A. gordonii, A.distyla, germination, O. flocktoniae, seedbank, temperature, viability

1. The effect of storage temperature on the germinability of NSW native seeds
Thomas Suri Taisa
Abstract: There are more than 13 million floral species distributed worldwide, but only 1.8 million is known.
The floral diversity supports our economy, maintains environment and provides nutritional requirement to
humans and animals. However, world plant diversity is threatened by changing environment due to climate
change and increasing human activities, prompting collection and conservation of most endangered species in ex
situ repository collections. In Australia, A. gordonii, A. distyla and O. flocktoniae, are threatened by natural
calamities such as fire, and are warned of extinction due to rapid urban and agricultural developments. Thus,
collection and conservation of these species in ex situ seedbanks is one of the priorities of Australia’s
conservation program. Seed storage in seedbanks requires prior knowledge of seed’s moisture retention, ambient
storage temperature and drying facilities to maintain viability of the seed. This study investigated the
germinability of three native plant species stored for 9-10 years at low optimal temperatures (2.5⁰C and -20⁰C).
It was found that there was no effect of the two temperature regimes on the germination while differences in
germination were highly significant between and within the species. A high proportion of germination was
observed in A. gordonii (96-97%), followed by A. distyla (80-95%) and O. flocktoniae (66-80%).
Additional keywords: A. gordonii, A.distyla, germination, O. flocktoniae, seedbank, temperature, viability.
Introduction
There are more than 13 million flora species distributed throughout the world, which includes plants,
fungi, fern and mosses (Australia Flora Statistics 2009). It is estimated that 1.8 million is known
worldwide (Offord and Makinson 2009). In Australia, the total number of floral species is difficult to
estimate as many of them are unidentified and lost in the ecosystems including forests, grasslands,
deserts and tundra. In 2009, it was estimated around 20 000 (93.5%) native flowering plants occur
across bioregions, and many demonstrate good adaptability to different environments (Offord and
Makinson 2009). Other than the known species, there are many bryophytes (~2 200), algae (~3 000),
ferns/allies (~525) and gymnosperms (~120, Australian Flora Statistics 2009). These species form the
megadiverse system and contributes to the biodiversity of Australia (Offord and Makinson, 2009).
The floral diversity supports our economy, maintains the environment, and above all it
provides food, shelter, fuel, fibre and medicine (Nderitu et al. 2008; Turpie et al. 2003). However,
many of these species are exposed to changing environment, facing a multitude of anthropogenic
treats, especially habitat fragmentation and degradation (Offord and Makinson 2009; Reed and
Sarasan 2011). Consequently, many plant species are threatened with extinction because of the
gradual disappearance of the terrestrial natural ecosystems for various human activities (Reed et al.
2011). It is estimated that more than 50% of the world’s plant species are endemic to 34 global
biodiversity hotspots, which once covered 15.7% of the earth’s land surface but reduced to 2.3%
(Reed et al. 2011). In Australia, it is estimated that at least 10% of the plant species are under treat of
extinction (Offord and Makinson 2009). The treats are associated with rapid human activities,
particularly large exploration and mining, logging, land clearing for urbanization and agricultural
activities (Hamilton et al. 2011; Offord and Makinson 2009; Woinarski 2010).
The floral biodiversity is also threatened by climate change, which affects the future
distribution of native species and ecosystems (Crossman et al. 2012). The combination of these
changes affects food security, the economy and the environment (Tscharntke et al. 2012). While a
number of species and ecosystems have demonstrated some capacity to adapt to climate change
(Harder and Aizen 2010), many are suffering negative consequences (Crossman et al. 2012).
Reducing the vulnerability of the native species and ecosystems to the climate change and human
activities is an increasingly important conservation objective (Crossman et al. 2012; Tscharntke et al.

