new ethylene receptors during storage in association with endogenous ethylene production could initiate flower and leaf abscission from 1-MCP pre-treated tissue. Sprigs pre-treated with STS may retain Ag+ in and around flower abscission zones that binds to newly formed receptors thereby reducing flower abscission for the duration of vase life (Chapter 4). Nonetheless, the data clearly indicate that 1-MCP pre-treatment of C. uncinatum can be effective at a range of temperatures. This 1-MCP treatment system when operated at 2oC caused little disruption to the normal postharvest handling process for C. uncinatum because flowering bunches could be cooled to around 2oC in a coolroom whilst being hydrated in buckets of water and receiving 1-MCP treatment.

Pre-treatment of ‘Paddy’s Late’ bunches with 150 nL 1-MCP/L for 15 hours at 2oC inside a coolroom was

2 effective in reducing ethylene-induced flower abscission (Figure 5.14) and the loss in vase life (Table 5.7). This further confirms that 1-MCP pre-treatment is effective when applied at high concentrations at low temperature. 1-MCP did not extend the vase lives of sprigs not exposed to ethylene (cf. Chapter 3). 1-MCP pre-treatment did not reduce the loss of weight or abscission of flowers and leaves from bunches that were held dry for 6 days at 20oC in cartons after pre-treatment (Table 5.8).

Application of 1-MCP by forced-air cooling into cartons containing ‘Purple Pride’ bunches was equally effective as treating bunches standing in buckets of water inside a coolroom. Sprigs sampled from different positions within cartons sustained minimal flower abscission (Figure 5.16) and had similar vase lives (Table 5.9). Thus, movement of air containing 1-MCP through cartons was uniform. Based on these results C. uncinatum bunches could be packed into cartons and rapidly cooled by forced-air cooling while receiving 1-MCP treatment.

Injection of 200 nL 1-MCP/L into sealed cartons containing ‘Lollypop’ bunches was not effective in preventing ethylene-induced flower abscission (Figure 5.8). It is possible that 1-MCP diffused out through the carton walls before it effectively spread within the carton. Increasing the concentration of 1- MCP injected into cartons containing ‘Purple Pride’ bunches to 2 µL/L only provided protection against ethylene to sprigs sampled closest to the injection point (Table 5.6). However, unlike forced-air cooling where air containing 1-MCP is drawn through the carton, diffusion of 1-MCP applied as a single injection appears to be poor. It is possible that by making multiple injections of 1-MCP at different locations on cartons the efficacy of this system will improve. However, this process will involve added handling and may prove time consuming.

1-MCP treatment by sustained release from tubes inside cartons containing ‘Alba’ bunches was only fully effective at protecting flowers from ethylene when three tubes were placed into each carton (Table 5.10). However, protection against ethylene was afforded for some sprigs sampled adjacent to tubes in other treatments, as evidenced by reduced flower abscission and extended sprig longevity. This alternative system is based on principles devised by Saltveit (1978) and Poole and Joyce (1993) whilst investigating simple ethylene gassing systems. 1-MCP apparently diffused through the rubber septa in tubes and into the carton atmosphere slowly over the 6 day treatment period. According to Poole and Joyce (1993) this system can maintain relatively constant concentrations in the surrounding atmosphere for long periods of time. Furthermore, it is compatible with ventilation systems such as forced air cooling which limit CO2 and ethylene accumulation.

The sustained release treatment system may, upon refinement, improve the efficacy of 1-MCP treatment in providing longer term protection of C. uncinatum against ethylene. It is hypothesised that a sustained release of 1-MCP around flowers will permit 1-MCP binding to newly formed ethylene receptors in the abscission zones of C. uncinatum flowers. Thus, the period of protection against ethylene may be extended. A slow release treatment would be comparatively easy to use and allow rapid dispatch of cut flowers to markets by eliminating the need to pre-treat flowers. Additionally, it may be a viable treatment

3 for growers without suitable enclosed treatment structures and those concerned about handling chemicals such as 1-MCP.

In the present study, the efficacy of laboratory scale 1-MCP treatments with C. uncinatum were shown to be reproducible on a commercial scale. Consequently, several 1-MCP application systems can be recommended for use by the native Australian cut flower industry. The release of 1-MCP into sealed tents and coolrooms for short (i.e. 3 hours) or long (i.e. 15 hours) duration at a range of temperatures are highly effective in protecting C. uncinatum standing in water against ethylene. Similarly, application of 1-MCP to bunches in cartons via forced air cooling appears to be viable alternative approach. Sustained release of 1-MCP gas from inside cartons promises to be the most effective treatment which, after refinement, may provide extended protection against ethylene. While it has been shown that 1-MCP treatment of several C. uncinatum cultivars reduces ethylene damage, it is anticipated that these application systems would protect other sensitive native Australian cut flowers against ethylene.

4 5 BIBLIOGRAPHY

Abdi, N., McGlasson, W.B., Holford, P., Williams, M., Mizrahi, Y. (1998). Responses of climacteric and suppressed-climacteric plums to treatment with propylene and 1-methylcyclopropene. Postharvest Biology and Technology 14, 29-39. Abeles, F.B., Leather, G.R., Forrence, L.E., and Craker, L.E. (1979). Abscission: regulation of senescence, protein synthesis, and enzyme secretion by ethylene. HortScience 6, 371-82. Abeles, F.B., Morgan, P.W., and Saltveit, M.E. (1992). Ethylene in Plant Biology, 2nd ed. 398 pp. (Academic Press, New York). Adams, D.O., and Yang, S.F. (1977). Methionine metabolism in apple tissue: implication of S- adenosylmethionine as an intermediate in the conversion of methionine to ethylene. Plant Physiology 60, 892-96. Adams, D.O., and Yang, S.F. (1979). Ethylene biosynthesis: identification of 1-aminocyclopropane-1- carboxylic acid as an intermediate in the conversion of methionine to ethylene. Proceedings of the National Academy of Sciences of the United States of America 76, 170-74. Addicott, F.T. (1982). Abscission. 369 pp. (University of California Press, Berkeley). Aharoni, N. (1989). Interrelationship between ethylene and growth regulators in the senescence of lettuce leaf discs. Journal of Plant Growth Regulation 8, 309-17. Aharoni, N., Anderson, J.D., and Lieberman, M. (1979a). Production and action of ethylene in senescing leaf discs. Plant Physiology 64, 805-9. Aharoni, N., and Lieberman, M. (1979). Ethylene as a regulator of senescence in tobacco leaf discs. Plant Physiology 64, 801-4. Aharoni, N., Lieberman, M., and Sisler, H.D. (1979b). Patterns of ethylene production in senescing leaves. Plant Physiology 64, 796-800. Amrhein, N., and Wenker, D. (1979). Novel inhibitors of ethylene production in higher plants. Plant and Cell Physiology 20, 1635-42. Atta-Aly, M.A., Saltveit, M.E., and Hobson, G.E. (1987). Effect of silver ions on ethylene biosynthesis by tomato fruit tissue. Plant Physiology 83, 44-8. Baker, J.E., Wang, C.Y., Lieberman, M., and Hardenburg, R. (1977). Delay of senescence in carnations by a rhizobitoxine analog and sodium benzoate. HortScience 12, 38-9. Beal, P., Howell, J., Joyce, D., and Shorter, A. (1995). Maturity stages for harvesting Grevillea for cut flowers. Queensland Department of Primary Industries publication QL95007. Ben-Arie, R., and Sonego, L. (1985). Modified-atmosphere storage of kiwifruit (Actinidia chinensis Planch) with ethylene removal. Scientia Horticulturae 27, 263-73. Beyer, E.M. (1976). A potent inhibitor of ethylene action in plants. Plant Physiology 58, 268-71. Beyer, E.M., and Morgan, P.W. (1971). Abscission: the role of ethylene modification of auxin transport. Plant Physiology 48, 208-12. Biale, J.B. (1964). Growth, maturation and senescence in fruits. Science 146, 880-8. Bleecker, A.B., Estelle, M.A., Somerville, C., and Kende, H. (1988). Insensitivity to ethylene conferred

6 by a dominant mutation in Arabidopsis thaliana. Science 241, 1086-9. Boller, T., Herner, R.C., Kende, H. (1979). Assay for and enzymatic formation of an ethylene precursor, 1-amino-cyclopropane-1-carboxylic acid. Planta 145, 293-303. Borochov, A., and Faragher, J. (1983). Comparison between ultraviolet irradiation and ethylene effects on senescence parameters in carnation flowers. Plant Physiology 71, 536-40. Bradford, K.J., and Yang, S.F. (1980). Xylem transport of 1-aminocyclopropane-1-carboxylic acid, an ethylene precursor, in waterlogged tomato plants. Plant Physiology 65, 322-6. Brady, C.J., and Speirs, J. (1991). Ethylene in fruit ontogeny and abscission. In: The Plant Hormone Ethylene. (Eds. A.K Mattoo and J.C. Suttle). pp. 235-58. (CRC Press, Boca Raton). Bramlage, W.J., Greene, D.W., Autio, W.R., and McLaughlin, J.M. (1980). Effects of aminoethoxyvinylglycine on internal ethylene concentrations and storage of apples. Journal of the American Society for Horticultural Science 105, 847-51. Brown, K.M. (1997). Ethylene and abscission. Physiologia Plantarum 100, 567-76. Broun, R., and Mayak, S. (1981). Aminooxyacetic acid as an inhibitor of ethylene synthesis and senescence in carnation flowers. Scientia Horticulturae 15, 277-82. Burdon, J., and Sexton, R. (1990). The role of ethylene in the shedding of red raspberry fruit. Annals of Botany 66, 111-20. Burg, S.P. (1962). The physiology of ethylene formation. Annual Review of Plant Physiology 13, 265- 302. Burg, S.P., and Burg, E.A. (1962). Role of ethylene in fruit ripening. Plant Physiology 37, 179-89. Burg, S.P., and Burg, E.A. (1965). Relationship between ethylene production and ripening in bananas. Botanical Gazette 126, 200-4. Burg, S.P., and Burg, E.A. (1967). Molecular requirements for the biological activity of ethylene. Plant Physiology 42, 144-52. Burg, S.P., and Dijkman, M.J. (1967). Ethylene and auxin participation in pollen induced fading of Vanda orchid blossoms. Plant Physiology 61, 812-5. Cameron, A.C., and Reid, M.S. (1981). The use of silver thiosulfate anionic complex as a foliar spray to prevent flower abscission of Zygocactus. HortScience 16, 761-2. Cameron, A.C., and Reid, M.S. (1983). Use of silver thiosulfate to prevent flower abscission from potted plants. Scientia Horticulturae 19, 373-8. Cardinale, F.C., Jennings, J.C., and Anderson, J.C. (1995). Use of the ethylene action inhibitor 1- methylcyclopropene to study the role of ethylene in elicitor-induced ethylene biosynthesis in tomato leaves (abstract only). Plant Physiology 108, 140. Chang, C., Kwok, S.F., Bleecker, A.B., and Meyerowitz, E.M. (1993). Arabidopsis ethylene response gene ETR1-similarity of product to 2-component regulators. Science 262, 539-44. Christoffersen, R.E., and Laties, G.G. (1982). Ethylene regulation of gene expression in carrots. Proceedings of the National Academy of Sciences of the United States of America 79, 4060-3. Conover, W.J. (1980). Practical Nonparametric Statistics, 2nd ed. 493 pp. (John Wiley and Sons, New York). Cook, D., Rasche, M., and Eisinger, W. (1985). Regulation of ethylene biosynthesis and action in cut

7 carnation flower senescence by cytokinins. Journal of the American Society for Horticultural Science 110, 24-7. Cooley, A.C. (1988). Silver recovery using steel wool metallic replacement cartridges. Journal of Imaging Technology 14, 167-73. Costin, R., and Costin, S. (1988). Tropical Grevillea hybrids. Australian Plants 14, 335-43. CSIRO (1972). Banana Ripening Guide. (Banana Research Advisory Committee, Technical Bulletin 3). 13 pp. (CSIRO, Melbourne). Ecker, J.R. (1995). The ethylene signal transduction pathway in plants. Science 268, 667-75. Faragher, J.D. (1986). Post-harvest physiology of waratah infloresccences (Telopea speciosissima, Proteaceae). Scientia Horticulturae 28, 271-9. Faragher, J.D. (1989). A review of research on postharvest physiology and horticulture of Australian native flowers. Acta Horticulturae 261, 249-56. FECA (Flower Export Council of Australia Inc.) (1996). Export guide for Australian flowers and foliages. RIRDC Project - FEC-2A, Export Development R&D for Australian Wildflower and Proteaceae. 104 pp. (FECA, Nedlands). Fluhr, R., and Mattoo, A.K. (1996). Ethylene - biosynthesis and perception. Critical Reviews in Plant Sciences 15, 479-523. Fisher, F., and Applequist, D.E. (1965). Synthesis of 1-methylcyclopropene. Journal of Organic Chemistry 30, 2089-90. Fujino, D.W., Reid, M.S., and Yang, S.F. (1980). Effects of aminooxyacetic acid on postharvest characteristics of carnation. Acta Horticulturae 113, 59-64. Gepstein, S., and Thimann, K.V. (1981). The role of ethylene in the senescence of oat leaves. Plant Physiology 68, 349-54. Gilissen, L.J.W. (1977). Style-controlled wilting of the flower. Planta 133, 275-80. Gladon, R.J., and Spear, G.J. (1984). Postshipment treatment of bud carnation with aminooxyacetic acid, aminoethoxyvinylglycine, and silver thiosulfate (abstract only). HortScience 19, 567.

8 Glick, B.R., Jacobson, C.B., Schwarze, M.M.K., and Paternak, J.J. (1994). 1-Aminocyclopropane-1- carboxylic Acid deaminase mutants of the plant growth promoting rhizobacterium Pseudomonas putida GR12-2 do not stimulate canola root elongation. Canadian Journal of Microbiology 40, 911-5. Glick, B.R., Penrose, D.M., and Li, J. (1998). A model for lowering of plant ethylene concentrations by plant growth-promoting bacteria. Journal of Theoretical Biology 190, 63-8. Goeschl, J.D., Rappaport, L., and Pratt, H.K. (1966). Ethylene as a factor regulating the growth of pea epicotyls subjected to physical stress. Plant Physiology 41, 877-84. Goh, C.J., Halevy, A.H., Engel, R., and Kofranek, A.M. (1985). Ethylene sensitivity in cut orchid flowers. Scientia Horticulturae 26, 57-67. Golding, J.B., Shearer, D., Wyllie, S.G., and McGlasson, W.B. (1998). Application of 1-MCP and propylene to identify ethylene-dependent ripening processes in mature banana fruit. Postharvest Biology and Technology 14, 87-98. Graham, T.K., Veenstra, J.N., and Armstrong, P.R. (1998). Ethylene removal in fruit and vegetable storages using a plasma reactor. Transactions of the American Society of Agricultural Engineers 41, 1767-73. Halevy, A.H. (1986). Flower senescence. In: Processes and Control of Plant Senescence. (Eds. Y.Y. Leshem, A.H. Halevy, and C. Frenkel). pp. 142-61 (Elseiver, Amsterdam). Halevy, A.H., and Kofranek, A.M. (1977). Silver treatment of carnation flowers for reducing ethylene damage and extending longevity. Journal of the American Society for Horticultural Science 102, 76-7. Halevy, A.H., and Mayak, S. (1981). Senescence and postharvest physiology of cut flowers, Part 2. In: Horticultural Reviews, Vol. 3. (Ed. J. Janick). pp. 59-143. (AVI Publishing, Westport). Halevy, A.H., Porat, R., Spiegelstein, H., Borochov, A., Botha, L., and Whitehead, C.S. (1996). Short- chain saturated fatty acids in the regulation of pollination-induced ethylene sensitivity of Phalaenopsis flowers. Physiologia Plantarum 97, 469-74. Halevy, A.H., Whitehead, C.S., and Kofranek, A.M. (1984). Does pollination induce corolla abscission of Cyclamen flowers by promoting ethylene production? Plant Physiology 75, 1090-3. Hall, J.A., Peirson, D., Ghosh, S., and Glick, B.R. (1996). Root elongation in various agronomic crops by the plant growth promoting rhizobacterium Pseudomonas putida GR12-2. Israel Journal of Plant Sciences 44, 37-42. Hatton, T.T., and Reeder, W.F. (1972). Quality of ‘Lula’ avocados stored in controlled atmospheres with or without ethylene. Journal of the American Society of Horticultural Science 97, 339-41. Hoekstra, F.A., and Weges, R. (1986). Lack of control by early pistillate ethylene of the accelerated wilting of Petunia hybrida flowers. Plant Physiology 80, 403-8. Holm, R.E., and Abeles, F.B. (1967). Abscission: the role of RNA synthesis. Plant Physiology 42, 1094- 1102. Hua, J., Chang, C., Sun, Q., and Meyerowitz, E.M. (1995). Ethylene insensitivity conferred by Arabidopsis ERS gene. Science 269, 1712-4. Hua, J., and Meyerowitz, E.M. (1998). Ethylene responses are negatively regulated by a receptor gene

9 family in Arabidopsis thaliana. Cell 94, 261-71. Hua, J., Sakai, H., Nourizadeh, S., Chen, Q.G., Bleecker, A.B., Ecker, J.R., and Meyerowitz, E.M. (1998). EIN4 and ERS2 are members of the putative ethylene receptor gene family in Arabidopsis. The Plant Cell 10, 1321-32. Inaba, A., and Nakamura, R. (1988). Numerical expression for estimating the minimum ethylene exposure time necessary to induce ripening in banana fruit. Journal of the American Society for Horticultural Science 113, 561-4. Jackson, M.B. (1979). Is the diageotropica tomato ethylene deficient? Physiologia Plantarum 46, 347- 51. Jackson, M.B. (1985). Ethylene responses of plants to waterlogging and submergence. Annual Review of Plant Physiology 36, 145-74. Jahn, O.L., Chace, W.G., and Cubbedge, R.H. (1973). Degreening response of ‘Hamlin’ oranges in relation to temperature, ethylene concentration, and fruit maturity. Journal of the American Society for Horticultural Science 98, 177-81. Jobling, J., McGlasson, W.B., and Dilley, D.R. (1991). Induction of ethylene synthesizing competency in Granny Smith apples by exposure to low temperature in air. Postharvest Biology and Technology 1, 111-8. Johnston, M.E., Tisdell, J.G., and Simons, D.H. (1992). Influences of precooling and silver thiosulphate on leaf abscission of two forms of rice flower. Postharvest Biology and Technology 2, 25-30. Jones, R.B., Faragher, J.D., and Van Doorn, W.G. (1993). Water relations of cut flowering branches of Thryptomene calycina (Lindl.) Stapf. (Myrtaceae). Postharvest Biology and Technology 3, 57- 67. Joyce, D.C. (1988). Postharvest characteristics of Geraldton waxflowers. Journal of the American Society for Horticultural Science 13, 738-42. Joyce, D.C. (1989). Treatments to prevent flower abscission in Geraldton wax. HortScience 24, 391. Joyce, D. (1992). Waxflower: to STS or not? Australian Horticulture October, 52-7. Joyce, D.C. (1993). Postharvest floral organ fall in Geraldton waxflower (Chamelaucium uncinatum Schauer). Australian Journal of Experimental Agriculture 33, 481-7. Joyce, D.C., Beal, P., and Shorter, A.J. (1996). Vase life characteristics of selected Grevillea. Australian Journal of Experimental Agriculture 36, 379-82. Joyce, D.C., and Haynes, Y. (1989). Postharvest treatment and shipping of W.A. flora. In: Proceedings of the W.A. Flora Summit, Geraldton Mid-West Development Authority and Horticulture 2001, September 14-15, Geraldton, W.A., Australia. pp. 43-65. Joyce, D., Jones, R., and Faragher, J. (1993). Postharvest characteristics of native Australian flowers. Postharvest News and Information 4, 61-7N. Joyce, D.C., Macnish, A.J., Hofman, P.J., Simons, D.H., and Reid, M.S. (1998). Use of 1- methylcyclopropene to modulate banana ripening. In: Biology and Biotechnology of the Plant Hormone Ethylene II Conference, Proceedings, September 5 - 8, Thira, Greece. (in press). Joyce, D.C., and Poole, M.C. (1993). Effects of ethylene and dehydration on cut flowering stems of Verticordia spp. Australian Journal of Experimental Agriculture 33, 489-93.

10 Joyce, D.C., Reid, M.S., and Evans, R.Y. (1990). Silver thiosulfate prevents ethylene-induced abscission in Holly and Mistletoe. HortScience 25, 90-2. Joyce, D.C., Shorter, A.J., Joyce, P.A., and Beal, P.R. (1995). Respiration and ethylene production by harvested Grevillea ‘Sylvia’ flowers and inflorescences. Acta Horticulturae 405, 224-231. Kader, A.A. (1985). Ethylene-induced senescence and physiological disorders in harvested horticultural crops. HortScience 20, 54-7. Kende, H. (1983). Ethylene biosynthesis. Annual Review of Plant Physiology and Plant Molecular Biology 44, 283-307. Kende, H., and Baumgartner, B. (1974). Regulation of aging in flowers of Ipomoea tricolor by ethylene. Planta 116, 279-89. Kende, H., and Hanson, A.D. (1976). Relationship between ethylene evolution and senescence in morning-glory flower tissue. Plant Physiology 57, 523-7. Ketring, D.L. (1977). Ethylene and seed germination. In: The Physiology and Biochemistry of Seed Dormancy and Germination. (Ed. A.A. Khan). pp. 157-78. (Elsevier, Amsterdam). Ketsa, S., and Luangsuwalai, K. (1996). The relationship between 1-aminocyclopropane-1-carboxylic acid content in pollinia, ethylene production and senescence of pollinated Dendrobium orchid flowers. Postharvest Biology and Technology 8, 57-64. Kidd, F., and West, C. (1945). Respiratory activity and duration of life of apples gathered at different stages of development and subsequently maintained at a constant temperature. Plant Physiology 20, 467-504. Kieber, J.J., Rothenberg, M., Roman, G., Feldmann, K.A., and Ecker, J.R. (1993). CTR1, a negative regulator of the ethylene response pathway in Arabidopsis, encodes a member of the Raf family of protein kinases. Cell 72, 427-41. Klee, H.J., Hayford, M.B., Kretzmer, K.A., Barry, G.F., and Kishmore, G.M. (1991). Control of ethylene biosynthesis by expression of a bacterial enzyme in transgenic tomato plants. The Plant Cell 3, 1187-93.

Klee, H.J., and Tieman, D. (1997). Potential applications of controlling ethylene synthesis and perception in transgenic plants. In: Biology and Biotechnology of the Plant Hormone Ethylene. (Eds. A.K. Kanellis, C. Chang, H. Kende, D. Grierson). pp. 289-97. (Kluwer Academic, Dordrecht). Ku, V.V.V., Wills, R.B.H., and Ben-Yehoshua, S. (1999). 1-Methylcyclopropene can differentially affect the postharvest life of strawberries exposed to ethylene. HortScience 24, 119-20. Lanahan, M.B., Yen, H-C., Giovannoni, J.J., and Klee, H.J. (1994). The Never-ripe mutation blocks ethylene perception in tomato. The Plant Cell 6, 521-30. Larsen, P.B., Woltering, E.J., and Woodson, W.R. (1993). Ethylene and interorgan signaling in flowers following pollination. In: Plant Signals in Interactions with Other Organisms. (Eds. I. Raskin and J. Schultz). pp. 112-22. (American Society of Plant Physiologists, Rockville). Lelievre, J.M., Tichit, L., Dao, P., Fillion, L., Nam, Y.W., Pech, J.C., and Latche, A. (1997). Effects of

11 chilling on the expression of ethylene biosynthetic genes in Passe-Crassane pear (Pyrus communis L.) fruits. Plant Molecular Biology 33, 847-55. Lieberman, M. (1979). Biosynthesis and action of ethylene. Annual Review of Plant Physiology 30, 533- 91. Ligawa, J.K., Joyce, D.C., and Hetherington, S.E. (1997). Exogenously supplied sucrose improves the postharvest quality of Grevillea ‘Sylvia’ inflorescences. Australian Journal of Experimental Agriculture 37, 809-16. Liu, Y., Su, L.Y., and Yang, S.F. (1985). Ethylene promotes the capability to malonylate 1- aminocyclopropane-1-carboxylate and D-amino acids in preclimacteric tomato fruits. Plant Physiology 77, 891-5. Lovell, P.J., Lovell, P.H., and Nichols, R. (1987). The control of flower senescence in petunia (Petunia hybrida). Annals of Botany 60, 49-59. Lyons, J.M. (1973). Chilling injury in plants. Annual Review of Plant Physiology 24, 445-66. Masia, A., Ventura, M., Gemma, H., and Sansavini, S. (1998). Effect of some plant growth regulator treatments on apple fruit ripening. Plant Growth Regulation 25, 127-34. Magid, R.M., Clarke, T.C., and Duncan, C.D. (1971). An efficient and convenient synthesis of 1- methylcyclopropene. Journal of Organic Chemistry 36, 1320-1. Marynick, M.C. (1977). Patterns of ethylene and carbon dioxide evolution during cotton explant abscission. Plant Physiology 59, 484-9. Mathooko, F.M., Kubo, Y., Inaba, A., and Nakamura, R. (1995). Characterization of the regulation of ethylene biosynthesis in tomato fruit by carbon dioxide and diazocyclopentadiene. Postharvest Biology and Technology 5, 221-33. Mattoo, A.K., and Aharoni, N. (1988). Ethylene and plant senescence. In: Senescence and Aging in Plants. (Eds. L.D. Nooden and A.C. Leopold). pp. 241-80. (Academic Press Inc., Sydney). Mayak, S., Garibaldi, E.A., and Kofranek, A.M. (1977). Carnation flower longevity: Microbial populations as related to silver nitrate stem impregnation. Journal of the American Society for Horticultural Science 102, 637-9. Mayak, S., and Halevy, A.H. (1980). Flower senescence. In: Senescences in Plants. (Ed. K.V. Thimann). pp.131-56. (CRC Press, Boca Raton). McAfee, J.A., and Morgan, P.W. (1971). Rates of production and internal levels of ethylene in vegetative cotton plant. Plant and Cell Physiology 12, 839-47. McCullagh, P., and Nelder, J.A. (1989). Generalized Linear Models, 2nd ed. 511 pp. (Chapman and Hall, London). McGlasson, W.B., Dostal, H.C., and Tigchelaar, E.C. (1975). Comparison of propylene-induced responses of immature fruit of normal and rin mutant tomatoes. Plant Physiology 55, 218-22. McGlasson, W.B., and Pratt, H.K. (1964). Effects of ethylene on cantaloupe fruits harvested at various ages. Plant Physiology 39, 120-7. McMurchie, E.J., McGlasson, W.B., and Eaks, I.L. (1972). Treatment of fruit with propylene gives information about the biogenesis of ethylene. Nature 237, 235-6. McKeon. T.A., Fernandez-Maculet, J.C., and Yang, S.F. (1995). Biosynthesis and metabolism of

12 ethylene. In: Plant Hormones - Physiology, Biochemistry and Molecular Biology. (Ed. P.J. Davies). pp. 118-39. (Kluwer Academic Publishers, Dordrecht). Mor, Y., Reid, M.S., and Kofranek, A.M. (1984). Pulse treatments with silver thiosulfate and sucrose improve the vase life of sweet peas. Journal of the American Society for Horticultural Science 109, 866-8. Muller, R., Serek, M., Sisler, E.C., and Andersen, A.S. (1997). Poststorage quality and rooting ability of Epipremnum pinnatum cuttings after treatment with ethylene actions inhibitors. Journal of Horticultural Science 72, 445-52. Nakajima, N., Mori, H., Yamazaki, K., and Imaseki, H. (1990). Molecular cloning and sequence of a complementary DNA encoding 1-aminocyclopropane-1-carboxylate synthase induced by tissue wounding. Plant and Cell Physiology 31, 1021-9. Nakatsuka, A., Shiomi, S., Kubo, Y., and Inaba, A. (1997). Expression and internal feedback regulation of ACC synthase and ACC oxidase genes in ripening tomato fruit. Plant and Cell Physiology 38, 1103-10. Narula, S.C., and Levy, K.J. (1977). A Monte Carlo comparison of several methods for analyzing small sample binomial data in a two-factor experiment without replication. Journal of Statistical Computation and Simulation 5, 135-43. Ness, P.J., and Romani, R.J. (1980). Effects of aminoethoxyvinylglycine and countereffects of ethylene on ripening of Bartlett pear fruits. Plant Physiology 65, 372-6. New, S., and Marriott, J. (1974). Post-harvest physiology of tetraploid banana fruit: response to storage and ripening. Annals of Applied Biology 78, 193-204. Nichols, R. (1966). Ethylene production during senescence of flowers. Journal of Horticultural Science 41, 279-90. Nichols, R. (1968). The response of carnations (Dianthus caryophyllus) to ethylene. Journal of Horticultural Science 43, 335-49. Nichols, R. (1976). Cell enlargement and sugar accumulation in the gynoecium of the glasshouse carnation (Dianthus caryophyllus L.) induced by ethylene. Planta 130, 47-52. Nichols, R. (1977). Sites of ethylene production in the pollinated and unpollinated senescing carnation (Dianthus caryophyllus) inflorescence. Planta 135, 155-9. Nichols, R., Bufler, G., Mor, Y., Fujino, D.W., and Reid, M.S. (1983). Changes in ethylene production and 1-aminocyclopropane-1-carboxylic acid content of pollinated carnation flowers. Journal of Plant Growth Regulation 2, 1-8. Nooden, L.D. (1988). The phenomenon of senescence and aging. In: Senescence and Aging in Plants. (Eds. L.D. Nooden and A.C. Leopold). pp. 1-50. (Academic Press Inc., Sydney). Nowak, J., and Rudnicki, R.M. (1990). Postharvest Handling and Storage of Cut Flowers, Florist Greens and Potted Plants. 210 pp. (Chapman and Hall, London). Oeller, P.W., Min-Wong, L., Taylor, L.P., Pike, D.A., Theologis, A. (1991). Reversible inhibition of tomato fruit senescence by antisense RNA. Science 254, 437-9. Olde, P., and Marriott, N. (1994). The Grevillea book. volume 1. 255 pp. (Kangaroo Press, Kenthurst). Owens, L.D., Lieberman, M., and Kunishi, A.T. (1971). Inhibition of ethylene production by

13 rhizobitoxine. Plant Physiology 48, 1-4. Page, S., and Olds, M. (1997). Botanica. 1007 pp. (Random House Australia, Milsons Point). Peacock, B.C. (1972). Role of ethylene in the initiation of fruit ripening. Queensland Journal of Agricultural and Animal Sciences 29, 137-45. Pech, J.C., Latche, A., Larrigaudiere, C., and Reid, M.S. (1987). Control of early ethylene synthesis in pollinated petunia flowers. Plant Physiology and Biochemistry 25, 431-7. Philosoph-Hadas, S., Meir., and Aharoni, N. (1985). Autoinhibition of ethylene production in tobacco leaf discs: enhancement of 1-aminocyclopropane-1-carboxylic acid conjugation. Physiologia Plantarum 63, 431-7. Picton, S., Gray, J.E., and Grierson, D. (1995). Ethylene genes and fruit ripening. In: Plant Hormones - Physiology, Biochemistry and Molecular Biology. (Ed. P.J. Davies). pp. 372-94. (Kluwer Academic Publishers, Dordrecht). Poole, M.C., and Joyce, D.C. (1993). A simple and reliable ethylene gassing system for plant studies. Australian Journal of Experimental Agriculture 33, 507-10. Porat, R., Halevy, A.H., Serek, M., and Borochov, A. (1995a). An increase in ethylene sensitivity following pollination is the initial event triggering an increase in ethylene production and enhanced senescence of Phalaenopsis orchid flowers. Physiologia Plantarum 93, 778-84. Porat, R., Shlomo, E., Serek, M., Sisler, E.C., and Borochov, A. (1995b). 1-Methylcyclopropene inhibits ethylene action in cut phlox flowers. Postharvest Biology and Technology 6, 313-9. Porat, R., Weiss, B., Cohen, L., Daus, A., Goren, R., and Droby, S. (1999). Effects of ethylene and 1- methylcyclopropene on the postharvest qualities of ‘Shamouti’ oranges. Postharvest Biology and Technology 15, 156-63. Pratt, H.K., and Goeschl, J.D. (1969). Physiological roles of ethylene in plants. Annual Review of Plant Physiology 20, 541-81. Purvis, A.C., and Barmore, C.R. (1981). Involvement of ethylene in chlorophyll degradation in peel of citrus fruits. Plant Physiology 68, 854-6. Reid, M.S. (1985a). Ethylene and abscission. HortScience 20, 45-50. Reid, M.S. (1985b). Ethylene in postharvest technology. In: Postharvest Technology of Horticultural Crops. (Eds. A.A. Kader, R.F. Kasmire, F.G. Mitchell, M.S. Reid, N.F. Sommer, J.F. Thompson). pp. 68-74. (University of California, Berkeley). Reid, M.S. (1995). Ethylene in plant growth, development and senescence. In: Plant Hormones - Physiology, Biochemistry and Molecular Biology. (Ed. P.J. Davies). pp. 486-508. (Kluwer Academic Publishers, Dordrecht). Reid, M., Dodge, L., and Joyce, D. (1996). Environmental effects on the inhibition of ethylene action by 1-MCP (Abstract only). In: Biology and Biotechnology of the Plant Hormone Ethylene, NATO Advanced Workshop, June 9-13, Crete, Greece. Abstracts. p. 57. Reid, M.S., Evans, R.Y., and Dodge, L.L. (1989). Ethylene and silver thiosulfate influence opening of cut rose flowers. Journal of the Amercian Society for Horticultural Science 114, 436-40. Reid, M.S., Fujino, D.W., Hoffman, N.E., and Whitehead, C.S. (1984). 1-Aminocyclopropane-1- carboxylic acid (ACC) - the transmitted stimulus in pollinated flowers. Journal of Plant Growth

14 Regulation 3, 189-96. Reid, M.S., Mor, Y., and Kofranek, A.M. (1981). Epinasty of Poinsettias - the role of auxin and ethylene. Plant Physiology 67, 950-2. Reid, M.S., Paul, J.L., Farhoomand, M.B., Kofranek, A.M., and Staby, G.L. (1980). Pulse treatments with silver thiosulfate complex extend the vase life of cut carnations. Journal of the American Society for Horticultural Science 105, 25-7. Reid, M.S., and Pratt, H.K. (1970). Ethylene and the respiration climacteric. Nature 226, 976-7. Reid, M.S., and Wu, M.J. (1991). Ethylene in flower development and senescence. In: The Plant Hormone Ethylene. (Eds. A.K. Mattoo and J.C. Suttle). pp. 215-34. (CRC Press, Boca Raton). Rick, C.M., and Butler, L. (1956). Phytogenetics of the tomato. Advances in Genetics 8, 267-382. Roberts, J., Schlindler, C.B., and Tucker, G.A. (1984). Ethylene-promoted tomato flower abscission and the possible involvement of an inhibitor. Planta 160, 159-63. Robbins, J., Reid, M.S., Rost, T., Paul, J.L. (1985). The effect of ethylene in adventitious root formation of Mung bean (Vigna radiata) cuttings. Journal of Plant Growth Regulation 4, 147-57. Sakai, H., Hua, J., Chen, G.Q., Chang, C., Medrano, L.J., Bleecker, A.B., and Meyerowitz, E.M. (1998). ETR2 is an ETR1-like gene involved in ethylene signaling in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America 95, 5812-7. Saltveit, M.E. (1978). Simple apparatus for diluting and dispensing trace concentrations of ethylene in air. HortScience 13, 249-51. Saltveit, M.E. (1980). An inexpensive chemical scrubber for oxidizing volatile organic contaminants in gases and storage room atmospheres. HortScience 15, 759-60. Saltveit, M.E., Bradford, K.J., and Dilley, D.R. (1978). Silver ion inhibits ethylene synthesis and action in ripening fruits. Journal of the American Society of Horticultural Science 103, 472-5. Schaller, G.E., and Bleecker, A.B. (1995). Ethylene-binding sites generated in yeast expressing the Arabidopsis ETR1 gene. Science 270, 1809-11.

