African Journal of Biotechnology - 5 April, 2012 Issue by Academic Journals

African Journal of

Biotechnology Volume 11

Number 28

ISSN 1684-5315

5 April, 2012

ABOUT AJB The African Journal of Biotechnology (AJB) is published bi-weekly (one volume per year) by Academic Journals. African Journal of Biotechnology (AJB) a new broad-based journal, is an open access journal that was founded on two key tenets: To publish the most exciting research in all areas of applied biochemistry, industrial microbiology, molecular biology, genomics and proteomics, food and agricultural technologies, and metabolic engineering. Secondly, to provide the most rapid turn-around time possible for reviewing and publishing, and to disseminate the articles freely for teaching and reference purposes. All articles published in AJB are peerreviewed.

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Editors George Nkem Ude, Ph.D Plant Breeder & Molecular Biologist Department of Natural Sciences Crawford Building, Rm 003A Bowie State University 14000 Jericho Park Road Bowie, MD 20715, USA N. John Tonukari, Ph.D Department of Biochemistry Delta State University PMB 1 Abraka, Nigeria Prof. Dr. AE Aboulata Plant Path. Res. Inst., ARC, POBox 12619, Giza, Egypt 30 D, El-Karama St., Alf Maskan, P.O. Box 1567, Ain Shams, Cairo, Egypt Dr. S.K Das Department of Applied Chemistry and Biotechnology, University of Fukui, Japan Prof. Okoh, A. I Applied and Environmental Microbiology Research Group (AEMREG), Department of Biochemistry and Microbiology, University of Fort Hare. P/Bag X1314 Alice 5700, South Africa Dr. Ismail TURKOGLU Department of Biology Education, Education Faculty, Fırat University, Elazığ, Turkey Prof T.K.Raja, PhD FRSC (UK) Department of Biotechnology PSG COLLEGE OF TECHNOLOGY (Autonomous) (Affiliated to Anna University) Coimbatore-641004, Tamilnadu, INDIA. Dr. George Edward Mamati Horticulture Department, Jomo Kenyatta University of Agriculture and Technology, P. O. Box 62000-00200, Nairobi, Kenya.

Dr Helal Ragab Moussa Bahnay, Al-bagour, Menoufia, Egypt. Dr VIPUL GOHEL Flat No. 403, Alankar Apartment, Sector 56, Gurgaon122 002, India. Dr. Sang-Han Lee Department of Food Science & Biotechnology, Kyungpook National University Daegu 702-701, Korea. Dr. Bhaskar Dutta DoD Biotechnology High Performance Computing Software Applications Institute (BHSAI) U.S. Army Medical Research and Materiel Command 2405 Whittier Drive Frederick, MD 21702 Dr. Muhammad Akram Faculty of Eastern Medicine and Surgery, Hamdard Al-Majeed College of Eastern Medicine, Hamdard University, Karachi. Dr. M.MURUGANANDAM Departtment of Biotechnology St. Michael College of Engineering & Technology, Kalayarkoil, India. Dr. Gökhan Aydin Suleyman Demirel University, Atabey Vocational School, Isparta-Türkiye, Dr. Rajib Roychowdhury Centre for Biotechnology (CBT), Visva Bharati, West-Bengal, India. Dr.YU JUNG KIM Department of Chemistry and Biochemistry California State University, San Bernardino 5500 University Parkway San Bernardino, CA 92407

Editorial Board Dr. Takuji Ohyama Faculty of Agriculture, Niigata University

Dr. Mehdi Vasfi Marandi University of Tehran

Dr. FÜgen DURLU-ÖZKAYA Gazi Üniversity, Tourism Faculty, Dept. of Gastronomy and Culinary Art

Dr. Reza Yari Islamic Azad University, Boroujerd Branch

Dr. Zahra Tahmasebi Fard Roudehen branche, Islamic Azad University

Dr. Tarnawski Sonia University of Neuchâtel – Laboratory of Microbiology

Dr. Albert Magrí Giro Technological Centre

Dr. Ping ZHENG Zhejiang University, Hangzhou, China. Prof. Pilar Morata University of Malaga

Dr. Greg Spear Rush University Medical Center

Dr. Mousavi Khaneghah College of Applied Science and Technology-Applied Food Science, Tehran, Iran.

Prof. Pavel KALAC University of South Bohemia, Czech Republic.

Dr. Kürsat KORKMAZ Ordu University, Faculty of Agriculture, Department of Soil Science and Plant nutrition

Dr. Tugay AYAŞAN Çukurova Agricultural Research Institute, PK:01321, ADANA-TURKEY.

Dr. Shuyang Yu Asistant research scientist, Department of Microbiology, University of Iowa Address: 51 newton road, 3-730B BSB bldg.Tel：+319-3357982, Iowa City, IA, 52246, USA.

Dr. Binxing Li E-mail: Binxing.Li@hsc.utah.edu

Dr Hsiu-Chi Cheng National Cheng Kung University and Hospital.

Dr. Kgomotso P. Sibeko University of Pretoria, South Africa.

Dr. Jian Wu Harbin medical university , China.

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Regular articles All portions of the manuscript must be typed doublespaced and all pages numbered starting from the title page. The Title should be a brief phrase describing the contents of the paper. The Title Page should include the authors' full names and affiliations, the name of the corresponding author along with phone, fax and E-mail information. Present addresses of authors should appear as a footnote. The Abstract should be informative and completely selfexplanatory, briefly present the topic, state the scope of the experiments, indicate significant data, and point out major findings and conclusions. The Abstract should be 100 to 200 words in length.. Complete sentences, active verbs, and the third person should be used, and the abstract should be written in the past tense. Standard nomenclature should be used and abbreviations should be avoided. No literature should be cited. Following the abstract, about 3 to 10 key words that will provide indexing references should be listed. A list of non-standard Abbreviations should be added. In general, non-standard abbreviations should be used only when the full term is very long and used often. Each abbreviation should be spelled out and introduced in parentheses the first time it is used in the text. Only recommended SI units should be used. Authors should use the solidus presentation (mg/ml). Standard abbreviations (such as ATP and DNA) need not be defined.

Review Process All manuscripts are reviewed by an editor and members of the Editorial Board or qualified outside reviewers. Authors cannot nominate reviewers. Only reviewers randomly selected from our database with specialization in the subject area will be contacted to evaluate the manuscripts. The process will be blind review. Decisions will be made as rapidly as possible, and the journal strives to return reviewersâ&#x20AC;&#x2122; comments to authors as fast as possible. The editorial board will re-review manuscripts that are accepted pending revision. It is the goal of the AJB to publish manuscripts within weeks after submission.

The Introduction should provide a clear statement of the problem, the relevant literature on the subject, and the proposed approach or solution. It should be understandable to colleagues from a broad range of scientific disciplines.

Materials and methods should be complete enough to allow experiments to be reproduced. However, only truly new procedures should be described in detail; previously published procedures should be cited, and important modifications of published procedures should be mentioned briefly. Capitalize trade names and include the manufacturer's name and address. Subheadings should be used. Methods in general use need not be described in detail.

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Fees and Charges: Authors are required to pay a $650 handling fee. Publication of an article in the African Journal of Biotechnology is not contingent upon the author's ability to pay the charges. Neither is acceptance to pay the handling fee a guarantee that the paper will be accepted for publication. Authors may still request (in advance) that the editorial office waive some of the handling fee under special circumstances. Copyright: ÂŠ 2012, Academic Journals. All rights Reserved. In accessing this journal, you agree that you will access the contents for your own personal use but not for any commercial use. Any use and or copies of this Journal in whole or in part must include the customary bibliographic citation, including author attribution, date and article title. Submission of a manuscript implies: that the work described has not been published before (except in the form of an abstract or as part of a published lecture, or thesis) that it is not under consideration for publication elsewhere; that if and when the manuscript is accepted for publication, the authors agree to automatic transfer of the copyright to the publisher. Disclaimer of Warranties In no event shall Academic Journals be liable for any special, incidental, indirect, or consequential damages of any kind arising out of or in connection with the use of the articles or other material derived from the AJB, whether or not advised of the possibility of damage, and on any theory of liability. This publication is provided "as is" without warranty of any kind, either expressed or implied, including, but not limited to, the implied warranties of merchantability, fitness for a particular purpose, or non-infringement. Descriptions of, or references to, products or publications does not imply endorsement of that product or publication. While every effort is made by Academic Journals to see that no inaccurate or misleading data, opinion or statements appear in this publication, they wish to make it clear that the data and opinions appearing in the articles and advertisements herein are the responsibility of the contributor or advertiser concerned. Academic Journals makes no warranty of any kind, either express or implied, regarding the quality, accuracy, availability, or validity of the data or information in this publication or of any other publication to which it may be linked.

African Journal of Biotechnology Table of Contents:

Volume 11

Number 28 5 April, 2012,

International Journal of Medicine and Medical Sciences

ences

ARTICLES

. Review

Phytopharmacology and medicinal properties of Salix aegyptiaca L. Jinous Asgarpanah

7145

Research Articles GENETICS AND MOLECULAR BIOLOGY Regulation of the flowering time of Arabidopsis thaliana by thylakoid ascorbate peroxidase Liang Chai, Jian-mei Wang, Zhi-yong Fan, Zhi-bin Liu, Guo-qin Wen, Xu-feng Li and Yi Yang

Determination of genetic relatedness among selected rice (Oryza sativa, L.) cultivars using microsatellite markers Ali Etemad, Mahmood Maziah and Siti Khalijah Daud

Comparison of the G-174C polymorphism of interleukin (IL)-6 in different countries Yeqing Tong, Zuxun Lu, Yanwei Zhang, Jianjun Ye1, Faxian Zhan, Shuangyi Hou, Yang Li, Xuhua Guan, Jin-quan Cheng and Jiafa Liu

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PLANT AND AGRICULTURAL TECHNOLOGY Influence of cutting frequency and fertilizer-N application on tiller production and herbage yield distribution over time in a guinea grass (Panicum maximum) sown pasture Onyeonagu, C. C and Asiegbu, J. E.

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5 April, 2012

ences ARTICLES Expression of hepatitis B surface antigen (HBsAg) gene in transgenic cherry tomato Zheng-jun Guan, Bin Guo, Hao-yong Hao, Yan-lin Huo, Jia-kun Dai and Ya-hui Wei

Characterization and evaluation of Bacillus isolates for their potential plant growth and biocontrol activities against tomato bacterial wilt Abdlwareth A. Almoneafy, G. L. Xie, W. X. Tian, L. H. Xu, G. Q. Zhang and Muhammad Ibrahim

Study of the system of tuberous root induction in vitro from Rehmannia glutinosa Tao Xue, Lan Guo, Jian-ping Xue, Yun-xian Song, He-dong Lu, Ai-min Zhang and Wei Sheng

Cryopreservation and plant regeneration of anther callus in Hevea by vitrification Quan-Nan Zhou, Ai-Hua Sun, Zhe Li, Yu-Wei Hua, Ze-Hai Jiang, Tian-Dai Huang, Xue-Mei Dai and Hua-Sun Huang

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INDUSTRIAL MICROBIOLOGY Genotypic identification and technological characterization of lactic acid bacteria isolated from traditional Turkish Kargi tulum cheese Buket Kunduhoglu, Ozlem Elcioglu, Yekta Gezginc, Ismail Akyol, Sevil Pilatin and Asya Cetinkaya

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Number 28 5 April, 2012

ences ARTICLES Kinetics of exoglucanase and endoglucanase produced by Aspergillus niger NRRL 567 Mohammad Ishfaq Ghori, Sibtain Ahmed, Mohammad Aslam Malana and Amer Jamil

Detoxification of cyanides in cassava flour by linamarase of Bacillus subtilis KM05 isolated from cassava peel Kasi Murugan, Yashotha, Kuppusamy Sekar and Saleh Al- Sohaibani

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FOOD TECHNOLOGY Effect of Îł-irradiation on the physicochemical properties of mixed soy protein isolate/starch material Weihe Xu, Boye Liu, Hongshun Yang, Kunlun Liu, Suxian Jia and Fusheng Chen

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Effects of different factors on the forward extraction of soy protein in reverse micelle systems Guanhao Bu, Haiyuan Liu, Fusheng Chen, Kunlun Liu, Yingying Yang and Yanxiu Gao

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ENTOMOLOGY The hymenopterous pollinators of Himalayan foot hills of Pakistan (distributional diversity) Hussain A, Khan M. Rahim, Ghffar A, Hayat Alia and Jamil A.

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FISHERY SCIENCE Ultrastructural study of spermatogenic stages in the protandrous sparid fish Diplodus cervinus cervinus (Lowe, 1838) from the South Eastern Mediterranean coast Nevine M. Abou Shabana

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ences ARTICLES Evaluation of the productive performance characteristics of red tilapia (Oreochromis sp.) injected with shark DNA into skeletal muscles and maintained diets containing different levels of probiotic and amino yeast Samy Yehya El-Zaeem, Talaat Nagy Amer and Nader Ezzat El-Tawil

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BIOTECHNIQUES Effect of gene transfer of Chlorella vulgaris n-3 fatty acid desaturase on mouse breast cancer cells Meilan Xue, Yinlin Ge, Jinyu Zhang, Qing Wang and Yongchao Liu

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ANIMAL SCIENCE Selection of ligand peptides with the ability to detect antibodies in enzootic bovine leukosis Elizangela Maira dos SANTOS, Rone CARDOSO, Luiz Ricardo GOULART FILHO, Marcos Bryan HEINEMANN, Rômulo Cerqueira LEITE and Jenner Karlisson Pimenta dos REIS

Prediction of metabolisable energy of poultry feeds by estimating in vitro organic matter digestibility Dragan Palić, Djordje Okanović, Djordje Psodorov, Natalija Džinić, Slobodan Lilić, Vladislav Zekić and Dragan Milić

Identification and characterization of variants in the 5' flanking region of bovine growth hormone gene GENG Rong-qing, WANG Lan-ping and CHANG Hong

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Review

Phytopharmacology and medicinal properties of Salix aegyptiaca L. Jinous Asgarpanah Department of Pharmacognosy, Pharmaceutical Sciences Branch, Islamic Azad University (IAU), Tehran, Iran. Email: asgarpanah@iaups.ac.ir. Tel: 22640051, Fax: 22602059. Accepted 16 March, 2012

Salix aegyptiaca L. is known as Musk Willow. S. aegyptiaca extracts and essential oils are important areas in drug development with some pharmacological activities in the Middle East, especially in Iran. For a long time S. aegyptiaca has been used in traditional medicines for the relief of anemia and vertigo, as a cardiotonic agent, as well as a fragrance additive in the preparation of local candies. S. aegyptiaca has recently been shown to have antioxidant, anxiolytic activity and hypocholestrolemic effect. High amounts of phenols and flavonoids such as gallic acid, caffeic acid, myricetin, catechin, quercetin as well as salicin, are reported from the leaves of this plant. 1,4-dimethoxybenzene, phenylethyl alcohol, carvone, citronellol, methyleugenol, eugenol, n-tetradecane and 4-methoxyacetophenone were identified as the major constituents of the essential oil in leaves of S. aegyptiaca. Due to the easy collection of the plant, being widespread and also its remarkable biological activities, this plant has become both food and medicine in Iran. This review presents comprehensive analyzed information on the botanical, chemical and pharmacological aspects of S. aegyptiaca. Key words: Salix aegyptiaca, Salicaceae, Musk Willow, essential oil. INTRODUCTION Salix aegyptiaca commonly known as Musk Willow is a flowering plant and generally cultivated in some provinces of Iran for hedge and ornamental purposes (Sonboli et al., 2010). It belongs to Salicaceae family in the order of Malpighiales that contains about 55 genera and more than 1000 species. The species of the genus Salix are deciduous trees and shrubs with simple, stipulate leaves alternately arranged on woody stems. Based on several publications (Fang-Zhen, 1987; Argus, 2007; Ohashi, 2000), about 526 distinct species are recognized for the genus worldwide. Former Soviet Union includes 120 species, the New World 103, China 275, Europe 65, Pakistan 26 and Iran 31 species and 6 hybrids (Maassoumi, 2009). Salix species probably originates from the Middle East, especially in Egypt and somewhere in Turkey, Iran, Iraq, Armenia, Turkmenistan and Afghanistan, but has spread as an ornamental plant up to Europe, America and Australia. S. aegyptiaca has been known as “Bidmeshk” in Iran and distributed in many parts of Iran especially in Urmia, North West of Iran (Rabbani et al., 2011). S. aegyptiaca is a deciduous shrub growing to 4 - 5 m. It is a vigorous, fast growing,

bushy, deciduous small tree with purplish-red, thick branches (Figure 1). Leaves are oblong, serrated, deep green above, underside hairy, up to 15 cm long (Figure 2). The inflorescence is catkin; catkins are fragrant and grey. Individual flowers are either male or female, but only one sex is to be found on one plant, so both male and female plants must be grown if seed is required and are pollinated by bees. Male catkins are 4 cm long with yellow anthers and probably include one of the used parts of this plant (Figure 3). Female catkins are 7.5 cm long. The plant is not self-fertile (Zargari, 1988). The male inflorescences distillate of the plant has long been used in Iranian folklore medicine as cardiotonic, treatment of anemia and vertigo, as well as a fragrance additive. The aqueous extract and essential oil of these inflorescences are also being used in confectionary, flavorful syrups and especially in the preparation of a local candy (Noghl-e Urmia) (Karimi et al., 2011). Ethnobotanically, rheumatic pains, affecting mainly the elderly, can be relieved by a decoction or infusion of S. aegyptiaca bark (Leporatti and Impieri, 2007). The decoction of the leaves or barks has also been used as

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Figure 1. Salix aegyptiaca (Musk Willow).

an antihelminthic and vermifuge remedy. The effect of Salix leaves, along with clove bud and Nigella, in the treatment of common wart has been reported (Rezaei et al., 2008). In addition, S. aegyptiaca is used as laxative, cardioprotective, nervous, sedative, hypnotic, somnolent, aphrodisiac, orexigenic, carminative and gastroprotection. The decoction of S. aegyptiaca leaves in honey still is used as a nervonic functional food. This decoction plus sugar has been used among Iranian and Turkish people for maladies like depression, neuropathic pain and rheumatoid arthritis (Karimi et al., 2011). The Salix family is famous due to its endogenous salicylate compounds e.g., salicylic acid and acetyl salicylic acid (ASA, AspirinÂŽ). This class of compounds exerts anti-inflammatory effects. The anti-inflammatory and antinociceptive properties of extracts of Salix family may be related to its phytochemicals such as salicin, myricetin, kaempferol, quercetin, rutin and luteolin (Qin and Sun, 2005; Nahrstedt et al., 2007). These compounds have immunomodulatory and anti-inflammatory activities by inhibiting pro-inflammatory cytokine

production and their receptors (Qin and Sun, 2005; Nahrstedt et al., 2007). The considerable myricetin, rutin and catechin content of musk willow extracts could potentially contribute to the anti-inflammatory functions of willow extracts (Enayat and Banerjee, 2009). According to Unani medicine, S. aegyptiaca has warm humor nature and ethnic herbalists prescribed it for cholelithiasis, cholecystitis, arthritis and rheumatism. The essential oil of S. aegyptiaca is febrifuge and is dubbed among Iranian people for its calming effect on heart and possibly its antihypertensive effect (Karimi et al., 2011). S. aegyptiaca male catkins are rich in volatile components such as Ń&#x20AC;-methoxybenzene, eugenol, carvone, cedrene oxide, and geraniol (Salehi-Surmaghi, 2009). A number of chemical constituents such as flavonoids and volatile substances have been isolated from different parts of the plant (Karimi et al., 2011; Enayat and Banerjee, 2009). From current pharmaceutical studies, additional pharmaceutical applications of S. aegyptiaca have revealed antioxidant, antiinflammatory, analgesic, anxiolytic and antihyperchlosterolemic

Asgarpanah

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Figure 2. Salix aegyptiaca leaf.

effects among others (Sonboli et al., 2010; Rabbani et al., 2011; Karimi et al., 2011; Karawya et al., 2010). Since the review and systemic analysis of chemistry, pharmacology and clinical properties of S. aegyptiaca have not been reported. We prompted to provide the currently available information on traditional and local knowledge, ethno-biological and ethno-medicinal issues, identification of pharmacologically important molecules and pharmacological studies on this useful plant. The aim of the present article is to introduce S. aegyptiaca as a medicinal plant by highlighting its traditional applications, as well as the recent findings for novel pharmacological and clinical applications.

The essential oil of S. aegyptiaca leaves contains 1,4dimethoxybenzene (61.5%) as the main component accompany with methyleugenol (21%), phenylethyl alcohol (10.9%), citronellol (8%), carvone (6%), eugenol (6%) and 4´-methoxyacetophenone (3%) (Karimi et al., 2011). 1,4-Dimethoxybenzene also known as “hydroquinone dimethyl ether” is the para form of dimethoxybenzene, a volatile aromatic ether with a sweet floral odor. It occurs naturally in Salix species (Dötterl et al., 2005). The presence of high amounts of phenolics including gallic acid, caffeic acid, vanillin, р-coumaric acid, myricetin, catechin, epigallocatechin gallate and flavonoids such as rutin, quercetin and salicin are indicated in the leaves of S. aegyptiaca (Enayat and Banerjee, 2009).

CHEMICAL COMPOSITION The commonly known phytochemical compounds from S. aegyptiaca are volatile substances, flavonoids and phenolics (Sonboli et al., 2010). р-Methoxybenzene (60%), eugenol (21%), decanol (4%), cedrene oxide (2.5%) and ocimene (2.3%) were identified as the major constituents of S. aegyptiaca male catkins essential oil collected from Urmia, North West of Iran, while carvone (16%), cedrene oxide (16%), geraniol (10%), carvacrol (9%), citronellol (4%) and р-methoxybenzene (2.3%) were characterized as the major ones of the essential oil of S. aegyptiaca male catkins collected from Shiraz, South of Iran (Salehi-Surmaghi, 2009).

ANTI-INFLAMMATORY PROPERTIES

AND

ANALGESIC

Although a number of steroidal or non-steroidal antiinflammatory drugs have been developed, researchers are changing their focus to natural products to develop new anti-inflammatory agents due to the side-effects of chemical drugs (Hyun and Kim, 2009; Shokrzadeh and Saeedi Sarvari, 2009). As a result, the search for other alternatives seems necessary and beneficial. Many cells and mediators are involved in proceeding inflammation. For example, macrophages are representative

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Figure 3. Salix aegyptiaca male catkins.

inflammatory cells involved in acute or chronic inflamematory responses by over-production of pro-inflammatory cytokines [for example, tumor necrosis factor (TNF)-a, interleukin (IL)-1b and granulocyte/macrophage colony stimulating factor (GMCSF)] and inflammatory mediators (Rhee et al., 2009; Lundberg, 2003; Walsh, 2003). The Salix family is famous due to its endogenous salicylate compounds e.g., salicylic acid and acetyl salicylic acid (ASA, Aspirin速) (Karimi et al., 2011). This class of compounds exert anti-inflammatory effects throughout the inhibition of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) leading to the inhibition of prostaglandin synthesis (Yu et al., 2002; Mahdi et al., 2006). The anti-inflammatory and antinociceptive properties of extracts of Salix species may be related to their phytochemicals such as salicin, myricetin, kaempferol, quercetin, rutin and luteolin (Qin and Sun, 2005; Nahrstedt et al., 2007). These compounds have anti-inflammatory activities by inhibiting pro-inflammatory cytokine production and their receptors (Qin and Sun, 2005; Nahrstedt et al., 2007). The considerable myricetin, rutin and catechin content of S. aegyptiaca extracts could potentially contribute to the anti-inflammatory functions of other Salix species extracts (Enayat and Banerjee, 2009).

Salicin, the major phenolic glycoside present in the bioactive extracts in Salix species is considered to be the pharmacologically active principle due to its structure similarity to aspirin. In animal models, the extracts from S. aegyptiaca leaves and male flowers have shown antiinflammatory effects in carrageenan-induced paw edema and hot plate tests (Karawya et al., 2010; Rabbani et al., 2010). The aqueous extract of S. aegyptiaca male flowers at a dose of 0.3, 0.6 and 1.2 mg/kg showed a significant analgesic effect compared to control group treated with ASA. In addition, it is demonstrated that the analgesic effect of 0.6 mg/kg of the extract was higher than ASA 300 mg/kg (Karawya et al., 2010). ANTIOXIDANT ACTIVITY An antioxidant is defined as any substance that when present at low concentrations compared to those of an oxidizable substrate, significantly delays or prevents oxidation of that substrate (Rhee et al., 2009; Halliwell and Gutteridge, 1990; Wiseman et al., 1997; Mates et al., 1999). Antioxidants are of interest to biologists and clinicians because they help to protect the human body

Asgarpanah

against damages induced by reactive free radicals generated in atherosclerosis, ischemic heart disease, cancer, Alzheimer's disease, Parkinson's disease and even in aging process (Aruoma, 2003; Hemati et al., 2010). There are many evidences that natural products and their derivatives have efficient anti-oxidative characteristics, consequently linked to anti-cancer, hypolipidemic, anti-aging and anti-inflammatory activities (Rhee et al., 2009; Halliwell et al., 1990; Wiseman et al., 1997; Hogg, 1998; Mates et al., 1999; Aruoma, 2003; Cho et al., 2006). The anti-oxidative capacities of S. aegyptiaca male catkins were evaluated by determining its effect on 1,1diphenyl-2-picrylhydrazyl (DPPH) radical scavenging (Sonboli et al., 2010). In DPPH radical-scavenging activity assay, the IC50 value was 27.7 µg/ml, which was comparable to the synthetic antioxidant butylated hydroxytoluene (BHT) (IC50 value = 26.5 µg/ml) as standard. Furthermore, antioxidant potential of S. aegyptiaca bark was determined by IC50 value equal to19 µg/ml (Enayat and Banerjee, 2009). The barks and male catkins have the highest antioxidant activity. The molecular mechanism of radical scavenging activity of S. aegyptiaca could be attributed to the presence of polyphenolic compounds. It has already been exhibited that polyphenolic compounds are responsible for radical scavenging activity, due to the ease of their hydrogen atom donation to active free radicals (Ho et al., 1994). The potent antioxidant activity of S. aegyptiaca supports its possible use as a natural antioxidant in food industries and other pharmaceutical preparations (Sonboli et al., 2010). ANXIOLYTIC PROPERTIES Anxiolytic disorders are among the most prevalent disorders that are characterized by symptoms such as overriding apprehension or mental tension. Most anxiety disorders tend to run a long course and thus require longterm treatment (Berrios, 1999). A wide range of herbal anti-anxiety medicines has been used in the past to treat different forms of anxiety disorders. Although synthetic drugs such as benzodiazepines have the advantage of rapid onset of action, they have the potential to interfere with patient’s normal activity and are often difficult to stop once the therapy has started. These problems and also other side effects that exist with synthetic anxiolytic drugs have prompted people to seek natural and herbal medicines (Rabbani et al., 2011). The extract of the flowers of S. aegyptiaca on elevated plus-maze (EPM) model of anxiety in mice produced anxiolytic effects and reduced locomotor activity. Oral and intraperitoneal (i.p.) administration of the extract significantly increased the percentage of time spent in the open arms of the EPM at 200 and 100 mg/kg, respectively. The flowers extract at 100 and 200 mg/kg significantly decreased the animal's locomotor activity at

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10 and 15 min time intervals. These doses, however, did not affect the ketamine-induced sleeping time. The 100 mg/kg dose of the plant extract when given by i.p. route seemed to be the optimal dose in producing the anxiolytic effects (Rabbani et al., 2010). HYPERCHOLESTEROLEMIA EFFECT Studies in both animals and humans have demonstrated that prolonged high cholesterol concentration in the circulating blood positively correlates with developing atherosclerosis (Pratico, 2001; Kurosawa et al., 2005). These changes are associated with the phenomenon that excessive load of cholesterol to the liver, above the acceptable level of its normal physiological limit, causes the liver to be unable in metabolizing the lipids, thus resulting in high cholesterol return in the circulating blood (Kushi et al., 1996). In Iranian ethno-medicine, S. aegyptiaca has been prescribed for ailments like cholecystitis and cholelithiasis due to its inhibition of bile acid synthesis or antimicrobial effects of its active components like phenylethyl alcohol. With respect to the lipid profile, the results suggested a time-dependent increase in the plasma total cholesterol (TC) level. This increase in TC in S. aegyptiaca-treated mice could be mainly due to the lack of protective effects of the essential oil of S. aegyptiaca and not due to age, since the TC was also increased as early as the first week of essential oil administration. A significant increase in plasma level of HDL was also observed. This increase in HDL can solely be responsible for the observed increase in TC since the essential oil might be affecting the HDL metabolism in the liver (Eder and Gidez, 1982). However, the results support that essential oil of S. aegyptiaca has no therapeutic and/or prophylactic effects against incoming dyslipidemia in rabbits. The essential oil of S. aegyptiaca contains several serum lipid-improving phytochemical ingredients such as eugenol and citronellol (Holmes and DiTullio, 1962; Germán et al., 1998). However, the other chemical ingredients in essential oil of S. aegyptiaca might participate in the phenomena that hasten atherogenesis (Karimi et al., 2011). CONCLUSION The objective of this article has been to show the recent advances in the exploration of S. aegyptiaca as phytotherapy and to illustrate its potential as a therapeutic agent. With the current information, it is evident that S. aegyptiaca has pharmacological functions including antiinflammatory, analgesic and antioxidant activities, among others. As the current information shows, it is also possible that phenolic glycosides and flavonoids might be useful in the development of new drugs to treat various

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diseases. However, it must be kept in mind that clinicians should remain cautious until more definitive studies demonstrate the safety, quality and efficacy of S. aegyptiaca. For these reasons, extensive pharmacological and chemical experiments, together with human metabolism will be a focus for future studies. Finally, this article emphasizes the potential of S. aegyptiaca to be employed in new therapeutic drugs and provide the basis for future research on the application of transitional medicinal plants. REFERENCES Argus GW (2007). Salix distribution maps and synopsis of their classification in North America, north of Mexico. Harvard Papers in Botany, 12: 335-368. Aruoma OI (2003). Methodological considerations for characterizing potential antioxidant actions of bioactive components in plant foods. Mutat. Res. 523-524: 9-20. Berrios GE (1999). Anxiety disorders: a conceptual history. J. Affect. Disord. 56: 83-94. Cho JY, Prak SC, Kim TW, Kim KS, Song JC, Kim SK, Lee HM, Sung HJ, Park HJ, Song YB, Yoo ES, Lee CH, Rhee MH (2006). Radical scavenging and anti-inflammatory activity of extracts from Opuntia humifusa. Raf. J. Pharm. Pharmacol. 58: 113-119. Dötterl S, Füssel U, Jürgens A, Aas G (2005). 1,4-Dimethoxybenzene, a floral scent compound in willows that attracts an oligolectic bee. J. Chem. Ecol. 31: 2993-2998. Eder HA, Gidez LL (1982). The clinical significance of the plasma high density lipoproteins. Med. Clin. North. Am. 66: 431-440. Enayat S, Banerjee S (2009). Comparative antioxidant activity of extracts from leaves, bark and catkins of Salix aegyptiaca. Food Chem. 116: 23-28. Fang-Zhen F (1987). On the distribution and origin of Salix in the world. Acta Phytotax. Sinica, 25: 307-312. Germán C, Leticia G, Adrian S, Fermando L, Maria S, Elizdath M, Francisco D, Joaquin T (1998). Hypolipidemic activity of dimethoxy unconjugated propenyl side-chain analogs of α-asarone in mice. Drug Dev. Res. 43: 105-108. Halliwell B, Gutteridge JMC (1990). Role of free radicals and catalytic metal ions in human disease: An overview. Method. Enzymol. 186: 185. Hemati A, Azarnia M, Angaji AH (2010). Medicinal effects of Heracleum persicum (Golpar). Middle-East J. Sci. Res. 5: 174-176. Ho CT, Osawa T, Huang MT, Rosen RT (1994). Food Phytochemicals for Cancer Prevention II. Teas, Spices, and Herbs. American Chemical Society, Washington, pp. 132-143. Hogg N (1998). Free radicals in disease. Seminars in reproductive endocrinology, 16: 241-248. Holmes WL, DiTullio NW (1962). Inhibitors of cholesterol biosynthesis which act at or beyond the mevalonic acid stage. Am. J. Clin. Nutr. 10: 310-322. Hyun TK, Kim JS (2009). The pharmacology and clinical properties of Kalopanax pictus. J. Med. Plants Res. 3(9): 613-620. Karawya MS, Ammar MN, Hifnawy MS, Al-Okbi SY, Mohamed DA, ElAnssary AA (2010). Phytochemical study and evaluation of the antiinflammatory activity of some medicinal plants growing in Egypt. Med. J. Islamic World Acad. Sci. 18(4): 139-150. Karimi I, Hayatgheybi H, Shamspur T, Kamalak A, Pooyanmehr M, Marandi Y (2011). Chemical composition and effect of an essential oil of Salix aegyptiaca (musk willow) in hypercholesterolemic rabbit model. Braz. J. Pharmacog. 21(3): 407-414.

Kurosawa T, Itoh F, Nozaki A, Nakano Y, Katsuda S, Osakabe N, Tsubone H, Kondo K, Itakura H (2005). Suppressive effects of cacao liquor polyphenols (CLP) on LDL oxidation and the development of atherosclerosis in Kurosawa and Kusanagi-hypercholesterolemic rabbits. Atherosclerosis, 179: 237-246. Kushi LH, Folsom AR, Prineas RJ, Mink PJ, Wuv-Bustick RM (1996). Dietary antioxidant vitamins and death from coronary heart disease in postmenopausal woman. N. Engl. J. Med. 334: 1156-1162. Leporatti ML, Impieri M (2007). Ethnobotanical notes about some uses of medicinal plants in Alto Tirreno Cosentino area (Calabria, Southern Italy). J. Ethnobiol. Ethnomed. pp. 3: 34. Lundberg IE (2003). Clinical symptoms in patients with myositis- an acquired metabolic myopathy idiopathy inflammation myopathies: Why do the muscles bwcomw weak? Curr. Opin. Rheumatol. 15: 675-678. Mahdi JG, Mahdi AJ, Bowen ID (2006). The historical analysis of aspirin discovery, its relation to the willow tree and antiproliferative and anticancer potential. Cell Prolif. 39: 147-155. Maassoumi AA (2009). Experimental taxonomy of the genus Salix L. (Salicaceae) in Iran. Iran. J. Bot. 15(1): 3-20. Mates JM, Perez-Gomez C, Nunez de Castro I (1999). Antioxidant enzymes and human diseases. Clin. Biochem. 32: 595-603. Nahrstedt A, Schmidt M, Jäggi R, Metz J, Khayyal M (2007). Willow bark extract: the contribution of polyphenols to the overall effect. Wien Med Wochenschr, 157: 348-358. Ohashi H (2000). A systematic enumeration of Japanese Salix (Salicaceae). J. Jpn. Bot. 75: 1-41. Pratico D (2001). Lipid peroxidation in mouse models of atherosclerosis. Trend Cardiovasc. Med. 11: 112-116. Qin F, Sun HX (2005). Immunosuppressive activity of Pollen Typhae ethanol extract on the immune responses in mice. J. Ethnopharmacol. 102: 424-429. Rabbani M, Sajjadi SE, Rahimi F (2010). Anxiolytic effect of flowers of Salix aegyptiaca L. in mouse model of anxiety. J. Complementary Integr. Med. 7(1): 18-22. Rabbani M, Vaseghi G, Sajjadi SE, Amin B (2011). Persian Herbal Medicines with Anxiolytic Properties. J. Med. Plants. 10(39): 7-11. Rezaei K, Jebraeili R, Delfan B, Noorytajer M, Meshkat MH, Maturianpour H (2008). The effect of clove bud, Nigella and Salix alba on wart and comparison with conventional therapy. Eur. J. Sci. Res. 21: 444-450. Rhee MH, Park HJ, Cho JY (2009). Salicornia herbaceae: Botanical, Chemical and pharmacological review of halophyte marsh plant. J. Med. Plants Res. 3(8): 548-555. Salehi Surmaghi MH (2009). Medicinal plants and herbal medicines. rd 3 vol. Donyaye Taghzieh publications, Tehran, pp. 109-112. Shokrzadeh M, Saeedi Sarvari SS (2009). Chemistry, Pharmacoligy and clinical properties of Sambucus ebulus: A review. J. Med. Plants Res. 4(2): 95-103. Sonboli A, Mojarrad M, Nejad Ebrahimi S, Enayat S (2010). Free radical scavenging activity and total phenolic content of methanolic extracts from male inflorescence of Salix aegyptiaca grown in Iran. Iranian J. Pharmaceut. Res. 9(3): 293-296. Walsh LJ (2003). Mast cells and oral inflammation. Crit. Rev. Oral Biol. Med. 14: 188-198. Wiseman SA, Balentine DA, Frei B (1997). Antioxidants in tea. Crit. Rev. Food Sci. Nutr. 37: 705-718. Yu HG, Huang JA, Yang YN, Huang H, Luo HS, Yu JP, Meier JJ, Schrader H, Bastian A, Schmidt WE, Schmitz F (2002). The effects of acetylsalicylic acid on proliferation, apoptosis, and invasion ofcyclooxygenase-2 negative colon cancer cells. Eur. J. Clin. Invest. 32: 838-846. Zargari A (1988). Medicinal plants. Vol. 2. Tehran University Press, Iran, p. 619.

Full Length Research Paper

Regulation of the flowering time of Arabidopsis thaliana by thylakoid ascorbate peroxidase Liang Chai, Jian-mei Wang, Zhi-yong Fan, Zhi-bin Liu, Guo-qin Wen, Xu-feng Li and Yi Yang* Key Laboratory of Bio-resources and Eco-environment of Ministry of Education, College of Life Science, Sichuan University, Chengdu, 610064, China. Accepted 19 March, 2012

Flowering time of higher plants is precisely controlled by various exogenous and endogenous factors. Recent researches implied that H2O2 is a potential flowering initiation factor. In order to confirm this hypothesis, thylakoid ascorbate peroxidase (tAPX) overexpressing Arabidopsis, the mutant line containing a T-DNA insertion and the wild type have been analyzed in this study, since APX was an important enzyme scavenging H2O2 in plant cells. It was found that during the vegetative growth stage there was no phenotypic difference among the three lines under common conditions, but 3,3’diaminobenzidinetetrahydrochloride (DAB) staining showed that the endogenous H2O2 content varied: the mutant line had the highest content; the wild type took the second place, while the tAPXoverexpressing line had the lowest H2O2 content. This trend was in accordance with the bolting and flowering time during the following reproductive growth stage: the mutant bolted and flowered first, followed by the wild type, and the overexpressing line bolted and flowered last. This correlation confirmed the previous hypothesis that “H2O2 is a possible factor in flowering induction”. Keywords: Ascorbate peroxidase, Arabidopsis thaliana, flowering time, hydrogen peroxide.

INTRODUCTION Researches about ascorbate peroxidase (APX, EC 1.11.1.11) started from 1976 (Foyer and Hailiwell, 1976) and so far, more than 30 years has passed. Previous researches about APX from higher plants almost focused on its redox status and enhancing tolerances to various stresses. So far, it is known that APX catalyzed the reduction of H2O2 to H2O and O2 using ascorbic acid (ASA) as specific electron donor (Asada, 1999), and it is the most important H2O2-eliminating enzyme in chloroplast (Asada, 1992). Its activity was detected not only in higher plants and eukaryotic algae, but also in Cyanobacteria and some kinds of insects. Murgia et al. (2004) found that Arabidopsis overexpressing tAPX was

*Corresponding author. Tel/Fax: 86-28-85410957.

E-mail:

yangyi528@vip.sina.com.

