171 1. Introduction In agriculture, seeds are considered as starting points of plant growth. Assuming seeds are non-dormant, germina- tion is the key component of seedling emergence (Forcella et al., 2000). Germination may be defined as the process associated with the initiation and completion of embryo emergence; it refers to the progress of a seed from im- bibition through radicle emergence (Gniazdowska et al., 2010). Germination parameters are useful for estimating the conversion of seeds to seedlings and, thus, the suit- ability of a seed lot for commercial seedling production. Germination parameters are also useful in determining the type of seed pre-treatment as well as nursery management practices needed to attain a high level of germination (Ko- lotelo et al., 2001). Abscisic acid (ABA) induces dorman- cy during maturation, and gibberellins (GAs) play a key role in dormancy release and in the promotion of germina- tion (Kaur et al., 2006). In many cases, endogenous levels of these hormones (GA, ABA) are associated with the abil- ity of the seeds to germinate (Seo et al., 2006); seed dor- mancy must be broken to induce germination. In morpho- physiological dormancy, seeds must be exposed to cold, heat, gibberellic acid, or chemical materials for dormancy breaking (Hilton, 1984; Dehghanpour et al., 2011). The use of potassium nitrate has been an important seed treat- ment in seed-testing laboratories for many years without a good explanation for its action mechanism (Çetinbas and Koyuncu, 2006). Hydrogen cyanamide (HCN , Dormex®) has been identified as one of the most effective dormancy- breaking agents in many deciduous plant species (Siller- Cepeda et al., 1992) and has been exploited mainly as an artificial rest-breaking agent to stimulate budbreak in mild winter climates (Ben Mohamed et al., 2012). However, the emission of HCN has been detected during the pre-germi- nation period of many seeds (Gniazdowska et al,. 2010). Pistachio (Pistacia vera L.), a member of Anacardiaceae family (Khan et al., 1999), is a dioecious species and for this reason it has acquired large genetic diversity (Behboodi, 2002). Seed dormancy, oily nature of cotyledons leading to early spoilage, risk of heterozygosis, and production of male/female plantlets are major problems associated with pistachio sexual propagation (Jangali Baygi, 2012). There- fore, under natural conditions, an erratic and irregular ger- mination over a quite long period. On the other hand, Pis- tachio plants cannot be readily propagated from cuttings taken from mature trees and are currently propagated by grafting on seedling rootstocks (Behboodi, 2002). Further- Dormancy removal in pistachio nut: Influences of Hydrogen Cyanamid (Dormex®) as compared to ordinary seed chemical pre-treatments M. Jangali Baygi 1, M. Alizadeh 1 (*), F. Ghaderifar 2, M. Sharifani 1 1 Horticulture Department, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran. 2 Agronomy Department, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran. Key words: dormancy, germination, gibberellin, KNO 3 , hydrogen cyanamid, pistachio. Abstract: Seed germination is considered an important phenological stage of plant growth during which the viable, non- dormant embryos develop into seedlings under favorable conditions. Currently, various seed priming treatments may be used to enhance germination values (germination percentage, Lag phase, time to reach 50% germination) and breaking dormancy; gibberellins and KNO3 are applied as common treatments in this regard. In the present study, efficacy of Hydrogen Cyanamid (HCN, Dormex®) on seed germination of two cultivated pistachio varieties (Abasali, Shahpasand) were studied and the results were compared to ordinary ingredients, namely gibberellic acid (GA3) and KNO3. The ex- traction of parameters was carried out using four-parameter Hill function. The results reveal that HCN may significantly improve critical parameters involved in pistachio seed germination. In Shahpasand variety, seeds treated with HCN ger- minated prior to other treatments (150.53 hours after treatment) and achieved 50% germination earlier than other treat- ments (199.00 hours only). According to the results, utilization of HCN in seed testing experiments may be encouraged. Adv. Hort. Sci., 2015 29(4): 171-175 (*) Corresponding author: mahdializadeh@gau.ac.ir Received for publication 19 May 2015 Accepted for publication 23 June 2015 172 Adv. Hort. Sci., 2015 29(4): 171-175 more breeding programs and development of hybrid seed- lings/superior rootstocks require a rapid and reliable method for seed germination (Ghazaeian et al., 2012). Hence, in the present study, effects of HCN on dormancy removal and seed germination of two pistachio varieties were studied and the results were compared to widespread chemical treat- ments such as GA 3 and KNO 3 as well as cold stratification. 