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Environmental regulation of embryo growth, dormancy breaking and germination in Narcissus alcaracensis (Amaryllidaceae), a threatened endemic Iberian daffodil.

INTRODUCTION

Narcissus (Amaryllidaceae) is a genus of perennial geophytes, known as daffodils. In the wild they occur principally on the Iberian Peninsula and in Morocco but are also found around the Mediterranean Basin and along the Atlantic coasts of Europe. Underdeveloped, linear embryos, which must elongate to a critical length before radicle emergence are a common feature of Amaryllidaceae species (Baskin and Baskin, 1998). However, information on the types of dormancy and the conditions necessary for embryo growth and germination is particularly scarce in this family (Vandelook and Van Assche, 2008). Many Narcissus taxa showing underdeveloped embryos are endemic to specific locations in the Mediterranean Basin, and very few studies have been carried out on their germination ecology.

Descriptions of the genus Narcissus have presented taxonomic difficulties caused by weak reproductive barriers, high hybridization ability, pronounced morphological variability, and the effects of speciation arising from polyploidy (Zonneveld, 2008). Webb (1980) listed 26 Narcissus species in the Flora Europaea. Blanchard (1990) recognized 65 species, and more recently Zonneveld (2008) differentiated 36 species, some of them with several subspecies, based on variations in nuclear DNA content.

Narcissus alcaracensis belongs to the section Pseudonarcissi DC. That section is morphologically diverse and unstable, presumably as a result of recent and continuing speciation (Fernandez-Casas, 1983; Dorda and Fernandez-Casas, 1994). The centre of origin of the majority of the section lies in the southern mountain ranges of the Iberian Peninsula (Sierra Nevada, Subbeticas, and Cazorla) where over ten new taxa have been described during the four last decades. However, many of these species are hardly morphologically distinguishable because speciation is still occurring (Rios-Ruiz et al., 1999). Although all species readily propagate from bulbs (Banares et al., 2003), seed germination ecology is poorly known. More information on seed gennination is essential for ex situ plant production, both for ornamental aims to supplement wild populations (Cerabolini et al., 2004), given that propagation from seeds is critical for maintaining genetic variability. Understanding seed germination will also provide insight into Narcissus life history ecology and habitat requirements to improve management of natural populations (Ikeda and Itoh, 2001; Navarro and Guitian, 2003; Gimenez-Benavides et al., 2005; Martinez-Sanchez et al., 2011; Aguado et al., 2011).

Narcissus alcaracensis has been ranked as "endangered" (see IUCN Criteria, 2001) in the Spanish Vascular Flora Red Lists (Aizpuru et al., 2000; Moreno, 2008), in the Atlas and Red Book of Threatened Vascular Flora in Spain (Banares et al., 2003), and in the IUCN Red List (2011), because of its limited geographical distribution, the small size of its populations, and their vulnerability to habitat disturbance. In addition, the regional government of Castilla-La Mancha, the administrative region where the species lives, protects it within the same threatened category and has stressed the urgency of a Recovery Plan for the taxon (DOCM, 1999).

As a contribution to the Recovery Plan for Narcissus alcaracensis, we investigated several traits of seed dormancy and germination ecology. In addition, we also evaluated the effects of temperature, light regime and gibberellic acid (GA3) on embryo growth. Up to now, the germinative ecology of Narcissus has been analyzed only in three species: N. bulbocodium (Thompson, 1977), N. pseudonarcissus (Vandelook and Van Assche, 2008), and N. hispanicus (Copete et al., 2011a). In all cases, radicle emergence took place at cool temperatures following a previous warm period. As N. alcaracensis range is restricted to mountain cold locations, we hypothesise that radicle emergence only occurs after a long period of either solely cold stratification of seeds simulating winter temperatures, or cold stratification followed by incubation at cool temperatures, simulating the genuine sequence of temperatures in winter-early spring. In addition, we also tested the hypothesis that growth rate of the embryo increases with seed age, as suggested by the higher germination percentages registered in dry-stored seeds of N. hispanicus (Copete et al., 2011a).

In Narcissus alcaracensis the embryo is linear in type with the radicular end touching the base of the seed. In freshly matured seeds the embryo is 1.42 [+ or -] 0.03 mm long, and the seed measures 3.99 [+ or -] 0.04 mm in length (mean [+ or -] SE; n = 40), which strongly suggests the existence of underdeveloped embryos. On the other hand, preliminary observations showed that seeds did not germinate during one month of incubation at temperatures simulating those in the natural habitat throughout the year (see "germination testing procedures" section in methods), suggesting that seeds have morphophysiological dormancy (MPD). Assuming this is the case, the main goal of the present study was to elucidate which one of the nine levels of MPD (Baskin et al., 2008) occurs in N. alcaracensis seeds. This study is one of a series focused on the germination ecology of daffodils endemic to the Iberian Peninsula, whose general goal is to determine levels of MPD in the genus, and the evolutionary relationships between them.

Specifically, the goals of this study were to analyze:

(a) The effects of temperature and illumination conditions, as well as [GA.sub.3] on breaking of dormancy and embryo growth.

(b) The influence of seed age on embryo growth.

(c) The phenology of embryo growth and seedling emergence.

(d) The germination responses of seeds buried in soil and periodically exhumed.

(e) The influence of duration of cold-stratification, and light conditions (light/dark cycle), during the stratification treatment, and seed age on germination percentage.

(f) The effect of warm stratification preceding the cold stratification on germination percentage.

METHODS

PLANT MATERIAL AND SOURCE OF SEEDS

Narcissus alcaracensis Rios, Rivera, Alcaraz and Obon is a bulbous geophyte, 30-50 cm in height during flowering, with two 15-45 x 0.6-1.1-cm leaves, and flowering stems with one (sometimes 2-3) flowers at the apex. It is endemic to the mountains of southern Spain (Sierra de Alcaraz, province of Albacete), where it grows between 1200-1400 m.a.s.l, on the banks of streams, and the borders of calcareous peat bogs and seasonal lagoons covered by large sedge communities (Magnocaricion elatae W. Koch, 1926) and dominated by Carex hispida Willd., C. elata All., C. distans L., and Schoenus nigricans L. It was first described by Rios-Ruiz et al. (1999). Plants flower in Mar.-Apr., and pollination is allogamous, mainly by bees. Seeds ripen in early Jun. in capsules each containing 35-45 seeds (Martinez-Lirola, 2003).

We used seeds collected from the core N. alcaracensis population, of around 18,000 plants (Martinez-Lirola, 2003), located in the Sierra de Alcaraz (Penascosa, Albacete), 30SWH5978, at 1280 m.a.s.l., on the borders of a small seasonal pond near the Vidrio lagoon, colonized by Juncus grassland and sedge communities.

One hundred capsules ([approximately equal to] 4000 seeds) were collected for preliminary tests on 8 Jun. 2002. On 14 Jun. 2003, 1200 intact, apparently healthy capsules ([approximately equal to] 50,000 seeds) were collected (1-2 per plant) for germination assays and ex situ conservation in germplasm banks. At the laboratory, freshly matured seeds were randomized and left to dry at room temperature (21-22 C, 50-60% r.h.) until 1 Jul., when the first germination assays were performed (seed age = 0 mo). Seeds for subsequent assays were stored in paper bags at room conditions.

In 2002, preliminary assays showed that a high percentage of seeds germinated when subjected to a 5 C cold-stratification pre-treatment for 3-4 mo and then incubated at low (5, 15/4 C) or intermediate (20/7 C) temperatures in darkness. The percentage of seeds germinating also increased with seed age.

EMBRYO GROWTH IN LIGHT CONDITIONS

All assays described below were carried out using freshly matured seeds (age = 0 mo) placed on two sheets of filter paper moistened with distilled water in Petri dishes wrapped with plastic film and incubated in the light, commencing 1 Jul. 2003.

