physiology, morphology and phenology of seed dormancy break

Physiology, morphology and phenology of seed dormancy break and germinationin the endemic Iberian species Narcissus hispanicus (Amaryllidaceae)

Elena Copete1,*, Jose M. Herranz1, Pablo Ferrandis1, Carol C. Baskin2,3 and Jerry M. Baskin2

1ETSIA, Department of Plant Production and Agricultural Technology, University of Castilla-La Mancha, University Campus s/n,Albacete 02071, Spain, 2Department of Biology, University of Kentucky, Lexington, KY 40506, USA and 3Department of Plant and

Soil Sciences, University of Kentucky, Lexington, KY 40546, USA* For correspondence. E-mail [email protected]

Received: 16 November 2010 Returned for revision: 6 December 2010 Accepted: 10 January 2011 Published electronically: 17 February 2011

† Background and Aims Only very few studies have been carried out on seed dormancy/germination in the largemonocot genus Narcissus. A primary aim of this study was to determine the kind of seed dormancy in Narcissushispanicus and relate the dormancy breaking and germination requirements to the field situation.† Methods Embryo growth, radicle emergence and shoot growth were studied by subjecting seeds with andwithout an emerged radicle to different periods of warm, cold or warm plus cold in natural temperatures outdoorsand under controlled laboratory conditions.† Key Results Mean embryo length in fresh seeds was approx. 1.31 mm, and embryos had to grow to 2.21 mmbefore radicle emergence. Embryos grew to full size and seeds germinated (radicles emerged) when they werewarm stratified for 90 d and then incubated at cool temperatures for 30 d. However, the embryos grew only alittle and no seeds germinated when they were incubated at 9/5, 10 or 15/4 8C for 30 d following a moist coldpre-treatment at 5, 9/5 or 10 8C. In the natural habitat of N. hispanicus, seeds are dispersed in late May, theembryo elongates in autumn and radicles emerge (seeds germinate) in early November; however, if the seedsare exposed to low temperatures before embryo growth is completed, they re-enter dormancy (secondary dor-mancy). The shoot does not emerge until March, after germinated seeds are cold stratified in winter.† Conclusion Seeds of N. hispanicus have deep simple epicotyl morphophysiological dormancy (MPD), with thedormancy formula C1bB(root) – C3(epicotyl). This is the first study on seeds with simple MPD to show thatembryos in advanced stages of growth can re-enter dormancy (secondary dormancy).

Key words: Dormancy break, embryo growth, epicotyl morphophysiological dormancy, germination, Narcissushispanicus, phenology, secondary dormancy, shoot emergence.

INTRODUCTION

Seeds of many temperate plant species are dormant at the timeof seed dispersal, and specific temperature requirements mustbe met before they will come out of dormancy and germinate(Baskin and Baskin, 1998). Dormancy prevents germination attimes when conditions are appropriate for germination, but theprobability of them remaining favourable for successful seed-ling establishment is low (Bewley et al., 2006). Thus, dor-mancy is an adaptive trait that optimizes the distribution ofgermination over time in a population of seeds.

The circumstances that determine germination and survivalof seedlings have an over-riding importance for the expansionor decline of populations (Rasmussen and Whigham, 1998).Therefore, knowledge about specific environmental conditionsthat trigger dormancy break of a species is a key factor indesigning ex situ propagation protocols to reinforce wildplant populations. That is to say, if we know the ecologicallife cycle of plants we will be able to predict the most favour-able periods for seedling emergence and establishment innature (Harper, 1977; Lentz and Johnson, 1998;Gimenez-Benavides et al., 2005). This information isespecially important in the case of rare and endemic species(Galmes et al., 2006) such as Narcissus hispanicus, which is

very vulnerable to possible habitat alterations due to itssmall populations.

Studies on the germination ecology of Iberian endemics upto the present (e.g. Perez-Garcıa et al., 1995; Escudero et al.,1997; Albert et al., 2002; Herranz et al., 2002; Galmes et al.,2006; Lorite et al., 2007) have focused on the influence oftemperature and light conditions on seed germination capa-bility. However, other important aspects of the germinationprocess have yet to be researched, such as germination phenol-ogy, germination responses of seeds buried in the soil andeffects of temperature on embryo growth in species with mor-phological dormancy (MD) and morphophysiological dor-mancy (MPD).

According to Baskin and Baskin (1998), the Amaryllidaceaehave underdeveloped linear embryos that are fully differen-tiated, thus they need to grow before the seed germinates.Nevertheless, data on embryo growth requirements are particu-larly scarce in this family. Thus, in this study, special attentionwas paid to the analysis of environmental conditions thatpromote embryo growth and subsequent germination.

Seeds with underdeveloped embryos have MD, and theyalso can have physiological dormancy (PD). If embryogrowth and radicle emergence are completed in about 30 dunder suitable conditions, seeds have only MD. On the other

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Annals of Botany 107: 1003–1016, 2011

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hand, if germination is delayed for more than about 30 d andseeds require a dormancy-breaking treatment such as exposureto moist cold (0–10 8C) and/or to moist warm (≥15 8C) stra-tification to germinate, they have MPD (Nikolaeva, 1977;Baskin and Baskin, 1990, 1998). In a preliminary study,seeds of N. hispanicus incubated under a wide range oflight–temperature conditions (described below in the‘Laboratory experiments’ section) had not germinated after30 d.

The genus Narcissus belongs to the monocotyledon familyAmaryllidaceae, which includes about 900 species in 60genera (Mabberley, 2008). Narcissus mainly has aMediterranean distribution, with the centre of diversity in theIberian Peninsula, and it also occurs in south-west France,northern Africa and eastward to Greece (Grey-Wilson andMathew, 1981). Narcissus hispanicus is a bulbous species inSection Pseudonarcissus. It reaches 60 cm in height and pro-duces large yellow flowers 5–6 cm in diameter, and thecorona is very flared at the mouth. This species blooms inMarch and April, fruits are formed in April and May, andseeds are dispersed in May and June as the ripe capsulesopen. The above-ground parts subsequently die, and the under-ground bulbs remain dormant until about the followingJanuary, when buds begin to emerge. It inhabits shadyenvironments of riverbank deciduous forests, above 600 m alti-tude (a.s.l.). This taxon is endemic to the west central part ofthe Iberian Peninsula (Moreno and Sainz, 1992) and hasbeen classified as a ‘Special Concern Taxon’ in the List ofThreatened Species of Castilla-La Mancha (D.O.C.M., 2001).

