Plant Species Biology (2009) 24, 20–26

doi: 10.1111/j.1442-1984.2009.00232.x

Morphological dormancy in seeds of the autumn-germinating shrub Lonicera caerulea var. emphyllocalyx (Caprifoliaceae) SHYAM S. PHARTYAL,*1 TETSUYA KONDO,* YOICHIRO HOSHINO,† CAROL C. BASKIN‡§ and JERRY M. BASKIN‡ *Environmental Horticulture and Landscape Architecture, Research Faculty of Agriculture and †Field Science Center for Northern Biosphere, Hokkaido University, Sapporo 060-8589, Japan; and ‡Department of Biology and §Department of Plant and Soil Sciences, University of Kentucky, Lexington, Kentucky 40546, USA

Abstract To better understand the germination ecophysiology of the genus Lonicera, the dormancy class, temperature requirements for embryo growth and radicle emergence and phenology of seedling emergence were determined for Lonicera caerulea var. emphyllocalyx. At maturity, seeds have an underdeveloped embryo (approximately 28% of the length of full-grown embryos). Embryos in fresh seeds grew to full length at 15, 20, 20/10 and 25/15°C within 3 weeks, but failed to grow at ⱕ 10°C and at 30°C. Radicles emerged from 86–100% of freshly matured seeds in light at 15, 20, 20/10 and 25/15°C within 28 days, but failed to emerge at 10°C. Radicles emerged equally well in a 12 h photoperiod and in continuous darkness at 25/15°C. Rapid embryo growth and germination over a range of conditions indicate that seeds of this taxon have morphological dormancy (MD); this is the first report of MD in a species of Lonicera. Seeds are dispersed in summer, at which time high temperatures promote embryo growth. Embryos grow to the critical length for germination in approximately 1 month; the peak of seedling emergence occurs in early autumn. Radicles emerged within 2 months from 98% of seeds buried at soil depths of 2 cm and 10 cm in the field in August in Sapporo, Japan; thus, seeds have no potential to form a persistent soil seed bank. However, seeds sown too late in autumn for embryos to grow remained viable and germinated the following summer when temperatures were high enough to promote embryo growth. Keywords: Caprifoliaceae, Haskap, Lonicera caerulea, morphological dormancy, soil seed bank. Received 21 August 2008; accepted 15 November 2008

Introduction The genus Lonicera (Caprifoliaceae), commonly known as ‘Honeysuckles’, includes more than 200 species that are distributed in temperate and subtropical regions of North America, Europe, North Africa and Asia. Most of the species are small trees or shrubs (Zheng et al. 2004). Lonicera caerulea L. var. emphyllocalyx (Maxim.) Nakai is native to Japan and is referred to locally as ‘Haskap’ or

Correspondence: Shyam S. Phartyal Email: [email protected] 1 Present address: Department of Forestry, H.N.B. Garhwal University, Srinagar-Garhwal (UK), India.

‘Haskappu’. The taxon is a deciduous shrub distributed in the intermountain areas from central (Chubu) to northern Honshu to Hokkaido (Okuyama 1977), and has recently been domesticated (Nakajima 1996; Takada et al. 2003). Historically, wild-growing ‘Haskap’ was one of the few fruit species available to the indigenous ‘Ainu’ people of Hokkaido Island. According to Sato (1985), it is difficult to distinguish between L. caerulea var. emphyllocalyx and the closely related Lonicera caerulea var. edulis. Recently, Haskap was introduced to southern Canada and northern USA for commercial production (Thompson 2006). Although the availability of L. caerulea var. emphyllocalyx as a fruit crop is increasing, the dormancy breaking and germination requirements of this taxon have not been investigated.

© 2009 The Authors Journal compilation © 2009 The Society for the Study of Species Biology