Firstly, to determine embryo length at dispersal of seeds, twenty seeds were placed in a 9-cm Petri dish. When the seeds had imbibed at room temperature for 24 h, embryos were excised with a razor blade and their lengths measured using a dissecting microscope equipped with a micrometer.

Five Petri dishes containing 20 seeds each were placed in a germination chamber programmed for one of the following temperatures: 5, 15/4, 20/7, 25/10, 28/14, and 32/ 18 C. Constant 5 C simulates the mean temperature during winter months, whilst the fluctuating treatments simulate average fall, spring, and summer conditions. After 1, 2, 3, 4, and 5 mo (1 mo = 30 d), one dish was removed from each chamber and the embryos of the 20 seeds contained were excised and measured. Mean length and standard error were calculated for each 20-embryo sample. If a seed had germinated in the dish, embryo length was assumed to be equal to the critical embryo length. To assess the critical embryo length, 200 seeds were incubated at 15/4 C after 3 mo at 5 C. After 15/4 C incubation for 21 d, embryos of 40 seeds with split seed coat but no radicle protrusion, i.e., critical embryo length, sensu Vandelook et al. (2007a) and Vandelook and Van Assche (2008), were measured. The mean length [+ or -] standard error of the embryo sample was 3.30 [+ or -] 0.06 mm, n = 40, with a range of 2.7-4 mm. The ratio of embryo length to seed length (critical E:S ratio) was also determined. It was 0.85 [+ or -] 0.001, ranging from 0.73-0.93. The minimal E:S ratio measured in seeds at the time of germination (the "threshold E:S ratio" hereafter) was set at 0.73.

Treatments A and B: Five hundred seeds were placed in a 16-cm Petri dish and incubated at 5 C. After 3 mo of incubation, 20 apparently healthy, ungerminated seeds were placed in each of five 9-cm Petri dishes and placed in chambers programmed for 15/4, 20/7, 25/10, 28/14, and 32/18 C, respectively, for 30 d (Treatment A). After treatment, the mean length and standard error were calculated for each sample of 20 embryos. The protocol was repeated 1 mo later, i.e., after incubation for 4 mo at 5 C (Treatment B).

Treatment C: One hundred and fifty seeds were placed in a 9-cm Petri dish and incubated at 28/14 C. After 4 mo of incubation, 20 seeds were placed in each of five 9-cm Petri dishes and placed at 5, 15/4, 20/7, 25/10, and 32/18 C, respectively, for 30 d. Following the treatment, the mean length and standard error were calculated for each sample of 20 embryos.

Treatments D and E: Two hundred and fifty seeds were placed in a 9-cm Petri dish, and exposed sequentially to 20/7 C for 1 mo, 15/4 C for 1 mo, and 5 C for 2 mo (Treatment D). Every 30 d, a sample of 20 apparently healthy seeds was removed at random, the embryos excised and measured, and mean length and standard error assessed. After 4 mo (the full extent of the treatment), 20 apparently healthy ungerminated seeds were placed in each of six 9-cm Petri dishes and incubated in chambers programmed for 5, 15/4, 20/7, 25/10, 28/ 14, and 32/18 C for 30 d (Treatment E). Mean length and standard error were calculated for each sample, taking the critical embryo length to be the real embryo size in germinated seeds (Hidayati et al., 2000a; Vandelook et al., 2007a).

EMBRYO GROWTH IN DARK CONDITIONS

To determine embryo growth at different temperatures in darkness, we performed the same assays to those described in light conditions, except for the assessment of critical embryo size. Dark conditions were achieved by wrapping Petri dishes in a double layer of aluminum foil. Handling of dishes and seeds was carried out under a green safe lamp (Vandelook et al., 2007a, b).

EFFECTS OF [GA.sub.3] ON DORMANCY BREAKING AND EMBRYO GROWTH

On 1 Jul. 2003, 20 seeds were placed on two sheets of filter paper moistened with a solution of 1000 mg/1 of gibberellic acid ([GA.sub.3]) in distilled water, in each of ten 9-cm Petri dishes. Dishes were placed in a chamber at 20/7 C, five in the light and five in darkness. This thermoperiod was chosen because it was different enough from the conditions required for cold stratification, but within the range of temperatures used in similar studies (Baskin et al., 2000). After 1, 2, 3, 4, and 5 mo, one dish was removed from each illumination condition and embryos were excised and measured. The mean length for each 20-embryo sample was compared with that of control embryos incubated in distilled water at the same temperature for the same period.

INFLUENCE OF SEED AGE ON EMBRYO GROWTH

The second control essay started on 1 Mar. 2005 showed that 20-mo aged seeds reached much higher germination percentages when incubated at 15/4 or 20/7 C in darkness than 0-mo aged seeds in the control. We therefore tested the hypothesis that embryo growth rate increases with seed age. On 1 Jul. 2005, twenty 24-mo-old seeds were placed on two sheets of filter paper moistened with distilled water in each of fifteen 9-cm Petri dishes sealed with parafilm and wrapped in a double layer of aluminum foil. Five dishes were incubated in each of three chambers at 5, 15/4, and 20/7 C, respectively. One dish was removed from each chamber after 1, 2, 3, 4, and 5 mo, and the embryos of non-germinated seeds were excised and measured. Embryo length for germinated seeds was assumed to be 3.30-mm (critical embryo length). The mean length was assessed for each 20-seed sample, and compared with those of embryos from 0-mo-old seeds incubated in the same conditions.

PHENOLOGY OF EMBRYO GROWTH

On 1 Jul. 2003, 50 seeds were mixed with fine sand and placed in each of twelve fine-mesh polyester cloth bags. The bags were buried in a pot at 5 cm depth and placed in a non-heated metal framehouse near Albacete (100 km from the Sierra de Alcaraz). The pot was watered to full capacity once a week from 1 Oct. to 31 May, and twice a month during the rest of the year, except on winter days when the soil was frozen. The resulting soil humidity conditions simulated those in the natural habitat of Narcissus alcaracensis. Temperatures in the greenhouse were recorded continuously with a meteorological datalogger and mean monthly maximum and minimum temperatures were calculated. One bag was exhumed each month commencing 1 Aug. 2003, and the contents sieved through a 1-mm mesh to recover the seeds. The embryos of 20 apparently intact seeds were excised immediately and measured, to assess mean length and standard error. The critical embryo length (3.3 mm) was assumed for seeds germinated within the bag while buried, in a percentage of embryos similar to that in germinated seeds.

PHENOLOGY OF SEEDLING EMERGENCE

On 1 Jul. 2003, three replicates of 200 seeds each were sown at 3-4 mm depth in 40 x 30 x 5 cm flat plastic trays with drainage holes, containing a substratum formed by a sterile mixture of peat and sand (3:1 ratio). Trays were placed in the unheated framehouse mentioned above and subjected to the watering regime described in the previous section. They were examined at 7-day intervals, and emerging seedlings were counted and removed. The study ended on 1 May 2007. Data were expressed as a percentage of cumulative emergence.

DORMANCY BREAKING IN BURIED SEEDS

On 1 Jul. 2003, 200 seeds mixed with fine sand were placed in each of twelve fine-mesh polyester cloth bags, and buried at a depth of 5 cm in a pot placed in the non-heated framehouse and subjected to a watering regime as described above. One bag was recovered each month commencing 1 Aug. 2003.

Seeds recovered were classified into four categories: (1) seeds germinated within the bag while buried; (2) viable non-dormant seeds, i.e., those germinating after an incubation period of 30 d at 15/4 C in darkness; (3) viable dormant seeds, i.e., those which did not germinate under the conditions described above, but which contained a healthy embryo; and (4) unviable seeds, i.e., seeds found damaged or decomposed when exhumed, and ungerminated seeds with unhealthy embryos.