Abbreviations: APX, Ascorbate peroxidase; ASA, ascorbate; CTAB, cetyltrimethylammonium bromide; DAB, 3,3’diaminobenzidinetetrahydrochloride; H2O2, hydrogen peroxide; PCR, polymerase chain reaction; ROS, reactive oxygen species

more resistant to photo-oxidative stress induced by Paraquat (Pq) and reduced the symptoms of cell death induced by nitric oxide. Overexpression of thylakoid APX from Solanum lycopersicum (StAPX) in tobacco increased early seedling tolerance to salinity and osmotic stresses (Sun et al., 2010). Our previous studies found that BnAPX from Brassica napus enhanced salt and drought tolerances in Arabidopsis (Chai et al., 2011). Moreover, Xu et al. (2008) attributed the enhanced salttolerance of transgenic Arabidopsis carrying a peroxisomal APX gene (HvAPX1) from barley to the reduction of oxidative stress injury. Since APX eliminated H2O2, it could also change some other phenotype caused by H2O2. Recent researches showed that H2O2 was a possible factor in flower induction. Lokhande et al. (2003) studied four Arabidopsis ecotypes from different latitudes and found that flowering started from low-latitude to highlatitude ecotypes. Since the lower-latitude ecotypes suffered higher oxidative stress, the study proposed the hypothesis that “H2O2 is one of the possible factors in flower induction”. Furthermore, Moharekar et al. (2007)

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found that in Arabidopsis, flowering time was negatively correlated with irradiance, which led to oxidative stress. Hence they proposed that an increase in oxidative stress induces earlier flowering. So it can be inferred that if H2O2-eliminating gene APX was overexpressed or suppressed in Arabidopsis, the flowering time should be delayed or advanced compared with the wild type, respectively. Although some precious study reported increased ROS during the transition to flowering (Banuelos et al., 2008; Lokhande et al., 2003; Moharekar et al., 2007), studies on APX-transgenic plants or mutants affecting the flowering time seemed seldom. In this study, tAPX-overexpressing Arabidopsis, the wild type and the mutant line containing a T-DNA insertion were studied and it was found that the bolting and flowering time was negatively related with the endogenous H2O2 content. In other words, the higher the endogenous H2O2 content, the earlier the Arabidopsis bolted and flowered. This result confirms the hypothesis that H2O2 initiated flowering (Lokhande et al., 2003). MATERIALS AND METHODS

reagent according to the instructions. RNA samples were then treated with RNase-free DNase I. The quality and concentration of RNA were accurately determined by a UV–visible light spectrophotometer. Reverse transcription was performed by reverse transcriptase in accordance with the instructions. The transcription level of tAPX was determined by both semiquantitative and quantitative real-time PCR (qPCR) analysis using the specific primers for APX (APX-1: 5′- TCTGGTGTTACCCACTGATG -3′, APX-2: 5′- TTTCCCGTAGAATACTTTGC-3′). The β-ACTIN gene (ACT-1: 5′- TCTCGTTGTCCTCCTCACTT -3′; ACT-2: 5′- TATCATCAGCCTCAGCCATT -3′) was used as an internal control and also amplified simultaneously from each sample. The semi-quantitative PCR cycling procedure consisted of 3 min at 95°C, 27 cycles for 30 s at 95°C, 30 s at 55°C and 30 s at 72°C, and a final 5-min extension at 72°C. The qPCR cycling consisted of 1 min at 95°C, 40 cycles of 10 s at 95°C, 40 s at 56°C, 45 s at 72°C (data collection), followed by a melting curve procedure of 1 min at 95°C, 1 min at 56°C, 78 cycles for 56°C ramping to 95°C at the rate of 0.5°C/10 s. The qPCR was performed on a Bio-Rad iCycler fluorescence thermocycler (BioRad, Hercules, CA). The fluorescence master mix reagent for the reaction was Sybr Green (Toyobo). All of the cycle threshold (Ct) values of tAPX amplification were normalized by the corresponding β-ACTIN Ct values. Three parallel repeats were done and the results were summarized as averages and the standard deviation (SD). The data were analyzed and plotted using Microsoft Office software.

Plant material and growth conditions Arabidopsis thaliana, accession Columbia was used in this study: the homozygous transgenic Arabidopsis overexpressing thylakoidAPX (tAPX) gene was obtained from Professor I. Murgia (2004). The mutant Arabidopsis line containing a T-DNA insert in the exon of the tAPX gene (SALK_027804.54.20) was obtained from the Arabidopsis Biological Resource Center (ABRC). The wild type was kept in our laboratory. After three days jarovization at 4°C, seeds of Arabidopsis were first surface-sterilized in 75% ethanol for 1 min, followed by immersion in 0.1% HgCl2 for 10 min, and rinsed at least three times with sterile distilled water. Then the seeds were germinated in soils-mixture of vermiculite: peat (1:1) in an environmentally climatic chamber at 22°C with 16/8 h of light/dark cycle and 70% humidity. The screening of the homozygous mutant Arabidopsis line The primer designing and the protocol were according to information online (http://signal.salk.edu/tdnaprimers.2.html). Three primers (LBb1.3, LP and RP) were used for the screening. They were LBb1.3 (5’-ATTTTGCCGATTTCGGAAC-3’), LP (5’- ACAAGATCAAACCCACGAATG-3’) and RP (5’- TACTTCACCAAGATGGGATGG-3’). Amplification for wild type should produce a long band from LP to RP; homozygous lines should produce a small band from RP to LBb1.3, and for heterozygous lines they got both bands. Total DNA was extracted from three-week-old Arabidopsis with cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle, 1990) and used as the template for the polymerase chain reaction (PCR). The PCR cycling procedure consisted of 5 min at 95°C, followed by 36 cycles for 30 s at 95°C, 30 s at 61°C and 60 s at 72°C, a final 8-min extension at 72°C, and termination of the reaction at 4°C.

The semi-quantitative and quantitative PCR analysis of the level of transcriptional level of tAPX Total RNA was prepared from leaves of three-week-old over expressing line, the wild type and the mutant seedlings using Trizol

H2 O2 staining with 3,3’-diaminobenzidine tetrahydrochloride (DAB) The H2O2 content was determined by the modified DAB method (Thordal-Christensen et al., 1997; Guan and Scandalios, 2000; Zhou et al., 2000). Leaves of Arabidopsis were cut and submerged into test tubes containing about 10 ml DAB solution (1 mg/ml DAB with NaOH, pH 3.8), and then the petioles fully immersed in the solution were cut again. After about 12 h in dark, the leaves were washed with water and then transferred into clean test tubes with cotton plug. EtOH (95%) was added into the test tubes, and the tubes were heated in boiling water for about 18 min. And the leaves were transferred into fresh EtOH when chlorophyll was removed completely. Measuring the flowering time Seeds of the overexpressing line, the wild type and the mutant line were kept in 4°C of three days and the process of jarovization could remove the dormancy to make sure the seeds germinated and grew synchronously. The seeds grew in soil under normal conditions (22°C, 16/8 h light/dark and 70% humidity), and when the first bolting began, the inflorescence heights were recorded and measured day by day. Three replicates were carried out and standard deviations were calculated. The data were analyzed and plotted in Microsoft Office software.

RESULTS The homozygous mutant Arabidopsis line The seeds of mutant line (SALK_027804.54.20) were gotten from ABRC, which had a T-DNA insertion into the first exon of tAPX gene. So the homozygous lines could be identified with three specificity primers. Figure 1a shows that amplification for the wild type got a unique

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Figure 1. The screening of the homozygous mutant line. a) PCR for wild type produced a unique long product of 1200 bp from LP to RP, while for mutant 2 nothing was amplified; b) PCR for mutant 2 produced only a short band of 550 bp from RP to LBb1.3, while for the wild type there was no amplification. M, DNA marker DL 2000; col, wild type; mutant2, mutant line 2.

large product of 1200 bp from LP to RP, and amplification for mutant 2 got only a small band of 550 bp from RP to LBb1.3 (Figure 1b). PCR for some other mutant plants obtained both bands (data not shown), because they had one DNA chain with the T-DNA insertion and the other chain without it and they were identified as heterozygous. So according to the method of screening the homozygous line, mutant 2 was considered homozygous and its seeds were used in the following researches. tAPX regulated the H2O2 content of the leaves Semi-quantitative and quantitative real-time PCR (qPCR) were used to verify the tAPX transcriptional level. Figure 2a shows that the expression of tAPX was enhanced in overexpressing line, while it was knocked down in the mutant. The relative expression value of tAPX mRNA observed in overexpressing line was over 52-fold higher than that observed in wild-type plants, while tAPX was seldom expressed in the mutant line (Figure 2b). Therefore, it was assumed that the transformed sense sequence driven by CaMV 35S promoter was effectively overexpressed and the T-DNA insertion in the mutant line knocked out the tAPX. Although there were no remarkable phenotypic differences among the overexpressing line at the vegetative growth stage, the wild type and the mutant line (data not shown) under normal conditions, exhibited some internal changes since the transcriptional level of tAPX altered so enormously

among the three lines. In consideration of its main function which was to scavenge the H2O2, endogenous levels of H2O2 in overexpressing line, the wild type and the mutant line were determined. DAB was involved in the staining, which reacted with H2O2 in situ and bronzing spots consequently appeared (Guan and Scandalios, 2000; Zhang et al., 2009). Therefore, as shown Figure 2c, leaves from transgenic line had the lightest color, while leaves from mutant line were dyed the most. In other words, the transgenic lines suffered the least H2O2, while the mutant suffered the most H2O2. Even in normal physiological and biochemical process, ROS including H2O2 was produced (Asada and Takahashi, 1987), and in this study, the level of H2O2 was eliminated more or less effectively in the overexpressing or mutant line respectively. Since the H2O2 content differed among the three lines, so the bolting and flowering time should be determined to test the hypothesis H2O2 regulated flowering time. tAPX regulated the bolting and flowering time of Arabidopsis To test whether H2O2 regulated the flowering initiation, seeds germinating contemporarily after jarovization were grown in soil. In the vegetative growth stage, there seemed no phenotypic difference between the overexpressing line, the wild type and the mutant line. On the 29th day after germination, the first bolting appeared

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Figure 2. tAPX transcriptional level and H2O2 content. a) The determination of transcriptional level by semi-quantitative PCR. b) Quantitative real-time PCR analysis of tAPX transcriptional level in overexpressing line, the wild type and the mutant. Total RNA was prepared from three-weeks-old seedlings. Î˛-ACTIN was used as the internal control. The tAPX/ACTIN ratio of overexpressing line or the mutant was normalized to that of wild type. c) H2 O2 content determined by DAB method of the overexpressing line, the wild type and the mutant respectively. 14-2, The overexpressing line; col, wild type; mutant2, mutant line 2. Results represent the mean of three replicates. Bars correspond to the standard deviation. DAB, 3,3â&#x20AC;&#x2122;Diaminobenzidinetetrahydrochloride.

in the mutant line, and then followed by the wild type. As to the overexpressing line, there was no bolting until the 35th day. And from the 29th day on, the inflorescence heights were measured and recorded every day. It was indicated in Figure 3a and b that the overexpression of tAPX altered the flowering time more remarkably than the

repression of tAPX did. For example, it was on the 35th day after germination that the inflorescence height of mutant line reached 5 cm, only 1 day earlier than the wild type. However, the overexpressing line got that height till 42 day (5 days later than the wild type). As reported by Murgia at al. (2004), seeds of 14-2 line germinated a little

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Age (days) Figure 3. tAPX regulated the flowering time. a) Inflorescence heights of the overexpressing line, the wild-type and the mutant line in the 38th day after germination. b) Inflorescence height was measured for each seedling during growth under controlled conditions. 14-2, The overexpressing line; col, the wild type; mutant2, mutant line 2. Results represent the mean of three replicates. Bars correspond to the standard deviation.

late than the wild type and the mutant, but it was because of the insertion site rather than the function of the tAPX gene and it was almost unnoticeable. So it did not affect the experiment results. DISCUSSION Flowering process is a complex life phenomenon in higher plants. It is an important transition from vegetative growth stage to reproductive growth stage (Yong et al.,

2000). The correct timing of flowering is therefore necessary to maximize reproductive success (Bernier, 1988; Simpson and Dean, 2002). To explain this mysterious situation, researchers carried out a lot of studies physiologically and biochemically, and various hypotheses were proposed in succession; for example, the florigen hypothesis (Lang, 1952; Evans, 1971), the nutrient diversion hypothesis (Bernier, 1988) and the multifactorial control model (Koornneef et al., 1998; Mouradov et al., 2002; Simpson and Dean, 2002). And the last one was the most widely

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Figure 4. Outline of the proposed potential pathways of how H 2 O2 regulated the flowering time. Solid lines indicate the known pathways; dotted lines indicate the pathways proposed in this study. H1, Hypothetical pathway 1; H2, hypothetical pathway 2.

acknowledged nowadays, which regarded the nutrient accumulation as an aspect of floral induction, and held that there were kinds of inducing factors and suppressing factors besides in flowering process. Recent studies focused on model plant Arabidopsis obtained a series of genes controlling flowering time and confirmed this hypothesis. In Arabidopsis, there were four pathways controlling the flowering time: the photoperiod pathway, the vernalization pathway, the autonomous pathway and the gibberellin pathway (Komeda, 2004). Besides, factors such as overcrowding, nutrient deficiency, heat, drought, salicylic acid and microRNA were already reported regulating flowering time (Simpson and Dean, 2002; Zhang and Zuo, 2006). In addition, Lokhande et al. (2003) and Moharekar et al. (2007) found that flowering time was negatively correlated with H2O2 content, so they proposed a hypothesis that H2O2 is a possible factor in flower induction. But these regulations of H2O2 content were passive to a certain degree, for the alteration of H2O2 content was carried out by stress treatment or comparison among Arabidopsis ecotypes from different latitudes. In this study, we took measures to actively control the H2O2 content. The transgenic Arabidopsis overexpressing tAPX and mutant line deficient in tAPX were obtained and the transcriptional level analysis indicated that the relative expression ratio of tAPX was 52:1:0 in the overexpressing line, the wild type and the mutant line, respectively (Figure 2b). As a result, the H2O2 content was lowest in the overexpressing line and highest in the mutant line (Figure 2c) under common conditions, and

this alteration finally led to the difference in flowering time (Figures 3a and b). The significance of this study was that H2O2 content was regulated on purpose by upstream tAPX gene, rather than altered by environmental stress factors. Further, the hypothesis that H2O2 was one of the possible factors in flower induction (Lokhande et al., 2003) was confirmed. It was also indicated in Figure 3b that the number of delayed flowering days in tAPXoverexpressing line compared with the wild type was obviously more magnificent than the number of advanced flowering days in the mutant line. It was known that there were kinds of APX isozymes in the Arabidopsis, so the mutant line lost tAPX which accounted for a small part of the total APX; while the overexpressing line increased the total APX because the 35S promoter started the tAPX gene absolutely and strongly. So the decrement of APX in the mutant was less than the increment of APX in the overexpressing line; and we considered it as the reason why the delayed flowering time in overexpressing line was more than the advanced flowering time in the mutant.This study provided a method in delaying or advancing the flowering time in Arabidopsis. Applying this technology to ameliorating the ornamental plants will be more practically significant. As to the mechanism, how some kinds of plant hormones promote flowering was revealed. For example, the gibberellins (Blรกzquez et al., 1998) promoted the flowering of Arabidopsis by activating the promoter of LEAFY gene. So we hypothesized that H2O2 activated some floral induction genes or repress some genes that deterred flowering (Figure 4). On the other

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hand, according to the water-water cycle in chloroplasts, the ·O2 produced by photosystem was disproportionated to H2O2 and O2 by SOD, then the H2O2 was reduced to water by APX subsequently (Asada, 1999). Thus, APX established an indirect contact with the photosystem, and we proposed another hypothesis that H2O2 might have potential impacts on the photoperiod pathway (Figure 4). Further researches on this are currently going on in our laboratory. ACKNOWLEDGEMENT This work was supported by the National Natural Science Foundation of China (30971557, 30971816). REFERENCES Asada K (1992). Ascorbate peroxidase-a hydrogen peroxide scavenging enzyme in plants. Physiol. Plant. 85: 235-241. Asada K (1999). The water-water cycle in chloroplasts scavenging of active oxygens and dissipation of excess photons. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50: 601-639. Asada K, Takahashi M (1987). Production and scavenging of active oxygen in photosynthesis. In: Kyle DJ, Osmond CB, Arntzen CJ, eds, Photoinhibition. Elsevier Science Publishers, Amsterdam, pp. 227287. Banuelos GR, Argumedo R, Patel K, Ng V, Zhou F, Vellanoweth RL (2008). The developmental transition to flowering in Arabidopsis is associated with an increase in leaf chloroplastic lipoxygenase activity. Plant Sci. 174: 366-373 Bernier G (1988). The control of floral evocation and morphogenesis. Annu. Rev. Plant Physiol. Plant Mol. Biol. 39: 175-219. Blázquez MA, Green R, Nilsson O, Sussman MR, Weigela D (1998). Gibberellins Promote Flowering of Arabidopsis by Activating the LEAFY Promoter. Plant Cell. 10: 791-800. Chai L, Wang JM, Fan ZY, Liu ZB, Li XF, Yang Y (2011). Ascorbate peroxidase gene from Brassica napus enhances salt and drought tolerances in Arabidopsis thaliana. Afr. J. Biotechnol. 10 (79): 1808518091 Doyle J, Doyle J (1990). Isolation of plant DNA from fresh tissues. Focus, 12: 13-15. Evans LT (1971). Flowering induction and the florigen concept. Annu. Rev. Plant Physiol. Plant Mol. Biol. 22: 365-394. Foyer CH, Hailiwell B (1976). The presence of glutathione and glutathione reductase in chloroplasts: proposed role in ascorbic acid metabolism. Planta. 133: 21-25. Guan LM, Scandalios JG (2000). Hydrogen peroxide-mediated catalase gene expression in response to wounding. Free Radical Biol. Med. 28: 1182-1190.

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Komeda Y (2004). Genetic regulation of time to flower in Arabidopsis thaliana. Annu. Rev. Plant Biol. 55: 521-535. Koornneef M, Alonso-Blanco C, Peeters AJM, Soppe W (1998). Genetic control of flowering time in Arabidopsis. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49: 345-370. Lang A (1952). Physiology of flowering. Annu. Rev. Plant Physiol. 3: 265-306. Lokhande SD, Ogawa KI, Tanaka A, Hara T (2003). Effect of temperature on ascorbate peroxidase activity and flowering of Arabidopsis thaliana ecotypes under different light conditions. J. Plant Physiol. 160: 57-64. Moharekar S, Moharekar S, Tanaka R, Ogawa KI, Tanaka A, Hara T (2007). Great promoting effect of high irradiance from germination on flowering in Arabidopsis thaliana – a process of photo-acclimation. Photosynthetica, 45: 259-265. Mouradov A, Cremer F, Coupland G (2002). Control of Flowering Time: Interacting Pathways as a Basis for Diversity. Plant Cell. 14, Suppl: S111-S130. Murgia I, Tarantino D, Vannini C, Bracale M, Carravieri S, Soave C (2004). Arabidopsis thaliana plants overexpressing thylakoidal ascorbate peroxidase show increased resistance to Paraquatinduced photooxidative stress and to nitric oxide-induced cell death. Plant Physiol. 38: 940-953. Simpson GG, Dean C (2002). Arabidopsis: the Rosetta Stone of Flowering Time? Science, 296: 285-289. Sun W, Duan M, Shu D, Yang S, Meng Q (2010). Over-expression of StAPX in tobacco improves seed germination and increases early seedling tolerance to salinity and osmotic stresses. Plant Cell Rep. 29: 917-926. Thordal-Christensen H, Zhang Z, Wei Y, Collinge DB (1997). Subcellular localization of H2O2 in plants. H2O2 accumulation in papillae and hypersensitive response during the barley-powdery mildew interaction. Plant J. 11: 1187-1194. Xu WF, Shi WM, Ueda A, Takabe T (2008). Mechanisms of Salt Tolerance in Transgenic Arabidopsis thaliana Carrying a Peroxisomal Ascorbate Peroxidase Gene from Barley. Pedosphere, 18: 486-495. Yong WD, Chong K, Xu ZH, Tan KH, Zhu ZQ (2000). Gene regulation study of flowering-time determination in higher plants. Chin. Sci. Bull. Chinese, 45 (5): 455-466. Zhang SZ, Zuo JR (2006). Advance in the Flowering Time Control of Arabidopsis. Prog. Biochem. Biophys. Chinese, 33(4): 301-309 Zhang XL, Wang PC, Song CP (2009). Methods of Detecting Hydrogen Peroxide in Plant Cells. Chin. Bull. Bot. Chinese, 44: 103-106 Zhou F, Andersen CH, Burhenne K, Hertz Fischer P, Collinge DB, Thordal-Christensen H (2000). Proton extrusion is an essential signalling component in the HR of epidermal single cells in the barley-powdery mildew interaction. Plant J. 23: 245-254.

Full Length Research Paper

Determination of genetic relatedness among selected rice (Oryza sativa, L.) cultivars using microsatellite markers Ali Etemad1, Mahmood Maziah1* and Siti Khalijah Daud2 ¹Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. ²Department of Biology, Faculty of Science, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. Accepted 5 May, 2011

For plant improvement programs, genetic variation information among different cultivars is very important. Genetic variation among 26 rice (Oryza sativa, L.) accessions, consisting of 13 Iranian and 13 Malaysian cultivars was investigated using microsatellite markers distributed across the rice genome. All the 21 selected microsatellite primers were successfully amplified by polymerase chain reaction (PCR) of which 20 (95.2%) were found to be polymorphic. A total of 75 alleles were detected at 21 microsatellite loci. The allele frequencies per locus ranged from 1 in RM338 to 5 alleles in RM307, RM161, RM334 and RM271. The mean number of alleles per locus was 3.57. Amongst these microsatellite loci, the largest polymorphism information content (PIC) value was 0.74 in RM 161, while the lowest PIC value was 0.0 in RM 338. Dendrogram was constructed based on dissimilarity values, unweighted pair group with arithmetic average (UPGMA) and it separated all the cultivars into six clusters. All these polymorphisms could be further evaluated for rice marker assisted selection and developed PCR methodology would expedite screening for large numbers of rice required for association studies. Key words: Genetic variation, genetic dissimilarity, polymorphism information content (PIC), polymerase chain reaction (PCR). INTRODUCTION The increase of human population and the limited availability of agricultural resources defiantly forced the search for new resources to overcome these phenomena. Developing countries face the challenges to rapidly increase agricultural productivity and feed their growing populations based on genetic potentials. Rice is one of the most important staple crops in most part of the world from which 50 to 80% of people obtain their calories (Khush, 2003).

*Corresponding author. E-mail: maziahm@biotech.upm.edu.my. Tel: +60389466703. Fax: +60389450913. Abbreviations: SSR, Simple sequence repeat; PIC, polymorphic information content; PCR, polymerase chain reaction.

Conventional breeding has played an essential role in rice cultivar improvement over the past decades. However, progress was slow and remains with several barriers such as, time-consuming in selection process and difficulties based on appropriate genotype selection. The quantitative nature of most agronomic traits has encouraged breeders to apply biotechnological techniques and molecular markers for rice breeding, which is known as molecular breeding. There are different types of molecular markers which are potentially different in cost, facilities required, reliability and detecting differences between individuals (Schlötterer, 2004; Schulman, 2007). Molecular study of germplasm would provide information for plant improvement programs in terms of the level of genetic variation within and between species. This information would then be used to select diverse parents of the same species or to identify the most

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closely related parents for inter-species crossing, to increase heterosis and to include desirable genes from more diverse backgrounds into the best germplasm (Henry, 1997). Maximum potential for genetic achievement is related to phenotypic variation which is present in the original population and maintained in the following selection cycles. Phenotypic variation is directly associated with genetic diversity even though it has interaction with environmental factors (Moose and Mumm, 2008). The classification and quantification of genetic diversity in closely related crop germplasm has been a major objective for a long time which is needed for a balanced use of genetic resources. The study of genetic difference in breeding resources is of primary interest to plant breeders. This information is needed for the selection and monitoring of germplasm and for the prediction of possible genetic gains (Chakravarthy and Rambabu, 2006). Markers based on expressed gene products, proteins or isozymes are also influenced by the environment and reveal a low level of polymorphism and low abundance (Ravi et al., 2003). In contrast, DNAbased molecular markers have proven to be powerful tools for the evaluation of genetic variation. The explanation of genetic relationships within and among species had been characterized by many untouched environmental factors (Powell et al., 1996). Ravi et al. (2003) reported that, the unique simple sequence repeat (SSR) profiles in rice cultivars could be generated by using a few primers that covered all 12 chromosomes. Microsatellites are tandem repeated motifs of 1 to 6 bp which have a frequent occurrence in all prokaryotic and eukaryotic genomes analyzed to date (Zane et al., 2002). The existence of microsatellites was verified by Hamada et al. (1982) in various eukaryotes and Tautz and Renz (1984) confirmed most microsatellites in plants. SSRs are present in both coding and noncoding regions and are distributed throughout the nuclear genome (Kalia et al., 2011). These can also be found in the chloroplastic (Provan et al., 2001; Chung et al., 2006) and mitochondrial (Rajendrakumar et al., 2007) genomes. SSR show high degree of length polymorphism (Zane et al., 2002) which is due to different number of repeats in the microsatellite regions; therefore, they can be easily detected by polymerase chain reaction (PCR). Microsatellites have high throughput genotyping and have proven to be an extremely valuable tool for establishment of genetic relationships (Parida et al., 2009; Kalia et al., 2011). The objective of this study was to determine the genetic variation and relationships among selected Iranian and Malaysian rice cultivars.

MATERIALS AND METHODS All the Iranian rice cultivar were collected from Iran Rice Research Institute, Rasht city but the Malaysia rice cultivars were from

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different countries like the Philippine, West Africa and West Bengal (Table 1) which were imported to Malaysia for research purposes. Henceforth, we assumed this group as Malaysian cultivars because the majority of cultivars were originated in Malaysia and obtained from the Malaysian Agriculture Research and Development Institute (MARDI). A total of 26 Iranian and Malaysian rice cultivars were selected as experimental materials, the details are described in Table 1. The seeds were raised in the soil in plastic containers (15 × 20 cm) placed in a glass house for one month.

Genomic DNA isolation Genomic DNA was extracted from fresh leaves of rice seedlings using the method of Dellaporta et al. (1983). The quality and quantity of the extracted DNA were estimated using UV spectrophotometer in two wavelengths (260 and 280 nm). The DNA samples were diluted to 20 ng/µl concentration with TE (Tris-EDTA) for SSR analyses and kept at -20°C. Selection of primers and PCR amplification A total of twenty one microsatellite primer pairs (Table 2) were selected from those reported by Temnykh et al. (2000); McCouch et al. (2001). The PCR amplification mixture was prepared in 0.2 ml micro centrifuge tubes. Each reaction mixture contains 2.5 µl of 10X PCR buffer, 1.5 mM MgCl2 (25 mM), 0.2 mM of each dNTP (10 mM), 0.2 µM of each primer (forward and reverse), 100 ng of DNA, 1 unit of Taq DNA polymerase( Research Biolab) and water was added to make the final volume to 25 µl. The amplification program consisted of the following cycles: 94°C for 4 min, 30 cycles of 94°C for 45 s, 55 to 63°C (depending on the annealing temperature of primer) for 30 s and 72°C for 1 min, and a final extension at 72°C for 10 min. The reproducibility of amplified products was established twice for each of the selected primers in this study. Electrophoresis of PCR products The amplification products (10 µl) were mixed with 2 µl loading buffer (Promega, USA) and separated by electrophoresis on 4% metaphor agarose gel which submerged in 1X TBE buffer (10 mM tris-borate, 1 mM EDTA) at 100 V for one hour and stained in ethidium bromide (1 mg/ml). A molecular DNA ladder of 25 to 766 bp (N3233L, Sigma USA) was used to evaluate the detected bands. Ethidium bromide staining of agarose gels generally revealed a multiple number of bands (Pervaiz et al., 2009).

Data analysis The genotypes were directly counted and scored, based on the allele size and estimated by the low molecular weight DNA ladder and allele binning were also conducted. The polymorphic information content (PIC) was employed for each locus to assess the information of each marker and its discriminatory ability. The calculation of PIC (Weir, 1996) for the ith marker is PIC = 1 – ΣPij(j = 1,2,..., n), where Pij is the frequency of the jth pattern for the ith marker and the summation extends over (n) patterns (Peng and Lapitan, 2005). Data analysis was carried out using POPGENE (Version 1.31) (Yeh and Boyle, 1997). To measure the genetic dissimilarity between the different genotypes within a single population, simple mismatch coefficient was conducted (Kosman and Leonard, 2005). This is identical to the normalized squared Euclidean distance (m = e2/n), where n is the total number of markers used for the analysis. Other softwares such as UVIDoc

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Table 1. List of names and origins of the selected Iranian and Malaysian rice cultivars.

Number of cultivar 1 2 3 4 5 6 7 8 9 10 11 12 13

Name HASHEMY I SALARY MUSA TAROM KHAZAR BINAM DORFAK SEPID ROD DOM SIAH CHAMPA SADRY HASHEMY II KADOOS DOM SEFID

Origin Iran Iran Iran Iran Iran Iran Iran Iran Iran Iran Iran Iran Iran

Number of cultivar 14 15 16 17 18 19 20 21 22 23 24 25 26

Name DULAR SALUMPIKIT IR 20 IRAT 13 IRAT 140 MR 211 MR 219 MR 220 MR 232 MR 219 LINE 4 MR 219 LINE 9 MORO BEREKAN SIAM PILIHAN

Origin West Bengal Malaysia Philippines Philippines Philippines Malaysia Malaysia Malaysia Malaysia Malaysia Malaysia West Africa Malaysia

Table 2. List of 21 microsatellite primer pairs with their chromosome locations, sequences (forward and reverse), repeated regions, observed sizes, number of alleles per locus and polymorphism information content (PIC) values.

Chromosome no.

Primer

Forward and reverse primer sequence (5’-3’) GCGAAAACACAATGCAAAAA GCGTTGGTTGGACCTGAC

Repeat region

Observed size

No. of alleles

PIC

(AG)26

88-132

0.671

RM1

RM283

GTCTACATGTACCCTTGTTGGG CGGCATGAGAGTCTGTGATG

(GA)18

152-166

0.349

RM259

TGGAGTTTGAGAGGAGGG CTTGTTGCATGGTGCCATGT

(CT)17

136-180

0.663

RM312

GTATGCATATTTGATAAGAG AAGTCACCGAGTTACCTTC

(ATTT)(GT)9

92-110

0.530

RM5

TGCAACTTCTAGCTGCTCGA GCATCCGATCTTGATGGG

(GA)14

96-126

0.579

RM237

CAAATCCCGACTGCTGTCC TGGGAAGAGAGCACTACAGC

(CT)18

110-150

0.522

RM338

CACAGGAGCAGGAGAAGAGC GGCAAACCGATCACTCAGTC

(CCT)6

183

0.00

RM55

CCGTCGCCGTAGTAGAGAAG TCCCGGTTATTTTAAGGCG

(GA)17

220-240

0.590

RM307

GTACTACCGACCTACCGTTCAC CTGCTATTGCATGAACTGCTGC

(AT)14(GT)21

124-190

0.706

RM161

TGCAGATGAGAAGCGGCGCCTC TGTGTCATCAGACGGCGCTCCG

(AG)20

132-176

0.745

RM334

GTTCAGTGTTCAGTGCCACC GACTGTTTGATCTTTGGTGGACG

(CTT)20

126-192

0.736

No., Number.

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Table 2. Continued.

RM11

TCTCCTCTTCCCCCGATC ATAGCGGGCGAGGCTTAG

(GA)17

126-154

0.647

RM25

GGAAAGAATGATCCTTTTCATGG CTACCATCAAAACCAATGTTC

(GA)18

104-136

0.594

RM44

ACGGGCAATCCGAACAACC TCGGGAAAACCTACCCTACC

(GA)16

102-122

0.577

RM433

TGCGCTGAACTAAACACAGC AGACAAACCTGGCCATTCAC

(AG)13

220-246

0.530

RM316

CTAGTTGGGCATACGATGGC ACGCTTATATGTTACGTCAAC

(GT)8-(TG)9(TTTG)4(TG)4

168-210

0.698

RM105

GTCGTCGACCCATCGGAGCCAC TGGTCGAGGTGGGGATCGGGTC

(CCT)6

114-126

0.506

RM271

TCAGATCTACAATTCCATCC TCGGTGAGACCTAGAGAGCC

(GA)18

94-136

0.706

RM287

TTCCCTGTTAAGAGAGAAATC GTGTATTTGGTGAAAGCAAC

(GA)18

98-122

0.651

RM277

CGGTCAAATCATCACCTGAC CAAGGCTTGCAAGGGAAG

(GA)11

114-128

0.579

RM511

CTTCGATCCGGTGACGAC AACGAAAGCGAAGCTGTCTC

(GAC)7

129-141

0.648

(Version 98), PIC calculator, NTSYS and Office Excel were also employed for data analyses. The presence or absence of alleles was voted as 1 or 0, respectively. The dendrogram was constructed based on the genetic similarity values using the unweighted pair group with arithmetic average (UPGMA) method with the NTSYS computer software (Rohlf, 1993).

RESULTS From 21 microsatellite primer pairs, 20 showed polymorphic bands (Figure 1). A total of 75 microsatellite alleles were amplified from 26 cultivars and this demonstrates the considerable variability among selected cultivars. The number of alleles per locus ranged from 1 to 5, with an average of 3.57 per locus (Figure 2). Four microsatellite loci (RM307, RM161, RM334 and RM271) revealed 5 alleles per locus; many studies have also reported significant differences in allelic diversity among various microsatellite loci (Akagi et al., 1997; McCouch et al., 2001; Ravi et al., 2003), while only one microsatellite locus (RM338) showed monomorph pattern.

The PIC value is an evidence of allele diversity and frequency among the varieties (Pervais et al., 2009). The PIC value of each marker can also be evaluated on the basis of its alleles and diverse for all SSR loci. In this study, the PIC for the 21 microsatellite loci ranged from 0 to 0.74, with mean value of 0.578. The largest PIC value was observed for locus RM161. The results indicated that high levels of polymorphisms were detected in these rice cultivars (Table 2). The dendrogram (Figure 3) showed six clusters that were demarcated at different dissimilarity coefficient levels. Cluster I consisted of (HashemyІ and Salary) cultivars, while Clusters II comprised 10 cultivars, namely: Musa Tarom, Dom Siah, Champa, Khazar, Sadry, HashemyІІ, Dom Sefid, IRAT13, Dular and Salumpikit, also, in Cluster III, four cultivars (Binam, Dorfak, Sepid Rood and Kadoos) were presented. In cluster IV, IR20 cultivar was the only member and in clusters V and VI, each consisted of four and five cultivars known as (MR211, MR 219, MR220 and IRAT140) and (MR232, MR219 Line 4, MR219 Line 9, Moro Berekan and Siam Pilihan), respectively (Table 1).

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Figure 1. Polymerase chain reaction (PCR) products of microsatellite primer RM1 for 26 rice cultivars. Lanes 1 to 13, Iranian cultivars; lanes 14 to 26, Malaysian cultivars (Table 1); M, DNA LADDER (25 to 766 bp).

A llele F requen c y C las s

100% 80% Allele E

60%

Allele D Allele C

40%

Allele B 20%

Allele A

R M1 R M28 3 R M25 9 R M 312 R M5 R M23 7 R M33 8 R M55 R M30 7 R M1 6 1 R M3 3 4 R M1 1 R M2 5 R M4 4 R M4 3 3 R M3 1 6 R M10 5 R M27 1 R M28 7 R M27 7 R M51 1

Figure 2. The frequency distribution of 75 alleles in 26 rice accessions detected by microsatellite, ranging from one to five alleles per locus.

Dissimilarity coefficiency varied from 0.188 in cluster I to 0.282 in cluster III, which consisted of Iranian cultivars. The genetic dissimilarity of cluster II which contained a mixture of Iranian and Malaysian cultivars was 0.195. Clusters IV, V and VI, consisted of Malaysian cultivars and exhibited the dissimilarity of 0.201, 0.217 and 0.326, respectively (Table 3). DISCUSSION The results of this study based on the number of alleles per loci were in agreements with those of Akagi et al.

(1997), McCouch et al. (2001) and Ravi et al. (2003) in which there were significant differences in allelic diversity amongst a range of microsatellite loci, and was lower than those reported by Jaymani et al. (2007). This inconsistency might be due to selection of genotypes or primers with high score alleles. The microsatellite involves di-nucleotide repeat motifs particularly those with GA repeats that amplified relatively small number of bands (Pervaiiz et al., 2009). Markers with PIC values of 0.5 or higher as demonstrated in this study are greatly informative for genetic studies and useful as a marker at a specific locus (Dewoody et al., 1995). The high PIC

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ІІ

ІІІ ІV V

VІ

Figure 3. Dendrogram resulting from UPGMA cluster analysis of 26 rice cultivars, based on genetic dissimilarity distance, data derived from 21 microsatellite primer pairs.

values in this study maybe due to co-dominant expression and multiple alleles (Ferreira and Grattapaglia, 1998). Based on the differences among the selected rice cultivars, six distinct

clusters were observed which illustrated that the selected markers were consistent as they can detect advanced levels of variations among closely related lines (Ganesh et al., 2007).

Conclusion The rice cultivars used in the present study were diverse because of differences in their origins,

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Table 3. Composition and size of clusters along with genetic dissimilarity values among rice accessions.

Cluster number Cluster I Cluster II Cluster III Cluster IV Cluster V Cluster VI

Number of genotype 2 10 4 1 4 5

ecotypes and geographical ranges, resulting in low similarity index values between them. Cultivars with lowest genetic similarity can be selected and used in breeding programs and screening for higher grain quality rice accession by back-cross program. The results suggested that microsatellite markers are reliable markers for detecting genetic differences among cultivars that originated from different geographical areas. ACKNOWLEDGEMENTS The authors would like to express their appreciation to Mr R. Ostadsaraee and Mr Abdullah Mohd Zain of the Iran Rice Research Institute and Malaysian Agriculture Research and Development Institute (MARDI) for their cooperation in providing the seed samples. Special thanks to Professor Dr. Tan Soon Guan and Dr. A. Javanmard for their helpful comments which improved the manuscript. We acknowledge the Faculty of Biotechnology and Biomolecular Sciences of Universiti Putra Malaysia (UPM) for supplying the chemicals and laboratory equipment for this study. REFERENCES Akagi H, Inagaki YA, Fujimura T (1997). Highly polymorphic microsatellite of rice consist of AT repeats, and a classification of closely related cultivars with these microsatellite loci. Theor. Appl. Genet. 94: 61–67. Chakravarthy KB, Rambabu N (2006). SSR marker based DNA fingerprinting and diversity study in rice (Oryza sativa L.). Afr. J. Biotechnol. 5: 684–688. Chung AM, Staub JE, Chen JF (2006). Molecular phylogeny of Cucumis species as revealed by consensus chloroplast SSR marker length and sequence variation. Genome 49: 219–229. Dellaporta S, Wood L, Hicks JB (1983). A plant DNA mini preparation: version II. Plant Mol. Biol. Report, 1(14):19-21. Dewoody JA, Honeycutt RL, Skow LC (1995). Microsatellite markers in white-tailed deer. J. Hered. 86: 317–319. Ferreira ME, Grattapaglia D (1998). Introdução ao uso de marcadores moleculares em Análise Genética. 3. ed. Embrapa, Brasília: 220. Ganesh Ram S, Thiruvengadam V, Kurungari VK (2007). Genetic diversity among cultivars, landraces and wild relatives of rice as revealed by microsatellite markers. Theor. Appl. Genet. 48(4), 337– 345. Hamada HM, Petrino MG, Kakunaga T (1982). A novel repeated element with Z-DNAforming potential is widely found in evolutionarily diverse eukaryotic genomes. Proc Natl Acad Sci USA 79:6465–6469.

