annual international conference

ISSN: 2089-208X

CHAPTER

L ILFI FEE SS CCIIEEN NC ECSE A N N U A L I N T E R N AT I O N A L C O N F E R E N C E S Y I A H K U A L A U N I V E R S I T Y ( A I C U N S Y I A H ) AAC Dayan Dawood, Darussalam - Banda Aceh, Indonesia. November 29-30, 2011

PROCEEDINGS

Editors: Z. A. Muchlisin, Indonesia Andrew H. Beird, Australia Ahmad Nayaya, Nigeria Musri Musman, Indonesia Teuku Alvisyahrin, Indonesia Mudatsir, Indonesia Sugito, Indonesia

SYIAH KUALA UNIVERSITY PRESS 2011

EDITORIAL BOARD Editor-in-Chief: Dr. Z. A. Muchlisin (Ichthyology, Indonesia)

Editorial Members: Dr. Andrew H. Baird (Marine Sciences, Australia) Dr. Ahmad Nayaya (Biodiversity Science, Nigeria) Dr. Musri Musman (Marine Science, Indonesia) Dr. Mudatsir (Health Science, Indonesia) Dr. Sugito (Agriculture Science, Indonesia) Dr. Teuku Alvisyahrin (Agriculture, Indonesia)

Organizer: Syiah Kuala University Research Institute

Publisher: Syiah Kuala University Press Kopelma Darusalam - Banda Aceh 2311, Indonesia

Cover Design by: Syahabuddin, ST

NOTICE OF DISCLAIMER The authors and publisher of these proceedings have used their best efforts in preparing and collecting the materials. These efforts include the development, research and testing of these theories to determine their effectiveness. However, the publisher bears no responsibility for any damage resulting from any inadvertent omission or inaccuracy in the proceedings. Further, the authors and publisher make no warranty of any kind, expressed or implied, with regard to the documentation contained in these proceedings. The authors of papers published in these proceedings have transferred their copyright to Syiah Kuala University. The opinions expressed by the authors do not necessarily reflect those of Syiah Kuala University and are solely the responsibility of the authors. All manuscripts printed in these proceedings were reproduced directly from the softcopies submitted by the authors (unedited), with only minor editorial amendments. Although all efforts were made to ensure the accuracy and correctness of the format, the Committee of Annual International Conference Syiah Kuala University will not be responsible or liable for any unintentional errors made during the publication or any conflict of interest of theis proceedings. Individual readers of these proceedings and nonprofit libraries acting for them are freely permitted to make fair use of the material, such as to copy a paper for use in teaching or research. Permission is granted by Syiah Kuala University Press to quote from these proceedings with the customary acknowledgment of the source. Republication or systematic or multiple reproduction of any material in these proceedings is permitted only with written permission from Syiah Kuala University Press. Request to republish or reproduce papers from these proceedings should be addressed to Syiah Kuala University Press, Kopelma Darussalam, Banda Aceh 23111, Indonesia.

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MESSAGE FROM THE RECTOR

On behalf of Syiah Kuala University (Unsyiah), I would like to extend my warmest welcome to all participants to the Annual International Conference of Syiah Kuala University 2011 (AICUNSYIAH 2011). Syiah Kuala University is the largest and the oldest national university in Aceh Province, Sumatera Island, Indonesia. The university was established on June 21st, 1961. The establishment of UNSYIAH was driven by a spirit to form an institution to bring the Acehnese to become educated, knowledgeable and pious to God Almighty. UNSYIAH has a vision to establish itself as an innovative, independent, and outstanding university in terms of the development of science, technology, humanities, sport and arts, in order to produce qualified graduates who highly honor moral and ethical values. This year, UNSYIAH has hosted a series of events commemorating its 50th anniversary. As part of the celebration, the university held an interdisciplinary academic conference between of November 29-30, 2011. The conference included plenary addresses, oral & poster parallel sessions and a tsunami/historical sites tour. I would like to thank our keynote and invited speakers for their time and support for this conference. I am excited to witness such development in the research scene. Lastly, I would like to thank the Organizing Committee for their hard work in making this event successful. To all participants, I wish you fruitful interactions with your peers and our foreign friends as well as, an enjoyable stay in Banda Aceh. Thank you. Sincerely Professor Dr. Darni Daud, M.Ed Rector of Syiah Kuala University

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MESSAGE FROM THE CHAIRMAN

I would like to take an opportunity to warmly welcome all the participants of the Annual International Conference of Syiah Kuala University 2011 (AIC- UNSYIAH 2011) to Banda Aceh, Indonesia. This conference was organized by The Syiah Kuala University Research Institute. The institute is honored to host this distinguished and meaningful event. This conference signifies yet another achievement by the Research Centre in bringing together scholars from all over the world to promote scholastic work. It is also in line with the University’s commitment to the advancement of knowledge through pursuing scientific discoveries and technological innovation. The response for the conference has been overwhelming. We are glad to have received abstract submissions and approximately 180 papers from 9 countries, however due to limitation of time slots only about 107 papers will be able to be presented. Among the participants, there are not only experienced experts and researchers but also postgraduate students from emerging areas such as agriculture, marine and fisheries, biology and chemistry in general, health sciences, law and politic, education, history and economics. The presented papers have been published in three different chapters; Life Sciences, Sciences and Engineering, and Education and Social Sciences. We hope that participants will exploit the great opportunity provided by this conference to link with some of the world’s best minds and establish collaborative ties. We also hope that it will spur inspirations for more innovative ideas. We look forward to welcoming you in the Annual International Conference of Syiah Kuala University 2011 and wish you a pleasant and enjoyable stay in Banda Aceh. Thank you. Sincerely Dr. Musri Musman, M.Sc Chairman/Director of Syiah Kuala University Research Institute

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CONTENTS Page

EDITORIAL BOARD NOTICE OF DISCLAIMER MESSAGE FROM THE RECTOR MESSAGE FROM THE CHAIRMAN CONTENTS

i ii iii iv v

AGRICULTURAL SESSION 1

A new computerized program for grain yield stability analysis in wheat

1

2

Image processing and biometrical investigations for choosing the most ranked rice cultivars

5

An Efficient Agrobacterium-mediated Transformation Method for Sugarcane (Saccharum officinarum L.)

11

Performance of Broiler Marketing in Umuahia Area of Abia State, Nigeria

17

Ali Arminian and Mohammad Mehdi Rahimi

Asad Masoumi Asl, Reza Amiri Fahliani and Alireza Khoshroo

3

Efendi and M. Matsuoka

4

Obasi I. O and Okafor I. J

5

Survey and study on yield and quality of patchouli oil in Aceh Barat Daya District, Indonesia based on original area of raw materials, methods and length of distilation

22

Arpi N, Cut Erika and Dewi Ermaya

6

Antioxidant activities of curry leaves (Murayya koeniigi) and salam leaves (Eugenia polyantha)

28

Breeding better Flax (Linum usitatissimum L.) for agronomic and consumption values under different field treatments

33

Novi Safriani, Normalina Arpi, Novia Mehra Erfiza and Rini Ariani Basyamfar

7

Mohammad Mehdi Rahimi, Mohammad Amin Zarei, and Ali Arminian

8

Effect of planting density on morphological characteristics and yield components of soybean (Glycine maxL) cultivars

36

Microencapsulation of Omega-3 Fatty Acids: what it is, how it’s made, and challenges in food technology

39

10

Application of Tiens Golden Harvest Fertilizer in Hibiscus sabdariffa L.

48

11

Detection QTLs for grain quality traits and yield in two BILs population of rice

52

Keyvan Shamsi and Soheil Kobraee

9

Sri Haryani Anwar

Zuyasna, Marai Rahmawati and Nurmasyitah

Maryam Hosseini, Saadolah Houshmand, Shahram Mohamadi, Alireza Tarang , Mahmod Khodambashi and Hossein Rahimsouroush

12

Effect of NPK fertilizer and biochar application to soil chemical properties of irrigation paddy

55

Anticandidal in vitro test of crude ethanol extract of pliek u on the growth of Candida albicans

59

Effect of fermentation container and thickness of bean mass during fermentation process of cocoa bean (Theobroma cocoa L)

64

Sufardi, Khairun Nisa, Zaitun, Chairunas, Anischan Gani, Peter Slavich, and Malem McLeod

13

Nurliana , Rinidar, and Dian Masyitha

14

E. Indarti, H.P.Widayat and N. Zuhri

v

15

Flavor of arabica coffee grown in Gayo Palteau as affected by varieties and processing techniques

70

Yusya Abubakar, Abubakar Karim and Fachrizal Fahlufi

ZOOLOGY AND MICROBIOLOGY SESSION 16

The shallow water hard corals of Pulau Weh, Aceh, Indonesia

76

17

Impact of Climate Change on Coral Reef Fish Fisheries in Sabang Waters, Indonesia

83

Spawning ground of depik, Rasbora tawarensis (Teleostei, Cyprinidae) in Lake Laut Tawar, Indonesia

95

Growth of wild juvenile and hatcheries Tiger Grouper (Epinephelus fuscoguttatus) were reared by providing different feeds

100

Preliminary study on the morphology and digestive tract development of tomato clownfish (Amphiprion frenatus) under captive condition

103

Andrew H. Baird, Stuart J. Campbell, Nur Fadli, Andrew S. Hoey and Edi Rudi

Edi Rudi, Taufik Iskandar, Nur Fadli, Hidayati

18

Z.A. Muchlisin

19

Muhammadar A.A, Mazlan A.G, Abdulah Samat, Asmawati and Simon K.D.

20

Putra F. Dedi and A.B Abol Munafi

21

Diversity and distribution of fishes of Gaji river, Bauchi State, Nigeria

113

22

Characteristic of fish protein hydrolysates from fish waste

122

23

Isolation of gram-negative bacteria from metacarpal injury of Panthera tigris sumatrae trapped in Subulussalam, Indonesia

131

Nayaya, A. J., Siti Azizah M. N., Adikwu, I. A., Istifanus, W. A. and Ezra, A. G. Fahrizal, Novi Safriani, Murna Muzaifa

Darmawi, Darniati, Zakiah Heryawati Manaf, Syafruddin and Arman Sayuti

24

Characteristics of frozen-thawed semen on Simmental and Limousin bulls in Ungaran, Indonesia

136

Enzyme activity of Ascaridia galli larvae was inhibited by Phenil Methanyl methane sulfonyl fluoride

141

The combination effect of Naphthalene Acetic Acid (NAA) and Benzyl Amino Purine (BAP) in micro propagation of castrol oil plant (Jatropha curcas L.)

146

Triva Murtina Lubis, Prabowo Purwono Putro, Aris Junaidi and Hamny

25

Ummu Balqis, Darmawi, Muhammad Hambal

26

Meutia Zahara, Zairin Thomy and Essy Harnelly

27

The effect of sipatah-patah (Cissus quadrangularis Salisb) extract administration on osteoclast formation and osteoporosis treatment

152

Sabri M

HEALTH SESSION 28

Molecular pathogenesis of preeclampsia: microRNA hypothesis

156

29

Photo-voice as a qualitative research method in nursing and health sciences: A literature review

163

Late post partum hemorrhage - causes and defining aspects: case series in Zainoel Abidin General Hospital, Banda Aceh

172

Empowerment: A Concept Analysis

176

Mohd. Andalas, Harapan, Diky Mudhakir, Muhammad Ichsan, Natalia C. Pedroza and Saurabh Laddha

Asniar, Urai Hatthakit, Wantanee Wironpanich

30

Bram Pradipta and Cut Meurah Yeni

31

Dara Febriana

vi

32

Helminthiasis prevalence in brick makers at Lambada Peukan Village, Aceh Besar Region, Indonesia

183

Water quality and water borne diseases in lowland ecosystem of Banyuasin, South Sumatra

186

Cesarean myomectomy: a case report in Zainoel Abidin General Hospital, Banda Aceh, Indonesia

197

Oral health-related quality of life among pregnant women at Hospital Universiti Sains Malaysia

201

Spontaneous initiation of parturition after two-days-course of lung maturation and controversies in management of pre-eclampsia: a case report

207

Dedy Syahrizal, Cut Mustika, Ikhsan and Mulkan Azhary

33

Dianita Ekawati, Tan Malaka, Robiyanto H Susanto, M.T. Kamaluddin, Dwi Setyawan and Amar Muntaha

34

Bram Pradipta and Mohd Andalas

35

Ema Yunita Sari, Norkhafizah Saddki, Azizah Yusoff

36

Eva Febia and Rajuddin

37

Ovitrap use in epidemiology study of Aedes aegypti and Aedes albopictus in Kuta Alam sub-district Banda Aceh, Indonesia

213

The antimalarial activity of the extract of the neem leaves (Azadirachta indica, A. Juss) on Plasmodium falciparum in vitro

217

Athaillah Farida, Abu Hassan Ahmad, Upik K Hadi, Muhammad Hambal, Yudha Fahrimal and Rimaya Shafitri

38

Hanifah Yusuf, Suryawati and Maryatun

39

Can uterine rupture be prevented: case series in Zainoel Abidin Hospital, Banda Aceh, Indonesia

224

Factors affecting the level of health care worker’s stigmatized and discriminatory attitude towards people living with HIV: A study at the Dr. Zainoel Abidin General Hospital, Banda Aceh, Indonesia

230

Expression and the release of IL-18 profile in major organs of malaria-infected mice

234

Eva Febia and Mohammad Andalas

40

Harapan, Syarifah Feramuhawan, Hendra Kurniawan, Mohd. Andalas, Mohammad Bellal Hossain

41

Kartini Hasballah

42

Willingness to pay and ability to pay for health care services at Zainoel Abidin public hospital Banda Aceh, Indonesia

242

Trends antimicrobial resistance in urinary tract infections: a research at the Dr. Zainoel Abidin Teaching Hospital, Banda Aceh, Indonesia

247

The isolation and identification of antimalarial agents of the n-hexane fraction of the neem leaves (Azadirachta indica A. Juss)

253

HbA1C levels in adolescent obesity, overweight and normoweight catholic high school eagles in Makassar Rajawali

257

Success of vaginal deliveries among women in labor with previous cesarean section: Analysis of 50 cases

262

Rina S. And Rosminah M

43

Said A. Khalilullah, Devi Susanti, Ramadhan W. Saputra and Nurjannah

44

Suryawati, Hanifah Yusuf and Sofia

45

Hasanuddin, Ilhamjaya Patellongi, Irfan Idris, and Rosdiana

46

Eva Febia, Jimmy Toga Sitorus and Cut Meurah Yeni

47

Grave’s Disease in 32-34 weeks of pregnancy

265

48

Is vaginal breech delivery safe option?: analysis of 50 vaginal breech delivery in Zainoel Abidin and Ibu Anak Hospital Banda Ace in 2011

