Physiological Dormancy in Forbs Native to South–West Queensland: Diagnosis and Classification ⁎ G.L

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South African Journal of Botany 74 (2008) 208–213 www.elsevier.com/locate/sajb

Physiological dormancy in forbs native to south–west Queensland: Diagnosis and classification ⁎ G.L. Hoyle a, , K.J. Steadman b, M.I. Daws c, S.W. Adkins a

a Integrated Seed Research Unit, School of Land, Crop and Food Sciences, The University of Queensland, St. Lucia, QLD, 4072 Australia b School of Pharmacy, The University of Queensland, St. Lucia, QLD 4072, Australia c Seed Conservation Department, Royal Botanic Gardens Kew, Wakehurst Place, Ardingly, West Sussex RH17 6TN, United Kingdom Received 18 October 2007; received in revised form 29 October 2007; accepted 1 November 2007

Abstract

Seed physiological dormancy (PD) is reportedly the primary reason why many Australian native plants are not currently used for revegetation of degraded land. However knowledge of germination and dormancy of forb species from semi-arid environments is lacking. Consequently, we investigated germination of 15 Australian forb species from four families, particularly Asteraceae, native to south–west Queensland (Qld). Seeds were tested for viability using tetrazolium chloride (TZ) and sown at 5 to 35 °C. Nine species, including seven Asteraceae, achieved germination exceedingor not significantly lower (PN0.05) than TZ test results. Despite spring dispersal, the majority of species had optimal germination at temperatures reminiscent of winter months. Only six species exhibited low germination across all temperatures investigated when compared to TZ results (Pb0.05), i.e. low germination could not be attributed to low seed viability. Of these, Actinobole uliginosum (Asteraceae) had non-deep PD since seeds responded to gibberellic acid (GA3) and dry after-ripening. In contrast, Goodenia fascicularis appeared to exhibit deep PD since seeds did not respond to GA3 or dry after-ripening, and scarification led to germination of abnormal seedlings. It appears that, contrary to expectations, seeds of many forbs native tosouth– west Qld (9 of 15 in this study), possess negligible or no dormancy and may therefore be suitable for use in land rehabilitation. Other species e.g. G. fascicularis require further work to investigate dormancy mechanisms and develop reliable germination techniques before seeds can be used effectively. © 2007 SAAB. Published by Elsevier B.V. All rights reserved.

Keywords: Australia; Germination; Physiological dormancy; Seed; Temperature

1. Introduction timated if, in the absence of germination, seed dormancy is not diagnosed. In addition, reliable methods for alleviating (or at least Dormancy is a seed characteristic, the degree of which defines bypassing) dormancy are essential for effective use and con- the conditions necessary for seed germination. This block or servation of seeds. Once dormancy is diagnosed, classification of series of blocks(s) within a dormant seed prevents germination, the type and level of dormancy can aid investigation of ‘dormancy despite adequate water availability, temperature and gaseous breaking’ treatments and germination requirements (Baskin and conditions for germination (Benech-Arnold et al., 2000). There- Baskin, 2004). fore dormancy is not due to the absence of environmental factors It is often possible to bypass the block(s) to germination and required to initiate germination and must be considered separately stimulate PD seeds to germinate by applying agents such as from germination requirements (Vleeshouwers et al., 1995). For gibberellins, e.g. GA3 (Cohn et al., 1989; Foley, 1992). However, both conservation and restoration, it is crucial that dormant seeds such ‘germination inducing factors’ do not affect dormancy status are distinguished from non-viable seeds. Viability can be underes- (Vleeshouwers et al., 1995) and can result in seedling abnormalities. In contrast, PD can be alleviated altogether by applying treatments that regulate temperature and seed moisture content (Benech-Arnold et al., 2000; Vleeshouwers and Bouwmeester, ⁎ Corresponding author. 2001). Such approaches are also likely to promote the develop- E-mail address: [email protected] (G.L. Hoyle). ment of normal, healthy seedlings.