With respect to the reproductive biology of Narcissus, pol-lination (Sage et al., 1999), storage of anthers and pollen(Bowes, 1990), pollen morphology (Chen and Ueda, 1977)and seed development after pollination in vitro (Balatkovaet al., 1977) have been studied. However, there are very fewstudies on the germination ecology or physiology ofNarcissus. In a study by Thompson (1977), seeds ofN. bulbocodium var. conspicuous germinated (radicle emer-gence) at cool temperatures following a previous warmperiod under control temperatures in incubators. Recently,Vandelook and Van Assche (2008) reported that radiclesemerge in autumn and shoots in spring from seeds ofN. pseudonarcissus. However, they still concluded that theseeds do not have epicotyl MPD, because, first, its embryo,and, secondly, its seedling, grow continuously. Clearly, allspecies belonging to the same genus do not necessarily havethe same level of MPD; for example, species of Sambucus(Hidayati et al., 2000, 2010), Osmorhiza (Baskin et al.,1995) and Erythronium (Baskin et al., 1995; Walck et al.,2002) have different levels of MPD. Thus, seeds ofN. hispanicus may or may not have the same dormancy-breaking and germination requirements as those of the othertwo species of Narcissus previously studied.

Therefore, the main goal of the present study was to eluci-date which one of the nine known levels of MPD (Baskinet al., 2008) occurs in N. hispanicus seeds. Moreover,additional work on conditions required for embryo growth ofspecies with underdeveloped embryos and comprehensiveknowledge of seed germination characteristics are neededbefore evolutionary relationships among various types ofMPD can be established (Walck et al., 1999). The specific

aims of this study were to (a) describe the phenology ofembryo growth and germination; (b) characterize germinationresponses of seeds buried in the soil; (c) determine the temp-erature requirements for dormancy break and embryo growth;(d ) analyse the influence of warm stratification, light/dark con-ditions during incubation and seed age on germination; (e) testthe effect of low temperature on shoot emergence from seedswith an emerged radicle; and ( f ) determine if low tempera-tures can induce dormancy in non-dormant seeds.

MATERIALS AND METHODS

Seed material

Fruits of Narcissus hispanicus Gouan are ripe when the cap-sules change from green to yellow and begin to open forseed dispersal. Thus, fruits with the same level of ripenesswere collected on 12 May 2007 and 7 May 2008 from about280–300 apparently vigorous and healthy plants growing inwell-lit situations in Riofrıo, Puebla de Don Rodrigo(Ciudad Real, central Spain), 640 m a.s.l., 30SUJ6927. Here,N. hispanicus grows in a Betula pendula subsp. fontquerivar. parvibracteata (Peinado, G. Moreno & M. Velasco)G. Moreno & Peinado riparian forest that is a special protectedhabitat (D.O.C.M., 1999). Ripe capsules were spread out in thelaboratory to allow them to open and the seeds to fall out.Seeds were dried in the laboratory (22–23 8C) until theinitial germination tests began on 1 June 2007 and 2008,respectively, at which time seeds were considered to be0 months old.

Outdoor pot studies

The aim of these studies was to describe the phenology ofembryo growth and of root and shoot emergence fromN. hispanicus seeds kept under near-natural temperature con-ditions. Hence, these observations were made on seeds in anon-heated metal frame shadehouse, in which the temperaturewas recorded throughout the study. The growing medium inoutdoor studies was a mixture of sterilized peat and sand(2:1 v/v). Pots and trays containing the seeds were wateredto field capacity once a week throughout the year, with twoexceptions. First, they were watered only twice a month inJuly and August, to simulate the summer moisture stress thatis common in the Mediterranean area. Also, frequent wateringcombined with high summer temperatures greatly increases thepossibility of seed decay. Secondly, water was withheld whenthe substratum was frozen in winter. Thus, temperature andsoil moisture conditions were similar to those in the naturalhabitat of N. hispanicus.

Phenology of embryo growth. On 1 July 2007, six groups of 50N. hispanicus seeds each were mixed with fine-grained steri-lized sand. Each group was placed in a fine-mesh polyestercloth bag, and the six bags were buried 5 cm deep in a pot.Each bag was labelled, but the label was not buried, whichmade it easy to recover each bag individually. The pot wasplaced in the metal-framed shadehouse and watered as pre-viously described. One bag was removed monthly, and thenthe seeds were separated from the sand using a 1 mm sieve.Embryos were excised from 25 healthy looking seeds and

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measured. These values were used in two ways: (1) monthlymean embryo length was calculated to analyse embryo meangrowth throughout the experiment and (2) each month these25 values were grouped into size classes to study temporalchanges in the distribution of embryo size structure fromJuly to December. On 1 December 2007, the study describedabove without the warm season pre-treatment was initiatedagain, using 20 groups of 50 seeds to compare with thestudy initiated on 1 July 2007. Thus, the effect of exposingseeds to high temperatures during the summer months onembryo growth was tested.

Embryos from seeds from which the radicle had emergedduring burial were recorded as having a critical embryolength. The critical embryo length for germination is thelength of the embryo at the time the seed coat splits butbefore the radicle emerges (Vandelook and Van Assche,2008). In N. hispanicus, the critical embryo length for germi-nation was 2.21 mm (s.e. ¼ 0.05 mm, n ¼ 40, range ¼ 1.80–2.60 mm).

Phenology of shoot emergence. On 1 July 2007, three trays withdrainage holes were filled with the growing medium, and 100N. hispanicus seeds were sown equidistant from each other ineach tray to avoid contact between them; seeds were buried at adepth of 5 mm. Three replicates were placed in the shadehouseand watered as described above. From July 2007 to May 2009,seed trays were examined once a week, and emergent shootswere counted and removed. The study was repeated beginningon 1 December 2007.

Phenology and dormancy break of radicles in buried seeds. On 1July 2007, six groups of 100 N. hispanicus seeds each weremixed with fine sterilized sand. Each group was placed in afine-mesh polyester cloth bag, and the five bags were buried5 cm deep in a pot. One bag was exhumed on the first dayof each month for 6 months. Healthy ungerminated seedswere incubated in darkness at 15/4 8C (one of the most favour-able thermoperiods for germination) for 45 d. Recovered seedswere classified into four categories: (1) seeds germinated(radicle emerged) within the bag during the burial period;(2) viable non-dormant seeds that germinated at 15/4 8C; (3)apparently viable dormant seeds that did not germinate at15/4 8C and had healthy embryos; and (4) non-viable seedsthat were dead (they were soft and did not contain a firmwhite embryo). The study was repeated beginning on 1December 2007. In bags buried in July, germination was.90 % in November and December. Thus, there were notenough ungerminated seeds to perform a germination test forthese dates.