270

Jimmy Toga Sitorus and Cut Meurah Yeni

Eva Febia and Mohammad Andalas

vii

Proceeding of Annual International Conference Syiah Kuala University 2011 Banda Aceh, Indonesia. November 29-30, 2011

A new computerized program for grain yield stability analysis in wheat 1

Ali Arminian and 2Mohammad Mehdi Rahimi

1

Department of Agronomy and Plant Breeding, Ilam University, Ilam, Iran; 2Department of Agriculture, Yasooj Branch, Islamic Azad University, Yasooj, Iran. Corresponding author: [email protected]

Abstract. Advanced statistical codes we present in MINITAB statistical environment, produces comprehensive computational and graphical outputs for the best stability analysis, additive main and multiplicative interaction effect (AMMI). The experiment comprised of a population of doubled-haploid wheat lines at 2006-2009 ys. As lattice designs. The results of AMMI analysis of grain yield showed the significant (PEnable commands. Then, type in front of the command line editor MTB> as: %AMMI c11-c14. The macro then asks some questions, which should be responded well to it operate goodly. Although the macro depicts the cluster analyses and biplots, but it is suggested that draw given diagrams by MINITAB macro after achieving the IPCA scores and nominal means from Graph>plot pathway. Results and Discussion The present macro outputs have many results, including: printing the original data, ANOVA table, treatments (genotypes) mean, standard deviations (SD) and effects, CV of design, residuals of genotypes, environments mean and SD and their effects, the central part of AMMI or ANOVA table covering Gollob F (Gollob, 1968), and also Cornelius F or FR (Cornelius 1993, Dias and Krzanowski, 2003). Other than Gollob F, the FR test is an alternative to cross-validation tests and according to Gauch (personal communications) could be an alternative to it. The ANOVA table of AMMI model for 4 ys. Of grain yield is shown in Table 1. It can be seen from the Table 1 that years, genotypes, and their interaction effects were significant and accounted for 49.1, 7.80 & 20.63% of the total variation, respectively. Furthermore, the written macro partitioned the GEI effect to three IPCA scores, accounted for 40.70, 35.32 & 23.96% of the GEI, respectively. The macro also drew a biplot containing years and genotypes against IPCA1 and IPCA2 scores. As shown from the Figure 1, year 2009 is the most stable year among all the years. Moreover, the genotypes located in the center of the Biplot are the most stable ones. And also e.g. genotypes "g72" and "g89" and "g76" is sample most sensitive genotypes in this experiment. Another issue in this experiment is choosing six of all genotypes within each environment, which is an efficiency of this macro compared to other softwares like GENSTAT or IRISTAT. Furthermore, in our written macro for MINITAB, the Finlay-Wilkinson regression analysis is possible to fit the linear regression the mean of each treatment to all genotype's means and compute the regression equation. Of course many possibilities are possible and computable in these macros which are not discussed here. Some advantages of our macro compared to other softwares doing AMMI analysis: Our program has many applications in various fields of sciences. Some of its efficiencies are comparatively as: 1) Simplicity and ease of use, 2) having potential color graphical representation, especially for biplots, 3) applying the F or FR test for cross-validation instead of RMSPD, which is very tedious and hard in other windows-based softwares, 4) applicable when some observations and even treatments are missed, 5) evolving some important valuable statistical models like PCA, factor analysis, cluster categorization, 6) extracting 6 of the stable genotypes within each environment, 7) performing the FinlayWilkinson regression fitting for each genotype as discussed, 8) performing supplementary genotype analysis compared to other programs for the first time among AMMI analysis 2


programs. In whole, this macro is suggested for selection the stable and high yielding materials in stability analysis of biological and agricultural materials to whom want to apply AMMI analysis easier and faster with vast analyses. Table 1. ANOVA table of wheat grain yield of 103 DH lines, their parents and 5 Iranian varieties at 2006-2009 ys. G and C denote Gollob and Cornelius, respectively. SOV R SS MS P-value Years (Y) R(E) Genotype (G) G*Y IPCA1-G IPCA1-C IPCA2-G IPCA2-C IPCA3-G IPCA3-C Error

3 8 100 300 102 198 100 98 98 800

36.27 1.49 5.76 15.23 6.20 9.03 5.38 3.65 3.65 15.10

12.09 0.19 0.06 0.05 0.06 0.05 0.05 0.04 0.04 0.02

0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 -

Figure 1. Biplot of IPCA1 and IPCA2 for genetypes and years at 2006-2009.

References Arminian A., Kang. M.S., Kozak M., Houshmand S., Mathews P. 2008. MULTPATH: A Comprehensive Minitab program for computing path coefficients and multiple regression for multivariate analyses. Journal of Crop Improvement 22:82-120. Cornelius P.L. 1993. Statistical tests and retention of terms in the additive main effects and multiplicative interaction model for cultivar trials.: Crop Sci. 33:1186-1193. Dias C.T.D.S., Krzanowski W.J. 2003. Model selection and cross validation in additive main effect and multiplicative interaction models. Crop Sci. 43:865-873. 3


Gauch H.G. 1992. Statistical analysis of regional yield trials; AMMI analysis of factorial designs. Elsevier Science, New York. Gauch H.G., Furnas R.E. 1991. Statistical analysis of yield trials with MATMODEL. Agron. J. 83:916-920. Gauch H.G., Piepho H.P., Annicchiarico P. 2008. Statistical analysis of yield trials by AMMI and GGE: further considerations. Crop Science 48:866-889. Gollob H.F. 1968. A statistical model which combines features of factor analytic and analysis of variance techniques. Psychometrika. 33:73-115. Pacheco R.M., Duarte J.B., Vencovsky R., Pinheiro J.B., Oliveira A.B. 2005. Use of supplementary genotypes in AMMI analysis. Theor Appl Genet 110:812-818. Taghouti M., Gaboun F., Nsarellah N. et al. 2010. Genotype x Environment interaction for quality traits in durum wheat cultivars adapted to different environments. African Journal of Biotechnology 9:3054-3062. Yan W., Kang M.S., Ma B., Woods S., Cornelius P.L. 2007. GGE biplot vs. AMMI analysis of genotype-by-environment data. Crop Science 47:643-653. Zobel R.W., Wright M.J., Gauch H.G. 1988. Statistical analysis of a yield trial. Agron. J. 80:388-393.

4


Image processing and biometrical investigations for choosing the most ranked rice cultivars 1

Asad Masoumi Asl, 1Reza Amiri Fahliani and 2Alireza Khoshroo

1

Department of Agronomy and Plant Breeding, Faculty of Agriculture, Yasouj University, Yasouj, Iran; 2 Department of Agricultral engineering, Faculty of Agriculture, Yasouj University, Yasouj, Iran. Corresponding author: [email protected]

Abstract: Rice (Oryza sativa L.) is one of the vital food staples in our daily life, amongst the oldest cultivated crops worldwide, ranks as the most widely grown crop. It serves as a vital nutritional material to more than half of the world increasing crowd. This experiment was conducted for evaluation and selection of the most ranked rice cultivars: Kamfirooz, Yasouj, Dom Siah, Gharib, Dollar, Hassan saraei, 304, Lenjan, and Musa Tarom (MTA) as the well-known and prevalent varieties in the 'Fars' and 'Kogilouyeh-o-BoyreAhmad' provinces, Iran, along with other countries. Rough rice grains of the varieties were randomly selected and their principal dimensions were imaged using camera and a special box for light controlling and including Perimeter, Area, MajorAxis Length, MinorAxis Length, Solidity, Eccentricity, and Equiv Diameter, following with image analyzing by image processing software, in a labratroy experiment. The data were statistically analyzed and graphically plotted using SPSS v.17 and MINITAB v.16 software programs. The results of the image analysis indicated that there were significantly differences between most cultivars in the case of physical traits. The cluster categorization of the cultivars showed also that 9 cultivars clustered in 5 groups, i.e. Yasouj and Lenjan cultivars were located in different groups individually, where cultivars: Kamfirrooz, Dom Siah and Dollar in a unique cluster, but Gharib and 304 in the 4th group and MTA and Hassan Saraei in the 5th group. The results of this experiment showed the separateness of rice most recent cultivars according to agronomic and physical status, and leads to picking the most valuable cultivars for upcoming food nutritional values and also experiments. Key words: agronomic traits, image processing, physical traits, rice.

Introduction Rice (Oryza sativa L.) is among the oldest of cultivated crops and ranks as the most widely grown food grain crop, serving as the staple food for about half the world`s population. world rice production increased from 520 million tones in 1990 to 637.4 million tones in 2008, while in Iran rice production increased from 1.3 million tones in 1980 to 3.6 million tonnes in 2008 (FAOSTAT, 2008). Grain quality is dependent on cooked and uncooked grain size, width and shape (Fotoukian et al., 2007). Higher grain yield and quality are two major subjects for many breeding programs. Physical properties of rice grains includes grain length, width and shape, have direct effect on marketability, and therefore the commercial success of modern rice cultivars (Redona et al., 1998). Rice kernels are usually used to cook as a meal for the human, thus the main indices for evaluation of rice quality are cooking and eating quality and appearance. Important eating quality indicators are amylose content (AC), gelatinization temperature (GT), gel consistency (GC). Appearance features includes milling quality, percentage of chalky grains, chalkiness (often defined the opaque parts in the endosperm) and transparency. Rice consumers in most countries including Iran give more attention to the appearance and cooking quality. The appearance quality directly affects commercial values of rice, and the chalkiness is a key index of appearance quality (Jianchang et al, 2007, He et al., 1999). In most Asian countries, commercial cultivars belong to the medium or long grain class. Long and slender grained Basmati cultivars of Indian and Pakistan command premium prices in the international market, while short and bold grained cultivars are preferred in Japan and Sri Lanka (Redona et al., 1998). Image analysis has been used in discriminating among various types of cereal grains (Lai et al., 1989, Zayas et al., 1986) and among wheat classes and varieties (Zayas et al., 1985, Zayas et al., 1986). A digital image processing system was described by Sapirstein et al (1987) to determine composition of mixtures of wheat, oats, barley, and rye. Neuman et al (1987) used digital image analysis to classify wheat cultivars according to kernel type. Travis and Draper (1985) used image processing and statistical analysis to obtain principal axis and confidence regions for 49 crop and weed species (Zayas et al., 1989). Appearance is mainly determined by the grain shape as specified by grain length (GL), grain width (GW), aspect ratio (length-to-width ratio) and the translucency of 5


the endosperm (Tan et al., 2000). Genetic analyses of GL and GW of rice grains have shown that grain shape is quantitatively heritable (Kou & Hsieh 1982 , Chen & Zhu 1998). It has been shown that rice grain shape is simultaneously controlled by triploid endosperm genes, cytoplasmic genes, embryo and maternal plant genes and their genotype-environment interaction (Shi & Zhu 1996 , Shi et al., 2000). In Iran, different Iranian or foreign rice varieties are cultivated. Yasouj local cultivar is cultivated in Kohgilooyeh & Boyer-Ahmad province, but there is no information about it. Precise quantification of local rice variety shape and physical features by image processing techniques and comparing the features with other Iranian and foreign varieties is the first step toward breeding program for selecting the most suitable cultivars. The aim of this study is characterizing shape features of selected rice varieties and classifying these varieties based on the features. Material and Methods The rice grain used in this research was obtained from IRRI, Kamfirooz and yasouj cities in Fars and Kogilooye & Boyer-Ahmad province in Iran. The varieties (Kamfirooz, Yasouj, Dom Siah, Gharib, Dollar, Hassan saraei, 304, Lenjan, Mosa Tarom (MTA)) used in the current research are the famous and prevalent varieties in the "Fars" and "Kogilooye & BoyerAhmad" province. The randomly selected rough rice grains of these varieties were imaged by using camera and a special box for light controlling. Then the images were analyzed by MATLAB software to extract the features. Feature analysis of the rice grains includes determining geometric features and shape related features. Geometric features including perimeter, area, and major and minor axis length were measured from the binary images. The perimeter of a region is defined as the length of its boundary. The area is the number of pixels within the boundary. Major axis length is the distance between the end points of the longest line that can be drawn through the kernel. Minor axis length is the distance between the end points of the longest line that can be drawn through the object while keeping perpendicularity with the major axis. Equivalent diameter is the diameter of a circle with the same rice grain region.

Equivalent diameter =

4 × Area

π

The statistical data analysis were carried out using SAS 9.1 and clustering were plotted in Minitab 16 software. Results and Discussion Perimeter ranged from 19.244 (DS) to 22.468 mm (Yasouj), Area from 18.949 (142) to 24.016 mm (Yasouj), Major Axis Length from 7.955 (Kamfirooz) to 9.570 (Yasouj), Minor Axis Length from 2.715 (141) to 3.418 (304), Solidity from 0.957 (MTA) to 0.979 (DS), Eccentricity from 0.903 (304) to 0.958 (141) and equival Diameter from 57.887 (142) to 65.235 (Yasouj). Physical values of measured traits were significantly difference (P 6.6 mm and L/W > 3), long grain Japonica (GL > 6.6 mm and L/W= 2- 3), medium grain Japonica (GL 5.5-6.6 mm and L/W= 2-3) and round grain Japonica (GL < 5.5 mm and L/W < 2) (Council Regulation (EC), 1987, No. 3877). Based on the above criteria, 3 cultivars could be classified as

6


long grain Indica and 6 cultivar as long grain Japonica (Table 1), This results to be confirmed by Koutroubas et al for European rices (He et al., 1999). The application of digital image analysis was used for classification of wheat cultivars according to kernel type and identity. Perfect type classification was obtained for four durum wheat varieties in admixture with tencommon wheat's representing a broad range of kernel types. Likewise, samples of each of five Canada Western Red Spring (CWRS) wheat varieties were all correctly allocated to the CWRS class (Neumna et al, 1987). Also, computer imaging technique was used for classifying giant ragweed seeds by preparing digital images of the seed top and side views. Seed samples of 20 different giant ragweed plants (classes) were prepared and digitally scanned. Variation of area and perimeter was least within classes but most among classes, thus these features can discriminate among seeds of different plants in giant ragweed. This experiment showed that in giant ragweed, imaging technique can be used to identification of different species seeds (Yusako et al., 2001). In another research, digital image analysis was used to assay the quality parameters of six varieties of Triticum aestivum L. and one variety of Triticum duro-compactum L. The results of this research showed that image analysis an easy and rapid method for obtaining the physical parameters of wheat grain (Firatligi-Durmus et al., 2010). In India, the grain quality of 100 upland/ahu rice genotypes was tested. I this research, parameters such as grain length, grain width, cooked grain length, cooked grain width, grain elongation ratio after cooking, grain widening ratio after cooking, alkali spreading value, gel consistency, starch, amylose, amyl pectin and total soluble sugar contents were measured. Analysis of variance demonstrated highly significant differences between those characteristics of genotypes (Sunayana et al., 2010). On the other hand, the conventional methods for classifying cultivars of paddy rice relies on physical morphological observation. This method requires experiences to identifying cultivars using charts and illustrations of kernel characteristics. For example, to classify the different cultivars of paddy rice, the characteristics such as ratio of length to perimeter of the kernel, the existence of beards and the length of beards, the color of the hull, the color of the inner and outer awn, the floss growth situation, etc. must be observed. Except for those characters, categorizing the cultivars that have a similar appearance by using only physical observation so difficult and it was prone that it has human error and also time-consuming. In addition, remarkable time was required to train such proficiencies. Zayas et al (1986) classified 77–83% of hard red winter and soft red winter wheat by using machine vision. Barker et al (1992) showed that visage ratios supported 60–63% correct classification for eight wheat cultivars, compared with 52–55% for slices. Shatadal et al (1995) classified and separated kernels of hard red spring wheat, durum wheat, barley, oats, and rye with 95% correct kernel classification (Chang-chu et al., 2005). Clustering of cultivars showed that 9 cultivars were falling in 5 group. Yasouj and Lenjan cultivars maked one group sloley, Kamfirrooz, DS and 142 cultivars in one group, 144 and 304 in one group and MTA and 141 in another group (figure 2). Quantitative features were extracted from the seed images, including perimeter, area, majorAxisLength, minorAxisLength, solidity, eccentricity and equival diameter. Fisher's linear discriminant with UPGMA classification was used to classification of cultivars (Yusako et al., 2001). Luo et al (1999) reported on the classification of cereal grains using the k-nearest neighbor statistical classifier or multi- player neural network classifier with classification rates of 96.9– 99% (Chang-chu et al., 2005).

