Liyanage, Offord and Sommerville (2020). Seed Science and Technology, 48, 2, 159-165. https://doi.org/10.15258/sst.2020.48.2.03
Research Note
Techniques for breaking seed dormancy of rainforest species from genus Acronychia
Ganesha S. Liyanage*, Catherine A. Offord and Karen D. Sommerville
The Australian PlantBank, The Royal Botanic Gardens and Domain Trust, Mount Annan, NSW 2567, Australia *Author for correspondence (E-mail: [email protected])
(Submitted February 2020; Accepted March 2020; Published online April 2020)
Abstract
We tested for dormancy in three species of Acronychia (Rutaceae) occurring in the rainforest in eastern Australia, A. imperforata, A. laevis and A. oblongifolia, by incubating fresh intact seeds on 0.8% water agar for one month at 25/10°C. Four different techniques were then tested for their effect on dormancy: (i) incubation of intact seeds on agar incorporating gibberellic acid (GA3); (ii) seed coat removal (decoating); (iii) scarification near the radicle emergence point (scarification-emergence point); and (iv) scarification opposite the radicle emergence point (scarification-back). Imbibition tests were performed to determine whether dormancy was due to an impermeable seed coat. Germination differed among treatments, but all three species showed a similar pattern. Intact seeds showed < 6% germination after one month indicating the presence of dormancy. Highest germination (> 65%) was observed following scarification-emergence point treatment. Seed coat removal also resulted in increased germination (40-47%), in comparison with intact seeds, but GA3 and scarification-back treatments did not (< 12%). Though the seedcoats of all species were permeable, increased germination responses to decoating and scarification-emergence point treatments suggest scarification is required to clear the radicle emergence point. This may be a useful dormancy-breaking technique for Acronychia spp. and may be suitable for related Rutaceae species.
Keywords: Acronychia, dormancy-breaking, Rutaceae, scarification, seed biology, seedcoat
Experimental and discussion
Seed dormancy is a trait that prevents seed germination during unfavourable environmental conditions where subsequent seedling establishment is likely to be unsuccessful (Baskin and Baskin, 2014). Ecologically, this is a beneficial trait that spreads the risk of failure of seedling establishment over time in disturbance-prone environments (Philippi and
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159 GANESHA S. LIYANAGE, CATHERINE A. OFFORD AND KAREN D. SOMMERVILLE
Seger, 1989; Philippi, 1993). However, from a conservation perspective, seed dormancy constrains the implementation of ex situ seed banking and in situ plant restoration. Good seed germination is essential to the success of both conservation practices. For example, standard seed banking involves drying seeds to 3-7% moisture content and storing them in subzero (≤ −20°C) freezer conditions (CBD, 2012). Testing how species respond to this drying and freezing involves a series of germination tests. In addition, germination tests need to be performed regularly to assess the viability of seed bank collections over time. Performing those tests is a challenge when the seeds are difficult or slow to germinate. Plant species from the Rutaceae family have often been recorded as producing seeds that are difficult to germinate due to dormancy (Auld, 2001; Floyd, 2008), yet studies on dormancy breaking techniques for Rutaceae species of non-sclerophyllous ecosystems, such as rainforests, are very limited (Elliot and Jones, 1982; Martyn et al., 2009). The genus Acronychia J.R. Forst. and G. Forst. is one of 152 genera in the Rutaceae family (POWO, 2019) and nearly 20 Acronychia species are found in Australian rainforests (Atlas of Living Australia, 2020). This is a genus that contains nationally endangered species such as A. littoralis T.G. Hartley and J.B. Williams (Environment Protection and Biodiversity Conservation Act, 1999) and that occurs in habitats that are themselves under threat (Sommerville et al., 2018). The development of ex situ seed bank collections and techniques to relieve dormancy is therefore urgent. In this current study we investigated three Acronychia species occurring in Australian rainforest, A. imperforata F. Muell., A. oblongifolia (A. Cunn. ex Hook.) Endl. Ex Heynh and A. laevis J.R. Forst. and G. Forst., in order to understand the treatments required to break dormancy and facilitate germination. This knowledge will contribute to efforts to conserve the species in ex situ seed bank collections. Acronychia imperforata is an endemic species found in lowland and littoral rainforests. Acronychia oblongifolia and A. laevis are recorded as occurring in warmer rainforests and dry and subtropical rainforests respectively (Floyd, 2008). For each species, mature fruits were collected from more than three well-established plants growing in The Australian Botanic Garden, Mount Annan, Australia (34°03'22.3''S 150°46'29.0''E). Acronychia oblongifolia fruits were collected in May 2018; A. imperforata and A. laevis fruits were collected in August 2018. Seeds were extracted from the fruits by hand and healthy- looking