Laboratory experiments

Experiments were conducted in temperature- and light-controlled conditions, following the recommendations ofBaskin and Baskin (1998). Germination chambers (Ibercexmodel F-4, Madrid, Spain) were equipped with a digital temp-erature and light control system [+0.1 8C, cool white fluor-escent light, 25 mmol m22 s21 (1350 lux)]. Seeds were testedfor germination in a 12 h daily photoperiod (¼light hereafter)and in continuous darkness (¼darkness hereafter), which wasachieved by wrapping Petri dishes in a double layer of

aluminium foil, at constant temperatures of 5 and 10 8C and at12/12 h daily temperature regimes of 9/5, 15/4, 20/7, 25/10,28/14 and 32/18 8C. In the 12/12 h alternating treatments, thehigh temperature coincided with the light phase and the lowtemperature with darkness to simulate day/night conditions.

The fluctuating temperatures used in the germination testssimulated mean maximum and mean minimum monthlytemperatures that characterize the annual climate cycle inthe seed-source region (lowlands of the Toledo Mountains):15/4 8C, November and March; 20/7 8C, October and April;25/10 8C, September and May; 28/14 8C, August and June;and 32/18 8C, July. The 5 8C treatment simulated the meantemperature recorded during winter months: December,January and February. The other low temperatures (9/5 8C and10 8C) were chosen because they are within the effective temp-erature range for cold stratification, which is from around 0 to 108C, with around 5 8C being optimal for many species (Stokes,1965; Nikolaeva, 1969).

Percentages of germination were computed based on thenumber of apparently viable seeds, i.e. seeds whose embryoswere white and firm. More than 95 % of the seeds were viable.

Effect of temperature on embryo growth. The purpose of thisexperiment was to determine the effect of (a) a range of temp-eratures on embryo growth and (b) a warm and a cold stratifica-tion pre-treatment on embryo growth at low temperatures (9/5,10 and 15/4 8C) for 45 d.

First, we measured the mean length of embryos in freshlymatured seeds. Thus, 25 seeds were placed on two sheets offilter paper moistened with distilled water in a 9 cm Petridish at room temperatures for 24 h. Embryos were excisedfrom imbibed seeds with a razor blade and their lengthsmeasured using a dissecting microscope equipped with amicrometer.

Two hundred seeds were placed in each of seven 16 cm Petridishes on two sheets of filter paper moistened with distilledwater and sealed with Parafilm. Each dish was placed inlight at 5, 9/5, 10, 20/7, 25/10, 28/14 and 32/18 8C. After30, 60 and 90 d, 25 healthy seeds were extracted from eachtemperature treatment, and their embryos were excised andmeasured. Mean length and standard error were calculatedfor each sample of 25 embryos.

After 90 d, seeds at 5, 9/5, 10, 20/7, 25/10, 28/14 and 32/18 8C were transferred to three incubation thermoperiods(9/5, 10 and 15/4 8C) in light for 45 d. After incubation,embryos were excised from 25 seeds in each pre-treatmentat each of the three incubation temperatures, and theirlengths were measured; mean length and standard errorwere calculated for each sample. Embryos from seeds thathad germinated were recorded as fully elongated, i.e. thecritical length for germination.

The E:S ratio is the relationship between embryo (E) andendosperm (S) lengths, so the critical E:S ratio is the meanof the E:S ratios of 40 seeds at the time their seed coats hadsplit but the radicles had not yet emerged.

Effect of time at 28/14 8C on radicle emergence. This study deter-mined the optimum time of warm stratification and the effectof seed age on germination. In July 2007 and March 2008,1600 seeds that were 0 months old (freshly matured seeds)and 1600 seeds that were 8 months old (seeds were stored in

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paper bags under laboratory conditions: 22–23 8C and 50–60 %air humidity), respectively, were placed in 16 cm Petri dishesat 28/14 8C in light for 15, 30, 60 and 90 d and then transferredto 10 and 15/4 8C in light and in darkness for 45 d. For eachtemperature × light condition, four replicates of 25 seedswere placed in 9 cm Petri dishes on two sheets of filterpaper and moistened with distilled water. Seeds incubated inlight were checked at 4 d intervals, while those in darknesswere checked only after 45 d, at the end of theincubation period.

Temperature requirement for radicle emergence. The aim of thisexperiment was to determine the optimal temperature forradicle emergence after embryo growth had occurred at 28/14 8C. In July 2007, 600 seeds were placed on moist filterpaper at 28/14 8C in light for 90 d, and then four replicatesof 25 seeds each were transferred to 5, 9/5, 10, 15/4, 20/7and 25/10 8C in light for 45 d. This study followed the samemethodology described under ‘Effect of time at 28/14 8C onradicle emergence’.

Temperature requirement for shoot emergence. The purpose ofthis experiment was to determine if cold stratification isrequired for shoot emergence in radicle-emerged seeds and,if so, how many weeks of this treatment are required. Theexperiment also showed what is the most favourable tempera-ture for shoot growth.

Seeds with emerged radicles were placed in 9 cm Petridishes on two sheets of filter paper moistened with distilledwater and incubated in light. Eight groups of 50 seeds eachwith roots 2–3 cm in length were moist cold (5 8C) stratifiedfor 0, 4, 8 or 12 weeks, and after each cold stratificationperiod one group was transferred to 10 8C and another to 15/4 8C. Also, four groups of 50 germinated seeds were placedat 5 8C for 8 weeks and then incubated at 5, 10, 20/7 and25/10 8C to determine the best temperature for shoot growth.

Induction of dormancy. In this section, we ask if low tempera-tures that may occur in late autumn will induce secondary PDin seeds in which PD had been broken and embryos had begunto grow but were not fully elongated. In July 2008, three lotsof 200 seeds each were placed in 9 cm Petri dishes in light totest the effect of two different sequences of stratification(warm plus cold and warm plus warm) on embryo growth andgermination. First, the three lots were placed at 28/14 8Cfor 60 d. Secondly, one of the lots was transferred to 5 8C for30 d, another one to 20/7 8C for 30 d and the third one to25/10 8C for 30 d. Finally, 4 × 25 seeds from each lot wereused to test germination at 15/4 8C in light for 45 d. Embryogrowth was measured monthly throughout the stratificationperiod and at the end of the incubation period.

Statistical analysis

Means and standard errors were calculated for percentagesof radicle and shoot emergence and for embryo lengths. Theeffect of stratification temperature, duration of stratificationand incubation temperature on embryo length were analysedby a three-way analysis of variance (ANOVA).

