Effect of stratification and scarification treatments on the germination of oriental hornbeam (Carpinus orientalis) seeds

Transcription

1 GERMINATION OF ORIENTAL HORNBEAM SEEDS Merou, T., Takos, I., Varsamis, G. and Xofis, P. (2012), Seed Sci. & Technol., 40, Research Note Effect of stratification and scarification treatments on the germination of oriental hornbeam (Carpinus orientalis) seeds T. MEROU 1, I. TAKOS 1, G. VARSAMIS 1 AND P. XOFIS 2 1 Technological Education Institute of Kavala, Department of Forestry and Natural Environment Management, Drama, Greece ( thmerou@teikav.edu.gr) 2 Forestry Service of Drama, Ag. Konstantinou 1, Drama, Greece ( pxofis@damt.gov.gr) (Accepted May 2012) Summary Carpinus orientalis is a common species in the semi-mountainous regions of Greece and elsewhere, with significant value for goat nutrition and as a natural flame retardant. Little is known about the germination behaviour of the species. This study describes the effects of various germination treatments on the germination of fresh C. orientalis seeds which were found to have a non-deep physiological dormancy imposed both by the endosperm and the embryo. Different levels of cold and warm stratification were applied, alone and in combination with chemical and mechanical scarification treatments. In addition, an excised embryo test was conducted. Cold stratification for three months at 5 C was found to be adequate to release dormancy and allow germination of almost all viable seeds. Acid scarification reduced germination without damaging the embryo, while mechanical scarification was lethal. Experimental and discussion Carpinus orientalis Mill. is native in an area extending from southeastern Europe to northern Iran and occurs usually on hot dry sites at lower altitudes than C. betulus L. (Bobrov, 1970). It is widely distributed in the semi-mountainous regions of Greece forming a significant part of goat nutrition (Papachristou et al., 1999). Further, it is characterised by a thick bark which reduces plant mortality during intensive wildfires (Banj Shafiei et al., 2010). Its low combustibility acts as a natural flame retardant decreasing fire spread and intensity. This potential is of great importance, especially under the foreseen conditions of increased summer aridity and potentially increased frequency and intensity of wildfires (Liu et al., 2010). Despite the wide distribution, current use and future potential of the species, there is limited information about its seed germination. Generally, Carpinus spp. seeds are collected early autumn (September - October) (Hartmann et al., 1997) and sown either in autumn or spring. However, Dirr and Heuser (1987) did not record any germination in 265

2 T. MEROU, I. TAKOS, G. VARSAMIS AND P. XOFIS the autumn seeding without seed pre-treatment. Suzuki (2000) reported that one month of cold stratification was enough to break the dormancy of C. laxiflora (Siebold. & Zucc.) Blume and C. tschonoskii Maxim seeds, while a long-term (10 months) cold stratification was necessary for C. cordata seeds to germinate. However, Baskin and Baskin (2001) and Bretzloff and Pellet (1979) reported that cold stratification is not very effective in breaking seed dormancy in C. caroliniana Walter and C. betulus unless they receive a period of warm stratification It seems that seed moisture content also plays a significant role in the selection of the appropriate pre-treatment method. Hartmann et al. (1997) reported that if Carpinus spp. seeds are dried after their collection, their coat becomes hard and scarification is then needed before prechilling. However, there is no reference about the critical seed moisture content. The present study had two main objectives: (1) to investigate the type of dormancy in C. orientalis seeds; and (2) to determine the effects of cold stratification and scarification in dormancy breaking. The fruits were harvested in autumn (October) from the semi-mountainous region of Drama, Greece (41 09'N, 24 10'E, elevation 200 m a.s.l.). After extracting the seeds (initial seed moisture content 12.5% of fresh weight), they were dried, at room temperature (approximate temperature 25 C day/15 C night) for a week (seed moisture content 11.7%). The seeds were then divided into sub-samples and subjected to different germination treatments, namely: (a) Cold stratification for 1, 2 or 3 months at a constant temperature of 5 C. (b) Warm stratification at room temperature (25 C day/15 C night) for two months followed by cold stratification for 1, 2 or 3 months. (c) Chemical scarification in concentrated (98%) H 2 SO 4 for 15 or 30 minutes followed by cold stratification for 1, 2 or 3 months. (d) Mechanical scarification in a scarifier (Forsberg 56701) for 5 seconds followed by cold stratification for 1, 2 or 3 months. (e) No treatment (control). Each treatment was tested with four replicates of 100 seeds each, which were placed on moist paper in Petri dishes (diameter 19 cm) on top of moist sterilised river sand. The germinator was maintained at 25 C for eight hours with 1000 lux light (cold light bulbs) and at 20 C for 16 hours in the dark. Germinated seeds were counted once a week for four weeks. The presence of a 2-mm-long radicle was the criterion for germination. An excised embryo test involves the removal of the embryo from the seed and its subsequent placement under germination conditions (Flemion, 1948). The purpose of the test was to evaluate the existence of embryo dormancy. Embryos were excised and placed, as four replicates of 25 seeds for each treatment, on moist filter paper in 19-cm-diameter Petri dishes. They were then placed in the germinator where the conditions were the same as in the germination test. Embryos were considered viable if they remained firm and showed evidence of growth; non-viable embryos showed signs of decay. Embryos were examined daily and measurements taken after 5, 7, 11, 14, 18 and 21 days when the test was terminated. 266

