ISSN 1313-8820 Volume 5, Number 4 December 2013

ISSN 1313-8820 Volume 5, Number 4 December 2013 2013

Editor-in-Chief Tsanko Yablanski Faculty of Agriculture Trakia University, Stara Zagora Bulgaria Co-Editor-in-Chief Radoslav Slavov Faculty of Agriculture Trakia University, Stara Zagora Bulgaria Editors a Sections Genetics a Breeding Atanas Atanasov (Bulgaria) Ihsan Soysal (Turkey) Max Rothschild (USA) Stoicho Metodiev (Bulgaria) Nutrition a Physiology Nikolai Todorov (Bulgaria) Peter Surai (UK) Zervas Georgios (Greece) Ivan Varlyakov (Bulgaria) Production Systems Dimitar Pavlov (Bulgaria) Dimitar Panaiotov (Bulgaria) Banko Banev (Bulgaria) Georgy Zhelyazkov (Bulgaria) Agriculture a Environment Georgi Petkov (Bulgaria) Ramesh Kanwar (USA) Product Quality a Safety Marin Kabakchiev (Bulgaria) Stefan Denev (Bulgaria) Vasil Atanasov (Bulgaria) English Editor Yanka Ivanova (Bulgaria) Scope a policy of the journal Agricultural Science a Technology /AST/ an International Scientific Journal of Agricultural a Technology Sciences is published in English in one volume of 4 issues per year, as a printed journal a in electronic form. The policy of the journal is to publish original papers, reviews a short communications covering the aspects of agriculture related with life sciences a modern technologies. It will offer opportunities to address the global needs relating to food a environment, health, exploit the technology to provide innovative products a sustainable development. Papers will be considered in aspects of both fuamental a applied science in the areas of Genetics a Breeding, Nutrition a Physiology, Production Systems, Agriculture a Environment a Product Quality a Safety. Other categories closely related to the above topics could be considered by the editors. The detailed information of the journal is available at the website. Proceedings of scientific meetings a conference reports will be considered for special issues. Submission of Manuscripts All manuscripts written in English should be submitted as MS-Word file attachments via e-mail to ascitech@uni-sz.bg. Manuscripts must be prepared strictly in accordance with the detailed instructions for authors at the website http://www.uni-sz.bg/ascitech/iex.html a the instructions on the last page of the journal. For each manuscript the signatures of all authors are needed confirming their consent to publish it a to nominate on author for correspoence. They have to be presented by a submission letter signed by all authors. The form of the submission letter is available upon from request from the Technical Assistance or could be downloaded from the website of the journal. Manuscripts submitted to this journal are considered if they have submitted only to it, they have not been published already, nor are they uer consideration for publication in press elsewhere. All manuscripts are subject to editorial review a the editors reserve the right to improve style a return the paper for rewriting to the authors, if necessary. The editorial board reserves rights to reject manuscripts based on priorities a space availability in the journal. The articles appearing in this journal are iexed a abstracted in: EBSCO Publishing, Inc. a AGRIS (FAO). The journal is accepted to be iexed with the support of a project BG051PO0013.3.05-0001 Science a business financed by Operational Programme Human Resources Development of EU. The title has been suggested to be included in SCOPUS (Elsevier) a Electronic Journals Submission Form (Thomson Reuters). Internet Access This journal is included in the Trakia University Journals online Service which can be fou at www.uni-sz.bg. Address of Editorial office: Agricultural Science a Technology Faculty of Agriculture, Trakia University Student's campus, 6000 Stara Zagora Bulgaria Telephone.: +359 42 699330 +359 42 699446 http://www.uni-sz.bg/ascitech/ Technical Assistance: Nely Tsvetanova Telephone.: +359 42 699446 E-mail: ascitech@uni-sz.bg

Volume 5, Number 4 December 2013 ISSN 1313-8820 2013

