ACTA S8 AGRICULTURAE SLOVENICA Biotehniška fakulteta Univerze v Ljubljani Biotechnical Faculty University of Ljubljana Acta agriculturae Slovenica • ISSN 1581-9175 • 105 - 1 • Ljubljana, marec 2015 Acta agriculturae Slovenica Volume / Letnik 105 • Number / Številka 1 • 2015 VSEBINA / CONTENTS Članki / Articles Helena BAŠA ČESNIK, Dejan BAVČAR, Klemen LISJAK 5 Volatile profile of wine Teran PTP Aromatične spojine vina Teran PTP Abdollah GHASEMI PIRBALOUTI, Yazdan AHMADZADEH, Fatemeh MALEKPOOR 15 Variation in antioxidant, and antibacterial activities and total phenolic content of the bulbs of mooseer (Allium hirtifolium Boiss.) Spremenljivost antioksidacijskega in antibakterijskega delovanja celokupnih fenolnih izvlečkov iz čebulic perzijske šalotke (Allium hirtifolium Boiss.) Mansour GHORBANPOUR, Mehrnaz HATAMI, Mahmoud HATAMI 23 Activating antioxidant enzymes, hyoscyamine and scopolamine biosynthesis of Hyoscyamus niger L. plants with nano-sized titanium dioxide ... Aktiviranje aktivnosti antioksidacijskih encimov, biosinteze hiosciamina in skopolamina pri črnem zobniku (Hyoscyamus niger L.) z nano ... Yousef NASIRI and Nosratollah NAJAFI 33 Effects of soil and foliar applications of iron and zinc on flowering and essential oil of chamomile at greenhouse conditions Učinki talnega in foliarnega dodajanja železa in cinka na cvetenje in vsebnost eteričnih olj prave kamilice (Chamomilla recutita (L.) Rauschert), ... Ghader HABIBI 43 Exogenous silicon leads to increased antioxidant capacity in freezing-stressed pistachio leaves Tretiranje listov pistacije (Pistacia vera Ahmadaghaii') s silicijem poveča njihovo antioksidativno sposobnost v mraznem stresu Anna LENZI, Ada BALDI, Romano TESI 53 Artichoke (Cynara scolymus L.) as cash-cover crop in an organic vegetable system Artičoka (Cynara scolymus L.) kot prodajno zanimiva vrtnina v ekološki pridelavi zelenjave Tomaž PRUS, Nina ZUPANČIČ, Helena GRČMAN 61 Soil of the lower valley of the Dragonja river (Slovenia) Tla spodnjega dela doline reke Dragonje (Slovenija) Peyman SHARIFI 73 Genetic variation for seed yield and some of agro-morphological traits in faba bean (Vicia faba L.) genotypes Genetska variabilnost pridelka semen in nekaterih agronomsko-morfoloških lastnosti genotipov boba (Vicia faba L.) Sali ALIU, Imer RUSINOVCI, Shukri FETAHU, Bekim GASHI, Emilija SIMEONOVSKA, Ludvik ROZMAN 85 The effect of salt stress on the germination of maize (Zea mays L.) seeds and photosynthetic pigments Vpliv slanostnega stresa na kalivost in fotosintezne pigmente koruze (Zea mays L.) Tomaž JEVŠNIK, Zlata LUTHAR 95 Successful disinfection protocol for orchid seeds and influence of gelling agent on germination and growth Uspešna metoda razkuževanja semen orhidej in vpliv strjevalca na kalitev in rast Naser SABAGHNIA 103 Identification of the most stable genotypes in multi-environment trials by using nonparametric methods Določanje najbolj stabilnih genotipov v različnih okoljih z neparametričnimi metodami Tjaša POGAČAR, Domen IPAVEC, Janko VERBIČ, Lučka KAJFEŽ-BOGATAJ 111 Calibration of the LINGRA-N model to simulate herbage yield of grass monocultures and permanent grassland in Slovenia Umerjanje modela LINGRA-N za simulacijo pridelka posameznih vrst trav in trajnega travinja v Sloveniji Tanja ZADRAŽNIK, Jelka ŠUŠTAR-VOZLIČ 125 Preučevanje odziva na sušni stres pri metuljnicah (Fabaceae) s proteomiko Proteomic studies of drought stress response in Fabaceae Matjaž BEBER 135 Ukrepi za zaviranje rasti vrhov jablane 'GALA' Measures to inhibit the growth of apple tree top with the 'GALA' variety Rajko BERNIK 141 Preizkusno delovanje stroja za luščenje orehov Testing of a machine for walnut cracking Krajši prispevki/Short Communications JTIJSKENS L.M.M., SCHOUTEN R.E., UNUK T., SIMČIČ M. 157 Green mathematics: Benefits of including biological variation in your data analysis Zelena matematika: koristi od vključevanja biološke spremenljivosti v analizo podatkov Tomaž BARTOL, Karmen STOPAR 165 Content analysis of the papers in the Acta agriculturae Slovenica Vsebinska obdelava prispevkov v Acta agriculturae Slovenica let. 105 št. 1 169 Navodila avtorjem Notes for authors COBISS Code 1.01 DOI: 10.14720/aas.2015.105.1.01 Agrovoc descriptors: wines, red wines, volatile compounds, provenance, indigenous organisms, aromatic compounds, esters, smell, extraction Agris category code: f60, q04 Volatile profile of wine Teran PTP Helena BAŠA ČESNIK1, Dejan BAVČAR1, Klemen LISJAK1 * Received November 19, 2014; accepted December 18, 2014. Delo je prispelo 19. novembra 2014, sprejeto 18. decembra 2014. ABSTRACT Teran PTP is a protected wine with a recognized traditional denomination produced from a grapevine variety 'Refosk' in winegrowing district Kras in Slovenia (European Union, 2009; Pravilnik, 2008). The aromatic profile of 82 Teran PTP wines produced in years 2011, 2012 and 2013 was monitored. In total the content of 16 volatile compounds was determined. The volatile compounds from wine were extracted following the liquid-liquid extraction and determined with a GC-MS method. The odour activity values and relative odour contributions were calculated for each volatile compound identified. Among sensorial important volatiles the highest odour activity values were determined for ethyl octanoate, ethyl hexanoate, isoamyl acetate and ethyl butyrate. Other research papers also showed, that all red wines investigated except one contained ethyl octanoate, ethyl hexanoate, isoamyl acetate and ethyl butyrate above sensory thresholds. Key words: Teran PTP, wine, volatile compounds, esters, aroma, GC/MS IZVLEČEK AROMATIČNE SPOJINE VINA TERAN PTP Vino Teran PTP je zaščiteno vino s priznanim tradicionalnim poimenovanjem, ki ga pridelujejo iz grozdja sorte 'Refošk' v vinorodnem okolišu Kras v Sloveniji (European Union, 2009; Pravilnik, 2008). Aromatični profil vina Teran PTP smo spremljali tri leta v 82 vzorcih iz letnikov 2011, 2012 in 2013. Določevali smo vsebnost 16 hlapnih spojin. Za določitev hlapnih spojin smo uporabili ekstrakcijo tekoče-tekoče, kateri je sledila določitev z GC-MS. Za vseh 16 spojin smo izračunali aktivne vonjalne vrednosti in njihove relativne prispevke. Med senzorično pomembnimi hlapnimi spojinami smo največje aktivne vonjalne vrednosti določili za etil oktanoat, etil heksanoat, izoamil acetat in etil butirat. Drugi raziskovalni članki so tudi pokazali, da so vsa preiskovana rdeča vina z izjemo enega vsebovala etil oktanoat, etil heksanoat, isoamil acetat in etil butirat nad pragom zaznave. Ključne besede: Teran PTP, vino, hlapne spojine, estri, aroma, GC/MS 1 INTRODUCTION Teran PTP (Recognised Traditional Denomination) is a typical wine from the Kras winegrowing district of the Primorska wine growing region in Slovenia, where it is usually called just »Teran«. The production of this unique red wine has been known since the first century AD (Vodopivec, 1999). Teran PTP is a wine produced from a variety 'Refosk' and known for its deep reddish-violet colour, caused by an abundance of anthocyanins and its medium tannin content (Vanzo et al., 2012). Due to its favorable phenolic compounds (anthocyanins), it is known for having positive health effects and good nutritional value for consumers (Fornasario et al., 2012). Taste of wine teran is both astringent and sour, derived from its high phenolic content and high total acidity. The unique feature of this wine is its fruity odour, reminiscent of raspberries, strawberries and cherries. Fruity odour of Teran PTP is considered most pronounced in the first year after vinification (Vodopivec, 1999). The volatile fraction of wine determines to a great extent its aroma, which is one of the most Agricultural Institute of Slovenia, Hacquetova ulica 17, SI-1000 Ljubljana, Slovenia, PhD,* corresponding author: klemen.lisjak@kis.si important characteristics influencing wine quality and consumer preferences (García-Carpintero et al., 2012b). However, the wine volatile fraction is extremely complex, where more than 1000 aromatic compounds have been identified, originating from different chemical groups, mostly higher alcohols, aldehydes, ethyl esters of fatty acids, fatty acids, ketones, monoterpenes and volatile phenols (Andujar-Ortiz et al., 2009). A content of listed aromatic compounds in wines range from a few ng l-1 to hundreds of mg l-1 (Andujar-Ortiz et al., 2009). The presence, abundance and various combinations of volatile compounds can be greatly affected by viticultural practices (variety, cultivation, grape thinning etc.), environmental conditions (climate, soil) and oenological measures (fermentation, yeasts, postfermentation treatments etc.) (Ribéreau Gayon et al., 2006; Welke et al., 2014). Not all volatile compounds present in wine contribute to aroma (Welke et al., 2014). The influence of volatile compounds on the final aroma depends on their content and the specific perception threshold of each compound. The threshold of olfactory perception is defined as the lowest content capable of producing an olfactory sensation detected by the human nose, and that can be identified by at least 50 % of the judges on a sensory evaluation panel (Welke et al., 2014). In our research, esters were of particular interest as they are usually responsible for the typical aroma of most not-aromatic varieties of wine (Etievant 1993; Ferreira et al, 1995). One factor that supports the use of this approach is the typical fruity smell of Teran PTP wines, which is correlated to abundant contents of esters. The results of the present study are particularly interesting, since the aromatic profile of Teran PTP wine has never been investigated before and even less over a multi-year period. Previous studies of Teran PTP were mostly concentrated on phenolic compounds and their influence on wine technologies or wine quality (Novak, 2011; Vrscaj Vodosek and Kosmerl, 2004). To determine the volatile compounds in the wine, different analytical techniques were used: discontinuous or continuous liquid-liquid extraction (LLE) (Andujar-Ortiz et al., 2009), solid phase extraction (SPE) (García-Carpintero et al., 2012a; García-Carpintero et al., 2014), solid phase microextraction (SPME) (Revi et al., 2014) and stir bar sorptive extraction (SBSE) (Martínez-Gil et al., 2012; Kosmerl and Zlatic et al., 2009). It should be mentioned that the LLE technique is slowly being replaced by more manageable and solvent-free techniques. However, this type of extraction is still a reference technique used for wine aromatic compounds extraction. The main advantages of this technique are its capacity to extract a wide range of compounds of different volatilities (as long as they have an affinity to the solvent), its high repeatability level and the possibility of carrying out simultaneous extractions (Andujar-Ortiz et al., 2009). For determination, gas chromatography (GC) was used, coupled to a flame ionization detector (FID) (Pino and Queris, 2011; Moreno-Pérez et al., 2013) or a mass spectrometer (MS) (Callejón et al., 2009; Pino and Queris, 2011). The mass spectrometer is the most widely used, because it enables unequivocal qualitative and quantitative detection of substances. Quantitative detrermination of the impact of aromatic compounds on the overall aroma of wine can be done by calculating the odor activity value (OAV) and relative odour contribution (ROC). On the other hand, a qualitative evaluation can be done based on the odor descriptors of each component e.g., floral, fruity, green, solvent, plastic, toasted and others (Welke et al., 2014). The aims of the present work are to present a volatile profile of Teran PTP wine produced in the Primorska winegrowing region (Kras district) in vintages 2011, 2012 and 2013 and a differentiation of an obtained volatile profile from other red wines. We focused our research on the different groups of volatile compounds formed during alcoholic fermentation together with C6 compounds deriving from the grapes. Some wine physico-chemical characteristics were also determined for all samples and presented in the article. 2 MATERIALS AND METHODS 2.1 Samples The Teran PTP wine samples were collected from the winegrowing district Kras wine producers directly from stainless steel tanks and/or wooden barrels. During a three-year monitoring period, 82 wines were sampled from different producers; 39 samples from the 2011 vintage, 22 samples from the 2012 vintage and 21 samples from the 2013 vintage. The wines were sampled 9 months after fermentation, after the completion of malolactic fermentation and before bottling. Analyses of the volatile compounds and standard wine parameters were performed one month after sampling at the Central Laboratories of the Agricultural Institute of Slovenia. 2.2 Materials used for determining the volatile compounds The standard volatile compounds, with the highest available purity (minimum of 98 %), were obtained from Merck, Sigma Aldrich, Fluka and SAFC. Stock solutions of each of the standard volatile compounds in pure dichloromethane were prepared with contents ranging from 1.8 to 2.5 g l-1. From the stock solutions, one mixed solution of all the minor volatile compounds was prepared. The final standards were prepared with proper dilutions from this flask. Internal standard 4-nonanol (0.12 g dissolved in 100 ml dichloromethane) was added using a 0.05 ml Hamilton syringe to 10 ml of dichloromethane standard solution and mixed. 2.3 Extraction and determination of volatile compounds For the extraction of minor volatile compounds (^g l-1), discontinuous liquid-liquid extraction (LLE) with dichloromethane (Sigma-Aldrich) was used. The wine (100 ml) was then transferred into a 250 ml Erlenmeyer flask. To this, 23 ^g of 4-nonanol was added as an internal standard, using a 0.05 ml Hamilton syringe from the corresponding ethanol solution. Dichloromethane (40 ml) was added and the mixture was stirred at 350 rpm for 20 min. Then the mixture was centrifuged (8500 g, 10 min) and the organic phase was recovered. The aqueous phase was re-extracted twice, using the same method. Finally, the organic phases were combined and dried over sodium sulphate. They were concentrated to a final volume of 1 ml with a rotary evaporator and nitrogen gas flow prior to GC-MS analysis. This method is described in detail in other articles (Bavčar et al., 2011a; Bavčar et al., 2011b; Bavčar and Baša Česnik, 2011). 2.4 Calculation of odour activity value and relative odour contribution The OAV is a quantitative approach for determining the impact that volatile compounds have on the aroma of wine. The OAV is obtained from the ratio between the content of an individual compound and its perception threshold. A volatile compound contributes to aroma when its content in wine is above the perception threshold. Therefore, only compounds with an OAV>1 can be perceived. (Pino and Queris, 2011; Welke et al., 2014). Another quantitative factor is the relative odor contribution (ROC), which is the percentage of the impact of a particular aroma compound. It is the ratio of the OAV percentage of each individual compound and the sum of the OAV of compounds that showed OAV>1 (Welke et al., 2014). 2.5 Statistical analysis Data were collected and edited using Excel (Microsoft Office Professional Plus 2010) and analysis of variance (one-way ANOVA) was performed on physico-chemical characteristics and aromatic compounds data using Statgraphics® Centurion XVI statistical software package (StatPoint Technologies). 3 RESULTS AND DISCUSSION Over a three-year period, we also monitored the basic parameters of Teran PTP wine. The results of the wine physico-chemical characteristics were obtained by adhering to standard EEC methods (European Union, 1990). These results are presented in Table 1. Most of statistically different physico-chemical characteristics were found in wines from 2011 vintage. But differences between the 2011, 2012 and 2013 vintages are not extensive and most probably caused only by different climatic conditions (Ribereau Gayon et al., 2006; Vodopivec, 1999). We can confirm that Teran PTP wines contain moderate levels of alcohol (12 vol %), are high in total dry extract and acidity levels, and have surprisingly low contents of both free and total sulphur dioxide in correlation with regulation's demands (Pravilnik, 2004). Table 1: Average physico-chemical characteristics of Teran PTP wines for the 2011, 2012 and 2013 vintages Preglednica 1: Povprečne fizikalno-kemijske značilnosti vin Teran PTP letnikov 2011, 2012 in 2013 Vintage 2011 Vintage 2012 Vintage 2013 n=39 n=22 n=21 Alcohol (vol. %) 12.01±0.60 A 11.95±0.58 A 12.06±0.46 A Extract (g l-1) 30.0±2.4 B 27.3±1.7 A 27.1±2.6 A Total acidity (g l-1 as tartaric) 7.5±0.7 A 8.0±0.8 B 7.5±0.8 AB Volatile acids (g l-1as acetic) 0.62±0.17 B 0.45±0.11 A 0.73±0.13 C Free SO2 (mg l-1) 13±3 B 12±1 A 12±4 A Total SO2 (mg l-1) 43±6 B 40±9 B 35±7 A pH 3.37±13 B 3.26±0.12 A 3.33±0.14 AB Relative density 0.9958±0.0001 B 0.9948±0.0007 A 0.9946±0.0008 A Lactic acid (g l-1) 2.1±0.4 B 1.5±0.4 A 2.5±0.6 C Reducing sugar (g l-1) 2.5±0.7 B 1.2±0.3 A 1.3±0.7 A n = number of samples all data present mean value ± standard deviation significant differences between vintages are indicated A, B, C atp <0.05 The focus of our research was to determine the presence of certain volatile compounds in Teran PTP wines from the 2011, 2012 and 2013 vintages. The results are presented in Table 2. We found that Teran PTP wine contains high amounts of 1-hexanol (the mean value of the three vintages was 1292 pg l-1) and y-butyrolactone (the mean value of the three vintages was 12920 pg l-1). We also found significant amounts of 2-phenyl-ethyl-acetate (the mean value of the three vintages was 49 pg l-1), isoamyl acetate, benzaldehyde, benzyl alcohol, c7's-3-hexen-1-ol, ethyl butyrate, ethyl decanoate, ethyl dodecanoate, ethyl hexadecanoate, ethyl hexanoate and ethyl octanoate. Table 2: Contents (^g l-1) of volatile compounds in Teran PTP wines from years 2011, 2012 and 2013 and their threshold values according to ((a) Li et al., 2008; (b) Duarte et al., 2010; (c) García-Carpintero et al., 2012a; (d) Rocha et al., 2004, 2005, (e) Sánchez-Palomo et al., 2012; (f) García-Carpintero et al., 2014; (g) Welke et al., 2014) Preglednica 2: Vsebnosti (^g l-1) hlapnih spojin v vinih Teran PTP letnikov 2011, 2012 in 2013 in njihovi pragovi zaznave kot navedeno v ((a) Li et al., 2008; (b) Duarte et al., 2010; (c) García-Carpintero et al., 2012a; (d) Rocha et al., 2004, 2005, (e) Sánchez-Palomo et al., 2012; (f) García-Carpintero et al., 2014; (g) Welke et al., 2014) Vintage 2011 Vinta ge 2012 Vintage 2013 Sensory treshold min - max average±std min - max average±std min - max average±std ALDEHYDES n-Hexaldehide (Capronaldehide) n.d. n.d. n.d. n.d. n.d. n.d. Benzaldehyde n.d. - 91 8±18 A 3.0.CO;2-Q Fornasario S., Tramer F., Žiberna L., Passamonti S. 2012. Biološka uporabnost in aktivnost pigmentov grozdja pri živalih: implikacije za zdravje ljudi, Simpozij AGROTUR, Ljubljana, 28. November 2012, pp. 7-13 García-Carpintero E. G., Gómez Gallego M. A., Sánchez-Palomo E., González Viñas M. A. 2012a. Impact of alternative technique to ageing using oak chips in alcoholic or in malolactic fermentation on volatile and sensory composition of red wines. Food Chemistry, 134: 851-863, DOI: 10.1016/j.foodchem.2012.02.194 García-Carpintero E.G., Sánchez-Palomo E., Gómez Gallego M. A., González-Viñas M. A. 2012b. Free and bound volatile compounds as markers of aromatic typicalness of Moravia Dulce, Rojal and Tortosí red wines. Food Chemistry, 131: 90-98, DOI: 10.1016/j.foodchem.2011.08.035 García-Carpintero E. G., Sánchez-Palomo E., Oliveria González Viñas M. A. 2014. Volatile composition of Bobal red wines subjected to alcoholic/malolactic fermentation with oak chips. Food Science and Technology, 55: 586-594, DOI: 10.1016/j.lwt.2013.10.024 Imre S. P., Kilmartin P. A., Rutan T., Mauk J. L., Nicolau L. 2012. Influence of soil geochemistry on the chemical and aroma profiles of Pinot noir wines. Journal of Food, Agriculture and Environment, 10: 280-288 Li H., Tao Y. S., Wang H., Zhang L. 2008. Impact odorants of Chardonnay dry white wine from Changli County (China). European Food Research and Technology, 227: 287-292, DOI: 10.1007/s00217-007-0722-9 Kosmerl T., Zlatic E. 2009. Determination of 2-aminoacetophenone in wines using the stir bar sorptive extraction method coupled with GC-MS and GC-NPD. Mitteilungen Klosterneuburg, 59: 121-126 Martínez-Gil A. M., Garde-Cerdán T., Zalacain A., Pardo-García A. I., Rosario Salinas M. 2012. Applications of an oak extract on Petit Verdot grapevines. Influence on grape and wine volatile compounds. Food Chemistry, 132: 1836-1845, DOI: 10.1016/j.foodchem.2011.12.016 Moreno-Pérez A., Vila-López R., Fernández-Fernández J. I., Martínez-Cutillas A., Gil-Muñoz R. 2013. Influence of cold pre-fermentation treatments on the major volatile compounds of three wine varieties. Food Chemistry, 139: 770-776,DOI: 10.1016/j.foodchem.2013.01.052 Novak S. 2011. The influence of oenological tannins on quality of wine Teran PTP. Graduathion thesis, Biotehnical Faculty, Ljubljana 2011, 65 str. Pino J. A., Queris O. 2011. Analysis of volatile compounds of mango wine. Food Chemistry, 125: 1141-1146, DOI: 10.1016/j.foodchem.2010.09.056 Pravilnik o vinu z oznako priznanega tradicionalnega poimenovanja - teran, Uradni list Republike Slovenije 16/2008 z dne 15.2.2008, str 1166 Ramey D., Ough C.S. 1980. Volatile ester hydrolysis or formation during storage of model solutions and wines. Journal of Agricultural and Food Chemistry, 28, 5:28-934; DOI: 10.1021/jf60231a021 Revi M., Badeka A., Kontakos S., Kontominas M.G. 2014. Effect of packaging material on enological parameters and volatile compounds of dry white wine. Food Chemistry, 152: 331-339, DOI: 10.1016/j.foodchem.2013.11.136 Ribéreau Gayon, P., Dubourdieu, D., Doneche, B., Lonvaud, A. 2006 (2nd ed). Handbook of Enology. Volume 1. The Microbiology of Wine and Vinifications. John Wiley & Sons, Chichester Rocha S.M., Coutinho P., Delgadillo I., Cardoso A.D., Coimbra M. A. 2005. Effect of enzymatic aroma release on the volatile compounds of white wines presenting different aroma potentials. Journal of the Science of Food and Agriculture, 85, 2: 199-205; DOI: 10.1002/jsfa.1937 Rocha S.M., Rodrigues F., Coutinho P., Delgadillo I., Cardoso A.D., Coimbra M. A. 2004. Volatile composition of Baga red wine. Assessment of the identification of the would-be impact odorants. Analytica Chimica Acta, 513, 1: 257-262; DOI: 10.1016/j.aca.2003.10.009 Sánchez-Palomo E., Gómez García-Carpintero E., Gómez Gallego M. A., González Vinas M. A. 2012. Gas Chromatography in Plant Science, Wine Technology, Toxicology and Some Specific Applications. The Aroma of Rojal Red Wines from La ManchaRegion -Determination of Key Odorants. Salih B., Çelikbiçak O. (eds.). Published online, InTech: 147-170, DOI: 10.5772/2517 Vanzo A., Šuklje K., Jenko M., Čuš F., Bavčar D., Lisjak K. 2012. Polifenolni potencial terana. Bioaktivne spojine Terana, Simpozij AGROTUR, Ljubljana, 28. November 2012, pp. 29-50 Vodopivec M. 1999. Kraški teran, Ljubljana, ČZP Kmečki glas: 162 str. Vrščaj Vodošek T., Košmerl T. 2004. Določanje fenolnih spojin in barvnih parametrov v vinih refošk in teran različnega geografskega porekla. V: Jubilejni 10.slovenski kemijski dnevi 2004, Maribor, 23. in 24. september 2004. Maribor 1-12 Welke J. E., Zanus M., Lazzarotto M., Alcaraz Zini C. 2014. Quantitative analysis of headspace volatile compounds using comprehensive two-dimensional gas chromatography and their contribution to the aroma of Chardonnay wine. Food Research International, 59: 85-99, DOI: 10.1016/j.foodres.2014.02.002 COBISS Code 1.01 DOI: 10.14720/aas.2015.105.1.02 Agrovoc descriptors: allium, antioxidants, bulbs, antibacterial properties, antimicrobial properties, antimicrobials, phenolic compounds, phenolic content, extracts, indigenous organisms Agris category code: f60, q04 Variation in antioxidant, and antibacterial activities and total phenolic content of the bulbs of mooseer (Allium hirtifolium Boiss.) Abdollah GHASEMI PIRBALOUTI1,2, Yazdan AHMADZADEH1, Fatemeh MALEKPOOR1 Received April 24, 2014; accepted December 04, 2014. Delo je prispelo 24. aprila 2014, sprejeto 04. decembra 2014. ABSTRACT IZVLEČEK Allium hirtifolium Boiss. (mooseer) belonging to the family Alliaceae, is an endemic species of Iran which grows wild in the Zagros Mountains range, western and southwestern Iran. The bulb of A. hirtifolium has been used as a flavouring agent, especially dairy foods and pickles by the indigenous people, southwestern Iran. In this study, the bulbs of various populations of the plant were collected from the alpine regions in Chaharmahal va Bakhtiari province, Iran. The total phenolic content of the ethanol extract was determined by Folin-Ciocalteu method, the antioxidant activity was evaluated by measuring 1,1-diphenyl-2-picrylhydrazyl (DPPH), and the antibacterial activity of the extracts against four bacteria, including Bacillus cereus, Listeria monocytogenes, Proteus vulgaris, and Salmonella typhimurium was determined by serial dilution assay. Results indicated that the total phenolic content in the ethanol extracts of different populations of A. hirtifolium ranged between 34 to 44 mg gallic acid/g extract. In addition, the extracts of A. hirtifolium indicated moderate-to-good inhibitory activities (MICs = 0.062 to 0.250 mg/ml) against four bacteria, especially against B. cereus. The antioxidant activity of the bulbs of A. hirtifolium indicated the extract acted as an effective DPPH scavenger, but were not as effective as the BHT control. This finding suggests that the bulbs of A. hirtifolium may be considered as a natural source of antioxidants and antimicrobial agents. Key words: Alliaceae, biological activity; endemic herbs; mooseer SPREMENLJIVOST ANTIOKSIDACIJSKEGA IN ANTIBAKTERIJSKEGA DELOVANJA CELOKUPNIH FENOLNIH IZVLEČKOV IZ ČEBULIC PERZIJSKE ŠALOTKE (Allium hirtifolium Boiss.) Perzijska šalotka (Allium hirtifolium Boiss. (mooseer), Allium stipitatum Regel) spada v družino lukovk (Alliaceae), je endemična vrsta Irana, ki raste samoniklo v zahodnem in jugozahodnem delu države na območju gorovja Zagros. Prebivalci jugozahodnega Irana jo uporablja kot začimbo v mlečnih izdelkih in vlaganju zelenjave. V tej raziskavi so bile analizirane čebulice različnih populacij, nabrane v alpinskih predelih province Chaharmahal va Bakhtiari. Celokupna vsebnost fenolov je bila določena v etanolnem izvlečku po metodi Folin-Ciocalteu, antioksidativno delovanje je bilo ovrednoteno in izmerjeno z 1,1-difenil-2-pikrilhidrazilom (DPPH), antibakterijsko delovanje izvlečkov je bilo določeno proti štirim vrstam bakterij, Bacillus cereus, Listeria monocytogenes, Proteus vulgaris, in Salmonella typhimurium