Non-deep physiological dormancy and the effect of temperature on the ...

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Journal of Horticultural Science & Biotechnology (2012) 87 (1) 52–56

Non-deep physiological dormancy and the effect of temperature on the germination of Dipteronia dyeriana seed

By A. J. TANG*, M. H. TIAN and J. P. LIU College of Life Sciences, Chongqing Normal University, No. 12 Tianchen Road, Chongqing 400047, P. R. China (e-mail: [email protected]) (Accepted 6 September 2011) SUMMARY Dipteronia dyeriana Henry (Aceraceae), a plant native to southwest China, is being grown extensively in home gardens and along roadsides for its high ornamental value. However, in the field it is an endangered species due to low rates of seedling establishment. We found that the seed coat was water-permeable, and that pre-treatment with concentrated H2SO4 or hot water, or the partial or total removal of the seed coat, could effectively increase the frequency of seed germination. In particular, total removal of the seed coat enhanced the percentage germination to 97% at 15ºC/4ºC (light/dark) with a 12-h photoperiod. Cold stratification (at 4ºC) for 3 months, or a 3-month out-ofdoors stratification (ODS) at 3ºC – 17ºC significantly accelerated the release from dormancy. Among the various germination temperatures tested, 15ºC/4ºC (day/night) was optimum (97% germination), followed by 10ºC (day and night). Seedling emergence in this species increased gradually to 47% in an outdoor experiment. Thus, D. dyeriana seeds have non-deep physiological dormancy (PD) that can be broken in Winter, allowing seeds to germinate in the following Spring.

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n endangered species is defined as a species having a high possibility of extinction in the near future (Saeki, 2008). One of the major causes of the extinction of plant species is a failure to contribute offspring to the next generation (Washitani and Yahara, 1996; Saeki, 2008). Sexual reproduction in the field is essential for the continued existence of several plant species. In China, such species include Manglietiastrum sinicum (Zheng et al., 2008), Olimarabidopsis pumila (Tang et al., 2009), and Dipteronia sinensis, where hardly any seed germination occurs in the field. Seed dormancy and germination play important roles as causal factors for the limited abundance or restricted distribution of a species (Fiedler, 1986). Thus, to artificially expand plant populations, it is important to understand what controls the breaking of seed dormancy and seed germination in the field, and to clarify those factors that affect seedling establishment and survival (Schemske et al., 1994; Zhang et al., 2002). In many cases, dormancy is imposed upon a seed by the seed coat, by factors within the embryo, or by both (Groot and Karssen, 1987; Sanchez et al., 1990; Debeaujon et al., 2000; Foley, 2001). Seed coat-imposed dormancy (i.e., a mechanical restraint) is a widespread phenomenon and more common than true embryo dormancy, in which the embryo fails to initiate growth even when removed from the constraints imposed by the seed coverings (Bewley and Black, 1994; Bewley, 1997; Ogawa et al., 2003). Thus, weakening or removal of the seed coat, perhaps combined with an increase in the growth potential of the embryo, can result in the protrusion of the radicle (Nabors and Lang, 1971). For example, Arabidopsis thaliana seeds are dormant *Author for correspondence.