Seed germinability was evaluated by the final cumulativegermination percentage of the number of apparently viable

seeds, which was compared among treatments by multifactorANOVAs. The effects of four factors were analysed: durationof stratification (four levels), light conditions (two levels),seed age (two levels) and incubation temperature (twolevels). When the effect of a factor was significant, differenceswere compared by a multiple comparison Tukey test. Prior toanalyses, the normality (Cochran test) and homoscedasticity(David test) of data were checked. Values of the final cumulat-ive germination percentages were arcsine square-root trans-formed before analysis.

RESULTS

Outdoor pot experiments

Phenology of embryo growth. Embryos in seeds buried on 1 July2007 grew slowly between this date and September, duringwhich time mean maximum and minimum temperatureswere 32.6 and 14.4 8C, respectively (Fig. 1). The meanlength of embryos was 1.31+ 0.03 mm on 1 July and1.49+ 0.03 mm on 1 September. However, between 1September and 1 November, when mean maximum andminimum temperatures were 23.0 and 9.3 8C, respectively,embryos grew rapidly. On 1 November, embryos had grownto a mean length of 2.21 mm, which was 100 % of the meanlength of fully elongated embryos. In November, 96 % of theburied seeds had germinated (radicle emergence).

On the other hand, hardly any embryos in seeds buried on1 December 2007 (Fig. 1C) reached the critical length for ger-mination during the first 10 months of burial, and thus veryfew (5 %) germinated during this time. Germination occurredafter the following warm season, reaching a value of around78 % in November 2008.

All embryos from seeds buried on 1 July 2007 (Fig. 2) werein the three smallest size classes. However, embryos grew(at different rates) during the summer months, and inAugust, September and October a broad range of size classeswas represented. By November, 100 % of embryos were inthe largest (.2.05 mm) size class.

Phenology of shoot emergence. Although radicles emerged inNovember 2007 from seeds buried on 1 July 2007 (Fig. 1B),shoots did not emerge until March 2008 (83 %). During this4 month period (November–March), mean daily maximumand minimum temperatures were 13.01 and –0.37 8C, respect-ively. In contrast, for seeds buried on 1 December 2007(Fig. 1C), shoots did not emerge until March 2009, afterseeds were warm stratified during summer 2008 and thencold stratified during the following winter.

Phenology of dormancy break of radicles in buried seeds. Almosthalf of the seeds buried in July (Fig. 3A) and retrieved inAugust had become non-dormant after 1 month, duringwhich time mean daily maximum and minimum temperatureswere 33.6 and 13.9 8C, respectively. Seeds germinated (radi-cles emerged) to 49 % (s.e. ¼ 2.00) at 15/4 8C in darkness.In September, after two warm months of burial, the percentageof non-dormant seeds was 80 (s.e. ¼ 4.00). Finally, all appar-ently viable seeds retrieved in October were non-dormant, butno seeds germinated in the bag until November and December,when temperatures were lower (mean maximum and minimum


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were 15.4 and –2.1 8C, respectively) than those in October(mean maximum and minimum were 19.4 and 7.0 8C,respectively).

In contrast, most seeds buried in December (Fig. 3B)remained dormant (.82 %) from January to May (meandaily maximum and minimum temperatures were 15.5 and1.8 8C, respectively).

Laboratory experiments

Effect of temperature on embryo growth. The mean length ofembryos from freshly matured N. hispanicus seeds was1.31 mm (s.e. ¼ 0.03, n ¼ 25), while that of the endospermwas 2.84 mm (s.e. ¼ 0.05, n ¼ 25). Hence, the E:S ratio(mean embryo length/mean endosperm length) in matureseeds was 0.46. Seeds without an embryo were scarce (,2 %).The critical E:S ratio was 0.76 (s.e. ¼ 0.01, n ¼ 40, range ¼0.61–0.86), where 0.61 is the minimal E:S ratio recorded ingerminated seeds, ‘threshold E:S ratio’ hereafter.

The mean length of embryos in seeds stratified in light at 5,9/5 or 10 8C (cold temperatures) for 90 d ranged from 1.41(E:S ¼ 0.49) to 1.52 mm (E:S ¼ 0.53) (Table 1), and 8 % ofthe embryos achieved the critical length for germination.Therefore, the percentage of germination was very low (5 %)for cold-stratified seeds transferred to incubation temperatures.

On the other hand, embryos in seeds stratified for the sameamount of time at 20/7, 25/10, 28/14 and 32/18 8C (warmtemperatures) reached mean lengths of 1.60 (E:S ¼ 0.56) to1.82 mm (E:S ¼ 0.62). At 20/7, 25/10, 28/14 and 32/18 8C,52, 20, 28 and 56 %, respectively, of the embryos reachedthe threshold E:S ratio. At 20/7 8C in light, embryos grewfrom 1.31 to 1.72 mm. When these seeds were transferred to9/5 8C in light for 45 d, 94 % of them germinated; all ungermi-nated seeds had embryos that had grown to the threshold E:Sratio.

Effect of time at 28/14 8C on radicle emergence. Following warmstratification at 28/14 8C, the optimum conditions for radicleemergence were 10 8C in darkness. Prolonging the length ofthe warm stratification treatment increased germination inboth light and darkness, although mostly in light.Nevertheless, fresh seeds that were warm stratified for only15 d and then incubated at 10 8C in darkness germinated to76.6 % (s.e. ¼ 5.81). In general, 8-month-old seeds germinatedto higher percentages than 0-month-old seeds, although germi-nation patterns were similar for both age classes (Fig. 4). Therewere significant effects on final germination percentages ofduration of the warm stratification, light conditions duringincubation, seed age and incubation temperature (Table 2).

A

–5

5

15

25

35 Minimum

Maximum

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40

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0·0

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1·0

1·5

2·0

2·5

Shoot emergence

Radicle emergence

Embryo length

C

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40

60

80

100

0·0

0·5

1·0

1·5

2·0

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2007

A N D M AJ

2008

F M J S OJ A N D M AJ

2009

F M

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erge

nce

(%)

Em

erge

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(%)

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n da

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re (

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bryo length (mm

) E

mbryo length (m

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FI G. 1. Mean daily minimum and maximum air temperatures (A) and phenology of embryo growth and of root and shoot emergence from seeds sown on soil ina non-heated shadehouse in July 2007 (B) and December 2007 (C).


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Temperature requirement for radicle emergence. After a warmstratification period at 28/14 8C for 90 d, seeds transferred to5 and 25/10 8C did not have emerged radicles (Fig. 5),and those incubated at 20/7 8C germinated to low percentages(6 %, s.e. ¼ 3.46). In contrast, seeds transferred to 9/5, 10 and15/4 8C began to germinate within 20–30 d, and final radicleemergence percentages were 88, 91 and 53 %, respectively.