s serijskim razredčitvenim testom. Rezultati so pokazali, da so etanolni izvlečki celokupnih fenolov iz čebulic različnih populacij te vrste vsebovali od 34 do 44 mg galične kisline na g izvlečka. Izvlečki so pokazali zmerno do dobro inhibitorno aktivnost (MICs = 0.062 do 0.250 mg/ml) proti omenjenim štirim vrstam bakterij, še posebej proti vrsti B. cereus. Antioksidativno delovanje izvlečkov čebulic je pokazalo, da so izvlečki delovali kot učinkoviti lovilci DPPH, vendar so bili manj učinkoviti kot BHT v kontroli. Izsledki kažejo, da so lahko čebulice perzijske šalotke (A. hirtifolium) dober naravni vir antioksidantov in antimikrobnih snovi. Ključne besede: A. hirtifolium, Alliaceae, biološka aktivnost, antioksidant, antimikrobna snov Department of Medicinal Plants, Faculty of Agriculture and Food Science, Shahrekord Branch, Islamic Azad University, Shahrekord, 88148 (PO. Box: 166), Iran. E-mail: ghasemi@iaushk.ac.ir or aghasemipir@psis.umass.edu 2 Medicinal Plants Program, Stockbridge School of Agriculture, College of Natural Sciences, Massachusetts University, Amherst, 01003, MA, USA 1 INTRODUCTION Plant extracts are rich sources of natural antioxidant and antibacterial compounds. Phenolic compounds present in spice plants as dietary sources possess bioactive properties protecting cellular systems against oxidative stress (Ghasemi Pirbalouti et al., 2013a). Recently, interest in finding naturally occurring antioxidants to replace synthetic antioxidants in foods and medicines has increased considerably, primarily due to the possible carcinogenicity of the synthetic antioxidants (Velioglu et al., 1998). The genus of Allium L. is the largest and important representative genus of the Alliaceae family comprises 700 species; each with different tastes, forms and colors; nonetheless, they are close in biochemical, phytochemical, and nutraceutical properties (Tepe et al., 2005). Allium species are revered to possess antibacterial, antifungal, antiviral, antiprotozoal, and anthelmintic activities (Ariga and Seki, 2006; Benkeblia, 2005) and they contain the powerful antioxidants, sulphur and other numerous phenolic compounds which have aroused great interests for food industries. The Allium species have been used for a long time as a medicinal for the prevention and treatment of certain diseases such as diabetes, arthritis, colds and flu, stress, fever, coughs, headache, hemorrhoids, asthma, arteriosclerosis, cancer, respiratory, gastrointestinal, rheumatic, and inflammatory disorders (Najjaa, et al., 2009; Kojuri et al., 2007; Amin, 1991). Biological and medical functions of Allium species are due to their sulphur compounds, such as S-alk (en) yl-L'Cysteine sulfoxides (Fritsch and Keusgen, 2006), however, presence of phenolic compounds are also beneficial for human health (Corzo-Martinez et al., 2007). Mooseer (Allium hirtifolium Boiss.), is an endemic plant of Iran which wild grows in the alpine regions in Zagros Mountains range from Northwestern to Southwestern of Iran with the climate of very to moderate cold (Ghahreman, 1984; Rechinger, 1984). A. hirtifolium is a nutritive plant with special taste which its dried bulb slices are used as an additive to yogurt and also pickling mixtures, rice, meat, sauces and salads. The bulbs of A. hirtifolium have been used as a flavouring agent, especially dairy foods and pickles by the indigenous people, southwestern Iran (Ghasemi Pirbalouti, 2009). In Iranian folk medicine, mooseer has been successfully used for treating rheumatic and inflammatory disorders. In addition, different medicinal properties such as antitrichomonas, antiproliferative, and immunomodulatory activities have also been reported for the bulbs of mooseer (Mozaffarian, 2008; Ghodrati Azadi et al., 2008; Jafarian et al., 2003; Amin, 1991). Results of previous studies (Ashrafi et al., 2004; Ismail et al., 2013) indicated that the aqueous and methanol extracts of mooseer bulbs have antimicrobial properties. Mooseer (Allium hirtifolium Boiss.), is an endemic plant of Iran which wild grows in the alpine regions in Zagros Mountains range from Northwestern to Southwestern of Iran with the climate of very to moderate cold (Ghahreman, 1984; Rechinger, 1984). A. hirtifolium is a nutritive plant with special taste which its dried bulb slices are used as an additive to yogurt and also pickling mixtures, rice, meat, sauces and salads. The bulbs of A. hirtifolium have been used as a flavouring agent, especially dairy foods and pickles by the indigenous people, southwestern Iran (Ghasemi Pirbalouti, 2009). In Iranian folk medicine, mooseer has been successfully used for treating rheumatic and inflammatory disorders. In addition, different medicinal properties such as antitrichomonas, antiproliferative, and immunomodulatory activities have also been reported for the bulbs of mooseer (Mozaffarian, 2008; Ghodrati Azadi et al., 2008; Jafarian et al., 2003; Amin, 1991). Results of previous studies (Ashrafi et al., 2004; Ismail et al., 2013) indicated that the aqueous and methanol extracts of mooseer bulbs have antimicrobial properties. To our knowledge, there are no published reports on diversity of total phenolic content, antibacterial and antioxidant activities of various populations of A. hirtifolium. The main objective of this study was to evaluate content of phenolic compounds, antioxidants and antibacterial activities of the ethanol extracts from the bulbs of various populations of A. hirtifolium, and to evaluate them as potential sources of natural antioxidants and antimicrobial. 2 MATERIAL AND METHODS 2.1 Plant material The samples of the bulb of A. hirtifolium collected from wild populations of the plants growing in various alpine regions of southwestern Iran were used in this study. In total, three replicate samples of 30 plants were gathered from three natural habitats at the early flowering between April 30th to May 20th 2012. The slope and elevation information were obtained from the Digital Elevation Model (DEM) using two well-known GIS software packages ILWIS (3.0 Academic). This array was geo-referenced using a metric UTM coordinate system and the geometric correction were carried out in the GIS ILWIS (Table 1). Soil physical and chemical characteristics, including pH, electrical conductivity (EC), organic carbon (OC%), and soil texture were determined (Table 1). Climatic data of the locations were determined using data collected by the nearest meteorology stations (Table 1). Plant identity was confirmed by Prof. V. Mozaffarian, and a representative voucher specimen (No. 1265) was been placed in the Herbarium of Research Center of Natural Resources of Chaharmahal va Bakhtiari province, Shahrekord, Iran. 2.2 Extract preparation Immediately following collection, the leaves of A. hirtifolium from each plant sample were separated and bagged independently. The bulbs were cleaned with tap water and cut into small slices by using a kitchen mixer. The tissue samples were subsequently air-dried in a shaded room at 30 ± 5 °C. A 100 g sample was extracted with 250 ml ethanol (96%, Merck, Darmstadt, Germany) at 45 °C for 8 h followed by a Soxhlet apparatus. The ethanol was subsequently removed under reduced pressure on a rotary evaporator (Model Zirbus 302 W, Italy) at 40 °C. The extracts were filtered using a Whatman No. 2. The extract samples were stored in universal bottles and refrigerated at 4 °C prior to use. Table 1. Geographical and climate of natural habitats of Allium hirtifolium Region Altitude Latitude Longitude P* T pH E.C. O.C Sand Silt Clay (m) (UTM) (UTM) (mm) (°C) (dS/m) (%) (%) (%) (%) Samsami 2742 0435278 3565206 779.9 12.6 6.85 0.528 1.931 26 36 38 Khaki 2487 0448579 3587078 327.3 10.6 7.5 0.442 1.541 20 42 38 Dasht-e-Laleh 2336 0428599 3599942 1025.1 9.7 7.23 0.348 1.117 32 32 36 * P: Annual precipitation (mm), T: Average temperature (°C), E.C.: Electrical conductivity (dS/m), O.C.: Organic carbon (%). Meteorological information was obtained from weather stations located within the study area and the surrounding zone; each value in the mean of 10 to 15 year data. Soil characteristics are based on average of samples taken from three farms in each region. 2.3 Determination of total phenolic content (TPC) The total amount of phenolic compounds in each extract was determined using the Folin-Ciocalteu method following procedure of Singleton and Rossi (1965) with some modifications. Briefly, 0.5 ml of the sample was mixed with 2.5 ml of Folin- Ciocalteu's (Sigma-Aldrich Co., Steineheim, Germany) phenol reagent for 5 min at 37 °C, 2 ml of saturated ^COs (7.5%) (Merck Co., Darmstadt, Germany) was added, and the mixture was brought to 10 ml with the addition of deionized, distilled water. The mixture was maintained at room temperature in the dark for 120 min and then the absorbance was measured at 765 nm against a reagent blank using a Perkin-Elmer Lambda UV/Vis spectrophotometer. Gallic acid (Merck Co., Darmstadt, Germany) was used as the reference standard and the total phenolic content was expressed as mg of gallic acid equivalents per gram of each extract on dry basis (mg GAE/g extract). 2.4 Antioxidant test The DPPH radical scavenging activity of the ethanol extract was determined using the method proposed by Hung et al. (2005). The extracts (100 (L) at concentrations of 8, 16, 32, 62.5, 125, 250, and 500 (ig/ml were mixed with 3.9 mL an equal volume of 0.2 mM ethanol solution of DPPH (Sigma-Aldrich Co., Steineheim, Germany). The disappearance of the DPPH after 30 min of incubation at room temperature was determined using a Perkin-Elmer Lambda UV/Vis spectrophotometer at 515 nm against a blank, i.e. without DPPH. Ethanol was used to zero the spectrophotometer and the absorbance of the DPPH radical without antioxidant and measure daily served as the control. The amount of sample necessary to decrease the absorbance of DPPH by 50% (IC50) was calculated graphically and the percentage inhibition was determined according to the equation: % inhibition= IACCGJ- Aàte" « 100 where AC0 is the absorbance of the control at t = 0 min and AAt is the absorbance of the antioxidant at t = 30 min. The food preservative butylated hydroxytoluene (BHT) was used as positive control. All measurements were replicated three times. 2.5 Antibacterial test Antibacterial activity of the extracts were tested using clinical isolates of four bacteria strains, the Gram-positive bacteria (Bacillus cereus and Listeria monocytogenes) and the Gram-negative bacteria (Proteus vulgaris and Salmonella typhimurium). The bacteria, originally obtained from chicken meat samples, were provided by the Food Microbiology Laboratory, Veterinary Medicine Faculty, (I.A.U.) Iran and had been positively identified using PCR-RFLP along with conventional morphological and biochemical tests. The population of each bacterial strain was increased by culturing in an overnight Mueller Hinton broth (MHB) at 37 °C. To quantify the antibacterial activity of the extracts, bacteria populations were prepared for testing by adjusting each population to 1.0 McFarland standards (1.0 x 107 CFU/mL), using a spectrophotometer (Perkin-Elmer Lambda UV/Vis, USA). Minimum inhibitory concentrations (MIC) were determined using the broth-serial dilution method following standardized methods (CLSI, 2012). The extracts and the antimicrobial agents (ciprofloxacin, and flumequine) were each dissolved in 5 % dimethyl sulfoxide (DMSO) and then diluted to the highest test concentration (500 (g/mL). Subsequent test concentrations were made in a series of two-fold dilutions to develop concentration levels of 8 to 500 ( g/ml in sterile, 10 ml test tubes containing MHB. A population of bacteria was subsequently added to each tube containing an essential oil or antimicrobial agent and then incubated at 37 °C for 48 h. After the incubation period, the absorbance of each incubated solution was measured at 630 nm using a spectrophotometer (Perkin-Elmer Lambda UV/Vis, USA) as a measure of bacterial growth to indicate MIC values. The minimum bactericidal concentration (MBC) of each essential oil was determined according to the MIC values by transferring 5 ( L from MIC tubes to agar plates and incubating at 37 °C for 48 h. The MBC was recorded as the minimum concentration of extract in which no viable bacterial growth was observed. All experimental tests were replicated three different times. 2.6 Statistical analysis Data were analyzed by one-way analysis of variance with three replications using the SPSS 19.0 statistical software. Means were compared with Duncan test atp < 0.05 level. 3 RESULTS AND DISCUSSION 3.1 Extraction yield The color of the ethanol extract from the bulbs of A. hirtifolium was light yellow. Statistical analysis indicated that there was significant difference (p < 0.05) among various populations for extract yield (Table 2). The highest extract yield was obtained from the Samsami population with 14.6% w/w on dry weight basis (Table 2). The lowest value of extract yield was obtained from the bulbs of A. hirtifolium collected in Koohrang population with 8.2% w/w on dry weight basis (Table 2). An earlier study by Jafarian et al. (2003) reported the hydroalcohol extract yield from the bulbs of A. bulbs was 51.9 g extract obtained from 100 g hirtifolium collected from Khansar (Isfahan), Iran powder by polyphenolic fraction method. A was 34% using percolation method. In addition, comparison of our results with the previous reports results of a study by Kazemi et al. (2010) indicated suggests differences in the extract yield of the plant the hydroalcohol extract yield from A. hirtifolium material could be attributed to extraction methods. Table 2: Extract yield, antioxidant activity, and total phenolic content of the ethanol extracts from the bulbs of Allium hirtifolium Species Part used Populations Extract yield (% w/w) Total phenolic (mg GAE /g extract) IC50 (mg/g) A. hirtifolium Bulb Samsami 14.58 ± 3.94 a 38.11 ± 5.06 bc 3.09 ± 0.65 c A. hirtifolium Bulb Khaki 11.12 ± 0.75 b 34.50 ± 4.12 c 2.51 ± 0.61 bc A. hirtifolium Bulb Dasht-e-Laleh 8.17 ± 1.36 bc 44.28 ± 6.58 a 1.90 ± 0.31 b BHT - - - - 0.21 ± 0.03 a ANOVA p < 0.05 p < 0.05 p < 0.01 fValues in column having similar letter are not statistically different atp < 0.05 3.2 Total phenolic contents In present study, total phenolic content in each extract was determined spectrometrically according to the Folin-Ciocalteu method and calculated as gallic acid equivalent (GAE). A significant difference (p < 0.05) for total phenolic content was measured among the extracts. The maximum total phenolic content was obtained from the extract of the Dasht-e-Laleh population with 44.28 ± 6.58 mg GAE/g extract (Table 2). Results of an earlier study by Ghahremani-majd et al. (2012) indicated that the total phenolic content in the methanol extracts from the bulbs of A. hirtifolium populations ranged from 8.4 to 0.5 mg GAE/g sample. Results of a study by Parakesh et al. (2007) indicted that total phenolic contents in the extracts from four (red, violet, white and green) varieties of Allium cepa varied from 4.6 to 74.1 mg/g GAE. Within the vegetable family, the composition and quantity of the phenolic are vary significantly according to different intrinsic and extrinsic factors, such as plant genetics and cultivar, soil and growing conditions, maturity state and harvest conditions (Jaffery et al., 2003). 3.3 Antioxidant test Antioxidant properties are very important in counteracting the deleterious role of free radicals in food and biological systems. In our study, the antioxidant activity of the extract from the various populations of A. hirtifolium was expressed as IC50 with values from 1.90 to 3.09 mg/ml that indicating the extracts act as moderate to good DPPH scavenger (Table 2). Significant difference (p < 0.01) in IC50 values were found for the extracts and control (BHT). The extract from the Dasht-e-Laleh population with the highest total phenolic content showed the highest antioxidant activity. Ghahremani-majd et al. (2012) have observed a linear response between total phenolic and antioxidant capacity of the extracts from A. hirtifolium bulbs in FRAP, ABTS, and DPPH assays. The antioxidant activity of Alliums species was reported by numerous investigators (Velioglu et al., 1998). 3.4 Antibacterial test The antibacterial activity of the extract from the various populations of A.hirtifolium was tested against the four pathogenic bacteria (Bacillus cereus, Listeria monocytogenes, Proteus vulgaris, and Salmonella typhimurium) by using the serial-dilution method. Extracts demonstrated relatively inhibitory activities against the pathogenic bacteria tested, the MICs and MBCs of the tested samples are presented in Table 3. Results of present study indicated that the different bacteria species demonstrated different levels of sensitivity to the extracts. The MICs of the extracts were within concentration ranges from 0.062 to 0.25 mg/ml, and the respective MBCs were from 0.125 to > 0.50 mg/ml. Generally, the ethanol extracts from the bulbs of A. hirtifolium indicated moderate to good inhibitory activities against four bacteria. The highest antibacterial activity was obtained from the extracts of the bulbs of the Dasht-e-Laleh and Samsami populations against Listeria monocytogenes and Bacillus cereus, respectively. Similarly, results obtained from the measurements of MICs in a study by Ghahremani-majd et al. (2012) showed that B. subtilis was the most sensitive microorganism tested to the extracts from the bulbs of A. hirtifolium with the lowest MIC values from 1.87 to 15 mg/ml. In addition, they reported the methanol extract from the bulbs of the Isfahan mooseer population had the highest antibacterial and antifungal activities against six bacteria and two fungi. Probably, in present study the phenolic compounds are responsible of the antibacterial activity of the extracts from the bulbs of A. hirtifolium. In other study (Amin and Kapadnis, 2005), the extract of bulbs of A. hirtifolium had the high antimicrobial activity against wide range of pathogenic and nonpathogenic bacteria and fungi with the MIC values from 0.001 to 0.010 mg/ml . Table 3: Antibacterial activity (MICs and MBCs) of the ethanol extracts from the bulbs of Allium hirtifolium against four bacteria Species / Part populations Bacillus cereus Listeria monocytogenes Proteus vulgaris Salmonella typhimurium Antibiotics used MIC MBC MIC MBC MIC MBC MIC MBC Og/ml) Og/ml) Og/ml) Og/ml) Og/ml) Og/ml) Og/ml) Og/ml) A. hirtifolium Bulb Samsami 62.5 125 125 500 125 500 125 250 A. hirtifolium Bulb Khaki 125 250 250 500 125 250 125 250 A. hirtifolium Bulb Dasht-e-Laleh 62.5 250 125 250 125 250 125 250 Ciprofloxacin - - 32.2 125 32.2 62.5 62.5 125 62.5 125 Ampicillin - - 62.5 125 62.5 125 125 250 125 250 The mechanisms by which plant extracts can inhibit microorganisms vary (Ahmad and Beg, 2001; Rodriguez et al., 2009; Thormar, 2011). Phenolic compounds can act at two different levels: the cell membrane and cell wall of the microorganisms (Taguri et al., 2006). They can interact with the membrane proteins of bacteria by means of hydrogen bonding through their hydroxyl groups which can result in changes in membrane permeability and cause cell destruction (Ghasemi Pirbalouti et al., 2013b). 4 CONCLUSION The present study is apparently the first report of quantitative total phenol profile, antioxidant and antibacterial activities of the ethanol extracts from the bulbs of A. hirtifolium collected from southwestern Iran. The results of current study demonstrated that the ethanol extract from some populations of A. hirtifolium with the maximum total phenolic content had the highest antioxidant activity by the DPPH assay. Total phenolic compounds present in the plant are responsible for its effective free radical scavenging, antioxidant and antimicrobial activities. In total, significant antioxidant and antibacterial activities of the extract of the studied herb provide a scientific validation for the traditional use of the plant as an accessible source of natural antioxidants and antimicrobial with consequent health benefits. 5 ACKNOWLEDGMENT Authors would like to acknowledge the Deputy of Research and Technology of Shahrekord Branch, I.A.U., Iran for supporting this study. 6 REFERENCES Ahmad, I., Beg, A.Z. 2001. Antimicrobial and phytochemical studies on 45 Indian medicinal plants against multi-drug resistant human pathogens. Journal of Ethnopharmacology, 74: 113-123; DOI: 10.1016/S0378-8741(00)00335-4 Amin, G.R. 1991. Popular medicinal plants of Iran, Tehran: Iranian Ministry of Health publication, p. 37. Ariga, T., Seki, T. 2006. Antithrombotic and anticancer effects of garlic-derived sulfur compounds: a review, BioFactors, 26: 93-103; DOI: 10.1002/biof.5520260201 Ashrafi, F., Akhavan Sepahi, A., Kazemzadeh, A. 2004. Effect of aqueous extract of shallot (Allium ascalonicum) on inhibition of growth of Pseudomonas aeroginosa. Iranian Journal of Pharmaceutical Research, 2: 71-71. Benkeblia, N. 2005. Free-radical scavenging capacity and antioxidant properties of some selected onions (Allium cepa L.) and garlic (Allium sativum L.) extracts. Brazilian Archives of Biology and Technology, 48: 753-759; DOI: 10.1590/S1516-89132005000600011 CLSI (Clinical and Laboratory Standards Institute). 2012. Performance standards for antimicrobial disks susceptibility tests: approved standards -Eleventh edition. M02-A11, CLSI, USA. Corzo-Martinez, M., Corzo, N., Villamiel, M. 2007. Biological properties of onion and garlic. Trends in Food Science Technology, 18: 609-625; DOI: 10.1016/j.tifs.2007.07.011 Fritsch, R.M., Keusgen, M. 2006. Occurrence and taxonomic significance of cysteine sulphoxides in the genus Allium L. (Alliaceae). Phytochemistry, 67: 1127-1135; DOI: 10.1016/j.phytochem.2006.03.006 Ghahremani-majd, H., Dashti, F., Dastan, D., Mumivand, H., Hadian, J., Esna-Ashari, M. 2012. Antioxidant and antimicrobial activities of Iranian mooseer (Allium hirtifolium Boiss) populations. Horticulture, Environment, and Biotechnology, 53: 116-122; DOI: 10.1007/s13580-012-0131-2 Ghasemi Pirbalouti, A. 2009. Medicinal plants used in Chaharmahal and Bakhtyari districts, Iran. Herba Polonica, 55: 69-75. Ghasemi Pirbalouti, A., Setayesh, M., Siahpoosh, A., Mashayekhi, H. 2013a. Antioxidant activity, total phenolic and flavonoids contents of three herbs used as condiments and additives in pickles products. Herba Polonica, 59: 51-62. Ghasemi Pirbalouti, A., Fatahi-Vanani, M., Craker, L., Shirmardi, H. 2013b. Chemical composition and bioactivity of essential oils of Hypericum helianthemoides. Hypericum perforatum and Hypericum scabrum. Pharmaceutical Biology, 52(2): 175-181; DOI: 10.3109/13880209.2013.821663 Ghahreman, A., 1984. Color Atlas of Iranian Plants. Institute of Forestries and Grasslands, Botany Division, No. 5, 512 pp. Ghodrati Azadi, H., Ghaffari, S.M., Riazi, G.H., Ahmadian, S., Vahedi, F. 2008. Antiproliferative activity of chloroformic extract of Persian Shallot, Allium hirtifolium, on tumor cell lines. Cytotechnology, 56: 179-185; DOI: 10.1007/s10616-008-9145-0 Hung, D., Ou, B., Prior, R.L. 2005. The chemistry behind antioxidant capacity assay. J. Journal of Agricultural and Food Chemistry, 53: 1841-1856; DOI: 10.1021/jf030723c Jafarian, A., Ghannadi, A., Elyasi, A. 2003. The effects of Allium hirtifolium Boiss on cell-mediated immune response in mice. Iranian Journal of Pharmaceutical Research, 2: 51-55. Jaffery, E.H., Brown, A.F., Kurilich, A.C., Keek, A.S., Matusheski, N., Klein, B.P. 2003. Variation in content of bioactive components in broccoli. Journal of Food Composition and Analysis, 16: 323-330; DOI: 10.1016/S0889-1575(03)00045-0 Ismail, S., Jalilian, F. A., Talebpour, A. H., Zargar, M., Shameli, K., Sekawi, Z., Jahanshiri, F. 2013. Chemical composition and antibacterial and cytotoxic activities of Allium hirtifolium boiss. BioMed Research International, DOI: 10.1155/2013/ 696835. Kazemi, S., Asgary, S., Moshtaghian, J., Rafieian, M., Adelnia, A., Shamsi, F. 2010. Liver-protective effects of hydroalcoholic extract of Allium hirtifolium boiss. In rats with alloxan-induced diabetes mellitus. ARYA Atheroscler, 6: 11-15. Kojuri, J., Vosoughi, A. R., Akrami, M. 2007. Effects of Anethum graveolens and garlic on lipid profile in hyperlipidemic patients. Lipids Health Disease, 6: 1- 5; DOI: 10.1186/1476-511X-6-5 Mozaffarian, V. 2008. A pictorial dictionary of medicinal plants. Latin-English-French-Germany-Persian/Complied, Farahang Moaser, Tehran, pp. 522. Najjaa, H., Ammar, E., Neffati, M. 2009. Antimicrobial activities of protenic extracts of Allium roseum L., a wild edible species in North Africa. Journal of Food Agriculture and Environment, 7: 150-154. Prakash, D., Singh, B. N., Upadhyay, G. 2007. Antioxidant and free radical scavenging activities of phenols from onion (Allium cepa). Food Chemistry 102(4): 1389-1393; DOI: 10.1016/j.foodchem.2006.06.063 Rechinger, K.H. 1984. Flora Iranica, Alliaceae, vol. 76. Akademische Druck, Univ. Verlagsanstalt Graz, Austria, 85 pp. Rodriguez, H., Curiel, J.A., Landete, J.M., De las Rivas, B., De Felipe, F.L., Gomez- Cordoves, C., Mancheno, J.M., Munoz, R. 2009. Food phenolics and lactic acid bacteria. International Journal of Food Microbiology, 132: 79-90; DOI: 10.1016/j.ijfoodmicro.2009.03.025 Singleton, V.L., Rossi, J.A. 1965. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. American Society for Enology and Viticulture, 16: 144-158. Taguri, T., Tanaka, T., Kouno, I. 2006. Antibacterial spectrum of plant polyphenols and extracts depending upon hydroxyphenyl structure. Biological & Pharmaceutical Bulletin, 29: 22262235; DOI: 10.1248/bpb.29.2226 Tepe, B., Sokmen, M., Akpulat, H.A., Sokmen, A. 2005. In vitro antioxidant activities of the methanol extracts of five Allium species from Turkey. Food Chemistry, 92: 89-92; DOI: 10.1016/j .foodchem.2004.07.016 Thormar H. 2011. Lipids and essential oils as antimicrobial agents. John Wiley & Sons, Ltd, Chichester, UK. DOI: 10.1002/9780470976623. Velioglu, Y. S., Mazza, G., Gao, L., Oomah, B.D. 1998. Antioxidant activity and total phenolics in selected fruits, vegetables and grain products. Journal of Agricultural and Food Chemistry, 46: 4113-4117; DOI: 10.1021/jf9801973 COBISS Code 1.01 DOI: 10.14720/aas.2015.105.1.03 Agrovoc descriptors: hyoscyamus niger, alcaloids, antioxidants, particle size, dimensions, crop yield, enzymes, biochemistry Agris category code: f60 Activating antioxidant enzymes, hyoscyamine and scopolamine biosynthesis of Hyoscyamus niger L. plants with nano-sized titanium dioxide and bulk application Mansour GHORBANPOUR1*, Mehrnaz HATAMI1, Mahmoud HATAMI2 Received August 27, 2014; accepted October 13, 2014. Delo je prispelo 27. avgusta 2014, sprejeto 13. oktobra 2014. ABSTRACT Application of nanotechnology is now widely distributed overall the life, especially in agricultural systems. This study intended to indicate the impacts of nano-sized titanium dioxide particles (NT) and bulk (BT) on antioxidant enzymes activities including superoxide dismutase (SOD), peroxidase (POX) and catalase (CAT), and variations of two major tropane alkaloids such as hyoscyamine (HYO) and scopolamine (SCO) in Hyoscyamus niger L. Plants were treated with different concentrations of NT and BT (0, 20, 40 and 80 mg l-1). Alkaloids extracted were identified by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) analysis. Results showed that SOD activity increased with increasing titanium dioxide concentration in both nano-particles and bulk treated plants. However, the highest and the lowest POX activity were observed in plants exposed to NT at 40 mg l-1 and control, respectively. Generally, all tested enzymes activities were higher in NT treated plants that those of BT except CAT activity at 80 mg l-1. The highest alkaloids content values, HYO: 0.286 g kg-1 and SCO: 0.126 g kg1, were achieved in plants treated with NT at 80 and 20 mg l-1, respectively. The maximum and minimum plant biomass and subsequently total alkaloids yield were obtained in plants exposed to NT at 40 mg l-1 and controls, respectively. Our results suggest that NT in appropriate level (40 mg l-1) may act as an elicitor for biochemical responses and tropane alkaloids biosynthesis in H. niger plants. Key words: black henbane, tropane alkaloids, antioxidant enzymes, nano-anatase TiO2 IZVLEČEK AKTIVIRANJE AKTIVNOSTI ANTIOKSIDACIJSKIH ENCIMOV, BIOSINTEZE HIOSCIAMINA IN SKOPOLAMINA PRI ČRNEM ZOBNIKU (Hyoscyamus niger L.) Z NANO IN CELOKUPNIMI DELCI TITANOVEGA DIOKSIDA Uporaba nanotehnologije je v svetu danes zelo razširjena v znanostih o življenju, še posebej v kmetijstvu. V raziskavi je bil preučevan vpliv nano delcev (NT) in celukopnih delcev (BT) titanovega dioksida na antioksidacijsko aktivnost encimov kot so superoksid dismutaza (SOD), peroksidaza (POX) in katalaza (CAT) in vpliv tega obravnavanja na variabilnost vsebnosti dveh glavnih tropanskih alkaloidov, hiosciamina (HYO) in skopolamina (SCO) v črnem zobniku (Hyoscyamus niger L.). Rastline so bile tretirane z naslednjimi koncentracijami NT in BT delcev: 0, 20, 40 and 80 mg l-1. Ekstrahirani alkaloidi so bili analizirani in določeni s plinsko kromatografijo (GC) in plinsko kromatografijo povezano z masno spektroskopijo (GC-MS). Rezultati so pokazali, da se je aktivnost SOD povečala pri tretmajih z NT in BT delci z naraščanjem njihove koncentracije. Aktivnost POX pa je bila največja pri rastlinah izpostavljenih NT delcem pri 40 mg l-1 in najmanjša pri kontroli. Nasplošno so bile aktivnosti vseh testiranih encimov večje pri rastlinah tretiranih z NT delci kot pri tretmaju z BT delci, razen aktivnosti CAT pri tretmaju z 80 mg l-1. Največji vsebnosti alkaloidov, HYO: 0.286 g kg-1 in SCO: 0.126 g kg-1, sta bili doseženi pri rastlinah tretiranih z NT delci pri koncentracijah 80 in 20 mg l-1. Največja biomasa in največji pridelek alkaloidov sta bila dosežena pri rastlinah tretiranih z NT pri 40 mg l-1 in najmanjša pri kontroli. Rezultati kažejo, da NT delci v primernih koncentracijah (40 mg l-1) delujejo kot elicitorji za biokemične odzive in biosintezo tropanskih alkaloidov pri črnem zobniku. Ključne besede: črni zobnik, tropanski aklaloidi, antioksidacijski encimi, nano-delci TiO2 1 Department of Medicinal Plants, Faculty of Agriculture and Natural Resources, Arak University, Arak, Iran; * corresponding author: email: m_ghorbanpour@yahoo.com 2 Iran Technical and Vocational Training Organization 1 INTRODUCTION Alkaloids are a diverse group of low-molecular-weight, nitrogen-containing compounds found in about 20% of plant species. Solanaceous plants are regarded as rich sources of alkaloids, namely the pharmaceutical by interesting tropane derivatives. Tropane alkaloids, especially hyoscyamine (HYO) and scopolamine (SCO) are widely used in medicine for their mydriatic, antispasmodic, anticholinergic, analgesic and sedative properties (Zehra et al., 1998). SCO, which is the 6,7-epoxide of HYO, is the most valued of the two tropane alkaloids (due to fewer side effects on nervous system), its worldwide demand being 10 times higher than that for HYO and its racemic form, atropine (Hashimoto et al., 1993). The synthetic production of these alkaloids is more expensive than their extraction from plant materials and they are, therefore, currently industrially extracted from various solanaceous plants belonging to the genera Atropa, Duboisia, Datura, Scopolia and Hyoscyamus. Black henbane (Hyoscyamus niger L.) has a very long history of use as a medicinal plant. A cosmopolitan, strong-scented annual or biennial herb, which all it's parts (root, leaf, and seed) contain tropane alkaloids such as HYO and SCO (Cuneyt et al., 2004). These metabolites are synthesized in roots and then transported to the aerial parts of the plant (Oksman, 1987). It has been exhibited that signal molecules are very potential elicitors for induction of plant secondary metabolites. Recent years, the applications of signal components as elicitors have evolved an effective strategy for the production of target secondary metabolites in plant cell cultures. However, it is still uncommon for commercial application. It therefore, application of elicitors in vivo is an easy and direct channel to promote the yield of plant secondary metabolites at the whole plant scale. Nanonmaterials could act as signal compounds to make metabolic and physiological responses but the underlying mechanisms are not fully understood (Hatami and Ghorbanpour, 2014). The development of nanotechnology on physiology and biochemistry has expanded the application area of nanomaterials in different fields due to their unique characteristics (Scrinis and Lyons, 2007). Also, this technology could open up new approaches in plant sciences and in agricultural researches. In recent years, many scientists have studied the effects of nanomaterials on seed germination and plant growth with the aim to promote its use for agricultural productions. Most of these studies are focused on the potential toxicity of nanoparticles on higher plants and positive, negative or inconsequential effects were presented. Most recently it was revealed that the use of appropriate concentration of nano-TiO2 increased the seed germination parameters and early growth of some medicinal and aromatic plants (Hatami and Ghorbanpour 2014). According to Lu et al., (2002) treatment of soybean (Glycine max) plants with a mixture of nano Sio2 and Tio2 increase nitrate reductase activity, stimulate its antioxidant system, and accelerate germination and growth. It is reported that silver nanoparticle treatment of Brassica juncea seedlings induced the activities of specific antioxidant enzymes (Priyadarshini et al., 2012). However, the mechanism of these nanoparticles has not been completely established yet. Also, in the field of medicinal and aromatic plants, the use of nanomaterials is relatively new and needs more researches. However, some studies have reported negative effects of TiO2 nanoparticles (NT) on higher plants that varied between plant tissues, growth stages, plant species, applied concentrations, and specific properties of nanoparticles (Castiglione et al., 2011). Thus, the exploration of their extensive application in agriculture and plant science is still in debate (Kurepa et al., 2010). Therefore, the present study was carried out to elucidate the potential effects of nanosized TiO2 (NT) and bulk (BT) application on antioxidant enzymes including SOD, POX and CAT activity and elicitation of two main tropane alkaloids such as HYO and SCO on Hyoscyamus niger L. plants. 2 MATERIALS AND METHODS 2.1 Transmission electron microscopy (TEM) image of nano-dioxide titanium Nano-sized TiO2 were provided from the Iranian Nanomaterials Pioneers Company, NANOSANY (Mashhad, IRAN). The size of the TiO2 nanoparticles was estimated to be 10-15 nm in diameter. A transmission electron microscopy (TEM) image of the TiO2 particles is also provided (Fig. 1). The crystal properties of TiO2 nanoparticles were examined by X-ray diffraction (XRD), which showed that used TiO2 nanoparticles were all present in the anatase form (Fig. 2). Figure 1: Transmission electron microscopy (TEM) image of TiO2 nanoparticles. Distribution of particles size was estimated to be 10-15 nm, scale bar = 36 nm Figure 2: XRD (X-ray diffraction) pattern of Titanium oxide (TiO2). Nanopowder (TiO2 anatase)- size: 10-15 nm-purity: 99%- surface area: 200-240 m2 g-1- pH: 6-6.5- bulk density: 0.24 g cm-3- true density: 3.9 g cm-3-color: white 2.2 Plant growth conditions and treatments The experiment was carried out in greenhouse conditions (25 °C day/17 °C night temperature, natural light 16 h light: 8 h dark and 75% relative humidity). Henbane seeds generally have low germination rate under normal laboratory conditions. Therefore, seeds were treated with 250 mg L-1 gibberellic acid (GA3) for 48 h at room temperature (25 ± 0.5 °C) for breaking dormancy and accelerating germination. After that seeds were surface-sterilized in 70% ethanol for 2 min and then in 25% commercial bleach (containing 6% sodium hypochlorite) for 10 min and finally rinsed with sterile distilled water. Subsequently, seeds were placed in petri dishes on two layers of filter paper (Whatman No.1) moistened with 4 ml distilled water. After 3 days, 90% of seeds germinated steadily. After germination, individual, healthy and uniform seedlings (when they had three true leaves) were transplanted into experimental pots (25 cm diameter and 30 cm deep, containing 8 kg soil). The physical and chemical characteristics of employed soil are given in table 1. Deionised water was used to prepare 0, 20, 40 and 80 mg l-1 NT and BT solutions. Then, three month-old plants at flowering stage were treated with 50 ml of employed solutions. Both sides of the leaves and stems i.e., whole foliage of the plants were sprayed with equal amounts of 50 ml aqueous solution of NT and BT by hand atomizer. Control plants were only treated with deionised water. The study was set up as completely randomized design with three replicates. All pots were harvested at the end of flowering stage and subsequently plant dry matter was weighted with a precision of 0.0001 g scale and was finely powdered in an electronic blender for enzymes assays and alkaloids extraction. 2.3 Antioxidant enzymes assays A crude enzyme extract was prepared by homogenizing 0.5 gram of powdered leaf sample in extraction buffer containing 0.5% Triton X-100 and 1% polyvinyl pyrrolidone in 100 mM potassium phosphate buffer (pH 7.0) using a chilled mortar and pestle. The homogenate was centrifuged and the supernatant was used for the following enzyme assays. 2.3.1. Superoxide dismutase (SOD, EC 1.15.1.1) SOD activity was determined according to Beauchamp and Fridovich (1971). The reaction mixture contained 1.17*10-6 mol l-1 riboflavin, 0.1 mol l-1 methionine, 2x10-5 mol l-1 KCN and 5.6*10-5 mol l-1 nitroblue tetrazolium (NBT) salt dissolved in 3 ml of 0.05 mol l-1 sodium phosphate buffer (pH 7.8). 3 ml of the reaction medium was added to 1 ml of enzyme extract. The mixtures were illuminated in glass test tubes by two sets of Philips 40 W fluorescent tubes in a single row. The absorbance was read at 560 nm in the spectrophotometer against the blank. SOD activity is expressed in U mg-1 protein. (U = change in 0.1 absorbance h-1 mg-1 protein under assay conditions). 2.3.2. Catalase (CAT, EC 1.11.1.6) CAT activity was assayed according to the method of Chandlee and Scandalios (1984). The assay mixture contained 2.6 ml of 50 mmol l-1 potassium phosphate buffer (pH 7.0), 0.4 ml of 15 mmol l-1 H2O2 and 0.04 ml of enzyme extract. Changes in the absorbance were read at 240 nm. The enzyme activity was expressed in U mg-1 protein (U=1mM of H2O2 reduction min-1 mg-1 protein). The enzyme protein was estimated by the method of Bradford (1976). 2.3.3. Peroxidase (POX, EC 1.11.1.7) POX activity was determined by the method of Kumar and Khan (1982). Assay mixture of POX contained 2 ml of 0.1 mol l-1 phosphate buffer (pH 6.8), 1 ml of 0.01mol.L-1 pyrogallol, 1 ml of 0.005 mol l-1 H2O2 and 0.5 ml of enzyme extract. The solution was incubated for 5 min at 25 °C after which the reaction was terminated by adding 1 ml of 2.5 mol l-1 H2SO4. The amount of purpurogallin formed was determined by measuring the absorbance at 420 nm against a blank prepared by adding the extract after the addition of 2.5 mol l-1 H2SO4 at zero time. The activity was expressed in U mg-1 protein. One U is defined as the change in the absorbance 0.1 min-1 mg-1 protein. 2.4 Alkaloid extraction Leaf samples were air dried, grinded into fine powder and sieved with laboratory mesh (size 30, mesh opening 545 ^m). A subsample of one gram from each samples was added to appropriate volume of CHCl3: MeOH: NH4OH 25%, (15:5: 1), sonicated for 20 min and then kept at water bath (40 °C) for one hour. Subsequent sample preparation and alkaloids extraction were based essentially on the method described by Kamada et al., (1986). 2.4.1. Alkaloid analysis and quantification Alkaloids extracted were identified by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) analysis. GC analysis was performed using a GC system equipped with a flame ionization detector (FID) and HP-5MS capillary column (30 m x 0.25 mm, film thickness 0.25 pm). Injector and detector temperatures were set at 220 and 290 °C, respectively. The column temperature was initially kept at 50 °C for 5 min, then gradually increased to 300 °C at a rate of 3 °C/min and maintained for 3 min. The flow rate of gas He was 0.8 ml/min. Then, 1 pL of extract was directly injected into the gas chromatograph. Each extraction was replicated three times and the compound percentages are the means of the three replicates. GC-MS analysis was carried out on an Agilent 6890 gas chromatograph (Agilent Technologies, Palo Alto, USA) fitted with a fused silica HP-5MS capillary column (30mx0.25mmx0.25pm). Oven temperature was programmed from 50 °C to 285 °C at 3 °C/min, and helium was used as carrier gas (0.8 mL/min), Mass spectra were obtained in an Agilent 5973 system operating in electron impact mode (EIMS) at 70 eV, coupled to an GC system. The identification of alkaloids was based on the comparison of their GC retention time and mass spectra (MS) data with their standards substances (HYO. HCl and SCO. HBr, Merck). The total tropane alkaloids (HYO + SCO) yield was quantified by both alkaloid content and dry weight; Total alkaloid yield (g plant-1) = Alkaloid content (g kg-1) x Plant dry weight (g plant-1). 2.5 Statistical analysis The data were subjected to ANOVA based on a completely randomized design (CRD) with three replications and were analyzed by SAS and MSTAT-C program, and probabilities of significance were used to test for significance among treatments and interactions, and the Duncan's multiple range test (p <0.05) was used to compare means. Values obtained were expressed as mean ± SD (standard deviation) from three replications (n=3) of each treatment. 3 RESULTS 3.1 Plant biomass and antioxidant enzymes status Analysis of variance showed that the most measured traits of this study have been significantly (P < 0.05) affected by NT levels. Mean comparison of data revealed that increasing titanium dioxide concentration especially at nano-sized (10-15 nm) up to 40 mg 1-1 significantly improved the plant dry weight to 42% compared to the unexposed control plants (table 2). However, there were no significant differences among BT levels on plant biomass production. The maximum and minimum plant biomass, 7.53 and 3.24 g plant-1, were obtained in NT treated plants at 40 and 80 mg l-1, respectively. There were noticeably differences in antioxidant enzymes activities among the employed treatments. SOD activity increased with NT and BT levels, and TiO2 application played a significant role in adjusting the enzyme activity (Fig 3 and 4). SOD activity increased with increasing TiO2 concentration in both nano-sized and bulk treatments. On the other hand, the highest SOD activity was observed at the highest NT and BT supply. CAT activity increased with NT application up to 20 mg l-1 and then decreased compared to other NT level, whereas, BT at all concentrations enhanced the CAT activities (Fig 3). Table 1: The physical and chemical characteristics of soil used in current experiment Characteristic Quantity Characteristic Quantity Soil texture Sandy loam CEC (Cmol(c)kg-1) 11.23 Clay(%) 14.32 total nitrogen(%) 0.051 Silt(%) 16 available phosphate (mgkg-1) 9.12 Sand(%) 69.68 available potassium (mgkg-1) 175 pH 7.0 Fe (mgkg-1) * 8.4 EC (dS/m) 1.04 Mn (mgkg-1) * 10.15 CaCO3% 5.82 Cu (mgkg-1) * 0.84 OC% 0.81 Zn (mgkg-1) * 0.52 SP% 28.2 * DTPA-Extractable Table 2: Mean values for plant biomass, major tropane alkaloids including hyoscyamine (HYO) and scopolamine (SCO) content and yield (mean ± S.D., n=3) in H. niger plants treated with different nano-sized TiO2 (NT) and bulk (BT) levels Treatment (mg l-1) Biomass Alkaloid content ( g kg-1) Alkaloid yield (g plant-1) Total alkaloids yield (g plant-1) HYO SCO HYO SCO (g plant"1) Control 4.32±0.14c 0.168±0.006e 0.084±0.013el 0.725±0.014f 0.362±0.021f 1.087±0.025g NT 20 5.81±0.22b 0.216±0.004c 0.126±0.004a 1.254±0.012b 0.732±0.012b 1.986±0.022b NT 40 7.53±0.16a 0.252±0.005b 0.114±0.005b 1.897±0.014a 0.858±0.014a 2.755±0.014a NT 80 3.24±0.15d 0.286±0.003a 0.114±0.003b 0.926±0.016c 0.434±0.011e 1.360±0.012d BT 20 4.35±0.18c 0.176±0.005d 0.108±0.002c 0.765±0.011e 0.469±0.013d 1.234±0.021e BT 40 5.91±0.21b 0.142±0.008f 0.098±0.009de 0.839±0.021d 0.579±0.015c 1.418±0.022c BT 80 4.41±0.19c 0.161±0.009e 0.105±0.003cd 0.710±0.019f 0.463±0.021d 1.173±0.027f LSD 0.17 0.008 0.005 0.016 0.012 0.023 Means in each column with similar letters are not significantly (p <0.05) different through the Duncan's multiple range test The maximum CAT activity was observed in BT at 80 mg l-1 treatment. With regard to the effects of NT and BT on adjusting CAT activity, low NT and high BT application significantly increased CAT activity up to 50% compared to untreated control plants. However, POX activity significantly increased under employed NT up to 40 mg l-1, and then decreased with NT concentration (Fig 3). Application of the high NT and BT concentrations significantly decreased POX activity; however, the final value was not lower than that of control in NT treated plants. However, the highest and the lowest POX activity were observed in plants exposed to NT at 40 mg l-1 and control, respectively. Generally, all tested enzymes activities were higher in NT treated plants that those of BT except CAT activity at 80 mg l-1. Figure 3: Influence of Nano-sized titanium dioxide (NT) concentrations (0, 20, 40 and 80 mg l-1) on superoxide dismutase (SOD), peroxidase (POX) and catalase (CAT) activities in H. niger plants. Values are given as mean ± S.D., (n=3) 8 r ■ Control LjETZO u BT 4.0 ijBTSD SOD POX CAT Antioxidant enivrée Figure 4: Influence of bulk titanium dioxide (BT) concentrations (0, 20, 40 and 80 mg l-1) on superoxide dismutase (SOD), peroxidase (POX) and catalase (CAT) activities in H. niger plants. Values are given as mean ± S.D., (n=3) 3.2. Alkaloids biosynthesis The results demonstrated that the applied treatments affected the shoot HYO and SCO content of black henbane plants (table 2). At high NT concentration (80 mg l-1), the highest HYO content (0.286 g kg-1) was obtained. By contrast, low NT concentration, 20 mg l-1, resulted in high SCO content (126 g kg-1). The lowest content of HYO (142 g kg-1) and SCO (0.087 g kg-1) were observed in plants treated with BT at 40 mg l-1and control groups, respectively. The yield of both HYO and SCO in black henbane plants was increased with NT application at 40 mg l"1 as presented in table 2. However, the minimum HYO (0.710 g plant"1) and SCO (0362 g plant"1) yield were recorded with application of the highest BT dose and unexposed control plants, respectively. The largest total alkaloids (HYO+ SCO) yield (2.755 g plant"1) was achieved in medium NT application (40 mg l"1) mainly because of high dry weight under this situation in comparison with the other treatments (table 2). 4 DISCUSSION Nowadays, nanoparticles happen to interest, mostly because of their possible use in varied technologies. They can be defined as objects ranging in size from 1-100 nm that because of their size may differ in the properties from the bulk materials. This can result from the high surface to volume ratio that increases their reactivity, the ability to penetrate cell membranes and possible biochemical activity. Application of nanotechnology is now widely distributed overall the life, especially in agricultural systems. Nanoparticles because of their physicochemical characteristics e.g., large surface area to volume ratio, ability to engineer electron exchange and highly surface reactive capabilities, are among the potentially candidates for modulating the redox status and changing the growth, performance and quality of plants (Mukherjee and Mahapatra, 2009). The ninth most abundant element and the second most abundant transition metal in the earth's crust is titanium element (about 6.32 ppm). Metal oxide nanoparticles, represented by titanium dioxide (TiO2), is of great technological importance in the field of heterogeneous catalysis for catalytic support of a wide variety of metals (Biener et al., 2005). The most important effects of TiO2 compounds on plants are enhancement of the yield of various crops (~10-20%); an improvement of some essential element contents in plant tissues; an increase in the peroxidase, catalase, and nitrate reductase activities in plant tissues; and an enhancement of the chlorophyll content in paprika (Capsicum anuum L.) and green alga (Chlorella pyrenoidosa) (Hruby et al., 2002). In our current work, different responses of the examined traits to various nano-sized TiO2 dosages could be due to the following principal factors that previously reported by many researchers: concentration of nanoparticles, particle size and specific surface area, physicochemical properties of nanoparticles, plant species, plant age/life cycle stage, growth media conditions, nanoparticles stability, and dilution agent. In our current experiment, NT treated H. niger plants at proper concentration caused higher biomass production than that of bulk and the control untreated plants. Whereas, at the highest NT concentration caused no positive impacts, when compared to the control, indicating the potential toxicity of NT particles with this adverse effect. Yin et al., (2011) mentioned that increasing nanosilver concentration caused a decrease in plant root growth, which indicate an increase in phytotoxicity of nano particles. Also, in our present research activity of antioxidant enzymes, SOD, POX and CAT were affected differently under various employed NT and BT treatments. In both NT and BT treated plants, there was an increase in activity of SOD and POX at certain concentrations, however, a significant decrease was observed for CAT activity at the highest NT concentration when compared to the other NT and BT treatments. According to Priyadarshini et al., (2012) Nano-silver particles decreased H2O2 production and increased the efficiency of redox reactions. And also they reported that higher concentration of nano-silver enhanced the activity of H2O2 metabolizing enzymes. It is well known that SOD is an enzyme that catalyzes the conversion of the O-2 to O2 and H2O2 (Hafis et al., 2011). Enhanced SOD activity of leaves under employed treatments may be interpreted as a direct response to augmented O-2 formation. It is previously suggested that the overexpression of SOD, if this is accompanied by increment of H2O2 scavenging mechanisms like POX and CAT, has been considered as a strategy to cope with oxidative damage (Kohler et al., 2006). Our results also indicated significant role of NT, particularly application of moderate levels (40 mg.L-1), provides a protective mechanism by increasing the activity of defense enzymes. Similar result was reported by Krishnaraj et al., (2012) that high activity of CAT and POX were recorded from leaf samples of plants subjected to nanosilver treatment, implying less ROS formation, resulting in less toxicity to the plants. They also reported that CAT and POX are enzymes that plays major role in ensuring protection against oxidative damage in plants exposed to nanosilver particles treatments. Lei et al., (2008) stated that nanoparticles (TiO2) declined oxidative damage in spinach chloroplast by increasing APX, SOD, POX, and CAT activity. It is suggested that combined reduction of APX, SOD and CAT activity resulted in high generation of intercellular ROS concentrations, which may be directly or indirectly be involved in the lipid peroxidation, senescence and cell death of plant (Debasis et al., 2007). In our research, the decrease in antioxidant enzymes activity observed in control plants may be directly attributed to lower secondary metabolites, HYO and SCO, biosynthesis. The content of alkaloids in plants could be increased through genetic and or environmental manipulations. However, not much information is available on the effect of nano-sized material impacts on the Here, HYO was found as a main alkaloid in the content of tropane alkaloids in H. niger plants. aerial parts of black henbane plants. 5 CONCLUSION Our results suggest that nano-sized titanium dioxide particles in proper levels may act as elicitor for physiological and biochemical responses and tropane alkaloids biosynthesis pathway in H. niger plants. In addition, low NT concentration showed enhancement on the production of plant HYO and SCO yield. 6 REFERENCES Beauchamp, C., Fridovich, I. 1971. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem. 44: 276-287; DOI: 10.1016/0003-2697(71)90370-8 Biener, J., Farfan-Arribas, E., Biener, M., Friend, C.M., Madix, R.J. 2005. Synthesis of TiO2 nanoparticles on the Au (111) surface. J. Chem. Phys. 123:0947051-6; DOI: 10.1063/1.1999607 Bradford , M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Annu. Rev. Bioch. 72: 248-254; DOI: 10.1016/0003-2697(76)90527-3 Castiglione, M.R., Giorgetti, L., Geri, C., Cremonini, R. 2011. The effects of nano-TiO2 on seed germination, development and mitosis of root tip cells of Vicia narbonensis L. and Zea mays L. J. Nanopart. Res. 13: 2443-2449; DOI: 10.1007/s11051-010-0135-8 Chandlee, J.M., Scandalios, J.G. 1984. Analysis of variants affecting the catalase development program in maize scutellum. Theor. Appl. Genet. 