15 Scott, K.J., McGlasson, W.B., and Roberts, E.A. (1970). Potassium permanganate as an ethylene absorbent in polyethylene bags to delay ripening of bananas during ripening. Australian Journal of Experimental Agriculture and Animal Husbandry 10, 237-40. Scott, K.J., Wills, R.B.H., and Patterson, B.D. (1971). Removal by ultra-violet lamp of ethylene and other hydrocarbons produced by bananas. Journal of the Science of Food and Agriculture 22, 496-7. Scott, K.J., and Wills, R.B.H. (1973). Atmospheric pollutants destroyed in an ultra violet scrubber. Laboratory Practice 22, 103-6. Serek, M., Reid, M.S., and Sisler, E.C. (1994a). A volatile ethylene inhibitor improves the postharvest life of potted roses. Journal of the American Society for Horticultural Science 119, 572-7. Serek, M., Sisler, E.C., and Reid, M.S. (1994b). Novel gaseous ethylene binding inhibitor prevents ethylene effects in potted flowering plants. Journal of the American Society of Horticultural Science 119, 1230-3. Serek, M., Sisler, E.C., and Reid, M.S. (1995a). Effects of 1-MCP on the vase life and ethylene response of cut flowers. Plant Growth Regulation 16, 93-7. Serek, M., Sisler, E.C., Reid, M.S. (1995b). 1-Methylcyclopropene, a novel gaseous inhibitor of ethylene action, improves the life of fruits, cut flowers and potted plants. Acta Horticulturae 394, 337-45. Serek, M., Sisler, E.C., and Reid, M.S. (1996). Ethylene and the postharvest performance of miniature roses. Acta Horticulturae 424, 145-9. Serek, M., Sisler, E.C., Tirosh, T., and Mayak, S. (1995c). 1-Methylcyclopropene prevents bud, flower, and leaf abscission of Geraldton Waxflower. HortScience 30, 1310. Serek, M., Tamari, G., Sisler, E.C., and Borochov, A. (1995d). Inhibition of ethylene-induced cellular senescence symptoms by 1-methylcyclopropene, a new inhibitor of ethylene action. Physiologia Plantarum 94, 229-32. Sexton, R., Lewis, L.N., Trewavas, A.K., and Kelly, P. (1985). Ethylene and abscission. In: Ethylene and Plant development. (Eds. J.A. Roberts, and G.A. Tucker). pp. 173-96. (Butterworths, London).

Sexton, R., and Roberts, J.A. (1982). Cell biology of abscission. Annual Review of Plant Physiology 33, 133-62. Sherman, M. (1985). Control of ethylene in the postharvest environment. HortScience 20, 57-60. Sisler, E.C. (1977). Ethylene activity of some pi acceptor compounds. Tobacco Science 21, 43-5. Sisler, E.C. (1979). Measurement of ethylene binding in plant tissue. Plant Physiology 64, 538-42. Sisler, E.C. (1980). Partial purification of an ethylene-binding component from plant tissue. Plant Physiology 66, 404-6. Sisler, E.C. (1982). Ethylene-binding properties of a Triton X-100 extract of mung bean sprouts. Journal of Plant Growth Regulation 1, 211-8. Sisler, E.C. (1991). Ethylene-binding components in plants. In: The Plant Hormone Ethylene. (Eds. A.K. Mattoo, and J.C. Suttle). pp. 81-99. (CRC Press: Boca Raton). Sisler, E.C., and Blankenship, S.M. (1993a). Diazocyclopentadiene, a light sensitive reagent for the ethylene receptor in plants. Plant Growth Regulation 12, 125-32.

16 Sisler, E.C., and Blankenship, S.M. (1993b). Effect of diazocyclopentadiene on tomato ripening. Plant Growth Regulation 12, 155-60. Sisler, E.C., Blankenship, S.M., Fearn, J.C., and Haynes, R. (1993). Effect of diazocyclopentadiene (DACP) on cut carnations. In: Cellular and Molecular Aspects of the Plant Hormone Ethylene. (Eds. J.C. Pech, A. Latche, and C. Balague). pp. 182-7. (Kluwer Academic Publishers, Dordrecht). Sisler, E.C., Dupille, E., and Serek, M. (1996a). Effect of 1-methylcyclopropene and methylenecyclopropene on ethylene binding and ethylene action on cut carnations. Plant Growth Regulation 18, 79-86. Sisler, E.C., Goren, R., Huberman, M. (1985). Effect of 2,5-norbornadiene on abscission and ethylene production in citrus leaf explants. Physiologia Plantarum 63, 114-20. Sisler, E.C., and Lallu, N. (1994). Effect of diazocyclopentadiene (DACP) on tomato fruits harvested at different ripening stages. Postharvest Biology and Technology 4, 245-54. Sisler, E.C., and Pian, A. (1973). Effect of ethylene and cyclic olefins on tobacco leaves. Tobacco Science 17, 68-72. Sisler, E.C., Reid, M.S., and Fujino, D.W. (1983). Investigation of the mode of action of ethylene in carnation senescence. Acta Horticulturae 141, 229-34. Sisler, E.C., Reid, M.S., and Yang, S.F. (1986). Effect of antagonists of ethylene action on binding of ethylene in cut carnations. Plant Growth Regulation 4, 213-8. Sisler, E.C., Serek, M., and Dupille, E. (1996b). Comparison of cyclopropene, 1-methylcyclopropene, and 3,3-dimethylcyclopropene as ethylene antagonists in plants. Plant Growth Regulation 18, 169-74. Sisler, E.C., and Serek, M. (1997). Inhibitors of ethylene responses in plants at the receptor level: recent developments. Physiologia Plantarum 100, 577-82. Sisler, E.C., and Serek, M. (1999). Compounds controlling the ethylene receptor. Botanical Bulletin of Academia Sinica 40, 1-7. Sisler, E.C., and Yang, S.F. (1984a). Ethylene, the gaseous plant hormone. BioScience 34, 234-8. Sisler, E.C., and Yang, S.F. (1984b). Anti-ethylene effects of cis-2-butene and cyclic olefins. Phytochemistry 23, 2765-8. Song, J., Tian, M.S., Dilley, D.R., and Beaudry, R.M. (1997). Effect of 1-MCP on apple fruit ripening and volatile production (abstract only). HortScience 32, 536. Stead, A.D. (1992). Pollination-induced flower senescence: a review. Plant Growth Regulation 11, 13- 20. Steel, R.G.D., and Torrie, J.H. (1987). Principles and Procedures of Statistics, A Biometrical Approach, 2nd ed. 633 pp. (McGraw-Hill Book Company, Singapore). Stewart, I., and Wheaton, T.A. (1972). Carotenoids in citrus: their accumulation induced by ethylene. Journal of Agricultural and Food Chemistry 20, 448-9. Suttle, J.C., and Kende, H. (1980). Ethylene action and loss of membrane integrity during petal senescence in Tradescantia. Plant Physiology 65, 1067-72. Taylorson, R.B. (1979). Response of weed seeds to ethylene and related hydrocarbons. Weed Science 27,

17 7-10. Tian, M.S., Gong, Y., and Bauchot, A.D. (1997). Ethylene biosynthesis and respiration in strawberry fruit treated with diazocyclopentadiene and IAA. Plant Growth Regulation 23, 195-200. Tigchelaar, E.C., McGlasson, W.B., and Buescher, R.W. (1978). Genetic regulation of tomato fruit ripening. HortScience 13, 508-13. Trippi, V., and Paulin, A. (1984). Senescence of cut carnations: a phasic phenomenon. Physiologia Plantarum 60, 221-6. Tucker, G., and Brady, C.J. (1987). Silver ions interrupt fruit ripening. Journal of Plant Physiology 127, 165-9. Van Altvorst, A.C., and Bovy, A.G. (1995). The role of ethylene in the senescence of carnation flowers, a review. Plant Growth Regulation 16, 43-53. Van Doorn, W.G., and Reid, M.S. (1992). Role of ethylene in flower senescence of Gypsophila paniculata L. Postharvest Biology and Technology 1, 265-72. Veen, H. (1979a). Effects of silver on ethylene synthesis and action in cut carnations. Planta 145, 467- 70. Veen, H (1979b). Effects of silver salts on ethylene production and respiration of cut carnations. Acta Horticulturae 91, 99-103. Veen, H. (1983). Silver thiosulphate: an experimental tool in plant science. Scientia Horticulturae 20, 211-24. Veen, H. (1987). Use of inhibitors of ethylene action. Acta Horticulturae 201, 213-22. Veen, H., and van de Geijn, S.C. (1978). Mobility and ionic form of silver as related to longevity of cut carnations. Planta 140, 93-6. Vendrell, M., and McGlasson, W.B. (1971). Inhibition of ethylene production in banana fruit tissue by ethylene treatment. Australian Journal of Biological Science 24, 885-95. Vuthapanich, S., Simons, D.H., and Turnbull, L.V. (1993). Effects of harvest maturity and postharvest treatments on vase life of Grevillea cv. Majestic inflorescences. In: Australasian Postharvest Horticulture Conference, Proceedings, September 20 - 24, The University of Queensland, Gatton College, Qld, Australia. pp. 45-51.

18 Wade, N.L., and Satyan, S.H. (1997). Eastern Australian native plants as cut flower export products. In: Australasian Postharvest Horticulture Conference, Proceedings, September 28 - October 3, University of Western Sydney, Hawkesbury, NSW, Australia. pp. 327-8. Wang, C.Y. (1989). Relation of chilling stress to ethylene production. In: Low Temperature Stress Physiology in Crops. (Ed. P.H. Li). pp. 178-89. (CRC Press, Boca Raton). Wang, C.Y., Baker, J.E., Hardenberg, R.E., and Lieberman, M. (1977). Effects of 2 analogs of rhizobitoxine and sodium benzoate on senescence of snapdragons. Journal of the American Society for Horticultural Science 102, 517-20. Wang, C.Y., and Mellenthin, W.M. (1977). Effect of aminoethoxy analog of rhizobitoxine on ripening of pears. Plant Physiology 59, 546-9. Wang, H., and Woodson, W.R. (1989). Reversible inhibition of ethylene action and interruption of petal senescence in carnation flowers by norbornadiene. Plant Physiology 89, 434-8. Whitehead, C.S., Fujino, D.W., and Reid, M.S. (1983). Identification of the ethylene precursor, 1- aminocyclopropane-1-carboxylic acid (ACC) in pollen. Scientica Horticulturae 21, 291-7. Whitehead, C.S., and Halevy, A.H. (1989). Ethylene sensitivity: the role of short-chain fatty acids in pollination-induced senescence of Petunia hybrida flowers. Journal of Plant Growth Regulation 8, 41-54. Williamson, V.G. (1996). Physiological and microbiological processes of cut flower senescence in two Australian native genera, Acacia and Boronia. PhD thesis, University of New England. 229 pp. (+ Appendices). Wills, R.B.H., and Kim, G.H. (1995). Effect of ethylene on postharvest life of strawberries. Postharvest Biology and Technology 6, 249-55. Wojciechowski, J. (1989). Ethylene removal from gases by means of catalytic combustion. Acta Horticulturae 258, 131-9. Woltering, E.J. (1987). Effects of ethylene on ornamental pot plants: a classification. Scientia Horticulturae 31, 283-94. Woltering, E.J., and Somhorst, D. (1990). Regulation of anthocyanin synthesis in Cymbidium flowers. Effects of emasculation and ethylene. Journal of Plant Physiology 136, 295-9. Woltering, E.J., and Sterling, E.P. (1986). Design for studies on ethylene sensitivity and ethylene production of ornamental products. Acta Horticulturae 181, 483-8. Woltering, E.J., and Van Doorn, W.G. (1988). Role of ethylene in senescence of petals - morphological and taxonomical relationships. Journal of Experimental Botany 39, 1605-16. Woodson, W.R., Brandt, A.S., Itzhaki, H., Maxson, J.M., Wang, H., Park, K.Y., and Larsen, P.B. (1993). Ethylene regulation and function of flower senescence-related genes. In: Cellular and Molecular Aspects of the Plant Hormone Ethylene. (Eds. J.C. Pech, A. Latche, and C. Balague). pp. 291-7. (Kluwer Academic Press, Dordrecht). Woodson, W.R., Hanchey, S.H., and Chisholm, D.N. (1985). Role of ethylene in the senescence of isolated Hibiscus petals. Plant Physiology 79, 679-83. Woodson, W.R., and Lawton, K.A. (1988). Ethylene-induced gene expression in carnation petals. Plant Physiology 87, 498-503.

19 Worrall, R.J., Wade, N., and Johnson, K. (1999). Improving the quality of NSW Christmas bush (Ceratopetalum gummiferum) flowers. In: 5th Australian Wildflower Conference, Proceedings, April 14-17, Melbourne, Australia. pp. 126-8. Wrigley, J.W., and Fagg, M. (1997). Australian Native Plants, 4th ed. 696 pp. (Reed Books, Kew).

Yang, S.F. (1981). Biosynthesis of ethylene and its regulation. In: Recent Advances in the Biochemistry of Fruits and Vegetables. (Eds. J. Friend and M.J.C. Rhodes). pp. 89-106. (Academic Press, London). Yang, S.F., and Hoffman, N.E. (1984). Ethylene biosynthesis and its regulation in higher plants. Annual Review of Plant Physiology 35, 155-89.

20 Yang, S.F., Yip, W.K., Satoh, S., Miyazaki, J.H., Jiao, X., Liu, Y., Su, L.Y., and Peiser, G.D. (1990). Metabolic aspects of ethylene biosynthesis. In: Plant Growth Substances 1988. (Eds. R.P. Pharis, and S.B. Road). pp. 291-9. (Springer-Verlag, Berlin). Yen, H.C., Lee, S., Tanksley, S.D., Lanahan, M.B., Klee, H.J., and Giovannoni, J.J. (1995). The tomato never-ripe locus regulates ethylene inducible gene expression and is linked to a homolog of the Arabidopsis ETR1 gene. Plant Physiology 107, 1343-53. Yu, Y.B., Adams, D.O., and Yang, S.F. (1979). 1-Aminocyclopropanecarboxylate synthase, a key enzyme in ethylene biosynthesis. Archives of Biochemistry and Biophysics 198, 280-6. Yu, Y.B., Adams, D.O., and Yang, S.F. (1980). Inhibition of ethylene production by 2,4-Dinitrophenol and high temperature. Plant Physiology 66, 286-90. Yu, Y.B., and Yang, S.F. (1979). Auxin-induced ethylene production and its inhibition by aminoethoxyvinylglycine and cobalt ion. Plant Physiology 64, 1074-7. Zacarias, L., and Reid, M.S. (1990). Role of growth regulators in the senescence of Arabidopsis thaliana leaves. Physiologia Plantarum 80, 549-54. Zhang, X.S., and O’Neill, S.D. (1993). Ovary and gametophyte development are coordinately regulated by auxin and ethylene following pollination. The Plant Cell 5, 403-18. Zieslin, N., and Gottesman, V. (1983). Involvement of ethylene in the abscission of flowers and petals of Leptospermum scoparium. Physiologia Plantarum 58, 114-8. Zobel, R.W. (1973). Some physiological characteristics of the ethylene-requiring tomato mutant diageotropica. Plant Physiology 52, 385-9.

21 APPENDICES

APPENDIX A LITERATURE REVIEW

1.1 ETHYLENE IN PLANT BIOLOGY

Ethylene is a simple, gaseous hydrocarbon molecule comprised of two carbon and four hydrogen atoms with one double bond (Abeles et al. 1992). It is a plant hormone which has profound effects on processes of growth, development and senescence (Fluhr and Mattoo 1996). Active in trace amounts, the effects of ethylene on plants are often spectacular and commercially important (Pratt and Goeschl 1969). Changes in the rate of ethylene synthesis by plants is commonly associated with specific stages of their development. Additionally, ethylene biosynthesis can also be stimulated in response to a variety of abiotic and biotic stresses. These stresses include temperature extremes, water stress, mechanical wounding, pathogen infection and exposure to chemicals or other hormones (Abeles et al. 1992). This review examines the roles of ethylene in plant biology and the various approaches to regulate ethylene in postharvest horticulture.

1.1.1 General roles

Ethylene is responsible for eliciting and coordinating a range of processes in plants. These processes include seed germination (Ketring 1977; Taylorson 1979), geotropism (Zobel 1973; Jackson 1979) and altered shoot growth and differentiation, including the seedling ‘triple response’ (Goeschl et al. 1966) and petiole epinasty (Bradford and Yang 1980; Reid et al. 1981). Altered root growth and differentiation (Jackson 1985), including adventitious rooting (Robbins et al. 1985) and senescence of vegetative organs (Mattoo and Aharoni 1988) are also regulated by ethylene. Additionally, ethylene is associated with flower induction (Burg 1962), flower sex determination (Abeles et al. 1992), flower opening (Reid et al. 1989), flower senescence (Nichols 1966; Halevy and Mayak 1981; Woltering and Van Doorn 1988), abscission of plant organs (Sexton et al. 1985) and fruit ripening (Kidd and West 1945; Burg and Burg 1962). The involvement of ethylene in abscission, vegetative and flower senescence and fruit ripening are discussed below in more detail.

1.1.2 Abscission

Abscission is the process where plant organs, including leaves, fruit, flowers, flower parts and buds, are shed (Sexton et al. 1985). Abscission involves separation of a narrow arrangement of cells called the separation layer within a generally anatomically distinct abscission zone. The cells of abscission zones are typically smaller and less vacuolated than adjacent cells. The stele which branches in the zone normally lacks lignified fibres (Sexton et al. 1985). According to Sexton et al. (1985), the process of

22 abscission involves activation of cell wall hydrolases and/or mechanical rupture, such as in the shedding of bark, which weaken the walls of cells in the abscission zone. Enzymes most commonly associated with abscission are cellulases and pectinases (Sexton et al. 1985). Commencement of abscission is associated with increased respiration (Marynick 1977) and stimulation of RNA and protein synthesis (Holm and Abeles 1967; Abeles et al. 1979). Ethylene has been implicated in the induction and acceleration of abscission (Sexton et al. 1985; Reid 1985a). However, ethylene is probably not the universal regulator of abscission, as evidenced by a poor correlation between endogenous ethylene and natural abscission in some plant species (Addicott 1982; Abeles et al. 1992).

Current evidence suggests that abscission is a two stage process controlled by the interaction of auxin and ethylene (Beyer and Morgan 1971; Addicott 1982; Sexton and Roberts 1982; Reid 1985a; Brown 1997). In the first stage, or the lag phase, high auxin flux from the subtending organ (i.e. leaf, flower or fruit) across the abscission zone maintains it in a state that is insensitive to ethylene. Thus, abscission is inhibited. During the second stage, or the separation phase, a decline in the auxin flux from the subtending organ causes the abscission zone to become sensitive to ethylene. Accordingly, abscission of the organ occurs. It was deduced that the relative flux of auxin across the abscission zone influenced abscission more than the absolute auxin concentration. Decrease in auxin source or sink strength, and flux, have been reported to lead to greater sensitivity to ethylene and the abscission of flowers and fruit (Roberts et al. 1984; Brown 1997). Flower and leaf abscission from a number of plants is accelerated by exposure to exogenous ethylene (Abeles et al. 1992). An association of elevated endogenous ethylene production by flowers with floral organ abscission provides evidence that ethylene regulates natural abscission (Abeles et al. 1992). For example, during and following pollination of cyclamen flowers, the rate of endogenous ethylene production increased and corolla abscission occurred (Halevy et al. 1984). Woltering and Van Doorn (1988) suggest that the senescence process in flowers of horticultural importance can be classified into two groups, those that abscise or those that wilt. Plant families which contained flowers that abscised exhibited the greatest sensitivity to ethylene.

Abscission of some immature and mature fruit can be induced by exposure to exogenous ethylene. Moreover, Brady and Spiers (1991) reported that endogenous ethylene and/or 1-aminocyclopropane-1- carboxylic acid (ACC; the immediate precursor of ethylene) concentrations were often higher during periods of abscission. Exogenously supplied or endogenously produced ethylene synchronised separation at abscission zones in raspberries. Conversely, treatment with aminoethoxyvinylglycine (AVG), an inhibitor of ethylene synthesis, prevented raspberry fruitlet abscission (Burdon and Sexton 1990). Sexton and Roberts (1982) determined that the activity of several cell wall degrading enzymes, including cellulase and polygalacturonase, was regulated by ethylene.

Treatment of plant tissue with inhibitors of ethylene synthesis (e.g. rhizobitoxine) or perception (e.g. Ag+) suggests that ethylene is involved in natural abscission processes of some plant species. For instance, abscission of Zygocactus and snapdragon flowers was prevented by STS (Cameron and Reid 1981) and

23 rhizobitoxine analogues (Wang et al. 1977), respectively. Thus, inhibition was evident even in the absence of exogenously supplied ethylene.

Assertions that ethylene is not the sole regulator of abscission are based on observations that treatment of a number of plants with ethylene did not induce leaf or flower abscission and that abscission proceeded in the absence of ethylene (Addicott 1982; Abeles et al. 1992). Accordingly, it has been claimed that the role of ethylene in abscission is not clear (Sexton et al. 1985). However, Reid (1985a) and Brown (1997) propose that regulation of ethylene sensitivity by the auxin flux from the subtending organ could account for the absence of abscission in the presence of ethylene during stage 1.

1.1.3 Senescence of vegetative tissue

Vegetative tissue senescence is usually associated with loss of protein, starch and chlorophyll and leads to tissue death (Mattoo and Aharoni 1988). Evidence that ethylene is involved in vegetative senescence has been based on studies where exogenous or endogenous ethylene was correlated with leaf senescence both on intact plants and of detached leaf tissue. Ethylene is associated with senescence of vegetative tissue in a number of plant species (Mattoo and Aharoni 1988). For example, ethylene treatment of tobacco leaf discs stimulated increased respiration, ethylene production and chlorophyll degradation, and thus senescence (Aharoni and Lieberman 1979). Also, ethylene synthesis inhibitors such as AVG inhibited the rise in ethylene production and delayed chlorophyll degradation. Exogenous ethylene has also been reported to increase the rate of degradation of chlorophyll, RNA and protein in lettuce leaf tissue (Aharoni 1989). Aharoni and Lieberman (1979) suggested that endogenous ethylene may regulate senescence in combination with other plant hormones including, auxin and cytokinin.

Ethylene production by and respiration of tobacco leaf discs increased during the rapid phase of chlorophyll degradation (Aharoni et al. 1979b). Aharoni et al. (1979a) found that increased ethylene production coincided with the advanced stages of natural senescence of tobacco leaves, but it was unclear whether ethylene stimulated senescence. However, Gepstein and Thimann (1981) reported that increased ethylene production preceded chlorophyll degradation of oat leaf segments. The relationship between ethylene production and chlorophyll degradation appears to vary according to plant species and the experimental system (viz. intact vs. excised tissue). Nevertheless, it appears that endogenous ethylene does regulate a number of events during vegetative senescence (Aharoni and Lieberman 1979; Gepstein and Thimann 1981). However, in other species, including cotton plants and an ethylene resistant mutant of Arabidopsis thaliana, senescence of vegetative organs was shown to proceed in the absence of exogenous or endogenous ethylene (McAfee and Morgan 1971; Zacarias and Reid 1990). Thus, senescence may also be regulated by increasing sensitivity of the tissue to ethylene.

1.1.4 Flower senescence

Flower senescence, like senescence of other organs is a highly regulated developmental process. A series

24 of co-ordinated biochemical and physiological changes lead to termination of floral organ life (Nooden 1988; Van Altvorst and Bovy 1995). Senescence of floral organs is associated with the visual changes of petal wilting and discolouration, and attendant biochemical changes. Biochemical changes include increased activity of hydrolytic enzymes, degradation of starch and chlorophyll and loss of cellular compartmentation (Van Altvorst and Bovy 1995). In some flowers, physiological changes that include surges in respiration and ethylene biosynthesis accompany senesence (Halevy 1986). In many plant species, flower senescence is accelerated and regulated by ethylene (Woltering and Van Doorn 1988). Halevy (1986) suggested that flowers can be classified as climacteric or non-climacteric based on the presence or absence, respectively, of increased rates of respiration and ethylene production associated with senescence.

Most research into ethylene-mediated flower senescence has been on cut carnation (Dianthus caryophyllus), which is an excellent ‘model’ flower (Reid 1995). Senescence of cut carnation flowers is characterised by wilting of petals accompanied by a climacteric increase in autocatalytic ethylene production (Nichols 1966). Exposure of flowers to ethylene stimulates ethylene biosynthesis, initiates and hastens flower senescence (Nichols 1968) and induces the loss of proteins, phospholipids and polar fatty acids (Trippi and Paulin 1984). Attendant changes in membrane permeability of cut carnation flowers exposed to ethylene led to loss of cellular compartmentation, as evidenced by mixing of cytoplasmic and vacuolar contents (Trippi and Paulin 1984). Borochov and Faragher (1983) found that ethylene application to cut carnation flowers elicited an earlier increase in membrane permeability. Ethylene has also been implicated in stimulating the swelling of the ovary of cut carnations (Nichols 1968, 1976) and mobilisation of carbohydrates from the petals to the developing ovary.

Additional evidence that ethylene mediates cut carnation flower senescence is provided from studies using inhibitors of ethylene biosynthesis and perception. Treatment of cut carnation flowers with aminooxyacetic acid (AOA), an inhibitor of ethylene biosynthesis, delayed flower senescence and endogenous ethylene production (Fujino et al. 1980). Likewise, treatment with STS, an inhibitor of ethylene perception, also prevented flower senescence (Veen 1979a). Woodson and Lawton (1988) reported that several mRNAs expressed in senescing petals of cut carnation flowers exposed to ethylene appeared to be related to the increase in ethylene production. These mRNAs were found to be similar to those that accumulated during natural senescence. Woodson et al. (1993) speculated that these mRNAs encoded for ACC synthase and ACC oxidase enzymes which were functionally important to the biochemical changes that occurred during senescence.

Application of exogenous ethylene to a number of other ornamentals, including Ipomoea tricolor (Kende and Baumgartner 1974; Kende and Hanson 1976), Tradescantia (Suttle and Kende 1980), Petunia (Whitehead et al. 1983), Hibiscus rosa-sinensis (Woodson et al. 1985) and various orchids such as Vanda, Cattleya and Cymbidium (Goh et al. 1985), also stimulates endogenous ethylene production and flower senescence. In contrast, the senescence of some flowers proceeds in the absence of ethylene (Woltering and van Doorn 1988). These flowers include Chrysanthemums (Woltering 1987), Iris and

25 Gladiolus (Woltering and van Doorn 1988) and non-pollinated Cyclamen (Halevy et al. 1984) and Dendrobium orchids (Ketsa and Luangsuwalai 1996).

1.1.4.1 Pollination-induced senescence

Pollination of many flowers, including orchids, carnation and Petunia, is accompanied by a sudden rise in the rate of endogenous ethylene production. This surge induces rapid flower senescence (Stead 1992). Ethylene has been implicated in coordinating a range of postpollination development events, including petal senescence (Nichols 1977), pigment changes (Woltering and Somhorst 1990), floral organ abscission (Stead 1992) and ovary growth and development (Zhang and O’Neill 1993).

In many species of orchids, endogenous ethylene production rates increase after pollination, emasculation or exposure to ethylene and signal the onset of flower senescence (Stead 1992). Orchid flower senescence is characterised by rapid wilting and/or anthocyanin breakdown or synthesis depending upon the particular species. The pollination signal responsible for inducing increased rates of ethylene production was proposed by Burg and Dijkman (1967) to be auxin, on the basis that orchid pollen contains substantial amounts of auxin. Furthermore, application of auxin to the stigmas of Phalaenopsis flowers mimicked the effect of pollination on ethylene production (Zhang and O’Neill 1993). However, Reid et al. (1984) reported that auxin had no effect on the senescence of carnation flowers.

In cut carnation flowers, pollination induces irreversible petal wilting and stimulates a sequential increase in the rate of ethylene production by stigmas, ovaries, receptacles and finally petals (Nichols et al. 1983). This sequence is possibly due to ethylene and ACC translocation from the stigma to other flower organs. However, Reid et al. (1984) showed that ethylene produced by the stigmas of flowers was not involved in petal senescence. They proposed that ACC was the stimulus for the pollination response. Radioactively 2-14C-labelled ACC applied to the stigma of pollinated Petunia flowers was transported to the petals where radioactively 14C-labelled ethylene was produced (Reid et al. 1984). Whitehead et al. (1983) established that Petunia and carnation pollen contained ACC which could be converted to ethylene in the stigma. However, Hoekstra and Weges (1986) showed that AVG-treated Petunia stigmas produced less ethylene following pollination, indicating that ACC synthesis by the stigmatic tissue was necessary for increased ethylene production. This finding was confirmed by Pech et al. (1987), who reported that pollination led to an increase in ACC synthase activity in Petunia styles.

In a study of cut carnation flowers, Nichols et al. (1983) discovered that pollen produced little or no ethylene. They suggested that the increased rate of ethylene production may be due to an interaction between the pollen and stigmatic tissue, possibly as a wound response from the growing pollen tubes as reported by Gilissen (1977). Stead (1992) suggested that the quantity of pollen-borne ACC was not sufficient to sustain ethylene production at rates exhibited following pollination. Nevertheless, pollen- borne ACC may contribute to the initiation of autocatalytic ethylene production by the stigma. Larsen et al. (1993) speculated that the interorgan signalling responsible for triggering postpollination events may

26 be initiated by a recognition process between pollen tubes and stigmatic tissue, and not by wounding. Thus, ethylene may act in interorgan communication following pollination because of its capacity to diffuse through intercellular spaces (Larsen et al. 1993).

There is also an arguement that pollination stimulates the activity of other compounds such as short-chain fatty acids which, in turn, increase the sensitivity of flowers to ethylene (Whitehead and Halevy 1989). Increases in the endogenous concentrations of octanoic acid and other short-chain fatty acids were found in the gynoecia and perianths of Phalaenopsis flowers and corollas of Petunia following pollination (Whitehead and Halevy 1989; Halevy et al. 1996). Furthermore, application of octanoic acid to the stigmas of AOA-treated Phalaenopsis flowers enhanced their sensitivity to ethylene (Halevy et al. 1996). These observations support the view that these compounds are transported to floral organs where they enhance ethylene sensitivity leading to flower senescence (Whitehead and Halevy 1989; Halevy et al. 1996).

1.1.5 Fruit ripening and senescence

Fruit ripening is a special case of organ senescence during which a series of biochemical and physiological changes, such as loss of chlorophyll, softening, colouring and sweetening, take place (Pratt and Goeschl 1969). Fruit have been classified as climacteric or non-climacteric based on their respiratory activity during ripening (Biale 1964). Climacteric fruit, such as bananas and tomatoes, are characterised by elevated rates of respiration at the onset of ripening. This burst is accompanied by distinct structural and compositional changes. In contrast, non-climacteric fruit show no changes in respiration that can be associated with marked changes in structure and composition. Ethylene production by climacteric fruit increases at the onset of ripening. This increase is thought to regulate the initiation of biochemical and physiological changes associated with ripening, including the increase in respiration (Burg and Burg 1962). Burg and Burg (1962) and Reid and Pratt (1970) proposed that the capacity of climacteric fruit to produce ethylene autocatalytically following exposure to exogenous or endogenous ethylene further distinguished them from non-climacteric fruit.

Treatment of climacteric banana fruit with propylene (an ethylene analogue) was shown by McMurchie et al. (1972) to induce a rapid increase in the rate of respiration and ethylene production by fruit, and an acceleration of ripening. However, no increase in the rate of ethylene production by non-climacteric lemon fruit treated with propylene was observed, although the rate of respiration increased and degreening was accelerated. These findings led McMurchie et al. (1972) to propose that two systems regulate ethylene production in higher plants. System 1 operated in climacteric, non-climacteric fruit and vegetative tissue and was responsible for basal and wound-induced ethylene production. System 2 occurred in climacteric fruit only, and was proposed to be responsible for the autocatalytic increase in ethylene production during ripening.

The internal ethylene content of climacteric fruit such as bananas remains relatively constant throughout

27 fruit growth and development. However, at the onset of ripening, a sharp increase in ethylene production precedes or accompanies the climacteric increase in respiration (Burg and Burg 1965). The accelerated production rate may be required to raise the internal content of ethylene to a stimulatory level or to initiate an autocatalytic response once the tissue became sensitive to the still relatively low endogenous level of ethylene (Burg and Burg 1965). Treatment of climacteric fruit with ethylene or compounds which mimic ethylene action, prematurely induce increased respiration and ethylene production and accelerate ripening and senescence (Burg and Burg 1962, 1967; McMurchie et al. 1972). The threshold exogenous ethylene concentration required to initiate ripening of mature climacteric fruit is generally in the range of 0.1-1.0 µL/L (Burg and Burg 1962).

Treatment of apple fruit with AVG or tomato fruit with Ag+ delays ripening, further confirming that ethylene is responsible for initiating ripening (Masia et al. 1998; Saltveit et al. 1978; Atta-Aly et al. 1987). Additionally, in climacteric fruit, treatment with Ag+ halts the ripening process even after it is well advanced. Thus, the continual presence of ethylene is necessary for ongoing co-ordination of ripening (Tucker and Brady 1987). Some fruit become more sensitive to ethylene as they mature or ripen (Burg and Burg 1965; Peacock 1972). For example, tomato fruit do not ripen in the presence of ethylene unless they are close to full maturity (McGlasson et al. 1975). Conversely, cantaloupe and banana fruit ripen in the presence of ethylene at any maturity stage, although higher concentrations of ethylene are required to initiate ripening of immature fruit (McGlasson and Pratt 1964; Pratt and Goeschl 1969).

Ripening of non-climacteric fruit is generally ethylene-independent. Exposure of non-climacteric fruit to exogenous ethylene usually stimulates increased respiration. However, upon removal of ethylene, the respiration rate decreases rapidly (Biale 1964). Non-climacteric fruit do not produce ethylene in response to exposure to ethylene (McMurchie et al. 1972). Nevertheless, some non-climacteric fruit are still sensitive to ethylene. For example, ethylene treatment causes colour changes in the flavedo of citrus fruit, as evidenced by accelerated chlorophyll degradation (Purvis and Barmore 1981) and enhanced carotenoid synthesis (Stewart and Wheaton 1972). Similarly, softening of non-climacteric strawberry fruit was accelerated by low levels of ethylene that accumulated inside punnets sealed with polyethylene film (Wills and Kim 1995).

1.2 ETHYLENE BIOSYNTHESIS

Ethylene is produced by a variety of organisms, including bacteria, fungi and higher plants, as part of their development and/or in response to various environmental stimuli (Abeles et al. 1992). Determination of the sequence of events in ethylene biosynthesis by higher plants has been the focus of intense research over the past 20 years. Ethylene biosynthesis in higher plants has been reviewed in detail by Lieberman (1979), Yang and Hoffman (1984), Kende (1993) and Fluhr and Mattoo (1996).

