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Horse chestnut (Aesculus hippocastanum), a species with seeds that are recalcitrant (desiccation intolerant) in terms of storage behaviour (Tompsett & Pritchard, 1993), is a widely cultivated deciduous tree common in the northern temperate zone. Although unusual for recalcitrant species (Tweddle et al., 2003), freshly fallen seeds exhibit an embryo-based physiological dormancy that is relieved by a period of hydrated storage (stratification) at cold temperatures (Suszka, 1966; Tompsett & Pritchard, 1993, 1998; Pritchard et al., 1996, 1999). However, in the absence of cold stratification some, but not all, seeds in the population are able to germinate at high temperatures (31–36°C; Pritchard et al., 1999). Aspects of these responses are similar to the type 2 nondeep physiological dormancy described for numerous species, particularly summer annuals, such that seeds become able to germinate at lower temperatures as dormancy is lost during cold stratification (Baskin & Baskin, 1998). This response is well described qualitatively in many species, and ensures germination in the spring when successful establishment is most likely to occur.
We previously used horse chestnut to study the effects of a single cold stratification temperature (6°C) on dormancy release. We measured the effects of stratification on the temperature at which germination rate was zero, termed the base temperature (Tb). We established that Tb progressively reduced at a rate of 0.18°C d−1 for seedlots from three consecutive years, although the starting point, Tb at seed fall, differed between harvests (Pritchard et al., 1999). Thus dormancy release in horse chestnut seeds can be described simply in terms of a Tb reduction gradually allowing germination to occur at progressively lower temperatures (Pritchard et al., 1999). In Solanum physalifolium Tb apparently reduces during dormancy release at certain alternating temperatures (del Monte & Tarquis, 1997). A reduction in Tb could be inferred from dormancy release data for seeds of Alnus glutinosa (McVean, 1955) and Betula sp. (Joseph, 1929); the lowest temperature at which germination occurred reduced from 18 to 7°C and from 31 to 15°C, respectively, by 6 wk cold stratification (2–5°C). Batlla & Benech-Arnold (2003) recently described the same response in Polygonum aviculare seeds, in which a decrease in the lowest temperature for germination (denoted Tl by the authors) corresponded with dormancy loss, reducing from an estimated initial 18°C at harvest. Dormancy release was faster at cool than at warm stratification temperatures, and the relationship allowed the use of stratification thermal time to predict dormancy status based on the accumulation of thermal stratification units below a ceiling temperature (Tc), similar to that used previously for horse chestnut (Pritchard et al., 1996).
An alternative, theoretical, population-based model describing seed dormancy behaviour has been proposed by Bradford (1996, 2002) in relation to a moving water potential threshold for growth. The model requires estimates of the mean base water potential for germination (ψb(50)) and its standard deviation, and the hydrotime constant for each seed population. In applying this theory to germination following dormancy release (dry after-ripening) in the winter annuals Bromus tectorum and Elymus elymoides, an important assumption is that Tb remains constant and is set at 0°C while ψb(50) reduces during dormancy release (Bauer et al., 1998; Meyer et al., 2000). However, our previous work on cold stratification-induced dormancy loss in hydrated horse chestnut seeds demonstrated that accounting for the reduction in Tb may suffice for quantifying horse chestnut seed performance (Pritchard et al., 1999).
In the present study, we considerably extend our earlier work to encompass the influence of stratification temperatures between 2 and 21°C on dormancy release in terms of a gradually reducing base temperature for germination. A benefit of directly determining Tb, and allowing Tb to vary, is the subsequent ability to quantify germination performance in terms of thermal time, q, allowing quantification of the combined effect of temperature-induced dormancy loss and temperature-dependent germination. Thus the relative effects of different temperatures are integrated into a model for the response during both dormancy release and germination phases. Potential reasons for dispersion of the data around the model are discussed, and the model is used to compare the germination response of three other horse chestnut seedlots.
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The base temperature for germination (Tb) is defined as the temperature at which the rate of germination is zero (Garcia-Huidobro et al., 1982). Making a correct assessment of Tb is imperative, because this value is the basis for the calculation of thermal time (θ) for germination, with thermal time accumulating more quickly when the difference between the temperature experienced by a seed and Tb is widest (Garcia-Huidobro et al., 1982; Covell et al., 1986). Based on information derived from nondormant seedlots, Tb is believed to be a single value and to remain constant among seeds in a population irrespective of physiological status. For example, differences in seed quality produced by priming or ageing did not alter Tb in Allium cepa seeds (Ellis & Butcher, 1988), and ageing in horse chestnut seeds stratified at 21°C had no impact on Tb. However, we have observed a decrease in mean Tb associated with dormancy release at 6°C (Pritchard et al., 1999), and in this study at temperatures between 2 (fast) and 16°C (very slow). Batlla & Benech-Arnold (2003) introduced an additional term, Tl, to describe the lower limit of the temperature range permissible for germination during dormancy release in seeds of the summer annual P. aviculare. In the same way as we have described for Tb in horse chestnut, Tl was an index of dormancy status, reducing as dormancy was released by cold stratification. However, in modelling P. aviculare germination the assumption was made that Tb was zero and constant, irrespective of dormancy status, and θ for germination was calculated above this base. The fit of our model is drastically reduced if this approach is taken for horse chestnut seeds; calculating θ using a Tb of zero results in θ50 ranging from 100 to 800°Cd. Thus, based on our work with horse chestnut seeds, we believe that Tb varies with dormancy status. In modelling horse chestnut germination it is necessary to account for this variation by allowing Tb not only to reflect dormancy status, but also to influence directly the calculation of θ.