2. Materials and Methods The present study was undertaken in the physiology lab- oratory at the Horticulture Department, Gorgan University of Agricultural Sciences and Natural Resources, Golestan, Gorgan, Iran. Two Iranian cultivated pistachio varieties, (‘Abbasali’ and ‘Shahpasand’) were procured from a com- mercial orchard located in Damghan, Semnan Province. Initially, the healthy and bold seeds were selected and the extracted kernels were treated for 24 h with different chemicals, i.e. GA 3 (50, 100 mg/l), KNO 3 (1.0, 1.5 %), and Dormex® (50, 100 mg/l), all procured from Sigma. Dor- mex is a mineral component with chemical formula H 2 CN 2 (Siller-cepeda et al., 1992). The chemical component uti- lized in the present study was aqueous Hydrogen Cyanamid (H 2 CN 2, 187364 Sigma, F.W.= 42.04 g/mole). Initially the required amount of HCN was dissolved in a few drops of ethanol to prepare the stock solution from which the con- centrations of 50-100 mg/l HCN were prepared with dis- tilled water. The control seeds were also soaked in distilled water only and some seeds of the same lot were kept in a refrigerator (5°C) as cold stratification treatment. In order to prevent fungal contamination, the treated kernels were surface sterilized with sodium hypochlorite 10% and then rinsed at least three times with autoclaved water. These were then cultured in plastic containers filled with autoclaved sand (diameter 2 mm). The samples were observed daily for a period of 54 days and the number of germinated seeds was recorded. Radicle emergence (5 mm in length) was taken as germinated seed. The cumulative germination count of each pistachio variety seed lot was fitted to a four-parameter Hill function (4-PHF; Equation 1) already reported by El- Kassaby et al. (2008) as a mathematical representation and parameter extraction of seed germination: (1) y = y 0 + axb cb + xb In the equation 1, y is considered as the percentage of cumulative germination at time x, y 0 is the intercept on the y axis (≤0), a is the asymptote, or maximum cumulative germination percentage, which is equivalent to germina- tion capacity, b is a mathematical parameter controlling the shape and steepness of the germination curve and c is the “half-maximal activation level” measured in days and represents the time required for 50% of viable seeds to germinate (G 50% ). Following extraction of a, b, c and Y 0, from 4-PHF, the time at germination onset (lag) and the final germination percentage (G max ) were also computed using the following equations: (2) lag = b -y 0 cb a + y 0 √ (3) G max = y 0 + a The entire research work was designed as a completely randomized design (CRD) with factorial arrangement in- cluding seven treatments and three replications. The data were analyzed using SAS software. 3. Results and Discussion Pistachio plants cannot be readily propagated from cut- tings procured from mature trees and are currently propa- gated through budding/grafting on seedling rootstocks. Though tissue culture techniques were proposed as new and advanced methods for multiplication of this species (Beh- boodi, 2002) seedling development followed by budding is still considered a superior method for pistachio orchard es- tablishment. However, seed dormancy and erratic germina- tion cause significant trouble for growers, nurserymen, and fruit breeders (Jangali Baygi, 2012). Dormancy release is accomplished by diverse mechanisms that include complex interactions with the environment mediated by plant hor- mones and other small molecules, and is supposed to se- lect conditions for germination that are most favorable for plant survival (Finkelstein et al., 2008). Application of GA 3 . Thiourea, and KNO 3 as common priming agents were for- merly reported in dormancy removal of some nut species such as wild pistachio (Khan et al., 1999), walnut (Kaur et al., 2006), and pecan (Ghazaeian et al., 2012). Hence, the present study was also undertaken on two cultivated pista- chio varieties