GERMINATION TESTING PROCEDURES

(a) General conditions of germination tests

We performed both control assays (in which seeds were incubated at a given steady temperature) and assays in which seeds were incubated for 1 mo at different temperatures and light conditions, after 3-4 mo wet cold stratification (5, 9/5, 10 C), or 2 mo warm stratification (1 mo at 20/7 C [right arrow] 1 mo at 15/4 C) preceding 2 mo 5 C cold stratification. Stratifications were carried out with either a 12-h photoperiod (=light) or in darkness, using seeds of different ages.

Germination tests were carried out at a constant 5 C temperature and at the following fuctuating thermoperiods: 15/4, 20/7, 25/10, 28/14, and 32/18 C, where each temperature value in each thermoperiod alternated every 12 h. All seeds in each temperature regime were tested both in darkness and in light. In treatments including both light and fluctuating temperatures, the light phase was programmed to coincide with the higher temperature and the dark phase with the lower one.

The fluctuating temperatures used in the assays simulated mean maximum and minimum monthly temperatures, typical of the annual climatic cycles at low altitudes in the Sierra de Alcaraz, the natural habitat of Narcissus alcaracensis and the source of the seeds tested. Thus, 15/4 C corresponds to Nov. and Mar., 20/7 C to Oct. and Apr., 25/10 C to Sept. and May, 28/14 C to Aug. and Jun., and 32/18 C to Jul. The 5 C treatment simulates the mean temperature recorded during the winter months: Dec., Jan., and Feb.

One hundred seeds were assigned to each treatment, divided into four 25-seed replicates. Each replicate was incubated on a double layer of filter paper moistened with distilled water, in a 9 cm diameter Petri dish sealed with parafilm, placed in germination chambers (Ibercex F4 Model, Madrid, Spain). Germination chambers were equipped with a digital temperature and light control system [[+ or -] 0.1 C, cool white fluorescent light, 25 [micro]mol x [m.sup.-2] x [s.sup.-1] (1350 lux)]. Tests lasted 30 d, following the recommendations of Baskin and Baskin (1998). The dark stratification and dark incubation treatments were achieved by wrapping Petri dishes in a double layer of aluminum foil. In light treatments, seeds were checked for germination every 3-4 d. Seeds incubated in darkness were checked only at the end of the test. Ungerminated seeds were checked for viability on the basis of the appearance of their embryos, paying special attention to their color and turgidity. Germination percentages were calculated on the basis of the number of viable seeds.

(b) Control assays

The first control was started on 1 Jul. 2003 (seed age = 0 mo). Four 25-seed replicates were used for each test condition: 15/4, 20/7, 25/10, 28/14, and 32/18 C, in both light and darkness. Dishes were examined at monthly intervals and germinated seeds were counted and removed. Assays lasted 5 mo, in order to compare germination results with those obtained from seeds which were stratified for 4 mo and then incubated for 1 mo at other temperatures. Dishes incubated in darkness were handled trader a green safe lamp (Vandelook et al., 2007a). The second control was carried out using the same conditions from 1 Mar. 2005 (seed age = 20 mo).

(c) Effects of cold stratification treatments, illumination conditions, and seed age on seed germination

Effective temperatures for cold stratification range from 0 to 10 C, with about 5 C being optimal for many species (Stokes, 1965; Baskin et al., 1992, 1995). In the present study, temperatures of 5, 10, and 9/5 C were used.

On 1 Jul. 2003, 1400 seeds were placed on two layers of filter paper wetted with 10 [cm.sup.3] of distilled water, in each of eight 16-cm Petri dishes. Dishes were sealed with parafilm. They were then subjected to the following cold-stratification treatments: two dishes at 5 C for 3 mo, two dishes at 5 C for 4 mo, two dishes at 9/5 C for 4 mo (Treatment F), and two dishes at 10 C for 4 mo (Treatment G). In each treatment, one dish was stratified in the light and one in darkness. The degree of hydration of filter paper in the dishes was checked once a month (under a green safe lamp for seeds stratified in darkness; Vandelook et al., 2007a, b). After stratification, ungerminated seeds apparently in good condition were selected for germination testing at all temperatures, both in light and darkness.

To determine the effect of seed age on germination, the cold-stratification at 5 C for 3 mo treatment and subsequent germination tests were repeated on 1 Mar. 2004 (seed age = 8 mo) and 1 Jul. 2004 (seed age = 12 mo). In addition, the cold stratification at 5 C for 4 mo treatment was also repeated on 1 Nov. 2003 (seed age = 4 mo).

(d) Effect of warm stratification preceding cold stratification on seed germination

This assay was performed to discover whether warm stratification (simulating autumn temperatures in the natural habitat -20/7 C and 15/4 C) preceding cold stratification (simulating winter cold -5 C) might enhance the germination capacity of Narcissus alcaracensis seeds, as has been shown in other species whose MPD is overcome by cold stratification (Baskin et al., 1995; Walck and Hidayati, 2004a).

On 1 Jul. 2003 (seed age = 0 mo), 1400 seeds were placed on two layers of filter paper wetted with 10 [cm.sup.3] of distilled water, in each of two 16-cm Petri dishes. Dishes were sealed with parafilm and one was stratified in light and the other in darkness. Dishes were submitted to the following temperature sequence: 20/7 C for 1 mo, 15/4 C for I mo, and 5 C for 2 mo (Treatment D). Seeds were subsequently tested for germination at all temperatures. Results were compared with the control test performed on 0-mo-old seeds.

STATISTICAL ANALYSIS

The effects on the length of embryos of the temperature and the duration of incubation were analyzed using a two-way ANOVA. The effect of seed age on embryo growth at different temperatures was analyzed using a one-way ANOVA.

Seed germinability was evaluated according to the final cumulative germination percentage, which was compared between treatments using multifactor ANOVAs. Comparing the germination percentage after cold stratification at 5 C, the effects of five factors were analyzed: (1) temperature (six levels); (2) light (two levels); (3) seed age (four levels); (4) length of cold stratification (two levels); and (5) light conditions during cold stratification (two levels). In the study of the influence of other stratification treatments on seed germinability, the effects of four factors were analyzed: (1) temperature (six levels); (2) light (two levels); (3) treatment (three levels); and (4) light conditions during stratification (two levels).

Where significant effects were discovered, differences were detected using a multiple comparison Tukey test. Normality (Cochran test) and homoscedasticity (David test) of the data were checked prior to the analyses. Values of the final cumulative germination percentage were square-root arcsine transformed.

RESULTS

EMBRYO GROWTH AT SEVERAL TEMPERATURES AND AFTER STRATIFICATION OF SEEDS In the light.--Mean length of embryos from freshly matured seeds incubated at 15/4, 20/7, 25/10, 28/14, and 32/18 C for 5 mo ranged from 1.87 to 2.42 mm (Table 1A). The critical embryo lerigth (i.e., embryo size in germinated seeds; 3.30 [+ or -] 0.06 mm) was not

achieved in any treatment, and no seeds germinated. However, for seeds incubated at 5 C for 5 mo, the mean embryo length was 2.50 [+ or -] 0.13 mm and 25% of seeds germinated.

Embryo growth and the percentage of seeds germinating increased substantially following cold stratification at 5 C for 3 or 4 mo and subsequent transfer to 15/4 C and 20/7 C (Treatments A and B, respectively). Indeed, germination was 35-40% when the preceding cold stratification period lasted 4 mo. The highest germination percentages were recorded for seeds incubated at 15/4 C for 30 d after a warm stratification simulating autumn temperatures (1 mo at 20/7 C followed by 1 mo at 15/4 C), followed by a period of 2 mo cold stratification at 5 C (Treatment E). At these conditions, mean embryo length was 3.16 [+ or -] 0.06 mm, 75% of embryos reached the threshold E:S ratio, and 45% of seeds germinated (Table 1A).

Following warm stratification at 28/14 C for 4 mo and subsequent transference for 30 d (Treatment C), no embryo achieved the threshold E:S ratio at any temperature, and no seeds germinated.