69: 71-77; DOI: 10.1007/BF00262543 Cuneyt, C., Kudret, K., Birsen, S. 2004. Physical and Physiological Dormancy in Black Henbane (Hyoscyamus niger L.) seeds. Journal of Plant Biology. 47: 391-395; DOI: 10.1007/BF03030556 Debasis, C., Chatterjee, J., Datta, S.K. 2007. Oxidative stress and antioxidant activity as the basis of senescence in chrysanthemum florets. Plant Growth Regul. 53: 107-115; DOI: 10.1007/s10725-007-9208-9 Flores, H.E., Vivanco, J.M., Loyola-Vargas, V.M. 1999. 'Radicle' biochemistry: the biology of root-specific metabolism. Trends Plant Sci. 4: 220-226; DOI: 10.1016/S1360-1385(99)01411-9 Hafis, C., Romero-Puertas, M.C., Rio, L.A., Abdelly, C., Sandalio, L.M. 2011. Antioxidative response of Hordeum maritimum L. to potassium deficiency. Acta Physiol Plant. 33: 193-202; DOI: 10.1007/s11738-010-0537-3 Hashimoto, T., Yun, D.J., Yamada, Y. 1993. Production of tropane alkaloids in genetically engineered root cultures. Phytochemistry. 32: 713-718; DOI: 10.1016/S0031-9422(00)95159-8 Hatami, M., Ghorbanpour, M., Defense enzymes activity and biochemical variations of Pelargonium zonale in response to nanosilver particles and dark storage. Turkish Journal of Biology, 2014, vol. 38, pp. 130-139; DOI: 10.3906/biy-1304-64 Hatami, M., Ghorbanpour, M., Salehiarjomand, H. 2014. Nano-anatase TiO2 modulates the germination behavior and seedling vigority of the five commercially important medicinal and aromatic plants. Journal of Biological and Environmental Sciences. 8(22): 53-59 Hruby, M., Cigler, P., Kuzel, S. 2002. Contribution to understanding the mechanism of titanium action in plant. J Plant Nutr. 25: 577-598; DOI: 10.1081/PLN-120003383 Kamada, H., Okamura, N., Satake, M., Harada, H., Shimomura, K. 1986. Alkaloid production by hairy root cultures inAtropa belladonna. Plant Cell Rep. 5: 239- 242; DOI: 10.1007/BF00269811 Krishnaraj, C., Jagan, E.G., Ramachandran, R., Abirami, S.M., Mohan, N., Kalaichelvan, P.T. 2012. Effect of biologically synthesized silver nanoparticles on Bacopa monnieri (Linn.) Wettst. plant growth metabolism. Process Biochem. 47: 651-658; DOI: 10.1016/j.procbio.2012.01.006 Kumar, K.B., Khan, P.A. 1982. Peroxidase and polyphenol oxidase in excised ragi (Eleusine coracana cv. PR 202) leaves during senescence. Ind. J. Exp. Bot. 20: 412-416. Kurepa, J., Paunesku, T., Vogt, S., Arora, H., Rabatic, B.M., Lu, J.J., Wanzer, M.B., Woloschak, G.E., Smalle, J.A. 2010. Uptake and distribution of ultrasmall anatase TiO2 alizarin red S nanoconjugates in Arabidopsis thaliana. Nano Lett. 10: 2296-2302; DOI: 10.1021/nl903518f Lei, Z., Mingyu, S., Xiao, W., Chao, L., Chunxiang, Q., Liang, C., Hao, H., Xiao-qing, L., Fashui, H. 2008. Antioxidant stress is promoted by nano-anatase in spinach chloroplasts under UV-B radiation. Biol Trace Elem Res. 121: 69-79; DOI: 10.1007/s12011-007-8028-0 Lu, C.M., Zhang, C.Y., Wen, J.Q., Wu, G.R. 2002. Research on the effect of nanometer materials on germination and growth enhancement of Glycine max and its mechanism. Soybean Sci. 21: 68-171. Mukherjee, M., Mahapatra, A. 2009. Effect of coinage metal nanoparticles and zwitterionic surfactant on reduction of [Co(NH3)5Cl](NO3)2 by iron. Colloid Surface. 350: 1-7; DOI: 10.1016/j.colsurfa.2009.08.021 Oksman, K. 1987. Scopolamine and Hyoscyamine Production by Plants and Cell Cultures of Hyoscymus muticus. PhD thesis, University of Helsinki, Helsinki, Finland, Priyadarshini, S., Deepesh, B., Zaidi, M.G.H., Pardha-saradhi, P., Khanna, P.K., Arora, S. 2012. Silver Nanoparticle-Mediated Enhancement in Growth and Antioxidant Status of Brassica juncea. Appl Biochem Biotech. 167: 2225- 2233; DOI: 10.1007/s12010-012-9759-8 Scrinis, G., Lyons, K. 2007. The Emerging Nano-Corporate Paradigm: Nanotechnology and the Transformation of Nature, Food and Agri-Food Systems. Int J Soc Agric Food. 15: 22-44. Yin, L., Cheng, Y., Espinasse, B., Colman, B.P., Auffan, M., Wiesner, M., Rose, J., Liu, J., Bernhardt, E.S. 2011. More than the ions: the effects of silver nanoparticles on Lolium multiflorum. Environ Sci Technol. 45: 2360-7; DOI: 10.1021/es103995x Zayed, R., Wink, M. 2004. Induction of tropane alkaloid formation in transformed root cultures of Brugmansia suaveolens (Solanaceae). Z Naturforsch. 59: 863-867. Zehra, M., Banerjee, S., Naqvi, A.A., Kumar, S. 1998. Variation in Growth and Tropane Alkaloid Production Capability of the Hairy Roots of Hyoscyamus albus, H. muticus and their Somatic Hybrid. Plant Science. 136: 93-99; DOI: 10.1016/S0168-9452(98)00091-0 COBISS Code 1.01 DOI: 10.14720/aas.2015.105.1.04 Agrovoc descriptors: chamomilla recutita, flowers, fertilizer application, foliar application, iron, zinc, essential oils, soil types, greenhouses Agris category code: f04, f62 Effects of soil and foliar applications of iron and zinc on flowering and essential oil of chamomile at greenhouse conditions Yousef NASIRI1* and Nosratollah NAJAFI2 Received September 25, 2013; accepted October 28, 2014. Delo je prispelo 25. septembra 2013, sprejeto 28. oktobra 2014. ABSTRACT IZVLEČEK In order to study the effects of soil and foliar applications of iron (Fe) and zinc (Zn) on flowering, flower yield and essential oil production of German chamomile a pot experiment was conducted under greenhouse conditions at the Faculty of Agriculture, University of Tabriz, Iran in 2012. The experiment was arranged as completely randomized design with 12 treatments and three replications. Treatments were as follow: Tj: control - without Fe or Zn fertilizers, T2: 30 mg FeSO4.7H2O kg-1 dry soil, T3: 22 mg ZnSO4.7H2O kg-1 dry soil, T4: 30 mg FeSO4.7H2O + 22 mg ZnSO4.7H2O kg-1 dry soil, T5: foliar spraying of FeSO4.7H2O (3.5 g L-1), T6: foliar spraying of FeSO4.7H2O (7.0 g L-1), T7: foliar spraying of ZnSO4.7H2O (2.5 g L-1), T8: foliar spraying of ZnSO4.7H2O (5.0 g L-1), T9: T5+T7, T10: T5+T8, T11: T6+T7, T12: T6+T8. The foliar spraying was done two times during the growing period. The results revealed that the flower number, flower yield, essential oil content and essential oil yield were significantly increased by soil and foliar applications of Fe + Zn, compared with the control (untreated). The highest flower number (477 plant-1), flower yield (11.6 g pot-1), essential oil content (0.88 %) and essential oil yield (119 mg pot-1) were recorded for the soil application of Fe + Zn (T4) by 58, 68, 21.4 and 105 % increment compared to the control, respectively. Foliar application of Fe + Zn (T12) was placed at the next rank; however this treatment had no significant difference with the soil application of Fe + Zn (T4). Other treatments did not show significant differences with the control. Generally, the results showed that soil or foliar application of Fe + Zn can be effective on increase or improve of quantity and quality of chamomile yield. Moreover, use of foliar application as a low cost method especially in areas with alkaline or calcareous soils can be recommended. Key words: Application methods, Essential oil, Iron, Matricaria chamomilla, Zinc UČINKI TALNEGA IN FOLIARNEGA DODAJANJA ŽELEZA IN CINKA NA CVETENJE IN VSEBNOST ETERIČNIH OLJ PRAVE KAMILICE (Chamomilla recutita (L.) Rauschert), GOJENE V RASTLINJAKU Lončni poskus gojenja prave kamilice (Chamomilla recutita (L.) Rauschert) je bil izveden v rastlinjaku z namenom ugotavljanja talnega in foliarnega dodajanja železa (Fe) in cinka (Zn) na njeno cvetenje, pridelek cvetov in produkcijo eteričnih olj na Faculty of Agriculture, University of Tabriz, Iran, leta 2012. Poskus je bil izveden kot popoln naključni poskus z 12obravnavanji in tremi ponovitvami. Obravnavanja so bila: T1: kontrola - brez gnojenja s Fe ali Zn, T2: 30 mg FeSO4.7H2O kg-1 suhih tal, T3: 22 mg ZnSO4.7H2O kg-1 suhih tal, T4: 30 mg FeSO4.7H2O + 22 mg ZnSO4.7H2O kg-1 suhih tal, T5: škropljenje listov z FeSO4.7H2O (3.5 g L-1), T6: škropljenje listov z FeSO4.7H2O (7.0 g L-1), T7: škropljenje listov s ZnSO4.7H2O (2.5 g L-1), T8: škroplenje listov z ZnSO4.7H2O (5.0 g L-1), T9: T5+T7, T10: T5+T8, T11: Tô+Ty, T12: T6+T8. Škropljenje listov je bilo opravljeno dvakrat v rastni dobi. Rezultati so pokazali, da je talno in foliarno gnojenje z Fe + Zn značilno povečalo število cvetov, pridelek cvetov, vsebnost in pridelek eteričnih olj v primerjavi s kontrolo. Največje število cvetov (477 na rastlino), največji pridelek cvetov (11.6 g na lonec), največja vsebnost eteričnih olj (0.88 %) in največji pridelek eteričnih olj (119 mg na lonec) so bili izmerjeni pri talnem dodajanju Fe + Zn (T4), povečanje je bilo za 58, 68, 21.4 in 105 % glede na kontrolno obravnavanje. Učinek foliarnega dodajanja Fe + Zn (T12) je bil takoj za talnim dodajanjem Fe + Zn (T4), vendar se od njega ni značilno razlikoval. Druga obravnavanja niso dala značilnih odstopanj od kontrole. V splošnem so rezultati pokazali, da lahko tako talno kot foliarno dodajanje Fe + Zn učinkovito poveča ali izboljša količino in kvaliteto pridelka prave kamilice. Uporabo foliarnega dodajanja bi kot poceni način gnojenja še posebej priporočali na območjih , kjer so tla bazična ali apnenčasta. Ključne besede: metode gnojenja, železo, cink, Matricaria chamomilla, eterična olja Assistant Professor, Agronomy and Plant Breeding Department, Faculty of Agriculture, University of Maragheh, 5518183111, Maragheh, Iran; Corresponding author E-mail: ysf_nasiri@maragheh.ac.ir Associate Professor, Soil Science Department, Faculty of Agriculture, University of Tabriz, 5166616471, Tabriz, Iran; n-najafi@tabrizu.ac.ir 2 1 INTRODUCTION Today medicinal plants are one of the resources of drugs for treatment of many diseases. Matricaria chamomilla is an annual plant belonging to the Asteraceae family. It is widely used and well-documented medicinal plants in the world. It is included in the pharmacopoeia of 26 countries (Hendaway and Khalid, 2011). Chamomile has many pharmacological properties. It is a traditional treatment for numerous disorders, including sleep disorders, digestion/intestinal conditions, skin infections/inflammation (including eczema), wound healing, infantile colic, teething pains, and diaper rash. It has been also reported that chamomile has moderate antioxidant and antimicrobial activities (Simpson, 2001; McKay and Blumberg, 2006). In order to obtain high quality and yield of crop, nutrients must be sufficient in growing environment of plant. Micronutrients as iron (Fe) and zinc (Zn) are the trace elements that play essential role in plant growth and increasing crop yields. Moreover, they improve plant nutrition and increase soil productivity (Marschner, 1995). Many crops respond to foliar and soil applications of micronutrients in terms of growth and crop yields. It is widely reported that foliar application of micronutrients at active growth stages will improve plant growth and consequently yield and quality in various crops (Kalidasu et al., 2008). Iron is a cofactor for a large number of enzymes that catalyze several biochemical processes within the plant (Brittenham, 1994; Marschner, 1995). It plays a vital role in the chlorophyll formation, thylakoid synthesis and chloroplast development and also functions in the respiratory enzymes. Moreover, iron serves in the transportation of energy in the plant (Miller et al., 1995). Zinc is known to have an important role either as a metal component of enzymes or as a functional, structural or regulatory cofactor of many enzymes. Zinc also has many essential roles in the plant growth and development including production of biomass, chlorophyll production, pollen function, fertilization, metabolism of RNA, proteins and the DNA formation (Marschner, 1995; Pandey et al., 2006; Cakmak, 2008). It is also, required for the synthesis of tryptophan, a precursor of IAA (Indole-3-Acetic Acid) which acts as a growth promoting substance (Marschner, 1995; Miller et al., 1995). Generally plants obtain their nutrients requirements from the soil, but they are capable to absorb nutrients through the leaves. Foliar plant nutrition is one of the techniques that farmers use for plant nutrition since 1950s, when they were learned that foliar fertilization was effective and economic (Ebrahimian et al., 2010). Foliar fertilization is extensively used as a practice to accurate the nutritional deficiencies in plants caused by inappropriate deliver of nutrients to roots (Silberbush and Ling, 2002). The most important use of foliar sprays has been in the application of micronutrients (Havlin et al., 2004). Micronutrients are added to foliar fertilizers, in order to compensate their deficiencies especially in arid and semi-arid regions with calcareous soils (Nasiri et al., 2010). Many recent researches have shown that a small amount of nutrients as Zn, Fe and Mn, applied by foliar spraying increases significantly the yield of crops (Said-Al Ahl and Mahmoud, 2009 & 2010; Nasiri et al., 2010; Zehtab-Salmasi et al., 2008 & 2012; Saedh et al., 2009). Nasiri et al. (2010) reported that flower yield, essential oil percentage and essential oil yield of chamomile were increased by foliar application of Fe and Zn compared with the control at farm conditions. Also Said-Al Ahl and Mahmoud (2010) reported that the highest plant height, branches per plant, fresh and dry biomass and essential oil yield of basil plants were obtained by foliar application of Zn and/or Fe in normal soil. The highest seed yield, oil yield, oil percentage, thousand seed weight and protein percentage of sunflower were obtained from the soil and foliar applications of Fe + Zn (Ebrahimian et al., 2010). Foliar spraying of Zn (100 mg L-1) in blue sage enhanced the length of peduncle and main inflorescence, number of inflorescence and florets, and fresh and dry weight of inflorescences/plant (Abd El-Aziz and Balbaa, 2007). Application of micronutrients increased fresh and dry mater, leaf area of plant, bush and leaf essential oil percentage and essential oil yield of peppermint (Zehtab-salmasi et al., 2008). Although the importance of micronutrients (such as Fe and Zn) on the growth and production of herbs in many research presented; however, there is little information about effectiveness of application methods of Zn and Fe on the growth and development of chamomile. In our previous research we investigated the effects of only foliar application of Fe and Zn on German chamomile at field conditions (Nasiri et al., 2010). Therefore, the purpose of present investigation was to study the effects of Fe and Zn application methods (Foliar spraying and Soil application) and different concentrations of them on flowering, yield and essential oil content of German chamomile. 2 MATERIALS AND METHODS Chamomile plants (Matricaria chamomilla) were grown in a sandy loam alkaline soil in the greenhouse of the Faculty of Agriculture, University of Tabriz, Iran in 2012. The seeds obtained from Hungary were sown in plastic pots (30 cm diameter) filled with 6 kg of dry soil which according to Table 1 was deficient in Fe and Zn (Hazelton and Murphy, 2007). Physicochemical characteristics of the soil used in the study were measured by methods of Gee and Bauder (1986) and Sparks et al. (1996). Each pot was supplied with 450 mg NH4NO3, 44 mg Ca(H2PO4)2.H2O, 150 mg K2SO4, 8 mg CuSO4.5H2O, 85 mg MgCl2.6H2O and 20 mg MnSO4.4H2O per kg of dry soil according to the soil testing. Table 1. Physicochemical characteristics of the soil used as potting media. pH 7.81 ECe (dS m-1) 0.71 Organic carbon content (%) 0.11 Calcium carbonate equivalent (%) Negligible Sand(%) 70 Silt (%) 18 Clay (%) 12 Texture Sandy loam Total N (%) -1 0.08 Available-P (mg kg-1) 5.7 Available-K (mg kg-1) 250 Available-Mg (mg kg-1) 99.1 Available-Ca (mg kg-1) 1149 Available-Fe (mg kg-1) 1.8 Available-Mn (mg kg-1) 1.1 Available-Zn (mg kg-1) 0.42 Available-Cu (mg kg-1) 1.3 ECe= Electrical conductivity of saturated soil paste extract Before seed planting, the above mentioned nutrients were dissolved in enough water and then were mixed with the pot soil. After the emergence of the seedlings four plants were kept per pot. Treatments in the experiment were as follow: Ti: control, T2: 30 mg FeSO4.7H2O kg-1 dry soil, T3: 22 mg ZnSO4.7H2O kg-1 dry soil, T4: 30 mg FeSO4.7H2O + 22 mg ZnSO4.7H2O kg-1 dry soil, T5: foliar spraying of FeSO4.7H2O (3.5 g L-1), T6 foliar spraying of FeSO4.7H2O (7.0 g L-1), T7 foliar spraying of ZnSO4.7H2O (2.5 g L-1), T8 foliar spraying of ZnSO4.7H2O (5.0 g L-1), T9: T5+T7, T10: T5+T8, T11: T6+T7, T12: T6+T8. These 12 treatments were arranged in a completely randomized design (CRD) with three replicates. The volume of the spraying solution was maintained just to cover completely the plant foliage till drip. The plants were sprayed twice at stem elongation and flowering stages. The plant flowers were harvested eight times in 4-5 days intervals. After each harvest flowers were dried in a shady place and were kept in a convenient location for essential oil extraction. The data recorded were: number of flowers in plant, dry weights of flowers in each pot as flower yield, essential oil percentage, and essential oil yield. Five grams dry flowers were hydro-distilled in a modified Clevenger apparatus in 1000 mL round bottomed flask with 500 mL distilled water for 4 h (Hoelz and Demuth, 1975; Letchamo, 1993). Essential oil yield was determined by multiplying essential oil percentage x average of dry weights of flowers per pot. The results were statistically analyzed using MSTATC software. The graphs were plotted using Excel software and the Duncan's Multiple Range Test at 5 % level was used to compare the means of treatments. 3 RESULTS AND DISCUSSION 3.1 Flower number The results in Table 2 show that flower number was significantly (p < 0.05) affected by different micronutrient fertilizer treatments. Means comparison indicated that the highest number of flowers (average 462 per plant) was noticed with the soil application of Fe + Zn (30 mg FeSO4.7H2O and 22 mg ZnSO4.7H2O kg-1 dry soil) and foliar Table 2: Effects of iron and zinc on flower, flower yield and essential oil of chamomile Flower Flower yield Essential oil Essential oil yield Treatments number/plant* (g/pot)** content (%) ** (mg/pot)*** T1 Control- without Fe or Zn fertilizer 301±9.81c 6.9±0.28c 0.84±0.024b 58±2.43c T2 Fe (30 mg FeSO4.7H2O kg-1 dry soil) 349±42.2bc 8.19±1.25c 0.87±0.034b 72±13.51bc T3 Zn (22 mg ZnSO4.7H2O kg-1 dry soil) 354±14.16bc 8.14±0.52c 0.88±0.031b 71±6.2a T4 Fe + Zn (T2+ T3) 477±38.4a 11.6±0.56a 1.02±0.026a 119±8.5bc T5 Fe foliar spraying (3.5 g FeSO4.7H2O L-1) 371±20.42abc 8.17±0.44c 0.86±0.029b 70±4.02bc T6 Fe foliar spraying (7.0 g FeSO4.7H2O L-1) 382±23.58abc 8.44±0.54bc 0.85±0.027b 72±6.61bc T7 Zn foliar spraying (2.5 g ZnSO4.7H2O L-1) 329±25.35c 7.87±0.45c 0.86±0.033b 68±5.95bc T8 Zn foliar spraying (5.0 g ZnSO4.7H2O L-1) 339±6.62bc 7.3±0.13c 0.85±0.025b 62±2.74bc T9 T5 + T7 363±14.61bc 8.32±0.20bc 0.92±0.026ab 76±0.41bc Tk : T5 + T8 385±32.06abc 8.01±0.40c 0.93±0.024ab 74±4.04bc T1 : T6 + T7 333±42.89c 8.46±0.85bc 0.93±0.026ab 78±6.1b T12 : T6+ T8 447±28.91ab 10.53±0.47ab 1.01±0.019a 106±3.86a F Test * ** ** *** Value represents mean ± standard error of three replicates. F Test: *** p<0.001; ** p<0.01; *: p<0.05. Means followed by the same letter in each column are not significantly different according to Duncan's Multiple Range Test at 5 % level. spraying of Fe + Zn (7.0 g FeSO4.7H2O L-1 and 5.0 g ZnSO4.7H2O L-1). These treatments increased flower number by 58 and 48 % compared with the control, respectively. Although soil application of Fe and Zn increased flower number 6 % compared to the foliar spraying, but there was no significant difference between these two methods of fertilizer application. 3.2 Flower yield Data presented in Table 2 show that soil application of 30 mg FeSO4.7H2O + 22 mg ZnSO4.7H2O per kg of dry soil) or foliar application of 7.0 g FeSO4.7H2O + 5.0 g ZnSO4.7H2O L-1 significantly (p < 0.01) increased flower yield in pot. The increments on flower yield were by 68.1 and 52.6 % respectively for the Fe + Zn soil application (T4) and Fe + Zn foliar application (Ti2) compared to the control. The lowest dry flower yield (6.9 g pot-1) was recorded in control treatment. Increment of flower yield might be due to the increased number of flowers per plant as a result of positive effects of iron and zinc application that mentioned in the previous section. Increment of flowers number is directly responsible for higher flower yield in chamomile. 3.3 Essential oil content The response of essential oil (EO) content (%) of chamomile to soil or foliar application with Fe and Zn is available in Table 2. EO % was significantly (p < 0.01) increased as a result of soil and foliar applications of Fe + Zn (T4 and T12 treatments). The increments were 21.4 and 20.2 %, respectively compared to the control plant. Although other treatments also increased this parameter compared to the control in chamomile plants, but these increments were not significant. Chamomile essential oil changes affected by the different treatments of this study are shown in Figure 1. Figure 1: Mean comparison of essential oil content of chamomile in different treatments of iron and zinc application. The same letters in columns indicate no significant difference according to Duncan's Multiple Range Test at 5 % level. Error bars represent standard errors (n=3). Ti: Control, T2: 30 mg FeSO4.7H2O kg-1 dry soil, T3: 22 mg ZnSO4.7H2O kg-1 dry soil, T4: 30 mg FeSO4.7H2O + 22 mg ZnSO4.7H2O kg-1 dry soil, T5: foliar spraying of FeSO4.7H2O (3.5 g L4), T6: foliar spraying of FeSO4.7H2O (7.0 g L-1), T7: foliar spraying of ZnSO4.7H2O (2.5 g L-1), T8: foliar spraying of ZnSO4.7H2O (5.0 g L-1), T9: T5+T7, T10: T5+T8, T11: T6+T7 and T12: T6+T8. 3.4 Essential oil yield The obtained results in Table 2 show significant differences (p < 0.001) were manifested in the plant essential oil yield (EOY) of chamomile due to Fe and Zn application treatments. The highest values of this parameter were obtained from the Fe + Zn soil application (T4) (119 mg pot-1), Fe + Zn foliar application (T12) (106 mg pot-1), and T11 (78 mg pot-1) that were 105, 85 and 34.4 % greater than the control (58 mg pot-1), respectively. Although EO yield was increased by other treatments but these increments were not significant compared to the control (Figure 2). Figure 2: Mean comparison of essential oil yield of chamomile in different treatments of iron and zinc application. The same letters in columns indicate no significant difference according to Duncan's Multiple Range Test at 5 % level. Error bars represent standard errors (n=3). Tj; Control, T2: 30 mg FeSO4.7H2O kg-1 dry soil, T3: 22 mg ZnSO4.7H2O kg-1 dry soil, T4: 30 mg FeSO4.7H2O + 22 mg ZnSO4.7H2O kg-1 dry soil, T5: foliar spraying of FeSO4.7H2O (3.5 g L-1), T6: foliar spraying of FeSO4.7H2O (7.0 g L-1), T7: foliar spraying of ZnSO4.7H2O (2.5 g L-1), T8: foliar spraying of ZnSO4.7H2O (5.0 g L-1), T9: T5+T7, T10: Ts+T8, Tn: T6+T7 and T12: T6+T8. The results of this experiment show that flower number and flower yield affected by soil or foliar application of Fe and Zn treatments. These results were in consonance with the findings of Abd El-Aziz and Balbaa (2007) on blue sage, Kaldiasu et al. (2008) on coriander and Ravi et al. (2008) on safflower. They reported the beneficial effects of iron and zinc on flower production of different plants. This beneficial effect of Zn and Fe can be attributed to the role of Zn in the synthesis of IAA, photosynthesis and nitrogen metabolism and the role of iron in the chlorophyll synthesis and nitrogen fixation (Marschner, 1995; Miller et al, 1995). On the other hand, increment of flower yield might be due to the increased number of flowers per plant as a result of positive effects of iron and zinc application that mentioned in the previous section. Increment of flowers number is directly responsible for higher flower yield in chamomile. These results are in agreement with those obtained by Grejtovsky et al. (2006) and Nasiri et al. (2010) on chamomile, Said-Al Ahl and Omer (2009) on coriander and Said-Al Ahl and Mahmoud (2010) on basil. They stated that soil or foliar application of iron and zinc led to the increment of flowering parameters and plant yield. In the case of essential oil percent and essential oil yield of chamomile plant, results showed significantly addition in their amount with application of Fe and Zn. The maximum amount of these parameters was observed in Fe + Zn soil and foliar application treatments, respectively. The increase in essential oil due to zinc and /or iron was also reported in Japanese mint (Misra and Sharma, 1991), cumin (El-Sawi and Mohamed, 2002), peppermint (Akhtar et al., 2009) and sweet basil (Said-Al Ahl and Mahmoud, 2010) and chamomile (Nasiri et al., 2010). Previous studies indicated that biosynthesis of secondary metabolites is not only controlled genetically but also affected intensely by ecological effects (Naghdi-Badi et al., 2004; Said-Al Ahl and Mahmoud, 2010). Plant nutrition as an environmental variable affects essential oil of medicinal plants. CO2 and glucose are precursors of monoterpene biosynthesis. Carbohydrates are a resource of energy and reducing power for terpenoid synthesis. CO2 fixation, content of primary metabolites and sucrose metabolism are closely linked with essential oil accumulation (Srivastava et al., 1997). As zinc is involved in photosynthesis and carbohydrate metabolism and CO2 and glucose are the most likely sources of carbon utilized in terpenoid biosynthesis, the role of Zn in influencing of essential oil accumulation seems particularly important. Moreover, iron has important functions in plant metabolism, such as activating catalase enzymes associated with superoxide dismutase, as well as in photorespiration and the glycolate pathway (Marschner, 1995). Increase of EOY previously reported by Nasiri et al. (2010) in chamomile at field conditions and Said-Al Ahl and Mahmoud (2010) in sweet basil. They found that combined application of Fe + Zn gave the highest values of essential oil yield under normal soil conditions. This increment seems may be due to the raise of flower yield and essential oil percentage as a result of positive effects of Fe and Zn application. Since the EOY is directly associated with the flower yield and EO %, so any increase in these two traits led to the increase of essential oil yield. Micronutrients such as Fe, Cu, Mn and Zn are essential for growth and development of the living plants. As they are found in the most redox reactions and are fundamental for cellular processes and in proteins and enzymes for structural and catalytic enzyme activities (Hall and Williams, 2003). These nutrients are known to be required for all higher plants and shortage of them in culture media causes deficiency symptoms and reducing plant growth (Marschner, 1995). Fe and Zn act as metal components of various enzymes and also are associated with saccharide metabolism, photosynthesis, and protein synthesis. Iron has important functions in plant metabolism, such as activating catalase enzymes associated with superoxide dismutase, as well as in photorespiration, the glycolate pathway and chlorophyll content. Zinc is an essential micronutrient for synthesis of IAA, cell division and the maintenance of membrane structure and function. Zn deficiency reduces plant growth, pollen viability, flowering, number of fruits and seed production (Sharma et al., 1990; Marschner, 1995). Therefore, sufficient amount of these nutrients in the plant is necessary for normal growth and obtain a satisfactory product. Many studies have reported that micronutrients such as Fe, Mn and Zn have important roles in plant growth and yield of aromatic and medicinal plants (Abd El- Wahab, 2008). Since, the soil application of micronutrients fertilizers in the cultivation may not meet the crop requirement for growth and nutrient use, thus the alternative effective approach is to apply these micronutrients as a foliar application (Saedh et al., 2009). The positive influence of application of micronutrients on crop growth may be due to the improved ability of the crop to absorb nutrients, photosynthesis and better sink-source relationship as these play vital role in various biochemical processes (Kalidasu, et al, 2008). Fe and Zn are absorbed by plant root and shoot as Fe2+, Fe3+ and Zn2+, respectively. The mobility and remobilization of these micronutrients in plants are low. The Fe and Zn concentrations in the soil solution are very low. The availability and solubility of Fe and Zn in soils were dependent on pH, organic matter content, texture, redox potential, moisture content, calcium carbonate equivalent percent, interactions with other elements, climate conditions and plant factors. The availability and solubility of Fe and Zn decrease with increased soil pH. So, the Fe and Zn deficiencies in plants can be observed in alkaline calcareous soils. At alkaline pH, Fe and Zn fertilizers used in soils precipitate as insoluble ZnCO3, FeCO3, ZnFe2O4 and ZnSiO4. Fe and Zn adsorption on the surface of CaCO3, clay minerals and Al/Fe oxides could also reduce the availability and solubility of these nutrients. As a result the effectiveness of these fertilizers is low when applied to soils (Marschner, 1995; Towfighi and Najafi, 2001; Havlin et al., 2004). So, when problems of soil fixation of these nutrients exist, foliar spraying constitutes an effective means of fertilizer application. Foliar fertilization needs lower amounts of fertilizers and provides for more rapid utilization of nutrients and permits the correction of observed deficiencies in less time than would be required by soil treatments (Havlin et al., 2004). 