Ex Situ Conservation of Australian Citrus Species: Investigations on Seed Biology, Cryopreservation and in Vitro Culture

Author Hamilton, Kim Nicole

Published 2007

Thesis Type Thesis (PhD Doctorate)

School School of Biomolecular and Biomedical Sciences

DOI https://doi.org/10.25904/1912/1593

Downloaded from http://hdl.handle.net/10072/365585

Griffith Research Online https://research-repository.griffith.edu.au

EX SITU CONSERVATION OF AUSTRALIAN CITRUS SPECIES: INVESTIGATIONS ON SEED BIOLOGY, CRYOPRESERVATION AND IN VITRO CULTURE

Kim Hamilton

BAppSc (Hons) MTeach

School of Biomedical and Biomolecular Science Centre for Forestry and Horticultural Research Griffith University, Brisbane, Queensland

A thesis submitted in fulfillment of the requirements of the degree of Doctor of Philosophy

January, 2007

STATEMENT OF ORIGINALITY

This work has not been previously submitted for a degree or diploma in any university. To the best of my knowledge and belief, the thesis contains no material published or written by another person except where due reference is made in the thesis itself.

Kim Hamilton

January 2007

ACKNOWLEDGEMENTS

Firstly, I wish to express my gratitude to all those people who have contributed to the production of this thesis. In particular, I thank my supervisors Dr. Sarah Ashmore, Prof. Rod Drew and Dr. Hugh Pritchard. They have provided invaluable guidance, assistance, encouragement and advice throughout this research work.

I thank all the staff of the Millennium Seed Bank Project (Royal Botanic Gardens, Kew) for training in seed science research and their friendly support. I am appreciative of the training in Scanning Electron Microscopy to Dr. Deb Stenzel (AEMF QUT) and in Differential Scanning Calorimetry to Dr. Chris Wood (Royal Botanic Gardens, Kew), Dr Wayde Martens (QUT) and Dr. Greg Cash (QUT), as well as Dr. Cameron Mc Conchie (CSIRO) for his help in seed oil extraction.

A special thank you to Mr. Malcolm Smith (QDPI Bundaberg) for his assistance, advice and the supply of fruit throughout this project. I also wish to thank Roger Goebel (QDPI), Phil Boyle (Mt Coot-tha Botanic Gardens), Phil Cameron (Mt Coot-tha Botanic Gardens) and John Wrench (QLD Bushfood Association) for also kindly donating fruit.

I gratefully thank Dr. Barbara Reed (USDA), Dr. Florent Engelmann (IPGRI), Dr. Paul Forster (QLD Herbarium), Dr. Sue Lee (Griffith University) and Dr. Jacinta Zalucki (Griffith University) for their helpful advice.

I acknowledge the generous financial support of this PhD provided by the Student Steering Committee of the Millennium Seed Bank Project (Royal Botanic Gardens, Kew), the International Plant Genetic Recourses/Australian Centre for International Agricultural Research, the Centre for Forestry and Horticultural Research and Griffith University.

Finally, I thank Brian Reis for the permission to use copyrighted photos of wild habitats of north Queensland from the Len Webb Ecological Images Collection.

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CONTENTS

ACKNOWLEDGEMENTS ...... III

LIST OF TABLES ...... VII

LIST OF FIGURES...... IX

ABSTRACT ...... XIII

CHAPTER 1.0 INTRODUCTION...... 1

1.1 Background to sustainable conservation for socio-economically valuable biodiversity……………………………………………………………………….2

1.2 Ex situ conservation approaches……..……………………….…………...….9 . 1.3 Priority species…...... 20

1.4 Citrus biodiversity…………………………………………………….…...... 21

. 1.5 Ex situ conservation of Citrus...... 23

1.6 Citrus seed biology and storage……………………………………………..36

1.7 In vitro options in Citrus...... 52

1.8 Summary………………………………………………………………….…58

1.9 Aims and objectives…………………………………………………...….…59

CHAPTER 2.0 COMPARATIVE SEED MORPHOLOGY OF CITRUS AUSTRALASICA, C. INODORA AND C. GARRAWAYI...... …………………………..62

2.1 Introduction ...... 63

2.2 Materials and Method...... 64

2.3 Results ...... 65

2.4 Discussion...... 79

CHAPTER 3.0 EFFECT OF TEMPERATURE ON GERMINATION OF CITRUS AUSTRALASICA, C. INODORA AND C. GARRAWAYI SEEDS...... 89

3.1 Introduction ...... 90

3.2 Materials and Method...... 92

3.3 Results ...... 94

3.4 Discussion...... 104

CHAPTER 4.0 DISTINGUISHING CHARACTERISTICS OF SEED MATURITY IN CITRUS GARRAWAYI IN RELATION TO GERMINABILITY …………………..109 4.1 Introduction ...... 110

4.2 Materials and Method...... 111

4.3 Results ...... 113

4.4 Discussion...... 127

CHAPTER 5.0 DESICCATION AND CRYOPRESERVATION OF SEEDS OF CITRUS AUSTRALASICA, C. INODORA AND C. GARRAWAYI...... 130

5.1 Introduction ...... 131

5.2 Materials and Method...... 133

5.3. Results ...... 137

5.3.1 Effect of desiccation and liquid nitrogen exposure on seed germinability in Citrus australasica...... 137 5.3.2 Effect of desiccation and liquid nitrogen exposure on seed germination in Citrus inodora...... 146

5.3.3 Effect of desiccation and liquid nitrogen exposure on seed germination in Citrus garrawayi, including the effect of seed maturity 151

5.3.4 Analysis of responses to cryopreservation in C. australasica, C. inodora and C. garrawayi and the association with climatic range and seed oil thermal properties of each species...... 162

5.4 Discussion...... 168

CHAPTER 6.0 IN VITRO CULTURE AND CRYOPRESERVATION OF CITRUS AUSTRALASICA, C. INODORA AND C. GARRWAYI ……………………… ...... 183

6.1 Introduction ...... 184

6.2 Materials and Method...... 187

6.3 Results ...... 197

6.3.1 Embryogenesis……………………………………………………197 6.3.2 Micropropagation…………………………………………………220 6.3.3 In vitro seed germination………………………………………….226

6.4 Discussion...... 234

CHAPTER 7.0 GENERAL DISCUSSION ...... 253

7.1 Overview of main findings/outcomes...... 254

7.2 Future research studies ...... 258

7.3 Conservation options in Australian wild Citrus ...... 260

CHAPTER 8.0 REFERENCES...... 265

APPENDIX ...... 302

Appendix 1: Research dissemination……………………………………………....…….303

LIST OF TABLES

Table 1.1 List of Queensland rare and threatened edible plants and/or crop wild relatives.

Table 1.2 Summary of conservation approaches investigated in cultivated Citrus.

Table 1.3 List of major cultivated and wild species of Citrus.

Table 1.4 Ex situ collections of Citrus as listed by Bioveristy International.

Table 1.5 Distribution, habitat and climatic conditions of Australian wild Citrus species, C. australasica, C. inodora and C. garrawayi.

Table 1.6 Summary of reports of effect of desiccation and liquid nitrogen exposure on seed germination of cultivated and wild species of Citrus.

Table 2.1 Characteristics of seed lots harvested between 2004-2006 for Citrus australasica, C. inodora and C. garrawayi.

Table 2.2 Comparative seed characteristics of Citrus australasica, C. inodora and C. garrawayi.

Table 3.1 Effect of temperature on mean percentage on seedling germination in Citrus australasica, C. inodora and C. garrawayi.

Table 3.2 Characteristics of endothermic events in cotyledon tissue and lipid extract of seeds of Citrus australasica, C. inodora and C. garrawayi on warming from low temperatures (-80ºC).

Table 3.3 Climatic data of the natural distribution range during fruiting period in Citrus australasica, C. inodora and C. garrawayi in relation to end temperature of lipid melt.

Table 4.1 Characteristics of fruit of Citrus garrawayi harvested in February 2006 containing seeds of different maturities.

Table 4.2 Characteristics of maturity of seeds in Citrus garrawayi.

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Table 4.3 Seed coat and embryo morphology of seeds at different maturities in Citrus garrawayi.

Table 5.1 Effect of desiccation on mean radicle and epicotyl emergence (±se) and duration until 50% final germination of seed of Citrus garrawayi at different maturities.

Table 5.2 Thermal analysis of lipid endotherm in dry seeds at different maturities in Citrus garrawayi.

Table 5.3 Effect of equilibrium relative humidity (%RH) on seed water activity, melt endotherm presence and thermal characteristics of embryo and seed coat tissue in Citrus garrawayi.

Table 5.4 Comparative germination ±liquid nitrogen (LN) exposure of dry seeds (3%MC) of Citrus australasica, C. inodora and C. garrawayi in relation to seed thermal properties.

Table 6.1 Callus and somatic induction from immature ovules of Australian wild Citrus species and commercial cultivars.

Table 6.2 Effect of medium composition on somatic embryo formation in seed of Citrus australasica, C. inodora and C. garrawayi.

Table 6.3 Effect of medium composition (solid) on somatic embryo formation from callus initiates (liquid cultured) of Citrus inodora.

Table 6.4 Cryopreservation of shoot tips of C. australasica using a vitrification technique.

Table 6.5 Characteristics of seed lots of Citrus inodora and C. garrawayi.

Table 6.6 Effect of desiccation level on thermal events during cooling (-80ºC) and warming of desiccated seeds (near mature) of Citrus garrawayi.

Table 7.1 Summary of in situ and ex situ conservation strategies for C. australasica, C. inodora and C. garrawayi.

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LIST OF FIGURES Fig. 1.1 Rare and threatened native fruits of subtropical and tropical rainforests of Queensland.

Fig. 1.2 Citrus australasica tree growing in cultivation and whole fruit from a commercial harvest.

Fig. 1.3 Rainforest types of north Queensland in which Citrus inodora (Russell River lime) and C. garrawayi (Mt White lime) grow.

Fig. 1.4 Citrus inodora leaves and fruit growing on trees of a research collection of the Queensland Department of Primary Industries.

Fig. 1.5 Citrus garrawayi tree and fruit growing on tree at the research collection of the Queensland Department of Primary Industries.

Fig. 1.6 Fruiting trees of cultivated Citrus species growing at the Brisbane Botanic Gardens.

Fig. 1.7 Fruiting period of Australian wild Citrus species in relation to season for northern (C. inodora and C. garrawayi) and southern (C. australasica) distributed species.

Fig. 2.1 Whole fruits of Citrus australasica, C. inodora and C. garrawayi.

Fig. 2.2 Whole cut fruit of Citrus australasica, C. inodora and C. garrawayi.

Fig. 2.3 Whole seed of Citrus australasica, C. inodora and C. garrawayi.

Fig. 2.4 Scanning electron micrographs (x50) of seed Citrus australasica, C. inodora C. garrawayi and cultivated species, C. limon.

Fig. 2.5 Scanning electron micrographs (x200 – x1600) of surface topography of seed coats of Citrus australasica, C. inodora and C. garrawayi.

Fig. 2.6 Seed anatomy of longitudinally sectioned seeds, as viewed by light microscopy, illustrating seed coat layers in Citrus australasica, C. inodora and C. garrawayi.

Fig. 2.7 Scanning electron micrographs (x400) illustrating anatomical features of sectioned seed coats of Citrus australasica, C. inodora, C. garrawayi and cultivated mandarin.

Fig. 2.8 Scanning electron micrographs illustrating detail of structures of seed integuments.

Fig. 2.9 Embryonic axis morphology, as viewed by light microscopy, of three Australian Citrus species.

Fig. 2.10 Scanning electron micrographs (x200 - x6000) of cotyledon cells of seed illustrating storage bodies.

Fig. 3.1 Mean monthly maximum and minimum temperatures for distribution region of Citrus australasica, C. inodora and C. garrawayi.

Fig. 3.2 Effect of temperature on radicle and epicotyl emergence from seed incubation for two, four and eight weeks in Citrus australasica, C. inodora and C. garrawayi.

Fig. 3.3 Effect of temperature on mean radicle and epicotyl length (mm) of germinated seeds of Citrus australasica, C. inodora and C. garrawayi.

Fig. 3.4 Representative differential scanning calorimetric thermocurves of transition events in cotyledon tissue and lipid extract of seeds of Citrus inodora and C. garrawayi, on warming from ultra low temperatures.

Fig. 3.5 Comparative thermocurves of in vivo lipid phase transition events in cotyledon tissue of Citrus australasica, C. inodora and C. garrawayi on warming from ultra low temperatures .

Fig. 4.1 Citrus garrawayi at field location during time of harvest showing fruiting and flowering at the same time.

Fig. 4.2 Embryo and longitudinal section of seed coat of mature seed Citrus garrawayi.

Fig. 4.3 Typical morphology of seed coat and embryo of seeds of Citrus garrawayi at different maturities.

Fig. 4.4. Scanning electron micrographs illustrating the development of the radicle and shoot primordia in embryonic axes of Citrus garrawayi.

Fig. 4.5 Scanning electron micrographs of transverse seed coat sections of Citrus garrawayi illustrating the development of epidermal fibres.

Fig. 4.6 Seed coat morphology of Citrus garrawayi seeds at different maturities.

Fig. 4.7 Distinguishing characteristics of mature seed in Citrus garrawayi, as viewed by scanning electron microscopy.

Fig. 4.8 Effect of seed maturity on radicle and epicotyl emergence (>1mm) in Citrus garrawayi.

Fig. 4.9 Effect of seed maturity on mean radicle and epicotyl length in Citrus garrawayi.

Fig. 5.1 Mean moisture content of desiccated seeds of Citrus australasica.

Fig. 5.2 Effect of desiccation and liquid nitrogen exposure on seed germination (>1mm radicle emergence) in Citrus australasica.

Fig. 5.3 Germination post cryopreservation in Citrus australasica seeds at different moisture contents.

Fig. 5.4 Effect of desiccation and liquid nitrogen exposure on seeds of Citrus australasica mean radicle and epicotyl length (mm).

Fig. 5.5 Effect of desiccation and liquid nitrogen exposure on radicle and epicotyl emergence (>1mm) from Citrus australasica seeds after 14 and 60 days incubation.

Fig. 5.6 Representative differential scanning calorimetric thermocurves of melt transition events in cotyledon tissue of Citrus australasica after desiccation of intact seeds to various mean % moisture contents.

Fig. 5.7 Enthalpy of melt transition in Citrus australasica cotyledon tissue at different water contents on warming from low temperatures (-80ºC).

Fig. 5.8 Melt transition events in cotyledon tissue of Citrus australasica at different moisture contents (MC) achieved by silica drying of intact seeds for 18 hours.

Fig. 5.9 Mean % moisture content of seeds of desiccated over silica or in a controlled relative humidity (RH) cabinet (15%RH).

Fig. 5.10 Effect of desiccation and liquid nitrogen exposure on radicle and epicotyl emergence (>1mm) in Citrus inodora.

Fig. 5.11 Effect of desiccation and liquid nitrogen exposure on mean radicle and epicotyl length (mm) of seeds of Citrus inodora.

Fig. 5.12 Effect of desiccation and liquid nitrogen exposure on mean radicle and epicotyl emergence of Citrus inodora seeds after 14, 30 and 60 days incubation.

Fig. 5.13 Mean moisture content of embryo, seed coat and whole seed of Citrus garrawayi seeds at different maturities.

Fig. 5.14 Effect of seed maturity in Citrus garrawayi on moisture content after silica- drying.

Fig. 5.15 Effect of desiccation and liquid nitrogen exposure on radicle emergence and epicotyl emergence (>1mm) from premature and mature seeds of Citrus garrawayi.

Fig. 5.16 Effect of desiccation and liquid nitrogen exposure on mean radicle and epicotyl length (mm) from immature, premature and mature seeds of Citrus garrawayi.

Fig. 5.17 Enthalpy of phase transition in Citrus garrawayi cotyledon tissue at different moisture contents (%wb) on warming from low temperatures (-100ºC).

Fig. 5.18 Mean monthly rainfall of natural distributions of Citrus australasica, C. inodora and C. garrawayi.

Fig. 5.19 Representative differential scanning calorimetric thermocurves of transition events in cotyledon tissue and lipid extract of seeds of Citrus garrawayi and C. inodora on cooling and warming from ultra low temperatures ( -80ºC).

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Fig. 5.20 Comparative thermocurves of in vivo lipid phase transition events in cotyledon tissue of Citrus australasica, C. inodora and C. garrawayi on cooling and warming from ultra low temperatures (-80ºC).

Fig. 6.1 Immature fruits of cultivated and Australian wild Citrus species.

Fig. 6.2 Somatic embryos and embryogenic callus of the mandarin cultivars of Murcott and Orlando and the Australian wild Citrus species, C. inodora.

Fig. 6.3 Somatic embryo multiplication and germination in C. inodora.

Fig 6.4 Effect of media composition on survival and callus production of ovules from immature fruit of Tahitian lime and Citrus garrawayi cultured in vitro.

Fig. 6.5 Somatic embryo formation, multiplication and germination from seed derived embryogenic callus ±liquid nitrogen exposure in Citrus inodora.

Fig. 6.6 Premature fruits, whole seed and sectioned seed of C. australasica.

Fig. 6.7 Effect of medium composition on callus formation from premature seeds of Citrus australasica.

Fig. 6.8 Callus formation from premature seeds of Citrus australasica cultured on basal medium plus the inclusion of various additives.

Fig. 6.9 Comparative somatic and zygotic embryo development in Australian wild Citrus.

Fig. 6.10 Multiplication of seed derived callus by liquid culture.

Fig. 6.11 Embryogenic callus/cell clusters and differentiated globular structures (proembryos) observed from liquid cultured callus of Citrus inodora, as viewed by light and scanning electron microscopy.

Fig. 6.12 Callus initiates from liquid culture of Citrus inodora plated onto solid basal medium and various additives to induce callus multiplication and somatic embryo formation.

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Fig. 6.14 Callus and embryo formation from callus initiates from liquid cultured of Citrus inodora plated onto solid basal medium modified by the inclusion of elevated malt (1250mgl-1) and citric acid (250mgl-1).

Fig. 6.15 Effect of citric acid concentration on callus formation (multiplication) from callus initiates (liquid cultured) of Citrus inodora.

Fig. 6.16 Morphologically abnormal and normal asynchronous somatic embryo formation in Citrus inodora.

Fig. 6.17 Callus, globular proembryos and somatic embryos forming on solid basal medium in Citrus inodora and a mandarin cultivar (Murcott).

Fig. 6.18 Percentage callus formation (multiplication) of plated callus initiates of Citrus inodora after liquid culture in basal medium containing various concentrations of citric acid.

Fig. 6.19 Effect of pretreatment and desiccation over silica gel on moisture content of alginate beads.

Fig. 6.20 Effect of desiccation and liquid nitrogen exposure on growth of encapsulated callus of Citrus inodora.

Fig. 6.22 Callus growth (formation) and somatic embryo formation of alginate encapsulated callus of Citrus inodora.

Fig. 6.23 In vitro propagation of nodal cuttings in C. australasica, C. inodora and C. garrawayi.

Fig. 6.24 In vitro growth of nodal cutting of Citrus inodora at 10 month post initiation.

Fig. 6.25 Effect of nodal cuttings preculture on mean (±s.e.) percentage shoot-tip survival and growth in Citrus australasica.

Fig. 6.26 Shoot growth of excised shoot-tip after preculture of nodal cuttings on different sucrose concentrations.

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Fig. 6.27 Growth of shoot-tips of Citrus australasica after application of plant vitrification solution and liquid nitrogen exposure.

Fig. 6.28 Fruit and near mature seeds of Citrus garrawayi.

Fig. 6.29. Effect of desiccation and liquid nitrogen exposure on mean % germination (>5mm) in Citrus inodora.

Fig. 6.30. Effect of desiccation and liquid nitrogen exposure on root and epicotyl length in Citrus inodora.

Fig. 6.31 Seed moisture content of near mature seeds of Citrus garrawayi after drying over silica gel.

Fig. 6.32 Effect of desiccation and liquid nitrogen exposure on germination of Citrus garrawayi.

Fig. 6.33 Effect of desiccation and liquid nitrogen exposure (-196ºC) on epicotyl and root length of germinated seeds of Citrus garrawayi.

Fig. 6.34 Healthy acclimatised Citrus inodora and C. garrawayi seedlings from in vitro cultured seeds ± liquid nitrogen exposure.

Fig. 6.35 Representative cooling thermograms of seeds (near mature) of Citrus garrawayi after desiccation to various moisture contents.

ABSTRACT Many potentially economically important taxa of Australia are threatened in situ and are vulnerable to erosion of genetic diversity and extinction. In this study, over one hundred rare and threatened Queensland edible plants and/or crop wild relatives were identified.

Many of these species have subtropical to tropical distribution and may have non-orthodox seed storage behaviour, thus excluding standard seed banking approaches for long-term ex situ conservation. There is an urgent need to develop alternative ex situ conservation strategies to conserve this diversity. Establishment of ex-situ collections of this valuable germplasm in field collections would be prohibitive in cost and would be susceptible to environmental damage, including disease and pest attack. In vitro and cryopreservation techniques offer alternative strategies for medium and long-term storage of germplasm.

However, there have been very few attempts to apply in vitro storage and cryopreservation techniques to any wild Australian tropical or subtropical species. Moreover, limitations exist for the development of alternative ex situ storage techniques due to a lack of basic research on plant ecology or biology, including seed physiology and morphology. Further restrictions to the development of ex situ conservation of these species occurs because of a lack of supporting techniques needed for cryostorage, such as in vitro culture, germination protocols, propagation and acclimation.

The Australian wild species of the Citrus genus are a priority for investigation of ex situ conservation strategies because of their conservation priority, potential socioeconomic importance (e.g. novel genes and fruits), probable non-orthodox seed storage behaviour and lack of corresponding techniques for their long term ex situ conservation. This study reports on seed biology, cryopreservation and in vitro culture of three Australian wild Citrus species, C. australasica (finger lime), C. inodora (Russell River lime) and C. garrawayi

(Mount White lime), to facilitate germplasm storage and as a regeneration system.

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Descriptors of mature seed morphology and anatomy are reported in

C. australasica, C. inodora and C. garrawayi - vital to the development and application of effective seed storage protocols (i.e. seed lot quality). C. garrawayi seed shape and seed coat morphology was found to be different to C. australasica and C. inodora. In addition, seed topography, as viewed by scanning electron microscopy, was found to be a useful tool for taxa identification.

In terms of both germination and seedling growth, temperatures of 20ºC were found to be sub-optimal for germination. Germination from seeds of all the three species was optimal at 30ºC and above 80%. Both C. australasica and C. inodora displayed

‘essentially’ orthodox seed storage behaviour, i.e. desiccation and cryopreservation tolerance, whilst C. garrawayi displayed more complex seed storage behaviour. Overall, it appears that seed banking of germplasm of these species could be by standard orthodox protocols (i.e. 5%MC at -20ºC). However, because of variation in seed responses and other storage constraints in these species, cryopreservation is recommended as the safest storage option to prevent seed deterioration (loss of viability).

Cryobiology studies have allowed the determination of the unfrozen water content

(WCu) of C .australasica (11%), C. inodora (est. 8%MC) and C. garrawayi (14%).

Desiccation of seeds to well below the WCu resulted in high levels of germination (radicle emergence <1mm) after liquid nitrogen exposure, but negligible levels of germination were observed from seeds above the WCu. This data both supports other studies undertaken on cultivated citrus and provides evidence that seed of these species will tolerate cryostorage when below the WCu.

In addition, this study demonstrated in vitro culture systems for the micropropagation and medium term storage of C. australasica, C. inodora and

C. garrawayi, as well as shoot-tip cryopreservation in C. australasica using a vitrification- xvii

based method. In vitro embryogenic potential, using a range of culture media, was low to moderate in C inodora and low in C. australasica, whilst C. garrawayi was recalcitrant to in vitro embryogenesis. The addition of citric acid to the embryo induction medium resulted in the best quality and highest number of somatic embryos from callus proliferated through liquid culture in C. inodora. This is the first report of the promotive affect of citric acid on embryo formation in the Citrus genus. Cryopreservation of encapsulated C. inodora embryogenic callus gave high levels of recovery (69%). However, further optimisation of embryo formation and plantlet recovery is needed to improve efficiency to be suitable conservation purposes. Micropropagation provides a useful tool, for medium-term storage of rare and threatened germplasm and offers a valuable step in the implementation of horticultural and restoration programs. Establishment of an in vitro culture system for shoot-tips also provides a technique for producing virus free material for germplasm exchange or maintenance.

The findings of this study facilitate the development of ex situ conservation of

Australian wild Citrus, which is of significant interest to complement in situ conservation and secure sustainable access to this rich biodiversity.

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CHAPTER 1 INTRODUCTION

1 CHAPTER 1 Introduction

1.1 Background to conservation of socio-economically valuable biodiversity

Paul Ehrlich, respected ecologist, argues that the human population has reached its present size by exploiting, degrading, and depleting the Earth’s fossil fuels, minerals, soil, water and biological diversity. ‘It is the loss of biological diversity,’ he argues, ‘that may prove the most serious’ (Owen 1985).

The Convention on Biological Diversity (CBD) (www.biodiv.org) was ratified by

Australia on the 18th June 1993. The convention was in a response to an increasing rate of species extinction and genetic erosion worldwide. The main objectives are to conserve, sustainably use and justly share the benefits of biological diversity. A decade later, the

Global Strategy for Plant Conservation (GSPC) was developed to meet the challenge of preventing the loss of plant diversity. The five objectives of the GSPC are (a) understanding and documenting plant diversity, (b) conserving plant diversity, (c) using plant diversity, (d) promoting education and awareness and (e) building capacity for the conservation of plant diversity (CDB 2002).

To address these GSPC objectives, sixteen targets were set. Examples of targets under this objective of conserving plant diversity are target 7, 60 per cent of the world’s threatened species conserved in situ; target 8, 60 per cent of threatened plant species in accessible ex situ collections, preferably in the country of origin, and 10 percent of them included in recovery and restoration programs and target 9 70 per cent of the genetic diversity of crops and other major socio-economically valuable plant species conserved, and associated local indigenous knowledge maintained (CBD 2002).

The GSPC targets prioritise the conservation of threatened and/or crop plant diversity both in situ and ex situ. Nearly 20% of the plants presumed extinct worldwide come from Australia (Briggs and Leigh 1996). Additionally, 5031 Australian taxa are listed

2 as rare or threatened and they are mostly endemic (94.6%). In order to address GSPC targets, identification and ex situ germplasm collections of many of Australia's unique edible species and wild relatives of crop species listed as rare or threatened in wild populations is urgently required.

In Australia, there has been recognition of the need for ex situ conservation as an insurance against unpredictable events (Armstrong 1991, Australian Government 1996

Cochrane 2004, Ashmore and Drew 2005). Internationally, the International Plant Genetic

Resources Institute (IPGRI, recently renamed Bioversity) is an international network mandated to support conservation and use of plant biodiversity. Examples of Australian individuals, networks and organisations involved in plant biodiversity conservation (in situ and ex situ) include, land owners, Australian Network for Plant Conservation, local and national non government environment groups (e.g. Greening Australia), mining companies, governmental organizations (e.g. Environment Australia, Department of Environment and

Heritage, State Environment Protection Agencies and Parks and Wildlife) as well as

Botanic Gardens, Department of Primary Industries, CSIRO, universities and local councils.

Importance of Crop Wild Relatives (CRW)

As early as 1926, Vavilov recognised the value and raised early concerns of the loss of agricultural biodiversity in crop plants (Maunder et al 2004). Genetic erosion is an increasingly important issue for agricultural plant species (Pence 1995). Thus, crop wild relatives (CWR) are of interest as a source of useful traits for introduction into food crops, medicinal and forestry purposes. Examples of CRWs that have been investigated as a source genetic diversity are Lycopersicon (Tomato, Tanksley and McCouch 1997), Cornus mas (Cornelian cherry, Brindza et al 2005), Oryza (rice, Tanksley and McCouch 1997),

Hordeum (barley, Scholz et al 2005), Triticum (wheat, Millet et al 2005), Mangifera

3 (mango, Campbell 2005), Olea (olives, Cultrera et al 2005), as well as Australian indigenous species of Glycine (soybean, Brown and Brubaker 2000), Gossypium (cotton,

Brown and Brubaker 2000), Sorghum (Dillon and Lawrence 2002) and Citrus (Skyes

1997).

The first domestication of food crops was 10,000 years ago. Cultivation of these wild species over time has led to a progressive narrowing of the genetic base (genetic bottleneck) in crop plants, for example in soybean and wheat (Tanksley and McCouch

1997). In general, cultivated crops contain fewer than 10% of the total available genetic diversity, with the majority of diversity found in related wild species (Westwood 1989,

Reed et al 2004). A comparison of isozyme polymorphisms between ten crop species and their wild relatives revealed a higher variability in wild species, which had a larger and/or different spectra of alleles (Brown 1989). Therefore, attention is shifting to the wild relatives to further enlarge the genetic base available to the breeder (Brown 1989). CWR are an important resource of unique and potential agronomically useful alleles (Tanksley and McCouch, 1997). Thus, conservation of diverse wild germplasm for breeding of current and future food species is important. A growing recognition of these CWRs, as a vital source of genetic diversity for long-term security of economically important species, was demonstrated by the strong attendance (45 countries) at the First International

Conference on Crop Wild Relative Conservation and Use held in Italy, 2005 and the development of a Global Strategy for CWR conservation and use

(www.pgrforum.org/conference).

Queensland rare and threatened edible plants and CWR biodiversity

Australian native plants have been a food source for indigenous people for at least 40 000 years (Roberts et al 1990, Ahmed and Johnson 2000). Furthermore, Australia’s resource of

4 hundreds of edible native plants is now being recognised by an increasingly popular bush food industry, which has an annual value estimated at $10-12 million dollars (Ahmed and

Johnson 2000). Consequently, long-term ex-situ conservation of germplasm of these unique

CWR and/or edible plants is of significant interest to complement in situ conservation and secure sustainable access for breeding programs, or in the development of bushfood or new crop industries. In addition, Australia has CRWs of many worldwide economically important species. For example, Queensland has indigenous species of Oryza (rice),

Sorghum and Citrus (Henderson 2002).

Prioritisation for the ex situ conservation of germplasm needs to consider not only potential socio-economic importance, but also current conservation status in situ, i.e. vulnerability to genetic erosion. The highest concentrations of threatened and poorly known taxa of Australia occur in northern Queensland and southern Western Australia. Of the total rare and threatened taxa in Australia, 1368 (i.e. 27.2%) occur in Queensland (Briggs and

Leigh 1996). A review, undertaken as part of this PhD project, of CWRs and edible plants in Queensland reveals that over one hundred are rare or threatened (Table 1.1).

Table 1.1 includes many socio-economically important species and CWRs that are listed as rare (R), vulnerable (V), endangered (E) and extinct (X) in situ. For example,

Diploglottis (3 species), Citrus inodora (V), C. garrawayi (R) (previously Microcitrus),

Garcinia brassii (R), Ipomoea (3 species), Piper mestonii (R), Syzygium (10 species),

Macadamia (6 species), Musa fitzalanii (X) and M. jackeyi (R). These rare and threatened species are predominantly of subtropical and tropical distribution and examples of plants growing either in cultivation or in the wild are given Figure 1.1. Conservation studies are urgently needed as the development and implementation of conservation strategies, both in situ and ex situ, are hindered by a lack of knowledge of these species’ ecology and reproductive biology.

5 Table 1.1 Queensland rare and threatenedA edible plantsB and/or crop wild relatives Family Species Common name STATUS Anacardiaceae Buchanania mangoides R Apiaceae Trachymene geraniifolia R Apiaceae Trachymene glandulosa R Arecaceae Archontophoenix myolensis E Arecaceae Calamus aruensis R Arecaceae Calamus warburgii V Arecaceae Livistona drudei V Arecaceae Livistona fulva R Arecaceae Livistona lanuginosa V Capparaceae Capparis humistrata Australian caper E Capparaceae Capparis sp. (Gloucester Island) Australian caper R Capparaceae Capparis thozetiana Australian caper V Clusiaceae Garcinia brassii R Commelinaceae Cartonema brachyantherum R Convolvulaceae Ipomoea antonschmidii Bush potato R Convolvulaceae Ipomoea saintronanensis Bush potato R Convolvulaceae Ipomoea stolonifera Bush potato R Cyatheaceae Cyathea baileyana Tree fern R Cyatheaceae Cyathea celebica Tree fern R Cyatheaceae Cyathea cunninghamii Tree fern R Cyatheaceae Cyathea exilis Tree fern E Cyatheaceae Cyathea felina Tree fern E Cycadaceae Cycas brunnea Cycad R Cycadaceae Cycas cairnsiana Cycad V Cycadaceae Cycas couttsiana Cycad R Cycadaceae Cycas desolata Cycad V Cycadaceae Cycas media subsp.ensata Cycad R Cycadaceae Cycas megacarpa Cycad E Cycadaceae Cycas ophiolitica Cycad E Cycadaceae Cycas platyphylla Cycad V Cycadaceae Cycas semota Cycad V Cycadaceae Cycas silvestris Cycad V Cycadaceae Cycas tuckeri Cycad V Davidsoniaceae Davidsonia johnsonii E Elaeocarpaceae Elaeocarpus coorangooloo R

6 Family Species Common name STATUS Elaeocarpaceae Elaeocarpus johnsonii Johnson’s Quandong R Elaeocarpaceae Elaeocarpus stellaris R Elaeocarpaceae Elaeocarpus thelmae R Epacridaceae Acrotriche baileyana Heath R Epacridaceae Leucopogon grandiflorus Heath R Epacridaceae Leucopogon cicatricatus Heath R

Epacridaceae Leucopogon malayanus subsp. Heath R novoguineensis Epacridaceae Leucopogon recurvisepalus Heath E Moraceae Ficus melinocarpa R Musaceae Musa fitzalanii X Musaceae Musa jackeyi R Myrtaceae Syzygium aquem R Myrtaceae Syzygium argyropedicum R Myrtaceae Syzygium buettnerianum R Myrtaceae Syzygium hodgkinsoniae Lilly pilly V Myrtaceae Syzygium moorei Rose apple V Myrtaceae Syzygium macilwraithianum R Myrtaceae Syzygium malaccense R Myrtaceae Syzygium pseudofastigiatum R Myrtaceae Syzygium rubrimolle R Myrtaceae Syzygium velarum V Orchidaceae Diuris oporina Ground orchid R Orchidaceae Diuris parvipetala Ground orchid R Orchidaceae Gastrodia crebriflora Ground orchid R Orchidaceae Gastrodia queenslandica Ground orchid R Orchidaceae Gastrodia urceolata Ground orchid R Pandanaceae Pandanus gemmifer Pandanus R Pandanaceae Pandanus zea Pandanus R Phormiaceae Dianella fruticans Flax lily R Phormiaceae Dianella incollata Flax lily R Piperaceae Piper mestonii R Poaceae Panicum chillagoanum Native millet R Proteaceae Banksia conferta V Proteaceae Banksia plagiocarpa R Proteaceae Hicksbeachia pinnatifolia Monkey/ Red bopple nut V

7 Family Species Common name STATUS Proteaceae Macadamia claudiensis V Proteaceae Macadamia grandis R Proteaceae Macadamia integrifolia Macadamia nut V Proteaceae Macadamia jansenii Bulberin nut E Proteaceae Macadamia ternifolia Small fruited QLD nut V Proteaceae Macadamia tetraphylla Rough shelled bush nut V Proteaceae Persoonia amaliae Geebung R Proteaceae Persoonia daphnoides Geebung R Proteaceae Persoonia prostrata Geebung X Proteaceae Persoonia volcanica Geebung R Rutaceae Acronychia acuminata R Rutaceae Acronychia baeuerlenii R Rutaceae Acronychia eungellensis R Rutaceae Acronychia littoralis E Rutaceae Citrus garrawayi Mount White lime R Rutaceae Citrus inodora Russell River lime V Sapindaceae Diploglottis campbellii Small - leaved tamarind E Sapindaceae Diploglottis harpullioides R Sapindaceae Diploglottis pedleyi R Zamiaceae Macrozamia cardiacensis R Zamiaceae Macrozamia conferta V Zamiaceae Macrozamia cranei E Zamiaceae Macrozamia crassifolia V Zamiaceae Macrozamia fearnsidei V Zamiaceae Macrozamia lomandroides E Zamiaceae Macrozamia longispina R Zamiaceae Macrozamia machinii V Zamiaceae Macrozamia occidua V Zamiaceae Macrozamia parcifolia V Zamiaceae Macrozamia pauli-guilielmi Pineapple zamia E Zamiaceae Macrozamia platyrhachis E Zamiaceae Macrozamia viridis E Zingiberaceae Alpinia hylandii Native ginger R ASpecies conservation status sourced from Henderson (2002): X Extinct: E Endangered: R Rare: V Vulnerable. BEdibile plants were referenced from several sources: Cribb and Cribb 1974, Low 1991, Cooper and Cooper 2004.

8 A B

Fig.C 1.1 Rare and threatened native fruits of subtropical and tropical rainforests of Queenland. (A) Diploglottis campbellii (Small-leaved tamarind) shown in cultivation at the Brisbane Botanic Gardens (Mt Cootha, 2004). (B) B Wild banana (Musa) growing in native habitat of Bloomfield River (Cape York Peninsula, 1969). (C) Piper mestonii fruit growing on plant in wet tropical lowland rainforest (Cucania near Babinda, 1959). Photographs (B) and

(C) are reproduced with the kind permission from the Len Webb Ecological Images Collection (www.griffith.edu.au/ins/collections/webb).

1.2 Conservation approaches

Complementary to in situ conservation, ex situ storage is an important component of conservation of genetic diversity of threatened species (Pence 1995). Ex situ conservation reduces the risk of total loss of genetic diversity if a threatened species becomes extinct in the wild. The aim is to maintain germplasm in a state suitable for re-establishing self- sustaining wild populations if/when a suitable site exists (Touchell et al. 1997). Germplasm

9 may also be used in recovery programs where genetic erosion of a species occurs (e.g. in breeding – fragmented populations) from factors such as climate, disease or habitat loss.

Conservation of novel and diverse germplasm is also crucial for the breeding of current and future economically important species, particularly food crops.

An ex situ collection of a species should reflect the genetic diversity in situ and needs an understanding of the evolutionary and ecological processes leading to variation, for effective maintenance of diversity (Cochrane 2004). This diversity is best conserved by utilising strategies that include a combination of in situ and ex situ approaches. These could include:

Seed bank (seeds stored at low temperature and moisture content) Cryopreservation (seeds, pollen and plant tissue stored in liquid nitrogen) In vitro culture (using somatic tissue or seed explants) Cultivation in a dedicated conservation facility (e.g. glasshouse) Cultivation in a display or reference collections (e.g. botanic gardens) Field gene bank Commercial cultivation Community gardens (e.g. traditional agricultural plants and medicinal plants) Horticultural cultivation in near natural conditions (inter situ) Horticulturally managed in situ wild population (e.g. hand pollination of wild orchids) In situ (managed and unmanaged habitat of wild plants) Maunder et al (2004)

Ex situ conservation of germplasm is particularly appropriate for the conservation of crops and their wild relatives (Englemann and Engels 2002). Worldwide, based on the State of the World Report (FOA 1996), agricultural germplasm banks held approximately 6 million accessions (cultivated and to lesser extent wild species) in 1996, with the majority

(60%) stored at low temperature or very low temperatures and about 10% maintained in field collections, in vitro or cryopreservation (Engelmann and Engels 2002). Collections of germplasm of some Australian CWRs are held in institutions both internationally (e.g., US 10 Department of Agriculture (USDA) and nationally. These Australian collections include the

Indigenous Crop Relatives Collection (Commonwealth Scientific and Industrial Research

Organisation (CSIRO), ACT), Tropical Crops and Forage Collection (QLD Department of

Primary Industries (QDPI), QLD), Bundaberg Research Station (QDPI, QLD) and CSIRO

Division of Horticulture (Victoria).

The choice of ex situ storage approaches depends on the plant species and can be as whole plants (e.g., field collections) seed, pollen or vegetative material (e.g. in vitro minimal growth or shoot tip cryopreservation). The majority of germplasm collections worldwide are of seed (Altoveros and Roa 1995), as it is the simplest and safest option for desiccation tolerant seeds (‘orthodox’). Such long-term seed storage for orthodox seed uses standard methods, i.e. seed at 3-7% seed moisture content at -18ºC. This method is used to minimise loss of viability (FAO-IPGRI 1994) with high levels, of viability likely over many decades and possibly centuries (Pritchard and Dickie 2003). For example, the

Australian Tropical Crops and Forages Collection (QDPI) and Millennium Seed Bank

Project (Royal Botanic Gardens, Kew, UK) store seed of wild species at about -20ºC after drying at 15ºC and 15% relative humidity (RH) to moisture content of about 5%.

However, not all species produce orthodox seed and Roberts (1973) defined the category ‘recalcitrant’ for seeds that lose viability when desiccated below about 15% moisture content. Many tropical and subtropical plants produce such recalcitrant or ‘non- orthodox’ seeds. About 50% of rainforest trees may have desiccation sensitive (recalcitrant) seeds (Tweedle et al 2003). Seed moisture contents of freshly harvested recalcitrant seeded species ranges from 30-70%, depending on the species (Baskin and Baskin 1998).

Continuous metabolism in seeds of recalcitrant species also mean loss of viability with moisture content below a certain critical level before germination (Baskin and Baskin 1998;

Pammenter and Berjak 1999). A further category of seeds demonstrating an intermediate

11 seed storage behaviour (i.e. some desiccation tolerance and/or sensitivity to some subzero temperatures) has been described in genera such as Coffea and Citrus (Ellis et al 1990,

Hong and Ellis 1995).

Thus, knowledge of the seed storage behaviour of a species is essential for the development of appropriate ex situ conservation strategies. Estimation of likely seed storage behaviour is based upon taxonomy, plant ecology, fruit characteristics and seed characteristics (Hong and Ellis 1996). A prediction of desiccation sensitivity in seeds of woody species has been proposed using a probabilistic model based on two seed traits in

104 species of different Families/Genera (Daws et al 2006a). However, these tools only assist in prediction of seed response to storage and empirical studies of seed responses to storage are still required to understand the seed storage physiology of each taxa.

Many of the rare and threatened CRWs of Queensland (cf. Table 1.1) are of subtropical and tropical origin and so likely to be non-orthodox in seed storage behaviour.

Thus, traditional ex situ seed storage methods (e.g. 5%MC at -20ºC) are not applicable. One option for ex situ storage of germplasm of non-orthodox seeded species is cultivation in field collections. Ex-situ field collections are however vulnerable to pest attack, diseases and natural hazards, as well as being costly to maintain. For example, Macadamia is an economically important genus that has six species listed as rare or threatened (Table 1.1) and has likely non-orthodox seeds, remaining viable for approximately four months at room temperate (King and Roberts 1979). Ex-situ collections of macadamia species are currently maintained in the field collections such as the National Macadamia Germplasm Conservation

Program (CSIRO). The vulnerability of such field germplasm collections to environmental stresses was recently demonstrated when heavy frost and poor drainage resulting in the loss of over half the trees in the CSIRO collection (Fraser 2006). This local example highlights the

12 need for the development of other conservation strategies complementary to field collections for long-term germplasm security.

Ex situ storage of non-orthodox seeded or problem species

Engelmann and Engels (2002) state that for storage of non-orthodox seeded species,

‘it is clear cryopreservation, often coupled with in vitro culture, represents the only option.’

A FOA (1994) worldwide survey revealed that 37 600 accessions are conserved in vitro.

Cryopreservation is the long-term storage of material at ultra low temperatures (e.g. liquid nitrogen, -196ºC) at which metabolic activities cease (Kartha and Engelmann 1994).

Pritchard (2007) reviewed seed cryopreservation studies over the past ten years (1995-

2005) and found considerable interest, with over 60 socio-economically important species being researched. Examples of cryopreservation of seeds with some level of desiccation tolerance include Carica papaya (Beewar et al 1983), Citrus aurantifolia (Cho et al

2002a.), Coffea liberica (Normah & Vengadasalam 1992) and Coffea arabica (Dussert et al

1998). For recalcitrant species, Berjak et al (2000) suggest rapid dehydration of excised embryonic axes (or explants) prior to storage at -196ºC as a suitable approach for long- term germplasm conservation of problem species. For example, embryonic axes of:

Quercus spp. (Gonzalez-Benito & Perez 1992), Citrus trifoliata (Radhamani & Chandal

1992), Camellia sinensis (Kim et al 2002), Citrus aurantifolia (Cho et al 2002a),

Azadirachta indica (Berjak 1996) and Artocarpus integer (Hor et al 1990) have been cryopreserved by this method.

In addition to the simple desiccation and cryopreservation of seed material and embryo axes, other cryopreservation techniques, including a range of preculture, cryoprotectant and in vitro steps, can be used to store plant material such as embryonic axes, shoot tips and somatic embryos. However, these more complex methods are generally dependent on the

13 availability of an efficient and reliable in vitro culture system (Ashmore 1997, Mycock et al

2004). In vitro culture techniques have been demonstrated in a number of Australian wild species, including rare and endangered species (Bunn and Dixon 1996, Taji and Williams

1996). For example, micropropagation systems have been established for the endangered

Queensland tropical fruit Davidonsia pruriens (Nand et al 2004), as well as for a critically endangered Western Australian species, Symonanthus bancroftii (Panaia et al 2000). In addition, micropropagation of both cultivated varieties and wild relatives of papaya (non- orthodox seeded) has been demonstrated (Drew 2003) and the in vitro culture system adopted for the successful recovery of plantlets after cryopreservation of shoot tips

(Ashmore et al 2000, 2001).

The development of a micropropagation system for a plant species not only allows the development of alternative ex situ storage methods (e.g. cryopreservation) but also provides a useful system for plant regeneration for maintenance of ex situ collections

(multiplication and duplication of germplasm) and propagation purposes (e.g. horticulture and in situ restoration purposes). Perth’s Botanical Gardens and Parks Authority has developed an integrated ex situ conservation strategy for WA threatened taxa, incorporating in vitro culture. They use in vitro micropropagation and storage (cryopreservation) techniques (e.g. cell suspensions, callus culture, somatic embryogenesis, shoot tips) for rapid large-scale production and protection of germplasm for species recovery efforts

(Cochrane 2004).

In vitro and cryopreservation approaches are also needed for vegetavively propagated species. For example, germplasm collections of cryopreserved shoot-tips

(meristems) of potato (Federal Agricultural Research Centre, Germany) and pear (USDA-

ARS National Clonal Germplasm Repository/National Centre for Genetic Resources

Preservation, USA) have been established with a regeneration rate of 40% being an

14 acceptable level for storage (Reed et al 2004). Furthermore, cryostorage of vegetative

(shoot tips) and seed material of endangered taxa have been stored at Perth’s Botanical

Gardens and Parks Authority (WA). These collections demonstrate the incorporation of cryopreservation into an integrated conservation strategy complementary to in situ strategies, ‘providing a safe and economical means for long-term storage of an endangered species’ (Touchell 2001). However, the majority of non-orthodox seeded species are still poorly understood and long term ex situ germplasm storage is currently not possible.