and the following results were found. The results of analysis of variance of the effects of chem- ical treatments on dormancy removal and germination pa- rameters of pistachio varieties are shown in Table 1. The data demonstrate a sharp and considerable effect of chemi- cal priming on germination percentage, the time at germina- tion onset (lag), and time taken to 50% germination in each seed lot. Furthermore, the results demonstrate the effective- ness of Dormex® to release pistachio seeds from dormancy. The lag phase is an important parameter to determine quality of seed lots. In Abbasali variety the control seeds germinated 731.28 h after culture, while the chemical prim- ing considerably reduced this period. For example, seeds treated with 1.5% KNO 3 germinated at 263.26 h only. As shown in figure 1, application of Dormex® at the rate of 100 mg/l had the similar effect on lag phase and the germi- nation at onset was recorded at 385 h following treatment. In the case of Shahpasand variety, Dormex® was found to be the best treatment to remove seed dormancy and the lag period from 150.35 h in control seeds was reduced to 52.78 h only. This result indicates that the priming treat- ment with Dormex® may lead to precocious and early seed 173 Jangali Baygi et al., Dormancy removal in pistachio nut: Influences of Hydrogen Cyanamid (Dormex®) germination. An application of calcium cyanamide was previously proposed to overcome a lack of winter chilling in many plants (Walton et al., 1991). Ben-Mohamed et al., (2012) investigated the effect of HCN on dormancy release and carbohydrate metabolism in the buds of grapevine. Their results showed that application of HCN to dormant grape buds advanced budbreak to 10 days after treatment while the untreated buds opened within 14 days after treat- ment. Furthermore, HCN caused significant differences in the rate and percentage of budbreak (85% as compared to 42% budbreak in the controls). However, there are quite a few reports on the effect of this compound on seed dor- mancy. Gniazdowska et al. (2010) studied the role of HCN on breaking dormancy of apple embryos. Interestingly, they found that the deep dormancy of apple embryos may be removed by a quite short pre-treatment (3-6 h) of HCN. Furthermore, the development of abnormal seedlings was also lower than control, non treated seeds. Such effect of HCN in apple embryos is comparable with the response of Shahpasand pistachio variety in the present study (Fig. 2). The time taken to reach 50% seed germination is also considered as a remarkable parameter in seed germina- tion experiments. According to figures 3 and 4, applica- tion of chemical treatments significantly reduced this Table 1 - The results of analysis of variance of the effects of chemical treatments on dormancy removal and germination parameters of two cultivated pistachio varieties Sorces of variations d.f. Mean square D 50 G max lag Variety 1 2508961.5** 1138.38** 1328137.5** Treatments 6 66477.16** 911.36** 49107.3** Variety x treatment 6 33053.44* 528.47** 21142.09* Error 28 12569.26 87.34 17639.87 cv 19.68 12.28 52.83 G max= germination percentage. D 50 = time to reach 50% germination. Lag= time to germination onset. Fig. 1 - Effect of different priming agents on time to germination at onset (lag phase) of Abbasali pistachio variety. Fig. 2 - Effect of different priming agents on time to germination at onset (lag phase) of Shahpasand pistachio variety. Fig. 3 - Effect of different priming agents on time to reach 50% germi- nation (G 50% ) of Abbasali pistachio variety. co ntro l GA 50 m g/l GA 10 0 m g/l HCN 50 m g/l HCN 10 0 m g/l KNO 3 1% KNO 3 1. 5% co ntro l GA 50 m g/l GA 10 0 m g/l HCN 50 m g/l HCN 10 0 m g/l KNO 3 1% KNO 3 1. 5% co ntro l GA 50 m g/l GA 10 0 m g/l HCN 50 m g/l HCN 10 0 m g/l KNO 3 1% KNO 3 1. 