4 CONCLUSIONS In this study it was found that Fe and Zn had beneficial effect on yield and essential oil production of chamomile plant. The obtained results also showed that the application of these two elements in combination had more positive and significantly effects on yield and essential oil of chamomile compared to the their individual applications (Tab. 2). Although there was no significant effect between two methods of fertilizer application in any of the studied parameters, however, soil application of iron and zinc was slightly more effective than use of them by foliar application, but this difference was not significant. With this interpretation, since the foliar application is low-costly technique of feeding plants by applying liquid fertilizer directly to their leaves (Baloch et al., 2008; Yassen et al., 2010), so the use of this method to compensate of micronutrients deficiency like iron and zinc and to improve of chamomile performance especially in arid and semi-arid regions with calcareous soils would be justified. 5 REFERENCES Abd-El-Aziz N.G., Balbaa L.K. 2007. Influence of Tyrosine and Zinc on Growth, Flowering and Chemical Constituents of Salvia farinacea Plants. Journal of Applied Sciences Research, 3(11): 14791489. Abd-El-Wahab M.A. 2008. Effect of some trace elements on growth, yield and chemical constituents of Trachyspermum ammi L. (AJOWAN) plants under Sinai conditions. Research Journal of Agricultural and Biology Sciences, 4(6): 717-724. Akhtar N., Abdul-MatinSarker M., Akhter H., Katrun-Nada M. 2009. Effect of planting time and micronutrient as zinc chloride on the growth, yield and oil content of Mentha pipeta. Bangladesh Journal of Scientific and Industrial Research, 44: 125-130; DOI: 10.3329/bjsir.v44i1.2721 Brittenham G.M. 1994. New advances in iron metabolism, iron deficiency and iron overload. Current Opinion in Hematology, 1: 549-556. Baloch Q.B., Chachar Q.I., Tareen M.N. 2008. Effect of foliar application of macro and micro nutrients on production of green chilies (Capsicum annuum L.). Journal of Agricultural Technology, 4: 177- 184. Cakmak I. 2008. Enrichment of cereal grains with zinc: Agronomic or genetic bio-fortification. Plant Soil, 302:1-17; DOI: 10.1007/s11104-007-9466-3 Ebrahimian E., Bybordi A., Pasban-eslam B., 2010. Efficiency of zinc and iron application methods on sunflower. Journal of Food, Agriculture & Environment, 8 (3&4): 783-789. El-Sawi S.A., Mohamed M.A. 2002. Cumin herb as a new source of essential oils and its response to foliar spray with some micro-elements, Food Chemistry, 77: 7580; DOI: 10.1016/S0308-8146(01)00326-0 Gee, G.W., Bauder, J.W.C., 1986. Methods of soil analysis, Part1, Physical and mineralogical methods (2 Ed.). Am. Soc. Agron., USA. Grejtovsky A., Markusova K., Eliasova A. 2006. The response of chamomile (Matricaria chamomilla L.) plants to soil zinc supply. Plant, Soil and Environment, 52: 1-7. Hall J.L., Williams L.E. 2003. Transition metal transporters in plants. Journal of Experimental Botany, 54 (393): 2601-2613; DOI: 10.1093/jxb/erg303 Havlin J.L., Beaton J.D., Tisdale S.L., Nelson W.L. 2004. Soil fertility and fertilizers: an introduction to nutrient management. 7th Edition, Prentice Hall, USA. Hazelton P.A., Murphy, B.W. 2007. Interpreting soil test results: what do all the numbers mean? CSIRO Publishing, Collingwood, Australia. Hendaway S.F., Khalid K.A. 2011. Effect of chemical and organic fertilizers on yield and essential oil of chamomile flower heads. Medicinal and Aromatic Plant Science and Biotechnology, 5 (1): 43-48. Hoelz J., Demuth G. 1975. Influence of ecological factors on the composition of essential oil and flavonoids in Matricaria chamomilla of different origin. Planta Medica, 27: 46-52. Kalidasu G., Sarada C., Yellamanda-Reddy T. 2008. Influence of micronutrients on growth and yield of coriander (Coriandrum sativum) in rainfed Vertisols. Journal of Spieces and Aromatic Crops, 17 (2): 187189. Letchamo W. 1993. Nitrogen application effects on yield and content of active substances in chamomile genotypes. In Janick J. and Simon, E. (eds) New Crops. Willey. New York. Marschner H. 1995. Mineral nutrient of higher plants. Edition 2, Academic Press Limited. Harcourt Brace and Company Publishers, London. McKay D.L., Blumberg J.B. 2006. A review of the bioactivity and potential health benefits of chamomile tea (Matricaria recutita L.). Phytotherapy Research, 20: 519-530; DOI: 10.1002/ptr.1900 Miller G.W., Huang I.J., Welkie G.W., Pushmik J.C. 1995. Function of iron in plants with special emphasis on chloroplasts and photosynthetic activity. In: Abadia J. (ed.) Iron nutrition in soils and plants. Kluwer Academic Publishers, Dordrecht; DOI: 10.1007/978-94-011-0503-3_4 Misra A., Sharma S. 1991. Critical Zn concentration for essential oil yield and menthol concentration of Japanese mint. Fertilizer Research, 29: 261-265; DOI: 10.1007/BF01052394 Naghdi-Badi H., Yazdani D., Mohammad A.S., Nazari F. 2004. Effects of spacing and harvesting on herbage yield and quality/quantity of oil in thyme, Thymus vulgaris L. Industrial Crops and Products, 19: 231236; DOI: 10.1016/j.indcrop.2003.10.005 Nasiri Y., Zehtab-Salmasi S., Nasrullahzadeh S., Najafi N., Ghassemi-Golezani K. 2010. Effects of foliar application of micronutrients (Fe and Zn) on flower yield and essential oil of chamomile (Matricaria chamomilla L.). Journal of Medicinal Plants Research, 4: 1733-1737. Pandey N., Pathak G.C., Sharma C.P. 2006. Zinc is critically required for pollen function and fertilization in lentil. Journal of trace element and medical biology, 20: 89-96; DOI: 10.1016/j.jtemb.2005.09.006 Ravi S., Channal H.T., Hebsur N.S., Patil B.N., Dharamtti P.R. 2008. Effect of sulphur, zinc and iron nutrition on growth, yield, nutrient uptake and quality of safflower (Carthamus tinctorius L.). Karnataka Journal of Agricultural Sciences, 21: 382-385. Saedh S.E., EL-Abady M.I., EL-Ghomary A.M., Farouk S. 2009. Influence of micronutrients foliar application and nitrogen fertilization on wheat yield and quality of grain and seed. Journal of Biology Sciences, 9(8): 851-858; DOI: 10.3923/jbs.2009.851.858 Said-Al-Ahl H.A.H., Omer E.A. 2009. Effect of spraying with zinc and / or iron on growth and chemical composition of coriander (Coriandrum sativum L.) harvested at three stages of development. Journal of Medicinal Food Plants, 1: 30-46. Said-Al-Ahl H.A.H., Mahmoud A. 2010. Effect of zinc and / or iron foliar application on growth and essential oil of sweet basil (Ocimum basilicum L.) under salt stress. Ozean Journal of Applied Science, 3: 97-111. Sharma P.N., Chatterjee C., Agarwala S.C., Sharma C.P. 1990. Zinc deficiency and pollen fertility in maize (Zea mays). Plant and Soil, 124 (2): 221-225; DOI: 10.1007/BF00009263 Silberbush M., Ling F. 2002. Response of maize to foliar vs. soil application of nitrogen-phosphorus-potassium fertilizers. Journal of Plant Nutrition, 25(11): 23332342; DOI: 10.1081/PLN-120014698 Simpson B.B. 2001. Herbal remedies Economic botany plants in our world Pp. 39-43 Edition 3, McGraw-Hill, Boston Burr Ridge, IL Dubuque, IA Madison, Wl, New York. Sparks, D.L., Page, A.L., Helmke, P.A., Loeppert, R.H., Soltanpour, P.N., Tabatabaei, M.A., Johnson, C.T., Sumner, M.E., 1996. Methods of Soil Analysis. Part 3, Chemical Methods. Soil Science Society of American Book Ser. 5. SSSA, Madison, WI, USA. Srivastava N.K., Misra A., Sharma S. 1997. Effect of zinc deficiency on net photosynthetic rate, 14C Partition, and oil accumulation in leaves of peppermint. Photosynthetica, 33: 71-79; DOI: 10.1023/A:1022127305883 Towfighi H. and N. Najafi. 2001. Changes in recovery and availability of native and applied zinc in waterlogged and non- waterlogged conditions in paddy soils of north of Iran. pp. 382-384. In: Proceedings of the 7th Iranian Soil Science Congress, 26-29 August, Shahrekord University, Shahrekord, Iran. Yassen A., Abou El-Nour E.A.A., Shedeed S. 2010. Response of wheat to foliar spray with urea and micronutrients. Journal of American Science, 6: 1422. Zehtab-Salmasi S., Heidari F., Alyari H. 2008. Effects of microelements and plant density on biomass and essential oil production of peppermint (Mentha piperita L.). Plant Science Research, 1: 24-26. Zehtab-Salmasi S., Saeideh B., Ghassemi-Golezani K. 2012. Effects of foliar application of Fe and Zn on seed yield and mucilage content of psyllium at different Stages of maturity. Pp.63-65. In: International Conference on Environment, Agriculture and Food Sciences (ICEAFS), August 11-12, Phuket, Thailand. COBISS Code 1.01 DOI: 10.14720/aas.2015.105.1.05 Agrovoc descriptors: pistacia vera, antioxidants, enzymes, oxidation, damage, stress, freezing, fertilizer application, foliar application, leaf area, membranes, moisture content Agris category code: f62 Exogenous silicon leads to increased antioxidant capacity in freezing-stressed pistachio leaves Ghader HABIBI1 Received November 24, 2014; accepted Janury 19, 2015. Delo je prispelo 24. novembra 2014, sprejeto 19. januaija 2015. ABSTRACT IZVLEČEK Freezing stress limits photosynthesis and growth of plants. This may be attributed to the enhancement of freezing-associated oxidative damage. In this study, we followed precisely changes in the extent of lipid peroxidation and oxidative damage in leaves of pistachio (Pistacia vera 'Ahmadaghaii') plants exposed to foliar-applied silicon (Si) under freezing stress. The foliar-applied Si decreased significantly damaging effects of cold on relative water content (RWC), accompanied by an increase in shoot fresh mass (SFM). In addition, pre-Si treatment caused a significant reduction of the leaf area lost by freezing. There was a remarkable increase in phenylalanine ammonia-lyase (PAL) activity during recovery. Since leaf phenolic content was not affected by supplementary Si, the possibility that exogenously applied Si directly influences the activity of PAL seems thin. In the present work, freezing stress caused great membrane damage, as assessed by lipid peroxidation, but Si application significantly reduced the membrane damage because of an efficient scavenging by superoxide dismutase (SOD) and peroxidase (POD). Under freezing, despite the increasing POD activity, Si-supplied plants accumulated the highest levels of hydrogen peroxide (H2O2) may act as a signal for recovery ability from freezing injury. A positive correlation was found between the concentration of malondialdehyde (MDA) and the percentage of necrotic leaf area. This study suggests that the possible mechanisms for Si enhanced freezing resistance may be attributed to the higher antioxidant defense activity and lower lipid peroxidation through leaf water retention, in addition to its role as a mere physical barrier. Key words: antioxidant enzymes, cold stress, Evans dye, hydrogen peroxide, phenylalanine ammonia-lyase, Pistacia vera, malondialdehyde, relative water content TRETIRANJE LISTOV PISTACIJE (Pistacia vera 'Ahmadaghaii') S SILICIJEM POVEČA NJIHOVO ANTIOKSIDATIVNO SPOSOBNOST V MRAZNEM STRESU Mrazni stres omejuje fotosintezo in rast rastlin, kar lahko pripišemo povečanju oksidativnih poškodb zaradi zmrzovanja. V raziskavi sta bili spremljani peroksidacija lipidov in oksidativne poškodbe listov pistacije (Pistacia vera 'Ahmadaghaii') izpostavljenih mraznem stresu in foliarnem tretmaju s silicijem (Si). Foliarna uporaba silicija je značilno zmanjšala učinke mraza na ravni relativne vsebnosti vode (RWC), kar je povzročilo povečanje sveže mase poganjkov (SFM). Dodatno je predtretiranje s Si povzročilo značilno zmanjšanje izgube listne površine zaradi zmrzovana. Med okrevanjem po mraznem stresu je bila opazno povečana aktivnost fenilalanin amonik-liaze (PAL). Zaradi nespremenjene vsebnosti fenolov v listih po aplikaciji Si je maloverjetno, da bi foliarno dodani Si neposredno vplival na aktivnost PAL. Mrazni stres je povzročil velike poškodbe membran, ki so bile ocenjene s peroksidacijo lipidov, a jih je uporaba Si značilno zmanjšala zaradi učinkovitega antioksidativnega delovanja superoksid dismutaze (SOD) in peroksidaze (POD). Kljub povečanju aktivnosti POD v razmerah zmrzovanja, so s Si-obravnavane rastline kopičile največje količine vodikovega peroksida (H2O2), ki je lahko deloval kot signal za sposobnost okrevanja po poškodbah zaradi zmrzovanja. Ugotovljena je bila pozitivna korelacija med koncentracijo malondialdehida (MDA) in odstotkom nekrotične listne površine. Raziskava kaže, da je možen mehanizen preko katerega Si povečuje odpornost na zmrzovanje večja antioksidativna obramba in manjša peroksidacija lipidov, ki se odraža v večjem zadrževanju vode poleg delovanja Si kot čisto fizikalne prepreke. Ključne besede: antioksidativni encimi, mrazni stres, Evansovo modrilo-T-1824 ,vodikov peroksid, phenilalanin amoniak-liaza, Pistacia vera, malondialdehid, relativna vsebnost vode Department of Biology, Payame Noor University, I. R. of Iran; Email: gader.habibi@gmail.com 1 INTRODUCTION Low temperatures severely reduce photosynthetic capacity and growth of plants that may be due to increased production of reactive oxygen species (ROS). Accumulation of ROS is capable of inducing damage to almost all cellular macromolecules including DNA, proteins and carbohydrates (Ding et al, 2010; Miller et al., 2010). The plant cells respond to elevation in ROS levels by increasing the expression and activity of ROS-scavenging enzymes in order to maintain redox homeostasis (Apel and Hirt, 2004; Miller et al., 2010). In addition, some plants of tropical and subtropical regions, exhibit sensitivity to cold stress and usually lack the ability for cold acclimation (Zhu et al., 2007; Takahashi et al, 2013). Silicon (Si) has been proved to be beneficial for the healthy growth and development of many plant species, particularly from the Gramineae family (Marschner, 1995; Broadley et al, 2011). Si application to crops has been reported to enhance their tolerance of multiple stresses (Ma, 2004; Guntzer, 2011), including pests and pathogens (Garbuzov et al., 2011; Dallagnol et al., 2012), metal toxicity (Rizwan et al., 2012; Habibi, 2014a), salt and water stress (Hattori et al., 2005; Liu et al., 2014). The mechanisms underlying silicon's capacity to increase stress resistance are still poorly understood. It has been reported that Si causes an improvement of water use efficiency and stimulation of antioxidative defense system in winter wheat (Liang et al, 2008), Paspalum vaginatum (He et al., 2010) and cucumber leaves (Liu et al. 2009). Increase in production of antioxidants and decline of ROS generation mediated by Si causes reduction of photo-oxidative damage, maintenance of chloroplast membranes integrity and thus enhancement of plants stress tolerance (Liang et al., 2008; Waraich et al., 2011). One of the major problems arising in some pistachio cultivation areas includes different levels of injuries caused by lower temperatures in early spring. Because of the fact that the yield of pistachio was reduced due to frost damage, the understanding of the physiological and biochemical mechanisms improving freezing tolerance of this species is very important. We hypothesize that the possible mechanisms for Si enhanced freezing stress may be attributed to the higher antioxidant defense activity and lower membrane oxidative damage through better water retention in leaf tissues. To test this hypothesis, we examined the effect of Si on the growth parameters, leaf water retention, and the enzymatic and non-enzymatic antioxidants and the membrane lipid peroxidation of freezing-stressed pistachio plants. 2 MATERIALS AND METHODS 2.1 Plant growth and treatments: Seeds of pistachio (Pistacia vera 'Ahmadaghaii') were sown in top of the cylindrical plastic pots. Pots were 14 cm in diameter and 105 cm in depth, filled with 15 kg sandy loam soil (pH 7.6, EC 1.32 dS m-1, field capacity (FC) 23 %, organic carbon (OC) 1.09 %). After emergence, the seedlings were thinned to one plant per pot and irrigated with distilled water every 5 days to maintain at 90 % field capacity. plants were grown in a growth chamber located near the city of Miandoab, NW Iran (46°6' E and 36°46' N) with day/night temperature of 25 °C/18 °C, relative humidity of 45-55 % and daily photon flux density (PFD) of about 1100-1200 ^mol m-2 s-1 throughout the experimental period. Seven weeks after sowing, half of the plants were sprayed with 10 mM Si (as K2SiO3, pH adjusted to 5.8 with phosphoric acid). A drop of Tween 20 (0.05 %, v/v) as surfactant was added to 500 ml of the spray solutions. Five days after the treatment, half of the control (untreated with Si) and half of the Si-treated plants were placed to a controlled environment chamber under a 12 h (1±1 °C) light (at 300 ^mol m-2 s-1 photosynthetic photon flux)/12 h (-2±1 °C) dark cycle at 85 % relative humidity for 2 days. After the freezing treatment, all plants were returned to normal conditions as described above, to allow leaves to recover from freezing stress. Samples were taken 2, 48 and 96 h after recovery after cold treatment. Each measurement was done independently and experiments were repeated at least three times. 2.2 Analysis of growth parameters: Leaves and roots were separated and washed with distilled water, blotted dry on filter paper and after determination of fresh mass (FM) they were dried for 48 h at 70 °C for determination of dry mass (DM). Relative water content (RWC) was measured and calculated according to Lara et al. (2003). 2.3 Assay of enzymes activity and related metabolites: Activities of antioxidant enzymes were determined according to the methods described elsewhere (Habibi 20146). For the determination of superoxide dismutase (SOD, EC 1.15.1.1) activity, enzyme was extracted in 25 mM HEPES pH 7.8 with 0.1 mM EDTA and the supernatant was added to the reaction mixture contained 0.1 mM EDTA, 50 mM Na2CO3 pH 10.2, 13 mM methionine, 63 ^M nitroblue tetrazolium chloride (NBT), 13 ^M riboflavin. One unit of SOD was defined as the amount of enzyme which produced a 50 % inhibition of NBT reduction under assay conditions. For the determination of catalase (CAT, EC 1.11.1.6) activity, samples were homogenized with 50 mM phosphate buffer pH 7.0 and assayed spectrophotometrically by following the degradation of H2O2 at 240 nm. Reaction medium contained 50 mM phosphate buffer pH 7 and 10 mM H2O2. Peroxidase (POD, EC 1.11.1.7) activity was determined using the guaiacol test at 470 nm. The enzyme was extracted by 10 mM phosphate buffer pH 7.0 and assayed in a solution contained 10 mM phosphate buffer, 5 mM H2O2 and 4 mM guaiacol. Ascorbate peroxidase (APX, EC 1.11.1.11) activity was assayed by following reduction in absorbance at 290 nm as ascorbate was oxidized according to the method of Boominathan and Doran (2002). The reaction mixture contained 50 mM phosphate buffer pH 7, 0.2 mM EDTA, 0.5 mM ascorbic acid and 50 ^g bovine serum albumin (BSA). Lipid peroxidation was estimated from the amount of malondialdehyde (MDA) formed in a reaction mixture containing thiobarbituric acid (Sigma) at 532 nm. MDA levels were calculated from a 1,1,3,3-tetraethoxypropane (Sigma) standard curve. The hydrogen peroxide (H2O2) contents in the leaves were assayed according to the method of Velikova et al. (2000). Leaves were homogenized in ice bath with 0.1% (w/v) TCA. The extract was centrifUged at 12,000 x g for 15 min, after which to 0.5 ml of the supernatant was added 0.5 ml of 10 mM potassium phosphate buffer (pH 7.0) and 1 ml of 1 M KI, the reaction was improved for 1 h in the dark and measured spectrophotometrically at 390 nm. The content of H2O2 was given on a standard curve. The total soluble proteins were measured according to the Bradford protein assay (Bradford, 1976). To assay for PAL activity, leaf samples were ground in 50 mM sodium phosphate buffer (pH 7.0) containing 2 % (w/v) polyvinylpolypyrrolidon (PVPP), 2 mM EDTA, 18 mM ß-mercaptoetanol and 0.1 % (v/v) Triton X-100. After centrifugation (15000 g for 15 min at 4 °C), PAL was assayed in the supernatant by measuring the formation of cinnamic acid at 290 nm according to modified method of Zucker (1965). Enzyme extracts were incubated at 30 °C for 60 min with 5 mM L-phenylalanine in 60 mM sodium borate buffer (pH 8.8). One unit (U) of PAL activity was defined as the amount of the enzyme that produced 1 nmol cinnamic acid per h. Total phenolic content was determined using the Folin-Ciocalteau method as modified by Velioglu et al. (1998). Gallic acid was used for constructing the standard curve. Results were expressed as mg gallic acid (GA) per gram of the fresh weight. 2.4 Measurement of cell death: Cell death was measured according to the method described by Schützendübel et al. (2001). After cold and Si treatments, leaf tips were inserted in Evans blue solution (0.025 % (w/v) Evans blue in water) for 30 min, followed by washing with water for 15 min. The trapped Evans blue was released from the leaves by homogenizing leaf tips in 1.6 mL of 50 % (v/v) MeOH and 1 % (w/v) SDS, and then centrifuged for 15 min. The optical density of the supernatant was determined at 600 nm and expressed on the basis of fresh mass. The percentage of necrotic area was calculated by measuring separately green and necrotic leaf area according to Irigoyen et al. (1996). Leaves were prepared for determination of Si (Jaiswal 2004) using Inductively-Coupled Plasma-Atomic Emission Spectrometry (ICP-AES, INTEGRA XL2, GBC, Australia). 2.5 Statistical analysis: Experiment was undertaken in complete randomized block design with 4 pots as 4 independent replications. Statistical analyses were carried out using Sigma stat (3.5) with Tukey test (p < 0.05). Correlation analysis using Spearman Rank Order Correlation in Sigma Stat (3.5) were conducted to determine the relationship between the measurement metabolites and the percentage of necrotic area. 3 RESULTS As shown in Table 1, freezing alone significantly reduced the shoot fresh weight of pistachio plants. Addition of 10 mM Si under cold conditions significantly increased the shoot fresh weight of plants as compared with freezing alone. No significant changes of SDM, RFM and RDM were found by foliar application of Si under both freezing and normal temperatures. Cold alone decreased relative water content (RWC) by 13 % after treatment for 96 h recovery, but foliar-applied Si decreased significantly damaging effects of cold on RWC. Freezing alone increased necrotic leaf area by 7.6 % after treatment for 96 h recovery, but the increase was only 3.4 % when Si was applied. Si content was elevated by foliar application of Si, but it was not affected by freezing stress during all treatment periods. Table 1: Effect of Si supplementation on the shoot fresh mass (SFM), shoot dry mass (SDM), root fresh mass (RFM), root dry mass (RDM), relative water content (RWC), necrotic leaf area, and the content of Si in pistachio plants after 96 h recovery after freezing treatment. Data of each row indicated by the same letters are not significantly different (p < 0.05). Data are the mean ± SD (n = 4) Parameters Control -Si +Si 96 h Rec -Si +Si SFM (g plant-1) 10.4±2.21 a 10.5±1.86 a 7.00±1.11b 8.91±1.03 a SDM (g plant-1) 0.91±0.16 a 0.84±0.08 a 0.77±0.10 a 0.86±0.06 a RFM (g plant-1) 3.73±0.40 a 4.16±0.69 a 3.56±0.22a 3.81±0.43 a RDM (g plant-1) 1.07±0.36 a 0.98±0.21 a 0.87±0.27 a 0.92±0.13 a RWC (%) 70±3.2 a 73±1.8 a 57±3.0 b 69±2.4 a Necrotic leaf area (%) 00.0±00.0c 00.0±00.0 c 7.60±1.10 a 3.40±0.52 b Leaf Si (mg g-1 DM) 0.79±0.22b 2.16±0.85 a 0.86±0.33 b 2.48±0.52 a Freezing treatment dramatically increased the PAL activity. Compared with freezing treatment alone, the PAL activity was not affected after 2, 46 and 96 h recovery, by supplementary Si following the freezing treatment (Fig. 1). Similarly, the leaf phenolic content was not influenced by supplementary Si. The percentage of Evans dye uptake increased under freezing stress in the non-Si-treated plants. In Si-supplemented plants, however, uptake of Evans dye did not rise upon cold exposure during the experimental period. Figure 1: Changes in specific activity of phenylalanine ammonia lyase (PAL) (a), the concentration of total phenolics (b) and the percentage of uptake of Evans dye (c) in pistachio plants grown with (+Si) or without Si (-Si) supplementation after 2, 46 and 96 h recovery after freezing treatment. Bars indicated with the same letter are not significantly different (p < 0.05). Data are the mean ± SD (n = 4). Freezing caused a significant increase in the activities of all analyzed antioxidant enzymes with the exception of APX. As shown in Fig. 2, the activities of antioxidant enzymes increased under freezing stress while Si application caused further increase being significant for SOD and POD activities. Freezing stress induced membrane damage as was indicated by higher MDA concentration (Fig. 3). However, the addition of Si to the freezing treatment significantly decreased MDA content compared with the corresponding freezing-treatment with no Si added. Cold stress caused a significant accumulation of hydrogen peroxide, and the continuation of the recovery time with or without Si application caused a further accumulation of H2O2. A positive correlation was found between the concentration of MDA and the percentage of necrotic leaf area (r = 0.96, p < 0.01 in cold treatment; r = 0.76, p < 0.01 in cold+Si treatment, Fig. 4). There was not a significant correlation between the concentration of H2O2 and the percentage of necrotic leaf area. Figure 2: Effect of foliar-applied Si on (a) the specific activity of superoxide dismutase (SOD), (b) catalase (CAT) and (c) peroxidase (POD) in pistachio after 2, 46 and 96 h recovery after freezing treatment. Bars indicated with the same letter are not significantly different (p < 0.05). Data are the mean ± SD (n = 4). □ -Si .