Emery and Offord (2019). Seed Science and Technology, 47, 1, 107-112. https://doi.org/10.15258/sst.2019.47.1.12
Research Note
The effect of the endocarp, heat-shock and short-term storage on the germination of Persoonia hirsuta (Proteaceae) seeds
Nathan J. Emery* and Catherine A. Offord
The Australian PlantBank, the Royal Botanic Gardens and Domain Trust, the Australian Botanic Garden Mount Annan, NSW 2567 Australia * Author for correspondence (E-mail: [email protected])
(Submitted November 2018; Accepted February 2019; Published online April 2019)
Abstract
Persoonia are Proteaceous shrubs or small trees that have a woody endocarp as part of the dispersal unit. Seeds with this structure are often difficult to germinate. The endangered Persoonia hirsuta (hairy geebung) is a high- interest plant species for which no previous seed biology research has been conducted. In the present study we investigated the seed germination response to heat and short-term ex situ storage at low and room temperature.
The effect of pre- and post-storage endocarp removal on germination was also examined. Germination success was significantly reduced when the endocarp was removed prior to storage due to increased microbial contamination coming from within the seeds. There was no significant effect of heat-shock on germination. Based on these findings, it is recommended that the endocarp be retained when storing or direct-sowing P. hirsuta seeds to enhance the likelihood of producing viable seedlings.
Keywords: germination, hairy geebung, microbial contamination, Proteaceae, pyrene, seed biology
Experimental and discussion
A Persoonia fruit is a drupe which has an outer fleshy mesocarp and leathery pericarp layers, and a hard, woody endocarp within that encapsulates either one or two embryos that are surrounded by a thin, papery testa. The endocarp is a mechanical dormancy mechanism that requires weakening over time or manual removal before germination can occur (Mullins et al., 2002; Bauer et al., 2004; Chia et al., 2016). The effects of the presence or removal of endocarp on the viability of Persoonia seeds (in terms of germination and microbial contamination) during ex situ storage is not known. In addition to the mechanical
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107 NATHAN J. EMERY AND CATHERINE A. OFFORD dormancy imposed by the endocarp, the embryo is physiologically dormant, requiring specific temperatures and/or germination stimulants, such as gibberellic acid or smoke- water, to induce germination (Mullins et al., 2002). Severe microbial contamination of seeds is a common occurrence in many Persoonia laboratory germination experiments. Several studies have suggested that surface-sterilising seeds in a mild bleach solution and/or preparing seeds under aseptic conditions can control outbreaks of contamination (Bauer et al., 2004; Frith and Offord, 2010). However, contamination in several Persoonia species has been observed to emerge from within the seeds. Following the discovery of a chemical compound with anti-microbial properties in the fruits of a P. pinifolia hybrid, it is possible that this chemical or similar compounds remain present in the endocarp post-dispersal, effectively protecting the seed from contamination (MacLeod et al., 1997). However, the location of this anti-microbial compound in the fruit was not identified. Under ex situ conditions, contamination might also be controlled by exposing Persoonia seeds to a brief heat-shock treatment prior to incubation, thereby killing microbial spores within the seed. Persoonia hirsuta Pers. (hairy geebung) is an obligate-seeding, spreading to decum bent shrub, 0.3–1.5 m tall, with moderate to densely hairy young branchlets. The species occurs in a highly patchy distribution within the dry sclerophyll woodlands in the Sydney region of New South Wales (NSW), Australia. It is taxonomically split into two subspecies: P. hirsuta subsp. hirsuta occurs within 20 km of the coast, and P. hirsuta subsp. evoluta occurs further inland. Intergrades between the subspecies have been recorded along an east-west cline. Despite the large area of occurrence, almost all known populations have gradually declined over the past two decades, and most consist of <10 plants or as isolated individuals. Consequently, the species is listed as Endangered under the Federal Environment Protection and Biodiversity Conservation Act 1999 and NSW Biodiversity Conservation Act 2016. Coupled with population decline, P. hirsuta is also threatened by clearing, urban development and inappropriate fire management, rendering the species particularly prone to local population extinction. Preliminary genetic evidence indicated a moderate level of inbreeding in two of the larger P. hirsuta populations, but some differentiation was noticeable, likely due to restricted