5% 174 Adv. Hort. Sci., 2015 29(4): 171-175 period. In Shahpasnd variety, the time taken to G 50% in control seeds was recorded as 562 h while in the case of priming treatments it was effectively reduced to 199 h (Dormex® 100 mg/l), 234 h (GA 3 100 mg/l) and 286 h (KNO 3 1.5%). In the case of Abbasali, the same trend was observed and KNO 3 1.5% was found to be the most effective treatment. In both varieties, cumulative germination in control seeds was low but it was enhanced following chemical priming (Fig. 5 and 6). This means that G max in ‘Abbas- ali’ increased from 46.23% in control seeds to 86.30% in seeds treated with KNO 3 . The same variety had 81.91% of G max when treated with Dormex®. Application of KNO 3 in ‘Shahpasand’ seeds led to 99.25% germination. Hence, the results clearly reveal that Dormex® was effective in dormancy removal and germination of pistachio nuts and that in some cases it was comparable or even better than GA 3 and KNO 3 treatments. The effectiveness of gibberellins in seed germination has been reported in numerous studies (Bewley and Black, 1994; Seo et al., 2006). In vitro germination of walnut (Juglans regia L.) embryos was undertaken by Kaur et al. (2006) and they used components such as GA 3, Benzyl ad- enine, and kinetin to improve embryo growth and devel- opment. They reported GA 3, along with low temperature, as the best treatment for walnut in vitro embryo germina- tion. In another experiment for dormancy breaking of tulip seeds, Rouhi et al. (2010) also applied GA 3 at rates from 250 to 500 mg/l. They observed an enhancement of seed germination as GA 3 levels were increased. In general, two main functions have been proposed for gibberellins during seed germination process. First, GA increases the growth potential of the embryo. Secondly, it is necessary to over- come the mechanical restraint conferred by the seed-cov- ering layers, by weakening the tissues surrounding the radicle (Kucera et al., 2005). Gibberellins stimulate ger- mination by inducing hydrolytic enzymes that weaken the barrier tissues such as the endosperm or seed coat, induc- ing mobilization of seed storage reserves, and stimulating expansion of the embryo (Finkelstein et al., 2008). Potassium nitrate is considered to be one of the most effective and widely used components with respect to breaking seed dormancy (Çetinbas and Koyuncu, 2006). As far as pistachio seed germination is concerned, Khan et al. (1999) applied KNO 3 at the rate of 1% to wild pistachio and they observed 26% seed germination as compared to control with only 2%. In an experiment carried out on Prunus avium L., the stratified seeds were treated with GA 3, KNO 3 and thiourea (Çetinbas and Koyuncu, 2006). Treatments with 7,500 mg/l KNO 3 after 120 days of strati- fication were more effective, yielding 64.54% germination of seeds with coat. However, in case of seeds without coat (kernels), GA 3 was found to be more effective than other chemicals. The role of HCN in dormancy removal may be attrib- uted to its stimulatory effects on endogenous ethylene pro- duction. Ethylene increases respiration as well as starch degradation of seed reserves and the embryo then starts germination. A similar result was also reported for tobacco in which ethylene was involved in endosperm rupture and Fig. 4 - Effect of different priming agents on time to reach 50% germi- nation (G 50% ) of Shapasand pistachio variety. Fig. 6 - Effect of different priming agents on cumulative germination percentage (G max ) of Shahpasand pistachio variety. Fig. 5 - Effect of different priming agents on cumulative germination percentage (G max ) of Abbasali pistachio variety. co ntro l GA 50 m g/l GA 10 0 m g/l HCN 50 m g/l HCN 10 0 m g/l KNO 3 1% KNO 3 1. 5% co ntro l GA 50 m g/l GA 10 0 m g/l HCN 50 m g/l HCN 10 0 m g/l KNO 3 1% KNO 3 1. 5% co ntro l GA 50 m g/l GA 10 0 m g/l HCN 50 m g/l HCN 10 0 m g/l KNO 3 1% KNO 3 1. 5% 175 Jangali Baygi et al., Dormancy removal in pistachio nut: Influences of Hydrogen Cyanamid (Dormex®) high bGlu I expression during seed germination (Kucera et al., 2005). Ben Mohamed et al. (2012) also stated that starch concentration was high in dormant buds of grape- vine and declined rapidly to reach its lowest level fol- lowing HCN application. The same process may occur in treated pistachio seeds leading to 99.25% germination in Shahpasand variety (Fig. 6). Another reason for the role of HCN in improvement of seed germination can be the acti- vation of the pentose-phosphate pathway which is thought to be necessary for the breaking of dormancy in buds and seeds (Walton et al., 1991). They demonstrated that ap- plication of HCN to kiwifruit dormant buds led to produc- tion of high levels of proline amino acid. They stated that elevated levels of proline in HCN-treated plants could be associated with a greater stimulation of the pentose-phos- phate pathway, resulting in a greater percentage of bud- break and increased bud fruitfulness. In conclusion, it can be stated that HCN is an effec- tive compound for