S E E D D O R M A N C Y I N LONICERA CAERULEA As several species of Lonicera have underdeveloped embryos (Martin 1946; Hidayati et al. 2000) that must elongate to a species-specific critical length prior to radicle emergence (Baskin & Baskin 1998), we expected that L. caerulea var. emphyllocalyx would also have underdeveloped embryos. If embryo growth and radicle and cotyledon emergence in seeds are completed at suitable incubation temperatures in approximately 30 days without any dormancy breaking treatment, the seeds are considered to have morphological dormancy (MD). However, if the seeds require > 30 days for germination and warm and/or cold stratification to break the dormancy they are considered to have morphophysiological dormancy (MPD) (Baskin & Baskin 1998). Thus, one of the aims of our study was to determine if seeds of L. caerulea var. emphyllocalyx have an underdeveloped embryo, and if so what are the temperature requirements for dormancy break and of radicle and shoot emergence. This information would allow us to determine whether the seeds have MD or MPD, and the level of MPD if they have MPD. Hidayati et al. (2000) confirmed that in Lonicera maackii and Lonicera morrowii a portion of the seeds in a given seed lot had MD and the remainder had MPD. However, only MPD was reported in seeds of Lonicera fragrantissima and Lonicera japonica (Hidayati et al. 2000). An important aspect of understanding the germination ecology of a species is to determine what controls the timing of germination under natural conditions. However, information on germination phenology under natural temperature conditions is required to determine what controls the timing of germination. Thus, our second aim was to monitor the phenology of seedling emergence from seeds sown outside in Sapporo, Japan. Furthermore, when a new crop species is introduced into a region, it is important to have information on its ability to form a persistent soil seed bank, which is a characteristic of many weedy species (Baker 1974). Thus, the third aim of the present study was to monitor radicle emergence in seeds artificially buried in soil under natural temperature conditions to investigate their ability to form a persistent soil seed bank.

Methods

Seed collection and general methods Mature dark blue fruits (berries) of L. caerulea var. emphyllocalyx were collected on 24 July 2007 and 30 July 2007 from plants growing in cultivation on the campus of Hokkaido University (43°04′N, 141°20′E), Sapporo, Japan. The seeds were removed from the pulp immediately and dried at ambient room conditions (approximately 25°C) for 2–4 days before the study commenced. Seeds collected on 24 July were used to examine the rate of embryo growth Plant Species Biology 24, 20–26

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and the effects of temperature and light and dark on radicle emergence. Seeds collected on 30 July were used to examine the effect of temperature on radicle emergence following warm stratification, the phenology of seedling emergence in the field and the potential to form a persistent soil seed bank. In all laboratory experiments, three replicates of 30 seeds each were used, and the seeds were placed into 55 mm diameter ¥ 10 mm deep plastic Petri dishes on two sheets of Whatman No. 1 filter paper (Whatman Japan KK, Tokyo, Japan) moistened with distilled water. The Petri dishes were sealed with parafilm to reduce water loss during incubation. Unless otherwise stated, the seeds were incubated in a 12-h daily photoperiod. At alternating temperature regimes, the high temperature was given for 12 h in light each day and the low temperature for 12 h in darkness. Cool white fluorescent tubes were used to provide the light and the photon irradiance (400–700 nm) at seed level was approximately 15–20 mmol/m2/s1. Radicle emergence was monitored at 2-day intervals for 5 weeks and seeds with an emerged radicle were considered to have germinated.

Embryo growth On 26 July 2007, 10 fresh imbibed seeds were cut into thin sections (40 mm) using an auto microtome. The embryo length (initial embryo length) of each seed was measured using a dissecting microscope equipped with a micrometer. On the same day, five Petri dishes containing 50 seeds each were placed into 5, 10, 15, 20, 25, 15/5, 20/10, 25/15 and 30/20°C. At weekly intervals for 3 weeks, two seeds were removed at random from each of the five dishes at each temperature and the length of each embryo was measured. Embryo length was expressed as a percentage of the fully elongated embryos (critical length). To determine critical embryo length, the embryo was measured in 10 seeds in which the seed coat was split, but the radicle had not emerged.

Optimum temperature for radicle emergence Seeds were incubated at 10, 15, 20, 25, 15/5, 20/10, 25/15 and 30/20°C. Radicle emergence was monitored at 2-day intervals for 2 weeks, after which time the percentage of seeds with an emerged radicle was calculated.

Effect of light and dark on radicle emergence Seeds were incubated at a 12 h photoperiod and in continuous darkness at the optimum temperature regime of 25/15°C, and the germination percentages in light and darkness were determined after 2, 3 and 4 weeks. All

© 2009 The Authors Journal compilation © 2009 The Society for the Study of Species Biology

22 S . S . P H A R T YA L ET AL. dishes to be incubated in darkness were wrapped with two layers of aluminum foil. To determine the rate of germination in darkness, nine dishes were placed in darkness and after 2, 3 and 4 weeks of incubation three dishes were opened and the number of germinated seeds was counted and the dishes were discarded (i.e. destructive sampling).

Optimum temperature for radicle emergence following warm stratification Seeds were warm stratified at 25/15°C for 10 days and then they were moved to 10, 15, 20, 15/5, 25/15 or 30/20°C in a 12 h photoperiod. After 10 days of warm stratification, radicle emergence was monitored at 3-day intervals for 33 days.