seeds were selected for experiments that commenced within two weeks of collection. As low seed fill is commonly reported in Rutaceae species, seed fill was first assessed using 40-60 seeds per species (depending on seed availability). Seeds were dissected and observed under a microscope and scored as ‘filled’ if they contained a firm green/white endosperm and embryo. The presence of dormancy was tested by placing three replicates of 15 seeds per species on Petri dishes containing 0.8% water agar. The Petri dishes were sealed with cling wrap to control moisture loss and the seeds were incubated at alternating temperatures of 25/10°C with a 12/12 hour light/dark cycle. The incubation temperature represented the maximum and minimum temperatures recorded around the time of seed dispersal for the regions in which these species mainly occur. Germination was recorded every second day for one month; a seed was considered to have germinated when the emerging radicle was > 1 mm in length. At the end of the incubation period, ungerminated
160 DORMANCY BREAKING OF ACRONYCHIA seeds were checked for firmness by pressing the seed with forceps. Any remaining firm seeds were dissected to check for the presence of a green/white embryo and endosperm and assess whether the seeds remained viable. The final germination percentage was adjusted for empty/unfilled seeds and an adjusted germination < 50% was considered to indicate dormancy (Baskin and Baskin, 2014). Four dormancy-breaking treatments were then applied to each species: (i) incubation of intact seeds on agar incorporating gibberellic acid (GA3 treatment); (ii) full seed coat removal (de-coating); (iii) scarification near the radicle emergence point (scarification- emergence point); (iv) scarification opposite the radicle emergence point (scarification- back). Three replicates of 15 seeds per treatment per species were placed on either 0.8% water agar (treatments ii, iii and iv) or 0.8% water agar incorporating 0.25% gibberellic acid (treatment i). An additional three replicates of 15 intact seeds were placed on 0.8% water agar as a control. Treatment (i) was not performed for A. laevis due to low seed numbers. All Petri dishes were incubated and seeds monitored for germination as described above. An imbibition test was performed to determine whether seedcoat permeability may have a role in maintaining dormancy. Two samples of 10 manually scarified and intact seeds were weighed then placed on 0.8% water agar and re-weighed at 24 hour intervals until all the scarified seeds had undergone imbibition. The germination percentages of intact seeds were calculated for each species to determine the presence/absence of dormancy. Differences in germination among dormancy- breaking treatments for each species were compared using a one factor Generalised Linear Model with a binomial error structure and logit link function. Where germination responses were significantly different, Tukey’s tests were performed to compare differences among treatments (Hothorn et al., 2008). Differences in seed mass between intact and scarified seeds resulting from imbibition were compared using a t-test. All data were analysed using the R 3.5.1 statistical platform (R Core Team, 2018). Seed fill was > 87% for both A. imperforata and A. oblongifolia and 60% for A. laevis. Final viable seed numbers were, therefore, corrected for empty seeds before the comparison of germination data for A. laevis. Intact seeds incubated on water agar germinated poorly for all species (5.0, 5.6 and 1.7% for A. imperforata, A. laevis and A. oblongifolia, respectively; figure 1). Application of dormancy-breaking treatments produced a similar germination pattern for all three species, with significant differences among treatments (A. imperforata df = 4, χ2 = 82.7, P < 0.001; A. laevis df = 2, Z = 29.4, P < 0.001; A. oblongifolia df = 4, χ2 = 119.3, P < 0.001). Gibberellic acid had no effect on germination for any species. Highest germination was observed when the seedcoat was scarified near the radicle emergence point (67, 72.5 and 65% for A. imperforata, A. laevis and A. oblongifolia, respectively) and this was significantly greater than the germination of the controls (Tukey’s test: A. imperforata Z = 4.6, P < 0.001; A. laevis Z = 3.5, P = 0.001; A. oblongifolia Z = 4.3, P < 0.001). Germination of de-coated seeds was higher, similar to the germination of scarification-emergence point treatment for all species except A. laevis which showed a significantly lower germination (Z = 2.4, P = 0.035). Germination was significantly less in the scarification-back treatment than both scarification-emergence point (A. imperforata Z = 3.6, P = 0.002; A. oblongifolia Z = 5.2, P < 0.001) and de- coating (A. imperforata Z = 2.8, P = 0.03; A. oblongifolia Z = 3.7, P < 0.001) treatments.
161 GANESHA S. LIYANAGE, CATHERINE A. OFFORD AND KAREN D. SOMMERVILLE
100 Treatment Control De-coating Gibberellic acid Scarification-back Scarification-emergency point 75
50 Germination (%) Germination
25
0
Acronychia Acronychia Acronychia imperforata laevis oblongifolia Species Figure 1. Mean germination percentages of three Acronychia species in response to different dormancy breaking treatments.