In darkness.--In dark conditions, embryo growth and germination percentage showed a similar pattern to that in the light, but the final values of both parameters were higher. Although embryos hardly grew when incubated at 20/7, 25/10, 28/14, or 32/18 C for 5 mo, at 15/4 C mean embryo length was 2.93 [+ or -] 0.10 mm, 45% of embryos achieved the threshold E:S ratio, and 5% of seeds germinated (Table 1B).

After 3 mo of cold stratification at 5 C followed by seed transference at 15/4 C (Treatment A), mean embryo length was 3.03 [+ or -] 0.08 mm, 65% of embryos achieved the threshold E:S ratio, and 50% of seeds germinated. These values increased when the initial phase of cold stratification lasted 4 mo (Treatment B). Treatment E (i.e., warm stratification [right arrow] cold stratification [right arrow] seed transference at all temperatures) resulted in the highest embryo growth and seed germination. So, when seeds were transferred to 15/4 C for 30 d, mean embryo length was 3.30 [+ or -] 0.00 mm, threshold E:S ratio was achieved in 100% of seeds, and 85% of seeds germinated (Table 1B).

EFFECTS OF [GA.sub.3] ON DORMANCY BREAK AND EMBRYO GROWTH

[GA.sub.3] did not promote embryo growth. The mean embryo length of seeds incubated at 20/ 7 C for 5 mo with a 1000 ppm-[GA.sub.3] solution was 2.06 [+ or -] 0.08 mm in the light, and 2.33 [+ or -] 0.05 mm in darkness. No seeds germinated and no embryos reached the threshold E:S (data not shown).

INFLUENCE OF SEED AGE ON EMBRYO GROWTH

In 24-mo-old seeds incubated in darkness at 15/4 or 20/7 C for 5 mo, mean embryo lengths were higher after 2 mo incubation than those recorded for 0-mo-old seeds. At 5 C, the differences were not significant. In old seeds, the final mean embryo length was 3.17 [+ or -] 0.08, and 3.21 [+ or -] 0.05 mm, at 5 and 15/4 C, respectively (Fig. 1).

The germination rate of 24-mo-old seeds incubated at 15/4 C in darkness was 45, 70, and 80% at 90, 120, and 150 d, respectively. Germination percentages decayed (i.e., 15, 40, and 45% at 90, 120, 150 d, respectively) when the incubation temperature was 20/7 C. In contrast, freshly matured seeds reached 0, 0, and 5% germination at the same periods when incubated at 15/4 C in darkness, and did not germinate at all when the temperature was 20/ 7C (Fig. 1).

PHENOLOGY OF EMBRYO GROWTH AND SEEDLING EMERGENCE

In buried seeds, the embryo hardly grew between 1 Jul. and 1 Nov. 2003. In contrast, the mean embryo length increased from 1.90 [+ or -] 0.07 mm to 2.82 [+ or -] 0.08 mm during Nov., when the mean maximum and minimum temperatures were 14.3 and 4.1 C, respectively. Embryo growth slowed during Dec. and Jan. and by 1 Feb. 2004, the mean embryo length was 3.10 [+ or -] 0.04 mm, and 25% of seeds had germinated within the bags. By 1 Mar. 2004, mean length was 3.27 [+ or -] 0.02 mm, 75% of the seeds had germinated, and embryo length measurements in buried seeds ceased from that date (Fig. 2).

From 1 Jul. 2003 to 1 Feb. 2004, no seedling emergence was recorded in the flat trays. Emergence commenced during the second fortnight of Feb., 1 mo later than the first radicle-emergence of seeds within the buried bags. Seedling emergence increased throughout Mar. 2004, just after the completion of embryo growth. During Mar., cumulative emergence increased from 12.83 [+ or -] 1.21% to 82.00 [+ or -] 2.26%. Mean maximum and minimum temperatures during Mar. were 13.2 and 1.8 C, respectively.

In spring 2004, the last emergent seedlings were recorded on 6 May, at which time cumulative emergence was 84.5 [+ or -] 2.9%. From 6 May 2004 to 1 Mar. 2005 no further emergence was recorded. At the end of spring 2005, the cumulative emergence was 98.33 [+ or -] 1.16%. The last emergence was recorded on 1 May 2006 (Fig. 2).

DORMANCY BREAKING IN BURIED SEEDS

Of the seeds recovered in the first five exhumations (from 1 Aug. to 1 Dec. 2003), most were viable and dormant (Fig. 3). Of the seeds rescued on 2 Jan. 2004, 4% had germinated within the bag and 18% were viable non-dormant. These percentages increased to 24 and 57%, respectively, in the seedlot exhumed on 1 Feb. 2004. The seed fractions on 1 Mar. 2004 were: 72% germinated in the bag, 21.5% viable non-dormant, 2% viable dormant, and 4.5% unviable. On 1 Apr. 2004, the values were: 83% germinated, and 11.5% viable non-dormant. From 1 May 2004 onwards most of the seeds exhumed were decomposed.

EFFECT OF COLD STRATIFICATION TREATMENTS, ILLUMINATION CONDITIONS AND SEED AGE ON SEED GERMINATION

In controls with freshly matured seeds incubated in the light, germination was always [less than equal to] 2%. At 15/4 C in darkness, germination was 14%. In 20-mo-old seeds, germination was also low ([less than or equal to] 14%) when incubated in the light, but in darkness germination reached 80.00 [+ or -] 3.16 and 48.00 [+ or -] 2.45% at 15/4 C and 20/7 C, respectively (Table 2).

In freshly matured seeds, cold-stratification treatments promoted germination, both at 5 C for 3 or 4 mo (Fig. 4) and 9/5 C (Treatment F) or 10 C (Treatment G) for 4 mo (Table 2). Specifically, germination increased in the order: 10, 9/5, and 5 C. Therefore, in seeds cold-stratified at 5 C in the light for 3 mo, germination achieved 53.00 [+ or -] 4.36% when the seeds were subsequently incubated at 15/4 C in darkness. Final germination rate increased when the stratification period was extended to 4 mo in combination with low or middle incubation temperatures (5, 15/4, 20/7 C; Fig. 4).

Germination percentages were higher in seeds incubated in darkness than in the light, and cold stratification in darkness was also more effective in breaking dormancy than when performed in the light (Fig. 4, Tables 2 and 3). Hence, cold stratification at 10 C was generally quite unsuccessful, except when the seeds were stratified and incubated in dark conditions (Table 2).

Germination ability in seeds cold-stratified for 3 or 4 mo at 5 C also increased with seed age. Eighty-five percent of 12-mo-old seeds cold-stratified for 3 mo at 5 C in darkness germinated when incubated at 15/4 C, both in darkness and in the light (Fig. 4). In general, when 12-mo-old seeds that had been cold stratified at 5 C for 3 mo were incubated at various temperatures (15/4, 20/7, 25/10 C), the final germination percentages were higher than those recorded for freshly matured seeds (0 mo old) which had been cold stratified for 4 mo at 5 C (Fig. 4). Therefore, dry storage reduced the cold-stratification period required for breaking of dormancy.

EFFECT ON SEED GERMINATION OF WARM STRATIFICATION PRECEDING COLD STRATIFICATION

Compared to cold stratification for 4 mo at 5, 9/5, or 10 C, stratification of freshly matured seeds at autumn temperatures (1 mo at 20/7 C [right arrow] 1 mo at 15/4 C) preceding 2 mo of cold stratification at 5 C (Treatment D) showed a beneficial effect on percentage germination when seeds were incubated at 15/4 C, irrespective of light conditions during stratification or the incubation phase. In fact, the highest germination rates in this study (90.00 [+ or -] 3.32%) were achieved with seeds incubated at 15/4 C in darkness following Treatment D (Table 2, Fig. 4). However, when seeds were incubated at 5, 25/10, 28/14, and 32/18 C, cold stratification for 4 mo at 5 C was more effective than Treatment D in promoting germination. At 20/7 C incubation, similar germination percentages were achieved in both stratification treatments.

