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Effects of cold stratification and sulphuric acid pre- treatments on germination of three provenances of smoke-tree (Cotinus coggygria Scop.) seeds in greenhouse and laboratory conditions

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Effects of cold stratification and sulphuric acid pre- treatments on germination of three provenances of smoke-tree (Cotinus coggygria Scop.) seeds in greenhouse and laboratory conditions
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  African Journal of Biotechnology Vol. 8 (19), pp. 4964-4968, 5 October, 2009 Available online at http://www.academicjournals.org/AJB ISSN 1684–5315 © 2009 Academic Journals Full Length Research Paper Effects of cold stratification and sulphuric acid pre-treatments on germination of three provenances of smoke-tree ( Cotinus coggygria   Scop.) seeds in greenhouse and laboratory conditions Z. Olmez*, A. Gokturk, B. Karasah and H. Yilmaz Artvin Coruh University, Faculty of Forestry, 08000 Artvin Turkey. Accepted 6 August, 2009 This study was carried out to determine which pre-treatments should be preferred to overcome dormancy problems of Cotinus coggygria   seeds which were collected from three different provenances. Pre-treatments applied to the seeds were submersion in concentrated (98%) sulphuric acid (10, 20, 30, 40 and 50 min) and submersion in sulphuric acid (10, 20, 30, 40 and 50 min) followed by cold stratification (15, 30, 45 and 60 days). The seeds were sown in polyethylene pots in the greenhouse and sown at 22 ± 1°C under darkness in laboratory conditions. The statistical approach was a randomized complete block design with 3 replications. Germinated seeds were observed periodically for 50 days to determine germination percentages and germination rates. Submersion in H 2 SO 4 for 50 min with cold stratification for 15 days gave the highest germination percentage (88.1%) in the laboratory while, while the highest germination percentage (70.2%) was obtained from submersion in H 2 SO 4 for 10 min with cold stratification for 30 days pre-treatment in the greenhouse. The analyses also revealed that the seeds collected from Ardanuç resulted in the highest germination percentage value (44.6%) in the laboratory among the provenances. The highest germination percentage (53.2%) sown in the greenhouse conditions was determined from the seeds collected in Artvin. Key words:   Cotinus coggygria  , germination, pre-treatments, seed dormancy. INTRODUCTION Vegetation cover is one of the most important factors in preventing and controlling soil erosion. It gives long-term soil surface protection by providing leaf cover that reduces rain-drop effects. In addition, it helps better soil structure development through establishing a root sys-tem, thereby increasing infiltration and soil stability (Pritchett and Fisher, 1987; Balcı, 1996). Cotinus coggygria   Scop. growing in steep and rocky landscapes is drought-tolerant plant that is important in preventing soil erosion. This species is also important as an orna-mental plants in many countries. Several cultivars produce a long period of midsummer floral and fruit ornamentation, showy plumose inflorescences and vivid *Corresponding authour. E-mail: zaferolmez@yahoo.com. Fax: +90 4662126951. autumn foliage color (Rudolf, 1974; Dirr, 1990; Krüssmann, 1984; Koller and Shadow, 1991; Pijut, 2008). The genus Cotinus   widely distributed through Central and Southern Europe to the Himalayas, Southwest China and the Southeastern United States (Krüssmann, 1984). Seeds of many woody plant species cannot germinate even if they are sown under optimal moisture, oxygen and soil conditions (Ürgenç and Çepel, 2001). This pro-blem is called dormancy and their causes are a hard and impermeable seed coat, immature or dormant embryo, absence of endosperm, or thick, fleshy seed cover (ISTA, 1966, 1993). There is a great deal of variation in germi-nation ability of seeds even within the same species. Poulsen (1996) reported that dormancy among and within seed lots of the same species varies with provenance, crop year and individual trees. There are various germination obstacles in Cotinus   seeds (Rudolf, 1974; Dirr and Heuser, 1987; Takos and  Olmez et al. 4965 Table 1.  