2. 2012). Identifying areas that are likely to become important for vulnerable species is necessary to
assist their adaptation to the changes and conserve the biological diversity for future use. This can be
achieved by ex situ and in situ conservation practices (Offord et al. 2004; Offord and Tyler 2009).
The ex situ approach of seed banking is cost-effective (Offord et al., 2004), and captures a
diverse genetic seed bearing plant species for later use (Martyn et al. 2009; Offord et al. 2004). It is
economically easy to maintain, requires small space, easy to handle and can hold a large number of
genetic diversity (Martyn et al. 2009). However, longevity of the storage is strongly influenced by the
dormancy of the species (Turner and Merritt 2009), moisture content (Offord et al. 2004) and storage
condition (Martyn et al. 2009). Germinability in this instance is very crucial to determine the recovery
potential of the seed after long period in the seedbank. The germinability of three threatened
Australian native species; Acacia gordonii, Allocasuarina distyla and Olearia flocktoniae remain
elusive, although some authors reported (Offord et al. 2004) a specific temperature and storage
condition. A review by Offord et al. (2004) indicated that these species require 10% moisture
retention at 5⁰C storage temperature for up to 10 years without the loss of viability. However,
reducing the current storage temperature might increase longevity of the seeds, considering the need
for these seeds to be stored for more than 10 years (Hamilton et al. 2009; Martyn et al. 2009).
Germination test was conducted on the three species (A. gordonii, A. distyla and O. flocktoniae) to
identify their germinability and longevity after 9-10 years of storage at lower storage temperatures.
Materials and Methods
Seed materials
The seeds of A. gordonii, A. distyla and O. flocktoniae were collected and kept in ex situ seedbank
conservation at Mt Annan Botanical Gardens since 2002. Seeds were divided between two lower
temperatures of 2.5⁰C and -20⁰C, and were stored for 9-10 years. Germinability of the seeds was pre-
tested (80-100%) before keeping them in the repository seedbank. These seeds were used in this
experiment to determine their germinability.
Treatment and incubation
Nine petri dishes, filled with solid water agar, were sown with seeds of A. gordonii, A. distyla and O.
flocktoniae at a rate of 25 seeds per dish. The seeds of A. gordonii were scarified to remove the
impermeable testa before sowing. Seed-containing petri dishes were labelled and kept in the
incubation room at a constant temperature of 20⁰C with 12/12 h light/dark. Germination of the seeds
was monitored periodically for two weeks.
Experimental design
The seeds of A. gordonii, A. distyla and O. flocktoniae were completely randomised in a split plot
design. Each species containing two accessions were splitted between two temperature treatments
(2.5⁰C and -20⁰C) in four replicates. Of the two accessions of each species, one was stored in both
temperature treatments, while the other accession was stored alone at low temperature (-20⁰C,
Freeze). Thus, a total of 36 treatments, comprising 3 species x 6 accessions x 2 temperatures, were
tested for germinability.
Germination assessment and viability testing
Germination census was done periodically at Day7, Day11 and Day14, for two weeks. Data was
collected from number of seeds germinated during germination census. The seed viability assessment
was done by carrying out cut test under microscope. Data from the germination counts and cut tests
were entered in the Microsoft Excel Windows 2010 and summarized prior to statistical analysis.
Statistical analysis
A Two-way Anova was used to determine the effect of storage temperature, species, accession and
their interaction on the germination of the seeds. The analysis was done using the Genstat program
(14th Edition) at the University of Sydney, Australian Technology Park, New South Wales. Least of

3. significance differences at 5% level were used to differentiate the means in factors that did not show
any significance in the germination.
Results
Seed quality assessment
The quality of the seeds was assessed at the time of the final germination count, whilst the proportion
of the healthy seeds was compared with damage (unhealthy seeds), which is represented by ‘Healthy
seed’ (Table 1). Based on this category, proportion of healthy seeds were significantly higher
(P<0.001) in the two accessions of A. gordonii, where the germination was ranged between 93 and
100% in both storage conditions. In the Olearia accessions, high proportion of healthy seeds were
significantly higher under 2.5⁰C and -20⁰C storage conditions for accession number 900610 and
20080208, respectively (Table 1). Comparatively, 76-85% of the seeds were healthy for the
Allocasuarina accessions under the two temperature regimes (Table 1).