3 GERMINATION OF ORIENTAL HORNBEAM SEEDS ANOVA was employed for testing the effect of each treatment as well as their interaction on the germination process. The data were tested for normality using the Shapiro-Wilk W-test (Shapiro et al., 1968). In cases where data, in at least one treatment level, deviated from normality all data were arcsine-square-root transformed prior to analysis, while the calculated means and standard deviations were back transformed for the graphical presentation of the results (McDonald, 2009). Tukey s HSD test was employed for post-hoc comparisons among treatment means and detection of significant differences. Total germination was calculated as the percentage of germinating seeds in relation to the number of seeds sown after subtracting the number of seeds that were found to be empty. When no cold stratification or cold stratification for only one month was applied, there was no germination under any of the scarification treatments so those results were excluded from the analysis. A significant proportion of the seeds that were subjected to warm stratification decayed (more than 40%). Subsequently the treatment was considered unsuccessful and it was not included in the statistical analysis. 100 c Total germination % a a b b b Control SA_15 SA_30 MS Scarification treatment Figure 1. Effects of acid scarification for 15 (SA_15) or 30 (SA_30) minutes, mechanical scarification (MS) and cold stratification (CS) treatments on the germination of seeds of Carpinus orientalis. Different letters indicate significant differences among treatments (P < 0.05). a CS 2 months CS 3 months b Cold stratification for three months alone gave the highest germination percentage, reaching almost 100% among seeds capable of germination (i.e. non-empties), while germination exceeding 40% was achieved after two months of cold stratification without any scarification treatment (figure 1). Chemical or mechanical scarification reduced germination compared with non-scarified seeds, even when combined with cold stratification for three months. In the excised embryo test, germination was calculated as the percentage of germinating embryos in relation to the number of embryos sown. A significant proportion (48%) of excised embryos germinated without any cold stratification or scarification 267

4 T. MEROU, I. TAKOS, G. VARSAMIS AND P. XOFIS treatment. However, there was increased germination when embryos were excised after cold stratification for three months, reaching 70% (figure 2). Acid scarification had no effect on the germination of excised embryos since the germination percentages are similar to those of control seeds for equal durations of cold stratification, apart from acid scarification for 30 min and cold stratification for one month. Mechanical scarification, on the other hand resulted in particularly low germination, not exceeding 6%. These results suggest that acid scarification does not have any significant effect on the embryo which maintains its viability and germination ability. Germination % abc b b ce abc ab e abc ab CS 1 month CS 2 months CS 3 months 10 0 control SA_15 SA_30 MS Germination treatment Figure 2. Effects of acid scarification for 15 (SA_15) or 30 (SA_30) minutes, mechanical scarification (MS) and cold stratification (CS) treatments on the germination of excised embryos of Carpinus orientalis (P < 0.05). f ef Untreated C. orientalis seeds did not germinate; seeds were dormant at the time of maturity and seeding. The dormancy is progressively released during three months of cold stratification. This result indicates that the seeds have physiological dormancy. The fact that dormancy is completely removed after three months of cold stratification while a significant number of excised embryos (48%) germinate without any additional treatment, is a strong indication that the level of physiological dormancy is non-deep (Baskin and Baskin, 2004). Unlike C. caroliniana and C. betulus for which warm stratification was recommended (Bretzloff and Pellet, 1979; Baskin and Baskin, 2001), C. orientalis seeds do not need warm stratification for dormancy release, most likely because the seeds have already experienced the hot and dry Mediterranean summer. A significant proportion of the excised embryos (48%) germinated without any additional stratification treatment. However, germination was significantly increased (70%) when the embryos were excised after three months of cold stratification. It is possible that the removal of the mechanical restraint imposed by the endosperm and/ or testa is enough to promote germination for a significant proportion of viable seeds. In addition, it appears that cold stratification releases dormancy not only by weakening 268