In vitro and cryopreservation methodologies

Cryopreservation techniques vary considerably between species in terms of both explant (e.g. seed, somatic embryo, shoot tip) choice and technique. Generally, the following components are included: in vitro culture, pregrowth, cryoprotection, freezing, thawing, recovery and plant regeneration (Harding 1996). Any successful cryopreservation protocols need to achieve a high level of recovery of genetically stable plants. Reed et al

(2004) summerised the main protocol dependents for plant recovery from cryopreservation:

Plant culture system

Pretreatments

Cryoprotectant type

Cryoprotectant exposure duration

Freezing and thawing rates

Recovery medium

Many factors affect plant recovery either in vitro or seed such as environmental conditions

(e.g., temperature, photoperiod), plant hormones (exogenous (in vitro) and endogenous), complex organic components (in vitro), genotype, physiological variables and

15 many others factors, known and unknown. Cryopreservation adds to this complexity. Bajaj

(1995) states, ‘cryopreservation involving freezing-storage-thawing culture is a complicated, multiple event.’ Validation (e.g. morphology) is essential for the development of robust cryopreservation protocols. A robust protocol would need to be applicable to a range of genotypes and have high plant recovery after cryostorage that demonstrates genetic integrity. Many successful cryopreservation techniques have been developed and applied to a range of explant types and species (Ashmore 1997). The following is a brief summary of some of the main techniques:

Desiccation

This method involves simple dehydration of an explant prior to immersion in liquid

nitrogen. Prevention of cryo-injury is often achieved by the removal of ‘free water’ and

consequently the prevention of lethal ice formation. The reduction of ice formation is

dependent on the extent of water removal (Kartha and Engelmann 1994). Moisture

contents of between 10 to 20% (fresh weight basis) are often optimal for survival of

freezing (Engelmann 2004). Thus, this technique is only viable for somatic tissue (e.g.

somatic embryos), seeds and embryonic axes that will tolerate this level of desiccation

and as such is species/tissue dependent. Desiccation cryopreservation techniques have

been used for desiccation tolerant seeds (orthodox and intermediate) and embryonic

axes of recalcitrant seeded species, for example seed of citrus and coffee (intermediate)

and rare or endangered taxa (orthodox) (Kartha and Englemann 1994, Touchell and

Dixon 1994, Pritchard 1995, 2007; Gonzales-Benito et al 1999, Cochrane 2004).

Vitrification-based

Vitrification-based methods involve pretreatment of samples with concentrated cryoprotectant solutions and, on rapid freezing (direct immersion in LN,) a highly

16 vicious solid ‘glass’ forms, thus avoiding lethal ice injury. Vitrification-based techniques have been successfully used for the cryopreservation of shoot tips of many tropical plants (Takagi 2000). Examples of the application of the vitrification-based techniques to problem species include: Carica papaya (Ashmore et al 2000, 2001),

Citrus aurantium (Al-Ababneh et al 2002), Wasabia japonica (Matsumoto et al 1995),

Garcinia mangostana (Normah 2000), Sechium edule (Abdelnour-Esquivel and

Engelmann 2002) and Macropidia fuliginosa (Turner et al 2001a.). The most widely used vitrification solution, for cryopreservation of a range of diverse tissues, is plant vitrification solution two (PVS2) developed by Sakai et al (1990). PVS2 consists of

30% glycerol, 15% ethylene & 15% and dimethyl sulfoxide (DMSO).

Encapsulation-dehydration

This method was developed by Fabre and Dereurddre (1990) and involves the encapsulation of an explant in a calcium alginate bead, followed by pregrowth (e.g. on high sucrose medium) and desiccation prior to direct immersion in liquid nitrogen

(Benson 1999). This method has been applied to many ‘problem’ or non-orthodox seeded species using various explant sources including: embryonic axes, somatic embryos and shoot tips (Cho et al 2002c, Gonezalez-Arnao et al 2003, Fabre and

Dereurddre 1990, Plessis et al 1993, Teresa et al 2000, Wang et al 2002).

Preculture-desiccation

Explants are precultured on cryoprotectants such as DMSO or elevated osmotica (e.g. high sucrose, PEG) before desiccation and rapid freezing (Ashmore 1997). This

17 technique has been applied to many ‘problem’ plant species, as a method for long term

ex situ storage of various explant source material (Ashmore 1997; Dumet et al 1993).

Cryopreservation – biological mechanisms

Dumet and Benson (2000) investigated both biochemical and physical factors involved in cryo-injury. These included free radicals (membrane lipid perioxidation), colligative damage factors, metabolic uncoupling, secondary oxidation, cellular dysfunction and loss of compartmentalization. Membrane damage is implicated as one of the main factors of freezing injury (Towill 1991).

Cryoprotectants, such as amino acids, polyols, sugars and lyotropic salts, help protect membranes during freezing and protect proteins and nucleic acids from inactivation

(Towill 1991). Pretreatments (e.g. high sucrose) reduce cell size as well as the cytoplasm to vacuole ratio, enhance the ability of cells or tissues to take up cryoprotectants, and/or modify cell walls and membranes to resist dehydration and deformation during freezing

(Luo and Reed 1997).

Vitrification of water involves the formation of an amorphous phase, a ‘glassy state’, on rapid freezing without the formation of ice crystals (Fahy et al 1984).

Vitrification of water in the cells prevents injury via intracellular freezing (ice formation) and the lethal effects of excess dehydration (Fujikawa and Jitsuyama 2000). It is important to rapidly thaw the tissue to prevent devitrification events. Cryopreservation techniques that rely on vitrification of water into a glassy state include any method using rapid freezing, for example desiccation, encapsulation-dehydration and vitrification-based methods .

18 Genetic integrity of in vitro and cryopreserved tissue

In vitro culture systems can lead to genetic changes known as somaclonal variation, which can be the result of either heritable genetic mutations or epigenetic changes (not transmitted to the next generation) (Ashmore, 1997). Somaclonal variation is mainly associated with callus cultures (i.e. dedifferentiated cells) or embryogenic suspension cultures, but is uncommon using direct micropropagation of shoot material (e.g. shoot apices, nodal cuttings) (Reed et al 2004).

Many established gene banks of vegetatively propagated plants rely on in vitro culture and this has been found to be suitable for routine and genetically stable, medium term storage of germplasm (Harding 1996). However, monitoring of genetic stability of ex situ collections, especially in vitro, is important as genetic shifts can arise due to chromosomal changes and gene mutations leading to morphological and biochemical changes (Reed et al 2004).

Cryopreservation involves a series of stresses to plant material (Engelmann 2004).

Thus, research has been conducted to confirm the genetic stability of recovered plantlets.

Genetic stability of a large potato germplasm collection has been assessed with some of the

125 varieties or genotypes frozen and stored in liquid nitrogen. Plants from all varieties or genotypes phenotypically resembled those of the unfrozen stock plants. Genetic stability of plants was assessed by flow cytometric measurements and DNA-fingerprinting. In the 161 tested samples neither polyploid plants nor unusual banding patterns were found (Schafer-

Menuhr et al 1996).

A molecular study of post cryogenic storage stability of cryopreserved potato shoot-tips, using the encapsulation/dehydration technique, also found that the observed nuclear DNA fragments and the chloroplast genome were stable (Harding and Benson

19 2000). Plants derived from Dioscorea floribunda shoot tips, cryopreserved using the vitrification technique, were found to be genetically stable at the molecular (RAPD fingerprints), phenotypic and biosynthetic levels (Ahuja et al 2002). In Conifer plants derived from cryopreserved embryogenic cultures, somaclonal variation (RAPD) was detected in some in vitro embryogenic cultures, 2 and 12 months after they were re- established following cryopreservation, but not in the corresponding regenerated trees

(DeVerno and Park 1999).

These studies on genetic integrity post cryostorage in shoot tips demonstrate cryopreservation can be a valid ex situ storage method for conservation of germplasm.

However, the study by DeVerno et al (1999) reinforces the need for morphological and molecular analysis, as well as the need for short and long-term studies of genetic integrity on regenerated plants post cryostorage.

1.3 Priority Species

Research on seed physiology of possible intermediate and recalcitrant seeded species is crucial to ensure future long-term security of the biodiversity of many valuable species, especially those currently threatened in situ. The vast majority of wild species have uninvestigated seed physiology, including their seed storage behavior. Prioritisation for selection of wild species for ex situ conservation can be based on several criteria such as (1) crop’s vulnerability to genetic erosion, (2) potential to agriculture and breeding, (3) existing genetic diversity available elsewhere and (4) availability of germplasm (Reed et al 2004).

There is a lack of knowledge of Queensland’s rare or threatened CWRs, such as reproductive biology, seed physiology, regeneration systems, genetic diversity and population dynamics, which is needed for the development of effective ex situ conservation strategies in these species. After consideration of the above issues, the Australian wild

20 species of the Citrus genus were chosen for further study through this PhD project because of their conservation priority, potential socioeconomic importance (e.g. novel genes (CWR) and fruits), probable non-orthodox seed storage behaviour and lack of corresponding techniques for their long term ex situ conservation. In particular, three species,

C. australasica (finger lime), C. inodora (Russell River lime) and C. garrawayi (Mt White lime), which have subtropical and tropical rainforest distribution, are the focus of investigations. Citrus australasica is a commercially important minor crop in Australia.

C. inodora is vulnerable in situ and C. garrawayi is rare in situ.

1.4 Citrus Biodiversity

The Rutaceae family (includes Citrus genus) has 160 species listed as threatened, placing it in the top ten families of rare or threatened species in Australia (Briggs and Leigh

1996). Aurantioideae is a subfamily of Rutaceae and is indigenous to the monsoon regions

(29 genera) of West Pakistan, north-central China, East Indian Archipelago, New Guinea, the Bismarck Archipelago, Australia, New Caledonia, Melanesia and Polynesian islands and tropical regions (5 genera) of Africa (Morton et al 2003). The Citrus genus belongs to the sub tribe Citrineae within the subfamily Aurantioideae (Engler 1931) and has a predominantly tropical to semi tropical origin (Swingle and Reece 1967). Citrineae are morphologically characterised by hesperidium fruits with a leathery rind or hard shell and a juicy pulp. Citrineae includes the true citrus fruit trees, a group of six genera: Fortunella,

Eremocitrus, Poncirus, Clymenia, Microcitrus and Citrus (Spiegel-Roy and Goldschmidt

1996). The characters that distinguish these genera are orange or lemon like fruits and specialized pulp vesicles (Spiegel-Roy and Goldschmidt 1996). The true citrus fruit trees are generally sexually and graft compatible (Grieve and Scora 1980) and are believed to be monophyletic (Guerra et al 2000, Morten et al 2003).

21 Taxonomy and phylogeny in Citrus has been controversial and even molecular based studies show some contradiction (Mabberley 2004). In part, this stems from the obscurity of the origin of Citrus, due to the high level of domestication and hybridization over centuries, habitat disturbance and apomixis (Scora 1988; Herrero et al 1996ab;

Mabberley 2004). Barrett and Rhodes (1976), proposed from a detailed morphological study, that many Citrus species and cultivars are derived from complex hybrids of only three true species, C. medica (citron), C. reticulata (mandarin) and C. maxima (previously grandis - pummelo) (Spiegel-Roy and Goldschmidt 1996). Recent molecular analysis

(Isozymes, RFLP, RADP, SCAR and cpDNA) supports this hypothesis that cultivated citrus (e.g., lime, oranges and lemons) are complex hybrids including parents from three species clusters, i.e. citron, mandarin and pummelo (Herrero et al 1996b, Federici et al

1998, Nicolosi et al 2000). Herrero et al (1996b), using analysis of ten isoenzyme systems, defines two major groups: the orange-mandarin group and the lime-lemon-citron-pummelo group.

Many Citrus species are located in South East Asia, whilst the Microcitrus (now

Citrus) genus is found only in Australia and New Guinea (Herrero et al 1996b).

Microcitrus is a genus very closely related to Citrus. Australia is believed to have been isolated from other landmasses for 20 to 30 million years (Spiegel-Roy and Goldschmidt

1996). Five species have been described in Australia, M. australasica, M. australis,

M. garrawayi, M. gracilis, M. inodora and two species in New Guinea, M. papuana and

M. waburgiana. M. garrawayi occurs predominantly in Australia but has been cited on

Good Enough Island, New Guinea (Forster 1991). Additionally, the Australian wild desert lime, Eremocitrus glauca, was described as the only member of the genus.

A study of Citrus and related genera found intergeneric and interspecfic genetic variation (cpDNA) in Microcitrus (five species) that was suggestive of an early divergence

22 from each other and other true citrus fruit tree genera (Abkenar et al 2004). A comparative study based on analysis of ten isoenzymes in genera of the subfamily Aurantioideae, revealed Microcitrus had the highest genetic variability, along with Atalantia, compared with other genera, including many other taxa of Citrus (Herrero et al 1996a). Molecular analysis, using RFLP and RAPD analysis, also found Microcitrus species are genetically diverse with a low heterozygotisity index (Federici et al 1998).

Originally classified in the Citrus genus, Swingle (1915) placed Microcitrus into a separate genus. More recently, Mabberley (1998) reassessed the distinction of Microcitrus and Eremocitrus from the Citrus genera and recommended its reincorporation. This means that there are now six species of Citrus in Australia; C. australasica, C. australis, C. garrawayi, C. glauca, C. gracilis and C. inodora.

1.5 Ex situ conservation of Citrus

The Citrus genus contains many species of worldwide economic importance. The worldwide production of the citrus fruit crop has experienced continuous growth and was estimated at over 105 million tons for the period 2000 to 2004 (sourced UNCTAD from

Food and Agricultural Organization (FAO) data; http://www.unctad.org/infocomm/). Citrus has widespread cultivation (140 countries) in predominantly subtropical and tropical climates and fruit production exceeds that of banana, grapes and apples (Gmitter et al

1992). Cultivated species of Citrus include C. medica (citron), C. maxima (pummelo), C. reticulata (mandarins), C. sinensis (orange), C. limon (lemon) and C. aurantifolia (lime).

Conservation in the Citrus genus, including wild species, is particularly crucial as historically, citrus has been a crop particularly vulnerable to serious diseases and pests (e.g. greening, bacterial canker, tristeza and phytothora) (Reuther 1977, Saamin and Ko 1996) and pathogens of increasing virulence are emerging (Herrero et al 1996). Furthermore,

23 concerns of diminishing habitat (e.g. land clearing) of wild species have resulted in the urgent need for in situ and ex situ conservation of existing wild biodiversity (White 1922,

Reuther 1977, Mabberley 2004, Sharma et al 2004). The International Treaty on Plant

Genetic Resources has identified citrus as one of 35 food crops important to humanity for conservation and development of crop diversity (FAO 2005). Internationally, the Bioversity

International and the Citrus Germplasm Network (GCGN) support and advance conservation and utilization of citrus biodiversity.

Citrus germplasm has traditionally been conserved in ex situ field collections of botanic gardens and research stations (Pérez 2000), because of its complex seed storage behaviour (Hong and Ellis 1995). These collections are vulnerable to pests, disease and natural disasters. In 1977, Ruether highlighted several research needs to support citrus germplasm conservation including improved seed storage, development of long term preservation techniques (e.g. freezing) and development of aseptic in vitro techniques for exchange of germplasm. The need for the development of alternative conservation strategies (e.g. in vitro and cryopreservation methods) of Citrus germplasm has also been well recognised in more recent publications (Engelmann et al 1994, Duran-Vila 1995,

Pérez 2000).

Many research and storage advances have been achieved in predominantly cultivated Citrus species. The most promising potential long term ex situ technique appears to be cryopreservation of seeds, although high levels of recovery is species dependent and low recovery is still observed in the more desiccation sensitive species (Normah and Siti Dewi

Sermiala 1997, Lambardi et al 2004, Hor et al 2005, Makeen et al 2005 ). The Instituto

Valenciano de Investigaciones Agrarias Citrus Germplasm Bank (Spain) are trialing the use

24 of cryostorage of seed, ovules, embryos, nucellar callus and shoot tips in Citrus species

(Perez 2000).

Overall, cryopreservation has been achieved using seed and embryos (Normah and Siti Dewi Serimala 1997; Cho et al 2001, 2002a; Santos and Stushnoff 2002, 2003;

Lambardi et al 2004, Hor et al 2005, Makeen et al 2005), shoot tips (Gonzalez-Arnao et. al 1998, Wang et al 2002, Wang and Deng 2004), somatic embryos (Gonzalez-Arnao et al 2003), embryogenic callus (Englemann et al 1994, Perez et al 1999) and cell suspensions (Aguilar et al 1993, Hao et al 2002, Sakai et al 1991). These cryopreservation protocols have given varied results dependent on species and have been predominantly based on either vitrification-based and encapsulation/dehydration techniques for vegetative explant material (in vitro) or simple desiccation (e.g. silica dried) for seeds. Table 1.2 summerises the conservation options that have been investigated in cultivated Citrus species. However, there has been no reports of seed storage physiology, and very few reports of using alternative cryopreservation and in vitro techniques, in the wild Australian wild Citrus species. Thus, conservation strategies and research conducted predominantly in cultivated members of the Citrus genus will form the framework for the present study on investigating ex situ conservation in

Australian wild Citrus species.

Table 1.3 lists the major cultivated and wild Citrus species and their suggested native habitat, which covers four regions, India, SE Asia, China and Australasia. The need for in situ and ex situ conservation of the rich biodiversity of centres of origin is recognised in NE India (Rai et al 1996, Sharma et al 2004), SE Asia (Saamin and Ko

1996) China (Deng et al 1996) and Australia (Mabberley 2004).

25 Citrus genebanks, including indigenous wild species, are held in these centres of origin in India (Indian Council of Agricultural Research) (Rai et al 1996), China (Citrus

Germplasm Repository) (Deng et al 1996), Australia (CSIRO and QLD DPI) and SE Asian countries, i.e. Malaysia, (e.g. Malaysian Agricultural Research and Development Institute and University of Malaya); Indonesia (Punten Horticultural Branch); Philippines (Bureau of

Plant Industry)) and Thailand (Saamin and Ko 1996).

Worldwide, ex situ collections of citrus germplasm (mostly field collections) are extensive, with 20924 accessions held in 114 institutes of 60 countries, based on the

Bioversity Directory of Germplasm Collections (Table 1.4). For example, large collections are held in Australia, Argentina, Brazil, China, Japan, India, Malaysia, Thailand, Turkey,

Spain and USA). Only 52 accessions of four Australian wild Citrus species (C. australasica, C. australis, C. glauca and C. inodora) are held in nine institutes of six different countries (Table 1.4). The Bioversity Directory of Germplasm Collections has no reports of collections of the Australian species C. garrawayi and C. gracilis. Limited field material of C. garrawayi is held in Australia in research collections and Botanic Gardens

(e.g. CSIRO, Atherton; QLD DPI, Bundaberg and Fairhill Native Plants and Botanic

Gardens).

Table 1.2 Summary of conservation approaches investigated in cultivated citrus Short term Medium term Long term

Seed In situ on farms and gardens In situ natural habitat

Field genebanks Cryopreservation of: Seed In vitro slow growth Embryonic axes

Ovules

Embryogenesis callus Shoot tips Pollen

26 Table 1.3 List of major cultivated and wild species of Citrus Suggested Native Species Common name Species statusA HabitatA India C. limon (L.) Burm.f. lemon Hybrid (C. media x C. maxima x Microcitrus ?) c

India C. indica Tan. Indian wild orange Primitive mandarin? B

India C. medica L. citron Species

Malaysia C. aurantifolia Christm. lime Hybrid (C. media x C. maxima x Microcitrus [now Citrus])?c

Malaysia and C. halimii BC Stone Species Peninsular Thailand Malaysia and C. maxima Burm. pummelo Species Thailand SE Asia C. hystrix DC Mauritius papeda Species?

SE Asia C. latifolia Tan. Tahiti lime Hybrid

SE Asia C. madurensis Lour. musk lime Hybrid

C. paradisi Macf.. grapefruit Hybrid (C. maxima x C. sinesis) C

China C. aurantium L. sour orange Hybrid (C. reticulata x C. maxima) CD

China C. ichangensis Swing. Ichang papeda Species

China C. reticulata Blanco mandarin Species

China C. sinensis (L.) Osb. sweet orange Hybrid (C. reticulata x C. maxima) CD

China C. trifoliata L. (Poncirus trifoliate orange Species trifoliata (L.) Raf.) (root stock)

New Guinea C. papuana Wint. Species

New Guinea C. warburgiana FM Bailey New Guinea wild Species lime Australia (NSW, QLD) C. australasica F Mueller finger lime Species

Australia ( QLD) C. australis Planch round lime Species

Australia (SA, NSW, C. glauca Lindl. desert lime Species QLD) Australia (NT) C. gracilis Mabb. Humpty Doo lime Species

Australia (QLD) C. garrawayi FM Bailey Mount White lime Species

Australia (QLD) C. inodora FM Bailey Russell River lime Species

A Suggested species status/habitat cited Barret and Rhodes (1976), Scora (1988), Saamin and Ko (1996), Spiegel-Roy and Goldschmidt (1996), Araújo et al (2003), Mabberley (2004). Suggested species: BScora (1988) and hybrid parentage: CBarret and Rhodes.1976 and DNicolosi et al 2000. Bold font indicates ‘true species’ that account for the majority parentage of cultivated hybrids (i.e. lemons, limes, grapefruits and oranges) (Barret and Rhodes 1976). 27

As previously discussed, Australia’s six wild Citrus species (C. australasica,

C. australis, C glauca, C. gracilis, C. garrawayi and C. inodora) have been reclassified and put within Citrus and as such represent a valuable source of genetic diversity. This makes

Australia the country with the largest number of indigenous Citrus species (Mabberley

1998, 2004) (Table 1.3). The large genetic variability identified in Australia’s wild Citrus species most likely reflects their ecological adaptations to a wide range of environmental conditions from tropical rainforests to desert (xerophytic) habitats (Herrero et al 1996b).

Australian wild citrus (limes) are recognized as an important novel fruit, being one of 14 top commercially significant Australian edible plants (Skyes 1993, 1997, Ahmed and

Johnson 2000). Additionally, Australian wild limes are a novel source of genetic variation for use as rootstocks or in breeding, with many potentially useful traits not found in cultivated varieties, such as drought tolerance, shortened period to fruiting, salt tolerance, disease resistance (e.g. Phytopthora), dwarf habit and uniquely shaped and coloured fruit

(Skyes 1997, Davison 2001). The southern species of Australian limes (e.g. C. australasica and C. australis) form part of the bushfood industry and have also been hybridized with commercial cultivars (Sykes, 1997, Mabberley 1998). More recently, C. inodora has been crossed with a mandarin cultivar. The resulting hybrids produced larger fruit than the wild species and had a short juvenility of approximately two years (M Smith pers. comm. 2005).

Therefore, long-term ex-situ conservation of this unique wild germplasm is of significant interest to complement in situ conservation and secure sustainable access for breeding programs, or the development of bushfood industries.

28 Table 1.4 Ex situ collections of Citrus as listed by BioversityA

Species No. No. Institutes Countries accessions Institutes Albania, Algeria, Antigua, Australia, Citrus spp. 20924 114 Agricultural Barbados, Barbuda, Bolivia, Brazil, (cultivated and research institutes China Colombia, Costa Rica, Cuba, wild species) and universities Dominican Republic, Ecuador, Fiji, France, Gabon, Germany, Ghana, Greece, Honduras, India, Indonesia,. Italy, Jamaica, Japan, Kenya, Madagascar, Malawi, Mexico, Morocco, Mozambique, Nepal, New Zealand, Nicaragua, Nigeria, Panama, Papua New Guinea, Peru, Philippines, Portugal, Puerto Rico, Saint Lucia, Seychelles, Sierra Leone, South Africa, Spain, Sri Lanka, Sudan, Suriname, Taiwan, Tanzania, Thailand, Trinidad and Tobago, Tunisia, Turkey, USA, Venezuela, Vietnam Australian spp.

C. australasica 26 7 CSIRO, Citrus Australia (Merbein), China, Spain Research Institute, (Valencia), Turkey, USA (Orlando and Sciences, IVIA, Riverside) Univ. of Cukurova, Univ. of California, USDA –ARS

C. australis 8 3 IVIA, Univ. of Spain (Valencia), Turkey, USA Cukurova, Univ. of (Orlando and Riverside) California, USDA- ARS C. gracilis 0 0

C. glauca 8 8 CNPMP, CSIRO, Brazil, Australia (Merbein), France, SRA/INRA-CIRAD, Turkey, USA (Riverside and Orlando) IVIA, Univ. of Cukurova, Univ. of California, USDA –ARS

C. garrawayi 0 0 C. inodora 10 4 Univ. of Cukurova, Spain (Valencia), Turkey, USA Univ. of California, (Riverside)A USDA-ARS

ASourced from the Bioversity Directory of Germplasm Collection (http://web.ipgri.cgiar.org/germplasm/default.asp: accessed 11/11/06). Institutes: CSIRO (Commonwealth Scientific & Industrial Research Organisation), Instituto Valenciano de Investigaciones Agrarias (IVIA), US Department of Agriculture (USDA-ARS), Centro Nacional De Pesquisa de Manbioca e Fruitcultura (CNPMP), Agricultural Research Station (SRA)/National Institute of Agricultural Research and Centre International of Cooperative Research (INRA-CIRAD).

29 Understanding seed storage behaviour depends on a knowledge of a species taxonomy, plant ecology, fruit characteristics and seed characteristics (Hong and Ellis

1996). The six Australian Citrus species are mostly distributed in Queensland and northern

NSW (Table 1.3). Table 1.5 summerises characteristics and distribution of the target species of this project, C. australasica, C. inodora and C. garrawayi. They are morphologically (e.g. tree habit) and ecologically distinct species, with distribution ranging from warm subtropical to hot tropical. Citrus australasica (F Muell.) is commonly found across southern Queensland and northeastern New A South Wales. It is a tall shrub (5m) with small leaves and straggling habit that grows in dry rainforest (complex notophyll vineforest) (Figure

1.2, Table 1.5). Citrus australasica has been the focus of previous research, particularly the breeding of new hybrids and its promotion as novel minor crop (Skyes 1997).

K. Hamilton 2006 B

Fig. 1.2 Citrus australasica (A) tree growing in cultivation at the Brisbane Botanic Gardens (Mt

Coot-tha) and (B) whole fruit __ from a commercial harvest. 2cm

30 Table 1.5 Distribution, habitat and climatic conditions of Australian wild Citrus species C. australasica, C. inodora and C. garrawayi

Species Natural Habitat Characteristics Conservation Flowering Fruiting Mean Mean Mean Mean Distribution Status annual annual annual Daily min. max. rainfall %RH temp. temp. (mm) (ºC) (ºC)

Citrus australasica Southern QLD and Dry rainforest - Tall shrub to 5 Common Feb.-May May - 9-12 21-24 1000- 70 F.Muell. northeastern NSW complex notophyll metres; fruit Sept. 1600 (9am) – Tambourine vineforest lemon taste (Finger Lime) Mountain, 50-60 Lamington (3pm) National Park

Citrus inodora Very restricted Lowland rainforest, Shrub to small F.M. Bailey area of northeast complex mesophyll tree; leaf size Vulnerable Aug.-Sept March- 15-18 24-27 1600- 70-80 QLD – Mossman vineforest; best varies up to . Sept. 3200 (9am) (Russell River and Bellenden Ker growth in the shade 18cm in length and Dec. Lime) Ranges of heavy rainforest and 4cm in 60 width; fruit (3pm) agreeable acid taste

Citrus garrawayi North QLD – Foothills and Small tree up to Rare May and April – 18-21 27-30 1200- 70 F.M. Bailey Cook district, upland rainforest - 10m tall; fruit Nov.- Dec. Nov 1600 (9am) summit of Mount complex notophyll tart sour taste (Mount White White (restricted and microphyll 50-60 Lime) to Cape York vineforests and (3pm) Peninsula) and vinethickets, Goodenough including Island in New monsoonal Guinea rainforests Habitat, distribution and characteristics sourced Bailey (1904), Brophy et al (2001), Birmingham (1998), Cooper and Cooper (2004), Forster (1991, 2002, Pers comm. 2004) and White (1922). Flowering and fruiting sourced from Cooper and Cooper (2004) (C. inodora and C. garrawayi) and Macintosh (2004) (C. australasica). Mean annual climate data over a thirty period (1961-1900) sourced from the Bureau of Meteorology (2005).

31 A B

Fig. 1.3 Rainforest types of north Queensland in which (A) Citrus inodora (Russell River lime) and (B) C. garrawayi (Mt White lime) grow in the wild. (A) Typical complex mesophyll vine forest (tropical very wet lowland rainforest) near Innisfail (1969) and (B) C complex notophyll vine forest. (McDowall Range, 1969). (C) Mt. White in background (Coen, 1954) where the type specimen of Citrus garrawayi (FM Bailey, 1904) was sampled. Photographs are reproduced with the kind permission from the Len Webb Ecological Images Collection (www.griffith.edu.au/ins/collections/webb).

. C inodora and C. garrawayi grow in northern Queensland and are listed as vulnerable and rare respectively in situ. White (1922) quotes Swingle, ‘so far attempts to introduce Russell River Lime (C. inodora) into culture have failed and the rapid clearing up of land along the Russell River threatens to exterminate the species altogether. It is hoped that Australian botanists and fruit-growers will not permit this to happen’. Citrus inodora

(FM Bailey) is now listed as vulnerable, as it has a very restricted distribution growing in lowland rainforests of northeastern Queensland and is protected under the 2000 schedule of the Queensland nature Conservation Act 1992 (Queensland Nature Conservation (Wildlife)

Regulation 1994) (Forster 2002).

Citrus inodora is an under storey tropical A rainforest plant (complex mesophyll vine

forest (Figure 1.3A, Table 1.4). It has a dwarf

habit and large luxurious leaves (up to 18cm)

(Figure 1.4, Table 1.5).

Citrus garrawayi (FM Bailey) is

distributed further north in QLD growing in

the monsoon forests and rainforests of the

Cape York Peninsula, as well as in New

K. Hamilton 2005 Guinea (Forster 1991). It is listed as rare B Fig. 1.4 Citrus inodora and also protected under the 2000 schedule (A) leaves and (B) fruit growing on trees of a of the Queensland Nature Conservation Act research collection of the 1992 (Forster 2002). It grows in vineforest Queensland Department of Primary Industries. and vinethickets (complex notophyll and microphyll) (Figure 1.3B) and has smaller ____ leaves than Citrus inodora and a taller habit 2cm (up to 10m) than other Australian limes (Figure

1.5, Table 1.5). Figure 1.3C shows Mt White in Cape York Peninsula of northern

Queensland where the type specimen of C. garrawayi was taken.

These Australian wild Citrus species, C. australasica, C. inodora and C. garrawayi,

all have small uniquely ‘finger’ shaped fruits (Figure 1.2, 1.4 and 1.5), compared to the

large round fruits of cultivated species like lemons, mandarins and oranges (Figure 1.6).

Fig. 1.5 Citrus garrawayi (A) A tree and (B) fruit growing on tree at the research collection of the Queensland Department of Primary Industries.

__ 2cm K. Hamilton 2005

Fig. 1.6 Fruiting trees of A cultivated Citrus species growing at the Brisbane

Botanic Gardens (Mt Coot-tha): (A) C. limon,

(B) C. reticulata and (C) C. sinensis.

C. limon (lemon

B C

C. reticulata (mandarin) C. sinensis (orange) K. Hamilton 2006

35 There have been no reports of long-term seed storage for any Australian wild Citrus species. Seeds stored at 4ºC lost viability after storage for one year (M. Smith Pers. comm.

2004). Ecological factors, such as water availability and temperature, can be critical to seed physiology (e.g. seed quality), as well as seedling survival and growth post germination.

Thus, knowledge of rainfall and temperature of the distribution of a species can assist investigations of seed storage behaviour in an unknown plant species. Citrus australasica has a warm subtropical distribution, with a mean annual rainfall of 1000-1600mm (Table

1.5). Whilst C. inodora and C. garrawayi have a warm to hot tropical distribution and a high mean annual rainfall of 1600-3200 (C. inodora) or 1200-1600mm (C. garrawayi). The distribution and taxonomy of C. australasica, C. inodora and C. garrawayi suggest possible non-orthodox seed storage behaviour (e.g. intermediate).

1.6 Citrus seed biology and storage

Intermediate seeded species, such as papaya and oil palm, do not tolerate the combined effects of low moisture contents and low temperatures (Vertucci and Farrant

1995) and common challenges are often encountered in storage responses (Hong and Ellis

1995, 1998). Further, intermediate seeded species, such as coffee and citrus, characteristically show variation between seed lots (Pritchard 2004), with many species displaying ‘essentially’ orthodox seed storage behaviour. For example, Coffea arabica seeds have been stored at -20ºC for 3 years (Hong and Ellis 1992). King et al (1981) similarly showed three Citrus species (lemon, lime and orange) were ‘essentially’ orthodox in seed storage behaviour. However, due to variation and other storage constraints in coffee and citrus, most studies have focused on storage at very low temperature (i.e.

36 cryopreservation) as the safest option to prevent seed deterioration (loss of viability)

(Dussert et al 2001, 2006, Lambardi et al 2004, Hor et al 2005).

The reasons for varied response of seed storage behaviour in intermediate seeded species are still elusive. Factors that have been observed to affect germination of seed after drying and/or cooling include, variation in stage of maturity (neem, Sacandé et al 2000), imbibition injury (neem, Sacandé et al 2001) and thawing rate (slow is detrimental) (coffee,

Dussert and Engelmann 2006). In particular, seed quality (i.e. desiccation sensitivity) was found to be a key determinant in recovery after cryopreservation in coffee (Dussert and

Engelmann 2006). Such complex storage is discussed by Pritchard (2007) in regard to a range of species that ‘are essentially desiccation tolerant (i.e. certainly not recalcitrant) but nonetheless display a diverse range of physiological responses that might affect an interpretation of their storage behaviour, and this means that maximizing their storage potential through improved handling is particularly challenging’.

Seed morphology

In six species of Meliaceae, closely related to the Ruataceae family (Chase et al

1999), a range of seed storage behaviours (orthodox to recalcitrant) were found based on a combination of four criteria: seed weight, shape, moisture content (mature seed) and plant ecology (Hong and Ellis 1998). There have been many reports on the seed storage physiology in cultivated Citrus species but very little is known about the in situ ecology including seed dispersal and seedling establishment. Moreover, there have been no reports of seed physiology and ecology in any Australian wild Citrus species

37 Seed size and shape can relate to germination capacity and final seedling growth.

Matilla et al (2005), in a recent review, concluded that seed size and shape are key factors in determining seed fate and persistence in the soil. Small and rounded seeds have been reported to persist longer in the soil (more easily buried, Thompson et al 1993), have lower predation (Hulme 1998), and less exposure to germination-promoting stimuli (Baskin and

Baskin 1998, Milberg et al 2000) compared to large, elongated or flattened seeds (Matilla et al 2005).

Seed coat characteristics can also be critical in seed physiology responses. For example, the development of physical dormancy via a seed coat barrier to water uptake and gas exchange (Matilla et al 2005). Seed coats are often suggested as a contributing factor

(chemical or physical inhibitors) in the erratic, slow and reduced germination in response to desiccation and storage that has been observed in citrus seeds (Mumford and Grout 1979,

Mumford and Panggabean 1982, Soestinsa et al 1985). Additionally, seed coat patterns have been correlated with ecological factors. For example, a relationship between germinability and habitat has been observed in the genus Tulbaghia (Alliaceae) (Vosa

2003). Seeds from dry habitats had seed coats composed of loose cells capable of absorbing water quickly, whilst species from wet habitats had cells that were welded together and somewhat impermeable to water.

Thus, seed morphology and ecology can greatly assist in understanding seed responses to storage processes such as desiccation. Moreover, micromorphology (e.g. seed coat topography) can be a useful tool in taxa identification. Detailed seed morphology and anatomy has not been previously described in the northern Queensland Citrus species,

38 C. inodora and C. garrawayi. Clarke and Prakash (2001) found close floral and embryological similarities between cultivated Citrus species and the southern distributed

Australian wild species, C. australasica and C. australis.

Temperature

Citrus seeds germinate at a range of temperatures but the vast majority of studies conduct germination of seeds at a constant temperature of either about 25ºC (Mumford and

Grout 1979, Cho et al 2002a, Lambardi et al 2004, Hor et al 2005, Makeen et al 2005) or

30ºC (King et al 1981, Soetisna et al 1985, Saipari et al 1998), with germination usually completed by 28-48 days (Ellis et al 1985). Mobayben (1980) reported that in cultivated

Citrus species, temperature had little effect on the final percentage of germination (radicle protrusion >2mm) over a temperature range of 11-30ºC. However, the rate of germination increased linearly within this temperature range. Mobayben (1980) estimated a minimum temperature of about 6-10ºC and optimum temperature of 30ºC for the germination of citrus seeds. This germination temperature range is consistent with the climatic restriction

(subtropical to tropical) of Citrus species. One limiting factor in the distribution of cultivated Citrus trees is low temperature (reduced growth 13ºC), which restricts citrus cultivation to subtropical and tropical climates (Spiegel-Roy and Goldschmidt 1996).

Although previous studies have been published on seed storage and, more limited, optimum temperature of germination in cultivated species of Citrus, such physiological

39 Fig. 1.7 Fruiting period of Australian wild Citrus species in relation to season for northern (C. inodora and C. garrawayi) and southern (C. australasica) distribution. Fruiting periods

(ACooper and Cooper 2004, BMacintosh 2004).

characteristics are unknown in the Australian wild members of the genus. However, the climate of these geographically separate populations may be reflected in a difference in germinability at range of temperatures. The southerly-distributed C. australasica has a warm subtropical distribution with an annual minimum mean temperature of 9-12ºC and mean maximum of 21-24ºC. The northern Australian species, C. inodora and C. garrawayi, grow in warmer tropical climates with a higher annual minimum mean temperature (15-

18ºC) and maximum mean temperature (24-30ºC) (Table 1.5). Observation of fruiting time also varies between these three species: May to September (C. australasica), March to

September and December (C. inodora) and April to November (C. garrawayi) (Figure 1.7).

Figure 1.7 shows these fruiting months predominantly represent colder periods of the year i.e. late autumn winter and early spring in the south (C. australasica) and dry season in the north (C. inodora and C. garrawayi).

40 A recent study on the oily intermediate seeded Cuphea carthagenesis has revealed that seed oils (triaclyglyerols) affect seed responses. Crane et al (2006) found that imbibition of seeds of a species with intermediate seed storage behaviour (i.e. Cuphea cartagenensis), whilst triacylglycerols are in solid (i.e. crystalline state) was lethal. Crane et al (2006) suggest such interactions may account for the damage observed in intermediate species on long-term storage. Citrus seeds have characteristically high oil content (22% to

52% lipid content (Vaughan 1970, Hor et al 2005), predominantly triglycerides (65-68%)

(El-Aldawy et al, 1999). Therefore, this phenomenon could contribute to the variable and unusual seed physiology observed in citrus. Thus, analysis of seed oils may allow more accurate interpretation of survival responses in Citrus species after drying and freezing.

Crane et al (2003) reviewed studies on intermediate seeded species and found that they often had lipids with relatively high crystallization melting temperatures. Moreover,

Crane et al (2006) noted the common practice of stimulating germination of dry seeds using heat treatment in dormant intermediate seeds of warm tropical origin. Crane et al (2006) concluded, supported by their observations at the USDA National Centre for Genetic

Resources Preservation, that ‘seeds express sensitivity to temperature rather than desiccation and that the temperature range and accumulation of damage correspond to triacyclglycerol phase behaviour and crystallization rate’. Thus, the observed unusual physiologies in many studies for intermediates may result from the high melting point of lipids, such that lethal imbibition injury of seeds occurs if imbibed whilst lipids are still crystalline (i.e. solid phase).

41 These findings are of significant practical importance in the seed banking of germplasm of intermediate seeded species, such that Crane et al (2006) recommend standard orthodox protocols (i.e. 5%MC at -20ºC) are thus applicable to intermediate species. However, they caution that anomalies (seed deterioration) have been observed in

Cuphea seeds stored at temperatures within the range of lipid crystalline phase events and as such recommend seeds of a species should not be stored within this temperature range.

Differential Scanning Calorimetry (DSC) provides a useful tool for the non-invasive observation of thermal behaviour and seed oils in seed tissues (e.g. cotyledons) of oil rich seeded species (Crane et al 2003, Walters et al 2005, Lehner et al 2006). DSC has been used to investigate the characteristic thermal properties of phase transitions in lipids, for example in oil rich cultivated Citrus and Coffea and Phoenix (date) species (Hor et al 2005,

Dussert et al 2001, Besbes et al 2004). Phase transitions (e.g., melting from solid

(crystalline) to liquid state) result in heat being absorbed (endothermic event) or liberated

(exothermic event). DSC measures the difference in energy (i.e. the amount supplied to maintain the same temperature) on phase transitions between the sample and an inert reference on warming or cooling. Thus, thermocurves (heat flow) are produced illustrating the endothermic and exothermic changes during phase transitions in the sample. These thermocurves can be used to determine the characteristic lipid profile of a species on warming from low temperatures, including the onset temperature, end temperature (i.e. melt point) and enthalpy of the lipid phase transition. There have been no reports of either the seed oil characteristics (e.g. content and thermal properties) or temperature range for seed germination in Australian wild Citrus species.

42 Seed quality

Three main factors important to seed storage are moisture content, temperature and seed quality. Of these factors, seed quality is an ambiguous factor under environmental and genetic control (Walters 2003). Difficulties in collection and storage of wild species commonly result from the heterogeneous nature of seed lots. These quality issues are often due to differences in flowering time, dry matter accumulation, dormancy, and maturation date, all genetically regulated traits that affect seed longevity (Walters 1998).

Seed quality, in terms of seed desiccation sensitivity, has been demonstrated as a key determinant in recovery of seedlings from coffee seeds after cryopreservation (Dussert and Engelmann 2006). Additionally, seed quality was also hypothesised to be the reason for variability observed in wild Coffea arabica species (Vasquez et al 2005). In wild species, seed lot quality in relation to seed developmental status and handling method appears to affect seed responses to desiccation and storage (Pritchard 2004).

Seed maturity often relates to seed longevity and physiology such as vigour, for example the ability to withstand a combination of stresses. The level of desiccation tolerance changes during seed development in most species, such that embryos become more tolerant on maturation and less tolerant on germination (Vertucci and Farrant 1995,

Kermode and Finch-Savage 2002). Seed maturity status has been suggested as a possible reason for the conflicting reports in seed responses to desiccation in neem seeds (Sacandé et al 2000, Berjak et al 1995, Esswara et al 1998, Gamene et al 1996). Daws et al (2006a) suggests that such cases may be due to the use of seeds developed under sub-optimal conditions which consequently have not achieved a maximum potential level of desiccation tolerance (Daws et al 2004, 2006b).

Seed development can be divided into three stages: histodifferentiation, expansion

(reserve deposition) and maturation drying (reduced metabolism) (Kermode and Finch-

43 Savage 2002). Maximum desiccation tolerance is often acquired at mass maturity (max. dry weight accumulation) (Vertucci and Farrant 1995, Kermode and Finch-Savage 2002).

Identification of these seed developmental stages for seed collection is often by the use of maturity ‘markers,’ such as non-destructive visual indictors like fruit or seed size, colour and hardness (Hay and Smith 2003, Aiazzi et al 2006). Preliminarily experiments in this present study indicated C. garrawayi seeds might have heterogeneous seed lots with seeds at a range of maturities. Thus, an important step to investigating seed desiccation in this wild species needs to be the identification of maturity ‘markers’ to obtain seed lots of uniform maturity prior to testing and interpretation of seed responses to desiccation and freezing.

To the author’s knowledge a set of markers for the identification of morphological characteristics at different maturities has not been reported in any Citrus species. As it is likely that seed quality, including the presence of seeds that are not fully mature, contributes to the loss viability of some citrus seeds on desiccation and freezing for storage, it is important to have seed lots of high quality for storage purposes.

Response to seed desiccation

Tolerance to seed desiccation varies within and between species of Citrus from orthodox to recalcitrant and it has been considered difficult to store by standard seed storage methods (Ellis et al 1985, Hong and Ellis 1995, Flynn et al 2004). Barton (1943) raised early questions about the desiccation sensitivity of citrus seeds, finding that the method of drying affected seed viability.

Particular seed moisture contents can be achieved by equilibrating seeds for about 1

- 4 weeks, dependent on species and seed composition (e.g. starch/oil rich), at particular relative humidities (%RH). This can be achieved by various methods including

44 equilibration over saturated salts or in a controlled rooms/cabinets (e.g. 15ºC at 15%RH).

Other more rapid drying methods include drying under ambient conditions (e.g. 50%RH), over self-indicating silica (ca. 8%RH) or by air-drying (e.g. laminar airflow). The moisture content is often determined gravimetrically after oven drying and presented on either a fresh or dry weight basis. The additional measurement of water activity of seeds (eRH/100) is also important in understanding seed water relations and can be determined using non- destructive methods (e.g. using a Rotronic device).

Several studies around the early 1980s demonstrated that seeds of many species of

Citrus could tolerate desiccation to low moisture content without much loss of viability using predominantly silica based drying method (Mumford and Grout 1979, King and

Roberts 1980, King et al 1981, Mumford and Panggabean 1982). For example, Mumford and Grout (1979) showed that Citrus limon (lemon) seeds, with the seed coat removed and dried to 1.2% (silica-dried) had a high level of germination. King et al (1981) found that citrus seeds of three species behaved like orthodox seeds, having improved storage longevity as both storage temperature and seed moisture content decreased. Nevertheless, longevity was difficult to determine using standard viability equations. King et al (1981) also tested a range of temperatures (-20 to 40C) and duration of storage in three species (C. limon, C. aurantium and C. sinensis) that were dried over silica at 20ºC and found that these species were essentially ‘orthodox’.

Subsequently, Hong and Ellis (1995) found that despite desiccation tolerance in some species, many species of Citrus show high variability in storage responses and have complex ‘intermediate’ seed storage behaviour, with some loss of viability at lower moisture contents. Table 1.6 shows that dehydration of seed using various drying methods of predominantly hybribised polyembryonic Citrus species resulted in high levels of germinability at low moisture contents (2 to 10%MC), e.g. C. aurantifolia, C. limon and

45 C. aurantium. However, many of the monoembryonic species are more desiccation sensitive in comparison to these hybrids, for example C. maxima and C. madurensis.