Nature of origin Iran Iran and Malaysia Iran Malaysia Malaysia Malaysia

Genetic dissimilarity 0.188 0.195 0.282 0.201 0.217 0.326

Henry R (1997). Practical applications of plant molecular biology, Chapman & Hall, London. Jayamani P, Negrao S, Martins M, Macas B, Oiveira MM (2007). Genetic relatedness of Portuguese rice accessions from diverse origins as assessed by microsatellite markers. Crop Sci. 47: 879-886. Kalia RK, Rai MK, Kalia S, Singh R, Dhawan AK (2011). Microsatellite markers: an overview of the recent progress in plants. Euphytica, 177:309–334. Khush G (2003). Productivity improvements in rice. Nutri. Rev. 61: S114-S116. Kosman E, Leonard KJ (2005). Similarity coefficients for molecular markers in studies if genetic relationships between individuals for haploid, diploid, and polyploidy species. Mol Ecol, Oxford, 14: 415424. McCouch SR, Temnykh A, Lukashova J, Coburn G, De-Clerck S, Cartinhour S, Harrington M, Thomson E, Septiningsih M, Semon P, Moncada M, Li JM (2001). Microsatellite markers in rice: abundance, diversity, and applications. Rice Genet. 117-135. Moose SP, Mumm RH (2008). Molecular plant breeding as the st foundation for 21 century crop improvement. Plant Physiol. 147: 969-977. Parida SK, Kalia SK, Sunita K, Dalal V, Hemaprabha G, Selvi A, Pandit A, Singh A, Gaikwad K, Sharma TR, Srivastava PS, Singh NK, Mohapatra T (2009). Informative genomic microsatellite markers for efficient genotyping applications in sugarcane. Theor. Appl. Genet. 118:327–338. Peng JH, Lapitan N (2005). Characterization of EST-derived microsatellites in the wheat genome and development of eSSR markers. Fund. Int. Genom, 5: 80–96. Pervaiz ZH, Rabbani MA, Pearce SR, Malik SA (2009). Determination of genetic variability of Asian rice (Oryza sativa L.) varieties using microsatellite markers. Afr. J. Biotechnol. 8: 5641-5651. Powell W, Morgante M, Andre C, Hanafey M, Vogel J, Tingey S, Rafalski A (1996). The comparison of RFLP, RAPD, AFLP and SSR (Microsatellite) markers for germplasm analysis. Mol. Breed. 2: 225– 238. Provan J, Powell W, Hollingsworth PM (2001). Chloroplast microsatellites: new tools for studies in plant ecology and evolution. Trends Ecol. Evol. 16:142–147. Rajendrakumar P, Biswal AK, Balachandran SM, Srinivasarao K, Sundaram RM (2007). Simple sequence repeats in organellar genomes of rice: frequency and distribution in genic and intergenic regions. Bioinfo. 23:1–4. Ravi M, Geethanjali S, Sameeyafarheen F, Maheswaran M (2003). Molecular marker based genetic diversity analysis in rice (Oryza sativa L.) using RAPD and SSR markers. Euphytica, 133: 243–252. Rohlf FJ (1993). NTSYS-pc. Numerical taxonomy and multivariate analysis system, version 2.02. Setauket (New York): Exeter Publishing. Schlötterer C (2004). The evolution of molecular markers - just a matter of fashion. Nat. Rev. Genet. 5: 63-69. Schulman AH (2007). Molecular markers to assess genetic diversity. Euphytica 158: 313-321. Tautz D, Renz M (1984). Simple sequences are ubiquitous repetitive components of eukaryotic genomes. Nucleic Acids Res. 12:4127– 4138.

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Temnykh S, Park WD, Ayres N, Cartinhour S, Hauck N, Lipovtiesich L (2000). Mapping and genome organization of microsatellite sequences in rice (Oryza sativa L.). Theor. Appl. Genet. 100: 697– 712. nd Weir BS (1996). Genetic data analysis ІІ, 2 ed. Sunderland, Massachusetts, Sinauer Associates: 377. Yeh F, Boyle T (1997). Population genetic analysis of codominant and dominant markers and quantitative traits. Bel. J. Bot. 129: 157–163.

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Full Length Research Paper

Comparison of the G-174C polymorphism of interleukin (IL)-6 in different countries Yeqing Tong1,2, Zuxun Lu2, Yanwei Zhang3,, Jianjun Ye1, Faxian Zhan1, Shuangyi Hou1, Yang Li1, Xuhua Guan1, Jin-quan Cheng3 and Jiafa Liu1* 1

Hubei Center for Disease Control and Prevention, Wuhan 430079, People’s Republic of China. School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People’s Republic of China. 3 Shenzhen Center for Disease Control and Prevention, Shenzhen 518020, People’s Republic of China.

Accepted 16 March, 2012

Polymorphism of G-174C in the interleukin-6 (IL-6) promoter could affect both the transcription and secretion of IL-6 and may be involved in inflammation related to and the pathogenesis of infectious diseases and chronic diseases. However, IL-6 G-174C polymorphism may differ in various ethnic groups. We recruited 300 Chinese healthy subjects among two ethnic populations in order to assess the nature of the IL-6 G-174C polymorphism in different ethnic populations of China. Common polymorphic loci in the IL-6 G-174C were determined by TaqMan SNP genotyping assays. We found that the C allele frequency at the -174 promoter region of IL-6 was extremely low in both Han and Uyghur ethnic groups. Therefore, it was concluded that the G to C variant at -174 of IL-6 is extremely rare in Chinese Han and Uyghur healthy populations, which are different from other American and African countries. Key words: Interleukin-6 (IL-6), G-174C, polymorphism, Infecious diseases healthy population. INTRODUCTION Interleukin-6 (IL-6), one of the confirmed multifunctional pro-inflammatory cytokines which is demonstrated to be associated with infectious diseases (Jang et al., 2004; Nagabhushanam et al., 2003; Boulware et al., 2011; Sobti et al., 2010; Ruff et al., 2007; Newsom-Davis et al., 2004; Poudrier et al., 2001) and cardiovascular diseases (Pola et al., 2003; Lalouschek et al., 2006; Hojo et al., 2002; Plutzky, 2001; Tentolouris et al., 2004; Jenny et al., 2002), tuberculosis, AIDS and stroke, have been given more and more attentions by researchers. IL-6 demonstrates a crucial role in anti-viral immunity and in modulating T and B cell responses. Therefore, it can be conceived that the development of neutralizing antibodies against any of these cytokines as a consequence of autoimmunity affects the cellular functions and clearance of pathogens and predisposes the host to infectious

*Corresponding author. E-mail: l_jiafa@163.com. Tel/Fax: + 86 27 87652011

diseases. In addition, IL-6- deficient mice have been shown to be susceptible to various bacterial infections, including Mycobacterium, Streptococcus pneumoniae, Pseudomonas aeruginosa and Klebsiella pneumoniae (van der Poll et al., 1997; van Enckevort et al., 2001; Diao and Kohanawa, 2005) It is well established that IL-6 is also a key mediator in the inflammatory response associated with atherosclerosis and thus involved in many cardiovascular diseases, with many studies suggesting IL-6 may play a central role in the inflammatory response to cerebral ischemia, carotid artery atherosclerosis and coronary heart disease (Lalouschek et al., 2006; Hojo et al., 2002; Plutzky, 2001). The change from glycine (G) to cytosine (C) at position 174 of the IL-6 gene creates a potential binding site for the transcription factor NF-1, resulting in repressed gene expression. The G allele, on the other hand, is associated with higher circulating IL-6 levels (Fishman et al., 1998). IL-6 G-174C has been confirmed to be an important risk biomarker for some diseases such as stroke, cancer, mental retardation and ankylosing

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Table 1. IL-6 genotypes and allele distribution among two ethnic groups.

Parameter Age(years) Sex(M/F)

Han (n = 200) 63.20 ± 10.35 105/95

Uyghur (n = 100) 63.18 ± 10.19 52/48

P Value >0.05 >0.05

IL-6 (-174) GG GC CC

199 1 0

98 2 0

>0.05

IL-6 alleles G C MAF

399 1 0.25 %

198 2 1 .00%

>0.05

MAF, Minor allele frequency.

spondylitis (Pola et al., 2003; Jenny et al., 2002; Gangwar et al., 2009; Resch et al., 2009; ColladoEscobar et al., 2000). We detected this important polymorphism in Chinese Han and Uyghur populations, which are the first and sixth largest ethnic populations in China, respectively. The goal of the study was to determine whether the IL-6 G-174C polymorphism differ in various ethnic population in different countries. MATERIALS AND METHODS Study subjects This study was carried out with prior approval from the local Ethics Committee. Three hundred healthy subjects examined in hospitals with detailed medical routine check-up report were enrolled from Shenzhen and Xinjiang. All subjects were given informed consents. The health condition conformed to the national stipulation physical examination standard. Subjects with a history or family history of hypertension, diabetes, stroke, cancer and chronic inflammatory disorders were excluded from the study. Sample processing After obtaining informed consent from these groups, 5 ml blood samples were taken in ethylenediamine tetraacetic acid (EDTA) and plain vials. Genomic DNA was extracted from the peripheral blood leukocyte pellet using a DNA extraction kit (AXYGEN, California, USA). DNA samples were stored at -80°C before use.

Genotyping of the IL-6 promoter genomic variants The polymorphisms of IL-6 G-174C were determined using a TaqMan single nucleotide polymorphism (SNP) genotyping technique. The IL-6 -174G/C primers were forward, 5′AGCCTCAATGACGACCTAAG-3′ and reverse, 5′GGGGCTGATTGGAAACCTTA-3′. The TaqMan minor groove binding (MGB) probes for detection of G/C polymorphism (rs1800795) were FAM-AGTTGTGTCTTGCGATGC–MGB and HEX- AGTTGTGTCTTGCCATGC-MGB. The primers and probes

were commercially supplied by Shanghai GeneCore Biotechnologies. Thermal cyclings were performed on, and allele frequencies were determined by Stratagene Mx4000 systems (Stratagene, La Jolla, CA, USA). Comparisons between Han and Uyghur populations were made with the Fisher’s exact test (nominal data). The statistical analysis was carried out using SPSS software package SPSS12.0 (SPSS Inc., Chicago, Illinois, USA). A meta-analysis was done to test heterogeneity between different populations. The Q statistic test was used to assess heterogeneity, in which a P value greater than 0.05 suggested a lack of heterogeneity. All statistic analyses were done with Stata Statistical Package (version 10.0). To improve the genotyping quality and validate our results, a random selection of 10% of the samples were re-genotyped and ten randomly selected simples were re-sequenced by laboratory personnel not otherwise involved in the study, and the results were found to be reproducible with no discrepancies noted.

RESULTS AND DISCUSSION Table 1 includes the demographic and clinical characteristics of the patients and controls from the Han and Uyghur populations. The ratio of males to females was 105:95 in the Han subjects and 52:48 in the Uyghur subjects. There were no significant differences in sex between two populations (P>0.05). In addition, Table 1 also shows the distributions of the genotypes and allelic frequencies of IL-6 -174G/C polymorphisms in the Chinese Han and Uyghur population. There were no statistically significant differences in the distribution of IL6 G-174C polymorphism between two either ethnic group (P>0.05). The C allele frequencies at the -174 promoter region of IL-6 were extremely low in both the Chinese Han and Uyghur populations. Figure 1 compares the IL-6 G-174C genotype frequency distribution between various populations, including Asian population, Caucasians and Blacks from American, African, Gujarat and Spanish (Cox et al., 2001; Collado-Escobar et al., 2000). The results among Han and Uyghur are very different from Caucasians and Blacks (P<0.05). The results of meta-

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IL-6 -174G/C genotype frequency distribution populations. Compared with Han population; P<0.05. Compared with Uyghur population; P<0.05.

analysis were also featured with strong heterogeneity between Asians, Caucasians and Blacks (Total I2=0.81, P<0.001). IL-6, one of the most important mediators in in vivo inflammatory reactions, is implicated in the development of many inflammatory diseases (Jang et al., 2004; Nagabhushanam et al., 2003; Boulware et al., 2011; Sobti et al., 2010; Ruff et al., 2007; Newsom-Davis et al., 2004; Poudrier et al., 2001; Pola et al., 2003; Lalouschek et al., 2006; Hojo et al., 2002). There are also mounting evidences confirming that IL-6 is a key regulator of inflammatory mechanisms that play an important role in the pathophysiology and development of autoimmune disease (Maddur et al., 2010). A part of the singlenucleotide polymorphisms (SNPs) identified in the IL-6 gene, especially within the non-coding promoter sequence has been shown to have a powerful influence on the expression of the gene (Fishman et al., 1998). Our study shows the distribution of a significant SNP (G-174C) of IL-6 in Chinese Han and Uyghur population. The Uyghur population, a minority group originated from Turkish nomads living in Xinjiang having language, religious beliefs and lifestyles that may be very different from either Han population or American/European populations, is a special population presenting the typical admixture of Eastern and Western anthropometric traits in china. A study has shown that the Uyghur population has 60% European ancestry and 40% East Asian ancestry (Xu et al., 2008). Some previous studies have found a unique association between the CC genotype of the G-174C IL-6 polymorphism and many important diseases (Pola et al.,

between

various

2003; Jenny et al., 2002). Recently, the G-174C promoter polymorphism has been linked with increased risk of cervical cancer and developmental delay (Gangwar et al., 2009; Resch et al., 2009). However, we found the distribution of this functional polymorphism of G-174C in the IL-6 is different from other populations. Interestingly, we found that the C allele frequency at the -174 promoter region of IL-6 was extremely low in the both Han and Uyghur population. The meta-analysis findings also suggest to us that there may be genetic backgrounds variations between different countries that may affect the risk of diseases. However, the reason for the rarity of C allele in the Chinese population is unclear and needs further ethnicity-specific studies. From the current study, we conclude that the C allele frequency at the -174 promoter region of IL-6 is extremely low in the Chinese Han and Uyghur population, which are very different from Caucasians and Blacks (Cox et al., 2001; Collado-Escobar et al., 2000). These findings may provide some evidences for future associated research.

REFERENCES Boulware DR, Hullsiek KH, Puronen CE, Rupert A, Baker JV et al., (Provide Complete Name (2011). Higher levels of CRP, D-dimer, IL6, and hyaluronic acid before initiation of antiretroviral therapy (ART) are associated with increased risk of AIDS or death. J. Infect. Dis. 203: 1637-1646. Collado-Escobar MD, Nieto A, Mataran L, Raya E, Martin J (2000). Interleukin 6 gene promoter polymorphism is not associated with ankylosing spondylitis. J. Rheumatol. 27: 1461-1463. Cox ED, Hoffmann SC, DiMercurio BS, Wesley RA, Harlan DM,Kirk AD, Blair PJ (2001). Cytokine polymorphic analyses indicate ethnic

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differences in the allelic distribution of interleukin-2 and interleukin-6. Transplantation, 72: 720-726. Diao H, Kohanawa M (2005) Endogenous interleukin-6 plays a crucial protective role in streptococcal toxic shock syndrome via suppression of tumor necrosis factor alpha production. Infect. Immun. 73: 37453748. Fishman D, Faulds G, Jeffery R, Mohamed-Ali V, Yudkin JS, Humphries S, Woo P (1998) The effect of novel polymorphisms in the interleukin6 (IL-6) gene on IL-6 transcription and plasma IL-6 levels, and an association with systemic-onset juvenile chronic arthritis. J. Clin. Invest. 102: 1369-1376. Gangwar R, Mittal B, Mittal RD (2009) Association of interleukin-6 174G>C promoter polymorphism with risk of cervical cancer. Int. J. Biol. Markers, 24: 11-16. Hojo Y, Ikeda U, Takahashi M, Shimada K (2002). Increased levels of monocyte-related cytokines in patients with unstable angina. Atherosclerosis, 161: 403-408. Jang S, Uematsu S, Akira S, Salgame P (2004). IL-6 and IL-10 induction from dendritic cells in response to Mycobacterium tuberculosis is predominantly dependent on TLR2-mediated recognition. J. Immunol. 173: 3392-3397. Jenny NS, Tracy RP, Ogg MS, Luong le A, Kuller LH Arnold AM, Sharrett AR, Humphries SE (2002). In the elderly, interleukin-6 plasma levels and the -174G>C polymorphism are associated with the development of cardiovascular disease. Arterioscler Thromb. Vasc. Biol. 22: 2066-2071. Lalouschek W, Schillinger M, Hsieh K, Endler G, Greisenegger S, Marculescu R, Lang W, Wagner O, Cheng S, Mannhalter C (2006). Polymorphisms of the inflammatory system and risk of ischemic cerebrovascular events. Clin. Chem. Lab. Med. 44: 918-923. Maddur MS, Vani J, Lacroix-Desmazes S, Kaveri S, Bayry J (2010). Autoimmunity as a predisposition for infectious diseases. PLoS Pathog. 6: e1001077. Nagabhushanam V, Solache A, Ting LM, Escaron CJ, Zhang JY, Ernst JD (2003). Innate inhibition of adaptive immunity: Mycobacterium tuberculosis-induced IL-6 inhibits macrophage responses to IFNgamma. J. Immunol. 171: 4750-4757. Newsom-Davis T, Bower M, Wildfire A, Thirlwell C, Nelson M ,Gazzard B, Stebbing J (2004). Resolution of AIDS-related Castleman's disease with anti-CD20 monoclonal antibodies is associated with declining IL-6 and TNF-alpha levels. Leuk Lymphoma, 45: 19391941. Plutzky J (2001) Inflammatory pathways in atherosclerosis and acute coronary syndromes. Am. J. Cardiol. 88: 10K-15K. Pola R, Flex A, Gaetani E, Flore R, Serricchio M, Pola P (2003) Synergistic effect of -174 G/C polymorphism of the interleukin-6 gene promoter and 469 E/K polymorphism of the intercellular adhesion molecule-1 gene in Italian patients with history of ischemic stroke. Stroke, 34: 881-885.

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Poudrier J, Weng X, Kay DG, Pare G, Calvo EL Hanna Z, Kosco-Vilbois MH, Jolicoeur P. The AIDS disease of CD4C/HIV transgenic mice shows impaired germinal centers and autoantibodies and develops in the absence of IFN-gamma and IL-6. Immunity, 15: 173-185. Resch B, Radinger A, Mannhalter C, Binder A, Haas J, Muller WD (2009). Interleukin-6 G(--174)C polymorphism is associated with mental retardation in cystic periventricular leucomalacia in preterm infants. Arch Dis. Child Fetal Neonatal Ed. 94: F304-306. Ruff KR, Puetter A, Levy LS (2007). Growth regulation of simian and human AIDS-related non-Hodgkin's lymphoma cell lines by TGFbeta1 and IL-6. BMC Cancer, 7: p. 35. Sobti RC, Berhane N, Mahedi SA, Kler R, Hosseini SA, Kuttiat V, Wanchu A (2010) Polymorphisms of IL-6 174 G/C, IL-10 -592 C/A and risk of HIV/AIDS among North Indian population. Mol. Cell Biochem. 337: 145-152. Tentolouris C, Tousoulis D, Antoniades C, Bosinakou E, Kotsopoulou M, Trikas A, Toutouzas P, Stefanadis C. (2004). Endothelial function and proinflammatory cytokines in patients with ischemic heart disease and dilated cardiomyopathy. Int. J. Cardiol. 94: 301-305. van der Poll T, Keogh CV, Guirao X, Buurman WA, Kopf M (1997). Interleukin-6 gene-deficient mice show impaired defense against pneumococcal pneumonia. J. Infect. Dis. 176: 439-444. van Enckevort FH, Sweep CG, Span PN, Netea MG, Hermus AR, Kullberg BJ (2001). Reduced adrenal response and increased mortality after systemic Klebsiella pneumoniae infection in interleukin6-deficient mice. Eur. Cytokine Network, 12: 581-586. Xu S, Huang W, Qian J, Jin L (2008). Analysis of genomic admixture in Uyghur and its implication in mapping strategy. Am. J. Hum. Genet. 82: 883-894.

Full Length Research Paper

Influence of cutting frequency and fertilizer-N application on tiller production and herbage yield distribution over time in a guinea grass (Panicum maximum) sown pasture Onyeonagu, C. C* and Asiegbu, J. E. Department of Crop Science, University of Nigeria, Nsukka, Nigeria. Accepted 19 March, 2012

An experiment was carried out to evaluate the effects of cutting frequency and nitrogen rates on guinea grass (Panicum maximum) tiller production and herbage yield distribution over time. Plants were grown in the Department of Crop Science Research and Teaching Farm, University of Nigeria, Nsukka. A 4 × 4 factorial experiment replicated three times was set in a randomized complete block design. Treatments comprised four levels of nitrogen fertilizer at 0, 150, 300 and 450 kg ha-1 and four harvesting frequencies of three-, six-, nine- and 12-weekly intervals. Tiller number per square meter of ground significantly (P < 0.05) decreased with increasing interval of cuts, but significantly (P < 0.05) increased with incremental application of fertilizer N in all the years. The three-weeks interval of cuts significantly produced the highest tiller number when combined with the highest N rate of 450 kg N ha-1 in 2003 compared with six, nine- or 12-week intervals of cut. Grass tiller number significantly (P < 0.05) increased with frequent cutting of intervals earlier in the 2001 season (June to August) than later (September to November) when compared with the infrequent cutting intervals. In most periods of the years, dry matter yields of grass herbage and crop fractions were significantly increased with increase in interval between cuts and with incremental application of nitrogen. However, grass dry matter yields were significantly reduced with longer intervals, relative to the shorter intervals late in the 2001, 2002 and 2003 seasons. Key words: Nitrogen fertilizer, crop fractions, interval between cuts, seasons. INTRODUCTION In the tropics, supply of herbage for livestock during the dry months of the year declines substantially (Omaliko, 1983; Oloyo and IIelaboye, 2002). Enough herbage must be produced during the production period through intensive management techniques in order to make possible conservation for the lean season or off-season periods. Intensive management practices include the use of fertilizers, choice of forage species, cutting management, control of bushes and weeds and pasture establishment (Omaliko, 1983; Dev, 2001). The response to cutting of a forage plant depends upon its seasonal yield of carbohydrate storage, its growth habit and extent

*Corresponding author. E-mail: onyeonagu@yahoo.com.

of inflorescence development (Dev, 2001). Seasonal effects on tiller production, pasture growth and development and on the harvesting intervals have been reported (Omaliko, 1980; Romero et al., 1987; Venuto et al., 1998; Bamikole et al., 2004; Onyeonagu and Asiegbu, 2005). According to Omaliko (1980), some combinations of longer harvesting intervals early in the season with shorter intervals towards the end of the season increased the annual dry matter yield of a pasture sward in Nsukka, derived savanna zone of Nigeria compared with using either type of the regimes all through the year. Considering the production pattern throughout the growing season, Omaliko observed that harvesting every three weeks instead of once in six weeks gave lower dry matter yield early in the season. However, between August and the end of the growing season in October, the yield from

Onyeonagu and Asiegbu

six-weekly interval of cuts dropped, while those of threeweekly cuts increased, thereby reducing the difference between the two regimes. Similar results were obtained comparing the four-weekly cuts with eight-weekly cuts. In addition, similar seasonal effects have also been reported by Binnie et al. (1997). They observed that lengthening the re-growth interval by a single six-week interval increased the annual yield of herbage dry matter (DM) and digestible organic matter (DOM) of perennial ryegrass from 10.13 to 11.15 and from 6.73 to 7.47 t/ha, respectively. The effect increased with increasing length of re-growth period and was greatest in the early part of the season. The responses to initial cutting date and cutting frequency on dry matter yield production have been reported for Panicum maximum grown under Nsukka, derived savanna zone conditions of Nigeria (Omaliko, 1980). However, information is scanty on the effects of cutting frequency and nitrogen fertilizer application on tiller production, herbage dry matter yield and yields of crop fractions of P. maximum sown and maintained pastures at different seasons of the year in Nigeria. The objective of this work was to evaluate the effect of cutting frequency and nitrogen fertilizer application on tiller production and herbage yield distribution over time in a guinea grass (P. maximum) pasture established and maintained in Nsukka. MATERIALS AND METHODS The experiment was carried out in the Department of Crop Science Research and Teaching Farm, University of Nigeria, Nsukka, located at latitude 06° 52′ N and longitude 07° 24′ E, and on altitude of 447.2 m above sea level. The experiment was a 4 × 4 factorial experiment, laid out in a randomized complete block design with three replications. Treatments comprised four levels of nitrogen fertilizer at 0, 150, 300 and 450 kg ha-1 and four harvesting frequencies of three-, six-, nine- and 12-weekly intervals, resulting in sixteen treatment combinations per block. An area of land 21.2 × 11.2 m (226.24 m2) was marked out into three blocks of 19.2 × 2.4 m each. Each block was further divided into 16 plots of 2.4 × 1.2 m each, with a sampling area of 0.9 × 1.8 m. Each block was separated by one meter path-way. Basal application of 75 kg K ha-1 and 44 kg P ha-1 as muriate of potash and single superphosphate, respectively, was made by broadcasting. Rooted cuttings of P. maximum with height of 15 cm were planted in August 2000 at 20 × 30 cm spacing. The treatment combinations were allocated completely at random in each of the three blocks. Cutting was done at uniform height of about 15 cm with shears. Total fresh weight of the harvested grass material per plot was recorded and about 500 g fresh weight per plot was separated into leaf, stem and inflorescence fractions. The fractions were subsequently oven-dried and weighed. These were used to calculate the total dry weights of the crop fractions and the total herbage. The harvest intervals of three, six, nine and 12 weeks gave eight, four, two and two samples, respectively, in 2001, 2002, 2003 and 2004 seasons (that is 24 weeks from May to November). The required quantity of nitrogen as urea (46% N) was divided according to the number of cuts in a year for each harvest interval and evenly applied on the plot after each cut. The soil of the experimental site was a sandy loam and was acidic in reaction. The soil had low amounts of nitrogen content, potassium, magnesium

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and base saturation. The soil was also low in cation exchange capacity, and high in available phosphorus (Onyeonagu, 2010). Tiller counts were made in each plot using a 25 cm square quadrant. The mean of three throws per plot was used to calculate tiller population m-2. All data collected were statistically analysed using GenStat (1995) statistical package and employing the procedure for a randomized complete block design. Separation of treatment means for significant effects was done using the procedure for the least significant difference (LSD) as outlined by Carmer and Swanson (1971) and Obi (1986). Data from different years were combined for analyses after Bartlett’s test for homogeneity of variances. Square root transformation of the form x + 0.5, where x is the observation, was employed whenever there is zero value.

RESULTS Tiller number per square meter in most instances decreased with increasing interval of cuts in various years (Table 1). On the other hand, tiller number increased significantly with incremental application of fertilizer N in all the four years. The highest tiller number was obtained with three-weekly cuts when 450 kg N ha-1 was applied in 2003. Cutting frequency × nitrogen interaction effect on grass tiller number was not significant in 2001, 2002 and 2004. Moreover, tiller number always appeared to increase from the early period of May to July through the subsequent advancing periods in 2001 and 2002 (Table 2). In most of the periods, tiller number was greater at three- than at six-weekly interval of cut and with greater N-rate. Tiller number per square meter also increased significantly with intense than with lax cutting in all the periods for comparable intervals (six or 12 weekly cuts) in all the four years except for 2001 (Table 3). Tiller number was clearly increased by N application compared with where N was not applied in all the years except for the first and last harvest periods in 2001 and 2004, respectively. Tiller number seemed to increase with advancing season in both 2001 and 2002, but tended to decrease at the late season in both 2003 and 2004. On average of four year’s data, tiller number was lower with 12-weekly interval compared with threeor six-weekly interval while the values for nine- and 12weekly intervals did not differ significantly (Table 4). There was progressive significant (P < 0.05) increase in tiller population with increasing rates of N-fertilizer application. In most instances, six-weekly interval of cuts gave higher dry matter yield of grass than the three-weekly interval of cut in 2001 (Table 5). Cutting interval did not influence grass herbage yield in 2002 although yield tended to increase with increase in interval between cut. Also, N treatment significantly (P < 0.05) increased yield compared with where N was not applied except in the third and last periods of 2001 and during the last period in 2002 where nitrogen fertilizer treatment did not affect grass yield. Grass yield seemed to pick up early in 2001 (May to June) and decreased with advancing season up

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Table 1. Effects of cutting frequency and fertilizer N application on tiller number (per m2). -1

Cutting frequency (week)

Nitrogen fertilizer (kg N ha ) 150 300

450

Mean

Year 2001 3 6 9 12 Mean

1180.3 1148.5 893.3 1510.1 1183.1

1347.6 1393.7 994.7 1758.9 1373.7

1557.8 1504.5 1155.7 1912.8 1532.7

1656.9 1695.6 1123.5 1800.8 1569.2

1435.7 1435.6 1041.8 1745.7 1414.7

Year 2002 3 6 9 12 Mean

1939.7 1365.2 1129.1 837.3 1317.8

2396.7 1691.2 1295.2 1073.6 1614.2

2668.7 1820.9 1455.2 1254.1 1799.7

2891.3 1967.5 1457.6 1178.9 1873.8

2474.1 1711.2 1334.3 1086.0 1651.4

Year 2003 3 6 9 12 Mean

1891.0 1260.0 1036.6 600.8 1197.0

2219.9 1421.7 1122.7 817.6 1395.5

2461.1 1499.9 1189.3 831.7 1495.5

2651.1 1539.6 1204.3 925.3 1580.1

2305.8 1430.3 1138.1 793.9 1417.0

Year 2004 3 6 9 12 Mean

1860.1 1356.0 971.5 607.2 1198.7

1965.8 1454.3 1078.9 681.6 1295.1

2048.3 1540.3 800.0 1280.2 1363.6

2134.3 1644.8 1158.1 800.0 1434.3

2002.1 1498.8 1002.1 705.3 1302.1

2001 100.29 100.29 –

2002 125.76 125.76 –

2003 89.48 89.48 178.95

2004 54.30 54.30 –

LSD0.05 for 2 cutting frequency means (C) LSD0.05 for 2 nitrogen means (N) LSD0.05 for 2 C × N means –, Non-significant F-test at 5% probability level.

to about mid September and then increased. In 2002, grass yield appeared to increase with season up to mid September and then decreased. In 2003 and 2004, the 6weekly interval of cut significantly (P < 0.05) gave higher dry matter yield of grass than the three-weekly interval of cut except at the late season in 2003 where the threeweekly interval of cut significantly (P < 0.05) produced higher grass yield than the six-weekly interval of cut (Table 6). In 2003, grass dry matter yield was higher in the mid seasons of August to September and September to November compared with the earlier season of June to August, and was always drastically depressed in the final period of November/December. Furthermore, yield was generally higher during the 12 weeks of May to August compared with the harvest

period of August to November in 2001 and 2002 (Table 7). Similar trend in decrease in yield at late season occurred in 2003 but not in 2004. Over the two periods, yield distribution was better with 12-weekly cut than with the 6-weekly cut except at the late seasons of 2001 and 2002 where the 6-weekly cut had better yield distribution than the 12-weekly cut. Harvest made from May to August 2001 in the three- and six-weekly schedules gave no inflorescence fractions but produced progressively more inflorescence in the third and fourth periods of August/September and September/November (Table 8). Leaf yield was similar in the first two periods (May to June and June to August) than with the last two periods (August to September and September to November). The six-weekly schedule significantly (P < 0.05) produced

Onyeonagu and Asiegbu

7173

Table 2. Tiller number (per m2) at various periods of the year for 3- and 6- weekly intervals. -1

Cutting frequency (week)

Nitrogen fertilizer (kg N ha ) 150 300

Mean

450

Year 2001 May 17 to June 28 3 6 Mean

1184.0 1264.0 1224.0

1245.3 1370.7 1308.0

2301.3 1306.7 1804.0

1736.0 1370.7 1553.3

1616.7 1328.0 1472.3

June 28 to Aug. 9 3 6 Mean

1184.0 1141.3 1162.7

1296.0 1232.0 1264.0

1456.0 1333.3 1394.7

1464.0 1344.0 1404.0

1350.0 1262.7 1306.3

Aug. 9 to Sept. 20 3 6 Mean

1151.2 881.6 1016.4

1424.0 1306.7 1365.3

1610.9 1278.4 1444.7

1716.5 1562.7 1639.6

1475.7 1257.3 1366.5

Sept. 20 to Nov. 1 3 6 Mean

1201.9 1307.2 1254.5

1425.1 1665.6 1545.3

1756.3 2099.7 1928.0

2145.9 2505.1 2325.5

1632.3 1894.4 1763.3

Year 2002 May 20 to July 1 3 6 Mean

1797.6 1130.7 1464.1

2246.7 1445.9 1846.3

2373.3 1571.7 1972.5

2755.4 1724.3 2239.8

2293.2 1468.1 1880.7

July 1 to Aug. 12 3 6 Mean

1894.1 1319.5 1606.8

2397.3 1605.3 2001.3

2604.5 1797.3 2200.9

2817.6 1929.1 2373.3

2428.4 1662.8 2045.6

Aug. 12 to Sept. 23 3 6 Mean

1969.9 1475.2 1722.5

2278.9 1861.3 2070.1

2639.2 1990.9 2315.1

2763.5 2038.9 2401.2

2412.9 1841.6 2127.2

Sept. 23 to Nov.4 3 6 Mean

2097.1 1535.5 1816.3

2664.0 3057.6 1852.3 1923.7 2258.1 2490.7 Year 2001 2nd 3rd period period – 147.50 179.93 208.50 – –

3228.5 2177.6 2703.1

2761.8 1872.3 2317.0

LSD0.05 for 2 cutting frequency means (C) LSD0.05 for 2 nitrogen means (N) LSD0.05 for 2 C × N means –, Non-significant F-test at 5% probability level.

1st period – – –

4th period

1st period

184.57 261.02 –

176.20 249.18 –

Year 2002 3rd period 188.96 99.99 267.23 141.40 – – 2nd period

4th period 191.23 270.44 –

7174

Afr. J. Biotechnol.

Table 3. Tiller number (per m2) at various periods of the year for 6- and 12- weekly intervals. -1

Cutting frequency (week)

Nitrogen fertilizer (kg N ha ) 150 300

450

Mean

Year 2001 May 17 to Aug. 9 6 12 Mean

1202.7 1280.0 1241.3

1301.3 1408.0 1354.7

1320.0 1653.3 1486.7

1357.3 1456.0 1406.7

1295.3 1449.3 1372.3

Aug. 9 to Nov. 1 6 12 Mean

1094.4 1740.3 1417.3

1486.1 2109.9 1798.0

1689.1 2172.3 1930.7

2033.9 2145.6 2089.7

1575.9 2042.0 1808.9

Year 2002 May 20 to Aug. 12 6 12 Mean

1225.1 732.3 978.7

1525.6 974.4 1250.0

1684.5 1048.5 1366.5

1826.7 1106.1 1466.4

1565.5 965.3 1265.4

Aug. 12 to Nov. 4 6 12 Mean

1462.1 942.4 1202.2

1856.8 1172.8 1514.8

1957.3 1459.7 1708.5

2108.3 1251.7 1680.0

1846.1 1206.7 1526.4

Year 2003 June 30 to Sept. 22 6 12 Mean

1465.1 677.3 1071.2

1506.7 956.3 1231.5

1600.8 952.5 1276.7

1666.7 1049.1 1357.9

1559.8 908.8 1234.3

Sept. 22 to Dec. 15 6 12 Mean

1054.9 524.3 789.6

1336.8 630.9 983.9

1398.9 710.9 1054.9

1412.5 801.6 1107.1

1300.8 666.9 983.9

Year 2004 June 1 to Aug. 24 6 12 Mean

1422.1 690.1 1056.1

1529.9 732.3 1131.1

1661.3 753.6 1207.5

1699.5 798.4 1248.9

1578.2 743.6 1160.9

Aug. 24 to Nov.16 6 12 Mean

1289.9 524.3 907.1

1378.7 630.9 1004.8 Year 2001 1st 2nd period period 128.16 179.43 – 253.75 – –

LSD0.05 for 2 cutting frequency means (C) LSD0.05 for 2 nitrogen means (N) LSD0.05 for 2 C × N means –, Non-significant F-test at 5% probability level.

1419.2 1590.1 710.9 801.6 1065.1 1195.9 Year 2002 1st 2nd period period 104.81 91.10 148.23 128.84 – 182.21

1419.5 666.9 1043.2 Year 2003 st

1 period

2 period

81.93 115.86 –

126.96 179.55 –

Year 2004 1st 2nd period period 61.93 145.36 87.58 – – –

Onyeonagu and Asiegbu

7175

Table 4. Effects of cutting frequency and fertilizer N application on the tiller number (per m2), mean of 4 years data (2001, 2002, 2003 and 2004). -1

3 6 9 12 Mean

Nitrogen fertilizer (kg N ha ) 0 150 300 1717.8 1982.5 2184.0 1282.4 1489.4 1591.4 1007.5 1122.9 1150.1 888.9 1082.9 1182.7 1224.2 1419.4 1527.0

LSD0.05 for 2 cutting frequency means (C) LSD0.05 for 2 nitrogen means (N) LSD0.05 for 2 C Ă&#x2014; N means

59.93 59.93 119.87

Cutting frequency (week)

higher stem yields in all the harvest periods than the three-weekly cuts. Cutting frequency, however, showed no significant effect on leaf dry matter yield in any of the harvest periods in 2002 (Table 9). The stem and inflorescence dry matter yields were significantly (P < 0.05) increased with increase in interval between cuts in all the periods except at the second period, where the inflorescence yield remained similar for the three and sixweekly intervals. The 450 kg N ha-1 significantly (P < 0.05) produced higher leaf yield than the control in the first three periods but had similar effect with the other N rates. Nitrogen application did not significantly influence the stem and inflorescence yields except at the third harvest period where stem and inflorescence yields were significantly (P < 0.05) increased with N application compared with the control treatment. The six weeks cutting interval significantly (P < 0.05) produced higher leaf dry matter yield during the second period in 2003 than the three-weekly cuts (Table 10). Cutting treatment showed no significant effect on leaf yield at the first and third periods. The six weeks interval between cuts significantly (P < 0.05) gave lower leaf yield than the three-weekly cuts at the last period. Stem dry matter yield was significantly (P < 0.05) increased during the first three harvest periods with increase in interval between cuts. Cutting treatment effect on stem dry matter yield was not significant at the last period. Inflorescence dry matter yield was significantly (P < 0.05) increased only during the second period with increase in interval between cuts. Nitrogen application significantly (P < 0.05) increased leaf yield compared with the control except in the second period where N effect was not significant. Stem dry matter yield was significantly increased at the first two periods with N application over the control treatment but was not significantly affected by N treatment at the last two periods. Moreover, nitrogen application significantly increased the inflorescence dry matter yield only at the second and third periods compared with the control treatment but had no significant effect at the first and fourth periods.

450 2333.4 1711.9 1235.9 1176.3 1614.4

Mean 2054.4 1518.8 1129.1 1082.7 1446.2

In 2004, the six-weekly schedule always produced higher leaf blade and stem dry matter than the threeweekly cuts (Table 11). Fertilizer N application always gave higher (P < 0.05) leaf blade yield compared with where N was not applied except at the last period. Fertilizer application significantly increased stem yield only at the first and third periods. Leaf blade dry matter was highest at the July to August period followed by the June to July period, October to November and then the August to early October period. Stem dry matter yield seemed to increase with season. There were no records of inflorescence yield at the first and second periods of the year 2004. Inflorescence yield was higher at the August to early October period than the October to November period. Whether for six or 12 weeks intervals of cut, leaf dry matter yield was greatest earlier in the season in both 2001 and 2002 seasons than later (Table 12). Leaf yield was higher with six- weekly cuts than the 12-weekly cuts at the last period in 2001. Stem yield was also greatest earlier in the season (May to August) than later (August to early November) in 2001. The reverse was the case in 2002. There were no records of inflorescence dry matter during the first period of 2001. Inflorescence yield was significantly (P < 0.05) higher with six-weekly harvests than the 12-weekly cuts at the last period in 2001. Cutting treatment had no effect on inflorescence yield at the first and second periods in 2002. Leaf dry matter yield was higher earlier in the season in both 2003 and 2004 seasons than later (Table 13). Yield was more evenly distributed with 12-weekly cut than with the 6-weekly cut in both years. Stem dry matter yield was also greatest earlier in the season (June to September) than later (September to mid December) in 2003. In 2004, stem yield was higher at the August to November period than the June/August period. At all periods, harvest at six- or 12-weekly intervals allowed for development and harvest of inflorescence which was generally greater at August to November period compared with other periods.