The primary sequence of ethylene biosynthesis involves conversion of methionine to S- adenosylmethionine (AdoMet) which then forms ACC and finally ethylene (Figure 1.1). The ethylene

28 biosynthetic pathway also has links to other pathways. The methionine cycle is responsible for replenishing this sulfur containing amino acid in order to sustain metabolic pathways, including ethylene biosynthesis (Adams and Yang 1977). Adams and Yang (1977) found that methionine levels in apple tissue were low and suspected that it must be reformed in order to maintain ethylene biosynthesis. They confirmed this theory by showing that the sulfur group of methylthioadenosine, a degradation product of the conversion of methionine to AdoMet, was hydrolysed to methylthioribose and then cycled back to form methionine. The conjugation of ACC with malonate to form 1-(malonylamino) cyclopropane-1- carboxylic acid (MACC) is also closely linked to the ethylene biosynthetic pathway (Yang et al. 1990). It is still unclear what regulates the accumulation of MACC. Fluhr and Mattoo (1996) suggest that conversion of ACC to MACC may contribute to the regulation of ACC accumulation and, hence, ethylene formation.

Catalysis of each step in the ethylene biosynthetic pathway is enzyme mediated. The conversion of methionine to AdoMet is catalysed by AdoMet synthetase (Yu and Yang 1979). ACC synthase (ACS) is responsible for catalysing the conversion of AdoMet to ACC (Adams and Yang 1979). ACS plays a major role in regulating ethylene biosynthesis, particularly during autocatalysis (positive feedback) or autoinhibition (negative feedback) where ACS is stimulated or inhibited, respectively (Yang and Hoffman 1984). ACS is regarded as being the major rate-limiting enzyme in ethylene biosynthesis (Picton et al. 1995). The conversion of ACC to ethylene, is catalysed by an oxidative enzyme called ACC oxidase (ACO). ACO is also considered rate limiting. It was formerly known as the ethylene forming enzyme (EFE; Adams and Yang 1979).

Methionine AdoMet synthetase

Methionine S-adenosyl methionine cycle ACC synthase

Methylthioadenosine + 1-aminocyclopropane-1-carboxylic acid ACC oxidase MACC Ethylene

Figure 1.1. Metabolic pathway of ethylene biosynthesis in higher plants (after Fluhr and Mattoo 1996).

29 1.3 INHIBITORS OF ETHYLENE BIOSYNTHESIS

Two inhibitors of ethylene biosynthesis which have received considerable attention in literature are AVG and AOA. Both inhibit pyridoxal phosphate-dependent enzymes, such as ACS (Yang 1981). The conversion of ACC to ethylene by ACO has been shown to be inhibited by the presence of cobalt ions and by high temperature (Yang and Hoffman 1984). Treatment of mung bean hypocotyls (Yu and Yang 1979) and oat leaves (Gepstein and Thimann 1981) with cobalt ions greatly inhibited ethylene production and associated senescence. Accumulation of ACC in mung bean hypocotyls treated with cobalt ions corresponded to reduced ethylene production, indicating that ACO activity was inhibited (Yu and Yang 1979). ACO activity is also inhibited in mung bean and apple fruit tissue exposed to high temperatures (e.g. 35oC), as evidenced by accumulation of ACC and reduced ethylene production (Yu et al. 1980).

1.3.1 Aminoethoxyvinylglycine

Rhizobitoxine is a phytotoxin produced by the Rhizobium japonicum bacteria and inhibits ethylene biosynthesis (Owens et al. 1971). AVG is an analogue of rhizobitoxine that has been shown to be a very effective inhibitor of ethylene-related processes in plants (Amrhein and Wenker 1979; Lieberman 1979). AVG prevents the conversion of AdoMet to ACC by inhibiting ACS (Boller et al. 1979). Ripening of preclimacteric pear fruit was delayed by vacuum infiltration with AVG (Wang and Mellenthin 1977). This inhibition was reversed by exposure of fruit to ethylene. AVG was less effective in delaying ripening of more mature pear fruit, presumably as endogenous ethylene levels were high (Ness and Romani 1980). Vacuum infiltration of apple fruit with AVG delayed ripening and reduced ethylene production (Bramlage et al. 1980). Similarly, spraying apple fruit with AVG preharvest inhibited postharvest ethylene production and thereby delayed ripening (Masia et al. 1998). AVG can also extend the life of ethylene- sensitive cut flowers (Cook et al. 1985; Gladon and Spear 1984). It inhibits ethylene production by cut carnation flowers (Baker et al. 1977; Cook et al. 1985).

1.3.2 Aminooxyacetic acid

AOA prevents ethylene synthesis in plant tissue by inhibiting ACS and thereby blocking conversion of methionine to ethylene (Yu et al. 1979). Much research with AOA has focused on cut flowers, as it is effectively incorporated into flowers as a vase solution additive. Fujino et al. (1980), Broun and Mayak (1981), Gladon and Spear (1984) and Woltering and Sterling (1986) tested AOA as a vase solution additive for cut carnation flowers. They found that pulse or continuous AOA treatment extended flower longevity and delayed and reduced ethylene production and respiration. However, a practical drawback with using AOA or other inhibitors of ethylene biosynthesis is their ineffectiveness in inhibiting senescence when flowers were exposed to exogenous ethylene. Further, because AVG and AOA do not inhibit the conversion of ACC to ethylene, their effectiveness is limited by the level of ACC already

30 present in the tissue (Yang and Hoffman 1984).

1.4 ETHYLENE PERCEPTION

Burg and Burg (1967) first speculated that ethylene binds to a receptor in plant tissue. They suggested ethylene binds to a protein-bound transition metal on the basis that olefins such as ethylene are known to form complexes with metals. This hypothesis was further developed by Sisler (1977), who proposed that ethylene binds to a metal in the receptor and withdraws electrons. The consequent ligand substitution process releases ethylene from the receptor and initiates an action response. There is now substantial belief that ethylene action is by binding to high affinity, saturable and specific receptors (Sisler 1979; Bleecker et al. 1988; Sisler and Blankenship 1993a). However, attempts to isolate and purify these receptors have so far failed. This section will review evidence in support of the existence of ethylene receptors.

Ethylene binding to plant tissue has been demonstrated by displacing 14C-labelled ethylene with unlabelled ethylene (Sisler 1979, 1982). Sisler (1979) estimated the number of binding sites in tobacco leaves to be 4000/cell or a concentration of 3.5 x 10-9 M. Inhibitors of ethylene action or perception, such 14 as 2,5-NBD and silver nitrate (AgNO3), were shown to displace C-labelled ethylene in a detergent extract of mung bean sprouts (Sisler 1982).

Various compounds mimic ethylene action upon association with the putative ethylene receptor (Burg and Burg 1967). These analogues include, in order of biological activity, propylene, carbon monoxide and acetylene (Abeles et al. 1992). In mimicking ethylene action, they have been used to characterise ethylene perception and to facilitate measurement of endogenous ethylene synthesised by plants (McMurchie et al. 1972).

Some bound ethylene dissociates rapidly from receptors in plant tissue, whilst dissociation of remaining ethylene is slow (Sisler 1979). Sisler (1979) interpreted these kinetics to indicate that more than one type of receptor existed. Following its partial purification from mung bean sprouts, Sisler (1980) reported that the receptor appears to be a membrane-bound protein. Based on these observations, Sisler and Yang (1984a) reconsidered the ethylene action model proposed by Sisler (1977). Ethylene molecules were believed to associate with the receptor in a reversible manner and upon dissociation a signal is activated. This signal then mediates changes in gene transcription and in turn the synthesis of specific proteins. These proteins are predominantly catabolic enzymes associated with plant tissue senescence (Reid and Wu 1991). Synthesis of novel mRNAs, proteins and enzymes are associated with ethylene responses (Christoffersen and Laties 1982; Reid 1985a).

Researchers have recently adopted molecular and genetic approaches to identify the ethylene receptor. Identification of genes that are expressed in association with ethylene responses has been made possible through selection of ethylene-insensitive mutants of the ‘model’ plant, Arabidopsis thaliana, and tomato

31 plants. A number of mutants affecting ethylene responses in A. thaliana seedlings have been identified using the ‘triple response’ bioassay. Upon exposure to ethylene normal seedlings are characterised by having (i) a short and thick hypocotyl, (ii) a short root, and (iii) an exaggerated apical hook. Ethylene binding and signal transduction mutants show other phenotypes.

Bleecker et al. (1988) isolated etr1 mutant A. thaliana seedlings, which are dominant and insensitive to ethylene compared to wild type seedlings. They claimed that the ETR1 gene acted as the ethylene receptor. Four etr1-related ethylene insensitive mutants have also been isolated, and the associated genes (ETR2, EIN4, ERS1 and ERS2) all share sequence homology with ETR1 (Hua et al. 1995, 1998; Sakai et al. 1998). A further series of ethylene insensitive mutants (ein2, ein3, ein5, ein6 and ein7) have been recently isolated in A. thaliana, providing evidence that ethylene perception and signal transduction in plants is a highly regulated process (Ecker 1995). Alternatively, the recessive mutant, ctr1, which expressed the ‘triple response’ in the absence of ethylene was identified (Kieber et al. 1993). Thus, CTR1 was reported to repress ethylene responses as its mutant (ctr1) has a constitutive ethylene response. The CTR1 gene encodes a putative serine/threonine protein kinase which is closely related to the Raf protein kinase family found in animals (Kieber et al. 1993).

Evidence that ETR1, a membrane-bound protein, is the ethylene receptor is based on observations that etr1 mutants bound less 14C-labelled ethylene than wild type seedlings (Bleecker et al. 1988). Further evidence that the ETR1 gene codes for the ethylene receptor has been based on double mutant analysis between (i) ctr1 and ein1 mutants, in which the ctr1 phenotype was displayed, and (ii) ctr1 and ein3 mutants, where the ein3 phenotype was shown (Kieber et al. 1993). Kieber et al. (1993) and Ecker (1995) suggested that the CTR1 gene acts downstream of the EIN1 and ETR1 genes. The ETR1 gene encodes for a protein composed of an amino-terminal domain which was demonstrated to bind ethylene when the ETR1 gene was expressed in yeast (Schaller and Bleecker 1995). The carboxyl-terminal region of the ETR1 protein consists of a putative histidine kinase domain and a receiver domain, and exhibits sequence homology to the bacterial two component regulators (Chang et al. 1993). Such proteins are sensors and transducers of a variety of signals in response to environmental stimuli. Hua and Meyerowitz (1998) suggested that since the amino-terminal domains of ETR2, EIN4 and ERS2 are similar to that of ETR1, they may also bind ethylene and act as receptors. They also suggested that these receptors may act either independently or as a complex when binding ethylene in plants.

A genetic pathway for ethylene signal transduction has been deduced from double mutant analyses. It is proposed by Sakai et al. (1998) that ETR1, ETR2, ERS1, ERS2 and EIN4 act upstream of CTR1 which is upstream of EIN2, EIN3, EIN5, EIN6 and EIN7 (Figure 1.2). By analogy with the bacterial two- component regulators, the ethylene signal transduction pathway has been proposed to involve the phosphorylation of the receptor-related proteins in a cascade. It is thought that CTR1 is inactivated and, in turn, downstream proteins are activated and a response is elicited (Ecker 1995). Reid (1995) linked the ethylene perception models proposed by Sisler (1977) and Ecker (1995), in suggesting that binding of an ethylene molecule to the receptor activates protein phosphorylation, causing ligand substitution and

32 activation of responses.

ETR1 ETR2 EIN3 EIN5 Ethylene ERS1 CTR1 EIN2 Ethylene ERS2 EIN6 response EIN4 EIN7

Figure 1.2. Proposed sequence of gene action in the ethylene signal transduction pathway (Hua et al. 1998; Hua and Meyerowitz 1998).

In contrast to A. thaliana, a number of tomato mutants have been selected at later vegetative and reproductive stages (Rick and Butler 1956; Tigchelaar et al. 1978). Tomato mutants in which fruit ripening was delayed included the Never-ripe (Nr), ripening inhibitor (rin) and non-ripening (nor) mutants, while the epi mutant ripened in the absence of ethylene. The discovery of the putative ethylene receptor proteins in A. thaliana ethylene insensitive mutants has been reported by Lanahan et al. (1994) to be consistent with similar receptors in mutant tomato plants. In tomato seedlings the Nr mutants remain insensitive to ethylene. Thus, both the seedling triple response and flower abscission are not observed following ethylene treatment (Lanahan et al. 1994). However, rin and nor mutants display the normal seedling triple response when exposed to ethylene. These observations indicate that the Nr mutation affects a range of plant ethylene responses, while rin and nor mutations specifically affect fruit ripening. ETR1-homologous genes have been isolated from tomato and share sequence homology with the Nr mutation. Thus, the signalling components required for a range of ethylene responses may be similar in a variety of plants (Yen et al. 1995).

33 1.5 INHIBITORS OF ETHYLENE PERCEPTION

As outlined above, substantial evidence exists to support the concept that ethylene action inhibitors act by preventing ethylene perception by plant tissue. Signal transduction and gene activation are inhibited by binding to and blocking of ethylene receptors. Various inhibitors have been reported to bind to the ethylene receptors in either a reversible or irreversible manner (Sisler and Serek 1997).

1.5.1 Silver ions

Beyer (1976) was first to discover the inhibitory effects of Ag+ against ethylene action in plants. Ag+ applied as a foliar spray of AgNO3 prevented the ‘triple’ response in etiolated pea seedlings, leaf abscission from cotton plants and senescence of Cattleya orchid flowers exposed to exogenous ethylene.

New growth of pea seedlings was also protected from ethylene by treatment with 240 mg AgNO3/L, suggesting that Ag+ acted systemically (Beyer 1976).

Mayak et al. (1977) reported that Ag+ also has anti-microbial properties, as evidenced by reduced microbial populations in the vase solutions holding cut carnation flower stems pre-treated with AgNO3.

Halevy and Kofranek (1977) found that placing stem ends of cut carnation flowers into AgNO3 solution was not as effective in extending vase life as dipping or spraying flower heads with the same solution. + Veen and van de Geijn (1978) showed that a different form of Ag , the anionic STS complex (Ag(S2O3)2) was more mobile in plant tissue than the cationic AgNO3. STS is made by combining AgNO3 and

Na2S2O3 solutions. This complex moved rapidly up cut carnation flower stems to the receptacle and was effective in preventing ethylene-induced flower senescence. However, Veen and van de Geijn (1978) also reported that treatment of cut carnation flowers with high STS concentrations caused phytotoxicity.

Ag+ is believed to bind to ethylene receptors and thereby block ethylene action, as evidenced by lower ethylene binding in mung bean sprouts following their treatment with Ag+ (Sisler 1982). Sisler et al. (1986) showed that in vivo ethylene binding by cut carnation flower petals was inhibited by Ag+ provided in the form of STS. Veen (1979a, b) reported that STS treatment of cut carnation flowers prevented the typical climacteric increase in ethylene production and suggested that by blocking the ethylene receptor, Ag+ may inhibit the autocatalytic increase in ethylene production. In apparent contrast, STS treatment has been shown to enhance ethylene production of some plant tissue (Aharoni et al. 1979a), including Leptospermum scoparium flowers (Zieslin and Gottesman 1983). In such instances, it was proposed that Ag+ inhibited the negative feedback mechanism of ethylene biosynthesis.

Much of the applied research with STS has been conducted with ornamentals. Ag+ is a chronic poison and is, therefore, not suitable for practical treatment of edible fruit and vegetables. Reid et al. (1980) found that pulsing cut stems of carnation flowers with 1-4 mM Ag+ in the STS complex for just 10 minutes was effective in delaying their senescence. A minimum concentration of 0.5 µmol Ag+/stem was required for maximum vase life. Greater than 5 µmol Ag+/stem was toxic, resulting in discolouration of

34 flower petals. Reid et al. (1980) reported that STS treatment had commercial potential as the relationship between concentration and treatment time could be tailored to suit particular needs and to deliver an optimal level of Ag+ to the plant tissue.

STS is currently used widely on a commercial scale (Nowak and Rudnicki 1990). It prevents a range of ethylene-induced disorders in ornamentals (Veen 1983). These include: flower senescence (petal wilt) of Gypsophila paniculata (Woltering and Van Doorn 1988; Van Doorn and Reid 1992); floral organ abscission from cut sweet pea (Mor et al. 1984), Petunia (Lovell et al. 1987), Geraldton waxflower (Joyce 1989) and from potted plants including Geranium and Bougainvillea (Cameron and Reid 1983); leaf abscission from mistletoe (Joyce et al. 1990); and, leaf and berry abscission from English holly (Joyce et al. 1990). Cameron and Reid (1981) reported that the protective ability of STS against ethylene was retained for at least 4 weeks in the treatment of Zygocactus, thereby making commercial application useful for preventing ethylene responsiveness during transit and retail display. However, as mentioned earlier, a potential problem with using Ag+ is the risk of phytotoxicity. The usual effective concentrations can be close to those that are toxic (Cameron and Reid 1981).

35 1.5.2 2,5-Norbornadiene

2,5-NBD was the first cyclic olefin shown to be an effective inhibitor of ethylene responses in plant tissue (Sisler and Pian 1973). 2,5-NBD inhibits ethylene action in tobacco (Sisler and Pian 1973), pea hypocotyls (Sisler and Yang 1984b) and cut carnation flowers (Sisler et al. 1983; Sisler et al. 1986; Wang and Woodson 1989). Treatment of these tissues involved pipetting 2,5-NBD liquid onto filter paper which then vaporised into a sealed chamber (Sisler et al. 1985).

Sisler et al. (1985) found that higher concentrations of 2,5-NBD were required to prevent abscission of citrus leaf explants challenged with increased ethylene concentrations. For example, 2000 µL 2,5-NBD/L was sufficient to protect citrus leaf explants from 2 µL ethylene/L, while 8000 µL 2,5-NBD/L was required to afford similar protection when explants were subsequently exposed to 10 µL ethylene/L. Lineweaver-Burk plots constructed by Sisler et al. (1985) demonstrated that 2,5-NBD competed with ethylene for the ethylene receptor. However, binding was reversible, with the inhibitory effect only being maintained when 2,5-NBD was constantly present. Further, 2,5-NBD has an unpleasant odour and may be a carcinogen (Sisler et al. 1986).

Liu et al. (1985) reported that 2,5-NBD inhibited malonylation of ACC in tomato fruit. Ethylene production, ACC content and ACS and ACO activities were rapidly reduced when cut carnation flowers producing autocatalytic ethylene were exposed to 2,5-NBD (Wang and Woodson 1989). Removal of flowers from treatment with 2,5-NBD resulted in recovery of ethylene biosynthesis. Liu et al. (1985) and Wang and Woodson (1989) both proposed that in each situation, 2,5-NBD, by binding to the ethylene receptor, disrupted the feedback mechanism involved in ethylene biosynthesis. Conversely, 2,5-NBD was shown to enhance ethylene production in winter squash by stimulating ACS transcription (Nakajima et al. 1990).

1.5.3 Diazocyclopentadiene

Treatment of cut carnation flowers with DACP gas, a di-cyclic olefin, was reported by Sisler et al. (1993) to effectively inhibit ethylene-induced senescence. Irradiation of DACP under fluorescent lights was found to be around 5000 times more effective than non-irradiated DACP in the treatment of cut carnation flowers (Sisler et al. 1993). Similarly, Sisler and Blankenship (1993b) reported that the ripening of tomato fruit was delayed by DACP treatment in the presence of fluorescent light. Only a slight delay was observed for fruit treated with DACP in the dark. Sisler and Blankenship (1993a, b) and Sisler et al. (1993) suggested that DACP irradiated with visible light gives rise to active unidentified compounds that block ethylene action in an apparently irreversible manner. Serek et al. (1994a) reported that 1 µL DACP/L was as effective as STS in preventing leaf and flower bud abscission and extending the longevity of potted roses. Binding assays using rose petals also indicated that DACP binding to the ethylene receptor was permanent. According to Serek et al. (1994a), DACP was presumed to attach covalently to the receptor when the diazo group decomposed. Tomato fruit treated with DACP for 24 hours at 25oC

36 remained insensitive to ethylene for 10 days (Sisler and Blankenship 1993b). Fruit transferred to 14.5oC immediately after treatment did not respond to ethylene for 20 days. This difference was thought to be due to more rapid synthesis of new receptors at the higher temperature.

Sisler and Blankenship (1993b) also reported that DACP in delaying ripening, also reduced ethylene production by green mature tomato fruit. However, when fruit regained sensitivity to ethylene the surge in climacteric ethylene production was greatly enhanced. Sisler and Lallu (1994) found that DACP retarded ripening of tomato fruit even when applied at advanced ripening stages. Ethylene production of pink and red fruit treated with DACP initially decreased, then, after 3-4 days rose to elevated levels. Moreover, Mathooko et al. (1995) reported that ethylene production by pink stage tomato fruit rapidly decreased in response to DACP treatment due to the inhibition of ACS and ACO activities and slightly increased MACC content. Mathooko et al. (1995) proposed that DACP, by binding to the ethylene receptors, disrupted the positive feedback mechanism of ethylene biosynthesis. Conversely, Tian et al. (1997) found that ethylene production by the non-climacteric strawberry fruit was stimulated by DACP due to an increase in ACC content of tissues and the prevention of the negative feedback mechanism of ethylene production.

37 1.5.4 Cyclopropenes

Some synthetic gaseous cyclopropenes, including cyclopropene (CP), 3,3-dimethylcyclopropene (3,3- DMCP) and 1-methylcyclopropene (1-MCP) (Figure 1.3) have recently been shown to prevent ethylene responses in plant tissue by binding to the ethylene receptor (Sisler et al. 1996a). Complete protection of banana fruit against ethylene was afforded by treatment with 0.7 nL CP/L or 0.7 µL 3,3-DMCP/L for 24 hours (Sisler et al. 1996a). Likewise, treatment of cut carnation flowers with 1 nL CP/L or 0.5-1.0 µL 3,3-DMCP/L for 24 hours provided protection against ethylene (Sisler et al. 1996a). Banana fruit at 24oC remained insensitive to ethylene for 11-12 days after treatment with CP and for 5 days after treatment with 3,3-DMCP, before ripening normally (Sisler et al. 1996a). CP and 3,3-DMCP appear to bind strongly to the ethylene receptor, possibly as a result of the highly strained nature of the molecules (Sisler et al. 1996a). Sisler et al. (1996a) proposed that a stearic and/or inductive effect due to the two methyl groups of 3,3-DMCP may hinder binding and hence require higher concentrations. Both CP and 3,3-DMCP are relatively stable gases at room temperature, although 3,3-DMCP was reported to be comparatively more stable than CP (Sisler and Serek 1997). A number of ethylene-induced responses in cut flowers, potted flowering plants and fruit are prevented by treatment with the gaseous cyclopropene, 1-MCP (Sisler and Serek 1997). The rapidly expanding literature on the use of 1-MCP in postharvest horticulture is reviewed below.

CH3 CH3

CH3 cyclopropene 3,3-dimethylcyclopropene 1-methylcyclopropene

Figure 1.3. Chemical structure of cyclopropene, 3,3-dimethylcyclopropene and 1- methylcyclopropene.

38 1.5.4.1 1-Methylcyclopropene

1.5.4.1.1 Introduction

1-MCP is a recently developed gaseous ethylene binding inhibitor in plant tissue (Serek et al. 1994b). Sisler et al. (1996a) reported that 1-MCP is the first gaseous irreversible inhibitor of ethylene perception that is effective in the dark. 1-MCP is a simple organic compound, being comprised of four carbon and six hydrogen atoms (Figure 1.3). It is generally accepted as being non-toxic and is active at nanomolar concentrations, making it an extremely promising candidate for commercial use (Sisler and Serek 1997). 1-MCP is a very stable gas at room temperature.

1.5.4.1.2 Mode of action

1-MCP molecules bind to ethylene receptors in a competitive and irreversible manner (Sisler et al. 1996a). Whilst 1-MCP occupies the receptor, ethylene cannot bind and elicit an action (Sisler and Serek 1997). Low binding constants were obtained in competition assays between 1-MCP and ethylene in carnation [Kd = 2.1 nL/L (Serek et al. 1995a)] and rose petals [Kd = 8 nL/L (Serek et al. 1994b)]. Thus, ethylene receptors are effectively blocked by a very low 1-MCP concentration. Serek et al. (1994b) used Lineweaver-Burk plots to discern the effects of ethylene concentration relationships for bud and flower abscission from begonia plants treated with 0 or 5 nL 1-MCP/L. The plots confirmed that 1-MCP competes with ethylene for the ethylene receptor. Sisler et al. (1996a) treated cut carnation flowers with 1-MCP labelled with tritium. Efflux diffusion rates of this compound from flower tissues were very low, even after 7 days, suggesting that 1-MCP binds permanently to the ethylene receptor.

Sisler (1977, 1991) proposed that ethylene initiates an action response by binding to and subsequently being released from a metal in the receptor. On the other hand, it was claimed by Sisler and Serek (1997) that the highly strained 1-MCP molecule binds very strongly to the receptor. It remains bound to the metal in the receptor and an action response is not completed.

39 1.5.4.1.3 Synthesis and detection

1-MCP can be synthesised by reacting a solution of phenyllithium in a solvent of 70% cyclohexane and 30% ether with 3-chloro-2-methylpropene (Sisler and Serek 1997). The resulting solution contains lithium chloride as a precipitate and the lithium salt of 1-MCP in solution. This solution remains stable for several months if held in a sealed glass tube stored at -20oC (Sisler and Serek 1997). Earlier variations of this synthesis procedure are described by Fisher and Applequist (1965) and Magid et al. (1971). The concentration of 1-MCP can be quantified by gas chromatography using a hydrocarbon separation column and calibration against butane (Sisler and Serek 1997). No detection limits for the determination of 1- MCP concentration by gas chromatography are provided in the literature. Nakatsuka et al. (1997) indicated that 1-MCP concentrations of 10–20 nL/L used for treatment of tomato fruit were estimated by diluting samples from the stock solution.

1.5.4.1.4 Responses of plant tissue to 1-MCP

Numerous accounts in the literature show that 1-MCP is highly potent in preventing ethylene-related processes in plant tissues (Table 1.1). Sisler et al. (1996a) reported that the concentration of 1-MCP required to provide cut carnation flowers with full protection against ethylene is inversely related to treatment time. For example, treatment of cut carnation flowers at 24oC with as little as 0.5 nL 1-MCP/L for 24 hours or 250 nL 1-MCP/L for just 5 minutes afforded a similar level of protection against ethylene. However, the effective 1-MCP concentration appears to vary according to the plant tissue or organ. Banana fruit and cut carnation flowers required a pre-treatment of 0.5 nL 1-MCP/L for 24 hours at 24oC, while tomato fruit required 7 nL 1-MCP/L for 24 hours at 24oC for complete protection against ethylene (Sisler et al. 1996b). Higher concentrations may be needed for growing vegetative tissue. As demonstrated with pea seedlings, 40 nL 1-MCP/L for 6 hours at 24oC was required to completely inhibit ethylene action (Sisler and Serek 1997). Reasons for this variable response are still unclear. Several researchers have investigated the inhibitory properties of 1-MCP at higher (micromolar) concentrations (Abdi et al. 1998; Golding et al. 1998).

Table 1.1. Ethylene-related plant processes reported to be inhibited by 1-methylcyclopropene treatment.

Ethylene-related process Plant material Reference Flower abscission Potted flowering begonia Serek et al. (1994b) plants Cut Penstemon flowers Serek et al. (1995a) Cut phlox flowers Porat et al. (1995b)

Leaf, flower bud and flower Potted flowering rose plants Serek et al. (1994b) abscission Cut Geraldton waxflower Serek et al. (1995c) Potted flowering miniature Serek et al. (1996) rose plants

40 Leaf abscission and yellowing Epipremnum pinnatum Muller et al. (1997) cuttings

Flower senescence (flower wilt) Cut Phalaenopsis flowers Porat et al. (1995a) Cut Petunia flowers Serek et al. (1995d) Flower senescence (flower closure) Potted flowering Kalanchoe Serek et al. (1994b) blossfeldiana plants Flower senescence (petal in- Cut carnation flowers Serek et al. (1995a) rolling) Sisler et al. (1996a)

Fruit ripening Banana fruit Sisler et al. (1996b) Golding et al. (1998) Tomato fruit Sisler et al. (1996b) Nakatsuka et al. (1997) Apple fruit Song et al. (1997) Plum fruit Abdi et al. (1998) Strawberry fruit Ku et al. (1999) Orange fruit Porat et al. (1999)

Chilling-induced fruit ripening Pear fruit Lelievre et al. (1997)

Inhibition of growth Pea seedlings Sisler et al. (1996b) Epinasty Tomato plants Cardinale et al. (1995)

1.5.4.1.5 Influence of temperature on 1-MCP treatment efficacy

The efficacy of 1-MCP treatment of cut Penstemon flowers was reported by Serek et al. (1995a) to be temperature dependent. 1-MCP treatments of 5 or 20 nL 1-MCP/L for 6 hours were highly effective at 20oC. However, they did not protect Penstemon flowers when applied at 2oC. Reid et al. (1996) reported that higher concentrations of 1-MCP and/or longer treatment times at 2oC were required to achieve complete inhibition of ethylene action in Kalanchoe flowers. Sisler and Serek (1997) suggested that the binding of 1-MCP molecules to ethylene receptors is reduced at low temperature. However, no explanation of the possible mechanism(s) involved were offered. One can speculate that the reduced efficacy of 1-MCP treatment at low temperature is due to decreased rates of diffusion of 1-MCP gas into the plant tissue or poorer binding kinetics. From a scientific and commercial perspective, a greater understanding of this attribute of 1-MCP is needed.

1.5.4.1.6 Effect of 1-MCP on various physiological responses

Porat et al. (1995a) reported that application of 1-MCP to Phalaenopsis orchid flowers effectively inhibited the pollination-induced increase in ethylene production. Sisler et al. (1996a) showed that 1- MCP delayed the time to peak ethylene production by cut carnation flowers compared to flowers treated only with ethylene. Moreover, the rate of ethylene production was reduced compared to flowers that were not treated with ethylene or 1-MCP. Sisler et al. (1996a) proposed that 1-MCP treatment irreversibly prevented autocatalytic production of ethylene. Lelievre et al. (1997) examined Passe-Crassane pear fruit

41 which require chilling treatment to stimulate ACS and ACO activity and to produce ethylene for ripening. Treatment with 4 µL 1-MCP/L at 2oC reduced ethylene production, ACS and ACO activities. In addition, 1-MCP prevented chilling-induced accumulation of ACS and ACO mRNA transcripts. Nakatsuka et al. (1997) also found that 1-MCP prevented increases in abundance of ACS and ACO mRNAs in ripening tomato fruit, and thereby reduced the activity of ACS and ACO. Nakatsuka et al. (1997) demonstrated that 1-MCP interferes with the positive feedback mechanism of ethylene biosynthesis, as proposed earlier by Sisler et al. (1996a). Treatment of apple fruit with 1-MCP also prevented the typical climacteric rise of respiration and ethylene and volatiles production (Song et al. 1997). However, 1-MCP treatment was not effective in preventing ripening of the high ethylene producing feijoa fruit (Reid et al. 1996).

Abdi et al. (1998) demonstrated that 1-MCP delayed the onset of and reduced the rates of both respiration and ethylene climacterics in climacteric and in suppressed-climacteric varieties of plum treated with propylene. Golding et al. (1998) similarly reported delayed respiration and ethylene climacterics and also delayed onset of volatile production in banana fruit treated with 1-MCP. However, in contrast to the findings of Abdi et al. (1998) with plum fruit, 1-MCP treatment enhanced peak rates of ethylene production by banana fruit (Golding et al. 1998). Enhanced rates of ethylene production were taken by Golding et al. (1998) to indicate disruption of the negative feedback mechanism of ethylene biosynthesis. They suggested that 1-MCP may block the feedback regulation by binding irreversibly to receptors involved in feedback regulation and/or preventing malonylation of ACC. The enhanced ethylene production by 1-MCP-treated bananas may be due to over-stimulation of ACS gene transcription.

1.5.4.1.7 Duration of 1-MCP effects on plant tissue

It is has been claimed that 1-MCP molecules bind irreversibly to ethylene receptors in plant tissues (Sisler et al. 1996b). Evidence that 1-MCP binding is irreversible is based on binding studies with petal explants and the insensitivity of 1-MCP-treated tissue to ethylene. For example, Sisler et al. (1996b) found that cut carnation flowers treated with 5 nL 1-MCP/L for 6 hours were insensitive to ethylene applied 10 days later. Similarly, Song et al. (1997) found that apple fruit treated with 500 nL 1-MCP/L on day 0 were still insensitive to ethylene treatment on day 11. However, by virtue of the limited frequency of ethylene treatments, these experiments did not establish that flowers and fruit remain insensitive to ethylene permanently.

In more detailed investigations, 1-MCP-treated cut carnation flowers, banana and tomato fruit regained sensitivity to ethylene at various times after 1-MCP treatment (Sisler and Serek 1997). Cut carnation flowers treated with 1-MCP remained insensitive to ethylene for 12-15 days at 24oC. Sisler and Serek (1997) also stated that for many cut flowers, the period of protection against ethylene afforded by 1-MCP exceeds the normal display life. For these cut flowers, longevity is determined by deterioration processes mediated by factors other than ethylene. Banana and tomato fruits treated with 1-MCP regained their competency to respond to ethylene after 10-12 days (Sisler et al. 1996b). The ability of 1-MCP-treated plant tissue to regain the capacity to respond to ethylene was suggested to be due to the synthesis of new

42 ethylene receptors (Sisler and Serek 1997)1. Thus, one can conclude that the efficacy of 1-MCP treatment will vary according to genotype, possibly being most limited by the apparent synthesis of new receptors.

1.5.4.1.8 Effect of tissue age or development stage on 1-MCP efficacy

Sisler et al. (1996a) reported that older cut carnation flowers displaying early senescence symptoms (petal in-rolling) required a higher concentration of 1-MCP (5 nL/L for 6 hours at 24oC) than young flowers (2.5 nL/L for 6 hours at 24oC) to prevent ethylene-induced senescence. This may have been due to the presence of higher levels of endogenous ethylene in the tissue which competed with 1-MCP molecules for the ethylene receptors. Nakatsuka et al. (1997) reported that 1-MCP treatment of tomato fruit at the pink stage only slightly inhibited ripening, compared to treatment at the green stage, as evidenced by colour development and ethylene production. Ripening of banana fruit was not inhibited by 1-MCP once autocatalytic ethylene production had commenced (i.e. 24 hours after treatment with propylene) (Golding et al. 1998). Klee and Tieman (1997) claim that plant tissues produce new ethylene receptors in association with increased ethylene synthesis. This may help explain why higher concentrations of 1- MCP are required to completely inhibit ethylene-related responses in older tissues.

1.6 INTERACTION BETWEEN ETHYLENE BIOSYNTHESIS AND PERCEPTION

Binding of ethylene to its receptor was speculated by Aharoni et al. (1979) to elicit a feedback signal which would serve to stimulate or inhibit ethylene production (Figure 1.4). It is now established that ethylene biosynthesis is regulated by positive and negative feedback mechanisms (Kende 1993). Positive feedback regulation (autocatalysis) of ethylene biosynthesis is a common feature of fruit ripening and flower senescence (Yang and Hoffman 1984). Exposure of plant tissue to exogenous or endogenous ethylene stimulates an increase in ethylene production which is preceded and/or accompanied by increased ACS and ACO activity (Yang and Hoffman 1984). Negative feedback regulation (autoinhibition) of ethylene biosynthesis has been associated with a number of fruit and vegetative tissues (Yang and Hoffman 1984). In this case, exogenous ethylene suppresses endogenous ethylene production (Vendrell and McGlasson 1971) and the accumulation of ACS transcripts (Nakajima et al. 1990). Liu et al. (1985) reported that ethylene is also capable of negative feedback regulation of its own production by promoting conjugation of ACC to MACC in green mature tomato fruit.