AGRICULTURAL SCIENCE AND TECHNOLOGY, VOL. 5, No 4, pp 459-462, 2013 Comparative GC/MS analysis of laveer (Lavaula angustifolia Mill.) inflorescence a essential oil volatiles T. Zagorcheva1*, S. Stanev2, K. Rusanov1, I. Atanassov1 1 AgroBioInstitute, 8 Dragan Tsankov, 1164 Sofia, Bulgaria Institute of Roses a Aromatic Plants, 49 Osvobozhdenie, 6100 Kazanlak, Bulgaria 2 Abstract. The composition of laveer inflorescence volatiles a distilled essential oils is significantly influenced by a number of factors, which makes it difficult to perform comparative analysis for larger laveer genetic resource collections a segregating populations. In the present study we test a simple procedure for GC/MS analysis of inflorescence volatiles, including frozen inflorescence sample storage, milling a hexane extraction of volatiles. A comparative GC/MS analysis of volatiles extracted from inflorescence samples a present in the distilled laveer oils derived from single bushes of seven Bulgarian laveer cultivars was carried out. The GC/MS analysis identified a total of 32 iividual volatiles including all 11 volatiles included in the laveer ISO Staard 3515. The analysis of three parallel hexane extracts from the same inflorescence samples showed good reproducibility of the determined relative abuances of the analyzed volatiles. The relation of the GC/MS data on inflorescence volatiles with the composition of the distilled laveer essential oils was evaluated through analysis of the volatile recovery rates for the analyzed cultivars. The performed ANOVA test of the variances of the calculated recovery rates of iividual volatiles showed that they are significantly influenced by the studied cultivars. The possibilities for application of the applied extraction procedure a comparative analysis of flower a inflorescence volatile compositions for large sets of laveer accessions a segregating populations are discussed. Keywords: laveer, inflorescence volatiles, essential oil, GC/MS, solvent extraction Introduction The iustrial cultivation of laveer (Lavaula angustifolia Mill.) a production of laveer essential oil in Bulgaria have been rapidly expaing during the last decade (Stanev et al., 2013). The increased growing of laveer has raised the need of breeding new highly productive laveer cultivars which are better adapted to the local environmental coitions, with plant shape suitable for efficient mechanized cultivation a flower collection. The preservation of the high quality of the produced essential oil is an ultimate coition for each new breeding line a cultivar developed, in spite of the particular breeding target. The characterization of laveer genetic resources a breeding lines commonly involves gas chromatography analysis (GC/FID or GC/MS) of the volatile composition of the distilled essential oils, (Smigielski et al., 2009; Chograni et al., 2010; Hassiotis et al., 2010; Verma et al., 2010; Kara a Baydar, 2012; Machado et al., 2013). However, the compositions of laveer inflorescence volatiles a distilled essential oil are significantly influenced by a number of factors such as the stage of inflorescence development, environmental coitions, daytime period of harvesting, flower storage a processing, (Guitton et al., 2010a, 2010b; Smigielski et al., 2011; Turek et al., 2012; Turek a Stintzing, 2012; Maietti et al., 2013; Zheljazkov et al., 2013). This requires the inflorescences of all laveer lines a accessions characterized within a particular study to be collected a subjected to essential oil distillation simultaneously within a short period of time in order to minimize the influence of the external factors. All this poses a significant drawback to the correct comparative characterization of larger sets of laveer accessions a segregating populations a hampers the application of modern plant breeding methods. Here we describe the results from evaluation of a simple procedure for laveer inflorescence sample collection, processing, solvent extraction a GC/MS analysis of volatiles. The study involves a comparative GC/MS analysis of the volatiles in the inflorescence extracts a distilled essential oils from single bushes of Bulgarian laveer cultivars. Material a methods Plant material Laveer inflorescences from single bushes of seven Lavaula angustifolia cultivars including cv. Jubileina, Hemus, Hebar, Raya, Sevtopolis, Drujba a Karlovo were collected on 29 June 2012 between 1 pm a 3 pm from the experimental field of the Institute of Roses a Aromatic Plants, Kazanlak, Bulgaria. Ten raomly chosen inflorescences were immediately frozen in liquid nitrogen a stored in closed containers at -80 C for later processing. The rest of the