7176

Afr. J. Biotechnol.

Table 5. Grass herbage yield (kg Dm/ha) at various periods in 2001 and 2002 for 3- and 6-weekly intervals. -1

Cutting frequency (week)

Nitrogen fertilizer (kg N ha ) 150 300

450

Mean

Year 2001 May 17 to June 28 3 6 Mean

1346.2 2178.6 1762.4

2317.9 2772.0 2544.9

1845.5 4206.4 3026.0

2107.9 2997.9 2552.9

1904.4 3038.8 2471.6

June 28 to Aug. 9 3 6 Mean

1605.8 2191.3 1898.6

2199.2 2860.0 2529.6

2072.6 3063.1 2567.9

2266.0 3189.3 2727.6

2035.9 2825.9 2430.9

Aug. 9 to Sept. 20 3 6 Mean

1357.8 1535.8 1446.8

2177.1 1956.4 2066.8

1960.8 2233.1 2097.0

1520.2 2431.4 1975.8

1754.0 2039.2 1896.6

Sept. 20 to Nov. 1 3 6 Mean

1903.9 3759.9 2831.9

1849.1 3893.8 2871.5

2340.6 4389.3 3364.9

2324.8 4163.5 3244.2

2104.6 4051.6 3078.1

Year 2002 May 20 to July 1 3 6 Mean

1140.3 1026.6 1083.4

1744.9 1532.4 1638.7

1922.7 1493.4 1708.1

1966.6 2296.3 2131.4

1693.6 1587.2 1640.4

July 1 to Aug. 12 3 6 Mean

1063.6 1243.4 1153.5

1606.3 2207.8 1907.1

2014.6 2087.7 2051.2

2134.7 2303.0 2218.9

1704.8 1960.5 1832.6

Aug. 12 to Sept. 23 3 6 Mean

1040.8 1291.4 1166.1

1745.2 2066.7 1906.0

2090.6 2228.4 2159.5

2033.0 2054.0 2043.5

1727.4 1910.1 1818.8

Sept. 23 to Nov.4 3 6 Mean

1219.3 1353.8 1286.6

1679.2 2322.6 2000.9

1489.2 1788.6 1638.9

LSD 0.05 for 2 cutting frequency means (C) LSD 0.05 for 2 nitrogen means (N) LSD 0.05 for 2 C × N means –, Non-significant F-test at 5% probability level.

1st period 507.97 718.37 –

1502.4 2109.8 1806.1 2001 2nd 3rd period period 362.30 – 512.36 – – –

4th period

1st period

1283.63 – –

– 651.73 –

1472.5 1893.7 1683.1 2002 3rd 2nd period period – – 378.78 383.46 – –

4th period – – –

Onyeonagu and Asiegbu

7177

Table 6. Grass herbage yield (kg Dm/ha) at various periods in 2003 and 2004 for 3- and 6- weekly intervals. -1

Cutting frequency (week)

Nitrogen fertilizer (kg N ha ) 150 300

450

Mean

Year 2003 June 30 to Aug. 11 3 6 Mean

795.4 1011.4 903.4

970.7 1399.1 1184.9

1394.3 1577.1 1485.7

1322.2 1493.5 1407.8

1120.7 1370.3 1245.5

Aug. 11 to Sept. 22 3 6 Mean

939.1 1525.5 1232.3

1115.8 1747.1 1431.4

1391.0 2344.0 1867.5

1314.6 2065.9 1690.3

1190.1 1920.6 1555.4

Sept. 22 to Nov. 3 3 6 Mean

699.8 846.8 773.3

1299.6 1857.9 1578.8

1278.6 1546.6 1412.6

1420.8 1868.0 1644.4

1174.7 1529.8 1352.3

Nov. 3 to Dec. 15 3 6 Mean

211.6 234.4 223.0

519.5 425.2 472.4

558.9 408.4 483.6

530.8 365.8 448.3

455.2 358.4 406.8

Year 2004 June 1 to July 13 3 6 Mean

912.8 1381.0 1146.9

1435.4 2436.6 1936.0

1749.4 2638.9 2194.1

2058.0 2338.6 2198.3

1538.9 2198.8 1868.8

July 13 to Aug. 24 3 6 Mean

839.8 1628.7 1234.3

1482.9 2205.8 1844.3

1749.9 2820.1 2285.0

1944.0 2576.9 2260.5

1504.2 2307.9 1906.0

Aug. 24 to Oct. 5 3 6 Mean

892.3 1642.0 1267.1

1157 2401.1 1779.5

1629.9 3176.1 2403.0

1765.1 2579.0 2172.0

1361.3 2449.5 1905.4

Oct. 5 to Nov.16 3 6 Mean

910.0 1654.7 1282.3

LSD 0.05 for 2 cutting frequency means (C) LSD 0.05 for 2 nitrogen means (N) LSD 0.05 for 2 C × N means –, Non-significant F-test at 5% probability level.

1st period 195.94 277.11 –

1175.5 1482.3 2664.0 2766.0 1919.8 2124.1 Year 2003 2nd 3rd 4th period period period 267.13 342.15 76.44 377.78 483.88 108.10 – – –

1672.7 2047.2 1860.0 1st period 304.97 431.29 –

1310.1 2283.0 1796.6 Year 2004 3rd 2nd period period 487.54 234.98 689.48 332.32 – –

4th period 459.66 – –

7178

Afr. J. Biotechnol.

Table 7. Grass herbage yield (kg Dm/ha) at various periods of the year for 6- and 12-weekly interval. -1

Cutting frequency (week)

Nitrogen fertilizer (kg N ha ) 150 300

450

Mean

Year 2001 May 17 to Aug. 9 6 12 Mean

4369.9 6551.5 5460.7

5632.0 5829.5 5730.7

7269.5 8533.0 7901.3

6187.2 10632.0 8409.6

5864.7 7886.5 6875.6

Aug. 9 to Nov. 1 6 12 Mean

5295.7 2547.4 3921.6

5850.3 2983.5 4416.9

6622.4 3216.4 4919.4

6594.9 3678.4 5136.7

6090.8 3106.4 4598.6

Year 2002 May 20 to Aug. 12 6 12 Mean

2270.0 3428.9 2849.5

3740.3 4211.7 3976.0

3581.1 4432.5 4006.8

4599.3 3430.1 4014.7

3547.7 3875.8 3711.8

Aug. 12 to Nov. 4 6 12 Mean

2645.2 3721.0 3183.1

4176.5 3043.4 3610.0

4551.0 2606.9 3578.9

3842.6 2028.3 2935.5

3803.8 2849.9 3326.9

Year 2003 June 30 to Sept. 22 6 12 Mean

2536.9 3887.5 3212.2

3146.1 2348.5 2747.3

3921.1 3947.8 3934.4

3559.4 3538.7 3549.1

3290.9 3430.6 3360.8

Sept. 22 to Dec. 15 6 12 Mean

1081.2 2428.3 1754.8

2283.2 3070.2 2676.7

1954.9 3061.4 2508.2

2233.8 2795.3 2514.6

1888.2 2838.8 2363.5

Year 2004 June 1 to Aug. 24 6 12 Mean

3009.7 5415.3 4212.5

4642.3 7594.7 6118.5

5459.0 7956.8 6707.9

4915.6 6251.7 5583.6

4506.7 6804.6 5655.6

3296.7 5655.9 4476.3 Year 2001

5065.1 5942.1 7404.2 9259.9 6234.7 7601.0 Year 2002 1st 2nd period period

Aug. 24 to Nov.16 6 12 Mean

LSD 0.05 for 2 cutting frequency means (C) LSD 0.05 for 2 nitrogen means (N) LSD 0.05 for 2 C × N means –, Non-significant F-test at 5% probability level.

1st period 1401.79 1982.42 –

2nd period 1628.89 – –

– – –

4626.2 7341.2 5983.7 Year 2003 1st period

2nd period

– – –

492.01 – –

4732.5 7415.3 6073.9 Year 2004 1st 2nd period period 1061.30 1500.90 –

1021.84 1445.10 –

Onyeonagu and Asiegbu

7179

Table 8. Dry matter yields of leaf, stem and inflorescence fractions at various periods of the year for 3- and 6- weekly intervals in 2001. Leaf blade Cutting frequency (week)

Stem

Nitrogen fertilizer (kg N ha-1) 0

150

300

450

May 17 to June 28 3

1306.5

2256.4

1800.1

2032.3

2053.8

2570.1

3581.4

Mean

1680.2

2413.3

June 28 to Aug. 9 3

1599.3

Mean

Inflorescence

Nitrogen fertilizer (kg N ha-1)

Mean

150

300

450

Mean

47.5 (5.9) 294.9 (16.5) 171.2 (11.2)

14.8 (2.7)

39.7 (6.3)

17.3 (3.7)

22.3 (4.1)

34.1 (5.6)

27.3 (4.3)

31.5 (5.4)

44.3 (5.7)

34.3 (5.2)

24.5 (4.2)

33.5 (5.3)

24.4 (4.5)

30.8 (4.7)

28.3 (4.7)

357.8 1197.7 777.8

136.2 174.4 155.3

116.6 269.1 192.9

111.8 200.8 156.3

188.0 285.8 236.9

138.1 232.5 185.3

150

300

450

1848.8

39.7 (5.0)

2629.4

2708.7

124.8 (10.0)

61.5 (6.6) 201.9 (13.0)

2690.7

2330.9

2278.8

82.3 (7.5)

131.7 (9.8)

45.4 (6.7) 625.0 (24.9) 335.2 (15.8)

75.6 (8.4) 368.6 (18.4) 222.1 (13.4)

55.6 (6.7) 330.1 (16.6) 192.8 (11.6)

2189.6

2052.8

2222.5

2016.0

6.5 (2.0)

2031.1

2543.2

2608.5

2771.9

2488.7

160.2 (12.6)

9.6 (2.3) 316.8 (17.4)

Mean

1815.2

2366.4

2330.6

2497.2

2252.4

83.4 (7.3)

163.2 (9.8)

19.8 (3.9) 454.6 (21.3) 237.2 (12.6)

43.5 (6.4) 417.4 (20.4) 230.5 (13.4)

19.8 (3.6) 337.3 (17.9) 178.6 (10.8)

Aug. 9 to Sept. 20 3

1315.1

2082.5

1881.9

1457.6

1684.3

27.9 (4.5)

1285.5

1652.4

1887.3

2014.7

1710.0

216.2 (13.6)

Mean

13003

1867.5

1884.6

1736.2

1697.1

122.0 (9.1)

54.9 (6.2) 276.7 (16.6) 165.8 (11.4)

61.7 (7.6) 314.2 (17.6) 188.0 (12.6)

45.4 (5.4) 372.4 (18.0) 208.9 (11.7)

Sept. 20 to Nov. 1 3 6 Mean

1423.7 2612.7 2018.2

1438.6 2462.6 1950.6

1958.0 2966.7 2462.4

1614.3 2443.4 2028.8

1608.7 2621.4 2115.0

344.0 972.8 658.4

293.9 1162.1 728.0

270.7 1221.8 746.3

522.5 1434.3 978.4

Leaf blade

LSD0.05 for 2 cutting frequency means (C) LSD0.05 for 2 nitrogen means (N) LSD0.05 for 2 C × N means

Nitrogen fertilizer (kg N ha-1)

Stem

Inflorescence

1st period

2nd period

3rd period

4th period

1st period

2nd period

3rd period

4th period

1st period

2nd period

3rd period

4th period

413.73

357.51

412.13

805.52

4.09

2.10

3.76

529.14

–

585.10 –

– –

5.78 –

2.97 –

– –

NY NY

– –

–, Non-significant F-test at 5% probability level. The comparison is based on transformed means in parenthesis because of zero values in some instances. NY, No Inflorescence yield.

DISCUSSION The significant increase in grass tiller number per

square meter of ground observed with frequent cutting interval and with incremental application of fertilizer N in all the years, was reported by

Wilman and Asiegbu (1982) and Harris et al. (1996). Onyeonagu and Asiegbu (2005) obtained similar results in a degraded pasture typified by P.

7180

Afr. J. Biotechnol.

Table 9. Dry matter yields of leaf, stem and inflorescence fractions at various periods of the year for 3- and 6- weekly intervals in 2002. Leaf blade Cutting frequency (week)

Stem

Nitrogen fertilizer (kg N ha-1)

Mean

150

300

450

May 20 to July 1 3 6 Mean

1140.3 1006.1 1073.2

1744.9 1488.3 1616.6

1922.7 1474.0 1698.4

1966.6 2191.4 2079.0

July 1 to Aug. 12 3 6 Mean

1063.4 1193.0 1128.2

1604.4 2120.6 1862.5

2014.6 2032.7 2023.7

Aug. 12 to Sept. 23 3 6 Mean

1000.1 1162.9 1081.5

1667.0 1692.1 1679.6

Sept. 23 to Nov.4 3 6 Mean

1077.9 1072.5 1075.2

LSD 0.05 for 2 cutting frequency means (C) LSD 0.05 for 2 nitrogen means (N) LSD 0.05 for 2 C × N means

Inflorescence

Nitrogen fertilizer (Kg N ha-1) 0

150

300

450

1693.6 1539.9 1616.8

0.0 (0.7) 17.0 (4.1) 8.5 (2.4)

0.0 (0.7) 40.7 (5.9) 20.4 (3.3)

0.0(0.7) 16.9(2.9) 8.5(1.8)

0.0 (0.7) 98.2 (9.2) 49.1 (5.0)

2134.7 2079.2 2107.0

1704.3 1856.4 1780.3

0.0 (0.7) 50.4 (6.4) 25.2 (3.6)

1.9 (1.3) 87.2 (8.6) 44.6 (4.9)

0.0(0.7) 55.0(6.2) 27.5(3.4)

1996.5 1744.1 1879.6

1891.3 1564.9 1728.1

1638.8 1541.0 1589.9

29.6 112.0 70.8

52.4 327.5 189.9

1288.4 1505.1 1396.8

1529.6 1691.0 1610.3

1377.3 1379.1 1378.2

1318.3 1411.9 1365.1

100.9 242.7 171.8

1st period

Leaf blade 2nd 3rd period period

– 631.98 –

– 349.46 –

– 318.68 –

Mean

Nitrogen fertilizer (kg N ha-1)

Mean

150

300

450

0.0 (0.7) 43.2 (5.5) 21.6 (3.1)

0.0 (0.7) 3.6 (2.0) 1.8 (1.3)

0.0 (0.7) 3.4 (1.8) 1.7 (1.2)

0.0 (0.7) 2.5 (1.4) 1.3 (1.0)

0.0 (0.7) 6.7 (2.3) 3.4 (1.5)

0.0 (0.7) 4.1 (1.9) 2.0 (1.3)

0.0 (0.7) 221.3 (14.6) 110.6 (7.7)

0.5 (0.8) 103.5 (9.0) 52.0 (4.9)

0.1 (0.8) 0.0 (0.7) 0.1 (0.7)

0.0 (0.7) 0.0 (0.7) 0.0 (0.7)

0.0 (0.7) 2.5 (1.4) 1.3 (1.0)

0.0 (0.7) 0.6 (0.9) 0.3 (0.8)

69.3 414.9 242.1

100.4 440.9 270.7

62.9 323.8 193.4

11.1 (2.8) 16.5 (4.1) 13.8 (3.5)

25.7 (5.1) 47.2 (6.8) 36.5 (5.9)

24.8 (4.9) 69.4 (7.9) 47.1 (6.4)

41.3 (6.4) 48.2 (6.9) 44.7 (6.7)

25.7 (4.8) 45.3 (6.4) 35.5 (5.6)

153.7 505.5 329.6

105.5 522.4 314.0

77.5 336.8 207.1

109.4 401.9 255.6

40.5 38.6 39.6

60.2 99.1 79.7

44.1 109.1 76.6

34.5 72.7 53.6

44.8 79.9 62.4

4th period

1st period

2nd period

3rd period

4th period

1st period

– – –

1.93 – –

2.70 – –

69.98 98.97 139.96

135.32 – –

0.67 – –

Stem

Inflorescence 2nd 3rd period period – – –

1.34 1.89 –

4th period 27.86 – –

–, Non-significant F-test at 5% probability level. The comparison is based on transformed means in parenthesis because of zero values in some instances.

maximum where they obtained higher tiller number with the three-weekly interval (655 tillers per m2) com-pared with the nine-weeks interval of cuts (521 tillers per m2) in 2001. They also observed that nitrogen fertilizer increased grass tiller number from 558 tillers per m2 with zero N to 2 -1 1315 tillers per m with 450 kg N ha in 2002 season. In the present investigation, the highest tiller

number was obtained in 2002 season, with threeweekly interval (2474 tillers per m2) and the least tiller number with 12-weeks interval of cuts (1086 tillers per m2). Nitrogen fertilizer increased grass 2 tiller number from 1318 tillers per m with zero N 2 -1 to 1874 tillers per m with 450 kg N ha in 2002 season. It would, however, be noted that while Onyeonagu and Asiegbu (2005) worked on degraded pasture, the present study was on sown

and maintained pasture, and this could account for the great differences in tiller numbers obtained. Degraded pastures tend to be unstable and have shorter growing period when compared with improved and well maintained species (Kennett et al., 1992). Wilman and Asiegbu (1982) working with perennial ryegrass (Lolium perenne) also obtained the highest tiller number with threeweekly interval (691 tillers per 0.1 m2) and the

Onyeonagu and Asiegbu

7181

Table 10. Dry matter yields of leaf, stem and inflorescence fractions at various periods of the year for 3- and 6- weekly intervals in 2003. Leaf blade

Cutting frequency (week)

Stem

Nitrogen fertilizer (kgNha-1)

Mean

150

300

450

June 30 to Aug. 11 3 6 Mean

791.1 1011.4 901.3

965.3 1324.2 1144.8

1389.6 1420.2 1404.9

1300.3 1324.1 1312.2

Aug. 11 to Sept. 22 3

937.2

1115.8

1378.4

1482.8

1625.6

Mean

1210.0

Sept. 22 to Nov. 3 3 6 Mean Nov. 3 to Dec. 15 3 6 Mean

Inflorescence

Nitrogen fertilizer (KgNha-1)

Mean

150

300

450

1111.6 1270.0 1190.8

4.3 (1.7) 0.0 (0.7) 2.1 (1.2)

5.4 (1.8) 74.9 (7.3) 40.1 (4.6)

4.8 (1.7) 156.9 (12.2) 80.8 (7.0)

21.9 (3.9) 168.1 (13.0) 95.0 (8.4)

1272.6

1176.0

1.9 (1.3)

10.2 (2.3)

1874.7

1557.3

1635.1

39.3 (5.9)

1370.7

1626.5

1415.0

1405.6

20.6 (3.6)

0.0 (0.7) 118.7 (10.1) 59.4 (5.4)

616.4 782.2 699.3

1194.2 1381.7 1288.0

1098.2 1173.5 1135.9

1174.7 1272.6 1223.7

1020.9 1152.5 1086.7

62.2 53.4 57.8

211.6 234.4 223.0

519.5 425.2 472.4

557.4 408.4 482.9

529.3 360.1 444.7

454.5 357.0 405.7

0.0 (0.7) 0.0 (0.7) 0.0 (0.7)

1st period

2nd period

3rd period

4th period

1st period

2nd period

3rd period

– 244.24 –

225.41 – –

– 270.65 –

75.88 107.31 –

2.60 3.68 5.21

2.76 3.91 5.52

155.31 – –

Mean

150

300

450

9.1 (2.3) 100.0 (8.3) 54.5 (5.3)

0.0 (0.7) 0.0 (0.7) 0.0 (0.7)

0.0 (0.7) 1.3 (1.2) 0.7 (0.9)

0.0 (0.7) 0.3 (0.8) 0.2 (0.8)

34.3 (5.0

11.6 (2.3)

0.0 (0.7)

2.4 (1.4)

7.7 (2.4)

2.5 (1.3)

452.5 (21.0)

468.6 (21.6)

269.7 (14.6)

3.4 (1.6)

2.7 (1.5)

16.8 (3.6)

40.0 (5.9)

15.7 (3.1)

231.3 (11.7)

251.4 (13.3)

140.7 (8.5)

1.7 (1.1)

1.3 (1.1)

9.6 (2.5)

23.9 (4.1)

9.1 (2.2)

78.3 414.3 246.3

127.4 320.2 223.8

168.8 524.7 346.8

109.2 328.2 218.7

21.2 (4.5) 11.2 (3.0) 16.2 (3.8)

27.1 (5.2) 62.0 (7.9) 44.5 (6.5)

53.0 (7.3) 52.8 (7.3) 52.9 (7.3)

77.3 (8.6) 70.7 (8.2) 74.0 (8.4)

44.7 (6.4) 49.2 (6.6) 46.9 (6.5)

0.0 (0.7) 0.0 (0.7) 0.0 (0.7)

1.3 (1.2) 0.0 (0.7) 0.6 (0.9)

0.9 (1.1) 5.6 (1.9) 3.3 (1.5)

0.5 (0.9) 1.4 (1.0) 1.0 (0.9)

0.0 (0.7) 0.0 (0.7) 0.0 (0.7)

0.2 (0.9) 0.0 (0.7) 0.1 (0.8)

0.6 (1.0) 0.0 (0.7) 0.3 (0.8)

0.2 (0.8) 0.0 (0.7) 0.1 (0.8)

4th period

1st period

2nd period

3rd period

4th period

– – –

1.63 2.30 –

– 2.03 –

– – –

Leaf blade

LSD 0.05 for 2 cutting frequency means (C) LSD 0.05 for 2 nitrogen means (N) LSD 0.05 for 2 C × N means

Nitrogen fertilizer (kgNha-1)

Stem

Inflorescence

–, Non-significant F-test at 5% probability level. The comparison is based on transformed means in parenthesis because of zero values in some instances.

least tiller number with eight to 12 weeks interval of cuts (426 tillers per 0.1 m2). They observed that nitrogen fertilizer increased grass tiller number from 492 tillers per 0.1 m2 with zero N to 675 tillers per 0.1 m2 with 224 kg N ha-1. In the present study, the three-weeks interval of cuts produced the highest tiller number when combined with the highest N rate of 450 kg N ha-1 in 2003 compared with six-, nine- or 12-week intervals of cut. In other forage grass genera,

nitrogen application was also effective to increase tillering. Wilman and Pearse (1984) while working on perennial ryegrass (L. perenne), utilized nitrogen rates of 0, 66 and 132 kg ha-1 and observed number of tillers of 844, 988 and 1076 for an area of 0.1 m2, respectively. Harris et al. (1996) reported an increase in tiller density of 4072, 6295 and 6673 tillers m-2 and numbers of tillers per plant of 3.37, 4.10 and 4.26 when -1 nitrogen was applied at 0, 200 and 400 kg ha ,

respectively. Increases in total tiller density (tiller m-2) up to the rate of 480 kg ha-1 year-1 were observed by McKenzie (1998) when nitrogen rates ranging from 120 to 720 kg ha-1 year-1 were utilized. Herling et al. (1991) in a setaria grass evaluation study verified that tillering increases beginning with the absence of nitrogen application up to rates of 80 and 160 kg ha-1; values were 11.49, 14.74 and 15.06 tillers per pot, respectively. The reduction in tiller number with

7182

Afr. J. Biotechnol.

Table 11. Dry matter yields of leaf, stem and inflorescence fractions at various periods of the year for 3- and 6- weekly intervals in 2004. Leaf blade Cutting (week)

frequency

Nitrogen fertilizer (kg N ha-1) 0

150

300

450

June 1 to July 13 3 6 Mean

905.1 1283.9 1094.5

1435.4 2086.7 1761.1

1687.1 2100.1 1893.6

1687.1 1998.5 1969.7

July 13 to Aug. 24 3 6 Mean

836.7 1408.9 1122.8

1482.9 1973.5 1728.2

1746.7 2324.3 2035.5

Aug. 24 to Oct 5 3 6 Mean

836.1 1352.8 1094.5

1099.9 1706.4 1403.2

Oct 5 to Nov. 16 3 6 Mean

855.3 1447.3 1151.3

1130.7 2118.6 1624.7

LSD 0.05 for 2 cutting frequency means (C) LSD 0.05 for 2 nitrogen means (N) LSD 0.05 for 2 C × N means

Stem

Inflorescence

Nitrogen fertilizer (Kg N ha-1)

Mean

150

300

450

1492.1 1867.3 1679.7

7.7 (2.1) 97.1 (9.8) 52.4 (6.0)

0.0 (0.7) 349.8 (18.1) 174.9 (9.4)

62.3 (7.6) 538.8 (22.7) 300.6 (15.1)

117.1 (10.5) 340.1 (18.4) 228.6 (14.4)

1873.3 2115.7 1994.5

1484.9 1955.6 1720.2

3.1 (1.5) 219.8 (12.3) 111.5 (6.9)

0.0 (0.7) 232.3 (13.4) 116.1 (7.1)

3.2 (1.5) 495.8 (20.7) 249.5 (11.1)

1467.3 2212.6 1839.9

1491.1 1685.3 1588.2

1223.6 1739.3 1481.4

41.1 229.8 135.4

42.7 573.5 308.1

1369.2 2144.2 1756.7

1535.5 1530.9 1533.2

1222.7 1516.4 1516.4

42.4 178.7 110.6

Mean

Nitrogen fertilizer (kg N ha-1)

Mean

150

300

450

46.8 (5.2) 331.5 (17.2) 189.1 (11.2)

70.8 (7.6) 461.2 (20.4) 266.0 (14.0)

19.3 (2.8) 352.3 (16.7) 185.8 (9.8)

119.5 757.4 438.5

212.6 696.5 454.6

104.0 564.3 334.1

15.1 59.4 37.2

15.4 121.1 68.2

43.1 206.1 124.6

61.4 197.1 129.3

33.7 145.9 89.8

34.5 420.6 227.5

86.5 530.1 308.3

103.0 424.8 263.9

66.6 388.6 227.6

12.3 28.8 20.5

10.3 124.9 67.6

26.6 91.7 59.1

34.3 91.5 62.9

20.9 84.2 52.5

Stem 2nd period

3rd period

4th period

1st period

Leaf blade 2nd period

3rd period

4th period

1st period

Inflorescence 2nd 3rd period period

225.62

353.97

232.88

350.14

2.44

4.45

141.03

131.81

51.30

35.55

319.08 –

500.59 –

329.34 –

– –

3.45 4.88

– –

199.45 –

186.41 –

NY NY

72.55 –

– –

4th period

–, Non-significant F-test at 5% probability level. The comparison is based on transformed means in parenthesis because of zero values in some instances. NY, No Inflorescence yield.

increasing intervals of cuts up to nine weeks as observed in 2001 season could be partly explained by the development of dense canopy and increased dead materials in the canopy in infrequently cut swards, and this has been reported (Bircham and Hodgson, 1983) to increase the shading of green living tissues and tiller bases. These effects were reported to reduce

tillering and the absorption of light energy to no useful purpose, thereby reducing light-use efficiency (Bircham and Hodgson, 1983). The present investigation revealed that grass tiller number increased with frequent cutting intervals earlier in the 2001 season (June to August) than later (September to November) when compared with the infrequent cutting intervals.

Hebblethwaite and Ivins (1977) reported similar differences in the seasonal production of tillers but however, showed that in severely lodged pastures of infrequently cut swards, secondary growth of vegetative tillers occurred during the last few weeks of the season and consequently increased the total number of tillers in infrequent than in frequently cut swards at the end of the season.

Onyeonagu and Asiegbu

7183

Table 12. Dry matter yields of leaf, stem and inflorescence fractions at various periods of the year for 6- and 12- weekly intervals in 2001 and 2002. Leaf blade Cutting (week)

frequency

Stem

Nitrogen fertilizer (KgNha-1) 0

150

300

450

4084.9 3990.5 4037.7

5113.3 4232.0 4672.6

6189.9 5899.6 6044.7

5401.3 6036.2 5718.8

3898.2

4115.0

4854.1

1985.3

2350.0

Mean

2941.7

Inflorescence

Nitrogen fertilizer (KgNha-1)

Mean

150

300

450

5197.3 5039.6 5118.5

285.1 2561.1 1423.1

518.7 1597.5 1058.1

1079.7 2633.4 1856.5

786.0 4595.7 2690.9

667.4 2846.9 1757.1

4458.2

4331.4

1189.0

1438.8

1536.0

1806.7

1492.6

1960.9

2539.0

2208.8

545.0

612.0

1193.7

1096.0

3232.5

3407.5

3498.6

3270.1

867.0

1025.4

1364.8

1451.3

2199.1

3608.9

3506.7

4270.6

3396.3

67.4 (7.6)

128.0 (10.5)

71.9 (6.9)

319.5 (17.4)

2855.9

3289.8

3409.9

2855.9

3102.9

Mean

2527.5

3449.3

3458.3

3563.2

3249.6

Aug. 12 to Nov.4 6 12 Mean

2235.4 1936.2 2085.8

3197.2 1946.8 2572.0

3435.1 1757.5 2596.3

2944.0 1336.1 2140.0

2952.9 1744.1 2348.5

2001 May 17 to Aug.9 6 12 Mean

Nitrogen fertilizer (KgNha-1) 0

150

300

450

861.7

208.5 (14.2) 17.1 (2.9)

296.5 (15.2) 21.5 (3.8)

232.3 (14.4) 61.8 (5.0)

1177.2

112.8 (8.5)

159.0 (9.5)

147.1 (9.7)

330.1 (16.4) 43.4 (4.3) 186.7 (10.3)

Mean

Aug. 9 to Nov. 1 266.8 (15.0) 35.9 (4.0) 151.4 (9.5)

2002 May 20 to Aug. 12

Leaf blade

LSD 0.05 for 2 cutting frequency means (C) LSD 0.05 for 2 nitrogen means (N) LSD 0.05 for 2 C × N means

1st period – 1457.77 –

2nd period 1085.45 – –

564.9 (23.1) 316.1 (15.4)

512.0 (19.3)

354.7 1581.1 967.9

833.0 995.0 914.0

2001 Stem 1st period 872.86 – –

996.6 (30.3) 534.3 (18.6)

896.0 (28.0)

566.0 (23.7) 442.7 (20.6)

937.3 797.1 867.2

777.7 627.5 702.6

Inflorescence 2nd period 629.79 – –

1st period NY NY NY

2nd period 5.30 – –

146.7 (10.6) 755.9 (26.3) 451.3 (18.4)

3.6 (2.0)

3.4 (1.8)

2.5 (1.4)

9.2 (2.8)

4.7 (2.0)

8.2 (2.1)

26.0 (4.4)

25.9 (5.0)

8.3 (2.6)

17.1 (3.6)

5.9 (2.1)

14.7 (3.1)

14.2 (3.2)

8.8 (2.7)

10.9 (2.8)

725.7 1000.1 862.9

55.1 203.7 129.4

146.3 101.5 123.9

178.5 52.3 115.4

121.0 64.7 92.8

125.2 105.6 115.4

2002 Stem

Leaf blade 1st period – – –

2nd period 673.63 – –

1st period 6.91 – –

Inflorescence 2nd period – – –

1st period – – –

2nd period – – –

–, Non-significant F-test at 5% probability level. The comparison is based on transformed means in parenthesis because of zero values in some instances. NY, No inflorescence yield.

This showed that the nature of growth pattern of the grass is important in determining the effect of inter-vals of cut on the tiller population. The pattern of growth in the grass used for the present

study is tufted. Results from the present study have shown that the highest yields were generally obtained with the longer cutting intervals as found by Omaliko

(1980), Man and Wiktorsson (2003) and Onyeonagu (2005). Possible explanations for the observed responses to defoliation are that the more mature grasses increased leaf area and

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Afr. J. Biotechnol.

Table 13. Dry matter yields of leaf, stem and inflorescence fractions at various periods of the year for 6- and 12- weekly intervals in 2003 and 2004. Leaf blade Cutting (week)

frequency

Stem

Nitrogen fertilizer (kg N ha-1) 0

150

300

450

2003 June 30 to Sept. 22 6 12 Mean

2494.2 2724.0 2609.1

2949.8 1757.6 2353.7

3294.9 2629.6 2962.2

2881.4 2112.6 2497.0

Sept. 22 to Dec. 15 6 12 Mean

1016.6 1935.3 1476.0

1806.9 2266.6 2036.8

1581.9 2176.7 1879.3

2004 June 1 to Aug. 24 6 12 Mean

2692.8 3638.8 3165.8

4060.2 5194.4 4627.3

Aug. 24 to Nov.16 6 12 Mean

2800.0 2981.7 2890.9

3825.0 4032.5 3928.8

Mean

150

300

450

2905.1 2305.9 2605.5

39.3 1082.0 560.6

193.6 551.2 372.4

609.4 1201.1 905.2

636.7 1335.7 986.2

1632.7 1903.2 1767.9

1509.5 2070.5 1790.0

53.4 467.7 260.6

414.3 759.3 586.8

320.2 838.9 579.6

4424.4 5270.1 4847.2

4114.2 3629.0 3871.6

3822.9 4433.1 4128.0

316.9 1741.3 1029.1

582.1 2349.5 1465.8

4356.8 5353.9 4855.3

3216.3 3961.1 3588.7

3549.5 4082.3 3815.9

408.5 2474.2 1441.3

994.1 3167.4 2080.8

2003 Stem

Leaf

LSD 0.05 for 2 cutting frequency means (C) LSD 0.05 for 2 nitrogen means (N) LSD 0.05 for 2 C × N means

Inflorescence

Nitrogen fertilizer (kg N ha-1)

1st period 401.14 – –

2nd period 325.26 – –

1st period 260.30 368.13 –

Mean

150

300

450

369.7 1042.5 706.1

3.4 (1.6) 81.5 (8.9) 42.4 (5.2)

2.7 (1.4) 39.7 (5.3) 21.2 (3.4)

16.8 (3.7) 117.1 (10.6) 67.0 (7.2)

41.4 (5.9) 90.4 (9.5) 65.9 (7.7)

16.1 (3.2) 82.2 (8.6) 49.1 (5.9)

530.4 843.1 686.7

329.6 727.3 528.4

11.2 (3.0) 25.3 (5.1) 18.2 (4.0)

62.0 (7.9) 44.3 (6.6) 53.1 (7.2)

52.8 (7.3) 45.8 (6.8) 49.3 (7.0)

70.7 (8.2) 49.1 (6.8) 59.9 (7.5)

49.1 (6.6) 41.1 (6.3) 45.1 (6.5)

1034.6 2566.1 1800.3

801.4 2558.9 1680.1

683.8 2303.9 1493.8

0.0 (0.7) 35.1 (5.7) 17.6 (3.2)

0.0 (0.7) 50.8 (6.0) 25.4 (3.3)

0.0 (0.7) 120.7 (8.7) 60.3 (4.7)

0.0 (0.7) 63.8 (7.9) 31.9 (4.3)

0.0 (0.7) 67.6 (7.1) 33.8 (3.9)

1287.5 3643.6 2465.6

1121.4 3181.6 2151.5

952.9 3116.7 2034.8

88.1 200.0 144.1

246.0 204.3 225.1

297.8 262.4 280.1

288.6 198.6 243.6

230.1 216.3 223.2

Inflorescence 2nd period 208.98 295.55 –

Nitrogen fertilizer (kg N ha-1)

1st period 2.25 3.19 –

2nd period – 1.87 –

2004 Stem

Leaf 1st period – 995.28 –

2nd period – 787.99 –

1st period 484.24 – –

2nd period 617.94 – –

Inflorescence 1st period 3.09 – –

2nd period – – –

–, Non-significant F-test at 5% probability level. The comparison is based on transformed means in parenthesis because of zero values in some instances.

photosynthesis, thus resulting in higher dry matter production (Man and Wiktorsson 2003). Reducing the interval between cuts reduced herbage production, the effect being greatest in the early part of the season, which concurs with the findings of Chestnutt et al. (1977) that frequent

cutting reduced herbage production to a greater degree in the early part of the season. This effect has been attributed to the high growth rates associated with uninterrupted growth during the reproductive phase of growth in the early part of the season (Binnie et al., 1997).

The reduced yields of longer intervals, relative to the shorter intervals late in the 2001, 2002 and 2003 seasons as observed in the present investigation is of interest. Similar seasonal effects of harvesting intervals have been reported on perennial ryegrass by Chestnutt et al. (1977).

Onyeonagu and Asiegbu

They attributed such seasonal effects of harvesting intervals to the inability of grass in the latter part of the season to produce any appreciable stem elongation leading to a sward which produces a dense leaf canopy with increasing senescence, lodging and a decreased net rate of dry matter accumulation as the growth interval lengthened. The results from the present investigation suggest that adopting a long interval of cut early in the season and a short interval of cut towards the end of the season may increase the annual dry matter yield compared with employing either type of regime all through the year. For example, in the case of the six-weekly cuts compared with the 12-weekly cuts in the 2001 and 2002 harvest years, or the three-weekly cuts compared with the six-weekly cuts in the 2003 harvest year, it was apparent that a combination of long (nine- or 12 -weekly) harvesting interval early in the season with the shorter (three- or six-) intervals towards the end of the season would make for an advantage in increasing the annual dry matter yield of grass herbage compared with using either of the two regimes all through the year. Conclusion The present information showed that cutting management and nitrogen application strongly affect yields of grasses. The results from the study suggest that adopting a longer interval of nine or 12 weeks interval of cut early in the season and a short interval of three or six weeks interval of cut towards the end of the season may increase the annual dry matter yield of pasture swards compared with employing either type of regime all through the year. Tiller production per square meter, grass dry matter production and yields of crop fractions were generally increased with incremental application of fertilizer-N in most of the periods of the years considered. REFERENCES Bamikole MA, Akinsoyinu AO, Ezenwa I, Babayemi OJ, Akinlade J, Adewumi K (2004). Effect of six-weekly harvests on the yield, chemical composition and dry matter degradability of Panicum maximum and Stylosanthes hamata in Nigeria. Grass Forage Sci. 59(4): 357 363. Binnie RC, Kilpatrick DJ, Chestnut DMB (1997). Effect of altering the length of re-growth interval in early, mid and late season on the productivity of grass swards. Journal of Agricultural Science, Cambridge, 128: 303-309. Bircham JS, Hodgson J (1983). The influence of sward condition on rates of herbage growth and senescence in mixed swards under continuous stocking management. Grass Forage Sci. 38: 323-331. Carmer SG, Swanson MR (1971). Detection of Differences Between Means. A Monte Carlo Study of Five Pairwise Multiple Comparison Procedures. Agron. J. 63: 940-945. Chestnutt DMB, Murdock JC, Harrington FJ, Binnie RC (1977). The effect of cutting frequency and applied nitrogen on production and digestibility of perennial ryegrass. J. Br. Grassland Soc. 32: 177-183 Dev I (2001). Problems and prospects of forage production and utilization of Indian Himalaya. ENVIS Bulletin: Himalayan Ecol. Dev. 9(2): 1-13.