1 In a recent publication obtained at the time of going to press, Sisler and Serek (1999) also suggested that 1-MCP may diffuse from receptors. 43 Methionine AdoMet synthetase Methionine S-adenosyl methionine cycle ACC synthase

Methylthioadenosine Regulation of + ethylene biosynthesis 1-Aminocyclopropane-1-carboxylic acid MACC transferase ACC oxidase MACC Ethylene

Perception by ethylene receptor

Signal transduction

Altered gene expression

Protein synthesis

Response

Figure 1.4. Schematic representation of the interaction between ethylene biosynthesis and perception in higher plants (after Fluhr and Mattoo 1996; Picton et al. 1995; Van Altvorst and Bovy 1995).

The interaction between ethylene biosynthesis and perception has been studied through the use of ethylene action inhibitors. The binding of ethylene action inhibitors to ethylene receptors is thought to stimulate autocatalysis of ethylene production by blocking negative feedback signals (Aharoni et al. 1979; Atta-Aly et al. 1987), thereby preventing malonylation of ACC (Liu et al. 1985) or stimulating ACS transcription (Nakajima et al. 1990). Alternatively, ethylene action inhibitors, by binding to the ethylene receptors, can cause autoinhibition of ethylene production by blocking positive feedback regulation signals which otherwise stimulate the stimulation of ACS and ACO activity (Wang and Woodson 1989) or reduce malonylation of ACC to MACC (Philosoph-Hadas et al. 1985). Despite increasing knowledge, the interaction between ethylene biosynthesis and perception is not fully understood.

1.7 ETHYLENE IN POSTHARVEST HORTICULTURE

1.7.1 Gas ripening and degreening

Ethylene treatment has been widely and effectively used by commercial operators to accelerate peel

44 degreening of early season citrus fruit since the 1920s (Sherman 1985). Citrus fruit, despite being non- climacteric, are sensitive to ethylene as evidenced by enhanced chlorophyll degradation (Purvis and Barmore 1981) and synthesis of carotenoid pigments (Stewart and Wheaton 1972). There is evidence that low endogenous ethylene levels are associated with natural peel degreening (Jahn et al. 1973). Treatment of citrus fruit with at least 5 µL ethylene/L in commercial ripening rooms promotes rapid and uniform degreening (Jahn et al. 1973).

Soon after the discovery that ethylene treatment accelerated degreening of citrus fruit, commercial operators started to use the same technology to ripen a range of climacteric fruit, including tomatoes, bananas and mangoes (Abeles et al. 1992). Treating climacteric fruit with ethylene is an effective means of inducing rapid, uniform and predictable ripening. Treating fruit usually involves injecting ethylene gas from a pressurised cylinder as a ‘shot’ or ‘trickle’ into a specially constructed ripening room or generating ethylene gas inside the room by catalytic conversion of ethanol (Sherman 1985). For example, banana fruit are generally treated with up to 1000 µL ethylene/L at 14.5 to 20oC for 24 hours or on two or three consecutive days until fruit show the first sign of colour change (CSIRO 1972; New and Marriott 1974). According to Inaba and Nakamura (1988), such high ethylene concentrations saturate the fruit and induce fruit of variable maturity and ripening stage to ripen evenly. The ethylene concentration or temperature of treatment can be manipulated to control the rate of fruit ripening (CSIRO 1972). The clear benefit of using ethylene treatments in commercial postharvest horticulture is facilitation of the orderly marketing of citrus, tomato, banana and mango fruit (Sherman 1985). 1.7.2 Acceleration of deterioration

Ethylene stimulates a range of responses in fruit, vegetables and ornamentals at concentrations as low as 0.1-1.0 µL/L (Burg and Burg 1962). Ethylene is a component of the ambient atmosphere and can range in concentration from 0.001-0.005 µL/L in rural areas to around 0.5 µL/L in urban environments (Abeles et al. 1992). The main sources of ethylene are anthropogenic (e.g. exhaust fumes from combustion engines, ballasts of fluorescent lights, and leaks from ripening rooms) or biogenic (e.g. ripening and senescing fruit and vegetables) (Reid 1985b).

Due to the enclosed nature of postharvest handling environments and retail outlets, ethylene tends to accumulate to levels which are biologically active. Exogenous ethylene can, therefore, induce a range of detrimental responses which reduce the postharvest life of fruit, vegetables and ornamentals (Abeles et al. 1992). Exposure of climacteric fruit to ethylene hastens ripening and, in turn, senescence and deterioration (Burg and Burg 1962). In many fruit and vegetables, unintentional exposure to ethylene accelerates chlorophyll degradation, abscission of leaves, softening and undesirable flavours, and creates conditions suitable for further deterioration by pathogens (Kader 1985). Exposure of many cut flowers of horticultural importance to ethylene stimulates rapid deterioration processes such as abscission or senescence of floral organs and attached leaves (Woltering and Van Doorn 1988). Accordingly, techniques which remove, avoid or inhibit ethylene are of considerable commercial importance in preventing the deleterious effects of ethylene (Reid 1985b). Some of these techniques are described

45 below.

1.7.3 Ethylene removal

Removal of ethylene gas from the postharvest environment is a strategy to protect sensitive produce from exogenous ethylene. Ethylene can be removed from storage areas by periodic or slow constant ventilation with fresh air or by scrubbing it from the storage atmosphere with compounds that trap or convert ethylene to inactive products (Sherman 1985). Potassium permanganate (KMnO4) sorbed onto a suitable large surface area carrier, such as vermiculite, perlite, silica gel, expanded glass or a commercial formulation  using aluminium oxide pellets (Purafil ), delayed ripening of banana (Scott et al. 1970), avocado (Hatton and Reeder 1972) and kiwifruit (Ben-Arie and Sonego 1985). KMnO4 based scrubbers oxidise ethylene to carbon dioxide (CO2) and water. In this process, the colour changes from purple to brown as MnO4 is reduced to MnO2 (Reid 1985b). However, the disadvantages of using KMnO4 based scrubbers are relatively high cost (Saltveit 1980), low effectiveness at very low or high ethylene concentrations (Ben- Arie and Sonego 1985) and toxicity concerns regarding its safe handling and disposal (Abeles et al.

1992). Ethylene can also be sorbed onto porous beds and oxidised to CO2 in the presence of a platinum catalyst and elevated temperature (Sherman 1985; Wojciechowski 1989). However, a major problem with this approach is the energy costs associated with removing heat generated by the platinum catalyst (Abeles et al. 1992).

Ultraviolet (UV) light can initiate the destruction of ethylene and other hydrocarbons produced by ripening bananas (Scott et al. 1971). Scott and Wills (1973) demonstrated that irradiating air with UV light at wavelengths of 185 nm and 254 nm generated ozone that oxidised the ethylene in the air. However, few commercial scale UV scrubbers have been developed. Alternatively, the recent development of a liquid electrode plasma system which reduces ethylene into simple and safe by-products may have commercial potential (Graham et al. 1998). This system was shown to reduce up to 75% of ethylene in an air stream containing 1 µL ethylene/L.

Ethylene production by plants can also be lowered by a number of plant growth promoting rhizobacteria that contain ACC deaminase, which catalyses the cleavage of ACC (Glick et al. 1994, 1998). Most research has focused on Pseudomonas putida, which effectively lowers endogenous ethylene levels in plant roots by hydrolysing plant ACC. This in turn, can promote longer root growth in various agronomic crops (Hall et al. 1996). Another soil bacterium, Mycobacterium paraffinicum, has been reported to remove ethylene from the soil by oxidation (Abeles et al. 1992). Application of ethylene-consuming bacteria to remove ethylene from postharvest horticulture environments has yet to be developed. Sherman (1985) speculated that the function of such organisms could possibly be enhanced by genetic engineering.

1.7.4 Biosynthesis inhibition

Effective prevention of a range of ethylene responses in plants through treatment with AOA and AVG has

46 been discussed earlier in this review. AOA is currently used as a commercial anti-ethylene treatment for cut carnations in The Netherlands (W. van Doorn, pers. comm.). A more recent approach to inhibiting ethylene biosynthesis in plants has involved isolation and cloning of genes in the ethylene biosynthetic pathway (Kende 1993; McKeon et al. 1995). ACS and ACO genes have been cloned from various plants. An ACS antisense gene expressed in tomato plants was found to prevent normal fruit ripening in air by inhibiting ethylene biosynthesis (Oeller et al. 1991). Likewise, fruit from tomato plants genetically transformed by insertion of antisense ACO genes produced lower rates of ethylene and displayed delayed ripening. As an alternative approach, Klee et al. (1991) cloned the gene encoding ACC deaminase from Pseudomonas sp. and expressed it in tomato plants. The ripening of fruit from these plants was delayed and their ethylene production was reduced considerably. The use of molecular techniques to inhibit ethylene biosynthesis may have profound commercial implications in the future.

1.7.5 Binding inhibition

As discussed earlier, a number of compounds that inhibit ethylene binding and, thus, its downstream actions have been developed for use in commercial horticulture. Some have already seen commercial application. STS is widely used as a commercial anti-ethylene treatment for a range of cut flowers and potted flowering plants (Veen 1983; Nowak and Rudnicki 1990). However, because the active ingredient of STS is Ag+, a heavy metal, its commercial use is restricted to ornamentals. Legislators in some countries are considering restricting the commercial use of STS because of environmental concerns (Serek et al. 1994a). Ag+ is a recognised environmental pollutant which makes handling and disposal of STS solutions and of treated tissues problematical (Serek et al. 1994a). The use of Ag+ retrieval systems similar to those used in film processing (Cooley 1988), whereby the Ag+ can be recovered from used STS solution, may have potential for application in the cut flower industry (Veen 1987).

Researchers have been recently seeking alternative anti-ethylene strategies suitable for commercial use. The discovery by Sisler and Pian (1973) of the gaseous cyclic olefin 2,5-NBD, has led to the investigation of several similar compounds as possible alternatives to STS. 2,5-NBD is an effective inhibitor, but its unpleasant odour and toxicity have prevented practical use (Sisler et al. 1986). DACP showed promise as an STS alternative (Sisler and Blankenship 1993a; Serek et al. 1994a). From a commercial perspective, DACP treatment has several drawbacks. It is explosive at high concentration, unstable at room temperature, only completely effective when treatment is conducted in the presence of light, and relatively high concentrations are required to overcome ethylene responses (Serek et al. 1995b).

Prevention of ethylene-induced responses in fruit and ornamentals treated with synthetic cyclopropenes has been recently reported (Sisler et al. 1996a). A gaseous cyclopropene, 1-MCP, is particularly effective at low concentrations and is generally accepted as being non-toxic. 1-MCP is a most likely candidate for  commercial use in the future (Sisler and Serek 1997). A commercial preparation of 1-MCP, EthylBloc  has been recently produced. EthylBloc is being evaluated by the Environmental Protection Agency in  the USA. It is anticipated that EthylBloc will soon be registered for use on ornamentals (J. Daly, pers.

47 comm.). However, as discussed earlier in this review, apparent limitations are associated with the use of 1-MCP as an anti-ethylene treatment. Namely, the sometimes limited duration of its inhibitory effects (Sisler et al. 1996b) and its apparently poor binding at low temperatures (Serek et al. 1995a; Reid et al. 1996; Sisler and Serek 1997). These issues are among those investigated in the present study on native Australian cut flowers.

48 49 APPENDIX B SUPPORTING AND STATISTICAL DATA

Appendix 2.1. Gas chromatogram of a 1-methylcyclopropene sample. Peaks 1 and 4 are unknown compounds. Peak 2 represents 1-methylcyclopropene, while peak 3 is cyclohexane.

50 Appendix 2.2. Gas chromatogram of a 1-methylcyclopropene sample reacted with 0.1 g elemental iodine in 10 mL absolute ethanol for 1 hour. Note: peak 2 is no longer present (cf. Appendix 2.1), indicating the usual location of the 1-methylcyclopropene peak.

51 Appendix 2.3. Time in days to > 10% flower abscission (score = 2), moderate flower discolouration (score = 2) and wilting (score = 2) of G. ‘Sylvia’ inflorescences treated on day 0 with 0, 5, 10 or 20 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Data followed by the same letter are not significantly different at P = 0.05 (n = 10). LSDs = 0.7 (abscission) and 0.6 (wilting) days.

Treatment Abscission Discolouration Wilting No ethylene (0 µL/L) Control (0 nL 1-MCP/L) 4.1 ± 0.1 b 3.7 ± 0.2 4.7 ± 0.3 a 5 nL 1-MCP/L 5.3 ± 0.3 cd 4.1 ± 0.1 5.7 ± 0.2 b 10 nL 1-MCP/L 4.7 ± 0.3 bc 3.8 ± 0.1 5.5 ± 0.3 b 20 nL 1-MCP/L 5.3 ± 0.3 cd 4.1 ± 0.2 5.6 ± 0.3 b Plus ethylene (10 µL/L) Control (0 nL 1-MCP/L) 2.1 ± 0.1 a zz 5 nL 1-MCP/L 5.3 ± 0.2 cd 4.0 ± 0.0 5.7 ± 0.2 b 10 nL 1-MCP/L 5.2 ± 0.1 c 3.9 ± 0.1 5.9 ± 0.1 b 20 nL 1-MCP/L 5.9 ± 0.3 d 4.0 ± 0.0 6.0 ± 0.0 b z Treatments were excluded from analysis as all flowers had abscised.

Appendix 2.4. ANOVA table for wilting of flowers on G. ‘Sylvia’ inflorescences treated on day 0 with 0, 5, 10 or 20 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P Treatment 6 10.8857 1.8143 4.07 0.002 Error 63 28.1000 0.4460 Total 69 38.9857

Appendix 2.5. ANOVA table for flower abscission from G. ‘Sylvia’ inflorescences treated on day 0 with 0, 5, 10 or 20 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP concentration (A) 3 75.737 25.246 42.57 0.000 Ethylene (B) 1 1.013 1.013 1.71 0.195 A x B 3 22.038 7.346 12.39 0.000 Error 72 42.700 0.593 Total 79 141.488

52 Appendix 2.6. ANOVA table for relative fresh weight of G. ‘Sylvia’ inflorescences treated on day 0 with 0, 5, 10 or 20 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 3 102450.8 34150.3 64.57 0.000 Ethylene (B) 1 7098.6 7098.6 13.42 0.000 A x B 3 24403.1 8134.4 15.38 0.000 Rep (A B) 72 38079.2 528.9 6.70 0.000 Day (C) 6 375997.3 62666.2 794.45 0.000 A x C 18 26399.8 1466.7 18.59 0.000 B x C 6 5302.2 883.7 11.20 0.000 A x B x C 18 26441.0 1468.9 18.62 0.000 Error 432 34076.2 78.9 Total 559 640248.2

Appendix 2.7. ANOVA table for vase life of G. ‘Sylvia’ inflorescences treated on day 0 with 0, 5, 10 or 20 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP concentration (A) 3 18.1375 6.0458 46.81 0.000 Ethylene (B) 1 3.6125 3.6125 27.97 0.000 A x B 3 9.3375 3.1125 24.10 0.000 Error 72 9.3000 0.1292 Total 79 40.3875

Appendix 2.8. ANOVA table for discolouration of flowers on G. ‘Sylvia’ inflorescences treated on day 0 with 0, 5, 10 or 20 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P Treatment 6 1.3714 0.2286 1.71 0.132 Error 63 8.4000 0.1333 Total 69 9.7714

Appendix 2.9. Summary of chi-square test for an association between treatments (1-MCP concentrations and ethylene) and opening scores (scores: 1 = < 5% to 3 = > 25%) for G. ‘Sylvia’ inflorescences treated on day 0 with 0, 5, 10 or 20 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Days Chi-square df P 0 z 14.359 7 0.114 1 z 16.314 14 0.397 2 24.557 14 0.039 3 31.183 14 0.005 4 16.435 12 0.172 5 z 22.418 12 0.001 6 8.889 6 0.180 7 z 7.368 5 0.416 z Fisher’s exact test was performed where the chi-square test was invalid.

53 Appendix 2.10. ANOVA table for vase solution uptake by G. ‘Sylvia’ inflorescences treated on day 0 with 0, 5, 10 or 20 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP concentration (A) 3 0.303517 0.101172 17.51 0.000 Ethylene (B) 1 0.258505 0.258505 44.73 0.000 A x B 3 0.014804 0.004935 0.85 0.469 Rep (A B) 72 0.416095 0.005779 3.28 0.000 Day (C) 6 1.847170 0.307862 174.75 0.000 A x C 18 0.125643 0.006980 3.96 0.000 B x C 6 0.337070 0.056178 31.89 0.000 A x B x C 18 0.063954 0.003553 2.02 0.008 Error 432 0.761087 0.001762 Total 559 4.127847

Appendix 2.11. Time in days to > 10% flower abscission (score = 2), moderate flower discolouration (score = 2) and wilting (score = 2) from G. ‘Sylvia’ inflorescences treated on day 0 with 10 nL 1-MCP/L for 0, 3, 6, 9 or 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Data followed by the same letter are not significantly different at P = 0.05 (n = 10). LSDs = 1.1 (abscission), 0.7 (discolouration) and 1.3 (wilting) days.

Treatment Abscission Discolouration Wilting No ethylene (0 µL/L) Control (0 nL 1-MCP/L) 3.5 ± 0.5 b 3.9 ± 0.1 ab 4.6 ± 0.6 b 10 nL 1-MCP/L, 3 hr 4.6 ± 0.5 bc 4.5 ± 0.4 b 5.3 ± 0.6 b 10 nL 1-MCP/L, 6 hr 4.7 ± 0.4 c 4.2 ± 0.1 ab 4.8 ± 0.5 b 10 nL 1-MCP/L, 9 hr 4.5 ± 0.3 bc 4.1 ± 0.2 ab 4.1 ± 0.3 b 10 nL 1-MCP/L, 12 hr 4.5 ± 0.6 bc 4.9 ± 0.3 b 4.5 ± 0.4 b Plus ethylene (10 µL/L) Control (0 nL 1-MCP/L) 1.1 ± 0.1 a z 1.0 ± 0.0 a 10 nL 1-MCP/L, 3 hr 3.8 ± 0.4 bc 4.3 ± 0.3 b 5.1 ± 0.5 b 10 nL 1-MCP/L, 6 hr 4.7 ± 0.4 c 3.5 ± 0.2 a 4.6 ± 0.5 b 10 nL 1-MCP/L, 9 hr 5.0 ± 0.4 c 4.2 ± 0.2 ab 5.1 ± 0.5 b 10 nL 1-MCP/L, 12 hr 5.7 ± 0.3 c 4.6 ± 0.4 b 4.8 ± 0.3 b z Treatment excluded from analysis as all flowers had abscised.

Appendix 2.12. ANOVA table for flower abscission from G. ‘Sylvia’ inflorescences treated on day 0 with 10 nL 1-MCP/L for 0, 3, 6, 9 or 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP contact time (A) 4 99.440 24.860 16.13 0.000 Ethylene (B) 1 2.250 2.250 1.46 0.230 A x B 4 38.200 9.550 6.20 0.000 Error 90 138.700 1.541 Total 99 278.590

54 Appendix 2.13. ANOVA table for wilting of flowers on G. ‘Sylvia’ inflorescences treated on day 0 with 10 nL 1-MCP/L for 0, 3, 6, 9 or 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP contact time (A) 4 67.840 16.960 8.33 0.000 Ethylene (B) 1 7.290 7.290 3.58 0.062 A x B 4 63.360 15.840 7.78 0.000 Error 90 183.300 2.037 Total 99 321.790

Appendix 2.14. ANOVA table for relative fresh weight of G. ‘Sylvia’ inflorescences treated on day 0 with 10 nL 1-MCP/L for 0, 3, 6, 9 or 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 4 148995.4 37248.9 55.89 0.000 Ethylene (B) 1 20687.9 20687.9 31.04 0.000 A x B 4 106777.2 26694.3 40.06 0.000 Rep (A B) 90 59978.8 666.4 8.75 0.000 Day (C) 6 445555.0 74259.2 974.48 0.000 A x C 24 16253.7 677.2 8.89 0.000 B x C 6 8873.6 1478.9 19.41 0.000 A x B x C 24 30012.3 1250.5 16.41 0.000 Error 540 41149.9 76.2 Total 699 878283.9

Appendix 2.15. ANOVA table for vase life of G. ‘Sylvia’ inflorescences treated on day 0 with 10 nL 1-MCP/L for 0, 3, 6, 9 or 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP contact time (A) 4 42.4600 10.6150 14.15 0.000 Ethylene (B) 1 5.2900 5.2900 7.05 0.009 A x B 4 18.8600 4.7150 6.29 0.000 Error 90 67.5000 0.7500 Total 99 134.1100

55 + Ethylene 3

- Ethylene 3 Discolouration score Discolouration 2

01234567 Time (days)

Appendix 2.16. Discolouration (scores: 1 = none/slight to 3 = advanced) of flowers on G. ‘Sylvia’ inflorescences treated on day 0 with 10 nL 1-1-MCP/L for 0 (z), 3 (), 6 (▲), 9 (▼) and 12 (◆) hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. The other half of the inflorescences were held in air without exogenous ethylene. Vertical bars represent standard errors of means (n = 10). Where no vertical bars appear, the standard error was smaller than the size of the symbol. LSD is presented in Appendix 2.11.

Appendix 2.17. ANOVA table for discolouration of flowers on G. ‘Sylvia’ inflorescences treated on day 0 with 10 nL 1-MCP/L for 0, 3, 6, 9 or 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P Treatment 8 13.2222 1.6528 2.33 0.026 Error 81 57.4000 0.7086 Total 89 70.6222

56 + Ethylene 3

- Ethylene 3 Opening score

01234567 Time (days)

Appendix 2.18. Opening (scores: 1 = < 5% to 3 = > 25%) of flowers on G. ‘Sylvia’ inflorescences treated on day 0 with 10 nL 1-1-MCP/L for 0 (z), 3 (), 6 (▲), 9 (▼) and 12 (◆) hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. The other half of the inflorescences were held in air without exogenous ethylene. Vertical bars represent standard errors of means (n = 10). Where no vertical bars appear, the standard error was smaller than the size of the symbol. Significant differences (P < 0.05) between treatments existed on days 0, 1 and 2 (Appendix 2.19).

Appendix 2.19. Summary of chi-square test for an association between treatments (1-MCP treatment duration and ethylene) and opening scores (scores: 1 = < 5% to 3 = > 25%) for G. ‘Sylvia’ inflorescences treated on day 0 with 10 nL 1-MCP/L for 0, 3, 6, 9 or 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Days Chi-square df P 0 37.500 9 0.000 1 55.692 18 0.000 2 53.301 18 0.000 3 11.380 16 0.785 4 z 13.942 16 0.642 5 7.619 8 0.471 6 9.643 8 0.410 7 z 16.364 8 0.101 z Fisher’s exact test was performed where the chi-square test was invalid. Appendix 2.20. ANOVA table for vase solution uptake by G. ‘Sylvia’ inflorescences treated on day 57 0 with 10 nL 1-MCP/L for 0, 3, 6, 9 or 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 4 0.00027057 0.00006764 3.41 0.012 Ethylene (B) 1 0.00011154 0.00011154 5.63 0.020 A x B 4 0.00054112 0.00013528 6.83 0.000 Rep (A B) 90 0.00178334 0.00001981 0.56 1.000 Day (C) 6 0.00577529 0.00096255 27.07 0.000 A x C 24 0.00134213 0.00005592 1.57 0.042 B x C 6 0.00019856 0.00003309 0.93 0.472 A x B x C 24 0.00042040 0.00001752 0.49 0.981 Error 540 0.01920429 0.00003556 Total 699 0.02964725

Appendix 2.21. ANOVA table for flower abscission from G. ‘Sylvia’ inflorescences treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 0, 5, 10 or 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Flower abscission score data were arcsine transformed for ANOVA.

Source of variation DF SS MS F P Temperature (A) 3 0.04451 0.01484 0.36 0.780 1-MCP (B) 1 21.30883 21.30883 520.29 0.000 Ethylene (C) 1 16.28499 16.28499 397.62 0.000 A x B 3 0.20754 0.06918 1.69 0.178 A x C 3 0.04151 0.01384 0.34 0.798 B x C 1 16.38125 16.38125 399.97 0.000 A x B x C 3 0.27306 0.09102 2.22 0.094 Rep (A B C) 64 2.62117 0.04096 5.65 0.000 Day (D) 7 17.39076 2.48439 342.69 0.000 A x D 21 0.08774 0.00418 0.58 0.934 B x D 7 7.12472 1.01782 140.40 0.000 C x D 7 5.95978 0.85140 117.44 0.000 A x B x D 21 0.19655 0.00936 1.29 0.175 A x C x D 21 0.06973 0.00332 0.46 0.982 B x C x D 7 6.05461 0.86494 119.31 0.000 A x B x C x D 21 0.23704 0.01129 1.56 0.056 Error 448 3.24783 0.00725 Total 639 97.53162

58 Appendix 2.22. ANOVA table for relative fresh weight of G. ‘Sylvia’ inflorescences treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 0, 5, 10 or 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P Temperature (A) 3 5674.5 1891.5 3.06 0.034 1-MCP (B) 1 221828.0 221828.0 358.83 0.000 Ethylene (C) 1 156003.8 156003.8 252.35 0.000 A x B 3 2808.0 936.0 1.51 0.219 A x C 3 3105.2 1035.1 1.67 0.181 B x C 1 173195.1 173195.1 280.16 0.000 A x B x C 3 3671.8 1223.9 1.98 0.126 Rep (A B C) 64 39564.5 618.2 10.28 0.000 Day (D) 6 71375.2 11895.9 197.75 0.000 A x D 18 2627.9 146.0 2.43 0.001 B x D 6 36431.0 6071.8 100.94 0.000 C x D 6 28273.7 4712.3 78.33 0.000 A x B x D 18 1052.6 58.5 0.97 0.491 A x C x D 18 2138.5 118.8 1.97 0.010 B x C x D 6 33989.2 5664.9 94.17 0.000 A x B x C x D 18 1076.6 59.8 0.99 0.465 Error 384 23099.8 60.2 Total 559 805915.3

Appendix 2.23. ANOVA table for vase life of G. ‘Sylvia’ inflorescences treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 0, 5, 10 or 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P Temperature (A) 3 5.850 1.950 0.88 0.458 1-MCP (B) 1 72.200 72.200 32.45 0.000 Ethylene (C) 1 45.000 45.000 20.22 0.000 A x B 3 5.700 1.900 0.85 0.470 A x C 3 1.500 0.500 0.22 0.879 B x C 1 42.050 42.050 18.90 0.000 A x B x C 3 10.850 3.617 1.63 0.192 Error 64 142.400 2.225 Total 79 325.550

59 + 1-MCP + Ethylene + 1-MCP - Ethylene 3

- 1-MCP + Ethylene - 1-MCP - Ethylene 3 Discolouration score 2

0123456701234567 Time (days)

Appendix 2.24. Discolouration (scores: 1 = none/slight to 3 = advanced) of flowers on G. ‘Sylvia’ inflorescences pre-treated with 0 or 10 nL 1-MCP/L for 12 hours at 0 (z), 5 (), 10 (▲) and 20oC (▼). Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. The other half of the inflorescences were not exposed to exogenous ethylene. Vertical bars represent standard errors of means (n = 5). Where no vertical bars appear, the standard error was smaller than the size of the symbol.

Appendix 2.25. ANOVA table for discolouration of flowers on G. ‘Sylvia’ inflorescences treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 0, 5, 10 or 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Flower discolouration score data were converted to a binary score for ANOVA.

Source of variation DF SS MS F P Temperature (A) 3 0.7556 0.2519 2.48 0.065 Treatment (B) 2 0.4111 0.2056 2.03 0.137 A x B 6 1.4111 0.2352 2.32 0.039 Rep (A B) 48 9.0667 0.1889 1.86 0.005 Day (C) 2 20.8444 10.4222 102.79 0.000 A x C 6 0.5778 0.0963 0.95 0.464 B x C 4 0.5222 0.1306 1.29 0.280 A x B x C 12 1.6556 0.1380 1.36 0.198 Error 96 9.7333 0.1014 Total 179 44.9778

60 + 1-MCP + Ethylene + 1-MCP - Ethylene 3

- 1-MCP + Ethylene - 1-MCP - Ethylene

Wilt score 3

0123456701234567 Time (days)

Appendix 2.26. Wilting (scores: 1 = none/slight to 3 = advanced) of flowers on G. ‘Sylvia’ inflorescences pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 0 (z), 5 (), 10 (▲) and 20oC (▼). Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. The other half of the inflorescences were not exposed to exogenous ethylene. Vertical bars represent standard errors of means (n = 5). Where no vertical bars appear, the standard error was smaller than the size of the symbol.

Appendix 2.27. ANOVA table for wilting of flowers on G. ‘Sylvia’ inflorescences treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 0, 5, 10 or 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Flower wilting score data were converted to a binary score for ANOVA.

Source of variation DF SS MS F P Temperature (A) 3 4.85000 1.61667 25.03 0.000 Treatment (B) 2 0.22500 0.11250 1.74 0.179 A x B 6 3.17500 0.52917 8.19 0.000 Rep (A B) 48 15.10000 0.31458 4.87 0.000 Day (C) 3 6.18333 2.06111 31.91 0.000 A x C 9 0.55000 0.06111 0.95 0.487 B x C 6 0.74167 0.12361 1.91 0.082 A x B x C 18 1.72500 0.09583 1.48 0.104 Error 144 9.30000 0.06458 Total 239 41.85000

61 + 1-MCP + Ethylene + 1-MCP - Ethylene 3

- 1-MCP + Ethylene - 1-MCP - Ethylene 3 Opening score Opening

0123456701234567 Time (days)

Appendix 2.28. Opening (scores: 1 = < 5% to 3 = > 25%) of flowers on G. ‘Sylvia’ inflorescences pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 0 (z), 5 (), 10 (▲) and 20oC (▼). Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. The other half of the inflorescences were not exposed to exogenous ethylene. Vertical bars represent standard errors of means (n = 5). Where no vertical bars appear, the standard error was smaller than the size of the symbol. No significant differences (P > 0.05) between treatments were observed (Appendix 2.29).

Appendix 2.29. Summary of chi-square test for an association between treatments (1-MCP treatment temperature and ethylene) and opening scores (scores: 1 = < 5% to 3 = > 25%) for G. ‘Sylvia’ inflorescences treated on day 0 with 10 nL 1-MCP/L for 12 hours at 0, 5, 10 or 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Days Chi-square df P 0 z 14.873 15 0.719 1 z 29.559 30 0.308 2 39.910 30 0.107 3 22.975 22 0.403 4 z 10.000 22 0.993 5 z 23.043 22 0.331 6 z 16.000 22 0.971 7 z 19.000 22 0.924 z Fisher’s exact test was performed where the chi-square test was invalid. Appendix 2.30. ANOVA table for vase solution uptake by G. ‘Sylvia’ inflorescences treated on day 62 0 with 0 or 10 nL 1-MCP/L for 12 hours at 0, 5, 10 or 20oC. Half of the inflorescences from each treatment were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P Temperature (A) 3 0.068376 0.022792 4.46 0.007 1-MCP (B) 1 0.172462 0.172462 33.76 0.000 Ethylene (C) 1 0.199333 0.199333 39.02 0.000 A x B 3 0.021723 0.007241 1.42 0.246 A x C 3 0.066269 0.022090 4.32 0.008 B x C 1 0.406493 0.406493 79.57 0.000 A x B x C 3 0.038562 0.012854 2.52 0.066 Rep (A B C) 64 0.326958 0.005109 1.11 0.282 Day (D) 6 0.410745 0.068457 14.82 0.000 A x D 18 0.209392 0.011633 2.52 0.001 B x D 6 0.134000 0.022333 4.83 0.000 C x D 6 0.259320 0.043220 9.35 0.000 A x B x D 18 0.100980 0.005610 1.21 0.246 A x C x D 18 0.177141 0.009841 2.13 0.005 B x C x D 6 0.057149 0.009525 2.06 0.057 A x B x C x D 18 0.090905 0.005050 1.09 0.357 Error 384 1.774132 0.004620 Total 559 4.513940

Appendix 2.31. ANOVA table for flower abscission from G. ‘Sylvia’ inflorescences treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each treatment were then exposed on day 1 to 100 µL propylene/L for 12 hours at 20oC. Flower abscission score data were arcsine transformed for ANOVA.

Source of variation DF SS MS F P 1-MCP (A) 1 8.17554 8.17554 1618.71 0.000 Propylene (B) 1 5.40153 5.40153 1069.47 0.000 A x B 1 5.87952 5.87952 1164.11 0.000 Rep (A B) 20 0.10101 0.00505 1.69 0.042 Day (C) 7 3.46264 0.49466 165.10 0.000 A x C 7 2.81713 0.40245 134.32 0.000 B x C 7 2.10954 0.30136 100.59 0.000 A x B x C 7 2.10477 0.30068 100.36 0.000 Error 140 0.41945 0.00300 Total 191 30.47113

Appendix 2.32. ANOVA table for relative fresh weight of G. ‘Sylvia’ inflorescences treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each treatment were then exposed on day 1 to 100 µL propylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 54580.3 54580.3 200.51 0.000 Propylene (B) 1 42663.8 42663.8 156.73 0.000 A x B 1 43963.1 43963.1 161.50 0.000 Rep (A B) 20 5444.2 272.2 10.89 0.000 Day (C) 6 33581.9 5597.0 223.94 0.000 A x C 6 8056.0 1342.7 53.72 0.000 B x C 6 8839.3 1473.2 58.95 0.000 A x B x C 6 9037.7 1506.3 60.27 0.000 Error 120 2999.1 25.0 Total 167 209165.4

63 Appendix 2.33. ANOVA table for vase solution uptake by G. ‘Sylvia’ inflorescences treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each treatment were then exposed on day 1 to 100 µL propylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 0.002236 0.002236 0.21 0.652 Propylene (B) 1 0.025098 0.025098 2.35 0.141 A x B 1 0.000111 0.000111 0.01 0.920 Rep (A B) 20 0.213859 0.010693 11.44 0.000 Day (C) 6 0.069928 0.088234 94.36 0.000 A x C 6 0.113838 0.011655 12.46 0.000 B x C 6 0.004909 0.018973 20.29 0.000 A x B x C 6 0.112213 0.000818 0.88 0.516 Error 120 1.774132 0.000935 Total 167 1.071599

Appendix 2.34. ANOVA table for vase life of G. ‘Sylvia’ inflorescences treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each treatment were then exposed on day 1 to 100 µL propylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 32.667 32.667 98.00 0.000 Propylene (B) 1 24.000 24.000 72.00 0.000 A x B 1 24.000 24.000 72.00 0.000 Error 20 6.667 0.333 Total 23 87.333

64 3

2 score

Discolouration 1

1 Opening score

2 Wilt score Wilt 1

01234567 Time (days)

Appendix 2.35. Flower discolouration (scores: 1 = none/slight to 3 = advanced), opening (scores: 1 = < 5% to 3 = > 25%) and wilting (scores: 1 = none/slight to 3 = advanced) from G. ‘Sylvia’ inflorescences treated with (●) 0 nL 1-MCP/L and 0 µL propylene/L, (■) 0 nL 1-MCP/L and 100 µL propylene/L, (▲) 10 nL 1-MCP/L and 0 µL propylene/L or (▼) 10 nL 1-MCP/L and 100 µL propylene/L. 1-MCP and propylene treatments were each conducted for 12 hours at 20oC on day 0 and 1, respectively. Vertical bars represent standard errors of means (n = 6).

