Seed Storage and Longevity of Australian Plant Species

Home , Corymbia eremaea, Eucalyptus incrassata, Eucalyptus orbifolia, Eucalyptus salmonophloia, Geleznowia, Hakea dohertyi

SEED STORAGE AND LONGEVITY OF AUSTRALIAN PLANT SPECIES

Andrew David Crawford

BSc.Hort (Hons) University of Western Australia

This thesis is presented for the degree of

Doctor of Philosophy

The University of Western Australia

School of Plant Biology

Faculty of Science

2013

Summary

Ex situ seed storage is an important tool helping to conserve the world’s plant biodiversity. The basic principles of long-term seed storage, reducing storage temperature and seed moisture content to increase seed longevity, have been known for a long time. These principles have been applied to develop genebank standards for the storage of plant species with orthodox seeds. Developed predominantly for the storage of agricultural species, they have increasingly been applied to a wide range of wild plants. Seeds of Australian species have been collected and stored since the earliest days of colonisation, however there is still scant information relating to the storability of Australian species. Despite the widespread adoption of the genebank standards at seed banks throughout Australia, there has been a suggestion that the recommended storage conditions may not be suitable for all Australian species.

The Western Australian Department of Environment’s Threatened Flora Seed Centre holds seed collections of the State’s conservation significant plants in long-term storage. These collections provide a representative sample of the flora of not only Western Australia, but Australia as a whole. The suitability of applying international standards for long-term seed storage to the Australian flora was examined by reviewing seed-storage data from the Western Australian Threatened Flora Seed Centre. A high proportion of collections, representative of some of the most common genera in Australia, maintained viability in the short (< 5 years) and medium (5–12 years) term. Declines in germination were evident for a small number of collections, however many of the declines were collection-specific and other collections of the same taxon did not decline. The longevity of two contrasting Australian species, Eucalyptus erythrocorys and Xanthorrhoea preissii, can be modelled using the improved seed viability equation using a one-step method. The viability constants obtained were KE = 8.81, CW = 4.97,

CH = 0.0412 and CQ = 0.000379 for E. erythrocorys and KE = 8.77, CW = 5.29, CH =

0.0382 and CQ = 0.000473 for X. preissii. The universal temperature constants could not be used without a significant increase in error. The storage behaviour of these two Australian species is in keeping with that of orthodox species from around the world. Predictions are that E. erythrocorys will be long-lived under genebank conditions, whilst X. preissii would be moderately long-lived.

The seed viability equation assumes that the standard deviation of seed deaths in time () is constant and hence KE, CW, CH and CQ are constant within a species, with changes in  the product of changes to the storage temperature and/or seed moisture content. This assumption was examined in relation to serotiny in three Banksia species (B. attenuata, B. hookeriana and B. leptophylla subsp. melletica) and the data supported this hypothesis. Seeds collected from older cones generally had lower initial viability than seeds from younger, mature cones. The only exception was the youngest cohort of seeds (1 year old) collected from Banksia hookeriana which had a lower initial viability than the next oldest cohort (2-3 years old), likely due to an immature fraction in the collection due to the long flowering period of the species. Secondly, seeds from cones of all ages could be attributed a single value for  within a species and contrary to expectations, all species in the study could be attributed a common value of .

This thesis has shown that the improved seed viability equation can be applied to Australian plants. One of the assumptions of this model, that  is constant within a species, has been shown to hold when applied to seeds of different ages extracted obtained from cones from three Banksia species. The findings illustrate the successful application of current long-term genebank storage standards to the seeds of Australian plants.

Table of contents Summary ...... i

Table of contents ...... iii

List of Figures ...... v

List of Tables...... vii

Acknowledgements ...... ix

Declaration ...... x

CHAPTER ONE - General Introduction ...... 1

Thesis structure ...... 2 Publications from this thesis ...... 2 General introduction ...... 3 CHAPTER TWO - Literature Review ...... 5

Plant conservation ...... 6 Plant conservation in Western Australia ...... 6 Ex situ seed conservation ...... 7 Seed viability testing ...... 8 Seed dormancy ...... 10 Seed germination ...... 12 Serotiny ...... 14 The principles of seed storage ...... 15 The seed viability equations ...... 15 Genebank standards ...... 18 Seed conservation in Australia ...... 19 Research objectives...... 20 CHAPTER THREE - Analysis of seed-bank data confirms suitability of international seed-storage standards for the Australian flora...... 21

CHAPTER FOUR - One-step fitting of seed viability constants for two Australian plant species ...... 34

CHAPTER FIVE - The influence of cone age on the relative longevity of Banksia seeds...... 45

CHAPTER SIX - General Discussion ...... 53

REFERENCES ...... 60 iii

APPENDIX ONE - Poster: Successful storage of seed from threatened west Australian plants ...... 75

List of Figures CHAPTER FOUR

Figure 1 Moisture sorption isotherms for (a) Eucalyptus erythrocorys page 38 and (b) Xanthorrhoea preissii determined at 45°C. FW, fresh weight. Figure 2 Relationship between seed moisture content (% fresh weight, page 39 FW) and seed longevity (σ, days) for individual seed survival curves of (a) Eucalyptus erythrocorys and (b) Xanthorrhoea preissii seeds in hermetic storage at 15 (■), 25 (□), 35 (●), 45 (○), 50 (▲), 60 (△) or 70°C (▼). The lines represent the

regression where the slope (CW) has been constrained to a common value. Figure 3 Seed survival data for Eucalyptus erythrocorys seeds stored at page 40 (a) 15, (b) 25, (c) 45, (d) 50 and (e) 60°C and the moisture contents (% fresh weight, FW) shown. Seed survival curves have been plotted using parameters derived from the one-step model fitting of the seed viability equation (Table 3). Figure 4 Seed survival data for Xanthorrhoea preissii seeds stored at page 41 (a) 15, (b) 25, (c) 35, (d) 45, (e) 50, (f) 60 and (g) 70°C and the moisture contents (% fresh weight, FW) shown. Seed survival curves have been plotted using parameters derived from the one-step model fitting of the seed viability equation (Table 3).

CHAPTER FIVE

Figure 1 Number of closed or open follicles per cone (mean ± s.e.) for page 48 three species of Banksia (A, B. leptophylla var. melletica; B, B. attenuata; and C, B. hookeriana) in relation to cone age class. Different letters indicate significant differences in total follicles (P = 0.05). Figure 2 Proportional fate of seeds (mean ± s.e.) in relation to cone page 49 age class for three Banksia species (A, B. leptophylla var. melletica; B, B. attenuata; and C, B. hookeriana). Seeds were counted as shed if the follicles were open.

Figure 3 Seed survival curves for various cone age classes of three page 50 Banksia species (A, B. leptophylla var. melletica; B, B. attenuata; and C, B. hookeriana) aged at 50°C and 63% relative humidity. Parameters for the lines fitted by the use of probit analysis are given in Table 1 (Model 1).

Figure 4 Viability (Ki; expressed as a percentage) calculated from page 51 seed ageing analysis of filled seeds in relation to cone age (years) for three Banksia species.