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GenStat (1995). Genstat Release 7.22 DE, Discovery Third Edition, Lawes Agricultural Trust Rothamsted Experimental Station, U.K. England. Harris SL Thom ER, Clark DA (1996). Effect of high rates of nitrogen fertilize on perennial ryegrass growth and morphology in grazed dairy pasture in northern New Zealand. N. Zealand J. Agric. Res. 39: 159169. Hebblethwaite PD, Ivins JD (1977). Nitrogen studies in lolium perenne growth for seed. I. level of application. J. Br. Grassland Soc. 32: 199204. Herling VR Zanetti MA, Gomide CA, Linia CG (1991). The influence of potassium and Nitrogen fertilization level and harvest intervals on Setaria grass (Setaria. Anceps cv. Kazungula) I. Dry matter yield and tillering physiology. Revista da Sociedade Brasileira de Zoontecnia (Brazil), 20(6): 561-571. Kennett GA, Lacey JR, Butt CA, Olson-Rutz KM, Haferkamp MR (1992). Effect of defoliation, shading and competition on spotted knapweed and blue bunch wheatgrass. J. Range Manage. 45(1): 363-369. Man NV, Wiktorsson H (2003). Forage yield, nutritive value, feed intake and digestibility of three grass species as affected by harvest frequency. Trop. Grasslands, 37: 101-110. McKenzie FR (1998). Influence of applied nitrogen on Vegetative, reproductive, and aerial tiller densities in Lolium perenne L. during the establishment year. Aust. J. Agri. Res. 49: 707-711. Obi IU (1986). Statistical Methods for Detecting Differences Between Treatment Means. SNAAP Press Limited, Enugu, Nigeria. p. 45. Oloyo RA, Llelaboye NOA (2002). Nutritive quality evaluation of seeds of some lesser-known crops. J. Anim. Prod. Res. 18(1&2): 11-18. Omaliko CPE (1980). Influence of initial cutting date and cutting frequency on yield and quality of star, elephant and guinea grass. Grasslands Forage Sci. 35: 139-145. Omaliko CPE (1983). Stockpiling of three tropical forage grass species. Agron. J. 75(2): 677-679. Onyeonagu CC (2005). Reclamation of a run-down pasture through improved management. M.Sc. Thesis submitted to the Faculty of Agriculture University of Nigeria, Nsukka, p. 65. Onyeonagu CC Asiegbu JE (2005). Effects of cutting management and N- fertilizer application on plant height, tiller production and percentage dry matter in a run-down Panicum maximum pasture. Agro-Science, 4(2): 28-33. Onyeonagu CC (2010). Studies on fertilizer nitrogen and cutting management of sown grass and legume pastures in pure and mixed swards. Ph.D Thesis Faculty of Agriculture University of Nigeria, Nsukka, p. 203. Romero F, Van Horn HH, Prine GM, French EC (1987). Effect of cutting interval upon yield, composition and digestibility of Florida, 77 alfalfa and Florigraze rhizoma Peanut. J. Anim. Sci. 65: 786-796. Venuto BC, Redfearna DD, Pitman WD (1998). Rhizona peanut responses to harvest frequency and nitrogen fertilization on louisiana coastal plain soil. Agron. J. 90: 826-830. Wilman D, Asiegbu JE (1982). The effect of clover variety, cutting interval and nitrogen application on herbage yields, proportions and heights in perennial ryegrass. White clover swards. Grasslands Forage Sci. 37: 1-13. Wilman D, Pearse PJ (1984). Effects of applied nitrogen on grass yield, nitrogen content, fillers and leares in field swards. J. Agric. Sci. 103: 201-211.

Full Length Research Paper

Expression of hepatitis B surface antigen (HBsAg) gene in transgenic cherry tomato Zheng-jun Guan1,2#, Bin Guo1#, Hao-yong Hao3, Yan-lin Huo3, Jia-kun Dai4 and Ya-hui Wei1* 1

Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, Northwest University, Xi’an Shaanxi 710069, People’s Republic of China. 2 Department of Life Sciences, Yuncheng University, Yuncheng, Shanxi 044000, People’s Republic of China. 3 Centre of Biological and Chemical Experiment, Yuncheng University, Yuncheng, Shanxi 044000, People’s Republic of China. 4 Enzyme Engineering Institute of Shaanxi, Academy of Sciences, Xi’an Shaanxi 710600, People’s Republic of China. Accepted 16 March, 2012

The plasmid carrying the gene (adr subtype) encoding the hepatitis B surface antigen (HBsAg) was constructed. Cotyledonary leaves of cherry tomato (Lycopersicon esculentum Mill.) were transformed using Agrobacterium tumefaciens strain LBA4404 harboring pCAMBIA-1301/HB constructs to express HBsAg. The transformed nature of the plants was confirmed by polymerase chain reaction (PCR) analysis and Southern blot hybridization. Expression of HBsAg was confirmed by western blotting and levels of expression were assayed by enzyme-linked immunosorbent assay (ELISA). Southern blot hybridization confirmed the stable integration of the target genes into the genomes of cherry tomato, while western blotting showed high levels of biologically active HBsAg in transgenic plants. ELISA assay showed that HBsAg in transgenic cherry tomato was 100.36 ng/g fresh weight (FW) in leaves and 127.54 ng/g FW in fruits, implying that recombinant HBsAg had natural epitope. This study indicates the feasibility of the expression of foreign antigens in cherry tomato plant for possible use as a raw and edible vaccine. Key words: Lycopersicum esculentum var. cerasiforme, cherry tomato, HBsAg gene, transgenic, expression.

INTRODUCTION Hepatitis B virus (HBV) infection is one of the most widespread viral infections of humans and causes acute and chronic hepatitis and hepatocellular carcinoma (Kane, 1995; Torbenson and Thomas, 2002). The number of chronic HBV carriers all over the world is estimated to be 350 million and the annual HBV morbidity

*Corresponding author. E-mail: weiyahui@nwu.edu.cn guanzj722822@126.com. Tel/Fax: +86-029-88302199.

Abbreviations: HBsAg, Hepatitis B surface antigen; HBV, hepatitis B virus; FW, fresh weight; 6-BA, 6-benzylaminopurine; CaMV35S, cauliflower mosaic virus 35S; ELISA, enzyme-linked immuno sorbent assay; HBsAg S, hepatitis B virus small antigen; IAA, indoleacetic acid; MS, Murashige and Skoog medium; PCR, polymerase chain reaction. #Both authors contributed equally to this work.

rate is more than 1 million (Michel, 2002). Vaccination is the best method to prevent HBV infection. The major envelope protein, hepatitis B surface antigen (HBsAg), which represents the major component of the HBV subunit vaccines, induces a protective immune response against infection. At present, the development of “edible” vaccines based on transgenic plants is one of the most promising directions in novel types of vaccines. HBsAg gene has been mainly expressed in tobacco (Mason et al., 1992; Thanavala et al., 1995), potato (Richter et al., 2001; Liu , 1999), banana (Sunil Kumar et al., 2005b; Elkholy et al., 2009), tomato (Salyaev et al., 2007; Srinivas et al., 2008) and other fruits and vegetables (Marcondes and Hansen 2008; Pniewski et al., 2006). Cherry tomato (Lycopersicon esculentum Mill.), a subspecies of common tomato varieties, is an annual or perennial herb of the Nightshade (Solanaceae), tomato genus (Lycopersicon) and tomato species. It has delicious taste and rich nutrition, and especially can be

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eaten raw as a fruit. Therefore, it is an ideal candidate crop for the production and delivery of oral vaccines. There have been some reports of antigen expression in tomato (L. esculentum). The hybrid gene that encoded the chimerical protein consisting of antigenic determinants of HIV-1 and HBV protective proteins was expressed in transgenic tomato fruit (Shchelkunov et al., 2004). Six plant expression cassettes (pHBS, pHER, pEFEHBS, pEFEHER, pSHER and pEFESHER) were used to assay the possible expression levels of hepatitis B surface antigen in tomato by agroinfiltration (Srinivas et al., 2008). The gene encoding the modified HBV large surface antigen under the control of a fruit-specific promoter was constructed and expressed in transgenic tomato plants (Lou et al., 2007). Hepatitis B virus surface antigen was synthesized in tomato plants transgenic for the preS2-S gene (Salyaev et al., 2007). The subtype adw of HBsAg was studied in the above-mentioned researches. However, Hepatitis B surface antigen (HBsAg/adr) expression in tomato plants, has not previously reported in detail. In this study, we constructed a plant expression vector carrying hepatitis B surface antigen gene (HBsAg/adr) transformed cherry tomato mediated by Agrobacterium tumefaciens, and obtained transgenic plants with an inserted HBsAg gene encoding the small protein. The novelty and priority of the research is that the object of this study was cherry tomato, a sub-variety of common tomato, which can be easily used as a raw and edible vaccine.

MATERIALS AND METHODS Strains and plasmids Escherichia coli DH5α strain and A. tumefaciens strain LBA4404 were employed. The plasmid pBI121/HB were previously constructed and maintained in our laboratory. The plasmid pCAMBIA1301 was kindly presented by CAMBIA Institute of Australian.

Plant materials Seeds of cherry tomato (L. esculentum Mill.) was kindly provided by Deng J. J. (Shannxi, Xi’an, Vegetable Research Institute). After immersion in water for 2 h, the seeds were sterilized with 70% alcohol for 1 min and were washed with sterile distilled water several times. Then they were sterilized with 10% NaClO for 5 min and rinsed several times. Finally, the seeds were placed to germinate on 1/2 Murashige–Skoog (MS) (Murashige and Skoog, 1962) medium without any hormone. Cotyledonary segments from 10-day-old seedlings were excised and used for Agrobacteriummediated transformation.

Construction of plasmids for plant transformation The HBsAg-carrying genetic constructs for plant transformation were obtained by the standard methods of gene engineering (Sambrook, and Russell 2002). In this study, we used the plasmid

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pBI121/HB, which carried the synthetic 681-bp gene encoding the recombinant hepatitis B surface antigen of the adr serotype (HBsAg/adr). The HBsAg gene fragment containing CaMV35S promoter and nitric oxide synthetase (NOS) terminator was cut out from the plasmid pBI121/HB, cloned into pCAMBIA1301 after digestion with both Hind III and EcoR I endonucleases, and transformed into DH5α and screened for recombinant plasmid named pCAMBIA1301/HB.

Plant transformation Agrobacterium strain LBA4404 cells were transformed by the direct method (Horsch et al., 1985) with the plasmids prepared from E. coli. clones after the structure of the plasmid had been verified by restriction digestion. The cherry tomato plants (L. esculentum Mill.) growing under sterile conditions were used. The leaf discs were transformed by co-cultivating with Agrobacterium strains transformed with pCAMBIA1301/HB (Horsch et al., 1985; Draper et al., 1988). Shoots were generated from transformed callus selected on MS medium containing 20 mg/L Hyg and 300 mg/L cefotaxime. Shoots were rooted in medium containing 20 mg/L Hyg and 150 mg/L cefotaxime, and eventually transplanted to soil. The following identification and detection was conducted by using 20 subcultured plants every group after transformation.

DNA isolation and PCR identification Isolation of DNA from cherry tomato leaves was conducted as described by Edwards et al. (1991). Polymerase chain reaction (PCR) conditions were those described by the Taq polymerase manufacturer (Life Technologies, Gaithersburg, MD). PCR conditions of reaction mixtures were as follows: an initial denaturation at 94°C for 5 min; 35 cycles at 94°C for 30 s, 59°C for 40 s, and 72°C for 1 min; followed by a 10-min incubation at 72°C. Primer sequences designed to amplify the HBsAg gene was 5′ AACGGGATCCCGCACC ATGGAGAACACAACATCA 3′ and the reverse primer 5′ CCCGGAATTCCGGCTTAAATGTAT ACCCAAAGAC 3′.

Southern analysis of transgenic plants The digested genomic DNA after gel separation was blotted into Hybond N+ nylon membranes (Amersham Pharamacia Biotech, UK). The 681 bp BamHI fragment from pCAMBIA1301/HB containing the HBsAg S gene was labeled using North2South® Direct HRP Labeling and Detection Kit from PIERCE (Pierce Biotechnology, Inc., Rockford, IL, USA) according to the manufacturer’s instructions, and used for hybridization. The Southern blotting and subsequent hybridization were carried out as described in Sambrook and Russell (2002). Results were detected by the exposure of X-ray film.

ELISA analysis Total protein was extracted from the single leave and fruit of untransformed control and transgenic plants as previously described (Mason et al., 1992). Each group contained 20 plants. The extracts were clarified and assayed in triplicates for the levels of HBsAg expression, and the mean values were taken as an estimate of the amount of HBsAg. An HBsAg enzyme-linked immunosorbent assay (ELISA) kit was purchased from Wantai Biotechnology Co., Ltd. (Beijing, China), and HBsAg standards were provided by Huamei Biotechnology Company. The positive control (human serum derived HBsAg) as a standard and negative

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Figure 1. The construction of plant expressive vector. Plasmid pCAMBIA1301/HB with inserted HBsAg under the control of the cauliflower mosaic virus promoter p35S (CaMV 35S).

control (protein extracted from un-transformed control) were used. Total soluble proteins containing expressed HBsAg were detected by ELISA and quantified with the standard curve established by series dilution of HBsAg standards.

Protein extraction and western blot Total protein of both untransformed and transgenic plants was obtained by centrifugation at 2000×g, and 5-fold concentration. Protein concentration was estimated by the procedure of Bradford (1976). Western blotting was carried out as described in Sambrook et al. (1995). Proteins were separated in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE, 15% acrylamide), and transferred to a Polyvinylidene fluoride (PVDF) membrane (Amersham) using a semi-dry transfer apparatus (Bio-Rad). The membranes were blocked for 1 h at room temperature with 150 mM NaCl, 10 mM Tris/HCl pH 8.0, 0.1% Tween 20 and 1% Triton X100, and 5% (w/v) skimmed milk. Blots were incubated with specific rabbit anti-Fab IgG polyclonal antibodies, prepared as previously described (Ayala et al., 1995), and diluted to 10 ug ml -1 in Tris buffered saline (TBS) containing 1% skimmed milk (1%TBS). After

2 h at 37°C, the membranes were washed and a goat anti-rabbit IgG alkaline phosphate conjugate (Boehringer Mannheim), after diluted 1:1600 in 1%TBS was added for 1 h at 37°C. The enzymatic reaction was developed with nitroblue tetrazolium (0.1 mg ml-1), and 5-bromo-4-chloro-3-indolyl phosphate (0.06 mg ml-1) in 100 mM NaCl, 5 mM MgCl2, and 100 mM Tris/HCl pH 9.5 was used. Apparent molecular weight of proteins was estimated with prestained markers (Rainbow Cold Markers, Amersham).

RESULTS Identification of Agrobacterium transformation with pCAMBIA1301/HB The HBsAg S gene fragment was amplified by PCR from plasmid pBI121/HB containing hepatitis B virus (adr subtype). Plant expression vector pCAMBIA1301/HB (Figure 1) in which HBsAg S gene was driven by the CaMV35S promoter and flanked downstream by the NOS-terminator was constructed. The vector was then

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To further confirm the integration of the HBsAg S gene in the transgenic cherry tomato genome, chromosomal DNA prepared from the PCR-positive plants transformed with pCAMBIA1301/HB were digested with BamHI/SacI followed by Southern blotting analysis. Plasmid pCAMBIA- 1301/HB DNA and genomic DNA of untransformed plant were also digested by HindIII/SacI and used as positive and negative control respectively. Southern hybridization showed the band with expected size in HBsAg S-transgenic lines, which confirmed the stable integration of the HBsAg S fragment into the cherry tomato genome. No hybridization band was detected in untransformed plant (Figure 3).

ELISA analysis

Figure 2. PCR products of pCAMBIA1301/HB. Lane 1, Digestion of pCAMBIA1301/HB with EcoR I + hind III; lane 2, digestion of pCAMBIA1301/HB with SacI + BamHI; lane M, λDNA/EcoRI + HindIII Marker.

mobilized into A. tumefaciens LBA4404 by freeze-thaw method. Kanamycin-resistant Agrobacterium cells were confirmed by PCR and prepared for the transformation of cherry tomato. Plasmids in A. tumefaciens LBA4404 were extracted for double restriction enzyme digestion. Around 700 and 1900 bp bands were found, which were respectively consistent with HBsAg S gene and CaMV35S + HBsAg + NOS. The results show that the pCAMBIA1301/HB was successfully transformed into A. tumefaciens LBA4404 (Figure 2). The cherry tomato leaf-sections transfected by A. tumefaciens LBA4404 were placed on selection medium of MS agar with hormones (2.0 mg/L 6-benzyladenine (6BA) and 0.1 mg/L indole-3-acetic acid (IAA)) and antibiotics (Hyg 20 mg/L and cefotaxime 300 mg/L). Shoots began to develop on callus at the edge of the leaf sections two weeks later. The shoots 1 to 2 cm in height were cut off from the callus and transferred to MS agar medium supplemented with 0.2 mg/L of IAA and 20 mg/L of Hyg for rooting.

PCR and Southern transformed plants

blotting

analysis

the

The transgenic plants transformed with HBsAg were successfully obtained. The presence of the HBsAg S gene in the genomic DNA of the putative transgenic plants was initially examined by PCR. The transformed plants showed the expected HBsAg S gene band of around 700 bp in size. No band was amplified from wildtype plant. Figure 3 presents partial results of the PCR.

ELISA assay was performed on crude protein extracts prepared from leaves and fruits of transgenic cherry tomato plants. Cutoff values were calculated for leaves of untransformed plant, where cutoff = OD450/630 (negative average) × 2.1. A sample was positive when the OD450/ 630 was higher than the cutoff value and was negative when less than the cutoff value. Results show that the samples of transgenic plants was positive for HBsAg S protein (Figure 4), which indicated that there was HBsAg S protein expression only in transformed cherry tomato plants compared to the control pants. ELISA analysis of the protein extracted from fruit and leaf tissues of the transgenic cherry tomato plants demonstrated that fruits and leaves of transgenic plants had much higher OD values than the untransformed plant control. The expression level reached 100.36 ng/g FW in leaves and 127.54 ng/g FW in fruits, respectively. This indicated that hepatitis B surface antigen could be expressed very well in cherry tomato, although these are not the maximal measures in single organs compared to relevant published data.

Western analysis Western analysis was carried out to confirm the HBsAg expression in transformed cherry tomato. A 24 kDa HBsAg specific band was noted in the transformed cherry tomato plants, whereas it was absent in the control untransformed plants (Figure 5). The result is consistent with the results obtained by Ganapathi et al., (2007) and Shekhawat et al., (2007). Development of stable transgenic plants Transformed cherry tomato leaf discs showed the development of shoots on hygromycin selection medium (Figure 6A and B). The shoots were excised and cultured on rooting medium. The transformed shoots developed roots and grew into complete plantlets on the selection

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Figure 3. Result of PCR detection and Southern hybridization of partial transgenic cherry tomato plants transformed with pCAMBIA1301/HB containing HBsAg S fragment. Lane M, Marker; lane 1, PCR production of untransformed plants; lanes 2 and 3, PCR production of transformed plants; lane 4, Southern blotting of DNA of untransformed plants; lanes 5 to 8, Southern blotting of DNA of transformed plants.

Figure 4. HBsAg-ELISA results of partial cherry tomato plants transformed with pCAMBIA1301/HB and un-transgenic plants (OD value). HBsAg S protein extracted from leaves and fruits of untransformed plant and transgenic cherry tomato plants. Cutoff (C.O.) value equals to multiplying the average OD450 value of non-transgenic plants by 2.1; 1. Blank; 2, negative control; 3, positive control; 4, negative control plants; 5, transgenic plant leaves; 6, transgenic plant fruits.

medium (Figure 6C). The 65 sterile transgenic seedlings with well-developed root system were obtained and transferred to the soil after culture of 28 days (Figure 6D and E). The transgenic tissue-cultural plantlets showed no significant differences compared to the control plants, apart from more green leaves. More obvious phenotypic alterations were gradually observed in the transgenic plants after their transplantation to the field, such as fleshy and dark green leaves, distinct notches on leaf

edges, shorter and thicker internodes, more adventitious roots, round fruits, long-shaped or oval-shaped fruits with normal seeds (Figure 6F to H).

Quality of the transgenic cherry tomato fruit We investigated the qualities of the transformed cherry tomato fruit after transplanted to the field (Table 1).

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Compared with the wild cherry tomato fruit, the transgenic fruit had higher mean fruit weight, lycopene content, organic acids content, reducing sugar content and starch content. Although there were some changes of qualities in the transgenic fruit, both of the transgenic fruit and the wild one had no significant difference.

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At the same time, although there was no significant quality difference between the transgenic fruit and the wild one, the transformed cherry tomato fruit had some higher quality parameters. It is speculated that the insertion of exogenous DNA led to the related changes of transcription, translation and metabolism in the plants, thus showing differences in plant morphology. And its mechanisms of variation are now being explored.

DISCUSSION According to the different epitopes, HBsAg can be divided into eight pure subtypes and two mixed subtypes. Every subtype has different geographical distribution. The adr subtype mainly exists in Asia and the Pacific, especially in China. The current vaccine in domestic and international research is mainly adw subtype; therefore the research on the adr subtype of HBsAg in this study is of great practical value. The data in this study proved that a human HBV surface small antigen gene could be expressed in cherry tomato efficiently. Our results add a new example to the successful reports of HBsAg S gene expression in transgenic plants. In recent years, plants have been used as a safe, convenient and economical alternative to the expression system for bioreactor including antibodies, vaccines, biopolymers and so on (Schillberg et al., 2005). Due to its palatable fruit, attractive nutrition and the fact that it can be eaten raw, cherry tomato holds its own advantages in this research area. Transgenic plants expressing recombinant antigens have successfully been in progress since the transformation method was first described by Mason et al. (1992). A deduction of this research is to develop virus vaccines which are produced in edible plants, so that the plant-derived vaccine can be ingested directly without purification or processing. And it appears to be a very promising alternative for expressing recombinant protein. However, a number of questions still remain to be solved. Its main disadvantages are low expression levels of antigen, potential impacts on the environment and human health. Therefore, much work need to be done such as studying the immunogenicity of the recombinant small surface antigen, the stability of foreign genes in future generations, the security of transgenic plants, and so on, some of which are going on. In the process of callus induction and subculture, a variety of mediums of hormone combinations were tried, which caused the transformed tomato calluses seriously browning. Nevertheless, in the MS medium supplemented with 2.0 mg/L 6-BA + 0.2 mg/L IAA, transformed callus regenerated shoots very well. When reaching a certain height, shoots could be transferred to rooting medium after cutting and be rooting. After transplanted to the field, the transformed seedlings grew well and had obvious changes compared to non-transgenic plants. The transgenic cherry tomato plants showed short internodes, corpulent and dark green leaves, more inflorescence and flower buds, and rounded fruits.

ACKNOWLEDGEMENTS This work was financially supported by the National Natural Science Foundation of China (no. 31000144); Opening Foundation of Key Laboratory of Resource Biology and Biotechnology in Western China (Northwest University) (08JZ72); Specialized Foundation of Department of Education of Shaanxi Province, China (09JK746); Northwestern University Graduate Selfinnovation Project (10YZZ36); and the Yuncheng University Doctor Scientific Research Project (YQ-2011042). We are grateful to Deng Jun-jun for providing the seeds of cherry tomato, CAMBIA Institute of Australian for providing plasmid pCAMBIA1301, and all members of our laboratory for numerous valuable discussions and advice about this paper. REFERENCES Ayala M, Fernﾃ｡ndez-de-Cossﾃｭo ME, Canaﾃ｡n-Haden L, Balint RF, Larrick JW, Gavilondo JV (1995). Variable region sequence modulates periplasmic export of a single chain Fv antibody fragment in E. coli. Biotechniques, 18: 832-842. Bradford MM (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein窶電ye binding. Anal. Biochem. 72: 248-254. Draper J, Scott R, Hamil J (1988). Transformation of Dicotyledonous Plant Cells Using the Ti Plasmid of Agrobacterium tumefaciens and the Ri Plasmid of A. rhizogenes. Plant Genetic Transformation and Gene Expression: A Laboratory Manual, Eds., Oxford: Blackwell Sci. pp. 69-160. Edwards K, Johnstone C, Thomson C (1991). A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Res. 19: 1349-1358. Elkholy SF, Ismail RM, Bahieldin A, Sadik AS, Madkour MA (2009). Expression of Hepatitis B surface Antigen (HBsAg) gene in transgenic banana (Musa Sp.). Arab J. Biotech. 12: 291-302. Ganapathi TR, Sunil Kumar GB, Srinivas L, Revathi CJ, Bapat VA (2007). Analysis of the limitations of hepatitis B surface antigen expression in soybean cell suspension cultures. Plant Cell Rep. 26: 1575-1584. Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT (1985). A simple and general method for transferring genes into plants. Science, 227: 1229-1231. Kane M (1995). Epidemiology of Hepatitis B infection in North America. Vaccine, 13 (Suppl 1): S16-S17. Liu DH (1999). Plant as a system for production of pharmaceutical proteins. Biotechnol. Bull. 15: 1-6. Lou XM, Yao QH, Zhang Z, Peng RH, Xiong AS, Wang HK (2007). Expression of the Human Hepatitis B Virus Large Surface Antigen gene in transgenic tomato plants. Clin. Vaccine Immunol. 4: 464-469. Marcondes J, Hansen E (2008). Transgenic lettuce seedlings carrying hepatitis B virus antigen HBsAg. Brazilian J. Infect. Dis. 12: 469-471. Mason HS, Lamd MK, Arntzen CJ (1992). Expression of Hepatitis B surface antigen in transgenic plants. Proc. Natl. Acad. Sci. 89: 11745-

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Shekhawat UKS, Ganapathi TR, Sunil Kumar GB, Srinivas L (2007). Sucrose-inducible expression of hepatitis B surface antigen using potato granule-bound starch synthase promoter. Plant Biotechnol. Rep. 1: 199-2067. Srinivas L, Sunil Kumar GB, Ganapathi RT, Revathi CJ, Bapat VA (2008). Transient and stable expression of hepatitis B surface antigen in tomato (Lycopersicon esculentum L.). Plant Biotechnol. Rep. 2: 16. Sunil Kumar GB, Ganapathi TR, Revathi CJ, Srinivas L, Bapat VA (2005b). Expression of hepatitis B surface antigen in transgenic banana plants. Planta, 222: 484-493. Thanavala Y, Yang YF, Lyons P (1995). Immunogenicity of transgenic plant derived Hepatitis B Surface Antigen. Proc. Natl. Acad. Sci. 92: 3358-3361. Torbenson M, Thomas DL (2002). Occult hepatitis B. Lancet Infect. Dis. 2: 479-486.

Full Length Research Paper

Characterization and evaluation of Bacillus isolates for their potential plant growth and biocontrol activities against tomato bacterial wilt Abdlwareth A. Almoneafy1,2, G. L. Xie1*, W. X. Tian1, L. H. Xu1, G. Q. Zhang1 and Muhammad Ibrahim1 1

State Key Laboratory of Rice Biology, Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Ministry of Agriculture, Institute of Biotechnology, Zhejiang University, Hangzhou 310029, China. 2 Laboratory of Botany and Microbiology, Department of Plant Protection, Faculty of Agriculture, Sana’a University, Sana’a, Yemen. Accepted 3 February, 2012

About 200 Bacillus isolates were isolated from tomato and potato rhizosphere and examined for their antagonistic activities against Ralstonia solanacearum T-91, the causal agent of tomato bacterial wilt (TBW), in vitro and in vivo. Four strains, AM1, D16, D29 and H8, have shown high potential of antagonistic activity against the pathogen in laboratory and greenhouse experiments. In greenhouse, 81.1 to 89.0% reduction of disease incidence of TBW was recorded in treated tomato plants with 4 isolates, which also significantly (p > 0.05) increased plant height by 22.7 to 43.7% and dry weight by 47.93 to 91.55% compared with non-treated control. 16SrRNA gene sequence, the biochemical and physiological tests and fatty acid methyl esters analysis assigned strains AM1 and D29 as Bacillus amyloliquefaciens, while strains D16 and H8 as Bacillus subtilis and B. methylotrophicus, respectively. In addition, the 4 strains showed ability to inhibit growth of the three soil-borne fungi, produce indole-3acetic acid, siderophores and also with exception of strain D16, the other 3 strains were capable of solubilizing phosphate. Therefore, these results suggest that out of 200 isolates, Bacillus stains AM1, D16, D29 and H8 support good antagonistic activity and could be applied as biocontrol agents against TBW under greenhouse conditions beside their potential to promote tomato plants growth. Key words: Tomato, Ralstonia solanacearum, Bacillus spp, biological control, plant growth promotion activities. INTRODUCTION Tomato (Lycopersicon esculentum) is one of the most popular vegetables in the world. It is the second most important vegetable crop next to potato. The world production was about 141,400,629 tons fresh fruit produced on 4,980,424 hectares in 2009 (http://faostat.fao.org/). Tomato crop has suffered from several biotic and abiotic stresses during its growing season. Among those stresses, bacterial wilt of tomato caused by Ralstonia solanacearum (Yabuuchi et al., 1995) is one of the most devastating and wide-spread

*Corresponding author. E-mail: glxie@zju.edu.cn. Tel/Fax: +86 0571 88982710.

diseases of crops worldwide (Poussier et al., 1999). R. solanacearum infects more than 200 species in 50 families (Hayward, 1991), including tomato, potato, eggplant, pepper, tobacco, banana, chilli and peanut (French and Sequeira, 1970). This aforementioned reason therefore makes this soil-borne pathogen difficult to control. Many methods like soil solarization, field sanitation, crop rotation and use of bactericides have been applied to control the disease, but with only limited success (Ciampi-Panno et al., 1989). Resistance cultivars have been used as an important component of integrated disease management. However, it is generally agreed that breeding for resistance is not completely effective, producing only limited gains and often lacking stability or durability (Boucher et al., 1992; Hayward,

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1991). Furthermore, the high variability of strains of R. solanacearum combined with the influence of environmental factors on host–pathogen interactions often restricts the expression of resistance to specific regions (Hayward, 1991). Many studies report that biological control is a promising alternative strategy to be used in the integrated control of various bacterial plant diseases (Weller, 1988). Biological control strategies may either help development of alternative management measures or be integrated with other practices for effective disease management at the field level. Plant growth-promoting rhizobacteria (PGPR), a group of root associated bacteria, intimately interact with the plant roots and consequently influence plant health and soil fertility. They offer an excellent combination of traits useful in disease control and plant growth promotion. This group can produce bioactive substances to promote plant growth and/or protect them against pathogens (Harish et al., 2009). Some PGPR may influence plant growth by synthesizing plant hormones or facilitating uptake of nutrients from the soil through different direct mechanisms such as atmospheric nitrogen (N) fixation, solubilization of phosphate, and synthesis of siderophores for iron sequestration, thus making nutrients more available to plants (Glick et al., 2007). Among PGPR cluster, Bacillus is one of the most potential genera due to their spore forming ability, thereby increasing the adaptation of Bacillus strains to commercial formulation and field application (Liu and Sinclair, 1993). Some species of Bacillus suppress plant pathogens and insect pests by producing antibiotic metabolites, while others stimulate plant host defenses prior to pathogen infection (van Loon, 2007), which indirectly contributes to increase crop production. Published reports on endophytic colonization and biofilm formation by Bacillus and Paenibacillus spp. have suggested that the endophytic colonization and biofilm formation improve the bacterium’s ability to act as a biocontrol agent against plant pathogens (Davey and O'toole, 2000; Hallmann et al., 1997; Timmusk et al., 2005). So applying cultivated plants and soil with these microorganisms will be important in preventing the environmental pollution that results from pesticides and chemical fertilizers. Hence, this study aimed to isolate and characterize Bacillus spp. from cultivated soil by solanaceous vegetables, screen them against R. solanacearum under laboratory conditions, evaluate their plant growth promotion activities in vitro and finally screen their efficacy against R. solanacearum under greenhouse conditions.

sample was collected from Hangzhou, China. Each sample was placed in plastic bag during sample collection in the field and 100 g were taken from each sample for isolation and maintained in suitable plastic cans at 4°C until use. Serial dilution method was applied to isolate rhizobacteria from soil, where 5 g of soil sample were added to 50 ml of sterile distilled water then shaken in rotary shaker at 160 rpm until homogenization of soil suspension. The suspensions were plated in Luria-Bertani agar (LBA) medium incubated at 28°C for 48 h. The appeared colonies of each representative isolate were picked and streaked in new LBA plates and incubated at 28°C for 48 h. Typical single colony for every acquired isolates was sub-cultured in nutrient agar slant for further study. For long term preservation, obtained isolates were stored in Eppendorf tubes containing LB with 20% glycerol at -20ºC.

MATERIALS AND METHODS

Evaluation of antagonistic isolates in greenhouse

Isolation of potential antagonistic isolates

To determine whether the potential selected antagonistic isolates can suppress tomato bacterial wilt (TBW) and enhance plant growth under greenhouse conditions or not, this experiment was carried out in greenhouse of the Agriculture and Biotechnology College during the period 25th May to 5th August, 2010. Ten selected

A total of 200 isolates have been isolated from ten rhizosphere soil samples of healthy tomato and potatoes collected from three locations in Yemen (Amran, Sana’a and Thamar areas), and one

Plant pathogen and culture conditions R. solanacearum strain T-91 belonging to race 1 biovar 3, which is a standard virulent strain on tomato (Li et al., 2010), was provided by the Institute of Biotechnology, Zhejiang University, China. It was grown on yeast extract peptone glucose agar (YPGA) medium (Xue et al., 2009). Preliminary screening for antagonistic activity in vitro This test was performed to screen antagonistic activity of obtained isolates from soil against R. solanacearum according to the method described by Li et al. (2008b). Cultures of R. solanacearum was grown overnight in nutrient broth; 0.5 ml of the liquid culture was mixed with 15 ml lukewarm molted nutrient agar in sterile plates and allowed to solidify. Afterwards, each tested isolate was spotted on the surface of agar plates and incubated them at 28°C for 72 h. Consequently, those bacteria that displayed positive inhibition activity were considered as antagonistic isolates and selected for further investigations. Laboratory in vitro assay for studying antagonism Antagonistic activity of selected isolates from preliminary test was evaluated against 3 plant pathogenic fungi (Fusarium graminearum, Pythium aphanidermatum and Rhizoctonia solani) by applying two techniques; dual culture method described by Ganesan and Gnanamanickam (1987) and double layer agar method given by Pawar and Puranik (2008). To check the antagonism ability against R. solanacearum, YPGA medium was seeded by adding 10 ml of Ralstonia suspension (overnight liquid culture of Ralstonia centrifuged and then the cell pellets were re-suspend with sterilized saline solution (0.85% NaCl) to final concentration~108 CFU/ml) and 5 ml of 2,3,5-triphenyl tetrazolium chloride (TZC) to 1 L of melted YPGA and pouring into sterilized plates. Thereafter, each test antagonist was transferred to surface of agar using sterile toothpicks and incubated at 28°C. Plates were checked after 72 h and clear haloes without pinkish coloration around the tested isolates indicating inhibition of Ralstonia growth were measured (Adesina et al., 2007). All experiments were done under completely randomized design (CRD) with three replicates.

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isolates which offer inhibition diameter (15 to 20 mm) during in vitro test were used for this investigation. Seeds of Tomato Cv. “Hezuo” (susceptible to infection by R. solanacearum) were surfacesterilized by immersing in 2% sodium hypochlorite solution for 2 min, and then washed thoroughly three times by sterilized water. Pots of 18 cm diameter filled by sterilized potting mixture (1 soil: 1 peat moss), were used in study. Cultivated pots were maintained under greenhouse conditions with range of temperature of 25 to 30°C and relative humidity between 70 to 90%. Two control treatments were considered; control one (C1) was treated only with pathogen and control two (C2) was not treated either with pathogen or tested isolates. Pots were arranged in completely randomized block design with four replicates for each treatment and 16 plants for each replicates.

the effect of tested antagonist on plant growth was measured in term of plant height. Thereafter at the end of experiment, above ground parts of plants were cut into small pieces and shoot fresh weight was recorded, then those parts were dried at 60ºC for three days and dry weight of shoot pieces was recorded. Moreover growth promotion efficacy was calculated to clarify the relative effect of tested antagonist on plant biomass compared with C1 or C2 treatments according to following formula:

GT-GC GPE (%) =

× 100 GC

Where, GPE refers to growth promotion efficacy, GT refers to growth parameter in antagonist-treated group and GC refers to growth parameter in control group.

Application by antagonistic strains Tomato plants were applied with tested antagonist twice; the first was before cultivation, when seeds were treated by tested isolates through overnight soaking in bacterial suspension adjusted to 108 CFU/ml and seeds that were immersed with sterilized saline served as control. Then bacterized seeds were transferred to dry sterilized filter paper and allowed to dry in laminar flow for 8 h. Before planting the microbiolized seeds into the pots, they were pregerminated on moistened filter paper with sterilized distilled water for 5 days. Each pot was sowed with the 4 pre-germinated seeds. The second application with tested antagonist was carried out before one week of inoculation with pathogen by soil drenching. About 50 ml of antagonist suspension (1.0 × 108 CFU ml-1) were poured in each pot and pots with 50 ml of saline served as control. Inoculation of R. solanacearum Ralstonia was cultured in yeast extract peptone glucose (YPG) broth medium and incubated at 30°C in rotary shaker with 160 rpm for 18 h and centrifuged at 10000 rpm for 10 min and afterward cell pellets were harvested and diluted to obtain the final concentration of 1.0 × 109 CFUml-1. Thereafter, tomato plants in all treatments except C2 were inoculated with the pathogen at the third to fourth leaf stage by punching each plant with sterilized needle at the base of stem above upper secondary root, subsequently 80 ml of the suspension were poured in every pot over wounded area. Pots in C2 treatment were treated by pouring 80 ml of sterilized saline solution in each of them. After inoculation, all pots were covered by polyethylene bags for 24 h to maintain high humidity (Algam et al., 2010). Disease assessment Tomato plants were monitored for development of wilt symptoms. Disease index data were recorded according to the scale ranged from 0 to 4 (Park et al., 2007). Based on disease index collected data, two parameters; disease incidence and biocontrol efficacy of antagonistic isolates were estimated as follows (Xue et al., 2009): Disease index × number of diseased plants in this index Disease incidence = × 100% Total number of plants investigated × the highest disease index Disease incidence of control - Disease incidence of antagonist-treated group Biocontrol efficacy =

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× 100% Disease incidence of control

Detection of plant growth promotion activities for antagonistic isolates Indole acetic acid production Selected isolates were investigated for their ability to produce indole acetic acid (IAA). Each isolate was grown in LB media supplemented with (40 µg/ml) L-tryptophan and incubated in shaker with 30°C and 160 rpm for 48 h. Next, bacterial culture was centrifuged at 10000 rpm for 15 min, and 1 ml of culture filtrate was mixed with 1 ml of Salkowski’s reagent (1.5 ml of FeCl3.6H2 O 0.5 M solution, in 80 ml of 60% H2 SO4) and the mixture incubated at room temperature for 30 min; presence of pink color indicates that isolate can produce indole acetic acid (IAA). Meanwhile, IAA concentration for each tested strain was quantified colorimetrically in 550 nm by spectrophotometer comparing with IAA standard curve (Gordon and Weber, 1951). Phosphate solubilization Capacity of selected isolates to solubilize phosphate in form of calcium phosphate was checked qualitatively by using glucose yeast extract agar (GYA) medium containing per 1 L distilled water; 10 g glucose, 2 g yeast extract and 15 g agar. In addition, two other solutions were prepared separately; first 5 g K2HPO4 was dissolved in 50 ml distilled water and second 10 g CaCl2 in 100 ml distilled water. These two solutions were added to 1 L GYA just before pouring medium to plates (Beneduzi et al., 2008b). Each tested isolate was grown in GY broth for 24 h, and then 10 µL of bacterial culture were dropped in each plate and incubated for 7 days at 28ºC. Isolates which showed clear halos around their colonies were considered as phosphate solubilizers.