insofar as both the embryo and the seed coat have been implicated in the control of dormancy and germination (Bewley and Black, 1994; Finch-Savage and LeubnerMetzger, 2006; Müller et al., 2006). The process of loss of dormancy can be hastened or slowed by environmental conditions. As reported by some investigators, cold stratification increased the efficiency of seed germination in Acer platanoides (Pawlowski et al., 2004) and A. pseudoplatanus (Gregorová, 1977), and in six other Acer species with dormant seeds (Shi et al., 2006). Although D. dyeriana is a rare species, locally, and is assessed as “endangered” on the China Red List of Endangered Species (Wang and Xie, 2004), it is becoming an important ornamental plant. Although we know that this species flowers in April, and that fruits ripen in October and are shed from the mother plants in early November (Li et al., 2003), data on the germination of brown mature D. dyeriana seed are not available. In some cases, cold stratification and dry storage, physical or chemical scarification, or weathering in the soil can facilitate the germination of seeds that possess physical dormancy (PY) and/or physiological dormancy (PD; Foley, 2001; Baskin and Baskin, 2004). Thus, the effects of several of these treatments were applied to study dormancy breaking in D. dyeriana seed. At present, D. dyeriana occurs only in sub-tropical, southwest China (Gu, 2003). As reported elsewhere, fossils of D. dyeriana have been found in colder temperate zones in northeast China (Manchester, 1999; McClain and Manchester, 2001). Based on its fossil record and its current geographic range, we hypothesised that low temperatures would be required for seed germination. Thus, the effect of temperature regime on seed germination in this species was also tested.

A. J. TANG, M. H. TIAN and J. P. LIU MATERIALS AND METHODS Seed collection and measurement Mature samaras were collected on 14 November 2008 from D. dyeriana plants growing in a degraded woodland located in Mengzi county (23º 22.2752'N; 103º 47.7140'E; 2,127 m asl) with a sub-tropical climate, in south-eastern Yunnan Province, southwest China. After being brought back to the laboratory, the seeds were separated from the dry fruit by hand. Subsequently, we measured the lengths and widths of 20 seeds using vernier calipers. The initial water contents of ten seeds were determined by the high constant-temperature oven method at 103ºC for 17 h (ISTA, 1999), and calculated on a fresh weight (FW) basis. The FW of a random sample of 100 seeds was determined, based on four replicates of 25 seeds each. General germination test All germination tests were performed on moist filter paper (Whatman No. 1) previously moistened with distilled water in 90 mm-diameter glass Petri dishes placed in a temperature- and light-controlled incubator (HPG-280B Illuminating Incubators; Har’erbin Electronic Apparatus Manufactory, Har’erbin, P. R. China). A 12 h photoperiod was provided by warmwhite fluorescent tubes at a photosynthetically-active radiation (PAR) intensity of 40 µmol m–2 s–1, measured using a LI-1400 Data Logger (LI-COR, Inc., Lincoln, NE, USA). Before testing, fresh or variously pretreated seeds were surface sterilised in 1% (w/v) sodium hypochlorite solution for 10 min and rinsed three-times with de-ionised water to reduce fungus infestation. Four replicates of 25 seeds each were tested for germination on top of filter papers previously moistened with 5 ml distilled water. Seeds were considered germinated when the radicle had emerged 2.0 mm from the intact seed. Germination test of fresh seed One week after collection, some freshly-harvested intact seeds were incubated at a range of constant temperatures (i.e., 4ºC, 10ºC, 15ºC, or 20ºC) and at a daily altering temperature of 15ºC/4ºC (day/night). Germination was also tested at ambient temperatures (AT; varying between approx. 17ºC – 3ºC). After 35 d, all germination experiments were terminated. Effect of stratification on germination To determine whether a period of cold (4ºC) and damp [relative humidity (RH) 60 – 70%] treatment, referred to as stratification, or out-of-doors stratification (ODS; varying between approx. 3ºC – 17ºC) could break dormancy, 500 seeds were stored in a closed polyethylene bag (170 mm  250 mm) with a small volume of moist vermiculite (approx. 60 – 70% RH) at 4ºC. Each polyethylene bag was perforated five-to-ten times with a pin. For ODS, 500 seeds were stored similarly 1 week after collection. During these 3-month stratification treatments, we observed and counted the numbers of seeds that germinated every 2 weeks. At the same time, we removed 60 non-germinated seeds for incubation at 15ºC/4ºC (day/night) under the same photoperiod as described above. Twenty seeds were assessed per replicate, with three replicates.

