The Effect of Different Stratification Conditions on the Germination of Fraxinus angustifolia Vahl . and F . ornus L . Seeds

The aim of the paper was to assess the impact of different stratification treatments recommended by the literature on the shortening of manna ash (F. ornus L.) and narrow-leaved ash (F. angustifolia Vahl.) seed germination in order to identify a suitable pre-germination treatment for seedling production. Seeds of three Southern Romanian origins for both species were exposed to four stratification treatments (cold (3oC) and warm (20oC), with and without sand-peat medium). The highest percentage of germination (up to 87% for Fraxinus ornus and up to 71% for F. angustifolia) was recorded in the case of the stratified seeds with cold medium (3oC) and relatively constant seed moisture (between 45% and 65%) throughout the treatment.


Introduction
Among the broadleaves found in Romanian lowland forests, the species belonging to Fraxinus genus are remarkable both for their timber value and for their silvicultural role (Haralamb, 1967).The most common native European Fraxinus species (F.excelsior L., F. angustifolia Vahl.and F. ornus L.) grow naturally in the country and cover more than 60,000 ha together with two rarer species (F.pallisae Wilmot.and F. coriariifolia Scheele) (Fraxigen, 2005;Şofletea and Curtu, 2007).In the present socioeconomic and forest ownership context of Romania, these species are important both for conservation and forest development purposes (Ioras and Abrudan, 2006;Şofletea et al. 2007;Şofletea et al. 2007;Abrudan et al., 2009, Ioras et al., 2009;Stancioiu et al., 2010).
Seeds have an inherent high genetic variability, which determine a great heterogeneity in their behavior and in their germination.Seed dormancy was observed in several species of Fraxinus genus (Bewley and Black, 1994), the mature seeds of which are frequently deeply dormant.Fraxinus spp.seeds often have pericarps impermeable to oxygen, metabolic inhibitors in the endosperm and embryos, immature embryos or deficiencies of growthpromoting substances in embryos (Villiers and Wareing, 1965;Bonner, 1974;Tinus, 1982;Nowag, 1998).The degree of dormancy shows some variation related to the climate of origin and seed lot (Edwards, 1980).
Dormant seeds can be stimulated to germinate using treatments that satisfy certain physiological requirements.The recommendations for treating Fraxinus spp.seeds to break dormancy or stimulate germination are quite diverse and include stratification, after-ripening fol-lowed by stratification, water soaking, the treatment with a variety of growth regulatory chemical etc. (Villiers and Wareing, 1964;Marshall, 1981;Walle, 1987;Piotto and Piccini, 1998).In Fraxinus spp., the stratification treatment consisting of a period of warm moist stratification at 15-20 o C prior to a period of cold moist stratification at 3-5 o C is considered by many authors to be effective (Bonner, 1974;Tylkowski, 1990;Piotto, 1994;Nowag, 1998).The International Seed Testing Association (ISTA) prescribes a single pretreatment to overcome dormancy in all Fraxinus ssp.seeds: 2 months of warm stratification at 20 o C followed by 7 months of cold stratification at 3-5 o C (ISTA, 1993).However, this treatment would appear too long taking into consideration the usual practices followed in nurseries.
In nature the beneficial effect of cold stratification is obtained during the winter, and in the nursery the same result may be achieved by keeping seeds at low temperatures, generally ranging from 1 to 5° C, mixed with moist peat or sand (Bradbeer, 1988).
The main environmental factor for achieving high germination is the temperature at which the seeds resume growth, whether pretreated or not; in some species an alternating temperature stimulates germination (Gordon and Edwards, 1991).ISTA rules (ISTA, 1993) prescribe alternating temperatures of 8 h at 30° C / 16 h at 20° C, in darkness, during the germination tests in Fraxinus spp.However, a better response of Fraxinus spp.seeds of to a wider daily temperature range (i.e.alternating 20/3° C or 25/5° C) was reported by several authors (Suszka, 1978;Piotto, 1994;Piotto and Piccini, 1998).time, the seed moisture was measured with a thermo balance (MB 45).
• Treatment 2 (T2): Warm treatment (20ºC) with medium.The seeds (4x100) of each origin were kept at 20º C, in the sand/peat medium, placed in plastic containers.The assessment/monitoring procedure was the same as in the case of the cold treatment.
• Treatments 3 and 4 (T3 and T4): Cold (3 o C) and warm (20 o C) treatments without medium.Two groups of seeds (4x100 each) were kept in parallel in two climate rooms.In the first climate room the temperature was set at 20ºC and the relative humidity of air at 95% and in the second climate room a temperature of 3ºC was maintained at an 80% relative humidity of air.The maximum humidity allowed by the climate room was chosen in both cases because in previous tests it was observed that seeds lost a large amount of water when placed in the climate room.
After stratification, the germination tests were performed in darkness at a temperature of 3 o C. The germination percentage was calculated for each species and origin whilst the potential differences between origins were assessed by one-way ANOVA.