Cultivated Citrus species are characterized by slow germination that is further delayed by drying to low moisture contents and low temperatures (King and Roberts 1980,

King et al 1981, Soetisna et al 1985). For example, C. limon (lemon) seeds displayed drying induced dormancy (increased germination time required for viable seeds) and needed a higher germination temperature (i.e. 30ºC) and a longer germination time (5 weeks) post drying (King and Roberts 1980). Many germination tests in citrus had previously been conducted over shorter time periods and at a lower temperature (e.g. 20ºC)

(King and Roberts 1980). This may be a factor involved (i.e. insufficient time for germination) in some of the variable reports of desiccation sensitivity in citrus. A longer time for water uptake is suggested as the reason for the resultant delay in germination on drying of citrus seeds (Soestisna et al 1985).

Seed coat characteristics (e.g. chemical inhibitors or the physical barrier) have also been suggested as a contributing factor in the delayed germination in citrus as its removal both accelerates and increases the level of germination (Mumford and Grout 1979,

Mumford and Panggabean 1982, King et al 1981, Soestinsa et al 1985). Many subsequent studies remove the seed coat to improve the germination (cf. Table 1.6). However, King and Roberts (1980) found that in C. limon (lemon) seeds, seed coat removal was unnecessary as long as sufficient time was allowed for seeds to germinate. Thus, Ellis et al

1985 (BIOVERSITY guidelines), do not recommended seed coat removal, as it is a time consuming procedure that rarely results in greater cumulative percentage germination. They recommend allowing sufficient time for germination of intact seeds as preferable due to seed coat removal as embryo damage can be caused by seed coat removal.

Table 1.6 Summary of reports of effect of desiccation and liquid nitrogen exposure on seed germination of cultivated and wild species of Citrus Species SSBA Drying %MC Germination Seed Reference method (%) coat -LN2 +LN2 C. aurantifolia Intermediate LF air 7.1 93 85 - Cho et al (lime) 7.9 45 36 + (2002) Silica 7.7 85 58 - 3.3 73 60 - 3.6 22 15 +

Equil. ca.2% ca.80 ca.80 - Hor et al (2005)

C. aurantium Intermediate LF air 20.4 80 60 + Lambardi (sour orange) 10.0 80 94 + et al (2004)

C. halimii Intermediate? Ambient air 9.5 17 25 + Normah & Siti Dewi Serimala LF air 8.9 78 67 em. ax. (1997)

Citrus limon Intermediate Silica 5.4 33 34 + Mumford and (lemon) 1.6 73 51 - Grout (1979)

LF air 15.3 100 33 + Lambardi 10.3 90 67 + et al (2004)

C. maxima Intermediate Silica 8.2 64 ns + Saipari et al. (Pummelo) (1998)

Equil. ca.12 ca. 80 20 - Hor et al ca.9 WC50 ns - (2005)

C. reticulata Intermediate Equil. ca.12 50 20 - Hor et al (Mandarin) (2005)

C.sinensis Intermediate LF air 23.8 50 + Lambardi (sweet orange) 16.4 100 93 + et al (2004)

Citrus Intermediate? Equil. ca.10 WC50 0 + Makeen et al suhuiensis ca.6.5 <10 8 + (2005) LF air ca.9 >80 83 em ax.

C. trifoliata Silica 24.8 74 nd + Saipari et al. 8.8 0 + (1998)

C. madurensis Recalcitrant? Equil. ca.14 WC50 ca.20 - Hor et al (Musk lime) (2005)

C. hystrix Recalcitrant Ambient air 27.0 0 0 + Normah & Siti LF air 11.0 65 60 em ax. Dewi Serimala (1997) ASeed storage behaviour (SSB) cited from IPGRI Species Compendium (Thormann et al 2004), except Citrus suhuiensis (Makeen et al 2005).Abbreviations: %MC - moisture content, Equil. - equilibrium over saturated solutions, LF air -laminar flow air-dried, WC50 -50% loss of initial germinability, em. ax -excised embryonic axis and ns -not stated. 47 Cryopreservation of Citrus seeds

Table 1.6 shows that dehydration of seed of many Citrus species gives protection during immersion in liquid nitrogen. For example, C. aurantifolia, C. limon and C. aurantium had a germination percentage of at least 80% from seeds cryopreserved at low moisture contents (i.e. 2 to 10%MC). In contrast, many of the monoembryonic species had a lower germinability at low moisture contents, which was further reduced (ca.20%) post cryopreservation (e.g. C. maxima and C. madurensis).

Tolerance to cryopreservation varies between species and studies (Table 1.6).

However, even in species with desiccation sensitive seeds, it has been shown that the embryonic axis has some tolerance to both desiccation and cryopreservation, including an endangered South East Asian wild species (Normah and Siti Dewi Serimala 1997, Makeen et al 2005). In addition, the use of other cryopreservation techniques, i.e. vitrification-based and encapsulation-dehydration, have been used for embryonic axes in possible recalcitrant seeded C. madurensis (Cho et al 2001, 2002b, 2002c). Thus, cryopreservation of excised embryonic axis may be the best long term ex situ storage option in desiccation sensitive species of Citrus, as is suggested for many other recalcitrant seeded species (Pritchard

2004). In desiccation tolerant species of Citrus, seed cryopreservation after simple desiccation has been demonstrated as a viable option for C. aurantifolia (Cho et al 2002a,

Hor et al 2005), C. limon (Mumford and Grout 1979, Lambardi et al 2004) and

C. aurantium (Lambardi et al 2004).

Rapid cooling (e.g. direct immersion in liquid nitrogen) has been shown to be detrimental (e.g. coffee, Dussert et al 1998) to survival in some oily seeded species, however most oil seeds do not appear to need a slow cooling rate (Pritchard 2007). Most

48 Citrus species are cryopreserved using a simple desiccation cryopreservation technique, with both rapid cooling and thawing protocols (i.e. 37-40ºC for 2-5 mins)(Cho et al 2002a,

Lambardi et al 2004, Hor et al 2005, Makeen et al 2005).

Thermodynamics

Moisture content measures the amount of water in relation to mass, but does not indicate the water activity of a seed. Seeds are hygroscopic, meaning they absorb and desorb water at equilibrium with the ambient environment. Thus, seed relative humidity, and therefore water activity (RH/100), can be manipulated by equilibrating seeds at a particular RH. The equilibrium relationship between seed moisture content and RH, at a given constant temperature, is a ‘reverse sigmoid curve of a moisture desorption or adsorption isotherm’ (Probert and Hay 2000). Thus, isotherms can be used to define three regions, ‘which indicate how the water is held in the tissue and therefore its thermodynamic properties and the level of physiological activity which can occur’ (Probert and Hay 2000, p. 379). Seed storage physiology can be classified based on water sorption (i.e. tolerance to desiccation to the different regions of the isotherm) and storage responses (Pritchard 2004).

There are three types (phases) of water activity based on binding sites:

Multimolecular (bulk ‘free’ water)

Weakly binding (weak affinity sorption sites)

Strongly bound (high affinity sorption sites)

All three types of water binding are present at all moisture contents. However, at high moisture contents bulk ‘freezable’ water predominates and conversely at very low moisture contents mostly strongly bound water predominates (Leopold and Vertucci 1986, Pritchard

2004).

49 Seed chemical composition (e.g. lipid) affects the absorption and desorption of water (i.e. isotherm curve shape) (Pritchard 2004). Thus, the moisture content of a seed equilibrated over at a particular RH and temperature is a function of the seed lipid content

(LC) (Pritchard 2007). Pritchard (2007) illustrated this using the examples of pea

(1.5%LC), sunflower (33%LC) and sesame (47.5%LC), seeds of each, equilibrated at

18%RH, would have an estimated moisture content of 6.3, 4.3 and 3.4%, respectively.

Loss of seed viability after exposure to freezing temperatures, such as liquid nitrogen exposure (e.g. -196ºC), is often due to lethal ice formation. Thus, survival in desiccation tolerant species on exposure to liquid nitrogen is dependent on removal of water to below the high moisture freezing limit (HMFL) (Stanwood 1985, Pritchard 1995).

HMFL is the content above which seed viability is predicted to significantly decrease by exposure to liquid nitrogen temperatures (Pritchard 2007). Small amounts of ice may form during cryopreservation in seeds at their HMFL and the lipid thermal transitions in lipid rich seeds may enhance the coalescence of small ice crystals into larger more damaging ice crystals (Pritchard 2007). Thus, when seeds are close to their HMFL, cooling and warming rate of liquid nitrogen exposure becomes a critical factor for survival (Pritchard 2007).

Hor et al (2005) proposed that oil rich intermediate seeds do not withstand the presence of freezable water on freezing or thawing. They suggest that cryopreservation is thus optimal in these species at their unfrozen water content (WCu). Differential scanning calorimetry (DSC) can be used to determine the presence of ‘freezable’ (free) water and its relationship with the optimal moisture content for cryopreservation (Vertucci 1989a). In oily seeded species such as Citrus, distinguishing between the phase transitions of either lipid or water allows the determination of unfrozen water content by DSC (Vertucci 1989a) and has been applied in species of coffee (Dussert et al 2001) and cultivated citrus (Hor et al 2005).

50 In four species of cultivated Citrus, the unfrozen water content ranged between 0.09

-1 to 0.14 gH2Og dry weight (ca.10%MC) and correlated with the optimal moisture content for seedling recovery after cryopreservation (Hor et al 2005). Moreover, Hor et al (2005) found that the optimum moisture content was achieved after equilibrium at approximately

75% RH (approx. 10% MC), which coincided with the unfrozen water content. Although the moisture content for desiccation and freezing tolerance varies between species of Coffea and Citrus (oily seeded), it has been shown that in terms of water activity the hydration window lies between 75-85%RH (Dussert et al 2001, 2006; Hor et al 2005).

Both HMFL and WCu are inversely related to the lipid content of seeds (Pritchard

2007). The study by Hor et al (2005) of the optimal hydration status for cryopreservation of oily intermediate seeds found a negative relationship (R=0.99 P=0.0031) between the unfrozen water content and lipid content of seeds in four species of Citrus. The authors combined this data with the other oily non-orthodox seed species’ values of seven coffee species (Dussert et al 2001), neem (Sacandé et al 2000), pea (Vertucci 1989a), Quercus robur (Pritchard and Manger 1998), Quercus rubra (Sun 1999), soybean (Vertucci 1989b) and found a highly significant correlation (R2 0.9236; p>0.0001) with the predictive

-1 equation for WCu (g H2O g d.wt) based on seed lipid content:

WCu =23.4-0.28LC

This equation is similar to the one determined by Pritchard (1995) for seed high moisture freezing limit (HMFL; %wb), compiled from HMFL data of 13 species and their lipid content:

HMFL=23.1-0 .21LC

These equations provide a useful predictive tool, based on lipid content, to estimate suitable seed moisture contents for cryopreservation purposes.

51 1.7 In vitro options in Citrus

In vitro culture techniques in Citrus

As well as having likely complex seed storage behaviour, the seed supply of

Australian Citrus species can be limited and erratic. This means that the development of in vitro techniques (slow growth and cryopreservation) are important as complementary conservation strategies for medium and long-term ex situ germplasm storage. In vitro culture of different types of explant material (e.g. shoot tip, anthers) can be used to initiate different morphogenic pathways such as embryogenesis or organogenesis (e.g. root formation). Citrus has been one of the most widely studied woody perennial plants for in vitro culture (Gmitter et al 1992). However, there has been only one report of in vitro culture in one of the Australian species, C. australasica (Ling and Iwamasa 1997). This report showed a very low frequency of somatic embryo induction. Nevertheless, successful in vitro culture systems and cryopreservation protocols have been reported for many cultivated Citrus species (Engelmann et al 1994, Gonzalez-Benito et al 1994, Duran-Vila

1995, Normah and Siti Dewi Serimala 1997, Gonzalez-Arnao et al 2003).

The development of an in vitro culture system in wild Australian Citrus species would not only allow the investigation of in vitro and cryopreservation storage (e.g. shoot tips) but also provide a useful system for plant multiplication (e.g. horticultural and restoration purposes). Furthermore, in vitro culture is an ideal approach for the exchange of uncontaminated (e.g. viruses and bacteria) material in Citrus. Traditionally, Citrus germplasm exchange has been by bud wood (Ashmore 1997) and seed. Unfortunately, the exchange of bud wood in Citrus has inadvertently led to the detrimental spread of fungal, bacterial and viral diseases within and between countries (Khawale and Singh, 2005).

52 Shoot-tip grafting in vitro has been used for disease eradication and indexing in Citrus worldwide (Navarro 1992, Ashmore 1997, Nvijayakumari et al 2006). Subsequently, many countries have restricted bubwood exchange due to the risk to regional biodiversity

(Khawale ands Singh, 2005). Safer germplasm exchange in Citrus can be achieved by seed and in vitro culture. Shoot tips are an ideal candidate for virus control and elimination in ex situ collection using cryopreservation, as well as for international germplasm exchange

(Kartha 1985) and could be supported by cryopreservation techniques.

Embryogenesis in Citrus

Adventive embryogenesis occurs from the maternal ovule tissues (nucleus and inner tegument) in many angiosperms (Litz and Gray 1992). Many cultivated Citrus species produce in vivo adventive embryos derived from nucellus cells and these develop together with the zygotic embryo to produce multiple embryos per seed (Pérez 2000, Carimi and De

Pasquale 2003). These polyembryonic species include C. limon (lemons), C. aurantifolia

(limes) and C. sinensis (oranges). However, exceptions are the monoembryonic species

C. medica citron (mandarin), C. maxima (pummelo) as well as genotypes of C. reticulata

(mandarin), which can be either mono- or poly-embryonic. The Australian wild Citrus species, C. australasica and C. australis have been reported to be monoembryonic (Clarke and Prakash, 2001).

Induction of somatic embryogenesis is also possible and occurs by one of two pathways (1) indirect embryogenesis, whereby differentiation invariably occurs from callus or (2) direct embryogenesis with the formation of embryos directly from the explant

53 without an intermediate callus stage, including direct secondary embryogenesis that can occur from existing somatic embryos (Litz and Gray 1992).

In vitro plant regeneration via somatic embryogenesis has been achieved for mostly polyembryonic species, for example, C. reticulata, C. aurantium, C. aurantifolia,

C. paradisi, C. sinensis and applied to many cultivars (Perez 2000, Carimi and De Pasquale

2003). Cultures have been initiated using various explant materials including immature seed (Ling and Iwamasa 1997), embryos (Agulilar et al. 1993) anthers and styles (Carimi et al 1995; Gonzalez-Arnao et. al 2003), embryos (Englemann et al 1994) and ovules

(Gonzalez-Arnao et. al 2003, Perez et al 1999, Song et al 1991). Of these techniques, the use of immature ovules appears to have been widely used for polyembryonic cultivars

(Perez et al 1998,1999; Agulilar et al 1993, Song et al 1991, Duran-Vila 1995).

Embryogenesis is induced in most plants by the exogenous application of plant hormones (i.e. auxins) and subsequent removal of the auxin usually allows embryo development. Kunitake and Mii (1995) comment on the unique characteristic of Citrus of stimulation of in vitro embryogenesis using hormone free medium supplemented with relatively uncommon sugars (e.g. lactose), sugar alcohols (i.e. glycerol) and complex natural substances such as malt and casein hydrolysate. However, results have been genotype dependent. Many successful embryogenesis protocols for polyembryonic Citrus species have used media without the addition of plant growth regulators. For example, embryogenic callus and somatic embryos were induced in Citrus species from ovules initiated on basal medium consisting of Murashige and Skoog (1962) (MS) base salts, vitamins, amino acid (glycine), malt extract (500mg-1) and high sucrose (50gl-1) (Tissert and Murashige 1977, Perez et al 1999).

54 Tomaz et al (2001) trialed sugars and sugar alcohols (i.e. galactose, glucose, lactose, sucrose, maltose and glycerol) for their affect on embryo induction from callus in five Citrus genotypes of orange (C. sinensis), mandarin (C. reticulata Blanco) and lime

(C. limonia L. Osbeck). Large numbers of embryos formed in orange and one cultivar of mandarin on medium containing galactose, lactose and maltose, but embryos also formed at a lower frequency on other media treatments (i.e. glucose, sucrose and glycerol). Rangpur lime produced very few embryos and only on medium containing lactose, maltose and glycerol (1-13 embryos/replicate). Kayim and Koc (2006) found that glycerol at 3 to 5% was best for embryo induction from callus. They tested a range of carbohydrates (glycerol, sorbitol, mannitol, lactose, glucose and galactose at different concentrations of 1-5%) in

C. clementina (clementine mandarin), C. sinensis and C. limon. Culture medium containing glycerol at a concentration of 0.3M has been demonstrated to promote embryo formation in many polyembryonic species, C. sinensis (Valencia and Caipira), C. limonia (Rangpur lime) (Tomaz et al 2001) and C. paradisi (grapefruit), as well as monoembryonic

C. papuana, a species closely related to the Australian species (Hao et al 2002).

Embryogenic callus of C. sinensis was shown to use glycerol as a source of carbon for growth and differentiation (Vu et al 1995).

Plant hormones such as BAP (6-Benylaminopurine) have also been used in successful induction of somatic embryogenesis from style explants (including stigma) in polyembryonic Citrus species (Carimi et al. 1995). BAP has also been used in embryogenesis protocols to increase callus multiplication (not induction) for various citrus genotypes (Duran-Vila 1995, Ling and Iwamasa 1997).

55 Somatic embryogenesis, though widely applied to polyembryonic Citrus species

(Marin and Duran-Vila 1991, Perez 2000) has been limited in success in monoembryonic

Citrus species. Attempts to induce embryogenesis from immature ovules of monoembryonic citrus could not be achieved in the 1970’s to 80’s (Debergh and

Zimmerman 1990). Kobayashi et al (1981) suggest the difficulty in embryogenic induction in monoembryonic species results from the lack of nucellus derived embryos and primordium cells (Kumitake and Mii et al 1995). Embryogenesis has since been induced in

C. maxima (pummelo), a cultivated monoembryonic species, using

2, 4-dichlorophenoxyacetic acid (2, 4-D) as well as BAP (Song et al 1991). Embryogenesis in several wild monoembryonic Rutaceae genera has also been demonstrated (Ling and

Iwamasa 1997). Of the wild Rutaceae genera tested, 30% to 1.9% of seeds showed embryogenesis, depending on the species. The lowest level of embryogenesis was reported for the Australian C. australasica, whereby 1 of 52 seeds had somatic embryo formation

(Ling and Iwamasa 1997).

Somatic embryogenesis has been demonstrated as a suitable system for germplasm conservation, by cryopreservation of embryogenic callus and somatic embryos, in predominantly polyembryonic, species of Citrus (Marin et al 1993, Engelmann et al 1994,

Perez et al 1997, 1999; Hao et al 2002). In addition, Pérez et al (2000) reported the successful cryopreservation of C. papuana, a New Guinea species of Citrus closely related to the Australian species, during her PhD studies (Pérez 1995).

It is important to note that the choice of explant for embryogenesis (somatic or zygotic) will depend on the purpose of the work, i.e. clonal multiplication (somatic tissue such as ovule nucellus) or germplasm conservation (embryonic axis (zygotic), not true to

56 type). One disadvantage of embryogenic-based systems (i.e. dedifferentiated cells/callus) is the increased likelihood of somaclonal variation. However, Citrus callus cultures have the unique capacity to grow in an undifferentiated state without the presence of exogenous plant hormones (Jiménez et al 2001), thus reducing this likelihood.

Sakai et al (1991a) reported recovery of morphologically uniform plants regenerated for nucellar cells of C. sinensis after cryopreservation for one year and Pérez et al (1997) found no affect on growth of plants recovered from citrus embryogenic callus after two years cryostorage. Moreover, genetic stability after cryopreservation was confirmed in Citrus species (twelve genotypes) by cytological and molecular examination: no DNA sequence variation was detected by randomly amplified polymorphic DNA, although cryopreservation did change the methylation status (methylation sensitive amplified polymorphism) (Hao et al 2002). In addition, embryogenic callus of C. paradisi

(grapefruit) stored in vitro by slow growth was found to retain embryogenic capacity and was genetically stable by cytological and molecular analysis (no DNA sequence variation).

However, DNA methylation differences were observed between stored and control callus.

In vitro shoot culture in Citrus

In vitro culture of differentiated tissues (i.e. seeds and shoot tips) has the advantage of being more genetically stable than callus based systems (Bajaj 1991). In vitro regeneration via shoot tip and nodal explants of Citrus species has been reported for various cultivated genotypes (poly and monoembryonic), using various combinations of BAP and

NAA ([alpha]-naphthaleneacetic acid alpha]-naphthaleneacetic acid), alone or in combination with other growth regulators (Debergh and Zimmerman 1990, Normah et al

57 1997, Paudyal and Haq 2000, Al-Bahrany 2002). For example, basal medium (e.g. MS) supplemented with BAP, NAA and/or IBA (indole-3-butyric acid) has been used to induce root formation in Citrus species (Carimi and De Pasquale 2003).

Research on citrus shoot tip cryopreservation is more limited than embryogenic systems.

The first report of meristem cryopreservation was by Gonzalez-Arnao et al (1998) in C. trifoliata with 40 to 50% survival achieved. To date, cryopreservation of meristem has been demonstrated in C. aurantuim (Al-Ababeh et al 2002) and and C. trifoliata (Gonzalez-Arno et al

1998, Wang et al 2000, 2002) and C. sinensis (Wang et al 2004) using encapsulation - dehydration and encapsulation - vitrification techniques.

1.8 Summary

This project has had the broad aim of the development of sustainable conservation technologies for selected Australian native fruits, particularly ex situ conservation. As a foundation to the studies described in the subsequent chapters, the following activities were undertaken:

The compilation of a list of rare and threatened Queensland edible species of socioeconomic value as current or potential horticultural species or wild relatives of important cultivated species Identification of target species within this list of probable non-orthodox seed storage behaviour

A review of existing studies in the target species/genus on genetic diversity, ecology, seed physiology (including storage) and ex situ conservation strategies

58 As described in this chapter, Australian Citrus species were selected for further study and the development of ex situ conservation technologies from the identified rare and threatened Queensland taxa of socio-economic importance. Selection was based on following several criteria: (1) vulnerability to genetic erosion (conservation status), (2) economic importance (e.g. novel traits for breeding programs), (3) probable non-orthodox seed storage and lack of existing long term ex situ conservation methods and (4) availability of germplasm (in cultivation in field research collections). Very little is known about the seed physiology, ecology and potential ex situ conservation strategies in the target species, but research has been reported on both cultivated citrus and a number of intermediate seeded species. This literature has been reviewed in this chapter as a background to and to inform the studies undertaken in this project.

1.9 Aims and Objectives

Aim: to investigate seed physiology and ex situ conservation in three Australian wild Citrus species (C. australasica, C. inodora and C. garrawayi). The following investigations were conducted on:

Comparative morphology and anatomy of seed of C. australasica C. inodora and

C. garrawayi were examined as a foundation to understanding the physiological

responses of seed of these species to desiccation and storage, and as a potential tool

for taxa identification (Chapter 2).

59 Studies on seed oil characteristics (i.e. content and thermal properties) in C.

australasica C. inodora and C. garrawayi and correlation with (i) germination of

seeds at a range of temperatures and (ii) natural distribution (Chapter 3).

Seed development and physiology in C. garrawayi, using scanning electron

microscopy, in an attempt to provide simple visual markers to improve seed lot

quality for the development and application of seed storage protocols (Chapter 4).

Seed desiccation and cryopreservation tolerance in C. australasica, C. inodora and

C. garrawayi (Chapter 5).

Cryobiology of seed survival in C. australasica and C. garrawayi by thermal

analysis of phase transitions in seed tissue at different moisture contents. In

addition, analysis of responses to cryopreservation and association with climatic

range and seed oil thermal properties of C. australasica, C. garrawayi and C.

inodora were investigated (Chapter 5).

The development of alternative ex-situ storage techniques, including in vitro culture

and cryopreservation methods in C. australasica C. inodora and C. garrawayi:

o Investigation of somatic embryogenesis in both cultivated Citrus species

(mono- and polyembryonic) and Australian wild species: C. australasica, C.

inodora and C. garrawayi, using ovules and seeds, to facilitate both mass

propagation and the development cryopreservation storage options (Chapter

6.3.1).

60 o Investigations of micropropagation and shoot-tip cryopreservation as

regeneration and storage options in C. australasica, C. inodora and

C. garrawayi (Chapter 6.3.2).

o In vitro germination of cryopreserved seeds of C. inodora and C. garrawayi.

This option was investigated to improve recovery levels (e.g. viability loss

due to fungal infection) and to facilitate subsequent micropropagation (e.g.

in vitro nodal cuttings) of cryopreserved seeds in rare and threatened

Australian wild Citrus species (Chapter 6.3.3).

Assessment of sustainable conservation strategies for Australia’s valuable wild

Citrus diversity, in view of the findings of this study, to make recommendations

about application of techniques developed and the direction for future research

(Chapter 7).

CHAPTER 2 COMPARATIVE SEED MORPHOLOGY OF CITRUS

AUSTRALASICA, C. INODORA AND C. GARRAWAYI

62 Chapter 2 Comparative seed morphology of C. australasica, C. inodora and C. garrawayi

2.1 Introduction

Seed physiological responses (e.g. germinability over a range of conditions), is often linked to seed characteristics and natural distribution/ecology of a species (cf. 1.8).

Hong and Ellis (1996) suggest estimation of seed storage behaviour of a species can be based on the multiple criteria of taxonomy, plant ecology, fruit characteristics and seed characteristics (e.g. seed weight, shape and moisture content). Although there are extensive reports on the seed physiology and to a lesser extent, seed morphology in cultivated Citrus species, there have been no reports in Australian wild Citrus species. Additionally, seed morphological features, such as micromorphology (e.g. seed coat topography), can be a useful tool for taxa identification. Detailed seed morphology and anatomy has not been described previously for the northern Queensland Citrus species, C. garrawayi and

C. inodora. However, Clarke and Prakash (2001) found close floral and embryological similarities between cultivated Citrus species and the southerly distributed species of

Australia, C. australasica and C. australis.

Investigations of the comparative morphology and anatomy of species of Australian wild Citrus (C. australasica, C. inodora and C. garrawayi) were undertaken, as a foundation to understanding the physiological responses of seed of these species to desiccation and storage.

63 2.2 Materials and methods

Fruit and seed material

Fruits of Citrus australasica, C. inodora and C. garrawayi were harvested/sourced from a research field collection at the QLD Department of Primary Industries in southern

Queensland (SQ DPI) and northern Queensland (NQ DPI), Brisbane Botanic Gardens

(BBG) and a commercial supplier (Comm.). Fruit was stored at 15ºC (dark) until seeds were extracted for use. Fruit characteristics of each harvest were recorded from 20 to 50 randomly sampled mature fruits in terms of rind colour, size (length x width) and weight.

Microscopy

Scanning electron microscopy (SEM) was used to observe the surface topography

(n=8 to 10) and seed coat anatomy (n=8 to 10) of seeds of each species randomly selected from two separately harvested seed lots: C. australasica (SQ DPI 2006; BBG 2006),

C. inodora (NQ DPI 2005; SQ DPI 2005) and C. garrawayi (SQ DPI 2005, 2006). Seed was processed for SEM by drying over silica for ten days (ca. 5%MC). The seeds were then critical point dried and mounted on SEM stubs with double-sided adhesive and sputter coated in gold prior to observation using a FEI Quanta 200 Environmental SEM at 10kV

(FEI Company, USA). Ten seeds of each species were longitudinally sectioned and examined using a stereo light microscope. Characteristics were recorded in terms of colour and external and internal morphology.

Moisture content determination

The moisture content (MC) of seeds was determined gravimetrically after drying at

103ºC for 18 ±1h and presented on a fresh weight basis (%MC). Seed moisture content

64 measurements were determined for each seed lot, either from four replicates on 25 seeds or

10 individual seeds.

Oil extraction and content determination

Seed oils were extracted for 2 replicates of 8 seeds of each species (C. inodora and

C. garrawayi) after 10 days silica drying. Dry seeds were ground and oil extracted using a

FexIKA 200 solvent (petroleum ether) based system (IKA-Werke GmbH KG). Extracted oils were measured gravimetrically and the lipid content (%LC) was presented on a seed dry weight basis.

2.3 Results

Fruit characteristics

Fruit size and appearance for C. australasica, C. inodora and C. garrawayi are shown in

Figure 2.1. It can be seen that whilst fruit size characteristics were fairly similar across years and harvest locations for C. inodora and C. garrawayi, there was considerable variation in C. australasica (Table 2.1). Mean fruit weight was also most variable in

C. australasica ranging from 9 to 28g for fruits harvested from three separate locations

(2004-2006). All three species had ‘finger’ shaped fruits (Figure 2.1). Citrus. australasica was the most variable in terms of outer rind colouration, being either mixtures or individual colours of green, red, purple and yellow. Citrus inodora fruits were green and/or yellow and C. garrawayi fruits were green coloured. Figure 2.2 shows these fruits in section, illustrating some of the range of pulp colouration (cream to pink) that was observed in

Australian wild Citrus species.

Fig. 2.1 Whole fruits of (A) Citrus australasica (B) C. inodora

and (C) C. garrawayi

Table 2.1 Characteristics of seed lots harvested between 2004-2006 for Citrus australasica, C. inodora and C. garrawayi

Species Year Source Fruit characteristics Seed characteristics Mean Mean Mean Mean fresh Mean dry Mean length width weight (g) weight (mg) weight (mg), moisture (cm) (cm) content (%wb) C. australasica 2006 BBG 11.1 ±0.5 2.2 ±0.04 27.7 ± 1.1 32.6 ±0.7 20.2 ±0.4 37.8 ±1.3

2005 SQ DPI 5.1 ± 0.3 1.7±0.04 8.6 ± 0.8 19.9 ±2.0 11.2±1.5 44.5 ±1.6

2004 Comm. 8.5 ±0.1 1.6 ±0.03 14.1 ±0.5 29.6 ±1.5 18.0 ±.18 38.8 ±0.79

C. inodora 2006 SQ DPI 4.6 ±0.09 1.8 ±0.03 9.3 ±0.4 38.8 ±0.8 27.1 ±1.1 30.1 ±3.0

2005 SQ DPI 5.1±0.01 1.8 ±0.07 11.3 ±0.8 31.7 ±1.8 21.1±1.1 33.2 ±0.1

2005 NQ DPI 4.9 ±0.02 1.5±0.01 7.4 ±0.7 40.1 ±0.5 24.9 ±0.6 37.9±1.5

C. garrawayi 2006 SQ DPI 7.3 ±0.2 2.5 ±0.03 30.5 ±0.7 22.8±1.1 12.0 ±0.6 44.0±2.0

2005 SQ DPI 8.3 ±.0.2 2.6 ±0.02 34.6 ±0.7 26.9±2.2 17.0±.1.3 40.5±1.7

Fruit characteristics mean (±se) recorded for 20-50 fruits randomly selected mature fruits per harvest, except NQ only eight fruits. Seed characteristics mean (±s.e.) of ten replicates of individual seeds for seed each harvest, except C. australasica 2004 Comm. (four replicates of 25 seeds). Moisture content determined gravimetrically after oven drying (18h ±1) and presented on a fresh weight basis (%wb).

67 A B

Fig. 2.2 Whole cut fruit of (A) Citrus australasica, (B) C. inodora and (C) C. garrawayi

Whole seed morphology

The mean number of seeds per fruit varied between species at 15±3 for

C. australasica, 21±2 for C. inodora and 3±1 for C. garrawayi (Table 2.2). Citrus australasica showed the greatest variability in seed number per fruit from different harvest locations and years (data not shown). Four harvests of fruits of C. australasica yielded negligible seeds (i.e.

Mature seeds were whitish cream to cream in colouration, 5-6mm in length, ovoid in shape, with a woody seed coat in all three species (Table 2.2, Figure 2.3). The seeds

A B

Fig. 2.3 Whole seed of (A) Citrus australasica, (B) C. inodora and

of C. australasica and C. inodora had a flattened ovoid shape, with a rounded and textured upper surface and a flat, smooth lower surface (Figure 2.3A,B). Citrus garrawayi differed from this, having a rounded shape to the seeds (Figure 2.3C). Figure 2.3 illustrates the semi-textured (C. australasica), smooth (C. inodora) and textured (C. garrawayi) visual appearance of the seeds. The mean percentage moisture content of seed lots of harvests between 2004 to 2006 showed some variation for each species: 37.8±1.3 to 44.5±1.6

(C. australasica), 30.1±3.0 to 37.9±1.5 (C. inodora) and 40.5±1.7 to 44.0±2.0

(C. garrawayi) (Table 2.1). Seeds of all three species had a mean fresh weight range between harvest year and locations of 19.9±2.0 to 32.6±0.7 (C. australasica), 31.7±1.8 to

40.1±0.5 (C. inodora) and 22.8±1.1 to 26.9.0±2.2 (C. garrawayi) (Table 2.1). Citrus inodora and C. garrawayi had oily seeds, i.e. mean % lipid content of 54.3 ±0.2 and

30.2±0.3, respectively (Table 2.2). The lipid content of C. australasica was not determined

(lack of equipment availably). 69 Table 2. Comparative seed characteristics of Citrus australasica, C. inodora and C. garrawayi Structure Characteristic Species C. asutralasica C. inodora C. garrawayi Whole Seed Shape Flattened ovoid Flattened ovoid Ovoid Dimensions (mm)A 6 x 4 x 2 6 x 4 x 3 5 x 4 x 4 Mean (±se) number seeds/fruit B 15± 3 (n=10) 21±2 (n=10) 3±1 (n=45) Mean (±se) lipid content (%) nd 54.3 ±0.2 30.2 ±0.3 Seed coat Mean (±se) thickness (μm) CD 147±9 109±8 204±9

Seed coat colour/texture A Cream and semi-textured (rounded Beige cream, smooth and glossy Whitish cream and textured dimpled top, flat smooth appearance (rounded top, flat undersurface) undersurface)

Tegmen colour A Golden tan/brown Golden tan/brown Light golden tan sometimes with pinkish colouration Ultrastructure C Testa Thick pitted epidermal fibres, Thick pitted epidermal fibres, Thick pitted epidermal fibres, extensive mucilage and very small extensive mucilage and very small extensive mucilage and long (hair protrusions protrusions like) protrusions Protrusion length (μm) 2-19 (n=29) 3-10 (n=28) 102 -351 (n=17)

Tegmen Cellular endosperm (vestiges) Cellular endosperm filled with Cellular endosperm filled with filled with storage bodies storage bodies storage bodies Embryo Colour A Green to pale green Whitish cream Greenish cream to cream

Morphology AC Well-differentiated embryonic axis Differentiated plumule and well Well-differentiated embryonic axis (plumule and radicle) and differentiated radicle and cotyledon (plumule and radicle) and cotyledon cells packed with storage cells filled with storage bodies cotyledon cells packed with storage bodies (ca.5μm diameter) (ca.5μm diameter) bodies (ca.5μm diameter)

A Characteristics determined from light microscope examination of ten individual seeds (n=10) for each species, except C. garrawayi (n=20). BSeed number determined from fruits sourced, C. australasica (BBG 2006), C.inodora (SQ DPI 2006) and C. garrawayi (SQ DPI 2005). CCharacteristics determined from scanning electron micrographs of either external seed coat surface, sectioned seed coats or excised embryonic axes (n=3-10). Protrusion length determined without removal of mucilage layer. DThickness of sectioned seed coats measured outside the micropylar or chalaza region; mean seed coat thickness was significantly different between all three species (P=<0.01, LSD).

70 Seed topography

The surface topography differed between the three species as viewed by scanning electron microscopy (SEM). The differing textural appearance at reasonably low magnification (x50) being either semi textured (C. australasica, C. inodora and C. limon) and textured (C. garrawayi) (Figure 2.4). The surface topography of the cultivated speices,

C.limon was also included for comparison

A B

C D

Fig 2.4 Scanning electron micrographs (x50) of seed (A) C. australasica, (B) C. inodora (C), C. garrawayi and (D) cultivated species, C. limon .

71 Examination of seeds at a higher magnification (x300-x1500) revealed that epidermal fibres, longitudinally stretched along the axis of the seed, and protrusions account for the textured appearance as illustrated in Figure 2.5A-D.

A B

C D

E F

Fig. 2.5 Scanning electron micrographs of surface topography of seed coats of C australasica seeds (A x200, B x1500), C. inodora (C x300, D x1500) and C. garrawayi (E x200, F x1600). 72 The semi-textured appearance of C. australasica and C. inodora seed coats are explained by the presence of epidermal fibres and very small protrusions (>20μm in length) beneath a layer of mucilage (Table 2.2). Whilst, the highly textured seed surface of

C. garrawayi results from the numerous long hair like protrusions (102 to 351μm in length) covering the seed surface (Figure 2.5E-F, Table 2.2). The protrusions were either the ends of fibres or arose from the side (tangential) walls of the fibres (Figure 2.5E). Figure 2.4D illustrates the surfaces of the cultivated C. limon (lemon) with numerous very small protrusions similar to those observed in C. australasica and C. inodora.

Seed coat anatomy and morphology

The mature seed coats of C. australasica C. inodora, and C. garrawayi all display a distinct outer seed coat (outer integument) and inner seed coat (tegmen) (Figure 2.6, 2.7).

The mean thickness of the seed coat (outer and inner seed coat) was found to be significantly different between C. australasica C. inodora, and C. garrawayi at 147±9,

109±8 and 204±9μm, respectively (Table 2.2). The mature seed coat anatomy of all three was similar. The developed outer integument consisted of several layers of thin walled parenchyma cells and an outer protective layer of epidermal fibres from which emerged protrusions covered in a layer of mucilage (Figure 2.8). The mucilage layer was slippery and shiny when moist and hardened upon drying. One distinguishing characteristic of all three species was the thick pitted walled fibres of the epidermis (exotestal) (Figure 2.7,

2.8A-D). These epidermal fibres are similar to those observed in cultivated Citrus species, as illustrated in Figure 2.7E of a cultivated mandarin seed coat. Vascular structures were

73 observed in longitudinal sections of the seed coat in the chalazal region in all three species

(Figure 2.7B, 2.8E).

The tegmen was golden tan to brown in C. australasica and C. inodora and light golden tan (sometimes with pinkish tinge) in C. garrawayi (Figure 2.6, Table 2.2). The tegmen consisted of several layers of remnant inner integument, nucellus and endosperm.

The vestiges of the endosperm formed a final distinctive cellular layer (ca. 3 cells thick) of tegmen that surrounded the embryo (Figure 2.6, 2.8F). The endosperm cells were packed with spherical bodies (Figure 2.8G).

Fig. 2.6 Seed anatomy of A longitudinally sectioned seeds,

as viewed by light microscopy, A A illustrating seed coat layers in B

(A) C. australasica, (B) C. inodora (C) and

C. garrawayi.

B C

B C A

D E

Fig. 2.7 Scanning electron micrographs illustrating anatomical features of sectioned seed coats of Citrus australasica (A x400, B x800) C. inodora (C x400), C. garrawayi (D x400) and cultivated mandarin (E x400).

75 A B

Fig. 2.8. Scanning electron micrographs illustrating detail of structures of seeds: layers of integument with thick layer of mucilage overlying exotestal fibres and parenchyma cells (A)

Citrus australasica (side seed coat x800) and (B) C. inodora (micropylar region x1500); characteristic pitting of thick walled fibre cells in (C) C. australasica (x2500) and (D) C. garrawayi (x3000); (E) vascular structures of chalazal region (C. inodora, x12000); (F) endosperm layers of tegmen (inner seed coat) (C. garrawayi x1500) and (G) storage bodies in endosperm cellular layer (C. garrawayi, x1500).

76 Embryo morphology

Embryo colour varied between the three species: green (C. australasica), whitish cream (C. inodora) and greenish cream to cream (C. garrawayi) (Table 2.2). Seeds of all three species had a single minute embryonic axis (monoembryonic), between two large cotyledons, and located near the microplyar and extremely rarely (<1%), two seedlings were observed in growing from seeds of C. inodora. The embryonic axes were about 1mm in length and varied in shape, i.e. less elongated in C. inodora compared to C. australasica and C. garrawayi. Light microscopy examination of sectioned seeds revealed the embryo axes of all three species had well-differentiated radicle and plumule poles (Figure 2.9,

Table 2.2).

A B

C Fig. 2.9 B Embryonic axisD

morphology, as viewed by light

microscopy, of three Australian Citrus species: (A, B) C. australasica, (C) C. inodora C D and (D) C. garrawayi .

E D E

77 D Scanning electron microscopy examination of sectioned seeds revealed that the cotyledon cells of all three species were packed with spherical storage bodies (ca. 5μm diameter) that were most likely predominantly lipid bodies in these oily seeds (Figure 2.10, Table 2.2).

A B

C D

E F

Fig. 2.10 Scanning electron micrographs of cotyledon cells of seed illustrating

storage bodies (arrow): Citrus australasica (A x3000, B x6000) C. inodora (C x3000, D x200), and C. garrawayi (E x3000, F x6000). 78 2.4 Discussion

Fruit and seed characteristics

The Australian wild Citrus species, C. australasica, C. inodora and C. garrawayi had a unique ‘finger shape’ that was distinctive compared to the larger rounded fruits of cultivated citrus (cf. Figure 1.4). Seed morphology of Australian wild Citrus species exhibited both similarities and differences compared to cultivated species. Seeds of cultivated Citrus spp. are 8-15mm in length and frequently exhibit polyembryogeny

(Corner 1976). The seed coat of cultivated Citrus species have a characteristic epidermal layer consisting of thick walled, pitted and lignified fibres, covered by a mucilaginous layer

(Vaughan 1970, Corner 1976, Boesewinkel 1978). These seed characteristics were also observed in the seed coat of the three Australian Citrus species studied here. In addition, the vascular strands observed in the chalazal region of all three species is most likely the vascular supply of the raphe to the seed, as occurs in cultivated citrus (Vaughan 1970,

Boesewinkel 1978). However, the three Australian wild Citrus species studied differ from most cultivated Citrus species in being monoembryonic and smaller in seed size

(ca.6x4mm). The three ‘true species’ (C. medica, C. maxima and C. reticulata) of cultivated Citrus are also monoembryonic, but have a larger seed size than the Australian wild species (e.g. C. maxima (15x7mm) and C. reticulata (8x4mm) (Radhamani et al

1991).

The endosperm of cultivated Citrus species (final layer of tegmen) consists of a few cellular layers, with cells containing oil drops (bodies) and minute aleurone grains (protein bodies) (Vaughan 1970). The numerous spherical bodies observed in the endosperm cells in the three Australian species are most likely also lipid and protein bodies.

The tegmen may play an important enzymatic function in the germination processes in Citrus species. High peroxidase activity has been reported in the tegmen of cultivated citrus (grapefruit and tangerine), which was higher than that of other seed parts (i.e. cotyledons, testa and whole seed) (Davis 1942). One aspect of drying seed of citrus is that the tegmen becomes brittle and separates from its contact to the embryo. Thus, drying could potentially affect the physiological responses (e.g. germination). However, the role of the remnant endosperm layer in mature Citrus seeds and any possible interactions with the embryo are poorly understood making it difficult to speculate if the ‘damaged’ endosperm contributes to the unusual seed physiology of this genus.

Seed topography

Radhamani et al (1991) defined two major groups in cultivated Citrus species based on seed coat protrusions: (A) C. aurantifolia, C. limon, C. limonia, C. reticulata had glossy textured seed coats and very small protrusions arranged in parallel rows giving a beaded appearance (e.g. C. limon shown in Figure 2.4C) and (B) C. aurantium, C. maxima and

C. maderaspatana had many protrusions that were longer, forming a hairy appearance on the seed coat surface. Previously, it had been observed that C. sinensis had long developed protrusions (Boesewinkel 1978). Furthermore, Mumford and Panggabean (1982) reported that the more desiccation tolerant species of C aurantifolia (lime) and C. limon (lemon) had numerous small protrusions, whilst Citrus aurantium (sour orange) was characterised by numerous but much longer seed coat protrusions and had greater sensitivity to desiccation.

In this study, the surface topography of seeds of C. australasica and C. inodora were most like the desiccation tolerant cultivated species C. aurantifolia and C. limon.

Citrus australasica and C. inodora (6x4mm) were also most comparable in terms of seed size to C. aurantifolia (6x4mm) (Radhamani et al 1991), which has small seeds compared to other cultivated species (Radhamani et al 1991). In a study of genetic relationships among citrus and related genera, using ten enzymatic systems, it was found that three

Australian Citrus species (C. australasica, C. australis and C. inodora) and one New

Guinea species (C. warburgiana), were most closely related to the cultivated citrus of the citron-lime group (Herrero et al 1996b). Thus, seed size and topography studied here tend to confirm this relationship. Additionally, pollen morphology of C. australasica and

C. warburgiana was reported most similar to the cultivated citrus group, lime-lemon-citron

(Kozaki and Hirai 1981). Furthermore, detailed morphological studies have suggested that

C. aurantifolia and C. limon are of trihybrid origin, including a possible Microcitrus parent

(Barrett and Rhodes 1976, Spiegel-Roy and Goldschmidt 1996). Molecular analysis, using

RFLPs and RAPDs, has supported the proposal of Microcitrus (now Citrus) as a possible ancestor of C. aurantifolia (Federici et al 1998). Thus, the morphological similarities (seed size and small protrusions) observed both in the Australian wild species C. australasica,

C. inodora and the cultivated species C. aurantifolia and C. limon might reflect this closer affinity.

The surface topography of C. garrawayi seeds was shown to be similar to that of the cultivated species C. aurantium (Mumford and Panggabean 1982) and C. sinensis.

81 (Boesewinkel 1978), particularly the longer epidermal protrusions identified in the outer seed coat layer. However, the protrusion length appears longer in C. garrawayi (102-

351μm) compared to these cultivated species (Boeswinkel 1978, Mumford and Panggabean

1982). To the author’s knowledge, no phylogenetic studies of Citrus have included

C. garrawayi. The morphological feature of longer protrusion length in C. garrawayi and the cultivated species C. aurantium and C. sinensis confirm the value of investigations elucidating relationships between C. garrawayi and the cultivated species.

