IntroductionTop

Material and methodsTop

Plant material

Seeds from eight wild populations of different species of Festuca, all from northwestern Spain, were examined (Table 1). Given the difficulty in identifying the exact species of narrow-leaved fescues, this material was treated at the 'group' level as belonging to either Festuca gr. ovina or Festuca gr. rubra (Huff & Palazzo, 1998; Oliveira et al., 2008). Tall fescue (Lolium arundinaceum (Schreb) Darbysch syn. Festuca arundinacea Schreb and Schedonorus arundinaceus (Schreb.) Dumort) can be considered a species complex consisting of three major (continental, mediterranean and rhizomatous) morphotypes (Hand et al., 2010). In this work, the tall fescue population is he-reinafter named Lolium arundinaceum.

All the accessions studied were multiplied in the same year in isolated micro-greenhouses at the CIAM (López-Díaz & Calvete, 2016). Sowing was perfor-med in October-November 2015, and harvesting per-formed in June and September of 2016. The harvested seeds were stored in the dark at 5ºC until being used in germination assays.

Table 1. Identification codes and origin of the fescue accessions studied.

^[1] Accession number in the CIAM collection. [2]Internal code used by the Spanish National Inventory of Plant Genetic Resources.

Germination assays

Germination assays were performed in quadruplicate in 90 mm Petri dishes at the Centro de Recursos Fitogené-ticos (CRF-INIA) over January-May 2017; all replicates involved 25 seeds. Seeds were cleaned to leave only the grain, lemma and palea, and care was taken to avoid se-lecting empty seeds. Seeds were placed on 155 g m-2 paper within the dishes, moistened with 2.5 mL of distilled wa-ter. The dishes were then incubated in controllable light/temperature chambers, adding water whenever necessary. Chamber light was provided by cool white fluorescent tubes (36W).

Assays were performed under constant temperature conditions of 10ºC, 15ºC and 20ºC, as well as under al-ternating temperature conditions of 10/20ºC and 20/30ºC, all under a 8/16 h light/dark photoperiod. In these latter assays the higher temperature coincided with the 8 h pe-riod of light (ISTA, 2017).

Under the 20/30ºC conditions (those recommended by the ISTA for all species of Festuca (ISTA, 2017), germination assays were also performed 1) in total darkness, 2) with cold stratification, and 3) with the application of gibberellic acid (GA3). For the dark germination assays, the Petri dishes were wrapped in aluminium foil, with exposure to light minimised du-ring counting. The last two treatments were intended to break any dormancy. Cold stratification was performed maintaining the seeds at 5ºC on the same kind of paper as above (moistened in the same way) for seven days (ISTA, 2017). GA3 was applied with the first 2.5 mL of water (concentration 500 mg/L).

Counts of germinated seeds were made every 1-3 days. Seeds were deemed to have germinated when the emerged radicle reached ≥3 mm in length. In general, the assays ran for 21 days (ISTA, 2017), although at 10ºC they were ex-tended to 27 days when germination events were observed close to the 21 day. At the end of the assays, non-germinated seeds were gently pressed using forceps, and classified as empty, dead or fresh (ISTA, 2017). Seeds that remained firm and sound were considered viable -fresh- and soft or rotten seeds were accounted as dead seeds. The number of empty seeds (non- true seeds) were excluded from calcula-tion of final germination percentages.

Data analysis

A full parametric time-to event model for interval-cen-sored data was used to examine the results, employing a three parameter log-logistic function with a zero lower limit to fit the data (Ritz et al., 2013). This method of analysis, which requires no assumptions be made, is well adapted to the present type of data and the object of investigation (Onofri et al., 2010, 2011; McNair et al., 2012). Other methods traditionally used in such studies, including those to check whether assumptions have been respected, are generally inadequate (Scott et al., 1984; Warton & Hui, 2011; Sileshi, 2012).

For each population and treatment, maximum percentages of germination (Gmax) and the time to reach a percentage “n” of germinated seeds (tn) were estimated via the fitted functions; t50 was used in the temperature as-says and t25 for all other treatments in order to compare the largest possible number of curves (Soltani et al., 2015). Significance was set at p<0.05 for all pairwise comparisons.

