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Effects of cold stratification, sulphuric acid, submersion in hot and tap water pretreatments in the greenhouse and open field conditions on germination of bladder-Senna (Colutea armena Boiss. and Huet.) seeds

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Effects of cold stratification, sulphuric acid, submersion in hot and tap water pretreatments in the greenhouse and open field conditions on germination of bladder-Senna (Colutea armena Boiss. and Huet.) seeds
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  African Journal of Biotechnology Vol. 8 (13), pp. 2973-2977, 6 July, 2009 Available online at http://www.academicjournals.org/AJB ISSN 1684–5315 © 2009 Academic Journals Full Length Research Paper Effects of cold stratification, sulphuric acid, submersion in hot and tap water pretreatments in the greenhouse and open field conditions on germination of bladder-Senna ( Colutea armena Boiss. and Huet.) seeds Zafer Olmez* and Askin Gokturk Artvin Coruh University, Faculty of Forestry, 08000 Artvin, Turkey. Accepted 11 May, 2009 This study was carried out to determine which pre-treatments should be preferred to overcome dormancy problems of Colutea armena   seeds which were collected from three different provenances. Pre-treatments applied to the seeds were submersion in concentrated (98%) sulphuric acid for 30 min, floating in hot water (100°C) followed by continual cooling for 24 h in the same water, soaking in tap water (20 ±±±±  1°C) for 24 h and cold stratification for 20, 40 and 60 days. The seeds were sown in polyethylene pots in the greenhouse and in seedbeds under open field conditions. The statistical approach was a randomized complete block design with three replications. Germinated seeds were observed periodically during 70 days to determine germination percentages and germination rates. The highest germination percentages (52.2, 56.7 and 60.5%) were obtained in seeds which were submersed in sulphuric acid for 30 min and sown in the greenhouse for all three provenances. The best germination rates for provenances of Köprüba  ı and Derinköy were 14 and 16 days which were determined in seeds soaked in sulphuric acid for 30 min and sown in the greenhouse. On the other hand, the best germination rate (12 days) of provenance of Salkımlı was determined in seeds which were floated in hot water (100°C) followed by continual cooling for 24 h in the same water and sown under open field conditions. Key words: Colutea armena  , germination, seed, greenhouse, open field condition. 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 re-duces rain-drop effect. 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). The genus Colutea   L. (bladder-senna) includes about 26 species of deciduous shrubs and small trees with a distribution rang-ing from the Mediterranean region and South-Eastern *Corresponding author. E-mail:   zaferolmez@yahoo.com. Fax: +90 4662126951. Europe and North-West Africa (Browicz, 1963). Colutea armena   (Boiss. and Huet.) is a drought-tolerant plant occurring in rocky and steep landscapes and is known as an important species in preventing soil erosion (Dirr, 1990; Pijut, 2008). Colutea   species are cultivated in temperate climates primarily for ornamental purposes (Rudolf, 1974; Krüsmann, 1984). Colutea   L. is a genus of great rusticity and is valuable as forage. Its domestication is limited by the low germination rates and high suscep-tibility of the seeds to fungal attack (Aguinagalde et al., 1990). 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 its causes are a hard and  2974 Afr. J. Biotechnol. Table 1.  Provenances of the collected Colutea armena   seeds. Provenance Exposure Altitude (m) Latitude Longitude Artvin-Köprüba  ı NE 212 41°11' 14" N 41°49' 43" E Artvin-Salkımlı SE 550 41°11' 42" N 41°52' 13" E Artvin-Derinköy SW 860 41°05' 54" N 41°52' 50" E impermeable seed coat, immature or dormant embryo, absence of endosperm, or thick, fleshy seed cover (ISTA, 1966 and 1993). There is great deal of variation in germination 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 Colutea   seeds (Dirr and Heuser, 1987; Dirr, 1990; Olmez et al.,  2007, Olmez et al., 2008) resulting in propagation diffi-culties (Heit, 1967; Piotto et al., 2003). There have been few studies to determine different methods and techni-ques to overcome seed dormancy in Colutea   species. 