Provenances of the collected Cotinus coggygria   seeds. Provenances Exposure Altitude (m) Latitude Longitude Ardanuç S 700 41°35' 42" N 42°15' 32" E Artvin E 622 41°11' 37" N 41°48' 38" E Derinköy SW 860 41°05' 54" N 41°52' 50" E Efthimiou, 2002; Piotto et al., 2003; Olmez et al., 2007a; Olmez et al., 2008) resulting in propagation difficulties. A few studies have tried to determine different methods and techniques to overcome seed dormancy in Cotinus   spe-cies. Generally pre-treatments such as submersion in hot water, mechanical or chemical scarification and hot aera-tion are used for seed coat dormancy while the cold and warm stratifications are usually applied to dormancy caused by restrictions at the embryo level (Landis et al., 1996). Among these methods and techniques, especially cold stratification, submersion in concentrated H 2 SO 4  and steeping seeds in hot water (88 - 100 ° C) followed by 24 h chilling are used to increase germination percentage of Cotinus   seeds (Rudolf, 1974; Dirr and Heuser, 1987; Stilinovic and Grbic, 1988; Dirr, 1990; Piotto et al., 2003,  Olmez et al., 2007a, Olmez et al., 2008). The aim of this study was to examine the influence of cold stratification and submersion in sulphuric acid pre-treatments on dormancy of C. coggygria   seeds which were sown both in the laboratory and in the greenhouse conditions. MATERIALS AND METHODS The fruits were collected by hand as soon as they were ripe in late August 2007 from C. coggygria   individuals, representing 3 different provenances (Ardanuç, Artvin and Derinköy) located in the North-Eastern part of Turkey (Table 1). The seeds were separated from the fruit material, rinsed with tap water, dried in the shade and stored at 5 ±  1°C in plastic bags. The following pre-treatments were applied to determine their effects on germination percentage (GP) and germination rate (GR) of C. coggygria   seeds;: i. Submersion in concentrated (98%) sulphuric acid for 10, 20, 30, 40 and 50 min. Submersion in concentrated (98%) sulphuric acid for 10, 20, 30, 40 and 50 min + cold stratification (CS) for 15, 30, 45 and 60 days. Control. The seeds were stratified by putting layers of moistened sand and seeds on top of each other in closed boxes. Since there was a risk for some of the seeds to be mixed with the sand because of their small size, linen cloth was placed between the sand and the seeds. The mean temperature of the room where cold stratification was applied on the seeds was 5 ±  1°C and the moisture of the sand and the seeds were checked regularly against drying, heating and poor aeration. The seeds were sown in polyethylene pots in the greenhouse and into the petri dishes filled using filter paper at 22 ± 1°C under darkness in the spring (April) of 2008. Polyethylene pots were filled with growing medium composed of forest soil, creek sand and manure (3:1:1). The experimental design was a randomized complete block with three replications (30 seeds in each replication) for each treatment. The number of germinated seeds [evaluation done according to ISTA Rules (1993)] was recorded for 7 th , 10 th , 14 th , 21 st , 28 th , 35 th  and 42 nd days. The GP and GR values were determined for each pre-treatment. The formula used in determining GR values is as follows (Pieper, 1952); ( ) ( ) ( ) ( ) T tinit nt nt n GR  ×++×+×+× = ...332211  Where GR = Germination rate, n = number of days for each coun-ting of germinated seeds, t = number of germinated seeds at each counting day, and T = total number of germinated seeds. The experiment lasted for about 50 days when it was observed that the seeds had stopped germinating. Data from the pre-treat-ments were analyzed using the SPSS statistical software after arc-sinus transformation was applied to GP values to meet ANOVA assumptions. The multifactor ANOVA and Duncan tests were used to compare treatment groups to find out whether they showed any statistically significant differences with significance level (  ) set at 0.05.   