Temperature requirement for shoot emergence. With an increasein length of the cold treatment at 5 8C, the rate of shoot growthafter seeds with emerged radicles were transferred to 15/4 or10 8C generally increased. A cold temperature of 5 8C pro-moted shoot emergence from germinated seeds.Consequently, prolonging the cold pre-treatment time shor-tened the incubation time for shoot development. Thus, all ger-minated seeds exposed to 5 8C for 12 weeks and thentransferred to 15/4 8C developed a shoot in 21 d (Fig. 6). Onthe other hand, seeds incubated at 15/4 8C without a previouscold treatment took 104 d to emerge. This behaviour wassimilar at both incubation temperatures, 15/4 and 10 8C.

Following 8 weeks of stratification at 5 8C, nearly 100 % ofshoots had emerged after 50 d of incubation at 10, 15/4, 20/7and 25/10 8C; however, at 5 8C only 42 % of the shoots hademerged (Fig. 7). Shoot emergence was slightly more rapidat 25/10 8C than at 10, 15/4 and 20/7 8C, and 64 % of theshoots emerged at 25/10 8C in only 18 d.

Induction of dormancy. Seeds placed at 28/14 8C for 60 d, trans-ferred to 25/10 8C for 1 month and then incubated at 15/4 8Cgerminated to 98 %; obviously, the embryo length hadreached the critical size (2.21 mm) (Table 3). In contrast,seeds transferred from 28/14 to 5 8C and then incubated at15/4 8C germinated to only 4 3 % and embryo length was1.94 mm. Thus, a portion of the seeds did not completeembryo growth, and they did not germinate when transferredto a favourable (15/4 8C) germination temperature.

If we compare embryo size class distribution for seeds coldstratified and then transferred to 15/4 8C (Fig. 8.IB) with thatfor seeds warm stratified and then transferred to 15/4 8C(Fig. 8IIB, IIIB), we see that embryo growth is completed inthe latter but not in the former seeds. Thus, embryos fromseeds warm stratified (Fig. 8II, III) reached the largest lengthrange at the end of the incubation time. However, embryosfrom cold-stratified seeds had a length structure distributedin different length ranges (Fig. 8IB). Therefore, cold stratifica-tion stopped the embryo growth that had begun during the pre-vious warm stratification at 28/14 8C.

DISCUSSION

Fresh seeds of N. hispanicus have an underdeveloped embryoat the time of dispersal in late spring. Mean endosperm (seed)length was 2.84+ 0.05 mm and mean embryo length 1.31+0.03 mm (E:S ratio ¼ 0.46). Embryos had to grow until theyreached at least 1.80 mm to germinate (E:S ¼ 0.76+ 0.01,n ¼ 40, range ¼ 0.61–0.86). Therefore, seeds have MD, i.e.the embryo grows while still inside the mature seed beforethe seed can germinate. Nevertheless, since embryo growth

0

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<1·30 1·30–1·451·46–1·601·61–1·751·76–1·901·91–2·05 >2·05

Em

bryo

s (%

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mbr

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December

Size class (mm)

FI G. 2. Changes in size class distribution of embryos in seeds sown in July2007 and recovered in August, September, October, November and December.


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and radicle emergence were not completed at suitable tempera-ture, light/dark and moisture conditions in about 30 d, seedsalso had PD and required a dormancy-breaking treatment(Baskin and Baskin, 2005). Thus, seeds have a combinationof MD and PD, i.e. they have MPD (Nikolaeva, 1977; Fig. 9).

Under light conditions and warm stratification (20/7, 25/10,28/14 or 32/18 8C) for 90 d, followed by cool temperatures(9/5, 10 or 15/4 8C) for 45 d, the embryos grew and then the radi-cles emerged from the seeds. However, embryos grew only alittle and no seeds germinated if seeds were first cold stratifiedfor 90 d and then incubated at 9/5, 10 and 15/4 8C. Hence,embryo growth (loss of morphological dormancy) occurs onlyduring warm (≥15 8C) stratification (Table 1). This behaviourindicates that seeds of N. hispanicus have some level ofsimple MPD (Baskin and Baskin, 1998). PD is broken by hightemperatures of summer, and during (and after) the breakingof PD embryo growth occurs. However, lower temperaturesare required for embryo growth than for the breaking of PD.

Thus, the high summer temperatures provide the appropriateconditions that result in germination as temperatures decline inautumn or at the beginning of winter. Finally, shoot growthoccurs after seeds with an emerged radicle are subjected tolow winter temperatures. Thus, seedling emergence occurs inMarch, when temperatures are high enough for shoot growth(Fig. 1B). This behaviour is common in most species with epi-cotyl dormancy (Baskin and Baskin, 1998). Therefore, we con-clude that N. hispanicus seeds have deep simple epicotylMPD. The dormancy formula is C1bB(root) – C3(epicotyl),where C1bB(root) indicates that the root of the underdevelopedembryo (B) has PD (C) that is non-deep (subscript 1) andbroken by a period of warm stratification (subscript b).C3(epicotyl) indicates that epicotyl dormancy in the seedwith a fully developed embryo and with root emerged isdeep PD (C3), requiring a long period of cold stratificationto elongate (emerge) (Nikolaeva, 2001; Baskin and Baskin,2008). To summarize dormancy break: at warm temperatures(i.e. summer), embryos begin to grow; at cool temperatures

(autumn), embryos become fully elongated and the radicleemerges; at cold temperatures (winter), the growth potentialof the epicotyl increases; and at cool to warm (spring) temp-eratures the epicotyl elongates rapidly (Fig. 9).

Strictly speaking an epicotyl is not formed in seedlings ofmonocotyledon species (Muller, 1978; Vandelook and VanAssche, 2008). Consequently, it may be more appropriate tospeak of ‘shoot dormancy’ in referring to monocots with thiskind of dormancy. However, a name change could produceconfusion since the term ‘epicotyl dormancy’ has been usedin reference to monocots throughout the seed germination lit-erature (Barton, 1936, 1944; Kondo et al., 2002, 2004). In fact,the monocotyledon family Liliaceae sensu lato includes manyspecies whose seeds have epicotyl dormancy (Baskin andBaskin, 1998).

Optimal conditions for germination were 90 d of warm stra-tification at 28/14 8C followed by incubation in light at 10 8Cfor 45 d, where the mean embryo length increased to2.16 mm (s.e. ¼ 0.03) and seeds germinated to 96 %(Table 1). Dry laboratory storage decreased the warm stratifica-tion time required to break dormancy (Fig. 4), i.e. PD waspartly broken by after-ripening prior to the time seeds weregiven a warm stratification treatment. Loss of non-deep PDoccurs in seeds of many species, especially those of winterannuals, as seeds after-ripen during the dry, hot conditionsof summer. Consequently, dormancy is broken duringsummer, and seeds can germinate in autumn (Baskin andBaskin, 1998).