Appendix 2.36. Time in days to moderate flower discolouration (score = 2) from G. ‘Sylvia’ inflorescences treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each treatment were then exposed on day 1 to 100 µL propylene/L for 12 hours at 20oC.

Treatment Discolouration 0 µL propylene/L 0 nL 1-MCP/L 6.0 ± 0.3 10 nL 1-MCP/L 6.5 ± 0.2 100 µL propylene/L 0 nL 1-MCP/L z 10 nL 1-MCP/L 6.3 ± 0.2 z Treatment excluded from analysis as all flowers had abscised. Appendix 2.37. ANOVA table for discolouration of flowers on G. ‘Sylvia’ inflorescences treated on 65 day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each treatment were then exposed on day 1 to 100 µL propylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P Treatment 2 0.7778 0.3889 1.21 0.327 Error 15 4.8333 0.3222 Total 17 5.6111

Appendix 2.38. Summary of chi-square test for an association between treatments (1-MCP and propylene) and opening scores (scores: 1 = < 5% to 3 = > 25%) for G. ‘Sylvia’ inflorescences treated on day 0 with 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each treatment were then exposed on day 1 to 100 µL propylene/L for 12 hours at 20oC.

Days Chi-square df P 0 yyy 1 2.667 3 0.446 2 4.200 6 0.650 3 1.667 4 0.797 4 2.500 4 0.645 5 z 3.154 4 0.811 6 2.400 2 0.301 7 2.400 2 0.301 z Fisher’s exact test was performed where the chi-square test was invalid. y Chi-square test could not be calculated as all inflorescences were of an equal score.

Appendix 2.39. ANOVA table for wilting of flowers on G. ‘Sylvia’ inflorescences treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each treatment were then exposed on day 1 to 100 µL propylene/L for 12 hours at 20oC. Flower wilting score data were converted to a binary score for ANOVA.

Source of variation DF SS MS F P Treatment (A) 2 0.05556 0.02778 0.50 0.616 Rep (A) 15 2.50000 0.16667 3.00 0.020 Day (B) 1 0.11111 0.11111 2.00 0.178 A x B 2 0.05556 0.02778 0.50 0.616 Error 15 0.83333 0.05556 Total 35 2.55556

Appendix 2.40. ANOVA table for ethylene production by G. ‘Sylvia’ inflorescences treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each treatment were then exposed on day 1 to 100 µL propylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 4.014 4.014 0.69 0.431 Propylene (B) 1 10.420 10.420 1.78 0.218 A x B 1 5.050 5.050 0.87 0.380 Rep (A B) 8 46.701 5.838 3.14 0.008 Day (C) 5 83.009 16.602 8.93 0.000 A x C 5 16.691 3.338 1.79 0.136 B x C 5 11.995 2.399 1.29 0.288 A x B x C 5 16.782 3.356 1.80 0.134 Error 40 74.400 1.860 Total 71 269.062

66 Appendix 2.41. ANOVA table for respiration by G. ‘Sylvia’ inflorescences treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each treatment were then exposed on day 1 to 100 µL propylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 3222.3 3222.3 4.20 0.074 Propylene (B) 1 4843.5 4843.5 6.32 0.036 A x B 1 12027.6 12027.6 15.69 0.004 Rep (A B) 8 6132.2 766.5 2.25 0.043 Day (C) 5 51257.4 10251.5 30.12 0.000 A x C 5 5978.1 1195.6 3.51 0.010 B x C 5 3563.4 712.7 2.09 0.086 A x B x C 5 5700.7 1140.1 3.35 0.013 Error 40 13612.6 340.3 Total 71 106337.7

Appendix 2.42. ANOVA table for flower abscission from G. ‘Sylvia’ inflorescences treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each treatment were then exposed on day 1 to 100 µL propylene/L for 12 hours at 20oC. Flower abscission score data were arcsine transformed for ANOVA.

Source of variation DF SS MS F P 1-MCP (A) 1 2.50204 2.50204 568.75 0.000 Propylene (B) 1 1.58024 1.58024 359.21 0.000 A x B 1 1.44227 1.44227 327.85 0.000 Rep (A B) 8 0.03519 0.00440 0.99 0.461 Day (C) 5 1.72636 0.34527 77.38 0.000 A x C 5 1.28078 0.25616 57.41 0.000 B x C 5 0.84655 0.16931 37.94 0.000 A x B x C 5 0.83258 0.16652 37.32 0.000 Error 40 0.17848 0.00446 Total 71 10.42449

Appendix 2.43. ANOVA table for ACC contents of flowers from G. ‘Sylvia’ inflorescences treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each treatment were then exposed on day 1 to 100 µL propylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P Treatment (A) 2 0.0299 0.0150 0.06 0.944 Rep (A) 6 1.5821 0.2637 1.02 0.459 Day (B) 2 8.3698 4.1849 16.13 0.000 A x B 4 0.2888 0.0722 0.28 0.886 Error 12 3.1130 0.2594 Total 26 13.3836

Appendix 2.44. ANOVA table for vase life of G. ‘Sylvia’ inflorescences used in the determination of flower ACC content, which were treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each treatment were then exposed on day 1 to 100 µL propylene/L for 12 hours at 20oC and then used to measure ACC.

Source of variation DF SS MS F P 1-MCP (A) 1 3.0000 3.0000 18.00 0.003 Propylene (B) 1 5.3333 5.3333 32.00 0.000 A x B 1 3.0000 3.0000 18.00 0.003 Error 8 1.3333 0.1667 Total 11 12.6667

67 3

2 score

Discolouration 1

1 Opening score

2 Wilt score 1

012345 Time (days)

Appendix 2.45. Discolouration (scores: 1 = none/slight to 3 = advanced), opening (scores: 1 = < 5% to 3 = > 25%) and wilting (scores: 1 = none/slight to 3 = advanced) of flowers from G. ‘Sylvia’ inflorescences used in the determination of flower ACC content, which were treated with (z) 0 nL 1-MCP/Land 0 µL propylene/L, () 0 nL 1-MCP/L and 100 µL propylene/L, (▲) 10 nL 1-MCP/L and 0 µL propylene/L, or (▼) 10 nL 1-MCP/L and 100 µL propylene/L. 1-MCP and propylene treatments were each conducted on day 0 and day 1, respectively, for 12 hours at 20oC. Vertical bars represent the standard errors of means.

Appendix 2.46. ANOVA table for discolouration of flowers from G. ‘Sylvia’ inflorescences treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each treatment were then exposed on day 1 to 100 µL propylene/L for 12 hours at 20oC. Flower discolouration score data were converted to a binary score for ANOVA.

Source of variation DF SS MS F P Treatment (A) 2 1.00000 0.50000 9.00 0.016 Rep (A) 6 3.00000 0.50000 9.00 0.009 Day (B) 1 0.05556 0.05556 1.00 0.356 A x B 2 0.11111 0.05556 1.00 0.422 Error 6 0.33333 0.05556 Total 17 4.50000

Appendix 2.47. ANOVA table for wilting of flowers from G. ‘Sylvia’ inflorescences treated on day 68 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each treatment were then exposed on day 1 to 100 µL propylene/L for 12 hours at 20oC. Flower wilting score data were converted to a binary score for ANOVA.

Source of variation DF SS MS F P Treatment (A) 2 2.88889 1.44444 19.50 0.000 Rep (A) 6 1.77778 0.29630 4.00 0.020 Day (B) 2 0.66667 0.33333 4.50 0.035 A x B 4 0.44444 0.11111 1.50 0.263 Error 12 0.88889 0.07407 Total 26 6.66667

Appendix 2.48. Summary of chi-square test for an association between treatments (1-MCP and propylene) and opening scores (scores: 1 = < 5% to 3 = > 25%) for G. ‘Sylvia’ inflorescences treated on day 0 with 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each treatment were then exposed on day 1 to 100 µL propylene/L for 12 hours at 20oC.

Days Chi-square df P 0 yyy 1 yyy 2 3.771 3 0.287 3 yyy 4 z 1.286 2 1.000 5 z 2.250 2 1.000 z Fisher’s exact test was performed where the chi-square test was invalid. y Chi-square test could not be calculated as all inflorescences were of an equal score.

Appendix 3.1. ANOVA table for perianth abscission from A. pinnatum inflorescences pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. (Experiment 1). Perianth abscission percentage data were arcsine transformed for ANOVA.

Source of variation DF SS MS F P 1-MCP (A) 1 0.012334 0.012334 0.60 0.442 Ethylene (B) 1 0.031237 0.031237 1.53 0.224 A x B 1 0.012746 0.012746 0.62 0.435 Rep (A B) 36 0.735143 0.020421 15.48 0.000 Day (C) 5 2.984423 0.596885 452.57 0.000 A x C 5 0.015361 0.003072 2.33 0.044 B x C 5 0.021893 0.004379 3.32 0.007 A x B x C 5 0.008388 0.001678 1.27 0.278 Error 180 0.237396 0.001319 Total 239 4.058920

69 Appendix 3.2. ANOVA table for relative fresh weight of A. pinnatum inflorescences pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. (Experiment 1).

Source of variation DF SS MS F P 1-MCP (A) 1 13.69 13.69 0.18 0.674 Ethylene (B) 1 523.31 523.31 6.89 0.013 A x B 1 38.16 38.16 0.50 0.483 Rep (A B) 36 2735.02 75.97 36.38 0.000 Day (C) 5 5086.49 1017.30 487.16 0.000 A x C 5 27.86 5.57 2.67 0.024 B x C 5 21.33 4.27 2.04 0.075 A x B x C 5 10.52 2.10 1.01 0.415 Error 180 375.88 2.09 Total 239 8832.26

Appendix 3.3. ANOVA table for vase solution uptake by A. pinnatum inflorescences pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. (Experiment 1).

Source of variation DF SS MS F P 1-MCP (A) 1 0.002060 0.002060 0.17 0.686 Ethylene (B) 1 0.000523 0.000523 0.04 0.839 A x B 1 0.000826 0.000826 0.07 0.798 Rep (A B) 36 0.447357 0.012427 6.72 0.000 Day (C) 5 1.180498 0.236100 127.71 0.000 A x C 5 0.004339 0.000868 0.47 0.799 B x C 5 0.100260 0.020052 10.85 0.000 A x B x C 5 0.001567 0.000313 0.17 0.974 Error 180 0.332774 0.001849 Total 239 2.070204

Appendix 3.4. ANOVA table for vase life of A. pinnatum inflorescences pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. (Experiment 1).

Source of variation DF SS MS F P 1-MCP (A) 1 0.225 0.225 0.15 0.699 Ethylene (B) 1 0.625 0.625 0.42 0.521 A x B 1 3.025 3.025 2.04 0.162 Error 36 53.500 1.486 Total 39 57.375

70 Appendix 3.5. ANOVA table for perianth abscission from A. pinnatum inflorescences pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 100 µL ethylene/L for 12 hours at 20oC. (Experiment 2). Perianth abscission percentage data were arcsine transformed for ANOVA.

Source of variation DF SS MS F P 1-MCP (A) 1 0.21949 0.21949 2.55 0.119 Ethylene (B) 1 0.20297 0.20297 2.36 0.133 A x B 1 0.12226 0.12226 1.42 0.241 Rep (A B) 36 3.09416 0.08595 19.06 0.000 Day (C) 6 4.76803 0.79467 176.19 0.000 A x C 6 0.03297 0.00549 1.22 0.298 B x C 6 0.01857 0.00309 0.69 0.661 A x B x C 6 0.01205 0.00201 0.45 0.848 Error 216 0.97423 0.00451 Total 279 9.44473

Appendix 3.6. ANOVA table for relative fresh weight of A. pinnatum inflorescences pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. (Experiment 2).

Source of variation DF SS MS F P 1-MCP (A) 1 645.55 645.55 7.53 0.009 Ethylene (B) 1 3.47 3.47 0.04 0.842 A x B 1 201.87 201.87 2.35 0.134 Rep (A B) 36 3086.86 85.75 11.84 0.000 Day (C) 6 9456.73 1576.12 217.56 0.000 A x C 6 84.28 14.05 1.94 0.076 B x C 6 28.06 4.68 0.65 0.694 A x B x C 6 22.12 3.69 0.51 0.801 Error 216 1564.82 7.24 Total 279 15093.75

Appendix 3.7. ANOVA table for vase solution uptake by A. pinnatum inflorescences pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. (Experiment 2).

Source of variation DF SS MS F P 1-MCP (A) 1 0.000000 0.000000 0.00 0.998 Ethylene (B) 1 0.000219 0.000219 0.02 0.886 A x B 1 0.011738 0.011738 1.12 0.296 Rep (A B) 36 0.376351 0.010454 7.16 0.000 Day (C) 6 1.590339 0.265057 181.64 0.000 A x C 6 0.012137 0.002023 1.39 0.221 B x C 6 0.103637 0.017273 11.84 0.000 A x B x C 5 0.003891 0.000649 0.44 0.848 Error 216 0.315201 0.001459 Total 279 2.413513

71 Appendix 3.8. ANOVA table for vase life of A. pinnatum inflorescences pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. (Experiment 2).

Source of variation DF SS MS F P 1-MCP (A) 1 13.225 13.225 12.11 0.001 Ethylene (B) 1 1.225 1.225 1.12 0.297 A x B 1 1.225 1.225 1.12 0.297 Error 36 39.300 1.092 Total 39 54.975

Appendix 3.9. ANOVA table for flower discolouration on flowering B. heterophylla stems pre- treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Flower discolouration score data were arcsine transformed for ANOVA.

Source of variation DF SS MS F P 1-MCP (A) 1 0.09825 0.09825 0.43 0.520 Ethylene (B) 1 0.53677 0.53677 2.32 0.139 A x B 1 0.03366 0.03366 0.15 0.706 Rep (A B) 28 6.47119 0.23111 14.12 0.000 Day (C) 12 36.97722 3.08144 188.27 0.000 A x C 12 0.04936 0.00411 0.25 0.995 B x C 12 0.43385 0.03615 2.21 0.011 A x B x C 12 0.14685 0.01224 0.75 0.704 Error 336 5.49939 0.01637 Total 415 50.24655

Appendix 3.10. ANOVA table for flower wilting on flowering B. heterophylla stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Flower wilting score data were converted to a binary score for ANOVA.

Source of variation DF SS MS F P 1-MCP (A) 1 0.00284 0.00284 0.14 0.712 Ethylene (B) 1 1.50284 1.50284 72.32 0.000 A x B 1 0.13920 0.13920 6.70 0.010 Rep (A B) 28 18.05682 0.64489 31.04 0.000 Day (C) 10 0.57955 0.05795 2.79 0.003 A x C 10 0.21591 0.02159 1.04 0.411 B x C 10 0.09091 0.00909 0.44 0.927 A x B x C 10 0.20455 0.02045 0.98 0.457 Error 280 5.81818 0.02078 Total 351 26.61080

72 Appendix 3.11. ANOVA table for relative fresh weight of flowering B. heterophylla stems pre- treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of the these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 417.8 417.8 0.25 0.624 Ethylene (B) 1 3361.6 3361.6 1.98 0.170 A x B 1 331.4 331.4 0.20 0.662 Rep (A B) 28 47527.9 1697.4 40.27 0.000 Day (C) 11 85878.5 7807.1 185.23 0.000 A x C 11 50.3 4.6 0.11 1.000 B x C 11 431.2 39.2 0.93 0.512 A x B x C 11 215.4 19.6 0.46 0.924 Error 308 112981.8 42.1 Total 383 151195.8

Appendix 3.12. ANOVA table for vase solution uptake by flowering B. heterophylla stems pre- treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 0.01873 0.01873 0.07 0.788 Ethylene (B) 1 1.06168 1.06168 4.19 0.050 A x B 1 2.18601 2.18601 8.62 0.007 Rep (A B) 28 7.09951 0.25355 9.83 0.000 Day (C) 11 11.87828 1.07984 41.85 0.000 A x C 11 0.10682 0.00971 0.38 0.965 B x C 11 0.25153 0.02287 0.89 0.554 A x B x C 11 3.95691 0.35972 13.94 0.000 Error 308 7.94786 0.02580 Total 383 34.50732

Appendix 3.13. ANOVA table for vase life of flowering B. heterophylla stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 5.168 5.168 3.77 0.062 Ethylene (B) 1 0.738 0.738 0.54 0.469 A x B 1 0.023 0.023 0.02 0.898 Error 28 38.357 1.370 Total 31 44.286

73 Appendix 3.14. ANOVA table for senescence of leaves on flowering C. adunca stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Leaf senescence percentage data were arcsine transformed for ANOVA.

Source of variation DF SS MS F P 1-MCP (A) 1 0.11354 0.11354 0.15 0.703 Ethylene (B) 1 0.16135 0.16135 0.21 0.650 A x B 1 1.68001 1.68001 2.18 0.148 Rep (A B) 36 27.70879 0.76969 36.17 0.000 Day (C) 12 23.16950 1.93079 90.74 0.000 A x C 12 0.06187 0.00516 0.24 0.996 B x C 12 0.17269 0.01439 0.68 0.775 A x B x C 12 0.11852 0.00988 0.46 0.935 Error 432 9.19249 0.02128 Total 519 62.37875

Appendix 3.15. ANOVA table for flower opening on flowering C. adunca stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Flower opening score data were arcsine transformed for ANOVA.

Source of variation DF SS MS F P 1-MCP (A) 1 5.57689 5.57689 3.25 0.080 Ethylene (B) 1 0.19217 0.19217 0.11 0.740 A x B 1 0.37659 0.37659 0.22 0.642 Rep (A B) 36 61.84831 1.71801 108.97 0.000 Day (C) 12 6.92869 0.57739 36.62 0.000 A x C 12 0.21237 0.01770 1.12 0.339 B x C 12 0.17362 0.01447 0.92 0.529 A x B x C 12 0.13305 0.01109 0.70 0.749 Error 432 6.81074 0.01577 Total 519 82.25242

Appendix 3.16. Time in days to moderate pedicel wilting (score = 2) for flowering C. adunca stems of pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Data followed by the same letters are not significantly different (LSD = 2.5) at P = 0.05.

Treatment Time (days) No ethylene (0 µL/L) 0 nL 1-MCP/L 7.8 ± 1.1 ab 10 nL 1-MCP/L 5.4 ± 0.8 a

Plus ethylene (10 µL/L) 0 nL 1-MCP/L 7.1 ± 0.8 ab 10 nL 1-MCP/L 9.1 ± 0.8 b

74 Appendix 3.17. ANOVA table for pedicel wilting of flowering C. adunca stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation df SS MS F P 1-MCP (A) 1 0.400 0.400 0.05 0.821 Ethylene (B) 1 22.500 22.500 2.94 0.095 A x B 1 48.400 48.400 6.32 0.017 Error 36 275.800 7.661 Total 39 347.100

Appendix 3.18. ANOVA table for peduncle wilting of flowering C. adunca stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Peduncle wilting score data were converted to a binary score for ANOVA.

Source of variation DF SS MS F P 1-MCP (A) 1 0.40833 0.40833 6.56 0.011 Ethylene (B) 1 0.20833 0.20833 3.34 0.068 A x B 1 2.40833 2.40833 38.66 0.000 Rep (A B) 36 29.13333 0.80926 12.99 0.000 Day (C) 11 41.04167 3.73106 59.90 0.000 A x C 11 0.54167 0.04924 0.79 0.650 B x C 11 0.84167 0.07652 1.23 0.266 A x B x C 11 0.74167 0.06742 1.08 0.374 Error 396 24.66667 0.06229 Total 479 99.99167

Appendix 3.19. ANOVA table for relative fresh weight of flowering C. adunca stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 810.2 810.2 0.87 0.357 Ethylene (B) 1 1058.4 1058.4 1.14 0.293 A x B 1 357.5 357.5 0.38 0.539 Rep (A B) 36 33494.8 930.4 14.40 0.000 Day (C) 5 39897.1 7979.4 123.54 0.000 A x C 5 221.0 44.2 0.68 0.636 B x C 5 201.5 40.3 0.62 0.682 A x B x C 5 402.4 80.5 1.25 0.289 Error 180 11626.3 64.6 Total 239 88069.3

75 Appendix 3.20. ANOVA table for vase solution uptake by flowering C. adunca stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 0.2329 0.2329 0.23 0.637 Ethylene (B) 1 0.2461 0.2461 0.24 0.628 A x B 1 0.8989 0.8989 0.87 0.357 Rep (A B) 36 37.1076 1.0308 7.64 0.000 Day (C) 5 52.5111 10.5022 77.87 0.000 A x C 5 1.0165 0.2033 1.51 0.190 B x C 5 0.1001 0.0200 0.15 0.980 A x B x C 5 2.1889 0.4378 3.25 0.008 Error 180 24.2753 0.1349 Total 239 118.5776

Appendix 3.21. ANOVA table for vase life of flowering C. adunca stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation df SS MS F P 1-MCP (A) 1 0.40 0.40 0.03 0.853 Ethylene (B) 1 2.50 2.50 0.22 0.645 A x B 1 28.90 28.90 2.50 0.123 Error 36 416.20 11.56 Total 39 448.00

Appendix 3.22. ANOVA table for flower abscission from flowering C. gummiferum stems pre- treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Flower abscission score data were arcsine transformed for ANOVA.

Source of variation DF SS MS F P 1-MCP (A) 1 2.95866 2.95866 23.96 0.000 Ethylene (B) 1 2.95866 2.95866 23.96 0.000 A x B 1 2.95866 2.95866 23.96 0.000 Rep (A B) 36 4.44541 0.12348 153.00 0.000 Day (C) 18 0.34808 0.01934 23.96 0.000 A x C 18 0.34808 0.01934 23.96 0.000 B x C 18 0.34808 0.01934 23.96 0.000 A x B x C 18 0.34808 0.01934 23.96 0.000 Error 648 0.52299 0.00081 Total 759 15.23668

Appendix 3.23. Time in days to moderate sepal wilting (score = 2) from flowering C. gummiferum stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene for 12 hours at 20oC. Data followed by the same letters are not significantly different (LSD = 3.3) at P = 0.05.

Treatment Time (days) No ethylene (0 µL ethylene/L) 0 nL 1-MCP/L 11.2 ± 1.1 a 10 nL 1-MCP/L 15.5 ± 0.9 b

Plus ethylene (10 µL ethylene/L) 0 nL 1-MCP/L 10.3 ± 1.4 a 10 nL 1-MCP/L 14.8 ± 1.1 b Appendix 3.24. ANOVA table for sepal wilting on flowering C. gummiferum stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these 76 treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation df SS MS F P 1-MCP (A) 1 193.60 193.60 14.35 0.001 Ethylene (B) 1 6.40 6.40 0.47 0.495 A x B 1 0.10 0.10 0.01 0.932 Error 36 485.80 13.49 Total 39 685.90

Appendix 3.25. ANOVA table for relative fresh weight of flowering C. gummiferum stems pre- treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 49727.1 49727.1 23.17 0.000 Ethylene (B) 1 11291.6 11291.6 5.26 0.028 A x B 1 9888.0 9888.0 4.61 0.039 Rep (A B) 36 77269.0 2146.4 46.08 0.000 Day (C) 17 98902.5 5817.8 124.90 0.000 A x C 17 6668.1 392.2 8.42 0.000 B x C 17 611.7 36.0 0.77 0.726 A x B x C 17 1041.9 61.3 1.32 0.176 Error 612 28507.2 46.6 Total 719 283907.2

Appendix 3.26. ANOVA table for vase solution uptake by flowering C. gummiferum stems pre- treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 0.39833 0.39833 4.23 0.047 Ethylene (B) 1 0.27984 0.27984 2.97 0.093 A x B 1 0.19511 0.19511 2.07 0.159 Rep (A B) 36 3.38961 0.09416 16.63 0.000 Day (C) 17 9.99075 0.58769 103.79 0.000 A x C 17 0.81776 0.04810 8.50 0.000 B x C 17 0.15300 0.00900 1.59 0.062 A x B x C 17 0.04931 0.00290 0.51 0.948 Error 612 3.46522 0.00566 Total 719 18.73893

Appendix 3.27. ANOVA table for vase life of flowering C. gummiferum stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 511.23 511.23 29.28 0.000 Ethylene (B) 1 126.03 126.03 7.22 0.011 A x B 1 75.62 75.62 4.33 0.045 Error 36 628.50 17.46 Total 39 1341.38

77 Appendix 3.28. ANOVA table for flower abscission from flowering C. uncinatum ‘Paddy’s Late’ sprigs pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the sprigs from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Flower abscission score data were arcsine transformed for ANOVA.

Source of variation DF SS MS F P 1-MCP (A) 1 35.6488 35.6488 1240.91 0.000 Ethylene (B) 1 25.7857 25.7857 897.59 0.000 A x B 1 20.1988 20.1988 703.11 0.000 Rep (A B) 36 1.0342 0.0287 5.81 0.000 Day (C) 13 4.9780 0.3829 77.46 0.000 A x C 13 2.8035 0.2157 43.63 0.000 B x C 13 2.1858 0.1681 34.01 0.000 A x B x C 13 2.2612 0.1739 35.19 0.000 Error 468 2.3135 0.0049 Total 559 97.2097

Appendix 3.29. ANOVA table for relative fresh weight of flowering C. uncinatum ‘Paddy’s Late’ sprigs pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the sprigs from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 58703.2 58703.2 118.98 0.000 Ethylene (B) 1 80636.4 80636.4 163.43 0.000 A x B 1 33051.4 33051.4 66.99 0.000 Rep (A B) 36 17762.3 493.4 20.07 0.000 Day (C) 12 125201.2 10433.4 424.49 0.000 A x C 12 4538.8 378.2 15.39 0.000 B x C 12 1072.8 89.4 3.64 0.000 A x B x C 12 6939.9 578.3 23.53 0.000 Error 432 10618.0 24.6 Total 519 338524.1

Appendix 3.30. ANOVA table for vase solution uptake by flowering C. uncinatum ‘Paddy’s Late’ sprigs pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the sprigs from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 0.70146 0.70146 8.60 0.006 Ethylene (B) 1 3.14143 3.14143 38.52 0.000 A x B 1 0.06191 0.06191 0.76 0.389 Rep (A B) 36 2.93610 0.08156 11.45 0.000 Day (C) 12 14.12827 1.17736 165.22 0.000 A x C 12 0.65356 0.05446 7.64 0.000 B x C 12 0.70162 0.05847 8.20 0.000 A x B x C 12 0.35038 0.02920 4.10 0.000 Error 432 3.07847 0.00713 Total 519 25.75320

78 Appendix 3.31. ANOVA table for vase life of flowering C. uncinatum ‘Paddy’s Late’ sprigs pre- treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the sprigs from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 254.47 254.47 131.83 0.000 Ethylene (B) 1 297.62 297.62 154.19 0.000 A x B 1 216.74 216.74 112.28 0.000 Error 36 69.49 1.93 Total 39 838.32

Appendix 3.32. ANOVA table for petal abscission from flowering E. scaber stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Petal abscission percentage data were arcsine transformed for ANOVA.

Source of variation DF SS MS F P 1-MCP (A) 1 0.007417 0.007417 0.26 0.610 Ethylene (B) 1 0.183779 0.183779 6.56 0.015 A x B 1 0.088798 0.088798 3.17 0.083 Rep (A B) 36 1.008317 0.028009 3.26 0.000 Day (C) 2 0.669872 0.334936 38.99 0.000 A x C 2 0.044362 0.022181 2.58 0.083 B x C 2 0.010266 0.005133 0.60 0.553 A x B x C 2 0.013856 0.006928 0.81 0.450 Error 72 0.618513 0.008590 Total 119 2.645181

Appendix 3.33. ANOVA table for relative fresh weight of flowering E. scaber stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 41.56 41.56 0.94 0.340 Ethylene (B) 1 9.68 9.68 0.22 0.643 A x B 1 8.49 8.49 0.19 0.664 Rep (A B) 36 1597.99 44.39 1.34 0.190 Day (C) 1 1792.12 1792.12 54.22 0.000 A x C 1 102.03 102.03 3.09 0.087 B x C 1 0.00 0.00 0.00 0.999 A x B x C 1 11.32 11.32 0.34 0.562 Error 36 1189.88 33.05 Total 79 4753.09

79 Appendix 3.34. ANOVA table for vase solution uptake by flowering E. scaber stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 0.09526 0.09526 3.35 0.076 Ethylene (B) 1 0.00208 0.00208 0.07 0.789 A x B 1 0.00442 0.00442 0.16 0.696 Rep (A B) 36 1.02516 0.02848 1.48 0.121 Day (C) 1 0.35726 0.35726 18.62 0.000 A x C 1 0.09285 0.09285 4.84 0.034 B x C 1 0.00002 0.00002 0.00 0.977 A x B x C 1 0.00970 0.00970 0.51 0.482 Error 36 0.69090 0.01919 Total 79 2.27764

Appendix 3.35. ANOVA table for vase life of flowering E. scaber stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 2.0385 2.0385 2.63 0.114 Ethylene (B) 1 0.0051 0.0051 0.01 0.936 A x B 1 0.1428 0.1428 0.18 0.670 Error 36 27.9048 0.7751 Total 39 30.0912

Appendix 3.36. Time in days to > 10% flower abscission (score = 2) from G. ‘Kay Williams’ inflorescences pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene for 12 hours at 20oC. Data followed by the same letters are not significantly different (LSD = 0.5) at P = 0.05.

Treatment Time (days) No ethylene (0 µL/L) 0 nL 1-MCP/L 4.3 ± 0.2 b 10 nL 1-MCP/L 4.4 ± 0.2 b

Plus ethylene (10 µL/L) 0 nL 1-MCP/L 1.0 ± 0.0 a 10 nL 1-MCP/L 4.3 ± 0.2 b

Appendix 3.37. ANOVA table for flower abscission from G. ‘Kay Williams’ inflorescences pre- treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation df SS MS F P 1-MCP (A) 1 22.321 22.321 110.29 0.000 Ethylene (B) 1 18.893 18.893 93.35 0.000 A x B 1 18.893 18.893 93.35 0.000 Error 24 4.857 0.202 Total 27 64.964

80 Appendix 3.38. ANOVA table for relative fresh weight of G. ‘Kay Williams’ inflorescences pre- treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 45245.4 45245.4 1479.17 0.000 Ethylene (B) 1 44377.5 44377.5 1450.80 0.000 A x B 1 36275.2 36275.2 1185.91 0.000 Rep (A B) 24 3836.2 159.8 5.23 0.000 Day (C) 4 18101.6 4525.4 147.94 0.000 A x C 4 1015.5 253.9 8.30 0.000 B x C 4 2317.4 579.3 18.94 0.000 A x B x C 4 2842.2 710.5 23.23 0.000 Error 96 2936.5 30.6 Total 139 156947.5

Appendix 3.39. ANOVA table for vase solution uptake by G. ‘Kay Williams’ inflorescences pre- treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 0.154340 0.154340 8.85 0.007 Ethylene (B) 1 0.041493 0.041493 2.38 0.136 A x B 1 0.036672 0.036672 2.10 0.160 Rep (A B) 24 0.418582 0.017441 3.40 0.000 Day (C) 4 0.201707 0.050427 9.82 0.000 A x C 4 0.062073 0.015518 3.02 0.021 B x C 4 0.061618 0.015405 3.00 0.022 A x B x C 4 0.029331 0.007333 1.43 0.230 Error 96 0.492812 0.005133 Total 139 1.498628

81 3

2 score

Discolouration Discolouration 1

Opening score 1

2 Wilt score Wilt 1

012345 Time (days)

Appendix 3.40. Flower discolouration (scores: 1 = none/slight to 3 = advanced), flower opening (scores: 1 = < 5% to 3 = > 25%) and flower wilting (scores: 1 = none/slight to 3 = advanced) from G. ‘Kay Williams’ inflorescences treated with 0 nL 1-MCP/L and 0 µL ethylene/L (z), 0 nL 1-MCP/L and 10 µL ethylene/L (), 10 nL 1-MCP/L and 0 µL ethylene/L (▲), or 10 nL 1-MCP/L and 10 µL ethylene/L (▼). 1-MCP and ethylene treatments were conducted for 12 hours at 20oC on days 0 and 1, respectively. Vertical bars represent the standard errors of means (n = 7). Where no vertical bars appear, standard errors are smaller than the size of the symbol.

Appendix 3.41. ANOVA table for flower discolouration from G. ‘Kay Williams’ inflorescences pre- treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Flower discolouration score data were arcsine transformed for ANOVA.

Source of variation DF SS MS F P Treatment (A) 2 0.57143 0.28571 3.00 0.075 Rep (A) 18 4.00000 0.22222 2.33 0.040 Day (B) 1 0.59524 0.59524 6.25 0.022 A x B 2 0.19048 0.09524 1.00 0.387 Error 18 1.71429 0.09524 Total 41 7.07143

82 Appendix 3.42. Summary of chi-square test for an association between treatment (1-MCP and ethylene) and opening scores (scores: 1 = < 5% to 3 = > 80%) for G. ‘Kay Williams’ inflorescences pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Days Chi-square df P 0 5.000 4 0.287 1 1.400 2 0.497 2 0.525 2 0.769 3a 2.100 2 0.541 4 bbb 5 bbb a Fisher’s exact test was performed where the chi-square test was invalid. b Chi-square test could not be calculated as all inflorescences were of an equal score.

Appendix 3.43. ANOVA table for flower wilting from G. ‘Kay Williams’ inflorescences pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Flower wilting score data were converted to a binary score for ANOVA.

Source of variation DF SS MS F P Treatment (A) 2 0.0476 0.0238 0.20 0.821 Rep (A) 18 3.5714 0.1984 1.67 0.144 Day (B) 1 1.5238 1.5238 12.80 0.002 A x B 2 0.3333 0.1667 1.40 0.272 Error 18 2.1429 0.1190 Total 41 7.6190

Appendix 3.44. ANOVA table for vase life of G. ‘Kay Williams’ inflorescences pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 22.321 22.321 133.93 0.000 Ethylene (B) 1 18.893 18.893 113.36 0.000 A x B 1 15.750 15.750 94.50 0.000 Error 24 4.000 0.167 Total 27 60.964

Appendix 3.45. Time in days to > 10% flower abscission (score = 2) from G. ‘Misty Pink’ inflorescences pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene for 12 hours at 20oC. Data followed by the same letters are not significantly different (LSD = 0.6) at P = 0.05.