All procedures were undertaken using R (R Core Team, 2017), employing the “drm” function of the “drc” package (Ritz et al., 2015).

Figures 1 to 4 show the results of the germination assays performed under the different conditions. All populations returned germination percentages of >85% under some set of conditions without additional dorman-cy breaking treatment. The fraction of dead seeds was small, with an overall average of 5.5%. Tables 2 and 3 show the estimated Gmax and tn values for the different germination temperatures and treatments respectively. In cases in which the germination rate had not stabilised by the end of the assay (Festuca gr. ovina 1300 - GA3, darkness; F. gr. ovina 2732A - 20/30ºC), the Tables 2-3 show the unadjusted values obtained in the assays (in such cases abnormally high germination percentages are predicted when extrapolating beyond the study period).

For Festuca gr. ovina, the highest Gmax values were achieved under the 10/20ºC and 15ºC conditions (Ta-ble 2), with no significant difference between them; nor was any difference seen in this respect between its three representative populations (1518, 2732A and 1300). Si-milar Gmax values were also achieved at 10ºC, although for population 2732A the difference between the 10ºC and 15ºC conditions (90.3% and 98%, respectively) was sig-nificantly different. At 20ºC the Gmax was significantly lower than at 15ºC in populations 1518 and 2732A. The 20/30ºC conditions were clearly unfavourable in all cases; in population 1300 no seeds germinated at all, and in 1518 and 2732A less than 50% did so (Fig. 1, Table 2). In all three Festuca gr. ovina populations, the t50 value fell as the temperature increased up to 15ºC, but rose significant-ly at 20ºC in Festuca gr. ovina 1300 and 2732A.

Both populations of Festuca gr. rubra (1300 and 1316) behaved in a manner very similar to the Festuca gr. ovina populations (Fig. 2, Table 2), with the highest Gmax values reached under the 10ºC, 10/20ºC and 15ºC conditions. In addition, the t50 values were clearly higher at 10ºC. At 20ºC the germination rate was significantly lower than at 15ºC in population 1316, while the 20/30ºC conditions were clearly unfavourable to both populations.

For the F. gigantea populations (1599 and 1652) no differences were seen in Gmax at 10ºC, 10/20ºC, 15ºC and 20ºC. Again, the value of t50 fell with rising tempe-rature. Under the 20/30ºC conditions, a significant reduc-tion in Gmax was seen, especially for population 1652 (Fig. 3, Table 2).

The single population of L. arundinaceum (2842) behaved differently to the other populations, reaching Gmax (96-98%) under all temperatures except the coolest (10ºC). The t50 was lowest under the 20ºC and 20/30ºC conditions. At 10ºC, the germination rate was clearly in-ferior (Fig. 4, Table 2).

The 20/30ºC/dark germination conditions had an in-hibitory effect on germination compared to the regular 20/30ºC conditions for the populations of F. gr. ovina and F. gr. rubra. Indeed, the germination rate was lower and t25 not reached until later (Table 3, Figs. 1 and 2). For the F. gr. rubra populations,the differences did not reach significance (Table 3). This is probably due to an imperfect performance of the parametric time-to event model and a high degree of uncertainty (wide confidence intervals) when germination rates are low. For the populations of F. gigantea and F. arundinacea, no significant differences were seen between the 20/30ºC/dark and 20/30ºC conditions with respect to Gmax or t25 values.

The dormancy-breaking treatments (GA3 and cold stratification) had a marked stimulatory effect on all the examined populations (Figs. 1-3). The population of L. arundinaceum was not subjected to these treatments since its germination rate was very high (95.9%) under the standard 20/30ºC conditions. In all cases the germination rate was higher with cold stratification, and the t25 values obtained were significantly lower (Table 3). The Gmax was similar under both sets of conditions, with significant differences seen only between F. gr. ovina 2732A and F. gigantea 1652.

Table 2. Maximum germination percentages (Gmax) and t50 values obtained for the seeds of different fescue accessions at different temperatures. Values were estimated from fitted curves.