List of Tables CHAPTER THREE

Table 1 Final germination (%), mean time to germination (MTG) page 24 and viability (%) of species before (initial) and after (re-test) medium-term storage (5–12 years) under genebank conditions for which no decline in germination was measured on the basis of chi-square analysis Table 2 Final germination (%), mean time to germination (MTG) page 26 and viability (%) of species before (initial) and after (re-test) short-term storage (<5 years) under genebank conditions for which no decline in germination was measured on the basis of chi-square analysis Table 3 Germination (%), mean time to germination (MTG) and page 30 viability (%) of species before (initial) and after (re-test) medium-term storage (5–12 years) under genebank conditions for which a decline in germination was seen on the basis of chi-square analysis Table 4 Ranked lists of the 20 most common genera, as measured by page 31 number of species or taxa for Australian flora (Crisp et al. 1999), Western Australian flora (Western Australian Herbarium 2005), Western Australian declared rare and priority flora (Atkins 2005), Western Australian commercial collections (Department of Conservation and Land Management 2005).

CHAPTER FOUR

Table 1 Ageing conditions (temperature, moisture content, sample page 37 number and estimates (s.e. in parentheses) of initial viability

(Ki) and seed longevity () derived from individual survival curves of Eucalyptus erythrocorys seeds. Table 2 Ageing conditions (temperature, moisture content, sample page 38 number and estimates (s.e. in parentheses) of initial viability

(Ki) and seed longevity () derived from individual survival

vii

curves of Xanthorrhoea preissii seeds.

Table 3 One-step estimation of the viability equation parameters (s.e. page 42 in parentheses) for two species, Eucalyptus erythrocorys and Xanthorrhoea preissii, using either independent estimates of

the temperature constants (CH and CQ) or fixing the

temperature constants to the universal values (CH = 0.0329;

CQ = 0.000478).

Table 4 Estimates of  (years) and p85 (years) of seeds of Eucalyptus page 42 erythrocorys and Xanthorrhoea preissii using the seed viability equation, with equation constants derived from a one-step fitting of viability data (Table 3).

CHAPTER FIVE

Table 1 Results of probit analysis of survival curves for seeds of page 51 various cone age classes from three Banksia species

viii

Acknowledgements

This thesis has been a long time in the making and my thanks go to my supervisors, Dr Kathryn Steadman, Professor Julie Plummer and Dr Robin Probert, for their support, understanding and encouragement during this period. I selected each of them as supervisors for the strengths I believed they could bring to my studies and they have exceeded my expectations.

Thanks to Anne Cochrane for the original suggestion to undertake this study and for securing the resources and support to make it happen. Thanks to the Department of Environment and Conservation, in particular the Science Division, where I have been employed for the duration of this study, for providing the time and resources which have allowed me to complete this project.

Thanks also go to the Royal Botanic Gardens, Kew and its Millennium Seed Bank Project, supported by the UK Millennium Commission, the Wellcome Trust and Orange plc. This study was undertaken as part of this international collaboration. It has been a rewarding project to be involved with. I have enjoyed my visits to the Millennium Seed Bank thanks to the warmth and friendliness of the staff and have made many good friends along the way. Special thanks to Fiona Hay for her assistance in data analysis and to John Adams for collecting the moisture sorption data when it was not possible for me to do so. Lastly, and certainly not least, I wish to thank my family for their support and understanding throughout the duration of my study. Most importantly my wife Leonie who has been there to provide encouragement throughout, as well as my children, Caitlin and Liam, who were born during the period this thesis was undertaken.

Declaration

This thesis contains published work and work prepared for publication, some of which has been co-authored. The bibliographical details of the work and where it appears in the thesis are outlined below.

 Crawford AD, Steadman KJ, Plummer JA, Cochrane A, Probert RJ. 2007. Analysis of seed-bank data confirms suitability of international seed-storage standards for the Australian flora. Australian Journal of Botany 55: 18-29. doi: 10.1071/BT06038. This paper forms Chapter 3 of this thesis. My research included germination testing as well as the compilation, analysis and interpretation of data provided by Anne Cochrane, manager of the Department of Environment and Conservation’s Threatened Flora Seed Centre. I wrote the manuscript and the other three co-authors are my supervisors who assisted with discussions of the research, its interpretation and editing.

 Crawford AD, Hay FR, Plummer, JA, Probert RJ, Steadman KJ. (2013) One- step fitting of seed viability constants for two Australian plant species, Eucalyptus erythrocorys (Myrtaceae) and Xanthorrhoea preissii (Xanthorrhoeaceae) Australian Journal of Botany. doi: 10.1071/BT12171. This paper has been accepted for publication and published online in The Australian Journal of Botany and forms Chapter 4 of this thesis. My research included the collection and processing of seeds of Eucalyptus erythrocorys and Xanthorrhoea preissii, as well as all seed ageing experimentation, analysis, interpretation and preparation of the manuscript. Seed oil content was conducted by the Western Australian Chemistry Centre and moisture sorption data was collected by staff at the Millennium Seed Bank. Dr Fiona Hay provided advice and assistance with the data analysis, and assisted with discussions of the research, its interpretation and editing. The other three co-authors are my supervisors who assisted with discussions of the research, its interpretation and editing.

 Crawford AD, Plummer JA, Probert RJ, Steadman KJ (2011) The influence of cone age on the relative longevity of Banksia seeds. Annals of Botany 107, 303- 309. doi: 10.1093/aob/mcq236. This paper forms Chapter 5 of this thesis. My research included the collection and extraction of seeds from Banksia cones, all experimental work, data analysis, x interpretation and preparation of the manuscripts. The three co-authors are my PhD supervisors who assisted with discussions of the research and editing.

Student Signature: ______

Andrew Crawford

Coordinating Supervisor Signature: ______

Professor Julie Plummer

CHAPTER ONE

General Introduction

Thesis structure

This thesis is composed of this chapter, the General Introduction, which outlines the structure of the thesis, the Literature Review, which provides background information and concludes with the hypotheses tested in this thesis. These hypotheses are examined in three research chapters; Chapter 3 examines the suitability of international seed- storage standards for the Australian flora, Chapter 4 examines one-step fitting of seed viability constants for two Australian plant species, Chapter 5 examines the influence of cone age on the relative longevity of Banksia seeds and the thesis concludes with Chapter 6, the General Discussion. Chapter 3 has been published in The Australia Journal of Botany, Chapter 4 has been submitted to the Australian Journal of Botany and Chapter 5 has been published in the Annals of Botany.

Publications from this thesis

 Journal papers

Crawford AD, Steadman KJ, Plummer JA, Cochrane A, Probert RJ. (2007) Analysis of seed-bank data confirms suitability of international seed-storage standards for the Australian flora. Australian Journal of Botany, 55: 18-29. doi: 10.1071/BT06038.

Crawford AD, Plummer JA, Probert RJ, Steadman KJ. (2011) The influence of cone age on the relative longevity of Banksia seeds. Annals of Botany, 107: 303-309. doi: doi: 10.1093/aob/mcq236.

Crawford AD, Hay FR, Plummer JA, Probert RJ, Steadman KJ (2013) One-step fitting of seed viability constants for two Australian plant species, Eucalyptus erythrocorys (Myrtaceae) and Xanthorrhoea preissii (Xanthorrhoeaceae) Australian Journal of Botany. doi: 10.1071/BT12171.

 Poster presentations

Crawford AD, Cochrane A, Steadman KJ, Plummer JA, Probert RJ (2005) Successful storage of seed from threatened west Australian plants. In '8th International workshop on seeds: germinating new ideas', 8-13th May 2005, Brisbane, p. 97

 Poster in Appendix 1

Crawford AD, Cochrane A, Steadman KJ, Plummer JA, Probert RJ (2005) Successful storage of seed from threatened west Australian plants. In 'Advances in plant conservation biology: implications for flora management and restoration: symposium', 25-27th October 2005, Kensington, p. 26

 Poster in Appendix 1

General introduction

Ex situ seed storage has become an important tool for helping conserve the genetic diversity of plant species around the world. Originally developed for storing seeds of agricultural species, the storage conditions recommended in germplasm conservation guidelines have increasingly been applied to the storage of wild plant species. During the last decade there has been a large increase in the number of facilities collecting and storing seeds of wild plant species long-term thanks largely to international conservation efforts such as the Millennium Seed Bank Project.