The patterns of embryo growth and radicle emergence inoutdoor experiments were in agreement with those carriedout under controlled conditions in the laboratory. This isimportant in determining the level of MPD in seeds ofN. hispanicus. The embryo, and consequently the radicle, isnot able to grow until the seed is warm stratified duringsummer followed by cool incubation temperatures that occurin autumn, which suggests a mechanism to prevent prematuregermination under extreme natural conditions in summer.

BA

0

20

40

60

80

100

A S O N D

See

d-s

tag

e ca

teg

ory

(%

)

J F M A M

InviableDormant apparently viableNon-dormant viableGerminated in the bag

FI G. 3. Changes in the percentage of dormant, non-dormant, inviable and germinated seeds of Narcissus hispanicus buried on 1 July 2007 (A) and 1 December2007 (B) and exhumed monthly for 5 months.


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Fifty-seven per cent of the seeds kept at 28/14 8C for 60 d toinitiate dormancy break and embryo growth, transferred to5 8C for 30 d (winter temperature) and then incubated at15/4 8C did not geminate, while those that were not cold stra-tified germinated to 98 % (Table 3). These data indicate thatlow temperatures induced non-dormant seeds into dormancy(Fig. 9). Our evidence for induction of secondary dormancyby exposure to cold stratification in seeds of N. hispanicus isthe first report of the induction of secondary dormancy inseeds with deep simple epicotyl MPD. Induction of secondarydormancy in seeds of Frasera caroliniensis (Threadgill et al.,1981) and Delphinium fissum subsp. sordidum (Herranz et al.,2010b), which have deep and intermediate complex MPD,respectively, was due to exposure of seeds to warm stratifica-tion. However, cold stratification induced secondary dormancyin seeds of the winter annuals Papaver rhoeas (Baskin et al.,2002) and Chaerophyllum tainturieri (Baskin and Baskin,1990), which have non-deep simple MPD.

The kind of dormancy induced in seeds of N. hispanicus,and the other four species with MPD, is PD, i.e. the PD partof MPD (Fig. 9). After PD is induced, germination is pre-vented until it is broken, either by cold stratification or bywarm stratification, depending on the species. In the seeds ofN. hispanicus, warm stratification would be required to breakthe PD. A high temperature requirement for loss of dormancyand low-temperature induction of non-dormant seeds into dor-mancy has also been found in seeds of the winter annualsAphanes arvensis (Roberts and Neilson, 1982), Lamiumamplexicaule (Baskin and Baskin, 1981), Thlaspi arvense(Baskin and Baskin, 1989) and Veronica arvensis (Baskinand Baskin, 1983a) that have only PD.

Light is not a requirement for embryo growth in seeds ofN. hispanicus, therefore the factor controlling embryo growthin autumn is temperature, i.e. although embryo growthbegins in summer it continues in autumn (in both light anddarkness) until embryos reach their full size. However, ifseeds of N. hispanicus are exposed to temperatures lowenough to stop embryo growth and induce secondary PD, thedormant seeds would have a range of embryo sizes (Fig. 8).In contrast, in seeds of C. tainturieri, both temperature andlight control embryo growth in autumn (Baskin and Baskin,1990). When seeds of C. tainturieri are exposed to favourableautumn temperatures (25/15 8C) for embryo growth and germi-nation, embryos do not grow unless seeds are exposed to light.Thus, if buried seeds of C. tainturieri fail to germinate and lowtemperatures induce PD, all of the embryos are small.

The induction of secondary PD in some seeds ofN. hispanicus would mean that the radicle does not emergeuntil the second autumn (Fig. 9). Embryo growth willresume the second autumn after PD is broken again thesecond summer. However, the relatively large embryo insome of the seeds induced into secondary PD may meanthey can germinate quite early the second autumn, beforethose produced in the current year. Almost 100 % of theseeds may germinate while buried in soil in darkness inautumn. However, seeds on the soil surface need a longerwarm stratification time to germinate than those in darkness(Fig. 4). Thus, low temperatures may induce some of theseseeds in light into dormancy before they can germinate(Table 3).

TA

BL

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Em

bry

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(mea

nle

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Incu

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Str

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90

d30

d60

d90

d9/58C

108C

15/48C

Cold

58C

1. 4

1+

0. 0

4A

BC

a(0

,0)

1. 4

1+

0. 0

4A

a(0

,4)

1. 4

4+

0. 0

3A

Ba

(0,

4)

1. 4

6+

0. 0

3A

a(0

,0)

1. 4

9+

0. 0

4A

a(0

,0)

1. 4

6+

0. 0

4A

a(0

,0)

9/58C

1. 3

2+

0. 0

4A

a(0

,4)

1. 3

7+

0. 0

5A

ab

(0,

4)

1. 4

1+

0. 0

3A

ab

(0,

4)

1. 5

0+

0. 0

4A

b(3

,8)

1. 5

3+

0. 0

5A

b(1

,8)

1. 5

0+

0. 0

4A

b(1

,4)

108C

1. 4

6+

0. 0

4A

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a(0

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9+

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(0,

4)

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War

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1. 3

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6B

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(0,

32)

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2+

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Db

c(0

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2. 1

9+

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1B

e(9

4,

100)

2. 0

3+

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Cd

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1. 5

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4B

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92)

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0+

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4B

Cb

(72,

88)

2. 0

5+

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6B

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(45,

76)

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1. 4

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(0,

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Db

(0,

28)

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92)

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92)

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1. 5

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80)

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)in

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Vandelook and Van Assche (2008) propose thatN. pseudonarcissus, which belongs to the same section ofthe genus as N. hispanicus, does not have a dormant shoot,i.e. does not have epicotyl dormancy. They considered seedsof N. pseudonarcissus to be non-dormant. The reason forthis, they argue, is that the time lag between germinationand emergence is a consequence of slow, continuous growthof the seedling at low temperatures. In contrast, in our studyon seeds of N. hispanicus with an emerged radicle, the shootdid not emerge until spring. Therefore, a total developmentalarrest occurred for about 4 months (in the phenology study),but only in the shoot since the radicle continued to grow(E. Copete, pers. obs.). This kind of dormancy is a phenologi-cal adaptation to the seasonal cycle in a temperate region(Baskin and Baskin 1983b, 1985a, b; Kondo et al., 2004).Thus, the above-ground parts of N. hispanicus are notexposed to freezing temperatures, and so the seedlings havea well-developed root system when the shoot expands inspring. Spring is often the most suitable period for seedlingestablishment in European woodlands (Grime, 2001).Further, as in many temperate nemoral species, the shoot ofN. hispanicus emerges at the end of the winter, well inadvance of tree canopy development. As such, the speciesbenefits from a high amount of sunlight for about 2months before canopy closure (Kondo et al., 2004; Herranzet al., 2010a).