■+Si a a a ■aba t (a) ab Ii b sb in 80 cm in the locations of probes 2 and 3 could be explained with inflow of the seawater. Salinity parameters in the profile 1690 could not be properly explained; they might be connected to excavation works for a local water supply. Higher ECe in the upper soil layers compared to the deeper soil layers is more likely the result of fertilization than to negative water balance or capillary action. The researched area has a high water table and due to capillary action water can rise through the soil matrix to the surface. However in winter, when precipitation is much higher than evapotranspiration (Table 5), salts move down through the soil profile. We assume that intensive leaching occurred also in the years 2013 and 2014, due to high precipitation rates (Table 5). Table 3: Parameters of cation exchange capacity and salinity Profile Horizon Soil depth Ca Mg K Na H CEC Base saturat. ESP SAR ECe cm mmolC/100g % % dS/m 1688 A 0 - 6 36.20 1.53 1.34 0.09 1.55 40.8 96.1 0.22 0.09 1.88 1688 P 6 - 23 37.68 1.32 1.12 0.08 0.75 41.0 98.0 0.20 0.09 1.43 1688 I(B) 23 - 50 37.85 1.26 0.55 1.25 0.70 41.6 98.3 3.00 1.85 3.77 1688 II(B) 50 - 80 38.35 1.56 0.41 0.81 0.75 41.9 98.1 1.93 1.13 2.52 1689 A 0 - 8 34.61 1.18 1.32 0.08 1.35 38.6 96.4 0.21 0.08 1.88 1689 P 8 - 42 37.03 0.95 0.70 0.67 0.50 39.9 98.7 1.68 1.02 2.52 1689 I(B) 42 - 65 36.85 1.11 0.37 0.33 0.45 39.2 98.7 0.84 0.57 1.52 1689 II(Go) 65 - 80 38.98 1.42 0.43 0.45 0.50 41.8 98.8 1.08 0.84 1.97 1690 A 0 - 8 38.35 1.30 0.72 1.67 1.25 43.3 97.0 3.86 3.75 6.12 1690 P 8 - 30 38.86 1.15 0.49 1.50 0.50 42.5 98.8 3.53 2.31 4.22 1690 I(B) 30 - 65 38.43 1.38 0.46 0.72 0.60 41.6 98.6 1.73 1.39 2.42 1690 II(Go) 65 - 80 39.03 1.66 0.45 1.73 0.60 43.5 98.6 3.98 3.24 4.22 1698 P1 0-29 33.04 1.12 0.65 0.07 0.95 35.9 97.2 0.19 0.10 1.17 1698 P2 29-57 33.71 1.12 0.51 0.09 1.05 36.4 97.3 0.25 0.30 1.17 1698 I 57-80 35.99 1.42 0.37 0.11 1.4 39.3 96.4 0.28 0.29 1.08 1698 II 80-100 34.32 1.49 0.32 0.13 0.55 36.8 98.6 0.35 0.61 1.08 Table 4: Electroconductivity of soil samples from soil probing Soil probe Altitude ECe (dS/m) [m a.s.l.] 0 - 30 cm 30 - 60 cm > 80 cm S1 1.4 1.35 1.35 2.52 S2 1.2 1.43 2.88 11.78 S3 1.5 0.90 1.35 3.97 S4 1.5 1.35 1.27 1.27 S5 1.9 1.43 1.43 1.35 S6 1.8 1.35 1.27 1.17 S7 1.5 1.35 1.27 2.78 S8 2.1 1.53 1.27 1.17 S9 2.7 1.35 1.27 1.35 S10 2.7 1.35 1.17 2.25 S11 3.5 1.17 1.08 1.17 S12 4.0 1.35 1.08 1.35 S13 3.9 1.17 1.08 1.27 S14 2.6 1.17 1.17 1.08 S15 2.4 1.27 1.17 1.08 S16 2.4 1.27 1.17 1.17 S17 2.2 1.08 1.43 1.08 S18 2.4 1.27 1.35 1.27 Exchangeable sodium percentage (ESP) and sodium adsorption ratio (SAR) were in the range from 0.2 to 3.8 % and from 0.08 to 3.75 %, respectively. ESP values in some soil horizons were higher compared to other soils in Slovenia where the share of sodium ions on adsorption complex is less than 1 % (Prus, 2007). However negative effects on soil structure are less probable; ESP below 10 % or SAR below 13 % does not structure was probably provided by high content of pose a risk to soil structure (Brady and Weil, 2002; Ca2+ ions in the soil. Rowell, 1994). Additional protection for the soil Table 5: Average monthly temperatures, precipitation, potential evapotranspiration and water balance for the period 1971-2000 and for the years 2012, 2013 and 2014 (Data source: Slovenian environment agency, 2014) 1971-2000 Month Jan Feb Mar Apr May June Jul Aug Sept Oct Nov Dec Avg/ Total Average temp. 4.1 4.5 7.4 11.6 16.4 20.1 22.5 21.7 17.6 13.6 8.4 5.1 12.8 Precipit. 56.3 47.1 61.3 65.3 68.8 85.8 57.6 78.1 123.8 120.5 91.3 75.3 931.2 Evapo-transpir. 30 41 66 90 125 142 163 149 98 64 38 29 1035 Water balance 26.3 6.1 -4.7 -24.7 -56.2 -56.2 -105.4 -70.9 25.8 56.5 53.3 46.3 -103.8 2012 Jan Feb Mar Apr May June Jul Aug Sept Oct Nov Dec Avg/ Total Average temp. 3.5 1.5 9.9 12.8 16.6 22.7 25.5 24.7 19.8 14.9 11.7 5.0 14.0 Precipit. 20.1 20.6 0.1 50.4 117.2 35.1 6.9 36.5 96.5 88.2 145.2 72.9 689.7 Evapo-transpir. 34.7 50.9 88.1 88.5 134.2 161.0 200.3 178.3 103.8 58.4 37.9 25.6 1161.7 Water balance -14.6 -30.3 -88.0 -38.1 -17.0 -125.9 -193.4 -141.8 -7.3 29.8 107.3 47.3 -472.0 2013 Jan Feb Mar Apr May June Jul Aug Sept Oct Nov Dec Avg/ Total Average temp. 5.6 4.8 7.4 13.2 16.5 20.5 24.3 23.2 18.9 15.3 1.4 6.9 13.2 Precipit. 89.6 99.2 166.2 75.1 118.5 63.8 5.2 53.1 77.8 95.3 190.2 21.1 1055.1 Evapo-transpir. 25.4 40.1 51.6 92.5 112.9 160.9 194.9 179.6 106.4 53.3 43.3 30.4 1091.3 Water balance 64.2 59.1 114.6 -17.4 5.6 -97.1 -189.7 -126.5 -28.6 42 146.9 -9.3 -36.2 2014 Jan Feb Mar Apr May June Jul Aug Sept Oct Nov Dec Avg/ Total Average temp. 9.4 9.8 10.8 13.9 16.2 21.6 21.7 21.5 17.9 15.4 13.0 7.8 14.9 Precipit. 87.6 171.7 47.4 124.1 89 55 264.7 94.5 208.5 115.4 139.3 65.2 1462.4 Evapo-transpir. 24.2 33.2 79.2 81.5 119.8 160.3 134.3 131.4 83 60.4 30.2 28.8 966.3 Water balance 63.4 138.5 -31.8 42.6 -30.8 -105.3 130.4 -36.9 125.5 55 109.1 36.4 496.1 3.4 Mineralogical characteristics of soil All the soil samples from different profiles and horizons consisted of the same minerals. Prevailing minerals were quartz, calcite, and muscovite/illite (Table 6). Small amount of plagioclases and vermiculite/chlorite group minerals were present in some samples. Muscovite and illite could not be distinguished with certainty due to their similar structure, and vermiculite/chlorite due to their low quantity. A semi-quantitative sample composition estimated by X'Pert HighScore software was controlled and calibrated by measurement of carbonate content (Table 2). Table 6: Mineralogical characteristics of soil: estimated mineral content in %, minerals presented in traces are marked with * Profile Horizon Quartz Calcite Muscovite/Illite Vermiculite/Chlorite Plagioclase 1688 P1 40 30 30 * * 1688 P2 30 30 30 * 10 1688 I 40 20 35 5 1688 II 45 35 20 * * 1689 A 45 30 15 * 10 1689 P 40 30 15 15 1689 I 30 35 25 10 1689 II 45 35 15 5 * 1690 A 45 35 17 3 1690 P 40 35 20 * 5 1690 I 30 40 15 5 10 1690 II 35 35 30 1698 P1 45 25 20 10 1698 P2 50 30 15 5 1698 I 40 30 15 5 10 1698 II 40 30 20 * 10 Diffractograms of different horizons from the same profile (Figure 2) clearly show that not only mineral composition but also ratios between minerals are similar. The influence of soil depth on mineral composition is minimal. Comparison of samples from the upper soil horizons (P2) of all profiles exhibits the same similarity (Figure 3), which indicates to the same soil forming factors for all the research area. The presence of swelling clay minerals, especially of montmorillonite, a member of the smectite group, was checked by careful examination of the XRD pattern. The presence of swelling clay mineral montmorillonite could not be confirmed in any of the soil samples. There is a possibility that the peak of montmorillonite was overlapped with vermiculite/chlorite, but even in that case the amount of montmorillonite would be small. view mufllt F" I muW , IL^ljuJ vUiwUL-JoUW-jA^A» * Poilton ("TTheta] (Copper (Cu>) Figure 2: Diffractograms for the profile 1698. Peaks of minerals are labelled with following abbreviations: v/chl vermiculite/chlorite, mu/ill - muscovite/illite, q - quartz, cc- calcite, pl - plagioclase Q , , -,—-,--—- 10 20 3d 40 50 OG 70 Ponton p7ThtU] (Copper (Cujl Figure 3: Diffractograms for the P2 horizons of profiles 1688, 1689, 1690 and 1698. Figure 2: Diffractograms for the profile 1698. Peaks of minerals are labelled with following abbreviations: v/chl - vermiculite/chlorite, mu/ill - muscovite/illite, q - quartz, cc - calcite, pl - plagioclase MLUil^UL^U. 3.5 Soil classification Soil was evaluated with the criteria of Slovenian Soil Classification (SSC, Soil map of Slovenia 1:25.000) and World reference base for soil resources (IUSS Working Group WRB, 2014). Three classification units of SSC and reference groups of WRB were discussed: (i) Alluvial soil/Fluvisol, (ii) Gleyic soil/Gleysol (iii) Cambic soil/Cambisol. Both soil classification systems have different criteria as well as different terminology to describe the same soil properties. The researched soil developed on the fine sediments of the river Dragonja, consequently the first idea was to classify it as Fluvisol. Fluvisols (IUSS Working Group WRB, 2014) have fluvic material > 25 cm thick and starting < 25 cm from the mineral soil surface; or from the lower limit of a plough layer that is < 40 cm thick, to a depth of > 50 cm from the mineral soil surface. Fluvic material refers to fluviatile, marine and lacustrine sediments that receive fresh material or have received it in the past and still show stratification. By the definition of WRB (IUSS Working Group WRB, 2014) stratification is evidenced by a layer that has > 0.2 % soil organic carbon and has a content of soil organic carbon > 25 % (relative) and > 0.2 % (absolute) higher than in the overlying layer. Alluvial soils by the definition of SSC develop on alluvial material and express either no or weak development of pedogenetic alteration. Soil horizons are difficult to recognise and soil layers are characterised and marked with roman numerals. The researched soil developed on silty clayey fluvic material with more than 0.2 % organic carbon and expresses weak development of pedogenetic alteration, i.e, a well-developed soil structure. We conclude that soil could be classified as alluvial soil according to SSC. However, the criterion of stratification for fluviatile material by WRB (IUSS Working Group WRB, 2014) is not entirely fulfilled as described above and as a consequence Fluvisol is not the appropriate reference group for the subject soil. Soil materials develop gleyic properties if they are saturated with groundwater (or were saturated in the past, if now drained) for a period that allows reducing conditions to occur. The vicinity of the river Dragonja and fine sediments as the parent material are factors that might lead to development of Gleysols. According to WRB (IUSS Working Group WRB, 2014) Gleysols have a layer > 25 cm thick and starting < 40 cm from the mineral soil surface, that has gleyic properties throughout and reducing conditions in some parts of every sublayer. Gleyic properties comprise of one or both of the following: (i) layer with > 95 % (exposed area) having colours considered to be reductimorphic (Munsell colour hue (moist) of N, 10Y, GY, G, BG, B, PB, or 2.5Y or 5Y with a chroma of < 2, or (ii) a layer with > 5 % (exposed area) mottles, the colour of which is considered to be oximorphic. Slovenian soil classification defines Gleysols similarly as defined by WRB (IUSS Working Group WRB, 2014); a hydromorphic surface humus horizon (Aa) must be present at depths less than 50 cm and followed immediately with both gleyic horizons (Go, Gr). Gr must start within 100 cm from the soil surface (Skoric, 1986). The researched soil did not express gleyic horizons starting < 40 cm from the mineral soil surface. In profile 1689, few mottles occur (Go horizon) at 80 cm. Munsell colour hue is mostly 10 YR. Soil from the profile 1690 has Munsell colour hue 2.5Y with the chroma (4/4 or 5/6). Such colour values could be linked to flysch material. In the study "Soils of the Slovenian Coastal region" (Stepancic et al., 1984) reported about several Cambisols developed on flysch material with a Munsell colour hue 2.5Y and chroma >2. Reductimorphic colour GLEY1 6/5GY has been found as matrix colour in the layer at 110 cm soil depth. Generally less intensive expression of gleyic properties in alluvial soils could be explained by the findings of Stepancic (Maticic, 1984), who reported that ground water, due to strong fluctuations, still contains plenty of oxygen and therefore the oxidation and reduction processes in the soil profile are much less pronounced. Nevertheless, due to the morphological properties of the studied soil, it cannot be classified as Gleysol neither according to WRB nor SSC. Soil profile structure with humus-accumulative or aric topsoil horizon and mineral subsurface is characteristic for cambic soil. Cambisols (IUSS Working Group WRB, 2014) have a cambic horizon starting < 50 cm from the soil surface and having its lower limit > 25 cm from the soil surface. The cambic horizon is a subsurface horizon showing evidence of pedogenetic alteration that ranges from weak to relatively strong. If the underlying layer has the same parent material, the cambic horizon usually shows higher oxide and/or clay contents than this underlying layer and/or evidence of removal of carbonates (at least > 5 % by mass, absolute, fine earth fraction). The pedogenetic alteration of a cambic horizon can also be established by contrast with one of the overlying mineral horizons that are generally richer in organic matter and therefore have a darker and/or less intense colour. In this case, some soil structure development is needed to prove pedogenetic alteration. Cambic soils, by the SSC definition consist of cambic horizon, which is a mineral soil horizon with well-expressed pedogenetic forms with less than 1 % organic matter. The soil in our research expressed homogeneity in most soil properties (clay content, carbonate content) but the stratification is evident in organic matter content and colour; organic matter content in the upper soil layer is higher. However, almost all soil layers have more than 1 % organic matter. Even though it was difficult to distinguish between alluvial and soil material, soil structure showed evidence of pedogenetic alteration. Considering all discussed criteria Cambisol is the appropriate reference group for the subject soil according to WRB (IUSS Working Group WRB, 2014). 4 CONCLUSIONS The researched soil has a silty clay loam texture with a high amount of silt. Soil structure is blocky or subangular with high aggregate stability. High amount of calcium carbonate content contributes to high aggregate stability. Soil has neutral or slightly alkaline pH, among base cations Ca2+ ions prevail (up to 99 %). Soil does not express intensive hydromorphic forms; few mottles occur only deeper in the soil profile. Exchangeable sodium percentage is ranging from 0.2 to 3.8 %. In most locations electroconductivity (ECe) does not exceed 2 ds/m; this happens only in some locations and in deeper soil layers, where ECe values exceed 4 ds/m; however in absence of morphological characteristic for salt affected soils (structure, concrete...) the soil could not be characterised as saline soil. Measured ECe and ESP values in soil from the lower valley of Dragonja are higher compared to other soils in Slovenia. In general, higher precipitation rates in Slovenia favour elluvial- illuvial processes and development of leached soils. Therefore, soils in the lower valley of the Dragonja River are rare and important for soil diversity in Slovenia. Prevailing minerals in the soil are quartz, calcite and muscovite/illite. Plagioclase and vermiculite/chlorite were found in small amounts. The presence of swelling clay mineral montmorillonite could not be confirmed in any of the soil samples. According to WRB soil classification, and based on morphological, chemical and mineralogical analyses, soil of the researched area could be classified as Calcaric Cambisol (aric, siltic). According to the Slovenian national soil classification, two soil types could be determined: (i) alluvial soil, calcaric and (ii) alluvial soil, calcaric, deeply gleyic. 5 ACKNOWLEDGEMENTS This work was funded by Slovenian Research Agency, Research Programme P4-0085. 6 REFERENCES Bašic I. 1976. Pedološko-melioraciona studija doline Dragonje i Drnice. Poljoprivredni inštitut Zagreb, Inštitut za pedologiju i poljoprivredne melioracije Zagreb: 60 p. Brady N.C., Weil R.R. 2002. The Nature and Properties of Soil. 13th edition. Pearson Education Inc: 960 p. ISO 10390, 2005. Soil Quality-Determination of pH. International Organization for Standardization, Genève, Switzerland. ISO 10693, 1995. Determination of Carbonate Content. Volumetric Method. International Organization for Standardization, Genève, Switzerland. ISO 11277, 2009. Determination of Particle Size Distribution in Mineral Soil Material-Method by Sieving and Sedimentation. International Organization for Standardization, Genève, Switzerland. ISO 11464, 2006. Soil Quality-Pretreatment of Samples for Physico-chemical Analysis. International Organization for Standardization, Genève, Switzerland. ISO 13878, 1987. Determination of Total Nitrogen Content by Dry Combustion ("Elemental Analysis"). International Organization for Standardization, Genève, Switzerland. ISO 14235, 1998. Soil Quality- Determination of Organic Carbon by Sulfochromic Oxidation. International Organization for Standardization, Genève, Switzerland. ISO 11274, 1998. Soil quality — Determination of the water retention characteristic — Laboratory methods. International Organization for Standardization, Genève, Switzerland. IUSS Working Group WRB. 2014. World Reference Base for Soil Resources 2014. International soil classification system for naming soils and creating legends for soil maps. World Soil Resources Reports No. 106. FAO, Rome:181 p. FAO. 2006. Guidelines for soil description. 4 thedition. Rome: 97 p. Matičič B. 1984. Izvajanje drenažnih sistemov. UEK v Ljubljani, VDO Biotehniška fakulteta, VTOZD za agronomijo, Katedra za melioracije in urejanje kmetijskega prostora: 358 p. Munsell soil color charts. 1971 and 2013. Munsel color division. Kollmorgen corporation Baltimore: 7 tabels. Ogorelec B., Mišič M., Šercelj A., Cimerman F., Faganeli J., Stegnar P. 1981. Sediment sečoveljske soline. Geologija, 24/2: 179-216 p. ÖNORM L 1087. 1993. Chemische Bodenuntersuchungen Bestimmung von pflanzenverfügbarem Phosphat und Kalium nach der Calcium-Acetat-Lactat (CAL)-Methode, Österreichisches Normungsinstitut: 4p. Pleničar M., Polšak A., Šikič D. 1973a. Osnovna geološka karta SFRJ Trst 1:100000. Tolmač lista Trst: L 33-88. Beograd. Zvezni geološki zavod Beograd: 68 p. Pleničar M., Polšak A., Šikic D.. 1973b. Tolmač za list Trst: L 33-88: Socialistična federativna republika Jugoslavija, osnovna geološka karta, 1:100 000: 68 p. Prus T. Zbijanje in zaslanjevanje tal v Sloveniji. 2007. In: Strategija varovanja tal v Sloveniji (Knapič M. ed): 103-110 p. Rowell L. D. 1994. Soil science. Methods & Applications. Longman group UK Limited: 350 p. Rupreht J. 2008. Pedološko poročilo o lastnostih tal na delu Sečoveljske doline: 13 p. Slovenian environment agency, 2014: http://meteo.arso.gov.si/met/en/climate/ (January, 2015). Soil map of Slovenia 1:25.000 (1999-2015); Biotechnical Faculty, University of Ljubljana -digital version Soil Survey laboratory methods manual, 1992. Soil Survey Investigations Report No. 42, Version 2.0. Stepančič D., Lobnik F., Prus T., Rupreht J. 1984. Tla slovenskega primorja. Biotehniška fakulteta, VTOZD za agronomijo, Katedra za pedologijo, prehrano rastlin in ekologijo: 47 p. Škoric A. 1986. Postanak, razvoj i sistematika tla. Fakultet poljoprivrednih znanosti Sveučilišta u Zagrebu: 172 p. Šporar M., Rupreht J., Lobnik F. (Ur.) 1994. Pedološka karta Slovenije 1:25000. PK4612. Buje. Ljubljana, Biotehniška fakulteta, Agronomija, Center za pedologijo in varstvo okolja. 1 zvd. barv. COBISS Code 1.01 DOI: 10.14720/aas.2015.105.1.08 Agrovoc descriptors: vicia faba, faba beans, varieties, crop yield, yield factors, seeds, genotypes, indigenous organisms, seed characteristics, plant breeding, selection, genes, environmental factors, heritability, agronomic characters Agris category code: f03, f62 Genetic variation for seed yield and some of agro-morphological traits in faba bean (Viciafaba L.) genotypes Peyman SHARIFI1 Received March 08, 2014; accepted February 23, 2015. Delo je prispelo 08. marca 2014, sprejeto 23. februarja 2015. ABSTRACT IZVLEČEK An investigation was carried out to select the most successful faba bean genotype(s) and to estimate the heritability for seed yield and some of agro-morphological traits. The results of analysis of variance indicated that the studied genotypes differed significantly for all of the traits. For 100-seed weight, two north's of Iran landraces (G1 and G2) and two improved breeding cultivars containing France (G4) and Barrakat (G10) possessed the heaviest seed weight, 161.33, 139, 119.67 and 166 g, respectively. G1 and G10 presented the highest values for dry seed weight (473.98 and 495.44 g m-2, respectively). G1 and G10 showed significantly higher magnitude values of the other traits. Broad sense heritability (h2) estimates were generally high to moderate for all of the studied traits. The highest estimates of broad sense heritability was inscribed as 98 % for pod length, dry seed length and dry seed width and 0.95 for hundred seed weight. The estimated broad-sense heritability was 0.80 for dry seed yield per m2. These results suggested that the environmental factors had a small effect on the inheritance of traits with high heritability. High estimates of heritability indicated that selection based on mean would be successful in improving of these traits. High heritability indicate an additive gene action for the traits, and hence, possible trait improvement through selection. Path coefficient analysis indicated that the traits containing day to harvesting, pod length, hundred seed weight and number of stems per plant play major role in seed yield determination of faba bean. Attention should be paid to these characters for augmentation of seed yield and these traits could be used as selection criteria in faba bean breeding programs. These findings indicate that selection for each or full of the above traits would be accompanied by high yielding ability under such conditions. Key words: additive gene action, breeding, faba bean, genetic variation, selection GENETSKA VARIABILNOST PRIDELKA SEMEN IN NEKATERIH AGRONOMSKO-MORFOLOŠKIH LASTNOSTI GENOTIPOV BOBA (Viciafaba L.) Raziskava je bila opravljena za izbor najdonosnejših genotipov boba z namenom oceniti heritabilnost oz. dednostni delež pridelka semen in nekaterih agronomsko-morfoloških lastnosti. Rezultati dobljeni z analizo variance so pokazali, da se vsi obravnavani genotipi značilno razlikujejo v vseh proučevanih lastnostih. Za maso 100-semen sta se izkazali najboljši dve akcesiji iz severnega Irana, (G1 in G2) in dve izboljšani sorti,'France' (G4) 'Barrakat' (G10), katerih masa je znašala, 161.33, 139, 119.67 in 166 g. Genotipa G1 in G10 sta imela največjo maso suhih semen na enoto površine (473.98 in 495.44 g m-2). Ista genotipa sta izkazala tudi večje vrednosti pri drugih analiziranih lastnostih. Ocene dednostnega deleža v širšem smislu (h2) so bile na splošno velike do zmerne za vse analizirane lastnosti. Največji delež k splošni dednosti so v vrednosti 98 % prispevali dolžina strokov, dolžina in širina suhega zrna in 0.95 masa stotih semen. Ocenjena splošna dednost za pridelek suhih semen na enoto površine je bila 0.80. Ti rezultati so pokazali, da so imeli okoljski dejavniki majhen vpliv na lastnost z velikim dednostnim deležem. Velike ocene dednostnega deleža so pokazale, da je selekcija, ki bi temeljila na povprečnih vrednostih lastnosti primerna za njihovo izboljšanje. Velik dednostni delež lastnosti označuje aditivni učinek genov in možno izboljšanje lastnosti s selekcijo. Analiza združevanja lastnosti je pokazala, da so imele lastnosti, ki so vključevale število dni do žetve, dolžino stroka, maso stotih semen in število stebel na rastlino največjo vlogo pri določanju pridelka semen boba. V žlahniteljskih programih s selekcijo bi bilo potrebno posvečati pozornost tistim lastnostim boba, ki prispevajo k povečanju pridelka semen. Izsledki te raziskave kažejo, da mora biti izbrana in spremljanja vsaka ali vse analizirane lastnosti, ki imajo vpliv na velikost pridelka v danih razmerah. Ključne besede: aditivno delovanje genov, žlahtnjenje, bob, genetska variabilnost, selekcija 1 Department of Agronomy and Plant Breeding, Rasht Branch, Islamic Azad University, Rasht, Iran; email: kadose@yahoo.com 1 INTRODUCTION Faba bean (Vicia faba L.), an autogamous plant with partial outcross ranging from 20 to 80 %, is a diploid plant with a relatively few number of large chromosomes (2n = 2x = 12) (Al-Barri and Shtaya, 2013; Basheer et al., 2013; Terzopoulos et al., 2008). It is originated in between the oriental Mediterranean countries and Afghanistan (Cubero, 1974) and most commonly included in the diets of inhabitants of the Middle East, the Mediterranean region, China and Ethiopia, and it can be used as a vegetable, green or dried, fresh or canned (Bond et al., 1985). The species is genetically isolated, tolerating no exchange of genes with any other species including its close relative Vicia narbonensis (Hawtin and Hebblethwaite, 1983). Genetic variability of faba bean is quite large. The great part of variability may be due to the presence of intermediate crossing system between autogamy and allogamy (Hanelt and Mettin, 1989). Yield improvement is a major breeding objective of most crop improvement programs in faba bean. The success of an autogamous plant breeding program depends on the choice of