Despite genebank standards being introduced in the mid 1990’s, and widely taken up throughout the world, there is still relatively little information available about the expected longevity of species held under these conditions. Detailed studies enabling the modelling of predicted seed longevity under a range of storage conditions are still scarce, particularly in relation to Australian plants. It has also been suggested that the recommended storage conditions may not be suitable for all Australian species, therefore, the determination of storage conditions should be conducted on a species by species basis.

Western Australia possesses a rich and diverse flora comprising over 12000 native plant taxa, about half Australia’s total flora. Associated with this high level of diversity is a high number of threatened species. This combination of high species diversity, with a high level of threat, has resulted in the South West Botanical Province of Western Australia being listed as one of the world’s 34 biodiversity hotspots. Currently in Western Australia over 400 species are gazetted as being threatened with extinction. The Department of Environment and Conservation is the agency responsible for the management of Western Australia’s flora and in 1993 it established the Threatened Flora Seed Centre. This facility was set up for long-term seed storage of conservation significant plant species, utilising storage conditions consistent with international guidelines.

The aim of this thesis was to improve the knowledge and understanding of seed storage in relation to Australian plant species. Specifically the study examines three main topics; the storage performance of Western Australian species currently held under genebank conditions, the application of the improved seed viability equation to Australian species and testing one of the assumptions of the viability equation, that the standard deviation of seed deaths in time () is constant within a species.

CHAPTER TWO

Literature Review

Plant conservation

The Earth’s biodiversity is being lost at an unprecedented rate due to human activities that degrade or encroach on habitats, increase pollution, and contribute to climate change (Zedan, 2005). Of the estimated 300,000 flowering plant species in the world (Pitman and Jorgenson, 2002; Paton et al., 2008) it is thought that between 20 and 40% are in danger of extinction (Pitman and Jorgenson, 2002). Preventing the loss of plant biodiversity is important for a number of reasons including direct values such as the economic value of plants now and in the future and indirect values such as the ecosystem services provided by plants. There is also an argument for the conservation of plants based on their aesthetic and intrinsic values (Given, 1994). Whilst in situ conservation measures should always be the priority of conservation actions, ex situ conservation can complement and assist these actions (Glowka et al., 1994).

Plant conservation in Western Australia

Western Australia possesses a rich and diverse flora comprising over 12000 native taxa (Western Australian Herbarium, 2012), with most species (60%) being endemic to the state (Beard et al., 2000). The south west region is the most diverse, containing over half the state’s species, half of which are endemic to the region (Hopper and Gioia, 2004). Associated with this species richness is a high number of conservation significant species; currently Western Australia has over 3000 taxa listed as rare, threatened or poorly known (Smith, 2012), with a further 14 presumed to be extinct (Marmion, 2012). Of these conservation taxa, 405 are protected under state legislation (Marmion, 2012) with 148 taxa listed as critically endangered (Smith, 2012). The high numbers of threatened species, more than other Australian states and most countries (Hopper and Gioia, 2004), in combination with high species endemism, has resulted in the south western botanical region being listed as one of the world’s 34 biodiversity hotspots (Mittermeier et al., 2004).

In order to combat the potential loss of so many species, a range of recovery actions are undertaken including translocations (eg. Monks and Coates, 2002; Cochrane et al., 2007), morphological and genetic studies to aid taxonomy (eg. Coates and

Hamley, 1999; Wege and Coates, 2007), and disease susceptibility (eg. Shearer et al., 2004; Shearer et al., 2010) and prevention studies (eg. Barrett et al., 2003; Shearer and Crane, 2012). One conservation action which underpins many of these conservation actions is ex situ storage of seeds.

Ex situ seed conservation

An ex situ seed storage facility aims to store and maintain viable and genetically representative collections of species at the population, individual and allelic level (Offord and Makinson, 2009). These seed banks aim to act as insurance policies against extinction of populations or species in the wild (Linington and Pritchard, 2001). The importance of ex situ plant conservation is illustrated by its inclusion in international conservation strategies, such as Article 9 of the Convention on Biological Diversity (Williams et al., 2003) and the resulting Target 8 of the Global Strategy for Plant Conservation (James, 2004)

In order for seed banks to be able to fulfil this promise, they need to meet the following criteria (Crawford and Monks, 2009):

1. the seeds must be genetically representative of the species, 2. the seed quantities must be adequate for their intended purpose, and 3. the seeds must be viable and germinable. Guidelines for sampling the genetic diversity of threatened plant species have been developed that take into account both intra- and inter-population variation (Guerrant et al., 2004) and now form the basis of collection guidelines for ex situ collections of non-domesticated species in Australia and elsewhere (eg. Way, 2003; Cochrane et al., 2009). According to Cochrane et al (2009) a conservation seed collection should be of sufficient size to:

 conduct initial viability or germination tests,  monitor the viability of the collection over time,  duplicate the collection with another seed bank for safekeeping,  translocate or reintroduce viable population/s into the wild, and  provide sufficient material for other intended or potential other uses.

A collection size of 10,000 to 20,000 seeds is the recommended target to achieve these goals (Way, 2003; Cochrane et al., 2009), however some uses, such as habitat restoration, will require far greater seed numbers. The reality is that for many non- domesticated species, particularly those that are rare or threatened, collections often fall short of these targets and ongoing collection of the species may be required to ensure adequate material is conserved for future recovery work (Cochrane et al., 2007).

Seed viability testing

The aim of a genebank is to maintain the viability of seed collections above a minimum standard (FAO/IPGRI, 1994). In order to ensure this is the case, the viability of a seed collection needs to be determined prior to storage, and then checked periodically through time to ensure it is being maintained. A range of tests are available for determining the viability of seeds, the most common being by seed x-ray, cut test, tetrazolium test, excised embryo test or seed germination (Gosling, 2003; Martyn et al., 2009; ISTA, 2012).

A seed x-ray allows internal examination to provide a quick method of checking whether seeds are filled. Seeds that are completely filled are potentially viable while seeds that are empty, insect- or physically-damaged are considered non-viable. Advantages of seed x-rays are that it is a quick and non-destructive method of assessing seed fill (Gosling, 2003; ISTA, 2012), whilst disadvantages are that it only gives an indication of the potential viability of a seed lot (Gosling, 2003) and the accuracy of the technique is reliant on the ability of the user to interpret the results correctly (Martyn et al., 2009).

A cut test is another simple method for assessing seed fill, to gain an indication of the potential viability of a seed collection. Using this technique, seeds are cut in half and the appearance of the internal tissues examined. Seeds are classed as potentially viable if they contain all tissues required for germination, and these tissues appear to be intact, firm and fresh (Gosling, 2003). A cut test can be used by itself on a sample of seeds or conducted at the conclusion of a germination test to determine the potential viability of ungerminated seeds (ISTA, 2012). Advantages of the cut test are that it is quick, taking only minutes to hours, and simple. Disadvantages are that it is destructive,

8 it only gives an indication of viability, and categorisation of the viability is open to interpretation (Gosling, 2003).

