1. The effect of storage temperature on the germinability of NSW native seeds Thomas Suri TaisaAbstract: 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 tohumans and animals. However, world plant diversity is threatened by changing environment due to climatechange and increasing human activities, prompting collection and conservation of most endangered species in exsitu repository collections. In Australia, A. gordonii, A. distyla and O. flocktoniae, are threatened by naturalcalamities 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’sconservation program. Seed storage in seedbanks requires prior knowledge of seed’s moisture retention, ambientstorage temperature and drying facilities to maintain viability of the seed. This study investigated thegerminability 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 ingermination were highly significant between and within the species. A high proportion of germination wasobserved 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.IntroductionThere 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 knownworldwide (Offord and Makinson 2009). In Australia, the total number of floral species is difficult toestimate 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 occuracross bioregions, and many demonstrate good adaptability to different environments (Offord andMakinson 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 themegadiverse system and contributes to the biodiversity of Australia (Offord and Makinson, 2009). The floral diversity supports our economy, maintains the environment, and above all itprovides 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 anthropogenictreats, especially habitat fragmentation and degradation (Offord and Makinson 2009; Reed andSarasan 2011). Consequently, many plant species are threatened with extinction because of thegradual 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 globalbiodiversity 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 ofextinction (Offord and Makinson 2009). The treats are associated with rapid human activities,particularly large exploration and mining, logging, land clearing for urbanization and agriculturalactivities (Hamilton et al. 2011; Offord and Makinson 2009; Woinarski 2010). The floral biodiversity is also threatened by climate change, which affects the futuredistribution of native species and ecosystems (Crossman et al. 2012). The combination of thesechanges affects food security, the economy and the environment (Tscharntke et al. 2012). While anumber 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 humanactivities 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 toassist their adaptation to the changes and conserve the biological diversity for future use. This can beachieved 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 adiverse genetic seed bearing plant species for later use (Martyn et al. 2009; Offord et al. 2004). It iseconomically easy to maintain, requires small space, easy to handle and can hold a large number ofgenetic diversity (Martyn et al. 2009). However, longevity of the storage is strongly influenced by thedormancy of the species (Turner and Merritt 2009), moisture content (Offord et al. 2004) and storagecondition (Martyn et al. 2009). Germinability in this instance is very crucial to determine the recoverypotential of the seed after long period in the seedbank. The germinability of three threatenedAustralian native species; Acacia gordonii, Allocasuarina distyla and Olearia flocktoniae remainelusive, although some authors reported (Offord et al. 2004) a specific temperature and storagecondition. A review by Offord et al. (2004) indicated that these species require 10% moistureretention 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 needfor 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) toidentify their germinability and longevity after 9-10 years of storage at lower storage temperatures.Materials and MethodsSeed materialsThe seeds of A. gordonii, A. distyla and O. flocktoniae were collected and kept in ex situ seedbankconservation at Mt Annan Botanical Gardens since 2002. Seeds were divided between two lowertemperatures 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 thisexperiment to determine their germinability.Treatment and incubationNine 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 theimpermeable testa before sowing. Seed-containing petri dishes were labelled and kept in theincubation room at a constant temperature of 20⁰C with 12/12 h light/dark. Germination of the seedswas monitored periodically for two weeks.Experimental designThe seeds of A. gordonii, A. distyla and O. flocktoniae were completely randomised in a split plotdesign. 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 bothtemperature 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, weretested for germinability.Germination assessment and viability testingGermination census was done periodically at Day7, Day11 and Day14, for two weeks. Data wascollected from number of seeds germinated during germination census. The seed viability assessmentwas done by carrying out cut test under microscope. Data from the germination counts and cut testswere entered in the Microsoft Excel Windows 2010 and summarized prior to statistical analysis.Statistical analysisA Two-way Anova was used to determine the effect of storage temperature, species, accession andtheir 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 showany significance in the germination.ResultsSeed quality assessmentThe quality of the seeds was assessed at the time of the final germination count, whilst the proportionof the healthy seeds was compared with damage (unhealthy seeds), which is represented by ‘Healthyseed’ (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 and100% in both storage conditions. In the Olearia accessions, high proportion of healthy seeds weresignificantly higher under 2.5⁰C and -20⁰C storage conditions for accession number 900610 and20080208, respectively (Table 1). Comparatively, 76-85% of the seeds were healthy for theAllocasuarina 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 germinationThe effect of two temperatures was tested in 2012 to determine the viability of the seeds after 9-10years of storage (Table 2). The mean germination at 2.5⁰C was 83%, which was 3% lower than 86% at20⁰C (LSD, 5% = 1.8). However, the storage temperatures had no effect on the germination of theseeds (Table 3).Table 2. Proportion (%) of germination in accessions at two storage temperatures Germination (%)Species*** Accession*** 2.5⁰C -20⁰C DifferenceAcacia 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 germinationThe 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, seedgermination was higher in 842963 (97.3%) of A. gordonii, than it was observed for 877412 (89%) and900610 (63%) of the A. distyla and O. flocktoniae species, respectively (see previous Table 2). Atfreezing 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 germinationThe native plant species had high significant effect (P<0.001) on the seed germination from bothstorage temperatures (see above Table 3). At 2.5⁰C storage (fridge condition), a high number