collected inflorescences were used for immediate distillation of laveer essential oil. Essential oil distillation Freshly harvested inflorescence material (100 g) was subjected to steam distillation for 1 h for the extraction of the essential oil. The oil was dried over anhydrous sodium sulfate a stored at -80 C prior analysis. Flower metabolite extraction a sample preparation for GC/MS analysis The laveer inflorescence material stored at -80 C was grou into fine powder in Teflon jars using liquid nitrogen a the Qiagen Tissue Lyser II Mill (1 min at 30 Hz). Eight hured mg of the grou inflorescence samples were transferred to 20 ml glass vials * e-mail: tzvetelina.zagorcheva@gmail.com 459

a extracted with 4 ml hexane (Sigma-Aldrich) containing 160µl/l 4-methyl-2-pentanone (Sigma-Aldrich) as internal staard at room temperature in a vortex (Vortex Genius 3, VWR) at 2000 rpm for three hours. The samples were centrifuged at 3000 g for 15 min. The clear supernatant was transferred into 5 ml vials, dried over anhydrous sodium sulfate a stored at -80 C prior analysis. The grou inflorescence sample from each laveer cultivar was used to carry out three parallel extractions a GC/MS analysis. The laveer essential oil samples were prepared for GC/MS analysis by diluting 1 μl of essential oil in 1 ml of hexane containing 160µl/l 4methyl-2-pentanone as internal staard (Sigma-Aldrich, Canada). GC/MS analysis The GC/MS analysis of the prepared flower extracts a diluted laveer oil samples was carried out on Agilent 7890A/5975C GC/MS system equipped with Agilent 7683B Series autosampler. All compous were separated on DB-WAXetr polar column (Agilent Technologies) utilizing polyethylene glycol (PEG) as a stationary phase with helium 5.0 as a carrier gas at a septum purge flow of 3 ml/min, splitless injection of 1 μl a injector temperature 250 C. Oven program: from 65 C to 170 C at 1.5 C/min. The massselective detector was operated at electron impact ionization voltage of 70 ev. GC/MS Data a statistical analysis Iividual chemical compous in all analyzed samples were identified using the AMDIS ver. 2.69 deconvolution software (National Institute of Staards a Technology (NIST), USA) by comparing the obtained mass spectra of iividual compous with the NIST 2008 mass spectral library a a custom-built MS library consisting of compous identified previously in laveer oils. In order to minimize scoring of false positive signals the deconvolution parameters in AMDIS were set to resolution (low), sensitivity (very low) a shape requirements (low). Retention iex was calculated for each compou using C10-C40 n-alkane mixture (SigmaAldrich). The relative abuances of all identified compous in the analyzed samples were calculated as pick areas of the analyzed compous were normalized to the pick area of the internal staard 4-methyl-2-pentanone. Data analysis a single factor ANOVA testing was performed using Microsoft Excel software (Microsoft Corporation). Results a discussion The composition of volatiles extracted from laveer inflorescences a present in the distilled essential oils were subjected to comparative GC/MS analysis. Inflorescence samples from single bushes of seven Bulgarian laveer cultivars were frozen in liquid nitrogen, milled a extracted with hexane. The obtained extracts were GC/MS analyzed in parallel with the essential oil samples distilled from the inflorescences collected from the same bushes. The preliminary testing of extractions with different solvents including chloroform, ethyl acetate a hexane a different periods of extraction produced similar GC/MS spectra a demonstrated that the applied hexane extraction for three hours produced the best results in terms of peak quality, (data not shown). Custom built AMDIS MS library consisting of 32 iividual compous identified in different inflorescence a essential oil samples was constructed. The total number of library hits detected within analysis of iividual inflorescence samples varied from 24 to 460 26 a correspoingly from 24 to 29 for the analyzed laveer oils (Table 1). Nineteen volatiles were detected in all inflorescence samples, 21 in all essential oil samples a 17 in all analyzed samples. All eleven volatiles included in the laveer ISO Staard 3515, ISO3515 (2002) were detected within the applied GC/MS analysis (Table 1). The comparison of the