pistachio seed germination. Moreover, the role of ordinary chemical seed pre-treatments such as GA 3 and KNO 3 to improve pistachio seed germination was reconfirmed. Application of HCN also leads to precocious germination and early seedling development. This param- eter may be useful for nurseries or fruit breeders as a use- ful measure to develop hybrid seedlings in a shorter time. Overall, although more experiments with other species should be carried out to confirm the results of the present research work, it can be said that utilization of HCN in seed testing experiments should be encouraged as an ef- fective pre-treatment and dormancy breaking agent. References BEHBOODI B. SH., 2002 - Tissue culture results of all wild pistachio species and some cultivars in Iran. - Acta Horticul- turae, 591: 399-409. BEN MOHAMED H., VADEL A.M., GEUNS J.M.C., KHEMIRA H., 2012 - Carbohydrate changes during dor- mancy release in superior seedless grapevine cuttings fol- lowing hydrogen cyanamide treatment. - Scientia Horticul- turae, 140: 19-25. BEWLEY J.D., BLACK M., 1994 - Seeds: Physiology of devel- opment and germination. - Plenum, NY, USA, pp. 367. ÇETINBAS M., KOYUNCU F., 2006 - Improving germination of Prunus avium L. seeds by gibberellic acid, potassium ni- trate and thiourea. - HortScience., 33(3): 119-123. DEHGHANPOUR FARADHAH H., TAVAKKOL AFSHARI R., SHARIFZADEH F., CHAVOSHINASAB S., 2011 - Ger- mination improvement and α-amylase and β-1,3-glucanase activity in dormant and non-dormant seeds of Oregano (Origanum vulgare). - Australian Journal of Crop Science, 5(4): 421-427. EL-KASSABY Y.A., MOSS I., KOLOTELO D., STOEHR M., 2008 - Seed germination: Mathematical representation and parameters extraction. - Forest Science, 54(2): 220-227. FINKELSTEIN R., REEVES W., ARIIZUMI T., STEBER C., 2008 - Molecular aspects of seed dormancy. - Annu. Rev. Plant Biol., 59: 387-415. FORCELLA F., BENECH ARNOLD R.L., SANCHEZ R., GH- ERSA C.M., 2000 - Modeling seedling emergence. - Field Crop Res., 67: 123-139. GHAZAEIAN M., ZAMANI S., SAFARNEJAD A., KHERAD- MAND G., 2012 - Effects of stratification treatments on ger- mination of pecan seeds (Carya illinoensis). - International Journal of AgriScience, 2(7): 588-591. GNIAZDOWSKA A., KRASUSKA U., BOGATEK R., 2010 - Dormancy removal in apple embryos by nitric oxide or cyanide involves modifications in ethylene biosynthetic path- way. - Planta., 232: 1397-1407. HILTON J.R., 1984 - The influence of light and potassium ni- trate on the dormancy and germination of Avena fatua L. (wild oat) seed and its ecological significance. - New Phy- tol., 96: 31-34. JANGALI BAYGI M., 2012 - Analysis of seed germination us- ing Hill function: A case study of Pistachio. - MSc thesis, Horticulture Dep., Gorgan University of Agricultural Sci- ences and Natural Resources, Gorgan, Golestan, Iran. KAUR R., SHARMA N., KUMAR K., SHARMA D.R., SHAR- MA S.D., 2006 - In vitro germination of walnut (Juglans re- gia L.) embryos. - Scientia Horticulturae, 109: 385-388. KHAN J., RAUF M.A., HARUN-UR-RASHID A.Z., KHAT- TACK M.S., 1999 - Different stratification techniques on seed germination of pistachio cv. Wild. - Pakistan Journal of Biological Sciences, 2(4): 1412-1414. KOLOTELO D., VAN STEENIS E., PETERSON M., BEN- NETT R., TROTTER D., DENNIS J., 2001 - Seed handling guidebook. - BC Ministry of Forests, Tree Improvement Branch, Victoria, BC, Canada, pp. 106. KUCERA B., COHN M.A., LEUBNER-METZGER G., 2005 - Plant hormone interactions during seed dormancy release and germination. - Seed Science Research, 15: 281-307. ROUHI H.R., SHAKARAMI K., TAVAKKOL AFSHARI R., 2010 - Seed treatments to overcome dormancy of waterlily tulip (Tulipa kaufmanniana Regel.). - Australian Journal of Crop Science, 4(9): 718-721. SEO M., HANADA A., KUWAHARA A., ENDO A., OKA- MOTO M., YAMAUCHI Y., NORTH H., MARION-POLL A., SUN T., KOSHIBA T., KAMIYA Y., YAMAGUCHI S., NAMBARA A., 2006 - Regulation of hormone metabolism in Arabidopsis seeds:phytochrome regulation of abscisic acid metabolism and abscisic acid regulation of gibberellin metabolism. - The Plant Journal, 48: 354-366. SILLER-CEPEDA J.H., FUCHIGAMI L.H., CHEN T.H.H., 1992 - Hydrogen Cyanamide-induced Budbreak and Phy- totoxicity in ‘Redhaven’ Peach Buds. - Hortscience., 27(8): 874-876. WALTON E.F., CLARK C.J., BOLDINGH A.L., 1991 - Effect of Hydrogen Cyanamide on amino acid profiles in kiwifruit buds during budbreak. - Plant Physiol., 97: 1256-1259.