The rate of shoot emergence was faster when germinatedseeds were exposed to a previous cold temperature forseveral weeks. Growth of shoots of N. hispanicus from seedswith an emerged radicle was promoted by a cold pre-treatmentat 5 8C for 8–12 weeks (Fig. 6). This can be considered as evi-dence of shoot dormancy. Gagea lutea has a similar behaviourin Japan (Kondo et al., 2004). According to Mondoni et al.(2009), Anemone ranunculoides has weak epicotyl MPDbecause shoot emergence in its seeds is not dependent onexposure to winter temperatures, since they can develop atcooler autumn temperature (10 8C). Something similar hap-pened in N. hispanicus, i.e. shoots emerged in germinatedseeds exposed to 10 8C, but much more slowly than in thosewith a pre-treatment at lower temperatures (Fig. 7).

In species that have a long delay between time of radicleemergence (autumn) and shoot emergence (spring), wewould like to know if the winter cold period is required forshoot emergence or if the low temperature just delays shootemergence. A complicating factor in trying to answer thisquestion is that the optimum spring temperature for shootemergence is quite low, e.g. 15/4 and 25/10 8C in darkness.Thus, if seeds with a developing root system are incubated at

0

20

40

60

80

100

15 d 30 d 60 d 90 d 15 d 30 d 60 d 90 d

8 Months0 Months

15 d 30 d 60 d 90 d 15 d 30 d 60 d 90 d

8 Months0 Months

10 °C 15/4 °C

Ger

min

atio

n (%

) LightDarkness

FI G. 4. Influence of the length of the stratification period (15, 30, 60 or 90 d) at 28/14 8C in light and seed age (0 and 8 months) on cumulative germinationpercentages (mean+ s.e., s.e .2 %) of Narcissus hispanicus seeds. Incubation in light and dark, as indicated, at 10 8C and at 15/4 8C for 45 d. Seeds collected in

2007.

TABLE 2. Effects of several factors on seed germination ofNarcissus hispanicus seeds collected in 2007

Factor d.f. F P Categories

Duration of stratification 3 65.84 0.0000 15a, 30b, 60bc, 90c

Light conditions (incubation) 1 314.64 0.0000 Light ,darknessIncubation temperature 1 57.08 0.0000 15/4 ,10Seed age 1 41.79 0.0000 0 ,8

The main effects on germination of the length of the stratification period at28/14 8C, light conditions during incubation, incubation temperature and seedage in a multifactor analysis of variance. The table shows degrees of freedom(d.f.), F-ratio values and categories of factors where germination differenceswere significant. Residual d.f. ¼ 121.

Values followed by different lowercase letters are significantly different(P , 0.05).

0

20

40

60

80

100

0 10 20 30 40Time (d) after transfer from 90 d at 28/14 ºC to

various temperatures

Rad

icle

em

erge

nce

(%)

5 ºC

9/5 ºC10 ºC

15/4 ºC20/7 ºC

25/10 ºC

FI G. 5. Effects of a 90 d treatment at 28/14 8C in light on cumulative radicleemergence (mean+ s.e., s.e .2 %) in Narcissus hispanicus seeds sub-sequently transferred to various temperatures in light. Seeds collected in 2007.


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normal spring temperatures they will receive some cold strati-fication (≤10 8C at night). On the other hand, if seeds are incu-bated at temperatures high enough to avoid cold stratification(at night), the temperature (.10 8C) would not have any eco-logical meaning and may even kill the seed/root. For thisreason, it is impossible to separate completely the effect of aprevious cold stratification period from the subsequent incu-bation period under a simulated spring temperature regime.The highest incubation temperature we tested was 25/10 8C(Fig. 10), so seeds were cold stratified (at 10 8C) for 12 hd21. Hence, after 100 d the shoot had emerged from 76 % ofthe seeds incubated continuously at 25/10 8C, while shootemergence was 96 % from seeds that had received 8 weeks

(56 d) cold and then 44 d incubation at 25/10 8C (totaltime ¼ 100 d). The cold-stratified seeds reached 76 % shootemergence in only about 31 d after they were transferredfrom 5 to 25/10 8C (total time ¼ 87 d). Thus, it is clear thatcold stratification increased the rate at which shoots emergedwhen the germinated seeds were exposed to spring tempera-tures. Shoots slowly emerged at 25/10 8C, but slow emergenceat the beginning of spring would not allow enough time forshoots to emerge before the arrival of summer. Thus, wesuggest that cold stratification is of great benefit because itincreases the ability of shoots to respond to spring tempera-tures. Also, if the shoot emergence curve of seeds with rootemerged incubated continuously at 25/10 8C is examinedclosely, it can be seen that at about 8 weeks (¼ 4 weekscold stratification at 10 8C) the angle of the curve shiftsupward. This increase in rate of shoot emergence after 8weeks suggests that after (4 weeks) cold stratification, theshoot in many seeds had gained the ability to grow rapidly.

Narcissus hispanicus seeds are dispersed in late May, a timesimilar to that of other temperate deciduous forest herbs withepicotyl dormancy, whose main period of seed dispersal isbetween May and July, e.g. Asarum canadense (Barton,1944), Hydrophyllum appendiculatum (Baskin and Baskin,1985a), H. macrophyllum (Baskin and Baskin, 1983b),Erythronium japonicum (Kondo et al., 2002) and Hexastylisheterophylla (Adams et al., 2003). This dispersal phenologyallows seeds to be warm stratified during summer, which isessential for subsequent radicle emergence at slightlydecreased temperatures in autumn. Another temperate

0

20

40

60

80

100

0 20 40 60 80 100

Time (d) at 15/4 ºC

See

ds w

ith s

hoot

gro

wth

(%

) y = 1·2119x – 21·193R

2 = 0·9165

0 weeks at 5 °C

y = 1·4864x – 13·264R

2 = 0·9298

4 weeks at 5 °C

y = 2·1224x – 2·9898R

2 = 0·9668

8 weeks at 5 °C

y = 4·0345x + 25·77R

2 = 0·921

12 weeks at 5 °C

0

20

40

60

80

100

0 20 40 60 80 100Time (d) at 10 ºC

See

ds w

ith s

hoot

gro

wth

(%

) y = 1·3926x – 28·654R

2 = 0·9227

0 weeks at 5 °C

y = 1·9127x – 21·228R

2 = 0·9222

4 weeks at 5 °C

y = 2·7406x – 18·65R

2 = 0·936

8 weeks at 5 °C

y = 2·5783x + 4·8499R

2 = 0·9312

12 weeks at 5 °C

FI G. 6. Shoot growth in germinated seeds of Narcissus hispanicus at 15/4 8C or at 10 8C following 0–12 weeks of cold treatment at 5 8C.