7


142 141 MTA Kamfirooz Lenjan 144 DS 304 Yasouj

Figure 1. Image of 9 rice cultivar

Similarity

4 4 .9 3

6 3 .2 9

8 1 .6 4

1 0 0 .0 0 141

MTA

Le n j a n

Yasouj

142

DS

K a m fir o o z

144

G e n o ty p e s

Figure 2. Dendrogram of 9 rice cultivars by physical traits.

8

304


Table 1. Physical traits of 9 rice cultivars and their mean comparisons. The dimension of some traits is mm.

Traits Perimeter

Area ea

MajorAxis Length

MinorAxis Length

Solidity

Eccentricity

Equiv Diameter

MajorAxisLen gth/MinorAxis Length

Genotypes 142

20.22cde

18.94d

8.63c

2.82c

0.97ab

0.94bc

57.88d

3.05

141

21.81ab

20.03cd

9.63a

2.71c

0.96bc

0.95a

59.58cd

3.54

MTA

21.29abc

19.75cd

9.29ab

2.80c

0.95c

0.95ab

59.15cd

3.31

Kamfirooz

19.26e

19.77cd

7.95e

3.18ab

0.97a

0.91ef

59.19cd

2.49

Lenjan

20.97bc

21.18bc

8.76bc

3.11b

0.96bc

0.93cd

61.22bc

2.81

144

20.62bcd

22.73ab

8.54cd

3.41a

0.97a

0.91ef

63.46ab

2.50

DS

19.24e

19.57cd

8.07de

3.10b

0.97a

0.92de

58.86cd

2.60

304

19.50de

21.32bc

7.97e

3.41a

0.97a

0.90f

61.47bc

2.33

yasouj

22.46 a

24.01a

9.57a

3.24ab

0.97ab

0.94bc

65.23a

2.94

LSD0.05

1.1972

2.2050

0.5489

0.2679

9

0.0109

0.0138

3.2580

-


References Chang-Chun L., Jai-Tsung Shaw, Keen-Yik Poong, Mei-Chu Hong, and Ming-Lai Shen. 2005. Classifying paddy rice by morphological and color features using machine vision. Cereal Chem. 82(6):649–653. Chen, J.G., Zhu, J., 1998. Genetic effects and genotype* environment interactions for appearance quality traits in Indica–Japonica crosses for rice (Oryza sativa L.). Sci. Agric. Sinica 31, 1–7. Firatligil-Durmuş, E., Sýkorová, A., Šárka, E., Bubník, Z., Schejbal1, M., Příhoda J. 2010. Geometric parameters of wheat grains using image nalysis and FEM approach.Cereal Research Communications Biotechnology and Agronomy. Volume 38, Number 1: 122-133. for rice panicle and grain characteristics. Theor. Appl. Genet. 96, 957–963. Fotoukian M.H.,Taleei Ali Reza,Gharahyazi Behzad. 2007. Genetic analysis of grain quality in rice (Oryza sativa l.) Using microsatellite markers. Journal of Agricultural Science; 13(2):415-425. He, P., Li, S.G., Qian, Q., Ma, Y.Q., Li, J.Z., Wang, W.M., Chen, Y., Zhu, L.H., 1999. Genetic analysis of rice grain quality. Theor. Appl. Genet. 98, 502–508. Jian-chang Y., Er-hua Ch., Cheng T., Hao Zh. And Zhi-qin W. 2007. Relationship of ethylene evolution rate and 1- Aminocyclopropane-1-Carboxlic acid concentration in grains during filling period with appearance quality of rice. Rice Science. 14(1): 33-41. Kuo, Y.C., Hsieh, S.C., 1982. Genetical studies on grain characters in rice. J. Agric. Res. China 31, 177–186. Lai, F. S., Zayas, I., and Pomeranz, Y. 1986. Application of pattern recognition techniques in the analysis of cereal grains.Cereal Chem.63:168. Neuman, M., Sapirstein, H.D., Shwedyk E. and Bushuk. W. 1987. Discrimination of wheat class and variety by digital image analysis of whole grain samples. Journal of Cereal Science.Volume 6, Issue 2, 125-132. Redona, E.D., Mackill, D.J., 1998. Quantitative trait locus analysis Shi, C.H., Zhu, J., 1996. Genetic analysis of endosperm, cytoplasmic and maternal effects for exterior quality traits in Indica rice. Chin. J. Biomath. 11, 73–81. Shi, C., Zhu, J., Wu, J., Fan, L., 2000. Genetic and genotype* environment interaction effects from embryo, endosperm, cytoplasm and maternal plant for rice grain shape traits of Indica rice. Field Crops Res. 68, 191–198. Sunayana Rathi; Raj Narain Singh Yadav; Ramendra Nath Sarma. 2010. Variability in grain quality characters of upland rice of Assam, India. Rice Science, 2010, 17(4): 330-333. Tan, Y.F., Xing, Y.Z., Li, J.X., Yu, S.B., Xu, C.G., Zhang, Q., 2000. Genetic bases of appearance: quality of rice grains in Shanyou 63, an elite rice hybrid. Theoret. Appl. Genet. 101, 823–829. Yusako S. Emilie E. Regnier, Tim D. Kikuo Fujimura, S. Kent Harrison and Miller B. McDonald.2001. Computer Image Analysis and Classification of Giant Ragweed Seeds. Weed Science. Vol. 49, No. 6, pp. 738-745. Zayas, I., Pomeranz, Y., and Lai, F. S. 1985. Discrimination between Arthur and Arkan wheats by image analysis. Cereal Chem. 62:478. Zayas, I., Lai, F. S., and Pomeranz, Y. 1986. Discrimination between wheat classes and varieties by image analysis. Cereal Chem. 63:52. Zayas, I., Pomeranz, Y. and Lai F. S.1989. Discrimination of wheat and nonwheat components in grain samples by image analysis.Cereal Chem. 66(3):233-237.

10


An Efficient Agrobacterium-mediated Transformation Method for Sugarcane (Saccharum officinarum L.) 1

Efendi and 2M. Matsuoka

1

Departmen of Agrotechnology, Faculty of Agriculture, Syiah Kuala University, Banda Aceh 23111, Indonesia; 2Japan International Research Center for Agriculture Science (JIRCAS), Okinawa Prefecture, Japan. Email: [email protected]

Abstract. Investigation of transformation method for sugarcane was carried out by using Agrobacterium tumefaciens with a vectors pMLH7133 that contained CaMV35S promoter and marker genes. Embryogenic callus and cell aggregates of suspension culture were used as the target materials for transformation. The aggregates cell was sonicated, and co-cultured with Agrobacterium tumefaciens. The cells were cultured in N6-2 medium containing an appropriate antibiotic to eliminate bacterial contamination. Embryogenic callus was co-cultured with Agrobacterium, and was also cultured in N6-2 medium as same as suspension culture. Selection was carried out by culturing the treated suspension culture and embryogenic callus with MS medium containing geneticin. The transformed calli were transferred to MS-R9s for shoot formation. The transformed cells were analyzed for the distribution of GUS activity histochemically. After histohemical staining with X-glux, light microscopy observation revealed that the transformed calli derived from the NiF4, Ni9, and NCo310 cultivars had blue coloration in its tissue. Thus, the gene for GUS appeared to have been transferred and to be expressed in the calli. The construct of pCL4 was also integrated in the bacteria. Transient expression of GUS gene was successfully confirmed in the transformed Agrobacterium. When we use suspension culture, the proportion of the calli showing transient GUS expression was 4.7-fold greater with the vector in pMLH7133. We also successfully produced transformed calli with higher level of transient GUS expression. The percentage of the calli showing the best transient GUS expression is pCL4. The embryogenic callus was more competent for transfer of T-DNA into sugarcane cells. Analysis of GUS activity indicated that the gene was expressed into the calli of sugarcane. The Results indicate that the promoter can serve as an effective regulatory element to produce strong expression in callus of sugarcane. When we inoculated embryogenic callus with Agrobacterium harbored binary vector pCL4, we also successfully produced transformed calli with higher level of transient GUS expression. Thus, the gene for GUS appeared to have been transferred and to be expressed into the calli. Putative transformed plants were tested by performing PCR and Southern Hybridization to confirm the integration and expression of the introduced genes.

Key words: Transformation, sugarcane, Agrobacterium, embryogenic callus.

Introduction Conventional plant breeding techniques have been extensively employed by selecting improved varieties. However, some important traits such as low resistance to insect and to herbicides appear to be limited for yield increasing. The Agrobacterium-mediated transformation of plants has become important in efforts aimed at the improvement of crops and may have an important impact on sugarcane yields in future. Plant transformation mediated by Agrobacterium tumefaciens has to be the most used method for the introduction of foreign genes into plant cells and the subsequent regeneration of transgenic plants (Efendi et al., 1996, Efendi et al., 1998). However, the lack of a reproducible methodology for stable transformation of sugarcane was an important obstacle for genetic manipulation during many years. Agrobacterium-mediated gene transfer into monocotyle-donous plants was difficult. However, in recent years, several monocotyledonous plants (rice, maize, wheat, barley, onion, soybean) were successfully transformed (Hiei et al., 1994; Ishida at al., 1996; Cheng at al.., 1997; Tingay at al., 1997; Eady at al.., 2000; Efendi et al., 2000; Efendi, 2001). Agrobacterium-mediated transformation method and recovery of the first transgenic plants have been reported in sugarcane (Arencibia at al., 1998). Recently, sugarcane cell cv. NiF4 and NiF9 was also successfully transformed using Agrobacterium tumefaciensmediated transformati-on with vector pMLH7133 and pIG121 containing genes of GUS, NPTII, and HPT (Arifin et al., 2002 and Matsuoka et al., 2002; Efendi, 2003; Efendi and Matsuoka, 2003). Although, transformation of sugarcane have been carried out in many studies, but only a few successful studies of stable Agrobacterium-mediated transformation of sugarcane have been reported. The problem of transferring Agrobacterium-mediated gene to plants is related with poor survival rate of the target cells or necrosis. The inoculated 11


cells were being traumatic due to the infection of Agrobacterium. Oxidative burst, phenolization and subsequent cell death have been described as frequent phenomena during the interaction of Agrobacterium with monocotyledonous plant cells (De la Riva et al., 1998). Thus, we need an improve method of transformation to solve the problem of phenolization. Recently, we reported that the use of ultrasound enhanced the efficiency of Agrobacterium-mediated transformation on sugarcane. Sonication-assisted Agrobacteriummediated transformation (SAAT) consists of subjecting the target tissue to ultrasound before immersing in an Agrobacterium suspension. The enhanced transformation rates using SAAT result from microwounding, where the energy released by cavitation causes small wounds both on the surface of and deep within the target tissue (Efendi and Matsuoka, 2004). Unfortunately, the use of ultrasound was not enough to establish a reproducible transformation method in sugarcane. Some improvements such as the use of different types of promoters and target materials are important for development of Agrobacterium-mediated transformation method for sugarcane. The promoter is a key DNA regulatory element that directs appropriate strength and patterns of gene expression in a constitutive or specific manner, and therefore, plays a crucial role in successful transformation studies. Moreover, the number and types of promoters that drive strong and constitutive expression of transgenes are relatively few in sugarcane. The viral Cauliflower Mosaic Virus 35S (CaMV35S) promoter has been widely used in the transformation of many dicot and monocot crops, but activity in sugarcane has been low as demonstrated in various studies (Elliott et al., 1998). The rice actin 1 and the Emu elements have shown higher activity than CaMV35S in different sugarcane tissues (Gallo-Meagher and Irvine, 1993), however, they have not been widely utilized in subsequent studies. Two sugarcane ubiquitin promoters, ub4 and ub9, were recently used to drive transient β-glucuronidase (GUS) expression in sugarcane calli, but GUS expression was not detected in regenerated plant tissue (Wei et al., 2001). The maize ubiquitin promoter Ubi-1 has been used to drive constitutive transgenes expression in sugarcane studies (Fako et al., 2000). The rice RUBQ2 polyubiquitin promoter, containing 5`upstream and intron regions, was identified from a rice bacterial artificial chromosome (BAC) genomic library (Wang et al., 2000). Transient GUS activity driven by RUBQ2 in rice suspension cells was ten to 15 times higher than with constructs containing the CaMV35S, and two to three times higher than the maize Ubi-1 promoter (Liu et al., 2003). In other hand, the target material such as embryogenic callus is another important factor that can improve the efficiency of Agrobacterium-mediated transformation for sugarcane. Liu et al. (2003) reported that the use of embryogenic callus showed many calli stained blue color after co-cultivation with Agrobacterium containing binary vector pCL4 harbored RUBQ2 promoter. Agrobacterium-mediated transformation of calli with the pCL4 resulted in significantly improve the efficiency of embryogenic callus transformation. Zhang et al. (2004) also successfully transformed embryogenic callus of sugarcane using Agrobacterium-mediated transformation. In this paper, we reported comparison of transient GUS gene expression in calli driven by RUBQ2 promoter of pCL4 and CaMV35S promoter of pMLH7133 via Agrobacterium-mediated transformation in sugarcane embryogenic callus and aggregates cells of suspension culture. The aggregates cell was sonicated, and co-cultured with the bacteria of Agrobacterium tumefaciens. Materials and Methods Suspension culture and callus formation Embryogenic callus and cell aggregates of suspension culture of three sugarcane cultivars i.e. NiF4, Ni9, and NCo310, the leading commercial cultivar of sugarcane in Japan, were used as the target materials of Agrobacterium-mediated transformation in the present experiment. Spindle sections from healthy plant taken from field were used for callus induction. Explants sized 5-10 mm were cultured in MS-1 medium (Table 2) containing 2 mg/l 2,4-D and the formed calli were transplanted into a new medium every four weeks three times. The well growing and compact calli were chosen for preparation of suspension culture and embryogenic callus. Calli were suspended in a flask filled with 30 ml liquid N6-2 (Table2) containing 2 mg/l 2,4-D. Cultures were incubated on a rotary shaker at 150 rpm 12


