Impaginato 511 Adv. Hort. Sci., 2018 32(4): 511-516 doi: 10.13128/ahs-20537 Effects of cold stratification and chemical treatments on seed germination in four hazelnut cultivars Z. Bakhtshahi-Dizgahi 1, M. Alizadeh 1 (*), E. Seifi 1, D. Javadi 2, A. Hosseinpour 1 1 Gorgan University of Agricultural Sciences and Natural Resources, Faculty of Plant Production, Department of Horticulture, Gorgan, Iran. 2 Guilan Research Center of Agriculture and Natural Resources, Rasht, Iran. Key words: chemical treatment, cold stratification, Corylus avellana l., filbert, germination. Abstract: Propagation of European hazelnut by seed is influenced by some seed treatments. In this investigation, effect of stratification period and some chemi- cals on seeds of four hazelnut cultivars were studied. GA3 and four months of stratification, each individually resulted in the highest germination percentage at 82.73% and 83.75%, respectively. There were significant differences between cultivars and treatments in terms of germination percentage and rate. The highest germination percentage and rate were observed in the local cultivar Gerd under GA3 treatment at 100 mg/L and also after four months of stratifica- tion. 1. Introduction propagation by seeds is a conventional method to produce new plants. this is one of the most recognized efficient methods which is widely applied for different plant species. Although sexual reproduction do not result in true to type plants, in breeding programs it is inevitable to apply it to grow hybrid seedlings. for example, in hazelnut, interspecific hybridization is necessary to transfer superior characters from wild species to the commercial european hazelnut (Corylus avellana l.) (erdogan and mehlenbacher, 2000). Seed germination is a main step in plant life cycle, and is influenced by various biotic and abiotic factors (Yuan and Wysocka-diller, 2006). it is essential to investigate different aspects in sexual propagation for all plant species. However, there are some common difficulties in using such approach to propagate many plants such as hazelnut, including seed dor- mancy and inconsistent seed germination which make some problems and retard improvement programs and sometimes end in hybrids loss. therefore, studying beneficial treatments to remove dormancy and sub- sequent uniform seed germination is considered of great importance (*) Corresponding author: mahdializadeh@gau.ac.ir Citation: bAkHtSHAHi-dizgAHi z., AlizAdeH m., Seifi e., jAvAdi d., HoSSeinpour A., 2018 - Effects of cold stratification and chemical treatments on seed germination in four hazelnut cultivars. - Adv. Hort. Sci., 32(4): 511-516 Copyright: © 2018 bakhtshahi-dizgahi z., Alizadeh m., Seifi e., javadi d., Hosseinpour A. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting information files. Competing Interests: the authors declare no competing interests. received for publication 14 April 2017 Accepted for publication 22 july 2018 AHS Advances in Horticultural Science Adv. Hort. Sci., 2018 32(4): 511-516 512 (Wang and berjak, 2000; Copeland and mcdonald, 2001). generally, germination process is controlled through a balance between inducing and inhibiting factors. provided that concentration of inducers is higher than inhibitors, seed dormancy will predomi- nate. Some stimulants such as temperature and light are necessary to lower the effect of inhibitors in seed. in such case, an inducing factor such as gib- berellic acid (gA3) could have cumulative influence so that germination process will commence (bradbeer, 1988). in many temperate zone species, dormancy pre- vents the seeds from germinating (derkx, 2000). A dormant seed would not germinate even in favorable environmental conditions. Several approaches have been suggested in literature to overcome this phe- nomenon, including cold stratification (bewley and black, 1994), and seed treatment by some chemicals such as gi bberel l i c aci d (gA 3), pol yami nes and thiourea (frankland, 1961; Çetinbaş and koyuncu, 2006; mello et al., 2009). Cold stratification plays a major role as a stimulant to break seed dormancy. Also its effect is accelerated in combination with chemicals or physical removing of seed coat (bewley and black, 1994). this technique is usually performed at temperatures between 0 and 10°C; which vary depending on different species. However, the best reported temperature for this kind of seed treatment is 5°C (bewley and black, 1994). Aygun et al. (2009) suggested that hazelnut seeds need two to six months of pre-germination cold stratification. dormancy in hazelnut seeds is diminished through cytological, hormonal and biochemical changes dur- ing cold stratification period. for example, mobiliza- tion of phytic acid and phosphate was observed dur- ing this treatment (vasilios et al., 2005). gA3 treatments could remove various seed physi- ological dormancies and induce germination of dor- mant seeds (frankland, 1961). Aygun et al. (2009) showed that in hazelnut seeds treated by gA3 at con- centrations from 0 to 200 mg/l, the highest seed ger- mination percentage was obtained by 100 mg/l gA3. the main polyamines existing in plant cells are putrescine, spermine and spermidine (davies, 2004). based on some evidences, polyamines have a role in seed dormancy process. Sinska and lewandowska (1991) found that putrescine, spermine and spermi- dine decreased in apple seeds during cold stratifica- tion. in