Table 1 use of native seed in land revegetation (Merritt et al., 2007). – Forb species and their collection sites in south west Queensland Furthermore, the majority of previous studies have focused on Family Authority Species Collection site Collection native grasses, hard-coated PY seeds, and species native to tem- GPS co-ordinates date perate and Mediterranean-type regions within Australia. Con- SE sequently, this study aimed to identify seed dormancy in a range Asteraceae (A. Gray) Actinobole − 28°06′ 145°76′ 22.10.04 of previously unstudied Australian forb species spanning four H. Eichler uliginosum 87.0′′ 39.2′′ families native to the semi-arid tropical region of south–west Qld. Asteraceae Sond. and Brachyscome − 28°06′ 145°81′ 22.10.04 ′′ ′′ F. Muell. melanocarpa 10.7 88.6 2. Materials and methods ex Sond. Asteraceae R. Br. Calotis − 27°98′ 148°33′ 21.10.04 cuneifolia 00.5′′ 37.8′′ 2.1. Seed collecting and processing Asteraceae N.T. Burb. Camptacra − 28°02′ 148°43′ 21.10.04 barbata 49.4′′ 69.5′′ Seeds of 15 species across four families of native forbs were − ′ ′ Asteraceae Turcz. Gnephosis 28°00 146°39 09.12.04 collected in south–west Qld between October and December arachnoidea 48.8′′ 33.4′′ Asteraceae (F. Muell.) Leiocarpa − 28°06′ 145°81′ 22.10.04 2004 (see Table 1). Where possible, at least 10,000 seeds/ Paul G. brevicompta 10.7′′ 88.6′′ species were collected from N50 individual plants. Only mature Wilson. seeds close to the point of natural dispersal were collected. Asteraceae F. Muell. Leptorhynchos − 28°00′ 146°39′ 09.12.04 Following collection, seeds were treated as if they were to be ′′ ′′ baileyi 48.8 33.4 used in land revegetation by being stored in paper envelopes at Asteraceae (DC.) Minuria − 26°98′ 146°03′ 23.10.04 Benth. integerrima 57.6′′ 26.6′′ 15 °C and 15 to 20% relative humidity (RH) for between 10 and Asteraceae (Schltdl.) Pycnosorus − 28°02′ 148°46′ 07.12.04 16 weeks before being used in germination experiments. Non- Sond. chrysanthes 70.0′′ 05.4′′ seed material was removed by hand, aspirator or by rubbing Asteraceae (DC.) Paul Rhodanthe − 28°00′ 146°39′ 09.12.04 seed capsules/flowers through a sieve using a rubber bung. G. Wilson floribunda 48.8′′ 33.4′′ Asteraceae (DC.) Paul Rhodanthe − 28°06′ 145°76′ 22.10.04 G. Wilson moschata 87.0′′ 38.2′′ 2.2. Seed viability and germination testing Asteraceae (F. Muell. Vittadinia − 27°97′ 148°01′ 08.12.04 ex Benth.) pterochaeta 90.3′′ 36.1′′ Viability of 3 replicates of 20 seeds/species was assessed using J.M. Black the tetrazolium chloride (TZ) staining technique (ISTA, 2003). − ′ ′ Campanulaceae P.J. Sm. Wahlenbergia 28°06 145°81 22.10.04 Seeds were initially hydrated on plain agar for 24 h at room timidifructa 10.7′′ 88.6′′ Goodeniaceae F. Muell. Goodenia − 28°06′ 145°81′ 24.10.04 temperature before being scarified (away from the embryo axis) and Tate. fascicularis 10.7′′ 88.6′′ and placed in TZ solution at 30 °C and darkness for 24 h. Seeds Plantaginaceae Decne. Plantago − 28°06′ 145°81′ 22.10.04 were then cut in half and examined. Only uniformly stained red/ cunninghamii 10.7′′ 88.6′′ dark pink embryos were considered ‘viable’. Germination tests used 3 replicates of 15 seeds each sown The most recent attempt to classify the observed diversity of into 9 cm diameter plastic Petri dishes containing 1% agar- dormancy types and levels (Baskin and Baskin, 2004) builds upon water. Petri dishes were sealed inside plastic bags to avoid agar Nikolaeva's