Phenology of seedling emergence On 2 August, 1 September, 1 October and 1 November 2007, three replicates of 100 seeds that had been stored dry at 5°C since they were collected on 30 July 2007 were sown on soil (1:1 v/v mixture of vermiculite and leaf mold) in pots and covered by 2–3 mm of sieved soil. The pots were placed into a non-temperature controlled metal framehouse in Sapporo, Hokkaido, Japan, until September 2008. The framehouse was covered by black, finemesh shade cloth, except from the last week of October 2007 to mid-June 2008. Snow covered the pots from 21 November 2007 to 3 April 2008. Soil in the pots was kept moist by daily watering, except when the pots were covered by snow and not watered. The temperature at the soil surface in the pots was measured at 15-min intervals throughout the study using thermo data loggers (RT-30S; Espec Mic, Aichi, Japan) located on the surface. The daily maximum, minimum and mean temperatures were calculated from these data. Seedlings that emerged above the soil surface were counted and removed from the pots at intervals of 3–4 days, except when the pots were covered by snow.

Statistical analyses A Kruskal–Wallis test was used to investigate whether temperature affected the final percentages of radicle emergence and seedling emergence. A one-way anova was then used to test significant differences between the different temperature treatments using a Student–Neuman– Keuls’ test.

Results

Embryo growth The embryos in freshly matured seeds were 0.53 ⫾ 0.02 mm (mean ⫾ standard error) long, which is approximately 27.8 ⫾ 1.29% of the length of fully elongated embryos (1.89 ⫾ 0.05 mm), that is, the critical length for germination (Fig. 1). Embryos grew to their full length (1.85–1.95 mm) within 3 weeks, at which time the radicle had begun to emerge from > 50% of the seeds at 15, 20, 20/10 and 25/15°C. Thus, embryo length increased approximately 360% between seed maturity and germination (Fig. 2).

Optimum temperature for radicle emergence Radicles emerged from 97–100% of fresh seeds within 28 days at 15, 20 and 25/15°C, from 33–86% of fresh seeds at 25, 15/5, 20/10 and 30/20°C and from 0% at 10°C (Fig. 3). The highest percentages of radicle emergence were for seeds incubated at 15, 20 and 25/15°C (c2 = 22.13, d.f. = 7, P = 0.002; Kruskal–Wallis test followed by a Student–Neuman–Keuls’ test). Thus, the 25/15°C temperature regime was used for subsequent experiments.

Effect of light and dark on radicle emergence After 2 weeks, radicles had emerged from 27.8% of the seeds incubated in a 12 h photoperiod, but no radicles had emerged from continuous darkness. However, radicles had emerged from 97.8 and 86.7% of seeds in light and continuous darkness, respectively, after 3 weeks, and from 100 and 96.7%, respectively, after 4 weeks.

Radicle emergence from buried seeds On 3 August 2007, 100 seeds were placed in each of 20 fine-mesh polyester bags and 10 bags each were buried at soil depths of 2 cm and 10 cm in a field in Sapporo. At monthly intervals, two bags were chosen randomly from each of the two depths. The bags were monitored for both germinated (with emerged radicle) and non-germinated (without emerged radicle, but viable) seeds. The temperatures at the two soil depths were recorded at 15-min intervals as described above.

Optimum temperature for radicle emergence following warm stratification Following 10 days of warm stratification, radicles emerged from 91–100% of the seeds at 15, 20, 25/15 and 30/20°C and from 40–57% of the seeds incubated at 10 and 15/5°C (Fig. 4). These percentage differences in radicle emergence were significant (c2 = 15.57, d.f. = 5, P = 0.008).

© 2009 The Authors Journal compilation © 2009 The Society for the Study of Species Biology

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Fig. 1 Embryo (arrow) growth in seeds of Lonicera caerulea var. emphyllocalyx at 25/15°C in a 12 h light/dark photoperiod.

Phenology of seedling emergence Cotyledons emerged from > 90% of seeds sown on 2 August and 1 September 2007, with a high percentage of emergence occurring between 10 September and 14 October 2007, during which time the mean daily Plant Species Biology 24, 20–26

maximum and minimum temperatures were 16.8°C and 11.4°C, respectively (Fig. 5). No seedlings emerged from seeds sown on 1 October or 1 November 2007 until June 2008. Seedling emergence began in the second week of June and by 17 September 2008 a seedling had emerged from 70% and 67% of the seeds sown on 1 October and 1

© 2009 The Authors Journal compilation © 2009 The Society for the Study of Species Biology

24 S . S . P H A R T YA L ET AL.

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November 2007, respectively. Differences were significant for the seeds sown in August and September (c2 = 8.6, d.f. = 3, P = 0.035).