Embryo morphology

As previously discussed, Citrus species can be either monoembryonic or polyembryonic. Adventive embryos arise in vivo from embryo initial cells of the nucellus in polyembrynonic Citrus species (Wakana and Uemto 1987 1988, Prakash and Lim 1996,

Carimi and De Pasquale 2003). The Australian species, C. australasica has been reported as monoembryonic (Clarke and Prakash 2001). The present study confirmed this finding and finds that both C. garrawayi and C. inodora also display monoembryony. Although single embryos were predominantly observed in C. inodora embryos, a small number

(<1%) of seeds were observed to grow two seedlings. Prakash and Lim (1996), in a review of reproduction in Rutaceae, also discuss the occurrence of polyembryony in monoembryonic species from the occurrence by: (i) budding or cleavage of the zygotic embryo in some Citrus cultivars (Frost 1926, 1938) and in monoembryonic cultivars such as C. reticulata cv. Clementine (Cameron and Garber 1968) or (ii) potential false polyemebryony through the presence of double or multiple embryo sacs in ovules as observed in monoembryonic C. grandis (Bacchi 1943, Banerji 1954).

82 Cotyledons of cultivated Citrus species are rich in fatty substances (Boesewinkel

1978), composed of oil drops (oil bodies) and aleurone grains (protein bodies) (Vaughan

1970). The SEM of cotyledonary cells of Australian wild Citrus species in the present study revealed numerous spherical bodies, most likely protein and oil storage bodies. Cultivated citrus seeds have been reported to contain between 22 - 52% lipid and 7-16% crude proteins (Vaughan 1970, Hor et al 2005). The two northern Australian species, C. inodora and C. garrawayi also contained high lipid content (LC), 30 and 54% respectively. Thus the storage bodies that were observed are most likely predominantly oil bodies.

The storage bodies observed in this current study were comparable in size (i.e. ca.

5m in dry seeds) to oil bodies observed in the oil rich seeds (52% LC) of neem

(Azadirachta indica) (Sacandé et al 2000). Neem belongs to the Meliaceae family, which is closely related to Rutaceae and has been classified as intermediate (Hong and Ellis 1996) in terms of seed storage behaviour. However, as occurs in citrus (Hong and Ellis 1995), reports of seed responses to desiccation have been variable (Gamene et al 1996, Berjak et al 1995, Esswara et al 1998, Sacandé et al 2000). The oil bodies in seeds of neem remained stable on extreme drying (6%MC) or after storage at 4ºC for two months at room temperature. Similarly, the storage bodies (most likely oil) were intact after drying to low moistures (ca.5%MC) in C. australasica, C. inodora and C. garrawayi. Although oil bodies were intact on drying and storage in neem, imbibition lead to the coalescence of oil bodies and resulted in loss of cellular integrity (Leprince et al 1998).

This is consistent with the proposal of Crane et al (2006) that intermediate oily seed physiology is partly attributable to imbibition injury on hydration of crystallized (i.e. solid phase) lipids rather than to sensitivity to desiccation (cf. 1.8.3). Thus, intermediate oily

83 seeded species, such as citrus, may not tolerate imbibition when lipids are crystalline

(solid). The melting point of seed oils is species dependent and can be determined using differential scanning caliormetry. The thermal properties of C. australasica, C. inodora and

C. garrawayi were investigated and reported in Chapter 3.

Eco-physiology and seed morphology

There have been many reports on seed storage physiology in cultivated Citrus species but very little is known about the in situ ecology, including seed dispersal and seedling establishment. Moreover, there have been no reports of seed physiology and ecology in any Australian wild Citrus species.

This present study reported that Australian wild Citrus species are oil rich, as previously reported in cultivated Citrus seeds (Vaughan 1970). The oil reserves are stored in the cotyledons in cultivated Citrus seeds and are mobilised on the second day of imbibition, with the rapid lipolysis of 58% of the total lipid occurring from day 2-16

(Garcia-Agustin et al 1992). Thus, lipids are an important seed reserve early in the sequence of germination events after imbibition. This suggests their main role in Citrus relates to early germination and seedling development as opposed to a reward for seed dispersal agents (e.g. small mammal), although other attributes, such as the fleshy edible fruits, of both wild and cultivated species may act as inducement to seed dispersers. The wild populations of Australian Citrus species offer an opportunity to future researchers to investigate the seed dispersal ecology of the Citrus genus.

84 Seeds have adaptations that facilitate germination and seedling growth. Seed coats are often suggested as a contributing factor (chemical or physical inhibitors) in the erratic, slow and reduced germination in response to desiccation and storage that has been observed in citrus seeds (Mumford and Grout 1979, Mumford and Panggabean 1982, King and

Roberts 1980). Additionally, seed coat patterns have been correlated with ecological factors. For example, a relationship between germinability and habitat has been observed in the genus Tulbaghia (Alliaceae) (Vosa 2003). Seeds from dry habitats had seed coats composed of loose cells capable of absorbing water quickly, whilst species from wet habitats had cells that were welded together and somewhat impermeable to water (Vosa

2003). In this present study, all three Australian Citrus species were covered in a thick mucilage layer. The purpose of the mucilage (pectinaceous substance) layer, which readily absorbs water (Boseswinkel 1978), and seed coat protrusions in Citrus are unknown. The albedo (pectin rich) of immature fruitlets of C. limon acts as a reservoir of moisture for juice sacs, seeds and leaves in times of drought and has been suggested as a possible adaptation to dry conditions (Schröder et al 2004). Thus, the mucilage of the outer seed coat layer in citrus (cultivated and wild species) may also be an adaptive feature for dispersal in dry conditions. This appears to fit with the limited knowledge of seed dispersal in C. garrawayi and C. inodora fruiting as the fruiting period occurs predominantly during the dry season in northern Queensland (cf. Figure 1.5).

Barrett and Rhodes (1976) used a multitude of characters to elucidate affinity relationships in cultivated citrus and related genera, distinguishing two main groups: (1)

C. aurantium, C. maxima, C. paradisi (grapefruit), C. reticulata, C. sinensis and (2)

C. aurantifolia, C. limon, C. medica. Additionally, a third group containing the Australian

85 wild Citrus species (previously Eremocitrus and Microcitrus species) was characterised.

Molecular studies have supported these affinity relationships (Herrero et al 1996b, Federici et al 1998, Nicolosi et al 2000). Interestingly, Citrus species from Group 1 have longer seed coat epidermal protrusions i.e. C. aurantium (Mumford and Panggabean 1982) and

C. sinensis (Boesewinkel 1978). Citrus reticulata have been reported to have ‘beaded’

(very small) protrusions (Radhamani et al 1991), although somewhat long (intermediate length) protrusions were observed in a mandarin cultivar in this present study. Thus, the presence of long developed protrusions may be indicative of either a common ancestor/parentage or an adaptive characteristic to common ecological conditions (e.g. dry vs. moist habitat). Either or both of these proposals are feasible as the Citrus genus is monophyletic nature and in the case of Australian Citrus, an early divergence (isolation) from the rest of the genus occurred (i.e. ecological adaptations) (cf. Chapter 1.4).

An overall tendency of the Citrus species in affinity Group 1, with their longer seed coat protrusion, is towards reduced desiccation tolerance (Barton 1943, Mumford and

Panggeabean 1982, Hor et al 2005, Saipari et al 1998), compared to Group 2, with very short protrusions (Barton 1943, Mumford and Grout 1979, Mumford and Panggeabean

1982, Cho et al 2002a, Hor et al 2005). However, it should be noted that the responses to desiccation reported in Citrus species in both groups have been variable and additionally there are no reports of C. medica (group 2) seed coat topography or desiccation tolerance.

Even so, protrusion length and/or mucilage layer, like fruit pectin, may be an ecological adaptation to dry conditions in citrus and appears to be linked with existing phylogenetic groupings (molecular and morphological studies).

86 Functions of mucilage, other than water retention, include regulation of germination by forming a barrier to oxygen (Boesewinkel and Bouman 1995). Alternatively, the mucilage layer may play a role in seed dispersal (e.g., assist transition through an animal gut) or adherence to soil/animals as observed in other seeds (Boesewinkel and Bouman

1995). Mumford and Panggabean (1982) mention the lack of a mechanism for the release of seeds from the fruit in cultivated citrus and suggest that microorganisms may play a role as the mucilage layer is very conducive to fungal growth. In particular, they propose the mucilage as a medium for microflora colonisation and subsequent breakdown of the seed coat and/or inhibitory substances allowing germination of the embryo. Little is currently known of ecology and distribution in any Citrus species. Further studies are needed on natural dispersal of seeds to determine the function of the seed mucilage, importance of protrusions and other structural features of seeds of the Citrus genus.

Seed size and shape can relate to germination capacity and final seedling growth. Matilla et al (2005), in a recent review, concluded that seed size and shape are key factors in determining seed fate and persistence in the soil. Small and rounded seeds have been reported to persist longer in the soil (more easily buried, Thompson et al 1993), have lower predation (Hulme 1998) and less exposure to germination-promoting stimuli (Baskin and

Baskin 1998, Milberg et al 2000) compared to large, elongated or flattened seeds (Matilla et al 2005). Citrus garrawayi seeds were rounded and somewhat smaller than the flattened ovoid shaped seeds of C. australasica and C. inodora. It is likely that the morphological distinctions observed in this present study are associated with these and/or other ecological adaptations. For example, the rounded seed shape and longer protrusion length in

C. garrawayi would result in an increased surface of seeds which could facilitate microflora

87 colonisation and germination when seeds are moist. This proposal is supported by the much thicker seed coat (e.g. restrictive to radicle emergence) observed in C. garrawayi seeds compared to C. australasica and C. inodora.

A wider study on seed coat structure and ecology in the six Australian wild Citrus species and cultivated species, such as C. medica, would prove valuable in the interpretation of phylogenetic relationships and seed storage behaviour in citrus. The wide range of ecological adaptations (wet rainforest through to desert environments) in the

Australian wild Citrus species offer a valuable opportunity to gain better taxonomic and ecological understanding of this economically important genus.

CHAPTER 3 EFFECT OF TEMPERATURE ON GERMINATION OF

CITRUS AUSTRALASICA, C. INODORA AND C. GARRAWAYI SEEDS

89 Chapter 3 Effect of temperature on germination of Citrus australasica, C, inodora and

C. garrawayi seeds

3.1 Introduction

Citrus seeds have been reported to germinate at a range of temperatures (11-35ºC)

(Mobayben 1980, Soetisna et al 1985) but 30ºC is recommended as the best temperature, with germination usually completed by 4-7 weeks (Ellis et al 1985). There is an estimated minimum temperature of about 6-10ºC (Mobayben 1980). This germination temperature range is consistent with the climatic restriction (subtropical to tropical) of Citrus species.

As reported in Chapter 2, C. inodora and C. garrawayi seeds have high lipid content of 54% and 30%, respectively. Investigation of the physico-chemical characteristics of seed oils of three cultivated Citrus species (C. aurantium, C. sinensis and C. mitis) revealed a uniform melting point of 7.0±0.1ºC (El-Adawy et al 1999). Interestingly, the lipid melt point (7ºC, El-Adawy et al 1999) coincides with the estimated minimum germination temperature (6-10ºC, Mobayben 1980) in cultivated citrus seeds.

The thermal behaviour of seed oils (i.e. storage reserves) differs based on their fatty acid composition, such that saturated fats have a higher melting point (i.e. solid to liquid phase) than unsaturated fats. Differential Scanning Calorimetry (DSC) provides a useful tool for the non-invasive observation of thermal behaviour in seed tissues (e.g. cotyledons) of oil rich seeded species (Crane et al 2003, Walters et al 2005, Lehner et al 2006). Phase transitions (e.g. melting from solid to liquid state) result in heat being absorbed

(endothermic event) or liberated (exothermic event). DSC thermocurves (heat flow)

illustrate the endothermic and exothermic changes during phase transitions in the sample.

These thermocurves can be used to determine the characteristic lipid profile of a species on warming from low temperatures, including the onset temperature, end melt point temperature and enthalpy of the lipid phase transition.

Optimum temperature of germination is unknown in the wild Australian species of the Citrus genus. Moreover, there have been no reports of germination behaviour in any

Citrus species at a range of temperatures and correlation with their natural distribution (e.g. climate data) and thermal properties of their seed oils. The climatically distinct and geographically separate distribution (cf. Table 1.5) of C. australasica (warm subtropical),

C. inodora (warm tropical) and C. garrawayi (hot tropical) means comparative differences in germinability and seed oil thermal properties may be reflected in germinability at a range of temperatures. Ecology of seed dispersal is unknown in Citrus, but seeds are short lived.

Thus, the monthly mean temperatures of fruiting (cf. Figure 1.5) most likely represent the seed dispersal period and were used to relate likely temperature of germination in situ.

The objectives of this study were two fold: (1) to determine the optimum temperature for germination in C. australasica C. inodora and C. garrawayi, (2) to relate germinability, at a range of temperatures, to species distribution (i.e. climate data) and thermal behaviour of seed oils (i.e. melt point).

91 3.2 Materials and methods

Fruit and seed material

Fruits of Citrus australasica, C. inodora, and C. garrawayi were sourced and handled as detailed in Materials and Methods section 2.2.

Seed Moisture content determination and oil extraction

Seed moisture content determination and seed oil extraction was as described in section 2.2. Moisture content was determined from ten individual seeds for each seed lot.

Climatic data of natural distribution range

Temperatures over a thirty year period (1961-1990) were compiled from data sourced from the Bureau of Meteorology (Australian Government 2005). The mean monthly minimum and maximum temperatures were determined for distribution locations in each species and then related to their fruiting period (cf. Table 1.5, Figure 1.5).

Whenever the temperature zones overlapped within the species’ natural distribution range, the lowest mean minimum temperature or highest mean maximum temperature value was used.

Seed germination and vigour

Seeds were incubated at a range of temperatures (10, 15, 20, 25 and 30ºC) on 0.6% agar in plastic vials. Emergence of the radicle and epicotyl (>1mm) was recorded at two, four and eight weeks after incubation for seeds of each treatment. For each temperature, five replicates of 10 seeds (n=50) were initiated for each species. Seedling growth was

92 determined by measurement of the epicotyl and root lengths (mm) of germinated seeds at the end of germination testing (8 weeks).

Differential Scanning Calorimetry (DSC)

Analysis of thermal events during warming of cotyledon tissue (ca. 5%MC) from ultra low temperatures (-80ºC) was performed using a DSC Q1000 V8.1 (TA Instruments).

The DSC machine was calibrated (melt onset/enthalpy) using indium (156.6ºC/28.54J-g), cyclohexane (6.54ºC/31.25J-g), docedcane (-9.65ºC/216.73J-g) and n-decane

(-29.66ºC/202.09J-g) and an empty aluminum pan was used as a reference. A section of cotyledon tissue was excised from four to seven intact seeds after silica drying from each seed lot. Cotyledon tissue was sealed in aluminum pans and then individually analysed using a cooling and warming rate of 20ºCmin-1. Warming thermograms were analysed using Universal V3.9A software to determine the temperature of melt end of endotherm

(intersection with baseline) and the enthalpies (calculated from area of endothermic peak under the baseline) and presented in joules per gram fresh weight (Jg-1 FW). Confirmation of lipid endothermic peaks was achieved by analysis of extracted seed oils of C. inodora and C. garrawayi.

Statistics

Germination and DSC data was analysed by an ANOVA (analysis of variance) with a least significance difference (Tukey test) test at the 0.05 level, using SPSS software version 12.0.1. ANOVA of germination data was performed on arcsin transformed germination proportions.

93 3.3 Results

Climatic data

Figure 3.1 shows the mean monthly minimum and maximum temperatures (1961-

1990) of the natural distribution ranges of C. australasica, C. inodora and C. garrawayi

(data sourced Bureau of Meteorology 2005). During the fruiting period (based on observations in the geographic range) the mean monthly minimum and maximum temperature range differed between C. australasica (6 to 9ºC and 18 to 24ºC), C. inodora

(12 to 18ºC and 24 to 30ºC) and C. garrawayi (15 to 21ºC and 27 to 33ºC).

Effect of temperature on radicle and epicotyl emergence between species

Interspecific variation was observed for radicle and epicotyl emergence in

C. australasica, C. inodora and C. garrawayi over a 20 - 30ºC temperature range (Figure

3.2). However, negligible radicle and epicotyl emergence was observed in all three species for seeds incubated at either 10 or 15ºC.

Mean % radicle emergence levels increased to 85% at 30ºC in C. australasica,

C. inodora and C. garrawayi. Additionally, early (2wk) and final (8 wk) mean % radicle emergence was not significantly different (P=>0.1) at 30ºC between all three species and ranged from 69 to 95% and 85 to 99%, respectively. Mean % radicle emergence was high

(close to 100%) at 25ºC and not significantly different (P=0.7) between C. australasica

(warm subtropical) and C. inodora (warm tropical). In contrast, C. garrawayi mean % radicle emergence was significantly less (P=<0.01), almost half the level (53%), than the other two species at 25ºC. Final levels (8 wk) of radicle emergence at 20ºC were suboptimal (ca.60%) and not significantly different (P=>0.6). between all three species.

94 Dec A Nov Oct Sep Aug Jul Jun

Month May Apr Mar Feb Jan 0 5 10 15 20 25 30 35 B Mean temperature (C)

Dec Nov Oct Sep Aug Jul Jun

Month May Apr Mar Feb Jan 0 5 10 15 20 25 30 35 Mean temperature (C)

Dec C Nov Oct Sep Aug Jul Jun

Month May Apr Mar Feb Jan 0 5 10 15 20 25 30 35 Mean temperature (C)

Fig. 3.1 Mean monthly maximum (red bar) and minimum (blue bar) temperatures for the distribution area of (A) C. australasica (B) C. inodora and (C) C. garrawayi. Mean monthly data values over a thirty period (1961-1990) sourced from the Bureau of Meteorology (2005).

95 2wk 100 A 100 B

80 80

60 60

40 40

20 20 Radicle emergence (%) Epicotyl emergence (%) 0 0

10 15 20 25 30 100 10 15 20 25 30 100 4wk C Temperature D 10-30C 80 80

60 60

40 40

20 20 Radicle emergence (%) Epicotyl emergence (%) 0 0

10 15 20 25 30 10 15 20 25 30 100 100 8wk E Temperature (ºC) F Temperature 80 80

60 60

40 40

20 20 Radicle emergence (%) Epicotyl emergence (%) 0 0

10 15 20 25 30 10 15 20 25 30 Temperature (ºC) Temperature (ºC)

Fig. 3.2 Effect of temperature on radicle and epicotyl emergence from seeds incubation for (AB) two, (C,D) four and (EF) eight weeks in C. australasica (___) C. inodora (__....) and C. garrawayi(----). For each species mean (± se) of five replicates (n=50) for each temperature. 96 Initial epicotyl emergence was much lower than radicle emergence after 2 weeks incubation. However, similar trends in effect of temperature on both radicle and epicotyl emergence between species was observed. For example, epicotyl emergence (4 and 8 wk) was close to complete and not significantly different (P=>0.2) between all species at 30ºC.

One notable difference was the affect of temperature on epicotyl emergence at 25ºC. Citrus australasica (96%) had significantly higher mean % epicotly emergence from seeds than both C. inodora (40%) and C. garrawayi (22%) after 4 weeks incubation at 25ºC. However, final levels (8wk) of epicotyl emergence were not significantly different (P=0.657) between

C. australasica (98%) and C. inodora (84%) at 25ºC, but C. garrawayi was significantly different (P=<0.001) and much lower (32%) than the two other species. The low mean % radicle and epicotyl emergence recorded in C. garrawayi in seeds incubated at 25ºC was partly attributable to the extensive fungal infection observed at this temperature.

Effect of temperature on seedling germination

The mean seedling germination (radicle and epicotyl emergence), after eight weeks incubation, differed at temperatures between 20 to 30ºC in C. australasica, C. inodora and

C. garrawayi (Table 3.1). Mean % gemination from C. australasica seeds was above 95% from seeds incubated at either 25 or 30 ºC, but incubation at 20ºC significantly reduced mean % germination to 66%. Mean % seedling germination in C. inodora and

C. garrawayi seeds incubated at 30ºC was best and significantly reduced within each species at either 20 or 25ºC. However, this represented only a 12% reduction between

C. inodora seeds incubated at 25ºC compared to those at 30ºC. In contrast, mean %

97 seedling germination from C. garrawayi seeds was only 32% after incubation at 25ºC, a large and significant 48% reduction compared to seedling germination at 30ºC (80%).

Table 3.1 Effect of temperature on mean percentage of seedling germination in Citrus australasica , C. inodora and C. garrawayi Temperature Germination (%) (ºC) C. australasica C. inodora C. garrawayi 20 66±17a 42±6a 32.0 ±4a 25 98±2b 84±2b 31.7±16a 30 95±2b 96±4c 80±8b Different lettering represents significant difference within column (P<0.05, Tukey test). Seedling (whole) germination (radicle and epicotyl emergence >1mm) recorded after 8 weeks incubation, five replicates for each temperature and species (n=50).

Effect of temperature on seedling growth

An overall trend of an increasing mean epicotyl and radicle length was seen for all three species as incubation temperatures increased from 20 to 30ºC, except for mean epicotyl length in C. australasica, which did not vary with temperature (Figure 3.3). Mean radicle length of C. australasica seeds incubated at 20ºC (27±3mm) was about half that of seeds incubated at either 25 (53±6mm) or 30ºC (61±7mm). Both mean epicotyl and radicle length in C. inodora was approximately doubled with each five degree increase in incubation temperature (20-30ºC), ranging from 7±1 to 24±2 mm and 6±1 to 19±1mm for radicle and epicotyl length, respectively. Similarly both mean radicle and epicotyl length was considerably shorter from C. garrawayi seeds incubated at 20ºC (4±1 and 11±2 mm), compared to those incubated at either 25ºC (24±4 and 17±3 mm) or 30ºC (47±4 and

37±20.mm).

70 C. australasica 40 A B 35 60 50 30 25 40 20 30 15 20

Radicle length (mm) Epicotyl length (mm) length Epicotyl 10 10 5

20 25 30 20 25 30 Temperature (ºC) Temperature (ºC) 70 C. inodora C 40 D 60 35

50 30

25 40 20 30 15 20 Radicle length (mm) length Radicle Epicotyl length (mm) length Epicotyl 10 10 5

20 25 30 20 25 30 Temperature (ºC) Temperature (ºC) 70 40 C. garrawayi E F 60 35

50 30

25 40 20 30 15 20 Radicle length (mm) Epicotyl length (mm) length Epicotyl 10 10 5

20 25 30 20 25 30 Temperature (ºC) Temperature (ºC)

Fig. 3.3 Effect of temperature on mean radicle and epicotyl length (mm) of germinated seeds of (AB) C. australasica, (CD) C. inodora and (EF) C. garrawayi. For each species, 5 replicates of 10 seeds were initiated for each

temperature (n=50) and germination was recorded after 60 days incubation.

Bars represent standard error.

Thermal analysis of seed oils

Differential scanning calorimetry was used to analyse the thermal events of seed storage oils (cotyledon tissue) in desiccated seeds of all three Citrus species. Extracted seed oils were also analysed for C. inodora and C. garrawayi, confirming that the endothermic peaks observed in cotyledon tissue represented lipid phase transitions (Figure 3.4).

There was some variation both within (i.e. different seed lots) and between species in the thermal characteristics of in vivo seed oils (cotyledon tissue) (Table 3.2). However, the mean temperatures of lipid melt end of the combined seed lots of C. australasica and

C. inodora were similar and not significantly different (7.5 and 8.4ºC respectively) (Table

3.3). In contrast, cotyledon tissue of C. garrawayi (most northerly distributed species) had a lipid melt end temperature of 10.9ºC, which was about 3ºC higher and significantly different to the other two species (Table 3.3). Thus, the mean end temperature of the lipid melt was positively associated with decrease in latitude (NSW to NQLD) and increase in temperature (warm subtropical to hot tropical). Comparative endothermic curves of lipid melt events in C. australasica, C. inodora and C. garrawayi, illustrated this shift in profile

(i.e. onset and end temperature of lipid phase transition) associated with changes in geographic distribution (Figure 3.5).

100

0.0 A

Lipid

Cotyledon

-1.0

(W/g) Heat Flow

C. inodora -2.0 -60 -40 -20 0 20

0 B

) Lipid

Cotyledon -1

Heat(W/g Flow

C. garrawayi -2 -60 -40 -20 0 20

Temperature (°C) Exo Up Fig. 3.4 Representative differential scanning calorimetric thermocurves of transition events in cotyledon tissue and lipid extract of seeds of (A) Citrus inodora and (B) C. garrawayi on warming from ultra low temperatures (-80ºC). Intact seeds were silica- dried for 10 days (ca.5%MC) prior to thermal analysis and seed oil extraction. 101

Table 3.2 Characteristics of endothermic events in cotyledon tissue and lipid extract of seeds of Citrus australasica , C. inodora and C. garrawayi on warming from low temperatures (-80ºC). LIPID MELT Species Sample Sourced Year Mean end Mean n temp. enthalpy (ºC) (Jg-1FW)

C. australasica Cotyledon SQ BBG 2006 7.11 ±0.32 36.41 ±0.50 6

Cotyledon SQ DPI 2006 7.97 ±0.27 37.18 ±1.40 4

C. inodora Lipid SQ DPI 2006 5.73 ±0.15 45.82 ±0.04 2

Cotyledon SQ DPI 2005 7.91 ±0.65 48.11±1.06 5

Cotyledon SQ DPI 2006 8.18 ±0.53 36.49 ±1.29 4

Cotyledon NQ DPI 2005 8.94 ±0.70 42.39 ±2.7 7

C. garrawayi Lipid SQ DPI 2006 10.13 ±0.57 43.37 ±2.55 2

Cotyledon SQ DPI 2005 10.58 ±0.81 35.20 ±3.37 7

Cotyledon SQ DPI 2006 11.56 ±0.75 33.98 ±1.19 4

Mean (±se) melt end temperature and enthalpy was determined using differential scanning calorimetry of cotyledon tissue excised from 4-7 seeds for each seed lot. Seeds were silica-dried (10 days) prior to analysis

102 0

Au In Ga -1

Heat Flow (W/g)

C. australasica (Au) C. inodora (In)

C. garrawayi (Ga) -2 -60 -40 -20 0 20 Exo Up Temperature (°C)

Fig. 3.5 Comparative thermocurves of in vivo lipid phase transition events in cotyledon tissue of C. australasica, C. inodora and C. garrawayi on warming from ultra low temperatures ( -80ºC). Intact seeds silica-dried for 10 days (ca. 5%MC) prior to cotyledon tissue extraction and thermal analysis.

Table 3.3 Climatic data of natural distribution range during fruiting period in Citrus australasica , C. inodora and C. garrawayi in relation to end temperature of lipid melt.

Species Natural Mean Mean Mean (±se) Distribution monthly monthly lipid melt end minimum maximum temp. temp. temp. (ºC)B range(ºC)A range(ºC)A

C. australasica SEQLD/ 6-9 18-24 7.5±0.3a NE NSW

C. inodora NE QLD 12-18 24 -30 8.4±0.4a

C. garrawayi N QLD 15-21 27-33 10.9±0.6b

A Mean climatic data over a thirty period (1961-1900) sourced from the Bureau of Meteorology (Australian Government 2005). BDifferent lettering represents a significant difference in mean lipid melt end temperature (P<0.05LSD). 103

3.4 Discussion

As previously discussed (cf. Chapter 1.5), many Citrus species display variable and often intermediate seed storage behaviour (Hong and Ellis et al 1995). The main limiting factor in the geographic distribution of cultivated Citrus species is low temperature

(reduced growth 13ºC), which restricts citrus cultivation to subtropical and tropical climates (Spiegel-Roy and Goldschmidt 1996). The present study suggests a relationship between monthly mean climatic data (i.e. temperature during fruiting period) and the physiological responses of seeds at a range of temperatures. Such investigations are difficult in cultivated species because of the high level of hybridisation and ambiguity of origin, even in true species (cf. Chapter 1.4).

Effect of temperature on germination and epicotyl and radicle growth

The optimum temperature for germination in all three species was 30ºC. The negligible germination in all three species at 15ºC suggests that this is the minimum temperature for germination. An increased tolerance to germination at a temperature between 20 to 25ºC was observed in C. australasica (warm subtropical) compared to

C. inodora (warm tropical) and C. garrawayi (hot tropical) and was consistent with their natural distribution range (i.e. climatic data).

Seeds imbibe water as an initial step towards germination (radicle protrusion).

Soetisna et al (1985) suggest that the decreased rate of germination at lower temperatures

(ca.20ºC) in cultivated citrus seeds was a result of a reduced rate of initial water uptake and the subsequent steps of germination Although a long germination period was employed (8 weeks) in this present study, the lack of germination at temperature

104 15 ºC may possibly reflect a reduction in water uptake capacity, rather than incapacity to germinate (i.e. delayed germination).

Epicotyl and radicle lengths increased with incubation temperature (20 to 30ºC) in all three species, except for mean epicotyl length in C. australasica. This is consistent with the maximum mean monthly temperatures during fruiting of C. australscia (18 to 24ºC),

C. inodora (24 to 30ºC) and C. garrawayi (27 to 33ºC) in the natural distribution range.

Thus, as with germination capacity, Citrus australasica seed vigour reflected a greater tolerance to cooler temperatures than the two northerly distributed species, C. inodora and

C. garrawayi.

It was also observed that seedling habit reflected that of the tree habit. Epicotyl length, after 8 weeks incubation, being shorter in the shrubs, C. inodora and C. australasica

(19mm long) compared to C. garrawayi, which has a tree habit (37mm long).

Optimal germination levels and seedling length were observed in all three species at

30ºC. These results were consistent with studies in cultivated Citrus species where germination is most rapid and complete between 30 to 35ºC (Ellis et al 1985). However,

30ºC was the recommended germination temperature in cultivated species, as at higher temperature (e.g. 35 ºC) seedling abnormalities have been observed (Soetinsa et al 1985,

Ellis et al 1985).

One unexpected observation from this present study was the extensive fungal infection observed in seeds of C. garrawayi incubated at 25ºC. An increase in temperature also resulted in an increasing fungal problem in cultivated citrus (Monselise 1962). In this study, considerably increased fungal infection, was observed at 25ºC, compared to either higher or lower temperature between 10 to 30 ºC in C. garrawayi. However, no significant

105 fungal infection was observed in either C. australasica or C. inodora seeds incubated at any temperature between 20 to 30ºC. Thus, C. garrawayi seeds appear to be more susceptible to fungal infection compared to the other two test species and fungal infection when was maximal at 25ºC. It is possible that this phenomenon increases germination in

C. garrawayi in the wild through the breakdown of the seedcoat (Cf. Chapter 2). However, regardless of any possible advantage of fungal invasion under field conditions, it is recommended that laboratory based germination testing of C. garrawayi be done at 30ºC to avoid potential losses to fungal infection.

Thermal analysis of seed oils

Results from this study indicate a relationship between lipid thermal properties and geographic distribution (i.e. climatic zone). The temperature of lipid melt end was higher in

C. garrawayi compared to C. inodora and C. australasica. However, it is difficult to associate this with germination behaviour observed in this present study, as germination was negligible at 15ºC, which is well above the lipid melt end temperatures. It appears that other factors (e.g. water uptake or other physio-chemical processes) may be delaying/affecting germination in the Australian wild Citrus species at temperatures above the temperature of their lipid melt end (ca. 10ºC). The Australian wild Citrus species,

C. glauca (desert lime) has a desert distribution and as such tolerates both low temperatures

(-2 to -4ºC) and hot temperatures (45ºC) (Macintosh 2004). An interesting avenue of future investigation would be to characterise the thermal characteristics of the seed oils of

C. glauca in relation to germinability at a range of temperatures to compare it with the subtropical and tropical Australian Citrus species in this present study.

106 Saturated fatty acids have a higher melt end temperature than unsaturated fatty acids

(cf.1.8.3). Besbes et al (2004) studied two Phoenix (date) cultivars, and suggested that the difference in melt onset temperature, -19ºC (Deglet Nour cultivar) compared to -22ºC

(Allig cultivar) could be attributed to higher levels of unsaturated fatty acids (i.e. Allig seed oil). Comparative endothermic curves of lipid melt events in cotyledon tissue of

C. australasica, C. inodora and C. garrawayi revealed a shift in melt onset and end temperature. These thermal profiles suggest that C. garrawayi may have a higher proportion of saturated fatty acids, compared to unsaturated fatty acid than C. australasica and C. inodora. Additionally, across all three species, an increased mean temperature of lipid melt end coincided with decreased latitude and increased temperature of their natural distributions (warm subtropical to hot tropical). Plants grown in cool climates tend to have a different fatty acid composition, with more unsaturated fatty acids, compared to those grown in warmer climates (Bewely and Black 1994). This is consistent with the apparent link between geographical distribution (i.e. climatic zone, warm tropical to hot tropical) and lipid end melt temperature (i.e. fatty acid composition) observed in seeds of Australian wild species of Citrus in this present study.

The chemical composition of seeds, such as the characteristic fatty acid composition of seed oils, is largely genetically determined (Bewley and Black 1994). Thus, the interspecific variability in seed oil thermal characteristics observed in this study could be an ecological adaptation of these Australian wild Citrus species to different climatic zones of their natural distribution. The similarity of melt end temperature (7ºC) in cultivated citrus seed oils (El-Aawdy 1999), is consistent with other biochemical and morphological similarities observed in many studies, and most likely reflects their narrow genetic base (cf.

Chapter 1.5). Whilst, the interspecific variation observed in lipid profiles of Australian

107 Citrus species in this present study may reflect their higher level of genetic diversity

(cf. Chapter 1.4). The seeds in this study were sourced from ex situ cultivated plants which experience different climatic conditions to plants growing in situ. Both the natural distribution (origin) and the environmental conditions during seed development and dispersal (i.e. ex situ) may have influenced the seed oil thermal behaviour observed in this study. Further investigations on seeds harvested in situ are needed to clarify the possible contribution of in situ growth conditions on seed oil composition and thermal behaviour.

The ecological advantage (physiological imperative) of a species having a lipid melt end point (i.e. entire liquid faction of oil reserves) near to or above the minimum temperature of natural distribution, as observed in this present study in C. australasica,

C. inodora and C. garrawayi, could be explained by the ideas of Crane et al (2006). In their study on imbibition of seeds of a species with intermediate seed storage behaviour (Cuphea cartagenensis), it was found that when seed oils were crystallized (i.e. solid phase), this was lethal (Crane et al 2006). Thus, these findings suggest a possible reason for the apparent link between geographic distribution (i.e. minimum temperature) and the thermal profiles of seed oils observed in Australian wild Citrus species.

A review of intermediate seeded species showed that they often had lipids with relatively high melt end temperatures (Crane et al 2003). It could be speculated that the characteristic delay in germination, observed in cultivated citrus seeds and linked to water uptake characteristics, is an adaptive response. That is, at lower temperatures, close to the lipid end melt point, seeds may have developed mechanisms to reduce water uptake that could lead to lethal injury. Although tentative, the physiological responses, i.e. lack of germination at low temperatures close to the lipid melt end, of Australian wild Citrus species supports this speculation.

108

CHAPTER 4 DISTINGUSHING CHARACTERISITICS OF SEED

MATURITY IN CITRUS GARRAWAYI IN RELATION TO

GERMINABILITY

109 CHAPTER 4 Distinguishing characteristics of seed maturity in Citrus garrawayi in relation to germinability

4.1 Introduction

Three main factors important to seed storage are moisture content, temperature and seed quality. Of these factors, seed quality is an ambiguous factor under environmental and genetic control (Walters 2003). Seed quality, in terms of seed desiccation sensitivity, has been demonstrated as a key determinant in recovery of seedlings from coffee seeds after cryopreservation (Dussert and Engelmann 2006). In wild species, seed lot quality in relation to seed developmental status and handling method appears to effect seed responses to desiccation and storage (Pritchard 2004).

Differential fruiting in C. garrawayi was observed over a three-year harvest period

(2004-2006). Consequently, seed lots contained a wide variation in seed developmental stages from a single harvest, and this is consistent with observations in many other wild species (Hay and Smith 2003). Heterogeneity in maturity within a seed lot and lack of descriptors of seed maturity are hindrances to the development and application of seed storage protocols in C. garrawayi.

Seed development can be divided into three stages: histodifferentiation, expansion

(reserve deposition) and maturation drying (reduced metabolism) (Kermode and Finch-

Savage 2002). Identification of these seed developmental stages for seed collecting is often by the use of maturity ‘markers’ such as non-destructive visual indicators like fruit or seed size, colour and hardness (Hay and Smith 2003, Aiazzi et al 2006).

110 In order to gain a foundation for understanding the response of C. garrawayi seeds to desiccation and storage, this current study investigates: (1) embryonic axis and seed coat development using scanning electron microscopy and (2) the use of the combinational traits of seed size and coat hardness as markers of seed morphological and physiological maturity.

4.2 Materials and methods

Fruit and seed material

Fruits of C. garrawayi were harvested from a research field collection, Queensland

Department of Primary Industries, in February 2005, May 2005 and March 2006. After extraction, seeds were graded into four categories of maturity based on seed length and seed coat hardness: approx. 2mm and soft (very immature), 3 4mm and soft (immature),

4 5mm and firm (premature), 5 7mm and woody (mature). Seed hardness was determined by lightly applying pressure to seeds between forceps and observing the level of resistance and thus woodiness of the coat. The fruit characteristics of 45 randomly sampled fruits from one harvest (200 fruits) in 2006 were recorded in terms of colour, size and internal morphology in order to assess relationships between fruit characteristics and seed maturity.

Moisture content determination

Seed moisture content measurements were determined on duplicates of ten individual seeds for each seed maturity from the two separate fruit harvests (n=20) as described in section 2.2.

111

Microscopy

Seeds were longitudinally sectioned and examined using a dissecting microscope for ten seeds of each maturity, duplicated by a second fruit harvest (n=20). Characteristics were recorded in terms of colour and external and internal morphology (e.g., embryonic axis differentiation and seed coat texture) in order to assess relationships between these characteristics and seed maturity stage.

Scanning electron microscopy (SEM) was used to observe the external morphology of the seed coats and embryos of graded seeds, as well as seed coat anatomical features. In order to examine embryonic axes the testa was removed and the embryonic axis excised from cotyledonary material. Embryonic axes and seeds were fixed in 4% glutaraldehyde buffered with 0·1molL–1 cacodylate and left overnight at 4ºC. Samples were then rinsed in two changes of buffer solution (0·1molL–1 cacodylate) followed by dehydration through a graduated ethanol series, exchanged with amyl acetate and then critical point dried. At a later stage, extra seeds (mature) were processed for SEM by drying over silica for ten days

(3% MC). Fixed and silica-dried samples were mounted on SEM stubs with double-sided adhesive and sputter coated in gold prior to observation using a FEI Quanta 200

Environmental SEM at 10kV (FEI Company, USA).

Germination and seedling growth

Seeds at three maturities (immature, premature and mature) were placed on 0.6% agar in sealed plastic containers and incubated at 30ºC with a 16h photoperiod for 60 days.

Germination was recorded as whole seedlings (radicle and epicotyl emergence >1mm).

112 Three replicates of ten seeds were initiated for each seed maturity in 2005 (February), and repeated by two replicates in 2005 (May) and three replicates in 2006 (March) (n=80 for each seed maturity). ANOVA with a Tukey (HSD) test at the 0.05 level was performed on arcsin transformed germination proportions using SPSS software version 12.0.1. Time until

50% of the final percentage germination was determined by measurements every 3 to 4 days of radicle and epicotyl emergence (>1mm) over a 60 day period for three replicates

(10 seeds/replicate), duplicated by two replicates for each seed maturity stage (n=50). The epicotyl and root length were measured at the end of the germination test (60 days).

4.3 Results

Fruit characteristics and relationship to seed maturity

Fruits of C. garrawayi were harvested from trees of a research collection during periods of substantial natural shedding of fruit. Fruits at different developmental stages and flowers were present on the same tree, with seeds of all maturities available throughout the fruiting season (Figure 4.1).

One hundred and forty five seeds were extracted from forty-five fruits, randomly sampled from a harvest of 200 fruits (2005) of all maturities. Based on the characteristics of seed size and seed coat hardness described in the materials and methods, only 36% (51) of these were mature seeds, the remaining 38% and 26% of seeds were immature and premature, respectively. Seedlessness, a desirable commercial trait, was observed in 18% of fruits (8/45).

Fruit characteristics (size, colour and internal morphology) gave some indication of the percentage of seed at each maturity that was present, with the greatest percentages of

113 mature seeds found in larger fruits (Figure 4.1B; Table 4.1). The majority (77%) of seeds extracted from small fruits (approx. 4cm in length) were immature, whilst the majority of seeds extracted from the medium (6cm) and large fruits (7cm) were premature (52%) and mature (76%), respectively (Figure 4.1B; Table 4.1).

Fig. 4.1 (A) Citrus garrawayi at field location during time of harvest

showing fruiting and flowering at the same time (B) Fruit of Citrus

garrawayi of different sizes and maturity collected from a single harvest. 114

Table 4.1 Characteristics of fruit of Citrus garrawayi harvested in February 2006 containing seeds of different maturities.

Fruit size Rind Pulp Pulp Seed Maturity (%) (±s.d.) length x width colour colour consistency (cm) Immature Premature Mature

4.4 ±1.3 x Green cream tightly packed 1.8 ±0.2 small juice sacs 77 10 3 or loose angular plump juice sacs

6.1 ±1.3 x Green cream or loose angular 23 52 23 2.1 ±0.3 pinkish cream plump juice sacs

7.2 ±1.0 x Green cream, pinkish loose angular 3 21 76 2.3 ±0.3 cream or pink plump juice sacs

Seeds were extracted from forty-five randomly selected fruits from a harvest of approximately 200 fruits in 2006. Eight fruits were seedless.

Whole seed anatomy and embryo morphology

Mature seeds were whitish cream to cream, 5-7mm in length, ovoid in shape, with a woody seed coat and % moisture content of 40.3±1.1. A single embryonic axis was located between two thick cotyledons (Figure 4.2 A, B). Seeds less than 5mm in length with a softer testa were less developed and had higher % moisture contents of 82.2±1.3

(immature) and 74.8±2.0 (premature) (Table 4.2). Mature seed coats consisted of a distinct outer testa (outer integument) and inner testa and often had a flattened beak at the

115 microplyar region and a distinct purplish brown chalazal spot (Figure 4.2C). The inner testa was cream to golden tan in colour.

A B B

Fig. 4.2 (A) Mature embryo of Citrus garrawayi with the seed coat removed. (B) Embryo with one cotyledon excised to reveal a developed embryonic axis. (C) Longitudinal section of mature seed coat.

The seeds were covered in a slippery layer of mucilage overlying epidermal protrusions visible at maturity (Figure 4.2C). An accumulation in dry weight was observed in seeds graded for maturity based on length and seed coat hardness. Immature, premature

116 and mature seeds had a mean dry weight of 2.4±0.2, 5.5±0.6 and 17.0±0.9mg, respectively

(Table 4.2).

Embryo maturity

Light microscopy examination of sectioned seeds revealed that it was only in

mature grade seeds (5-7mm in length and woody seed coat hardness) that the embryo

completely filled the seed (Figure 4.3). The embryo changed from translucent green though

to a cream or greenish cream colour at maturity (Table 4.3). The development of the

embryonic axes of C. garrawayi was observed using scanning electron microscopy of seeds

at different maturities.

A B C

Fig. 4.3 Typical morphology of seed coat and embryo of seeds of Citrus garrawayi at different maturities, (A) immature, (B) premature and (C) mature. Seeds were longitudinally sectioned and examined under a stereo light microscope (n=20). 117

Mean embryonic axis length was 310±61, 682±34 and 934±69μm in immature, premature and mature seeds, respectively (Table 4.2). Early developmental stages of cotyledon, radicle and plumule formation were observed in embryonic axes excised from immature seeds (34 soft seed coat) (Figure 4.4 A, B). Axes from premature seeds (45 firm seed coat) were more developed, with a differentiated radicle and plumule pole of 383±23 and

391±32μm in width (57mm woody seed coat), respectively (Figure 4 C, D; Table 4.3).

The embryonic axes in mature seeds had well-differentiated radicle and shoot primordia

(Figure 4.4 E, F), with a wider radicle pole compared to plumule pole (509±38 x

382±12μm) (Figure 4.4 E, F; Table 4.3).

Table 4.2 Characteristics of maturity of seeds in Citrus garrawayi

Seed Mean seed Mean seed dry Mean seed Mean Mean (±s.e.) maturity fresh weight weight (mg),A moisture content embryonic germination (mg)A (%wb)A axis (%)C length(m)B

Immature 13.3 ±0.5 2.4 ±0.2 82.2±1.3 310±61 4.4±4.4*

Premature 21.1 ±1.1 5.5 ±0.6 74.8±2.0 682±34 51.3±9.5*

Mature 28.7±1.4 17.0 ±0.9 40.3±1.1 934±69 88.0±3.2*

AMean (±s.e.) of ten replicates of individual seeds for each seed maturity, duplicated by a second fruit harvest (n=20). Moisture content determined gravimetrically and presented on fresh weight basis (%wb). BMean (±s.e.) of 3-5 excised embryonic axes examined by scanning electron microscopy CMean (±s.e.) of 2-3 replicates of ten seeds per experiment in triplicate for each seed maturity (n=80). Seeds sourced from three separate fruit harvests in 2005 and 2006. Germination (radicle and epicotyl emergence >1mm) recorded 60 days post incubation. *Represents a significant difference at P=<0.001 level (Tukey HSD).

118

Table 4.3 Seed coat and embryo morphology of seeds at different maturities in Citrus garrawayi

Seed maturity Structure Characteristic Immature Premature Mature Embryo Colour A Translucent green Green, pale green Greenish cream to to pale green to greenish cream cream Morphology A B Early cotyledon Differentiation and Well-differentiated and axis growth of shoot primordia and differentiation cotyledons and embryo fills seed within liquid filled embryonic axis sac Mean (±s.e.) plumule x - 391 ±32 x 383 ±23 382 ±12 x 509 ±38 radicle axis width (m)B Seed coat Colour/texture A Translucent to Whitish cream and Whitish cream to whitish cream and semi-smooth to cream and textured smooth textured Morphology A B Thin walled Sparse to extensive Compression of cell epidermal fibres mucilage coverage layers, thick pitted and none to few and many epidermal fibres, emerging protrusions extensive mucilage protrusions and numerous protrusions Protrusion length (μm) B 4 - 70 15 - 117 102 -351

A Characteristics determined from light microscope examination of 10 individual sectioned seeds for each seed maturity, duplicated from second fruit harvest (n=20). BCharacteristics determined from scanning electron micrographs of either external seed coat surface, sectioned seed coats or excised embryonic axes (n=3-6).

119 A B

C D

E F

Fig. 4.4 Scanning electron micrographs illustrating the development of the radicle and shoot primordia in embryonic axes of Citrus garrawayi. The embryonic axes were excised from (AB) immature, (CD) premature and (EF) mature seeds.