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Effect of removing the seed coat on germination Two days after collection, the seed coat was removed from 100 seeds. Peeling-off the seed coat left the embryo (i.e., the embryonic axis and cotyledons) as a single unit, hereafter referred to as a “de-coated seed”. Seed coats were removed using forceps and a scalpel. De-coated seeds were incubated at 4ºC, 10ºC, 15ºC, 20ºC, 15ºC/4ºC (day/night), or at AT. Intact seeds were used as controls. The percentage germination in each treatment was calculated each week for 35 d. Impact of hot water or concentrated sulphuric acid treatment on seed germination To ascertain whether it was possible to improve the percentage germination of D. dyeriana seed, two physical treatments were applied to 100 seeds per treatment 7 d after collection. The treatments consisted of : (1) dipping intact seed into hot water (HW; 80ºC) for 0.5, 1.0, 5.0, or 10.0 min; or (2) soaking intact seed in concentrated sulphuric acid [CSA; 98% (v/v) H2SO4] for 0.5, 1.0, or 5.0 min. Untreated seed (n = 100) were used as controls. Four replicates of each treatment, each with 25 seeds, were then allowed to germinate (as above) for 35 d at 15ºC/4ºC in the incubators described above. Out-of-doors seedling emergence To determine the timing of germination in D. dyeriana seed left out-of-doors (OD) 3 d after collection, 200 seeds were sown at a depth of 30 mm in each of two small flats in an experimental field at the Kunming Institute of Botany, P. R. China. Based on our records, ambient temperatures varied from 3ºC (the lowest temperature during the 3-month experimental period) to 17ºC (the highest temperatures during the 3-month experimental period) which was in the dry season with approx. 200 mm precipitation, similar to that in the natural habitat of this species. Germination was observed each week, and seedling emergence was counted. Imbibition Following the method of Baskin et al. (2006), tests were carried out to determine whether or not the seed coats were water-permeable 4 d after harvest. Ten nonscarified seeds and ten seeds of D. dyeriana mechanically-scarified using small forceps to make a hole in the seed coat were weighed individually. Three replicates, each of ten scarified seeds or ten non-scarified seeds were used. All seeds were placed in 10 ml distilled water at room temperature (18º ± 3ºC) for 1, 2, 4, 8, 12, 24, 32, 40, 48, 72, 96, or 120 h. The seeds were then taken out, blotted dry, and weighed individually. The amount of water taken up by each seed was determined as the increase in seed mass, based on the following equation: Wn = Wi – Wd where Wn = net increase in mass, Wi = mass of the imbibed seed, and Wd = mass of the dry seed. Data analysis Percentages of germination were calculated and analysis of variance (ANOVA) was used to determine the effects of the various factors tested on the percentage germination (arcsine transformed for linearity).

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Dormancy and germination of Dipteronia dyeriana seed 100

Increase in mass (g FW)

0.7

Control

Germination (%)

0.8 Scarified

0.6 0.5 0.4

80 60 40 20 0

0.3

Control

HW-0.5

HW-1

0.2

HW-5

HW-10

CSA-0.5

CSA-1

CSA-5

Physical treatment

0.1 0 1

2

4

8

16

24

32

40

48

72

96 120

Imbibition time (h) FIG. 1 Water uptake in control (non-scarified) seeds and scarified seeds of Dipteronia dyeriana during imbibition at room temperature (18º ± 3ºC). Each datum point is a mean (n = 3) of three replicates (± SD).

Differences between the means of replicates were determined using the least significant difference (LSD) test at P < 0.05, or by standard errors of the mean values.