calculated relative abuances of the volatiles detected in GC/MS analysis of the three parallel extractions from each inflorescence sample showed good reproducibility of the volatile extraction a analysis, with relative staard deviations /RSD/ varying from 4% to 38% for the iividual compous with average RSD values below 20%. The further comparison of the results from GC/MS analysis of the inflorescence extracts a essential oil samples for each cultivar reveals three groups of volatiles detected only in part of these samples. The compous from the first group, including cis-geraniol a transgeraniol, were well detected in all essential oil samples but not in the hexane extracts. This is most possibly due to the less efficient hexane extraction of these compous from inflorescence samples a/or increase of their relative abuances in the volatile pools following the essential oil distillation. This group could also be expaed including camphor but the obtained data don't allow to correctly estimate whether this compou is inefficiently extracted from fluorescence samples or is present in low abuance uer the GC/MS detection limit in the majority of the tested samples. The volatiles from the seco group, including β-sesquiphellarene a α-bergamotene, were detected in higher abuances in the inflorescence extracts rather than in the essential oil samples possibly due to their more efficient hexane extraction than essential oil distillation. The third group of volatiles is more complex a includes compous detected only in inflorescence or essential oil samples of single but not all analyzed cultivars, for example the absence of 1-Octen-3-ol in the essential oil from cv. Hebar or αterpineol in inflorescence extract of cv. Jubileina (Table 1). The reasons for such lack of detection of a particular compou in only one type of the samples, inflorescence extract or essential oil originating from the same bush of iividual laveer cultivar, are not clear. The parallel collection of inflorescences for preparation of both types of samples from each single bush/cultivar should largely suppress the variation in their volatile composition caused by the environmental factors. Thus, one of the possible causes could be that the volatile composition of the inflorescence sample used for hexane extraction differs from the volatile composition of the entire inflorescence of the sampled bush. In a recent study Guitton et al. (2010) reported that the abuances of most of the iividual laveer volatiles strongly depe on the stage of single flower development. From the other side the laveer flower development is related to uneven development of the flowers within a single inflorescence a uneven development of the separate inflorescences within the same bush (Upson a Arews, 2004; Guitton et al., 2010; Lane et al., 2010). All this makes it possible to speculate that at least some of the observed differences of the volatile pools of the inflorescence extracts a essential oils originating from a single laveer bush are a result from the pointed uneven flower a inflorescence development within the sampled bushes. The possible depeence of the recovery rate of a particular compou from its relative abuance in the volatile pool of the tested laveer genotype could be another source for the observed discrepancies between volatile compositions of the inflorescence extracts a essential oils. The GC/MS data from the present study allow to evaluate the possible influence of abuances of particular compous in the inflorescence volatile pool over their recovery rate in the distilled essential oils. To do this we calculated the ratio

Table 1. Relative abuances of GC/MS analyzed volatiles in inflorescence extracts a distilled essential oils of seven laveer cultivars. Compou 1-Octen-3-ol* 3-Carene* 3-Octanol* 3-OctanoneISO* 4-Hexen-1-ol* α -Bergamotenent α -Santalene* α-terpineoliso* Benzaldehyde* β -cis-ocimeneiso* β-farnesene* β -LinaloolISO* β Phellarene* β Pinene* β-sesquiphellarenent β -trans-ocimeneiso* Borneol* Borneol acetate* CamphorISO nt Caryophyllene* cis-geraniolnt EucalyptolISO* γ-terpinenent Geranyl acetate* Germacrene D* D-LimoneneISO* Lavaulyl acetateiso* Linalool acetateiso * Nerol acetate* Octen-1-ol, acetate* trans-geraniolnt Terpinen-4-olISO nt RSD range (average)2 Relative abuances of the compous in the inflorescence extracts1 