RESULTS AND DISCUSSION Statistical analyses showed that the pre-treatments used in this study affected seed GP and GR both in the green-house and laboratory conditions significantly. Submersion in H 2 SO 4 for 50 min with CS for 15 days gave the highest GP (88.1%) in the laboratory while, the highest GP (70.2%) was obtained from submersion in H 2 SO 4 for 10 min with CS for 30 days pre-treatment in the greenhouse. Increasing the duration of CS when it was used with H 2 SO 4 resulted in a decrease in GPs both in the green-house and laboratory (Table 2). Previous studies that used H 2 SO 4 application and the combinations of CS with H 2 SO 4 reported successful outcomes to overcome dor-mancy of C. coggygria   seeds (Dirr and Heuser, 1987; Takos and Efthimiou, 2002; Piotto et al., 2003; Olmez et  al., 2007a; Olmez et al., 2008). Smoke-tree seeds have both a hard seedcoat and an internal dormancy, thus causing slow and irregular germination. Seeds can be stimulated to germinate more uniformly by sulphuric acid scarification followed by cold stratification (Rudolf, 1974; Dirr and Heuser, 1987; Stilinovic and Grbic, 1988). Takos and Efthimiou (2002) reported a higher germina-tion (73%) in the laboratory than nursery conditions (19%) in C. coggygria seeds when submersion in H 2 SO 4 for 30 min followed by CS for 60 days pre-treatment was  4966 Afr. J. Biotechnol. Table 2.  Results of statistical analyses showing the relationship of the germination percentage and rate with different pre-treatments for laboratory and greenhouse conditions. Pre-treatments in the laboratory F-Ratio GP (%) F-Ratio GR (days) 10 min H 2 SO 4  + 60 day cold stratification 0.00a 0a 20 min H 2 SO 4  + 60 day cold stratification 0.00a 0a 30 min H 2 SO 4  + 60 day cold stratification 39.983* 0.00a 5.274* 0a 50 min H 2 SO 4  + 60 day cold stratification 0.00a 0a 30 min H 2 SO 4  0.37a 4abcd 20 min H 2 SO 4  0.74a 8bcde 50 min H 2 SO 4  0.74a 9cde 10 min H 2 SO 4  1.11a 4abcd Control 2.22a 2abc 40 min H 2 SO 4  2.59a 18f 40 min H 2 SO 4  + 60 day cold stratification 6.67a 1ab 20 min H 2 SO 4  + 45 day cold stratification 56.27b 11def 50 min H 2 SO 4  + 45 day cold stratification 56.64b 9de 30 min H 2 SO 4  + 30 day cold stratification 57.36b 12ef 10 min H 2 SO 4  + 15 day cold stratification 57.5b 15ef 10 min H 2 SO 4  + 45 day cold stratification 58.11b 12ef 40 min H 2 SO 4  + 45 day cold stratification 62.57bc 10de 20 min H 2 SO 4  + 15 day cold stratification 62.59bc 12ef 30 min H 2 SO 4  + 45 day cold stratification 68.88bcd 10def 50 min H 2 SO 4  + 30 day cold stratification 69.22bcd 12ef 40 min H 2 SO 4  + 30 day cold stratification 69.93bcd 11def 30 min H 2 SO 4  + 15 day cold stratification 73.29bcde 10de 10 min H 2 SO 4  + 30 day cold stratification 73.76bcde 12ef 20 min H 2 SO 4  + 30 day cold stratification 78.12cde 11def 40 min H 2 SO 4  + 15 day cold stratification 83.68de 11def 50 min H 2 SO 4  + 15 day cold stratification 88.11e 11def Pre-treatments in the greenhouse Control 2.80a 28c 10 min H 2 SO 4  6.47a 23bc 20 min H 2 SO 4  22.989* 11.82a 3.921* 29c 40 min H 2 SO 4  12.79a 27c 50 min H 2 SO 4  15.27a 17ab 30 min H 2 SO 4  16.80 a 30c 20 min H 2 SO 4  + 45 day cold stratification 36.98b 14a 30 min H 2 SO 4  + 45 day cold stratification 37.67b 14a 40 min H 2 SO 4  + 45 day cold stratification 50.29bc 12a 50 min H 2 SO 4  + 45 day cold stratification 51.64bcd 15ab 10 min H 2 SO 4  + 45 day cold stratification 54.61cde 14a 20 min H 2 SO 4  + 30 day cold stratification 63.51cde 14a 10 min H 2 SO 4  + 15 day cold stratification 63.80cde 17ab 20 min H 2 SO 4  + 15 day cold stratification 64.99cde 16ab 50 min H 2 SO 4  + 30 day cold stratification 65.00cde 14a 40 min H 2 SO 4  + 30 day cold stratification 65.50cde 12a 30 min H 2 SO 4  + 15 day cold stratification 67.30de 13a 40 min H 2 SO 4  + 15 day cold stratification 67.98e 14a 50 min H 2 SO 4  + 15 day cold stratification 68.86e 13a 30 min H 2 SO 4  + 30 day cold stratification 69.79e 13a 10 min H 2 SO 4  + 30 day cold stratification 70.18e 14a Means in column with the same letter are not significantly different at   = 0.05. * VS: Pre-treatments, significantly different at α  = 0.05.    Olmez et al. 4967 Table 3.  