Siderophores production Evaluation for Siderophores production of selected isolates was carried in LBA medium supplemented with chrome azurol S (CAS) complex (Schwyn and Neilands, 1987). Each isolate was grown in LB medium for 24 h, then one drop of culture was spotted in CAS plates and incubated in 28°C for 3 days; presence of orange halos around the colonies indicated that bacterial isolate were able to produce siderophores.

Plant growth parameters assessment

Statistical analysis

After one month from second application by antagonistic isolates,

Obtained data were subjected to analysis of variance (ANOVA) test

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Figure 1. The inhibition effect expressed by inhibition area diameter (mm) of ten antagonistic bacterial strains on R. solanacearum growth on tetrazolium chloride (TZC) medium. Columns with the same letters are not significantly different based on LSD test (p < 0.05), Error bars in each column represent the standard error within same treatment.

by using SAS software (SAS Institute, Cary, NC). General linear model (GLM) procedure was used to check the significant differences among main treatments. Individual comparisons between mean values were performed by using the least significant differences (LSD) test (P = 0.05). Correlation analysis was performed by CORR procedure.

according to the recommendations of the commercial identification system, Microbial identification System (MIDI). FAMEs profiles were compared with the MIDI identification database TSBA50, version 5.00 (MIDI Inc., Newark, DE, USA). Biochemical and physiological characterizations

Identification of selected bacterial strains Sequence analysis for 16S rRNA gene Total DNA from each bacterial strain was extracted using TIANamp Bacteria DNA Kit (Tiangen Biotech (Beijing) co., Ltd.). Thereafter, the 16S rRNA gene was amplified using universal primers; 16sP0 (5´-GAA GAG TTT GAT CCT GGC TCA G - 3´) and 16sP6 (5´CTA CGG CTA CCT TGT TTAC GA - 3´). The amplified products were purified using DNA gel extraction kit. Purified products were submitted to Shanghai Sangon, China, for sequencing. Obtained sequenced data of 16S rRNA were compared with attainable sequences in GENBANK by using BLAST sequence search to determine the phylogenetic affiliation; consequently identification of bacterial isolates was performed according to the similarity of 16SrRNA genes. Furthermore, a phylogenetic tree was created based on partially sequence of 16S rRNA and closely related sequences using MEGA5 (version 5.03) (Kumar et al., 2004). Sequences of four isolates were submitted to GenBank to get accession number for isolates H8, D16, AM1 and D29 which were assigned as JN411089, JN411090, JN411091 and JN411092, respectively.

Fatty acid methyl esters (FAMEs) analysis Respective isolates were grown in TSB agar plates at 28°C for 24 h, and then each isolate was subjected to saponification, methylation and purification processes of FAMEs. Purified esters were analyzed

The four selected isolates were characterized based on their biochemical and physiological reactions according to Bergey's Manual of Systematic Bacteriology (Vos et al., 2009) to confirm the results of 16S rRNA and FAME analysis. Four isolates were checked for gram reaction, motility, spore formation, colony color, cell shape, anaerobic growth, growth in different pH values, growth in different NaCl concentrations, hydrolysis of starch and gelatin, catalase reaction, nitrate reduction, citrate utilization and utilization of some carbon sources including sucrose, L-rhamnose, arginine, glycerol, lactic acid, inositol, D-rhafinos and D-Sorbitol.

RESULTS In vitro screening of bacterial antagonistic activity Among 200 rhizobacteria isolates screened during preliminary test, ten isolates AM1, AM2, AM5, AM50, AM47, D11, D12, D16, D29 and H8 presented inhibition activity against R. solanacearum. Therefore, these 10 bacterial isolates were considered as bacteria with antagonistic characteristics. Among them, four isolates Am1, D16, D29 and H8 gave the highest means of inhibition diameter of 9.33, 8.33, 9.33 and 8.66 mm, respectively, while the other isolates offer a wide range of antagonistic activity from 5 to 7 mm in laboratory in vitro test (Figure 1).

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Table 1. Reduction of tomato bacterial wilt incidence and biocontrol efficacy of ten antagonistic bacterial isolates against Ralstonia solanacearum.

Treatment AM1+ R.s AM2 + R.s AM47 + R.s AM5 + R.s AM50 + R.s D11 + R.s D12 + R.s D16 + R.s D29 + R.s H8 + R.s R. s.

Disease incidence (%) 9.40 ± 7.65c 81.11 ± 16.59a a 83.37 ± 14.80 a 88.43 ± 10.05 a 82.09 ± 18.81 79.86 ± 17.49a 52.26 ± 12.36b 16.48 ± 7.03b 14.12 ± 10.12b b 12.96 ± 3.21 a 78.89 ± 19.66

Biocontrol efficacy (%) 88.98 ± 9.69a 9.66 ± 19.79c c 8.10 ± 7.02 c 1.39 ± 14.63 c 9.83 ± 12.38 12.15 ± 10.67c 41.21 ± 18.47b 81.14 ± 9.77a 83.68 ± 11.88a a 85.35 ± 4.98 -

Data are presented as mean value ± standard error of four replicates, and each replicate contains four plants. Values with same letters within each column indicate no significant difference according LSD test at p < 0.05. R.s = Ralstonia solanacearum.

Greenhouse study Antagonistic bacterial strains showed significant differences for their abilities to suppress TBW. Isolate AM1 showed the lowest value of disease incidence as well as the highest value of biocontrol efficacy against R. solanacearum of 9.40 and 88.98%, respectively. While isolate Am5 exhibited the highest disease incidence and lowest value of biocontrol efficacy with 88.43% and 1.39% respectively. Isolates D16, D29 and H8 sustained significantly lower disease incidence and biocontrol efficacy compared with control. In contrast, the rest isolates failed to suppress bacterial wilt in greenhouse despite their offering antagonistic activity against Ralstonia in the in vitro assay (Table 1). Moreover, noteworthy significant differences among treatments regarding plant height and biomass were noted. Plants treated with isolates D16 and D29 presented the highest values of plant fresh weight by 34.38 and 35.34 g, respectively, with high significant differences, compared with pathogen infected control and plants treated by isolates AM2, AM5, AM50, AM47 and D11. For dry weight, isolates D16, D29 and H8 significantly increase the plant dry weight (g) compared with other treatments with 5.71, 5.42 and 5.48 g respectively. Likewise high GPE (%) increase was recorded with plants treated with isolates D16, D29 and H8 by 56.43, 60.78 and 40.01% and 91.55, 81.77 and 84% for fresh and dry weight, respectively. However, treatments by other isolates did not show high increasing of biomass compared with control except treatment with isolate AM1 which was 47.93% higher than control (Table 2). In addition, in terms of plant height, significant differences were also noted with treatments of D16 and D29 which significantly increased the plant height with 61.33 and 61.28 cm, respectively with GPE (%)

increasing by 43.67 and 43.57%, respectively compared with control (Table 2). According to their high antagonistic activity against R. solanacearum (in vitro and in vivo assays) and their high performance regarding to enhance plant growth. The four isolates (AM1, D16, D29 and H8) were selected for further investigations. Plant growth promotion traits and inhibition activity against fungi Plant growth promotion traits for selected strains and their activity against three plant pathogenic fungi presented in Table 3 showed that all four strains gave significance levels of IAA ranging from 44.25 µg/ml by strain AM1 to 59.56 µg/ml for strain H8, with significant difference between four strains. The four strains have ability to produce siderophores as evidenced formation of orange halo around the colony, although isolate H8 showed bigger halo than others. For phosphate solubilization, all the strains were able to solubilize phosphate by giving a clear halo around bacterial colony in GYA medium except isolate D16, where its colony did not surround by any clear area in GYA medium. All strains presented inhibition activity against all of three fungi F. graminearum, P. aphanidermatum and R. solani in dual culture test. However, isolate D29 displayed the highest inhibition percentage against F. graminearum which was significantly higher than other isolates, while strains D16 and D29 gave also high inhibition percentage 95.19 and 87% against P. aphanidermatum in double layer test. Moreover, all four strains totally suppressed the growth of R. solani in double layer test. Bacterial strains identification The results of fatty acid analysis for isolates Am1, D29,

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Table 2. Effect of application of antagonistic bacterial isolates and Ralstonia solanacearum on tomato plants fresh, dry weight and plant height.

Treatment AM1+ R.s AM2 + R.s AM47+ R.s AM5 + R.s AM50 + R.s D11 + R.s D12 + R.s D16 + R.s D29 + R.s H8 + R.s R. s. Saline solution

Plant fresh weight (g) Mean GPE (%) bcd 26.24 ± 3.13 19.40 f 13.80 ± 6.14 ef 16.18 ± 5.28 f 10.50 ± 0.95 ef 17.14 ± 6.72 cde 23.47 ± 2.82 6.76 abcd 27.33 ± 1.51 24.33 a 34.38 ± 5.38 56.43 35.34 ± 6.91a 60.78 abc 30.77 ± 3.25 40.01 21.98 ± 6.15de ab 31.83 ± 1.87 -

Plant dry weight (g) Mean GPE (%) b 4.41 ± 0.37 47.93 d 3.02 ± 0.61 1.42 cd 3.32 ± 0.25 11.30 d 2.42 ± 0.48 cd 3.31 ± 0.30 11.11 3.09 ± 0.51cd 3.84 bc 4.03 ± 0.34 35.35 a 5.71 ± 0.86 91.55 a 5.42 ± 0.77 81.77 a 5.48 ± 0.69 84.00 2.98 ± 0.86d ab 4.99 ± 0.22 -

Plant height (cm) Mean GPE (%) bcd 52.36 ±2.88 22.65 ef 46.53 ± 1.79 8.99 bcd 53.14 ± 2.17 24.48 de 48.28 ± 2.11 13.09 f 46.28 ± 4.02 8.40 53.47 ± 5.80bc 25.26 dce 50.19 ± 2.27 17.58 a 61.33 ± 0.55 43.67 61.28 ± 1.63a 43.54 ab 56.44 ± 1.45 32.22 42.69 ± 5.01f cde 48.89 ± 2.27 -

Data are presented as a mean value ± standard error of four replicates, and each replicate contains four plants. Values with same letters within each column indicate no significant difference according LSD test (p < 0.05). R.s = Raistonia solanacearum.

D16 and H8, showed that low similarity percentage 0.27, 0.28 and 0.28 with Bacillus lentus for isolates Am1, D29 and D16, respectively and 0.32 similarity percentage with Paenibacillus lentimorbus for isolate H8. Although these results showed closest matches to the above mentioned bacillus species, their low similarity values were non considerable and inadequate to identify bacterial strains to species level. However, acquired FAMEs profiles could be suggestible to identify those strains within Bacillus genus. The obtained sequences (1200 to 1500 bp) of the 4 isolates (AM1, D16, D29 and H8) revealed 100, 99, 100 and 100% homology with 16S rRNA sequence of Bacillus amyloliquefaciens (accession number JN582030), B. subtilis (accession number EF472266), B. amyloliquefaciens (accession number JF460733) and B. methylotrophicus (accession number HQ662588) in GenBank, respectively. For biochemical and physiological characteristic tests, data in Table 4 revealed that the 4 selected isolates are gram positive, rod shaped, motile, aerobic growth and able to form spores. All strains gave positive reaction for catalase test and starch hydrolysis. Moreover, they were positive for utilization of lactic acid and D-sorbitol. The results of biochemical and physiological tests were therefore guidable to discriminate four isolates from each other. DISCUSSION Bacterial species which inhabit in rhizosphere area have an impressive effect to protect the plant roots from soilborne pathogens as well as to improve plant growth (Glick et al., 2007; Harish et al., 2009; Singh et al., 2011). In this study, rhizobacterial isolates belonging to genus

Bacillus were isolated from tomato and potato fields with the objective of obtaining the efficient strains representing high disease control performance against TBW, and also to offer several plant growth promoting activities contributing positively to improve health and growth of plant. In vitro laboratory test, all of 10 isolates represent remarkable antagonistic activity against R. solanacearum. The four isolates (B amyloliquefaciens AM1, B. subtilis D16, B amyloliquefaciens D29 and B. methylotrophicus H8) represented the highest inhibition effect against pathogen. These results are similar with previous studies reporting the antagonistic activity of B. subtilis (Leifert et al., 1995; Lemessa and Zeller, 2007; Pinchuk et al., 2002) and B. amyloliquefaciens (Li et al., 2008a; Yoshida et al., 2001) against plant pathogenic bacteria and fungi. In this study, YPGA medium was considered for in vitro screening assay because this medium was reported as suitable for R. solanacearum growth, especially when YPGA was amended with TZC which makes the medium more selective to R. solanacearum (Xue et al., 2009). Moreover, tested antagonistic isolates demonstrated an abundant growth in this medium and this is in agreement with other studies which emphasize that the type of culture medium strongly affects antagonistic activity by mediating the production of substances responsible for inhibition (Montesinos et al., 1996). Meanwhile, Nguyen and Ranamukhaarachchi (2010) reported that the best antagonistic activity is obtained with culture medium containing 2.5% sucrose and 2% peptone (w/v) with 28°C and initial pH value of 7. The mechanism of antagonistic effect in the plate may be due to antibiosis or production of siderophores or both of them (Adesina et al., 2007; Lemessa and Zeller, 2007). Despite a large proportion of potential biocontrol agent,

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Table 3. IAA and siderophores production and phosphor solubilization ability of the four bacilli selected strains.

Strain AM1 D16 D29 H8

IAA productiona (µg/ml)

Siderophore productionb

Phosphor c solubilization

44.25 ± 3.52c b 53.21 ± 2.30 b 51.54 ± 1.72 59.56 ± 4.60a

+ + + +

+ + +

Fusarium graminearum d a Dual culture Double layer b + 68.22 b + 70.89 a + 83.11 + 66.82b

Inhibition activity against fungi (%) Pythium aphanidermatum a Dual culture Double layer b + 55.00 a + 95.19 a + 87.04 + 63.52b

Rhizoctonia solani Dual culture Double layer + 100 + 100 + 100 + 100

( ): values followed by same letter means no significant difference based on LSD test (p < 0.05); ( ): +: able to produce Siderophore; ( ): + : able to solubilize Phosphor, - : not able to solubilize; d ( ): + : able to inhibit fungal growth.

strains present considerable inhibition activity against targeted pathogen in vitro, and a few of them kept this ability while applying them in greenhouse or in field (Lugtenberg and Kamilova, 2009). Several soil-borne plant pathogens co-exist in the same field, while potential biocontrol agents was selected based on their ability to control a certain targeted pathogen (Xue et al., 2009). Therefore, selected isolates from in vitro and in vivo test were further checked for their ability to suppress the growth of some soil-borne plant pathogenic fungi to increase their chance by offering sufficient protection to the host plant against wide spectrum of soil-borne pathogens. In greenhouse study, application of tomato plants by strain B. amyloliquefaciens AM1, B. subtilis D16, B. amyloliquefaciens D29 and B. methylotrophicus H8 gave the best suppression of disease incidence of TBW and this suppression was significantly higher than other studied isolates. Furthermore, plants treated with the four strains supported high values of plant height and biomass (fresh and dry weight) compared with the control treatment. The in vivo inhibitory activity of B. amyloliquefaciens against R. solanacearum in this study is in line with that of Hu et al. (2010) who reported that antibacterial activity of suspension of B. amyloliquefaciens against Capsicum bacterial

wilt caused by R. solanacearum in greenhouse and field. Furthermore, Jetiyanon (2007) has found that a mixture of B. amyloliquefaciens strain IN937a and B. pumilus strain IN937b induced the production of defense related enzymes against R. solanacearum and other plant pathogens. Regarding B. methylotrophicus H8, this strain maintains stable antagonistic activity against R. solanacearum in the in vitro and in planta tests, and according to Madhaiyan et al. (2010), this species is a novel species in genus of Bacillus. So far our study is the first report that emphasizes the antagonistic effect of B. methylotrophicus (isolate H8) against TBW. Four Bacillus strains, AM1, D16, D29 and H8 have demonstrated a high ability to produce IAA, with their production ranging from 44.25 µg/ml of AM1 to 59.56 µg/ml of H8. In general, poor correlation (0.423, 0.473) (p < 0.05) was observed between the amount of IAA with plant height and fresh weight. However, relatively strong correlation value (0.801) was noted between IAA amount and dry weight of plants; this could explain to some extent why the plant growth parameters in case of isolate AM1 had low values compared to the other three isolates, inspite of its high antagonistic activity in vitro and in vivo. Likewise, Idris et al. (2007) reported that inactivation of gene

responsible for IAA biosynthesis in B. amyloliquefaciens FZB42 cause reduction in IAA concentration and consequently resulting in low plant growth promotion activity. Regarding phosphate solubilization, all the 4 strains except D16 showed capability to solubilize inorganic phosphate. Bacillus species have been documented for their capacity to increase availability of phosphorus in soil; for example B. amyloliquefaciens can increase the availability of phosphorus from insoluble myoinositol hexaphosphate or phytate by producing an extracellular phytase which can catalyze sequential hydrolysis of phytate to less-phosphorylated myoinositol derivatives and inorganic phosphate (Jorquera et al., 2008). In addition, obtained results showed that the four strains were able to produce siderophores in vitro. There are many reports indicating that bacilli can produce siderophores (Beneduzi et al., 2008a; Yadav et al., 2011), although a mechanism of biocontrol activity of Bacillus spp. mostly refers to their ability to produce a wide spectrum of antimicrobial compounds as well as elicitation of induced systemic resistance of plants (Haas and Defago, 2005). However, Yu et al. (2011) found that B. subtilis CAS15 which can produce siderophores was able to suppress Fusarium wilt

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Table 4. Differential biochemical characteristic of four Bacilli isolates AM1, D29, D16 and H8 with three other related Bacilli species.

Biochemical characteristic Pigmentation Shape Spore formation Anaerobic growth Motility Gram reaction pH 4 pH 5 pH 6 Growth in 1%NaCl Growth in 4%NaCl Growth in 8%NaCl Nitrate reduction Tri-sodium citrate Catalase Gelatin hydrolysis Starch hydrolysis L-Rhamnose Arginine Inositol Glycerol Lactic acid Sucrose D-Sorbitol D-rhafinos

1 Opaque Rod + + + + + + + + + + + + + + +

2 Opaque Rod + + + + + + + + + + + + + + + +

3 Opaque Rod + + + + + + + + + + + + + + + -

4 Creamy white Rod + + + + + + + + + + + + + + + + + +

5 Opaque Rod + + + + + + + + + + + + + + + + + + -

6 Opaque Rod + + + + + + + + + + + + + + + +

7 Creamy white Rod + + + + + + + + + + + + + + + + +

1: D29; 2:AM1, 3:D16 and 4:H8, 5: Bacillus subtilis, 6: B. amyloliquefaciens, 7: B. methylotrophicus , +: positive reaction or growth, -: negative reaction or no growth.

incidence in greenhouse, and this suppression was negatively affected after iron supplementation. Furthermore, ability of tested four isolate to inhibit the growth of the three soil born fungi means these isolates have several mechanisms to suppress plant pathogenic microorganisms, and have a high chance to keep their activity for protection of host plants in greenhouse and field. Finally, we can conclude that the four Bacilli strains AM1, D29, D16 and H8 have proved to be potential biocontrol agents against TBW, in addition to their capability to improve tomato growth under greenhouse conditions. Further studies are, however, needed to investigate the mode of action of these strains in terms of inducing systemic resistance and enhancing their antibiosis activity against plant pathogens, as well as confirming the antagonistic ability of these strains in field trials at different locations. ACKNOWLEDGMENT This project was supported by the Special Fund for Agroscientific Research in t he Public Interest (201003029,

201003066), National Natural Science Foundation of China (30871655) and Zhejiang Provincial Agricultural Departments of China (2008C22072, SN200811). REFERENCES Adesina MF, Lembke A, Costa R, Speksnijder A, andSmalla K (2007). Screening of Bacterial Isolates from Various European Soils for in Vitro Antagonistic Activity Towards Rhizoctonia Solani and Fusarium Oxysporum: Site-Dependent Composition and Diversity Revealed. Soil Biol. Biochem. 39: 2818-2828. Algam SAE, Xie G, LB, Yu S, Su T, Larsen J (2010). Effects of Paenibacillus Strains and Chitosan on Plant Growth Promotion and Control of Ralstonia Wilt in Tomato. Eur. J. Plant Pathol. 92(3): 593600. Beneduzi A, Peres D, da Costa PB, Zanettini MHB, Passaglia LMP, (2008a). Genetic and Phenotypic Diversity of Plant-GrowthPromoting Bacilli Isolated from Wheat Fields in Southern Brazil. Res. Microbiol. 159(4): 244-250. Beneduzi A, Peres D, Vargas LK, Bodanese-Zanettini MH, Passaglia LMP (2008b). Evaluation of Genetic Diversity and Plant Growth Promoting Activities of Nitrogen-Fixing Bacilli Isolated from Rice Fields in South Brazil. Appl. Soil Ecol. 39(3): 311-320. Boucher CA, Gough CL, Arlat M (1992). Molecular Genetics of Pathogenicity Determinants of Pseudomonas Solanacearum with Special Emphasis on Hrp Genes. Annu. Rev. Phytopathol. 30(1): 443-461.

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Full Length Research Paper

Study of the system of tuberous root induction in vitro from Rehmannia glutinosa Tao Xue, Lan Guo, Jian-ping Xue*, Yun-xian Song, He-dong Lu, Ai-min Zhang and Wei Sheng Anhui Key Laboratory of Plant Resources and Biology, School of Life Sciences, Huaibei Normal University, Huaibei 235000 Anhui, People’s Republic of China. Accepted 19 March, 2012

This study investigated the induction system of tuberous root in vitro from Rehmannia glutinosa. The roles of plant growth substance, carbohydrates, and minerals were evaluated for induction and development of tuberous root in vitro. The results show that Murashige and Skoog (MS) contributed greatly to induction of tuberous root in vitro, followed by α-naphthalene acetic acid (NAA), sucrose and 6-benzyladenine (BA). The optimal medium was ¼ MS supplemented with 1.5 mgL-1 BA, 0.15 mgL-1 NAA and 5% sucrose. In addition, paclobutrazol (PP333) and methyl jasmonate (MeJA) also played an important role in induction of tuberous root in vitro, the most appropriate concentrations were 1 mgL-1 and 10 µmolL-1 respectively. Key words: Rehmannia glutinosa, tuberous root, tissue culture in vitro. INTRODUCTION Rehmannia glutinosa, commonly known as ‘‘di-huang’’ in Chinese, is a perennial herb endemic to China, Japan and Korea. The tuberous root of R. glutinosa (the medicinal part of the herb) is very commonly used in Traditional Chinese Medicine in fresh, dried or steamed forms in terms of the processing methods. Traditionally, R. glutinosa has been used as tonics for replenishing Yin deficiency. Over the past two decades, considerable progresses have been made on the pharmacology of R. glutinosa. It has been found that R. glutinosa and its active components have wide pharmacological actions on the blood circulation, immune capability, endocrine balance, cardiovascular regulation and the health of nervous system (Zhang et al., 2008). However, R. glutinosa was susceptible to virus as its roots were constantly used for propagation, thus the yield and quality of R. glutinosa are decreasing (Matsumoto et al., 1989; Wen et al., 2001). To solve the problem, there were attempts for in vitro approaches; Xu and Davey (1983) reported shoot regeneration from mesophyll protoplasts and leaf explants of R. glutinosa. Later, different culture conditions for R. glutinosa plantlets were

*Corresponding author. E-mail: xuejp2000@yahoo.com.cn. Tel: +86-561-3802025.

investigated (Cui et al., 2000). Subsequently, Chen et al. (2004) reported callus induction and plant regeneration from various organs such as leaf, stem-segment, and petiole. Also, cultivation of virus-free seedlings could be a way for solving this problem (Shao et al., 2008), yet, it is difficult to apply it in agriculture production due to its unsatisfactory coefficient of propagation, long cycle of breeding and inconvenient transportation. In view of this situation, in vitro abnormal organs were successfully induced from potato (Sarkar et al., 2006) and yam (Olivier et al., 2011), which could substitute virus-free seedling in production. Induction of tuberous root in vitro from R. glutinosa was also conducted (Xue et al., 2002), but the system of induction was imperfect. So far, the efficiency of induction on tuberous root in vitro could not achieve the desired yields. As the average number of tuberous root obtained in vitro was small, the formation time was long, and the physiological mechanisms that controlled the morphogenesis of tuberous root were unclear. Therefore, it is necessary to search for an ideal system for high frequency tuberous root induction from R. glutinosa, which would also be a direct and convenient system for studying the mechanism that drives the formation of tuberous root.

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Table 1. The design form of the orthogonal test L9 (34).

Level

Factor -1

-1

A (MS) 1/4

B (BA, mgL ) 1.5

C (NAA, mgL ) 0.05

D (Sucrose, gL ) 40

1/2

2.0

0.1

2.5

0.15

MATERIALS AND METHODS

RESULTS

Plant materials and culture conditions

Effects of MS, sucrose, BA and NAA on induction of tuberous root in vitro from R. glutinosa

Explants were collected from the tuberous roots of R. glutinosa grown at the experimental fields of Wenxian County, Henan Province, People’s Republic of China (R. glutinosa was identified by Prof. Jianping Xue. A voucher specimen of the plant was deposited at the School of Life Sciences, Huaibei Normal University). They were cultured in sand at 25°C in light incubator. One week later, shoots formed in the tuberous roots were cut with one or two leaves, which were first washed thoroughly with tap water for 2 h. The shoots were immersed in 70% (v/v) ethyl alcohol and surface sterilized with 0.1% (w/v) mercuric chloride solution (HgCl2) for 6 min, and then rinsed six times with sterile deionized water. The explants were sectioned from the sterilized shoots and incubated in 150 ml Erlenmeyer flasks at 2 to 3 explants per container each with 40 ml Murashige and Skoog (MS) solid medium (Murashige and Skoog, 1962), and supplemented with 1 mgL-1 BA and 0.1 mgL-1 NAA. The pH of the medium was adjusted to 5.8 before adding 7% (w/v) agar and autoclaved at 121°C for 15 min. Explants were incubated under 25 to 26°C and illuminated at 30 to 40 µmol/m-2s-1 with a photoperiod regime of 12 h light and 12 h dark cycle. Tuberous root induction from R. glutinosa One month later, seedlings cultured in the initial medium were cut into 2 to 3 cm long stems with one or two leaves and transferred to ½ MS medium supplemented with 1 mgL-1 indolebutyric acid (IBA) and 3% sucrose (Xue et al., 2002). Six days later, adventitious roots were formed in the seedlings, which were transferred to media with different treatments of orthogonal design when the length of adventitious roots was between 0.5 and 1 cm (Table 1). Effects of paclobutrazol (PP333) and methyl jasmonate (MeJA) on the induction of tuberous root On the basis of the suitable medium for induction of tuberous root in vitro which was selected by orthogonal design, the effects of paclobutrazol (PP333) and methyl jasmonate (MeJA) on the induction of tuberous root in vitro were studied respectively. With the same method, stems with adventitious roots were transferred to the media supplemented with different concentrations of PP333 (0, 1, 2, 3 mgL-1) or MeJA (0, 0.1, 1, 10, 50 µmolL-1). Statistical analysis The experimental design was completely randomized with three replications per treatment, each treatment consisting of a total of 30 explants. Average weight of tuberous roots in vitro per seedling was recorded which were cultured for 20 days and the data were analyzed by using Minitab 15.

The K value (average of certain factor and level) can be used to determine the global optimal condition for induction of tuberous root in vitro. Experimental results showed that the K value by magnitude was K1>K2>K3 (Table 2), which illustrated that ¼ MS played a good role in promoting root enlargement. With the increase of the concentration of major element in MS medium, the average weight of tuberous root per seedling decreased accordingly. On the other hand, as the concentration of NAA in this range (0.05 to 0.15 mgL-1) increased, the average weight of tuberous root per seedling was enhanced; the average weight of tuberous roots in vitro formed in the medium supplemented with 0.15 mgL-1 NAA was 1.16 times as heavy as that in the medium supplemented with 0.05 mgL-1 NAA. The R value (range of K) can show the effect of a certain factor on the tuberous root induction. Based on the R values, the effect orders of the four factors were not the same: A>C>D>B. The results of analysis of variance of average quality of tuberous roots per seedling among the four selected factors are presented in Table 3. Obviously, MS, NAA and sucrose all played significant roles in the formation of tuberous root in vitro due to Pvalues, which were all less than 0.05 but it was not significantly influenced by BA. Comparing the K value of sucrose, K2 was higher than the others, which showed that 5% sucrose was beneficial to the formation of tuberous root in vitro. Also, it indicated that sucrose promoted induction of tuberous root in vitro and development when its concentration increased from 4 to 5%, but an opposite effect appeared with the concentration of sucrose increasing between 5 and 6%. With visual analysis according to Table 2, optimal medium for tuberous root induction from R. glutinosa could be obtained, which was A1B1C3D2: ¼ MS supplemented with 1.5 mgL-1 BA, 0.15 mgL-1 NAA and 5% sucrose .This combination was not included in Table 2. 20 bottles of the medium from A1B1C3D2 were prepared for verification experiment. The average weight of tuberous roots per seedling was 0.1594 g and was slightly higher than those from nine combinations of the orthogonal

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Table 2. Results of tuberous roots in vitro induced in different media of orthogonal design.

Factors and their codes

Number 1 2 3 4 5 6 7 8 9 K1 K2 K3 R

A (MS) 1/4 1/4 1/4 1/2 1/2 1/2 1 1 1 0.362 0.332 0.289 0.073

-1

B (BA, mgL ) 1.5 2.0 2.5 1.5 2.0 2.5 1.5 2.0 2.5 0.333 0.325 0.324 0.009

-1

C (NAA, mgL ) 0.05 0.1 0.15 0.1 0.15 0.05 0.15 0.05 0.1 0.303 0.328 0.351 0.048

-1

D (Sucrose, gL ) 40 50 60 60 40 50 50 60 40 0.302 0.357 0.323 0.055

Average fresh quality of tuberous roots per seedling (g) Replication 1 Replication 2 Replication 3 0.106 0.098 0.113 0.132 0.141 0.117 0.129 0.117 0.132 0.119 0.102 0.112 0.126 0.083 0.119 0.109 0.107 0.118 0.137 0.109 0.102 0.081 0.095 0.082 0.092 0.081 0.087

Table 3. Result of one-way analysis of variance.

Sources of variance A) MS B) BA C) NAA D) Sucrose Error

Sum of squares 0.0025407 0.0000507 0.0013287 0.0025513 0.0014105

df 2 2 2 2 18

F value 8.96 0.18 4.69 4.90

P value 0.002** 0.838 0.023* 0.020*

*Significantly different at P<0.05 among A, B, C and D; **significantly different at P<0.01 among A, B, C and D.

experiment, Figure 1a.

golden tuberous roots with new buds were formed in this medium about two months later though plantlets almost died (Figure 1d, e).

Effects of PP333 on the tuberous roots formation Plantlets were transferred to the ¼ MS medium supplemented with 1.5 mgL-1 BA, 0.15 mgL-1 NAA, 5% sucrose and different concentrations of PP333 (0 to 3 mgL 1 ) when the length of adventitious roots was between 0.5 and 1 cm. Three days later, adventitious roots of the plantlet transferred to the medium without PP333 began to enlarge. Yet, the amount of time the adventitious roots became inflated in the medium supplemented with 1 to 3 mgL-1 PP333 was put off 7 days. With the concentration of -1 PP333 increasing from 1 to 3 mgL , the lagged effect on tuberous roots induction in vitro was visible. At 25 days, the tuberous roots induced in the medium without PP333 formed a small quantity of callus (Figure 1b) while that in the medium supplemented with PP333 continued expanding without callus. The medium supplemented with 1 mgL-1 PP333 was suitable for the formation of tuberous roots in vitro (Figure 1c); moreover, some

Effects of MeJA on the tuberous roots formation Plantlets were subcultured onto the ¼ MS medium -1 -1 supplemented with 1.5 mgL BA, 0.15 mgL NAA, 5% sucrose and different concentrations of MeJA (0 to 50 µmolL-1) when the length of adventitious roots was about 0.5 to 1 cm. Adventitious roots began to swell after 5 days in the media supplemented with MeJA with 0 to 10 µmolL-1. Significantly, more tuberous roots were observed -1 in the medium containing 10 µmolL MeJA after 10 days and no tuberous roots developed in the medium -1 containing 50 µmol.L MeJA. When the concentration of MeJA increased to 10 µmolL-1, the average weight of tuberous roots was 5.5 times as heavy as that of the control group (Figure 1f, g). However, while the concentration of MeJA changed from 10 to 50 µmolL-1, a sharp decline trend could be observed according to Figure 2. Obviously, high concentrations of MeJA could

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Figure 1. Development of tuberous roots of R. glutinosa in vitro in different media. A) tuberous roots of R. glutinosa formed in the screening medium: ¼ MS supplemented with 1.5 mgL-1 BA, 0.15 mgL-1 NAA and 5% sucrose; b) callus formed on the tuberous roots in the screening medium with further culture; c) tuberous roots formed in the screening medium supplemented with 1 mgL-1 PP333; d and e) tuberous roots developed in the screening medium supplemented with 1 mgL-1 PP333 for two months; f and g) tuberous roots formed in the screening medium supplemented with 10 µmolL-1 MeJA.

inhibit tuberous root induction and development. Thus, among the five concentrations, medium supplemented with 10 µmolL-1 MeJA was the best one for tuberous root induction in vitro. DISCUSSION The metamorphosis of plant organs is a genetic trait, which largely depends on environment, plant growth substances, and nutritional conditions (Jose and Satheeshkumar, 2010). Usually, with cytokinin and auxin synergy, cells could divide rapidly and organ could be induced and developed. It was reported that no microtubers in vitro of Dioscorea nipponica could be induced in MS medium supplemented with either BA or NAA (Chen et al., 2007). In this study, the results show that NAA played a significant role in promoting tuberous roots formation in vitro and adventitious roots in the medium supplemented with BA but not NAA only elongated without swelling. It was reported that the pH

value of cell wall matrix was reduced by auxin, so as to activate some enzyme (Hager et al., 1991), and the cell wall could be relaxed under the action of the enzyme, which would lead to cell expansion for organ development. As one of the auxins, NAA could also stimulate cell divisions. With increasing number and volume of cell, tuberous root generated naturally. Major element concentration in MS medium plays an important role in the growth of plant in vitro and the synthesis and accumulation of metabolites, which could change ionic strength and osmotic pressure of the medium. In this trail, results show that adventitious roots in the ¼ MS medium swelled rapidly and the quality of tuberous roots formed in the medium was higher than those in the other media and there is a general agreement with the reports of Huang et al. (2010). However, it was the first time to study the effect of major element of MS on the induction of tuberous root in vitro and the concrete role of major element needs a further research. In a similar manner to the phytohormones, sucrose has

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Average weight of tuberous roots per plantlet (g)

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1 0. 8 0. 6 0. 4 0. 2 0 0

0.1

-1

Concentration of MeJA (µmolL ) Figure 2. Effects of MeJA on the tuberous roots induction in vitro.

been shown to play an important role in the in vitro tuberous root formation of R. glutinosa. As a carbon source, sucrose could not only provide carbon for synthesis of new cell compounds, but also played a decisive role in regulating the water absorption of plantlets in tissue culture (Du et al., 2009). Indeed, higher sucrose concentration (8 to 12%) in growth media was shown to promote bulb induction (Zel et al., 1997) or potato tuberisation (Prat, 2010) and formation of tubers was delayed on media containing lower sucrose concentration in contrast to media with 3% sucrose as already observed by Lawrence and Barker (1963) on potato. Results of this experiment show that tuberous roots in medium containing 50 g/L sucrose developed well however, tuberous roots could not develop well in the medium either with low or high concentration of sucrose. Perhaps, low concentration of sucrose could not provide enough osmotic pressure for tuberous roots induction, and when the concentration of sucrose increased, it was difficult for plantlet to absorb water for the reduction of medium water potential, which was not conductive for photosynthesis. Therefore, the development of tuberous roots was negatively correlated with a lower or higher concentration of sucrose. The previous studies suggested that gibberellic acid (GA3) played a negative role in the formation of potato tubers in vitro (Hussey and Stacey, 1984). In this experiment, different concentrations of PP333 were added to the medium, which could inhibit the generation of endogenous GA3, and the results showed that low concentrations of PP333 would be helpful in the formation of tuberous roots but high concentrations of PP333 would play a negative role in the development of tuberous roots. Our results were in partial agreement with indications given by Sheng et al. (1991), who noted that appropriate concentration of PP333 could promote tubers of potato formation and enlargement. Also, Zhang et al. (2004)

pointed out that low concentration of PP333 promoted earlier potato formation, and increased microtuber fresh weight and mean diameter. It was reported that PP333 could increase chlorophyll content which could enhance leaf photosynthetic rate and inhibit the vegetative growth (Sheng et al., 1991). Meanwhile, PP333 inhibited plant producing endogenous GA3. This could be the reason why appropriate concentration of PP333 would be facilitative for tuberous roots development. However, high concentration of PP333 would inhibit vegetative growth severely, which might inhibit tuberous roots development indirectly. The trend was the same with PP333. It could obviously be observed that low concentrations of MeJA promoted the development of tuberous roots while high concentrations of MeJA caused inhibition instead. It has been reported that appropriate concentrations of MeJA might increase the accumulation of storage material and then promote the development of tuberous root (Du et al., 2009). However, high concentrations of MeJA might promote premature aging of leaves (Chou and Kao, 1992; Chen et al., 2004), which would affect absorption of nutrients of plantlets and inhibit the enlargement of tuberous roots indirectly. Yet, the mechanism of MeJA on tuberous roots induction and development is still unclear and further studies in this direction would provide further insight into this aspect. To sum up, the appropriate medium for tuberous root induction from R. glutinosa was ¼ MS supplemented with -1 -1 -1 1.5 mgL BA, 0.15 mgL NAA, 5% sucrose and 1 mgL -1 PP333 or 10 µmolL MeJA. It was reported that GA and abscisic acid (ABA) was directly related to tubers development (Okazawa, 1960; Xu et al, 1998), and the influences of many other plant growth regulators on tubers mainly through an endogenous regulation of GA: ABA balance. We guess that the effects of those factors on tuberous root induction might depend on their roles in

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regulating endogenous hormones, which might be related to regulation between vegetative and reproductive growth. Conclusion In conclusion, we studied induction of tuberous roots in vitro from R. glutinosa systematically and established an effective protocol for tuberous roots induction. It would provide materials for artificial seeds, which might be a new method for propagation of R. glutinosa. Meanwhile, it could be a new model for further studies on R. glutinosa. ACKNOWLEDGEMENTS This study was sponsored by the National Natural Science Foundation of China (no. 30973963), the Natural Science Foundation of Anhui Province (no. 090413252) and the Key Project of Natural Science of Anhui Provincial University (no. KJ2009A160). REFERENCES Chen CT, Su YS, Kao CH (2004). Changes in soluble sugar content and respiration rate in methyl jasmonate treated rice leaves. Botanical Bullet. Acad. Sin. 45(3): 197-202. Chen FQ, Fu Y, Wang DL, Gao X, Wang L (2007). The effect of plant growth regulators and sucrose on the micropropagation and microtuberization of Dioscorea nipponica Makino. J. Plant Growth Regul. 26: 38-45. Chen MY, Liang SZ, Wang ZZ, Yan JT (2004). Tissue culture and plantlet regeneration of Rehmannia glutinosa. Acta Botanica BorealiOccidentalia Sin. 24(6): 1083-1087. Chou CM, Kao CH (1992). Methyl jasmonate, calcium, and leaf senescence in rice. Plant Physiol. 99(4): 1693-1694. Cui YY, Hahn EJ, Kozai T, Paek KY (2000). Number of air exchanges, sucrose concentration, photosynthetic photon flux, and differences in photoperiod and dark period temperatures affect growth of Rehmannia glutinosa plantlets in vitro. Plant Cell Tissue Organ Cult. 62: 219-226. Du HM, Tang DM, Huang DF (2009). Effects of Methyl Jasmonate on in vitro tuberization of Taro. J. Shanghai Jiaotong Univ. Agric. Sci. 27(5): 481-484. Hager A, Debus G, Edel HG, Stransky H, Serrano R (1991). Auxin induces exocytosis and the rapid synthesis of a high-turnover pool of + plasma-membrane H -ATPase. Planta, 185: 527-537. Huang T, Gao W, Wang J, Cao Y (2010). Effects of culture conditions on biomass and active components of adventitious roots culture in Panax ginseng. J. Chin. Mater. Med. 35(1):13-17. Hussey G, Stacey NJ (1984). Factors affecting the formation of in vitro tubers of potato (Solanum tuberosum L.). Ann. Bot. 53(4): 565-578. Jose B, Satheeshkumar K (2010). Tuberous roots an ideal system for high frequency in vitro regeneration in Plumbago rosea L. Plant Tissue Cult. Biotech. 20(2): 203-209. Lawrence CH, Barker WG (1963). A study of tuberisation in the potato (Solanum tuberosum). Am. Pot. J. 40: 349-356.