DISCUSSION

Seed dormancy (or blocks to germination) includes a diverse range of mechanisms including time required for embryo growth (example of morphological dormancy), hormone/biochemical cues and environmental cues (examples of physiological dormancy). It is clear that Narcissus alcaracensis seeds show morphophysiological dormancy (MPD) because: (1) they have embryos that are differentiated but underdeveloped (mean embryo length of 1.42 [+ or -] 0.03 mm versus mean seed length of 3.99 [+ or -] 0.04 mm) ; (2) the embryo does not achieve the critical length (i.e., 3.30 [+ or -] 0.06 mm) unless seeds are placed on a moist substrate in the most favorable light and temperature conditions; and (3) seeds do not germinate until a minimum period of 90 d elapses. In addition, (1) cold stratification for a 90 d period, preferably at 5 C in darkness, was the only requirement for breaking dormancy, and initiating embryo growth and germination; and (2) [GA.sub.3] did not substitute for stratification, which suggests that seeds of this species show deep complex MPD (Baskin and Baskin, 2004a, 2005; Walck and Hidayati, 2004a; Vandelook et al., 2007b). However, some of our results do not fully match the MPD classification. Firstly, dry storage at laboratory conditions can shorten the cold stratification period required for the dormancy breaking. Second, exposure to a slightly warm stratification (1 mo at 20/7 C [right arrow] 1 mo at 15/4 C) had similar effects to dry storage. These characteristics are typical of intermediate physiological dormancy in seeds with fully-developed embryos, which are often (but not always) sensitive to [GA.sub.3] (Baskin et al., 2002; Baskin and Baskin, 2004b). These results suggest that the physiological dormancy in N. alcaracensis is of an intermediate level and that their seeds show intermediate complex MPD. Intermediate complex MPD is frequent in Ranunculaceae and bas been observed in Araliaceae, Papaveraceae, and Caprifoliaceae, but never in the genus Narcissus or in any other Amaryllidaceae (Baskin and Baskin, 1998).

Germination ecology of Narcissus alcaracensis differs substantially from that of N. pseudonarcissus (Vandelook and Van Assche, 2008) and N. hispanicus (Copete et al., 2011a), since in these species embryo growth is continuous throughout summer, and radicle protrusion occurs in early autumn, although the timing of seedling emergence is delayed up to late winter. Seeds of N. hispanicus have deep simple epicotyl MPD (Copete et al., 2011a). That dormancy level however was not assigned to N. pseudonarcissus by Vandelook and Van Assche (2008), as seedling growth is continuous. In the study on N. bulbocodium (Thompson, 1977) there is no reference to embryo size in relation to the endosperm, so future analyses are needed to determine whether their seeds are physiological or morphophysiologically dormant.

In the unheated framehouse, the peak of seedling emergence occurred in Mar. 2004. At the beginning of that month, mean embryo length in buried seeds was 3.27 [+ or -] 0.02 mm, very close to the critical embryo length, so that once embryo growth was completed seedling emergence was more rapid. The number of hours of cold stratification (0-10 C) from 1 Sept. to 15 Feb. 2004 (the date of commencement of seedling emergence) was c. 1932, some 11.5 wk of continuous cold stratification. Narcissus alcaracensis seeds were also subjected to a warm stratification pre-treatment (temperature [greater than or equal to] 15[degrees]C) in dark conditions during the summer and autumn months of 2003. Although such a warm stratification is not essential for germination, it could have a beneficial effect, as revealed in the laboratory, where the highest germination percentages were achieved after stratification Treatment D followed by incubation at 15/4 C (Table 2). Moreover, Treatment D increased embryo growth in the light compared to cold stratification for 4 mo at 5 C (Table 1A).

Although a warm stratification pre-treatment preceding cold stratification is only strictly necessary for the germination of seeds with nondeep complex MPD (Baskin and Baskin, 1998), it could stimulate germination in seeds with intermediate complex MPD, as observed in Delphiniumfissum subsp, sordidum (Herranz et al., 2010b), and even in taxa with deep complex MPD such as Osmorhiza occidentalis, O. chilensis, Erythronium grandiflorum (Baskin et al., 1995), Anthriscus sylvestris (Baskin et al., 2000), and Osmorhiza depauperata (Walck and Hidayati, 2004a). Baskin et al. (1995) have postulated that deep complex MPD may derive from nondeep complex MPD. However, Wen et al. (2002) analyzed the types of dormancy found in Osmorhiza and concluded that the derived condition was nondeep complex MPD. Walck and Hidayati (2004a) concluded that "the stimulatory effect from a warm pre-treatment in species needing only cold stratification for dormancy breaking is a pre-adaptation that initiated the development of an absolute warm requirement in species needing both warm and cold stratification." In our opinion, future studies on the germination ecology of other species of Narcissus, which probably show individual levels of complex MPD (as suggested by preliminary observations in our lab), could contribute to solving these issues.

Results in the non-heated framehouse strongly suggest that dormancy break and embryo growth in Narcissus alcaracensis seeds occur during late autumn and winter in nature (Fig. 2), so they would become ready to germinate from late winter to early spring (Feb.-Apr.) at low (5, 15/4 C) and middle (20/7 C) temperatures (Fig. 4, Table 2), including the cold-stratification temperature (5 C), as recorded in other species with different levels of complex MPD: Osmorhiza claytonii (nondeep level; Baskin and Baskin, 1991), Sambucus racemosa (intermediate level; Hidayati et al., 2000b), and Chaerophyllum temulum (deep level; Vandelook et al., 2007b). The ability to germinate at low temperatures is widespread in Mediterranean species (Thompson, 1968; Bell et al., 1993). After treatments that break dormancy, the germination ability of N. alcaracensis seeds deceases considerably at temperatures [greater than or equal to] 25/10 C, because embryo growth is interrupted at such temperatures.

The main ecological consequence when cold stratification is required for breaking seed dormancy is obviously the minimization of seedling mortality. Seeds cannot germinate during summer and autumn because they are still dormant, so massive death of small, cold-sensitive seedlings due to winter frosts is avoided. Seedlings emerging during autumn would not have enough time to form a large enough perennating structure (bulb), to survive over winter. Germination during late winter-early spring provides cold-intolerant seedlings with a 3-mo period of optimal light and soil moisture conditions to grow before the onset of summer drought, common in mesic woodland herbaceous habitats (Baskin and Baskin, 1994).

The highest germination percentage observed in the freshly matured seed controls (14%) was achieved when seeds were incubated at 15/4 C in darkness (Table 2). Some seeds germinated because during the 5-mo assay period they were exposed to 10.2 wk of effective cold stratification at 4 C, and a similar period of warm stratification at 15 C. However, in the control using 20-mo-old seeds, germination percentage at 15/4 C in darkness was 80% (Table 2). This increase results from the increase in embryo growth rate in older seeds (Fig. 1). The differences in embryo growth rate, and also in germination ability, were more pronounced at 20/7 C in darkness (Fig. 1). However, in 20-mo-old seeds cold stratified for 3 and 4 mo at 5 C, embryo growth was similar to that of freshly matured seeds (Fig. 1), although it is probable that in cold-stratified, old seeds embryo growth would be higher than in cold-stratified 0-mo-old seeds when transferred to several incubation temperatures. This might explain the increase in germination ability with seed age after dry storage in laboratory conditions for 8 or 12 mo followed by 3 mo cold stratification at 5 C (Fig. 4; Table 3). Although enhanced germination percentages with seed age are frequent in seeds with nondeep physiological dormancy (Baskin and Baskin, 1998; Copete et al., 2005), and even in seeds with deep physiological dormancy such as Euonymus europaeus (Baskin and Baskin, 1998), it is a seed trait little known in seeds with MPD, apart from Aconitum napellus subsp, castellanum (Herranz et al., 2010a), Delphinium fissum subsp, sordidum (Herranz et al., 2010b), Narcissus hispanicus (Copete et al., 2011a), and Merendera montana (Copete et al., 2011b). Although in the first species cited above, changes in embryo growth rate with seed age were not studied, in M. montana seeds embryo growth rate increased with seed age (Copete et al., 2011b). In future studies, it would be interesting to determine at what age dry-stored seeds exhibit their highest embryo growth rate, although a decrease would be expected before seeds lose their viability.