and limited pollen and seed dispersal (Haynes, 2015). A lack of viable fruit-set has also hampered seed biology research on this species. With a future need to carry out conservation plantings, P. hirsuta is a model species for other high-interest plants displaying similar problems. Furthermore, optimising ex situ storage techniques is important for species that may experience poor fruit-set over multiple seasons. In the present study, the germination percentage was examined following the removal of endocarps before and after short-term low (−20°C) and room temperature storage. In addition, since P. hirsuta occurs in fire-prone ecosystems, we also tested the effect of a heat-shock treatment on germination. In June 2017, a population of 20 P. hirsuta plants was surveyed in Glenorie, in the Hills Shire district northwest of Sydney, NSW. The geographic location of the population meant that plants were most likely intergrades between the two subspecies. A collection of approximately 1,000 fruits was made from a number of these plants in November 2017. Fruits were brought to the laboratory at the Australian PlantBank at the Australian
108 GERMINATION OF PERSOONIA HIRSUTA SEEDS
Botanic Garden Mount Annan (ABGMA), and the fleshy layers were left to break down for three weeks before being cleaned and removed using tepid deionised water. The remaining pyrenes (endocarp and seed within) were then transferred to a drying room (15°C and 15% relative humidity) to equilibrate for four weeks until the commencement of experiments in February 2018. Three batches of 320 pyrenes were separated and assigned to either a no-storage control, low temperature storage, or room temperature storage treatment (figure 1). The endocarp was removed from 160 seeds from each batch by initially soaking pyrenes in deionised water for one hour and then cracking using a bench-mounted hand vice, and then re-dried for 24 hours at 15% RH and 15°C. For each storage treatment, the 160 extracted seeds and the remaining 160 pyrenes were vacuum-sealed in foil packets. Packets were then placed in a −20°C freezer (low temperature) or in the laboratory (room temperature) for six months. Thermocron iButton data loggers were placed in the laboratory and freezer to continually record the ambient temperature (figure 1). Following storage treatments, 80 pyrenes and 80 extracted seeds were placed in an aluminium tray and heat-treated in a fan-forced oven at 80°C for 10 minutes. This treatment was adopted as it was shown to increase germination success in several Grevillea species (Morris, 2000). The remaining pyrenes from each batch were then processed to extract the seeds, as described before. For each experimental treatment combination, four replicates of 20 seeds were sown into sterile plastic cell culture plates with 24 flat-bottom wells (Corning® Costar® TC- -1 ® treated). Around 2 ml of sterile water agar (9 g L ; Sigma-Aldrich ) was pipetted into each 3.4 mL well. Well-plates were preferred over traditional Petri plates as any microbial contamination would be isolated within a well. Seeds were then incubated at a 15/25°C alternating temperature regime with a 12-hour diurnal light cycle. These temperatures have been effective in maximising germination in six other Persoonia spp. from NSW (Catelotti and Offord, 2017). Plates were checked weekly for 12 weeks, with germination recorded when there was > 2 mm visible radicle growth. After this time, the number of non-germinated seeds with a visibly white and firm embryo per treatment combination were recorded as viable. Any seeds or seedlings with visible microbial growth were recorded as contaminated. All data were converted from percentages to proportions, adjusted for viability and then square-root-transformed to satisfy assumptions of normal distribution prior to analysis in R (version 3.4.0). The data were analysed using an ANOVA to examine the interactive effects of heat shock, storage conditions and endocarp removal on germination. Tukey’s HSD analyses were applied to identify significant differences among treatments. The final germination in the no-storage control seeds was independent of pre-treatment combinations, ranging from 66 to 90% (table 1). However, a significant interaction was detected among storage, heat and endocarp treatments from the statistical model (P = 0.019). Final germination percentage was significantly reduced when seeds were stored for six months without an endocarp and treated with heat compared with the control
(P < 0.001). Retaining the endocarp resulted in statistically similar germination success in both storage treatments. Comparatively, pre-storage endocarp removal gave lower final germination percentages following storage than when the endocarp was retained; however, these results were not statistically significant (table 1).