comprehensive system (Nikolaeva, 1969). Physio- desiccation. Seeds of all species were placed in germination logical dormancy (PD), the most common form of dormancy incubators at 15, 20, 25 and 30 °C, with constant light provided (Baskin and Baskin, 2004), is thought to be caused by a physio- by fluorescent tubes (ca.50μmol m− 2 s− 1). Five of the species logical state of the embryo and possibly a decreased gas perme- (A. uliginosum, Brachyscome melanocarpa, Camptacra bar- ability of seed covering structures (Baskin and Baskin, 2001). bata, Plantago cunninghamii and Goodenia fascicularis) were Three levels of PD are recognized: non-deep, intermediate and also sown at 5, 10 and 35 °C. Germination, defined as radicle deep (see Baskin and Baskin, 2004). Physical dormancy (PY) is emergence by at least 1 mm, was scored every 7 d, germination due to a water impermeable seed or fruit coat, morphological tests ran for 60 d and any non-germinated seeds were assessed dormancy (MD) requires that an underdeveloped embryo grows using a cut test. Empty or necrotic seeds were excluded when before germination can commence and combinations of PD and calculating percentage germination. MD also exist (MPD) (Baskin and Baskin, 2001). In recent years, partly due to increasing demands for biolo- 2.3. Treatment conditions gically diverse land rehabilitation, seed dormancy of Australian native seeds has received considerable attention. It is frequently Based on high viability (results of TZ tests) and low germi- reported that the majority of Australian plant species possess seed nation percentages, two species (A. uliginosum and G. dormancy mechanisms of some sort (Bell, 1999; Koch and fascicularis) underwent further investigation. Gibberellic acid Dixon, 2000; Tieu et al., 2002; Merritt et al., 2007), and that (GA3) was applied to seeds via incorporation into the agar species in diverse families, including Euphorbiaceae, Asteraceae germination medium; seeds received either constant application and Goodeniaceae (i.e. herbaceous, non-grassy species), have of 250, 125 or 62.5 mg/L throughout the germination test, or a unknown dormancy alleviation and germination requirements. As pulsed application of 250 mg/L for 24 h before seeds were a result, dormancy is cited as the greatest obstacle to the effective moved to plain agar for germination. Mechanical scarification 210 G.L. Hoyle et al. / South African Journal of Botany 74 (2008) 208–213 was applied to seeds using a scalpel blade; G. fascicularis seeds were carried out at constant 20 °C (G. fascicularis)or15°C were completely de-coated, or a small piece of seed coat re- (A. uliginosum), 12/12 h light/dark. moved from the vicinity of the radicle or cotyledons, before seeds were moved to germination test conditions. Seeds re- 2.4. Statistical analysis ceiving a dry after-ripening (DAR) treatment were stored in paper envelopes inside a drying oven at 40 °C, 15–20% RH A 2-sample t-test was carried out/species to assess whether and darkness for four weeks before being moved to germina- there was a difference between observed maximum percentage tion test conditions. Imbibition curves for scarified and non- germination and seed viability. To assess differences between scarified seeds were obtained by periodically weighing seeds combinations/concentrations within treatments upon A. uligi- as they imbibed on agar at room temperature. Germination tests nosum and G. fascicularis, one-way ANOVAwas carried out on