and were maintained by transferring one ml volume of cell aggregates to a fresh N6-2 medium every week. Embryogenic callus was performed using aggregates cell. The cells were culture onto MS medium containing 1.5 mg/L 2, 4-D for one month. Bacterial strain and vectors We have investigated transformation method in sugarcane by using Agrobacterium tumefaciens. Transformation of Agrobacterium was conducted by introducing pCL4 molecule C into E. coli JM109. The construct was transferred into Agrobacterium LBA4404 by the freeze-thaw method using CaCl. The Agrobacterium was constructed separately with two binary vectors (pMLH7133, pCL4). The vector of pMLH7133 was introduced in strain EHA101 of the Agrobacterium, the vector of pCL4 was transferred into strain LBA4404. The vector of pMLH7133 contained genes of GUS, NPTII, and HPT, and the vectors of pCL4 contained genes for NPTII and GUS. For inoculation of embryogenic callus and suspension culture, the bacteria Agrobacterium were grown in LB medium containing 25 mg/l rifampicin and 50 mg/l kanamycin for 12-16 hours. The bacterial cells were collected by centrifugation 5000 rpm for 5 minutes. Transformation of aggregate cell The cell aggregates of sugarcane collected from 2-3 months suspension culture in liquid N62 medium were co-cultivated at 22OC, 150 rpm with Agrobacterium strain EHA101 contained binary vector pMLH7133 and strain LBA4404 contained pCL4. Elimination of the overgrowth bacteria was done in 5 day after co-cultivation in liquid N6-2 containing 50 mg/l acetocyringon by washing with sterilized water and 5 minutes sonication at 45 KHz. Then, the cells were cultured in N6-2 medium containing 250 mg/l carbenicilin for two days to eliminate bacterial contamination. The culture medium was replaced with MS solid medium containing 250 mg/l carbenicilin for a week. Transformation of embryogenic callus The Embryogenic callus was co-cultured with Agrobacterium for five days on MS medium containing 2, 4-D 1.5 mg/l and acetocyringon 50 mg/l at 22OC. The cells were washed with sterilized water by shaking with Vortex and then cultured in N6-2 medium containing 250 mg/l carbenicilin for two days to eliminate bacterial contamination. Screening of transformed callus was done four weeks with selective MS medium containing 50 mg/l geneticin and carbenicilin 250 mg/l. For regeneration, the calli were transferred to MS-R9s medium containing 1 mg/l BA, 0.2 mg/l IAA, 50 mg/l geneticin and 250 mg/l carbenicilin. The cultures were incubated in the dark for 3 days, and then incubated under fluorescent light (26oC and 16 h light). Histohemical GUS assays were performed 2 weeks after transfer of the calli into the medium containing 50 mg/l geneticin and carbenicilin 250 mg/l. Calli were placed in a GUS assay mix (Jefferson 1987) and incubated overnight at 28°C. The GUS assay mix was removed, and the tissue was rinsed twice with 70% ethanol to stop reaction. The number of GUS positive was observed by light microscope. Results and Discussion Molecule of pCL4 contained RUBQ2 promoter was successfully introduced into E. coli JM109. The construct was transferred into Agrobacterium LBA4404 by the freeze-thaw method using CaCl. A transformed colony of Agrobacterium was tested by spreading the cells on a LB agar plate containing appropriate antibiotic selection (25 mg/l rifampicin and 50mg/l kanamycin). Transient of GUS gene expression of the Agrobacterium was performed by incubating the bacteria with X-Gluc mix overnight at 28°C. This result showed that the transformed Agrobacterium had blue coloration (Figure 1.A) and indicated that the construct of pCL4 was integrated in the bacteria. Transient expression of GUS gene was successfully confirmed in the transformed Agrobacterium. Knowledge that the T-DNA could be transferred from the Ti plasmid of A. tumefaciens into plant cells and integrated into the nuclear DNA certainly raised the possibility that it could be used as a vector to transfer genes of interest into plant cells and, ultimately, into fertile plants that could transmit the genes to their progeny. Several findings emerged that were crucial in the endeavor to generate transgenic plants. The first of these were studies on tumors induced by T-DNA variants, either naturally occurring or isolated through mutagenesis.

13


A

B

C

.

.

.

C

T

T

Figure 1. A. Transient expression of GUS gene in the transformed colony of Agrobacterium. C, control, non-transformed Agrobacterium; T, transformed Agrobacterium with vector pCL4. B. Selected calli of embryogenic callus cultivar Ni9 after screening the inoculated callus during four weeks with selective MS medium containing 50 mg/l geneticin and 250 mg/l carbenicilin. C. GUS gene activity in inoculated callus of sugarcane cultivar NiF4 after the Agrobacterium-mediated transformation and selection with selective MS medium containing geneticin 50 mg/l and carbenicilin 250 mg/l for two week.

The results showed that it was produced many selected calli after screening the inoculated callus during four weeks with selective MS medium containing 50 mg/l geneticin and 250 mg/l carbenicilin (Figure 1.B). After GUS assay, light microscopy observation revealed that the inoculated calli derived from NiF4, Ni9, and NCo310 had blue coloration in its tissue (Figure 1.C). In consequence, we got many selected sugarcane calli, and found that the binary vector pCL4 carried RUBQ2 promoter significantly enhanced the efficiency of sugarcane transformation, especially in cultivar NiF4. When we use suspension culture of the agregates cell, the proportion of the calli showing transient GUS expression was 4.7fold greater with the pCL4 containing the RUBQ2 promoter than with the CaMV35S promoter in pMLH7133 (Table 1). When we inoculated the embryogenic callus with Agrobacterium harbored binary vector pCL4, we also successfully produced transformed calli with higher level of transient GUS expression. Thus, the gene for GUS appeared to have been transferred and to be expressed into the calli. The percentage of calli showing transient GUS expression was 12.1-fold greater with the pCL4 than with the pMLH7133 (Table 1). This result showed that the embryogenic callus was more competent for transfer of T-DNA into sugarcane cells. It is suggested that the embryogenic callus was less sensitive to necrosis, oxidative burst, or phenolization that caused by bacterial infection. Analysis of GUS activity indicated that the gene was expressed into the calli of sugarcane. Results from this GUS activity indicate that RUBQ2 can serve as an effective regulatory element to produce strong expression in callus of sugarcane, especially for cultivar NiF4. DNA constructs containing RUBQ2 promoter produced higher levels of transient GUS expression by Agrobacterium-mediated transformation in calli of sugarcane. Table 1. Frequency of GUS activity in transformed calli of sugarcane that using CaMV35S & RUBQ2 promoters Types of materials

Promoters

GUS positive of sugarcane cultivars (%) NiF4

Ni9

NCo310

Suspension culture

CaMV35S

2.2

3.1

1.2

Embryogenic callus

CaMV35S

5.8

8.6

3.3

Suspension culture

RUBQ2

10.3

9.1

4.5

Embryogenic callus

RUBQ2

70.4

10.3

20.8

CaMV35S: Cauli Mosaic Virus 35S promoter. RUBQ2

14

: Rice Ubi Quitin2 promoter


High GUS gene expression levels driven by RUBQ2 in sugarcane described in this report suggest that the rice polyubiquitin promoter would function efficiently in other monocotyledonous plants as well. This result showed that the use of RUBQ2 promoter could improve the efficiency of Agrobacterium-mediated transformation for sugarcane, especially in the calli of sugarcane. Liu et al. (2003) reported that the use of embryogenic callus showed many calli stained blue color after co-cultivation with Agrobacterium containing binary vector pCL4 harbored RUBQ2 promoter. The method of Agrobacterium-mediated transformation of calli with the pCL4 resulted in significantly improve the efficiency of embryogenic callus transformation. Zhang et al. (2004) also successfully transformed embryogenic callus of sugarcane using Agrobacterium-mediated transformation. However, to obtain whole transformed plant, we need further experiments for regeneration and selection of transformed plants. Continuous analysis of putative transformed plants should be carried out by performing PCR and Southern Hybridization to confirm the integration and expression of the introduced genes from intact transgenic plants. The use of RUBQ2 promoter was effective to enhance the transient GUS expression in calli of sugarcane and could contribute to set up an efficient transformation method for sugarcane. However, further investigation of detailed conditions, such as variations of pH, temperature, and period of co-culture should be carried out to establish an efficient and reproducible protocol. Introduction of useful genes also needed to be studied to make the method practical. Conclusions Embryogenic callus and cell aggregates were used for sugarcane transformation. The materials was sonicated, and co-cultured with Agrobacterium tumefaciens. Selection was carried out by culturing the treated materials with MS medium containing geneticin. The transformed calli were transferred to MS-R9s for shoot formation. The transformed cells were analyzed for the distribution of GUS activity histochemically. Light microscopy observation revealed that the transformed calli derived from the NiF4, Ni9, and NCo310 cultivars had blue coloration in its tissue. Tthe gene for GUS appeared to have been expressed in the calli. When we use suspension culture, the proportion of the calli showing transient GUS expression was 4.7-fold greater with the vector in pMLH7133. We also successfully produced transformed calli with higher level of transient GUS expression. The percentage of the calli showing the best transient GUS expression. This result showed that the embryogenic callus was more competent for transfer of T-DNA into sugarcane cells. Analysis of GUS activity indicated that the gene was expressed into the calli of sugarcane. Results from this results indicate that the promoter can serve as an effective regulatory element to produce strong expression in callus of sugarcane. Acknowledgements This research was supported by the Japan International Research Center for Agriculture Science Okinawa Subtropical Station, Japan 2003-2004. We gratefully acknowledge James H. Oard, Department of Agronomy, Louisiana Agricultural Experiment Station, Louisiana State University Agricultural Center, 104 Sturgis Hall, Baton Rouge, LA 70803, USA for providing construct pCL4. References Arencibia, A., E. Carmona, P. Tellez, M-T. Chan, S-M. Yu, L. Trujillo., and P. Oramas. 1998. An efficient protocol for sugarcane (Saccharum spp. L.) transformation mediated by Agrobacterium tumefaciens. Trans. Res., 7:213-222. Arifin, N.S., T. Terauchi, Y. Tamura, M. Tanio , and Matsuoka, M. 2002. Development of method for sugarcane transformation mediated by Agrobacterium tumefaciens. Breeding Research, 4 (2):318 Cheng, M, Fry, J.E., Pang, S.Z., Zhou, H.P., Hironaka, C.M., Duncan, D.R., Conner, W. and Wan, Y.C. (1997). Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiology, 115:971-980. De la Riva, G.A., J.Gonzalez-Cabrera, R. Vazquez Padron, and C. Ayra-Pardo. 1998. Agrobacterium tumefaciens: a natural tool for plant transformation. Journal of Biotechnology, 1 (3):118-133 15


Eady, C.C., Weld, R.J., Lister, C.E.2000. Agrobacterium tumefaciens-mediated transformation and transgenic-plant regeneration of onion (Allium cepa L.). Plant Cell reports, 19(4):376-381. Efendi. A. Kisaka, T. Kameya. 1996. Production of transgenic soybean (Glycine max (L.) Merr) using Agrobacterium tumefaciens and analysis of the transgenic plant. Journal Breeding Science. Vol. 46 (2) Thn.1996. Hlm 127. Efendi. A. Kisaka, T. Kameya. 1998. Transformation of soybean and harricot bean by injecting germinating with Agrobacterium. Journal Breeding Science 48(2): 137. Efendi. A. Kisaka, T. Kameya. 2000. Transformation of Soybean by Infecting embryogenic calli with Agrobacterium tumfaciens and that of soybean and kidney bean by injecting the bacteria into germinating seed. International Journal for Plant Biotechnology 17(3): 187 -194 Efendi. 2001. Transformation of soybean (Glycine max L.) with LEA gene and analysis of the transgenic plants tolerance to water deficit and salt stress. Jurnal Agrista 6(4): 1219. Efendi. 2003. Develoment of Agrobacterium-mediated transformation by Sonication for sugarcane, ISTEC Journal 2(3): 56- 63. Efendi, Matsuoka, M. 2003. Effects of time and period of sonication on Agrobacterium ellimination, Japanese Breeding Science Journal 12 (3): 79-87. Efendi, Matsuoka, M. 2004. Comparison of Promoter in Agrobacterium-mediated transformation for sugarcane. Japan International Research Center for Agriculture Science, JIRCAS Working Report, Edisi September 2004. Elliott, A.R., J.A. Campbell, R.I.S. Brettell, C.P.L. Grof. 1998. Agrobacterium-mediated transformation of sugarcane using GFP as a screenable marker, Aust. J. Plant Physiol. 25:739-743. Fako, M.C., A.T. Neto, and E.C. Ulian. 2001. Transformation and expression of a gene for herbicide resistance in a Brazilian sugarcane, Plant Cell Rep. 19:1188-1194. Gallo-Meagher, M., J.E. Irvine. 1993. Effects of tissue type and promoter strength on transient GUS expression in sugarcane following particle bombardment. Plant Cell Rep. 12:666-670. Hiei, Y., Ohta, S., Komari, T. and Kumashiro, T. (1994). Efficient transformation of rice (Oriza sativa) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. The Plant Journal 6:271-282 Ishida, Y., Saito, H., Ohta, S., Hiei, Y., Komari, T., and Kumashiro, T. (1996). High efficiency transformation of maize (Zea mayz L.) mediated by Agrobacterium tumefaciens. Nature Biotechnology, 4:745-750. Matsuoka, M., N.S.Arifin, T.Terauchi, Y.Tamura, M.Tanio, A. Hayakawa, and H.Miwa. 2002. Transformation of sugarcane cell mediated by Agrobacterium and subsequent shoot regeneration. Japanese Journal of Tropical Agriculture 46(1):11-12 Tingay, S. McElroy, D., Kalla, R., Fieg, S., Wang, M., (1997) Agrobacterium tumefaciensmediated barley transformation. The Plant Journal 11:1369-1376. Wei, H., H. Albert, and P. Moore. 2001. Characterization of two sugarcane polyubiqutin gene promoters and putative matrix attachment regions for gene regulation in monocots, in: XI International Plant and Animal Genome Conference, San Diego, USA, p178. Wang, J., J. Jiang, and J.H. Oard. 2000. Structure, expression and promoter activity of two polyubiquitin genes from rice (Oryza sativa L.). Plant Sci. 156:201-211. Zhang, S., B. Yang, J. Luo, and Ishiki. 2004. Efficient transformation method of sugarcane mediated by Agrobacterium. Breeding Research 6(1):253.