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Matsumoto M, Shoyama Y, Nishioka I, Iwai H, Wakimoto S (1989). Identification of viruses infected in Rehmannia glutinosa Libosch. Var purpurea Makino and effect of virus infection on root yield and iridoid glycoside contents. Plant Cell Rep. 7: 636-638. Murashige T, Skoog F (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473-479. Okazawa Y (1960). Studies on the relation between the tuber formation of potato plant and its natural gibberellin content. Proc. Crop Sci. Soc. Jpn. 29: 121-124. Olivier KA, Konan KN, Anike FN, Agbo GN, Dodo HW (2011). In vitro induction of minitubers in yam (Dioscorea cayenensis-D. rotundata complex). Plant Cell Tissue Organ Cult. doi: 10.1007/s11240-0110084-7. Prat S (2010). Hormonal and daylength control of potato tuberisation. Plant Hormones, pp. 574-596. Sarkar D, Pandey SK, Sharma S (2006). Cytokinins antagonize the jasmonates action on the regulation of potato (Solanum tuberosum) tuber formation in vitro. Plant Cell Tissue Organ Cult. 87: 285-295. Shao CY, Gao SL, Chen F, Zhang XX, Ren B (2008). Virus-free culture and rapid propagation of Rehmannia glutinosa Libosch. Pharmaceut. Biotechnol. 15(4): 258-261. Sheng ZM, Shen YQ, Sheng YH, Liu ZL, Cao HF, Tong YW, Shen YG, Gu HQ (1991). Stimulation effect of met on growth of potato tubers. Acta Agric. Shanghai, 7(1): 69-73. Wen XS, Li XE, Yang SL (2001). Viral diseases of Rehmannia glutinosa and problems demanding prompt solution. Chinese, Tradit. Herbal Drugs, 32(7): 662-665. Xu X, Lammeren AAM, Vermeer E, Vreugdenhil D (1998). The role of gibberellin, abscisic acid, and sucrose in the regulation of potato tuber formation in vitro. Plant Physiol. 117: 575-584. Xu XH, Davey MR (1983). Shoot regeneration from mesophyll protoplasts and leaf explants of Rehmannia glutinosa. Plant Cell Rep. 2: 55-57. Xue JP, Shi LY, Zhang AM (2002). Microtuber induction in vitro from Rehmannia glutinosa. J. Chin. Mater. Med. 27(11): 824-82. Zel J, Debeljak N, Ucman R, Ravnikar M (1997). The effect of jasmonic acid, sucrose and darkness on garlic (Allium sativum L.cv. Ptujski jesenski) bulb formation in vitro. vitro Cell Dev. Biol Plant. 33: 231235. Zhang RX, Li MX, Jia ZP (2008). Rehmannia glutinosa: review of botany, chemistry and pharmacology. J Ethnopharmacol. 117: 199214. Zhang ZJ, Li HZ, Yao HL, Zhou WJ (2004). Effect of paclobutrazol on explant growth and tuberization in potato. J. Zhejiang Univ. Agric. Life Sci. 30(3): 318-322.

Full Length Research Paper

Effects of the nitrogen and phosphorus fertilization on the yield and quality of the hairy vetch (Vicia villosa Roth.) and barley (Hordeum vulgare L.) mixture Emine Budakli Carpici and Mukerrem Melis Tunali Department of Field Crops, Faculty of Agriculture, Uludag University, Gorukle Campus, 16059 Bursa, Turkey. Accepted 19 March, 2012

This study was intended to determine the effects of nitrogen (0, 30, 60 and 90 kg N ha-1) and phosphorus rates (0, 30, 60, 90 and 120 kg P2O5 ha-1) on dry matter yield, hairy vetch ratio, crude protein yield, acid detergent fiber (ADF), neutral detergent fiber (NDF) and total digestible nutrient (TDN) of hairy vetch-barley mixture (50:50) in Bursa-Turkey in 2009 to 2010 and 2010 to 2011 growing years. Dry matter and crude protein yields were affected by nitrogen rates, and the highest dry matter and crude protein yields were determined at 30 kg N ha-1. Phosphorus rates significantly affected most of the components determined in this study. The highest values of dry matter and crude protein yields, hairy vetch ratio and TDN and the lowest value of ADF content were obtained at 30 kg P2O5 ha-1. 30 kg N ha-1 -1 and 30 kg P2O5 ha may be recommended to be applied on hairy vetch-barley mixture in the similar experimental ecologies in order to produce economically high and quality forage product. Key words: Hairy vetch, barley, mixture, nitrogen, phosphorus, dry matter yield, crude protein yield. INTRODUCTION Hairy vetch is an annual legume cultivated under rainfed conditions in the semi-arid regions of Mediterranean countries, integrated into the conventional cereal-fallow crop rotation system (Turk et al., 2009). Legume-grass mixtures increases not only forage yield, but also provide nursing and physical support for the companion legume (Soya, 1994). Thus, the potential benefits of legumecereal mixtures over their monocultures might be due to their higher yield, protein and forage quality, yield stability and reduced incidence of pests, weeds, and diseases (Carr et al., 1998). Intercropping of vetch with barley grown for forage and grain may improve forage quality and yield. Many researches have showed that vetch-barley mixture exhibited greater production than respective sole vetch or barley (Bugdaycıgil et al., 1996; Yılmaz et al.,

1996; Dhima et al., 2007; Lithourgidis et al., 2007; Bayram et al., 2009; Ansar et al., 2010). Bingol et al. (2007) found that all of the mixtures of vetch with barley had significantly higher digestible dry matter and crude protein yield. Intercropping barley with common vetch improved forage quality and increased protein yield of barley without reducing dry matter yield (Thompson et al., 1992). On the other hand, there is no enough study evaluating the effects of nitrogen and phosphorus fertilization on dry matter yield and forage quality of vetch-barley mixture. However, this matter requires investigation scientifically and the presentation of the findings to the services of growers. The objective of this study was to determine the effects of different rates of nitrogen and phosphorus fertilization on the dry matter yield, hairy vetch ratio, crude protein yield, acid detergent fiber (ADF), neutral detergent fiber (NDF) and total digestible nutrient (TDN) of hairy vetchbarley mixture.

*Corresponding author. E-mail: ebudakli@uludag.edu.tr. Abbreviations: ADF, Acid detergent fiber; NDF, neutral detergent fiber; TDN, total digestible nutrient.

MATERIALS AND METHODS Field trials were conducted during 2009 to 2010 and 2010 to 2011

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Table 1. Monthly precipitation and temperature in 2009 to 2010, 2010 to 2011 and in long years (1975-2008) in Bursa.

Month November December January February March April May Total Mean

Monthly precipitation (mm) Long years 2009-2010 2010-2011 85.4 80.6 24.0 96.4 119.1 152.6 80.3 149.7 72.4 66.2 178.9 18.4 62.7 115.3 67.4 65.2 63.4 76.8 43.4 29.4 27.3 499.6 736.4 438.9 -

growing seasons on clay loam soil at the Agricultural Research and Experiment Center of Uludag University, Bursa (40° 11′ N, 29° 04′ E). Soil test values indicated a pH of 7, with none saline, low values in lime and organic matter and rich in potassium. Precipitation distribution and amount differed markedly between experimental years (Table 1). Total precipitation in 2009 to 2010 growing period was 236.8 mm over long years. These higher precipitation occurred in December, January, February and March. Precipitation in the second year was 60.7 mm less than that of the long years mean (Table 1). There were almost no differences between mean temperatures of the experimental and long years. Hairy vetch cv. Selcuklu 2002 and barley cv. Akhisar-98 were used as plant entries. Ammonium nitrate (34%) was used as nitrogen source and four levels (0, 30, 60 and 90 kg ha-1) were applied. The second factor of the experiment was phosphorus and was applied as triple super phosphate (42 to 44% P2O5) with 0, 30, 60, 90 and 120 kg ha-1 rates. Fertilizers were incorporated into the soil by hand broadcasting. Fertilizer applications were done at sowing. Fertilizer treatments were arranged randomly in complete block experiment design with three replications. Plot size was 1.2 × 4 m = 4.8 m2 and in each plot, six rows were formed. Row spacing among rows was 20 cm. In the mixtures, seeds of species were blended before sowing and then sown in the same rows. Seeding rates for the pure stands were 80 and 200 kg ha-1 for hairy vetch and barley, respectively. The ratio between hairy vetch and barley was 50:50 in the mixture and the seed rate of each species in the mixture was determined by multiplying pure seed rate and proportion of each species. The ratio 50:50 between hairy vetch and barley, and harvesting time at 50% flowering stage used in this study was determined in a previous research (Bayram et al., 2009). Sowing was done by hand on November in 2009 and 2010, respectively. Furthermore, stands were harvested when hairy vetch reached 50% flowering stage. After harvest, fresh forage samples from plots were randomly taken and put into cotton bags. They were ovendried at 78°C for 48 h and weighed, then dry weight percentages were calculated. Dry matter yield of each plot was calculated by multiplying the fresh weight of each plot with its dry weight percentages. The contribution of hairy vetch to the dry matter yield in a mixture of each fertilizer treatment plot was determined as percentage. Also, 1 g ground sample was used for the total nitrogen determination by Kjedahl method, while 0.5 g was used for ADF and NDF. ADF and NDF were analyzed by sequential detergent analysis method (Goering and Van Soest, 1970). Crude protein yield was calculated by multiplying dry matter yield of each plot with its crude protein content and converted to yield per hectare. The components studied in this research were dry matter yield, hairy vetch ratio, crude protein yield, ADF, NDF and TDN. Variance analysis were conducted over two-year data of components by

Monthly Temperature (°C) Long years 2009-2010 2010-2011 10.3 10.1 15.5 7.1 9.5 9.8 5.4 7.0 5.6 5.9 9.3 6.0 8.5 9.0 8.3 13.0 13.4 10.5 17.7 19.2 16.7 9.7 11.1 10.3

using randomized complete block experimental design, and 1 and 5 % were used for significance levels. LSD of 5% was used to determine the different groups of fertilizer treatments when variance analyses of a component was found significant. All calculations were made by using MINITAB and MSTAT-C programs.

RESULTS AND DISCUSSION Data averaged over two years and subjected to variance analysis (ANOVA) are given in Table 2. Results of ANOVA indicated that the effects of nitrogen rates were of significance only on dry matter and crude protein yields, but the effects of phosphorus rates were determined on most components. In addition, the effects of interaction between nitrogen and phosphorus on hairy veth ratio was observed significant (Table 2). The effects of nitrogen rates on dry matter yields were significant (p < 0.05) and 30 kg N ha-1 rate produced the highest dry -1 matter yield (10779 kg ha ), while the lowest dry matter yields (8439 and 8794 kg ha-1) were produced by plots untreated or treated with 90 kg N ha-1 (Table 2). Numerous workers have determined different nitrogen rates for maximum dry matter yield in common vetchbarley or hairy vetch- barley mixture (Cimrin et al., 2001; Karaca and Cimrin, 2002; Mohsenabadi et al., 2008). These are natural results due to the different ecologies, cultivars of vetch and barley, and mixture rates. Nitrogen rates had no effects on hairy vetch ratios and they ranged from 43.36 to 46.95% (Table 2). Similar results were reported by Karaca and Cimrin (2002). However, reverse results were reported by some other workers (Cimrin et al., 2001). The response of crude protein yield to nitrogen fertilization was statistically significant (p < 0.01). All rates of nitrogen increased crude protein yield but there were no differences among their effects (Table 2). Mohsenabadi et al. (2008) reported that nitrogen fertilizer increased the crude protein yield of vetch-barley mixture. The values of ADF, NDF and TDN unaffected by nitrogen rates ranged from 33.73 to 35.39%, 49.60 to 58.32% and 61.34 to 62.62%, respectively (Table 2). The effects of phosphorus rates on dry matter yields

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Table 2. Effects of nitrogen and phosphorus rates on dry matter yield (kg ha-1), hairy vetch ratio (%), crude protein yield (kg ha-1), ADF (%), NDF (%) and TDN (%) of hairy vetch-barley mixture (mean of two years).

Nitrogen Rate -1 (kg ha ) 0 30 60 90

Dry matter yield -1 (kg ha ) b 8439 10779a ab 9915 b 8794

Hairy vetch ratio (%)

Crude protein yield (kg ha-1)

ADF (%)

NDF (%)

TDN (%)

33.73 34.05 35.10 35.39

49.60 54.25 51.60 58.32

62.62 62.38 61.56 61.34

46.95 45.43 46.60 43.36

925.4 1342.3a a 1215.3 a 1249.7

55.94 a 70.81 bc 61.94 ab 64.53 65.90ab

946.4 a 1613.2 b 1323.4 ab 1381.0 1362.5ab

35.26 c 31.74 a 39.88 ab 38.76 33.36bc

57.91 52.56 53.40 55.65 53.91

61.43 a 64.18 c 57.84 bc 58.71 62.92ab

* ns ** ns * * ns

ns ** ** ns ns ns ns

ns ns * * ns ns ns

ns ns ns ns ns ns ns

ns ns * ns ns ns ns

-1

Phosphorus rate (kg ha ) d 0 8429 a 30 11256 b 60 10140 c 90 9199 120 9239c Years (Y) Nitrogen (N) Phosphorus (P) Y×N Y×P N×P Y×N×P

ns * ** ns ns ns ns

abc

Means of the same column followed by the same letter were not significantly different at the 0.05 level using LSD test. *, **: F-test significant at p ≤/0.05, and p ≤/0.01, respectively; ns,not significant; ADF, acid detergent fiber; NDF, neutral detergent fiber; TDN, total digestible nutrient.

were significant (p < 0.01) and the highest dry matter yield (11256 kg ha-1) was obtained at 30 kg P2O5 ha-1, while the lowest dry matter yield (8429 kg ha-1) was obtained at 0 kg P2O5 ha-1. As the rates of phosphorus increased, the dry matter yields decreased (Table 2). Cimrin et al. (2001) reported that phosphorus fertilizer significantly increased dry matter yield and they found the highest dry matter yield at 80 and 120 kg P2O5 ha-1 levels. However, some researchers reported that phosphorus rate had no effect on dry matter yield of vetch-barley mixture (Karaca and Cimrin, 2002). Phosphorus rates increased hairy vetch ratio in the mixture significantly, but -1 the highest ratio was obtained at 30 kg P2O5 ha (Table 2). Phosphorus application also affected crude protein yields of sowings. The lowest crude protein yield was obtained at the untreated plots and the highest was at 30 -1 kg P2O5 ha treated plots (Table 2). Similar results were reported by Cimrin et al. (2001) and Karaca and Cimrin (2002). Moreover, the ADF content of the mixture changed significantly with the phosphorus rates. However, the effects of rates were not of consistent rule. The lowest -1 ADF content (31.74%) was obtained at 30 kg P2O5 ha treatment, and the other rates yielded forages that contained higher than or equal to those of the untreated plots (Table 2). In this study, phosphorus rates had no effect on NDF contents and ranged from 52.56 to 57.91% (Table 2). Meanwhile, TDN values were affected by

phosphorus fertilizer applications but there were no regulated rules seen about the effects of fertilizer rates. The lowest and the highest TDN were observed at 60 and 30 kg P2O5 ha-1 rates, respectively, and the values of other rates took place between the highest and the lowest TDN values (Table 2). Conclusıons This study was conducted under rainfed conditions of Southern Marmara Region in order to determine the nitrogen and phosphorus needs of hairy vetch and barley mixtures sown at 50:50 mixed ratio. For this purpose, 0, -1 30, 60 and 90 kg ha rates of pure nitrogen and 0, 30, 60, 90 and 120 kg ha-1 rates of pure phosphorus were used. Dry matter yield and some quality parameters of the mixtures were measured and the average values of two-year experiment were evaluated and discussed. The results extracted from the study were as follows: (a) Nitrogen fertilization exhibited its effects only on dry matter and crude protein yields, (b) The dry matter yield at 30 kg N ha-1 rate was the -1 -1 highest, followed by 60 kg N ha rate. The 90 kg N ha rate made no difference from unfertilized plots, (c) The effects of nitrogen rates on crude protein yield were same but higher than that of unfertilized plants,

Budakli Carpici and Tunali

(d) Phosphorus fertilization did affect all parameters but NDF contents, (e) Dry matter yields at all phoshorus rates were higher than that of unfertilized plants, and the 30 kg P2O5 ha-1 rate produced the highest yield followed by 60 kg P2O5 ha-1 rate, (f) Hairy vetch ratio was the lowest level at unfertilized -1 plots, and the highest at 30 kg P2O5 ha rate, (g) Appearances of the effects of phosphorus fertilization on crude protein yields were very similar to those on hairy vetch ratios. Plots not treated with phosphorus produced less dry matter yield than plots treated with different phosphorus rates. The highest crude protein yield was obtained at 30 kg P2O5 ha-1 rate, (h) 30 kg P2O5 ha-1 rate produced forage lower in ADF and higher in TDN, but 60 kg P2O5 ha-1 rate produced -1 forage completely different than that of 30 kg P2O5 ha rate. Depending on the two-year results, 30 kg ha-1 rates of nitrogen and phosphorus may be recommended to be applied on hairy vetch-barley mixture blended at 50:50 ratio and grown under the Southern Marmara Region in order to produce economically higher and quality forage product. REFERENCES Ansar M, Ahmed ZI, Malik M A, Nadeemi M, Majeed A, Rischkowsky BA (2010). Forage Yield and Quality Potential of Winter Cereal-Vetch Mixtures under Rainfed Conditions. Emir. J. Food Agric. 22(1): 25-36. Bayram G, Carpici EB, Celik N (2009). A Research on Effects of Different Seeding Ratios and Cutting Stages on Hay Yield and Quality in Mixtures of Barley (Hordeum vulgare L.) and Hairy Vetch th (Vicia villosa Roth.). 8 Field Crops Congress, Turkey-Hatay, 19-22 October, 501-504. Bingol NT, Karsli MA, Yılmaz IH, Bolat D (2007). The Effects of Planting Time and Combination on the Nutrient Composition and Digestible Dry Matter Yield of Four Mixtures of Vetch Varieties Intercropped with Barley. J. Vet. Anim. Sci. 31: 297-302. Bugdaycıgil M, Sabancı CO, Ozpınar H, Enginlioglu G (1996). The effect of Various Vetch and Barley Mixtures on Forage Yield and Quality. Third Range-Pasture and Forage Congress, Turkey, 17-19 June, 316-320.

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Carr PM, Martin GB, Caton JS, Poland WW (1998). Forage and Nitrogen Yield of Barley–pea and Oat–pea Intercrops. Agron. J. 90: 79-84. Cimrin KM, Karaca S, Bozkurt MA (2001). The Effect of Fertilization on the Chemical Composition and Hay Yield in Vetch + Barley Mixtures. J. Agric. Sci. 7(4): 32-36. Dhima KV, Lithourgidis AS, Vasilakoglou IB, Dordas CA (2007). Competition Indices of Common Vetch and Cereal Intercrops in Two Seeding Ratio. Field Crops Res. 100: 249-256. Goering MK, Van Soest PJ (1970). Forage Fibre Analysis. USDA Agricultural Handbook, USA. 379-1-20. Karaca S, Cimrin KM (2002). Effects of the Nitrogen and Phosphorus Fertilization on the Yield and Quality of the Common Vetch (Vicia sativa L.) and Barley (Hordeum vulgare L.) Mixture. J. Agric. Sci. 12(1): 47-52. Lithourgidis AS, Dhima KV, Vasilakoglou IB, Dordas CA, Yiakoulaki MD (2007). Sustainable Production of Barley and Wheat by Intercropping Common Vetch. Agron. Sustain. Dev. 27: 95-99. Mohsenabadi GhR, Jahansooz MR, Chaichi MR, Rahimian Mashhadi H, Liaghat AM, Savagheb GhR (2008). Evaluation of Barley–Vetch Intercrop at Different Nitrogen Rates. J. Agric. Sci. Technol. 10: 2331. Soya H (1994). Effect on the Seed Yield and Yield Characteristics of Common Vetch (Vicia sativa L.) of the Percentage of Barley (Hordeum vulgare L.) as a Nurse Crop in Mixture and Row Spacing. Field Crop Abst. No 8229. Thompson DJ, Stout DG, Moore T (1992). Forage Production by 4 Annual Cropping Sequences Emphasizing Barley under Irrigation in Southern Interior British Columbia. Can. J. Plant Sci. 72: 181-185. Turk M, Albayrak S, Yuksel O (2009). Effects of Fertilisation and Harvesting Stages on Forage Yield and Quality of Hairy Vetch (Vicia villosa Roth.). New Zealand J. Agric. Res. 52: 269-275. Yılmaz S, Günel E, Saglamtimur T (1996). A Research to Determination of the Most Suitable Seeding rates and Cutting Times of Common Vecth (Vicia sativa L.) + Barley (Hordeum vulgare L.) Mixture under Hatay Ecological Condition. Third Range-Pasture and Forage Congress, Turkey, 17-19 June, 355-360.

Full Length Research Paper

Cryopreservation and plant regeneration of anther callus in Hevea by vitrification Quan-Nan Zhou, Ai-Hua Sun, Zhe Li*, Yu-Wei Hua, Ze-Hai Jiang, Tian-Dai Huang, Xue-Mei Dai and Hua-Sun Huang* Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences, State Center for Rubber Breeding, Danzhou 571737, China, Key Laboratory of Rubber Biology, Ministry of Agriculture, State Engineering and Technology Research Center for Key Tropical Crops, Danzhou 571737, China. Accepted 16 March, 2012

Callus induced from anther of Hevea brasiliensis was successfully cryopreserved in liquid nitrogen (LN) by vitrification method and subsequently regenerated into plants. The effects of different preculture time, loading and dehydration duration on callus viability after cryopreservation were evaluated. The effective cryopreservation protocol involved preculturing on modified Murashige and Skoog (MS) medium containing 5 % sucrose (w/v) and 5% dimethyl sulfoxide (DMSO) (v/v) for 3 days, loading with 60% plant vitrification solution 2 (PVS2) for 20 min at 0°C and dehydration with ice-cold PVS2 for 40 min. Dehydrated samples were directly immersed into LN, stored for 24 h and re-warmed in a water bath at 40°C. Using this protocol, H. brasiliensis callus showed 71.7% viability after cryopreservation. In conclusion, we developed a simple and effective method for the cryopreservation of H. brasiliensis callus, which allows long-term maintenance of valuable genotypes. Key words: Callus, cryopreservation, Hevea brasiliensis, regeneration, vitrification. INTRODUCTION Rubber tree (Hevea brasiliensis Muell. Arg.) from tropical and subtropical countries supplies more than 30% of the elastomer market demand. Work under way in different rubber research institutes is focused on agricultural exploitation and on creating superior clones for rubber and wood production, and for disease resistance. The fact that rubber trees have long life cycle and cross-pollinated, makes the breeding of rubber trees more difficult. In recent years, the study on the tissue culture of H. brasiliensis Muell. Arg. has been developed (Nayanakantha and Seneviratne, 2007). Wang et al. (1980) successfully obtained the first H. brasiliensis plantlet from anther using

*Corresponding author. E-mail: lizhecn@yahoo.cn and xjshhs@163.com. Tel: +86-898-23300585. Fax: +86-89823300315. 2,4-D, 2,4-Dichlorophenoxyacetic acid; 6-BA, 6Benzylaminopurine; DMSO, Dimethyl Sulfoxide; IAA, Indole3-acetic acid; KT, Kinetin; LN, liquid nitrogen; MS, Murashige and Skoog (1962) medium; NAA, α-Naphthalene acetic acid; PVS2, plant vitrification solution 2; GA3, Gibberellic acid.

tissue culture techniques. Cryopreservation has become a very important tool for the long-term storage of plant germplasm with minimal space and maintenance (Sakai and Engelmann, 2007). At 196°C, metabolic processes and growth activity of cryopreserved cells are almost stopped (Stanwood, 1985; Benson, 2008). Theoretically, the plant materials are in a stable state for an infinite period of time, provided ice formation inside the cells is low enough to allow plant recovery. Cryopreservation of H. brasiliensis has only been reported by a few researchers. Normah et al. (1986) found that embryonic axes could withstand cryopreservation after partial desiccation. Veisseire et al. (1993) successfully freeze embryogenic cell suspensions of one commercial clone. Subsequently, Engelmann et al. (1997) developed two effective cryopreservation protocols for embryogenic callus of two commercial clones of H. brasiliensis but only somatic embryos not plantlets were established. Lardet et al. (2007) succeeded in obtaining the commercial clone plantlets after cyropreservation for H. brasiliensis. Their study mainly focused on the effect of different CaCl2 concentrations in preculture medium.

Zhou et al.

The experiments of Engelmann et al. (1997) and Lardet et al. (2007) both applied an average cooling rate of 0.2°C/min down to -40°C controlled by a programmable freezer or placed in -80°C freezer. The slow cooling procedure needs the use of expensive devices and a careful manipulation of plant materials and cooling rates. In comparison, vitrification is a simple cryopreservation procedure which does not required the use of expensive cooling equipments. By vitrification, detrimental intracellular ice formation is avoided by using a highly concentrated cryoprotective solution to dehydrate and penetrate cells (Sakai and Engelmann, 2007). The plant vitrification solution 2 (PVS2) removed cellular water, changed cellular freezing behavior and limited ice crystallization in mint and garlic shoot tips during cryoprotection (Volk and Walters, 2006). However, the solution was also lethal with extended exposure times (Volk et al., 2006). Until now, the vitrification method has been successfully used in many species (Volk and Caspersen, 2007; Turner et al., 2001; Fahy et al., 2004). However, there is no report of it being applied to the callus of H. brasiliensis anther. The optimal preculture time, loading and dehydration duration are the keys of producing a high level of survival and successful cryopreservation by vitrification. In this study, we investigated the effects of preculture time, loading and dehydration duration on the viability of H. brasiliensis callus after cryopreservation. A simple vitrification protocol for cryopreservation of H. brasiliensis callus was established with a commercial clone. MATERIALS AND METHODS The callus regenerated from H. brasiliensis Reyan 7-33-97 anthers, a line cultivated in the experimental farm of Rubber Research Institute, Chinese Academy of Tropical Agriculture Sciences was used. The induction of the callus was performed using a modified method of Wang et al. (1980). The anthers were cultured on MS medium supplemented with 1.5 mg l-1 2, 4-dichlorophenoxyacetic acid (2,4-D), 1.5 mg l-1 Kinetin (KT) and 1.5 mg l-1 α-naphthalene acetic acid (NAA) and incubated at 28 ± 2°C under darkness for 4 weeks. The callus was transferred into fresh medium every 4 weeks under the same culture conditions. Three experiments were conducted to evaluate the effects of different preculture period, loading time in 60% PVS2 and dehydration time in ice-cold PVS2.

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Different loading time Callus precultured for 3 days was loaded with 60% PVS2 at room temperature for 10, 15, 20, 30 and 40 min and then treated with icecold PVS2 for 40 min, and immersed directly into LN for 24 h. The callus was recovered from cryopreservation as described previously.

Different dehydration duration The callus precultured for 3 days was loaded with 60% PVS2 at room temperature for 20 min and treated with ice-cold PVS2 for 10, 20, 30, 40 and 60 min, respectively. Subsequently, the callus was rapidly immersed in LN for 24 h and then recovered from cryopreservation as described previously.

Regrowth assessment and plant regeneration In all experiments, about 10 to 20 calli were used for each treatment and the experiments were repeated three times. The regrowth rate of the calli was expressed relative to the number of cryopreserved ones. The viability of the calli after cryopreservation was assessed by visual observation of growth. Callus precultured for 3 days were loaded with 60% PVS2 at room temperature for 20 min, treated with ice-cold PVS2 for 40 min and immersed directly into LN for 24 h. After thawing, the callus was resumed and then transferred into the embryo-induction medium which comprised Murashige and Skoog (MS) medium supplemented with 1.0 mg l-1 6-benzylamino purine (6-BA), 3.0 mg l-1 KT, 0.2 mg l-1 NAA and 0.05 mg l-1 gibberellic acid (GA3) and cultured in darkness at 28 ± 2°C for 4 weeks. Mature somatic embryos were transplanted into test tubes containing 30 ml MS medium supplemented with 0.5 mg l-1 KT, 0.2 mg l-1 indole-3-acetic acid (IAA), 0.3 mg l-1 GA3 and incubated in a growth room under the conditions of 28 ± 2°C, humidity (60/70%), 12-h light:12-h dark photoperiod and transferred into fresh medium every 4 weeks. About 8 weeks later, the plantlets were obtained in the test tubes.

Statistical analysis All experiments described here were repeated three times. The data were analyzed by one-way analysis of variance (ANOVA) on the statistical package of statistical analysis system (SAS) program (Version 9.0). Significant differences between means were assessed by Duncan’s test at P = 0.05.

RESULTS

Different preculture time

Evaluation of different preculture time

Callus was transferred into the preculture medium [modified Murashige and Skoog (1962) medium with sucrose (5% w/v), dimethyl sulfoxide (DMSO) (5% v/v)] and cultured for 0, 1, 2, 3, 4, 5 and 7 days. Ten (10) calli were transferred into 1.8 ml cryotube, respectively. They were then loaded with 60% PVS2 at room temperature for 20 min, treated with ice-cold PVS2 for 40 min, and immersed directly into liquid nitrogen (LN) for 24 h (Sakai et al., 1990). The callus was then recovered from cryopreservation, warmed in a water-bath by thermostat at 40°C and then plant regeneration was carried out according to the modified methods of Wang et al. (1980).

The length of the preculture time was critical to the viability of H. brasiliensis callus after cryopreservation. As shown in Figure 1, with the prolonging of preculture time, the viability of callus dramatically increased and reached a maximum at 3 days, followed by decline after 3 days. A significant difference was observed when precultured for 3 day compared with 0, 1, 2, 4, 5 or 7 days. However, no significant difference was examined when precultured for 2 or 4 days.

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Precultured time (days) Figure 1. Effect of preculture time on the viability of Hevea brasiliensis callus after cryopreservation. Callus were precultured for 0, 1, 2, 3, 4, 5 and 7 days, respectively loaded with 60% PVS2 for 20 min, dehydrated with ice-cold PVS2 for 40 min, immersed in LN and bathed in 40°C water. Data are presented as mean values ± standard error. Values followed by the same letters are not significantly different at the 5% level (Duncan’s multiple range test). Each value is the mean of three replicates. The bars represent standard errors.

Figure 2. Effect of loading time on the viability of Hevea brasiliensis callus after cryopreservation. Callus were precultured with 3 days, loaded with 60% PVS2 for 10, 15, 20, 30 and 40 min, respectively, dehydrated with ice-cold concentrated PVS2 for 40min, immersed in LN and bathed in 40°C water. Data are presented as mean values ± standard error. Values followed by the same letters are not significantly different at the 5% level (Duncan’s multiple range test). Each value is the mean of three replicates. The bars represent standard errors.

Optimization of loading and dehydration duration The length of the loading and dehydration duration was also very important to the viability of H. brasiliensis callus after cryopreservation. As shown in Figure 2, the viability of the callus increased considerably when callus precultured

for 3 days was loaded with 60% PVS2 at room temperature between 15 and 30 min and reached the highest (about 70%) at 20 min. Nevertheless, a significantly lower viability was examined when callus was loaded for 10 or 40 min. Figure 3 shows the effect of different dehydration duration on the viability of H. brasiliensis callus after cryopre-

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Figure 3. Effect of dehydration duration on the viability of Hevea brasiliensis callus after cryopreservation. Callus were precultured with 3 days, loaded with 60% PVS2 for 20 min, dehydrated with ice-cold concentrated PVS2 for 10, 20, 30, 40 and 60 min respectively, immersed in LN and bathed in 40°C water. Data are presented as mean values ± standard error. Values followed by the same letters are not significantly different at the 5% level (Duncan’s multiple range test). Each value is the mean of three replicates. The bars represent standard errors.

servation. The viability of the callus largely increased with dehydration duration prolonging and reached a maximum when treated with ice-cold PVS2 for 40 min and then significantly decreased at 60 min. The viability of callus treated with PVS2 for 20 or 30 min was not significantly different. Plant regeneration After thawing, the callus stored in LN for 24 h showed a 2 to 3 days lag phase and recovered completely and grew into somatic embryos after 40 days of incubation. The embryos were induced into regenerated plantlets (Figure 4). DISCUSSION In this study, we reports for the first time the successful cryopreservation of H. brasiliensis anther callus using a simple vitrification protocol. The effective cryopreservation protocol involved preculturing on modified MS medium containing 5% sucrose (w/v) and 5% DMSO (v/v) for 3 days, loading with 60% PVS2 for 20 min at 0°C and dehydration with ice-cold PVS2 for 40 min before dehydrated callus was directly plunged into LN, stored for 24 h and re-warmed in a water bath at 40°C. Studies of several groups found that preculture had significant influences on the survival rate of plant material after cryopreservation (Charoensub et al., 1999, Lambardi

et al., 2000). Preculture could induce the stress hardening and enhance the freeze tolerance and survival rates (Göldner et al., 1991, Takagi et al., 1997). It was reported that preculture before cryopreservation on a medium with or without calcium was necessary for post-thaw callus growth recovery (Kohmura et al., 1992, Lardet et al., 2007). In this experiment, it was observed that the viability of H. brasiliensis callus after cryopreservation reached the highest when precultured for 3 days. Preculturing for 0, 1 or 2 days all resulted in the lower viability, which may be due to an insufficient acquisition of desiccation tolerance or cryoprotection (Turner et al., 2001b). Preculturing for longer than 3 days also showed the lower viability, this was probably attributed to the tissue growth and changes in physiological condition. Using Picea mariana (black spruce) embryogenic cultures, Touchell et al. (2002) obtained the highest survival by preculturing embryogenic masses for 2 days followed by incubation in PVS2 solution at 0°C for 30 min and plunging directly into LN. In many species, preculture appears to be insufficient to produce a high level of survival by vitrification. Direct exposure to highly concentrated vitrification solutions is toxic. Therefore, samples cryopreserved by vitrification need to be loaded with a cryoprotective solution (Sakai and Engelmann, 2007). In the present study, the viability of H. brasiliensis callus after cryopreservation reached a maximum when loaded with 60% PVS2 for 20 min at room temperature. In Anigozanthos viridis, the optimal loading time was also 20 min at room temperature (Turner et al., 2001a), while in Asparagus officinalis L., the time was 10

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Figure 4. Plant regeneration of Hevea brasiliensis callus after cryopreservation. (a) Callus developed after cryopreservation and culturing for 20 days. (b) Somatic embryos developed from callus after cryopreservation and culturing for 60 days. (c) The amplified embryos. (d) (e) Plantlets obtained from somatic embryos. Scale bars represent 1 (a, b, d, and e) and 0.2 cm (c), respectively.

min (Nishizawa et al., 1993). The dehydration duration is also very important to produce high regrowth rate of the callus after cryopreservation by vitrification. In the experiment, the highest viability of H. brasiliensis callus after cryopreservation was obtained when exposed to PVS2 solution for 40 min. The dehydration duration shorter than 40 min showed the lower viability which may be due to inadequate desiccated callus. The dehydration duration longer than 40 min also resulted in lower viability, most likely attributed to the injury from chemical toxicity or excessive desiccation (Sakai et al., 2007; Touchell et al., 2002). In sweet potato and black spruce, the optimal exposure time to PVS2 solution was reported to be 60 and 30 min at 25°C, respectively (Hirai and Sakai, 2003; Touchell et al., 2002). In the present study, it was shown that the cell recovery of cryopreserved callus delayed by 2 to 3 days compared to non-cryopreserved ones. A similar observation was reported in lychee (Xie et al., 2008). The callus developed after cryopreservation was transferred into proliferation medium for 40 days which were longer than the non-

cryopreserved callus, then transferred into embryoinduction medium, and the plantlets were observed in the cuvettes after 60 days. The plantlets had no phenotypic difference with those derived from non-cryopreserved callus. In conclusion, this experiment successfully obtained the plantlets of the line of large-scale cultivated H. brasiliensis Reyan 7-33-97 anther callus after cryopreservation. The method that the callus immersed in LN directly from room temperature is simpler and more convenient than CIRAD that achieved an average cooling rate of 0.2°C/min down to -40°C controlled by a programmable freezer (Engelmann et al., 1997; Lardet et al., 2007).

Acknowledgements Financially supported by the Key Scientific and Technological Item of Hainan Province “Studies on cryopreservation of embryogenic callus from anther and post-thaw plant regeneration in Hevea” (070105), and the

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International Scientific and Technological Cooperation Item “Studies on friable callus long-term subculture, cryopreservation and plant regeneration in Hevea” (2008DFA32020). REFERENCES Benson EE (2008). Cryopreservation Theory. In Plant Cryopreservation: A Practical Guide, (Ed.) BM Reed, Springer Press, Germany. Charoensub R, Phansiri S, Sakai A and Yongmenitchai W (1999). Cryopreservation of cassava in vitro-grown shoot tips cooled to −196℃ by vitrification. CryoLett. 20: 89-94. Engelmann F, Lartaud M, Chabrillange N, Carron MP, Etienne H (1997). Cryopreservation of embryogenic calluses of two commercial of Hevea brasiliensis. CryoLett. 18: 107-116. Fahy GM, Wowk B, Wu J, Paynter S (2004). Improved vitrification solutions based on the predictability of vitrification solution toxicity. Cryobiology, 48: 22-35. Göldner EM, Seitz U, Reinhard E (1991). Cryopreservation of Digitalis lanata Ehrh. cell cultures: Preculture and freeze tolerance. Plant Cell Tissue Org. Cult. 24: 19-24. Hirai D, Sakai A (2003). Simplified cryopreservation of sweet potato [Ipomoea batatas (L.) Lam.] by optimizing conditions for osmoprotection. Plant Cell Rep. 21: 961-966. Kohmura H, Sakai A, chokyu S, Yakuwa T (1992). Cryopreservation of in vitro-cultured multiple bud clusters of asparagus (Asparagus officinalis L. cv Hiroshimagreen (2n=30) by the techniques of vitrification. Plant Cell Rep. 11: 433-437. Lambardi M, Fabbri A, Caccavale A (2000). Cryopreservation of white poplar (Populus alba L.) by vitrification of in vitro-grown shoot tips. Plant Cell Rep. 19: 213-218. Lardet L, Martin F, Dessailly F, Carron MP, Montoro P (2007). Effect of exogenous calcium on post-thaw growth recovery and subsequent plant regeneration of cryopreserved embryogenic calli of Hevea brasiliensis (Müll. Arg.). Plant Cell Rep., 26: 559-569. Murashige T, Skoog F (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant, 15: 473-497. Nayanakantha NMC, Seneviratne P (2007). Tissue culture of rubber: past, present and future prospects. Cey. J. Sci. (Bio. Sci.) 36: 116-125. Nishizawa S, Sakai A;Amano Y, Matsuzawa T (1993). Cryopreservation of asparagus (Asparagus officinalis L.) embryogenic suspension cells and subsequent plant regeneration by vitrification. Plant Sci. 91: 67-73. Normah MN, Chin HF, Hor YL (1986) Desiccation and cryopreservation of embryogenic axes of Hevea brasiliensis Muell.-Arg. Pertanika, 9: 299303. Sakai A, Engelmann F (2007). Vitrification, encapsulation-vitrification and droplet-vitrification: a review. CryoLett. 28: 151-172. Sakai A, Engelmann F (2007). Vitrification, encapsulation-vitrification and droplet-vitrification: a review. CryoLett. 28: 151-172. Sakai A, Kobayashi S, Oiyama I (1990). Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification. Plant Cell Rep., 9: 30-33. Stanwood PC (1985). Cryopreservation of seedgermplasm for genetic conservation. In: Cryopreservationof Plant Cells and Organs (Ed. Kartha KK). CRC Press, Inc. Boca Raton, Florida. Takagi H, Thinh NT, Islam OM (1997). Cryopreservation of in vitro grown shoot tips of vitrification procedure. Plant Cell Rep. 16: 594-599.