Radicle emergence from buried seeds After 1 month, 32 and 38% of the seeds buried on 6 August 2007 at soil depths of 2 and 10 cm in the field, respectively, had emerged radicles, and after 2 months radicles had emerged from > 98% of the seeds at both depths (Fig. 6). The mean daily maximum and minimum temperatures during the 2-month period were 23.4°C and 16.6°C at a depth of 2 cm and 20°C and 18.2°C at 10 cm.

Discussion Seeds of L. caerulea var. emphyllocalyx have an underdeveloped spatulate embryo (0.53 mm in length) at maturity

Fig. 3 Effect of (a) constant and (b) alternating temperature regimes on radicle emergence of Lonicera caerulea var. emphyllocalyx. The final percentages of radicle emergence among the incubation temperatures followed by different letters indicate significant differences using a Kruskal–Wallis test followed by a Student–Neuman–Keuls’ test. Error bars are the standard error.

that grew to full length (1.89 mm) within 2–3 weeks at warmer temperatures; 20 and 25/15°C were optimal temperatures. In addition, radicles and cotyledons emerged from 100% of the seeds at these temperatures within 30 days. Embryos in seeds with MD are not physiologically dormant, thus seeds do not require a dormancy breaking treatment to germinate. Only incubation at suitable conditions for a short period of time is needed for the embryo to grow to full size, and then the radicle emerges immediately (Nikolaeva 1977; Baskin & Baskin 1998, 2004). At appropriate temperature, moisture and light conditions, embryos in these seeds begin to grow (elongate) within a period of a few days to 1–2 weeks, and seeds germinate within approximately 30 days (Baskin & Baskin 1998). Thus, based on the rapid rate of embryo growth and germination L. caerulea var. emphyllocalyx seeds have only MD.

© 2009 The Authors Journal compilation © 2009 The Society for the Study of Species Biology

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Fig. 4 Effect of warm (25/15°C) stratification on radicle emergence of Lonicera caerulea var. emphyllocalyx. Seeds were warm stratified for 10 days before being moved to 10, 15, 20, 15/5, 25/15 or 30/20°C. WS, warm stratification. The final percentages of radicle emergence among the incubation temperatures followed by different letters indicate significant differences using a Kruskal–Wallis test followed by a Student–Neuman–Keuls’ test. Error bars are the standard error.

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Fig. 5 Daily maximum, minimum and mean temperatures and seedling emergence (%) of Lonicera caerulea var. emphyllocalyx sown outdoors in Sapporo, Japan, on 2 August, 1 September, 1 October and 1 November 2007 (arrows). The final percentages of seedling emergence among the seeds sown in the different months followed by the same letter are not significantly different using a Kruskal–Wallis test followed by a Student–Neuman– Keuls’ test.

Luken and Goessling (1995) reported that most seeds of L. maackii do not have a well-developed dormancy mechanism, and that seeds germinated easily in warm, moist conditions, implying that they had only MD. However, a detailed study on L. maackii and L. morrowii by Hidayati Plant Species Biology 24, 20–26

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Fig. 6 Radicle emergence (%) from Lonicera caerulea var. emphyllocalyx seeds buried at soil depths of (a) 2 cm and (b) 10 cm in the field on 6 August 2007. Daily maximum, minimum and mean temperatures at soil depths of 2 and 10 cm outside in Sapporo, Japan, from 6 August to 10 October 2007 are shown.

et al. (2000) revealed that approximately half of the freshly matured seeds of both species had MD, and the remaining seeds had MPD. Lonicera morrowii seeds with MPD required 6 weeks of warm stratification to germinate, and those of L. maackii with MPD required 12 weeks of cold stratification to germinate (Hidayati et al. 2000). All seeds of L. fragrantissima and L. japonica had MPD, that is, the embryo was underdeveloped and physiologically dormant (Hidayati et al. 2000). Baskin and Baskin (1998) concluded that seeds of L. oblongifolia and L. hirsuta have MPD based on data from Brinkman (1974). Thus, seeds of L. caerulea var. emphyllocalyx differ from those of the other Lonicera species that have been investigated to date because they have only MD. The presence of only MD in seeds of L. caerulea var. emphyllocalyx and the maturation/dispersal of seeds in July–August play an important role in the timing of germination. That is, following dispersal in late July–early August, the temperatures are high enough for embryo

© 2009 The Authors Journal compilation © 2009 The Society for the Study of Species Biology