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Performance of broiler marketing in Umuahia area of Abia State, Nigeria I. O. Obasi and I. J. Okafor Department of Agric Economics, Micheal Okpara University of Agriculture, Umudike, Abia State, Nigeria. Email- [email protected] Abstract. This study examined the performance of broiler marketing in Abia State of Nigeria.The specific objectives of the study were to examine the performance of broiler marketing in terms of the marketing cost and returns, marketing margin and marketing efficiency, as well as identify factors affecting the income of broiler marketers in the study area. Primary and secondary data were used for this stud. Forty- five respondents from each category; producer-marketers and sole markers were selected both purposively and randomly from the population across the major markets and production areas in Umuahia North and Umuahia South Local Government Areas of the state. The result of the study showed that the business was profitable though with high marketing margin .In terms of economic efficiency, the marketing was efficient. The significant variables influencing the income of the producer-marketers were marketing experience, purchase cost, feed cost, and other variables such as electricity, depreciation and rent. For the sole marketers, the significant variables influencing their income were marketing experience, ages, experience, feed cost and level of formal education. It is recommended that Government should put into consideration the significant variables in policy formulations and provide conducive environment for the private sector to invest in this business in order to address the meat demand of the citizenry. Keywords: Performance, broiler, marketing

Introduction One of the most fundamental challenges facing Nigeria today is ensuring that Nigerians have ample food supply to sustain rural and urban livelihoods. However, this seems to be seriously endangered by the ever increasing demand for food which stems from the fast growth in the population of the country. Most studies have shown that domestic food production on the aggregate has been growing and at the rate of 2.5% per annum, while demand for food on the other hand has been growing at the rate of 3.5% per annum. (Ojo, 2003). There is a wide gap between domestic food supply and food demand (Ajibefun, 2003). A large proportion of the population in developing countries are living under poverty line whose problems apart from getting three “Square Meals” per day include, shelter, clothing, minimum nutritional requirements and of course optimal health care. The growing scarcity and cost of animal protein gradually getting out of the reach of many Nigerians, leading to several steps to increase the rate of agricultural food production by the government. To this end, the poultry industry in Nigeria has played and has continued to play an important role in producing ample protein for the growing population in order to solve malnutrition problem. (Effiong and Onuekwusi, 2006). Poultry business has witnessed great change in Nigeria. It has graduated from subsistence to commercial poultry farming. In Abia State, most poultry farms established are small scale, while the few large scale farms are predominantly owned by corporate bodies and wealthy individuals. The primary motive of any business is to maximize profit. The success of any poultry farm depends on the management efficiency, the market situations amongst other influences. Maximum poultry production depends partly on the environment, technical know-how and the quality of resources employed in the production process. (Nayer, 1989). Due to Government programmes in the last decades on livestock development in Nigeria, many poultry farms producing meat and eggs were established. This development brought about the emergence of broiler farming raised specifically for meat production. Modern commercial broilers, typically known as Cornish crosses or Cornish – Rocks are specifically bred for large scale, efficient meat production and grow much faster than egg or tradition dual purpose breeds. They are noted for having very fast growth rates, a high feed conversion ratio and low levels of activating. Broilers often reach a harvest (slaughter) weight of 4-5 pounds (1.5 – 3kg), dressed in only eight weeks (Nayer, 1989).

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Fatuga (1996) observed that broiler contributes about 10% of the national meat production. According to him, poultry have ranked the fourth major source of animal proteins consumed in Nigeria. This is largely due to the fact that in comparison to other livestock enterprises, broiler production has the advantage of fast growth rate, cheap, high feed conversion efficiency, can be eaten by one family, and is not forbidden by any culture or religion in Nigeria. The production level of broiler meat is currently on the rise, and if given proper attention, it can be relied upon in a short run for ameliorating the deficit in protein supply as well as the poor means of livelihood for most farmers in the country. Therefore, boosting production of broilers can be encouraged when the entrepreneurs have adequate information on the marketing of poultry products in Nigeria. The American marketing association (AMA), defined marketing as the performance of all business activities that direct the flow of goods and services as they move from produces to consumers. Marketing plays a crucial role in a market economy, (Mejeha et al., 2000). Its roles become more important in areas where there is high level of commercial activities and high rate of urbanization (Olukosi and Isitor, 1990). Increasing marketing activities enhances the provision of more and better poultry products at low prices, to increasing numbers of people. The marketing process enables poultry farmers and other people who engage in agricultural marketing to generate income, thereby increasing their welfare. In trying to explain the role of marketing, Busch and Huston (1985) propounded the gap theory which is based on the premise that marketing need not exist until a social economy reached the point where producers of economic goods are not the consumers of the same goods. This situation creates a separation or gap. It is in response to the need to bridge this gap that we have marketing. In a competitive economy, agricultural development cannot occur without improved marketing. This is because agricultural marketing is concerned with all the economic activities involved in the production and distribution of agricultural products (Odii and Obih 2000). In Nigeria, the huge costs involved in the marketing of broiler products have drastically reduced the margin realizable from the enterprise. Considerable improvements in broiler production have been made by the application of modern techniques. However, there have been significant failures within developing countries to understand the interrelationship between broiler production and broiler marketing, since efficient marketing stimulates production. The objectives of the study were to estimate the marketing costs and returns, marketing margin as well as marketing efficiency, and to identify factors affecting the income of broiler marketer Materials and Methods The research was conducted in Umuahia Zone of Abia State, Nigeria, comprising of Umuahia North and South Local Government Areas. The study covered two categories of broiler marketers in Umuahia zone of Abia State, Nigeria. The first category included those in the production and marketing of broilers, while the second group involves those who market broilers only. This study employed purposive sampling techniques in selecting the respondents. A total of Ninety respondents were selected purposively from the study area. Forty five respondents will be from both categories as mentioned above. Data collected were analyzed some statistical tools. To analyze the performance of broiler marketing, net return, marketing margin and economic efficiency models were used. They are stated as follows: NET RETURN = TOTAL RETURN – TOTAL COST MARKET MARGIN

=

SELLING PRICE – SUPPLY PRICE X 100 SELLING PRICE 1 The formula for marketing efficiency as given by Odii and Obih, (2002), is as follows; ECONOMIC EFFICIENCY = TOTAL REVENUE (N) TOTAL COST (N) The activities are said to be efficient if the operations in which these ratios are computed are greater than one and inefficient when it is less than one (Odii and Obih, 2002). Factors influencing the income of broiler marketers were analyzed using multiple repressions. The model specification for the regression is as follows: Y = f (X1, X2, X3, X4, X5, X6, X7, X8, ) 18


Where Y = income from broiler sales in naira, X1 = Age in years, X2 = Marketing Experience in years, X3 = Cost of broiler purchase in naira, X4 = Transportation cost in naira, X5 = Cost of Feed in naira, X6 = Incidence of Disease (yes=1, 0 = otherwise), X7 = Level of Education in Years, X8 = Other Variables (Electricity Costs, Sanitation Costs, etc) Results and Discussion This section discusses the net returns, marketing margin, technical and economic efficiencies. It also discusses factors influencing the income of broiler farmers as well as the major challenges limiting against broiler marketers in the study area. Table 1. Cost and Returns Analysis for Both Categories of Respondents.

Average Supply Price (N) Average Selling Price (N) Average Total Cost (N) Average Income (N) Marketing margin (%) Net Returns (N) Economic Efficiency

Producer-Marketers

Marketers only

170.4 1,218.8 280,658.7 359,422.2 86 78,763.5 1.28

1,232.2 1,487.8 361,428.7 529,480 17.18 68,051.3 1.47

Table 1.0 above showed a net return of (N) 359,422.2 and (N) 529,480.0 for producer-marketers and sole marketers respectively implying that the business was profitable. Thus, the sole broiler marketers made more profit than the producer-marketers. Marketing margins were high compared to the acceptable standards, (Scarborough and Kydd, 1992) .The economic efficiency for these group were1.28 and 1.47, showing that they were economically efficient in their operations, as the ratios were greater than one. Therefore, the business could be said to be profitable, viable and economically efficient (Salako et al, 2007). Factors influencing the income of broiler marketers The multiple regression result of factors influencing marketing efficiency of broiler marketing is presented in Table 2 below: Table 2.Multiple Regression Result for Factors Affecting Producer- Marketers. Variables Constant

Linear 210.348 (9.322)***

Exponential 11.471 (47.569) ***

Double-Log 1.605 (1.809)*

Semi-Log 11.471 (47.569)***

X1 (Age)

- 990.886 (-.456)

-.004 (-. 472)

- 174 (.840)

- . 004 (-.472)

X2 (Mktg Experience)

4303.743 (1.371)

.031 (2.617)***

.075 (1.205)

. 031 (2.617)***

X3 (Purchase Cost)

.300 (. 371)

4.29E -006 (1.432)

.147 (1.733)*

4.29E -006 (1.432)

X4 (Trans Cost)

66.460 (3.227)*** 1.351 (6.238)***

-3.34E -005 (-.438) 2.35E – 006 (2.928)***

.029 (.400) . 617 (7.163)***

-3.34E -005 (-. 438) 2.35E – 006 (2.928)***

R2

- . 411 (- 1.288) .005 (.133) 3.695 (1.853)* .960

- 4.94E - 007 (- .418) 1.53E -008 (.110) 1.53E – 005 (2.068) ** .923

- . 008 (- . 742) - .019 - 1. 471 .133 (1.846)* .970

-4.94E -007 (- . 418) 1.53E – 008 (.110) 1.53E – 005 (2.068)** .923

R-2 – ratio

..952 109.149***

.906 53.729***

.963 143.159***

.906 53.729***

X5 (Feed Cost) X6 (Incid of Disease) X7 (Education) X8 (other cost)

*** = Significant at 1%, ** = Significant at 5%, * equation. The figures in parenthesis are t-ratios. 19

= Significant at 10%,+ = lead


From Table 2 above, based on the number of significant variables, the semi log regression model was chosen as the lead equation. The F- ratio and the value of R2 conform to apriori expectation. The value of F-ration was significant which indicates the overall significance of the study result. The value of R2 was 0.923, which implies that about 92% of the explanatory variable in the income of broiler marketing was as a result of the explanatory variable, while only 0.08 or 8% was attributed to error or variables not included in the model. The result further showed that marketing experience, purchase cost, feed cost and other cost variable such as electricity, depreciation of equipment, rent were the significant variables that influenced the income of broiler marketers in the study area. The number of years spent in the business had a direct relationship on the income of the marketers meaning that greater experience brings about greater marketing income. The cost of purchase of broiler chicks or broiler for resale and feed cost had a direct negative relationship on marketing income as the higher the cost of purchase of broiler chicks and feed, the lower the income of marketers. This conforms to apriori expectation as a higher cost of inputs brings about reduced income of marketers. The marketing experience and cost of cost of feed were significant at 1%, while other costs were significant at 5%. Other variables like incidence of disease, education, and age were not significant determinants of marketer’s income. Table 3. Multiple Regression Result for Factors Affecting Marketers Only. Variables Constant

Linear -41421.802 (-.605) 743.777 ( .370)

Exponential 11.569 (48.763)*** .012 (1 . 761)*

Double-Log - . 116 (- . 147) .106 (.766)

Semi-Log 4933857.3 (6.319)*** -99907.870 (-3.728)***

X2

55.366 (.020)

-.005 (- .491)

-.031 (- .786)

1012.255 (.026)

X3 (Purchase Cost)

1.205 (23.065)***

2.36E 006 (13.O40) ***

.976 (27.928)**

426783.97 (12.308)***

X4 (Trans Cost)

16.281 (.758) .180 (.180)

2.13E – 005 (.286) 5.93E – 006 (.774)

-.6.43E -006 (- .183) .029 (.753)

13.237 (.381) - 304.441 (-2.580)***

X6 (Incidence of Disease)

2031.784 (1.920)*

.003 (.897)

. 036 (1.213)

24174.380 (.816) 4020.631 (2.739)***

X7 (Education)

.675 (.206)

3.32E – 006 (.292)

- . 002 (-.182)

X8 (other cost)

-

2.857 (-.800) .954

-1.73E – 005 (1.395) .884

- .003 (- . 053) .970

32163.832 (.537)

.944 93.923***

. 854 34.277***

.963 143.458***

.827 27.258***

X1 (Age)

X5 (Feed Cost)

R2 -2

R F – ratio

*** = Significant at 1%,** = Significant at 5%, * equation. The figures in parenthesis are the t –ratios.

.858

= Significant at 10%, + = lead

The result in Table 3.0 for sole broiler marketers only showed that the variables of significance were age of the marketers, purchase cost, feed cost, and level of formal education acquired by the marketer. These entire significant variables had direct relationship to the income of broiler marketers. The F- ratio was significant at 1% which showed the overall significant of the result. The marketing experience, age, cost of feed, and levels of education attained by marketers were all significant at 1%. the value of R2 was 0.97, meaning that 97% of the variation in the income earned by the respondents who engage in marketing of broiler only was attributed to the explanatory variables, while the other remaining 3% was due to the error term. The result also showed that education was a necessity for improved marketing, as earlier stated by Oni and Yusuf, (1999). 20