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 well-known and used to increase germi-nation percentage of Colutea   seeds (Allue Andrade, 1983; Dirr and Heuser, 1987; Piotto et al., 2003; Olmez et  al., 2007). The aim of this study was to examine the influence of four pre-treatments (cold stratification, submersion in sulphuric acid, submersion in hot water followed by 24 h chilling and submersion in tap water for 24 h) on dor-mancy of C. armena   seeds which were sown both in the greenhouse and under open field conditions. MATERIALS AND METHODS Ripe fruits were collected in August, 2004 from 15 C. armena   indivi-duals, representing three different provenances (Derinköy, Salkımlı and Köprüba  ı) in Artvin 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 after the ratios of filled seeds to all the collected seeds were determined. The following pre-treatments were applied to determine their effects on germination percentage (GP) and germination rate (GR) of C. armena seeds; Cold stratification (CS) for 20, 40 and 60 days, Floating in tap water (20 ±  1°C) for 24 h, Floating in hot water (100°C) followed by continual cooling for 24 h in the same water, Submersion in concentrated (98%) sulphuric acid for 30 min, Control (no treatment). The seeds were stratified by putting layers of moistened sand and seeds on top of each other. 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. The moisture of the sand and the seeds were checked continuously against drying, heating and poor aera-tion. The seeds were sown in polyethylene pots in the greenhouse and in seedbeds under open field conditions in the spring (March) of 2005. Polyethylene pots were filled with growing medium com-posed of forest soil, creek sand, and manure (1:1:1). The experi-mental design was a randomized complete block with three repli-cations (30 seeds per 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  and 21 st days and in every week (7 days) after the the 21 st  day counting. Germination percentage (GP) and germination rate (GR) values were determined for each pre-treatment and filled seed ratios were used to determine GP. The following formula was used for deter-mining GR (Pieper, 1952); ( ) ( ) ( ) ( ) T tinit nt nt n GR  ×++×+×+× = ...332211  Where GR: Germination rate; n: Number of days for each counting of germinated seeds;  t: Number of germinated seeds at each counting day; and T: Total number of germinated seeds The experiment lasted for about 70 (80) days when it was observed that the seeds stopped germinating. Data from the treat-ments were analyzed using the SPSS statistical software after arc-sinus transformation was applied to GP values to meet ANOVA assum-ptions. 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 highest germination percentages (52.2, 56.7 and 60.5%) for all three prove-nances (Derinköy, Köprüba  ı and Salkımlı, respectively) were obtained in seeds which were submersed in sul-phuric acid for 30 min and sown in the greenhouse (Table 2). Similar findings were reported by Dirr (1990) who found that soaking Colutea seeds in sulphuric acid for 30 - 60 min resulted in good germination. According to Dirr (1990), some Colutea seeds did not germinate easily un-less the impermeable seed coat was ruptured by mech-anical or chemical scarification. Olmez et al. (2008) stated that soaking C.   armena   seeds in sulphuric acid for 30 min and sown in the greenhouse resulted in good GP value (77.2%). In addition, Olmez et al. (2007) reported  Olmez and Gokturk 2975 Table 2.  Relationship of the germination percentage and rate of C. armena   seeds with different pre-treatments for three provenances in the greenhouse (G) and under open field (OF) conditions. Pre-treatments F-Ratio GP (%) F-Ratio GR (day) Provenance of Derinköy  Cold stratification for 60 days (OF) 11.11a 39de Floating in tap water for 24 h (OF) 12.22a 20abc Control (OF) 14.44a 25abcd Cold stratification for 40 days (OF) 16.67a 43de Control (G) 20.00a 44de Cold stratification for 20 days (OF) 20.00a 29abcde Floating in tap water for 24 h (G) 21.11a 42de Cold stratification for 40 days (G) 22.22ab 48e Submersion in sulphuric acid for 30 min (OF) 24.44ab 28abcd Cold stratification for 60 days (G) 28.89ab 39cde Floating in hot water (100°C) followed by continual cooling for 24 h in the same water (OF) 28.89ab 27abcd Cold stratification for 20 days (G) 4.07* 1.17** 3.59*** 31.11ab 17.62* 1.11** 3.21*** 37bcde Floating in hot water (100°C) followed by continual cooling for 24 h in the same water (G) 41.11bc 20ab Submersion in sulphuric acid for 30 min (G) 52.22c 16a Provenance of Köprüba  ı  Cold