Results and discussions
The initial seed viability varied between 69.75% and 88,25% for F. angustifolia and between 81.5% and 90% for F. ornus (Tab.2).These relatively high values indicated that seeds would be capable of germination under favorable conditions and that failure to germinate would be attributable mainly to seed dormancy.
The results of the imbibition test resulted in similar water absorption curves (Fig. 1) for each origin of the two species; in the first days, a fast water absorption was recorded, followed by a period of low absorption.Based on these results, all seeds from treatments 3 and 4 (without medium, in the climate rooms) were soaked for 48 hours in water in order to reach a moisture of around 50% before being placed in the climate rooms.
Keeping the seeds in containers with sand/peat medium at high temperature (20ºC) resulted in their decay after 15 weeks of treatment, so this method proved to be ineffective.
For nursery practice purposes it is important to identify an effective stratification method that allows for seed sowing next spring after harvesting.In this paper we assessed the impact of four different stratification conditions on the germination of Fraxinus ornus and Fraxinus angustifolia seeds, in order to identify a suitable pre-germination treatment for seedling production.

Materials and methods
Seeds of both species were collected in the period 26-29 October 2009 from three origins in Southern Romania (Tab.1).
A tetrazolium test was performed in order to assess the initial viability of seeds from each origin, according to ISTA provisions (4 repetitions of 100 seeds) (ISTA, 1993;Draghici and Abrudan, 2011), after the manual grading of seeds and the removal of the sterile ones at the beginning of November 2009.
In order to establish a suitable moisture for germination, seed imbibition was evaluated by gravimetric method at room temperature (Parascan et al., 1975), using for each origin of the two species three repetitions of 50 seeds, as described by Draghici and Abrudan (2011).
The seeds were kept for 10 weeks after harvesting in the refrigerator at a relatively constant temperature of 4 ºC; afterwards four stratification conditions/treatments were applied for each species and origin: • Treatment 1 (T1): Cold treatment (3 o C) with medium.Four repetitions of 100 seeds were mixed with a sand/peat medium in a volume ratio of 1:3 and were placed in plastic containers.The medium consisted of an equal proportion of sand and peat, having a relative humidity of 29% (wetting degree U6=wet) (Târziu, 1997), which was measured with the ECH2O EC-5 sensors for soil moisture.
After weighing, the containers were kept at a temperature of 3ºC in the seed conservation centre of ICAS Braşov (Draghici and Abrudan, 2011).Medium moisture was assessed daily, by weighing and filling with water the deficit that occurred from the previous day.Once a week the seeds were removed from containers and medium aeration and homogenization were performed.At the same Tab. 1.The origin of manna ash (F.ornus L.) and narrow-leaved ash (F.angustifolia Vahl. ) seeds Unfavourable results (no germination) were also recorded when applying the two treatments without sand/ peat medium (in climate rooms at 3ºC / 80% air humidity and 20ºC / 95% air humidity respectively).In both treatments, seed humidity decreased very sharply (Fig. 2), possibly due to the air ventilation in the climate rooms.
Seed moisture remained relatively constant throughout the cold treatment with medium, ranging between 56% and 64% for F. angustifolia and between 45% and 53% for F. ornus.
The cold treatment with medium was the only one which provided satisfactory seed germination (Fig. 3).
The treatment period was relatively shorter for F. ornus and the first seed sprouted in the same period (after 14 weeks of cold treatment) for all three origins, despite the fact that significant differences (p<0.05) in germination percentage were recorded between Răcari (87%) and Drăgăşani (69%) origins.F. angustifolia seeds started to germinate after 22 weeks and for unknown reasons only the seeds of one origin (i.e.Răcari) germinated (71%).

Conclusions
Keeping the seeds of F. ornus and F. angustifolia in a sand/peat medium, at low temperature (3 o C) and a relative constant humidity level (between 45% and 65%) could be an efficient and relatively simple treatment to overcome seed dormancy.Under such conditions, the seeds of F. ornus could start to germinate up to 7 weeks earlier than those of F. angustifolia.
Warm treatment is not recommended for breaking seed dormancy in Fraxinus spp. as the seeds decay after 3-4 months of stratification.

Fig. 3 .
Fig. 3. Evolution of seed germination in the cold treatment (3ºC) with medium