Conclusion This work has been able to identify that the marketing of broiler in the study area is efficient to a good extent, yet it is pertinent to solve the problems facing the marketing of this commodity Based on findings from the study it is recommended that the government should cooperate with the private sector in order to set up poultry hatcheries in the South Eastern part of the country in order to eliminate the problem of delays arising from the transportation of day old chicks from hatcheries in the South West of the country. The private sector should be encouraged and provided with incentives to invest in commercial feed formulation and distribution in order to reduce the cost of feed. References Ajibefun, I.A. 2003. Determinants of technical efficiency in traditional agricultural production: Application of stochastic frontier modeling to food crop farmers in south western Nigeria. African Journal of Economic Policicy 10(2): 3-5. Busch P.S. and Huston M.J. 1985. Marketing strategic foundations. Richard D. Irwin Inc. Homewood, Illinois. Effiong E.O. and Onuekwusi G.C. 2006. Comparative analysis of small and large scale broiler farms in Uyo metropolis of Akwa Ibom State of Nigeria. Proceedings of the 40th Conference of Agricultural Society of Nigeria, held at NRCRI, Umudike, October, 16th20th, 2006. pp. 25-30. Fatuga B.C. 1996. Animal production in Nigeria and feed supplies. Nigerian Journal of Animal Production 4(1):56-58. Mejeha R.O., Nwosu A.C. and Ifenkwe G.E. 2000. Analysis of markets for staple food in Umuahia zone: Implications for food security in Umuahia urban. Unpublished report, Michael Okpara University of Agriculture, Umudike, Abia State, Nigeria. Nayer J.P. 1989. Economic trend of broiler in Benzhal. India Poultry Review 17:22-29. Odii M. and Obih U. 2000. Optimising cassava marketing margin and efficiency for agricultural development in the 21st century. Proceedings of a National Conference in Honour of Professor Martin O Ijere, Federal University of Agriculture, Umudike, Abia State, Nigeria, 24th-26th August, 1999, pp. 46-49. Ojo M.O. 2003. Food policy and economic development in Nigeria. Publishers Services United, Lagos, Nigeria. Olukosi J.O. and Isitor S.U. 1990. Introduction to agricultural marketing and prices: Principles and Applications. G.U. Publications, Abuja. Oni O.A. and Yusuf S.A. 1999. The effects of farmers’ Socio-Economic characteristics on livestock production in Ibadan Metropolis. Proceedings of the 4th Conference of Animal Science Association, sept 14th-16th, 1999, IITA, Ibadan, Nigeria, pp. 245-248. Salako B.A., Adedalu O.A. and Adesiyan O.I. 2007. An analysis of cost and returns to broiler marketing in Ogbomosho Local Government Area of Oyo State, Nigeria. Nigerian Journal of Poultry. Science 1: 26-30. Scarborough V. and Kydd J. 1992. Economic analysis of agricultural markets: A manual, marketing series,Vol.5, National Resource Institute(NRI), Chatham, UK.

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Survey and study on yield and quality of patchouli oil in Aceh Barat Daya District, Indonesia based on original area of raw materials, methods and length of distilation Arpi N, Cut Erika and Dewi Ermaya Department of Agricultural Product Technology, Syiah Kuala University, Banda Aceh, Indonesia. Corresponding Author: [email protected] Abstract. Patchouli oil from patchouli plant (Pogostemon cablin Benth) is one of important essential oils as a source of Indonesian foreign exchange. It is about 90% of world patchouli oil from Indonesia (Suara Merdeka, 2006). The objectives of this research were to assess the yield and quality of patchouli oils from four different villages in the sub district of Kuala Batee, Aceh Barat Daya based on the original area of raw material and the method of distillation, also based on length of distillation. The nested design was used in this research with the treatment of two factors combination, with the level of one factor similar but not identical. The treatments were the original area of raw material and the distillation method used in the original area of the raw material (BM1-4), and the length of distillation (5, 6, and 7 hours). Parameter observed were yield, specific gravity, refractive index, alcohol solubility, the concentration of patchouli alcohol, ester number, acid number, and the sensory test on color and clarity. The results indicated that the original area of raw material and distillation method (BM) had a significant effect on yield, refractive index, clarity, and acid number. The yield was 2.85%-4.5%, and patchouli oil from BM4 and BM2 gave higher yield but lower patchouli alcohol concentration, and clarity. The results also indicated that the longer time of distillation the higher patchouli oil yield, specific gravity, and patchouli alcohol concentration. However, it affected the lower alcohol solubility and clarity, the higher ester number, and the darker color of the patchouli oil. The concentration of patchouli alcohol in this study ranges from 21.36% to 34.03%. Patchouli oil yielded in this research have complied the SNI 06-2385-2006. Key words: yield, quality, patchouli oil, patchouli alcohol.

Introduction Patchouli oil from the plant (Pogostemon cablin Benth) is one of the important resources which contribute greatly to Indonesian foreign exchange reserve. Approximately 90% of world’s patchouli oil is produced in Indonesia (Suara Merdeka, 2006). Indonesian patchouli’s field in total take up to 16639 ha, recorded in 2004, and produced 2424 ton of patchouli oil. Patchouli oil industry Indonesia, especially the patchouli oil industry from Aceh, has contributed up to 70% of Indonesian patchouli oil production, equivalent to 1696.8 ton/year from 2876 ha wide of field (Ditjenbun, 2005). Patchouli world market developments nowadays has started to reach stalemate, since the world’s patchouli demand of 2300-2400 tons/year can already be met by some patchouli producer countries. Other common constraints in patchouli agribusiness are the low level of concentration of the produced patchouli oil, low oil quality and homogeneity of oil, unsteady supply of product, and the fluctuation in product’s price (Satriana, 2008). Preliminary survey in four villages in the district of Kuala Batee, Southwest Aceh, shows that the distillation process in each distillation plant from the four villages are different. This is due to the different distillation method and tools used, as well as different raw materials sourced from different areas. Aceh’s patchoulis produce high concentration of oil which varies from 2.5-5%. In addition, the distillation time is determined based on whether or not there is still oil to be distilled. Distillation is stopped whenever the distilled liquid coming out of the instrument does not contain oil anymore. It is expected that the distillates will also vary, thus affecting the yield and quality of the distillates. The yield obtained from the distillation plants of the sample village on average is 2.8% or 0.56 kg of oil for every 20 kg distilled raw material. The produced patchouli oil generally has brownish color. The research is aimed to assess yield and quality of patchouli oil from 4 (four) different villages in Kuala Batee sub district, Aceh Barat Daya based on various raw materials, methods, and length of distillation time. 22


Materials and Methods Raw material Leaves, stems, and branches (twigs) of patchouli plant cv. Aceh (Pogostemon cablin) was obtained from Aceh province. Water source was supplied from each village where the experiment was conducted. Chemicals Aquadest, khloroform, acetic acid, potassium iodide, sodium thiosulphate 0.1 N, alcohol 95 %, KOH 0.1 N, HCl 0.5 N, starch, and phenolphthalein. Distillation process The raw materials, patchouli leaves, twigs, and stems are dried until the moisture content reach ± 14% (± 3 days). Then the material is cut into pieces with a machete (knife) with a length of ± 5 cm. 20 kg of dried patchouli materials which have been cut into pieces (per unit of nested trial) is put into the refiner kettle and treated with appropriate distillation treatment time according to W1, W2 and W3. Distillation is carried out continuously with a pressure of ± 1 atm. after W1 is reached (5 hours) the product is taken and the distillation is continued for W2 and W3.The product of distillation, which are the refined patchouli oil and water are stored in the reservoir container, and patchouli oil is separated. Some of patchouli oil that has been distilled for 5 hours are taken and used as a sample for W1. The rest of W1 is mixed with the distilled product of W2 and used as W2. The mix of W1 and W2 is then mixed with distilled product of W3 and used as sample of W3. Next, the patchouli oil’s yield is calculated and physical and chemical analysis as well as organoleptic test (sensory analysis) is conducted. Analytical methods Yield was calculated and is made to satisfy the Indonesian national standard (SNI 062385, 2006). The specific gravity, refractive index, ethanol solubility, GC-MS method, ester and acid number were also measured and compared with the standard of SNI 06-2385, 2006. Whereas sensory test on color and clarity analysis was determined using sensory test method adapted from Soekarto, 1985. Experimental design The nested design was used in this research with the treatment of two factors combination, with the level of one factor similar but not identical. The first treatment factor is the original area of raw material (B) with distillation method nested in the original area of raw material (BM), and the second factor is the length of distillation (W) nested on each distillation method. The first factor, BM consist of 4 levels which is the original area of raw material and distillation method Jeumpa village (BM1), Krueng Panto village (BM2), Gudang village (BM3), and Blang Panyang village (BM4). The second factor, W consists of three levels which are W1 = 5 hours starting from the time when the water evaporates, W2 = 6 hours, and W3 = up until there is no more oil to be distilled (± 7 hours). The analysis is repeated two times, so there are 4x3x2 = 24 experimental units. The distillation method are, M1= distillation method using water and vapor in a kettle, with flowing cooling water, M2= vapor method, with cooling water in a stagnant (not flowing) tub, M3 = water and vapor method, with cooling water in a stagnant tub, and M4 = vapor method, with flowing cooling water. Result and Discussion The yield of patchouli oil ranges from 2.85% -4.5%, with a general average value of 3.73%. High yield of patchouli oil (3.95%) comes from the village of Blang Panyang (BM4) and from the village of Krueng Panto (BM2), whereas that of a village Jeumpa (BM1= 3.71%) and Gudang village (BM3 =3.29%) has lower yield (P ≤ 0.05) (Figure1). The yield of patchouli oil increased with increasing distillation time (P ≤ 0.5%), which are 3.31%, 3.71% and 4.15% for 5, 6, and ± 7 hours of distillation respectively (Figure 2). This is because the longer the distillation is, the more heat is received by the material to vaporize the oil fraction which is difficult to be vaporized before. Patchouli oil fraction which is hard to be evaporated is expected to have higher boiling point.The yield of patchouli oil in this research range 3.31% - 4.15% indicating the yield agrees with the reference, which is 2.5 - 5% (Suyono, 2001).

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Figure 1. Yield, acid number, and clarity value of Patchouli oil from four different villages.

Figure 2. The effect of distillation length towards yield, solubility in alcohol, ester number, and colour of Patchouli oil. Figure 3 indicated that the longer the distillation time is the higher (P≤0.05) specific gravity of the patchouli oil will be(0.939-0.955). It is expected that the longer the time and the higher temperature of distillation is the more patchouli oil component with high specific gravity will vaporize. Specific gravity is influenced by constituent of essential oil which is influenced by the nutrients in the soil where the plant grows. Patchouli oil with high specific gravity has high weight fraction of patchouli oil, which usualy contain more oxygenated terpene component. However, according to Ginting (2004), the increases of specific gravity is due to the long distillation time. This causes polymerization of chemical components with high boiling point, which is the patchouli alcohol, in patchouli oil, thus causing the purity of patchouli oil to decrease caused by the formation of higher molecular weight polymers. The refractive index of each treatment ranges between 1.5010 - 1.5056 (Figure 4). Compared to Indonesian National Standard of patchouli oil, refraction index found in this study were lower and does not satisfy the SNI 06-2386-2006 standard of 1.507 – 1.515. Refractive index of essential oil is strongly related to the composition of the oil; however the composition depends on the nutrients contained in the soil. Just like the specific gravity, the composition of the essential oil affects the refractive index of the oil. The more long chained components such as sesquiterpene or oxygenated components being distilled the higher medium density of the essential oil will be, thus it becomes difficult for light to be refracted and lead to higher refractive index of the essential oil. Patchouli oil with high refractive index has better quality than the lower counterpart.

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Figure 3. Spesific gravity of Patchouli oil influenced by distillation length.

Figure 4. Refractive index of Patchouli oil from four different villages. The solubility of patchouli oil in 90% ethanol with different distillation time (W) varies from 3.68 to 6.02 ml. Figure 2 showed that the fastest solubility rate was obtained at the treatment of 5 hours-distillation time (3.68 ml ethanol was used to dissolve 1 mL patchouli oil). The longer distillation time is the more ethanol quantity needed to dissolve the patchouli oil (P≤0.05). This is due to chemical component in patchouli oil is polimerized due to increase of temperature during distillation. Moreover the solubility of essential patchouli oil in ethanol is determined by the amount of terpene and oxygenated terpene. Essential oil with a lot of oxygenated terpene compound has lower solubility in ethanol. While on the other hand, essential oil with more oxygenated terpene compound in its composition is easier to dissolve in ethanol. Quality of patchouli oil is influenced by its patchouli alcohol compound. The higher the patchouli alcohol concentration is the better the oil quality will be. Patchouli alcohol concentration level which is determined by using Gas Chromatography-Mass Spectrometry (GC-MS) shows that patchouli oil produced using the raw material from Krueng Panto which has been distilled using vapor method, with stagnant cooling water in a tub, and distillation time of 5 hours (BM2W1), contains 21.36% of patchouli alcohol compound as its main composition while the other 72.511% are non-patchouli alcohol compound. However when the distillation time is increased to 7 hours (BM2W3), the patchouli alcohol compound in the oil also rose to 28.11%. Patchouli oil coming from Jeumpa village which has been distilled using water and vapor method, with condenser in flowing water, and distillation time of 7 hours (BM1W3) has higher composition of patchouli alcohol compound that reach up to 34.03%. Patchouli alcohol concentration does not only depend on the raw material source, but also the time used for distillation process. The longer the distillation is conducted the higher the temperature inside the distillation kettle, thus increasing patchouli alcohol concentration in 25


the oil. This is due to the high boiling point of patchouli alcohol fraction, thus it will only vaporizes at high temperature. The ester number increases along with the increase in distillation time (P≤0.05). Figure 2 shows that ester number range from 8.023 – 14.990 g/ml which still fit the quality criteria of SNI 06-2385-2006 that state the maximum ester number of 20 g/ml. With a longer distillation time there will be more chemical compound with high boiling point inside the patchouli’s leaf that get polymerized, thus resulting in higher ester number, darker oil’s color, and lower oil’s quality. There are differences in patchouli oil’s acid number due to the difference in the source of the raw materials and methods of distillation used between each treatment site. Acid numbers of the oil from each of the different sources and methods of distillation ranges from 2.40 to 4.17 mg/ml, which still satisfy the quality requirements of SNI 06-2385-2006 that states the maximum number of 8 mg/ml. Patchouli oil acid number which the raw material comes from Jeumpa village (BM1) and the Blang Panyang village (BM4) were lower (P ≤ 0.05) than the two other villages. Essential oils contain small amounts of organic acids that formed naturally or are produced from the oxidation and hydrolysis of esters (Ketaren, 1985). In addition, according to Guenther (1970), patchouli leaves that are dried and protected from air and light have a relatively low number of free acids. Since during the drying process of the leaves the organic acids present in the leaves are evaporated. The results of the research show that the distillation time gives a significant influence (p ≥ 0.01) to the color of patchouli oil. The highest color number is found in the patchouli oil that has been distilled for 5 hours is with color number found as 3.9 which tends to show dark yellowish color, while the lowest one is the one that has been distilled for 7 hours, which gives 2.2 color number and with light brown color. This is presumably due to the long distillation time, which increases the content of weight fraction of patchouli oil, which in turn causes the color to darken. In addition, the changes in color also occur due to the rise in temperatures as the length of distillation time increases, causing the oil that is close to the wall of the kettle become easily damaged. The color of patchouli oil found in this research is yellow to light brown color which is still within the range of SNI 06-2385-2006 quality requirement of yellow to reddish brown patchouli oil’s color. Patchouli oil with high clarity value is obtained from the oil produced in Jeumpa village (BM1) and Gudang village (BM3) by using water and vapor distillation methods (Figure 1). This value is higher than the clarity value of patchouli oil obtained from the village of Blang Panyang (BM4) which use vapor distillation method. Clarity of patchouli oil is also influenced by the time used for distillation, the longer the distillation process is the less clear the color of the oil produced (Figure 2). Conclusions The source of raw materials and distillation method used(vapor versus water and vapor, condenser in a stagnant water bath or inside flowing water) will affect the yield, refractive index, patchouli alcohol content, acid number, and the clarity of the patchouli oil produced. Patchouli oil produced in general has yield range from 2.85% -4.5%, refractive index of 1.5010-1.5056, and acid number of 2.40 to 4.17 g/ml. The longer the time used for the distillation process is, the higher the yield of patchouli oil, the higher the specific gravity (reach up to 0.955), and the higher the patchouli alcohol concentration is (34.03%). On the other hand, the lower the solubility of patchouli oil in alcohol, the less clear, the higher the ester number (14 990 g / ml), and the darker the color of the produced patchouli oil will be. References Ginting, S. 2004. Pengaruh Lama Penyulingan Terhadap Rendemen dan Mutu Minyak Atsiri Daun Sereh Wangi. FP. USU, Sumatera Utara. Guenther, E. 1970. The essential oils, volume I. Van Nostrand Reinhold Company, New York. 87-226. 1949.55 Ketaren, S. 1985. Pengantar Teknologi Minyak atsiri. Balai Pustaka, Jakarta. 27-33, 191204 26