26 S . S . P H A R T YA L ET AL. growth; however, growth depends on the favorability of the soil moisture conditions. If embryos grow during August, seeds germinate in September (Fig. 5). After embryos grow, seeds germinate over a wide range of temperatures in light and darkness. A 10-day period at warm (25/15°C) temperatures increased the ability of seeds to germinate at low (10°C) temperatures, but this was not because of the breaking of any physiological dormancy in the seeds. In contrast, at 25/15°C for 10 days, embryos began to grow and their length increased to approximately 58% of the critical length required for radicle emergence. When moved from 25/15°C to 10°C, embryo growth continued and 40% of the seeds germinated within 33 days. Thus, if seeds are exposed to relatively short periods of favorable moisture conditions in summer, some of them might be able to germinate in autumn because embryo growth was initiated during summer. If seeds are sown in late summer–early autumn, when conditions are not warm (and moist) long enough for embryos to grow, germination is delayed until the following summer. Significantly, seeds remained viable when sown outside on 1 October and 1 November, and they began to germinate the following June, after minimum daily temperatures reached ⱖ10°C. Lonicera caerulea var. emphyllocalyx seeds germinated equally well in both light and continuous darkness; however, more seeds of L. japonica, L. maackii and L. morrowii germinated in light than in continuous darkness (Hidayati et al. 2000). The non-dependence of L. caerulea var. emphyllocalyx seeds on light for germination helps to explain why radicles emerged from > 98% of seeds after burial for 2 months. In addition, during the 2-month period of burial the soil temperatures were in the optimal range for embryo growth and radicle emergence. Thus, we found no evidence that seeds of L. caerulea var. emphyllocalyx have the potential to form a persistent seed bank. Hidayati et al. (2000) also concluded that seeds of four invasive species in eastern North America, L. fragrantissima, L. japonica, L. maackii and L. morrowii, did not form a soil seed bank. However, before L. caerulea var. emphyllocalyx is introduced to other countries as a new crop, its ability to form a soil seed bank should be determined in the new environment.

References Baker H. G. (1974) The evolution of weeds. Annual Review of Ecology and Systematics 5: 1–24. Baskin C. C. & Baskin J. M. (1998) Seeds: Ecology, Biogeography, and Evolution of Dormancy and Germination. Academic Press, San Diego. Baskin J. M. & Baskin C. C. (2004) A classification system for seed dormancy. Seed Science Research 14: 1–16. Brinkman K. A. (1974) Lonicera L. In: Schopmeyer C. S. (Technical Coordinator) (ed.). Seeds of Woody Plants in the United States. USDA Forest Service Agriculture Handbook No. 450. Forest Service, USDA, Washington, pp. 515–519. Hidayati S. N., Baskin J. M. & Baskin C. C. (2000) Dormancy breaking and germination requirements of seeds of four Lonicera species (Caprifoliaceae) with underdeveloped spatulate embryos. Seed Science Research 10: 459–469. Luken J. O. & Goessling N. (1995) Seedling distribution and potential persistence of exotic shrub Lonicera maackii in fragmented forests. The American Midland Naturalist 133: 124–130. Martin A. C. (1946) The comparative internal morphology of seeds. The American Midland Naturalist 36: 513–660. Nakajima F. (1996) Small fruit growing in Hokkaido: No 1. Hasukappu. Hokkaido Prefecture Agriculture Extension Service, Extension Publication, Sapporo. (In Japanese.) Nikolaeva M. G. (1977) Factors controlling the seed dormancy pattern. In: Khan A. A. (ed.). The Physiology and Biochemistry of Seed Dormancy and Germination. North-Holland Publishing Company, Amsterdam, pp. 51–74. Okuyama S. (1977) Terasaki’s Illustrated Flora of Japan. Heibonsha, Tokyo. (In Japanese.) Sato T. (1985) About Lonicera caerulea var. emphyllocalyx and L. caerulea var. edulis in Hokkaido. Wildlife Report 2: 47–51. (In Japanese.) Takada M., Nakano H., Hoshino Y. & Sato H. (2003) Evaluation of eating qualities and some horticultural characteristics for selection of elite lines in Lonicera caerulea L. Research Bulletin of Hokkaido University Farm 33: 21–38. (In Japanese with English summary.) Thompson M. M. (2006) Introducing haskap, Japanese blue honeysuckle. Journal of the American Pomological Society 60: 164– 168. Zheng H., Wu Y., Ding J., Binion D., Fu W. & Reardon R. (2004) Invasive Plants of Asian Origin Established in the United States and Their Natural Enemies. USDA Forest Service-FHTET-200405, pp. 98–103.

Acknowledgment The senior author thanks the Japanese Society for the Promotion of Science for a Postdoctoral Fellowship (P06195).

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