120 Seed coat development

Changes in the development of the seed coat were observed in seeds of

C. garrawayi graded by length and hardness using scanning electron microscopy and observations are summerised in Table 4.3. One distinguishing marker of seed coat maturation was the change in the thickness and structure of the epidermal fibres. These were beginning to form in very immature seeds and the fibres had a cell width of about

7μm and wall thickness of 1μm (Figure 4.5A). In contrast, mature seeds (5-7 mm in length with a woody seed coat), had mature thick pitted fibres approximately 20μm wide with a cell wall thickness of 4μm (Figure 4.5B).

Fig. 4.5 Scanning electron micrographs of transverse seed coat

sections of Citrus garrawayi illustrating the development of epidermal fibres. (A) Epidermal fibres of an very immature seed with early thickening of cell walls. (B) Thick, pitted cell walls of mature fibres. 121 The morphological characteristics of seeds at different maturities are presented in

Figure 4.6A-D. The increase in mucilage coverage and protrusions during seed coat maturation was discernable unaided or at low magnification (Figure 4.6 A-D). SEM examination revealed that the epidermal fibres were visible as the outer seed coat layer, longitudinally stretched along the axis of the seed, in immature and premature seeds (Figure

4.6 E, F, G). In mature seeds a thick layer of mucilage had formed that covered these epidermal fibres (Figure 7H). In the early stages of seed coat development the emergence of protrusions, 4-15μm in length, was observed (Figure 4.6 E, F; Table 4.3). The protrusions were often the actual end of fibre cells but they also appear to arise from the tangential walls of the fibres. The length and number of protrusions increased in more developed premature seeds (Figure 4.6 G; Table 4.3). The seed coats of mature seeds displayed the longest protrusions (102-351μm), and these were seen to be densely covering the seed coat surface (Figure 4.6 H, L). Longitudinal sections of the seed coat revealed the compression and absorption of cell layers in the mature seed coat compared to immature and premature seeds (Figure 4.6 I-L).

SEM of mature seed morphology and anatomy

SEM of mature seed was undertaken to generate clear descriptors of the major morphological characteristics of these seeds. The embryonic axis from mature seeds was located near the micropylar region with the radicle closest to the seed coat wall (4.7 A, C).

The outer integument consisted of several layers of thin walled parenchyma cells and then a protective layer of fibres from which emerged protrusions covered in a layer of mucilage

(Figure 4.7D). Both the fibres and protrusions had thick-pitted cell walls (4.7B). The chalazal region of the seed was flattened with many bulges covered by a thick layer of mucilage (Figure 7A). The vascular structures that were observed in longitudinal sections of the seed coat in the chalazal region are illustrated in Figures 4.7E, F.

122 A E

B F J

G K C

D H L

Fig. 4.6 Seed coat morphology of Citrus garrawayi seeds at different maturities; (AEI) very immature, (BFJ) immature, (CGK) premature, (DHL) mature. (A-D) Morphological features of the outer seed coat including mucilage coverage and texture (light microscope). (E-H) Emergence and growth of epidermal protrusions during seed coat development viewed by scanning electron microscopy (SEM; x800 magnification). (I-L) Longitudinal section of seed coat, observed by SEM, showing the development of cell layers (x300 magnification).

123

A B

C D

E F

Fig. 4.7 Distinguishing characteristics of mature seed in Citrus garrawayi as viewed by scanning electron microscopy. (A) External morphology and (B - F) sectioned seeds illustrating mature structures: (B) thick pitted wall of epidermal protrusion and fibre cells overlaid by a mucilage layer, (C) mature embryonic axis located near microplyar region of seed coat, (D) cell layers of outer integument, (E) vascular strands, protrusions and mucilage of the124 chalazal region and (F) detail of vascular structures of the chalazal region.

Effect of seed maturity on germination and seedling growth

Figure 4.8 shows the % 100 radicle and epicotyl emergence in 80 immature, premature and mature 60 seeds of C. garrawayi over a 60 40 day period, with monitoring every 20 radicle emergence(%) few days. This allows both the rate 0 6131016 21 24 28 32 39 51 60 of germination as well as the final 100 B germination levels to be 80 determined for each maturity. 60

Immature seeds, with incomplete 40

embryo and seed coat Epicotyl (%) emergence 20

0 histodifferentiation, had very low 61312345678910111016 21 24 28 32 39 51 60 Time (days) final germination (4±4%), even Fig. 4.8 Effect of seed maturity on (A) radicle and after 60 days of incubation, and (B) epicotyl emergence (>1mm) in Citrus this was significantly lower than garrawayi. Seeds graded into three categories based on maturity: immature (), premature (), premature seeds (51±10%) (Figure mature ( ). Data points are the mean of three replicates of 4.8, Table 4.2). The final 10 seeds, duplicated by two replicate for each maturity stage (n=50). Bars represent one s.e. of mean. percentage of germination from mature seeds was significantly higher (88±2%) than premature seeds. Thus, seed maturity has a large impact on germination. The time until 50% of the final percentage of radicle emergence involved a small lag of approximately six days from immature and premature 125

seeds (16 days) compared to mature seeds (10 days) (Figure 4.8A). The time until 50% of the final percentage of epicotyl emergence occurred approximately 16 days from initiation in all seed maturities (Figure 4.8B). Seedling growth was also affected by seed maturity, with an increase in seedling size with seed maturity (Figure 4.9). Root length, after sixty days incubation, was longer at 40±2mm from mature seeds, compared to 16±2 and 4±1mm from premature and immature seeds, respectively (Figure 4.9A). Seed maturity was also indicative of increased mean epicotyl length at l3±4, 18±2 and 30±1mm in immature, premature and mature seeds, respectively (Figure 4.9B).

A B 50 50

40 40

30 30

20 20

10 10 Radicle length (mm) Epicotyl length (mm)

0 0 Immature Premature Mature Immature Premature Mature Seed maturity Seed maturity Fig. 4.9 Effect of seed maturity on mean (±se) (A) radicle and (B) epicotyl length in

Citrus garrawayi. Mean (±s.e.) of germinated seeds of triplicate experiments (2-3 replicates of ten seeds per experiment) for each seed maturity in 2005 and 2006. Radicle and epicotyl length recorded 60 days after seeds were incubated.

126 4.4 Discussion

The results of this study show, overall, that the combinational, and easily observable, seed traits of size and coat hardness (i.e. woody) correlated well with morphological, anatomical and physiological seed maturity in C. garrawayi. For example, decreased moisture content and an increased level of dry weight, embryo differentiation, seed coat differentiation and germinability coincided with increased seed size and woodiness.

In simalar studies on the evaluation of the effect of dispersal unit size and seed colour on germination of Atriplex cordobensis, it was reported that these characteristics related to the proportion of mature embryos (seed quality) (Aiazzi et al 2006). Seed colour in Atriplex cordobensis was distinct, ranging from light brown to reddish. Seed colour changes in C. garrawayi were not always indicative of seed maturity. Consequently, seed colour should only be a guide in combination with other seed characteristics of maturity in

C. garrawayi. Examples of good visual indicators of seed maturation were a thick layer of mucilage (clear and slippery) and the presence of numerous seed coat protrusions that could be seen unaided or with low magnification. These seed characteristics, together with seed size and hardness, can be used as a non-destructive practical means for estimation of seed maturity in C. garrawayi to assess seed lot quality for both the development and application of seed storage protocols.

The stages of seed development in most species are marked by distinct changes in seed fresh weight, dry weight and moisture content (Kermode and Finch-Savage 2002). A drop in seed moisture content to around 40% and an increase in dry weight were both seen to mark seed maturation in C. garrawayi. Measurements of seed moisture content and dry

127 weight are both destructive, but are straightforward and standard methods to determine seed maturity in most species (Hay and Smith 2003). At maturity, the embryonic axis of

C. garrawayi was nearly 1mm in length with a well-differentiated radicle and shoot primordia. Additionally, the radicle pole was wider than the shoot pole so that the axis was a triangular cone shape, similar to that of other cultivated and wild species of Citrus.

Maturation of the embryo and seed coat appeared to be synchronous in this study, such that the thickening of the cell walls of the epidermal fibres to form the woody seed coat coincided with increased embryo filling of the seed, embryonic axis differentiation and germinability. Germination levels of immature seeds, with incomplete embryo and seed coat histodiffereniation, were very low. In contrast, premature seeds had an embryonic axis with a differentiated plumule and radicle pole that was capable of much higher levels of germination (51%) prior to attainment of seed maximum dry weight. Mature seeds had a well-differentiated embryo that filled the seed (maximum dry weight) and had the significantly highest level of germination (88%).

Both low germination and a lag in germination were observed in premature Citrus embryos of five cultivars (Wakana et al 2004). A similar lag in radicle emergence was observed from immature and premature seeds compared to mature seeds in

C. garrawayi. The lag in radicle emergence may be due to the incomplete embryo development (especially axis) that was observed in immature and premature seeds.

Detailed seed morphology and anatomy during development has not been described previously in C. garrawayi. Characterization of seed coat development in a species is important in the correct interpretation of the mature seed coat (Boesewinkel and Bouman

1995). In this present study, microscopic examination of seeds at different maturities

128 revealed the origin of epidermal fibres and the formation of epidermal fibres (obscured by mucilage layer in mature seeds). Citrus garrawayi seed morphology was comparable to other species of the genus Citrus as previously discussed (cf. Chapter 2).

Difficulty of access, limited material (conservation listed) and the occurrence of differential maturity within individual seed collections provide challenges to ex situ storage of seed in Citrus garrawayi. The morphological and physiological characterisation of seed maturity, previously unknown in this species, will aid in both improving seed lot quality and interpretation of seed responses to storage processes such as desiccation and cryopreservation.

129

CHAPTER 5 DESICCATION AND CRYOPRESERVATION OF SEEDS

OF CITRUS AUSTRALASICA, C. INODORA AND C. GARRAWAYI

130 CHAPTER 5 Desiccation and cryopreservation of seeds of Citrus australasica,

C. inodora and C. garrawayi

5.1. Introduction

Cryopreservation of seed after drying to low moisture contents has been demonstrated as a potential strategy for long-term storage of seed in many cultivated Citrus species (Mumford and Grout 1979, Normah and Siti Dewi Serimala 1997, Cho et al 2002a,

Lambardi et al 2004, Hor et al 2005). However, tolerance to desiccation and cryopreservation has varied in the Citrus species studied (cf. Table 1.6). In four species of

-1 cultivated Citrus, the unfrozen water content (WCu) ranged between 0.9 to 0.14 gH2Og dry weight (ca.10%MC) and this correlated with the optimal moisture content for seedling recovery after cryopreservation (Hor et al 2005, Pritchard 2007). Differential scanning calorimetry (DSC) has been used to determine the WCu (i.e. removal of ‘freezable’ free water) and its relationship with the optimal moisture content for cryopreservation (Vertucci

1989a, Dussert et al 2001, Hor et al 2005). The germination characteristics, including tolerance to desiccation and cryopreservation, have not been previously reported in any

Australian wild Citrus species.

Seed tolerance to desiccation (e.g. for storage purpose) is often dependent on the developmental status of a seed and is acquired at completion of morphological development, i.e. around mass maturity (completion of reserve accumulation) (Golovina et al 2001, Kermode and Finch-Savage 2002, Hay and Smith 2003, Pritchard 2004).

Moreover, seeds capable of a high level of germination prior to maturity may not have attained their maximal tolerance to desiccation and storage (Pritchard 2004). It was reported in Chapter 4 that seeds of C. garrawayi at different developmental stages varied

131 greatly in germination capacity and had maximal germination (88%) when seeds were mature. Premature seeds with differentiated embryos (cotyledonary) at about 30% dry weight accumulation, had significantly reduced germination level (51%) (cf. Chapter 3).

The acquisition of desiccation tolerance is often associated with the onset of accumulation of protein and oil bodies (dry weight accumulation of seed reserves), which Vertucci and

Farrant (1995) suggest may minimise the mechanical stresses of desiccation.

DSC analysis can be used for the non-invasive observation of thermal behaviour and modifications in oil reserves in seed tissues (e.g. cotyledons) (Crane et al 2003, Walters et al 2005, Lehner et al 2006). Thermal analysis of seeds of C. garrawayi, at different morphological stages, is reported here to assess the level of oil reserve accumulation of seeds of different maturities and the relationship with physiological responses to desiccation and cryopreservation (i.e. germination capacity). DSC analysis has also been used to analyse the seed oil thermal properties in C. australasica, C. inodora and

C. garrawayi to correlate this to their cryopreservation tolerance.

The overall aim of this study was to investigate cryopreservation of seeds of

C. australasica, C. garrawayi and C. inodora and determine its potential application for ex situ conservation. The following aspects were studied:

(1) The effect of desiccation and cryopreservation of seed on germination and seedling growth.

(2) The effect of seed maturity on desiccation and cryopreservation tolerance in

C. garrawayi.

(3) Thermal analysis of phase transitions in cotyledon tissue at different moisture contents and its correlation with seed germination after cryopreservation.

132 (4) Seed oil thermal properties on cooling and warming from ultra low temperatures and correlation with survival after cryopreservation.

5.2 Materials and methods

Plant material

Fruits of Citrus australasica, C. garrawayi and C. inodora were sourced from different locations and years as detailed in Chapter 2 (Material and Methods 2.2; Table

2.1). Seeds of C. garrawayi of different maturities (immature, premature and mature) were as described in section 4.2.

Seed desiccation

A range of moisture contents for germination testing was achieved in seeds of

C. australasica (Comm. 2004 and BBG 2006) and C. inodora (SQ DPI 2006) by incubation under various drying conditions: silica gel at room temperature (ca.8%RH) for either 18 hours or 10 days (2006 only) or in a environmentally controlled cabinet at 15% RH and

15ºC for 7-10 days. The same desiccation protocol was used to prepare seeds of C. australasica for thermal analysis using DSC. Seeds of C. garrawayi (SQ 2005 Feb./May;

SQ2006) were desiccated to a range of moisture contents by drying over silica gel for periods from 18h to 5 days. Additionally, seeds of C. garrawayi were equilibrated to a range of relative humidities, either over lithium chloride solutions (60-100%RH) for 10 days or bench dried (ca.50%RH) for 5 days to prepare seeds at range of moisture contents/water activities for thermal analysis (DSC).

133

Seed cryopreservation

Desiccated seeds of C. australasica, C. inodora and C. garrawayi were placed in cryotubes and directly immersed in liquid nitrogen. After storage in liquid nitrogen for at least two days, seeds were rapidly thawed by plunging cryotubes into 40ºC water for one minute. Post cryopreservation, seeds were surface disinfected by rinsing in 2% sodium hypochlorite (30 seconds), followed by two rinses in tap water before germination testing.

Germination and seedling growth in C. australasica and C. inodora

For each treatment (desiccation ±liquid nitrogen exposure), seeds were either placed on moist filter papers in petri dishes (C. australasica, 2004 only) or 0.6% agar in plastic containers. Seeds were incubated at 30ºC with an 8h (C. australasica, 2004 only) or 16h photoperiod. Seed germination was recorded as radicle emergence (>1mm) after 30 days

(C. australasica) or 60 days (C. inodora and C. garrawayi) incubation. Replications were as indicated on tables and figures. Seed vigour was assessed by seedling growth (length) and duration until radicle and epicotyl emergence. Emergence of the epicotyl and radicle

(>1mm) was recorded at 14, 30 and 60 days after incubation for seeds of each treatment

(n=50; 5 replicates). Epicotyl and root length (mm) of germinated seeds was measured at the end of the germination testing (60 days incubation).

Germination and seedling growth of seeds of C. garrawayi at different maturities

Seeds of Citrus garrawayi at different maturities (immature, premature and mature) were placed on 0.6% agar in sealed plastic containers and incubated at 30ºC, with a 16h photoperiod for 60 days. For each treatment, 5 replicates of 10 seeds were initiated for each

134 seed maturity (n=50). Radicle and epicotyl emergence (>1mm) was recorded every 3-4 days in seeds of C. garrawayi and germination curves were used to determine the time to

50% final radicle and epicotyl emergence (>1mm) for each desiccation level and seed maturity. The epicotyl and root lengths of germinated seeds were measured at the end of each germination test (60 days).

Moisture content determination

Seed moisture content measurements were determined, as described in section 2.2, from 4 replicates of 25 seeds (C. australasica 2004 only) or ten individual seeds.

Thermal analysis (DSC)

C. australasica seeds

Analysis of thermal events during freezing and thawing of seeds of C. australasica was performed using a TA Instruments (Q1000 V8.1) DSC machine as described in section

3.2. Cotyledon tissue was excised from 4 to 5 seeds for each desiccation treatment and was individually analysed.

C. garrawayi seeds

Analysis of thermal events during freezing down to -100ºC and thawing of seeds of

C. garrawayi was performed using a Perkin-Elmer (TAC7/DX7 Series) DSC machine. The

DSC was calibrated (melt onset/enthalpy) using cyclohexane (6.54ºC/31.25J-g) docedcane

(-9.65ºC/216.73J-g), n-octane (-56.76ºC/182J-g) and n-decane (-29.66ºC/202.09J-g).

Cotyledon tissue was excised from at least 5 seeds from each treatment.

135

DSC Analysis

Tissues of both species were analysed by placing individual samples in aluminum pans, with an empty aluminum pan used as a reference. A cooling and warming rate of

20ºCmin-1 was used. Warming thermograms were analysed using Universal V3.9A software to determine the enthalpies (calculated from the area of the endothermic peak under the baseline) and presented in joules per gram fresh weight (Jg-1FW), of melt transitions and onset temperatures of the samples. Sample pans were punctured after analysis and the moisture content of each sample was determined gravimetrically after oven drying.

Climatic data of the natural distribution range of each species

Mean monthly rainfall over a thirty year period (1961-1990) were compiled from data sourced from the Bureau of Meteorology (Australian Government 2005). The mean monthly rainfall (mm) was determined for distribution locations of each species and plotted against the known fruiting period for each species (cf. Table 1.5, Figure 1.5).

Statistics

Germination and seedling length (radicle and epicotyl) data were analysed using

ANOVA (analysis of variance) with a Tukey test at the 0.05 level of significance using

SPSS software version 12.0.1. Germination proportions were arcsine transformed prior to statistical analysis.

136 5.3 Results

5.3.1 Effect of desiccation and liquid nitrogen exposure on seed germination in Citrus australasica

Effect of desiccation and cryopreservation on seed germination

Seeds of C. australasica harvested in 2004 and 2006 from different locations, were found to have similar moisture contents after exposure to the same desiccation treatments

(Figure 5.1). The mean % moisture contents for fresh and desiccated seeds of the combined seed lots from 2004 and 2006 were 38.4±0.7 (Fresh), 17.3 ±0.6 (18h silica- dried), 4.6 ±0.4 (7-10d @15%RH) and 3.1±0.3 (10d silica, 2006 only) (Figure 5.1). Thus, seed at a range of desiccation levels was available for germination testing.

2006 Seed MC 2004 Seed MC Silica (10d)

15%RH/15ºC (10d)

Silica (18h)

Desiccation treatment

Fresh

0 5 10 15 20 25 30 35 40 Seed moisture content (% wb)

Fig. 5.1 Mean moisture content of desiccated seeds of Citrus australasica. Determined gravimetrically and presented on a fresh weight basis (%wb) from either 25 seeds replicated by four (2004) or 10 replicates of individual seeds (2006). Bars represent ±s.e.

137

A B 100 100

80 80

60 60

40 40

Germination (%) Germination 20 (%) Germination 20

0 0 38 17 5 3 38 17 5 3 Seed moisture content (%wb) Seed moisture content (%wb)

Fig. 5.2 Effect of (A) desiccation and (B) liquid nitrogen exposure on seed germination (>1mm radicle emergence) in Citrus australasica. Mean (±s.e.) of five replicates of 10 seeds

(2006) and four replicates of 25 seeds (2004). Germination recorded after 30 days incubation.

The results of seed germination of C. australasica at range of moisture contents and post cryopreservation are presented in Figure 5.2. Mean percent germination from seeds desiccated to low moisture contents (17, 5 or 3%) was over 95% and not significantly different (P=0.3) from fresh seeds (Figure 5.2). Seed germination post cryopreservation was 94% at both 5 and 3%MC and not significantly different to uncryopreserved seed at the same moisture contents. Liquid nitrogen exposure reduced the percentage of seed germination to 10±3 for seeds at 17% moisture content and no germination was observed from fresh seeds.

Examination of seeds at 17%MC that did not germinate after liquid nitrogen exposure, showed differential colouration (tissue damage) across the embryo, after 30 days incubation, this included white/cream (tissue death) and green (living tissue) regions of colouration. Figure 5.3 (A) shows an example of unviable seed with differential colouration

(injury) across the embryo tissue. Figure 5.3 (B, C) shows the germination of seeds at 8 and

60 days post cryopreservation following desiccation to 5%MC.

138

A C

Fig. 5.3 Germination post cryopreservation in Citrus australasica seeds at different moisture contents. (A) An unviable seed at 17% moisture content (MC) with differential tissue damage across the embryo after 30 days incubation. (B) Seed at 5%MC germinating after 30 days incubation and (C) a healthy seedling from seeds at 5%MC after 60 days incubation.

139 120 A 25 B

100 20

80 15 60 10 40

Radicle length (mm) 5 20 Epicotyl length (mm)

0 0 38 17 17 17 5 5 5 3 3 3 38 17 5 3 Seed moisture content (%wb) Seed moisture content (%wb)

Fig. 5.4 Effect of desiccation and liquid nitrogen exposure on seeds of Citrus australasica mean (A) radicle and (B) epicotyl length (mm). Mean (±s.e.) of germinated seeds of five replicates of 10 seeds. Radicle and epicotyl length (>1mm) was recorded 60 days after incubation. No significant difference (ANOVA) between seeds at all moisture contents ±liquid nitrogen exposure in either mean radicle (P=0.5) or epicotyl length (P=0.2).

Effect of desiccation and cryopreservation on seedling growth

Radicle and epicotyl lengths of seedlings, after incubation for 60 days on germination media, were not significantly different regardless of initial seed desiccation

(Figure 5.4 A, B). Radicle and epicotyl lengths were also not significantly different for seedlings post cryopreservation from seeds that germinated at moisture contents of 17% or less.

Effect of desiccation and cryopreservation on seed vigour

Radicle emergence, after 14 and 60 days incubation, was similar across all seed desiccation levels. Mean % radicle emergence ranged from 88 to 98 after 14 days incubation and was close to 100% by 60 days, regardless of desiccation level (Figure 5.5

A, C).

140

A B

100 14 d -LN100 14 d -LN +LN +LN 80 80

60 60

40 40

20 Radicle emergence (%) 20 (%) Epicotyl emergence

0 0 38 17 5 3 38 17 5 3 C 100 Seed moisture content (%wb)60 d -LN100 Seed moisture content (%wb)60 d D-LN +LN +LN 80 80

60 60

40 40

Radicle emergence (%) 20 20 Epicotyl emergence (%)

0 0 38 17 5 3 38 17 5 3 Seed moisture content (%wb) Seed moisture content (%wb)

Fig. 5.5 Effect of desiccation and liquid nitrogen exposure on radicle and epicotyl emergence (>1mm) from Citrus australasica seeds after (A, B) 14

and (C, D) 60 days incubation. Mean (±s.e.) of five replicates of 10 seeds for each treatment.

Figure 5.5 B and D shows the time to and % epicotyl emergence following desiccation and cryopreservation of seed at 14 and 60 days post incubation. The mean % epicotyl emergence, after 14 days incubation, was delayed based on seed moisture content, i.e. 42±7, 87±3, 5±3 and 14±4 from seeds at 38, 17, 5, and 3 %MC, respectively. However, by 60 days of incubation, seeds at all moisture contents had a similar mean % epicotyl

141 emergence of close to 100%, i.e. 94±2, 100, 88±4 and 92±4 from seed at 38, 17, 5, and

3%MC, respectively. In addition, liquid nitrogen further delayed the mean % epicotyl emergence from seeds at low moistures below 5% by 14 days incubation . Thus, overall, the final mean % epicotyl emergence (i.e. seedling recovery) was close to 100 for all desiccation levels, but the time to epicotyl emergence was delayed at moisture contents of

5% or less and liquid nitrogen exposure.

Very few seedlings were recovered from cryopreservation at moisture levels of 17% or above. Seeds at 5 and 3%MC had the best seedling recovery of close to 80% epicotyl emergence at 60 days, only about 20% below the fresh seed germination levels.

Effect of seed moisture content on phase transitions

Figure 5.6 shows the representative endothermic events on warming from low temperatures (-80ºC) in cotyledon tissue excised from intact seeds of Citrus australasica desiccated to different moisture contents. Differential scanning calorimetry of all seeds revealed the presence of a major endothermic peak at around –250C (Figure 5.6 a-d).

Thermal analysis of fresh seeds (38%MC) and seeds silica-dried for 18 hours (17%MC), revealed the presence of an additional much larger endothermic peak at around -50C

(Figure 5.6 a, b). This additional peak decreased in size as seed moisture content decreased.

The mean enthalpy and onset temperature of these two endothermic peaks, on warming from -80ºC, differed. The first endothermic peak had an onset temperature of -26.3±0.2 ºC and enthalpy of 35.6±0.5 Jg-1FW. The additional endothermic peak (tissue at 16%MC) had an onset temperature of -6.8±0.7 ºC and enthalpy of 68.8±4.2 Jg-1FW. These peaks were attributed to phase transitions of lipid (first peak) and water (second peak).

142

Fig. 5.6 Representative differential scanning calorimetric thermocurves of melt transition events in cotyledon tissue of Citrus australasica after desiccation of

intact seeds to mean % moisture contents of: (a) 3 (silica-dried for 10 days), (b) 5

(15% relative humidity for 10 days), (c) 17 (silica-dried 18 hours), and (d) 38

(untreated).

The unfrozen water content of the seeds can be determined as the x-intercept of two regression lines of the least square best fit of seeds at moisture contents with (i) the presence and (ii) the absence of endothermic peak 2 (water phase transition) as previously described by Vertucci 1989a and Dussert et al 2001. By this method, the unfrozen water

-1 content of C. australascia was determined as 11%MC (ca. 0.10gH2Og dry weight) (Figure

5.7). This was determined as the intercept of regression lines of cotyledon tissue at moisture

2 contents of either 15% (R2= 0.79, p=0.003) or <15% (R = 0.24, p=0.1).

143

100

-1 80

Melt enthalpy (jg FW) 40

0 5 10 15 20 25 30 Moisture content (%wb)

Fig. 5.7 Enthalpy of melt transition in Citrus australasica cotyledon tissue at different water contents on warming from low temperatures (-80ºC). Regression lines are the least square

best fit for seeds with the presence or absence of a water melt endothermic peak: either 15%MC 2 2 (R =0.79; p=0.003) or <15%MC (R =0.24; p=0.1)

This correlated well with the high recovery observed in seeds of C. australasica at low moisture contents, i.e. below the unfrozen water content of 11%, and low recovery in seeds at 17% or greater moisture content.

144

Fig. 5.8 Melt transition events in cotyledon tissue of Citrus australasica

at different moisture contents (MC) achieved by silica drying of intact seeds for 18 hours. Labels represent small peak (SP) and large peaks (LP)

recorded for cotyledon tissue at 15% MC and >18%MC, respectively.

In order to further refine the phase transition characteristics of seed at a mean 17% mean (i.e. 18h silica-dried), DSC analysis on seed at between 15%MC and above 18%MC were compared (Figure 5.8). This shows that a large peak (LP) for water phase transition was observed in seeds at >18% MC but a much smaller peak (SP) was observed in the seed at 15% MC, after non-equilibrium silica drying for only 18 hours. This may explain why there was a low level of germination (10%) observed in the p±opulation of seeds post cryopreservation with a mean moisture content of 17% (Figure 5.2 B). It is likely that 15% moisture content is the upper limit for survival after liquid nitrogen exposure in

C. australasica seeds.

145 5.3.2 Effect of desiccation and liquid nitrogen exposure on seed germination in Citrus inodora

Effect of desiccation and cryopreservation on seed germination

Figure 5.9 shows the mean % moisture contents of fresh seeds (30.1±3.0) of

C. inodora, and seed desiccated for 18h over silica (12.0±2.0), 10d over silica (3.2±0.34) and 10d in an incubator at 15%RH (2.4±0.15). Seeds at these moisture contents were used for subsequent germination testing and cryopreservation trials.

15%RH (10d)

Silica-dried (10d)

Silica-dried (18h)

Desiccation treatment Fresh

0 10203040 Moisture content (%wb)

Fig. 5.9 Mean % moisture content of Citrus inodora

seed desiccated over silica gel or in a controlled relative humidity (RH) cabinet (15%RH). Determined

gravimetrically and presented on a fresh weight basis (%wb) from 10 replicates of individual seeds for each treatment. Bars represent ±s.e.

146

A B

Fig. 5.10 Effect of desiccation and liquid nitrogen exposure on (A) radicle and (B) epicotyl emergence (>1mm) in Citrus inodora. Mean (±s.e.) of five replicates (n=50) for each

treatment and was recorded 60 days after incubation.

Seed radicle and epicotyl emergence after desiccation and liquid nitrogen exposure is presented in Figure 5.10. Both radicle and epicotyl emergence were highest (88-96%) in fresh seeds (30%MC) or seeds at 12%MC and not significantly different from one another

(p=>0.8). However, % epicotyl emergence was significantly (P=<0.05) reduced (71%) in seeds desiccated to low moisture contents (3%) compared to seeds at higher moisture contents, i.e. 12 and 30%MC. Similar results were observed for radicle emergence, except a significant reduction was not observed until desiccation reached a very low moisture content of 2%.

Radicle and epicotyl emergence from seeds after liquid nitrogen was best (>70%) in seeds at a low moisture content of 3% and was comparable (P=>0.9) to non-cryopreserved seeds at 3%MC. Some survival was observed at 12%MC, but radicle and epicotyl emergence levels were negligible (6%).

147 Effect of desiccation and cryopreservation on seed growth

Figure 5.11 shows the results of measurement of radicle and epicotyl lengths, after 60 days germination incubation, at a range of seed desiccation levels and post cryopreservation.

Radicle length was not significantly different between fresh seeds (30%MC) and seeds desiccated to 12 and 3%MC (P=>0.9) and ranged between 24±2 to 27±3mm. In addition, liquid nitrogen exposure did not significantly reduce radicle length (21±3mm) from seeds surviving at 3%MC (P=>0.9). However, desiccation to a very low 2%MC resulted in a significantly reduced (P=<0.05) mean radicle length (14±2mm), compared to seeds at higher moisture contents i.e. 3%MC (Figure 5.11A). Mean epicotyl length was about

20mm (15±1 to 22±2mm) and not significantly different for all seeds regardless of desiccation level and liquid nitrogen exposure (Figure 5.11B). Thus, overall radicle and epicotyl length was similar between germinated cryopreserved and non-cryopreserved seeds (i.e. 2 and 3%MC), except for radicle length in seeds at 2%MC.

30 A 30 B

25 25

20 20

15 15

10 10

5 5 Radicle length (mm) Epicotyl length (mm) 0 0 30 12 3 3 2 2 30 12 3 3 2 2 Moisture content (%wb) Moisture content (%wb)

Fig. 5.11 Effect of desiccation and liquid nitrogen exposure on mean (A) radicle and (B) epicotyl length (mm) of seeds of Citrus inodora. Mean (±s.e.) of germinated seeds of five replicates of 10 seeds for each treatment and was recorded 60 days after incubation.

148

Effect of desiccation and cryopreservation on seed duration until germination

Seed vigour was assessed by measuring the rates of both radicle and epicotyl emergence. Figure 5.12 shows radicle emergence, after 14, 30 and 60 days incubation, across all desiccation levels ± liquid nitrogen exposure. A delay in mean % radicle emergence was observed after 14 days incubation, as a linear decrease with decreasing moisture content, i.e. 92±5 (30%MC) to 15±7 (2%), which was amplified by liquid nitrogen exposure. However, close to final (60 day) levels, within treatments, were observed by 30 days regardless of desiccation level or liquid nitrogen exposure. A considerable delay was observed in epicotyl emergence in seeds on desiccation and liquid nitrogen exposure after 30 days incubation, compared to levels observed after 60 days incubation (Figure 5.12). Particularly notable was both the delay and subsequent recovery of seeds at 3%MC after 30 (52% -LN and 8 % +LN) and 60 days (71% -LN and 72% +LN) incubation. Thus, mean % radicle and epicotyl emergence was particularly delayed at moisture contents of 3% or less and this was further exacerbated by liquid nitrogen exposure.

149

100 14d 100 14d

80 80

60 60

40 40

Radicle emergence (%) 20 20 Epicotyl emergence (%) 0 0 100 30 12 3 230d 100 30 12 3 230d Seed moisture content (%wb) Seed moisture content (%wb) 80 80

60 60

40 40

Radicle emergence(%) 20 20 Epicotyl emergence (%)

0 0 100 30 12 3 260d 100 30 12 3 260d Seed moisture content (%wb) Seed moisture content (%wb)

80 80

60 60

40 40

Radicle emergence (%) 20 20 (%) Epicotyl emergence 0 0 30 12 3 2 30 12 3 2 Seed moisture content (%wb) Seed moisture content (%wb)

Fig. 5.12 Effect of desiccation and liquid nitrogen exposure on mean radicle and epicotyl emergence of Citrus inodora seeds after 14, 30 and 60 days incubation. Mean (±s.e.) of five replicates of 10 seeds for each treatment (n=50).

150 5.3.3 Effect of desiccation and liquid nitrogen exposure on seed germination in Citrus garrawayi, including the effect of seed maturity

Effect of desiccation on seed moisture content in relation to seed maturity

Figure 5.13 shows that moisture content decreased with increased seed maturity - immature (82.2±1.3%), premature (74.8±2.0%) and mature (40.3±1.1%). In addition, the embryo had a lower mean moisture content than the seed coat in both premature (64.8±3.2 and 74.3±1.5, respectively) and mature seeds (37.9±6.5 and 45.5±2.6, respectively).

Embryos of immature seeds were too small (incomplete histodifferentiation) to determine their moisture content, whilst both premature and mature seeds had much larger differentiated embryos (cotyledonary). Figure 5.14 shows that C. garrawayi seeds of different maturities (immature, premature and mature) had a similar drying rate when dried over silica for 18 to 240 hours. However, as the initial seed moisture content decreased with seed maturity, the final seed moisture content levels were also much lower after 240h silica drying; immature (21.9±3.4), premature (10.6±2.1) and mature (4.6±0.4).

Whole seed 90 Seed coat 80 Embryo 70 60 50

40 30 20 Moisture(%wb) content 10 0

Immature Premature Mature Seed maturity Fig. 5.13 Mean moisture content of embryo, seed coat and whole seed of Citrus

garrawayi seeds at different maturities. Determined gravimetrically after oven drying, from ten replicates of individual tissue samples (until constant weight at 80ºC) and whole seed (18h at 103 ºC). 151

240h Immature Premature Mature 120h

48h

18h

Fresh Desiccation duration (silica)

0 20406080100

Moisture Content (% wb)

Fig. 5.14 Effect of seed maturity in Citrus garrawayi on seed moisture content after silica-drying. Mean (±s.e.) of ten replicates of individual seeds for each desiccation level. Determined gravimetrically and presented on a wet weight basis

(%wb).

Effect of desiccation on seed germination in relation to seed maturity

Table 5.1 shows the effect of desiccation on radicle and epicotyl emergence of seeds at different maturities. Mean radicle and epicotyl emergence was negligible (<10%) from immature seeds at 82.2% (fresh) and 43.1% moisture content. Fresh premature (76.8%MC) and mature (40.5%MC) had much higher % of radicle and epicotyl emergence of 70±15 and 57±19 (premature) and 96±3 and 84±4 (mature), respectively. However, desiccation of premature seeds to 13.1%MC reduced the mean radicle and epicotyl emergence to a much lower 49±7 and 28±14%, respectively. In contrast, mature seeds tolerated desiccation to much lower moisture contents of 6.5 and 2.9%MC, with high levels of both mean % radicle

(91±4 to 96±3) and epicotyl (69±5 and 74±6) emergence.

152

Table 5.1 Effect of desiccation on mean radicle and epicotyl emergence (±se) and duration until 50% final germination of seed of Citrus garrawayi at different maturities.

Seed Maturity Moisture Emergence (%) Time until 50% of Content final emergence (% wb) (days) Radicle Epicotyl Radicle Epicotyl

Immature 82.2 8±8 8±8 16 16

43.1 9±7 6±4 21 26

Premature 74.8 70±15 57±19 15 16

30.1 64±7 55±15 16 28

13.1 49±7 28±14 12 27

Mature 40.3 96±3 84±4 10 18

6.5 95±2 74±6 12 23

2.9 91±4 69±5 11 15

Mean (±se) of radicle and epicotyl emergence (>1mm), three replicates and seed lot two by two replicates (n=50), except premature 13.1%MC no duplicate (n=40). Germination on 0.6% agar at 30ºC, 16 hour photoperiod, recorded two months post initiation. Desiccation of seeds was over silica for 18 to 120h. Mean moisture content presented on a fresh weight basis determined from ten individual seeds for each maturities (n=10, one seed lot), except fresh seeds (n=20, two seed lots).

Time until 50% of final percent of radicle and epicotyl emergence differed between seeds at different maturities and was further affected by desiccation (Table 5.1). As previously reported in Chapter 4 (section 4.3), time until 50% of final levels for radicle emergence was 16 days from fresh immature and premature seeds, compared to only 10 days from mature seeds. Desiccation had little effect on the rate of radicle emergence in mature seeds, which ranged from 10 to 11 days in seeds from 40.3 to 2.9%MC. In contrast, mean radicle emergence from immature seeds was delayed from 16 to 21 days by

153 desiccation from 82.2% (fresh seeds) to 43.1%MC. Desiccation of premature seeds to 30.1 and 13.1%MC did not delay however radicle emergence

The time until 50% of final levels of epicotyl emergence was about 16 days for fresh seeds at all maturities. Desiccation delayed epicotyl emergence to 23 days after desiccation of seeds of all maturities. In mature seeds desiccated to a very low moisture content of 2.9%MC, time until 50% final levels of epicotyl emergence was, however, similar to control levels (ca.16 days). Overall, duration until radicle and epicotyl emergence was least affected by desiccation in mature seeds.

Thermal analysis of seeds at different maturities

The decrease in moisture content with seed maturity coincided with a seven-fold increase in dry seed weight (Table 5.2). In order to determine whether this increase in dry weight is at least partially attributable to the accumulation of seed oil reserves, the thermal events in seed tissue at different maturities were analysed using DSC. Because the seed tissue

(cotyledon) was dry, any phase transition events on warming from low temperature could be attributed to lipid melt events (endotherm) and not water (cf. Chapter 5.3.1). Table 5.2 shows the lack of a lipid endotherm in immature seeds. In contrast, a lipid endotherm was detected in some premature (40%) and all mature (100%) seed samples. The thermal characteristics of the lipid phase transitions differed between premature seeds and mature seeds. Most notable was the much lower mean enthalpy values observed from immature

(2.2±0.3 Jg-1FW) compare to mature seeds (28.5±1.7 Jg-1FW). Thus, the presence of a lipid endotherm and the energy level (i.e. enthalpy Jg-1FW), determined using DSC analysis, increased substantially from premature to mature seeds and corresponded with the increase in seed mean dry weight.

154

Table 5.2 Thermal analysis of lipid endotherm in dry seeds at different maturities in Citrus garrawayi Seed MC Dry Thermal analysis Maturity (%) weight Endotherm Endotherm Melt onset (mg) A presence enthalpy temperature n (%) (Jg-1FW) (ºC)

ImmatureB 21.9 2.4 ±0.2 0 - - 6

PrematureB 10.6 5.5 ±0.6 40 2.2 ±0.3 -7.7 ±2.2 5

MatureC 4.6 17.0 ±0.9 100 28.5 ±1.7 -10.0 ±1.2 8

AMean (±s.e.) of 8-10 individual seeds/cotyledon tissue. BThermal analysis on whole seeds that were silica dried for ten days. CThermal analysis on cotyledon tissue after intact seeds bench dried for five days.

Effect of desiccation and cryopreservation on premature and mature seed germination

Seed germination results after desiccation and cryopreservation for premature and mature seeds are presented in Figure 5.15 (desiccation only data as presented in Table 3.1). Both desiccation and liquid nitrogen exposure considerably reduced radicle emergence from premature seeds compared to untreated seeds. The best mean % radicle emergence after liquid nitrogen exposure was observed from seeds at 30.1%MC, at 64±7 (-LN) and 42±14

(+LN). This was reduced on further desiccation to 13.1%MC to only 49±7% (-LN) and

23±5% (+LN). Mean % epicotyl emergence (i.e. whole seedling recovery) was approximately halved with each decrease in moisture content and further reduced after liquid nitrogen exposure. For example, premature seeds at 13.1%MC had a low mean epicotyl emergence of 28±14%, that was further reduced to only 14±6% by liquid nitrogen exposure. Interestingly, premature seeds at a rather high moisture contents, i.e. 30.1%, still had relatively high levels of recovery after liquid nitrogen exposure. 155

A Premature B Premature 100 -LN +LN 100 -LN +LN 80 80 60 60

40 40

Radicle emergence (%) 20 Epicotyl emergence (%)

0 0 74.8 30.1 13.1 74.8 30.1 13.1 Moisture content (%wb) Moisture content (%wb)

Mature Mature

C D 100 -LN +LN 100 -LN +LN 80 80

60 60

40 40

20 Radicle emergence (%) 20 Epicotyl emergnece (%)

0 0 40.3 6.5 2.9 40.3 6.5 2.9 Moisture content Moisture content (%wb)

Fig. 5.15 Effect of desiccation and liquid nitrogen exposure on radicle emergence and epicotyl emergence (>1mm) from (A, B) premature and (C, D) mature seeds of Citrus garrawayi. Mean (±s.e.) of three replicates of 10 seeds, duplicated by two replicates (n=50) for each treatment and was recorded after 7 weeks incubation.

156 In contrast, mature seeds had a much greater tolerance, as assessed by radicle and epicotyl emergence, to the combinational stresses of desiccation and liquid nitrogen exposure. Mean % radicle and epicotyl emergence from mature seeds desiccated to low moisture contents (3%) was not significantly different (P=0.6) to control levels and was about 70 to 80% (Figure 5.15 C, D). However, liquid nitrogen exposure significantly

(P=0.01) reduced mean % radicle emergence in seeds at 6.5 and 2.9%MC compared to seeds 7%MC without liquid nitrogen exposure. Seeds at 2.9%MC did not show significantly different (P=0.08) radicle emergence ±LN exposure. Mean % radicle and epicotyl emergence after liquid nitrogen exposure was comparable from seeds at low moisture contents (i.e. 6.5 and 2.9), about 70% and 40% for radicle and epicotyl emergence respectively (Figure 5.15).

Effect of desiccation and cryopreservation on seedling growth

Figure 5.16 shows data, which assesses seed vigour in terms of seedling growth, i.e. measuring radicle and epicotyl lengths of seeds of different maturities following desiccation and liquid nitrogen exposure. Mean radicle and epicotyl lengths (mm) for both desiccation levels were not significantly different for immature (p=>0.6) seeds, regardless of desiccation level and liquid nitrogen exposure, after 60 days incubation. This was also the case for radicle and epicotyl lengths (mm) of premature seeds which were not significantly (P=>0.2) different for any desiccation level ±liquid nitrogen exposure. Mature seeds at 40.3%MC (fresh seeds) and 6.5%MC had a radicle length (mm) of 40±2 and 37±3 that was not significantly different (P=0.9). However, further desiccation to 2.9% or liquid nitrogen exposure of seeds at 6.5% and 2.9%MC resulted in a significant reduction

(p=<0.02) in mean radicle lengths (17±2 to 25±3mm), compared to fresh seeds. Mean epicotyl length (mm) was not significantly different (P=>0.1) from mature seeds at

40.3%MC (30±1mm) and seeds desiccated to 6.5%MC±liquid nitrogen exposure.

157 However, epicotyl length was significantly different (P=0.01) in seeds at 2.9%MC ±liquid nitrogen exposure (23±1 and 20±1mm), compared to fresh seeds (40.3%MC). Thus, overall, radicle and epicotyl lengths were similar between seeds desiccated to 6.5%MC and fresh seeds. However, further desiccation to to very low moistures contents (2.9%MC) or seed desiccation and liquid nitrogen reduced radicle (6.5%MC) and epicotyl (2.9%MC) lengths. Immature ABImmature 40 30

30 20 20

10 10 Radicle length (mm) Epicotyl length (mm) Epicotyl length 0 0 82.2 43.1 43.1 82.2 43.1 43.1 PrematureMoisture content (%) C PrematureMoisture content (%) D 40 30

30 20

10 10 Radicel length (mm) Epicotyl length (mm) Epicotyl length 0 0 74.8 30.1 30.1 13.1 13.1 74.8 30.1 30.1 13.1 13.1 MatureMoisture content (%) E MatureMoisture content (%) F 40 30

30 20 20

10 10 Radicle length (mm) Epicotyl length (mm) 0 0 40.3 6.5 6.5 2.9 2.9 40.3 6.5 6.5 2.9 2.9 Moisture content (%) Moisture content (%)

Fig. 5.16 Effect of desiccation and liquid nitrogen exposure on mean radicle and epicotyl length (mm) from (A,B) immature, (C,D) premature and (E,F) mature seeds of Citrus garrawayi. Mean (±s.e.) of germinated seeds of five replicates of 10 seeds. Radicle and epicotyl length (>1mm) recorded 60 days after incubation. 158

Effect of seed moisture content on phase transitions in mature seed tissues

Table 5.3 shows the results of DSC analysis of either cotyledon or seed coat tissue of C. garrawayi, equilibrated over a series of relative humidities (50 to 100%RH), to a range of moisture contents/water activities. The main difference in the thermal behaviour of these two tissues was the presence (cotyledon tissue, i.e. embryo) and absence (seed coat) of a melt endotherm that was attributable to the lipid phase transitions, in samples at low mean moisture contents <14%MC. Although seed coat tissue, at low moisture contents showed no melt endothermic events, endothermic peaks were detected in seed coat tissue at higher moisture contents and these were characteristic of water melt phase transitions. That is, seed coat tissue from seeds with a very high water activity (0.96) had a melt onset mean temperature of 0.2±0.9ºC and mean enthalpy of 179.7±7.4 Jg-1FW.

The main endothermic event in dry cotyledon tissue, with a seed water activity of

0.79, was attributed to a lipid phase transition and had a melt onset temperature of -100C and enthalpy of 39.9 Jg-1FW. In comparison, cotyledon tissue excised from seeds with a high water activity of 0.96 displayed a different main endotherm. This endothermic peak was observed in seeds 30%MC and increased in size (i.e. enthalpy) with seed mean moisture content (Figure 5.17) and was attributable to water phase transition with an onset temperature of -2.90C and enthalpy of 124.9±14.7 Jg-1FW.