Manoi, F. 2007. Perkembangan teknologi pengolahan dan penggunaan minyak nilam serta pemanfaatan limbahnya. Balai penelitian Tanaman Obat dan Aromatherapy. Satriana, D. 2008. Nilam. http./www. benss.budidaya tanaman.htm. Soekarto, S. T. 1990. Dasar-dasar pengawasan dan standarisasi mutu pangan. PAU IPB, Bogor. Suyono, A. H. 2001. Nilam, tanaman semak pencetak dolar. http:/www.indomedia.com/intisari/2001/Sept/khas_flona.htm. Suara Merdeka. 2003. Harga Minyak Nilam Meroket. www.indonesia.com/intisari. Jawa Timur. (22/3/2008).

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Antioxidant activities of curry leaves (Murayya koeniigi) and salam leaves (Eugenia polyantha) Novi Safriani, Normalina Arpi, Novia Mehra Erfiza and Rini Ariani Basyamfar Department of Agriculture, Syiah Kuala University, Banda Aceh 23111, Indonesia. Corresponding Author: [email protected] Abstract. This study aimed to extract the active antioxidant compounds from curry leaves (Murayya koeniigi) and salam leaves (Eugenia polyantha) using three types of solvent; water, ethanol (50%) and hexane, and determine the total polyphenols contents, activity of free radicals scavenging using DPPH (1,1-diphenyl-2-picrylhydrazyl) and ferric reducing power of the extract of those materials. The result showed that curry leaves extracted using water contain a higher amount of polyphenols than other solvent extracts, while for the salam leaves, ethanol (50%) extracts give a higher polyphenol content than others. Total polyphenols extracts had a positive correlation with antioxidant activity in both DPPH radical scavenging and ferric reducing power. Extracts that contain a high amount of polyphenols also exhibit high antioxidant activity. The result indicated that the polarity level of the solvent will determine extraction result and its antioxidant activity. Keywords: Curry leaves (Murayya koeniigi), salam leaves (Eugenia polyantha), antioxidant activity, total polyphenol contents.

Introduction Lipid oxidation is a major cause of quality deterioration in color, flavor, texture, and nutritive values during food processing and storage. One effective way to prevent such oxidative damage is the use of antioxidants. Antioxidant can inhibit the lipid oxidative damage, so it can prolong the shelf life of foods, especially those rich in poly-unsaturated fats. However, it can not fix the food products that have been oxidized (Wong et al., 2006; Lee et al.,2007; Sarastani et al., 2002; Pokorny, 1991). The addition of synthetic antioxidants, such as propyl gallate, butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT) has been used in the food industry to control lipid oxidation in foods. However, the use of these synthetic antioxidants has potential health risks and toxicity (Wong et al., 2006; Sarastani et al., 2002). Therefore, consumers tend to search for natural antioxidants which is considered more safe because the extracts obtained from natural ingredients. This encourages researchers and food industry to search for antioxidants from natural sources to replace synthetic ones. Vitamins A, C and E, carotenoids and flavonoids are antioxidants derived from the diet. flavonoids, also called polyphenols, commonly occur as glycosides in plants (Pietta, 2000). As antioxidants, flavonoids have been reported to be able to inhibit lipid peroxidation, to scavenge free radicals and active oxygen, to inactivate lipoxygenase, and to chelate iron ions (Yen et al., 1997). Several potential local plant products, such as curry leaves and salam leaves, are known to contain active polyphenolic compounds and these compounds are potential antioxidants. Wong et al. (2006) reported that curry leaves obtained from the market in Singapore contain high total phenol, but showed low antioxidant activity while salam leaves contain high total phenol and showed high antioxidant activity as well. Nowadays, both curry leaves and salam leaves only used as vegetable and flavoring food that still have low economic values. The purpose of this study is to extract the active antioxidant compounds from the curry leaves (Murayya koeniigi) and salam leaves (Eugenia polyantha) grown in Aceh using the solvent of water, ethanol and hexane, and determine the total polyphenol contents, free radical scavenging activity using DPPH (1,1 - diphenyl-2-picrylhydrazyl) and reducing power of extracts of those materials. Results from this study will provide information on the antioxidant activity of those plant extracts so that the active antioxidant compounds from the curry leaves (Murayya koeniigi) and salam leaves (Eugenia polyantha) can be used further as a functional food or natural antioxidants used in the food products processing.

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Materials and Methods Materials Plant materials were curry leaves (Murayya koeniigi) and salam leaves (Eugenia polyantha) were obtained fresh from Penyeurat village, Banda Aceh, Indonesia. Chemicals used were ethanol, hexane, Folin-Ciocalteu phenol reagent, gallic acid, Sodium carbonate (Na2CO3), potassium ferricyanide [(K3Fe(CN)6], trichloroacetic acid, ferric chloride (FeCl3), phosphate buffer, and 1,1-diphenyl-2-picryhydrazyl free radical (DPPH). Extraction The samples were ground using a domestic dry blender. Extraction of samples were performed according to the method of Wong et al. (2006) and Leong and Shui (2001). Each sample (2,5 g) was extracted using 25 ml of water (1:10 w/v). The mixture was allowed to stand at room temperature for 1 hr in the dark, then the mixture was centrifuged at 2000 rpm for 5 minutes. The obtained extract was filled in sealed small bottles and stored in a refrigerator at 4⁰C until ready for analysis (Wong et al.,2006). For extraction using ethanol and hexane, each sample (2,5 g) was extracted with 25 ml of solvent (50% ethanol and hexane) (1:10 w/v). Then the mixture was stirred for 60 s using a vortex and centrifuged at 2000 rpm for 5 minutes (Leong and Shui, 2001). The obtained extract was filled in sealed small bottles and stored in a refrigerator at 4⁰C until ready for analysis. Total polyphenol contents determination The total polyphenol contens of the extracts was determined using the Folin-Ciocalteu assay according to the method described by Hung and Yen (2002). A 0,1 ml extract was mixed with 0,1 ml of aquadest and 0,1 ml of Folin-Ciocalteu reagent 50%. The mixture was stirred for 3 minutes using a vortex and added 2 ml of Na2CO3 2%. Then the solution was shaken by using a vortex and allowed to stand for 30 minutes in the dark. The absorbance of the reaction mixture was read at λ = 750 nm. The total polyphenols contents of the extract was expressed as mg gallic acid equivalents per g of plant material. Antioxidant activity determination using DPPH free radical scavenging method The DPPH free radical scavenging activity of each sample was measured using Spectrophotometer (UV-Vis 1700 Pharma Spec, Shimadzu) according to the method of Burda and Oleszek (2001), which modified. Briefly, a 0,1 mM solution of DPPH in ethanol was prepared. Each extract (1 ml) was added to 2 ml of ethanolic DPPH solution until the color of sample became purple. Then, the mixture was shaken using a vortex and left to stand at room temperature for 30 minutes in a dark place. Furthermore, it was stirred again using a vortex. The absorbance of the solution was measured at 517 nm. The degree of decoloration of the solution indicates the scavenging efficiency of the added substance. The free radical scavenging activity was calculated as a percentage of DPPH decoloration using the following equation: Free radical scavenging activity=100 x(1 – absorbance of sample/absorbance of reference) Reducing Power Determination The reducing power of sample extracts was assayed according to the method of Yen and Chen (1995) modified from the method of Oyaizu (1986). Sample extracts were mixed with phosphate buffer (2,5 ml, 0,2 M, pH 6,6) and potassium ferricyanide [(K3Fe(CN)6] (2,5 ml, 1%). The mixture was shaken using a vortex and incubated at 50⁰C for 20 min. Then it was cooled. Trichloroacetic acid (2,5 ml, 10%) was added to the mixture, which was then stirred using a vortex and centrifuged at 3000 rpm for 10 min. The solution (2,5 ml) was mixed with distilled water (2,5 ml) and ferric chloride (FeCl3) (0,5 ml, 0,1%). Then the mixture was shaken again using a vortex and the absorbance of the solution was measured at 700 nm. Statistical Analysis The research was conducted using randomized complete block design with two treatments (the source of natural antioxidant and the type of solvent for the sample extraction) and three replications. The obtained data were then statistically analyzed using Analisys Of Variance (ANOVA). If the test result indicates significant differences between the treatments, it will be proceed with the advanced test Smallest Real Difference (LSD) (Sugandi and Sugianto, 1994).

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Results and Discussion Total polyphenol contents Extraction was performed using three kinds of solvent with different polarity, to obtain every active component in the curry leaves and salam leaves, which are polar, semipolar and non-polar, as a potential antioxidant compounds. The level of polarity will determine extraction result and antioxidant activity contained in the extract. The Folin-Ciocalteu phenol reagent is used to obtain a crude estimate of the amount of phenolic compounds present in the plant extracts. The result of the total polyphenol analysis (Figure 1) shows that the phenolic content of aqueous extract of curry leaves is higher than salam leaves and curry leaves extracted using other solvent, while the phenolic content of the ethanol extract of the salam leaves is higher than those of curry leaves and other solvent extract of salam leaves. Wong et al. (2006) reported a similar trend where the total polyphenol of water extract of curry leaves was also higher than those of salam leaves. This indicates that the phenolic compounds of curry leaves extract are more polar than salam leaves extract. According to Larson (1988) in Lubis et al. (2007), phenolic components known as primary antioxidants derived from plants are polar.

Figure 1. The effect of natural antioxidant extracts and solvent types interaction on total polyphenol content (values followed by the same letter indicate no significant differences) Antioxidant activity DPPH free radical scavenging activity The determination of DPPH free radical scavenging activity is based on the reduction of DPPH radicals in ethanol which causes an absorbance drop at 515 nm (wong et al.,2006). The color of solution changes from purple to yellow. This change occurs when DPPH was captured by antioxidants which remove H atoms to form a stable DPPH-H (Frankel, 1998; Nenadi dan Tsimidou, 2002) The DPPH free radical scavenging activity of curry leaves and salam leaves are shown in Figure 2. It indicates that the type of solvent gave a different antioxidant activity of extracts. Aqueous extracts of curry leaves and salam leaves showed higher antioxidant activity and significantly different than the extract using ethanol and hexane solvent. According to Chang et al. (1997), the polarity will determine the extraction result and antioxidant activity contained in the extract. Both the water extract and the ethanol extract were not significantly different for each source of raw materials, except the hexane extracts of curry leaves has antioxidant activity higher than the salam leaves.

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Figure 2. The effect of natural antioxidant extracts and solvent types interaction on antioxidant activity (values followed by the same letter indicate n o significant differences) Reducing power In the reducing power determination, the reductant (antioxidant) in the sample will reduce Fe3⁺ ions (potassium ferricyanide complex [(K3Fe(CN)6]) to the ions Fe2⁺ (ferrous form)). Therefore, Fe2⁺ can be monitored by measuring the formation of Perl’s Prussian blue at 700 nm. Increased absorbance indicated an increase in reducing power (Lai et al., 2001; Yen and Chen, 1995). As shown in Figure 3, Aqueous extracts of Curry leaves has higher reducing power than ethanol and hexane extracts, whereas for the salam leaves, ethanol extracts exhibit a higher reducing power than other extracts. These results correlated positively with the total polyphenol content. High content of total polyphenols showed a high reducing power of curry leaves and salam leaves extracts. The results reveal that both curry leaves and salam leaves are electron donors and could react with free radicals, convert them to more stable products, and terminate radical chain reaction.