The use of biochemical stains is another method by which the viability of seeds can be assessed, the most common being 2,3,5-triphenyl tetrazolium chloride or bromide, known more simply as a TZ test (Gosling, 2003; ISTA, 2012). The principle of the TZ test is that when the colourless TZ solution is absorbed by cells, it will react, in living cells, with dehydrogenase enzymes produced through the process of respiration. The result of this reaction is a red, non-diffusible substance, triphenyl formazan. The living parts of seeds will therefore be stained red, whilst non-viable portions will be colourless (ISTA, 2012). Whilst simple in theory, the application of the TZ test in practice can be problematic. Well documented procedures have been developed for a range of species (eg. Paynter and Dixon, 1990; Bell et al., 1995; Ooi et al., 2004; ISTA, 2012), but where procedures have not been developed the application of sub-optimal staining treatments may lead to erroneous estimates of seed viability (Bell et al., 1995; Ooi et al., 2004). Other disadvantages of the TZ test are that it is destructive and can be very labour intensive to apply. Also, as with x-ray and cut tests, interpretation of results can be subjective. Advantages of the TZ test are that it can provide results in a matter of days, so is relatively quick, and can be used on species with high levels of seed dormancy (Gosling, 2003).

An excised embryo test is used to determine the viability of seeds which are slow to germinate or exhibit seed dormancy requiring long periods (months) to overcome. The test involves the manual excision of embryos from the seed, then incubating them under conditions suitable for growth (Gosling, 2003; ISTA, 2012). The main advantage of this technique is that it can provide a more rapid estimate of viability than might be possible if a germination test was conducted (Gosling, 2003) or can elicit germination where standard germination tests prove unsuccessful (eg. Paynter and Dixon, 1990; Plummer et al., 1995). Another advantage, is that although the technique is destructive, the germinants can potentially be used for other purposes subsequent to the test such as research or translocations (Martyn et al., 2009). A major disadvantage with the technique is the time involved and the high level of skill required to perform the test (Gosling, 2003).

Arguably the best method for determining the viability of a seed collection is through the use of a germination test. Whilst viability tests aim to determine the

9 proportion of a collection that is either alive or dead, a germination test determines the potential of a collection to produce normal healthy seedlings (Gosling, 2003). Where the optimal seed germination conditions for a species are known and seeds are non- dormant, the germinability of a seed lot will provide a good estimate of the viability of that collection. Conversely, sub-optimal germination conditions or seed dormancy can lead to an underestimation of seed viability (Martyn et al., 2009). As the ability to produce healthy plants from stored seeds is the ultimate purpose of genebanks, the germination test provides a realistic measure, given the knowledge of a species’ germination/dormancy breaking requirements at a point in time.

For most species stored in agricultural, horticultural or silvicultural genebanks, standard germination treatments have been developed (e.g. Gunn 2001; International Seed Testing Association 2005). Unfortunately, to gain this information large numbers of seeds are needed to undertake the required germination/dormancy studies. When dealing with collections of rare plants, seed quantity is often a limiting factor. Information from related taxa may be useful in determining treatments to overcome dormancy and optimise germination; however published information on the germination of many Australian genera is limited.

Seed dormancy

Dormancy is a seed characteristic, the degree of which defines the conditions that are required for seed germination. The wider the range of conditions under which a seed will germinate the lower the level of dormancy (Vleeshouwers et al., 1995). Five categories of seed dormancy are currently recognised: physical, physiological, morphological, morphophysiological and combinational dormancy (Baskin and Baskin, 2004b). A dichotomous key has been developed allowing the classification of seed dormancy types (Baskin and Baskin, 2004a).

Physical dormancy is caused by the seed or fruit coat being impermeable to water and is maintained until the seed/fruit coat becomes permeable (Baskin et al., 2000). Physical dormancy has been recorded within 15 plant families from around the world (Baskin and Baskin, 2004a) many of which are represented in the Australian flora eg. Fabaceae (Bell, 1999), Malvaceae (Wilkins et al., 2009), Rhamnaceae (Turner et al.,

2005) and Sapindaceae (Cook et al., 2008). In nature physical dormancy may be broken by environmental factors such as high, fluctuating temperatures or fire. Physical dormancy can also be broken by a range of artificial means including scarification (physical or chemical), wet heat or dry heat (Baskin and Baskin, 2001). The problem with many of these treatments is that the optimal treatment can be species specific. Without prior testing on a species by species basis dormancy breaking treatments may be sub-optimal (eg. Crawford et al., 2003). Manual nicking of the seed coat using a scalpel or other precision instrument provides a reliable means of breaking physical dormancy and is arguably the best method where repeatable results are required. The disadvantage of this technique is that it can very time consuming compared to other treatments (Baskin and Baskin, 2001).

Physiological dormancy is the most common type of seed dormancy (Baskin and Baskin, 2004a) and is well represented in the Australian flora (eg. Baker et al., 2005; Ainsley et al., 2008; Hoyle et al., 2008a). Seeds exhibiting physiological dormancy are prevented from germinating until a chemical change takes place within the seed (Fenner and Thompson, 2005). This change is regulated by temperature and seed moisture content (Benech-Arnold et al., 2000). Physiological dormancy has three levels: non- deep, intermediate and deep. Seeds with non-deep physiological dormancy produce normal seedlings if the embryos are excised from the seed. Non-deep physiological dormancy can be broken by the application of gibberellic acid, or depending on the species, by scarification of the seed coat. The dormancy can also be broken by after- ripening in dry storage or by cold (c. 0-10°C) or warm (15°C) stratification. The embryos of seeds with intermediate physiological dormancy will also produce normal seedlings when excised and gibberellic acid can promote germination of some, but not all species exhibiting this type of dormancy. Species with intermediate physiological dormancy require 2-3 months of cold stratification to break the dormancy. Dry after- ripening can shorten the cold stratification period. Seeds with deep physiological dormancy produce abnormal seedlings if the embryos are excised from the seed and gibberellic acid does not promote germination. Deep physiological dormancy requires at least 3-4 months of warm or cold stratification before germination can take place (Baskin and Baskin, 2004b).

Seeds with morphological dormancy have small, but differentiated embryos, and require a period of time for the embryo to fully develop before germination can occur. Seeds with dormancy of this type require no special pre-treatment for germination to 11 occur. The dormancy period is the time taken from imbibition to radicle emergence, usually within a month (Baskin and Baskin, 2001). Morphological dormancy has been recorded for a number of plants families in the Australian flora (eg. Ooi, 2007).

Morphophysiological dormancy is a combination of both morphological and physiological dormancy (Baskin and Baskin, 2001). Morphophysiological dormancy has been recorded for a range of plant families in Australia (eg. Baker et al., 2005; Ooi et al., 2006; Ooi, 2007; Hidayati et al., 2012)

A final dormancy class, combinational, is a combination of both physical and physiological dormancy (Baskin and Baskin, 2004b). This dormancy type has been reported in families present in the Australian flora (Baskin and Baskin, 2001; Turner et al., 2006).

Seed germination

Seed germination consists of the events that occur between a seed imbibing water and the emergence of the embryonic axis through its surrounding structures (Bewley, 2006). In practice, there a range of ways in which germination is measured ranging from radicle emergence (Steadman et al., 2003; Ooi et al., 2006), often by a defined amount (Baker et al., 2005; Hoyle et al., 2008b), to complete emergence of both roots and shoots (Northam and Callihan, 1994; ISTA, 2012).