Treatment Time (days) No ethylene (0 µL/L) 0 nL 1-MCP/L 4.1 ± 0.2 b 10 nL 1-MCP/L 4.2 ± 0.2 b Plus ethylene (10 µL/L) 0 nL 1-MCP/L 1.0 ± 0.0 a 10 nL 1-MCP/L 4.0 ± 0.2 b

83 Appendix 3.46. ANOVA table for flower abscission from G. ‘Misty Pink’ inflorescences pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each treatment were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation df SS MS F P 1-MCP (A) 1 24.025 24.025 59.65 0.000 Ethylene (B) 1 27.225 27.225 67.59 0.000 A x B 1 21.025 21.025 52.20 0.000 Error 36 14.500 0.403 Total 39 86.775

Appendix 3.47. ANOVA table for relative fresh weight of G. ‘Misty Pink’ inflorescences treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 43642.7 43642.7 805.27 0.000 Ethylene (B) 1 44791.2 44791.2 826.46 0.000 A x B 1 37935.8 37935.8 699.97 0.000 Rep (A B) 36 14337.8 398.3 7.35 0.000 Day (C) 4 54037.5 13509.4 249.27 0.000 A x C 4 3447.1 861.8 15.90 0.000 B x C 4 5507.2 1376.8 25.40 0.000 A x B x C 4 4508.3 1127.1 20.80 0.000 Error 144 7804.3 54.2 Total 199 216012.0

Appendix 3.48. ANOVA table for vase solution uptake by G. ‘Misty Pink’ inflorescences pre- treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 0.017429 0.017429 2.10 0.156 Ethylene (B) 1 0.127602 0.127602 15.38 0.000 A x B 1 0.062640 0.062640 7.55 0.009 Rep (A B) 36 0.298662 0.008296 9.92 0.000 Day (C) 4 0.154118 0.038529 46.08 0.000 A x C 4 0.015191 0.003798 4.54 0.002 B x C 4 0.021846 0.005462 6.53 0.000 A x B x C 4 0.003903 0.000976 1.17 0.328 Error 144 0.120413 0.000836 Total 199 0.821803

84 3

2 score

Discolouration 1

2 Wilt score Wilt 1

Opening score 1

012345 Time (days)

Appendix 3.49. Flower discolouration (scores: 1 = none/slight to 3 = advanced), wilting (scores: 1 = none/slight to 3 = advanced) and opening (scores: 1 = < 5% to 3 = > 25%) from G. ‘Misty Pink’ inflorescences treated with 0 nL 1-MCP/L and 0 µL ethylene/L (z), 0 nL 1-MCP/L and 10 µL ethylene/L (), 10 nL 1-MCP/L and 0 µL ethylene/L (▲), or 10 nL 1-MCP/L and 10 µL ethylene/L (▼). 1-MCP and ethylene treatments were conducted for 12 hours at 20oC on days 0 and 1, respectively. Vertical bars represent the standard errors of means (n = 10). No significant differences (P > 0.05) between treatments existed for flower opening data except on day 1 (Appendix 3.52).

Appendix 3.50. ANOVA table for flower discolouration from G. ‘Misty Pink’ inflorescences pre- treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Flower discolouration score data were arcsine transformed for ANOVA.

Source of variation DF SS MS F P Treatment (A) 2 0.2889 0.1444 1.21 0.307 Rep (A) 27 8.0333 0.2975 2.48 0.002 Day (B) 2 7.4889 3.7444 31.27 0.000 A x B 4 0.0444 0.0111 0.09 0.984 Error 54 6.4667 0.1198 Total 89 22.3222

85 Appendix 3.51. ANOVA table for flower wilting from G. ‘Misty Pink’ inflorescences pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Flower wilting score data were converted to a binary score for ANOVA.

Source of variation DF SS MS F P Treatment (A) 2 0.2333 0.1167 1.03 0.370 Rep (A) 27 8.4500 0.3130 2.77 0.005 Day (B) 1 2.0167 2.0167 17.85 0.000 A x B 2 0.4333 0.2167 1.92 0.166 Error 27 3.0500 0.1130 Total 59 14.1833

Appendix 3.52. Summary of chi-square test for an association between treatment (1-MCP and ethylene) and opening scores (scores: 1 = < 5% to 3 = > 25%) for G. ‘Misty Pink’ inflorescences pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Days Chi-square df P 0 2.219 6 0.855 1 8.459 3 0.037 2 2.222 3 0.528 3 zzz 4 zzz 5 zzz z Chi-square test could not be calculated as all inflorescences were of an equal score.

Appendix 3.53. ANOVA table for vase life of G. ‘Misty Pink’ inflorescences pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each treatment were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 21.025 21.025 59.60 0.000 Ethylene (B) 1 27.225 27.225 77.17 0.000 A x B 1 21.025 21.025 59.60 0.000 Error 36 12.700 0.353 Total 39 81.975

Appendix 3.54. ANOVA table for flower abscission from G. ‘Sandra Gordon’ inflorescences on the last day of the experiment. Inflorescences were pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Flower abscission score data were arcsine transformed for ANOVA.

Source of variation DF SS MS F P 1-MCP (A) 1 7.94622 7.94622 1766.52 0.000 Ethylene (B) 1 7.35716 7.35716 1635.56 0.000 A x B 1 7.35716 7.35716 1635.56 0.000 Rep (A B) 36 1.12206 0.03117 6.93 0.000 Day (C) 7 3.35789 0.47970 106.64 0.000 A x C 7 2.97023 0.42432 94.33 0.000 B x C 7 2.70733 0.38676 85.98 0.000 A x B x C 7 2.70733 0.38676 85.98 0.000 Error 252 1.13356 0.00450 Total 319 36.65893

86 Appendix 3.55. ANOVA table for relative fresh weight of G. ‘Sandra Gordon’ inflorescences pre- treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 45132.6 45132.6 1253.65 0.000 Ethylene (B) 1 44232.0 44232.0 1228.64 0.000 A x B 1 32114.0 32114.0 892.03 0.000 Rep (A B) 36 8480.5 235.6 6.54 0.000 Day (C) 6 11500.7 1916.8 53.24 0.000 A x C 6 8594.7 1432.5 39.79 0.000 B x C 6 7535.6 1255.9 34.89 0.000 A x B x C 6 6448.4 1074.7 29.85 0.000 Error 216 7776.2 36.0 Total 279 171814.7

Appendix 3.56. ANOVA table for vase solution uptake by G. ‘Sandra Gordon’ inflorescences pre- treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 0.00174 0.00174 0.10 0.753 Ethylene (B) 1 0.00045 0.00045 0.03 0.872 A x B 1 0.00749 0.00749 0.43 0.514 Rep (A B) 36 0.62169 0.01727 1.38 0.085 Day (C) 6 1.63618 0.27270 21.79 0.000 A x C 6 0.17391 0.02898 2.32 0.035 B x C 6 0.10293 0.01716 1.37 0.227 A x B x C 6 0.04274 0.00712 0.57 0.755 Error 216 2.70262 0.01251 Total 279 5.28974

87 3

2 score

Discolouration 1

Opening score 1

2 Wilt score 1

01234567 Time (days)

Appendix 3.57. Flower discolouration (scores: 1 = none/slight to 3 = advanced), opening (scores: 1= < 5% to 3 = > 25%) and wilting (scores: 1 = none/slight to 3 = advanced) from G. ‘Sandra Gordon’ inflorescences treated with 0 nL 1-MCP/L and 0 µL ethylene/L (z), 0 nL 1-MCP/L and 10 µL ethylene/L (), 10 nL 1-MCP/L and 0 µL ethylene/L (▲) or 10 nL 1-MCP/L and 10 µL ethylene/L (▼). 1-MCP and ethylene treatments were conducted for 12 hours at 20oC on days 0 and 1, respectively. Vertical bars represent the standard errors of means (n = 10). LSD for flower discolouration data is presented in Appendix 3.57. No significant differences (P > 0.05) between treatments existed for flower opening data, except on day 2 (Appendix 3.60).

88 Appendix 3.58. Time in days to moderate flower discolouration (score = 2) for G. ‘Sandra Gordon’ inflorescences pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene for 12 hours at 20oC. Data followed by the same letters are not significantly different (LSD = 0.3) at P = 0.05.

Treatment Time (days) No ethylene (0 µL/L) 0 nL 1-MCP/L 6.0 ± 0.1 ab 10 nL 1-MCP/L 6.4 ± 0.2 b Plus ethylene (10 µL/L) 0 nL 1-MCP/L z 10 nL 1-MCP/L 5.8 ± 0.1 a z Treatment was excluded from statistical analysis as all flowers had abscised.

Appendix 3.59. ANOVA table for flower discolouration from G. ‘Sandra Gordon’ inflorescences pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation df SS MS F P Treatment 2 1.8667 0.9333 4.20 0.026 Error 27 6.0000 0.2222 Total 29 7.8667

Appendix 3.60. Summary of chi-square test for an association between treatment (1-MCP and ethylene) and opening scores (scores: 1 = none/slight to 3 = advanced) for G. ‘Sandra Gordon’ inflorescences pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Days Chi-square df P 0 bbb 1 2.165 3 0.539 2a 12.894 6 0.018 3a 3.077 3 1.000 4a 3.077 3 1.000 5 bbb 6 bbb 7 bbb a Fisher’s exact test was performed where the chi-square test was invalid. b Chi-square test could not be calculated as all inflorescences were of an equal score.

Appendix 3.61. ANOVA table for flower wilting from G. ‘Sandra Gordon’ inflorescences pre- treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Flower wilting score data were converted to a binary score for ANOVA.

Source of variation DF SS MS F P Treatment 2 0.4667 0.2333 2.10 0.142 Error 27 3.0000 0.1111 Total 29 3.4667

89 Appendix 3.62. ANOVA table for vase life of G. ‘Sandra Gordon’ inflorescences pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 44.100 44.100 264.60 0.000 Ethylene (B) 1 52.900 52.900 317.40 0.000 A x B 1 28.900 28.900 173.40 0.000 Error 36 6.000 0.167 Total 39 131.900

Appendix 3.63. ANOVA table for petal abscissiona from flowering L. petersonii stems of pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 12.500 12.500 8.38 0.007 Ethylene (B) 1 1.125 1.125 0.75 0.392 A x B 1 4.500 4.500 3.02 0.093 Error 28 41.750 1.491 Total 31 59.875 a Petal abscission data is equivalent to vase life data.

Appendix 3.64. ANOVA table for relative fresh weight of flowering L. petersonii stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 595.76 595.76 15.87 0.000 Ethylene (B) 1 1455.44 1455.44 38.77 0.000 A x B 1 17.90 17.90 0.48 0.491 Rep (A B) 28 12504.35 446.58 11.90 0.000 Day (C) 5 32718.26 6543.65 174.30 0.000 A x C 5 58.16 11.63 0.31 0.906 B x C 5 255.89 51.18 1.36 0.242 A x B x C 5 201.21 40.24 1.07 0.379 Error 140 5255.84 37.54 Total 191 53062.81

Appendix 3.65. ANOVA table for vase solution uptake by flowering L. petersonii stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 0.05956 0.05956 0.53 0.474 Ethylene (B) 1 0.81489 0.81489 7.22 0.012 A x B 1 0.13850 0.13850 1.23 0.278 Rep (A B) 28 3.16180 0.11292 3.14 0.000 Day (C) 5 33.40368 6.68074 185.69 0.000 A x C 5 0.51971 0.10394 2.89 0.016 B x C 5 0.94294 0.18859 5.24 0.000 A x B x C 5 0.15634 0.03127 0.87 0.504 Error 140 5.03681 0.03598 Total 191 44.23423

90 Appendix 3.66. ANOVA table for flower abscission and senescencea from flowering L. scoparium ‘Winter Cheer’ stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 1.8360 1.8360 2.41 0.133 Ethylene (B) 1 0.8332 0.8332 1.09 0.306 A x B 1 0.0489 0.0489 0.06 0.802 Error 24 18.2619 0.7609 Total 27 20.9800 a Flower abscission and senescence data is equivalent to vase life data.

Appendix 3.67. ANOVA table for relative fresh weight of flowering L. scoparium ‘Winter Cheer’ stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation df SS MS F P 1-MCP (A) 1 0.0000009 0.0000009 0.01 0.944 Ethylene (B) 1 0.0023418 0.0023418 13.69 0.001 A x B 1 0.0001472 0.0001472 0.86 0.363 Error 24 0.0041059 0.0001711 Total 27 0.0065957

Appendix 3.68. ANOVA table for vase solution uptake by flowering L. scoparium ‘Winter Cheer’ stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 0.00200 0.00200 0.09 0.764 Ethylene (B) 1 0.11198 0.11198 5.15 0.032 A x B 1 0.01720 0.01720 0.79 0.382 Rep (A B) 24 0.52149 0.02173 4.64 0.000 Day (C) 3 4.11011 1.37004 292.78 0.000 A x C 3 0.00204 0.00068 0.15 0.932 B x C 3 0.07014 0.02338 5.00 0.003 A x B x C 3 0.01117 0.00372 0.80 0.500 Error 72 0.33692 0.00468 Total 111 5.18305

Appendix 3.69. Time in days to moderate pedicel (score = 2) and peduncle wilting (score = 2) and > 10% flower opening (score = 2) on flowering O. diosmifolius stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Data followed by the same letters are not significantly different at P = 0.05. LSD for peduncle wilting = 1.1.

Assessment No ethylene (0 µL ethylene/L) Plus ethylene (10 µL ethylene/L) 0 nL 1-MCP/L 10 nL 1-MCP/L 0 nL 1-MCP/L 10 nL 1-MCP/L Pedicel wilting 9.0 ± 0.6 8.8 ± 0.5 8.7 ± 0.7 9.7 ± 0.5 Flower opening 6.1 ± 1.0 6.0 ± 0.3 5.8 ± 0.6 5.2 ± 0.4 Peduncle wilting 6.0 ± 0.5 b 5.3 ± 0.4 ab 4.6 ± 0.3 a 4.7 ± 0.4 a

91 Appendix 3.70. ANOVA table for pedicel wilting of flowering O. diosmifolius stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation df SS MS F P 1-MCP (A) 1 1.600 1.600 0.50 0.485 Ethylene (B) 1 0.900 0.900 0.28 0.600 A x B 1 3.600 3.600 1.12 0.297 Error 36 115.800 3.217 Total 39 121.900

Appendix 3.71. ANOVA table for flower opening on O. diosmifolius stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation df SS MS F P 1-MCP (A) 1 1.225 1.225 0.29 0.596 Ethylene (B) 1 3.025 3.025 0.71 0.406 A x B 1 0.625 0.625 0.15 0.705 Error 36 154.100 4.281 Total 39 121.900

Appendix 3.72. ANOVA table for leaf senescence on flowering O. diosmifolius stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Leaf senescence percentage data were arcsine transformed for ANOVA.

Source of variation DF SS MS F P 1-MCP (A) 1 0.08598 0.08598 2.61 0.107 Ethylene (B) 1 0.05530 0.05530 1.68 0.196 A x B 1 1.84812 1.84812 56.02 0.000 Rep (A B) 36 43.12407 1.19789 36.31 0.000 Day (C) 14 41.92761 2.99483 90.79 0.000 A x C 14 0.23734 0.01695 0.51 0.926 B x C 14 0.08921 0.00637 0.19 0.999 A x B x C 14 0.73948 0.05282 1.60 0.075 Error 504 16.62565 0.03299 Total 599 104.73274

Appendix 3.73. ANOVA table for leaf abscission from flowering O. diosmifolius stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 3.101 3.101 0.34 0.560 Ethylene (B) 1 35.445 35.445 3.89 0.050 A x B 1 64.351 64.351 7.07 0.008 Rep (A B) 36 2372.816 65.912 7.24 0.000 Day (C) 7 2348.837 335.548 36.87 0.000 A x C 7 11.755 1.679 0.18 0.988 B x C 7 66.812 9.545 1.05 0.398 A x B x C 7 93.405 13.344 1.47 0.180 Error 252 2293.409 9.101 Total 319 7289.930

92 Appendix 3.74. ANOVA table for relative fresh weight of flowering O. diosmifolius stems pre- treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 100.27 100.27 2.75 0.099 Ethylene (B) 1 191.38 191.38 5.25 0.023 A x B 1 55.50 55.50 1.52 0.218 Rep (A B) 36 4848.80 134.69 3.70 0.000 Day (C) 6 33073.28 5512.21 151.29 0.000 A x C 6 201.06 33.51 0.92 0.482 B x C 6 84.25 14.04 0.39 0.888 A x B x C 6 626.79 104.47 2.87 0.010 Error 216 7869.91 36.43 Total 279 47051.25

Appendix 3.75. ANOVA table for peduncle wilting from flowering O. diosmifolius stems pre- treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation df SS MS F P 1-MCP (A) 1 0.900 0.900 0.57 0.454 Ethylene (B) 1 10.000 10.000 6.36 0.016 A x B 1 1.600 1.600 1.02 0.320 Error 36 56.600 1.572 Total 39 69.100

Appendix 3.76. ANOVA table for vase solution uptake by flowering O. diosmifolius stems pre- treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 0.15768 0.15768 2.18 0.149 Ethylene (B) 1 0.00560 0.00560 0.08 0.783 A x B 1 0.16920 0.16920 2.33 0.135 Rep (A B) 36 2.60898 0.07247 4.32 0.000 Day (C) 6 25.12580 4.18763 249.62 0.000 A x C 6 0.65128 0.10855 6.47 0.000 B x C 6 2.20046 0.36674 21.86 0.000 A x B x C 6 0.08305 0.01384 0.83 0.552 Error 216 3.62356 0.01678 Total 279 34.62561

Appendix 3.77. ANOVA table for vase life of flowering O. diosmifolius stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation df SS MS F P 1-MCP (A) 1 0.025 0.025 0.00 0.955 Ethylene (B) 1 0.225 0.225 0.03 0.864 A x B 1 27.225 27.225 3.58 0.066 Error 36 273.500 7.597 Total 39 300.975

93 Appendix 3.78. ANOVA table for relative fresh weight of flowering P. lanceolata stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 4.68 4.68 0.60 0.438 Ethylene (B) 1 169.08 169.08 21.77 0.000 A x B 1 159.03 159.03 20.48 0.000 Rep (A B) 36 5412.26 150.34 19.36 0.000 Day (C) 9 3933.33 437.04 56.28 0.000 A x C 9 62.45 6.94 0.89 0.531 B x C 9 133.02 14.78 1.90 0.051 A x B x C 9 40.88 4.54 0.58 0.809 Error 324 2516.00 7.77 Total 399 12430.72

Appendix 3.79. ANOVA table for vase solution uptake by flowering P. lanceolata stems pre- treated with 0 or 10 nL 1-MCP/L for 12 hours at 20oC on day 0. Half of the stems from each of these treatments were then exposed to 10 µL ethylene/L for 12 hours at 20oC on day 1.

Source of variation DF SS MS F P 1-MCP (A) 1 0.5993 0.5993 1.24 0.272 Ethylene (B) 1 1.7425 1.7425 3.62 0.065 A x B 1 0.2716 0.2716 0.56 0.458 Rep (A B) 36 17.3506 0.4820 5.66 0.000 Day (C) 10 199.8068 19.9807 234.65 0.000 A x C 10 1.6186 0.1619 1.90 0.044 B x C 10 1.5734 0.1573 1.85 0.051 A x B x C 10 1.2355 0.1235 1.45 0.156 Error 360 30.6543 0.0852 Total 439 254.8525

Appendix 3.80. ANOVA table for vase life of flowering P. lanceolata stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation df SS MS F P 1-MCP (A) 1 0.625 0.625 0.10 0.756 Ethylene (B) 1 3.025 3.025 0.47 0.496 A x B 1 18.225 18.225 2.85 0.100 Error 36 229.900 6.386 Total 39 251.775

Appendix 3.81. ANOVA table for perianth abscissiona from clonally propagated T. speciosissima ‘Shady Lady’ inflorescences pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 3.025 3.025 3.000 0.092 Ethylene (B) 1 4.225 4.225 4.19 0.048 A x B 1 1.225 1.225 1.21 0.278 Error 36 36.300 1.008 Total 39 44.775 a Perianth abscission data is equivalent to vase life data.

94 Appendix 3.82. ANOVA table for relative fresh weight of clonally propagated T. speciosissima ‘Shady Lady’ inflorescences pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 18.23 18.23 2.04 0.157 Ethylene (B) 1 59.84 59.84 6.71 0.012 A x B 1 139.52 139.52 15.64 0.000 Rep (A B) 36 1794.46 49.85 5.59 0.000 Day (C) 2 5423.71 2711.85 304.02 0.000 A x C 2 39.58 19.79 2.22 0.116 B x C 2 62.39 31.20 3.50 0.036 A x B x C 2 61.71 30.85 3.46 0.037 Error 72 642.23 8.92 Total 119 8241.67

2 Opening score 1 0.5

0.4

0.3 Solution uptake 0.2 (mL/g initial FW/2 days)(mL/g initialFW/2 0246 Time (days)

Appendix 3.83. Flower opening (scores: 1 = < 5% to 3 = 25%) and vase solution uptake for clonally propagated T. speciosissima ‘Shady Lady’ inflorescences treated with 0 nL 1-MCP/L and 0 µL ethylene/L (●), 0 nL 1-MCP/L and 10 µL ethylene/L (■), 10 nL 1-MCP/L and 0 µL ethylene/L (▲), or 10 nL 1-MCP/L and 10 µL ethylene/L (▼). 1-MCP and ethylene treatments were each conducted for 12 hours at 20oC on days 0 and 1, respectively. Vertical bars represent standard errors of means (n = 10). LSD for vase solution uptake data = 0.054 mL/g initial FW/2 days.

95 Appendix 3.84. Summary of chi-square test for an association between treatment (1-MCP and ethylene) and opening scores (scores: 1 = < 5% to 3 = 25%) for clonally propagated T. speciosissima inflorescences pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Days Chi-square df P 0 5.518 6 0.479 2 3.436 6 0.752 4 2.222 3 0.528 6 3.077 3 0.380

Appendix 3.85. ANOVA table for vase solution uptake by clonally propagated T. speciosissima ‘Shady Lady’ inflorescences pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 0.00913 0.00913 0.40 0.533 Ethylene (B) 1 0.00852 0.00852 0.37 0.547 A x B 1 0.02023 0.02023 0.88 0.356 Rep (A B) 36 0.83149 0.02310 2.25 0.002 Day (C) 2 0.12585 0.06293 6.13 0.003 A x C 2 0.00271 0.00136 0.13 0.876 B x C 2 0.04749 0.02375 2.31 0.106 A x B x C 2 0.02771 0.01386 1.35 0.266 Error 72 0.73895 0.01026 Total 119 1.81210

Appendix 3.86. ANOVA table for perianth abscissiona from seed-grown T. speciosissima inflorescences pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 10.0000 10.0000 11.84 0.001 Ethylene (B) 1 0.4000 0.4000 0.47 0.496 A x B 1 3.6000 3.6000 4.26 0.046 Error 36 30.4000 0.8444 Total 39 44.4000 a Perianth abscission data is equivalent to vase life data.

Appendix 3.87. ANOVA table for relative fresh weight of seed-grown T. speciosissima inflorescences pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments on day 1 were then exposed to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 484.429 484.429 457.63 0.000 Ethylene (B) 1 8.536 8.536 8.06 0.006 A x B 1 30.786 30.786 29.08 0.000 Rep (A B) 36 630.005 17.500 16.53 0.000 Day (C) 2 1598.538 799.269 755.06 0.000 A x C 2 15.743 7.871 7.44 0.001 B x C 2 8.643 4.321 4.08 0.021 A x B x C 2 4.821 2.411 2.28 0.110 Error 72 76.216 1.059 Total 119 2857.715

96 5

2 Opening score 1

0.5

0.4

0.3 Solution uptake 0.2 (mL/g initial FW/2 days) FW/2 (mL/g initial 0246 Time (days)

Appendix 3.88. Flower opening (scores: 1 = < 5% to > 25%) and vase solution uptake for seed- grown T. speciosissima inflorescences treated with 0 nL 1-MCP/L and 0 µL ethylene/L (●), 0 nL 1- MCP/L and 10 µL ethylene/L (■), 10 nL 1-MCP/L and 0 µL ethylene/L (▲), or 10 nL 1-MCP/L and 10 µL ethylene/L (▼). 1-MCP and ethylene treatments were each conducted for 12 hours at 20oC on days 0 and 1, respectively. Vertical bars represent standard errors of means (n = 10). No significant differences (P > 0.05) between treatments for flower opening data existed except on day 2 (Appendix 3.89). LSD for vase solution uptake data = 0.021 mL/g initial FW/2 days.

Appendix 3.89. Summary of chi-square test for an association between treatment (1-MCP and ethylene) and opening scores (scores: 1 = < 5% to > 25%) for seed-grown T. speciosissima inflorescences pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Days Chi-square df P 0 5.583 6 0.471 2a 28.092 9 0.001 4 12.229 6 0.057 6 9.730 6 0.137 a Fisher’s exact test was performed where the Chi-square test was invalid.

97 Appendix 3.90. ANOVA table for vase solution uptake by seed-grown T. speciosissima inflorescences pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the inflorescences from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 0.0087845 0.0087845 1.72 0.198 Ethylene (B) 1 0.0081173 0.0081173 1.59 0.215 A x B 1 0.0006846 0.0006846 0.13 0.716 Rep (A B) 36 0.1834766 0.0050966 8.49 0.000 Day (C) 2 0.2589631 0.1294815 215.61 0.000 A x C 2 0.0008777 0.0004388 0.73 0.485 B x C 2 0.0004721 0.0002361 0.39 0.676 A x B x C 2 0.0000999 0.0000499 0.08 0.920 Error 72 0.0432393 0.0006005 Total 119 0.5047150

Appendix 3.91. ANOVA table for flower abscission from flowering T. calycina stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Flower abscission percentage data were arcsine transformed for ANOVA.

Source of variation DF SS MS F P 1-MCP (A) 1 0.004819 0.004819 3.85 0.057 Ethylene (B) 1 0.009479 0.009479 7.58 0.009 A x B 1 0.029175 0.029175 23.33 0.000 Rep (A B) 36 1.012007 0.028111 22.48 0.000 Day (C) 1 0.016710 0.016710 13.36 0.001 A x C 1 0.000075 0.000075 0.06 0.808 B x C 1 0.005367 0.005367 4.29 0.046 A x B x C 1 0.000076 0.000076 0.06 0.807 Error 36 0.045027 0.001251 Total 79 1.122734

Appendix 3.92. ANOVA table for closure of flowers from flowering T. calycina stems treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Flower closure percentage data were arcsine transformed for ANOVA.

Source of variation DF SS MS F P 1-MCP (A) 1 0.000029 0.000029 0.00 0.954 Ethylene (B) 1 0.045650 0.045650 5.45 0.025 A x B 1 0.038608 0.038608 4.61 0.039 Rep (A B) 36 2.579372 0.071649 8.55 0.000 Day (C) 1 0.561463 0.561463 67.03 0.000 A x C 1 0.103394 0.103394 12.34 0.001 B x C 1 0.002476 0.002476 0.30 0.590 A x B x C 1 0.003919 0.003919 0.47 0.498 Error 36 0.301527 0.008376 Total 79 3.636438

98 Appendix 3.93. ANOVA table for relative fresh weight of flowering T. calycina stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 297.52 297.52 13.82 0.000 Ethylene (B) 1 333.37 297.52 15.48 0.000 A x B 1 207.26 297.52 9.63 0.003 Rep (A B) 36 4336.46 120.46 5.59 0.000 Day (C) 2 7814.04 3907.02 181.44 0.000 A x C 2 113.92 56.96 2.65 0.078 B x C 2 38.93 19.46 0.90 0.410 A x B x C 2 52.36 26.18 1.22 0.302 Error 72 1550.42 21.53 Total 119 14744.28

Appendix 3.94. ANOVA table for vase solution uptake by flowering T. calycina stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 1.18093 1.18093 2.75 0.106 Ethylene (B) 1 0.31580 0.31580 0.74 0.396 A x B 1 1.14391 1.14391 2.67 0.111 Rep (A B) 36 15.43458 0.42874 4.80 0.000 Day (C) 2 3.56050 1.78025 19.91 0.000 A x C 2 0.25092 0.12546 1.40 0.252 B x C 2 0.15106 0.07553 0.84 0.434 A x B x C 2 0.44877 0.22438 2.51 0.088 Error 72 6.43745 0.08941 Total 119 28.92391

Appendix 3.95. ANOVA table for vase life of flowering stems of T. calycina pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 2.5000 2.5000 2.59 0.117 Ethylene (B) 1 2.5000 2.5000 2.59 0.117 A x B 1 0.1000 0.1000 0.10 0.750 Error 36 34.8000 0.9667 Total 39 39.9000

99 Appendix 3.96. ANOVA table for flower abscission from flowering V. nitens stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Flower abscission score data were arcsine transformed for ANOVA.

Source of variation DF SS MS F P 1-MCP (A) 1 12.17359 12.17359 5360.52 0.000 Ethylene (B) 1 12.17359 12.17359 5360.52 0.000 A x B 1 12.17359 12.17359 5360.52 0.000 Rep (A B) 36 10.31738 0.28659 126.20 0.000 Day (C) 14 1.03250 0.07375 32.47 0.000 A x C 14 1.03250 0.07375 32.47 0.000 B x C 14 1.03250 0.07375 32.47 0.000 A x B x C 14 1.03250 0.07375 32.47 0.000 Error 504 1.14457 0.00227 Total 599 52.11271

Appendix 3.97. ANOVA table for relative fresh weight of flowering V. nitens stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 9760.6 9760.6 640.34 0.000 Ethylene (B) 1 7361.9 7361.9 482.97 0.000 A x B 1 8793.1 8793.1 576.87 0.000 Rep (A B) 36 24879.7 691.1 45.34 0.000 Day (C) 13 72947.3 5611.3 368.13 0.000 A x C 13 468.4 36.0 2.36 0.005 B x C 13 76.0 5.8 0.38 0.975 A x B x C 13 150.9 11.6 0.76 0.701 Error 468 7133.6 15.2 Total 559 131571.5

Appendix 3.98. ANOVA table for vase solution uptake by flowering V. nitens stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 0.421726 0.421726 14.63 0.001 Ethylene (B) 1 0.056679 0.056679 1.97 0.169 A x B 1 0.006583 0.006583 0.23 0.636 Rep (A B) 36 1.037974 0.028833 16.36 0.000 Day (C) 13 2.889912 0.222301 126.17 0.000 A x C 13 0.195215 0.015017 8.52 0.000 B x C 13 0.037035 0.002849 1.62 0.077 A x B x C 13 0.007638 0.000588 0.33 0.987 Error 468 0.824590 0.001762 Total 559 5.477352

100 Appendix 3.99. ANOVA table for vase life of flowering V. nitens stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation df SS MS F P 1-MCP (A) 1 207.03 207.03 35.21 0.000 Ethylene (B) 1 180.63 180.63 30.72 0.000 A x B 1 330.62 330.62 56.22 0.000 Error 36 211.70 5.88 Total 39 929.97

Appendix 3.100. ANOVA table for flower abscissiona from flowering Z. cytisoides stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation df SS MS F P 1-MCP (A) 1 11.250 11.250 3.44 0.082 Ethylene (B) 1 2.450 2.450 0.75 0.400 A x B 1 0.450 0.450 0.14 0.716 Error 16 52.400 3.275 Total 19 66.550 a Flower abscission data is equivalent to vase life data.

Appendix 3.101. ANOVA table for relative fresh weight of flowering Z. cytisoides stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 1402.59 1402.59 171.56 0.000 Ethylene (B) 1 170.74 170.74 20.88 0.000 A x B 1 307.98 307.98 37.67 0.000 Rep (A B) 36 1982.10 123.88 15.15 0.000 Day (C) 14 20911.64 1493.69 182.70 0.000 A x C 14 153.26 10.95 1.34 0.186 B x C 14 261.61 18.69 2.29 0.006 A x B x C 14 95.19 6.80 0.83 0.634 Error 224 1831.34 8.18 Total 299 27116.45

Appendix 3.102. ANOVA table for vase solution uptake by flowering Z. cytisoides stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 0.19699 0.19699 2.67 0.122 Ethylene (B) 1 0.06211 0.06211 0.84 0.373 A x B 1 0.15041 0.15041 2.04 0.173 Rep (A B) 16 1.18212 0.07388 7.65 0.000 Day (C) 14 11.03470 0.78819 81.64 0.000 A x C 14 0.55468 0.03962 4.10 0.000 B x C 14 0.43029 0.03073 3.18 0.000 A x B x C 14 0.23713 0.01694 1.75 0.047 Error 224 2.16251 0.00965 Total 299 16.01095

101 Appendix 3.103. ANOVA table for flower wilting from flowering B. heterophylla stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L or pulsed with STS (0.5 mM Ag+) for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 72 hours at 20oC. Flower wilting score data were converted to a binary score for ANOVA.

Source of variation DF SS MS F P Treatment (A) 2 16.4704 8.2352 120.55 0.000 Ethylene (B) 1 1.6667 1.6667 24.40 0.000 A x B 2 15.5444 7.7722 113.77 0.000 Rep (A B) 54 23.0889 0.4276 6.26 0.000 Day (C) 8 38.5926 4.8241 70.62 0.000 A x C 16 7.1296 0.4456 6.52 0.000 B x C 8 0.2667 0.0333 0.49 0.865 A x B x C 16 2.7222 0.1701 2.49 0.001 Error 432 29.5111 0.0683 Total 539 134.9926

Appendix 3.104. ANOVA table for relative fresh weight of flowering B. heterophylla stems pre- treated on day 0 with 0 or 10 nL 1-MCP/L or pulsed with STS (0.5 mM Ag+) for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 72 hours at 20oC.

Source of variation DF SS MS F P Treatment (A) 2 5903.0 2951.5 161.14 0.000 Ethylene (B) 1 23413.7 23413.7 1278.33 0.000 A x B 2 13243.1 6621.6 361.52 0.000 Rep (A B) 54 46640.9 863.7 47.16 0.000 Day (C) 11 150542.7 13685.7 747.20 0.000 A x C 22 2580.2 117.3 6.40 0.000 B x C 11 1685.5 153.2 8.37 0.000 A x B x C 22 1038.2 47.2 2.58 0.000 Error 594 10879.6 18.3 Total 719 255927.0

Appendix 3.105. ANOVA table for vase life of flowering B. heterophylla stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L or pulsed with STS (0.5 mM Ag+) for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 72 hours at 20oC.

Source of variation DF SS MS F P Treatment (A) 2 7.600 3.800 1.14 0.329 Ethylene (B) 1 4.267 4.267 1.28 0.264 A x B 2 32.133 16.067 4.80 0.012 Error 54 180.600 3.344 Total 59 224.600

102 Appendix 3.106. Time in days to > 50% flower discolouration (score = 3) on flowering B. heterophylla stems pre-treated on day 0 with 0 or 10 nL 1-MCP/L or pulsed with STS (0.5 mM Ag+) for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene for 72 hours at 20oC. Data followed by the same letters are not significantly different (LSD = 1.1) at P = 0.05.

Treatment Time (days) No ethylene (0 µL ethylene/L) 0 nL 1-MCP/L 4.6 ± 0.3 a 10 nL 1-MCP/L 4.8 ± 0.4 a 0.5 mM Ag+ 7.0 ± 0.5 b

Plus ethylene (10 µL ethylene/L) 0 nL 1-MCP/L 4.5 ± 0.4 a 10 nL 1-MCP/L 4.2 ± 0.2 a 0.5 mM Ag+ 5.1 ± 0.5 a

Appendix 3.107. ANOVA table for flower discolouration from flowering B. heterophylla stems pre- treated on day 0 with 0 or 10 nL 1-MCP/L or pulsed with STS (0.5 mM Ag+) for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation df SS MS F P Agent (1-MCP or STS) (A) 2 31.033 15.517 9.63 0.000 Ethylene (B) 1 11.267 11.267 6.99 0.011 A x B 2 8.633 4.317 2.68 0.078 Error 54 87.000 1.611 Total 59 137.933

Appendix 3.108. ANOVA table for vase solution uptake by flowering B. heterophylla stems pre- treated on day 0 with 0 or 10 nL 1-MCP/L or pulsed with STS (0.5 mM Ag+) for 12 hours at 20oC. Half of the stems from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 72 hours at 20oC.