Table 1. Proportion (%) of seed-quality assessment.
A. gordonii A. distyla O. flocktoniae
Accession 842963 970299 877412 20050491 900610 20080208
t-test
Storage 2.5⁰ probability
-20⁰C 2.5⁰C -20⁰C 2.5⁰C -20⁰C 2.5⁰C -20⁰C 2.5⁰C -20⁰C 2.5⁰C -20⁰C
temp. C
Viable 1.3 - 1.0 - 1.0 - 5.0 20.0 9.0 7.0 0.026*
Mushy 1.3 5.2 - 2.0 7.0 3.0 - 7.0 13.0 8.0 7.0 0.053*
Discolor - 4.0 - 4.0 - 2.0 - 1.0 0.375NS
Empty - - - - - 12.0 - 6.0 0.024*
Predated - - - 10.0 - - 0.53NS
Healthy 98. 93.0 - 100 85.3 76.0 - 85.0 96.0 88.0 - 96.0 0.001***
seeds1 9
* Significant (P<0.05). ***highly significant (P<0.001). NS, not significant (P>0.05).
1, proportion of healthy seeds determined in the final count after germination.
Effect of storage temperature on germination
The effect of two temperatures was tested in 2012 to determine the viability of the seeds after 9-10
years of storage (Table 2). The mean germination at 2.5⁰C was 83%, which was 3% lower than 86% at
20⁰C (LSD, 5% = 1.8). However, the storage temperatures had no effect on the germination of the
seeds (Table 3).
Table 2. Proportion (%) of germination in accessions at two storage temperatures
Germination (%)
Species*** Accession***
2.5⁰C -20⁰C Difference
Acacia gordonii 842963 97.3 95.0 2.3
970299 - 97.0 -
Allocasuarina distyla 877412 89.0 93.0 4.0
20050491 - 84.0 -
Olearia flocktoniae 900610 63.0 69.0 6.0
20080208 - 80.0 -
3.2
83.1 86.3
*** Highly significant (P<0.001). LSD (5%) = 1.8

4. Table 3. Tests of significance effect of species, accessions, storage and their interaction on the seed
germination.
Average germination (%) t-test probability and significance
Storage 84.0 0.387NS
Species 85.2 0.001***
Accession 72.3 0.001***
Species x Access x Storage Interaction 84.8 0.001***
***highly significant (P<0.001). NS, not significant (P>0.05)
Effect of accession on germination
The effect of accessions on the seed germination was tested, where germination was highly (P<0.001)
affected by accessions (see above Table 3). Under fridge condition of 2.5⁰C at storage, seed
germination was higher in 842963 (97.3%) of A. gordonii, than it was observed for 877412 (89%) and
900610 (63%) of the A. distyla and O. flocktoniae species, respectively (see previous Table 2). At
freezing condition of -20⁰C, high germination percentage was observed for the same accession,
842963 (95%) of A. gordonii, followed by 877412 (93%) of A. distyla and 900610 (69%) of O.
flocktoniae. Under the same storage, the accession 970299 of A.gordonii had high proportion (97%)
of seeds germinated compared to the 20050491 (84%) of A. distyla and 20080208 (69%) of the O.
flocktoniae.
Effect of species on germination
The native plant species had high significant effect (P<0.001) on the seed germination from both
storage temperatures (see above Table 3). At 2.5⁰C storage (fridge condition), a high number of seeds
were germinated in the A. gordonii species, with 97% and 96% at each temperature regimes
respectively (Table 4). The germination in A. distyla was 89% and about 88% at 2.5⁰C and -20⁰C,
respectively. Comparatively, O. flocktoniae species had the lowest germination percentage at both
temperatures (Table 4). Further, the germination difference of this species was relatively higher
(9.5%), compared to A. gordonii and A. distyla.