Figure 3. The effect of natural antioxidant extracts and solvent types interaction on reducing power (values followed by the same letter indicate no significant differences)

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Conclusions The result showed that aqueous extracts of curry leaves provide a higher amount of polyphenols and antioxidant activity in both DPPH radical scavenging and ferric reducing power than other solvent extracts, while for the salam leaves, ethanol (50%) extracts give a higher polyphenol content and reducing power than others. Total polyphenols extracts had a positive correlation with antioxidant activity in both DPPH radical scavenging and ferric reducing power. Further research is required to isolate and identify the antioxidative components in curry leaves and salam leaves. It is also necessary to test the heat stability and its application in the food system. Acknowledgements The authors thank Ms. Ade Irma Selphia for her technical assistance. The author acknowledges that the research was supported by Research Grant from Syiah Kuala University, Ministry of National Education, Indonesia. References Burda, S., and W. Oleszek. 2001. Antioxidant and antiradical activities of flavonoids. J. Agric. Food Chem, 49: 2774-2779. Frankel, P. 1998. Polyphenol content and total antioxidant potential of selected italian wines. J. Agric. Food Chem, 45: 1152-1155. Hung, C.Y and Yen, G.C. 2002. Antioxidant activity of phenolic compounds isolated from Mesona Procumbens Hemsl. J. Agric. Food Chem. 50:2993-2997. Lai, L.S., Chou, S.T., Chao, W.W. 2001. Studies on the antioxidative activities of Hsian-tsao (Mesona procumbens Hemsl) leaf gum. J. Agric. Food Chem, 49:963-968. Lee, J.M., Chung, H., Chang, P.S., Lee, J.H. 2007. Development of a method predicting the oxidative stability of edible oils using 2,2-diphenyl-1-picrylhydrazyl (DPPH). Food Chemistry, 103:662-669. Leong, L. P and G. Shui. 2001. An Investigation of antioxidant capacity of fruit in Singapore markets. J. Agric. Food Chem, 76:69-75. Lubis, Y. M., Aisyah, Y., Erfiza, N.M. 2007. Potensi biji pinang (Areca catechu L.) sebagai sumber antioksidan. Laporan Penelitian. Fakultas Pertanian, Universitas Syiah Kuala. Nenadis, N., and M. Tsimidou. 2002. Observations on the estimation of scavenging activity of phenolic compounds using rapid DPPH test. J. Am. Oil. Chem. Soc. 79: 11911195. Pietta, P.G. 2000. Flavonoids as antioxidants. Journal of Natural Products, 63:1035-1042. Pokorny, J. 1991. Natural antioxidant for food use. Trens Food Sci. Techno 9:223-327. Sarastani, D., Soekarto, S.T., Muchtadi, T.R., Fardiaz, D., Apriyantono, A. 2002. Aktivitas antioksidan ekstrak dan fraksi ekstrak biji atung. Jurnal Teknologi dan Industri Pangan, vol. XIII, No. 2. Sugandi, E., Sugiarto. 1994. Rancangan percobaan, teori dan aplikasi. Andi Ofset, Yogyakarta. Wong, S.P., Leong, L.P., Koh, J.H.W. 2006. Antioxidant activities of aqueous extracts of selected plants. Food Chemistry, 99:775–783. Yen, G.C dan Chen, H.Y. 1995. Antioxidant activity of various tea extracts in relation to their antimutagenicity. J. Agric. Food Chem, 43:27-32. Yen, G.C., Chen, H.Y., Peng, H.H. 1997. Antioxidant and pro-oxidant effects of various tea extracts. J. Agric. Food Chem, 45:30-34.

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Breeding better Flax (Linum usitatissimum L.) for agronomic and consumption values under different field treatments 1

Mohammad Mehdi Rahimi, 2Mohammad Amin Zarei and 3Ali Arminian

1

Department of Agriculture, Yasooj Branch, Islamic Azad University, Yasooj, Iran; Department of Agriculture, Bushehr Branch, Islamic Azad University, Bushehr, Iran; 3 Department of Agronomy and Plant Breeding, Ilam University, Ilam, Iran. Corresponding author: [email protected] 2

Abstract. We studies some best levels of N fertilizer (0 to 150 kgha-1) and cultivation dates on grain yield its related characters and 5 food consumption compositions on 8 Linseed varieties at Yasooj Azad University, Iran, during 2009 and 2010 in field experiments as RBD split-plot designs. As a result, early sowings (14th March) along with moderate-to-high N fertilizer (100-150 kgha-1) led to highest yields. Significant positive associations found between agronomic and biochemical; oil percentage and Linoleic fatty acids. Path coefficients analysis revealed interestingly that Capsule number and primary branch per plant, plant height and 1000-seed weight had the most positive direct effects on seed yield. According to PCA, first two traits, along with height and 1000-seed weight, oleic and Linoleic acids and dry weight had the most contributions, interpreting almost all the variation. Thus, to simultaneous breeding of high oil and yielding flax varieties, capsule number per plant, primary branch per plant, plant height and 1000-seed weight should firstly be considered followed by oleic and Linoleic fatty acids. Key words: Flax (Linseed), grain yield, food values, multivariate analysis,

Introduction Flax (Linum usitatissimum L.; n=15), or linseed, is an important oilseed crop which is the only species in the Linaceae family with economic and agronomic values (Tadesse et al., 2009). It has nutrients and pharmaceutical uses and used for edible and lightening purposes and also in animal fat and poultry diets (Khan et al., 2010). Flax seeds contain 30-45% oil, making it an important industrial crop. Due to rapid drying off, flax oil is quite valuable in dye industry. It has high unsaturated fatty acids, especially Linolenic acid (Khan et al., 2010). The yield of flax is related to e.g. number of plant per unit area, number of capsules per plant and weight of seeds per capsule. Obtaining varieties with high yield and quality for linseed depend upon applying fertilizers like N. For most crop plants, seed yield is determined (multiplied) by the product of seed number and intact seeds which are influenced by N application denoting the importance of N fertilizer. Seed yield depends majorly on plant height, seed number per capsule, capsule number per plant, 1000-seed weight and primary branch number, respectively (Nie et al., 1995). Due to less impression of direct selection for yield, more efforts should be over indirect selection for yield components. Proper understanding of association of different traits, provide more reliable selection criterion to achieve a high seed yield (Akbar et al., 2001). Simple correlation coefficients and ANOVA tables (even in multiple-year trials) may not evolve satisfactory results in uncovering the real interrelationships among agro-biochemical characters. Nevertheless, selection for yield via highly correlated characters becomes easy if the contribution of different characters to yield is quantified using path coefficient analysis (Dewey & Lu, 1959) or PCA method a branch of multivariate approaches. The present study was conducted to study the associations among yield, yield related and some biochemical characters (protein, oil and fatty acids compositions) in linseed under different nitrogen and planting dates to determine the best cultivars for enhance seed yield and food consumption components of flax. Materials and methods Eight flax (linseed) varieties viz: Atlante, Bionda, Raulinus, Astamm2.764, Somaco, Linda, Olay-ozon, and Saidabad grown in the Department of Agronomy, Yasooj Azad University, Iran (N 30˚50 َ , E 51˚41َ , 1831 a.s.l), spring 2008-09 and 2009-10. Experimental design was a split-plot with 4 replication as RB design combined over 2 years. Following to agronomic managements, amount of seeds oil measured using Soxhlet apparatus (Soxhlet, 33


1879; Dorina et al., 2008), and fatty oleic, linoleic and Linolenic acids extracted using gas chromatography (GC). In addition to oil and fatty acids, seed yield (kgha-1), harvest index (HI), 1000-seed weight (g), plant height (PH), primary branches number per plant, capsules number per plant, and dry weight (biomass, g) were recorded following to harvest. Then mean of 10 samples were used for statistical analyses. The data were analyzed by ANOVA to determine significant differences between the 8 genotypes using SAS (Ver. 9.1, SAS Institute Inc., Cary, NC) and Minitab (version 15, LEAD Technologies, Inc, USA). ANOVA for all the traits performed as combined analysis, and path coefficients were estimated according to Dewey and Lu (1959), where seed yield (kgha-1) was kept as final dependent variable and other contributing characters (Tadesse et al., 2009) as causal variables. Furthermore, PCA analysis performed using Minitab software to determine the best relationships among characters. Results and Discussion ANOVA results indicated significant differences (P 8 mm) , while the MB is 60


classified as moderate and inactive for MS. Types of antimicrobials and microbes and also test methods are greatly affect antimicrobial activity (Maguire 2000). Antimicrobial activity was tested using paper disk is strongly influenced by the type and size of paper discs, concentration of media properties and the ability of antimicrobials in diffuse media. Addition of other materials carried by the compound and the type of microbes that are used also affects the antimicrobial activity. Table 1. Anti-candida activity against C. albicans Type of anti-candidal

Mean± SD of diameter clear zone (mm)

MS

2,00±0

MB

8,00±0,80

E

10,67±0,47

Candistin* * nystatin 10.000 IU/100 µl

13,67±1,24

Figure 1. Growth inhibition of C. albicans (Ca) caused by M (minyeuk simplah), MB (minyeuk Brok), E (ethanol extract of pliek u) and Cd (candistin). Killing time activity of Ethanol Extract of pliek u The affect of ethanol extract of pliek u (E) and incubation time on the number of C. albicans shown on Figure 2. Addition of 3,36 mg / ml of E and incubation times were very significantly (P 1 cm/1 hour), either in frank breech or complete breech presentation, and footling presentation who came on the second stage of labor so that it was impossible to pursue cesarean section. We defined the outcome of vaginal breech delivery to be successful, failed, as well as poor. Successful outcome if the baby in breech presentation can be delivered vaginally. From those who were successful we also analyzed whether the baby needed to be treated in neonatal intensive care, its duration of stay in neonatal intensive care, or whether the baby was rooming-in with his mother, We defined failed outcome if there was dystocia or failure to progress in active phase of labor so that we decided to undergo emergency 270


cesarean section. Poor outcome was defined as mortality of the baby because of intrapartal hypoxia caused by head entrapment. Result and Discussion There were 50 subjects who underwent vaginal breech delivery. The age of subject ranged from 16 years old until 41 years old. There was one neonatal mortality caused by head entrapment. The case was Mrs N, 29 years old, Gravida 2 Para 1 40 weeks of gestational age. She had delivered baby girl 2200 grams on her first child. The progress of labor run smoothly but on the second stage of labor there was dry labor and head entrapment. Lack of amniotic fluid became one important factor in selecting patients who underwent vaginal breech delivery. The baby boy was born died after more than 15 minutes head entrapment, male baby, 3000 grams. From 50 vaginal breech delivery, there was one neonatal mortality case, there were two failed vaginal breech delivery that proceeded to cesarean section, and 47 cases were delivered vaginally. From 47 cases delivered vaginally there were three cases where the baby was treated to neonatal intensive care unit (NICU) or more than 24 hours. The data was shown on the Table 1. Table 1. Data among subject with successful vaginal breech delivery, failed vaginal delivery and with neonatal mortality Demographic data

Gravida Primigravida Multigravida Gestational age ≤ 40 wga >40 wga Baby birthweight 1500 g-2000 g 2001 g-2500 g 2501 g-3000 g 3001 g-3500 g 3501 g-3999 g ≥4000 g Presentation Frank breech or complete breech Footling (2nd phase of labor)

Successful vaginal breech delivery (n=47) Rooming-in baby Observed in (n=45) NICU (n=2)

Failed vaginal delivery (underwent emergency cesarean section) (n=2)

Neonatal mortality caused by head entrapment (n=1)

Total case (n=50)

7 38

2 0

0 2

0 1

9 (18%) 41 (82%)

39 6

1 1

0 2

1 0

41 (82%) 9 (18%)

3 11 21 10 0 0

0 1 0 1 0 0

0 0 0 0 0 2

0 0 0 1 0 0

3 (6%) 12 (24%) 21 (42%) 11 (22%) 0(0%) 0 (0%)

42

2

2

1

47 (94%)

3

0

0

0

3 (6%)

The result showed that most of subjects who underwent vaginal breech delivery were multigravida. Only 9 subjects (18%) were primigravida. It might be caused by strict selection vaginal breech delivery which selected primigravida to undergo elective cesarean section than planned vaginal breech delivery. All of subjects who failed vaginal breech trial were more than 40 weeks of gestational age, with baby weight ≥ 4000 grams. Dystocia or lack of progress in vaginal breech delivery was caused by fetopelvic disproportion. We should reviewed and re-analyzed our estimated fetal weight if there is suspicion fetopelvic disproportion in the case of lack of progress in labor. Most of subjects who succeeded vaginal breech delivery were 40 weeks of gestational age or less. No induction was recommended so if the pregnancy was more than 42 weeks, the management would be likely by elective cesarean section. Two subjects who failed vaginal breech delivery in this study were more than 40 weeks of gestational age. It might be hypothesized that if there is occurrence of fetopelvic disproportion, labor would be longer than 40 weeks of gestational age. Most of the subjects who succeeded in vaginal breech delivery were those with fetal birth-weight ranged from 2501-3000 grams (42%) and 2001-2500 grams (24%). 271


The success and failure of vaginal breech delivery was based on fetal birth-weight, instead of parity. From this study, two subjects who failed vaginal breech delivery were multipara with birth-weight of 4000 grams. Multiparity women can fail vaginal breech delivery when the history of previously born baby was smaller than this pregnancy. Those two subject who failed had delivered babies less than 3500 grams in the past, but somehow in this pregnancy, the baby birth-weight was 4000 grams and that resulted in vaginal breech delivery failure. Pparity might influenced the outcome of the baby where all baby who needed to be treated in NICU were delivered from Primigravida. One baby weight 3250 grams and other baby weight 2280 grams. The two babies were delivered from primigravida patients who asphyxiated at the first minutes and observed in NICU for several days. It was also similar with the case of neonatal mortality, where it happened in multiparity woman with history of delivering baby smaller than this pregnancy. We can summarize that the safest vaginal breech delivery happened in multigravida women with fetal birthweight smaller than previous cephalic or breech vaginal delivery. The fetal weight which was safest in vaginal breech trial ranged from 2000-3000 grams, so that primigravida can undergo safe vaginal breech delivery if the fetal birthweight ranged from 2000 grams-3000 grams. Conclusion Vaginal breech delivery was still a safe option for primigravida with fetal birth weight ranged from 2000 grams until 3000 grams and multigravida with the history of delivering larger baby vaginally. The progress of labor should be assessed. When there is lack progress of labor, suspicion of fetopelvic disproportion should be thought and estimated fetal weight should be re-calculated. References Alarab M, Regan C, O’Connell M, Keane D, Herlihy C, Foley M. 2004. Singleton vaginal breech delivery at term: still a safe option. Obstet Gynecol 103:407-12. Goffinet F, Carayol M, Foidart J, Alexander S, Uzan S, Subtil D, et al. 2006. Is planned vaginal delivery for breech presentation at term still an option? Results of an observational prospective survey in France and Belgium. Am J Obstet Gynecol 194;1002-11. Hannah ME, Hannah WJ, Hewson SA, Hodnett ED, Saigal S, Willan AR. 2000. Planned cesarean section versus planned vaginal birth for breech presentation at term: a randomised multicentre trial. Term Breech Trial Collaborative Group. Lancet 356:137583. Kostaka A. 2004. Inappropriate use of randomized trials to evaluate complex phenomena: case study of vaginal breech delivery. Brit Med J 329:1039-42. Kostaka A. 2009. Vaginal delivery of breech presentation. Society of Obstetricians and Gynecologists of Canada (SOGC). No 226. Michel S, Drain A, Closset E, Deruelle P, Ego A, Subtil D. 2011. Evaluation of a decision protocol for type delivery of infants in breech presentation at term. European Journal of Obstetrics and Gynecology and Reproductive Biology 158:194-198. Molkenboer J, Bouckaert P, Roumen F. 2003. Recent trends in breech delivery in the Netherlands. Br J Obstet Gynaecol 110:948-51. Royal College of Obstetricians and Gynecologists. The management of breech presentation. December 2006 Guideline No. 20 b.

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