Source of variation DF SS MS F P Agent (1-MCP or Ag+) (A) 2 0.44624 0.22312 5.71 0.006 Ethylene (B) 1 0.00039 0.00039 0.01 0.920 A x B 2 1.43496 0.71748 18.36 0.000 Rep (A B) 54 2.10978 0.03907 12.31 0.000 Day (C) 11 13.37318 1.21574 383.06 0.000 A x C 22 0.60994 0.02772 8.74 0.000 B x C 11 0.02513 0.00228 0.72 0.720 A x B x C 22 0.35653 0.01621 5.11 0.000 Error 594 1.88522 0.00317 Total 719 20.24139

Appendix 3.109. Ethylene production by detached flowers or flowering stems of B. heterophylla at different stages of wilting. The number of measurements at each wilting stage or score are shown in parentheses. Data followed by the same letters are not significantly different at P = 0.05. LSD for ethylene production by individual flowers and flowering stems = 1.8 and 1.3 µL/kg FW/hr, respectively.

Flower wilt score Ethylene production (µL/kg FW/hr) Individual flowers Flowering stems 1 (none/slight) 1.3 ± 0.5 a (n = 59) 2.0 ± 0.4 a (n = 54) 2 (moderate) 5.8 ± 1.1 b (n = 4) 2.9 ± 0.2 ab (n = 3) 3 (advanced) 5.2 ± 1.1 b (n = 7) 3.4 ± 0.5 b (n = 8) Appendix 3.110. Ethylene production by detached flowers or flowering stems of B. heterophylla at different stages of discolouration. The number of measurements at each discolouration stage or 103 score are shown in parentheses. Data followed by the same letters are not significantly different at P = 0.05. LSD for ethylene production by individual flowers and flowering stems = 2.3 and 1.3 µL/kg FW/hr, respectively.

Flower discolouration score Ethylene production (µL/kg FW/hr) Individual flowers Flowering stems 1 (0-25%) 0.9 ± 0.2 a (n = 16) 2.2 ± 0.5 a (n = 17) 2 (26-50%) 1.2 ± 0.6 a (n = 18) 1.8 ± 0.4 a (n = 19) 3 (51-75%) 2.1 ± 0.7 ab (n = 20) 2.6 ± 1.1 a (n = 29) 4 (76-100%) 3.6 ± 1.3 b (n = 16) z z Flowers did not reach a discolouration score of 4.

Appendix 3.111. ANOVA table for ethylene production by detached B. heterophylla flowers sampled at different stages of wilting on a 3 point scale.

Source of variation df SS MS F P Wilting 2 159.56 78.78 41.38 0.000 Error 67 129.17 1.93 Total 69 288.73

Appendix 3.112. ANOVA table for ethylene production by detached B. heterophylla flowers sampled at different stages of discolouration on a 4 point scale

Source of variation df SS MS F P Discolouration 3 71.04 23.68 7.18 0.000 Error 66 217.69 3.30 Total 69 288.73

Appendix 3.113. ANOVA table for ethylene production by flowering B. heterophylla stems when flowers were at different stages of wilting on a 3 point scale

Source of variation df SS MS F P Wilting 2 14.293 7.146 7.31 0.001 Error 62 60.589 0.977 Total 64 74.881

Appendix 3.114. ANOVA table for ethylene production by flowering B. heterophylla stems when flowers were at different stages of discolouration on a 4 point scale.

Source of variation df SS MS F P Discolouration 2 8.03 4.01 3.72 0.030 Error 62 66.85 1.08 Total 64 74.88

104 Appendix 4.1. Change in flower abscission (scores: 1 = < 10% to 5 = > 80%) after exposure to ethylene for G. ‘Sylvia’ inflorescences pre-treated on day 0 with 10 nL 1-MCP/L for 12 hours at 2 or 20oC. Different sub-samples of these inflorescences were then treated daily until day 5 with 10 µL ethylene/L for 12 hours at 20oC. Data followed by the same letters are not significantly different (LSD = 1.7) at P = 0.05 (n = 5). A logistic transformation of flower abscission data was performed prior to ANOVA.

Days between 1-MCP pre-treatment and Flower abscission exposure to ethylene

1-MCP at 2oC1-MCP at 20oC 1 2.1 ± 0.0 d -5.3 ± 0.0 a 2 2.1 ± 0.0 d -3.4 ± 1.2 b 3 2.1 ± 0.04 d -2.1 ± 1.4 b 4 1.9 ± 0.10 cd 0.3 ± 0.5 c 5 1.7 ± 0.08 cd 0.8 ± 0.4cd

Appendix 4.2. Loss of relative fresh weight after exposure to ethylene from G. ‘Sylvia’ inflorescences pre-treated on day 0 with 10 nL 1-MCP/L for 12 hours at 2 or 20oC. Different sub- samples of these inflorescences were then treated daily until day 5 with 10 µL ethylene/L for 12 hours at 20oC. Data followed by the same letters are not significantly different (LSD = 15%) at P = 0.05 (n = 5). A logistic transformation of relative fresh weight data was performed prior to ANOVA.

Days between 1-MCP pre-treatment and Loss of fresh weight (%) exposure to ethylene

1-MCP at 2oC1-MCP at 20oC 1 69 ± 2.1 cd 2 ± 1.4 a 2 75 ± 3.6 d 11 ± 7.1 a 3 72 ± 2.0 cd 34 ± 11 b 4 61 ± 3.3 cd 45 ± 5.0 bc 5 59 ± 3.7 c 38 ± 5.0 b

Appendix 4.3. ANOVA table for flower abscission from G. ‘Sylvia’ inflorescences pre-treated with 10 nL 1-MCP/L for 12 hours at 2 or 20oC. Different sub-samples of these inflorescences were then treated daily until day 5 with 10 µL ethylene/L for 12 hours at 20oC. A logistic transformation of flower abscission data was performed prior to ANOVA.

Source of variation DF SS MS F P Temperature (A) 1 190.109 190.109 101.09 0.000 Treatment (B) 4 57.137 14.284 7.60 0.000 A x B 4 71.906 17.977 9.56 0.000 Error 40 75.220 1.881 Total 49 394.372

105 Appendix 4.4. ANOVA table for the loss of relative fresh weight from G. ‘Sylvia’ inflorescences pre-treated with 10 nL 1-MCP/L for 12 hours at 2 or 20oC. Different sub-samples of these inflorescences were then treated daily until day 5 with 10 µL ethylene/L for 12 hours at 20oC. A logistic transformation of relative fresh weight data was performed prior to ANOVA.

Source of variation df SS MS F P Temperature (A) 1 2.09408 2.09408 155.68 0.000 Time of ethylene treatment (B) 4 0.21605 0.05401 4.02 0.008 A x B 4 0.55457 0.13864 10.31 0.000 Error 40 0.53804 0.01345 Total 49 3.40274

Appendix 4.5. ANOVA table for vase solution uptake by G. ‘Sylvia’ inflorescences pre-treated with 10 nL 1-MCP/L for 12 hours at 2 or 20oC. Different sub-samples of these inflorescences were then treated daily until day 5 with 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P Treatment (A) 4 0.061322 0.015330 7.43 0.000 Temperature (B) 1 0.045499 0.045499 22.04 0.000 A x B 4 0.031394 0.007848 3.80 0.010 Rep (A B) 40 0.082558 0.002064 3.41 0.000 Day (C) 4 0.584569 0.146142 241.51 0.000 A x C 16 0.243258 0.015204 25.13 0.000 B x C 4 0.032175 0.008044 13.29 0.000 A x B x C 16 0.062012 0.003876 6.41 0.000 Error 160 0.096819 0.000605 Total 249 1.239606

Appendix 4.6. ANOVA table for vase life of G. ‘Sylvia’ inflorescences pre-treated with 10 nL 1- MCP/L for 12 hours at 2 or 20oC. Different sub-samples of these inflorescences were then treated daily until day 5 with 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P Temperature (A) 1 8.820 8.820 13.57 0.001 Treatment (B) 4 9.800 2.450 3.770 0.011 A x B 4 11.88 2.970 4.570 0.004 Error 40 26.00 0.650 Total 49 56.50

106 Control treatments Sequential treatments 3

1 o 2oC 2 C Control treatments Sequential treatments

Wilt score 3

1 20oC 20oC

01234560123456 Time (days)

Appendix 4.7. Flower wilting (scores: 1 = none/slight to 3 = advanced) on G. ‘Sylvia’ inflorescences pre-treated with 10 nL 1-MCP/L at 2 or 20oC. Different sub-samples of these inflorescences were then sequentially exposed to 10 µL ethylene/L at 20oC on days 1 (●), 2 (■), 3 (▲), 4 (▼) or 5 (◆). Control inflorescences were treated with 0 nL 1-MCP/L at 2 or 20oC and 0 µL ethylene/L at 20oC (●), 0 nL 1-MCP/L at 2 or 20oC and 10 µL ethylene/L at 20oC (■) or 10 nL 1-MCP/L at 2 or 20oC and 0 µL ethylene/L at 20oC (▲). 1-MCP and ethylene treatments were each conducted for 12 hours on days 0 or 1, respectively, unless otherwise stated. Vertical bars represent standard errors of means (n = 5).

107 Control treatments Sequential treatments 3

1 2oC 2oC Control treatments Sequential treatments 3 Opening score Opening

1 o o 20 C 20 C

01234560123456 Time (days)

Appendix 4.8. Flower opening (scores: 1 = < 5% to 3 = > 25%) on G. ‘Sylvia’ inflorescences pre- treated with 10 nL 1-MCP/L at 2 or 20oC. Different sub-samples of these inflorescences were then sequentially exposed to 10 µL ethylene/L at 20oC on days 1 (●), 2 (■), 3 (▲), 4 (▼) or 5 (◆). Control inflorescences were treated with 0 nL 1-MCP/L at 2 or 20oC and 0 µL ethylene/L at 20oC (●), 0 nL 1-MCP/L at 2 or 20oC and 10 µL ethylene/L at 20oC (■) or 10 nL 1-MCP/L at 2 or 20oC and 0 µL ethylene/L at 20oC (▲). 1-MCP and ethylene treatments were each conducted for 12 hours on days 0 or 1, respectively, unless otherwise stated. Vertical bars represent standard errors of means (n = 5).

108 Control treatments Sequential treatments 3

1 2oC2oC Control treatments Sequential treatments 3 Discolouration score Discolouration 2

1 20oC 20oC

01234560123456 Time (days)

Appendix 4.9. Flower discolouration (scores: 1 = none/slight to 3 = advanced) on G. ‘Sylvia’ inflorescences pre-treated with 10 nL 1-MCP/L at 2 or 20oC. Different sub-samples of these inflorescences were then sequentially exposed to 10 µL ethylene/L at 20oC on days 1 (●), 2 (■), 3 (▲), 4 (▼) or 5 (◆). Control inflorescences were treated with 0 nL 1-MCP/L at 2 or 20oC and 0 µL ethylene/L at 20oC (●), 0 nL 1-MCP/L at 2 or 20oC and 10 µL ethylene/L at 20oC (■) or 10 nL 1- MCP/L at 2 or 20oC and 0 µL ethylene/L at 20oC (▲). 1-MCP and ethylene treatments were each conducted for 12 hours on days 0 or 1, respectively, unless otherwise stated. Vertical bars represent standard errors of means (n = 5).

109 Appendix 4.10. Change in flower abscission (%) after exposure to ethylene from C. uncinatum ‘Lollypop’ sprigs pre-treated on day 0 with 10 nL 1-MCP/L or STS (0.5 mM Ag+) for 12 hours at 2 or 20oC. Different sub-samples of sprigs from each of these treatments were then treated daily until day 9 with 10 µL ethylene/L for 12 hours at 20oC. Data followed by the same letters are not significantly different (LSD = 0.9) at P = 0.05 (n = 5). A logistic transformation of flower abscission data was performed prior to ANOVA.

Days between 1-MCP or 10 nL 1-MCP/L on day 0 0.5 mM Ag+ on day 0 STS pre-treatment and exposure to ethylene

2oC20oC2oC20oC

1 -3.5 ± 0.2 a -3.3 ± 0.1 a -3.5 ± 0.1 a -2.2 ± 0.6b 2 -2.2 ± 0.6 de -3.6 ± 0.1 a -3.5 ± 0.1 a -3.4 ± 0.1 a 3 -2.2 ± 0.5 b -3.5 ± 0.2 a -3.3 ± 0.2 ab -1.9 ± 0.6 b 4 3.4 ± 0.2 e -3.6 ± 0.1 a -3.4 ± 0.1 a -3.6 ± 0.1 a 5 3.3 ± 0.1 e -3.1 ± 0.6 ab -3.6 ± 0.2 a -3.1 ± 0.1 ab 6 3.2 ± 0.3 e -3.3 ± 0.1 a -3.5 ± 0.1 a -3.1 ± 0.4 ab 7 1.4 ± 0.4 d 0.4 ± 0.6 c -3.2 ± 0.4 ab -3.2 ± 0.4 a 8 3.3 ± 0.3 e 0.9 ± 0.3 cd -2.4 ± 0.4 b -3.0 ± 0.3 ab 9 3.7 ± 0.1 e 2.9 ± 0.3 e -3.5 ± 0.1 a -2.9 ± 0.1 ab

Appendix 4.12. Change in flower abscission (%) after exposure to ethylene from C. uncinatum ‘Alba’ sprigs pre-treated on day 0 with 10 nL 1-MCP/L or STS (0.5 mM Ag+) for 12 hours at 2 or 20oC. Different sub-samples of sprigs from each of these treatments were then treated daily until day 10 with 10 µL ethylene/L for 12 hours at 20oC. Data followed by the same letters are not significantly different (LSD = 1.6) at P = 0.05 (n = 5). A logistic transformation of flower abscission data was performed prior to ANOVA.

Days between 1-MCP or 10 nL 1-MCP/L on day 0 0.5 mM Ag+ on day 0 STS pre-treatment and exposure to ethylene

2oC20oC2oC20oC

1 -3.3 ± 0.2 ab -3.2 ± 0.1 ab -3.4 ± 0.1 ab -3.5 ± 0.1 ab 2 -1.7 ± 0.6 bc -2.8 ± 0.7 ab -3.6 ± 0.1 ab -3.2 ± 0.3 ab 3 0.5 ± 1.2 cd -3.1 ± 0.4 ab -3.3 ± 0.2 ab -3.2 ± 0.4 ab 4 -4.2 ± 0.8 c -2.8 ± 0.5 ab -3.0 ± 0.1 ab -3.3 ± 0.1 ab 5 0.8 ± 0.2 cd 0.2 ± 0.3 cd -1.9 ± 0.4 bc -3.2 ± 0.3 ab 6 0.9 ± 1.1 cd -1.7 ± 0.3 bc -2.8 ± 0.1 ab -3.6 ± 0.1 ab 7 -0.5 ± 0.7 c -2.4 ± 0.3 ab -2.8 ± 0.9 ab -2.1 ± 0.6 b 8 -1.3 ± 0.5 bc -0.8 ± 0.9 bc -2.4 ± 0.7 ab -3.8 ± 0.1 a 9 0.2 ± 1.3 cd 0.9 ± 0.6 cd -3.4 ± 0.1 ab -3.4 ± 0.1 ab 10 1.3 ± 1.0 d -0.7 ± 1.2 bc -3.8 ± 0.1 a -3.7 ± 0.1 a

110 Appendix 4.13. Change in flower abscission (%) after exposure to ethylene from C. uncinatum ‘Mid Pink’ sprigs pre-treated on day 0 with 10 nL 1-MCP/L or STS (0.5 mM Ag+) for 12 hours at 2 or 20oC. Different sub-samples of sprigs from each of these treatments were then treated daily until day 8 with 10 µL ethylene/L for 12 hours at 20oC. Data followed by the same letters are not significantly different (LSD = 1.0) at P = 0.05 (n = 5). A logistic transformation of flower abscission data was performed prior to ANOVA.

Days between 1-MCP or 10 nL 1-MCP/L on day 0 0.5 mM Ag+ on day 0 STS pre-treatment and exposure to ethylene

2oC20oC2oC20oC

1 -2.7 ± 0.4 b -3.6 ± 0.1 ab -3.8 ± 0.1 a -3.6 ± 0.2 ab 2 -2.1 ± 0.5 bc -3.7 ± 0.1 ab -3.8 ± 0.2 a -3.7 ± 0.2 a 3 3.1 ± 0.4 f -3.3 ± 0.1 ab -3.8 ± 0.2 a -3.6 ± 0.1 ab 4 2.7 ± 0.3 ef -1.2 ± 0.7 c -3.6 ± 0.1 ab -3.6 ± 0.2 ab 5 3.3 ± 0.4 f 2.0 ± 0.6 e -3.5 ± 0.1 ab -3.7 ± 0.1 a 6 3.2 ± 0.6 f 1.5 ± 1.0 de -3.7 ± 0.2 a -3.0 ± 0.2 ab 7 2.1 ± 0.7 e 0.9 ± 0.3 d -3.3 ± 0.4 ab -2.9 ± 0.3 ab 8 3.7 ± 0.1 f 2.9 ± 0.5 ef -3.9 ± 0.2 a -3.4 ± 0.3 ab

Appendix 4.14. Loss of relative fresh weight after exposure to ethylene from C. uncinatum ‘Lollypop’ sprigs pre-treated on day 0 with 10 nL 1-MCP/L or STS (0.5 mM Ag+) for 12 hours at 2 or 20oC. Different sub-samples of sprigs from each of these treatments were then treated daily until day 9 with 10 µL ethylene/L for 12 hours at 20oC. Data followed by the same letters are not significantly different (LSD = 13.5%) at P = 0.05 (n = 5). A logistic transformation of relative fresh weight data was performed prior to ANOVA.

Days between Loss of fresh weight (% of initial FW) 1-MCP or STS pre- treatment and exposure to ethylene

1-MCP at 2oC1-MCP at 20oCSTS at 2oCSTS at 20oC

1 -2.6 ± 0.3 ab -0.1 ± 0.5 ab 0.2 ± 2.1 b 15.3 ± 4.8 c 2 24.8 ± 2.4 cd 1.4 ± 0.8 b 1.9 ± 1.1 bc 10.6 ± 1.6 bc 3 4.3 ± 2.9 bc 2.0 ± 0.5 bc 0.9 ± 0.4 b 10.1 ± 2.2 bc 4 54.5 ± 3.6 e 5.7 ± 1.3 bc 0.8 ± 0.3 b 2.9 ± 2.0 bc 5 52.7 ± 1.5 e 10.8 ± 4.7 bc 5.3 ± 1.5 bc 3.3 ± 1.4 bc 6 47.4 ± 6.4 e 3.1 ± 2.2 bc 1.4 ± 0.9 b 1.7 ± 2.2 b 7 40.2 ± 4.6 de 30.6 ± 7.0 d 9.1 ± 1.5 bc 6.6 ± 1.7 bc 8 44.1 ± 8.5 e 18.4 ± 13.3 cd 9.2 ± 6.8 bc 2.2 ± 13.8 bc 9 44.0 ± 4.3 de 32.9 ± 5.2 de 5.1 ± 2.9 bc 9.5 ± 9.5 bc

111 Appendix 4.15. Loss of relative fresh weight after exposure to ethylene from C. uncinatum ‘Alba’ sprigs pre-treated on day 0 with 10 nL 1-MCP/L or STS (0.5 mM Ag+) for 12 hours at 2 or 20oC. Different sub-samples of sprigs from each of these treatments were then treated daily until day 10 with 10 µL ethylene/L for 12 hours at 20oC. Data followed by the same letters are not significantly different (LSD = 12.5%) at P = 0.05 (n = 5). A logistic transformation of relative fresh weight data was performed prior to ANOVA.

Days between 1- Loss of fresh weight (% of initial FW) MCP or STS pre- treatment and exposure to ethylene

1-MCP at 2oC1-MCP at 20oCSTS at 2oCSTS at 20oC

1 -0.9 ± 0.7 a 0.7 ± 0.4 ab -0.2 ± 0.2 ab 3.5 ± 0.8 ab 2 14.0 ± 4.9 bc 4.4 ± 3.0 ab -0.5 ± 0.6 a 0.8 ± 1.2 ab 3 31.0 ± 6.5 cd 8.2 ± 2.0 ab 1.5 ± 1.0 ab 6.4 ± 1.2 ab 4 24.1 ± 7.5 bc 8.4 ± 3.3 ab 0.2 ± 0.6 ab 8.2 ± 4.9 ab 5 28.8 ± 3.4 c 2.7 ± 5.7 c 10.0 ± 3.1 ab 6.4 ± 1.8 ab 6 29.8 ± 7.7 c 5.7 ± 2.1 ab 3.3 ± 1.4 ab 0.3 ± 0.9 ab 7 25.6 ± 6.4 c 0.2 ± 0.8 ab 6.4 ± 4.8 ab 3.6 ± 2.6 ab 8 12.1 ± 6.7 b 19.7 ± 8.1 bc 0.8 ± 2.9 ab 1.6 ± 3.2 ab 9 19.4 ± 9.8 bc 32.8 ± 6.2 cd 0.9 ± 1.0 ab 3.4 ± 1.3 ab 10 42.5 ± 9.4 d 15.8 ± 9.5 bc 2.9 ± 0.5 ab 4.0 ± 1.0 ab

Appendix 4.16. Loss of relative fresh weight after exposure to ethylene from C. uncinatum ‘Mid Pink’ sprigs pre-treated on day 0 with 10 nL 1-MCP/L or STS (0.5 mM Ag+) for 12 hours at 2 or 20oC. Different sub-samples of sprigs from each of these treatments were then treated daily until day 8 with 10 µL ethylene/L for 12 hours at 20oC. Data followed by the same letters are not significantly different (LSD = 10.1%) at P = 0.05 (n = 5). A logistic transformation of relative fresh weight data was performed prior to ANOVA.

Days between Loss of fresh weight (% of initial FW) 1-MCP or STS pre-treatment and exposure to ethylene

1-MCP at 2oC1-MCP at 20oCSTS at 2oCSTS at 20oC

1 0.7 ± 2.0 a 0.6 ± 0.8 a -1.6 ± 0.4 a 0.7 ± 0.8 a 2 6.0 ± 1.1 ab 4.7 ± 1.8 ab 1.7 ± 0.5 ab 4.8 ± 2.2 ab 3 37.1 ± 2.5 d 2.0 ± 0.2 ab 1.0 ± 0.3 a 4.2 ± 1.0 ab 4 53.0 ± 2.9 e 17.1 ± 4.6 bc 2.4 ± 1.0 ab 6.4 ± 4.1 ab 5 51.7 ± 3.5 e 45.2 ± 4.9 de 0.6 ± 1.8 a 0.8 ± 0.4 a 6 48.1 ± 4.5 e 41.4 ± 10.9 de 7.3 ± 3.3 ab 12.0 ± 8.2 b 7 40.6 ± 6.5 de 36.1 ± 4.1 d 9.2 ± 5.4 ab 23.0 ± 5.9 c 8 52.6 ± 0.8 e 50.7 ± 3.5 e 19.5 ± 3.5 bc 12.4 ± 5.0 bc

112 Appendix 4.17. ANOVA table for flower abscission from C. uncinatum ‘Lollypop’ sprigs pre- treated on day 0 with 10 nL 1-MCP/L or STS (0.5 mM Ag+) for 12 hours at 2 or 20oC. Different sub-samples of sprigs from each of these treatments were then treated daily until day 9 with 10 µL ethylene/L for 12 hours at 20oC. A logistic transformation of flower abscission data was performed prior to ANOVA.

Source of variation df SS MS F P Time of ethylene treatment (A) 8 168.232 21.029 40.92 0.000 Agent - 1-MCP or STS (B) 1 417.666 417.666 812.74 0.000 Temperature 1 107.103 107.103 208.41 0.000 A x B 8 202.257 25.282 49.20 0.000 A x C 8 102.320 12.790 24.89 0.000 B x C 1 166.483 166.483 323.96 0.000 A x B x C 8 75.087 9.386 18.26 0.000 Error 144 74.002 0.514 Total 179 1313.151

Appendix 4.18. ANOVA table for flower abscission from C. uncinatum ‘Alba’ sprigs pre-treated on day 0 with 10 nL 1-MCP/L or STS (0.5 mM Ag+) for 12 hours at 2 or 20oC. Different sub-samples of sprigs from each of these treatments were then treated daily until day 10 with 10 µL ethylene/L for 12 hours at 20oC. A logistic transformation of flower abscission data was performed prior to ANOVA.

Source of variation df SS MS F P Time of ethylene treatment (A) 9 81.292 9.032 5.50 0.000 Agent - 1-MCP or STS (B) 1 240.968 240.968 146.72 0.000 Temperature 1 30.099 30.099 18.33 0.000 A x B 9 73.288 8.143 4.96 0.000 A x C 9 22.092 2.455 1.49 0.154 B x C 1 12.999 12.999 7.91 0.006 A x B x C 9 35.807 3.979 2.42 0.013 Error 160 262.769 1.642 Total 199 759.314

Appendix 4.19. ANOVA table for flower abscission from C. uncinatum ‘Mid Pink’ sprigs pre- treated on day 0 with 10 nL 1-MCP/L or STS (0.5 mM Ag+) for 12 hours at 2 or 20oC. Different sub-samples of sprigs from each of these treatments were then treated daily until day 8 with 10 µL ethylene/L for 12 hours at 20oC. A logistic transformation of flower abscission data was performed prior to ANOVA.

Source of variation df SS MS F P Time of ethylene treatment (A) 7 226.539 32.363 46.06 0.000 Agent - 1-MCP or STS (B) 1 677.120 677.120 963.80 0.000 Temperature 1 39.295 39.295 55.93 0.000 A x B 7 191.779 27.397 39.00 0.000 A x C 7 36.590 5.227 7.44 0.000 B x C 1 60.975 60.975 86.79 0.000 A x B x C 7 32.901 4.700 6.69 0.000 Error 128 89.927 0.703 Total 159 1355.126

113 Appendix 4.20. ANOVA table for relative fresh weight of C. uncinatum ‘Lollypop’ sprigs pre- treated on day 0 with 10 nL 1-MCP/L or STS (0.5 mM Ag+) for 12 hours at 2 or 20oC. Different sub-samples of sprigs from each of these treatments were then treated daily until day 9 with 10 µL ethylene/L for 12 hours at 20oC. A logistic transformation of relative fresh weight data was performed prior to ANOVA.

Source of variation df SS MS F P Time of ethylene treatment (A) 8 0.81886 0.10236 8.58 0.000 Agent - 1-MCP or STS (B) 1 1.40085 1.40085 117.45 0.000 Temperature 1 0.43381 0.43381 36.37 0.000 A x B 8 0.91681 0.11460 9.61 0.000 A x C 8 0.54858 0.06857 5.75 0.000 B x C 1 0.75540 0.75540 63.33 0.000 A x B x C 8 0.26109 0.03264 2.74 0.008 Error 144 1.71753 0.01193 Total 179 6.85294

Appendix 4.21. ANOVA table for relative fresh weight of C. uncinatum ‘Alba’ sprigs pre-treated on day 0 with 10 nL 1-MCP/L or STS (0.5 mM Ag+) for 12 hours at 2 or 20oC. Different sub- samples of sprigs from each of these treatments were then treated daily until day 10 with 10 µL ethylene/L for 12 hours at 20oC. A logistic transformation of relative fresh weight data was performed prior to ANOVA.

Source of variation df SS MS F P Time of ethylene treatment (A) 9 0.48182 0.05354 5.24 0.000 Agent - 1-MCP or STS (B) 1 1.02065 1.02065 99.83 0.000 Temperature 1 0.10217 0.10217 9.99 0.002 A x B 9 0.29406 0.03267 3.20 0.001 A x C 9 0.27492 0.03055 2.99 0.003 B x C 1 0.16940 0.16940 16.57 0.000 A x B x C 9 0.24971 0.02775 2.71 0.006 Error 160 1.63578 0.01022 Total 199 4.22850

Appendix 4.22. ANOVA table for relative fresh weight of C. uncinatum ‘Mid Pink’ sprigs pre- treated on day 0 with 10 nL 1-MCP/L or STS (0.5 mM Ag+) for 12 hours at 2 or 20oC. Different sub-samples of sprigs from each of these treatments were then treated daily until day 8 with 10 µL ethylene/L for 12 hours at 20oC. A logistic transformation of relative fresh weight data was performed prior to ANOVA.

Source of variation df SS MS F P Time of ethylene treatment (A) 7 2.03998 0.29143 37.33 0.000 Agent - 1-MCP or STS (B) 1 2.29529 2.29529 294.02 0.000 Temperature 1 0.07156 0.07156 9.17 0.003 A x B 7 0.95987 0.13712 17.57 0.000 A x C 7 0.20970 0.02996 3.84 0.001 B x C 1 0.21144 0.21144 27.08 0.000 A x B x C 7 0.24264 0.03466 4.44 0.000 Error 128 0.99925 0.00781 Total 159 7.02975

114 Appendix 4.23. ANOVA table for vase life of C. uncinatum ‘Lollypop’ sprigs pre-treated on day 0 with 10 nL 1-MCP/L or STS (0.5 mM Ag+) for 12 hours at 2 or 20oC. Different sub-samples of sprigs from each of these treatments were then treated daily until day 9 with 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P Temperature (A) 1 46.006 46.006 20.27 0.000 Time of ethylene treatment (B) 8 75.111 9.389 4.14 0.000 Agent - 1-MCP or STS (C) 1 101.250 101.250 44.61 0.000 A x B 8 77.644 9.706 4.28 0.000 A x C 1 211.250 211.250 93.08 0.000 B x C 8 59.400 7.425 3.27 0.002 A x B x C 8 101.400 12.675 5.59 0.000 Error 144 326.800 2.269 Total 179 998.861

Appendix 4.24. ANOVA table for vase life of C. uncinatum ‘Mid Pink’ sprigs pre-treated on day 0 with 10 nL 1-MCP/L or STS (0.5 mM Ag+) for 12 hours at 2 or 20oC. Different sub-samples of sprigs from each of these treatments were then treated daily until day 10 with 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P Temperature (A) 1 1.225 1.225 0.71 0.401 Time of ethylene treatment (B) 7 30.400 4.343 2.51 0.019 Agent - 1-MCP or STS (C) 1 7.225 7.225 4.18 0.043 A x B 7 6.175 0.882 0.51 0.825 A x C 1 8.100 8.100 4.69 0.032 B x C 7 52.175 7.454 4.31 0.000 A x B x C 7 27.900 3.986 2.31 0.030 Error 128 221.200 1.728 Total 159 354.400

Appendix 4.25. ANOVA table for vase life of C. uncinatum ‘Alba’ sprigs pre-treated on day 0 with 10 nL 1-MCP/L or STS (0.5 mM Ag+) for 12 hours at 2 or 20oC. Different sub-samples of sprigs from each of these treatments were then treated daily until day 8 with 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P Temperature (A) 1 2.645 2.645 0.62 0.432 Time of ethylene treatment (B) 9 87.905 9.767 2.29 0.019 Agent - 1-MCP or STS (C) 1 93.845 93.845 22.02 0.000 A x B 9 28.905 3.212 0.75 0.659 A x C 1 83.205 83.205 19.52 0.000 B x C 9 68.705 7.634 1.79 0.074 A x B x C 9 41.545 4.616 1.08 0.378 Error 160 682.000 4.263 Total 199 1088.755

115 Appendix 4.26. ANOVA table for vase solution uptake by C. uncinatum ‘Lollypop’ sprigs pre- treated with 10 nL 1-MCP/L or STS (0.5 mM Ag+) for 12 hours at 2 or 20oC. Different sub-samples of sprigs from each of these treatments were then treated daily until day 9 with 10 µL ethylene/L for 12 hours at 20oC.

Source of variation df SS MS F P Time of ethylene treatment (A) 8 1.12132 0.14016 2.93 0.005 Agent - 1-MCP or STS (B) 1 21.38160 21.38160 447.18 0.000 Temperature (C) 1 1.31512 1.31512 27.50 0.000 Rep (A B C) 144 6.88529 0.04781 5.47 0.000 A x B 8 1.28480 0.16060 3.36 0.001 A x C 8 0.80220 0.10027 2.10 0.040 B x C 1 1.53361 1.53361 32.07 0.000 A x B x C 8 1.90354 0.23794 4.98 0.000 Day (D) 8 50.31703 6.28963 719.06 0.000 A x D 64 4.75692 0.07433 8.50 0.000 B x D 8 0.70690 0.08836 10.10 0.000 C x D 8 0.31651 0.3956 4.52 0.000 A x B x D 64 1.16415 0.01819 2.08 0.000 A x C x D 64 0.61191 0.00956 1.09 0.292 B x C x D 8 1.28136 0.16017 18.31 0.000 A x B x C x D 64 0.65813 0.01028 1.18 0.168 Error 1162 10.07650 0.00875 Total 1619 106.11690

Appendix 4.27. ANOVA table for vase solution uptake by C. uncinatum ‘Alba’ sprigs pre-treated with 10 nL 1-MCP/L or STS (0.5 mM Ag+) for 12 hours at 2 or 20oC. Different sub-samples of sprigs from each of these treatments were then treated daily until day 10 with 10 µL ethylene/L for 12 hours at 20oC.

Source of variation df SS MS F P Time of ethylene treatment (A) 9 1.81399 0.20155 1.62 0.114 Agent - 1-MCP or STS (B) 1 0.43212 0.43212 3.47 0.064 Temperature (C) 1 5.79699 5.79699 46.51 0.000 Rep (A B C) 160 19.94435 0.12465 11.83 0.000 A x B 9 0.76460 0.08496 0.68 0.725 A x C 9 1.58034 0.17559 1.41 0.188 B x C 1 0.69302 0.69302 5.56 0.020 A x B x C 9 1.36267 0.15141 1.21 0.289 Day (D) 9 35.78733 3.97637 377.27 0.000 A x D 81 4.11083 0.05075 4.82 0.000 B x D 9 1.97092 0.21899 20.78 0.000 C x D 9 0.82213 0.09135 8.67 0.000 A x B x D 81 1.95518 0.02414 2.29 0.000 A x C x D 81 0.95858 0.01183 1.12 0.222 B x C x D 9 0.22993 0.02555 2.42 0.010 A x B x C x D 81 1.44872 0.01789 1.70 0.000 Error 1440 15.17749 0.01054 Total 1999 94.84919

116 Appendix 4.28. ANOVA table for vase solution uptake by C. uncinatum ‘Mid Pink’ sprigs pre- treated with 10 nL 1-MCP/L or STS (0.5 mM Ag+) for 12 hours at 2 or 20oC. Different sub-samples of sprigs from each of these treatments were then treated daily until day 8 with 10 µL ethylene/L for 12 hours at 20oC.

Source of variation df SS MS F P Time of ethylene treatment (A) 7 0.95267 0.13610 2.29 0.031 Agent - 1-MCP or STS (B) 1 4.80866 4.80866 80.84 0.000 Temperature (C) 1 2.57648 2.57648 43.81 0.000 Rep (A B C) 128 7.61377 0.05948 7.86 0.000 A x B 7 1.36385 0.19484 3.28 0.003 A x C 7 0.45686 0.06527 1.10 0.369 B x C 1 0.00002 0.00002 0.00 0.985 A x B x C 7 0.22596 0.03228 0.54 0.801 Day (D) 7 36.66761 5.23823 692.20 0.000 A x D 49 4.09401 0.08355 11.04 0.000 B x D 7 1.91722 0.27389 36.19 0.000 C x D 7 0.41008 0.05858 7.74 0.000 A x B x D 49 0.59695 0.01218 1.61 0.006 A x C x D 49 0.34889 0.00712 0.94 0.590 B x C x D 7 0.01714 0.00245 0.32 0.944 A x B x C x D 49 0.27883 0.00569 0.75 0.895 Error 896 6.78047 0.00757 Total 1279 69.10946

Appendix 5.1. ANOVA table for flower abscission from C. uncinatum ‘CWA Pink’ sprigs on bunches pre-treated on day 0 with 200 nL 1-MCP/L, STS (0.2 mM Ag+) or 0 nL 1-MCP/L and 0 mM Ag+ for 6 hours at 20oC. Sprigs from each of these treatments were then exposed on day 0 to 10 µL ethylene/L for 12 hours at 20oC. Flower abscission score data were arcsine transformed for ANOVA.