Table 4. Proportion (%) of germination in species at two storage temperatures
Species*** Germination (%)
2.5⁰C -20⁰C Difference
Acacia gordonii 97.3 96.0 1.3
Allocasuarina distyla 89.0 88.5 1.5
Olearia floctoniae 63.0 74.5 9.5
*** highly significant (P<0.001)
Interaction effect on germination
The interaction effect of species, accession and the storage was tested (see previous Table 3). The
effect of the combination of these factors had high significant effect (P<0.001) on the germination of
all seeds. Over all, the three native plant species and the six accessions had high significant effect on
the germination of the seeds than the effect of the two storage temperatures.
Stability of seed viability

5. The germination of the seeds in the recent test is compared with the earlier experiment results using
the six accessions of the three species (Table 5). Generally, germination of the seeds in A. gordonii
species remains relatively high compared to the first test result. Comparatively, germination in
900610 of the O. flocktoniae species was dropped by 24%, while the decline in the accession
20080208 was 14% after ten years of storage. Seed germination in the 877412 of A. distyla was
reduced by 9%.
Table 5. Current germination test is compared to the previous germination result.
Germination (%)
Species Family Accession No. First test Second test
Difference
(year) (2012)
Accacia gordonii Fabaceae 842963 82 (2002) 96 4
970299 100 (2002) 97 3
Allocasuaina distyla Casuarinaceae 877412 100 (2002) 91 9
20050491 80 (2005) 84 4
Olearia flocktoniae Asteraceae 900610 80 (2002) 66 24
20080208 94 (2009) 80 14
Discussion
Storing seeds in a seed bank is a cost-effective method of conserving a wide range of seed bearing
plant diversity in a repository collection (Offord et al. 2004). However, the extent of a seed to be kept
in storage depends on the longevity potential of the species. Longevity is an important trait of all seed
bearing species to determine whether they could survive and viable after long period of storage
(Walters et al. 2004). The survival of the seeds in an ex situ collection is influenced by other factors
including the past history of the seed during collection, moisture retention, storage condition and
importantly the dormancy of the species (Martyn et al. 2009). Little is known about the longevity of
many seeds in Australia (Offord et al. 2004). Collection and testing of a large number of species is
important to rescue threatened species including A. gordonii, A. distyla and O. flocktoniae.
The main finding of this study is that all species retained viability between 80-97% at an
optimal storage condition, with variation among the plant species and accessions. High proportion,
between 96-97%, of viable seed was observed in Acacia gordonii and its accessions (Table 2). The
maximum viability of the seeds in the A. gordonii was attributed to the dormancy traits that
characterize the species, as it was indicated in the viability assessment (Table 1). The Acacia species
are belong to the Fabaceae family and seeds are characterised by hard seedcoat and impermeable testa
that prevents uptake of water and hence ensuring seed dormancy (Auld 1996). Consequently, the
seeds of this species remain viable, although they were kept at an optimum temperature during
storage. Increasing the optimum temperature in storing A. gordonii species did not show any
difference in germination compared to the earlier test result, while seed viability narrowly dropped by
average of 3% in the second test (Table 5). This suggests two possibilities, where the first possible
approach is to increase the storage time at the current storage condition, if needed to maintain the
viability above 90%. And the second possibility is to increase optimum storage condition to determine
the optimum temperature until germination is seen to be reduced, exceeding the recommended
minimal viability level. Generally, most species of the Acacieae and Mirbelieae genera showed high
90-100% dormancy levels at moisture retention between 10-15% (Auld 1996). More of the seeds of
Acacia species in this study were viable as indicated by high proportion (over 95%) of seeds
germinated with minimal seed damage whilst suggesting a further extension of the storage time.
