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 © 2019 Emery and Offord. This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: https://creativecommons.org/licenses/by-nc/4.0 107 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 develop ment 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