60 (A) Treatment 60 (B) 60 (C) intact scarified
40 40 40
20 20 20 Seed mass increase (%) mass increase Seed
0 0 0
0 25 50 75 100 125 0 25 50 75 100 125 0 25 50 75 100 125 Days Days Days Figure 2. Cumulative mean seed mass increase (± s.e.) for intact and scarified seeds of (A) Acronychia imperforata, (B) Acronychia laevis and (C) Acronychia oblongifolia placed on 0.8% water agar.
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The imbibition test showed a similar seed mass increase (20-40%) within the first 24-48 hours for both intact and scarified seeds (figure 2; Welch’s t test A. imperforata df = 58.5, t = 0.9, P = 0.35; A. laevis df = 74.3, t = 1.3, P = 0.19; A. oblongifolia df = 77.8, t = 1.5, P = 0.13). Intact seeds that failed to achieve > 50% germination within a month under favourable germination conditions, as seen in the three Acronychia species studied here, are considered to be dormant (Baskin and Baskin, 2014). In the wild, such a trait allows species to avoid germination where subsequent seedling establishment may be unsuccessful (Cohen, 1966; Philippi, 1993; Venable, 2007). In a nursery or seedbank laboratory, such seeds require treatment to break dormancy and facilitate germination (Turner and Merritt, 2009). The response to the dormancy-breaking treatments applied here varied among treatments, but all three Acronychia species showed a similar pattern. Both treatments that involved scarification of the seedcoat near the radicle emergence point (scarification-emergence point and de-coating) led to significantly greater germination than the untreated seeds, with scarification near the radicle emergence point producing the highest germination percentages for all three species. The lower germination percentage achieved by de- coating may have been the result of inadvertent damage to the embryo during removal of the full seedcoat. Scarification near the radicle emergence point therefore appears to be the best technique to break dormancy in A. imperforata, A. laevis and A. oblongifolia, and potentially other dormant species within the Acronychia genus. When germination is achieved by scarification, it indicates a potential role of the seedcoat in maintaining dormancy. The seed coat can impose dormancy by excluding water or oxygen (Brits and Manning 2019), mechanically restricting embryo growth, chemically inhibiting embryo growth, or preventing the leaching of growth inhibitors from the embryo itself (Baskin and Baskin 2014). In this study, the imbibition test showed a similar water imbibing capacity in both intact and scarified seeds, confirming permeability of the seedcoat to water. Scarification opposite the radicle emergence point showed a very low germination response, indicating there could be other factors influencing dormancy than the permeability of the seedcoat to either water or oxygen. Only the treatments that cleared the radicle emergence point (scarification-emergence point and de-coating) led to good germination. This confirmed the absence of germination inhibitors in the seedcoat (as seeds were able to germinate in the presence of part of the seed coat), but may be an indication of the embryo’s inability to push through the seedcoat or may indicate the role of a localised part of the seedcoat in controlling seed germination. Rutaceae is not a family that contains underdeveloped embryos (Baskin and Baskin, 2014) and the embryo to seed length ratio of the three species tested here confirmed the presence of a developed embryo in each (data not shown). This characteristic indicates that dormancy in these species is not likely to be the result of an inability to push through the seedcoat due to an underdeveloped embryo. Interestingly, Briggs et al. (2016) reported a similar dormancy- breaking mechanism to that used here for Grevillea juniperina, which germinated up to 95% when the seedcoat was removed over the micropylar end where the radicle emerges. In this case, the author suggested that the seedcoat constrained embryo elongation. This could also be the mechanism behind the dormancy of Acronychia species. Baskin and Baskin (2014) discussed the lack of push power in an embryo which plays a role in
163 GANESHA S. LIYANAGE, CATHERINE A. OFFORD AND KAREN D. SOMMERVILLE delaying germination of intact seeds, and in successful germination of excised embryos, of non-deep physiologically dormant species. Based on current study results, therefore, non-deep physiological dormancy can be proposed as the dormancy type present in the three Acronychia species. Closer observation of the embryo during germination and how this type of dormancy is broken in natural environments requires further study but, at this stage, we can confirm that scarification near the radicle emergence point can be used as a successful dormancy breaking technique for species from genus Acronychia for both ex situ and in situ conservation purposes.
Acknowledgements
The work of G.L. and K.S. is funded by the supporters of the Rainforest Seed Conservation Project including HSBC Bank Australia, TransGrid, the Klorane Botanical Foundation, The Ian Potter Foundation and several generous individuals.
Reference
Atlas of Living Australia (2020). Website at
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R Core Team (2018). R: A Language and Environment for Statistical Computing, R Foundation for Statistical Computing, Vienna, Austria.
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