versão On-line ISSN 0717-6643
Gayana Bot. v.64 n.1 Concepción jun. 2007
Gayana Bot. 64(1): 40-45, 2007
The effect of chilling on seed germination of placea species (Asparagales: Amaryllidaceae), an endemic genus to central Chile
Efecto del frío sobre la germinación de semillas en especies de Placea (Asparagales: Amaryllidaceae), un género endémico de Chile central
Pablo C. Guerrero1, Ana C. Sandoval2 & Pedro León-Lobos2,3
1Departamento de Ciencias Ecológicas, Facultad de Ciencias, Universidad de Chile. Las Palmeras 3425, Ñuñoa, Santiago, Chile. email@example.com
2 Banco de Base de Germoplasma, Centro Regional Intihuasi, Instituto de Investigaciones Agropecuarias, INIA, Casilla 73, Vicuña, Chile. firstname.lastname@example.org
3Centro de Estudios Avanzados en Zonas Aridas (CEAZA), Casilla 599, La Serena, Chile. email@example.com
The effect of chilling on germination was examined for six Placea species, a genus in the Amaryllidaceae endemic to the Andes and Coastal Range of central Chile. Based on their natural environment, we hypothesized that low temperatures would be important for germination in theses species. We addressed two questions (i) do Placea seeds require chilling for seed germination? (ii) if they required chilling, does thermal history before chilling affect their responsivity to chilling? At 4ºC seeds of all species germinated faster and to a higher level than at 25ºC. After 28 days, when seeds held at 25ºC were moved to 4ºC, germination increased significantly, however at the end of the assay, their germination was still lower than those seeds initially at 4ºC. Thus, Placea seeds required chilling for germinate. Seeds of Placea species might have physiological dormancy or might be non-dormant but with narrow and low range of temperature that are optimal for germination.
Keywords: Seed physiology, dormancy, biodiversity.
Se examinó el efecto del frío en la germinación de seis especies de Placea, un género en las Amaryllidaceae endémico de los Andes y Costa de Chile central. Basándonos en su ambiente natural, hipotetizamos que las bajas temperaturas debieran ser importantes para la germinación de estas especies. Se analizaron dos preguntas, (i) ¿Necesitan las semillas de Placea frío para poder germinar? (ii) si requieren frío, ¿afecta la historia térmica, antes del frío en su posterior respuesta al frío? A 4°C las semillas de todas las especies germinaron más rápidamente y en mayor cantidad que a 25°C. En el día 28, cuando las semillas a 25°C fueron movidas a 4°C, la germinación aumentó significativamente, sin embargo al final del ensayo, su germinación siguió siendo inferior que aquellas inicialmente a 4°C. Por lo tanto, las semillas de Placea requirieron frío para germinar. Las semillas de Placea tendrían latencia fisiológica o podrían ser no latentes pero con un acotado rango de temperatura óptima para su germinación.
Palabras claves: Fisiología de semillas, latencia, biodiversidad.
Low temperatures, less than 5°C, within highland habitats can be a strong selective pressure which drives the ecology and the evolution of plants (Bliss 1971; Chabot & Billings 1972; Cavieres & Arroyo 2000; Fenner & Thompson 2005). As seed germination is an adaptative process in plants (Harper 1977), selective pressures should guide the evolution of traits that increase establishment success of individuals. As cold temperatures, less than 10°C, are commonly produced in winter, plant species have evolved differential germination responses to cold requirements, thus temperature is a signal that seeds perceive and respond (Baskin & Baskin 1998). For example, some seeds can only germinate after cold stratification which produces the loss of dormancy. For other species, low temperatures might serve to slow germination in non-dormant seeds. Both mechanisms would enable germination after winter representing a natural mechanism which ensures that germination occurs in the spring, finally other species have seeds that germinate only under narrow and low temperature ranges (Bliss 1971; Baskin & Baskin 1998; Probert 2000; Sacandé et al. 2004). Since temperatures regimes vary across altitudinal and latitudinal ranges, it is interesting to ask if this climatic variation results in the evolution of differential germination requirements (i.e. chilling) of sister species.
The genus Placea Miers ex Lindley (Amaryllidaceae) are geophytes endemic to Coastal and Andean mountains to the central Chile between 31º00'-34º25'S; all species are characterized by narrow distributions (Traub & Moldenke 1949). Most Placea species are threatened by overexploitation of their bulbs due to their ornamental value and habitat destruction, nevertheless, their seed physiology, ecology and evolution are largely unknown (Meerow et al. 1999). The Mediterranean climatic region where species occur, present hot and dry summers with temperatures between 20ºC and 35ºC, and with wet and cold winters with temperatures ranging between 0ºC and 10ºC (di Castri & Hajek 1976).