5 GERMINATION OF ORIENTAL HORNBEAM SEEDS the seed covering layers, but also by promoting embryo growth. In fact embryo- and endosperm-imposed dormancy are both components of physiological dormancy and their sum and interaction determine the degree of the whole seed physiological dormancy (Finch-Savage and Leubner-Metzer, 2006). The duration of seed exposure to chemical scarification is crucial for embryo viability and varies with the species (Baskin and Baskin, 2001). Hartmann et al. (1997) recommended scarification for dried seeds of Carpinus spp. before germination. In the current study, acid scarification applied on fresh seeds dramatically decreased seed germination showing that there is no need for chemical scarification. However, embryos excised after exposure to acid scarification and cold stratification present similar total germination to those excised after cold stratification alone for equal durations. Thus, despite the negative impact of acid exposure on the germination of fresh seeds, there is no embryo damage. A possible hypothesis is that acid is likely to increase and strengthen the resistance of endosperm and/or testa without affecting the embryo, which remains viable, requiring perhaps a longer period of cold stratification for weakening the endosperm. This increased resistance might be adequate to ensure survival and viability of seeds eaten by herbivores and subsequently exposed to the action of digestive fluids when eaten by herbivores (Mancilla-Leyton et al., 2011). However, this is just a hypothesis and additional data are needed to support it. Mechanical scarification, on the other hand, led to the almost total loss of embryo viability which was probably the result of extensive endosperm damage that possibly negatively affected the rate and path of water entry. Conclusions Fresh seeds of oriental hornbeam (Carpinus orientalis) seem to have both embryo and endosperm dormancy which is successfully broken by a three month prechilling at a 5 C temperature. Acid scarification has a negative effect on seed germination without damaging the embryo, while mechanical scarification has a lethal effect. References Banj Shafiei, A., Akbarinia, M., Jalali, G. and Hosseini, M. (2010). Forest fire effects in beech dominated mountain forest of Iran. Forest Ecology and Management, 259, Baskin, C.C. and Baskin, J.M. (2001). Seeds: Ecology, Biogeography and Evolution of Dormancy and Germination. Academic Press, San Diego. Baskin, J.M. and Baskin, C.C. (2004). A classification system for seed dormancy. Seed Science Research, 14, Bobrov, E.G. (1970). Carpinus L., In Flora of the U.S.S.R., (ed. N. Landau), vol. 5, pp , Keter Press, Jerusalem, Israel. Bretzloff, L.V. and Pellett, N.E. (1979). Effect of stratification and gibberellic acid on the germination of Carpinus caroliniana Walt. HortScience,14, Dirr, M.A. and Heuser, C.W. Jr. (1987). The reference Manual of Woody Plant Propagation: from Seed to Tissue Culture, p. 239, Varsity Press, Athens, GA. Finch-Savage, W.E. and Leubner-Metzger, G. (2006). Seed dormancy and the control of germination. New Phytologist, 171,

6 T. MEROU, I. TAKOS, G. VARSAMIS AND P. XOFIS Flemion, F. (1948). Reliability of the excised embryo method as a rapid test for determining the germinative capacity of dormant seeds. Contributions of the Boyce Thompson Institute, 15, Hartmann, H., Kester T., Davies D.E. and Geneve R.L. (1997). Plant Propagation. Principles and practices. 5 th Edition, Prentice-Hall International. Liu, Y., Stanturf, J. and Goodrick, S. (2010). Trends in global wildfire potential in a changing climate. Forest Ecology and Management, 259, Mancilla-Leyton, J.M., Fernandez-Ales, R. and Martın Vicente, A. (2011). Plant- ungulate interaction: goat gut passage effect on survival and germination of Mediterranean shrub seeds. Journal of Vegetation Science, 22, McDonald, J.H. (2009). Handbook of Biological Statistics. 2 nd Edition, Sparky House Publishing, Baltimore, Maryland. Mouillot, F., Rambal, S. and Joffre, R. (2002). Simulating climate change impacts on fire frequency and vegetation dynamics in a Mediterranean-type ecosystem. Global Change Biology, 8, Papachristou, T.G., Platis, P.D., Papanastasis, V.P. and Tsiouvaras, C.N. (1999). Use of deciduous woody species as a diet supplement for goats grazing mediterranean shrublands during the dry season. Animal Feed Science and Technology, 80, 3-4. Shapiro, S.S., Wilk, M.B. and Chen, H.J. (1968). A comparative study of various tests of normality. Journal of the American Statistical Association, 63, Suzuki, W. (2000). Germination traits and adaptive regeneration strategies of the three Carpinus species. Journal of Forest Research, 5,