stratification for 60 days (OF) 13.89a 35abcd Control (OF) 15.05a 23abc Cold stratification for 40 days (OF) 3.88*** 16.20a 6.23* 29abcd Floating in hot water (100°C) followed by continual cooling for 24 h in the same water (OF) 21.99ab 2.08*** 19abc Floating in tap water for 24 h (OF) 24.31ab 27abcd Cold stratification for 20 days (OF) 28.94abc 33abcd Submersion in sulphuric acid for 30 min (OF) 28.94abc 16ab Cold stratification for 20 days (G) 33.56abcd 39cd Cold stratification for 40 days (G) 42.82bd 36abcd Floating in hot water (100°C) followed by continual cooling for 24 h in the same water(G) 45.14bd 23abc Control (G) 50.92cd 37bcd Cold stratification for 60 days (G) 52.08cd 47d Submersion in sulphuric acid for 30 min (G) 56.71d 14a Floating in tap water for 24 h (G) 56.71d 28abcd Provenance of Salkımlı  Cold stratification for 20 days (OF) 4.94a 22abcd Floating in tap water for 24 h (OF) 8.64a 28abcd Control (OF) 9.88a 30abcd Cold stratification for 60 days (OF) 3.18* 17.28ab 5.58* 39abcd Cold stratification for 40 days (OF) 12.21** 17.28ab 2.02*** 40bcd Control (G) 8.47*** 28.39bc 45cd Floating in tap water for 24 h (G) 29.63bc 44cd Cold stratification for 60 days (G) 32.09bcd 50d Cold stratification for 40 days (G) 33.33bcd 37abcd Floating in hot water (100°C) followed by continual cooling for 24 h in the same water (OF) 35.80cd 12a Cold stratification for 20 days (G) 37.04cd 42bcd Submersion in sulphuric acid for 30 min (OF) 40.74cd 20abc Floating in hot water (100°C) followed by continual cooling for 24 h in the same water (G) 49.38de 29abcd Submersion in sulphuric acid for 30 min (G) 60.49e 15ab *VS: Greenhouse (Treatment), significantly different at α  = 0.05, **VS: Open Field (Treatment), significantly different at α  = 0.05, ***VS: Greenhouse*Open Field (Treatment), significantly different at α = 0.05. Means in column with the same letter are not significantly different at   = 0.05.    2976 Afr. J. Biotechnol. Table 3.  Relationship of the germination percentage and rate with different pre-treatments and provenances in the greenhouse (G) and under open field (OF) conditions. Pre-treatments F-Ratio GP (%) F-Ratio GR (days) Control (OF) 13.26a 26abc Cold stratification for 60 days (OF) 14.04a 38cde Floating in tap water for 24 h (OF) 15.20a 25ab Cold stratification for 40 days (OF) 16.76a 28bcd Cold stratification for 20 days (OF) 18.32ab 38cde Floating in hot water (100°C) followed by continual cooling for 24 h in the same water (OF) 28.85bc 19ab Submersion in sulphuric acid for 30 min (OF) 31.19c 40de Cold stratification for 20 days (G) 32.75c 38cde Control (G) 33.14c 39de Cold stratification for 40 days (G) 33.92cd 42e Floating in tap water for 24 h (G) 35.87cd 45e Cold stratification for 60 days (G) 37.82cd 22ab Floating in hot water (100°C) followed by continual cooling for 24 h in the same water (G) 45.22d 24ab Submersion in sulphuric acid for 30 min (G) 4.418* 56.53e 5.484* 15a Provenances Derinköy 7.898** 25.89a 1.214 33 Salkımlı 27.40a 32 Köprüba  ı 35.17b 29 * VS: Pre-treatments, significantly different at α = 0.05, ** VS: Provenances, significantly different at α  = 0.05. Means in column with the same letter are not significantly different at   = 0.05. that scarification of C.   armena   seeds by soaking in sulphuric acid for 30 min and sown in the laboratory conditions gave high GP (82.8%) and a short time to maximum GR (7 days). The lowest average GP (13.3%) was determined in control seeds which were sown under open field con-ditions (Table 3). Dirr and Heuser (1987) reported that untreated Colutea   seeds could be sown in the autumn, but scarification was required for spring-sowing. It can be also stated that there is an affirmative effect of green-house condition on GP (Table 3). The best germination rates for provenances of Köprüba  ı and Derinköy were 14 and 16 days which were determined in seeds soaked in sulphuric acid for 30 min and sown in the greenhouse. On the other hand, the best germination rate (12 days) of provenance of Salkımlı was determined in seeds which were floated in hot water (100°C) followed by continual cooling for 24 h in the same water and sown under open field conditions (Table 2). While sulphuric acid treatment gave the best average germination rate at 15 days in the greenhouse, it took 45 days for seeds cold stratified for 60 days to reach the slowest germination rate (Table 3). The hot water pre-treatment resulted in higher GP and shorter time to maximum GR (45.2% and 24 days in the greenhouse and 