fact, putrescine and spermidine had inducing effect and spermine had inhibiting effect on apple seed germination. Although thiourea is not applied commonly in seed germination experiments, it is able to enhance germination of some kinds of seeds (gul and Weber, 1998; Çetinbaş and koyuncu, 2006). Stidham et al. (1980) showed that thiourea had an inducing effect on germination of 18 shrub species. According to Cetinbaş and koyuncu (2006), this prop- erty of thiourea is attributed to its cytokinin-related effect in removing inhibitors. this investigation aimed to study the effects of some treatments including some chemicals and cold stratification on percentage and rate of seed germi- nation in four hazelnut cultivars grown in iran. 2. Materials and Methods Seeds of four hazelnut cultivars including a local c u l t i v a r g e r d a n d t h r e e i n t r o d u c e d c u l t i v a r s barcelona, ronde (= ronde du piemont) and Segorbe were collected from Astara Hazelnut research Station, in Astara, guilan province, iran. defected seeds were discarded and proper seeds were sepa- rated to study. Treatments treatments applied in this study included gA3 (100 and 200 mg/l), putrescine (0.01 and 0.1 mm), thiourea (1000 and 2000 mg/l) and stratification for two and four months. the control treatment was dis- tilled water. Germination test the seeds were soaked for 24 hours in gA3 (100 and 200 mg/l), putrescine (0.01 and 0.1 mm) and thiourea (1000 and 2000 mg/l), and also some seeds were soaked in distilled water. in order to prevent seeds from rotting, the treated seeds were surface sterilized with sodium hypochlorite (10% v/v for 5 min.) and then rinsed by sterilized water. Afterwards, the seeds were cultured in plastic pots containing pre-autoclaved sand as the medium (diameter of 2 mm). germination test was conducted in a factorial completely randomized design with three treatments including “no stratification”, “two months of stratifi- cation” and “four months of stratification”. emerging radical was considered as the index of germination. in chemical treatments, the number of germinated seeds up to 40 days from the culture date, and in stratification treatments, the number of germinated seeds up to 40 days from outing from refrigerator were recorded. Stratification treatments were just performed by keeping them in refrigerator (5°C) for two to four months. Cultured seeds were inspected regularly and in case of moisture decrease of media, Bakhtshahi-Dizgahi Z. - Seed germination in four halzenut cultivars 513 autoclaved water was sprayed on them. the mea- sured characteristics were percentage and rate of seed germination. the following formula was used to calculate germination percentage. germination percentage = (number of germinated seeds / total number of seeds) × 100 germination rate is defined as the time to reach 50 percent of germination, which was calculated by the formula below: germination rate = (1/time of reaching 50 percent of germination) for each cultivar, 210 seeds were used. the data were analyzed by analysis of variance (AnovA) and the software germin-g was applied to measure the target parameters (Soltani et al., 2004). 3. Results and Discussion According to the analysis of variance, there was a significant difference among cultivars and chemical treatments with regard to germination percentage (p < 0.01) (fig. 1). even so, no significant difference was observed among cultivars and chemical treatments regarding germination rate (fig. 2). in addition, effect of stratification on germination rate and percentage was significantly different between the cultivars (p<0.01) (fig. 3 and 4). f or germi n ati on p ercen tage, th e d i fferen ce between mutual effects of “cultivar × chemical treat- ment” (p<0.05) (fig. 1), “cultivar × stratification” (p<0.05) (fig. 4) and “chemical treatment × stratifica- tion” (p<0.01) (fig. 5) was significant. for germination rate, the mutual effect of “culti- var × chemical treatment” showed a significant dif- ference (p<0.05) (fig. 2). of the four hazelnut cultivars, ‘gerd’ had the high- est germination percentage (82.73%) at 100 mg/l gA3, which showed 50.76% increase compared to the control (31.97%). Although applying both gA3 con- centrations was resulted in higher germination per- centages, but no difference was observed between these two levels (fig. 1). between stratification and cultivar, the highest germination percentage was exhibited after four fig. 1 - effect of chemical treatments and cultivars on seed ger- mination percentage of hazelnut cultivars. fig. 2 - effect of chemical treatments and cultivars on seed ger- mination rate of hazelnut cultivars. fig. 3 - effect of stratification treatments on seed germination rate of hazelnut cultivars. fig. 4 - effect of stratification treatments and cultivar on seed germination percentage of hazelnut cultivars. fig. 5 - effect of stratification and chemical treatments on seed germination percentage of hazelnut cultivars. Adv. Hort. Sci., 2018 32(4): 511-516 514 months of stratification in the local cultivar gerd. no difference existed in four months of stratification between two local cultivars and barcelona (fig. 6). figure 5 reveals that between mutual effect of chemicals and stratification treatments, the highest germination percentage occurred through four months of stratification and in the control treatment. nevertheless, in treatments of