In order for germination to occur there are three basic requirements; water, oxygen, and a suitable temperature. For some species light may or may not be needed for germination to occur (Baskin and Baskin, 2001). In addition to these requirements for germination there are a range of environmental chemicals that can stimulate germination such as ethylene (Baskin and Baskin, 2001) and smoke (van Staden et al., 2000).

The first requirement for germination is a suitable seed moisture content, which is species specific (Baskin and Baskin, 2001). For laboratory testing, a range of moistened substrates such as paper, sand or organic matter (ISTA, 2012) or a medium containing water such as agar (Terry et al., 2003) can be used to provide seeds with adequate moisture for germination.

Oxygen is required by germinating seeds for respiration. Whilst not normally a limiting factor in laboratory testing of seed germination, some seeds may have covering structures that may prevent or limit the amount of oxygen getting to the embryo (Baskin and Baskin, 2001).

The precise temperatures required for seed germination will be species specific, with each species having maxima and minima, above or below which no germination will occur (Côme and Corbineau, 2006). The range of temperatures over which a species will germinate will also be affected by the dormancy status of the seed (Vleeshouwers et al., 1995). Whilst alternating temperatures can often produce better germination than constant ones (Baskin and Baskin, 2001), constant temperatures are often used successfully for laboratory germination (eg. Bell, 1999; Gunn, 2001; ISTA, 2012).

In the south west of Western Australia, a region with a Mediterranean climate, germination temperatures of between 10 and 20°C have been found to be optimal for most species. These temperatures coincide with those experienced during the onset of reliable rainfall in winter (Bell, 1999) and is similar to findings for species from Mediterranean environments in other parts of the world (eg. Thanos et al., 1995; Dousii and Thanos, 2002). Species specific optima are dependent on where the species are found and their specific microhabitats (Bell et al., 1993), however a germination temperature of 15°C has proved suitable for a wide range of species from the Western Australian flora (Cochrane et al., 2002), in particular for Banksia attenuata, B. leptophylla (Cowling and Lamont, 1987), B. hookeriana (Enright and Lamont, 1989; Barrett et al., 2005), Eucalyptus erythrocorys, and Xanthorrhoea preissii (Bell et al., 1995).

Many species germinate equally well in light or in the dark (Baskin and Baskin, 1988). There are however some species that germinate better in the light and others that do better in the dark (Bell et al., 1995; Bell, 1999; Baskin and Baskin, 2001). For routine germination testing in the laboratory, it is recommended that seeds are exposed to light, unless there is evidence that it is inhibitory, as it allows for better development of seedlings (ISTA, 2012). Where artificial lights are used, the photoperiod is often alternating (Gunn, 2001; Terry et al., 2003; Turner and Merritt, 2009).

For the species that will be studied in detail in this thesis (Banksia attenuata, B. hookeriana, B. leptophylla, Eucalyptus erythrocorys and Xanthorrhoea preissii), seed 13 dormancy is not an issue (Cowling and Lamont, 1987; Enright and Lamont, 1989; Bell et al., 1995; Barrett et al., 2005). For some of the other species for which seed storage performance will be examined seed dormancy may be an issue. As seed dormancy is not the focus of this study, germination treatments that will be used will be repeating treatments that have proven to be effective previously (eg. Cochrane et al., 2002), or will use treatments that have proven to be more effective than those previously used (Department of Environment and Conservation, unpublished data). This ‘best bet’ approach of germination testing, using current published and unpublished data to guide the selection of germination treatments, can provide reasonable results in many instances (eg. Cochrane et al., 2002; Godefroid et al., 2010).

Serotiny

Serotiny is a plant adaptation where seeds are retained in the canopy of a plant for a prolonged period, rather than either being dispersed and germinating, or forming a soil seed bank. Serotinous plants therefore retain seeds produced in a number of different years as an aerial seed bank (Lamont et al., 1991). Many Banksia species exhibit some degree of serotiny. An index of the degree of serotiny of banksias is calculated as the inverse slope of the linear regression of the percentage of open follicles against cone age and so is a function of both the proportion of seeds and the length of time of retention on the plant (Cowling and Lamont, 1985). Species with a degree of serotiny < 4 are considered weakly serotinous, whilst those > 20 are strongly serotinous (Lamont, 1991). The degree of serotiny is not always consistent within a species; for example, serotiny tends to decrease with increasing rainfall (Cowling and Lamont, 1985). Serotinous seeds gradually lose viability while on the plant. Decreases in seed germination with increasing cone age have been recorded for a number of serotinous Banksia species (e.g. Cowling and Lamont, 1985; Cowling et al., 1987; Enright et al., 1996; Barrett et al., 2005). The rate of viability loss with time on the plant is species specific (e.g. Cowling et al., 1987). Previous Banksia studies examining cone age and seed viability have focused on the viability of seeds at the time of collection. There is currently no information on how seeds collected from different age classes from serotinous species respond when stored under controlled conditions.

The principles of seed storage

The basic principles of long-term seed storage, reducing storage temperature and seed moisture content to increase seed longevity, have been known for a long time (eg. Crocker, 1938; Roberts, 1972). The principle of reducing seed moisture content to increase seed longevity only applies to orthodox seeds, that is, seeds that maintain viability after drying to low (c. 5%) moisture contents. Seeds which rapidly lose viability when bulk water is removed by drying are termed recalcitrant (Roberts, 1973). Many recalcitrant species, particularly those from tropical regions are also chilling sensitive and cannot be stored below a temperature of around 15°C (Pammenter and Berjak, 1999). A third class of seed storage behaviour, intermediate seeds, can tolerate partial drying to c. 10% moisture content but further drying reduces the viability of seeds (Ellis et al., 1990). In contrast to orthodox seeds, reducing the storage temperature of intermediate seeds below 0°C results in a decrease in seed viability, with tropical species showing a decline in viability below c. 10°C (Hong and Ellis, 1996). Species with recalcitrant seeds appear to be relatively uncommon in nature (Dickie and Pritchard, 2002) and are more likely to be found in aseasonal, moist habitats, such as evergreen rainforests, with the proportion of species possessing desiccation sensitive seeds declining as habitats become drier (Tweedle et al., 2003). It is thought that over 1500 plant species from Eastern Australia may possess recalcitrant seeds, with most of these coming from rainforest habitats (Ashmore et al., 2011). Information on the occurrence of recalcitrance in the seeds of Western Australian plants species is lacking.

The seed viability equations

For orthodox seeds, seed life spans are normally distributed (Roberts, 1960) and longevity can be increased in a predictable way by decreasing seed moisture content and storage temperature (Ellis and Roberts, 1980). In the early 1960s a series of three equations, the basic seed viability equations, were developed to help predict the longevity of crop seeds under constant storage conditions (Roberts, 1960). The first

15 equation described the normal distribution of seed deaths in time for the whole population:

( ̅)

( √ ) (1)

where y is the relative frequency of deaths occurring at time , ̅ is the mean viability period, and  is the standard deviation of the distribution of seed deaths in time (Roberts, 1972). The second equation showed that  is proportional to ̅ , thus:

̅ (2) where is constant for the species (Roberts, 1960). The third equation related ̅ to storage temperature and seed moisture content:

̅ (3) where m is the seed moisture content on a fresh weight basis (%), t is the storage temperature (°C) and , and are constants (Roberts, 1972).