28.9% and 19 days under open field conditions, respectively) compared to the tap water pre-treatment (35.9% and 38 days in the greenhouse and 15.2% and 25 days under open field conditions, respect-tively) (Table 3). These findings are supported by similar studies done on Colutea seeds (Allue Andrade, 1983; Dirr and Heuser, 1987; Olmez et al., 2008). Dirr (1990) stated that soaking Colutea   seeds initially in 88°C-water and allowing them to remain in that water for 24 h result- ed in good GP values. In addition, Piotto et al. (2003) reported that scarification of Colutea   seeds follow-ed by floating in hot water (80°C) gave high GP and a short time to maximum GR. The maximum average GP value among the CS pre-treatments was 37.8% for seeds that were cold stratified for 60 days and sown in the greenhouse. The analyses also revealed that the seeds collected from Köprüba  ı resulted in the highest GP value (35.2%) among the pro- venances (Table 3). Consequently, among all the pre-treatments applied to the C. armena   seeds, soaking in sulphuric acid for 30 min resulted in the highest GP (56.5%) and the shortest time before maximum GR (15 days). It is followed by the pre-treatment of floating in hot water (100°C) followed by continual cooling for 24 h in the same water with 45.2% of GP. Therefore, these results indicate that the pre-treat-ment by submersion in sulphuric acid for 30 min and    sowing in greenhouse condition should be used to over-come dormancy of the C. armena   seeds. REFERENCES  Aguinagalde I, Perezgarcia F, Gonzalez AE (1990). Flavonoids in Seed Coats of 2 Colutea Species-Ecophysiological Aspects. J. Basic Microbiol, 30(8): 547-553. Allue Andrade JL (1983). Morfoligia Clases, Atributos, Dificultades Tratamientos en la Produccion Germinacion de Las Semillas de Colutea arborescens L. (Morphology, Types, Attributes, Difficulties and Treatments in Production and Germination of Seeds of Colutea arborescens L.). Anales Del Instituto Nacional De Investigaciones Agrarias Seria Forestal, 7: 129-154. Balcı AN (1996). Toprak Koruması.  stanbul Üniversitesi, Yayın No: 439,  stanbul. Browicz K (1963). The Genus Colutea   L. Monograph. Monographie Botanicae  , 14 : 1-136. 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 Pres, Athens. Heit CE (1967). Propagation from Seed, Successful Propagation of Six Hardseeded Group Species. Am. Nurseryman, 125(12): 10-12, 37-41, 44-45. 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: 1-259. Krüsmann G (1984). Manual of Cultivated Broad-leaved Trees and Shrubs. Vol. 1, Timber Pres, Beaverton, USA. Landis TD, Barthell A, Loucks D (1996). Seed Treatments to Overcome Dormancy. Forest Nursery Notes, United States, Department of Agriculture, Forest Services, July, pp. 9-12, USA. Olmez Z, Gokturk A, Temel F (2007). Effects of Cold Stratification, Sulphuric Acid, Submersion in Hot and Tap Water Pretreatments on Germination of Bladder-Senna ( Colutea armena Boiss. & Huet.) Seeds. Seed Sci. Technol. 35(2): 266-271. Olmez Z, Yahyaoglu Z, Temel F, Gokturk A (2008). Effects of Some Pretreatments on Germination of Bladder-Sena ( Colutea armena   Boiss. and Huet.) and Smoke-Tree ( Cotinus coggygria   Scop.) Seeds. J. Environ. Biol. 29(3): 319-323. Pieper A (1952). Das Saatgut, P. Parey Verlag Berlin, Hamburg, Germany. Olmez and Gokturk 2977 Pijut PM (2008). Colutea L., Bladder-Senna. USDA Forest Service Hardwood Tree Improvement and Regeneration Centre, USA, www.nsl.fs.fed.us/wpsm/Colutea.pdf, 04.06.2008. Piotto B, Bartolini G, Bussotti F, Asensio A, García C, Chessa I, Ciccarese C, Ciccarese L, Crosti R, Cullum FJ, Noi AD, García P, Lambardi M, Lisci M, Lucci S, Melini S, Carlos J, Reinoso M, Murranca S, Nieddu G, Pacini E, Pagni G, Patumi M, Garcia FP, Piccini C, Rossetto M, Tranne G, Tylkowski T (2003). Fact Sheets on the Propagation of Mediterranean Trees and Shrubs from Seed. In (eds. Piotto B and Noi AD), Italy, Seed Propagation of Mediterranean Trees and Shrubs APAT, I.G.E.R. srl., pp. 11-51. Poulsen K (1996). Case Study: Neem ( Azadirachta indica A. Juss.) Seed Research. In Proceedings of an International Workshop on Improved Methods for Handling and Storage of Intermediate/Recalcitrant. (eds. Ouedraogos AS, Poulsen K and Stubsgaard F), Humlebaek, Denmark. June 8-10, Trop. Forest Tree Seeds, pp. 14-22. Pritchett WL, Fisher RF (1987). Properties and Management of Forest Soils. Second Edition, John Wiley and Sons, New York, USA. Rudolf PO (1974). Colutea arborescens L. 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