^* Unadjusted value (i.e., as recorded) (see Results). Values with the same letter within accessions are not significantly different (p>0.05).

Figure 1.  Effect of different temperatures (left), darkness, and dormancy breaking treatments (right) on the seed germination rate of three populations of Festuca gr. ovina.

Figure 2.  Effect of different temperatures (left), darkness, and dormancy breaking treatments (right) on the seed germination rate of two populations of Festuca gr. rubra.

Figure 3.  Effect of different temperatures (left), darkness, and dormancy breaking treatments (right) on the seed germination rate of two populations of Festuca gigantea.

The use of adequate germination protocols is essential in germplasm bank management. Only then can viability monitoring be reliably performed. Ideally, all the viable seeds in a test sample should germinate, which demands that the germination conditions be optimum and any dormancy phenomena overcome. Given the large number of assays that have to be performed, and the generally limited resources of germplasm banks, it is essential that procedures require the least effort possible. Moreover, ra-pid germination reduces the possibility of contamination by saprophytic microorganisms. Given that the number of seeds preserved is commonly quite small, especially for wild species, material is not wasted if assays doomed to failure can be avoided.

Temperature is one of the most relevant factors that regu-late seed germination and dormancy. In the present work, the reduction in germination at 20/30ºC was common for all the narrow-leaved fescues (F. gr. rubra, F. gr. ovina) and for F. gigantea This results are similar to those previously reported. Kearns & Toole (1939), and Williams (1983) both obtained higher germination rates for Festuca rubra at 10-20ºC than at higher temperatures. The different behaviour of tall fescue (L. arundinaceum), which showed a clear fall in germination at 10ºC, also agrees with studies reporting an optimum germination temperature ranging from 20ºC to 30ºC (Hill et al., 1985; Lu et al., 2008; Sharifiamina et al., 2016).

Many cool-season grass seeds have a light requirement for optimum germination. The light-absorbing pigment phytochrome can detect the difference in light quality and maintain seed dormancy under a leafed canopy until gaps in the vegetation are encountered (McDonald et al., 1996). In contrast, in several grass species, e.g. Festuca hallii, the prolonged exposure to light can decrease germination rates and ability of seeds to germinate, especially at low water potentials (Mollard & Naeth, 2014). In our study, the presence of light under the standard 20-30ºC condi-tions appeared to be a positive influence on germination in the narrow-leaved fescues. A stimulatory effect of light is also reported in F. rubra and F. arundinaca (Danielson & Toole, 1976; Williams, 1983). Therefore, for seed ger-mination testing, photoperiod conditions are advisable for most fescues and related species.

Although high percentages of germination were obtai-ned under some temperature conditions, F. gr. ovina, F. gr. rubra and F. gigantea showed non-deep physiological dor-mancy at suboptimal temperatures. The existence of seed dormancy limited to a range of conditions is usually termed conditional or relative dormancy; its function is to prevent germination until conditions are more suited to plant deve-lopment (Baskin & Baskin, 2004). In the above taxa, GA3 and stratification at 5ºC for 7 days efficiently promoted germination at 20/30ºC. The positive effect of cold strati-fication on germination has previously been reported for F. rubra (Kearns & Toole, 1939), F. gigantea (Chorlton et al., 1997) and F. arundinacea (Boyce et al., 1976).

The taxa examined in the present work belong to the 'cold season grasses'. The seeds produced by the members of this group commonly show after-ripening or post-harvest dormancy, which prevents them from germinating soon after their dispersal or collection at the start of summer. This dormancy fades over the next few months, allowing germination after the dry season has passed. The exposure of seeds to high after-ripening temperatures favours their exit from this kind of dormancy (Baskin & Baskin, 1998; Steadman et al., 2003). Post-harvest dormancy among members of Festuca has been known for decades (Kearns & Toole, 1939). Stanisavljević et al. (2010) reported F. arundinacea, F. pratensis and F. rubra to show high germination percentages three to four months after seed collection. In the present work, the populations of F. gr. ovina and F. gr. rubra still showed relatively high percentages of dormant seeds at 20/30ºC even at 4-11 months post-harvest (when the assays were performed); their storage at low temperature could have contributed to the maintenance of dormancy (Chorlton et al., 1997). It should also be noted that in the above work performed by Stanisavljević et al. (2010), the seeds were subjected to cold stratification for 5 days, which very probably reduced the intensity of their dormancy. It is not improbable that the seeds of the present population of tall fescue, which showed no reduction in germination at 20/30ºC, experienced a degree of post-harvest dormancy before the assay was performed.