The fact that most Narcissus alcaracensis seeds (>80%) germinated within bags while buried from Jan. to Apr. (Fig. 3) reveals that there are not inhibitory causes of germination (e.g., low oxygen and/or high carbon dioxide levels, and volatile products of anaerobic respiration; Bloom et al., 1990) apart from unfavourable temperature conditions in buried non-dormant seeds. This of course impeded the study of dormancy cycling. However, the

study of seedling emergence (Fig. 2) showed that at the end of the first vegetative period, 15.5% seeds remained ungerminated in the soil (cumulative seedling emergence 84.5 [+ or -] 2.90%) and most seeds germinated during the second post-sown spring (cumulative seedling emergence 98.33 [+ or -] 1.16%). The lack of germination in the late autumn of 2004, may be due to the induction of dormancy by high summer temperatures in seeds ungerminated at the end of spring 2004, even though favourable temperatures for germination and emergence were recorded (i.e., 20/7, 15/4, and 5 C). This has been observed in other species with intermediate complex MPD (e.g., Delphinium fissum subsp, sordidum; Herranz et al., 2010b) or deep complex MPD (e.g., Frasera caroliniensis; Threadgill et al., 1981).

Seeds of Narcissus alcaracensis incubated in the dark following cold stratification exhibited higher germination percentages than those incubated in photoperiod conditions (Fig. 4, Table 2 and 3), as recorded in other monocots such as Schoenolirion croceum (Walck and Hidayati, 2004b) and Trillium camschatcense (Kondo et al., 2011). The promotion of germination in dark conditions may have important ecological consequences. Firstly, it makes difficult for seeds to persist in the soil seed bank (Walck et al., 1998; Milberg et al., 2000). Second, the ability to germinate in darkness with adequate underground soil moisture would ensure seedling survival and growth, in contrast to seed germination in the light, i.e., on the soil surface, where dry periods may occur in early spring and severely damage establishing seedlings (Thanos et al., 1991; Bell et al., 1999).

Although the ability of Narcissus alcaracensis seeds to germinate in darkness restricts their ability to form persistent soil seed banks, our assay of seedling emergence (Fig. 2) revealed that 13.83% seeds sown on 1 Sept. 2003 produced seedlings in the second post-sown spring. This suggests that the species could have the ability to form small persistent seed banks in the natural habitat due to temperature-mediated restrictions to germination, providing with important ecological advantages for population resilience (Baskin and Baskin, 1978; Milberg, 1994).

RECOMMENDATIONS FOR POPULATION-RESTORATION PROGRAMS

Our results suggest the following protocols for the production of plants for future population restoration (reintroduction and reinforcement) programs in the native habitat of this species. On 1 Sept., during the vegetative cycle 1, 2-mo-old seeds should be submitted to stratification Treatment D (for a period of 4 mo) in darkness. Subsequently, seeds should be incubated at 15/4 C in darkness for 1 mo. Early in the following Feb., seeds with an emerged radicle should be sown in pots filled with sterile peat-sand substratum (proportion 2:1); the cotyledons of most seeds will complete emergence within 1 mo. During Mar.-May seedlings will produce a small bulb (2 mm diameter), the aerial part will whiter in early Jun. and the plant will enter in a vegetative dormancy phase. At that time watering should be stopped to avoid decomposition until next early Nov., in the second vegetative cycle. As cycles 2 and 3 progress, bulbs will increase in size. Around Feb. in the fourth cycle, bulbs will have reached 1.5 cm diameter, and be ready to produce flower stems, at which time the plants will be ready to move into the natural habitat.

Acknowledgments.--The authors thank E. Molina and R. Herranz for their assistance with laboratory work. We are grateful to K. Walsh for checking the English. Funds kindly provided by the local Government of Castilla-La Mancha (Plan Regional de I+D+i; Consejeria de Educacion y Ciencia) are gratefully acknowledged: "Creation of a germplasm bank of threatened wild flora in the Botanical Garden of Castilla-La Mancha" (PAI07-0088-0300) and "Germination ecology of twelve singular or threatened plant species with morphophysiological dormancy" (PEII10-0170-1830). Comments from three anonymous reviewers contributed substantially to enhance the quality of the paper.

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SUBMITTED 9 JANUARY 2012

ACCEPTED 17 MAY 2012

J. M. HERRANZ, M. A. COPETE (1) AND P. FERRANDIS

Institute of Botany, University of Castilla-La Mancha, University campus s/n, Albacete 02071, Spain

(1) Corresponding author: Telephone: +34 967 599294; e-mail: miguel.copete@uclm.es


TABLE 1.--Influence of temperature, incubation period and light
conditions (Table IA: incubation in light; Table 1B: incubation in
darkness) on  embryo growth in Narcissus alcaracensis seeds

                        Incubation thermoperiods ([degrees]C)
                              in light conditions

                             5                        15/4

Table 1A
  Incubation  1   1.76 [+ or -] 0.04 (Abc)  1.92 [+ or -] 0.06 (Ac)
    period        (0,0)                     (0,0)
    (mo)      2   1.87 [+ or -] 0.05 (Aa)   2.12 [+ or -] 0.08 (ABb)
                  (0,0)                     (0,0)
              3   1.94 [+ or -] 0.07 (Aa)   2.21  [+ or -] 0.05 (Abb)
                  (0,0)                     (0,0)
              4   2.08 [+ or -] 0.07 (Abc)  2.27  [+ or -] 0.06 (ABb)
                  (5,5)                     (0,0)
              5   2.50 [+ or -] 0.13 (Bb)   2.42 [+ or -] 0.08 (BCb)
                  (25,30)                   (0,15)
  Treatments  A   2.08 [+ or -] 0.07 (Aa)   2.44 [+ or -] 0.13 (BCa)
                  (5,5)                     (25,25)
              B   2.50 [+ or -] 0.13 (Ba)   2.67 [+ or -] 0.12 (Ca)
                  (25,30)                   (35,35)
              C   2.08 [+ or -] 0.05 (Aa)   2.10 [+ or -] 0.06 (ABa)
                  (0,0)                     (0,0)
              E   2.80 [+ or -] 0.09 (Ba)   3.16 [+ or -] 0.06 (Db)
                  (5,45)                    (45,75)

Table 1B
  Incubation  1   1.73 [+ or -] 0.05 (Aa)   1.99 [+ or -] 0.07 (Ab)
    period        (0,0)                     (0,0)
    (mo)      2   2.09 [+ or -] 0.04 (Ab)   2.26 [+ or -] 0.05 (Abb)
                  (0,0)                     (0,0)
              3   2.55 [+ or -] 0.09 (Bbc)  2.53 [+ or -] 0.07 (BCb)
                  (0,35)                    (0,15)
              4   2.95 [+ or -] 0.14 (Cb)   2.65 [+ or -] 0.09 (CDb)
                  (30,60)                   (0,35)
              5   3.13 [+ or -] 0.07 (Cb)   2.93 [+ or -] 0.10 (DEb)
                  (60,75)                   (5,45)
  Treatments  A   2.95 [+ or -] 0.14 (Cbc)  3.03 [+ or -] 0.08 (EFc)
                  (30,60)                   (50,65)
              B   3.13 [+ or -] 0.07 (Cb)   3.12 [+ or -] 0.05 (EFb)
                  (60,75)                   (60,85)
              C   2.09 [+ or -] 0.04 (Aab)  2.25 [+ or -] 0.04 (ABb)
                  (0,0)                     (0,0)
              E   3.16 [+ or -] 0.07 (Cbc)  3.30 [+ or -] 0.00 (Fc)
                  (40,80)                   (85,100)