These equations were later refined to take into account the differences in longevity that can occur between seed lots of the same species, as well as providing a better fit to data when a wide range of temperatures or seed moisture contents were considered, resulting in what is known as the improved viability equation (Ellis and

Roberts, 1980). The term Ki, the initial viability of a seed collection (probit %), was introduced to take into account differences in longevity that can occur between seed collections caused by genotype and pre-storage effects on longevity (Ellis and Roberts, 1980). The seed survival curve for a seed collection under a given set of storage conditions, when plotted as probit percentage viability against time p, produces a straight line with negative slope which can be described by 1/ (Finney, 1971). The viability of a seed collection, v (probit %) can be predicted by the equation:

v = Ki – p /  (4)

It is assumed that the relative longevity of seed collections is maintained in all storage environments and the slope (1/) is not affected by genotype nor seed quality (Ellis and Roberts, 1980).

When a wide range of temperature or seed moisture contents was considered it was found that the temperature and moisture terms from the basic viability equations 16 needed to be modified to better fit the data (Ellis and Roberts, 1980), resulting in what is known as the improved viability equation:

E W (5) where E is the theoretical value of at 1% moisture content, W is a species’ moisture constant, and and are species’ temperature constants (Ellis and Roberts, 1980). This equation can be combined with equation (4) to give:

2 KE – CW log10m – CHt – CQt (6) v = Ki - p / 10

The constants KE and CW, CH and CQ are all specific to a species, however it has been suggested that universal values for the temperature constants CH and CQ (0.0329 and 0.000478 respectively) can be applied across species (Dickie et al., 1990).

There is an assumption that  is also constant within a species, with changes in  the product of changes to the storage temperature and/or seed moisture content (Ellis and Roberts, 1980). However, exceptions to this relationship have been found in relation to seed developmental age (Kameswara Rao et al., 1991; Hay et al., 1997), genotype (Zanakis et al., 1994; Kochanek et al., 2009), pre-zygotic growth environment (Kochanek et al., 2010) and seed priming (Probert et al., 1991).

The quadratic function relating storage temperature to seed longevity was introduced into the viability equation to better fit storage data at sub-zero temperatures, however there are limits to the range of temperatures to which it can be applied and it is not recommended to use the equation below temperatures of -20°C (Pritchard and Dickie, 2003). This is because the quadratic function predicts a reversal in the relationship between temperature and seed longevity at the point tL where:

⁄( ) (7)

If the universal temperature constants are used, tL is -34.4°C, indicating that longevity of seeds decreases at temperatures below this point, however this is not the case (Dickie et al., 1990). It is therefore not recommended to use the equation to extrapolate longevity below -20°C (Pritchard and Dickie, 2003).

The viability equation assumes that there is a negative linear relationship between the logarithms of seed moisture content and . Low seed moisture limits to this linear relationship have been reported (Ellis et al., 1988; Ellis et al., 1989). Reducing 17 moisture contents below this critical level may have no beneficial effect on seed longevity (Ellis et al., 1988) or may actually have a detrimental effect (Vertucci and Roos, 1990). There is also an upper limit to this relationship, above which, in the presence of oxygen, longevity improves with increasing seed moisture content until full hydration is reached (Roberts and Ellis, 1989).

Genebank standards

The principles that underlie the seed viability equation have led to the development of genebank standards (FAO/IPGRI, 1994). These standards outline the preferred conditions for long-term storage of seeds, with the recommendation that seed moisture content be in the range of 3-7% depending on the species and related seed oil content, achieved by drying seeds at 10-25ºC and 10-15% relative humidity. Seeds should then be placed into containers that are moisture-proof and sealable (Gómez- Campo, 2002; Gómez-Campo, 2006; Walters, 2007), then stored at temperatures of - 18ºC or below (FAO/IPGRI, 1994). Despite these conditions being developed predominantly for the storage of agricultural species, they have been applied to the storage of wild plants species around the world (e.g. Guerrant and Raven, 2003; Walsh et al., 2003; Cochrane, 2004; Probert et al., 2009; Godefroid et al., 2010).

Even when species are stored under these conditions there is a large amount of variation in longevity between species. For example, seeds of Anemone nemorosa are predicted to survive less than a year under genebank conditions (Ali et al., 2007) whilst Arabidopsis thaliana is estimated to survive in excess of a thousand years under similar conditions (Hay et al., 2003). Correlates between a range of ecological characteristics and seed longevity have been suggested. For example endospermic seeds have been found, in general, to be shorter lived than non-endospermic seeds; seeds from hot, dry environments have been found to be longer lived than seeds from cool, wet environments (Probert et al., 2009); and seeds from alpine plants are shorter lived than those from lowland population or related taxa (Mondini et al., 2011).

Seed conservation in Australia

Seeds of Australian native plants species have been collected and stored since the early exploration of the country (Duchess of Hamilton and Bruce, 1998). A review of seed banking in Australia in 1993 (Morse et al., 1993) found that although facilities involved with the storage of seeds existed in all states and mainland territories, very few were storing to the standard required for long-term storage. Of those using conditions suitable for long-term storage, most were storing material of agricultural or horticultural crops, with only a couple conserving native Australian species. At this time Western Australia’s Department of Environment and Conservation’s Threatened Flora Seed Centre was the only facility established with the primary purpose of collecting and storing seeds of rare or threatened plants for conservation purposes under genebank conditions (Morse et al., 1993; Cochrane, 2004; Underwood, 2011). Many seed banks, like the NSW Seedbank, evolved their storage conditions over time to eventually achieve conditions suitable for long term storage (Offord et al., 2004). In 2001 a collaborative international seed conservation effort, the Millennium Seed Bank Project, managed by the Royal Botanic Gardens, Kew, commenced (Smith et al., 2003; van Slageren, 2003). This project resulted in considerable investment into the seed banking of Australian native plant species and resulted in long-term ex situ seed conservation facilities being established in all Australian States and mainland territories.

Despite this history of seed storage, there is very little information on the longevity of Australian native species, particularly under genebank conditions. Some species, particularly those exhibiting physical dormancy such as Acacia, have been recorded to have viable seeds after up to 50 years storage at ambient conditions (Ewart, 1908) and a number of species can maintain reasonable viability when stored in an air- conditioned room (18 - 22°C) for periods of up to 10 years (Gunn, 2001). Some of the limited information available for seeds of Australian species stored under genebank conditions shows some species storing as well, or better, than other storage conditions (Gunn, 2001; von Richter et al., 2001; Merritt et al., 2003; Offord et al., 2003; Offord et al., 2004) whilst other species have stored poorly (Merritt et al., 2003; Offord et al., 2004). This final finding has led to the suggestion that normal, long term seed bank conditions may not be appropriate for all Australian species (Merritt and Dixon, 2003; Merritt et al., 2003) and that investigations into storage conditions for Australian species be carried out on a species by species basis (Coates and Dixon, 2007). If valid, 19 these conclusions have implications for the way ex situ seed collections of Australian plant species are managed.

Research objectives

The aim of this thesis was to investigate seed storage of Australian native plant species. Chapter 3 outlines a review into the storage performance of seed collections held in the short- and medium-term at the Western Australian Department of Environment and Conservation’s Threatened Flora Seed Centre, testing the hypothesis that conditions currently used for the storage of seeds are not suitable for Australian species. Chapter 4 outlines the application of the improved seed viability equation for two contrasting, endemic Western Australian species: a eudicot, Eucalyptus erythrocorys F.Muell. (Myrtaceae) and a monocot, Xanthorrhoea preissii Endl. (Xanthorrhoeaceae). Three hypotheses are tested in Chapter 4: that E. erythrocorys and X. preissii show orthodox seed survival characteristics; that the improved seed viability equation can be used to model seed survival of each species; and that the universal temperature constants can be applied to each species. One of the assumptions of the seed viability equation is that within a species, at a given storage temperature and moisture content, the standard deviation of the distribution of seed deaths in time () is not affected by genotype or initial seed quality. Chapter 5 tests the hypothesis that this assumption holds for serotinous Banksia species, as well as testing the hypothesis that there is a relationship between the degree of serotiny and the time for viability to fall by one probit (). These research chapters are followed by a General Discussion chapter of the research undertaken.