The differences seen between L. arundinaceum and the remaining taxa might be associated with the environmental conditions under which natural emergence and germination occur. In temperate climates, the germination of fescues usually takes place in autumn; the seed bank that builds up in the soil during the summer is practically absent by the winter (Thompson & Grime, 1979; Gibson & Newman, 2001). The capacity of L. arundinaceum seeds to germinate at higher temperatures than those of the other taxa might be related to its better adaptation to hot, dry conditions (Cross et al., 2013). This might favour earlier emergence and aid in the colonisation of disturbed ground, which is usually warmer.

The present results not only reveal inter-specific variation in terms of germination and dormancy, but also differences between populations of the same species. This might be due to differences in the environmental conditions of the mother plants during seed development or to genetic factors associated with selection pressures at work in their habitats (Lord, 1994; Chorlton et al., 1997; Anderson & Milberg, 1998). However, since all the seeds used in the present work were produced by multiplication in the same place and in the same year, the differences seen between the populations of F. gr. ovina and F. gigantea are more likely to have a genetic explanation. Compared to the other members of their groups, the smaller percentages of germination recorded at high temperatures for F. gr. ovina 1300 and F. gigantea 1652 might be explained by their adaptation to their place of origin (further from the coast and at higher altitude, Table 1). However, the coastal and montane populations of F. gr. rubra showed very little difference in their germination behaviour.

The rules published by ISTA (2017) for all species of Festuca indicate that germination assays be performed at 15/25º or 20/30°C, with cold stratification if needed. However, in the present work, and with the exception of L. arundinaceum, the 20/30ºC conditions were those associated with the smallest Gmax values when no cold stratif ication was provided. Indeed, even when such stratification was provided, only 55% of the F. gr. ovina 1300 seeds assayed had germinated by day 21. The present Gmax results indicate the optimum temperature for the germination of fescue seeds to be around 15ºC, with no significant differences between the constant 15ºC and the alternating 10/20ºC conditions. In the narrow-leaved fescues, the t50 values were generally smaller at 15ºC although similar to those obtained at 20ºC. For F. gigantea, germination was slightly earlier under the 20ºC conditions that at 15ºC. Finally, L, arundinaceum germinated most rapidly under the 20ºC and 20/30ºC conditions.

The present results could help germplasm banks improve the viability monitoring of their pasture grass seeds. The optimum germination temperature for wild fescue seeds would appear to be 15 or 20ºC depending on the species. However, only a limited number of populations was examined; it may be that greater variation in this and in the degree of dormancy would be seen across a larger number. Cold stratification is recommended when apparently viable but dormant seeds (“fresh seeds”) remain at the end of an assay. Indeed, this could be performed a posteriori for the non-germinated seeds, thus avoiding the need to repeat the entire assay.

Figure 4.  Effect of different temperatures (left), darkness, and dormancy breaking treatments (right) on the seed germination rate of a population of Lolium arundinaceum.

Table 3. IMaximum germination percentages (Gmax) and t25 values obtained for the seeds of different fescue accessions germinated at 20-30ºC, with different treatments to break dormancy. Values were estimated from the fitted curves.

^* Unadjusted value (recorded directly during the assays (see Results). Values with the same letter within accessions are not significantly different (p>0.05). Strat.: cold stratification at 5ºC for 7 days; GA3: exposure to gibberellic acid; Dark: germination without light; Control: no treatment.

The authors thank the CIAM and especially Julio Enrique López Díaz and Gonzalo Flores Calvete, for providing the seeds used in this work, and for information regarding their multiplication. Adrian Burton is thanked for editing the English manuscript.