109 NATHAN J. EMERY AND CATHERINE A. OFFORD
LOW TEMPERATURE ROOM TEMPERATURE CONTROL STORAGE STORAGE
320 pyrenes 320 pyrenes 320 pyrenes
160 160 160 160 160 160 endocarps intact endocarps intact endocarps intact removed pyrenes removed pyrenes removed pyrenes
NO STORAGE 6 MONTHS STORAGE 6 MONTHS STORAGE
-18°C 25°C 24°C 23°C -19°C 22°C 21°C -20°C 20°C Feb Mar Apr May June July Feb Mar Apr May June July
endocarps endocarps endocarps removed removed removed
80 80 80 80 80 80 80 80 80 80 80 80 heat- not heat- not heat- not heat- not heat- not heat- not treated treated treated treated treated treated treated treated treated treated treated treated
SEEDS INCUBATEDSEEDS INCUBATED SEEDS INCUBATED
Figure 1. Experimental framework to examine the effect of the endocarp, heat-shock and short-term storage on the germination and microbial contamination of Persoonia hirsuta seeds. Storage temperature data are daily averages ± standard error (s.e.). Low-temperature and room temperature storage were approximately −20°C and 22°C, respectively.
110 GERMINATION OF PERSOONIA HIRSUTA SEEDS
Table 1. Germination percentage of Persoonia hirsuta seeds. Different lower-case letters indicate a significant
(P < 0.05) difference among different fruit treatment combinations within the same storage method (column); data for the different storage methods that significantly differed from the control within the same fruit treatment are shown in bold.
Fruit treatment Ex situ storage treatment
Endocarp Heat None (control) Low temperature Room temperature Retained No heat 90a 62a 71a Heat 66a 77a 84a
Removed No heat 79a 46a 38ab Heat 90a 35a 6b
The highly sensitive response of P. hirsuta seeds to pre-storage endocarp removal was also reflected in the occurrence of microbial contamination. Seeds stored without an endocarp had higher contamination frequency (61-91%) than those stored with an endocarp (16-26%). This increase highlights the need to store P. hirsuta seeds with intact endocarps to maintain viability in the short-term. Furthermore, post-storage heat-shock treatment appeared to have no effect on the prevalence of contamination. However, it is possible that a short 10-minute 80°C treatment was not long enough to suitably reduce microbial prevalence. Seed moisture content was not examined in this study, and it is possible that briefly soaking endocarps to extract seeds prior to storage would reduce viability during short-term storage. All endocarps had previously been subjected to moisture during the defleshing process and there is an equal possibility that, given the permeability of Persoonia endocarps (Norman and Koch, 2008), the embryos may have imbibed at this stage. It is unknown whether germination might be initiated during these processes, but it seems unlikely given the results of this work. Testing different soaking times on seed longevity would provide further useful insights into this question. Results from this study demonstrate the effectiveness of the woody endocarp in maintaining the viability of P. hirsuta seeds following short-term storage at low temperature and room temperature. While endocarp removal eliminates the mechanical dormancy constraint on a Persoonia seed, it is also important to consider other possible ecological roles of the endocarp (Bauer et al., 2004; Chia et al., 2016). If anti-microbial compounds occur in the endocarp, then mechanically extracting fresh seeds within could increase the likelihood of microbial growth, and therefore limit and bias germination results compared with germination in situ (MacLeod et al., 1997). In species such as P. hirsuta that display very poor fruit set and natural recruitment, it is important that propagation methods are interrogated to maximise seed germination and viability following ex situ storage. The difficulties in successfully germinating Persoonia species ex situ, particularly the rare and threatened species, emphasises the concern for ensuring persistence in situ. The results from this study show that retaining the endocarp prior to short-term storage can maintain germination while reducing the occurrence of microbial contamination. Further studies should investigate whether these results are upheld following longer storage times, and in other Persoonia species. For on-ground conservation management, it may be preferable to direct-sow Persoonia pyrenes in situ, even though an endocarp may take
111 NATHAN J. EMERY AND CATHERINE A. OFFORD several years to breakdown enough to allow germination to commence (Mullins et al., 2002; Chia et al., 2016). The potential of direct-sowing P. hirsuta pyrenes as part of restoration programs is under investigation.
Acknowledgements
Persoonia hirsuta fruits were collected in accordance with the scientific license conditions issued by the Office of Environment and Heritage, NSW (SL100569). The authors thank Lisa Willock for assistance in locating and accessing plants in the Hills Shire; Christie Foster for assisting with fruit collecting; and Veronica Viler for laboratory and nursery support. This work was funded by an Australian Coal Industry ACARP scientific grant (Project C24013).
References
Bauer, L.M., Johnston, M.E. and Williams, R.R. (2004). Fruit processing, seed viability and dormancy mechanisms of Persoonia sericea A. Cunn. ex R. Br. and P. virgata R. Br. (Proteaceae). Seed Science and Technology, 32, 663-670.
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