                        Incubation thermoperiods ([degrees]C)
                              in light conditions

                            20/7                       25/10

Table 1A
  Incubation  1   1.52 [+ or -] 0.05 (Aa)    1.60 [+ or -] 0.04 (Aab)
    period        (0,0)                      (0,0)
    (mo)      2   1.75 [+ or -] 0.04 (ABa)   1.72 [+ or -] 0.04 (Aa)
                  (0,0)                      (0,0)
              3   1.85 [+ or -] 0.05 (ABCa)  1.80 [+ or -] 0.05 Aba
                  (0,0)                      (0,0)
              4   1.95 [+ or -] 0.04 (BCab)  1.85 [+ or -] 0.05 (ABCab)
                  (0,0)                      (0,0)
              5   2.07 [+ or -] 0.05 (BCa)   2.02 [+ or -] 0.05 (BCa)
                  (0,0)                      (0,0)
  Treatments  A   2.18 [+ or -] 0.13 (CDa)   2.11 [+ or -] 0.08 (Ca)
                  (15,20)                    (5,5)
              B   2.45 [+ or -] 0.15 (Da)    2.44 [+ or -] 0.12 (Da)
                  (40,40)                    (25,25)
              C   2.05 [+ or -] 0.05 (BCa)   2.03 [+ or -] 0.06 (BCa)
                  (0,0)                      (0,0)
              E   2.99 [+ or -] 0.09 (Eab)   2.87 [+ or -] 0.06 (Eab)
                  (25,60)                    (5,35)

Table 1B
  Incubation  1   1.73 [+ or -] 0.05 (Aa)    1.72 [+ or -] 0.05 (Aa)
    period        (0,0)                      (0,0)
    (mo)      2   1.80 [+ or -] 0.04 (ABa)   1.82 [+ or -] 0.05 (ABa)
                  (0,0)                      (0,0)
              3   1.85 [+ or -] 0.04 (ABa)   1.88 [+ or -] 0.05 (ABa)
                  (0,0)                      (0,0)
              4   2.19 [+ or -] 0.05 (CDa)   1.98 [+ or -] 0.05 (ABa)
                  (0,0)                      (0,0)
              5   2.22 [+ or -] 0.05 (CDa)   2.13 [+ or -] 0.05 (Ba)
                  (0,0)                      (0,0)
  Treatments  A   2.50 [+ or -] 0.12 (DEa)   2.55 [+ or -] 0.09 (Cab)
                  (25,30)                    (15,15)
              B   2.78 [+ or -] 0.13 (Eab)   2.82 [+ or -] 0.13 (CDab)
                  (50,50)                    (50,65)
              C   2.11 [+ or -] 0.04 (BCab)  2.11 [+ or -] 0.05 (Bab)
                  (0,0)                      (0,0)
              E   3.16 [+ or -] 0.06 (Fbc)   3.11 [+ or -] 0.07 (Dbc)
                  (50,80)                    (40,70)

                        Incubation thermoperiods ([degrees]C)
                              in light conditions

                            28/14                      32/18

Table 1A
  Incubation  1   1.63 [+ or -] 0.03 (Aab)   1.60 [+ or -] 0.04 (Aab)
    period        (0,0)                      (0,0)
    (mo)      2   1.78 [+ or -] 0.05 Aba     1.75 [+ or -] 0.05 (ABa)
                  (0,0)                      (0,0)
              3   1.83 [+ or -] O.04 (Aba)   1.79 [+ or -] O.04 (ABa)
                  (0,0)                      (0,0)
              4   2.00 [+ or -] 0.05 (BCab)  1.79 [+ or -] 0.04 (ABa)
                  (0,0)                      (0,0)
              5   2.03 [+ or -] 0.05 (BCa)   1.87 [+ or -] 0.05 (ABCa)
                  (0,0)                      (0,0)
  Treatments  A   2.16 [+ or -] 0.06 (Ca)    2.15 [+ or -] 0.08 (Ca)
                  (0,0)                      (0,0)
              B   2.60 [+ or -] 0.12 (Da)    2.46 [+ or -] 0.11 (Da)
                  (25,45)                    (20,35)
              C   2.03 [+ or -] 0.05 (BCa)   2,02 [+ or -] 0.05 (BCa)
                  (0,0)                      (0,0)
              E   2.74 [+ or -] 0.07 (Da)    2,74 [+ or -] 0.08 (Da)
                  (5,40)                     (0,40)

Table 1B
  Incubation  1   1.64 [+ or -] 0.03 (Aa)    1,63 [+ or -] 0.04 (Aa)
    period        (0,0)                      (0.0)
    (mo)      2   1.76 [+ or -] 0.07 (ABa)   1,74 [+ or -] 0.06 (Aa)
                  (0,0)                      (0.0)
              3   1.92 [+ or -] 0.04 (ABCa)  1,91 [+ or -] 0.07 (Aba)
                  (0,0)                      (0.0)
              4   1.99 [+ or -] 0.04 (BCa)   1,93 [+ or -] 0.04 (ABa)
                  (0,0)                      (0.0)
              5   2.11 [+ or -] 0.04 (Ca)    2,09 [+ or -] 0.07 (BCa)
                  (0,0)                      (0.0)
  Treatments  A   2.56 [+ or -] O.11 (Dab)   2,37 [+ or -] 0.08 (CDa)
                  (5,25)                     (0.15)
              B   2.51 [+ or -] 0.14 (Da)    2,64 [+ or -] 0.09 (DEa)
                  (30,35)                    (20.20)
              C   2.11 [+ or -] 0.04 (Cab)   2,06 [+ or -] 0.04 (Ba)
                  (0,0)                      (0,0)
              E   2.99 [+ or -] 0.07 (Eab)   2.81 [+ or -] 0.09 (Ea)
                  (20,60)                    (5,60)

                  Incubation thermoperiods
                    ([degrees]C) in light
                         conditions

                           Treat.D

Table 1A
  Incubation  1   1.52 [+ or -] 0.05 (Aa)
    period        (0,0)
    (mo)      2   1.85 [+ or -] 0.06 (Ba)
                  (0,0)
              3   2.24 [+ or -] 0.06 (Cb)
                  (0,0)
              4   2.57 [+ or -] 0.05 (Dd)
                  (0,15)
              5

  Treatments  A

              B

              C

              E

Table 1B
  Incubation  1   1.73 [+ or -] 0.05 (Aa)
    period        (0,0)
    (mo)      2   2.15 [+ or -] 0.06 (Bb)
                  (0,0)
              3   2.82 [+ or -] 0.08 (Cc)
                  (0,35)
              4   2.89 [+ or -] 0.06 (Cb)
                  (10,60)
              5

  Treatments  A

              B

              C

              E

Seed age at the beginning of the assay = 0 mo. Values are shown in mm
(mean [+ or /] SE, n = 20). Values followed by different uppercase
letters within columns  or different lowercase letters within rows are
significantly different ([alpha] = 0.05). The first number in brackets
is the percentage of seeds germinating, and the  second is the
percentage o seeds reaching the threshold E:S ratio. Additional
treatments: Treatment A: seeds transferred at all temperatures after 3
mo at 5 C;  Treatment B: seeds transferred at all temperatures after 4
mo at 5 C; Treatment C: seeds transferred at all temperatures after 4
mo at 28/14 C; Treatment D:  seeds exposed for 1 mo at 20/7 C,
followed by 1 mo at 15/4 C, and 2 mo at 5 C; Treatment E: seeds
transferred at all temperatures after Treatment D

TABLE 2.--Influence of incubation temperature and light conditions
during seed stratification and/or incubation on germination
percentages in  Narcissus alcaracensis seeds

                                          Control

                Test                    Seed age (mo)
            temperature
            ([degrees]C)           0                     20