Fig. 1. Percentage germination at day 60 (mean±S.E.) of Asteraceae (a–l), Campanulaceae (m), Goodeniaceae (n) and Plantaginaceae species (o) at 10, 15, 20 and 25 °C. Hashed bars indicate the percentage viability as determined by tetrazolium chloride staining (mean±S.E.). G.L. Hoyle et al. / South African Journal of Botany 74 (2008) 208–213 211 arcsine transformed data (Zar, 1984). ANOVA pair-wise com- and P. cunninghamii (Fig. 1o). The remaining six species achieved parisons were made using Fisher's l SD test at 5% significance significantly lower maximum germination (2-sample t-test: level. Statistical analysis was carried out in Minitab 15 (Minitab P≤0.001) compared with viability, at the temperatures investi- Inc., 2007). gated; A. uliginosum (Fig. 1a), Gnephosis arachnoidea (Fig. 1e), Rhodanthe floribunda (Fig. 1j), Rhodanthe moschata (Fig. 1k), 3. Results Vittadinia pterochaeta (Fig. 1l) and G. fascicularis (Fig. 1n). A. uliginosum and G. fascicularis exhibited the highest viability 3.1. Seed viability and germination coupled with the lowest germination and were therefore chosen for further investigation. Seed viability of all species ranged from 65 to 100% ac- cording to TZ staining (Fig. 1). Seeds of all 15 species responded to 3.2. G. fascicularis at least one of the constant temperatures, to achieve between 10% (A. uliginosum at 15 °C) and 100% (C. barbata at 15 °C) final Intact and scarified G. fascicularis seeds imbibed at the same germination (Fig. 1). The majority of species reached maximum rate and to the same extent (Fig. 2a) and it was observed that seeds germination at 15 °C (9 species in total). However B. melanocarpa contained large spatulate embryos and relatively little endosperm. responded better to 10 °C and G. fascicularis responded better to There was little response to constant application of any concen- 20 °C (Fig. 1). Of the five species sown at the full range of tration of GA3 at 20 °C for 60 days (one-way ANOVA: F=2.39, germination temperatures (5 to 35 °C) four germinated at 5 °C; A. d.f.=3, P=0.144,); maximum germination was only 18% with uliginosum (5%), B. melanocarpa (38%), C. barbata (100%) and 125 mg/L GA3 (Fig. 2b). Similarly, there was little response to the P. cunninghamii (100%), and there was no germination of any DAR either with or without GA3 in the germination media (one- species above 25 °C. In relation to seed viability, a total of 9 species way ANOVA: F=1.62, d.f.=3, P=0.260); DAR followed by achieved germination that exceeded, equaled or was not signi- constant application of 125 mg/L GA3 achieved only 22% ficantly lower (2-sample t-test: PN0.05) than results from TZ germination (Fig. 2c). Seeds responded to mechanical scarifica- testing: B. melanocarpa (Fig. 1b), Calotis cuneifolia (Fig. 1c), C. tion and de-coating prior to germination testing (one-way barbata (Fig. 1d), Leiocarpa brevicompta (Fig. 1f), Leptor- ANOVA: F=6.94, d.f.=3, P=0.013), for example scarification hynchos baileyi (Fig. 1g), Minuria integerrima (Fig. 1h), Pycno- above the radicle stimulated 71% germination after 60 days at sorus chrysanthes (Fig. 1i), Wahlenbergia timidifructa (Fig. 1m) 20 °C (Fig. 2d). However de-coating seeds caused seedlings to