genotypes capable of producing progeny with desired trait combinations. Yield in faba bean, similar to the other crops, is a complex trait and constitute by many of morphological and physiological traits that correlated each other. Plant height, number of stems and pods per plant, biological yield, harvest index, 100-seed weight, days to flowering and maturity are the most important traits in faba bean improvement for increasing seed yield due to direct and indirect correlation with seed yield (Loss and Siddique, 1997) The genetic improvement of crop desired traits depends on the nature and magnitude of genetic variability and interactions involved in the inheritance of these traits which can be estimated using experimental design techniques. Many researchers studied heritability for seed yield, yield components and the other agro-morphological traits in faba bean. Seed yield is a complex trait that is quantitatively inherited with low heritability value (Bond, 1966). The low heritability and consequent limited genetic advance for yield in response to selection had led many scientists to search for characters which are associated with yield but which are more highly heritable (De Pace, 1979). Toker (2004) grown eight faba bean genotypes at in order to estimate the broad-sense heritability, and the heritability for plant height, number of stems and pods per plant, seed yield, biological yield, 100-seed weight, days to flowering and maturity estimated as 83, 63, 43, 62, 52, 99, 97 and 97 %, respectively. Alghamdi (2007) carried out a research in order to determine the genetic behavior of six faba bean genotypes and the results revealed that the studied genotypes significantly differed for all of the traits including plant height, number of pods per plant, number of seeds per plant, seed weight per plant and seed yield. The highest estimates of broad sense heritability were obtained for flowering date (0.986), number of pods per plant (0.96), number of seeds per plant (0.957) and maturity date (0.905), respectively. Hanna and Hayes (1966) showed low heritability for number of pods per plant (0.24), number of seeds per plant (0.23) and seed weight (0.46), respectively. El-Kadi (1968) indicated that the broad-sense heritability ranged from 6.6 to 52.1 % for seed yield, 48.1 to 65.1 % for number of stems per plant, 42.8 to 63.9 % for number of pods per plant, 51.6 to 62.2 % for plant height and 48.0 to 86.2 % for seed index. El-Kady and Khalil (1979) revealed that broad-sense heritability estimates ranged from 36.2 to 90.6 %, 10.6 to 50.9 % and 27.1 to 62.0 % for seed yield, number of seeds per plant and seed weight per plant, respectively. Abo El-Zahab et al. (1980) studied broad-sense heritability for number of pods per plant, number of seeds per pod, seed weight per plant and seed yield. Their findings indicated that heritability values were 88.4, 99.9, 84.3 and 21.3 % respectively. Bora et al. (1998) stated that the high heritability was followed by high genetic advance for fruiting stems per plant, number of pods per plant and seed yield per plant, indicating the scope for their improvement through selection. Ibrahim (2010) indicated narrow-sense heritability was high for 100-seed weight and low for seed yield per plant. Kalia and Sood (2004) revealed high broad-sense heritability estimates (0.97) along with high genetic advance (126 %) for pod yield. The present investigation aimed at the agronomic performance of ten faba bean genotypes in order to employ the most successful genotype(s) in a breading program. Heritability via variance components for seed yield and some of agro- bean genotypes in lowland of the north region of morphological traits were also determined on faba Iran. 2 MATERIALS AND METHODS This study was carried out during 2011 and 2012 in Shanderman, Iran (longitude, 49° 55' E; latitude, 37° 27' N; altitude, 71 m above sea level; climate, wet). Experimental material comprised 10 genotypes of faba bean that some of their features is given in Table 1. 2.1 Experimental field area The sowing of seeds was conducted in 8 December 2011 by hand. Field experiments were conducted in a randomized complete block design with three replications. Each plot consisted of four rows with 6 m long and distance between rows was 50 cm. The seeding rate was 15 plants per m2. Forty five kilogram nitrogen, phosphorus and potassium per hectare were applied as compose fertilizer including 15 kg ha-1 from any of fertilizers (15-1515) prior to sowing. All recommended agronomic practices were followed to raise good crop. The following statistical model was adopted for experimental design: Yj = M + Gi + r +Sj measured on ten plants selected randomly from all plots. Dry seed yield per m2 (SY) and dry seed weight per plant were weighed in maturity harvesting stage. 2.3 Statistical analysis The analysis of variance was carried out according to Steel and Torrie (1980) in data after collecting and means comparison of any traits in genotypes were performed by the least significant difference (LSD) test. The genotypic and phenotypic variances ( jg and Jp ) were calculated from the partitioning mean squares expectation (Table 2) as follows: 2 MSg - MSe ag _--- g r a2p =a2g + a2/ r Where, general mean; Rj: effect of jth block (j = 1, 2, 3); Gil effect of i genotype (i = 1, experimental Error. 2.2 Estimated characters 2 ... 10) and eijk: Fifteen plants of each plot were harvested by hand and weight of seed and pod per plant were measured before and at physiological maturity stage for all of the genotypes. The characters via day to pod initiation (DP), day to harvesting (DH) and plant height (PH) were also calculated before harvesting. The remainder of plants in each plot were harvested by hand at harvest maturity stage and pod length (PL), number of dry seeds per pod (DSP), dry seed length (LS), dry seed width (SW), hundred seed weight (HSW), number of stems per plant (StP), number of pods per plant (PP), number of pods per stem (PSt), number of seeds per stem (SSt) and number of seeds per pod (SP) were Where, g and r are number of genotype and replication, respectively; J2p, J2g and J2e are components of variance for phenotypic, genotypes and error, respectively. Broad sense heritability (h2B) was estimated as: genotypic variance/phenotypic variance (Roy, 2000): H 2 H Entire analyses were performed with the Statistical Analysis System (SAS) Software and Excel. GGE (genotype main effect plus genotype-by-environment interaction) biplots are commonly used to analyze two-way data where rows and columns represent different experimental units (e.g. genotypes and traits). The mean values for genotypes across traits are used to form a symmetrical data matrix from which the first two PC are extracted (Yan and Hunt 2002). All biplots presented in this paper were generated using the software GGE biplot package that runs in a windows environment, an earlier version of which Table 1. Information of some of important traits on stu was described in Yan (2001). GGE-biplot was carried out on seven genotypes (g1, g2, g3, g5, g6, g9 and g10) and three remained genotypes (g4, g7 and g8) were excluded from biplot analysis according to their similarity to the other genotypes. i genotypes Genotype number Genotype name 1 - 2 - 3 France 4 Filip3 5 Filip5 6 - 7 - 8 - 9 - 10 Barrakat Breedng status Seed structure Autochthonous Large landrace Autochthonous Large landrace Improved breeding Intermediate cultivars Improved breeding Small cultivars Improved breeding Small cultivars Autochthonous Small landrace Autochthonous Small landrace Autochthonous Small landrace Autochthonous Small landrace Improved breeding Large cultivars Origin_ North of Iran (Guilan) North of Iran (Mazandaran) France Syria Syria Lorestan (Khorramabad) Lorestan (Borujerdl) Lorestan (Borujerd2) Lorestan (Borujerd3) Iran/ Gurgaon 3 RESULTS AND DISCUSSION 3.1 Analysis of variances Significance of mean squares due to different sources of variability for studied traits is summarized in Table (2). Results indicated that the studied genotypes differed significantly for all of the traits except plant height, weight of pod per plant before physiological maturity stage, number of stem per plant, number of pod per plant and ratio of pod weight per plant before physiological maturity stage. 3.2 Genotypes' mean performance The mean performances for different traits of ten faba bean genotypes are given in Table (3). Mean comparison were performed by least significant differences (LSD). Data revealed that the genotype 5 and 8 possessed the earliest pod initiation (140.33 day) and maturity date (167.67 day), respectively. On the other hand, G9 and G1 took 146 and 175.33 days to pod initiation and maturity date, respectively. Respecting to plant height, G10 possessed the tallest plants (88.5 cm) whereas, G2 presented the shortest plants (76.21 cm). With regards to pod length, G10 owned the tallest pods (15.49). On the other side, G6 possessed the shortest ones (5.65). For weight of seed per plant before physiological stage, G2 revealed heaviest seed weight (33.22 g). For ratio of seed length to width, G1 showed the highest ratio (1.51). For number of stems per plant, number of pods per plant and number of pods per stem, G1 recorded 4.46, 12.19 and 2.77, respectively. Results indicated that the G10 possessed the highest values for number of seeds per pod, dry seed length, dry seed width, weight of pod per plant at physiological maturity stage, weight of seed per plant at physiological maturity stage, weight of pod per plant before physiological maturity stage, hundred seed weight, number of seed per plant and number of seed per stem. For the ratios of seed per pods before and at the physiological stages and ratio of seeds per plant before physiological stage G9 had the highest ratio values 0.59, 0.64 and 0.27, respectively. G2 possessed the highest values of pod per plant ratio before physiological stage. For 100-seed weight, G1, G2, G3 and G10 possessed the heaviest seed weight 161.33, 139, 119.67 and 166 g, respectively. On the other hand, the other genotypes possessed the lowest values. G1 and G10 presented the highest value for dry seed yield, 473.98 and 495.44 g m-2, respectively. On the other hand, G7 exhibited the lowest value for dry seed yield (106.63 g m-2). From the above mentioned results, it could be deduced that G10 followed by G1 indicated the tallest plants and pods, highest number of pods, seeds, seeds weight per plant and dry seed yield (g m-2). These results express that the selection prospects within this genotype to improve the performance through breeding program. Table 2: Analysis of variance for some of morphological traits in some of faba bean genotypes SOV df DP DH PH (cm) PL (cm) DSP LS (cm) WS (cm) R 2 0.4ns 24.7* 38.75ns 1.09ns 0.04ns 0.008ns 0.0008ns G 9 16.03** 18.01* 88.49ns 28.80** 1.82** 0.806** 0.35** E 18 3.18 5.63 116.86 0.7_0.15 0.02 0.007 CV_1.24 1.39 13.45 10.23 11.44 8.92 7.35_ SOV df SY WPo/Pl(ph) WSe/Pl(ph) WPo/Pl(bph) WSe/Pl(bph) HSW L/W _(g/m2) (g/ plant) (g/ plant) (g/ plant) (g/ plant) (g)_ R 2 5661.45ns 458.88ns 111.73ns 0.033** 0.0068ns 295.43ns 0.0053ns G 9 63438.15** 8173.14** 1527.88** 0.009ns 0.0106* 7671.87** 0.0152** E 18 12450.91 932.6_271.28 0.011_0.0039_409.14 0.0029 CV_47.66_41.39_45.20_24.08_36.37_22.52 3.94 SOV df StP PP PSt SSt Se/Po(Ph) Se/Po(bPh) Se/Pl(bph) Po/Pl(bph) R 2 0.03ns 20.29ns 0.77ns 42.18* 0.0022ns 0.014ns 0.0068ns 0.03ns G 9 1.23ns 14.68ns 1.25** 28.85* 0.005** 0.068** 0.0106* 0.0089ns E 18 0.77 10.98 0.39 9.9 0.0011 0.013_0.0039 0.0103 CV_24.98 38.15 24.77 43.6 6.5_27.68_36.37_24.08 SY: Dry Seed Weight per m2; DP: Day to Pod initiation; DH: Day to Harvesting; PH: Plant Height; PL: Pod Length; LS: Dry Seed Length; WS: Dry Seed Width; DSP: Number of Seed per Plant; WPo/Pl(ph): Weight of Pod per Plant at Physiological maturity Stage; WSe/Pl(ph): Weight of Seed per Plant at Physiological maturity Stage; WPo/Pl(bph): Weight of Pod per Plant before Physiological maturity Stage; WSe/Pl(bph): Weight of Seed per Plant before Physiological maturity Stage; HSW: Hundred Seed Weight; L/W: Ratio of Seed Length to Width; StP: Number of Stem per Plant; PP: Number of Pod per Plant; SP: Number of Seed per Plant; PSt: Number of Pod per Stem; SSt: Number of Seed per Stem; Se/Po(Ph): Ratio of Seed Weight per Pod at Physiological maturity Stage; Se/Po(bPh): Ratio of Seed Weight per Pod before Physiological maturity Stage; Se/Pl(bph): Ratio of Seed Weight per Plant before Physiological maturity Stage; Po/Pl(bph): Ratio of Pod Weight per Plant before Physiological maturity Stage. ns, not significant *, significant at the 0.05 probability level **, significant at the 0.01 probability level Table 3: Averaged performance for some of morphological traits in faba bean genotypes Genotype number DP DH PH (cm) PL DSP LS (cm) WS (cm) 1 142.67 175.33 86.33 8.70 2.40 2.30 1.52 2 140.67 172.33 76.21 9.80 3.22 2.12 1.51 3 141.00 173.33 77.33 10.17 3.46 1.82 1.38 4 144.33 171.33 77.67 5.93 3.12 1.13 0.81 5 140.33 170.00 74.33 8.07 3.46 1.55 1.21 6 144.67 169.67 84.83 5.65 2.76 1.18 0.90 7 147.00 171.00 74.25 6.30 3.37 1.17 0.85 8 144.33 167.67 85.25 5.85 2.81 1.13 0.86 9 146.00 168.00 76.79 5.81 3.43 1.14 0.80 10 142.00 173.33 88.50 15.49 5.30 2.37 1.66 Mean 143.30 171.20 80.15 8.18 3.33 1.59 1.15 SE 0.731 0.774 1.717 0.979 0.246 0.164 0.108 LSD(1%) 4.19 5.57 25.41 1.97 0.89 0.34 0.19 Genotype number SY (g/ m2) WPo/Pl(ph) WSe/Pl(ph) Wpo/Pl(bph) (g/ plant) (g/ plant) (g/ plant) WSe/Pl(bph) (g/ plant) HSW (g) L/W 1 315.9867 128.00 64.60 100.89 26.11 161.33 1.51 2 162.2253 92.80 47.37 114.00 33.22 139.00 1.40 3 204.0678 68.97 32.22 51.33 11.44 119.67 1.32 4 107.4393 56.23 30.27 70.78 27.56 48.67 1.39 5 88.15763 61.27 27.83 38.33 14.67 65.33 1.29 6 112.1333 34.83 18.73 33.67 17.89 48.00 1.30 7 71.08951 35.22 19.78 50.83 29.00 52.87 1.38 8 87.13008 33.07 19.13 31.56 17.22 50.00 1.32 9 82.35512 33.48 19.43 25.33 18.56 47.33 1.43 10 330.2933 193.97 85.03 175.89 30.67 166.00 1.42 Mean 156.0878 73.78 36.44 69.26 22.63 89.82 1.38 SE 45.985 16.506 7.136 15.133 2.382 15.995 0.022 LSD(1%) 262.25 71.77 38.71 0.23 0.15 47.54 0.13 Genotype number StP PP SP PSt SSt Se/Po(Ph) Se/Po(bPh) Se/Pl(bPh) Po/Pl(bPh) 1 4.46 12.19 43.11 2.77 9.22 0.50 0.27 0.11 0.41 2 4.31 9.69 43.44 2.19 8.19 0.51 0.34 0.17 0.50 3 3.31 5.18 15.67 1.67 4.48 0.47 0.23 0.10 0.44 4 2.76 10.27 33.11 3.72 11.32 0.54 0.41 0.19 0.48 5 2.64 6.03 12.11 2.35 5.64 0.45 0.39 0.18 0.45 6 3.86 8.67 16.44 2.19 4.08 0.56 0.52 0.18 0.35 7 3.23 10.69 23.39 3.19 6.79 0.57 0.53 0.23 0.42 8 3.17 7.04 17.33 2.18 5.18 0.56 0.56 0.21 0.37 9 3.24 9.58 17.33 3.03 4.36 0.59 0.64 0.27 0.35 10 4.16 7.50 50.78 1.85 12.91 0.44 0.19 0.09 0.47 Mean 3.51 8.68 2.95 2.51 7.22 0.52 0.41 0.17 0.42 SE 0.203 0.700 0.426 0.204 0.980 0.017 0.048 0.018 0.017 LSD(1%) 2.06 7.78 3.12 1.46 7.4 0.08 0.27 0.15 0.24 SE: Standard error; LSD: Least significant differences 3.3 Genetic parameter Estimates of phenotypic and genotypic variances and broad sense heritability from the partition of mean squares are presented in Table (4). Data indicated that the extent of phenotypic and genotypic variances varied from trait to another. Broad sense heritability (h2) estimates were generally high to moderate for all of studied traits. The highest estimates of broad sense heritability was inscribed as 98 % for pod length, dry seed length and dry seed width and 0.95 for hundred seed weight. The estimated broad-sense heritability was 0.80 for dry seed yield per m2. These results suggested that the environmental factors had a small effect on the inheritance of traits with high heritability. High estimates of heritability indicated that selection based on mean would be successful in improving these traits. The lowest value of h2 was observed for number of stems and pods per plant with 037 and 0.25, respectively. The estimated traits before maturity stage containing weight of pod per plant at physiological maturity stage, weight of seed per plant at physiological maturity stage and weight of seed per plant before physiological maturity stage had moderate heritability. The moderate value of heritability for number of pods per stem, number of seeds per stem and number of seeds per pod were 0.69, 0.66 and 0.68, respectively. In agreement with the results of this work, Toker (2004) recorded high to moderate heritability for traits containing days to flowering and maturity, 100-seed weight and seed yield and low heritability for number of stems and pods per plant. The results of this research are also similar to the results of Alghamdi (2007), Hanna and Hayes (1966), El-Kadi (1968), El-Kady and Khalil (1979), Bora et al. (1998), Ibrahim (2010) and Kalia and Sood (2004). The magnitude of heritability was affected by the type of genetic material and yield level of environment due to the fact that the plant height, number of stems and pods per plant, dry seed yield, 100-seed weight, days to pod initiation and maturity of plants are created by the effects of genes and environment. Table 4: Estimated some of genotypic and phenotypic parameters for studied traits in faba bean Traits Genotypic Phenotypic Broad-sense variance variance Heritability DP 4.28 5.34 0.80 DH 4.13 6.00 0.69 PL (cm) 9.37 9.60 0.98 DSP 0.56 0.61 0.92 LS (cm) 0.26 0.27 0.98 WS (cm) 0.11 0.12 0.98 SY (g/m2) 16995.75 21146.05 0.80 WPo/Pl(ph) 2413.51 2724.38 0.89 WSe/Pl(ph) 418.87 509.29 0.82 WSe/Pl(bph) 0.0022 0.0035 0.63 HSW (g) 2420.91 2557.29 0.95 L/W 0.0041 0.0051 0.81 StP 0.15 0.41 0.37 PP 1.23 4.89 0.25 PSt 0.29 0.42 0.69 SSt 6.32 9.62 0.66 SP 1.23 1.82 0.68 3.4 Path coefficient analysis The results of correlation coefficients between seed yield per m2 and were positively significant via the traits containing DH, PL, HSW, L/W, NStPl, NSeSt and NSePl. The significant negative correlation coefficient was detected between seed yield and days to pod initiation. The advantage of path analysis is that it permits the partitioning of the correlation coefficient into its components. One component is the path coefficient (or standardized partial regression coefficient) that measures the direct effect of a predictor variable upon its response variable. The other component is the indirect effect(s) of a predictor variable on the response variable through the predictor variables (Dewey and Lu, 1959). The results showed that the coefficient of determination were 54 % (Table 5). It represents the influence of the traits involved in the study on total variability of dry seed yield. Path coefficient analysis indicated that the traits containing day to harvesting (p=0.244), pod length (p=0.303), hundred seed weight (p=0.206) and number of stems per plant (p=0.216) play major role in seed yield determination of faba bean. This result concur with Alghamdi (2007) detected significant positive correlations between seed yield and each of number of pods per plant, number of seeds per plant, seed weight per plant and biological yield. In confirming with we results, Berhe et al., (1998) indicated the number of seeds per plant and 100-seed weight were the major direct contributors to seed yield per plant. These results also agree with those of Tadesse et al. (2011) and Ulukan et al. (2003) that found out days to maturity, number of pod per plants, seed per pod, thousand seed weight and plant height, pod length, and grain number per pod had high positive direct effect on seed yield per plot. Table 5: Path coefficients for seed yield components in faba bean. The diagonal under line numbers is direct effects of any trait on seed yield Traits DP DH PH PL HSW L/W StP SSt SP Overall effects (r) DP -0.019 0.005 0.003 0.011 0.009 -0.003 0.007 0.003 0.008 -0.43 DH -0.073 0.244 0.059 0.121 0.144 0.099 0.107 0.132 0.115 0.650 PH -0.006 0.007 0.032 0.009 0.004 0.004 0.016 0.008 0.010 0.330 PL -0.166 0.151 0.089 0.303 0.229 0.068 0.120 0.149 0.236 0.697 HSW -0.098 0.122 0.027 0.156 0.206 0.075 0.095 0.080 0.125 0.719 L/W 0.014 0.037 0.013 0.020 0.033 0.092 0.020 0.044 0.028 0.372 NStPl -0.084 0.095 0.111 0.085 0.099 0.049 0.216 0.062 0.077 0.578 NSeSt 0.006 -0.017 -0.008 -0.015 -0.012 -0.015 -0.009 -0.032 -0.026 0.453 NSePl 0.002 -0.002 0.001 0.004 0.003 0.002 0.002 0.005 0.006 0.581 R2=0.54 V1 - R2 =0. 56 The symbol of traits is the same as in Table 2 3.5 Biplot analysis The polygon view of the biplot will be displayed (Figure 1). This view helps identify cultivars with the highest values for one or more traits. The scores of three traits containing PH, NStPl and LW fell in the genotype 10 sector, suggesting that this cultivar had highest or near-highest values for these three traits. Genotype 1 had the highest values for PH, NStPl and L/W. Since the biplot did not explain all the variation, these predictions may not exactly reflect the observed numbers. Nonetheless, cultivars that are among the top with regard to a trait can be identified with confidence. Yan and Kang (2003) demonstrated the numerous utilities of genotype and genotype-by-environment (GGE) biplot in visual analysis of genotype-by-trait data for evaluating cultivars based on traits and comparing cultivars as packages of traits. Sharifi and Safari Motlagh (2011) and Sharifi (2012) were also used biplot techniques for analysis the data obtained from diallel crosses in rice. Mohammadi and Amri (2011) examined the performance of 13 durum (Triticum turgidum L. var. durum Desf.) genotypes on the basis of multiple traits by biplot method. The tester vectors (Figure 2) are the lines that originate from the biplot origin and reach markers of the traits. Since the cosine of the angle between the vectors of any two traits approximates the correlation coefficient between them, this view of the biplot is best for visualizing the interrelationship among traits. Figure 2 suggests close positively associations among PL, L/W, HSW, DH, NStPl and dry seed yield per m2. Plant height indicated a weak but positive correlation with dry seed yield. GGE biplot were also used for identifying traits that are closely associated with, and therefore can be used in indirect selection for, a target trait (Yan and Kang, 2003). Oladejo et al. (2011) used biplot method to determine the interrelationships among physiological traits of thirty cowpea cultivars and identify suitable traits for indirect selection for improved crop yield. 1.40- 0.84- 0.56- p 0.28 c 2 0.00- -0.28- -0.56- -0.84- -1.12- PC1 = 70.3%, PC2 = 12.3 fc. Sum = 82.6% / PH gl - g3 StP L/W 810 SY % / g2 \ DH HSW / PL 'h "1 r -0.84 -0.56 -0.28 0.00 0.28 0.56 0.84 PC 1 ~r i r .12 1.40 1.68 Figure 1: Biplot analysis indicating polygon view for some of traits in faba bean genotypes g1: Autochthonous landrace (Guilan); g2: Autochthonous landrace (Mazandaran); g3: France; g5: Filip5; g6: Autochthonous landrace (Khorramabad); g9: Autochthonous landrace (Borujerd); g10: Barrakat 1.40- 1.12- 0 84- 0.56- p 0.28-C 2 nnn. -0.28- -0.56- -0.84- -1.12- PC1 = 70.3%, PC2 = 12.3%, Sum = 82.6% g6 /PH / S1 StP --■—.—.--._SY g10 g2 DH S5 ^-PL g3 1 I I I I T -0.84 -0.56 -0.28 0.00 0.28 0.56 O.i P C 1 "i i r 1.12 1.40 1.68 Figure 2: Biplot analysis indicating relationships between some of traits in faba bean genotypes. g1: Autochthonous landrace (Guilan); g2: Autochthonous landrace (Mazandaran); g3: France; g5: Filip5; g6: Autochthonous landrace (Khorramabad); g9: Autochthonous landrace (Borujerd); g10: Barrakat 4 CONCLUSION This study indicated that agro-morphological traits were significantly differed in the investigated faba bean genotypes. Among the 10 faba bean genotypes used in this study, G1 and G10 showed significantly higher magnitude values than other genotypes. G1 and G10 are an autochthonous landrace from Guilan, Iran and improved breeding variety, naming Barrakat, respectively. The variability among the faba bean genotypes was expected because of their different origins. Heritability of large number of traits was high and moderate. High estimates of heritability indicated that selection based on mean would be successful in improving these traits. High heritability indicate an additive gene action and, hence, possible trait improvement through selection. Some of genotypes such as G5, G7, G8 and G9 can be crosses with G1 and G10 and used their offspring's for breeding programs such as QTL mapping, diallel analysis and generation mean analysis. 5 ACKNOWLEDGMENTS This work was the part of first author's research project "Study yield and some of agronomic traits in faba bean (Vicia faba L.) genotypes on Guilan and Lorestan province" which supported by "Islamic Azad University, Rasht Branch, Youngh Researchers Club" in Guilan province. 6 REFERENCES Abo-El-Zahab A.A., Ashor A.M., El-Hadeedy K.H. (1980). Comparative analysis of growth, development and yield of five field bean cultivars (Vicia faba L.) Z. Acker-und Pflanzenbau. 149: 113. Alghamdi S.S. (2007). Genetic Behavior of Some Selected Faba Bean Genotypes. African Crop Science Conference Proceedings. 8: 709-714. Al-Barri T., Shtaya M.J.Y., (2013). Phenotypic characterization of faba bean (Vicia faba L.) landraces grown in Palestine. Journal of Agricultural Science; 5: 110-117. Basheer-Salimia R., Shtaya M., Awad M., Abdallah J., Hamdan Y. (2013). Genetic diversity of Palestine landraces of faba bean (Vicia faba) based on RAPD markers. Genetics and Molecular Research 12 (3): 3314-3323; DOI: 10.4238/2013.September.3.8 Berhe, A., Bejiga, G., Mekonnen, D., (1998). Associations of some characters with seed yield in local varieties of Faba bean. African Crop Science Journal. 6: 197-204. Bond D.A., Lawes D.A., Hawtin G.C., Saxena M.C., Stephens J.H. (1985). Faba bean (Vicia faba L.). In: Summerfield RJ, Roberts EH (Eds) Grain Legume Crops, Collins, London: 199-265. Bond D.A. (1966). Yield and components of yield in diallel crosses between inbred lines of winter beans (Vicia faba). Journal of Agricultural Science of Cambridge. 67: 335-336; DOI: 10.1017/S0021859600017329 Bora G.C., Gupta S.N., Tomer Y.S., Singh S. (1998). Genetic variability, correlation and path analysis in faba bean (Vicia faba). Indian Journal of Agricultural Sciences. 68(4): 212-214. Cubero, J.I. (1974). The evolution of vicia faba. Theoretical and Applied Genetics, 45: 47-51; DOI: 10.1007/BF00283475 De Pace C. (1979). Characteristics with significant correlation to seed yield and broad bean population grown in Southern Italy. In: Semi Current research on Vicia faba in Western Europe. Ed BABND, GR Scarascia Mugnozza and M.H. Poulsen, Pub. EECEUR, 6244 En., Luxembourg: 144-167. Dewey D.R., Lu K.H. (1959). A correlation and path coefficient analysis of components of crested wheat grass seed production. Agronomy Journal. 