two months of stratifi- cation and no stratification, both levels of gA3 result- ed in the highest germination percentage. in all cultivars, the highest germination rate was recorded in ‘gerd’ (fig. 6). four months of stratifica- tion had the greatest influence on germination rate (fig. 3). of chemical treatments, the fastest germina- tion was obtained by using the first level of gA3 (100 mg/l) in the cultivar gerd (fig. 2). various studies have demonstrated a direct rela- tionship between cold stratification length and increasing in germination percentage. As can be clearly seen in figures 4 and 5, when stratification period increased, as a result, germination percentage of hazelnut seeds increased in all treatments and all cultivars. Similar results were observed by Aygun et al. (2009) which suggested that 120 days of cold stratification ended in higher germination percentage i n h a z e l n u t s e e d s i n c o m p a r i s o n w i t h c o n t r o l . furthermore, bradbeer (1988) reported that three months of cold stratification led to a rise in hazelnut seed germination percentage. in other studies, three m o n t h s o f s t r a t i f i c a t i o n w i t h o u t a n y w a r m i n g i m p r o v e d g e r m i n a t i o n p e r c e n t a g e i n s e e d s o f Jasminum fruticans (pipinis et al., 2009). Another example for such impact has been reported by Chin et al. (1992) in kiwifruit. in addition, there are many other studies that support the influence of applying some chemicals and plant growth regulators on rising germination percentage of seeds, which could be used singly or in combination with chilling. As a matter of fact, these chemicals are considered as substitutes for chilling requirement of seeds, and also can decrease length of chilling period. overall, gibberellins and cytokinins are able to promote seed germination in plants (davies, 2004; miransari and Smith, 2014). in contrast, abscisic acid (AbA) plays an inhibiting role in seed germination (miransari and Smith, 2014). Among types of gib- berellins, gA3, gA4 and gA7 have the most impact on germination enhancement. However, cytokinins and auxins have so lower effect compared to gibberellins and cytokinins in terms of stimulating germination. in f a c t , t h e e f f e c t o f a l l t h e s e g r o w t h r e g u l a t o r s depends upon other factors such as light, tempera- ture and oxygen; and also there are some mutual effects between them. gibberellins and cytokinins are capable of neutralizing the inhibiting effect of abscisic acid. Since all these growth regulators natu- r a l l y e x i s t i n s e e d i n d i f f e r e n t r a t i o s , s o t h e i r observed effects on seed germination could be inter- preted by the state of hormone balance (kucera et al., 2005). gibberellins are able to enhance seeds of differ- ent species to germinate through different ways. that is, external use of gibberellins could induce ger- mination in seeds in which lack of germination is due to seed coat (e.g. legumes), or seed dormancy is because of seed embryo (e.g. apple, birch, hazelnut) (davies, 2004; miransari and Smith, 2014). besides, in seeds that their germination depends on exposure to light (e.g. Arabidopsis, lettuce), gA3 could promote seed germination even in the dark (Cao et al., 2005). based on the results obtained in this study, the high- est germination percentage and rate were gained through gA3 treatment at 100 mg/l (fig. 1, 2), which corresponded with results reported by Aygun et al. (2009) that showed higher germination percentage after treatment by 100 mg/l gibberellin. in addition, there are some reports on gibberellin application on hazelnut seeds in order to enhance germination results (bradbeer and pinfield, 1967; jarvis and Wilson, 1977; pinfield and Stobart, 2006). in addition to gibberellins, other substances such as thiourea can also overcome seed dormancy. Several investigations have demonstrated the effect of thiourea on dormancy breaking and increasing germination percentage of seeds in different plant s p e c i e s ( g u l a n d W e b e r , 1 9 9 8 ; Ç e t i n b a ş a n d koyuncu, 2006). Also ojha et al. (2010) showed that after using four treatments including gibberellin, potassium nitrate, ascorbic acid and thiourea on seeds of Abrus precatorius, despite increasing germi- nation percentage in all treatments, the highest and fig. 6 - Seed germination rates in four hazelnut cultivars. Bakhtshahi-Dizgahi Z. - Seed germination in four halzenut cultivars 515 lowest germination percentage were obtained by gibberellin and thiourea treatments, respectively. polyamines are also a group of growth regulators and could improve seed germination in some species (Szczotka and lewandowska, 1989; Sinska and lewandowska, 1991). furthermore, in this research, putrescine had a significant difference compared to the control, in terms of germination percentage and germination rate of hazelnut seeds (fig. 2). 4. Conclusions the results of this study revealed that the hor- mone gA3 and four months of cold stratification resulted in the highest germination percentage in hazelnut seeds at 82.73% and 83.75%, respectively. the cultivars and chemical treatments had significant effect on seed germination which were the highest in the local cultivar gerd under gA3 treatment at 100 mg/l and also after four months of cold stratification. 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