0

20

40

60

80

100

0 10 20 30 40 50Time (d) incubation

See

ds w

ith s

hoot

gro

wth

(%

) 5 ºC

10 ºC

15/4 ºC

20/7 ºC

25/10 ºC

FI G. 7. Shoot growth in germinated seeds of Narcissus hispanicus at differenttemperatures following 8 weeks of cold treatment at 5 8C.


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deciduous forest herb with epicotyl dormancy, Cimicifugaracemosa, disperses its seeds in September, but they neverthe-less receive a sufficiently long period of warm stratification forthe radicle to emerge in autumn (Baskin and Baskin, 1985b).In contrast, the temperate deciduous forest shrub Viburnumacerifolium is an exceptional species with epicotyl MPD,since its natural seed dispersal occurs in October–December

(Hidayati et al., 2005); thus, the seeds do not germinate(root emergence) until the following autumn. In one of theoutdoor experiments (Fig. 1C), N. hispanicus seeds that wereburied in December did not germinate until the followingautumn. Ungerminated seeds were exposed to cold stratifyingtemperatures in the first winter, but dormancy in the epicotylcan be broken only after radicles have emerged (Baskin and

TABLE 3. Effect of warm stratification (28/14 8C) for 60 d followed by cold (5 8C) or warm stratification (20/7 8C or 25/10 8C) for30 d and then incubation at 15/4 8C for 45 d on embryo growth (mean+ s.e.) in 12-month-old seeds of Narcissus hispanicus

Previous common stratification at 28/14 8C: 60 dSubsequent different stratification temperatures:

30 d Incubation at 15/4 8C: 45 d

1.66+0.03a (0, 24) 5 8C 1.78+0.05Aab (10, 48) 1.94+0.05Ab (43, 64)20/7 8C 1.86+0.05Ab (0, 72) 2.19+0.01Bc (96, 100)

25/10 8C 1.84+0.05Ab (0, 76) 2.20+0.00Bc (98, 100)

Values followed by different uppercase letters within a column or different lowercase letters within a row are significantly different (P , 0.05). The firstnumber in parentheses is the percentage of germination, and the second one is the percentage of seeds with an E:S ratio greater than the threshold E:S ratio.

0

20

40

60

80

100

Em

bryo

s (%

)

0

20

40

60

80

100

Em

bryo

s (%

)

0

20

40

60

80

100

Em

bryo

s (%

)

IA:28/14 ºC + 5 ºC

IB:28/14 ºC + 5 ºC + 15/4 ºC

IIB:28/14 ºC + 20/7 ºC + 15/4 ºC

IIIB:28/14 ºC + 25/10 ºC + 15/4 ºC

IIA:28/14 ºC + 20/7 ºC

IIIA:28/14 ºC + 25/10 ºC

Size class (mm) Size class (mm)

<1·30 1·30–1·45 1·46–1·60 1·61–1·75 1·76–1·90 1·91–2·05 >2·05 <1·30 1·30–1·45 1·46–1·60 1·61–1·75 1·76–1·90 1·91–2·05 >2·05

FI G. 8. Changes in size class distribution of embryos throughout the induction of dormancy study. Seeds were stratified at 28/14 8C in light for 60 d before beingtransferred to 5 8C (IA), 20/7 8C (IIA) or 25/10 8C (IIIA) in light for 30 d, and then incubated at 15/4 8C for 45 d (IB, IIB, IIIB).


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Baskin, 1998), hence shoots did not develop until early in thesecond spring.

Some species of the genus Narcissus are used widely in gar-dening, and consequently they have a commercial interest.However, N. hispanicus is a species with a small area of dis-tribution and vulnerable to extinction from habitat alterations;thus, its conservation is of concern. As such, this research

contributes to the knowledge of the germination ecology ofthis species and thereby provides a guideline on how togrow plants from seeds to reinforce populations in case thisaction is necessary.

ACKNOWLEDGMENTS

This work was supported by the PAI07-0088-0300 (‘Creationof a plant germplasm bank of endangered species in theBotanical Garden of Castilla-La Mancha’) andPEII10-0170-1830 (‘Germination ecology of 12 singular and/or threatened species with morphophysiological dormancy’)projects, supported by the regional Government ofCastilla-La Mancha. During the study, E.C.C. held a grantfrom the regional Government (Consejerıa de Educacion yCiencia, Junta de Comunidades de Castilla-La Mancha) andthe European Social Found. We thank Carlos Guillen for lab-oratory assistance.

LITERATURE CITED

Adams CA, Baskin JM, Baskin CC. 2003. Epicotyl dormancy in the mesicwoodland herb Hexastylis heterophylla (Aristolochiaceae). Journal of theTorrey Botanical Society 130: 11–15.

Seed dispersal

Seed reenters PD (C1b)C1bB’(root) – C3(epicotyl)

C1bB(root) – C3(epicotyl)

1st winter (low temperatures)

Epicotyl dormancy broken

1st autumn (MODERATE temperatures)

Embryo completes growth

Shoot emergence

1st autumn (LOW temperatures)

Some embryos do not becomefully elongated (B’)

2nd summer

Embryo completes growth

2nd winter

Epicotyl dormancy is broken

Shoot emergence 2nd spring

1st spring (moderate temperatures)

Embryo begins to growB(root) – C3(epicotyl)

1st summer (high temperatures)PD (C1b) broken

1st spring

1st winter

Later 2nd autumn

Early 2nd autumn

PD(C1b) broken

B’(root) – C3(epicotyl)

Root emergence– C3(epicotyl)

Root emergence– C3(epicotyl)

C1bB’(root) – C3(epicotyl)

FI G. 9. Conceptual model of dormancy break in seeds of Narcissus hispanicus. Some seeds re-enter MPD [C1bB’(root)-, where B’ indicates embryos havegrown, but have not fully developed].

y = 1·201x – 37·408R

2 = 0·9563

y = 0·5307x – 3·1001R

2 = 0·9251

0

20

40

60

80

100

0 20 40 60 80 100

Time (d) at indicated temperatures

See

ds w

ith s

hoot

gro

wth

(%

)

25/10 ºC

5 ºC(8w) + 25/10 ºC

FI G. 10. Shoot growth in germinated seeds of Narcissus hispanicus at25/10 8C with or without a previous cold treatment at 5 8C.


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