51: 515— 518; DOI: 10.2134/agronj1959.00021962005100090002x El-Kady D.A. (1968). Genotypic and phenotypic variability and their implications in Vicia faba L. breeding. M.Sc. Thesis, Cairo University: 138 p. El-Kady M.A.K., Khalil S.A. (1979). Behaviour of seed yield components in cross between broad bean cultivars and selection for superior yield. Egyptian Journal of Agronomy. 4: 159-170. Hanelt P., Metin D. 1989. Biosystematics of the genus Vicia L. (Leguminosae). Annual review of ecological systems, 20: 199-223. Hanna A.S., Hayes J.D. (1966). Genetic studies in field beans, Vicia faba L. I. Genetic variability in flower, pod and seed characters in a cross between two inbred lines. Z. Pflanzenzuchtg. 56: 285-294. Hawtin G.C., Hebblethwaite P.D. (1983). The faba bean (Vicia faba L.).: A basis for improvement, (P.D. Hebblethwaite, ed). Butterworths, London, UK. pp 3-22. Ibrahim H.M. (2010). Heterosis, Combining Ability and Components of Genetic Variance in Faba Bean (Vicia faba L.). Meteorology, Environment and Arid Land Agriculture Environmental Science. 21(1): 35-50. Kalia P., Sood S.H. (2004). Genetic variation and association analysis for pod yield and other agronomic and quality characters in an Indian Himalayan collection of broad bean (Vicia faba L.). SABRAO Journal of Breeding and Genetics. 36(2): 55-61. Loss S.P., Siddique K.H.M. (1997). Adaptation of faba bean (Vicia faba L.) to dryland Mediterranean-type environments. I. Seed yield and yield components. Field Crops Research. 52: 17-28; DOI: 10.1016/S0378-4290(96)03455-7 Mohammadi R., Amri A. (2011). Graphic Analysis of Trait Relations and Genotype Evaluation in Durum Wheat. Journal of Crop Improvement. 25: 680-696. Oladejo A.S., Akinwale R.O., and Obisesan I.O. (2011). Interrelationships between grain yield and other physiological traits of cowpea cultivars. African Crop Science Journal. 19: 189 - 200. Roy D. (2000). Plant breeding analysis and exploitation of variation. Alpha Science. 798 p. Sharifi P. (2012). Graphic analysis of salinity tolerance traits of rice (Oryza sativa L.) using biplot method. Cereal Research Communications. 40(3): 416-424; DOI: 10.1556/CRC.40.2012.3.3 Sharifi P., Safari Motlagh M.R. (2011). Biplot analysis of diallel crosses for cold tolerance in rice at the germination stage. Crop and Pasture Science. 62(2): 169-176; DOI: 10.1071/CP10207 Steel R.G.D., Torrie J.H. (1980). Principles and Procedures of Statistics. McGraw-Hill Book Company, Inc., New York: 633 p. Tadesse T., Fikere M., Legesse T., Parven A. (2011). Correlation and path coefficient analysis of yield and its component in faba bean (Vicia faba L.) germplasm International Journal of Biodiversity and Conservation. 3: 376-382. Terzopoulos P.J., Bebeli P.J. (2008). Genetic diversity analysis of Mediterranean faba bean (Vicia faba L.) with ISSR markers. Field Crops Research. 108: 3944; DOI: 10.1016/j.fcr.2008.02.015 Toker C. (2004). Estimates of broad-sense heritability for seed yield and yield criteria in faba bean (Vicia faba L.). Hereditas. 140: 222-225; DOI: 10.1111/j.1601-5223.2004.01780.x Ulukan H., Mustafa G., Siddik K. (2003). A Path Coefficient Analysis Some Yield and Yield Components in Faba Bean (Vicia faba L.) Genotypes. Pakistan Journal of Biological Sciences. 6: 1951-1955; DOI: 10.3923/pjbs.2003.1951.1955 Yan W. (2001). GGE biplot - a Windows application for graphical analysis of multienvironment trial data and other types of two-way data. Agronomy Journal 93: 1111-1118; DOI: 10.2134/agronj2001.9351111x Yan W., Hunt L.A. (2002). Biplot analysis of diallel data. Crop Science. 42: 21-30; DOI: 10.1201/9781420040371 Yan W., Kang M. (2003). GGE Biplot Analysis: A Graphical Tool for Breeders, Geneticists, and Agronomists. CRC Press, 288 pages. COBISS Code 1.01 DOI: 10.14720/aas.2015.105.1.09 Agrovoc descriptors: zea mays, maize, soil salinity, site factors, stress, chemicophysical properties, seeds, germinability, genotypes, varieties, photosynthesis, pigments, carotenoids, chlorophyll Agris category code: f62 The effect of salt stress on the germination of maize (Zea mays L.) seeds and photosynthetic pigments Sali ALIU1, Imer RUSINOVCI1, Shukri FETAHU1, Bekim GASHI2, Emilija SIMEONOVSKA3, Ludvik ROZMAN4 Received February 12, 2015; accepted March 09, 2015. Delo je prispelo 12. februarja 2015, sprejeto 09. marca 2015. ABSTRACT IZVLEČEK The objective of this study was to investigate the effect of salinity stress on seed germination and chlorophyll content in maize. In the study, two maize hybrids were included (Bc 678 and Bc 408) originating from the Bc Institute at Rugvica near Zagreb (Croatia) and two maize populations (LMP-1 and LMP-2) originating from Kosovo. The experiment was conducted in four replicates of 100 seeds, which were germinated on top of double-layered papers, each with 10 ml of salt solution of NaCl and CaCl2 in Petri dishes. Germinated seeds were counted every 24 h for 15 days. The photosynthetic pigments, chlorophylls 'a' and 'b' as well as carotenoids were extracted with 80 % acetone. Chlorophyll and carotenoid contents were calculated using absorbance values at 662, 644 and 440 nm. The effects of the NaCl and CaCl2 concentrations accounted for a high proportion of the variance in all analyses. The results showed that both germination percentage and germination index decreased significantly in all cultivars at the highest salt concentrations. The significant differences between different concentrations of salinity were also found in all cultivars for the content of chlorophyll 'a' and 'b' and for the content of carotenoids. Key words: maize, salinity stress, germination, NaCl, CaCl2, chlorophyll, carotenoids VPLIV SLANOSTNEGA STRESA NA KALIVOST IN FOTOSINTEZNE PIGMENTE KORUZE (Zea mays L.) Namen raziskave je bil proučiti vpliv slanosti tal na kalivost zrnja ter vsebnost kolorofila in karotinoidov pri koruzi. V proučevanje sta bila vključena dva hibrida 'Bc 678' and 'Bc 408', vzgojena na Zavodu za koruzo Inštituta za žlahtnjenje rastlin v Zagrebu ter dve domači populaciji (LMP-1 and LMP-2) s Kosova. Poskus je bil izveden v 4 ponovitvah in sicer za kalivost v petrijevkah po 100 zrn, za vsebnost klorofila in karotinoidov pa v lončkih po 2 rastlini z 1 kg substrata. Vsak genotip je bil, poleg kontrole, tretiran s 4 različnimi koncentracijami (50, 100, 200 in 400 mMol NaCl in CaCl2 ). Kalivost smo ugotavljali prvih 15 dni vsakih 24 ur. Klorofil 'a' in 'b' in karotinoide smo ekstrahirali z 80 % acetonom. Vsebnost klorofila in karotinoidov smo računali s pomočjo absorpcijske vrednosti pri 662, 644 and 440 nm. Pri največji slanosti (400 mMol NaCl in CaCl2) je pri vseh kultivarjih ugotovljen statistično značilno manjši odstotek in indeks kalivosti. Prav tako so pri vseh kultivarjih ugotovljene statistično značilne razlike med različnimi koncentracijami slanosti tudi za vsebnost klorofila 'a' in 'b' ter vsebnost karotinoidov. Ključne besede: koruza, slanost tal, kalivost, NaCl, CaCl2, klorofil, karotinoidi 1 University of Prishtina, Faculty of Agriculture, Department of Crop Science, Prishtina, Kosovo; e-mail: sali.aliu@uni-pr.edu 2 University of Prishtina, Faculty of Natural Science, Department of Biology, Prishtina, Kosovo; e-mail: bekim.gashi@uni-pr.edu 3 The Faculty of Agriculture Science and Food, Skopje, Macedonia University of Ljubljana, Biotechnical Faculty, Ljubljana, Slovenia; e-mail: ludvik.rozman@bf.uni-lj.si 4 1 INTRODUCTION Salinity stress negatively impacts agricultural yields throughout the world, affecting production, whether for subsistence or economic gain. At present, about 20 % of the world's cultivated land and approximately half of all irrigated land and 2.1 % of the dry agriculture land is affected by salinity (FAO, 2000). Salinization is spreading more rapidly in irrigated lands because of inappropriate management of irrigation and drainage. Moreover, rain, cyclones and wind add NaCl to coastal agricultural lands (FAO, 2008). Maize (Zea mays L.) is the important cereal crop, providing basic food and oil for human cosumption, as well as feed for livestock throughout the world, but this crop is normally submissive to salt stress. Maize, a plant with a C4 metabolism, is also classified as moderately sensitive to salinity (Katerji et al., 1994). The rapid increase in the world's population requires an expansion of crop areas to raise food production. In this context, a significant part of agricultural crops is cultivated on low quality soils, which are sometimes affected by salinity (Allen et al., 1983). Different strategies for diminishing the adverse effects of salinity stress on plants are currently in practice. Salinity due to the over-accumulation of NaCl is usually of great concern and is the most damaging factor in arid and semi-arid regions. Saline soils are widespread throughout the world, and their genesis may be natural or accelerated by irrigated agriculture, the intensive use of water resources combined with high evaporation rates and human activity (Lambers, 2003; Arzani, 2008). The osmotic adjustment, i.e. the reduction of cellular osmotic potential by net solute accumulation, has been considered to be an important mechanism of salt and drought tolerance in plants. This reduction in osmotic potential in salt-stressed plants can be a result of inorganic ion (Na+, Cl-, and K+) and compatible organic solute (soluble carbohydrates, amino acids, proline, betaines, etc.) accumulations (Hasegawa et al., 2000). Salinity-induced crop yield reduction takes place due to a number of physiological and biochemical disfunctions in plants grown under salinity stress, which have been listed in a number of comprehensive reviews (Kaya et al., 2013). Salinity is considered to be a major abiotic stress affecting germination, seedling growth, and crop production in arid and semi-arid regions (Yohannes and Abraha, 2013). Moreover, salinity has an adverse effect on seed germination of many crops, by creating an osmotic potential outside the seed, thereby inhibiting the absorption of water, or by the toxic effect of Na+ and Cl- (Khajeh-Hosseini et al., 2003). Therefore, salinity is one of the most significant abiotic factors limiting crop productivity (Munns, 1993; Gama et al., 2007). The ability of seeds to germinate at high salt concentrations in the soil is of crucial importance for the survival of many plant species. Although salinity stress mostly reduces the germination percentage and delays the onset of germination, its effects are modified by interactions with other environmental factors, such as temperature and light (Bojovic et al., 2010). In saline habitats, satisfactory seed germination takes place after high precipitation, when the soil salinity is reduced (Khan and Rizvi, 1994). Seed priming stimulates many of the metabolic processes involved in the early phases of germination, and it has been observed that seedlings from primed seeds grow more vigorously, and perform better in adverse conditions (Cramer, 2002). It has been shown that soil salinity increases P, Mn, and Zn and decreases K and Fe concentrations in plant tissues (Turan et al., 2010). The present study was to investigate the response of maize seed germination, the content of chlorophyll 'a', 'b' and carotenoid content to different salinity concentration of NaCl and CaCl2. 2 MATERIAL AND METHODS 2.1 Plant material The plant material that was included in our study was two maize hybrids ('Bc 678' and 'Bc 408') originating from the Maize Dept. of Bc Institute at Rugvica near Zagreb (Croatia) and two maize populations (LMP-1 and LMP-2) originating from Kosovo. The experiment was done in the Department of Crop Science, Laboratory of Plant Breeding, University of Prishtina. The seeds were disinfected in NaOCl 1% for 60 minutes and then rinsed three times with distilled and sterilized water. Maize seeds were germinated on moistened filter paper. The prepared seeds were placed on the germinator for germination (after addition of 10 ml H2O) for ten days in temperature 25 °C. Pots were filled with compost (minimum 1 kg/pot) for each cultivar and for each treatment. In total, 32 pots were prepared for salt treatment including NaCl and CaCl2, and a control. During the experiment, solutions with different concentrations for each salt treatment was prepared in the growth period. For two salts (NaCl and CaCl2), the concentrations were 50, 100, 200 and 400 mMol. After 20 days of exposure, the following parameters were determined in different parts of the plants: chlorophyll pigments and concentration, and seed germination. After disinfection, the seeds were divided into nine treatment groups for each salt solution: H2O (Control), 50, 100, 200, 400 mMol NaCl and 50, 100, 200, 400 mMol CaCl2. 2.2 Soil material The compost consisted of pH (CaCl2)=5.8; salt concentration (g L-1 KCl=0.9; Nitrogen (NH4+NO3)=155 mg L-1, CaCl2=120; Phosphorus (P2O5) mg L-1 CAL=150 and potassium (K2O) mg L-1 CAL=200. The maize seedlings were transfered to compost in 1 kg weight pots in controlled environment cabinets with 12-hour photoperiods and temperatures of 25/19 °C day/night and 75 % relative humidity. 2.3 Seed germination assays Germination and early seedling growth were compared at 25 °C (optimum temperature) in the dark. The filter papers were moistened with 20 ml distilled water. For all the seeds groups, the experiment was conducted with four replicates of 100 seeds. Seeds were germinated on top of double-layered papers (ISTA, 1996) with 10 ml of each of the salt solutions of NaCl and CaCl2 in 10 cm Petri dishes (4 Petri dishes x 25 seeds = 100 seeds x 4 replications = 400 seeds per treatment). These Petri dishes were placed in sealed plastic bags to avoid moisture loss. Seeds were allowed to germinate at 24±1 °C and for 16 hours on the light (day) and 8 hours in the dark (night). The germination percentage is an estimate of the viability of seeds. Germinated seeds were counted every 24 hours for 15 days. According to Sharma (2010), seeds were considered to have germinated upon the emergence of radicles (> 2 mm). Full Germination Percent (FGP) was calculating according to equation: FGP = ^rlQQ where N - is total seed number; n - number of germinated seeds. The Mean Germination Time (MGT) was calculated for each lot, using the daily counts, according to the equation (Moradi et al., 2008): MGT = ZnB where n= number of seeds newly germinated at day D, D - days from the beginning of the germination test, £ n -number of all germinated seeds (final germination). Germination Index (GI) was calculated as described in the Association of Official Seed Analysis (AOSA, 1983) according to the following formula: where GI - is Germination Index, G1, G2, .... Gx - is germination at 1, 2, .. ..x day, 1, 2, .... x - day of counting of germinated seeds. 2.4 Pigments analysis Pigments were extracted by grinding 60-80 mg freshly sampled leaves. At the time of sampling, the plants reached the stage of five leaves; we took the third leaf and put in an 80% acetone/water solution containing MgCO3 (0.5% w/v), at room temperature for 24 hours in the dark. Photosynthetic pigments of all samples were extracted in triplicate to minimize experimental errors. Concentrations of chlorophyll and carotenoid contents were measured by using absorbance recorded at 662, 644 and 440 nm for maximum absorption of chlorophyll 'a' (Chl 'a'), chlorophyll 'b' (Chl 'b') and carotenoids, respectively. The extinction coefficients were determined by a UV-Vis spectrophotometer (SECOMAM, Anthelie Advanced 5). Pigment contents were calculated in mg g-1 fresh leaf weight (FW) by applying the absorption coefficient equations, described by Lichtenthaler (1986); Aliu et al. (2013 and 2014); Gashi et al. (2013): Chl 'a' (mg g-1 FW) = [9.784 (OD662) - 0.99 (OD644)] x V/FW, Chl 'b' (mg g-1 FW) = [21.426 (OD644) - 4.65 (OD662)] x V/FW, Carotenoids (mg g-1 FW) = [4.695 (OD440) - 0.268 (Chl a + Chl b)] x V/FW. Where is: FW - fresh leaf weight, OD - optical density, V - volume of sample. 2.5 Statistical analysis SPSS version 19 was used for analysis of variance for all parameters and to compare of treatment means with Duncan's Multiple Range Test. Relationships among the traits were estimated with Pearson correlation analysis. 3 RESULTS AND DISCUSSION Analyses of variance showed a wide range and highly significant effects of NaCl and CaCl2 concentrations on the parameters of seed germination. The effects of the NaCl and CaCl2 concentrations accounted for a high proportion of the variance in all analyses include Full Germination Percent (FGP), Mean Germination Time (MGT) and Germination Index (GI) (Table 1). The FGP at all maize genotypes ranged from 14 to 100%, depending on treatments. FGP for the hybrid 'Bc 678' was low (16 and 44 %) after treatments with 400 mMol CaCl2 and 400 mMol NaCl, respectively. When comparing these values (16 and 44%) to the any other values (Control, 50, 100 and 200 mMol CaCfe and mMol NaCl), they were significantly lower. In the case of hybrid 'Bc 408', between different treatments the same differences for FGP are also present, while lower values (20 and 74%) were recorded in treatment with 400 mMol NaCl and CaCl2. In comparison with 'Bc 678', the hybrid 'Bc 408' had the highest (87.8%) average values of FGP. The applied high concentrations with NaCl and CaCl2 for FGP on treatments, as well as 400 mMol in seed Local Maize Populations (LMPs) had negative effects or inhibited the physiological processes. LMP-1 and LMP-2 had significantly lower values of FGP after the treatments of 200 and 400 mMol CaCl2 and NaCl than the control. In both hybrids, the differences in FGP, in treatments with 50, 100, 200 mMol, both for NaCl and CaCl2 concentrations, resulted in no significant differences, except in the case of the hybrid 'Bc 678' after the treatment with 200 mMol CaCl2. In both populations (LMP-1 and LMP-2) significant differences exist almost among all treatments. Therefore, the populations could be more susceptible to salinity stress than hybrids. However, the seed germination percentage of populations decreased at the highest level of salinity, but the hybrid 'BC 678' expresses significantly higher values of FGP at 50 and 100 mMol CaCl2 and at 50, 100 and 200 mMol NaCl than the control does. Many authors (Amzallag et al., 1990; Djanaguiraman et al., 2006) found that plants' exposure to low level salinity activates an array of processes leading to an improvement of plant stress tolerance. High salt concentrations negatively affect maize growth. Rahman et al. (2000) reported that maize cultivars were significantly more tolerant to salt stress at germination than at later stages of growth. In order to determine the usefulness of Tripsacum in improving salt tolerance in maize, and the effects of NaCl, in vitro and in vivo, Pesqueira et al., (2006) evaluated an intergeneric hybrid obtained from crossing Zea mays L. with Tripsacum dactyloides L. The different levels of NaCl and CaCl2 concentrations also significantly affected the mean germination time (MGT) and germination index (GI) (Table 1). The significantly greater number of days for MGT at all genotypes were obtained from treatments in 400 mMol NaCl and CaCl2 concentrations; furthermore, the concentration of 400 mMol CaCl2 also resulted in a significantly higher number of days when compared to the same concentration of NaCl. Similar findings were also obtained for GI. For both parameters, the greatest differences between control and treatments in 400 mMol NaCl and CaCl2 concentrations for maize populations than for hybrids were obtained. For maize hybrids, the MGT at 400 mMol CaCl2 and NaCl concentration the 6.87 and 6.50 days were obtained, respectively; while for maize populations (LMP) the values 7.94 and 7.88 were obtained. On the basis of these results, we can conclude that the maize populations are more responsive to soil salinity than hybrids. Taiz and Zeiger (2002) concluded that the high concentration of NaCl in the salt solution increases its osmotic potential. In addition, the high absorption of Na and Cl ions during seed germination can be due to the cell toxicity that finally inhibits or slows the rate of germination and thus decreases the germination percentage. Moreover, the germination indices of all the cultivars decreased with increasing salt stress (Carpici et al., 2009). The leaf is a very important photosynthetic organ, in which light energy is transformed through the green pigment chlorophyll into the potential energy of asimilates. Our results show that the chlorophyll content concentration was significantly changed under different salinity concentrations (Table 2). In many cases, the significantly higher content of chlorophyll 'a' was found at lower salinity concentrations of only NaCl. The significantly higher content of chlorophyll 'a' than in control was obtained at concentrations of 100 mMol NaCl ('Bc 408' and LPM-1), at concentrations of 50 and 200 mMol NaCl ('Bc 678'), and at concentrations of 50, 100 and 200 mMol NaCl (LPM-2). Significantly lower contents of chlorophyll 'a' were found at higher concentrations of 100, 200 and 400 mMol CaCl2 ('Bc 678'), 200 and 400 mMol CaCl2 ('Bc 408' and LPM-2) and at 400 mMol CaCl2 (LMP-1). NaCl was affected on lower chlorophyll ' a' at all cultivars only at 400 mMol concentrations. Similar results for chlorophyll 'a' in different treatments of maize were obtained by Daughtry et al. (1999), ranging from 10.4 to 34.6 mg g-1. The content of chlorophyll 'b' is less variable than chlorophyl 'a' under different salinity concentrations. Significantly higher contents of chlorophyll 'b' were found only at 'Bc 678' (50 mMol CaCl2 and 200 mMol NaCl), at LMP-2 (50, 100 and 200 mMol NaCl); while the lowest chlorophyll 'b' contents at 200 and 400 mMol CaCl2 ('Bc 678', 'Bc 408' and LMP-2), at 400 mMol CaCl2 (LMP-1) and only at 400 mMol NaCl ('Bc 408', LMP-1 and LMP-2) were found. In general, the highest salinity concentrations reduced content of both chlorophyll 'a' and 'b' compared to the control. The significantly lower content of carotenoides was determined at higher concentrations of treatments, 400 mMol CaCl2, at all cultivars and at 400 mMol NaCl at both LMP (Table 2). The most responsive to salinity stress relating to carotenoides content was LMP-2; at both highest concentrations, it showed the lowest carotenoid content, while at 50, 100 and 200 mMol NaCl concentrations it showed significantly higher carotenoids content than the control. Table 1: The effect of salinity on seed germination in maize cultivars Treatment 'Bc 678' 'Bc 408' LMP-1 LMP-2 FGP * (%) MGT (days) GI % FGP (%) MGT (days) GI % FGP (%) MGT (days) GI % FGP (%) MGT (days) GI % Control 96b 4.0c 59.3ab 100a 4.0c 61.7a 100a 4.0cd 61.7a 88b 4.1de 53.4 50 mM CaCl2 100a 4.0c 61.7a 100a 4.0c 61.7a 100a 4.0cd 61.4a 82b 4.0e 50.5 100 mM CaCl2 100a 4.0c 61.7a 100a 4.0c 61.5a 98a 4.1bc 59.9a 96a 4.1de 57.9a 200 mM CaCl2 96b 4.1c 57.9b 96a 4.0c 55.8b 68b 4.7ab 35.3c 70c 4.2d 39.5 400 mM CaCl2 16d 6.9a 3.6d 20c 6.5a 5.2d 28c 7.9a 3.7e 14d 7.9a 2.0 50 mM NaCl 100a 4.0c 61.7a 100a 4.0c 61.7a 100a 4.0cd 60.5a 86b 4.0e 53.1 100 mM NaCl 100a 4.0c 61.7a 100a 4.0c 61.7a 94a 4.0cd 57.3a 94a 4.0e 58.0a 200 mM NaCl 100a 4.0c 61.7a 100a 4.0c 61.0a 72b 3.4d 46.1b 88b 4.6c 46.8c 400 mM NaCl 44c 4.6b 23.1c 74b 5.3b 33.6c 20c 5.0b 9.7d 16d 6.2b 5.9e Average 83.6 4.4 50.3 87.8 4.4 51.6 75.6 4.6 44.0 70.2 4.8 40.8 a a c e *FGP - final germination percentage; MGT - mean germination time; GI - germination index; * - values within individual columns indicated by at least one equal letter are not significantly different at 0.05 probability level The effect of salt stress on the germination of maize (Zea mays L.) seeds and photosynthetic pigments Table 2: Effect of salinity on photosynthetic pigments content (mg g-1 FW) of maize cultivars 'Bc 678' 'Bc 408' LMP-1 LMP-2 Treatment Chl a Chl b Carot Total Chl Ratio alb Chl a Chl b Carot Total Chl Ratio alb Chl a Chl b Carot Total Chl Ratio alb Chl a Chl b Carot Total Chl Ratio alb Control 38.8c* 8.8bc 5.6bc 47.5c 4.4bc 37.3b 10.1a 5.8ab 47.3c 3.8a 39.5bc 10.1a 6.2ab 49.6bc 4.0a 37.9b 8.9c 5.6c 46.7b 4.2a 50 mM CaCl2 44.8abc 10.9a 6.2ab 55.8ab 4.1ab 38.9b 9.5abc 5.7ab 48.4c 4.2a 38.8bc 10.8a 5.2b 49.6bc 3.7ab 41.1c 9.6c 5.9bc 50.7b 4.3a 100 mM CaCl2 31.2d 7.8cd 4.9cd 39.1d 4.0cd 37.3b 9 4abc 5.4ab 46.7c 4.0a 43.8b 10.1a 6.0ab 53.9bc 4.3a 23.4d 5.7e 3.2d 29.1d 4.1a 200 mM CaCl2 27.3d 6.8de 3.9d 34.1d 4.0d 30.6c 7.4c 5.6b 38.0d 4.2a 36.7c 8.6a 5.5ab 45.2c 4.3a 33.0c 7.5d 4.7c 40.5c 4.4a 400 mM CaCl2 20.1e 5.3e 3.8d 25.4e 3.8d 9.7d 3.5d 2.1c 13.2e 2.8b 6.1e 2.2b 1.3d 8.2e 2.8b 10.0e 2.8f 2.2d 12.9e 3.6b 50 mM NaCl 46.2ab 11.6a 7.1a 57.8 ab 4.0a 40.9ab 9.8ab 5.9ab 50.7bc 4.2a 38.5c 9.2a 5.8ab 47.7bc 4.2a 48.0a 11.3b 7.3a 59.3a 4.3a 100 mM NaCl 41.3bc 9 9ab 6.2ab 51.3bc 4.2ab 44.3a 10.6a 6.4a 55.2a 4.2a 47.0a 11.2a 6.9a 58.2a 4.2a 52.3a 12.6a 8.3a 64.9a 4.2a 200 mM NaCl 48.5a 11.1a 6.6ab 59.6a 4.3ab 42.3ab 10.9a 6.3a 5 3. 1ab 3.9a 39.2bc 9.7a 5.4b 49.0bc 4.1a 49.7a 11.9ab 7.1ab 61.7a 4.2 a 400 mM NaCl 21.5c 6.2ab 6.1ab 27.7d 3.5d 29.8c 7 7bc 4.9ab 37.5d 3.9a 15.1d 4.2b 2.7c 19.2d 3.6ab 20.0d 5.9e 3.4d 25.1d 3.9b Average 35.5 8.7 5.6 47.0 4.1 34.5 8.8 5.3 43.3 3.9 33.9 8.4 5.0 42.3 3.9 35.1 8.5 5.3 43.4 4.1 * - values within individual columns indicated by at least one equal letter are not significantly different at 0.05 probability level In general, the correlation coefficients between all the studied properties for most cultivars were positive and statistically significant, except for the MGT, for which there was a negative and significant correlation (Table 3). Only for hybrid 'Bc 678' were a lower value of correlation coefficients between the ratio of chlorophyll 'a' and 'b' and other properties obtained; statistically nonsignificant correlation coefficients were obtained only for MGT and chlorophyll 'a'. Wue et al. (2008) investigated similar issues and have obtained similar results. Table 3: The correlation coefficients between investigated traits in maize cultivars Treatment FGP MGT GI Chl 'a' Chl 'b' Carotenoids Chl (a+b) FGP 1 MGT -0.90** 1 GI 1 ** -0.90** 1 00 t-» vo Chl 'a' 0.50** -0.62** 0.51** 1 o PQ Chl 'b' 0.48* -0.58** 0.48* 0.95** 1 Carot. 0.50** -0.62** 0.51** 0.99** 0.96** 1 Total Chl 0.39* -0.48** 0.40* 0.92** 0.92** 0.93** 1 Ratio a/b 0.30 * -0.38 0.30 * 0.42 0.13 0.37 0.24 FGP 1 MGT -0.97** 1 GI 0.98** -0.99** 1 00 o Chl 'a' 0.92** -0.93** 0.92** 1 o PQ Chl 'b' 0.86** -0.89** 0.87** 0.93** 1 Carot. 0.91** -0.93** 0.92** 0.99** 0.95** 1 Total Chl 0.85** -0.87** 0.85** 0.93** 0.98** 0.95** 1 Ratio a/b 0.76** -0.72** 0.73** 0.75** 0.50** 0.71** 0.54** FGP 1 MGT -0.69** 1 GI 0.98** -0.80** 1 i Chl 'a' 0.88** -0.84** 0.92** 1 S -j Chl 'b' 0.86** -0.82** 0.90** 0.93** 1 Carot. 0.89** -0.85** 0.92** 0.99** 0.95** 1 Total Chl 0.85** -0.82** 0.88** 0.96** 0.91** 0.96** 1 Ratio a/b 0.55** -0.65** 0.58** 0.72** 0.45* 0.68** 0.69** FGP 1 MGT -0.91** 1 GI 0.99** -0.93** 1