The influence of temperature between 2 and 16°C on the reduction of Tb has been quantified, resulting in an equation that describes the change in Tb as dormancy is lost during stratification. Over the range of temperatures usually used for artificial stratification of seeds (5–10°C), the relationship between stratification temperature and dormancy loss rate is linear, which would allow the use of thermal stratification time to model dormancy release. This technique has been applied previously with this species (Pritchard et al., 1996), and similarly in P. aviculare (Batlla & Benech-Arnold, 2003). However, over the wider range of temperatures used in the present study there was a sigmoid relationship between the effect of temperature on the rate of dormancy loss in horse chestnut (Fig. 3). The lack of linearity negates the use of thermal stratification time as a means to model the dormancy release component, and in the present case the sigmoid equation has been directly incorporated into the final germination model. For ecological studies, in which the temperature experienced can vary widely, this approach would be appropriate for accurate prediction of horse chestnut dormancy release and germination.
The mechanism by which a change in base temperature for germination progression occurs is not clear. However, in developing sunflower seeds a correlation has been observed between the induction of dormancy and a decrease in oleoyl phosphatidyl choline desaturase (ODS) activity, which is associated with the enhancement of 18 : 2 fatty acids (Hilhorst, 1998). Moreover, sunflower achenes are able to desaturate oleate significantly only at low temperatures (García-Díaz et al., 2002). In addition, chilling-induced dormancy relief in apple buds is associated with enhanced activity of ODS as measured by an increase in linoleic acid content of the cell membranes (Erez, 2000). Thus cold-activated increases in polyunsaturated fatty acids may result in the progressive depression of membrane melting points, thereby permitting regulated metabolism at the lower temperature. In the case of cold-stratified horse chestnut seeds, this could explain their eventual germination at low temperatures.
At harvest only approximately half the population were capable of immediate germination at temperatures > 26°C. Germination performance at high temperatures (31 and 36°C) was improved following stratification even after the first sampling time, 43 d for the coldest temperature and 100 d for the 16°C treatment. This improvement in germinability during stratification at 16°C was unexpected because dormancy release (Tb reduction) was exceptionally slow (< 2°C by 3 months), suggesting that the observed physiological changes were not associated with dormancy release per se. It was previously noted that horse chestnut seeds required a combined development and cold stratification time of at least 150 d to achieve 80% germination at 26°C (Tompsett & Pritchard, 1993). Thus it is likely that some horse chestnut seeds are less mature than others at shedding and continue development during the early stages of stratification.
The poor high-temperature germination performance of 21°C stratified seeds, in which there was no apparent dormancy loss, was probably caused by reduced viability rather than maintenance of seed immaturity. Viability loss is slow for seeds in hydrated storage at 16°C, with more than one-third of seeds remaining germinable after 3 yr (Pritchard et al., 1996). On the other hand, freshly harvested, unstratified seeds are expected to be able to germinate eventually when imbibed at 26°C, a temperature marginally higher than Tb at harvest (25.3°C); however only a small proportion of UK populations do so (Tompsett & Pritchard, 1993; Pritchard et al., 1999). Thus, contrary to the performance of some dormant phenotypes of Avena fatua (Naylor & Fedec, 1978), horse chestnut seeds appear incapable of maintaining viability for long at around 21–26°C, probably dying in the germination test.
Dormancy release and germination of horse chestnut seeds has been predicted by estimating the q accumulation above a systematic, temperature-dependent reduction in Tb. Germination of two seedlots collected in different years from the same trees, and one population collected nearly 30 yr previously in Poland, was similar when calculated in terms of q using parameters measured for the present seedlot. However, sections of the population did not conform to the model, and further investigation is required. These were newly shed, unstratified seeds that exhibited reduced germinability (probably because of immaturity); seed with reduced viability; and seeds on the brink of germination caused by Tb being close to stratification temperature. Additionally, there was significant spread in q50 between treatments in which no consistent pattern was established at this stage. Some of the variation may be linked to imprecise temperature data in which small differences can become large over the extended period involved here. Accounting for the distribution in Tb among the population (Table 1), rather than using mean Tb, may also remove some of the dispersion.
The seeds of many temperate trees and spring-germinating annuals and perennials respond to stratification at cool temperatures (Baskin & Baskin, 1998). It remains to be assessed whether this approach, in which germination is modelled in terms of thermal time accumulation above a gradually reducing Tb, can adequately describe seed dormancy release patterns in other species. The relevance of the model to horse chestnut seedlots from southern European locations, closer to the natural origin of the species, also needs consideration.