RESULTS Seed information Each almost-round samara contained one or two nonendospermous seeds, and the fruit wing is thin and leathery. Three indices were measured: (i) seed moisture content (approx. 9.2 ± 0.6%); (ii) the 1,000-seed weight (170.1 ± 6.3 g); and (iii) seed length and seed width (10.1 mm  8.6 mm). The papery seed coat was found to be water-permeable. Water uptake by intact D. dyeriana seeds was rapid, irrespective of whether the seed coat had been scarified or not (Figure 1). Effect of temperature, seed coat, and pre-treatment on germination The time-course of germination for de-coated seeds showed that 15ºC/4ºC was optimum, the percentage germination being 97%. Constant higher or lower temperatures did not give such good results. In particular, the lowest and highest temperatures (≤ 4ºC and 20ºC) were least favourable for seed germination in 100

FIG. 3 Percentage germination data for intact Dipteronia dyeriana seeds kept at 15ºC/4ºC with a 12-h photoperiod for 30 d. Pre-treatments included being dipped in hot water (HW) at 80ºC for 0.5, 1.0, 5.0, or 10 min (shown as HW-0.5, HW-1, HW-5, and HW-10, respectively) or being soaked in concentrated H2SO4 (CSA) for 0.5, 1.0, or 5.0 min (shown as CSA-0.5, CSA-1 and CSA-5, respectively). Values were means (n = 4) of four replicates (± SD).

this species (Figure 2). For fresh intact seed of D. dyeriana, germination occurred at low levels (< 5%) within 35 d at any of the temperatures applied (data not shown). In contrast, freshly-harvested de-coated seeds germinated rapidly and to high values (83%, > 77%, and > 62%) at constant temperatures of 10ºC, > 15ºC, and at AT within 30 d (Figure 2). Differences in the final percentages of germination among the various temperatures were significant (P < 0.01). HW or CSA treatment improved the percentage germination, if the duration of treatment was appropriate. In particular, CSA applied to seeds for 1 min was most effective at improving germination (Figure 3). Total removal of the seed coat was most effective for promoting germination (P < 0.05; Figure 3). Effect of cold stratification on germination Intact seed of D. dyeriana did not germinate under any of the test conditions applied after 4-weeks cold stratification, or ODS. After being stratified, a portion of the seeds germinated naturally in moist vermiculite (Table I). After 10 weeks or more of cold stratification or ODS, seed dormancy was significantly reduced or eliminated. The difference between cold stratification and ODS was slight, and not significant (Table I). Seedling emergence A total rate of seedling emergence of 13% was scored on day-60, increasing to 47% after 3 months. No seedling

Germination (%)

4ć 80

10ć

60

15ć

TABLE I Percentage germination (mean ± SD) of Dipteronia dyeriana seed during cold stratification at 4ºC or out-of-doors (approx. 3ºC – 17ºC), and of stratified seeds incubated at 15ºC/4ºC (day/night) with a 12-h photoperiod for 35 d

20ć 40

Cold stratification at 4ºC

15ć/4ć AT

20

Stratification period (weeks)

0 0

7

14

21

28

30

Time (d) FIG. 2 Time-courses of percentage germination for freshly de-coated Dipteronia dyeriana seeds kept at a range of temperatures with a 12 h photoperiod. Values were means (n = 4) of four replicates (± SD). AT, ambient temperature (3ºC – 17ºC).

2 4 6 8 10 12

Out-of-doors stratification (ODS)

GPBS* (%)

FGAI** (%) ± SD

GPBS (%)

FGAI (%) ± SD

0 0 7 11 17 31

0 0 19 ± 3 c† 81 ± 2 b 93 ± 1 a 94 ± 1a

0 0 4 6 15 24

0 0 17 ± 4 c 74 ± 2 b 86 ± 3 a 91 ± 1 a

*GPBS, germination percentage (%) in a polyethylene bag during stratification. **FGAI, final germination percentage after 35 d incubation. † Mean values (n = 4) in each column followed by a different lower-case letter were significantly different by the LSD test at P ≤ 0.05.