159

Table 5.3 Effect of equilibrium relative humidity (%RH) on seed water activity, melt endotherm presence and thermal characteristics of embryo and seed coat tissue in Citrus garrawayi

Cotyledon Seed coat

Equilibrium Seed Mean MC Endotherm Mean Mean melt Mean Endotherm Mean Mean melt

RH (%) Water (%) presence (%) enthalpy onset temp. MC (%) presence (%) enthalpy onset temp.

activity (Jg-1FW) (ºC) (Jg-1FW) (ºC)

50 0.52 4.6±0.3 100 28.5 ±1.7 -10.0 ±1.2 9.6 ±0.6 0 - -

70 0.62 6.1±0.2 100 24.3 ±3.3 -15.3±1.5 11.3 ±0.4 0 - -

80 0.72 7.8±0.6 100 29.1 ±2.5 -18.6 ±2.3 13.7 ±0.3 0 - -

85 0.79 8.0±0.3 100 39.9 ±7.1 -11.1±1.7 14.8 ±0.8 20 0.18 ±0.01 -5.9 ±0

100 0.96 51.4±14.2 100 124.9±14.7 -2.9 ±0.7 72.4 ±2.8 100 179.7 ±7.4 0.2 ±0.9

Seeds were equilibrated either by air-drying (laboratory bench 5d) or over a range of concentrations of lithium chloride (60 to 100%RH) for 10 d. Seed water activity determined using Rotronic device.

160 The unfrozen water content of C. garrawayi seeds was calculated as the x-intercept of two regression lines of the least square best fit of cotyledon tissue at moisture contents with (i) the presence and (ii) the absence of lipid endotherm and presented in Figure 5.17.

By this method, the unfrozen water content of C. garrawayi was determined as 14%MC.

This was calculated from the intercept of regression lines of cotyledon tissue at moisture contents above and below 15% (fresh weigh basis), i.e. 30%MC (R2= 0.964; p=0.001) or <12%MC (R2= 0.012; p=0.57).

Fig. 5.17 Enthalpy of phase transition in Citrus garrawayi cotyledon tissue at different moisture contents (%wb) on warming from low temperatures (-100ºC). Regression lines

are the least square best fit for seeds either >30% MC (R2= 0.964, P=<0.001) or <12% 2 MC (R = 0.012, p=0.57).

161 5.3.4 Analysis of responses to cryopreservation in C. australasica, C. inodora and

C. garrawayi and association with climatic range and seed oil thermal properties of each species

Mean monthly rainfall of natural distribution

Figure 5.18 shows the mean monthly rainfall (mm) (1961-1990) of the natural distribution ranges of C. australasica, C. inodora and C. garrawayi (compiled for distribution from data sourced form the Bureau of Meteorology 2005). The distribution range of C. australasica has a fairly uniform rainfall pattern throughout the year (i.e. 25-

100mm/month), whilst the two tropical northern species have a distinct dry and wet season, with high levels of rainfall (up to 400mm) during the wet season months (October to April).

During the fruiting periods (cf. Figure 1.5), which are the predominantly during the drier periods, the mean monthly rainfall (mm) is 25 to 100mm in C. australasica, 25 to 400mm in C. inodora and 5 to 100mm in C. garrawayi.

450 Fig. 5.18 Mean monthly C. australasica rainfall of natural distributions 400 C. inodora C. garrawayi of Citrus australasica, 350

C. inodora and C. garrawayi. 300 250

200

150

Mean rainfall (mm) 100 50

n y v c pr a un Jul o e Ja Feb Mar A M J Aug Sep Oct N D Month

162 Thus, the lower limits of monthly rainfall (25mm) are the same in C. australasica and

C. inodora during the fruiting season (cf. Figure 1.7). For C. garrawayi much lower monthly rainfall (5 to 10mm/month) occurs during the 4 months of its fruiting period. A mean rainfall of 50mm/month was observed at about the end of the fruiting period, for all three speices and this then leads into the wettest period of the year (i.e. at least 5 months of

100mm rainfall/month).

Comparative seed germination after deisccation and cryopreservation

Table 5.4 shows a comparative summary of the germination and cryobiology data presented previously in this study. Mean % radicle and epicotyl emergence from seeds at

3%MC ± liquid nitrogen exposure in C. australasica, C. inodora and C. garrawayi.

Desiccation tolerance was demonstrated, to low moisture contents (3%MC), well below the

WCu, in seeds of all three species, i.e. mean % radicle emergence of 84%. However, lower levels of epicotyl emergence (i.e. seedling recovery) were observed from seeds at 3%MC in

C. inodora and C. garrawayi (ca.70%) compared to C. australasica (92%).

Tolerance to cryopreservation was also demonstrated in seeds at 3%MC in

C. australasica and C. inodora. Radicle emergence was at least about 80% and epicotyl emergence was above 70% from seeds at 3%MC in C. australasica and C. inodora, after liquid nitrogen exposure. In contrast, C. garrawayi seeds at 3% (and 6.5%) were less tolerant to cryopreservation. A mean % radicle emergence of 69% (22% reduction) was observed and a much lower mean % epicotyl emergence of 42±12 (i.e. seedling recovery) was observed from C. garrawayi seeds.

163

Table 5.4 Comparative germination ±liquid nitrogen (LN) exposure of dry seeds (3%MC) of Citrus australasica, C. inodora and C. garrawayi in relation to seed thermal properties Emergence (%) Lipid thermal characteristics –LN +LN

Species WCu (%wb) Lipid Melt onset Crystallization Melt content (ºC) onset (ºC) enthalpy Radicle Epicotyl Radicle Epicotyl (% d.wt) (Jg-1FW)

C. australasica 11 44A -26.3±0.2 -9.2±1.6 36.7±0.6 96±4 92±4 96±4 75±2

C. inodora 8 A 54 -22.7±0.7 -7.4±0.5 42.7±1.7 84±7 71±13 79±5 71±7

C. garrawayi 14 30 -13.7±2.2 -4.8±0.7 34.8±1.1 91±4 69±5 69±10 42±12

A WCu is unfrozen water content (presented on a fresh weight basis, %wb). Estimated using equation developed by Hor et al (2005), WCu =23.4-0.28LC. Lipid characteristics determined using differential scanning calorimetry of dry cotyledon tissue (ca.5%MC) for 10 to 16 individual samples randomly selected from 2 to 3 seed lots for each species. Germination recorded 8 weeks after seed initiation for five replicates per treatment (n=50).

164 Overall, some level of tolerance to desiccation and cryopreservation, in decreasing order, was observed in C. australasica, C. inodora and C. garrawayi. Additionally, the level of tolerance appears to reduce with a decrease in latitude (NSW to NQLD) and climatic conditions of natural distribution (i.e. subtropical to hot tropical) for these species.

Comparative thermal transitions in seed tissues

The unfrozen water content was close to 10%MC for all three species,

C. australasica (11%MC), C. inodora (est. 8%MC) and C. garrawayi (14%MC) (Table

5.4). Table 5.4 also shows the thermal characteristics on freezing (liquid to solid

(crystalline)) and thawing (solid to liquid) in dry seed of the three species. Phase transitions observed in seed tissue (cotyledon) below the unfrozen water content most likely represents that of lipids, as each of these species has a high lipid content (C. australasica, est.44%;

C. inodora, 54% and C. garrawayi, 30%). Extracted seed oils were analysed on cooling and warming (-80ºC to 30 ºC) for dry seeds of C. inodora and C. garrawayi, which confirmed that the thermal events (exo/endothermic peaks) observed in cotyledon tissue represented lipid phase transitions (Figure 5.19). The mean onset temperature of crystallization and melt events of lipids (cotyledon tissue) differed between the species (Table 5.4);

C. australasica (-9.2±1.6 and -26.3±0.2), C. inodora (-7.4±0.5 and -22.7±0.7) and

C. garrawayi (-4.8±0.7 and -13.7±2.2).

Figure 5.20 shows comparative exothermic (crystallization) and endothermic (melt) curves of lipid phase transition in the three species, illustrating this shift in profile (i.e. onset temperature) of the lipid phase transition. This shift in thermocurves (i.e. mean onset temperatures of lipid melt and crystallization events) between species was positively associated with a decrease in latitude (NSW to NQLD) and increased temperatures (i.e. the subtropical to hot tropical of the natural distribution range of these species).

165

2 A C. garrawayi

Cotyledon 2005 1 Cotyledon 2006 Lipid

0 -60 -40 -20 0

0.0 B

Heat Flow (W/g)

Lipid

Cotyledon

-1.0

C. inodora -2.0 -60 -40 -20 0 20

Fig. 5.19 Representative differential scanning calorimetric thermocurves of transition events in cotyledon tissue and lipid extract of seeds of (A) Citrus garrawayi on cooling to ultra low temperatures (-80ºC) and (B) Citrus inodora on warming from -80ºC. Intact seeds were silica-dried for 10 days (ca.5%MC) prior to thermal analysis and seed oil extraction.

166

2 A

C. australasica (Au) C. inodora (In) C. garrawayi (Ga)

Ga 1 In Au

W/ ( 0

-60 -40 -20 0

0 B

Heat Flow Heat Flow

Au In Ga -1

-2 -60 -40 -20 0 20 Exo Up Temperature (°C)

Fig. 5.20 Comparative thermocurves of in vivo lipid phase transition events in cotyledon tissue of C. australasica, C. inodora and C. garrawayi on (A) cooling and (B) warming from ultra low temperatures (-80ºC). Intact seeds silica-dried for 10 days 167 (ca. 5%MC) prior to cotyledon tissue extraction and thermal analysis. 5.4 Discussion

Seed desiccation

Barton (1943) raised early questions about the desiccation sensitivity of citrus seeds, finding that the method of drying affected seed viability. Many subsequent studies demonstrated a high level of desiccation tolerance in some Citrus species (e.g.

C. aurantifolia and C. limon) using rapid drying methods (i.e. silica or airflow) (Mumford and Grout 1979, Mumford and Panggabean 1982, Lambardi et al 2004). More recently,

Dussert et al (2006) showed that slow equilibrium drying (e.g. equilibration over 81% relative humidity) led to significant deteriorative processes in coffee. Dussert et al (2006) explains these findings as validation of the proposal by Walters et al (2001) that slow drying may induce oxidative stress because of the longer time spent at intermediary hydration levels where the down regulation of metabolism is uncoordinated (Leprince et al

1999, 2000). This supports the predominant use of rapid drying methods (e.g. silica and laminar airflow) in studies of Citrus seed storage (Mumford and Grout 1979, King and

Roberts 1980, King et al 1981, Mumford and Panggabean 1982, Soetisna et al 1985,

Saipari et al 1998, Cho et al 2002a, Lambardi et al 2004). However, Dussert et al (2006) assert the need for equilibrium drying in species with a narrow hydration window for cryopreservation.

In this present study, rapid drying methods (8-15%RH for about 5-10 days) resulted in high levels of germination from seeds of C. australasica (92%) and slightly lower levels in C. inodora and C. garrawayi (ca.70%) at very low moisture contents (<5%MC). Thus, rapid drying methods may be the best in these species given their tolerance to very low

168 moisture contents i.e. well below WCu indicating that there is no narrow hydration window for cryopreservation purposes.

Both the northern Queensland tropical species of wild Citrus showed a significant reduction in either epicotyl emergence (C. inodora) or seedling length (C. garrawayi) on desiccation to very low moisture contents (3%). In contrast, significant reductions were not observed between untreated and desiccated seeds of the southerly-distributed subtropical species (C. australasica). However, overall the decreasing order of species tolerance to rapid seed desiccation, without a significant reduction in seedling length and percentage radicle emergence, was C. australasica (<3%MC), C. inodora ( 3%MC) and C. garrawayi

(7%MC).

Mumford and Panggabean (1982) found that seed topography of three cultivated

Citrus species, as determined by scanning electron microscope, were different and reflected the contours of the underlying epidermal cells. In particular, the length of protrusions on the outer epidermal wall was found to be longer in the less desiccation tolerant species (loss of viability below 8%MC), compared to the more desiccation tolerant species (remained viable at 3%MC or below). Differentiation in protrusion length in the three Australian wild

Citrus species studied may also be associated with differences in desiccation tolerance.

However, the differentiation in desiccation tolerance was observed mainly in terms of seed vigour (i.e. seedling length) in this study rather than in viability loss, as had been found in cultivated Citrus species. Loss of vigour was seen at <3%MC in C. australasica and

3%MC in C. inodora. Both these species have very small protrusions on the seed coat.

169 C. garrawayi has much longer epidermal protrusions (size) and loses seed vigour at

7%MC.

Rainfall of natural distribution in relation to desiccation tolerance

Ecological factors, such as water availability and temperature, can be critical to seed physiology (e.g. seed quality), as well as seedling survival and growth post germination. In particular, knowledge of rainfall and temperature within the natural distribution of a species can assist investigations of seed storage behaviour. The subtropical distribution range of

C. australasica has a uniform rainfall pattern throughout the year (i.e. 25-100mm/month).

The two tropical northern species (C. inodora and C. garrawayi) experience distinct, dry and wet seasons, with very high rainfall throughout the wet season (300-400mm/month).

Although there was some variation in the fruiting months of the three species, this occurred predominantly in the drier (and cooler) period of the year. Interestingly, the end of the fruiting period occurred at about the beginning of the wettest period of the year in all three species. It is likely that seed dispersal occurs during this time and as such would coincide with the wettest periods of the year. However, seed dispersal periods/mechanisms of both

Australian wild and cultivated Citrus have not been described and it is difficult to relate rainfall data to the physiological responses of the seeds (i.e. germinability). There is a need for an in situ study of the seed ecology of Australian wild Citrus species in order to understand seed physiological responses and morphology in relation to climatic data (e.g. seed dispersal period).

170 Effect of seed maturity on desiccation and cryopreservation tolerance in C. garrawayi

Variation in the stage of seed maturity has been suggested as an important factor in the complex seed responses (e.g. on desiccation and cryopreservation tolerance) of oil rich seeded species such as neem (Sacandé et al 2000) and coffee (Vasquez et al 2005, Dussert and Engelmann 2006). It has also been suggested as a possible reason for the conflicting reports of seed desiccation responses in neem seeds (Sacandé et al 2000, Berjak et al 1995,

Esswara et al 1998, Gamene et al 1996). There have been no previous reports on the importance of seed maturity on seed viability losses in citrus.

Seed lot quality in relation to seed developmental status and handling methods appears to affect seed responses to desiccation and storage, particularly in wild species

(Pritchard 2004). Citrus garrawayi was observed to have more heterogeneity in seed lots in terms of seed maturity, than either C. australasica or C. inodora (data not shown). Seed quality differences are often due to differences in flowering time, dry matter accumulation, dormancy and maturation, all of which are genetically regulated traits that affect seed longevity (Walters 1998). The variation in developmental status of seeds within seed lots of

C. garrawayi appears to be attributable to differential timing of flowering and fruiting, as has been observed in many wild species (Hay and Smith 2003). Thus, as detailed in

Chapter 4.4, seed maturity ‘markers’ were developed to reduce loss of seed viability on application of stress conditions (e.g. desiccation and cryopreservation) in seed lots, by reducing variation in maturity of seed lots.

In the present chapter, comparative tolerance to desiccation between seeds at difference maturities in C. garrawayi was related to seed physiology (i.e. germinability). It was found, for example, that in premature seeds germination levels were reduced by about

171 half, down to 28% germination on desiccation to only 13%MC, whilst, mature seeds desiccated to much lower moisture contents (3%MC) retained high levels of germination

(ca.70%). Germination was further reduced by a combination of stresses (i.e. desiccation and cryopreservation) to about half again in premature seeds (i.e. only 14% germination from seeds at 13%MC). Germination from immature seed was negligible regardless of treatment, most likely because of incomplete embryo histodifferentiation. Overall, the level of desiccation tolerance increased during seed development in C. garrawayi. This has been previously described as common to many species, with desiccation tolerance maximal at about full seed maturity (Vertucci and Farrant 1995, Kermode and Finch-Savage 2002).

As previously discussed (cf. Chapter 1.6). maximum desiccation tolerance is often acquired at mass maturity (Kermode and Finch-Savage 2002), i.e. at completion of dry matter accumulation, including reserve deposition. This reserve accumulation (e.g. oil and protein) may minimise the mechanical stresses of desiccation (Vertucci and Farrant 1995).

In the present study, dry weight accumulation in premature seeds of C. garrawayi was only

32% of that of mature seeds. A large proportion of dry weight accumulation in

C. garrawayi seeds would be attributable to the accumulation of oil reserves, which comprise 30% of mature seed composition. DSC analysis of seeds at different maturities showed that seed oils were detectable in only 40% of premature seed, whereas seed oils were always detectable in mature seeds. The 40% of premature seeds with detectable seed oils may correspond to the proportion (ca.30%) of seeds that survived desiccation to

13%MC. This is consistent with the findings of Golvina et al (2001), who found that desiccation tolerance was more cellular based such that, ‘acquisition of desiccation tolerance, as assessed by germination, was associated with an upsurge in cytoplasmic

172 viscosity, the onset of accumulation of protein and oil bodies, and retention of membrane integrity upon dehydration/rehydration’.

Cryopreservation of seeds of Australian wild Citrus species

Desiccation of seed in many Citrus species has been shown to give protection to immersion in liquid nitrogen. A review of these studies are given in Table 1.6. For example, a germination percentage of at least 80% was demonstrated from seeds of

C. aurantifolia, C. limon and C. aurantium at low moisture contents (2 to 10%MC).

However, some species (e.g. C. maxima and C. madurensis) had a lower germinability from seeds at low moisture contents and this was further reduced (ca.20%) following cryopreservation.

Hor et al (2005) found that, in four Citrus species, the removal of bulk ‘free’ water

(unfrozen water content) occurred at about 10%MC and this correlated with the optimal moisture content for seedling recovery after cryopreservation. In the present study, the unfrozen water content (WCu) of C. australasica and C. garrawayi seeds was determined, using DSC analysis, to be 11 and 14%MC, respectively, which is close to the 10%MC determined by Hor et al (2005). WCu has been shown to be inversely related to the lipid content of seeds (Hor et al 2005, Pritchard 2007). It was thus possible to estimate the WCu

C. inodora (54%LC) to be 8%MC.

Seed oils of cultivated citrus are stored in the cotyledons as oil bodies (Vaughan

1970) and are predominantly triglycerides (65-68%) (El-Adawy et al 1999). Vertucci

(1989b) studied the relationships between thermal transitions in oil rich soybean seeds and concluded that triglycerides might interact with water to cause sensitivity to freezing

173 damage. Hor et al (2005) confirmed that cultivated citrus seeds do not appear to withstand the presence of freezable water on freezing or thawing. The present study also supports this proposal, as seeds above the WCu in C. australasica (17%MC) and C. inodora (12%MC) were observed to have negligible germination after cryopreservation. However, high levels of seedling recovery (>70%) were observed from seeds of these species at low moisture contents (3%MC), well below the unfrozen water content. Despite high levels of recovery from C. garrawayi on desiccation to low moisture contents (i.e. 3 and 7%MC), well below the WCu., seedling recovery after cryopreservation was the lowest (40%) of the three test species.

Citrus aurantifolia seeds are the most desiccation and freezing tolerant of the cultivated Citrus species (King et al 1981, Normah and Siti Dewi Serimala 1997, Mumford and Panggabean 1982, Hor et al 2005). Citrus aurantifolia seeds desiccated to 4.5% moisture content had 83% germination and 85% after cryopreservation (Cho et al 2002a).

The present study shows that C. australasica and C. inodora seeds had a similar response to C. aurantifolia, with >70% germination (seedling recovery) from seeds at 3-5% MC

±liquid nitrogen exposure. However, the high level of germination of C. aurantifolia seeds was only achieved by removal of the seed coat; much lower % germination (ca. 25) was observed from intact seeds after desiccation and cryopreservation at 8 weeks incubation

(Cho et al 2002a). Thus, although similar in terms of germination levels post desiccation and cryopreservation, it was unnecessary to remove the seed coat in order to achieve high levels of germination in C. australasica and C. inodora.

Seed coat characteristics (physical barrier), despite being thickest in C. garrawayi

(cf. Table 2.2), are unlikely to account for the lower levels of seedling recovery observed

174 after cryopreservation in C. garrawayi, compared to the other species. This is because, although epicotyl emergence was low, radicle emergence levels were much higher, and it is thus clear that seed coat rupture had already occurred in these seeds. Thus, the decreased levels of epicotyl emergence (ca.40%) observed from seeds at 7 and 3%MC on exposure to liquid nitrogen is more likely related to other factors. For example, physical restriction caused by the large cotyledons (plumule within) or injury to the plumule by cryopreservation and/or in combination with desiccation stresses is likely. In addition, fungal infection also appears to be partially responsible for loss of seed viability in

C. garrawayi. Thus, optimisation to improve the low seedling recovery in C. garrawayi should include the investigation of aseptic in vitro germination to reduce any losses in viability due to fungal infection.

A delay in epicotyl emergence was observed in seeds of the three Australian wild

Citrus species on desiccation (<7%MC) ±liquid nitrogen exposure. This is consistent with the characteristic delay in germination from dry seeds of cultivated citrus (King et al 1981).

Soetisna et al (1985) proposed this delay in citrus germination as attributable to both the rate of imbibition and the growth processes that follow. They found that fresh seeds at approximately 50% moisture content are close to the water content required for growth processes of germination to begin and suggest that extra time is required for dry seeds to imbibe water and initiate the growth phase of germination.

Overall, the seeds of the three Australian wild Citrus species showed a differential level of response in terms of epicotyl and the radicle emergence following desiccation to low moisture contents and cryopreservation. The radicle was robust to stress, i.e. radicle emergence rates and levels remained high following both desiccation and cryopreservation.

175 In contrast, epicotyl emergence was considerably delayed in seeds dried to low moisture contents (<5%) and was further delayed by cryopreservation. Additionally, seedling recovery was reduced to between 15 to 27% after liquid nitrogen exposure in seeds at low moisture contents. Mumford and Grout (1979) reported a comparable 22% reduction in viability after liquid nitrogen exposure in seeds of C. limon (seed coat removed) at very low moisture contents (1.6%). The reduced seed vigour (e.g. delay in epicotyl emergence) from seeds at low moisture contents after cryopreservation in Australian wild Citrus species may be due to the additional stresses of freezing on dry seeds (<5% MC). In particular, oil rich seeds have been reported to be more susceptible to freezing injury at low moisture contents on rapid cooling at a rate of 200ºC min-1 (i.e. direct immersion in liquid nitrogen), possibily due to phase transitions of seed oils (Vertucci 1989b, Dussert et al 2001, Hor et al 2005).

The endothermic phase transitions in cotyledon tissue in C. australasica were comparable to those observed in other studies of cultivated Citrus species (Santos and

Stushnoff 2003, Hor et al 2005). The melt endotherm of water observed in cotyledon tissue of C. australasica at high moisture contents (>18%) was similar to that observed in fully hydrated excised embryonic axes of C. sinensis (orange) (Santos and Stushnoff 2003), with an onset temperature of -5ºC. Furthermore, the y values at origin from the regression line of cotyledon tissue at low moisture contents (lipid endothermic events) were comparable to the values observed by Hor et al (2005) for two cultivated Citrus species. However, seeds of C. australasica appear to differ from cultivated species, such as C. reticulata (Hor et al

2005), with less overlap between the lipid and water phase transitions (endotherms).

The present study observed that different seed tissues, excised from seeds and equilibrated over a series of different relative humidities were at different moisture contents

176 after the same period of incubation. In particular, the seed coat equilibrated at higher moisture content than the embryo. Seed tissues from C. garrawayi seeds, with a water activity 0.72 (below WCu), had different thermal properties in tissue of the embryo

(cotyledon) and seed coat that could be attributed the presence (embryo) or absence (seed coat) of oils, as detected using DSC analysis. Makeen et al (2006) propose that in oily seeds of citrus, there are fewer water binding sites, that is a lower number of sorption sites as suggested by Vertucci and Leopold (1987), because of the hydrophobic properties of oily tissues and thus the increased water activity as a result of an increase in free water molecules. This is consistent with the thermal behaviour of oil rich (cotyledon) and poor

(seed coat) seed tissues in C. garrawayi seeds in the present study.

Thermal transitions of seed lipid phase

As previously discussed, all three Australian species of citrus were tolerant to desiccation to low moistures, well below the unfrozen water content. Thus, water phase transitions (e.g. ice formation) are unlikely to be a cause of any injury on exposure to liquid nitrogen. The major phase transitions, as recorded using DSC analysis, in embryo tissue of

C. australasica, C. inodora and C. garrawayi were the crystallization and melting of seed oils. Thus, differences in survival post cryopreservation between the three species may be partly attributable to the thermal properties of their seed oils.

The thermal transitions of seed oils (i.e. storage reserves) differ based on their fatty acid composition, with saturated fats having a higher melting point (i.e. solid to liquid phase) than unsaturated fats. This is because saturated fatty acids have a uniform shape that allows them to pack easily into a crystal, whereas in unsaturated fatty acids (e.g. linolenic

177 acid) the carbon–carbon double bonds introduce bends and kinks, making crystal formation difficult (McMurry 1994).

As discussed in Chapter 3, DSC analysis can reveal differences in saturated and unsaturated fatty acid levels between the seed oils of species. In the present study, the melt onset and crystal onset temperatures of seed oils differed between the three species. For example, melt onset was -26ºC, -23 ºC and -14 ºC in C. australasica, C. inodora and

C. garrawayi, respectively. These results suggest that C. garrawayi seeds have had a lower proportion of unsaturated fatty acids than C. australasica and C. inodora. Additionally, the decreased lipid melt and crystallization onset temperature (i.e. increased unsaturated fatty acids) coincided with a decrease in latitude of the natural geographic ranges of these three species (from warm subtropical southeastern Australia [C. australasica] latitudes to the northerly latitudes of warm tropical [C. inodora] and hot tropical [C. garrawayi]).

Plants that grow in cool climates tend to have a different fatty acid composition, with more unsaturated fatty acids, compared to those that grow in warmer climates (Bewely and Black 1994). This is consistent with the apparent association between geographic gradient and lipid melt and crystallization temperature (i.e. fatty acid composition) observed in seeds of Australian wild species of Citrus in this study. Other seed traits, such as seed size in Australian Glycine species, have been observed to be influenced by latitudinal gradient and climatic zones, i.e. seed size increased with proximity to the equator

(Murray et al 2003).

Although the thermal characteristics of seed oils have been linked to many environmental factors, the temperatures in the natural ranges of Australian wild Citrus species appear to have a strong association with differences in seed oil thermal properties.

Differences in latitude are greatest between the southerly distributed species C. australasica

178 and the northerly distributed species C. inodora and C. garrawayi. However, seed oil thermal transitions were most similar (e.g. -26 and -23ºC melt onset temperature) in

C. australasica (SE Australia) and C. inodora (NE Australia). Both C. australasica and

C. inodora grow in areas with a mean annual temperature in the mid twenties, i.e. warm subtropical and warm tropical climatic zone, respectively. In contrast, seed oils of

C. garrawayi (Far N Australia) have a higher melt onset (-14ºC) and grow in regions with a warmer mean annual temperature of about 30ºC, i.e. a hot tropical climatic zone. However, as discussed in Chapter 3 (p. 108), further investigations may be required to clarify the effects of the natural distribution (origin) and the environmental conditions during seed development and dispersal on seed oil thermal behaviour as ex situ sourced seed material

000was used in this study

Importantly, fatty acid composition may affect seed survival after cryopreservation

(Pritchard 2007). Dussert et al (2001) found a correlation between the percentage of unsaturated fatty acids and seedling recovery in nine species of oil rich Coffea species after liquid nitrogen exposure. In this study, DSC analysis revealed greater proportions of unsaturated fatty acids based on thermal transitions (cooling and warming) in cotyledon tissue of C. australasica and C. inodora, compared to C. garrawayi. This appears to correspond with seedling recovery post cryopreservation in these species. Seeds at <7%MC in C. garrawayi had the lowest seedling recovery of the three species at about 40% seedling recovered after liquid nitrogen exposure compared to above 70% in C. australasica and

C. inodora (all at MCs well below WCu). Thus, fatty acid composition appears to affect tolerance to cryopreservation in Australian Citrus species, such that a lower survival appears to be associated with decreased levels of unsaturated fatty acids.

Cultivated citrus species often have a 1:2 ratio of saturated (predominately palmitic acid, ca. 25-30%) to unsaturated fatty acids (predominantly oleic, ca.25%) and linoleic

179 acids (ca. 30-40%) (Seed Oil Fatty Acids database [www.bagkf.de/sofa]), El-Adawy 1999).

Species with large departures from a 1:2 ratio of saturated to unsaturated oils, include

C. maxima and C. trifoliata at about 1:1 (Kalayasiri et al 1996, Gorjaev et al 1977, cited www.bagkf.de/sofa/). Both these species have been reported to lose germination capacity on desiccation to 9%MC (Saipari et al 1998, Hor et al 2005), with low recovery from cryopreservation (ca.20%) reported in C. maxima (Hor et al 2005). In contrast, Citrus species with a 1:2 ratio of saturated to unsaturated fatty acids have a demonstrated tolerance to both desiccation and cryopreservation. For example, high levels of germination after liquid nitrogen exposure were recorded for seeds of different moisture contents in the range of 16% or below in C. aurantifolia (85% at 7%MC), C. limon (67% at 10%MC),

C. sinensis (93% at 16%MC) and C. aurantium (94% at 10%MC)(cf. Table 1.6). An exception to this are reports of low of recovery after cryopreservation in C. reticulata, which also has a 1:2 ration of saturated to unsaturated fatty acids. This species displays some desiccation sensitivity (<12%MC) and has low optimal levels of seed germination

(ca. 20%) after equilibrium drying and cryopreservation, using a rapid cooling protocol

(Hor et al 2005). Thus, overall there appears to be an association between the level of unsaturated fatty acids and survival after cryopreservation in both cultivated and Australian wild species of Citrus. However, inconsistencies (e.g. C. reticulata) suggest that further study is needed to confirm this association.

Seed storage of Australian subtropical and tropical wild Citrus species

The surface topography (cf. Chapter 2) and germination behaviour (e.g., desiccation/freezing tolerance) of seeds of C. australasica and C. inodora seeds are most similar to the desiccation tolerant cultivated species C. aurantifolia (common lime). The level of tolerance to both desiccation and cryopreservation indicates that these species are

180 ‘essentially’ orthodox in seed storage behaviour. However, C. garrawayi seeds display a more complex seed storage behaviour than C. australasica and C. inodora. Variation in seed developmental stages within seed lots and thermal behaviour of in vivo seed oils may be contributing factors in the differences in physiological (i.e. germinability) responses between seeds of C. garrawayi and the other species in this study and warrants further investigations.

Intermediate seeded species, such as coffee and citrus, characteristically show variation between seed lots (Pritchard 2004). In addition, these species can display

‘essentially’ orthodox seed storage behaviour. For example, Coffea arabica (Hong and Ellis

1992) and the seeds of three Citrus species (lemon, lime and orange) (King et al 1981) have been observed to display ‘essentially’ orthodox seed storage behaviour after storage at

-20ºC. In the present study, both C. australasica and C. inodora displayed ‘essentially’ orthodox seed storage behaviour, e.g. desiccation and cryopreservation tolerance.

Additionally, in preliminary experiments, C. australasica seeds were observed to have high levels of seedling germination (86±1%) from seeds after 4-month storage at -20ºC (data not shown). Thus, it appears that seed banking of germplasm of these species could be by standard orthodox protocols (i.e. 5%MC at -20ºC). However, because of variation in seed responses and other storage constraints in species such as coffee and citrus, most studies have focused on cryopreservation as potentially the safest storage option to prevent seed deterioration (loss of viability) (Dussert et al 2001, 2006, Lambardi et al 2004, Hor et al

2005).

Additionally, Crane et al (2006) observed unexplained seed deterioration, in oil rich intermediate seeded Cuphea as well as in other species (USDA seed bank), from seeds

181 stored at temperatures within the range of lipid crystalline phase transitions. Thus, they recommend that seeds should not be stored within the temperature range of oil thermal transitions. In this study, it was shown that the crystallization and melt onset temperatures of seed oils of three wild Citrus species ranged between -5 to -9ºC and -14 to -26ºC, respectively. Thus, storage of these species long term at -20ºC (i.e. close to or within seed oil phase transition temperatures) is not advisable and could lead to seed deterioration. This lends further support for the predominance of studies suggesting cryopreservation as the safest option for the long-term storage of Citrus seeds, even in desiccation tolerant species.

Differences in ‘storage’ behaviour in intermediate seeded species of citrus, compared to orthodox seeded species, was also supported by a recent study of water activity in five taxa of Citrus (Makeen et al 2006). Seed chemical composition (e.g. lipid) affects the absorption and desorption of water (i.e. isotherm curve shape) (Pritchard 2004). Makeen et al (2006) found correlations between lipid content and water sorption parameters and observed a hyperbolic isotherm curve in citrus, as opposed to the typical sigmoidal shape characteristic of orthodox seed isotherms.

182

CHAPTER 6 IN VITRO CULTURE AND CRYOPRESERVATION OF

CITRUS AUSTRALASICA, C. INODORA AND C. GARRAWAYI

183 Chapter 6 In vitro culture and cryopreservation of C. australasica, C. inodora and C. garrawayi

6.1 Introduction

Seed supply of Australian wild Citrus species can be limited and erratic, as well as heterogeneous in developmental status (e.g. Citrus garrawayi), which means conventional storage techniques (i.e. 5%MC at -20ºC) are not generally applicable. Thus, the development of alternative in vitro and cryopreservation techniques is important for medium and long-term ex situ germplasm storage. In addition, in vitro culture systems for

Australian wild Citrus species would allow the mass propagation of these species and assist breeding and conservation programs. In cultivated Citrus species, in vitro culture and cryopreservation protocols have been developed as a preferred conservation method because seeds display non-orthodox seed storage behaviour and for horticultural purposes

(e.g. storage and clonal proprogation of elite cultivars). However, there have been very few attempts to apply in vitro and associated cryopreservation techniques to Australian tropical and subtropical fruit species, including Citrus.

Cryopreservation of mostly polyembryonic cultivated citrus varieties has been reported using seed and embryos (Mumford and Grout 1979, Normah and Siti Dewi

Serimala 1997, Cho et al 2002bc, Lambardi et al 2004, Hor et al 2005, Santos and

Stushnoff 2002, 2003), shoot tips (Gonzalez-Arnao et. al. 1998, Wang and Deng 2004), somatic embryos (Marin et al 1993, Gonzalez-Arnao et al 2003) and embryogenic cells/callus (Aguilar et al 1993, Englemann et al 1994, Pérez et al 1999, Hao et al 2002,

Sakai et al 1991ab). These cryopreservation protocols have given varied results dependent

184 on genotype and have been predominantly based on either vitrification or encapsulation/dehydration techniques.

Embryogenesis

Somatic embryogenesis has been achieved in many polyembryonic Citrus species using media without the addition of plant growth regulators (Pérez 2000, Duran Vila 1995,

Carimi and De Pasquale 2003). For example, embryogenic callus and somatic embryos were induced in several polyembryonic mandarin cultivars from ovules initiated on basal medium that was a formulation of Murashige and Skoog (1962) base salts, modified vitamins, malt and high sucrose (50gl-1) (Pérez et al 1999). Malt is the most common medium supplement to induce embryogenesis in Citrus. However, embryogenic protocols for citrus have included many other medium additives such as, abscisic acid (ABA), 6- benylaminopurine (BA), casein hydrolysate, glycerol, lactose and maltose (Beloualy 1991,

Ben-Hayyim and Neumann 1983, Carimi et al 1998ab, Kochba et al 1978, Gmitter et al.

1990, Hao et al 2004, Tomaz et al 2001) (cf. review section 1.7).

Embryogenic cells are ideal material for cryopreservation, as they are highly cytoplasmic and as such contain less water for lethal ice formation (Finer 1994).

Cryopreservation of embryogenic callus has been demonstrated successfully in many cultivated Citrus species (Engelmann et al 1994, Perez et al 1999, Perez 2000).

Embryogenic callus/cells has been proliferated by culture on either solid (e.g. mandarin cultivars, Perez et al 1999) or liquid medium (Hao et al 2002), prior to cryopreservation and subsequent embryo induction for plant recovery on solid culture medium.

Citric acid is an organic acid found in plant tissues and gives citrus fruits their sour taste. Orange juice, that contains citric acid, has been reported to stimulate the growth of

185 callus in citrus (Einset 1978, Duran-Vila and Navarro 1989, Amo-Marco and Picazo 1994).

Amo-Marco and Picazo (1994) found that medium supplemented with orange juice stimulated callus growth in C. sinensis. However, there has been no previous report of the use of citric acid in somatic embryogenesis in citrus. Citric acid and other antioxidants (e.g.

PVP) have been found to improve callus formation (organogenesis), as well as shoot differentiation and root formation, by inhibiting tissue necrosis (i.e. tissue browning caused by oxidation of phenols) in pine (Tang et al 2004) and to prevent lethal browning of leaf explants in a critically endangered Australian wild species (Panaia et al 2000). Nicol et al

(1991) have also reported the use of citric acid to have promotive affect on embryo induction and embryo differentiation in alfalfa (Medicago sativa L.).

This present study investigates somatic embryogenesis in selected cultivated Citrus species (mono- and polyembryonic), as well as the Australian wild species: C. australasica,

C. inodora and C. garrawayi. Both ovules and seeds are used as explant material to facilitate both mass propagation and the development of cryopreservation storage options.

Modifications to medium formulation for embryo induction is also investigated using plant hormones (ABA, BAP), antioxidants (polyvinlypolyrrolidone, PVP), sugars (sucrose, lactose and maltose) and sugar alcohol (glycerol), amino acid derivative (betaine), casein hydrolysate and malt extract, which have been effective in embryogenesis in cultivated

Citrus or other species, as well as the novel application of citric acid in citrus.

Micropropagation and shoot-tip cryopreservation

Research on citrus shoot tip cryopreservation is more limited (Gonzalez-Arnao et al

1998, Wang et al 2002, 2004) than embryogenic systems. In vitro regeneration via shoot tip and nodal explants of Citrus species has been reported for various cultivated genotypes (poly and 186 monoembryonic), using various combinations of cytokinins and auxins (e.g. BAP and NAA). In addition, cryopreservation of shoot tips has been demonstrated in cultivated Citrus species using encapsulation-dehydration and vitrification techniques (cf. review section 1.7). The present study investigates in vitro shoot culture systems in C. australasica, C. inodora and C. garrawayi for micropropagation, medium–term storage and the generation of in vitro shoot-tips for cryopreservation.

In vitro seed germination

Cryostorage of seed after controlled drying to low moisture contents (<16%) and in vitro germination has been demonstrated as an alternative option for long-term ex situ storage of the cultivated Citrus species, C. aurantium, C. limon, C. sinensis (Lambardi et al

2004). In the present study, in vitro germination of C. garrawayi and C. inodora is investigated to test the use of in vitro germination following desiccation and cryostorage on germination seeds of both species. Both germination rates and seedling vigour are tested. In vitro germination may also reduce losses associated with fungal infection, which occurs using standard germination protocols.

6.2 Materials and Methods

Medium formulation and culture conditions for all experiments

Basal medium was used for all experiments, as described by Perez et al (1999). The basal medium was modified and consisted of half MS salts, myo-inositol (100mgl–1), thiamine (0.2mgl–1), pyridoxine-HCL (1mgl–1), nicotinic acid (4mgl–1), sucrose (50gl–1) and malt extract (250mg l-1). Modifications to this formulation are as indicated. Solidification of medium was achieved using 0.6% agar and pH was adjusted to 5.7±0.1 using NaOH and 187 HCL prior to autoclaving (121ºC for 20mins). Incubation of cultures for all experiments was at 251 C for a 16 hour photoperiod (25mol m-2s–1).

6.2.1 Embryogenesis

Initiation using immature ovules

Cultures were initiated on basal medium as described by Pérez et al (1999) for somatic embryo formation from ovules. Immature fruits were harvested from a research field collection of the Queensland Department of Primary Industries (QDPI), approximately six weeks after anthesis, from mature plants of Tahitian lime (3-4mm in width) and mandarin (C. reticulata) cultivars – Imperial (4-5mm), Murcott; (5-6 mm) and

Orlando (12-13mm), as well as the Australian wild species, C. australasica (1mm),

C. inodora (1-2mm) and C. garrawayi (3-6mm). Immature fruits were stored at 4ºC until use. Fruits were surface sterilsied in 1% sodium hypochlorite (10 mins) and a few drops of wetting agent (Tween). Ovules were then excised aseptically (laminar flowhood), using a hypodermic needle, from immature fruit and placed on basal medium. Somatic embryo formation on basal medium was tested for all seven species/cultivars. For each species/cultivar, 11 petri dishes with 10 ovules per dish (n=110) were used. Basal medium was modified in another experiment for ovules of C. garrawayi and Tahitian lime by the inclusion of one the following: 6-Benylaminopurine (BAP) (3.3 M, 6.6M, 13M and

26.6M), betaine (250 mg l-1 and 1000 mg l-1) and polyvinylpyrrolidone (PVP) (8g l-1). For both experiments, cultures were observed at regular intervals and results recorded at 3 months post initiation for growth or necrosis of ovules, production of callus and embryo formation.

188 Initiation using seed

Mature fruits of C. australasica, C. inodora and C. garrawayi were harvested from the QDPI for each test species and stored at 15ºC prior to use. Mature seeds were extracted from surface sterilised fruits (2% sodium hypochlorite, 10 mins), before being placed on a range of media to induce callus and embryo formation. Basal medium was modified by the addition (except elevated level of sucrose) of the following: 0.3M sucrose, 0.3M sucrose plus 0.2M glycerol, 15gl-1 maltose, abscisic acid (0.75μM), casein hydrolysate (500mg l-1 and 1000mgl-1). For each treatment, six petri dishes were initiated with five seeds per replicate (n=30). Callus and somatic embryo formation was observed and results recorded three months after culture initiation.

In a later experiment, immature seeds of C. australasica, from fruits harvested from the Brisbane Botanic Gardens (Mt Coot–tha), were used to initiate cultures (fruits ranged in size from 39 to 74mm long by 8 to 14mm wide and weighed between 1.7 to 7.6g). The seed extraction and sterilisation method was as for mature seed. Basal medium was modified by the inclusion (except elevated level of malt) of one of the following substances: glycerol

(0.3M), malt (1250mgl-1 malt), malt (1250mgl-1) plus citric acid (250mgl-1), lactose (5gl-1

[no sucrose]) and BAP (0.05μM). In addition, seeds were silica-dried for 1.5h prior to initiation on basal medium with BAP (0.05μM). For each treatment, eleven petri dishes were initiated with five seeds per replicate (n=55). Callus and somatic embryo formation was observed and recorded 3 months after culture initiation.

Preliminary encapsulation-dehydration of seed-initiated Citrus inodora embryogenic callus

Seed-initiated embryogenic callus was cryopreserved using an encapsulation – dehydration protocol. Alginate encapsulated embryogenic callus was precultured for 3 days

189

in liquid basal medium plus evaluated sucrose (0.5M), prior to drying under sterile air for 5 hours. Beads within cryovials were then directly immersed in liquid nitrogen. Beads recovered from liquid nitrogen were rapidly thawed (40ºC), placed on basal medium and incubated in the dark for one week before being placed in the light.

Multiplication of embryogenic callus of Citrus inodora in liquid culture

Small proliferations of white to yellow friable embryogenic callus, with a nodular appearance induced from seed of C. inodora, was removed and subcultured (solid medium) until enough was obtained to initiate liquid cultures (several months). Four to five clumps of callus of embryogenic callus were placed into 250ml glass flasks containing 100ml of basal medium, modified by the inclusion of evaluated sucrose (0.3M) and 0.3M glycerol.

Periodically (every 2-3 months) the liquid medium was supplemented with 0.1mg l-1 BAP to improve proliferation of embryogenic callus.

The effect of the addition of citric acid to the liquid culture medium on callus multiplication and somatic embryo formation on plating of suspension-cultured callus was tested. Citric acid at various concentrations (0, 250, 500 and 750mgl-1) was added to liquid basal medium (three replicates/concentration). After three weeks in liquid culture, callus masses (0.5 to 1mm in diameter) were plated (ca. 20 callus masses/plate) onto solid basal medium plus BAP (0.05μM). For each citric acid concentration, 10 replicates (petri dishes) were initiated and callus and somatic embryo formation was observed eight weeks after culture initiation.

190 Effect of medium composition (solid) on callus and somatic embryo formation

Modifications to the solid medium used for plating of liquid cultures of embryogenic callus were investigated to improve callus multiplication and embryo formation. Liquid cultured callus initiates (0.5 to 1mm in diameter) were plated onto basal medium, modified by the inclusion (except elevated level of malt) of one of the following: glycerol (0.3M), lactose (5gl-1 [no sucrose]), malt (1250mg l-1) and malt (1250mg l-1) plus citric acid (250 mgl-1). In addition, the combinatorial effect of malt and citric acid supplements to the basal medium at various concentrations on callus and embryo formation was investigated in a subsequent experiment. Citric acid at a concentration of between 60 to

500mgl-1 was added to either basal medium (i.e. malt 250mgl-1) or basal medium plus an elevated level of malt (1250mgl-1). For each medium composition, 15 replicates (petri dishes) of approximately 20 callus clumps (liquid cultured) were initiated. Callus and somatic embryo formation was observed eight weeks after culture initiation for both experiments.

Cryopreservation of C. inodora embryogenic callus by encapsulation – dehydration

Embryogenic callus of C. inodora multiplied in liquid culture was used for cryopreservation trials using an encapsulation – dehydration protocol. Embryonic callus masses were encapsulated in 3% sodium alginate (low viscosity) consisting of basal medium plus 0.4 M sucrose, without calcium. Each bead contained 2 - 4 embryonic callus masses (initiates) of between 0.5mm - 1mm diameter size. Beads were formed during incubation in 100mM calcium chloride for 30 minutes.

191 Encapsulated embryonic callus was then pretreated overnight in liquid basal medium (BM) plus 0.5M sucrose and 0.3M glycerol, prior to application of loading solution (BM plus 0.75M sucrose and 0.75M glycerol) for one hour. The pretreated beads were blotted onto filter paper and desiccated for one to six hours over silica gel (40g) on filter paper in sealed glass petri dishes. Dehydrated beads were then placed in cryovials, directly immersed in liquid nitrogen and stored overnight. After cryopreservation, beads were rapidly thawed (40ºC for 1 min), plated on solid basal medium and incubated in the dark for two days before being placed in the light. For each treatment, four petri dishes were initiated with ten beads per replicate (n=40). Survival and multiplication of embryogenic callus for each treatment was observed four weeks after culture. Moisture content (MC) for each treatment was determined from empty beads (3 replicates of 20 beads) on a fresh weight basis (%wb), after drying at 80ºC until a constant weight was recorded.