D Hb Hm J 5% - 34% (16%) 0.23 0.27 0.25 0.09 9% - 35% (19%) 0.24 0.52 0.15 5% - 28% (12%) 0.20 8% - 31% (14%) 1.44 1.55 0.66 0.69 5% - 30% (13%) 1.54 0.97 0.39 0.81 7% - 27% (14%) 0.46 0.31 0.11 0.23 6% - 30% (14%) 1.62 1.14 0.51 0.63 9% - 30% (15%) 0.58 0.12 0.20 7% - 36% (19%) 0.37 0.37 0.43 5% - 33% (15%) 3.15 14.75 1.90 3.65 9% - 34% (20%) 15.78 12.45 6.21 2.17 3% - 23% (14%) 41.93 46.78 38.43 22.16 7% - 33% (16%) 3.99 1.04 3.21 0.13 12% - 21%(16%) 0.89 1.09 1.20 0.55 0.58 0.38 9% - 34% (18%) 2.52 15.69 3.14 5.97 6% - 32% (18%) 1.16 2.80 1.33 0.79 5% - 38% (14%) 0.53 0.41 0.24 4% - 21% (12%) 11.60 10.03 6.43 5.14 4% - 32% (18%) 0.91 0.60 1.70 0.10 6% - 29% (16%) 1.07 2.10 1.51 0.95 5% - 29% (14%) 3.98 2.12 0.43 1.29 5% - 27% (15%) 1.56 0.91 1.39 0.27 9% - 28% (12%) 8.96 10.69 6.53 10.46 5% - 21% (10%) 52.94 44.47 60.81 46.83 7% - 30% (16%) 0.77 1.40 0.88 0.67 5% - 30% (17%) 0.79 1.50 0.81 0.73 7.76 1.83 Relative abuances of the compous in the essential oils3 K R S D Hb Hm J K 0.47 0.14 0.18 0.61 0.58 2.09 0.13 0.19 1.00 2.66 33.55 1.42 0.70 8.48 0.24 7.86 1.10 1.18 1.18 0.72 5.35 55.40 0.74 0.83 0.19 0.28 0.26 0.21 2.06 1.55 0.26 0.91 0.11 0.21 4.20 10.31 55.66 1.34 1.08 4.51 1.12 0.61 15.30 0.78 1.45 4.20 0.65 12.50 63.96 1.03 0.98 0.38 0.31 0.36 3.61 1.52 0.56 2.02 0.19 0.40 10.00 22.92 48.83 3.75 1.43 0.37 6.53 2.81 0.67 27.65 1.00 2.13 9.12 1.70 18.47 64.02 1.67 2.59 0.18 0.19 1.16 1.16 0.50 1.71 0.30 2.17 5.55 34.43 1.68 1.45 1.48 0.70 3.83 0.26 0.67 1.94 0.62 0.94 6.34 30.57 1.15 0.69 0.64 1.32 0.15 0.04 0.63 0.33 0.14 2.01 0.15 4.14 1.66 25.96 0.25 1.05 4.83 0.83 0.18 1.38 0.31 0.21 1.77 0.24 0.41 3.57 32.72 1.03 0.48 0.83 0.10 0.33 0.17 0.11 1.24 0.20 1.14 1.31 23.13 1.02 0.83 1.52 0.52 0.16 1.51 0.19 0.65 1.58 0.61 2.62 30.99 0.90 0.38 0.51 0.07 0.50 0.60 0.10 2.00 3.00 0.55 20.00 0.06 0.80 4.00 0.60 0.20 1.00 0.30 0.07 2.00 0.20 0.30 7.00 30.00 1.00 0.50 0.80 5.00 0.25 0.06 0.07 0.32 0.16 0.62 0.74 0.13 0.79 0.87 21.15 0.56 0.80 3.40 0.11 2.41 0.12 0.64 1.15 0.25 0.42 2.47 23.41 0.70 0.38 0.29 0.08 R S 0.08 0.11 0.04 0.05 0.08 0.10 0.80 0.80 0.47 0.39 0.10 0.12 1.51 1.26 0.10 0.12 1.64 1.72 1.18 1.77 26.07 18.74 0.34 0.51 0.74 0.62 1.56 1.15 0.41 0.61 0.23 0.14 0.12 1.94 2.43 0.24 0.20 0.34 0.23 0.07 1.29 1.12 0.43 0.48 0.29 0.38 4.05 3.77 22.45 20.68 0.78 0.69 0.35 0.63 0.63 0.51 Cultivar abbreviations: D Drujba, Hb Hebar, Hm Hemus, J Jubileina, K Karlovo, R Raya, S Sevtopolis Nd not detected 1 Relative abuances of the volatiles in the inflorescence extracts after normalization with the abuance of the internal staard. The presented values are average values of GC/MS analysis of three parallel extracted samples from the inflorescences collected from each cultivar. 2 Range (minimal a maximal) of the values a average (in brackets) value of the relative staard deviation calculated for the three relative abuances determined for the GC/MS analyzed volatiles for each cultivar. 3 Relative abuances of the volatiles in the essential oils distilled from single sampled cultivar bushes after normalization with the abuance of the internal staard. ISO Volatiles included in the laveer essential oil ISO Staard 3515 * Volatiles which recovery rates in the essential oils significantly vary between the cultivars after ANOVA test of variances of the ratios of relative abuances of the volatiles in the essential oils a inflorescence extracts nt not tested between relative abuances of the compous detected in the essential oil samples to their relative abuances in the correspoing inflorescence extracts. Assuming that the rates of recovery of the particular volatiles in the distilled essential oils do not depe significantly on the relative abuances of the volatiles in the analyzed inflorescence volatile pool, we evaluated such 'null hypothesis' using one way ANOVA. The results of the analysis demonstrate that the recovery rates of the volatiles present in both inflorescence extracts a essential oil samples significantly depe on the tested cultivar, i.e. on the volatile composition of the inflorescences (Table 1). The obtained results differ from the observed earlier relative stability of the volatile recovery rates for essential oil distilled from the oil bearing rose Rosa damascena (Rusanov et al., 2011). The present dataset doesn't allow to point out the cause(s) for the observed cultivar depeent variations of the volatile recovery rates. The possible causes could be the above pointed uneven flower a inflorescence development within single laveer bush a/or specific features of the laveer oil steam distillation procedure which differ from those used for rose oil distillation. However, the observed genotype depeence of the volatile recovery rates makes it unrealistic to extrapolate the GC/MS data from 461