CHAPTER THREE

Analysis of seed-bank data confirms suitability of international seed- storage standards for the Australian flora.

This Chapter has been published as a paper in the Australian Journal of Botany.

Crawford AD, Steadman KJ, Plummer JA, Cochrane A, Probert RJ. 2007. Analysis of seed-bank data confirms suitability of international seed-storage standards for the Australian flora. Australian Journal of Botany 55: 18-29.

My research included germination testing as well as the compilation and analysis and interpretation of data provided by Anne Cochrane, manager of the Department of Environment and Conservation’s (previously known as the Department of Conservation and Land Management) Threatened Flora Seed Centre. I wrote the manuscript and the other three co-authors are my PhD supervisors who assisted with discussions of the research, its interpretation and editing.

Table and figure numbers for this chapter relate to the publication.

CHAPTER FOUR

One-step fitting of seed viability constants for two Australian plant species

This Chapter was accepted for publication by the Australian Journal of Botany on the 22nd October 2012 and was published online on the 21st December 2012. doi: 10.1071/BT12171.

My research included the collection and processing of seeds of Eucalyptus erythrocorys and Xanthorrhoea preissii, as well as all seed ageing experimentation and analysis, interpretation and preparation of the manuscript. Seed oil content was conducted by the Western Australian Chemistry Centre and moisture sorption data was collected by staff at the Millennium Seed Bank and this is duly acknowledged in the acknowledgments. Dr Fiona Hay of the International Rice Research Institute provided advice and assistance with the data analysis and assisted with discussions of the research, its interpretation and editing. The other three co-authors are my PhD supervisors who assisted with discussions of the research, its interpretation and editing.

Table and figure numbers for this chapter relate to the publication.

CHAPTER FIVE

The influence of cone age on the relative longevity of Banksia seeds.

This Chapter has been published as a paper in the Annals of Botany.

Crawford AD, Plummer JA, Probert RJ, Steadman KJ (2011) The influence of cone age on the relative longevity of Banksia seeds. Annals of Botany 107, 303-309.

My research included the collection and extraction of seeds from Banksia cones, all experimental work, data analysis, interpretation and preparation of the manuscripts. The three co-authors are my PhD supervisors who assisted with discussions of the research and editing.

Table and figure numbers for this chapter relate to the publication.

CHAPTER SIX

General Discussion

General discussion

In the last decade there has been an increase in the number of ex situ seed conservation facilities set up to conserve germplasm of the world’s wild plant species, largely due to the inclusion of ex situ conservation in worldwide conservation strategies (Williams et al., 2003) and due to projects such as the Millennium Seed Bank Project (Smith et al., 2003; van Slageren, 2003). These conservation facilities aim to act as insurance policies against extinction of species or populations in the wild (Linington and Pritchard, 2001). In order to be able to fulfil this goal, viable seeds, genetically representative of their source population and species will be required.

The main factors affecting the longevity of seeds, storage temperature and seed moisture content, have long been known (Roberts, 1972) and have been manipulated to extend the storage life of seeds. This has led to the development of genebank standards for the storage of orthodox seeds (FAO/IPGRI, 1994). Originally developed for the storage of seeds of species for food and agriculture, these standards have now been applied to the seeds of a wide range of wild plant species (Linington, 2003).

The adoption of these standards by genebanks storing seeds of species from the Australian flora has come under scrutiny after it was found that two of four Western Australian plant species tested in a seed storage study showed poor storage performance after 18 months at conditions within the range outlined by these standards (Merritt et al., 2003). The conclusions drawn from this study were that the storage conditions recommended in the genebank standards may not be suitable for all Australian plant species and that storage parameters should be determined on a species by species basis (Merritt and Dixon, 2003; Coates and Dixon, 2007). Although a reasonable recommendation, it would be difficult to undertake in practice. For instance, in Western Australia there are over 400 species listed as being in danger of extinction (Marmion, 2012). To determine the storage parameters for even this small subset of the Australian flora would be a huge task considering that in the c. 50 years that seed storage longevity has been modelled, only about 60 species have so far been examined. The other factor which prevents this work being conducted is seed availability. For threatened species, those most in need of ex situ conservation, available seed is often limited (Cochrane et al., 2007) and would likely rule out the ability to conduct storage experiments.

In this thesis evidence was gathered to examine viability decline in ex situ seed collections held in the Western Australian Department of Environment and Conservation’s (previously known as the Department of Conservation and and Management) Threatened Flora Seed Centre, which had been stored in the short- (< 5 years) or medium- (5-12 years) term under conditions in line with the genebank storage standards (Chapter 3). A large proportion of collections held in this facility showed no significant decline in viability. Although the focus of the Threatened Flora Seed Centre’s collection and conservation activities is Western Australia’s threatened plant species, they can be considered representative of Australian plants in general, as the genera making up the collections represent some of the most common in the Australian flora.

Declines in the viability of some collections were found. In some cases other collections of the same species had been stored for the same period or longer with no decline in viability. This indicated it was not the species per se that responded poorly to the storage conditions but rather the specific collection of that species. This highlights a common problem with current information relating to the storage of wild species, which is that available information comes from only a limited number of collections, often a single collection. For instance of the over 30,000 species held in storage at the Millennium Seed Bank, only 1/3 are represented by more than one collection (van Slageren pers. comm.). Where more than one collection of a species has been examined variable storage responses are often seen (Walters et al., 2005; Godefroid et al., 2010).

In this study the only species to show declines in viability, which were represented by more than one collection of comparable storage period, were the closely related Stylidium coroniforme F.L.Erickson & J.H.Willis subsp. coroniforme and S. amabile Wege & Coates. In Chapter 3 it was found that the number of apparently viable seeds had not declined, just the germination of the collection. It was proposed that the change in germination treatment was probably responsible for the decline, rather than a decline in viability. Subsequent testing at the Threatened Flora Seed Centre has shown this to be the case, with germination results indicating there was no decline in viability for the two S. amabile collections, nor for one of the S. coroniforme subsp. coroniforme collections.

In Chapter 4 the seed storage behaviour of the two endemic Western Australian species Eucalyptus erythrocorys and Xanthorrhoea preissii, was found to be consistent

55 with other orthodox species from around the world. Seed viability parameters were determined from these ageing studies, enabling the modelling of seed survival using the improved seed viability equation (Ellis and Roberts, 1980). The only aspect of this model fitting that did not conform to what has previously been found is that the universal values for the temperature constants CH and CQ (Dickie et al., 1990), could not be used without a significant increase in the error. This does not appear to be due to anything intrinsically different about the Australian species examined, as the values for the temperature constants derived in this study were within the broad range of values obtained for the species originally used to obtain the universal temperature constants (Dickie et al., 1990). The reason is more likely due to the different statistical approach used in this study, a one-step approach, which takes into account the full variability of the data, resulting in smaller standard errors of parameter estimates (Hay et al., 2003).