The aim of our work was to evaluate the effects of chilling on the germination of six Placea species. Our hypothesis was that if low temperatures effectively represent a selective pressure, we should expect that Placea species should have dependence of chilling in their germination process. Two questions were addressed to examined chilling effects on germination; (i) do Placea seeds require chilling for seed germination? (ii) And, if they required chilling, does thermal history (pretreatment temperature) before chilling affect their responsivity to chilling? A pretreatment was used because for some other geophytes a period of warm temperatures (c. 25ºC) is necessary before seeds can germinate at cooler temperatures (Pritchard et al. 1993; Baskin & Baskin 1998).
MATERIALS AND METHODS
The species used in our study were Placea amoena Phil., P. ornata Miers ex Lindley, P. grandiflora Lem., P. lutea Phil., P. arzae Phil. and P. davidii Ravenna (Traub & Moldenke 1949). Only P. germaini was not included in the assay because it was not found at the expected locality; we presume that this species might be synonym of P. grandiflora, as their morphological descriptions are highly similar and they have sympatric distributions (Traub & Moldenke 1949). The other species studied have allopatric distributions. Nomenclature is based on Marticorena and Quezada (1985) and IPNI (2005).
Placea ornata, P. arzae and P. davidii grow within open areas on hills, whereas P. grandiflora grows within gaps of sclerophyllous forest and, P. lutea and P. amoena grow in shrublands. Flowering of P. ornata, P. grandiflora, P. amoena. P. lutea and P. arzae occurs between September and early October, while seed dispersion occurs from mid-October until November. Only P. davidii flowers between November and December, dispersing their seeds from January up to February.
All species were collected from natural populations in central Chile (Table I), between October 2004 and February 2005 (spring and summer) from more than 50 individuals and then pooled. Once collected, seeds were dried in a forced air room at 15% R.H. and 15-17ºC to around 5% moisture content and stored in a long term conservation chamber (-18ºC and 15% of relative humidity) to avoid premature germination and the loss of viability before the assay.
To assess the effect of chilling on seed germination, 300 seeds per species were sown in Petri dishes on filter paper and then stored in germination chambers with two constant temperature conditions at 4ºC and 25ºC with controlled relative humidity (60%), and a photoperiod of 14:10 (day/night respectively). After 28 days, the seeds at 25ºC were moved to 4ºC and the test continued until 70 days. For each treatment, 10 replicates with 15 seeds each were utilized. Germination was monitored daily and dishes were periodically watered with distilled water and the filter papers changed if fungal contamination was observed.
To asses the effect of temperature in the seed germination, an Analysis of Variance with repeated measures (ANOVAr) was conducted; due to the temporal dependence of germination, also species and temperature were considered as fixed factors. The repeated measure (i.e. time) consisted of a comparison between the total germination at 28 days (4 weeks), the moment of temperature change from 25ºC to 4ºC, and total germination at the end of the test (70 days). All data were arcsine transformed for a best fit to normality assumptions (Zar, 1999). In addition, two a priori tests of planned comparisons were performed at 28 and 70 days comparing differences between temperature treatments. If seeds require chilling to germinate, the 4ºC condition should present higher total germination. Moreover, if seeds at the 4ºC condition germinate more at 28 days but after the 70 days the total germination does not vary between temperature treatments we can presume that the effect of temperature is reversible, and for the contrary if after 70 days significant differences between the total germination are detect we can presume that seeds responses are not reversible, thus the 25ºC treatment could be inhibitory rather than neutral in effect. In other words if seeds are placed at supraoptimal temperatures their germination will decrease but the effect is only neutral if the seeds perform as predicted when returned to the sub-optimal range.
Significant differences on seed germination between Placea species were detected (ANOVAr, F5, 108 = 18.04, P < 0.001) (Figure 1). Furthermore, the effect of temperature on seed germination was strongly significant (ANOVAr, F1, 108 = 1085.44, P < 0.001), observing that at 4ºC all species germinated more rapidly and to a higher level than the treatment initially at 25ºC (Figure 1). In fact, seed germination of species at 28 days strongly varied between temperature treatments (ANOVAr planned comparisons, F1, 108 = 1979.63, P < 0.001) (Figure 1), indicating that chilling produced early differences in seed germination performance. In addition, an interaction between species and temperature was detected (ANOVAr, F5, 108 = 132.2, P = 0.038), meaning that the effect of temperature produced differential changes on seed germination between species, nevertheless all species showed a positive effect of low temperatures in their germination (Figure 1).