inflorescence extract analysis for preliminary assessment a prediction of the composition of essential oil distilled from the same inflorescence pool. It further largely hampers the possible application of the GC/MS analysis of solvent extracts from small inflorescence samples for comparative characterization of a larger set of laveer accessions or iividual bushes from segregating populations in respect to their essential oils. The above described variations of the volatiles recovery rates also make it difficult to apply GC/MS analysis of laveer essential oil for correct characterization of the impact of particular genotype a allele configuration, necessary for application of modern plant breeding. Considering the reported by Guitton et al. (2010) changes of the volatile pool composition during laveer flower development a the above discussed problems it is possible to suggest that an efficient strategy for correct comparative characterization of flower volatile composition of a set of laveer genotypes has to include at least three separate analyses of iividual flowers collected from each genotype. The proposed three analyses have to be carried out with solvent extracts from flowers at stages 1, 3 a 5 of flower development, according to Guitton et al. (2010) correspoing to early flower development, fully open flowers a seed formation stages. Thus analyses of the pointed flower stages will represent all three groups of volatiles possessing different accumulation pattern within the flower development a therefore the complexity of the volatile pools of the laveer flower a inflorescence with impact on the distilled essential oil. Conclusion The procedure for solvent extraction of laveer inflorescences a GC/MS analysis of extracted volatiles tested in the present study identifies nearly all volatiles detected in the distilled essential oil, including those from the ISO3515:2002 laveer oil staard. However, the performed ANOVA test of variances of the ratios between relative abuances of iividual volatiles in the distilled essential oils a inflorescence extracts from single bushes of the tested cultivars showed significant influence of the cultivars over the calculated volatile recovery rates. This suggests that a detailed characterization a comparative analysis of volatile pools of laveer genetic resources collections a segregating populations will require collection, solvent extractions a GC/MS analysis of iividual flowers at several stages of flower development related to accumulation of specific volatile subsets. Acknowledgements The authors would like to thank Ivaila Dincheva (ABI) for the excellent technical assistance a the National Science Fu at the Ministry of Education, Youth a Science, Bulgaria for supporting the presented research via project DO 02-105. References Chograni H, Zaouali Y, Rajeb C a Boussaid M, 2010. Essential Oil Variation among Natural Populations of Lavaula multifida L. (Lamiaceae). Chemistry a Biodiversity, 7, 933-942. Guitton Y, Nicole F, Moja S, Benabdelkader T, Valot N, Legra S, Jullien F a Legere L, 2010a. Laveer inflorescence: a 462 model to study regulation of terpenes synthesis. Plant Signaling a Behavior, 5, 749-751. Guitton Y, Nicole F, Moja S, Valot N, Legra S, Jullien F a Legere L, 2010b. Differential accumulation of volatile terpene a terpene synthase mrnas during laveer (Lavaula angustifolia a L. x intermedia) inflorescence development. Physiologia Plantarum, 138, 150-163. Hassiotis CN, Tarantilis PA, Daferera D a Polissiou MG, 2010. Etherio, a new variety of Lavaula angustifolia with improved essential oil production a composition from natural selected genotypes growing in Greece. Iustrial Crops a Products, 32, 77-82. ISO3515:2002, Oil of laveer (Lavaula angustifolia Mill.) Staard. http://www.iso.org. Kara N a, Baydar H, 2012. Essential oil contents a composition of laveers a lavains cultivated in Turkey. Research on Crop, 