Incubation
  Light          5                 --                    --
                15/4      2 [+ or -] 1.0 (Aa)   14 [+ or -] 3.3 (Bab)
                20/7      1 [+ or -] 0.9 (Aa)    3 [+ or -] 0.9 (Aab)
               25/10      2 [+ or -] 1.0 (Aab)   0 [+ or -] 0 (Aa)
               28/14      1 [+ or -] 0.9 (Aab)   0 [+ or -] 0 (Aa)
               32/18      0 [+ or -] O (Aa)      0 [+ or -] 0 (Aa)
  Darkness       5                 --                    --
                15/4      14 [+ or -] 3 (Ba)    80 [+ or -] 3.2 (Dca)
                20/7       2 [+ or -] 1.0 (Aa)  48 [+ or -] 2.5 (Ccd)
               25/10       0 [+ or -] 0 (Aa)     2 [+ or -] 1 (Aa)
               28/14       0 [+ or -] 0 (Aa)     0 [+ or -] 0 (Aa)
              32 / 18      0 [+ or -] 0 (Aa)     0 [+ or -] 0 (Aa)

                          Stratification in light
                Test
            temperature
            ([degrees]C)         Treat. D

Incubation
  Light          5         5 [+ or -] 0.9 (Aba)
                15/4      48 [+ or -] 6.0 (DEc)
                20/7      26 [+ or -] 3.3 (CDc)
               25/10       6 [+ or -] 3.0 (Aabc)
               28/14       7 [+ or -] 1.7 (ABbcd)
               32/18       2 [+ or -] 1.0 (Aabc)
  Darkness       5        50 [+ or -] 3.2 (DEd)
                15/4      90 [+ or -] 3.3 (Fd)
                20/7      60 [+ or -] 2.5 (Ed)
               25/10      51 [+ or -] 3.6 (Ed)
               28/14      21 [+ or -] 2.2 (BCd)
              32 / 18      1 [+ or -] 0.9 (Aa)

                          Stratification in light
                Test
            temperature
            ([degrees]C)         Treat. F

Incubation
  Light          5        18 [+ or -] 2.2 (CAb)
                15/4      26 [+ or -] 4.1 (Cbc)
                20/7      22 [+ or -] 3.3 (Cc)
               25/10      12 [+ or -] 1.4 (BCc)
               28/14       2 [+ or -] 1.0 (Aabc)
               32/18       1 [+ or -] 0.9 (Aab)
  Darkness       5        20 [+ or -] 1.4 (Cb)
                15/4      62 [+ or -] 4.6 (Db)
                20/7      58 [+ or -] 2.2 (Dd)
               25/10      26 [+ or -] 2.2 (Cbc)
               28/14      13 [+ or -] 1.7 (BCcd)
              32 / 18      5 [+ or -] 1.7 (ABab)

                          Stratification in light
                Test
            temperature
            ([degrees]C)         Treat. G

Incubation
  Light          5         4 [+ or -] 1.4 (ABa)
                15/4       5 [+ or -] 3.3 (ABa)
                20/7       5 [+ or -] 1.7 (ABab)
               25/10       5 [+ or -] 0.9 (Ababc)
               28/14       1 [+ or -] 0.9 (Aab)
               32/18       0 [+ or -] 0   (Aa)
  Darkness       5         3 [+ or -] 0.9 (ABa)
                15/4      26 [+ or -] 2.2 (Ca)
                20/7      12 [+ or -] 1.4 (BCb)
               25/10       2 [+ or -] 1.0 (ABa)
               28/14       3 [+ or -] 0.9 (ABb)
              32 / 18      3 [+ or -] 0.9 (ABab)

                             Stratification in
                Test             darkness
            temperature
            ([degrees]C)         Treat. D

Incubation
  Light          5         3 [+ or -] 0.9 (Aa)
                15/4      50 [+ or -] 4.1 (Ec)
                20/7      21 [+ or -] 4.6 (CDc)
               25/10       9 [+ or -] 2.6 (ABCbc)
               28/14      10 [+ or -] 2.2 (ABCcd)
               32/18       5 [+ or -] 0.9 (ABc)
  Darkness       5        40 [+ or -] 3.7 (DEcd)
                15/4      85 [+ or -] 2.2 (Fcd)
                20/7      52 [+ or -] 3.2 (Ecd)
               25/10      42 [+ or -] 3.3 (Ecd)
               28/14      19 [+ or -] 2.6 (BCd)
              32 / 18      4 [+ or -] 1.4 (Aab)

                            Stratification in
                Test            darkness
            temperature
            ([degrees]C)        Treat. F

Incubation
  Light          5        17 [+ or -] 1.2 (BCb)
                15/4      36 [+ or -] 2.5 (Dbc)
                20/7      15 [+ or -] 0.9 (Bbc)
               25/10      14 [+ or -] 2.2 (Bc)
               28/14      14 [+ or -] 2.2 (Bd)
               32/18       4 [+ or -] 1.4 (Abc)
  Darkness       5        30 [+ or -] 2.2 (CDbc)
                15/4      54 [+ or -] 2.2 (Eb)
                20/7      40 [+ or -] 2.5 (DEc)
               25/10      40 [+ or -] 3.2 (DEcd)
               28/14      15 [+ or -] 1.7 (Bcd)
              32 / 18      9 [+ or -] 1.7 (ABb)

                             Stratification in
                Test             darkness
            temperature
            ([degrees]C)         Treat. G

Incubation
  Light          5         7 [+ or -] 1.7 (BCDab)
                15/4       6 [+ or -] 1.0 (BCDa)
                20/7       3 [+ or -] 1.7 (ABCa)
               25/10       2 [+ or -] 1.0 (ABCab)
               28/14       1 [+ or -] 0.9 (Abab)
               32/18       0 [+ or -] 0 (Aa)
  Darkness       5        22 [+ or -] 2.2 EFb
                15/4      71 [+ or -] 1.7 (Gbc)
                20/7      39 [+ or -] 3.0 (Fc)
               25/10      18 [+ or -] 2.2 (DEb)
               28/14       9 [+ or -] 1.7 (CDEc)
              32 / 18      4 [+ or -] 1.4 (ABCab)

Results are shown as means [+ or /] SE. Values followed by different
uppercase letters within columns or different lowercase letters within
rows are significantly  different ([alpha] = 0.05). Controls: length =
5 mo; seed age at the beginning of the assay = 0 and 20 mo.
Stratification treatments: Treatment D: seeds exposed  for 1 mo at 20/
7 C, followed by 1 mo at 15/4 C, and 2 mo at 5 C; Treatment F: seeds
exposed for 4 mo at 9/5 C; Treatment G: seeds exposed for 4 mo at 10 C

TABLE 3.--Effects of various factors on seed germination of Narcissus
alcaracensis

Factor                              df      F        P

Incubation temperature               5    196.28   <0.001

Light conditions (incubation)        1    294.25   <0.001
Light conditions (stratification)    1    24.34    <0.001
Seed age                             3    129.76   <0.001
Duration of stratification           1    193.80   <0.001

Factor                                        Categories *

Incubation temperature              32/18 (a), 28/14 (b), 25/10 (c),
                                      5 (c), 20/ 7 (d), 15/4 (e)
Light conditions (incubation)       Light < Darkness
Light conditions (stratification)   Light < Darkness
Seed age                            0 (a), 8 (b), 4 (b), 12 (c)
Duration of stratification          3 < 4

The principal effects on germination of incubation temperature, light
conditions during incubation  and cold stratification, seed age, and
duration of cold stratification as revealed by multifactor analysis of
variance. The table shows degrees of freedom (df), F-ratio values, and
categories of factors where  differences in germination were
significant. Residual degrees of freedom: 468

* Categories with different letters are significantly different
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Author:Herranz, J.M.; Copete, M.A.; Ferrandis, P.
Publication:The American Midland Naturalist
Article Type:Report
Geographic Code:1USA
Date:Jan 1, 2013
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