On the other hand, within subjects, time was strongly significant (ANOVAr, F1, 108 = 557.79, P < 0.0001) observing that seed germination after 70 days was higher than at 28 days (Figure 1). However, the interaction between time and the temperature treatments was also strongly significant (ANOVAr, F1, 108 = 512.02, P < 0.0001), detecting that species placed initially at 4ºC, after 70 days, presented higher germination than species initially placed at 25ºC (ANOVAr planned comparisons, F1, 108 = 228.58, P < 0.001). This strongly suggests that the period in which species were at 25ºC (during 28 days) had irreversible consequences on Placea spp. seed germination, because after the change of temperature (25ºC to 4ºC) at 28 days no species reached to the maximum of germination detected for the 4ºC treatment (Figure 1). In addition the interaction between species and time was significant (ANOVAr, F5, 108 = 11.75, P < 0.0001), meaning that species differed in their germination patterns through time, particularly P. davidii which had the smallest difference between germination at 28 and 70 days compared to other species. Finally, the triple interaction between time, species and treatment was also significant (ANOVAr, F5, 108 = 16.95, P < 0.0001); this interaction suggested that some species had differential capacity to achieved high germination when moved from warm to cool temperature. This is the case in P. ornata with cool germination of 97% and 81% germination when moved from warm to cool conditions, P. davidii were 85% and 23% respectively (Figure 1).
In our study we found that, consistent with the hypothesis proposed, all species strongly required chilling for germination. In fact, in the 4ºC treatment species seed germination began sooner and progressed faster compared to the 25ºC treatment. Moreover, germination significantly increased when seeds initially at 25ºC were transferred to 4ºC after 28 days. Nevertheless, the magnitude of the increase on seed germination after the reduction of temperature (25ºC to 4ºC) was significantly inferior to the germination showed of all species in the treatment initially at 4ºC, meaning that seed responses is not reversible, although this study shows that species varied in their capacity of improvement of seed germination after warm temperature. The later could be a consequence of seed losing viability after 28 days wet at 25ºC, rather than a possible imposition of secondary dormancy or thermoinhibition. Indeed, all seed that did not germinate at the end of the experiment were mouldy. The chilling effects observed in Placea spp. are in general similar to those obtained for other Alpine and Andean species, such that chilling or cold stratification is required for maximum rates of germination (Reynolds 1984; Baskin & Baskin 1998; Cavieres & Arroyo 2000).
Overall, the physiology and seed germination of Chilean Amaryllidaceae is poorly known, however morphological or morphophysiological dormancy have been suggested for the family due to the presence of underdeveloped embryos in their seeds (Baskin & Baskin 1998), but the later has not been determined. Placea species might have physiological rather than morphophysiological dormancy where seeds are dispersed with mature embryo and the seeds require a period of chilling for the best germination. Alternatively, Placea seeds may be non-dormant, but with narrow and low range of optimal temperature for germination as detected in Prunus africana (Hook.f.) Kalkman (Sacandé et al. 2004). Comparatively, seeds initiate rapidly their germination at 4ºC reaching their maximum in less than 7 days after the experiment started, indicating that cold conditions effectively stimulate germination. Theses results are similar to those described by Schiappacasse et al. (2002) where 85% of seeds of P. arzae germinated at 7ºC, but with reduced germination at 15ºC. Placea seed could have nondeep physiological dormancy taking in account their fast germination at 4ºC. Further studies should seek to clarify the existence, kind and degree of dormancy (physiological or morphophysiological) in Placea spp. and the underlying mechanism. Both physiological and morphophysiological dormancy prevents germination during summer where germination might be followed by high mortality in the cohort due to the extreme dry conditions in central Chile (Fuentes et al. 1984). We presume that particularly those species which occurs in the Coastal Range, germination might occur during autumn and winter when temperature and moisture are adequate for the process. However, rapid germination of Placea seeds due to moisture and chilling responses could increase the risk of seedlings being exposed to frost once snow had melted in their natural habitats at mid to high elevation in the Andes of Central Chile during winter. Then during spring survival seedlings have the opportunity to intake energy, while their roots are burying in to the soil for creating the new bulbs. In summer, the foliage dry but the bulbs remains in the underground "waiting" for the growing season.
Apparently the trait of germination under chilling conditions might be not exclusive to Placea species within Chilean Amaryllidaceae, as suggested from results of Schiappacasse et al. (2002). Other Amaryllidaceae, like Phycella australis Ravenna and Rhodophiala rhodolirion (Baker) Traub (Andes) required cold temperature (7ºC) to germinate, but others like R. montana (Phil.) Traub, R. bagnoldii (Herb.) Traub and R. splendens (Renjifo) Traub, did not. More detailed studies are needed to determine patterns of seed germination in Amaryllidaceae species from temperate and alpine region.
We are grateful with L. Arriagada and N. García for their help in founding Placea species and with Marcos Acosta for their valuable help in field. We would also like to thank H.W. Pritchard for his constructive comments on an earlier version of the manuscript. PCG acknowledges the support of the CONICYT doctoral fellowship D-21070301. This research was supported by the Millennium Seed Bank Project from The Royal Botanic Gardens, Kew.
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Recibido: 09.08.06, Aceptado: 26.10.06