Results of statistical analyses showing the relationship of the germination percentage and rate with different provenances for laboratory and greenhouse conditions. Provenances in the laboratory F-Ratio GP (%) F-Ratio GR (days) Derinköy 6.529* 35.15a 4.273* 7a Artvin 39.21a 9ab Ardanuç 44.55b 10b Provenances in the greenhouse Derinköy 8.082  NS  48.15 0.959 NS  13 Ardanuç 48.46 17 Artvin 53.16 18 Means in column with the same letter are not significantly different at   = 0.05. * VS: Provenances, significantly different at α  = 0.05, NS: Nonsignificant at α  = 0.05. used. In general, the present results for C.   coggygria   seeds were parallel to the above studies’ findings since immersing in H 2 SO 4 followed by CS resulted in early, uniform and high GP in the laboratory and greenhouse. Olmez et al. (2007b) stated that the highest GP (44.2%) was obtained in C. coggyria   seeds that were cold stra-tified for 60 days and sown under greenhouse condi-tions. In addition, scarification with sulphuric acid (20-80 min) followed by cold stratification for 60-80 days pre-treatments were suggested by Piotto et al. (2003) for C. coggyria   seeds. Olmez et al. (2008) reported that while submersion in sulphuric acid for 20 min with cold stratifi-cation for 60 days gave the highest germination (82.77%) in the greenhouse, 80 min sulphuric acid with 60 day CS pretreatment gave the highest germination percentage (12.47%) under open field conditions for C. coggyria  . The maximum GP value among the H 2 SO 4 pre-treat-ments was 2.6% for seeds that were submersed for 40 min and sown in the laboratory and was 26.8% from 30 min H 2 SO 4 pre-treatment in the greenhouse. CS with sub-mersion in H 2 SO 4 pre-treatments gave higher GP values both in the greenhouse and in the laboratory than H 2 SO 4 pre-treatment alone (Table 2). According to Dirr (1990), Cotinus seeds did not germinate easily unless the hard seed coat was ruptured by mechanical or chemical scari-fication. The analyses also revealed that the seeds collected from Ardanuç resulted in the highest GP value (44.6%) in the laboratory among the provenances. The highest average GP (53.2%) was determined from the seeds collected in Artvin and sown in the greenhouse conditions (Table 3). The best germination rate (12 days) was determined in seeds soaked in H 2 SO 4 for 40 min followed by 30 and 45 day CS pre-treatments and sown in the greenhouse. The GR was 11 days for the highest GP (88.1%) that were obtained from submersion in H 2 SO 4 for 50 min with CS for 15 days pre-treatment in the laboratory condition (Table 2). It was observed that  C. coggygria   seeds were germinating while the stratification treatment was con-tinuing after 45 th  day. This may explain why we could not obtain any germination from the seeds that were cold stratified for 60 days and sown in the laboratory. Consequently, among all the pre-treatments applied to the C. coggygria   seeds, submersion in H 2 SO 4 for 50 min with 15 day-CS in the laboratory and submersion in H 2 SO 4 for 10 min with 30 day-CS resulted in the highest GPs, respectively 88.1% and 70.2%. The results for indi-cate that the duration of pre-treatment of submersion in H 2 SO 4 between 10 and 50 with CS up to 45 days could be preferably used to overcome germination dormancy of C. coggygria   seeds. ACKNOWLEGEMENT The authors would like to thank the Kafkas University for its financial support of this research (2007-OF-019). REFERENCES Balcı AN (1996). Toprak koruması.  stanbul Üniversitesi, Yayın No: 439,  stanbul, Türkiye. Dirr MA (1990). Manual of woody landscape plants, their identification, ornamental characteristics, culture, propagation and uses. Stipes Publishing Company, Champaign, IL. Dirr MA, Heuser CW (1987). The reference manual of woody plant propagation from seed to tissue culture. Varsity Press, Athens, GA. ISTA (International Seed Testing Association) (1966). Internationale Vorschriften für die Prüfung von Saatgut. Vol. 31/4, Wageningen, The Netherlands. ISTA (1993). Rules for testing seeds. Seed Sci. Technol. 21(1): p. 259. Koller GL, Shadow DO (1991). 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