Source of variation DF SS MS F P Treatment (A) 2 16.61774 8.30887 1516.06 0.000 Rep (A) 27 2.48890 0.09218 16.82 0.000 Day (B) 9 1.67999 0.18667 34.06 0.000 A x B 18 0.87126 0.04840 8.83 0.000 Error 243 1.33178 0.00548 Total 299 22.98968

Appendix 5.2. ANOVA table for relative fresh weight of C. uncinatum ‘CWA Pink’ sprigs on bunches pre-treated on day 0 with 200 nL 1-MCP/L, STS (0.2 mM Ag+) or 0 nL 1-MCP/L and 0 mM Ag+ for 6 hours at 20oC. Sprigs from each of these treatments were then exposed on day 0 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P Treatment (A) 2 8925.93 4462.96 178.33 0.000 Rep (A) 27 5867.48 217.31 8.68 0.000 Day (B) 9 25183.71 2798.19 111.81 0.000 A x B 18 658.47 36.58 1.46 0.105 Error 243 6081.43 25.03 Total 299 46717.02

117 Appendix 5.3. ANOVA table for vase solution uptake by C. uncinatum ‘CWA Pink’ sprigs on bunches pre-treated on day 0 with 200 nL 1-MCP/L, STS (0.2 mM Ag+) or 0 nL 1-MCP/L and 0 mM Ag+ for 6 hours at 20oC. Sprigs from each of these treatments were then exposed on day 0 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P Treatment (A) 2 0.33085 0.16543 14.74 0.000 Rep (A) 27 0.83938 0.03109 2.77 0.000 Day (B) 9 7.50511 0.83390 74.31 0.000 A x B 18 0.42397 0.02355 2.10 0.007 Error 243 2.72704 0.01122 Total 299 11.82635

Appendix 5.4. ANOVA table for vase life of C. uncinatum ‘CWA Pink’ sprigs on bunches pre- treated on day 0 with 200 nL 1-MCP/L, STS (0.2 mM Ag+) or 0 nL 1-MCP/L and 0 mM Ag+ for 6 hours at 20oC. Sprigs from each of these treatments were then exposed on day 0 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P Treatment 2 248.27 124.13 39.15 0.000 Error 27 85.60 3.17 Total 29 333.87

Appendix 5.5. ANOVA table for weight loss from C. uncinatum ‘CWA Pink’ bunches pre-treated on day 0 with 200 nL 1-MCP/L, STS (0.2 mM Ag+) or 0 nL 1-MCP/L and 0 mM Ag+ for 6 hours at 20oC. Bunches were then held inside commercial flower cartons for 6 days at 20oC.

Source of variation DF SS MS F P Pre-treatment 2 417.97 208.98 7.21 0.006 Error 15 435.08 29.01 Total 17 853.05

Appendix 5.6. ANOVA table for relative fresh weight of C. uncinatum ‘CWA Pink’ sprigs on bunches pre-treated on day 0 with 200 nL 1-MCP/L, STS (0.2 mM Ag+) or 0 nL 1-MCP/L and 0 mM Ag+ for 6 hours at 20oC. Bunches were then held inside commercial flower cartons for 6 days at 20oC.

Source of variation DF SS MS F P Pre-treatment (A) 2 3815.54 1907.77 129.24 0.000 Rep (A) 27 9336.46 345.79 23.42 0.000 Day (B) 4 5636.05 1409.01 95.45 0.000 A x B 8 112.27 14.03 0.95 0.479 Error 108 1594.30 14.76 Total 149 20494.61

Appendix 5.7. ANOVA table for solution uptake by C. uncinatum ‘CWA Pink’ sprigs on bunches pre-treated on day 0 with 200 nL 1-MCP/L, STS (0.2 mM Ag+) or 0 nL 1-MCP/L and 0 mM Ag+ for 6 hours at 20oC. Bunches were then held inside commercial flower cartons for 6 days at 20oC.

Source of variation DF SS MS F P Pre-treatment (A) 2 0.072098 0.036049 5.62 0.005 Rep (A) 27 0.389678 0.014433 2.25 0.002 Day (B) 4 1.377567 0.344392 53.71 0.000 A x B 8 0.090946 0.011368 1.77 0.090 Error 108 0.692519 0.006412 Total 149 2.622809

Appendix 5.8. ANOVA table for vase life of C. uncinatum ‘CWA Pink’ sprigs on bunches pre- treated on day 0 with 200 nL 1-MCP/L, STS (0.2 mM Ag+) or 0 nL 1-MCP/L and 0 mM Ag+ for 6 118 hours at 20oC. Bunches were then held inside commercial flower cartons for 6 days at 20oC.

Source of variation DF SS MS F P Pre-treatment 2 40.067 20.033 10.86 0.000 Error 27 49.800 1.844 Total 29 89.867

Appendix 5.9. ANOVA table for relative fresh weight of C. uncinatum ‘Fortune Cookie’ sprigs on bunches pre-treated on day 0 with 200 nL 1-MCP/L, STS (0.2 mM Ag+) or 0 nL 1-MCP/L and 0 mM Ag+ for 14 hours at 2 or 20oC. Sprigs from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P Treatment (A) 2 92355.0 46177.5 2806.89 0.000 Temperature (B) 1 1408.2 1408.2 85.60 0.000 A x B 2 842.8 421.4 25.62 0.000 Rep (A B) 24 10104.5 421.0 25.59 0.000 Day (C) 9 31831.2 3536.8 214.98 0.000 A x C 18 6854.3 380.8 23.15 0.000 B x C 9 454.3 50.5 3.07 0.002 A x B x C 18 207.7 11.5 0.70 0.808 Error 216 3553.5 16.5 Total 299 147611.5

Appendix 5.10. ANOVA table for solution uptake by C. uncinatum ‘Fortune Cookie’ sprigs on bunches pre-treated on day 0 with 200 nL 1-MCP/L, STS (0.2 mM Ag+) or 0 nL 1-MCP/L and 0 mM Ag+ for 14 hours at 2 or 20oC. Sprigs from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P Treatment (A) 2 0.29234 0.14617 2.84 0.061 Temperature (B) 1 0.30728 0.30728 5.96 0.015 A x B 2 0.02030 0.01015 0.20 0.821 Rep (A B) 24 2.15356 0.08973 1.74 0.021 Day (C) 9 40.14767 4.46085 86.54 0.000 A x C 18 5.41051 0.30058 5.83 0.000 B x C 9 0.47184 0.05243 1.02 0.427 A x B x C 18 0.46094 0.02561 0.50 0.958 Error 216 11.13423 0.05155 Total 299 60.39867

Appendix 5.11. ANOVA table for vase life of C. uncinatum ‘Fortune Cookie’ sprigs on bunches pre-treated on day 0 with 200 nL 1-MCP/L, STS (0.2 mM Ag+) or 0 nL 1-MCP/L and 0 mM Ag+ for 14 hours at 2 or 20oC. Sprigs from each of these treatments were then exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P Treatment (A) 2 249.800 124.900 59.48 0.000 Temperature (B) 1 0.000 0.000 0.00 1.000 A x B 2 1.800 0.900 0.43 0.656 Error 24 50.400 2.100 Total 29 302.000

Appendix 5.12. ANOVA table for weight loss from C. uncinatum ‘Fortune Cookie’ bunches pre- treated on day 0 with 200 nL 1-MCP/L, STS (0.2 mM Ag+) or 0 nL 1-MCP/L and 0 mM Ag+ for 14 hours at 2 or 20oC. Bunches were then held inside commercial flower cartons for 6 days at 20oC.

Source of variation DF SS MS F P Pre-treatment (A) 2 16.698 8.349 1.44 0.253 119 Temperature (B) 1 62.647 62.647 10.81 0.003 A x B 2 236.339 118.170 20.39 0.000 Error 30 173.867 5.796 Total 35 489.551

Appendix 5.13. ANOVA table for relative fresh weight of C. uncinatum ‘Fortune Cookie’ sprigs on bunches pre-treated on day 0 with 200 nL 1-MCP/L, STS (0.2 mM Ag+) or 0 nL 1-MCP/L and 0 mM Ag+ for 14 hours at 2 or 20oC. Bunches were then held inside commercial flower cartons for 6 days at 20oC.

Source of variation DF SS MS F P Pre-treatment (A) 2 590.42 295.21 17.16 0.000 Temperature (B) 1 45.27 45.27 2.63 0.108 A x B 2 2914.97 1457.48 84.73 0.000 Rep (A B) 24 18336.39 764.02 44.41 0.000 Day (C) 4 7361.65 1840.41 106.99 0.000 A x C 8 183.65 22.96 1.33 0.236 B x C 4 441.13 110.28 6.41 0.000 A x B x C 8 39.51 4.94 0.29 0.969 Error 96 1651.43 17.20 Total 149 31564.42

Appendix 5.14. ANOVA table for solution uptake by C. uncinatum ‘Fortune Cookie’ sprigs on bunches pre-treated on day 0 with 200 nL 1-MCP/L, STS (0.2 mM Ag+) or 0 nL 1-MCP/L and 0 mM Ag+ for 14 hours at 2 or 20oC. Bunches were then held inside commercial flower cartons for 6 days at 20oC.

Source of variation DF SS MS F P Pre-treatment (A) 2 0.56653 0.28326 45.09 0.000 Temperature (B) 1 0.00546 0.00546 0.87 0.354 A x B 2 0.13475 0.06737 10.73 0.000 Rep (A B) 24 1.12908 0.04704 7.49 0.000 Day (C) 4 3.94153 0.98538 156.86 0.000 A x C 8 0.12936 0.01617 2.57 0.014 B x C 4 0.06893 0.01723 2.74 0.033 A x B x C 8 0.07223 0.00903 1.44 0.191 Error 96 0.60306 0.00628 Total 149 6.65092

Appendix 5.15. ANOVA table for vase life of sprigs from C. uncinatum ‘Fortune Cookie’ bunches pre-treated on day 0 with 200 nL 1-MCP/L, STS (0.2 mM Ag+) or 0 nL 1-MCP/L and 0 mM Ag+ for 14 hours at 2 or 20oC. Bunches were then held inside commercial flower cartons for 6 days at 20oC.

Source of variation DF SS MS F P Pre-treatment (A) 2 6.0667 3.0333 3.31 0.054 Temperature (B) 1 1.2000 1.2000 1.31 0.264 A x B 2 0.6000 0.3000 0.33 0.724 Error 24 22.0000 0.9167 Total 29 29.8667

120 Appendix 5.16. ANOVA table for flower abscission from C. uncinatum ‘Lollypop’ sprigs on bunches pre-treated on day 0 with 0 or 200 nL 1-MCP/L in sealed flower cartons for 24 hours at 2oC. Sprigs from each of these treatments were sampled from 4 positions within cartons and exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Flower abscission score data were arcsine transformed for ANOVA.

Source of variation DF SS MS F P 1-MCP (A) 1 0.085841 0.085841 39.49 0.000 Position (B) 3 0.311065 0.103688 47.70 0.000 A x B 3 0.364608 0.121536 55.91 0.000 Rep (A B) 32 0.626030 0.019563 9.00 0.000 Day (C) 3 0.046461 0.015487 7.12 0.000 A x C 3 0.028614 0.009538 4.39 0.006 B x C 9 0.103688 0.011521 5.30 0.000 A x B x C 9 0.121536 0.013504 6.21 0.000 Error 96 0.208677 0.002174 Total 159 1.896521

Appendix 5.17. ANOVA table for relative fresh weight of C. uncinatum ‘Lollypop’ sprigs on bunches pre-treated on day 0 with 0 or 200 nL 1-MCP/L in sealed flower cartons for 24 hours at 2oC. Sprigs from each of these treatments were sampled from 4 positions within cartons and exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 84.89 84.89 5.51 0.021 Position (B) 3 2159.18 719.73 46.70 0.000 A x B 3 1618.63 539.54 35.01 0.000 Rep (A B) 32 6733.51 210.42 13.65 0.000 Day (C) 3 3737.44 1245.81 80.83 0.000 A x C 3 174.36 58.12 3.77 0.013 B x C 9 296.86 32.98 2.14 0.033 A x B x C 9 525.85 58.43 3.79 0.000 Error 96 1479.57 15.41 Total 159 16810.29

Appendix 5.18. ANOVA table for vase solution uptake by C. uncinatum ‘Lollypop’ sprigs on bunches pre-treated on day 0 with 0 or 200 nL 1-MCP/L in sealed flower cartons for 24 hours at 2oC. Sprigs from each of these treatments were sampled from 4 positions within cartons and exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 0.1512 0.1512 3.59 0.061 Position (B) 3 0.3178 0.1059 2.51 0.063 A x B 3 0.4660 0.1553 3.68 0.015 Rep (A B) 32 1.3696 0.0428 1.01 0.461 Day (C) 3 99.8431 33.2810 788.94 0.000 A x C 3 0.2224 0.0741 1.76 0.161 B x C 9 0.2739 0.0304 0.72 0.688 A x B x C 9 0.6663 0.0740 1.75 0.087 Error 96 4.0497 0.0422 Total 159 107.3601

121 Appendix 5.19. ANOVA table for flower abscission from C. uncinatum ‘Purple Pride’ sprigs on bunches pre-treated on day 0 with 0 or 2 µL 1-MCP/L in sealed flower cartons for 24 hours at 2oC. Sprigs from each of these treatments were sampled from 4 positions within cartons and exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC. Flower abscission score data were arcsine transformed for ANOVA.

Source of variation DF SS MS F P 1-MCP (A) 1 2.42006 2.42006 888.17 0.000 Position (B) 3 0.32405 0.10802 39.64 0.000 A x B 3 1.58031 0.52677 193.33 0.000 Rep (A B) 32 4.69412 0.14669 53.84 0.000 Day (C) 6 0.23516 0.03919 14.38 0.000 A x C 6 0.06949 0.01158 4.25 0.000 B x C 18 0.04396 0.00244 0.90 0.584 A x B x C 18 0.13465 0.00748 2.75 0.000 Error 192 0.52316 0.00272 Total 279 10.02496

Appendix 5.20. ANOVA table for relative fresh weight of C. uncinatum ‘Purple Pride’ sprigs on bunches pre-treated on day 0 with 0 or 2 µL 1-MCP/L in sealed flower cartons for 24 hours at 2oC. Sprigs from each of these treatments were sampled from 4 positions within cartons and exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 4005.11 4005.11 479.51 0.000 Position (B) 3 984.10 328.03 39.27 0.000 A x B 3 7808.11 2602.70 311.61 0.000 Rep (A B) 32 12555.56 392.36 46.98 0.000 Day (C) 6 17906.65 2984.44 357.31 0.000 A x C 6 125.22 20.87 2.50 0.024 B x C 18 439.28 24.40 2.92 0.000 A x B x C 18 338.25 18.79 2.25 0.004 Error 192 1603.69 8.35 Total 279 45765.97

Appendix 5.21. ANOVA table for vase solution uptake by C. uncinatum ‘Purple Pride’ sprigs on bunches pre-treated on day 0 with 0 or 2 µL 1-MCP/L in sealed flower cartons for 24 hours at 2oC. Sprigs from each of these treatments were sampled from 4 positions within cartons and exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 0.000018 0.000018 0.01 0.938 Position (B) 3 0.120206 0.040069 13.60 0.000 A x B 3 0.013253 0.004418 1.50 0.216 Rep (A B) 32 0.438521 0.013704 4.65 0.000 Day (C) 6 1.566605 0.261101 88.65 0.000 A x C 6 0.076975 0.012829 4.36 0.000 B x C 18 0.120193 0.006677 2.27 0.003 A x B x C 18 0.070901 0.003939 1.34 0.168 Error 192 0.565501 0.002945 Total 279 2.972172

122 Appendix 5.22. ANOVA table for vase life of C. uncinatum ‘Purple Pride’ sprigs on bunches pre- treated on day 0 with 0 or 2 µL 1-MCP/L in sealed flower cartons for 24 hours at 2oC. Sprigs from each of these treatments were sampled from 4 positions within cartons and exposed on day 1 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 28.900 28.900 10.80 0.002 Position (B) 3 10.600 3.533 1.32 0.285 A x B 3 42.500 14.167 5.30 0.004 Error 32 85.600 2.675 Total 39 167.600

Appendix 5.23. ANOVA table for flower abscission from C. uncinatum ‘Paddy’s Late’ sprigs on bunches pre-treated on day 0 with 0 or 150 nL 1-MCP/L for 15 hours at 2oC. Half of the sprigs from each of these treatments were then exposed on day 0 to 10 µL ethylene/L for 12 hours at 20oC. Flower abscission score data were arcsine transformed for ANOVA.

Source of variation DF SS MS F P 1-MCP (A) 1 11.21337 11.21337 2591.40 0.000 Ethylene (B) 1 12.63087 12.63087 2918.98 0.000 A x B 1 8.25373 8.25373 1907.43 0.000 Rep (A B) 36 3.19554 0.08877 20.51 0.000 Day (C) 6 0.34577 0.05763 13.32 0.000 A x C 6 0.03826 0.00638 1.47 0.188 B x C 6 0.04166 0.00694 1.60 0.147 A x B x C 6 0.15128 0.02521 5.83 0.000 Error 216 0.93466 0.00433 Total 279 36.80514

Appendix 5.24. ANOVA table for relative fresh weight of C. uncinatum ‘Paddy’s Late’ sprigs on bunches pre-treated on day 0 with 0 or 150 nL 1-MCP/L for 15 hours at 2oC. Half of the sprigs from each of these treatments were then exposed on day 0 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 48961.5 48961.5 2127.87 0.000 Ethylene (B) 1 18598.5 18598.5 808.29 0.000 A x B 1 19223.5 19223.5 835.45 0.000 Rep (A B) 36 31685.1 880.1 38.25 0.000 Day (C) 6 44834.1 7472.4 324.75 0.000 A x C 6 1570.0 261.7 11.37 0.000 B x C 6 3646.0 607.7 26.41 0.000 A x B x C 6 3372.7 562.1 24.43 0.000 Error 216 4970.1 23.0 Total 279 176861.7

123 Appendix 5.25. ANOVA table for vase solution uptake by C. uncinatum ‘Paddy’s Late’ sprigs on bunches pre-treated on day 0 with 0 or 150 nL 1-MCP/L for 15 hours at 2oC. Half of the sprigs from each of these treatments were then exposed on day 0 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 2.32577 2.32577 118.41 0.000 Ethylene (B) 1 0.20430 0.20430 10.40 0.001 A x B 1 0.11606 0.11606 5.91 0.016 Rep (A B) 36 2.91789 0.08105 4.13 0.000 Day (C) 6 5.24303 0.87384 44.49 0.000 A x C 6 1.71561 0.28594 14.56 0.000 B x C 6 0.19786 0.03298 1.68 0.127 A x B x C 6 0.11165 0.01861 0.95 0.462 Error 216 4.24246 0.01964 Total 279 17.07463

Appendix 5.26. ANOVA table for vase life of C. uncinatum ‘Paddy’s Late’ sprigs on bunches pre- treated on day 0 with 0 or 150 nL 1-MCP/L for 15 hours at 2oC. Half of the sprigs from each of these treatments were then exposed on day 0 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 9.452 9.452 7.41 0.010 Ethylene (B) 1 21.674 21.674 16.99 0.000 A x B 1 5.297 5.297 4.15 0.049 Error 36 45.922 1.276 Total 39 82.345

Appendix 5.27. ANOVA table for weight loss from C. uncinatum ‘Paddy’s Late’ bunches pre- treated on day 0 with 0 or 150 nL 1-MCP/L for 15 hours at 2oC. Bunches were then held inside commercial flower cartons for 6 days at 20oC.

Source of variation DF SS MS F P 1-MCP 1 9.86 9.86 0.56 0.464 Error 18 316.90 17.61 Total 19 326.76

Appendix 5.28. ANOVA table for abscised flowers and leaves from C. uncinatum ‘Paddy’s Late’ bunches pre-treated on day 0 with 0 or 150 nL 1-MCP/L for 15 hours at 2oC. Bunches were then held inside commercial flower cartons for 6 days at 20oC.

Source of variation DF SS MS F P 1-MCP 1 0.6502 0.6502 2.09 0.286 Error 2 0.6235 0.3118 Total 3 1.2737

Appendix 5.29. ANOVA table for flower abscission from C. uncinatum ‘Paddy’s Late’ bunches pre-treated on day 0 with 0 or 150 nL 1-MCP/L for 15 hours at 2oC. Bunches were then held inside commercial flower cartons for 6 days at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 0.01091 0.01091 0.43 0.519 Rep (A) 18 0.90827 0.05046 2.00 0.076 Day (B) 1 1.29908 1.29908 51.46 0.000 A x B 1 0.00085 0.00085 0.03 0.857 Error 18 0.45439 0.02524 Total 39 2.67350 Appendix 5.30. ANOVA table for relative fresh weight of C. uncinatum ‘Paddy’s Late’ bunches pre-treated on day 0 with 0 or 150 nL 1-MCP/L for 15 hours at 2oC. Bunches were then held inside commercial flower cartons for 6 days at 20oC. 124 Source of variation DF SS MS F P 1-MCP (A) 1 36.65 36.65 2.22 0.154 Rep (A) 18 6231.70 346.21 20.95 0.000 Day (B) 1 1379.49 1379.49 83.47 0.000 A x B 1 0.00 0.00 0.00 0.988 Error 18 297.49 16.53 Total 39 7945.32

Appendix 5.31. ANOVA table for vase solution uptake by C. uncinatum ‘Paddy’s Late’ bunches pre-treated on day 0 with 0 or 150 nL 1-MCP/L for 15 hours at 2oC. Bunches were then held inside commercial flower cartons for 6 days at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 0.01710 0.01710 1.40 0.252 Rep (A) 18 1.16493 0.06472 5.31 0.000 Day (B) 1 0.70562 0.70562 57.88 0.000 A x B 1 0.01133 0.01133 0.93 0.348 Error 18 0.21943 0.01219 Total 39 2.11841

Appendix 5.32. ANOVA table for vase life of C. uncinatum ‘Paddy’s Late’ bunches pre-treated on day 0 with 0 or 150 nL 1-MCP/L for 15 hours at 2oC. Bunches were then held inside commercial flower cartons for 6 days at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 0.0500 0.0500 0.36 0.556 Error 18 2.5000 0.1389 Total 19 2.5500

125 Appendix 5.33. ANOVA table for flower abscission from C. uncinatum ‘Purple Pride’ sprigs on bunches pre-treated on day 0 with 0 or 200 nL 1-MCP/L for 3 hours at 2oC in buckets of water or in cartons against a forced-air cooler. Half of the sprigs from each of these treatments sampled from 4 positions within cartons or on bunches were then exposed on day 0 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 20.71325 20.71325 2858.98 0.000 Ethylene (B) 1 21.27670 21.27670 2936.75 0.000 Position (C) 3 0.40014 0.13338 18.41 0.000 A x B 1 18.27129 18.27129 2521.93 0.000 A x C 3 0.19607 0.06536 9.02 0.000 B x C 3 0.06526 0.02175 3.00 0.030 A x B x C 3 0.05030 0.01677 2.31 0.075 Rep (A B C) 32 2.00833 0.06276 8.66 0.000 Day (D) 9 7.15053 0.79450 109.66 0.000 A x D 9 5.11952 0.56884 78.51 0.000 B x D 9 5.23844 0.58205 80.34 0.000 C x D 27 0.15053 0.00558 0.77 0.794 A x B x D 9 4.45400 0.49489 68.31 0.000 A x C x D 27 0.11415 0.00423 0.58 0.956 B x C x D 27 0.08063 0.00299 0.41 0.997 A x B x C x D 27 0.07480 0.00277 0.38 0.998 Error 768 5.56414 0.00724 Total 959 90.92808

Appendix 5.34. ANOVA table for relative fresh weight of C. uncinatum ‘Purple Pride’ sprigs on bunches pre-treated on day 0 with 0 or 200 nL 1-MCP/L for 3 hours at 2oC in buckets of water or in cartons against a forced-air cooler. Half of the sprigs from each of these treatments sampled from 4 positions within cartons or on bunches were then exposed on day 0 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 46100.6 46100.6 707.77 0.000 Ethylene (B) 1 69234.5 69234.5 1062.94 0.000 Position (C) 3 2594.2 864.7 13.28 0.000 A x B 1 47883.2 47883.2 735.14 0.000 A x C 3 1467.4 489.1 7.51 0.000 B x C 3 2308.1 769.4 11.81 0.000 A x B x C 3 2379.0 793.0 12.17 0.000 Rep (A B C) 32 19752.5 617.3 9.48 0.000 Day (D) 8 48133.8 6016.7 92.37 0.000 A x D 8 661.1 82.6 1.27 0.257 B x D 8 685.6 85.7 1.32 0.232 C x D 24 804.3 33.5 0.51 0.975 A x B x D 8 813.3 101.7 1.56 0.133 A x C x D 24 665.1 27.7 0.43 0.993 B x C x D 24 36.9 1.5 0.02 1.000 A x B x C x D 24 625.4 26.1 0.40 0.996 Error 688 44812.8 65.1 Total 863 288957.9

126 Appendix 5.35. ANOVA table for vase solution uptake by C. uncinatum ‘Purple Pride’ sprigs on bunches pre-treated on day 0 with 0 or 200 nL 1-MCP/L for 3 hours at 2oC in buckets of water or in cartons against a forced-air cooler. Half of the sprigs from each of these treatments sampled from 4 positions within cartons or on bunches were then exposed on day 0 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 0.17680 0.17680 15.48 0.000 Ethylene (B) 1 0.52569 0.52569 46.02 0.000 Position (C) 3 0.63255 0.21085 18.46 0.000 A x B 1 0.01533 0.01533 1.34 0.247 A x C 3 0.08644 0.02881 2.52 0.057 B x C 3 0.24510 0.08170 7.15 0.000 A x B x C 3 0.11914 0.03971 3.48 0.016 Rep (A B C) 32 1.12292 0.03509 3.07 0.000 Day (D) 8 26.96017 3.37002 295.00 0.000 A x D 8 0.12117 0.01515 1.33 0.227 B x D 8 2.53858 0.31732 27.78 0.000 C x D 24 0.28168 0.01174 1.03 0.427 A x B x D 8 0.02107 0.00263 0.23 0.985 A x C x D 24 0.17129 0.00714 0.62 0.919 B x C x D 24 0.15696 0.00654 0.57 0.951 A x B x C x D 24 0.17494 0.00729 0.64 0.909 Error 688 7.85952 0.01142 Total 863 41.20935

Appendix 5.36. ANOVA table for vase life of C. uncinatum ‘Purple Pride’ sprigs on bunches pre- treated on day 0 with 0 or 200 nL 1-MCP/L for 3 hours at 2oC in buckets of water or in cartons against a forced-air cooler. Half of the sprigs from each of these treatments were then exposed on day 0 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 119.260 119.260 105.39 0.000 Ethylene (B) 1 128.344 128.344 113.42 0.000 Bucket/Carton (C) 1 7.594 7.594 6.71 0.011 A x B 1 142.594 142.594 126.01 0.000 A x C 1 0.010 0.010 0.01 0.924 B x C 1 5.510 5.510 4.87 0.030 A x B x C 1 0.260 0.260 0.23 0.633 Error 88 99.583 1.132 Total 95 503.156

Appendix 5.37. ANOVA table for vase life of C. uncinatum ‘Purple Pride’ sprigs on bunches pre- treated on day 0 with 0 or 200 nL 1-MCP/L for 3 hours at 2oC in buckets of water or in cartons against a forced-air cooler. Half of the sprigs from each of these treatments sampled from 4 positions within cartons or on bunches were then exposed on day 0 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 1 60.750 60.750 45.56 0.000 Ethylene (B) 1 40.333 40.333 30.25 0.000 Position (C) 3 3.417 1.139 0.85 0.475 A x B 1 65.333 65.333 49.00 0.000 A x C 3 1.750 0.583 0.44 0.728 B x C 3 1.167 0.389 0.29 0.831 A x B x C 3 7.833 2.611 1.96 0.140 Error 32 42.667 1.333 Total 47 223.250 Appendix 5.38. ANOVA table for flower abscission from C. uncinatum ‘Alba’ sprigs on bunches pre-treated on day 0 in cartons with no 1-MCP (control), 1, 2 or 3 tubes of 1-MCP gas for 6 days at 127 20oC. Half of the sprigs from each of these treatments sampled from 4 positions within cartons were then exposed on day 6 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 3 12.38681 4.12894 1030.08 0.000 Ethylene (B) 1 0.17897 0.17897 44.65 0.000 Position (C) 3 25.10022 8.36674 2087.32 0.000 A x B 3 3.98533 1.32844 331.42 0.000 A x C 9 11.44005 1.27112 317.12 0.000 B x C 3 0.37479 0.12493 31.17 0.000 A x B x C 9 4.42532 0.49170 122.67 0.000 Rep (A B C) 64 11.12957 0.17390 43.38 0.000 Day (D) 6 4.89576 0.81596 203.56 0.000 A x D 18 1.74876 0.09715 24.24 0.000 B x D 6 0.07713 0.01285 3.21 0.004 C x D 18 4.36811 0.24267 60.54 0.000 A x B x D 18 0.54992 0.03055 7.62 0.000 A x C x D 18 1.66714 0.03087 7.70 0.000 B x C x D 54 0.13708 0.00762 1.90 0.015 A x B x C x D 18 0.69440 0.01286 3.21 0.000 Error 384 1.53921 0.00401 Total 671 84.69857

Appendix 5.39. ANOVA table for relative fresh weight of C. uncinatum ‘Alba’ sprigs on bunches pre-treated on day 0 in cartons with no 1-MCP (control), one tube, two tubes or three tubes of 1- MCP gas for 6 days at 20oC. Half of the sprigs from each of these treatments sampled from 4 positions within cartons were then exposed on day 6 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 3 22522.4 7507.5 605.40 0.000 Ethylene (B) 1 3307.6 3307.6 266.72 0.000 Position (C) 3 65215.8 21738.6 1753.00 0.000 A x B 3 4610.8 1536.9 123.94 0.000 A x C 9 29282.7 3253.6 262.37 0.000 B x C 3 1810.0 603.3 48.65 0.000 A x B x C 9 22444.0 2493.8 201.10 0.000 Rep (A B C) 64 61908.5 967.3 78.00 0.000 Day (D) 5 53445.9 10689.2 861.98 0.000 A x D 15 1116.0 74.4 6.00 0.000 B x D 5 94.9 19.0 1.53 0.180 C x D 15 1563.7 104.2 8.41 0.000 A x B x D 15 192.2 12.8 1.03 0.420 A x C x D 45 1175.8 26.1 2.11 0.000 B x C x D 15 820.3 54.7 4.41 0.000 A x B x C x D 45 998.4 22.2 1.79 0.002 Error 320 3968.3 12.4 Total 575 274477.3

128 Appendix 5.40. ANOVA table for vase solution uptake by C. uncinatum ‘Alba’ sprigs on bunches pre-treated on day 0 in cartons with no 1-MCP (control), one tube, two tubes or three tubes of 1- MCP gas for 6 days at 20oC. Half of the sprigs from each of these treatments sampled from 4 positions within cartons were then exposed on day 6 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 3 0.048191 0.016064 4.45 0.004 Ethylene (B) 1 0.144698 0.144698 40.08 0.000 Position (C) 3 0.090478 0.030159 8.35 0.000 A x B 3 0.024570 0.008190 2.27 0.081 A x C 9 0.103899 0.011544 3.20 0.001 B x C 3 0.516064 0.172021 47.65 0.000 A x B x C 9 0.666172 0.074019 20.50 0.000 Rep (A B C) 64 2.234972 0.034921 9.67 0.000 Day (D) 5 3.006910 0.601382 166.57 0.000 A x D 15 0.047425 0.003162 0.88 0.592 B x D 5 0.027907 0.005581 1.55 0.175 C x D 15 0.070689 0.004713 1.31 0.197 A x B x D 15 0.025689 0.001713 0.47 0.952 A x C x D 45 0.164407 0.003653 1.01 0.456 B x C x D 15 0.176254 0.011750 3.25 0.000 A x B x C x D 45 0.177900 0.003953 1.09 0.322 Error 320 1.155336 0.003610 Total 575 8.681562

Appendix 5.41. ANOVA table for vase life of C. uncinatum ‘Alba’ sprigs on bunches pre-treated on day 0 in cartons with no 1-MCP (control), one tube, two tubes or three tubes of 1-MCP gas for 6 days at 20oC. Half of the sprigs from each of these treatments sampled from 4 positions within cartons were then exposed on day 6 to 10 µL ethylene/L for 12 hours at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 3 20.708 6.903 3.04 0.035 Ethylene (B) 1 10.667 10.667 4.70 0.034 Position (C) 3 11.542 3.847 1.69 0.177 A x B 3 12.250 4.083 1.80 0.156 A x C 9 19.375 2.153 0.95 0.491 B x C 3 3.750 1.250 0.55 0.650 A x B x C 9 46.333 5.148 2.27 0.028 Error 64 145.333 2.271 Total 95 269.958

Appendix 5.41. ANOVA table for weight loss from C. uncinatum ‘Alba’ bunches pre-treated on day 0 in cartons with no 1-MCP (control), one tube, two tubes or three tubes of 1-MCP gas for 6 days at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 3 1146.22 382.07 22.18 0.000 Carton (B) 2 319.15 159.57 9.26 0.000 A x B 6 255.01 42.50 2.47 0.034 Error 60 1033.59 17.23 Total 71 2753.97

129 Appendix 5.42. ANOVA table for abscised flowers and leaves from C. uncinatum ‘Alba’ bunches pre-treated on day 0 in cartons with no 1-MCP (control), one tube, two tubes or three tubes of 1- MCP gas for 6 days at 20oC.

Source of variation DF SS MS F P 1-MCP (A) 3 27.0195 9.0065 22.14 0.000 Error 8 3.2548 0.4069 Total 11 30.2743

130 APPENDIX C SUMMARY TABLE OF PROJECT ACHIEVEMENTS

Milestone Achievement indicator

1. Review literature Review of relevant 1-MCP and ethylene papers (Appendix A).

2. Manufacture and quantification of 1-MCP Gas chromatograms showing 1-MCP (Appendices 2.1 and 2.2).

3. Testing 1-MCP on native cut flowers (conduct Data presented in Chapter 2 showing 1-MCP dosage experiments) dosage (concentration, duration and temperature) effects on Grevillea ‘Sylvia’ inflorescences. Additional data on the effect of temperature on the duration of efficacy of 1-MCP treatment on Grevillea ‘Sylvia’ and Chamelaucium uncinatum (Chapter 4).

4. Testing 1-MCP on native cut flowers (conduct Data presented in Chapter 3 showing 1-MCP and screening experiments on a range of native cut ethylene treatment effects on a variety of native cut flowers) flowers.

5. Design and test commercial application system Data presented in Chapter 5 showing efficacy of for 1-MCP treatment commercial scale application of 1-MCP treatment to Chamelaucium uncinatum flowers.

6. Prepare thesis and publications Presentation and/or preparation of material intended to update research and industry personnel of 1-MCP effects on native cut flowers (see Communications Strategy)

131