6.2.2 Micropropagtation and shoot-tip cryopreservation

In vitro culture of nodal cuttings

For each test species, Citrus australasica, C. garrawayi and C. inodora, seeds were surface sterilised (2% sodium hypochlorite) and germinated in vitro as source material for nodal cuttings. Nodal cuttings, 2-3 cm in length, were initiated on basal medium (25gl-1 sucrose) modified by the inclusion of either 1μM (C. australasica) or 0.5 μM (C. inodora and C. garrawayi) naphthalene acetic acid (NAA). At least 10 replicates of ten cuttings were initiated for each species (n=>110).

192 Preculture of nodal cuttings of C. australasica

Nodal cuttings were precultured on various concentrations of sucrose prior to axillary shoot-tip excision to test for improved tolerance to cryopreservation, based on a modified method of Dr Barbara Reed (pers comm. 2004). Nodal cuttings, about 1 cm in length, were precultured on solid basal medium plus sucrose (0.1 to 0.75M); 6 replicates of

10 cuttings were initiated for each sucrose concentration. After one week, of preculture axilliary shoot-tips were excised (ca. 1mm) and cultured on basal medium (25gl-1 sucrose) plus 0.05μM BAP. Excised shoot-tip survival and growth was recorded after one month’s culture.

Cryopreservation of shoot-tips of C. australasica

A vitrification based protocol was investigated using Plant Vitrification Solution 2

(Sakai et al 1990) as a cryoprotectant and modified from the method described by Ashmore et al (2000) for papaya shoot tips. Axillary shoot-tips (ca. 1mm) of C. australasica were excised from in vitro multiplied cuttings (as previously described). Excised shoot-tips were precultured overnight in liquid basal medium (50gl-1 sucrose) containing 0.05μM BA, and then incubated for 60mins in loading solution (20% PVS2 at RT), prior to 20 minute incubation in 100% PVS2. Droplets of PVS2 containing a single meritsem were then pipetted onto aluminum foil strips (ca. 4 x 2cm), directly immersed in liquid nitrogen (at least 30 mins) and thawed in 37ºC thawing medium (liquid basal medium plus 1.2M sucrose) for 30 minutes. After cryopreservation, shoot-tips were cultured in the dark on solid basal medium (25gl-1 sucrose) plus BAP 0.05μM for 2 days prior to incubation under light conditions. Shoot-tip survival and growth was assessed 1 month post cryopreservation; 3 replicates of 10 shoot-tips were initiated for each treatment (n=30) and

193 this was replicated in a later experiment. In addition, to test the sensitivity of shoot-tips to

PVS2, the duration of exposure (10, 15 and 20mins) was tested (3 replicates of 10 shoot- tips for each treatment (n=30)).

100% PVS2 solution contained 30% glycerol, 15% ethylene glycol and 15% DMSO

(Dimethyl sulfoxide) in 49.1ml liquid basal medium, 30g glycerol, 15g ethylene glycol and

13.6ml of DMSO. All the components of the PVS2 solution were mixed and autoclaved, except DMSO, which was added after sterilisation. The loading solution consisted of 20ml of sterile 100% PVS2 added to 80ml of sterile liquid basal medium. Fresh PVS2 solutions were prepared for each experiment.

6.2.3 In vitro seed germination

Plant material and seed extraction

Fruits of C. inodora and C. garrawayi were harvested from QDPI research field collections, located in either northern or southeastern Queensland. Fruit of both species was stored at 15ºC prior to testing. Fruits were rinsed in tap water, surface sterilised with 5% sodium hypochlorite (10 minutes) and then the seeds were extracted under aseptic conditions.

Seed desiccation

C. inodora seeds (mature) were desiccated over sterile activated silica gel (30g) within a parafilm sealed glass petri dish for 18 hours. Three replicates of ten seeds of

C. inodora were initiated from each of the seed lots. Seeds (near mature) of C. garrawayi were also desiccated over silica gel for 3, 4, 5.5 or 18 hours. Six replicates of 5 seeds of

C. garrawayi were tested for each desiccation level and duplicated by 3 replicates of 10

194 seeds from a second fruit harvest. Moisture content (MC) was determined gravimetrically, after drying seeds for 18 hours at 103 ºC and presented on a fresh weight basis (%MC).

Seed moisture content measurements of C. inodora were done in duplicate using ten individual seeds from each seed lot. The moisture content of near mature seeds of

C. garrawayi, at each desiccation level, was determined from three replicates of ten seeds for each seed lot.

Seed cryostorage

Desiccated seeds of both species were directly immersed in cryotubes into liquid nitrogen. Thawing was achieved by plunging into water at 40 ºC. Seeds were stored in liquid nitrogen for at least 24 hours prior to thawing.

Seed surface sterilisation

After desiccation and cryopreservation, seeds were surface sterilised by rinsing in

2% sodium hypochlorite (30 seconds), followed by a rinse in 70% ethanol (30 seconds), followed by two rinses in sterile deionised water before germination testing.

In vitro germination

Seeds of both species were germinated in sterile glass jars on basal medium containing 25g/l sucrose. Percentage germination (radicle emergence >5mm) and seedling growth (epicotyl and root lengths) was assessed after eight weeks for C. inodora and approximately seven weeks for seeds of C. garrawayi. Epicotyl and radicle emergence

(>1mm) from seeds of C. inodora were recorded periodically (3-4 days) over an eight week period and time until 50% of final emergence levels was determined.

195

Seedling acclimatisation

Seedlings germinated in vitro after both desiccation and liquid nitrogen exposure in

C. inodora and C. garrawayi were acclimatised to shade house conditions. In vitro grown seedlings were thoroughly cleaned (i.e. culture medium removed) under tap water prior to transfer to vermiculite. The seedlings were then grown in a humidity tent at almost 100% relative humidity (one week) and then approximately 70% relative humidity (one week) prior to final transfer to the shade house. The seedlings were periodically (fortnightly) treated with a liquid fungicide and fertilizer (Aquasol). Survival of acclimatised seedlings was assessed after five weeks of culture in the shade house.

Differential Scanning Calorimetry (DSC)

Analysis of thermal events during freezing (-80ºC) and thawing of near mature seeds of C. garrawayi was performed using a DSC Q1000 V8.1 (TA Instruments), as previously described in section 5.2. Except, five intact seeds from each desiccation treatment were individually analysed using a cooling and warming rate of 10ºC/minute.

Statistical Analysis

Effects of treatments were tested by Analysis of Variance with least significant difference (LSD) calculated at 0.05 level of significance using SPSS 12.1 software.

Proportions were arcsine transformed prior to statistical analysis. Results in figures and tables were presented as untransformed percentages.

196 6.3 Results

6.3.1 Embryogenesis

Initiation using immature ovule Fig. 6.1 Immature fruits of A Somatic embryogenesis Australian wild Citrus species, (A) C. australasica, was investigated in both mono- (B) C. inodora and (C) C. and poly-embryonic cultivated garrawayi, and commercial cultivars, (D) Tahitian limes, varieties of local importance and (E) Imperial mandarin, (F) C. australasica Murcott and (G) Orlando. monoembryonic Australian wild C B Citrus species, using ovules excised from immature fruits

(Figure 6.1).

Growth of ovules on basal C. inodora C. garrawayi medium varied between the D E cultivars and species tested

(Table 6.1). The polyembryonic commercial cultivars (Murcott and Orlando) produced large Tahitian lime Imperial di amounts of multiplying globular F G callus ranging from 12.7% to

49.0% of ovules cultured.

Australian monoembryonic species, C. australasica, Murcott Orlando C. inodora and C. garrawayi,

197 and monoembryonic cultivars (Imperial Mandarin and Tahitian Lime) produced little callus, with the exception of C. inodora, which did not readily form somatic embryos.

Murcott ovules produced the most prolific white and yellow globular embryogenic callus, from which green (up to 5mm) somatic embryos formed from 35% of ovules.

Orlando also formed many somatic embryos from 15% of ovules cultured (Figure 6.2A-C).

White and cream coloured globular callus was induced in 4.6% of Imperial mandarin

(monoembryogenic) ovules cultured, with no somatic embryo formation within the experimental period. Tahitian lime produced green translucent torpedo structures in 18.9% of ovules cultured on basal medium, which developed no further and eventually became transparent.

Table 6.1 Callus and somatic induction from immature ovules of Australian wild Citrus species and commercial cultivars

Genotype Percentage Percentage Percentage ovules ovules showing ovules producing somatic growth1 producing embryos callus Wild species C. australasica 0.0 ± 0.0 0.0± 0.0 0.0± 0.0 C. inodora 99.1±0.9 10.0±3.0 1.8±1.2 C. garrawayi 91.8 ± 8.2 42.7±11.2 0.0± 0.0 Cultivars Tahitian lime 95.5±4.6 46.4± 12.0 0.0± 0.0 Imperial mandarin 77.0 ±10.7 4.6± 2.7 0.0± 0.0 Murcott mandarin 68.0 ± 6.1 49.0± 3.1 35.0±5.8 Orlando mandarin 32.7±10.3 12.7± 3.8 15.0±4.1 Mean± s.e. of eleven replicates (10 ovules/replicate, n=110). Ovules from immature fruit were placed on half strength basal medium (Perez et al 1999). All genotypes are monoembryonic except Murcott and Orlando. Observations recorded 3 month post initiation.

198 A B C

D Fig 6.2 Somatic embryos and embryogenic callus of the mandarin cultivars of (A,B) Murcott and (C) Orlando (5-7mm long), and Australian wild Citrus species (D) C. inodora (1-2 mm long).

Similar torpedo structures were also observed forming from callus induced from

Murcott ovules (Figure 6.2B). No somatic embryo formation was seen in C. australasica and C. garrawayi and the only Australian citrus species to produce somatic embryos was

Citrus inodora, with only 1.8% of ovules forming embryos (Figure 6.2D). Subsequent subculture onto the same medium to multiply the C. inodora somatic embryos resulted in thousands of embryos being produced, including many which have germinated (Figure 6.3).

After the experimental period, limited formation of proembryos and embryo formation was observed from the globular embryogenic callus produced from ovules of the monoembryonic Imperial mandarin.

199

The effect of medium composition was trialed in an attempt to induce embryogenesis in

C. garrawayi and Tahitian lime from excised immature ovules. Basal Fig 6.3 Somatic embryo multiplication and germination in C. inodora medium was modified by the inclusion of one the following Ovules A 100 surviving substances, benylaminopurine 90 80 Ovules -1 70 producing (3.3μM to 26 μM), betaine (250mgl , callus 60 -1 50 1000mgl ) and polyvinlypyrrolidone 40 Percentage 30 (8gl-1). Figure 6.4 shows that 20 10 0 regardless of these medium Percentage A P BA B aine M BA M t μ 100 3μM μ g/l PV B 6 8 3. 3.3 2 /l be modification only small amounts of 90 6.7 μM BA1 g m 0 80 25 1000 mg/l betaine 70 callus were produced (fewer than 60 50 40% of ovules cultured) and no 40 Percentage 30 20 treatment induced somatic embryo 10 0

e formation in either test species. In A ne P ium B in i V d M M BA ta μ μ l-1 P l Me 6 beta a 3.3μM BA 2 1 g 6.7 l-1 be 8 addition, all media modifications 13.3 μM BA g Bas m mg l- 0 50 0 2 0 1 reduced callus production to less than Treatment media half that of control levels (basal Fig. 6.4 Effect of media composition on survival and medium only) in both species - except callus production of ovules from immature fruit of (A) from ovules of C. garrawayi cultured Tahitian lime and (B) Citrus garrawayi cultured in vitro. Mean (± s.e) of six replicates (10 ovules/replicate; n=60); BM- half on basal medium including betaine at strength basal medium (Perez et al 1999); BA- benylaminopurine; 250mgl -1. PVP – polyvinylpyrrolidone. 200

Seed-initiated somatic embryo induction A Embryogenesis was further investigated in C. australasica,

C. inodora and C. garrawayi, using mature seeds of all three, and premature seeds in

C. australasica, to initiate cultures. Table 6.2 shows the effect of media composition on somatic embryo formation. Citrus australasica and C. garrawayi mature seed did not form somatic embryos on any of the media trialed. The best result for somatic embryo formation for C. inodora was observed on basal medium, with 16.7% of seeds forming somatic embryos. Citrus inodora seed formed somatic embryos at lower frequencies on basal medium containing either 0.3M sucrose (3.3%) or 500 mg/l casein hydrosylate (6.7%).

Table 6.2 Effect of medium composition on somatic embryo formation in seed of Citrus australasica, C. inodora and C. garrawayi

Medium Seeds producing somatic embryos (%) Citrus australasica Citrus inodora Citrus garrawayi

Basal medium (BM) 0 16.7 0

BM + 0.3M sucrose 0 3.3 0

BM + 0.3M sucrose + 0.2M glycerol 0 0 0

BM+ 15gl-1 maltose 0 0 0

BM+ 0.75μM abscisic acid 0 0 0

BM+ 500 mgl-1 casein hydrosylate 0 6.7 0

BM+ 1000 mgl-1 casein hydrosylate 0 0 0

Five replicates of five seeds (n= 30) initiated for each media treatment. Cultures observed 3 month post initiation.

201

A B

Fig 6.5 (A) Somatic embryo formation, multiplication and

germination of seed initiated cultures of C. inodora on basal medium. (B) Somatic embryo formation from seed initiated callus at 3 months post cryopreservation

Embryogenic callus and somatic embryos of C. inodora were multiplied on subculture (Figure 6.5A). In a preliminary experiment, seed-initiated embryogenic callus of

Citrus inodora was observed to form somatic embryos that germinated into healthy plants post cryopreservation, using an encapsulation-dehydration protocol (Figure 6.5B).

However, sufficient levels of embryogenic callus or somatic embryos at a suitable uniform stage of development was not obtained using this culture system for either experimental or germplasm conservation purposes.

The effect of medium composition on induction of callus and somatic embryo formation was also investigated using premature seeds of C. australasica (Figure 6.6A).

The inclusion of various media additives affected the mean % of seeds observed to have callus formation, as shown in figure 6.7. The greatest % of callus formation (63.6±8.5) was observed from premature seeds cultured on basal medium plus glycerol (0.3M) almost doubled the % for seeds on basal medium only (34.0±3.1). The addition of BAP (0.05μM)

202

resulted in a lower mean % callus formation from seeds (20.0±2.0), further reduced by seed desiccation (1.5h silica-dried) prior to culture (4.4±2.9). The use of lactose instead of sucrose resulted in callus formation on only one seed (2.0±2.0%), whilst increasing malt levels from 250mgl-1 (BM) to 1250 mgl-1 produced comparable levels of callus formation

(34.0±3.1 and 28.0±10.2 respectively). The addition of citric acid (250 mgl-1) with high malt (1250 mgl-1) resulted in no seeds showing callus formation. The callus produced was whitish cream in colour for all media treatments (Figure 6.8A-E). Embryo formation was not observed in any media treatment, except from one seed of 55 seeds initiated on basal medium containing both malt (1250 mgl-1) and citric acid (250 mgl-1). This occurred in the absence of callus formation (Figure 6.6F). This embryo formation in C. australasica appeared similar to that observed from ovule derived embryogenic callus in the monembryonic Imperial mandarin cultivar (Figure 6.8G).

In all the experiments to induce somatic embryo formation in Australian wild Citrus species, a substantial level of embryogenesis was only observed in C. inodora. For both ovule and seed initiated cultures (solid medium) production of embryos in C. inodora was unsynchronised and some embryos were observed to have abnormal morphology, e.g. multiple cotyledons. However, subculture resulted in multiplication of embryos (secondary direct embryogenesis) and most appeared normal and moved quickly through formation to germination of healthy plantlets with normal root and shoot morphology (Figure 6.2E,

Figure 6.5A)

203

A B

Fig. 6.6 (A) Premature fruits, (B) whole seed and

Callus formation (%)

0 Basal BM + BM + BA BM + BA BM + 5% BM + malt BM + malt medium glycerol (0.05μM) (0.05μM) Lactose (1250mg/l) (1250mg/l) (BM) (0.3M) andand silica-seed and citric dried(1.5h)siliclia dired acid (1.5h) (250mg/l) Treatment Fig. 6.7 Effect of medium composition on callus formation from premature seeds of Citrus australasica. Mean (±s.e.) percentage of 11

replicates of five seeds (n=55) for each treatment.

204

A B

A C D

E F

G Fig. 6.8 Callus formation from premature seeds of Citrus australasica cultured on basal medium plus the inclusion of various additives: (A) none, (B) glycerol (0.3M), (C) BAP (0.05μM), (D) lactose (5 gl-1) and (E) malt (1250 mg l-1). (F) Somatic embryo formation on basal medium containing malt (1250 mg l-1) and citric acid (250 mg l-1) and (G) similar embryo formation from ovule- initiated callus of monembryonic mandarin cultivar, Imperial Mandarin.

205 Somatic embryos of C. inodora followed a similar pattern in later developmental stages to their zygotic counterparts (cf. Chapter 4 – embryo development in C. garrawayi).

Similarities in embryo development between the Australian wild Citrus zygotic embryos and the somatic embryos of C. inodora included the development of globular structures followed by cotyledonary and plumule differentiation as illustrated in figure 6.9. Mature zygotic and somatic embryos consisted of both differentiated cotyledons and shoot primodoria, however cotyledon enlargement observed in zygotic embryos was not observed in somatic embryos.

Multiplication of embryogenic callus of C. inodora

The proliferation of callus masses of C. inodora was investigated in liquid culture with a view to obtaining sufficient embryogenic material for mass propagation and germplasm storage. Seed-initiated embryogenic callus was initially multiplied on solid basal medium (several months/subcultures) until enough was obtained to initiate liquid cultures consisting of basal medium supplemented with 0.3M sucrose and 0.3M glycerol

(Figure 6.10).

A B

Fig. 6.10 (A) Seed-intiated somatic embryos and embryogenic callus in Citrus inodora. (B) Multiplication of seed derived callus (solid medium) suitable to initiate (C) liquid cultures for mass multiplication. 206

(B) SOMATIC (A) ZYGOTIC

Cotyledon and embryo axis differentiation

______1mm

Mature ______embryo 1mm

Fig 6.9 Comparative (A) zygotic and (B) somatic embryo development in Australian wild Citrus

207 Liquid culture of this callus lead predominantly to the proliferation of proembryogenic cell masses of compact rounded cells, as observed by scanning electron microcopy (Figure 6.11A, B). However, differentiated globular structures (proembryos) were also observed as shown in Figure 6.11 (C, D), but these did not develop further, even after transfer to liquid basal medium without glycerol supplementation (data not shown).

Using the liquid based proliferation system, large amounts of embryogenic callus masses were obtained, as illustrated in Figure 6.10. Thus, further investigations were undertaken to try to improve the induction of somatic embryos by plating callus masses

(intiates) derived from liquid culture onto a range of solid media.

A B

C D

Fig. 6.11 (A,B) Embryogenic callus/cell clusters and (CD) differentiated globular structures (proembryos) observed from liquid cultured callus of Citrus

inodora as viewed by (A,C) light and (C,D) scanning electron microscopy. 208 Effect of medium composition on callus and somatic embryo formation

Liquid cultured callus masses (initiates) of C. inodora were plated onto solid basal media containing different additives. Callus formation (multiplication) of liquid cultured callus initiates differed between media treatments trialed. The proliferation of white to cream callus was observed on all medium trialed, however formation of numerous green structures (proembryos) was observed only on basal medium containing malt and citric acid (Figure 6.12). Mean % callus formation was significantly increased

(P=<0.02) from callus initiates plated onto solid basal medium containing either 0.3M glycerol (72.0±7.7), 1250mg l-1 malt (64.5±6.2) or 1250mg l-1 malt plus 250mg l-1 citrus acid (84.0±6.3), compared to levels observed on basal medium only (44.1±5.3) (Figure

6.13). However, the substitution of lactose (5%) for sucrose in basal medium did not significantly (P=0.97) affect mean % callus formation (44.4±4.6). After two months of culture, somatic embryo formation was only observed from callus cultured on basal medium containing either lactose (5%) or both malt

(1250mg l-1) and citric acid

(250mgl-1) (Table 6.3A).

Basal medium containing both malt and citric acid gave rise to 2-10 somatic embryos Fig. 6.13 Effect of various additives added to basal medium on callus initiates of Citrus inodora from liquid on 41% of plates and in culture. Mean (±s.e.) of 15 replicates. addition had the formation of numerous proembryos (Figure 6.14).

209

Fig. 6.12 Callus initiates from liquid culture of Citrus

inodora plated onto solid basal medium and various additives to induce callus multiplication and somatic

embryo formation, (A) basal medium only (BM), (B) BM plus lactose (5% (no sucrose)), (C) BM containing malt (1250mg l-1) and (D) BM containing malt (1250mg l-1) and citric acid (250 mg l-1); 3 plates of each treatment are shown. 210

Table 6.3 Effect of medium composition (solid) on somatic embryo formation from callus initiates (liquid cultured) of Citrus inodora.

A. Experiment 1 - various medium additives SE formation Number of (% plates) SEs/plate

Basal medium (malt 250mgl-1) (BM) 0.0 -

BM + lactose (5gl-1(no sucrose)) 22.2 2

BM+ glycerol (0.3M) 0.0 -

BM + malt (1250mg/l) 0.0 -

BM + malt (1250mgl-1) and citric acid (250mgl-1) 41.2 2 to 10

B. Experiment 2 - malt and citric acid combinations

BM (malt 250mg/l) 0.0 -

BM + citric acid (60mg/l) 0.0 -

BM + citric acid (125mgl-1) 0.0 -

BM + citric acid (500mgl-1) 18.8 2 to 17

BM + malt (1250mgl-1) 4.8 68

BM + malt (1250mgl-1) and citric acid (60mgl-1) 13.3 7 to 17

BM + malt (1250mgl-1) and citric acid (125mgl-1) 0.0 -

BM + malt (1250mgl-1) and citric acid (250mgl-1) 25.0 6 to 31 For each medium composition, 15 replicates (petri dishes) of approximately 20 callus masses (ca.1mm) were initiated. The percentage of replicates showing somatic embryo formation was recorded 2 months after culture initiation.

211 A second experiment was conducted to test the effect of various combinations of malt and citric concentrations, on embryogenic callus formation and embryo induction. The addition of 60, 125 or 500mgl-1 citric acid only / resulted in reduced proliferation of B embryogenic callus, compared to the control

(basal medium only) (Figure 6.15A). The mean percentage callus formation of 88.3±2.1 Fig. 6.14 Callus and embryo formation from observed for basal medium was reduced to callus initiates from liquid cultured of C. -1 -1 15.9±1.7 (60mgl ), 33.0±10.6 (125mgl ) and inodora plated onto solid basal medium 48.1±9.1 (500mgl-1). In contrast, % callus modified by the inclusion of elevated malt (1250mgl-1) and citric acid (250mgl-1). formation on basal medium modified by the inclusion of higher concentrations of malt (1250mgl-1malt), as well as citric acid at a range of concentrations (60, 125 and 250mgl-1), was not significantly (P=>0.3) different to control treatment (no citric acid) and remained at high levels (ca. 80%) (Figure 6.15B).

In terms of embryo formation, none was observed from plated callus on basal medium plus 60 or 150mgl-1citric acid only (Table 6.4B). Somatic embryo formation was observed on basal medium plus 500mgl-1citric acid only (18.8% of plates), basal medium plus elevated malt (1250mgl-1malt) only (4.8%) and basal medium plus elevated malt

(1250mgl-1malt) and either 60mgl-1 (13.3%) or 250mgl-1(25.0%) citric acid. As observed in the first experiment, the combination of 1250mgl-1malt and 250mgl-1 citric acid produced the greatest % plates with somatic embryos.

212

250mgl-1 malt A

1250mgl-1 malt B

Callus formation (%)

Fig. 6.15 Effect of citric acid concentration on callus formation (multiplication) from callus initiates (liquid cultured) of Citrus inodora added to (A) basal medium

(malt 250mgl-1) or (B) basal medium and elevated malt -1 (1250mgl ). Mean % (±se) of 15 replicates (ca. 20 callus initiates/replicate) for each concentration level.

B Fig. 6.16 Morphologically (A, B, C) A abnormal and (D) normal asynchronous somatic embryo formation in C. inodora. Cultures initiated from liquid proliferated callus onto solid (A, B)

basal medium and (C, D) basal medium C D and citric acid (500mgl-1). Note: somatic embryos and callus were subcultured onto basal medium plus BAP (0.05μM) after two month culture on treatment media.

E Although citric acid appears key to embryo formation, there was variation in the % of plates that formed somatic embryos for the best trial medium (1250mgl-1malt and

250mgl-1 citric acid) between the first and second experiments, i.e. 41 and 25% respectively.

On subculture (basal medium plus BAP 0.05μM), callus and proembryos derived from the range of media treatments resulted in further embryo formation and multiplication, including from callus cultured on basal medium only (data not shown). However, regardless of media treatment, abnormal embryos were frequently observed (Figure 6.16A-

C). Nevertheless, somatic embryo developmental stages were similar to those observed in the ovule derived somatic embryos from cultivated mandarin, i.e. the formation of globular, heart, torpedo and cotyledonary embryos. Figure 6.17 shows these structures from ovule derived callus of a polyembryonic mandarin cultivar (Murcott) and from C. inodora callus, including the production of spherical globular structures (proembryos) and a mixture of white and yellow friable callus.

214

A Fig. 6.17 Callus, globular proembryos and somatic embryo formation on solid

basal medium in (A) Citrus inodora, and (B,C) mandarin

cultivar, Murcott .

B C

The addition of citric acid at various concentrations (250, 500 and 750mgl-1) to liquid basal medium, prior to the plating of callus initiates onto solid basal medium, was also attempted in order to try to improve embryo formation. However, figure 6.18 shows that the mean % callus formation (multiplication) was negligible (<1%) for all citric acid concentrations tested, compared to control levels (77.3±8.5%). In addition, no embryos or globular structures were observed to form during liquid culture in any of the media containing citric acid.

215 Overall, phenotypic differences (increased morphological abnormalities) and

decreased regenerative capacity was observed from indirect embryogenesis (i.e. callus

derived) compared to direct embryogenesis in C. inodora. However, optimisation of culture

medium components did result in improved embryo formation, frequency and development

from indirect embryogenesis. Callus initiates plated onto citric acid in conjunction with

high levels of malt resulted in numerous proembryos being produced. In terms of embryo

formation, this was optimal on basal medium plus elevated malt (1250mgl-1malt) and citric

acid (250mgl-1).

Fig. 6.18 Percentage callus ABCD formation (multiplication) of plated callus initiates of C. inodora, after liquid culture in basal medium containing various concentrations of citric acid: (A) 0mg l-1 250mg l-1 500mg l-1 750mg l-1 none, (B) 250mg l-1, (C) 500mg l- 100 and (D) 750mg l-1. Callus initiates plated onto solid basal medium 80 plus BA (0.05μM). Mean (±se) of ten replicates (petri dishes) for each 60 concentration. 40

20 Callus formation (%)

0 0 250 500 750 Citric acid (mg/l)

216 Cryopreservation of embryogenic callus of C. inodora by encapsulation-dehydration

Cryopreservation of embryogenic callus (liquid cultured) of C. inodora, using an encapsulation-dehydration technique, gave high recovery rates after liquid nitrogen exposure. The moisture content of alginate encapsulated callus was reduced by preculture overnight (liquid basal medium with 0.3M sucrose and 0.3M glycerol) and 1 hour in a loading solution (0.75M sucrose, 0.75M glycerol), followed by drying over silica for 1 to 6 hours. The moisture content of empty beads was 70% after pretreatment that was reduced by drying over silica gel to 52% (1h), 36% (2h), 25% (3h), 22% (4h) and 19% (6h) (Figure

6.19). The growth of encapsulated callus at this range of moisture contents was investigated pre and post cryopreservation and the data is shown in Figure 6.20. Reduction of bead moisture content to 22.3±0.1 - 24.9±0.2%MC (3 to 4h silica dried), resulted in recovery and growth post cryopreservation of callus from above 60% of beads and was not significantly different (P=>0.1) to control levels (i.e.± desiccation and liquid nitrogen exposure).

90 Fig. 6.19 Effect of pretreatment and desiccation over silica gel on 80 moisture content of alginate 70 beads. Moisture content determined 60 gravimetrically and presented on a fresh 50 weight basis (%wb) for three replicates 40 of 20 empty beads (n=60); prior to silica drying beads were first precultured 30 (0.5M sucrose + 0.3M glycerol 20 overnight) and pretreated in loading Miosture Content (%wb) 10 solution (0.75Msucrose + 0.75M

N P P 1 2 3 4 5 6 glycerol for 1 h). o r C h h h h h n e h e cu + S S S S S S i i i i i i lt P li lica lica lic lic lica u re ca a a re t re (P a C tm ) e n t 217 Treatment

100 Series1-LN2 90 Series2+LN2 80 25 70 60 50

40 30 20

(% beads) Growth Callus 10 0 701234567 52 36 25 22 20 19 Bead miosturecontent (%wb)

Fig. 6.20 Effect of desiccation and liquid nitrogen exposure of

encapsulated callus of Citrus inodora on callus growth. Mean percentage (±s.e.) of callus growth from alginate beads of four replicates of 10 beads (n=40) for each treatment, recorded one month post culture initiation.

The best mean % of beads (69%) showing callus growth after liquid nitrogen exposure was observed at 25%MC. Silica drying of beads for 5 hours resulted in a significantly (P=0.01) reduced mean % of bead observed to have callus growth from

82.5±4.8 to 45.8±8. However, liquid nitrogen exposure did not significantly (P=0.7) further reduce this level (40.0±4.1). At higher (>36%) and lower (19%) moisture contents, the mean % of beads showing callus growth post cryopreservation was significantly reduced

(P=0.05).

218 Thus, reduction of bead moisture content to between 20 to 25% moisture content gave enough protection from liquid nitrogen exposure to result in recovery levels comparable to controls. Somatic embryo formation was observed (>2month post culture) from callus ±liquid nitrogen exposure (Figure 6.22). However, embryo formation was erratic and many embryos had abnormal morphology, as previously observed from plated liquid cultured callus.

A C

Fig. 6.22 Callus growth (formation) and somatic embryo formation of alginate encapsulated callus of Citrus inodora, (A) after pretreatment on a combination of high sucrose and glycerol

and (B,C) after pretreatment, desiccation (20%MC) and liquid nitrogen exposure. Cryopreserved callus was transferred to basal medium containing 5% lactose (no sucrose) to induce somatic embryo formation (pictured in B).

219 6.3.2 Micropropagation and shoot-tip cryopreservation

In vitro culture of nodal cuttings

Micropropagation from in vitro nodal cuttings was achieved in C. australasica,

C. inodora and C. garrawayi. Preliminary investigation of auxin type and level revealed that 2.7μM NAA resulted in a high frequency of root formation in C. inodora (data not shown). Subsequent experimentation revealed much lower levels of NAA were also effective. Over 77% of nodal cuttings of C. australasica, C. inodora and C. garrawayi gave root formation, little or no callus formation and healthy apically dominant plants, when initiated onto basal medium containing 0.5-1μM NAA (Figure 6.23).The cultures could be maintained for 10 to 12 months without sub culture (medium-term storage) (Figure 6.24).

In addition, this provided a regeneration system and material (mass propagation) for trials of cryopreservation techniques in vitro on shoot-tip material.

Fig. 6.24 In vitro growth of nodal cuttings (ca. 10 plantlets) of Citrus

inodora at 10 months post intiation (medium term storage).

220

B 100 90 80 70 60 50

40 30 Root Formation (%) 20

10 0 C. australasica C. inodora C.garrawayi

Citrus species

Fig. 6.23 In vitro propagation of nodal cuttings in

C. australasica, C. inodora and C. garrawayi. (A)

In vitro plantlets and (B) percentage (±s.e.) root formation.

221 A Fig. 6.25 Effect of nodal cutting (ca. 1cm in length) preculture (one week) on mean (±s.e.) percentage shoot-tip (A) survival, (B) growth and (C) shoot length (mm) on excision and culture on solid basal medium (25gl-1) plus 0.05μM BA. Growth and survival recorded after 1 month’s culture - 6 replicates of 5 shoot-tips for each sucrose concentration.

B C

Sucrose concentration (M)

Preculture of nodal cuttings of C. australasica

As a preliminary to undertaking shoot-tip cryopreservation, the culture of excised shoot-tips was investigated. This included the preculture of nodal cutting on various concentrations of sucrose prior to axillary shoot-tip excision to try to improve tolerance to cryopreservation stresses (e.g. cryoprotectant toxicity and desiccation), based on a method of B. Reed (pers comm. 2004). Mean % shoot-tip survival and growth (i.e. shoot development) was significantly reduced (p=<0.001), when excised from nodal cuttings precultured on 0.3 to 0.75M sucrose (10% to 43%), compared to medium concentration at

0.1M sucrose (93%)(Figure 6.25A,B). Figure 6.25C shows that mean shoot length (mm) was similar across sucrose concentrations from 0.1 to 0.5M and ranged from 4±1 to

5±1mm.

222 A B Fig. 6.26 Shoot growth of excised meristem after

preculture of nodal cutting on (A) 0.1M and (B) 0.3M sucrose. (C) Survival of

meristematic tissue and necrosis of surrounding C excised tissue after

preulture of nodal cutting, prior to shoot-tip excision,

on 0.75M sucrose.

In contrast, shoot-tips excised from nodal cuttings precultured on higher sucrose concentrations (0.75M) were significantly (p=<0.05) shorter (2±1mm) than control (0.1M sucrose) shoots (Figure 6.26). Thus, overall preculture of nodal cuttings on higher sucrose concentrations, prior to shoot-tip excision, resulted in both reduced recovery and growth in

C. australasica and so basal medium containing low sucrose concentration (25gl-1) was employed in subsequent cryopreservation experiments.

Cryopreservation of shoot-tips of C. australasica

Investigations of shoot-tip cryopreservation in C. australasica were undertaken using a vitrification based technique. In addition, the duration of exposure to 100% plant vitrification solution 2 (PVS2) was investigated. Table 6.4 shows the results of these experiments in mean percentage survival of shoot-tips, after one month’s culture on basal medium (25gl sucrose) plus 0.05μM BAP. A high level of mean % survival (93%) occurred in excision controls and was not significantly different (P=0.5) to control shoot-tips

223 precultured overnight. In the first experiment, the addition of 60min exposure to 20% PVS2 did not result in significantly different (P=>0.6) mean % survival of shoot-tips compared to control levels. In contrast, in the second experiment mean % survival was significantly

(P=<0.001) reduced compared to control levels, at only 34%.

Thus, shoot-tips from the second experiment showed greater sensitivity to PVS2 pretreatment (i.e. 20% PVS2 for 60mins). In addition, shoot-tips from the second experiment were more sensitive (almost a third less survival) to the application of the 100%

PVS2, prior to liquid nitrogen exposure. The application of 100% PVS2 significantly

(P=<0.001) reduced the mean percentage survival of shoot-tips, compared to control levels, in both the first (49±5.2%) and second experiment (20±7.1%). However, mean % shoot-tip survival was not significantly different (P=>0.15) after exposure to 100% PVS2 for shorter durations, i.e. 10 (29.5±5.3) and 15 (36.9±7.2) minutes.

Table 6.4 Cryopreservation of shoot tips of C. australasica using a vitrification technique

Treatment Experiment Mean (±s.e) Survival (%) -LN +LN Control 1 (excised only) 1 93.3±6.7 n.d 2 93.3±3.3 n.d

Control 2 (O/N incubation) 2 89.0±1.0 n.d

Control 3 (20% PVS2) 1 91.6±8.3 n.d 2 33.0±11.5 n.d

100% PVS2 - 20 min 1 61.9±4.3 40.7±9.3 2 20.0±7.1 0.0±0.0 100% PVS2 - 15 min 2 36.9±7.2 n.d 100% PVS2 - 10 min 2 29.5±5.3 d.d

For each experiment the mean (±s.e.) of 3 replicates of ten meristems for each treatment was recorded after one month of culture; n.d. no data.

224

A C

B Fig. 6.27 Growth of meristems of Citrus australasica after (A) application of plant vitrification solution 2 (PVS2) and (B) liquid nitrogen exposure. (C) Plant regeneration of excised meristem that survived PVS2 application after

subculture onto basal medium plus auxin (0.5μM NAA). Letter T denotes remains of thorn (excised prior to culture).

Shoot-tip survival after exposure to liquid nitrogen gave different results for the two experiments conducted. In the first experiment, a mean % survival of 40.7±9.3 was recorded after cryopreservation, which was not significantly different (P=>0.24) to the control (100% PVS2). Growth of shoot-tips after the application of PVS2 ±liquid nitrogen exposure is shown in figure 6.27 (A, B). In contrast, shoot-tips cryopreserved in the second experiment, using the same PVS2 protocol, recorded no survival after cryopreservation.

Plant regeneration of the shoot-tips that survived was achieved by subculture onto basal medium containing 0.5μM NAA (Figure 6.27C).

225 6.3.3 In vitro seed germination

Seed lot characteristics

A B

Fig. 6.28 (A) Fruit and leaves of Citrus garrawayi. (B) Cut near mature seed of Citrus garrawayi, illustrating trhe developed embryo cotyledons and axis.

Seeds of Citrus inodora were extracted from mature fruits harvested from QDPI research collections in northern and southeastern Queensland (cf. Table2.1). Seeds of

C. garrawayi were extracted from green fruits, five to six centimeters long (Table 6.5,

Figure 6.28A). The seeds used in this study were not quite mature, as insufficient mature seeds were available. Near mature seeds were approximately 5mm in length with a firm, white to cream seed coat. The moisture content of seeds from two separate harvests were

48.7 % ± 0.5 and 52.5 % ±2.5 (Table 6.5). The embryonic axis was well developed with shoot primordia and radicle visible (Figure 6.28B).

Table 6.5 Characteristics of seed lots of C. garrawayi

Fruit dimensions Fruit weight (g) Seed fresh Seed dry Moisture width x length (cm) weight weight content (mg) (mg) (% wb) 5.4±0.1 x 2.1±0.3 14.4±0.4 21.7±0.8 11.1±0.4 48.7±0.5

5.7±0.1 x 2.2±0.3 16.7±0.4 20.5±1.2 9.6±0.6 52.5±2.5

Mean (±s.e.). Fruit measurements determined from 25 to 50 replicates. Moisture content determined gravimetrically three replicates of ten seeds in and 15 replicates in 2004 and was presented on a fresh weight basis (%wb). 226 Desiccation and Cryostorage

Citrus inodora 90 Series1-LN The mean moisture content of 80 Series2+LN 70 ) % freshly extracted seeds from the two ( 60 50 seed lots was 35.4% ±1.0. The seed 40 30 moisture content was reduced to 10.7% Germination 20 ±0.8 by desiccation for 18 hours over 10 0 silica gel. In vitro germination 35.4%35.4%MC35.4 (fresh) (fresh) 10.7%MC 10.710.7% Seed Moisture Content (%fwb) percentages of fresh (35.4%MC) and Fig. 6.29 Effect of desiccation and liquid desiccated seeds (10.7%MC) were nitrogen exposure (LN) on the mean % in comparable at 78% ±5 and 72% ±10, vitro germination (>5mm) in Citrus inodora. Columns are the mean of 3 replicates of 10 seeds and respectively (Figure 6.29). duplicate (n=60). Bars represent one s.e. of mean.

Mean % germianation post liquid nitrogen exposure of seeds at

10.7%MC was 50% ±6. Radicle emergence (>1mm) was delayed from seeds pre and post liquid nitrogen treatment, compared to fresh seeds. The time until 50% of the final mean percentage of radicle emergence was approximately 18 days after incubation from fresh seeds, but was about 4 weeks in desiccated seeds ±liquid nitrogen exposure. The time until

50% of the final mean percentage of epicotyl emergence was the same (ca. five weeks) for both fresh and desiccated seeds, although delay of a futher week was observed in the emergence of the epicotyl from desiccated seeds after cryostorage

After eight weeks incubation, the mean root length was similar from fresh and desiccated seeds, 19.5mm ±2.0 and 20.4mm ±2.2 respectively (Figure 6.30A).

227 A B 30 30 Series1-LN +LNSeries2 25 25 ) )

20 mm ( 20 mm ( th th g g 15 15 l len y 10 10 icot Root len Root p E 5 5

0 0 35.435.4% (Fresh) 10.70% 10.7 35.435.4% (Fresh) 10.70%10.7 Seed Moisture Content (%fwb) Seed Moisture Content (%fwb)

Fig. 6.30 Effect of desiccation and liquid nitrogen exposure on (A) root and (B) epicotyl length in Citrus inodora. Mean of germinated seeds of three replicates of 10 seeds and duplicate after 8 weeks incubation. Moisture content mean of 10 individual seeds for each seed lot (n=20) and presented on a fresh weight basis (%fwb). Bars represent one s.e. of mean.

However, there was a reduction in the mean root length to 15.6mm ±1.5 in seeds after liquid nitrogen exposure. The mean epicotyl length was also reduced after liquid nitrogen exposure (Figure6.30B).

Citrus garrawayi

The mean moisture content of freshly extracted seeds (near mature) of two seed lots of C. garrawayi, harvested in 2004 and 2005, was 51.9% ±2.1. The seed moisture content was reduced to between 27.5 % ±2.2 and 8.2% ±0.7 after drying over silica gel from 3 to 18 hours (Figure 6.31). Fresh seeds had a relatively low germination percentage of 46±9

(Figure 6.32). Germination levels were similar to that of fresh seeds for seeds desiccated down to 8.2% MC. Germination post cryostorage was best (47±8) for seed at 8% MC, with only 18% germination at 19.6%MC and there was negligible germination at moisture contents above 19.6% (Figure 6.32).

228 60

Seed Moisture Content (%fwb) Content Seed Moisture 0 0345.518 Desiccation (hours)

Fig. 6.31 Seed moisture content of near mature seeds of

Citrus garrawayi, after drying over silica gel. Moisture content determined gravimetrically and presented on fresh weight basis (%wb). Mean (± s.e.) of three replicates of ten seeds in 2004, duplicated in 2005 for each desiccation level (n=60), except seeds dried for 120 in 2005 only (n=30).

80 -LN 70 +LN 60

50 40

(%) Germination 20 10 0

51.9 (fresh) 27.5 29.1 19.6 8.2 Seed Moisture Content (%fwb) Fig. 6.32 Effect of desiccation and liquid nitrogen exposure on

in vitro germination of Citrus garrawayi seeds. Germination recorded as radicle length >5mm. Mean (±s.e.) six replicates of five seeds (2004), duplicated (2005) by 3 replicates of ten (n=60).

229 A 30 -LN +LN 25

Epicotyl length (mm) length Epicotyl 5

B 51.9 (fresh) 27.5 29.1 19.6 8.2 45 Seed Moisture Content (%fwb)

40 35 30

25 20 15

(mm) length Root 10 5 0 51.9 (fresh) 27.5 29.1 19.6 8.2 Seed Moisture Content (%fwb)

Fig.. 6.33. Effect of desiccation and liquid nitrogen exposure (-196ºC) on (A) epicotyl and (B) root length of

germinated seeds of Citrus garrawayi. Mean (±s.e.) six replicates of five seeds (2004), duplicated (2005) by 3 replicates of ten (n=60).

Mean root length from germinated seeds was similar regardless of seed treatment, ranging from 23.2±3.5 to 35.5mm±3.7mm (Figure 6.33A). Epicotyl growth was also similar for all desiccation levels for germinated seeds plus and minus liquid nitrogen exposure (Figure 6.33B).

230

A Fig. 6.34 (A) Healthy

acclimatised Citrus -LN2 +LN2 garrawayi seedlings from in

vitro culture after 3 to 5h silica drying (ca.20%MC) ±

liquid nitrogen (LN2) exposure of seeds. (B) Growth of acclimatised C. C. garrawayi inodora seedlings from seed

B germinated in vitro after +LN2 silica dying for 18h and

liquid nitrogen exposure.

C. inodorai

Acclimatisation of in vitro germinated seeds

Importantly, in vitro cultured plants of Australian wild Citrus were amenable to acclimatisation to the shade house. Plantlets were acclimatised by a progressive reduction of the relative humidity and maintenance of plant health (i.e. plant nutrition and fungal control) as shown in figure 6.34. The majority of seedlings were successfully acclimatised from control (ca.80) and cryopreserved seeds (ca. 60%) germinated in vitro in both

C. inodora and C. garrawayi. Plants transplanted to pots and the shade house were observed to be phenotypically normal and developed both a healthy shoot and root system.

231 DSC of Citrus garrawayi seeds

Differential scanning calorimetric (DSC) thermograms of freezing and thawing events of seeds (near mature) of C. garrawayi revealed that there were significantly lower mean enthalpy values for endothermic and exothermic phase transitions in seeds silica- dried to low moisture contents (<20%MC) (Table 6.6). Seeds at high moisture contents

(silica-dried 3 or 4h) had mean enthalpy values of above 71Jg-1FW whilst, seeds desiccated for 5.5 and 18 hours had enthalpy values below 36 Jg-1FW in both cooling and warming thermograms. There was a significant difference in enthalpy values between seeds at higher compared to lower moisture contents.

Dry seed at a very low water contents of 3.3%MC were used to determine the characteristic lipid peaks on thermograms of seeds (cf. Chapter 5.3). In cooling thermograms of seeds at higher moisture contents, the beginning of lipid phase transition was often seen as a shoulder (arrow) of water freezing peaks (Figure 6.35). Dry seeds

(3.3%MC, silica- dried 120h) had a mean enthalpy of melt transition of 11.0 ±2.9Jg-1FW and onset temperature of - 7.4 ºC ±1.4 in warming thermograms. Cooling thermograms of dry seeds resulted in a mean transition enthalpy of 6.8 ±1.3Jg-1FW and an onset temperature of -11.4ºC ± 0.9 (data not shown).

Overall, C. inodora seeds showed tolerance to desiccation down to around 11% MC and Citrus garrawayi seeds (near mature) to 8%MC and cryopreservation, using in vitro germination system. In addition, DSC analysis of C. garrawayi seeds revealed that the absence of phase transitions attributable to ice formation and melting in seeds silica-dried to low moisture contents coincided with the physiological responses of seeds, i.e. optimal germination post cryostorage associated with seed desiccation to low moisture contents .

232

Fig. 6.35 Representative cooling thermograms of seeds (near mature) of Citrus garrawayi: (A) fresh seed (52% MC), (B) desiccated seed (8%MC) and (C) dry seed (3%MC). Thermal analysis by differential scanning calorimetry using a cooling rate of 10ºC/minute.