13, 675-681. Lane A, Boecklemann A, Woronuk GN, Sarker L a Mahmoud SS, 2010. A genomics resource for investigating regulation of essential oil production in Lavaula angustifolia. Planta, 231, 835845. Machado MP, Ciotta MN, Deschamps C, Zanette F, Cocco LC a Biasi LA, 2013. In vitro propagation a chemical characterization of the essential oil of Lavaula angustifolia cultivated in Southern Brazil. Ciencia Rural, 43, 283-289. Maietti S, Rossi D, Guerrini A, Useli C, Romagnoli C, Poli F, Bruni R a Sacchetti G, 2013. A multivariate analysis approach to the study of chemical a functional properties of chemo-diverse plant derivatives: laveer essential oils. Flavour a Fragrance Journal, 28, 144-154. Rusanov KE, Kovacheva NM a Atanassov II, 2011. Comparative Gc/Ms Analysis of Rose Flower a Distilled Oil Volatiles of the Oil Bearing Rose Rosa Damascena. Biotechnology a Biotechnological Equipment, 25, 2210-2216. Smigielski K, Prusinowska R, Raj A, Sikora M, Wolinska K a Gruska R, 2011. Effect of Drying on the Composition of Essential Oil from Lavaula angustifolia. Journal of Essential Oil Bearing Plants, 14, 532-542. Smigielski K, Raj A, Krosowiak K a Gruska R, 2009. Chemical Composition of the Essential Oil of Lavaula angustifolia Cultivated in Pola. J Essent Oil Bear Plants, 12, 338-347. Stanev S, Zagorcheva T a Atanassov I, 2013. Iustrial cultivation of Lavaula angustifolia a laveer oil production in Bulgaria in the 21st century: challenges a possible molecular genetics a breeding solutions. Biotechnology a Biotechnological Equipment, (in press). Turek C, Kirschmann N a Stintzing FC, 2012. Quality monitoring of selected essential oils upon storage at different temperature regimes. Zeitschrift für Arznei u Gewürzpflanzen, 17, 73-79. Turek C a Stintzing FC, 2012. Impact of different storage coitions on the quality of selected essential oils. Food Research International, 46, 341-353. Upson T a Arews S, 2004. The genus Lavaula. KEW BOOKS, Royal Botanic Gardens, ISBN, 1842460102. Verma RS, Rahman LU, Chanotiya CS, Verma RK, Chauhan A, Yadav A, Singh A a Yadav AK, 2010. Essential oil composition of Lavaula angustifolia Mill. cultivated in the mid hills of Uttarakha, Iia. Journal of the Serbian Chemical Society, 75, 343-348 Zheljazkov VD, Cantre CL, Astatkie T a Jeliazkova E, 2013. Distillation Time Effect on Laveer Essential Oil Yield a Composition. Journal Oleo Science, 62, 195-199.

AGRICULTURAL SCIENCE AND TECHNOLOGY, VOL. 5, No 4, 2013 CONTENTS 1/2 Genetics a Breeding Investigation on the possibility to efficiently use Ukrainian cultivars for developing of early winter wheat lines I. Grain productivity N. Tsenov, T. Petrova, E. Tsenova 351 Combinig ability for grain yield of late maize lines N. Petrovska 358 Use of recurrent selection in middle late synthetic maize population I. Results of the first cycle in synthetic 1/2005 N. Petrovska, V. Valkova 362 Genetic diversity a distance between two Bulgarian local sheep breeds assessed by microsatellite markers S. Georgieva, E. Todorovska, D. Hristova, I. Dimitrova, N. Stancheva, Ts. Yablanski 367 Testing of new Bulgarian sunflower hybrids uer the coitions of North-East Bulgaria I. Productivity a traits related to productivity G. Georgiev, P. Peevska, E. Penchev 371 Comparative morphological study of new Burley tobacco lines T. Radoukova, Y.Dyulgerski 376 Effect of genotypic a environmental factors on the inheritance of the main characters in chickpea a relationships between them R. Sturzu, T. Nistot, Cr. Melucă, Fl. Bodescu, A. Stoilova 380 Evaluation of double haploid lines of winter malting barley using selection iices B. Dyulgerova, D. Valcheva 384 Evaluation of the combining ability of grain yield of mutant maize lines M. Ilchovska 388 Comparative study of some biochemical iicators in Karakachan a Copper-Red Shumen sheep breeds G. Angelov, I.Dimitrova, T. Mehmedov, P. Stamberov, N. Stancheva, S. Georgieva, Zh. Nakev 391 Nutrition a Physiology Impaired pancreatic function in mulard ducks with experimental aflatoxicosis I. Valchev, N. Grozeva, D. Kanakov, Ts. Hristov, L. Lazarov, R. Binev, Y. Nikolov 394 Comparative investigations on feeding efficiency in growing a fattening DanBred a Topigs hybrid pigs G. Ganchev, A. Ilchev 400 Blood parameters in yearling sheep fed Paulownia (Paulownia spp.) leaves I. Varlyakov, V. Radev, T. Slavov, G. Ganchev 405