Fig. 2. Imbibition of intact and scarified G. fascicularis seeds upon agar (a). Final mean percentage germination (±S.E.) of G. fascicularis at 20 °C, 12/12 h light/dark, after constant application of various concentrations of GA3 (b), dry after-ripening (DAR)±GA3 (c) and decoating and mechanical scarification (MS) in the vicinity of the cotyledons (cot end) or radicle (rad end) (d). 212 G.L. Hoyle et al. / South African Journal of Botany 74 (2008) 208–213

Many Australian plant species synchronise seed germination with the reliable winter–rainfall period in their native habitat (Bellairs and Bell, 1990; Curtis, 1996). While semi-arid tropical Qld does not experience a ‘drought’ season in the strict sense (rainfall is distributed throughout the year), summer temperatures are high (mean min. and max. temperatures of 22 and 35 °C respectively). Consequently, lower mean min. and max. autumn (14 to 28 °C) and winter (7 to 20 °C) temperatures (BoM, 2000), which will result in reduced evapo-transpiration, may signal optimal germination conditions and increased chances of successful seedling establishment. In support of this we found that maximum germination of many of the forbs investigated was dependent upon cooler temperatures (ca. 15 °C) that are not necessarily present at the time of seed dispersal. Thus, rather than possessing seed dormancy, germination in these species may simply be temperature dependent (Baskin and Baskin, 2001), i.e. seeds can only germinate in autumn/winter despite being dispersed in summer. Low germination when compared to TZ results suggests that seeds of six of the species investigated possess dormancy mechanisms of some sort, although with the exception of G. fascicularis and A. uliginosum for which there was further investigation, we are unable to confirm dormancy type. Imbibition curves for intact and scarified seeds of G. fascicularis revealed that low germination was not a result of seed impermeability or PY (Fig. 2a), results that are supported by previous studies that have found no evidence of PY in this family (Baskin et al., 2006). After observing the large embryo size relative to seed size, MD and MPD were also ruled out. Fig. 3. Final percentage germination (±S.E.) of A. uliginosum at 15 °C, 12/12 h Germination of G. fascicularis was slow and gradual and continued light/dark with constant and pulsed application of 250 mg/L GA3 (a) or a pre- to increase beyond 4 weeks (data not shown). Water permeability, germination test dry after-ripening (DAR) treatment (b). low embryo length: seed length ratio and slow germination are grow abnormally long radicles that did not penetrate the agar, common characteristics of seeds exhibiting PD (Baskin and Baskin, before seedlings became stunted and died. Scarification above the 2001). The fact that these seeds also failed to respond to GA3,dry cotyledons resulted in stunted radicle growth and scarification after-ripening and exhibited seedling abnormalities post-scarifica- close to the radicle meant that cotyledons were not able to fully tion suggested deep PD (see Baskin and Baskin, 2004). Similarly, emerge from the seed coat (results not shown). PD has been diagnosed for a number of species belonging to this family (Sugimoto and Lidbetter, 2002; Cochrane and Probert, 3.3. A. uliginosum 2006) with studies suggesting that a combination or series of factors may be required to achieve maximum germination of other Good- Constant application of 250 mg/L GA3 at 15 °C achieved 100% eniaceae species. germination of A. uliginosum, and a 24 h pulse achieved 38% Germination of A. uliginosum remained low at all temperatures germination (Fig. 3a). Seeds were also responsive to the 4 week (Fig. 1a). Constant application of 250 mg/L GA3 bypassed PD to DAR treatment (one-way ANOVA: F=23.6, d.f.=2, P=0.001); achieve 100% germination (Fig. 3a). In addition, the DAR germination increased gradually to 63% at 15 °C (Fig. 3b). treatment alleviated PD to achieve N60% germination (Fig. 3b). These results suggest that A. uliginosum possesses non-deep PD, 4. Discussion a conclusion that is supported by reports of germination requirements for other Australian Asteraceae genera (Bunker, 1994; Physiological dormancy (PD) is reported to be common in seeds Plummer and Bell, 1995; Peishi et al., 1999). of Australian native plants including species in the Asteraceae and The role of dormancy is to prevent germination during favorable other forb genera (Merritt et al., 2007). Despite this, 9 species out of conditions, when the resulting plant is not likely to survive and 15, including 7 Asteraceae species, achieved germination that reproduce (Vleeshouwers et al., 1995). We conclude that the spe- matched viability, without application of germination stimulants or cies investigated in this study exhibit strategies for avoiding ger- dormancy alleviating treatments (Fig. 1). If dormancy alleviation mination during superficially suitable spring and early summer occurred in the seed storage conditions, it was likely to have been conditions. Some species exhibit dormancy mechanisms that are very slow due to low seed moisture content and low temperature likely to exist to postpone germination until after the summer. (Steadman et al., 2003). Therefore we concluded that seeds of these However other species simply have a germination requirement for species possessed negligible or no dormancy upon dispersal and did cool temperatures that are more indicative of autumn/winter. not have a requirement for alternating temperatures. Since the majority of species studied germinated readily without G.L. Hoyle et al. / South African Journal of Botany 74 (2008) 208–213 213

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