of seedswere germinated in the A. gordonii species, with 97% and 96% at each temperature regimesrespectively (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 bothtemperatures (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 germinationThe interaction effect of species, accession and the storage was tested (see previous Table 3). Theeffect of the combination of these factors had high significant effect (P<0.001) on the germination ofall seeds. Over all, the three native plant species and the six accessions had high significant effect onthe 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 usingthe six accessions of the three species (Table 5). Generally, germination of the seeds in A. gordoniispecies remains relatively high compared to the first test result. Comparatively, germination in900610 of the O. flocktoniae species was dropped by 24%, while the decline in the accession20080208 was 14% after ten years of storage. Seed germination in the 877412 of A. distyla wasreduced 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 14DiscussionStoring seeds in a seed bank is a cost-effective method of conserving a wide range of seed bearingplant diversity in a repository collection (Offord et al. 2004). However, the extent of a seed to be keptin storage depends on the longevity potential of the species. Longevity is an important trait of all seedbearing 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 factorsincluding the past history of the seed during collection, moisture retention, storage condition andimportantly the dormancy of the species (Martyn et al. 2009). Little is known about the longevity ofmany seeds in Australia (Offord et al. 2004). Collection and testing of a large number of species isimportant 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 anoptimal 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). Themaximum viability of the seeds in the A. gordonii was attributed to the dormancy traits thatcharacterize the species, as it was indicated in the viability assessment (Table 1). The Acacia speciesare belong to the Fabaceae family and seeds are characterised by hard seedcoat and impermeable testathat prevents uptake of water and hence ensuring seed dormancy (Auld 1996). Consequently, theseeds of this species remain viable, although they were kept at an optimum temperature duringstorage. Increasing the optimum temperature in storing A. gordonii species did not show anydifference in germination compared to the earlier test result, while seed viability narrowly dropped byaverage of 3% in the second test (Table 5). This suggests two possibilities, where the first possibleapproach is to increase the storage time at the current storage condition, if needed to maintain theviability above 90%. And the second possibility is to increase optimum storage condition to determinethe optimum temperature until germination is seen to be reduced, exceeding the recommendedminimal viability level. Generally, most species of the Acacieae and Mirbelieae genera showed high90-100% dormancy levels at moisture retention between 10-15% (Auld 1996). More of the seeds ofAcacia species in this study were viable as indicated by high proportion (over 95%) of seedsgerminated 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 plantorgans including seeds, which varies among plant species (Walters et al. 2004). Some traits of seedmorphology, such as seed size and seed coat, are the important characteristics that determine toleranceto extreme temperatures (Baskin and Baskin 2004). Compared to A. gordonii, the low germination inseeds of A. distyla and O. flocktoniae were low between 80-90% (Table 5). Low germination in thesespecies might be due to factors associated with seed morphology traits, as it was represented by highproportion (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 theoptimum storage temperature badly affected the seeds or possibly some biological activity might haveoccurred, exhausting storage carbohydrate, and consequently resulted in low germination. Someinformation of this species confer that seeds do not have an effective dormancy, whilst seedsgerminate when sufficient moisture is available and temperature is suitable (NSW National Parks andWildlife 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 excessmoisture 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 ischaracterized by small size, and vulnerable to extreme conditions (National Parks and WildlifeServices 2004). Many seeds of O. flocktoniae were relatively affected, where more than 7% of theseeds were found mushy. In addition, because of the seeds were smaller in size, compared to theAcacia and Allocasuarina species, large proportion of seeds were found 12% empty, particularly at2.5⁰C. Empty seeds can be a source of contamination, as they could become the breeding ground ofpathogens that could affect seed longevity and germinability (Martyn et al., 2009; Offord andMakinson 2009). Generally, seed viability of O. flocktoniae species did not change much from 10years in storage and the germination remained at 80% (Table 5). A similar result was reported in 2004by National Parks and Wildlife Services (2004), where the germination of the seeds conserved from1990 was found to be 80% when tested in 2003. The compelling result suggests an extension of timein storage for O. flocktoniae at current storage condition, while continue to monitor the viabilityperiodically. And also further option is suggested increasing the optimum temperatures to limitbiological activity to increase longevity and improve viability since high proportion of seeds damagedduring storage.Conclusion and recommendationThis study found that germination was ranged between 80-97% across plant species, but variationexists between and within species. Temperature had no effect on the germination of the seed acceptchanges that occurred due to species and accessions. The insignificance effect of storage temperatureon germination could suggest further storage options, or otherwise to maintain seeds at currentoptimal condition. However, longevity and viability of the seeds are crucially important, which arevoluntarily hampered by fluctuation in storage conditions, particularly temperature, and foreigncontaminants 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 toincrease optimal temperature for further investigation on germinability. Further monitoring andevaluation of the viability of the seeds is crucial. However, 10-years interval might be too longconsidering aging of the seeds while in seedbank. To date, specific information on germinationremains 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 understandtheir storage requirement.AcknowledgementFinally, 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 aminute. Nevertheless, this study has been a stepping stone in understanding what the nature holds forhuman kind. I am pleased to thank Catherine A. Offord and Amelia J. Martyn for the effort in gettingthe principles of the conservation across to us, the students of Horticultural Science. With my duerespect, I wish you both a good luck in your future endeavour.
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