AGRICULTURAL SCIENCE AND TECHNOLOGY, VOL. 5, No 4, 2013 CONTENTS 2/2 Changes in some blood parameters in yearling rams fed diets with different protein a lipid levels V. Radev, T. Slavov, I. Varlyakov 410 Production Systems Effect of the sowing norm a nitrogen fertilization on the yield from dry bean (Phaseolus vulgaris L.) cultivar Beslet G. Milev 415 Evapotranspiration of corn crop for silage R. Bazitov, A. Stoyanova 420 Productivity a economic traits of winter oilseed rape (Brassica napus var. biennis) uer the coitions of Dobrudzha G. Georgiev, G. Georgiev, P. Chamurliyski 424 Feasibility of the use of heat energy from alternative sources for air coitioning in sows facility K. Peichev, R. Georgiev 428 Productivity of green beans, irrigated at different pre-irrigation soil moisture R. Petrova, A. Matev, K. Koumanov, B. Harizanova-Petrova 432 Agriculture a Environment Comparative assessment of plant resources as substrates for bioshlam production Z. Shiarska, V. Kirov, G. Kostadinova, B. Baykov 438 The influence of organic carbon on bioremediation process of wastewater originate from aquaculture with use of microalgae from genera Botryococcus a Scenedesmus I. Sirakov, K. Velichkova, G. Beev, Y. Staykov 443 Sanitary hygienic assessment of drinking water from uergrou source at a pig farm G. Kostadinova 448 Product Quality a Safety Study of bee honey by spectral analysis in the near infrared spectrum I. Zhelyazkova, S. Atanasova, K. Elencheva Karaneycheva 455 Comparative GC/MS analysis of laveer (Lavaula angustifolia Mill.) inflorescence a essential oil volatiles T. Zagorcheva, S. Stanev, K. Rusanov, I. Atanassov 459 Influence of key factors on the time of initial coagulation of cow's milk using milk-clotting enzyme of camel origin P. Panayotov, K. Yoanidu, P. Boyanova, B. Milenkov 463

Instruction for authors Preparation of papers Papers shall be submitted at the editorial office typed on staard typing pages (A4, 30 lines per page, 62 characters per line). The editors recomme up to 15 pages for full research paper ( including abstract references, tables, figures a other appeices) The manuscript should be structured as follows: Title, Names of authors a affiliation address, Abstract, List of keywords, Introduction, Material a methods,results, Discussion, Conclusion, Acknowledgements (if any), References, Tables, Figures. The title needs to be as concise a informative about the nature of research. It should be written with small letter /bold, 14/ without any abbreviations. Names a affiliation of authors The names of the authors should be presented from the initials of first names followed by the family names. The complete address a name of the institution should be stated next. The affiliation of authors are designated by different signs. 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Edition, name of publisher, place of publication. Example: Oldenbroek JK, 1999. Genebanks a the conservation of farm animal genetic resources, Seco edition. DLO Institute for Animal Science a Health, Netherlas. Book chapter or conference proceedings: Author(s) surname a initials, year. Title. In: Title of the book or of the proceedings followed by the editor(s), volume, pages. Name of publisher, place of publication. Example: Mauff G, Pulverer G, Operkuch W, Hummel K a Hidden C, 1995. C3variants a diverse phenotypes of unconverted a converted C3. In: Provides of the Biological Fluids (ed. H. Peters), vol. 22, 143-165, Pergamon Press. Oxford, UK. Todorov N a Mitev J, 1995. Effect of level of feeding during dry period, a body coition score on reproductive performance in dairy cows,ixth International Conference on Production Diseases in Farm Animals, Sept.11 14, Berlin, Germany, p. 302 (Abstr.). Thesis: Penkov D, 2008. Estimation of metabolic energy a true digestibility of amino acids of some feeds in experiments with muscus duck (Carina moshata, L). Thesis for DSc. Agrarian University, Plovdiv, 314 pp. The Editorial Board of the Journal is not responsible for incorrect quotes of reference sources a the relevant violations of copyrights. Ethics Studies performed on experimental animals should be carried out according to internationally recognized guidelines for animal welfare. That should be clearly described in the respective section Material a methods.

Volume 5, Number 4 December 2013