A. J. TANG, M. H. TIAN and J. P. LIU emergence from the soil was observed in the remaining seeds.

DISCUSSION Each mature seed of D. dyeriana has a developed embryo and a permeable seed coat (Figure 1), but freshly-harvested intact seed did not germinate under the conditions tested here. In contrast, total removal of the seed coat, or partial abrasion of the seed coat around the radicle, resulted in high percentages of germination within 30 d (Figure 2; Figure 3). These results suggest that, in order to penetrate the seed coat, the embryo must exert a certain minimum force. Thus, it was inferred that the embryos in D. dyeriana seed have enough force to break through the seed coat. The time-courses of germination in Figure 2 showed that temperature affected germination. The optimum was 15ºC/4ºC (day/night), followed by a constant temperature of 10ºC (Figure 2). However, de-coated or coat-weakened seed could only germinate at lower temperatures. Clearly, temperature had a direct effect on germination. This could be explained as follows: in the field, dormant seeds are commonly subjected to low (fluctuating) temperatures after dispersal. Such temperature fluctuations are frequently effective at breaking dormancy (Baskin and Baskin, 1998). In the present study, the relatively higher constant temperatures apparently had inhibitory effects on the germination of D. dyeriana seed. Similarly, physiologically dormant seeds of Eriastrum diffusum (Polemoniaceae) and Eriogonum abertianum (Polygonaceae) only germinated at low temperatures (Baskin et al., 1993). Schütz (2000) also reported that almost all Carex species investigated responded positively to diurnal temperature fluctuations, and only a few species germinated at temperatures below 10ºC. Similar conclusions were drawn by Probert (2000) and Baskin and Baskin (2004) in other species. For seeds of D. dyeriana, weakening of the seed coat by short-term immersion in hot water (HW) or concentrated H2SO4 (CSA) was effective at promoting germination. In addition to such artificial treatments, Amen (1966) found that germination of Arnebia benthamii seeds in the field occurred after the seed coat had been sufficiently weathered, and when environmental factors were non-limiting. The long period required for weakening of the seed coat could also explain why seedlings of several wetland Carex spp. (i.e., species of temperate and cold region sedges)

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emerge only 2 – 3 years after seed shedding (Maas and Schopp-Guth, 1995). In the present study, our findings, including data for out-of-doors seedling emergence, showed that intact seeds could acquire the ability to germinate only after a certain period of cold stratification, which confirms that dormancy resides mainly in the embryos of D. dyeriana seed, and that there may be a soil seed-bank in the field. Germination of dormant seeds after the seed coat has been removed agrees with the concept of the ‘mechanical barrier’ model of coat-imposed dormancy. However, in many species, the picture appears more complex and it is likely that more than one factor operates simultaneously. Schütz (2000) suggested that it was likely that thick, water-permeable seed coats offered considerable resistance to germination, which could explain why scarification at the micropylar end promoted germination in Carex panacea, C. albonigra, and C. firm seeds. In our study, the embryos in D. dyeriana seed had physiological dormancy, thus their covering layers (e.g., seed coat) prevented the embryo from elongating and/or germinating. During cold stratification, however, the embryo acquired the ability to override this mechanical resistance (Table I). Consequently, seeds germinated rapidly after 10 weeks of cold stratification (93%) or ODS (86%). Similarly, Yang et al. (1999) found that cold stratification at 3ºC produced significantly greater germinability in all physiologically dormant seeds of Ariscae dracontium (Araceae). Thus, based on such a comparison, we concluded that D. dyeriana seed exhibit non-deep physiological dormancy, and were gradually released from dormancy during cold stratification. In this context, after investigating germination responses in three congeneric cactus species, Ramírez-Padilla and Valverde (2005) concluded that seed germination patterns may have a direct impact on the rarity of a species. For D. dyeriana growing in the field, rare successful seedling establishment may be related to the particular mechanism(s) of germination or the suitability of its habitat conditions. We thank Professors Carol C. Baskin and Jerry M. Baskin for their initial comments on the manuscript. This study was financially supported by the Particular Life Foundation of the Chinese Academy of Sciences (Project No. KSCX2-YW-Z-0925), the Chongqing Education Committee (Project No. KJ100610) and the Doctoral Start-up Foundation of Chongqing Normal University (Project No. 09XLB016).

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