Exo Up Temperature ºC

Table 6.6 Effect of desiccation level on thermal events during cooling (-80ºC) and warming of desiccated seeds (near mature) of Citrus garrawayi

Desiccation Mean moisture Enthalpy (Jg-1 fresh weight) duration (h) content (%) Warming Cooling 0 51.9 96.4 a 85.2 a 3 29.1 80.1 a 83.2 a 4 27.5 74.0 a 71.3 a 5.5 19.6 33.6 b 35.5 b 18 8.2 28.3 b 22.0 b 120 3.3 11.0 b 6.8 b Thermal analysis by differential scanning calorimetry using a cooling and warming rate of 10ºC/minute. Thermal values are the mean of four to seven separate assays of individual seeds. Desiccation was over silica gel. Moisture content determined from three replicates of ten for each of two seed lots. Different lettering represents a mean significant difference within columns at 0.05 level (LSD).

233 6.4 Discussion

6.4.1 Embryogensis

Ovule and seed initiated cultures

This study has confirmed that the polyembryonic commercial cultivars, Murcott and

Orlando, produce prolific embryogenic callus and somatic embryos that regenerate into plants, as previously demonstrated by Perez et al (1999). Although somatic embryo formation was not observed in Tahitian lime, some torpedo shaped structures formed from ovules. These structures may be proembryos, similar to those observed by Tomaz et al

(2001) in Rangpur lime. Even so, modification of basal media in this present study did not promote further development of these structures.

The monoembryonic Australian wild species and monoembryonic Imperial mandarin did not produce much callus and, with the exception of C. inodora, did not readily form somatic embryos. This is in line with other studies in which embryogenesis has been widely applied to polyembryonic genotypes but has found limited success in monoembryonic species. However, embryogenesis has been demonstrated (Ling and

Iwamasa, 1997) in several wild monoembryonic members of Rutaceae. Of the wild

Rutaceae genera tested, 30% to 1.9% of seeds showed embryogenesis, depending on the species. The lowest level of embryogenesis was reported for the Australian C. australasica

(the only citrus species tested), with only 1 of 52 seeds showing somatic embryo formation

(Ling and Iwamasa, 1997).

In the present study, similar levels of somatic embryo formation were observed in

C. australasica, from premature seed cultured on basal medium that was supplemented by malt extract and citric acid. However, no embryo formation occurred on other medium

234 modifications. These results confirm the previously reported difficulty of inducing embryogenesis from seeds in Citrus australasica. Necrosis of all of the C. australasica ovules cultured in vitro was observed in this study. The source tree had fruit blackening and dropping off from early in the season. This could account for the ovule death observed in cultured ovules in this particular experiment. Thus, further experimentation is needed to determine if immature ovules of C. australasica are amenable to in vitro embryogenesis.

Cytokinin (BAP) has induced somatic embryogenesis from stigma/style explants

(Carimi et al. 1998ab) and has also been used in embryogenesis protocols to increase callus multiplication in various citrus cultivars and species (Duran Vila et al 1995, Ling and

Iwamasa 1997). In this current study, modification of the Pérez et al (1999) method of embryogenesis, by the addition of various concentrations of BAP to basal medium did not induce somatic embryo formation and resulted in lower frequencies of callus induction from ovules in both Citrus garrawayi and Tahitian lime.

Kochba et al (1978) found ABA induced embryogenesis from ovular tissues of

C. sinensis. In the present study, ABA did not stimulate embryogenesis in seed initiated cultures of the three Australian Citrus species tested. Betaine, used to induce somatic embryo formation from mature seed of Camellia sinensis (Akula et al 2000), was also unsuccessful in inducing embryo formation in C. garrawayi ovules. However, basal medium supplemented with 250 mg l-1of betaine was the only modified medium to produce similar amounts of callus from C. garrawayi ovules as those cultured on basal medium alone. Although embryogenesis has been reported in some monoembryonic species (Ling and Iwamasa 1997, Carimi et al 1999), many studies have been unsuccessful in generating

235 somatic embryos from monoembryogenic genotypes of Citrus (Button and Kochba 1977,

Kobayashi et al 1981, 1982, Perez et al 1999).

Factors that are crucial to somatic embryogenesis include genotype, culture medium composition and explant type and developmental stage (Carimi and De Pasquale 2003). In the present study, both immature ovule and seed material resulted in no embryogenesis in

C. garrawayi, despite medium modification. Future studies could focus on investigation of explant type. For example, successful embryo induction was achieved in monoembryonic

C. medica using transverse thin cell layer sections of stigma and styles (Carimi et al 1999).

Some success was, however, achieved in the Australian monoembryonic species

C. inodora. Ovule and seed initiated cultures of C. inodora resulted in both the production of embryogenic callus and embryos. Callus formation was limited, and despite some embryos being observed arising from callus, embryos predominantly arose by direct or secondary direct embryogenesis, i.e. without intermediate callus formation.

As previously discussed (cf. Section 1.7), adventive embryos are generated from nucellar tissue (maternal tissue), as well as zygotic embryos in polyembryonic Citrus species and cultivars in vivo (Wakana and Uemto 1987, Perez 2000, Carimi and De

Pasquale 2003). The degree of in vivo adventive embryogensis of polyembryonic genotypes varies. For example, mandarin and orange cultivars have been reported to have a very high to high degree of in vivo adventive embryogenesis, but it is low to moderate in C. limon

(Wakana and Uemto 1987).

Differences in embryogenic potential between monoembryogenic species and polyembryogenic Citrus species are poorly understood. Pérez (2000) suggests that it may be due to different endogenous hormonal balances or unknown inhibitory components

236 found in the chlalazal region of monoembryonic seeds (Tissert and Murashige 1977,

Gmitter and Moore 1986). Widespread application of somatic embryogenesis has been reported in polyembryonic cultivars and species. Kobayashi et al (1981) suggested the difficulty in embryogenic induction in monoembryonic species results from the lack of nucellus derived embryos and primordium cells (Kumitake and Mii et al 1995), i.e. in vivo embryogenic potential. Citrus inodora appeared to have a greater tendency to in vitro embryogenesis than either C. australasica or C. garrawayi. Interestingly, C. inodora was both the only Australian species to show substantial embryogenesis in vitro, as well as the only test species to have more than one embryo observed in vivo (i.e. two seedlings observed from <1% of seeds [cf. Chapter 2.4]). Thus, C. inodora has the capacity to more readily form embryos in vitro than the other two test species and this may be related to a greater in vivo embryogenic potential, which has been observed in cultivated citrus.

C. garrawayi was the most recalcitrant test species to in vitro embryogenesis.

Embryogenic callus proliferation in C. inodora

Somatic embryos are an excellent material for germplasm storage via cryopreservation (Bajaj 1995). In this present study, somatic embryos initiated from both seed and ovules of C. inodora moved quickly but non-synchronously through induction to germination of numerous healthy plantlets. The asynchronous embryo formation and rapid acquisition of germinability meant that little material at a suitable (or the same) developmental stage was available for meaningful cryopreservation trials. An alternative material for cryopreservation was embryogenic callus, which has been cryopreserved in many cultivated Citrus species (Engelmann et al 1994, Perez 2000, Hao et al 2004, Carimi

237 and De Pasquale 2003). However, subculture of embryogenic callus of ovule and seed initiated cultures of C. inodora on solid medium resulted in only limited proliferation.

Thus, multiplication of seed initiated callus via liquid culture was investigated. Culture in liquid medium containing high levels of glycerol resulted in mass proliferation and differentiation of globular structures (proembryos) from seed-initiated embryogenic callus of C. inodora.

Glycerol has been previously reported to have a stimulatory effect on citrus somatic embryogenesis (Ben-Hayyim and Neumann 1983, Jimenez et al 2001, Tomaz et al 2001).

Hao et al. (2002) also used glycerol in solid basal medium for induction of somatic embryogenesis in Citrus papuana, a New Guinea species of Citrus closely related to the

Australian species. It has been suggested that such additives may act as an embryogenic stimulus through a stress response, which triggers increased production of endogenous hormone levels (Fehér et al 2003). Further, Jiménez et al (2001) observed an association link between glycerol promoted somatic embryogenesis in several commercial citrus cultivars and higher levels of auxin and cytokinin.

Embryo morphology

Somatic embryos of many plant species have been found to follow a similar pattern of development to their zygotic counterparts (Dhed'a et al 1991, Attree and Fowke 1993,

Dodeman et al 1997). Citrus sinensis somatic embryos have been reported to be morphologically similar to small in vivo adventive embryos (Mioseeva et al 2006,

Kultunow et al 1996). The Australian species do not form adventive ‘somatic’ embryos in vivo (monoembryonic) for such a comparison. However, somatic embryos of C. inodora

238 showed morphological similarities to zygotic embryo counterparts. In addition, they followed a similar pattern of differentiation to somatic embryos of cultivated citrus, i.e. globular, heart shaped and cotyledonary. Although, there was asynchronous embryo formation and some morphological abnormalities (e.g. multiple cotyledons) were observed.

This is consistent with observations of cultivated Citrus species where somatic embryo populations were both heterogeneous in size and numbers of cotyledons (Moiseeva et al

2006). In addition, Navarro et al (1985) reported a high level of abnormal plants regenerating from somatic embryos of monoembryogenic species of cultivated citrus.

The reason for the appearance of abnormal phenotypic characteristics in somatic embryo derived plantlets in monoembryonic species, but not polyembryonic varieties, is not understood (Navarro et al 1985, Carimi and De Pasquale 2003). However, the degree of in vivo polyembryony and the ability to regenerate somatic embryos in vitro has been linked (Mitra and Chaturvedi 1972). Thus, although poorly understood, the level of morphological abnormalities of somatic embryos in different species and genotypes of cultivated citrus appears to be linked to embryogenic potential. This was supported by histological examination of several cultivated citrus genotypes of both a high and low frequency somatic embryo production from callus. Two genotypes of C. sinensis and one genotype of C. reticulata (Blanco), with a high frequency of embryo formation, had normal histodifferentiation in embryos at all developmental stages. In contrast, another genotype of mandarin and one lime genotype (C. limonia L. Osbeck), that formed embryos at a low frequency, showed abnormalities such as lack of or dedifferentiation of the protoderm, as well as an absence of apical shoot-tips and procambial strands (Tomaz et al 2001). These findings in embryogenesis in cultivated citrus are consistent with the observations of the

239 present study, i.e. morphological abnormalities in C. inodora embryos, a species that demonstrated a low to moderate in vitro embryogenic potential.

In the present study, somatic embryos of C. inodora derived from proliferative direct embryogenesis were more morphologically normal and had a greater capacity for both formation and whole plant germination, when compared with those from indirect embryogenesis (i.e. callus derived). A genotype dependence for in vitro embryo production and histo differentiation in cultivated citrus has also been observed between different callus lines of the same genotype (Maul et al 2006). Citrus embryogenic callus cultures can differ in endogenous hormone levels, dependent on genotype and culture conditions (Jimenez et al 2001). Thus, Maul et al 2006 speculated that the differences between callus lines for early embryogenesis could also be attributable to endogenous hormone levels or slight differences in environmental conditions during in vitro cultures. Consequently, this could affect the timing of signalling factors that activate during morphogenic events. Thus, the difference in direct and indirect embryogenesis (i.e. morphogenic potential) observed in present study on C. inodora, may also be attributable to differences in endogenous hormone levels. Culture medium provides both nutrients and developmental signals (Pullman and

Buchanan 2006). However, the large number of possible variables associated with medium composition and endogenous hormone levels, make it very difficult to establish which factors were most crucial to normal embryo formation.

Effect of medium composition on callus and somatic embryo formation

Experiments were conducted to improve the synchronicity and embryo formation from embryogenic callus of C. inodora (proliferated by liquid culture) through media modification. Overall, most substances had little or no affect on callus or embryo induction, 240 compared to the control (basal medium only). However, glycerol was promotive, in liquid and solid medium, on callus proliferation, and malt or lactose (sucrose substitue) on embryo formation, which has been previously reported in many other Citrus species (cf. section 1.7).

Overall, the best medium modification trialed, giving the highest levels of somatic embryo formation (from callus) in C. inodora, was the addition of citric acid to solid basal medium, especially in combination with high concentrations of malt extract. Citric acid had a dramatic affect, inducing mass formation of proembryos and the highest number on embryos formed after two months culture. The use of malt extract to induce embryogenesis in the Citrus genus is well-established (Perez et al 1999, Perez 2000, Carimi and De

Pasquale 2003), but the use citric acid is novel.

Glycerol was promotive to the formation of globular structures (proembryos) in liquid cultured embryogenic callus. However, further development of proembryos resulted in embryos that had incomplete germination of shoots only (data not shown). Thus, it could be speculated that these embryos did not have proper early histodifferentiation of root and shoot poles, which has been previously observed in abnormal embryos of cultivated citrus

(Tomaz et al 2001). By contrast, addition of citric acid to media resulted in the most prolific embryo and proembryo formation, as well as producing many morphologically normal embryos that were capable of whole germination, unlike those induced on other trial media. Citric acid appears to have promoted proper histodifferentiation of both a root and shoot pole in C. inodora embryos. Such a promotive affect on embryo bipolarity by citric acid has been previously reported in alfalfa (Medicago sativa L.) (Nichol et al 1991).

241 However, callus proliferation in alfalfa was inhibited by a pre-treatment in citric acid, while total embryo yield and conversion to plantlets was substantially higher in the absence of pretreatment (Nicol et al 1991). Similar results were observed in this current study in C. inodora. Citric acid applied to liquid cultures of C. inodora callus, prior to plating of callus on solid medium, resulted in almost complete inhibition of callus proliferation. However, when used as a supplement to solid basal medium, and particularly in conjunction with high malt, citric acid induced the greatest level of embryo formation and whole plantlet conversion from liquid cultured callus initiates. Malt has complex constituents, so it is difficult to determine its role. However, high levels of malt resulted in good callus proliferation and some formation of embryos that often displayed abnormalities and germination of only shoots. So, it is speculated that citric acid’s role might be to contribute to proper embryo histodifferentiation. Overall, citric acid was observed to induce synchronous formation of numerous proembryos from callus that was not observed in any other media treatments trialed. In adddition, citric acid appeared to normalise proembryo production and histodifferentiation in C. inodora, but further histological study is needed to confirm these findings.

The concentrations of different organic acids (e.g. oxalate, citrate, malate and lactate) vary dependent on in vivo embryo developmental stage in cotton, coffee and pine

(Miernyk and Trelease 1981, Rogers 1999, Pullman and Buchanan 2006). However, the role of organic acid in plant embryo development is poorly understood (Pullman and

Buchanan 2006). Pullman and Buchanan (2006) quantitatively analysed the levels of organic acids at various developmental stages in zygotic embryos of pine, in an attempt to better optimise and understand somatic embryogenesis i.e. to produce a culture medium

242 that resembles the in vivo environment to develop vigorous embryos. Interestingly, citric acid was found to be at its highest concentration during early zygotic embryo developmental stages and subsequently declined with further development. Moreover, in whole grains of Coffea canephora and Coffea arabica, citric acid increased from mid seed development to maturation. Thus, addition of exogenous citric acid may be promotive on embryo formation and development in C. inodora, by mimicking in vivo conditions of embryo development, i.e. morphogenic cues of early embryo development.

This study adds support to the finding by Nicol et al (1991) in alfalfa that citric acid supplements to culture medium promotes embryo formation. However, a recent study in Pinus spatula somatic embryogenesis reported pretreatment of explants with antioxidants or incorporation into basal medium of antioxidants, including citric acid, had a negative effect on the initiation of embryogenic cultures, somatic embryo production and plantlet recovery (Malabadi and Van Staden 2005). However, Nicol et al (1991) observed that there was an optimal concentration of organic acid for embryogenesis in cell cultures of alfalfa, with higher concentrations toxic to further embryogenesis. Thus, this optimum may not have been achieved in the study on pine and/or exogenous addition of citric acid may not be as effective in pine.

Han et al (2002) found an association between embryo formation and endogenous hormone levels in sweet orange and satsuma mandarin from liquid cultured callus. The endogenous auxin level was found to decrease in callus that underwent embryo formation, and remained relatively in high in callus that had no embryo formation. This is consistent with earlier reports by Spiegel-Roy and Kochba (1980) that exogenous auxins (i.e. IAA,

NAA, and 2, 4-D) suppressed embryo formation from nucellar derived callus, whilst auxin

243 inhibitors had a promotive affect. Interestingly, the effect of the inclusion of both malt and citric acid at different concentrations triggered different morphogenic responses (i.e. callus proliferation or inhibition, as well as and/or embryo formation) in a manner suggestive of an assocication with endogenous plant hormones levels (i.e. cytokinin to auxin ratio) in C. inodora.

Nicol et al (1991) reported that they were the first to observe the affect of organic acid treatment on somatic embryo development. To the authors’ knowledge, the current study reports for the first time the promotive affect of exogenous citric acid on somatic embryogenesis in the Citrus genus and Rutaceae family. The effect of citric acid, especially in combination with malt, on embryo formation and endogenous hormone levels of in vitro culture embryogenic material, in both wild and cultivated Citrus species, warrants further investigation.

Cryopreservation of C. inodora embryogenic callus by encapsulation – dehydration

Encapsulation-dehydration treatments prior to cryopreservation improve recovery after cryopreservation by prompting vitrification (glass formation) on rapid cooling to

-196ºC; this avoids the formation of potentially lethal intra and extracellular ice (Dereuddre et al 1991, Sakai 2004). In the present study, encapsulation of C. inodora embryogenic callus and desiccation to 25% moisture content resulted in survival and growth rates comparable to control treatments. Block (2003) investigated the thermal activity of empty alginate beads after dehydration in 0.75M sucrose and evaporative air-cooling. It was found that for successful vitrification and glass formation that did not destabilize on rewarming, a bead moisture content of 26% was optimal, with much of the remaining water being osmotically inactive. This present study supports this finding with the optimal survival and

244 growth of embryogenic callus post cryopreservation observed in beads with a 25% moisture content. When the moisture content was further reduced to below 22%, the viability of embryogenic callus was significantly reduced prior to liquid nitrogen exposure. At higher moisture contents the large amount of freezable water is likely to cause lethal injury, explaining the significantly reduced regrowth rates of embryogenic callus of C. inodora at higher moisture contents post cryopreservation. Thus, the high recovery rates post cryopreservation observed from C. inodora embryogenic callus was most likely due to the lack of lethal ice formation resultant from the combination of cell morphology (i.e. highly cytoplasmic ) and glass formation at reduced moisture contents.

This study has demonstrated that cryopreservation of embryogenic callus of

C. inodora is feasible. However, morphological abnormalities of embryos derived from indirect embryogenesis and asynchronous development mean optimisation of the in vitro system is needed to establish a reliable synchronous system for germplasm conservation purposes.

6.4.2 Micropropagation and shoot-tip cryopreservation

In vitro culture of nodal cuttings

In this study, plant regeneration of C. australasica, C. garrawayi and C. inodora by in vitro nodal cuttings was achieved on basal medium containing naphthaleneacetic acid

(0.5 to 1μM NAA). These results are similar to those observed in Citrus halimii, an endangered wild species in Malaysia (Normah et al, 1997), with good root formation on culture medium containing a low concentration of NAA. The establishment of an in vitro culture system for three Australian Citrus species provides a basis for both

245 micropropagation, medium term storage (without subculture for 10-12 months) and the development of shoot-tip cryopreservation protocols.

Cryopreservation of shoot-tips of C. australasica

This study, to the author’s knowledge, reports for the first time the of recovery of shoot-tips of a species of Citrus using a PVS2 based technique, although further experimentation to optimise this system is needed. For example, there was a disparity between survival (i.e. 41% compared to zero survival) observed between the two experiments that cryopreserved shoot-tips of C. australasica. This may be partly due to differences in the physiological status of the parent material. Plant health is crucial to survival of cryopreservation, i.e. there are low survival rates from material in poor condition (Reed et al 2004). The greater sensitivity to stress, i.e. the lower survival after pretreatment (20% PVS2), suggests suboptimal physiological status in shoot-tips from the second experiment. Thus, physiological conditioning of C. australasica plants and excised shoot-tips appears to be crucial to improving survival to the extreme stress (e.g. dehydration) involved in cryopreservation techniques.

As well as initial plant health, recovery of plants from cryopreservation is dependent on many factors, such as pretreatment and cryoprotectant type, duration of exposure to cryoprotectant exposure, freezing and thawing rates and recovery medium

(Reed et al 2004). This complexity means many other factors, within both the in vitro culture and cryopreservation protocols may have an impact on recovery and make it difficult to determine the reason for limited success. The lack of reproducibly in protocol reported in this study and the relatively low survival post cryostorage from the first

246 experiment (i.e. 41%) suggest that this technique may not be ideal for germplasm storage purposes.

Lethal injury of shoot-tips most likely resulted from dehydration injury and/or the toxicity of PVS2. Preculture on elevated sucrose concentrations has been reported in successful cryopreservation studies of Citrus shoot-tips (Gonzalez-Arnao et al 1998, Al-

Ababneh et al 2002, Wang et al 2002). Wang et al (2-002) found preculture of excised shoot-tips of two Citrus species on increasing concentrations of sucrose (0.3M, 0.5, 0.75 and 1M), over about a one week period, resulted in a significantly improved survival.

However, attempts to preculture nodal cuttings on elevated sucrose ( 0.3M) concentrations, prior to shoot-tip excision in C. australasica, reduced survival levels to over half that of the controls (0.1M sucrose). Thus, shoot-tips of C. australasica appear to be more sensitive to elevated sucrose than other members of Citrus.

Shoot tip tolerance to preculture on elevated sucrose concentrations was found to be species dependent in coffee (Mari et al 1995). Coffea sessiliflora shoot-tips tolerated preculture in liquid medium containing 0.75M sucrose, whereas C. racemosa needed a progressive increase in sucrose concentration. Optimisation of both preculture and pretreatment of C. australasica shoot-tip could likewise involve a step wise increase in sucrose concentration in any future studies.

However, the application of in vitro culture and cryopreservation methodologies developed in cultivated Citrus species may not be straightforward due to the unique biology and ecology of the Australian wild species. Touchell and Dixon (1996) state that ‘existing cryostorage procedures developed for shoot tips of agricultural and horticultural species are not readily adaptable to native Australian species. The development of cryostorage

247 processes for highly endemic and specialised Australian flora needs to consider the unique characteristics of each species.’

For example, cryopreservation of shoot tips of Western Australian species, that showed sensitivity to PVS2, was significantly improved by using a modified PVS2 solutions with reduced or zero DMSO (Turner et al 2001). Thus, the use of a less toxic plant vitrification solution could prove beneficial in C. australasica. Other protocol modifications that have proven successful in Australian wild species (i.e. six

Haemodoraceae taxa) include preculture of shoot-tips in 0.4M sorbitol and the application

PVS2 at 0ºC (Tuner et al 2000b). Of the six species tested, five were successfully cryopreserved using this method with 8% to 35% of shoot tips surviving cryopreservation.

Moreover, Turner et al (2000b) found that the key to successful cryopreservation protocols for a range of Australian native taxa was the preculture of shoot tips on high concentrations of sorbitol prior to incubation in PVS2. In further studies, Tuner et al (2001) found that preculture duration and use of glycerol and modification of PVS2, by reduced or zero

DMSO, improved recovery of shoot-tips of Australian endangered taxa. These modified vitrification based techniques, that have proved successful in the cryopreservation of other

Australian taxa, warrant investigation in any future studies in Australian wild Citrus species.

Alternative cryopreservation techniques, such as encapsulation-dehydration, that have been demonstrated for shoot tips in other Citrus species, could also be investigated.

However, preliminary trials in the present study included an encapsulation-dehydration based protocol, which proved unsuccessful. Very low survival (<20%) of shoot-tips occurred after preculture in liquid basal medium with elevated sucrose and/or glycerol

248 concentrations (i.e. 0.3M - 0.5M) for 3 days, followed by overnight incubation in 0.75M sucrose, and silica-drying to 20%MC (data not shown). This is consistent with findings in other Australian native species, where vitrification based protocols have proved to be the most successful (Touchell and Dixon 1996, Turner et al 2001).

One morphological difference between cryopreserved and non-cryopreserved shoot- tips of C. australasica was the tissue whitening (death) of surrounding tissue after liquid nitrogen exposure. Microscopic observations revealed a similar response in banana (Musa spp.), where only the most meristematic tissue of proliferating shoot-tip clumps survived cryopreservation (Panis et al 1996). This is consistent with the current study in

C. australasica, where survival was also limited to meristematic zones (whitening of surrounding tissue) in cryopreserved shoot-tips.

Overall, this study has demonstrated that phenotypically normal plantlets can be recovered from cryopreserved shoot-tips of C. australasica, using a modified PVS2 technique. However, differences in survival levels between experiments and the relatively low recovery rate (41%) means further research is required to improve the efficiency for this technique to be suitable for germplasm conservation purposes. In vitro shoot tip culture has been reported in Australian wild species of the Rutaceae family, for example

Eriostemon australasius (Plummer and deFossard 1981). Although, to the author’s knowledge, this the first report of cryopreservation of shoot tips of Australian Rutaceae taxa.

In addition, an in vitro culture system is now available for micropropagation and medium term storage of germplasm of Australian wild Citrus species. This is particularly important to conservation efforts in the rare and vulnerable listed species of C. inodora and

249 C. garrawayi. In addition, establishment of an in vitro shoot-tip culture system in

Australian wild Citrus provides a useful system for producing virus free material (e.g. germplasm exchange/maintenance).

6.4.3 In vitro seed germination

In three cultivated Citrus species, Lambardi et al. (2004) found that desiccation of seeds to moisture contents of between 10% - 16% resulted in only a negligible reduction in their germinability. In the present study, C. inodora seeds similarly showed tolerance to desiccation to around 11% MC. However, cryostorage reduced germination by over 22%, with only 50% of seeds germinating after liquid nitrogen exposure. Lambardi et al. (2004) observed similar results in C. limon (lemon) seeds at 10%MC (air-dried 10 h), with a drop of 23% germination from cryostored seeds.

In chapter 5.3.2 of this study, it was shown that at very low moisture contents (3%) seeds of C. inodora had a high recovery level (ca. 80%) after cryopreservation. However, in vitro germination in the current study resulted in a lower level of recovery post cryopreservation (50%). This disparity between the recovery levels was probably not because of the differences of the culture systems. Ice formation is widely accepted as a major contributing factor in lethal injury during freezing. It is more likely that the reduced level of recovery was because of damaging ice formation in the seeds germinated in vitro, as these seeds were at a higher moisture content (11%), which was above the unfrozen water content of seeds of C. inodora (est. 8% (cf. section 5.3.4)). Thus, reduced recovery may be indicative of an intolerance in this species to the presence of ‘free’ water on

250 freezing, which is consistent with similar findings in cultivated citrus seeds (Hor et al 2005)

(cf. Chapter 5.4).

In previous studies, (cf. Chapter 2.4 and 5.4) it was proposed that low seedling recovery after desiccation and cryopreservation in C. garrawayi might have been partially due to fungal infection. However, in the present study, which was under aseptic conditions, higher levels of recovery both pre or post desiccation and cryopreservation were not observed compared to the ex vitro levels (cf. Chapter 5.3.3).

The best germination percentages post cryostorage of seeds (near mature) of

C. garrawayi was observed at the lowest moisture content tested (8%MC). Desiccation of seeds to 8% MC and cryostorage did not greatly affect either the level of germination or seedling vigour in this study. This correlated with a reduction or absence of ice formation in seeds at low moisture contents, as determined by DSC scans. Hor et al. (2005), in a study on cultivated Citrus species, reported the same water content (ca.8%) as optimal for cryostorage in two test species (C. aurantifolia and C. reticulata) and found this water content coincided with the unfrozen water content of the seeds.

Germination levels in both C. inodora and C. garrawayi after cryopreservation and in vitro germination was at between 40 to 50% (cf. Chapter 5). Overall, these levels were less than that observed in ex vitro germination studies. Optimisation of the culture environment (e.g. decreased sucrose concentration), and in the case of C. inodora, a lower seed moisture content, may improve on these germination levels. On the other hand, growth

(i.e. root and epicotyl lengths) was comparable after about two months incubation in both species between the two culture systems, i.e. in vitro and ex vitro germination.

251 This study demonstrated, using a straightforward system, that seeds of the two rare and threatened Australian Citrus species can be germinated in vitro after cryostorage, including successful acclimatisation of plants to the shade house. This protocol provides a method for the mass propagation (i.e. micropropagation of nodal cuttings, cf. section 6.2) of the limited seed material available in these species and as such would facilitate utilisation

(e.g. propagation for restoration programs) of seeds recovered from cryostorage.

252

CHAPTER 7.0 GENERAL DISCUSSION

253 Chapter 7.0 General Discussion

7.1 Overview of main findings/outcomes

This research study makes a significant contribution to our understanding of the seed biology, in vitro culture and cryobiology of Australian wild Citrus species. These findings facilitate the development of ex situ conservation strategies, including options for the long- term storage (seed cryopreservation) and mass propagation (in vitro culture) of this valuable and threatened genetic diversity. The key findings of this study were:

Over one hundred rare and threatened Queensland edible plants and/or CRWs were

identified, many of which have subtropical to tropical distribution and may have

non-orthodox seed storage behaviour (SSB).

The Australian wild species of the Citrus genus are a priority for the investigation

of ex situ conservation strategies because of their conservation priority, potential

socioeconomic importance (e.g. novel genes (CWR) and fruits), probable non-

orthodox seed storage behaviour and lack of corresponding techniques for their long

term ex situ conservation.

Descriptors of mature seed morphology and anatomy have been given for

C. australasica, C. inodora and C. garrawayi - vital to the development and

application of effective seed storage protocols (i.e. seed lot quality). This is

especially the case in C. garrawayi because of variation in fruiting that was

observed over a three-year harvest period (2004-2006). The combinational seed

traits of size and coat hardness (i.e. woody) were found to be useful indictors of

seed maturity, as they correlated well with morphological, anatomical and

physiological seed development. For example, differentiation of embryo and seed

254 coat, dry weight increase, presence of lipid (DSC endothermic event), high level of

germinability and desiccation tolerance in mature seeds of C. garrawayi.

Seed characteristics of Australian wild Citrus species were found to be similar to

cultivated species of the genera, for example, high seed oil content (ca. 30 to 50%),

green to cream cotyledons, mature seed moisture content (ca. 40%) and seed coat

anatomy (as determined by electron microscopy).

C. garrawayi was found to differ in seed shape (rounded to three-angular) and seed

coat (SC) morphology (i.e. SC thickness, much longer protrusion length) from

C. australasica and C. inodora (rounded surface and flat underside in shape). The

well-developed minute protrusions of C. garrawayi were most similar to those

observed in previous studies in the less desiccation tolerant cultivated species of

C. sinensis and C. aurantium. Surface topography of seeds of more desiccation

tolerant C. australasica and C. inodora were most like the desiccation tolerant

cultivated species C. aurantifolia and C. limon. Seed surface topography was also

found to be a useful tool for taxonomic identification.

In terms of both germination and seedling growth, 20ºC was found to be sub-

optimal for germination, whilst germination of seeds of all three species at 30ºC was

>80%. Tolerance to germination at a temperature range between 20 to 25ºC was

greatest in C. australasica (warm subtropical), reduced in C. inodora (warm

tropical) and lest in C. garrawayi (hot tropical) and was consistent with their natural

distribution ranges (i.e. climatic data).

255 Seed of C. australasica, C. inodora and C. garrawayi tolerated desiccation to low

moisture contents. Overall, the decreasing order of species tolerance to rapid seed

desiccation, without a significant reduction in seedling length and percentage

radicle emergence, was C. australasica (<3%MC), C. inodora (3%MC) and

C. garrawayi (7%MC). Thus, for cryopreservation purposes rapid drying methods

are recommended in these species, due to their tolerance to desiccation to low

moisture contents (i.e. well below WCu (ca.10%)).

Not withstanding the above finding, there was a differential level of response in

terms of epicotyl and radicle emergence following desiccation to low moisture

contents and cryopreservation. The radicle was robust to stress, i.e. radicle

emergence rates and levels remained high following both desiccation and

cryopreservation. In contrast, epicotyl emergence was somewhat delayed in seeds

dried to low moisture contents (<5%) and was further delayed by cryopreservation.

The tolerance to both desiccation and cryopreservation of seeds of C. australasica

and C. inodora indicates that these species are ‘essentially’ orthodox in seed storage

behaviour. However, C. garrawayi seeds displayed more complex seed storage

behaviour.

Thermal analysis of phase transitions in cotyledon tissue of C. australasica,

C. inodora and C. garrawayi revealed differences in the crystallization and melt

onset temperatures of seed oils between these species; C. australasica (-9.2±1.6 and

-26.3±0.2), C. inodora (-7.4±0.5 and -22.7±0.7) and C. garrawayi (-4.8±0.7 and

-13.7±2.2).

An association was found between seed thermal properties of Australian wild Citrus

species and the geographic gradient, i.e. an increased mean temperature of lipid

256 melt end coincided with decreasing latitude and increasing temperature of natural

distribution (warm subtropical to hot tropical). Additionally, the thermal behaviour

of seed oils (i.e. lower melt/crystallization onset temperature) corresponded to

seedling recovery after cryopreservation.

In vitro embryogenic potential, using a range of media, was low to moderate in

C inodora and low in C. australasica, whilst C. garrawayi was recalcitrant to in

vitro embryogenesis.

Somatic embryogenesis was demonstrated from both ovule and seed explant

material in C. inodora.

Glycerol in liquid and solid culture media promoted embryogenic callus

proliferation and malt or lactose (substituted for sucrose) promoted embryo

formation in C. inodora, which is similar to many previous reports in other Citrus

species.

The addition of citric acid to the medium resulted in the best quality and highest

number of somatic embryos from callus proliferated through liquid culture in

C. inodora. This is the first report of the promotive affect of citric acid on embryo

formation in the Citrus genus.

Cryopreservation of encapsulated C. inodora embryogenic callus gave high levels

of recovery (69%) after desiccation to 25% moisture content.

A straightforward in vitro culture system was established for C. australasica,

C. garrawayi and C. inodora by nodal cuttings. This provides a system for

micropropagation, germplasm exchange, medium term storage (without subculture

257 for 10-12 months) and the development of cryopreservation protocols for shoot tip

material.

Cryopreservation of shoot tips of C. australasica, using a vitrification-based

technique was demonstrated.

Seeds of the two rare and threatened Australian Citrus species can be germinated in

vitro after cryostorage, and this could be ultilised for ongoing micropropagation.

A successful acclimatisation protocol was demonstrated for in vitro grown plants of

C. inodora and C. garrawayi.

7.2 Future research studies

These research findings highlighted some key areas for future studies in citrus. These include:

A wider study on seed coat structure and ecology in the six Australian wild Citrus

species and cultivated species, such as C. medica, would prove valuable in the

interpretation of phylogenetic relationships and seed storage behaviour in citrus to

gain better taxonomic and ecological understanding of this economically important

genus (e.g. seed dispersal mechanisms). In particular, the morphologically unique

characteristics of seed of C. garrawayi suggests that further study may help to

improve our understanding of the relationships between Australian wild Citrus

species and the cultivated species.

A review by Crane et al (2003) of intermediate seeded species showed these often

have seed oil reserves with relatively high melt end temperatures (Crane et al 2003).

In a later study, it was found that imbibition of an intermediate seeded species,

when seed oil reserves were in a solid (crystalline) state, was lethal. The current

258 study speculates that the characteristic delay in germination, observed in cultivated

citrus seeds and linked to their water uptake characteristics (Soetinsa et al 1985), is

an adaptive response to prevent germination during cold (or dry) periods. That is at

lower temperatures, close to the lipid end melt, seeds may have developed

mechanisms to reduce their water uptake that could otherwise lead to lethal injury.

Although tentative, the physiological responses, i.e. lack of germination at low

temperatures close to the lipid melt end of Australian wild Citrus species, supports

this speculation and warrants further investigations.

Further study of the comparative thermal properties of seed oils and fatty acid

composition on a wider range of Australian wild Citrus species (e.g. most cold

tolerant species C. glauca) and cultivated species should prove useful in

understanding the physiological responses of seeds to cryopreservation in the Citrus

genus.

The seeds in this study were sourced from ex situ cultivated plants grown under

environmental conditions that may differ from the natural growing conditions in

situ. Thus, both natural distribution (origin) and the environmental conditions

during seed development and dispersal (ex situ) may have influenced the observed

seed characteristics. Hence, further investigations on seeds harvested in situ would

be valuable to study the contribution of growth conditions (ex situ and in situ) on

seed characteristics. However, such studies are difficult using seed harvested from

plants growing in their natural range (in situ) as seed supply is low (especially

C. inodora) and erratic fruiting occurs (pers comm. P. Forster 2007). Sites of natural

distribution are also sometimes very difficult to access (e.g. Far N QLD populations

of C. garrawayi).

259 The cryopreservation of embryogenic callus of C. inodora resulted in high levels of

recovery. However, morphological abnormalities of embryos derived from indirect

embryogenesis and asynchronous development requires further study and

optimisation of the in vitro system to establish a reliable synchronous system.

Citric acid appears to normalise proembryo production and histodifferentiation in

C. inodora and further studies are needed to confirm these findings. This could

include histological examination, as well as investigation of this phenomenon in

cultivated polyembryonic Citrus species to ascertain if exogenous citric acid has a

promotive affect on somatic embryogenesis throughout the genus.

The lack of reproducibly and the relatively low survival (41%) of C. australasica

shoot-tips post cryopreservation means optimistaion is required (e.g. physiological

conditioning) to improve the efficiency of the vitrification-based protocol

demonstrated in this study.

7.3 Conservation options in Australian wild Citrus

Ex situ collections are intended to reduce population extinction risk, preserve genetic diversity, and provide propagules for restoration and recovery programs (Havens et al 2004 p.454). Understanding of the ecology, biology and conservation genetics (e.g. population dynamics) is needed for the effective development and application of ex situ conservation techniques to maintain genetic diversity (i.e. low viability loss and genetic drift) for ex situ collections. A large knowledge deficit exists in the majority of wild taxa about most aspects relevant to the collection and security of in situ plant biodiversity, including complementary conservation strategies such as ex situ. Ex situ conservation of

260 germplasm is ‘particularly appropriate for the conservation of crops and their wild relatives’ (Englemann and Engels 2002).

Citrus is a crop of worldwide economic importance that has been historically vulnerable to genetic erosion from hybridisation (domestication over hundred’s of years), serious diseases, pests and pathogens, as well as land clearing. The urgent need for in situ and ex situ conservation of existing wild biodiversity is widely recognised as important to humanity for both conservation and development of crop diversity. The research findings of the present study on seed biology and in vitro culture of Australian wild Citrus contributes significantly to future efforts to conserve this valuable germplasm by providing alternative options such as cryopreservation and micropropagation. Previously, storage of germplasm of Australian wild Citrus species was limited to medium (field collections) and short term

(4ºC for one year, M. Smith pers comm.) techniques with little capacity for mass propagation. The rich Citrus diversity endemic to Australia, especially its rare and threatened species, are a priority for ex situ conservation under target 8 and 9 of the Global

Strategy for Plant Conservation (GSPC), to conserve 60 per cent of threatened plant species in ex situ collections (Target 8) and 70 per cent of the genetic diversity of crops and other socio-economically valuable plant species (Target 9).

Table 7.1 summerises the options that are available for the conservation of Australia wild Citrus species. Cryopreservation has been demonstrated as a potential strategy for long-term storage of seed in many cultivated Citrus species (Mumford and Grout 1979,

Normah and Siti Dewi Serimala 1997, Cho et al 2002a, Lambardi et al 2004, Hor et al.

2005) and is recommended in this study for the Australian wild species, C. australasica,

C. inodora and C. garrawayi. This present study finds cryopreservation as the safest option,

261 even in the desiccation tolerant species (C. australasica and C. inodora), as storage at standard temperature of -20ºC is within or close to the seed oil thermal transitions of these oily seeded species and thus may lead to seed deterioration. Walters et al (2004) predict a half-life of 3400 years for lettuce seeds stored by cryopreservation (6.5%MC). Pritchard

(2004) estimates comparable storage of lettuce seeds at -18ºC is 75 times less than this at between 46-70 years. Thus, cryopreservation also has the clear advantage of extending the longevity of stored seeds.

Cryobiology has become an important scientific discipline for the conservation of wild plant diversity (Touchell, 2000). Cryopreservation was shown to be an ex situ conservation option for a horticulturally significant rare and endangered native taxa of

Western Australian (Tuner et al 2000b, 2001). This study reports for the first time cryopreservation as a viable option for socio-economically important rare and threatened species of Queensland.

Seed supply in Australian wild Citrus species can be limited and erratic. This means that the in vitro techniques (slow growth and cryopreservation) reported in this study are needed as complementary conservation strategies for medium and long-term ex situ germplasm storage. The development of an in vitro culture system in wild Australian Citrus species facilitates cryopreservation storage (e.g. shoot tips) but also provide a useful system for plant multiplication (e.g. for horticultural and restoration purposes). Furthermore, in vitro culture is an ideal approach for germplasm exchange of uncontaminated (e.g. viruses and bacteria) material in Citrus.

262 Table 7.1 Summary of in situ and ex situ conservation strategies for C. australasica, C. inodora and C. garrawayi Material Method Requirements Applicable Duration

In situ Whole Protection of wild Existing legislation for protection + Long term plant/population/ populations in of rare and vulnerable species. habitat natural habitat Research needed on genetic characterization, ecology and biology of wild populations and need for implementation of complementary ex situ germplasm collections. Ex situ Whole plants Field gene bank Limited material currently held in + Medium- field collections nationally and term internationally. Plantlets Micropropagation High rates of root initiation of + Medium and slow growth nodal cuttings from seedlings and term minimal maintenance with 10-12 months subculture regime Seed Cryopreservation Viable option with high recovery + Long term rates

Shoot -tip Cryopreservation Plantlet recovery after +? Long term cryopreserved using vitrification based protocol, but efficiency needs improvement. Embryogenic Cryopreservation High rates of recovery and growth - Long term callus of callus post cryopreservation, (C. inodora) but erratic and abnormal somatic embryo formation. Further research on optimasation of embryogenesis system needed- not recommended for germplasm storage. 263 Australian wild Citrus diversity is best conserved by utilizing strategies that include a combination of both in situ and ex situ approaches and these could include:

Protection and management of in situ wild populations

Cultivation as commercial horticultural crops (raise awareness of north QLD species)

Field germplasm collections and reference species in botanic gardens

Ex situ storage of plant material long term by cryopreservation: o Seed o Shoot tips (further optimisation required)

In vitro storage through micropropagation (also facilitating multiplication of germplasm for horticultural purpose or restoration programs) In conclusion, micropropagation, in vitro storage and cryopreservation are now real options for Australian wild Citrus species, allowing the development for the first time of a comprehensive and effective ex situ conservation strategy for Australian wild Citrus. Long- term conservation of germplasm of these species is important to ensure future access to genetic material for plant improvement projects, as well as regeneration and restoration programs. However, there are still limitations to the implementation of effective ex situ conservation strategy by alternate storage techniques and these include (i) the need for sustainable funding and infrastructure for the ex situ storage of non-orthodox seeded species, such as Australian wild Citrus, by non conventional storage techniques (i.e. cryopreservation) and (ii) the lack of knowledge of in situ genetic diversity and population dynamics of these species to inform effective maintenance of diversity. In order to address these limitations, further investment and study is needed for Australian wild Citrus species as well as for other non-orthodox seeded tropical fruits of Australia. Education and awareness programs on the unique tropical fruits of Australia and their conservation needs are important for the support and development of any future research and conservation endeavours.

264

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APPENDIX

302 APPENDIX 1. RESEARCH DISSEMINATION

Refereed journal publications

1. Hamilton, K.N., S.E. Ashmore, R.A. Drew and H.W. Pritchard. (2007) Seed morphology and ultrastruture in Citrus garrawayi (Rutaceae) in relation to germinability. Australian Journal of Botany. 55 (6): in press

2. Hamilton, K.N., S.E. Ashmore & R.A. Drew. Citric acid improves embryo formation from embryogenic callus of a monoembryonic wild Citrus species (Citrus inodora). In Vitro Cellular and Developmental Biology – Plant (submitted to journal)

Refereed conference publications

3. Hamilton, K.N., S.E. Ashmore & R.A. Drew. (2005) Investigations on desiccation and freezing tolerance of Citrus australasica seed for ex situ conservation. In ‘Proceedings of the Fifth Australian Workshop on Native Seed Biology’. Brisbane, Queensland. 31-23 June 2004. (eds. Adkins, S.W., Ainsley, P.J., Bellairs, S.M., Coates, D.J. and Bell, L.C.). ACMER: Brisbane, 157-161

4. Hamilton, K.N., S.E. Ashmore, & R.A. Drew (2005) Development of conservation strategies for Citrus species of importance to Australia. In: ‘Proceedings of the International Symposium on Harnessing the Potential of Horticulture in the Asian- Pacific Region’. (ed. Drew, R.A.). Acta Horticulturae, 694: 111-115

Conference poster presentations

5. Hamilton, K.N., S.E. Ashmore & R.A. Drew. Seed conservation studies in rare and threatened wild relatives of citrus in Australia. First International Conference on Crop Wild Relative Conservation and Use, September 2005, Sicily ITALY

6. Hamilton, K.N., S.E Ashmore. & R.A. Drew. Investigations of ex situ conservation of Australian Citrus species: seed storage, embryogenesis and cryopreservation options. Eighth International Workshop on Seeds, 8-13 May 2005, Brisbane AUSTRALIA

7. Hamilton, K.N., R.A. Drew & S.E. Ashmore. Development of ex situ conservation techniques for Citrus inodora: a threatened rainforest species of northern Queensland. International Symposium on Biotechnology of Temperate Fruit Crops and Tropical Species, October 10-14 2005, Daytona Beach, Florida USA

303 In addition, research findings were disseminated via research meetings as well as to local and national public audiences. These included:

ABC Radio, 2007, SE Queensland

The Sunday Mail (Courier Mail), 2007, Queensland

Discovery (Griffith University science magazine), 2007, Brisbane

Australian Millennium Seed Bank Partners Science Meeting, February 2007, Sydney

Centre for Horticultural and Forestry Research Annual Meeting, 2006, Bribie Island (awarded best student presentation)

Queensland Subtropical Fruit Club (Guest speaker), 2006, Brisbane

Seed Conservation Department, Millennium Seed Bank (Royal Botanic Gardens, Kew), Research Meeting and Newsletter, October 2005 and July 2006, Wakehurst Place, UK

National science and education children’s television program (‘Totally Wild’), 2005

First Annual Meeting of the Bioversity-ACIAR project ‘Development of Advanced Technologies for Germplasm Conservation of Tropical Fruit Species’, December 2003, Bangkok, Thailand

304