The seed viability equation assumes that standard deviation of seed deaths in time () is constant and hence KE, CW, CH and CQ are constant within a species, with changes in  the product of changes to the storage temperature and/or seed moisture content (Ellis and Roberts, 1980). Exceptions to this relationship have been found in relation to seed developmental age (Kameswara Rao et al., 1991; Hay et al., 1997), genotype (Zanakis et al., 1994; Kochanek et al., 2009), pre-zygotic growth environment (Kochanek et al., 2010) and seed priming (Probert et al., 1991) and this was further examined for Australian species.

In Chapter 5 this assumption was examined in relation to serotiny in three Banksia species. The hypothesis was that within a species, seeds from cones of different ages would lose viability at the same rate (1/), with any differences in longevity being attributable to differences in initial viability. The data supported this hypothesis. Firstly, seeds collected from older cones had lower initial viability than seeds from younger, mature cones. The only exception was the youngest cohort of seeds (1 year old) collected from Banksia hookeriana which had a lower initial viability than the next oldest cohort (2-3 years old), likely due to an immature fraction in the collection due to the long flowering period of the species. Secondly, seeds from cones of all ages could be attributed a single value for  within a species and contrary to expectations, all species in the study could be attributed a common value of .

These findings highlight the broad applicability of the seed viability equations to a wide range of species. Despite the exceptions that have been found to the underlying

56 assumptions, the viability equation still provides a useful predictive tool to gauge the impact of controlled storage conditions on seed longevity.

In order to put all of these findings into context it must be remembered that the ex situ storage of seeds for conservation purposes is a risk management strategy. These collections aim to store genetically representative samples of a species, to be used at some indeterminate time in the future, to re-establish the species back into the wild. In order to fulfil this goal the seeds need to be viable and germinable when required. It has been suggested, in relation to the Australian flora, that the storage conditions currently recommended for long term storage may not be suitable for all species (Merritt et al., 2003). This is almost certainly going to be the case. It is already known that there is a broad category of seeds, recalcitrants, which would be killed by storage at the conditions used for the long-term storage of orthodox seeds. Even between species considered orthodox, there is going to be a wide range of seed longevities. For example, the estimated time for seeds to lose one probit of viability when stored at -20°C with a moisture content equilibrated at 15% relative humidity at 15°C has been found to range from 32 years for Ulmus carpinifolia to 6106 years for Sorghum bicolor (Hong et al., 1998). For the two species studied in Chapter 4, the comparable estimate was 825 years for Xanthorrhoea preissii and 4945 years for Eucalyptus erythrocorys (Ch. 4, Table 4).

There has also been disagreement over what is the most suitable seed moisture content for the long term storage of seeds (Ellis et al., 1991; Vertucci and Roos, 1991; Smith, 1992; Vertucci and Roos, 1993). Whilst many seed banks store seeds with moisture contents in equilibrium with 10-15% relative humidity at 10-25°C (FAO/IPGRI, 1994), there is a suggestion that the resultant seed moisture content may drop to sub-optimal levels when stored at sub-zero temperatures and that a higher relative humidity (19-27%) should be used (Vertucci and Roos, 1991; Vertucci and Roos, 1993). Unfortunately, due to the long time periods involved, it will be many years before enough empirical evidence is available for seeds stored under the various ‘optimal’ conditions proposed to determine which hypothesis is correct.

In reality, a standard set of storage conditions will never be optimal for all collections of all species. The optimum moisture content for storage will depend on the sorption properties of the seeds (Pritchard and Dickie, 2003) but the notion that optimal storage conditions need to be investigated on a species by species basis (Coates and Dixon, 2007) is not practical. Efforts have been made to find ecological correlates with

57 seed longevity to identify potentially short-lived species which can help guide decisions about collection re-test intervals (Probert et al., 2009). Even where the expected longevity of collections can be predicted, exceptions are always a possibility (Kameswara Rao et al., 1991; Probert et al., 1991; Zanakis et al., 1994; Hay et al., 1997; Kochanek et al., 2009; Kochanek et al., 2010). There is also the possibility that the seed storage container may be inadequate, faulty or damaged, which could allow moisture ingress and hence an increase in seed moisture content (Gómez-Campo, 2002; Manger et al., 2003; Hay et al., 2012).

Seed bank managers must therefore manage their collections in light of these uncertainties. The usual method for monitoring the viability of a collection is to perform germination tests at intervals, with a recommended initial period of 10 years for collections with a high initial viability, or five years for collections with poor initial viability or for species with known poor storage life (FAO/IPGRI, 1994). These re-tests aim to detect when a collection’s viability drops to 85% of its initial viability, at which point the collection should be regenerated and seeds collected for storage. Unfortunately the number of seeds required to conduct these tests with any confidence is large (Guerrant and Fiedler, 2004). As the size of ex situ seed collections can often be small, particularly for threatened species (Cochrane et al., 2007), the size of samples that might be able to be used for testing and the number of sampling points through time, will be limited.

The decision about when to conduct a re-test and how frequently is going to be a balance between the risk of losing valuable genetic material through viability loss and using up seed material unnecessarily if viability decline is not expected. The 10 year re- test frequency still appears to be a reasonable sampling period, despite the fact that some species/collections may still have lost a significant amount of viability in this period (Chapter 3; Walters et al., 2005; Probert pers. comm.). Analysis of 20 year re- test data across a diverse range of plant families stored under genebank conditions, has shown that a number of collections have a significant decline in viability (Probert et al., 2009), whilst analysis of another re-test data set consisting of range of collections with initial viabilities between 76 and 99% and an average storage time of 38 years, indicated that the average viability had dropped to 58% (with a range 0 to 100%) (Walters et al., 2005).

It is possible for a collection to have lost at least 50% of its viability within 10 years (Walters et al., 2005) and so it is proposed that a better way to manage ex situ collections, if it is not possible to conduct a germination re-test 10 years after the last test, is to assume that the collection has lost viability. A decision can then be made on the best course of action based on conservation priorities and available resources. Emphasis needs to be placed on identifying potentially short-lived species or groups of plants.

In conclusion, the hypothesis that current long-term storage practices are not suitable for Australian plant species cannot be supported. Rather the storage practices appear generally suitable, with a few exceptions, appearing to be related to seed quality rather than species characteristics. The hypothesis that E. erythrocorys and X. preissii show orthodox seed survival characteristics was supported. As a result the improved seed viability equation could be used to model seed survival of each species, supporting the second hypothesis. The hypothesis that the universal temperature constants could be applied to each species had to be rejected, however it is argued that from a practical standpoint it would be still acceptable to use them. Finally, the hypothesis that within a species, at a given storage temperature and moisture content, the standard deviation of the distribution of seed deaths in time () is not affected by initial seed quality was supported, however there was no support for the hypothesis that there is a relationship between the degree of serotiny and the time for viability to fall by one probit ().

This thesis has shown that the seeds of orthodox Australian species respond to adjustments in seed moisture content and storage temperature in the same way as orthodox species from other parts of the world. Whilst the current recommendations for the long-term storage of seeds may not be optimal for all species, it has been shown they can be applied to a diverse range of species resulting in significant improvements in longevity. Now the emphasis of seed storage research should be shifted to identifying potentially short-lived species and resources put into the adequate maintenance of existing and future collections.

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APPENDIX ONE

Poster: Successful storage of seed from threatened west Australian plants

Poster presented at:

 the 8th International Workshop on Seeds: Germinating new ideas, held in Brisbane on the 8-13th May 2005  the Advances in Plant Conservation Biology: Implications for Flora Management and Restoration Symposium, held at Kensington on the 25-27th October 2005