Extreme, low temperatures at storage are believed to stop biological activity of the plant
organs including seeds, which varies among plant species (Walters et al. 2004). Some traits of seed
morphology, such as seed size and seed coat, are the important characteristics that determine tolerance
to extreme temperatures (Baskin and Baskin 2004). Compared to A. gordonii, the low germination in
seeds of A. distyla and O. flocktoniae were low between 80-90% (Table 5). Low germination in these
species might be due to factors associated with seed morphology traits, as it was represented by high
proportion (range 3-7%). According to NSW National Park & Wildlife (2000), there is no empirical

6. information about seed longevity and viability of A. gordonii. However, it is believed that either the
optimum storage temperature badly affected the seeds or possibly some biological activity might have
occurred, exhausting storage carbohydrate, and consequently resulted in low germination. Some
information of this species confer that seeds do not have an effective dormancy, whilst seeds
germinate when sufficient moisture is available and temperature is suitable (NSW National Parks and
Wildlife Services 2000). In this instance, 4-7% of mushy seeds at 2.5⁰C storage temperature for A.
distyla (Table 1), suggested either seed had undergone biological activity or contained excess
moisture during storage that affected the germinability.
The O. flocktoniae species had the low germination percentage, which ranged 66-80%.
However, the germination was reduced by 14-24% in 10 years (Table 5). The seed of this species is
characterized by small size, and vulnerable to extreme conditions (National Parks and Wildlife
Services 2004). Many seeds of O. flocktoniae were relatively affected, where more than 7% of the
seeds were found mushy. In addition, because of the seeds were smaller in size, compared to the
Acacia and Allocasuarina species, large proportion of seeds were found 12% empty, particularly at
2.5⁰C. Empty seeds can be a source of contamination, as they could become the breeding ground of
pathogens that could affect seed longevity and germinability (Martyn et al., 2009; Offord and
Makinson 2009). Generally, seed viability of O. flocktoniae species did not change much from 10
years in storage and the germination remained at 80% (Table 5). A similar result was reported in 2004
by National Parks and Wildlife Services (2004), where the germination of the seeds conserved from
1990 was found to be 80% when tested in 2003. The compelling result suggests an extension of time
in storage for O. flocktoniae at current storage condition, while continue to monitor the viability
periodically. And also further option is suggested increasing the optimum temperatures to limit
biological activity to increase longevity and improve viability since high proportion of seeds damaged
during storage.
Conclusion and recommendation
This study found that germination was ranged between 80-97% across plant species, but variation
exists between and within species. Temperature had no effect on the germination of the seed accept
changes that occurred due to species and accessions. The insignificance effect of storage temperature
on germination could suggest further storage options, or otherwise to maintain seeds at current
optimal condition. However, longevity and viability of the seeds are crucially important, which are
voluntarily hampered by fluctuation in storage conditions, particularly temperature, and foreign
contaminants such as empty seeds or pathogens.
Generally, all seeds maintained viability after 10 years in low optimal storage temperatures.
Longevity of these species in storage can be sustained by proper storage conditions, unless needed to
increase optimal temperature for further investigation on germinability. Further monitoring and
evaluation of the viability of the seeds is crucial. However, 10-years interval might be too long
considering aging of the seeds while in seedbank. To date, specific information on germination
remains elusive as a wide range of Australian native seeds are not tested for longevity and viability.
Future efforts in germination studies are crucial to cover as many species as possible to understand
their storage requirement.
Acknowledgement
Finally, the study of plant biodiversity with particular focus on seed collection, purification, testing,
storing and plant recovery has been a great challenge, and quite difficult to digest everything in a
minute. Nevertheless, this study has been a stepping stone in understanding what the nature holds for
human kind. I am pleased to thank Catherine A. Offord and Amelia J. Martyn for the effort in getting
the principles of the conservation across to us, the students of Horticultural Science. With my due
respect, I wish you both a good luck in your future endeavour.

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