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Touchell DH, Chiang VL, Tsai CJ (2002). Cryopreservation of embryogenic cultures of Picea mariana (black spruce) using vitrification. Plant Cell Rep. 21: 118-124. Turner S, Senaratna T, Touchell D, Bunn E, Dixon K, Tan B (2001). Stereochemical arrangement of hydroxyl groups in sugar and polyalcohol molecules as an important factor in effective cryopreservation. Plant Sci. 160: 489-497. Turner SR, Krauss SL, Bunn E, Senaratna T, Dixon K, Tan B, Touchell D (2001a) . Genetic fidelity and viability of Anigozanthos viridis following tissue culture, cold storage and cryopreservation. Plant Sci. 161: 10991106. Turner SR, Senaratna T, Bunn E, Tan B, Dixon KW, Touchell DH (2001b) Cryopreservation of shoot tips from six endangered Australian species using a modified vitrification protocol. Ann. Bot. 87: 371-378. Veisseire P, Guerrier J, Coudret A (1993). Cryopreservation of embryogenic cell suspension of Hevea brasiliensis. CryoLett. 14: 295302 Volk GM, Caspersen AM (2007). Plasmolysis and recovery of different cell types in cryoprotected shoot tips of Mentha x piperita. Protoplasm, 231: 215-226. Volk, GM, Harris J, Rotindo KE (2006). Survival of mint shoot tips after exposure to cryoprotectant solution components. Cryobiology, 52: 305308. Volk, GM, Walters C (2006). Plant vitrification solution 2 lowers water content and alters freezing behaviour in shoot tips during cryoprotection. Cryobiology, 52: 48-61. Wang ZY, Zeng XS, Chen CQ, Wu HY, Li QY, Fan GJ, Lu WJ (1980). Induction of rubber plantlets from anther of Hevea brasiliensis Muell. Arg. in vitro. (In Chinese with English abstract). Chin. J. Trop. Crops, 1: 16-25. Xie YM, Zeng JW, Zhang QM, Yi GJ (2008). Cryopreservation of Litchi embryogenic suspension cells by vitrification technique. (In Chinese with English abstract). Chin. J. Trop Crops, 29: 622-625.

Full Length Research Paper

Genotypic identification and technological characterization of lactic acid bacteria isolated from traditional Turkish Kargi tulum cheese Buket Kunduhoglu1*, Ozlem Elcioglu2, Yekta Gezginc3, Ismail Akyol4, Sevil Pilatin1 and Asya Cetinkaya5 1

Department of Biology, Faculty of Science and Arts, University of Eskişehir Osmangazi, 26480-Eskişehir, Turkey. 2 Institute of Science, University of Eskişehir Osmangazi, 26480-Eskişehir, Turkey. 3 Department of Food Engineering, Faculty of Agriculture, University of Kahramanmaraş Sütçü Đmam, 46100Kahramanmaraş, Turkey. 4 Microbiology and Microbial Genetics Laboratory, University of Kahramanmaraş Sütçü Đmam, USKIM, 46100Kahramanmaraş, Turkey. 5 Kars Vocational School, University of Kafkas, 36100-Kars, Turkey. Accepted 2 March, 2012

Kargi tulum cheese is an artisanal cheese produced through the spontaneous fermentation of raw milk ripened inside a goat-skin bag. The objective of this study was to characterize the dominant species of natural lactic acid bacteria (LAB) found in Kargi tulum cheese. Some technologically important properties of LAB isolates were also determined. Seven samples of cheese of different levels of ripeness were taken from local producers and 97 isolates were obtained from these samples. Non-spore forming, Gram-positive, catalase- and oxidase-negative isolates were assessed as LAB. Based on genotypic characterization, the dominant LAB were identified as Lactobacillus paracasei (43.3%), Lactobacillus plantarum (23.7%), Enterococcus durans (6.2%), Streptococcus thermophilus (6.2%), Lactobacillus brevis (5.2%), Enterococcus faecium (5.2%), Lactobacillus fermentum (4.1%) and Lactobacillus pentosus (1%). Homofermentative strains such as L. paracasei, L. plantarum, S. thermophilus; E. durans and E. faecium were selected as probable starter cultures. These strains were tolerant to 6.5% salt. They are also characterized by acidification ability (from pH = 6.6 to 4.7 to 6.0, in 6 to 8 h), low proteinase and high peptidase, esterase, esterase-lipase, β-galactosidase and βglucosidase activities. They also produce diacetyl and H2O2. Key words: Lactic acid bacteria, genotypic characterization, technological characterization, tulum cheese. INTRODUCTION Kargi tulum cheese is manufactured as an organic food in the high plateau of Kargi, Çorum, in the Middle Anatolian region of Turkey. It is manufactured in summer months

*Corresponding author. E-mail: bkunduh@gmail.com. Tel: +90222-2393750. Fax: +90-222-2393578.

and marketed in public bazaars in autumn (Dinkci et al., 2007). Kargi tulum cheese is produced by small-scale dairies using raw whole sheep, goat, cow and buffalo milk or a mixture of these and no added lactic starters. The traditional production method of Kargi tulum cheese begins with fresh raw milk that is coagulated by adding rennin and salt (2 to 3% w/w). The indigenous microflora contained in raw milk increase the acidity of the curd (12

Kunduhoglu et al.

h). Afterwards, the curd is cut into small pieces by hand (in pea size, approximately 1 cm3) and put into small cotton bags. A weight is then put over the bags to drain the whey (at 20°C for 24 h). The drained curds are kneaded with salt and put into larger cotton bags. Any remaining whey is drained by putting a weight over the cotton bags. The cheese is removed from the big cotton bags at certain intervals (once every 15 to 20 days), kneaded and put into new cotton bags. This process is repeated until the end of October. Afterwards, the curds are compressed into goat-skin bags so that no entrapped air remains. Goat-skin bags are the material traditionally used to package tulum cheese. Kargi tulum cheese prepared in this way is then incubated for at least 3 months (at 4 to 6°C) in the uplands. Kargi tulum is a semi-hard cheese with a crumbly texture, white-cream color and a quite favorable taste (personal communication with local producer). Because of their unique taste, artisanal cheeses produced through spontaneous fermentation of unpasteurized milk receive a great deal of attention from consumers, not only in Turkey but also in other countries around the world. However, it is difficult to control spontaneous cheese fermentation by indigenous microflora in raw milk and the fermentation process and practically impossible to standardize the quality and shelflife of the product. Furthermore, pathogens, such as Staphylococcus aureus, Escherichia coli type I (Efe and Heperkan, 1995), Listeria monocytogenes and Salmonella spp. (Colak et al., 2007), have been isolated from tulum cheeses. While pasteurization eliminates several pathogens that threaten consumer health, it also eliminates a large part of the indigenous lactic acid bacteria (LAB) contained in raw milk and changes the cheese flavor (Torres-Llanez et al., 2006). Therefore, when producing traditional cheeses using pasteurized milk and modern production processes, it is very important to provide a taste and flavor similar to that of traditionally produced raw milk cheeses (Perez-Elortondo et al., 1998). To ensure this similarity, the cheese’s dominant LAB should be isolated, identified and positive technological characteristics determined. The main objective of this study was to characterize and identify the LAB that plays a predominant role in the spontaneous fermentation of Kargi tulum cheese. Also, technologically important characteristics such as acidification ability, enzymatic and proteolytic activities of these LAB strains were determined. The findings obtained from this study may aid in selecting LAB strains as potential starter cultures to improve the commercial value of Kargi tulum cheese, and in preserving the valuable bacterial strains as genetic resources of Turkey. To the best of our knowledge, this is the first report on the LAB composition of traditionally processed Kargi

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tulum cheese. MATERIALS AND METHODS Cheese samples Seven samples (samples 1 to 7) of Kargi tulum cheese at different stages of ripening (1, 2, 3 and 4 months) were obtained from local producers. Samples 1 and 2 (1 month old) were put into cotton bags. The other five samples (2 to 4 months old) were taken as being put into goat-skin bag. Maturation temperatures of cheeses were 4 to 12°C. All samples were transported to the laboratory at 4°C and analyzed within 24 h. Chemical analysis of cheese samples All Kargi tulum cheese samples were analyzed for moisture content (TS EN ISO 5534, 2006), titratable acidity as lactic acid-°SH and pH (TS 591, 2006), total N, total protein (TS EN ISO 8968-3, 2004), total fat (TS 3046, 1978), NaCl content and salt in the dry matter (TS EN ISO 5943, 2001). All chemical analyses were carried in the laboratories of the Ministry of Agriculture and Rural Affairs in accordance with the standard methods stated in parentheses. Isolation of LAB The cheese samples (25 g) were homogenized in sterile Ringer’s solution (225 ml) with a blender for 2 min. Decimal dilutions were made in sterile 0.85% (w/v) saline. From each dilution, 0.1 ml was plated in duplicate on MRS or M17 agar medium, incubated for 6 days under anaerobic conditions at 37 or 30°C, respectively. Five colonies were randomly picked from plates with 30 to 300 colonies and purified by streaking on the same isolation medium. All isolates were tested for cell morphology, Gram staining, catalase, activity, oxidase activity and spore formation. Gram-positive, catalasenegative, oxidase-negative and non-spore-forming isolates with rod or cocci shapes were considered LAB and selected for further examination. The LAB isolates were phenotypically identified (data not shown) by the API identificaticon system (API 50 CHL and API 20 Strep test kits, bioMérieux, France). The pure cultures of these strains were stored in MRS or M17 broth supplemented with 25% glycerol at −80°C. When required, cultures were activated by two consecutive transfers to MRS or M17 broth incubated at 37 or 30°C, respectively. Genotypic identification of LAB isolates Molecular identification of isolated LAB strains was carried out according to the methods as described by Tabasco et al. (2007) and Walter et al. (2000). Genomic DNA was extracted using the Genomic DNA Extraction Kit (Fermentas) according to the supplier’s specifications. DNA quantity and quality were determined by nano-drop and confirmed by agarose gel electrophoresis. Molecular identification of isolates was conducted using specific primers to amplify the 16S rRNA region. These primers were obtained from Iontek (Istanbul, Turkey) and sequences are listed in Table 1. The PCR amplification reaction was performed in a 40 µl solution containing 1 µl of each primer (20 pmol), 4 µl 10X reaction buffer, 1 µl of each dNTPs, 0.5 µl Taq DNA polymerase and 1 µl of

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Table 1. Species-specific primers (16S RNA) used to identify LAB strains isolated from Kargi tulum cheese samples.

Species

Primer Dur. F Dur. R

Sequence (5'→3') CCTACTGATATTAAGACAGCG GGGCGGTGTGTACAAGGC

Length of product (bp) 295

E. faecium

Ef. F Ef. R

GAAAAAACAATAGAAGAATTAT TGCTTTTTTGAATTCTTCTTTA

215

L. brevis

Lb. F Lb. R

CTTGCACTGATTTTAACA GGGCGGTGTGTACAAGGC

1340

L. fermentum

Ferm. F Ferm. R

GCCGCCTAAGGTGGGACAGAT CTGATCGTAGATCAGTCAAG

301

L. paracasei

Pcas. F Pcas. R

GCACCGAGATTCAACATGGAA GCCATCTTTCAGCCAAGAACC

142

L. pentosus

Pent. F Pent. R

CAGTGGCGCGGTTGATATC TCGGGATTACCAAACATCAC

218

L. plantarum

Plan. F Plan. R

GCCGCCTAAGGTGGGACAGAT TTACCTAACGGTAAATGCGA

318

S. thermophilus

Therm. F Therm. R

ACGCTGAAGAGAGGAGCTTG GCAATTGCCCCTTTCAAATA

157

E. durans

the isolated DNA. The PCR products were generated using an initial denaturation step of 4 min at 94°C, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 50°C for 1 min, elongation at 72°C for 1 min and final at 72°C for 5 min. PCR reactions were performed in an Eppendorf Mastercycler Personal system. All of the PCR amplification products were analyzed on 1% (m/v) agarose gels, and the separated fragments were stained with 0.5 µg/ml ethidium bromide (Sisto et al., 2009) and visualized under UV light. Technological properties of LAB Acidification ability The isolates activated in MRS or M17 broth (30°C for 24 h) were centrifuged at 7500 rpm for 5 min, and the pellets were washed with peptone water and re-suspended in the same medium. Tubes containing 10 ml of whole fat UHT milk (pH = 6.6) were inoculated with the cell suspension (1% v/v) and incubated at 30°C for 48 h. Then, pH changes in the milk were measured using a pH meter (WTW 735, Germany) during incubation (after 2, 4, 6, 8, 24 and 48 h) (Olasupo et al., 2001). Proteolytic activity Surface-dried plates of skim milk agar (10% skim milk + 2% agar

Reference Walter et al. (2000)

Walter et al. (2000)

Tabasco et al. (2007)

Walter et al. (2000)

Tabasco et al. (2007)

w/v) were used to determine the proteolytic activity (Gordon et al., 1973). The same cell suspensions discussed earlier were spotted (5 µl) into the plate surfaces. Plates were incubated at 30°C for 24 h and then examined for the appearance of halos of proteolysis around the spotted samples. Diacetyl production The production of diacetyl was performed qualitatively. Tubes containing 5 ml of whole-fat UHT milk were inoculated with the activated cell suspensions (1% v/v) and incubated at 30°C for 24 h. After incubation, 1 ml aliquot was transferred to another sterile test tube and reagents (0.5 ml of 1% alpha naphthol and 0.5 ml of 16% KOH) were added. Diacetyl-producing strains had a red ring at the top of the tube after incubation (King, 1948). Hydrogen peroxide (H2 O2) production LAB isolates were grown at 30°C for 48 h in 5 ml MRS broth and diluted with 5 ml of sterile distilled water. The cells were removed by centrifugation at 7500 rpm for 5 min and the supernatant fluid was filtered (0.45 µm, Millipore Corporation, USA). Then, 0.5 ml citric acid, 0.5 ml ammonium molybdate and 0.5 ml KI (in that order) were added to 4 ml of supernatant and mixed vigorously. The optical density of this solution was determined at 350 nm. In order to determine the H2 O2 concentration in the supernatants, standard curve of the H2 O2 (30%) was used (Whittenbury, 1964).

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Table 2. Chemical characteristics of Kargi tulum cheese at different ripening periods.

Age of Kargi tulum cheese samples Chemical Property Moisture content (%) Titratable acidity (°SH) pH Total Protein (%) Total Nitrogen (%) Fat (%) Fat in dry matter (%) NaCl in dry matter (%)

1 month 2

2 months 3

3 months

53.9 1.5 5.05 14.8 2.3 24.0 52.0 2.5

46.4 2.8 5.09 21.9 3.4 25.0 46.6 5.4

42.5 2.5 5.13 22.2 3.5 29.0 50.4 5.8

35.0 2.7 5.2 23.4 3.7 35.0 53.8 4.7

5 38.0 2.6 5.15 23.4 3.7 32.0 51.6 5.6

4 months 6

29.0 2.9 5.20 26.0 4.1 39.5 55.6 3.1

25.0 2.8 5.25 25.1 3.9 44.3 59.1 3.6

*1-7: Sample number of cheeses.

Enzymatic profiles of LAB isolates The enzymatic profiles of the LAB were assayed using API-zym galleries (bioMérieux, France) according to manufacturer’s instructions. These tests were replicated twice.

days), the dry matter, salt, protein, fat and °SH values of tulum cheese samples increased. The pH of tulum cheese during the ripening period usually varies from 4.8 to 5.2 (Hayaloglu et al., 2007a). Similar to these findings, in our study, the pH of cheese samples varied from 5.05 to 5.25.

RESULTS AND DISCUSSION Chemical properties of the cheese samples

Genotypic identification of LAB

Chemical properties of the 7 Kargi tulum cheese samples at different ripening periods are given in Table 2. As seen in the table, the moisture ratio in the younger cheese samples (samples 1 and 2) which were not put in goatskin bags was high. However, due to the porous texture of the goat-skin bags, the moisture ratio of the cheese sampled during the ripening period gradually decreased. While the amounts of dry matter in the fresh cheese samples were 46.1% (sample 1) and 53.6% (sample 2), the amounts of dry matter in the cheese samples that were ripened for 4 months were 71 and 75%. During the ripening period, an increase in the amount of salt in dry matter in the samples was observed. For tulum cheese, 3 to 6% (w/w) salt in dry matter is acceptable (TSE 3001, 2006). The amount of salt in dry matter in our cheese samples varied between 2.5 to 5.8%. Ripened tulum cheese contains a high proportion of fat. The lowest fat and fat in dry matter ratios were found in 1 to 2-month-old cheese samples. This ratio gradually increased towards the end of the ripening period. In parallel with the ripening period, the total protein and nitrogen ratios in the fresh cheese samples also increased. Except for cheese sample no. 1, there were no apparent changes in the °SH values of the samples. However, as the ripening period increased, a slight elevation in the °SH values was measured. Similar to our study, Bayar and Ozrenk (2011) have also reported that during ripening period (max 90

Gel images obtained as a result of the genotypic characterization are shown in Figure 1. Identification of 92 isolates was performed using species-specific primers, but five isolates could not be identified with the primers used. The distribution of the LAB species isolated from different cheese samples and their isolation frequencies are shown in Table 3. In our study, it was found that the dominant population in Kargi tulum cheese samples was lactobacilli (77.3%; 75/97). The predominant species in almost all cheese samples was Lactobacillus paracasei, followed by Lactobacillus plantarum. Other lactobacilli were found during various stages of the incubation period. Although, their isolation frequency was not high, Enterococcus faecium and Enterococcus durans were isolated from the 1-, 2- and 3-month-old cheese samples. Streptococcus thermophilus was isolated from the 3- and 4-month-old samples. The low isolation frequency of these species maybe due to cell autolysis during the cheese ripening period. Various studies conducted on fermentative microflora of other ‘tulum’ cheese types (manufactured with different milk types, under different conditions, or in different regions) have reported different dominant LAB species in cheeses of different levels of ripeness. Bostan et al. (1992) found that Lc. lactis subsp. lactis and Enterococcus faecalis were dominant in tulum cheese made from cow milk at the beginning of its ripening period, and E. faecium, Lc. lactis subsp. lactis,

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Figure 1. PCR amplification products obtained from pure culture. DNA sizes are enumerated according to the M: 100 bp DNA ladder. L. paracasei (lanes 2, 4, 13, 15, 17, 19, 20, 22, 23, 26, 27, 29, 30, 33, 34, 36, 38, 39, 41, 43, 44, 47, 49, 55 to 57, 60, 62 to 65, 77, 84, 85, 95, 98, 105, 111, 114, 115, 118 and 119), L. plantarum (lanes 1, 3, 5, 6 to 8, 10, 12, 14, 21, 40, 42, 45, 58, 68, 71, 80, 82, 108, 109, 110, 116 and 117), St. thermophilus (lanes 16, 52, 54, 59, 61 and 67), E. durans (lanes 9, 53, 101, 106, 112 and 113), L. brevis (lanes 11, 35, 37, 46 and 51), L. fermentum (lanes 18, 28, 48 and 50), E. faecium (lanes 24, 62, 99, 102) and L. pentosus (lane 83).

Lb. casei and Lb. plantarum became dominant over time. Similar to the findings in our study, Sengul and Cakmakci (2003) found that 92% of Savak tulum cheese isolates were Lactobacillus spp. They reported that the dominant species among such isolates were Lb . parabuchneri and Lb . bifermentans. In a study conducted by Oksuztepe et al. (2005) using Savak tulum cheese, the number of lactococci and lactobacilli was high at the beginning and at the end of the ripening period, respectively and Lb. casei subsp. casei, Lb. plantarum, Lc. lactis subsp. lactis, Lc. lactis subsp. cremoris and Leu. mesenteroides subsp. cremoris were the predominant species. Technological characterization of LAB In this part of the study, the potential of the LAB isolated from Kargi tulum cheese to be used as starter cultures was evaluated. Starters selected from among the native

LAB of Kargi tulum cheese could be used to produce Kargi tulum cheese safer in terms of human health and standardized in quality, while preserving its traditional aroma. Starters used in cheese production must have a high acidifying capacity and be able to develop at the salt concentration and temperature at which the cheese is produced. To avoid rapid ripening of and bitterness in the cheese, its proteolytic activity should be low and should produce a pleasant taste and smell at the desired dosage and combination (Halkman, 1991; Arora et al., 1990). The extent to which our isolates had these characteristics and their potential as starter cultures were determined by performing the tests shown in Tables 4 and 5. In addition to coagulation, rapid acidification of milk inhibits several pathogens and saprophytic organisms. Garabal et al. (2008) stated that the use of LAB which reduce the pH of milk below 6 within the first 6 h, as a starter culture in cheese production provides an advantage. In our study, Lb. plantarumâ&#x2C6;&#x2019;5, 7, 71 and Lb. paracaseiâ&#x2C6;&#x2019;30 reduced the pH of UHT milk to pH = 5.8 to 6.0 within 6 h. Lb.

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Table 3. Genotypic species characterization of the LAB isolates using species-specific primers and distribution of these species in cheese samples. Isolation percentages (%) are indicated in parenthesis.

Age of Kargi tulum cheese samples Genotypic identification

1 month

Lb. paracasei Lb. plantarum Lb. brevis Lb. fermentum Lb. pentosus E. faecium E. durans S. thermophilus Unidentified

n 42 23 5 4 1 5 6 6 5

1* 11 (42.3) 7 (26.9) 4 (15.4) 2 (7.7) − − 1 (3.8) − 1 (3.8)

Total

2 months 2 4 (33.3) 2 (16.7) − − − 2 (16.7) 1 (8.3) − 3 (25.0)

3 months

4 months

3 6 (46.2) 2 (15.4) − − 1 (7.7) 2 (15.4) 2 (15.4) − −

4 4 (66.7) − − 1 (16.7) − 1 (16.7) − − −

5 1 (25) 1 (25) − − − − − 1 (25) 1 (25)

6 7 (53.8) 1 (7.7) − − − − 1 (7.7) 4 (30.8) −

7 9 (39.1) 10 (43.5) 1 (4.3) 1 (4.3) − − 1 (4.3) 1 (4.3) −

*1-7: Sample number of cheeses. − : No isolation

plantarum−3, 7 and 14 and Lb. paracasei−15 reduced the pH of milk to 4.7 to 5.2 at the end of 8 h (Table 4). After 24 h, the milk pH decreased from 3.7 to 3.9 with Lb. plantarum−5, 7 and Lb. paracasei−39 and 40 (Table 4). It has been reported that lactobacilli with a high caseinolytic activity play a critical role in the ripening of cheese (Broome and Hickey, 1991). Almost all of our isolates hydrolyzed casein (Table 4). It has been reported that lactobacilli with a high caseinolytic activity play a critical role in the ripening of cheese (Broome and Hickey, 1991). Almost all of our isolates hydrolyzed casein (Table 4). The strains with the highest caseinolytic activity (21 to 24 mm in diameter) were E. faecium−104> S. thermophilus−54= E. durans−112 > 9 = Lb. plantarum−3 = Lb. brevis−46. Lb. plantarum−82 and Lb. paracasei−98 were the lowest caseinolytic strains (0 to 6 mm in diameter). Other strains that showed low caseinolytic activity (7 to 13 mm in diameter) were Lb. plantarum−7, 71 and 110 and Lb. paracasei−19, 27, 47, 85. It is known that LAB produces various natural antimicrobials such as organic acids (lactic acid, acetic acid, formic acid, etc.), CO2, H2O2, diacetyl and bacteriocins (Messens and De Vuyst, 2002). In our study, all of our LAB isolates produced varying amounts of H2O2. The isolates that produced the highest amount of H2O2 (1.5 to 3.0 µl/ml) were Lb. paracasei−27, 29, and 39 and Lb. plantarum−6 (Table 4). Citrate is the primary organic acid contained in milk, and diacetyl and acetoin are formed as a result of citrate fermentation by LAB such as Leu. citrovorum, Str. faecalis, Str. diacetylactis,

Lb. casei, Lb. plantarum, L. brevis and L. bulgaricus. Diacetyl is also a major substance that gives cheese a distinctive flavor and butter smell (Hegazi and AboElnaga, 1980). Except for the L. brevis and Lb. fermentum strains, all of our LAB strains produced diacetyl (Table 4). The enzymatic characteristics of the LAB used in cheese production are among the factors that most affect cheese flavor (McSweeney, 2004). For ripened hard cheeses, starter cultures with high proteolytic activity are used. However, low proteolytic activity is required for fresh unripened cheeses and semi-hard ripened cheeses. On the other hand, if proteases are not balanced with peptidases and are found in cheese at high ratios, this can cause bitterness and texture defects (Coskun , 2000; Halkman and Taşkın, 2001). In our study, the enzymatic profiles of LAB strains were assayed using the API zym galleries. Each LAB strain produced a wide spectrum of enzymes (Table 5). While the leucine arylamidase and valine arylamidase (peptidases) enzyme activities were observed in 97 to 100% of LAB strains, α-chymotrypsin activity was found in only 8.3% of the strains and none of the strains showed trypsin activity. In terms of proteolytic activity, the majority of our LAB strains will provide a positive contribution to flavor formation in cheese. In addition, for cheeses produced by ripening, starter species with low lipase but high esterase and esteraselipase enzyme activities are preferred for their flavor and texture formation (Arora et al., 1990). Controlled lipolysis during ripening positively affects the flavor development in tulum cheese (Hayaloglu, 2007b). In our study, the

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Strains identified Lb. paracasei Lb. plantarum S. thermophilus E. durans Lb. brevis Lb. fermentum

n 42 23 6 6 5 4

Mean 6. 3 6.2 6.4 6.3 6.2 6.4

Range 5.9− 6.5 5.8− 6.5 6.3− 6.6 6.1− 6.5 6.0− 6.4 6.2− 6.5

Mean 6.1 5.9 6.2 6.2 5.8 6.3

Range 5.2− 6.5 4.7− 6.5 5.8− 6.6 5.9− 6.5 5.4− 6.3 6.1− 6.4

Mean 4.8 4.7 4.6 5.2 4.8 4.8

Range 3.8− 6.5 3.7− 6.5 3.9− 6.4 4.5− 6.5 4.6− 5.1 4.5− 5.3

Proteolytic activity (zone of proteolysis in mm) Mean Range 13.0 6.0− 20 12.9 0− 21 15.3 12− 22 16.0 7.0− 22 15.0 11− 21 15.8 19− 14

E. faecium

6.2

6.1− 6.3

6.0

5.9− 6.0

4.5

4.5− 4.5

15.7

Lb. pentosus

6.4

6.1

4.7

Unidentified

6.1

5.9− 6.3

5.9

5.3− 6.1

4.5

4.0− 4.9

pH of UHT Milk (Acidifying activity) 6h

24 h

Production of H2O2 (µl/ml) Mean Range 1.1 0.3− 3.0 1.2 0.4− 2.9 1.2 1.0− 1.3 1.0 0.9− 1.1 0.8 0.5− 1.1 1.3 1.1− 1.6

10− 24

1.1

15.0

9.7

3− 14

Diacetyl producers

Table 4. Some technological characteristics of LAB strains isolated from the cheese samples.

18 7 3 4 − −

1.0− 1.1

1.0

0.7

0.5− 1.1

Table 5. Enzymatic profiles of LAB strains isolated from the cheese samples.

Number of strains showed enzyme activity

Enzyme name Alkaline phosphatase Esterase Esterase Lipase Lipase Leucine arylamidase Valine arylamidase Cystine arylamidase Trypsin α- Chymotrypsin Acid phosphatase Naphthol-AS-BIphosphohydrolase

Lb. par. (n=42) 9 30 34 3 42 42 14 − 3 39

Lb. pla. (n=23) 5 11 13 − 23 22 16 − 2 20

S. ther. (n=6) 1 5 4 − 6 6 2 − 1 6

E. dur. (n=6) − 4 6 − 6 6 1 − − 6

Lb. bre. (n=5) − 3 5 − 5 5 3 − − 5

Lb. fer. (n=4) 1 4 4 − 4 4 2 − 1 4

E. fae. (n=5) − 5 5 − 5 5 1 − − 5

Lb. pen.(n=1) − − − − 1 1 − − − −

Un-identified (n=5) − 5 5 − 5 5 1 − 1 5

Total 16 67 76 3 97 96 40 − 8 90

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Table 5 cont.

α- Galactosidase β- Galactosidase β- Glucuronidase α- Glucosidase β- Glucosidase

32 41 24 41 42

10 22 6 21 21

3 6 2 6 6

5 6 2 6 6

5 5 4 5 5

3 4 1 4 4

5 2 1 5 5

− 1 − 1 1

5 5 5 5 5

68 92 45 94 95

−: No activity.

majority of lactobacilli showed esterase (65%) and esterase-lipase (75%) enzyme activities within 24 h, but lipase activity was observed in only 3 (3.1%) isolates. While all enterococci had esterase-lipase activity, only 78% showed lipase activity. Esterase and esterase-lipase enzyme activities were observed in 60 and 90% of streptococci, respectively. When we address the situation in terms of Kargi tulum production on an industrial scale, a large number of isolates which are likely starter cultures and have low lipase, high esterase and esterase-lipase enzyme activities were obtained in our study. β-Galactosidase (lactase) is an enzyme found in milk that hydrolyzes lactose to galactose and glucose monosaccharides. Almost all of our isolates showe β-galactosidase and βglucosidase enzyme activities. Conclusions Kargi tulum cheese is an artisanal cheese produced by small family dairies using raw milk. It is consumed locally, after being ripened in the uplands for 3 to 4 months in a goat-skin bag. Such

products produced though natural fermentation are an important part of Turkey’s cultural heritage. However, no scientific research has been conducted on the indigenous LAB that play a role in the fermentation of this cheese. In our study, homofermentative strains isolated from Kargi tulum cheese samples such as L. paracasei−19 and 111; L. plantarum−10, 45 and 71; S. thermophilus−59; E. durans−9 and 106; and E. faecium−24 and 37 were selected as potential starter cultures. These strains were tolerant to 6.5% salt; had a fast acidifying capacity; produced diacetyl and H2O2; had low proteinase and lipase activities but high peptidase, esterase and esterase-lipase activities; and had βgalactosidase and β-glucosidase activities. Starter cultures prepared from various combinations of the aforementioned indigenous LAB strains can be used in Kargi tulum cheese production with pasteurized milk. The most appropriate starter culture combination to give the traditional texture, flavor and taste to artisanal Kargi tulum cheese should be determined and safe production of the cheese for consumers at industrial scale with standardized quality will be possible. However, further study is required to evaluate the selected

strains, individually and in mixed cultures, on a pilot scale. Before use in industrial production, it would also be beneficial to determine the phage resistance of the selected starter strains. ACKNOWLEDGEMENT This research was supported by the Scientific Research Projects Commission of Eskişehir Osmangazi University (Project number: 200719027). REFERENCES Arora G, Lee BH, Lamoureux M (1990). Characterization of enzyme profiles of Lactobacillus casei species by a rapid API ZYM system, J. Dairy Sci. 73: 264-273. Bayar N, Ozrenk E (2011). The effect of quality properties on Tulum cheese using different package materials, Afr. J. Biotechnol. 10: 1393-1399. Bostan M, Ugur M, Ciftcioglu G (1992). Tulum peynirinde laktik asit bakterileri ve kuf florasi. IU Veteriner Fakultesi Dergisi. 17: 111-118. Broome MC, Hickey MW (1991). Peptidase activity of nonstarter lactobacilli, Aust. J. Dairy Technol. 46: 19-23. Colak H, Hampikyan H, Bingol EB, Ulusoy B (2007). Prevalence of L. monocytogenes and Salmonella spp. in

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Tulum cheese. Food Control, 18: 576-579. Coskun H (2000). Peynirlerin olgunlaşmasında starterlerin rolü. Süt Mikrobiyolojisi ve Katkı Maddeleri. VI. Süt ve Süt Ürünleri Sempozyumu Tebliğler Kitabı, pp. 47-56, Tekirdağ. Dinkci N, Akalin AS, Gonc S, Unal G (2007). Isocratic Reverse-Phase HPLC for determination of organic acids in Kargi Tulum Cheese. Chromatographia Supplement. 66: S45-S49. Efe A, Heperkan D (1995). Tulum peynirlerinde patojen bakteriler, In: II. Gida Muhendisligi Ulusal Sempozyumu, Ankara, Turkey, pp. 45-54. Garabal JI, Rodríguez-Alonso P, Centeno JA (2008). Characterization of lactic acid bacteria isolated from raw cows’ milk cheeses currently produced in Galicia (NW Spain). Food Sci. Technol.-LEB, 41: 1452– 1458. Gordon RE, Haynes WC, Pang CHN (1973). The Genus Bacillus, Handbook United States Department of Agriculture,Washington, D.C. p 427. Halkman AK (1991). Tarim Mikrobiyolojisi. Ankara Üniversitesi Ziraat Fakültesi Yayınları No: 1214. Ankara Üniversitesi Baskı Ofset Ünitesi. Ankara, p. 82 Halkman AK, Taskın Y (2001). Süt Endüstrisinde Starter Kültür. Gıda Mühendisliği Dergisi. 5: 13-18. Hayaloglu AA, Cakmakci S, Brechany EY, Deegan KC, McSweeney PLH (2007a). Microbiology, biochemistry and volatile composition of Tulum cheese ripened in goat’s skin or plastic bags. J. Dairy Sci. 90: 1102–1121. Hayaloglu AA, Fox PF, Guven M, Cakmakcı S (2007b). Cheeses of Turkey: 1. Varieties ripened in goat-skin bags. Lait. 87: 79-95. Hegazi FZ, Abo-Elnaga IG (1980). Production of acetoin and diacetyl by lactic acid bacteria in skimmed milk with added citrate and pyruvate. Z. Lebensm. Unters. F. A. 171: 367-370. King N (1948). Modification of the Voges– Proskauer test for rapid colorimetric determination of acetylmethyl carbinol plus diacetyl in butter cultures. Dairy Ind. 13: 860-866. McSweeney PLH (2004). Biochemistry of cheese ripening. Int. J. Dairy Technol. 57: 127-144. Messens W, De Vuyst L (2002). Inhibitory substances produced by Lactobacilli isolated from sourdoughs-a review. Int. J. Food Microbiol. 72: 31-43. Oksuztepe G, Patir B, Calicioglu M (2005). Identification and distribution of lactic acid bacteria during the ripening of Savak Tulum cheese. Turk. J. Vet. Anim. Sci. 29: 873-879. Olasupo NA, Schillinger U, Holzapfel WH (2001). Studies on some technological properties of predominant lactic acid bacteria isolated from Nigerian fermented foods. Food Biotechnol. 15: 157-167. Perez-Elortondo FJP, Aldamiz EP, Albisu M, Barcina Y (1998). Indigenous lactic acid bacteria in Idizabal ewes’ milk cheese. Int. Dairy J. 8: 725-732. Sengul M, Cakmakci S (2003). Characterization of natural isolates of lactic acid bacteria from Erzincan (Savak) Tulum cheese. Milchwissenschaft . 58: 510-513.

Sisto A, Bellis P, Visconti A, Morelli L, Lavermicocca P (2009). Development of a PCR assay for the strain-specific identification of probiotic strain Lactobacillus paracasei IMPC2.1. Int. J. Food Microbiol. 136: 59-65. Tabasco R, Paarup T, Janer C, Pelaez C, Requena T (2007). Selective enumeration and identification of mixed cultures of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, L. acidophilus, L. paracasei subsp. paracasei and Bifidobacterium lactis in fermented milk. Int. Dairy J. 17: 1107-1114. Torres-Llanez MJ, Vallejo-Cordoba B, Diaz-Cinco ME, MazorraManzano MA, Gonzalez-Cordova AF (2006). Characterization of the natural microflora of artisanal Mexican Fresco cheese. Food Control, 17: 683-690. TS 591 (2006). Standard of White Cheese. Turkish Standards Institution, Ankara, Turkey. TS 3001 (2006). Tulum Cheese. Turkish Standards Institution, Ankara, Turkey. TS 3046 (1978). Cheese-Determination of Fat Content-Van Gulik Method. Turkish Standards Institution, Ankara, Turkey. TS EN ISO 5534 (2006). Cheese and processed cheese-Determination of the total solids content. Turkish Standards Institution, Ankara, Turkey. TS EN ISO 5943 (2001). Cheese and processed cheese products Determination of chloride content- Potentiometric titration method. Turkish Standards Institution, Ankara, Turkey. TS EN ISO 8968-3: ISO 8968-3 (2004). Milk. Determination of nitrogen content. Part 3: Block-digestion method. Turkish Standards Institution, Ankara, Turkey. Walter J, Tannock GW, Tilsala-Timisjarvi A, Rodtong S, Loach DM, Munro K, Alatossava T (2000). Detection and identification of gastrointestinal Lactobacillus species by using denaturing gradient gel electrophoresis and species-specific PCR primers. Appl. Environ. Microbiol. 66: 297-303. Whittenbury R (1964). Hydrogen peroxide formation and catalase activity in the lactic acid bacteria. J. Gen. Microbiol. 35: 13-26.

Full Length Research Paper

Kinetics of exoglucanase and endoglucanase produced by Aspergillus niger NRRL 567 Mohammad Ishfaq Ghori1, Sibtain Ahmed2,3, Mohammad Aslam Malana1 and Amer Jamil2* 1

Department of Chemistry, Bahauddin Zakariya University, Multan, Pakistan. Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad-38040, Pakistan. 3 School of Medicine, University of New Mexico, MSC10-5550, Albuquerque, NM 87131-000, USA.

Accepted 22 March, 2012

In this study we reported for the first time kinetics of exoglucanase (EXG) and endoglucanase (EG) from Aspergillus niger NRRL 567. The optimum pH and temperature for crude EXG and EG was found to be 3.5 and 30°C respectively. Zn2+, Ca2+, Mn2+ and Co2+ enhanced the crude activity of EXG and EG whereas Mg2+, Fe2+ and Hg2+ showed various degree of inhibitory effects. Cu2+ enhanced crude EXG activity and inhibited crude EG activity. The energy of activation (Ea) for the EXG and EG were 21.20 and 22.52 kJ mol-1, respectively. The Q10 values obtained for the EXG and EG were 1.38 and 1.4, respectively. These enzymes had lower Km value that shows their high affinity for the substrates. Overall, the studies demonstrate that these enzymes may be suitable for industrial use. Key words: Exoglucanase, endoglucanase, kinetics, characterization, Aspergillus niger NRRL 567. INTRODUCTION Cellulosic biomass is the largest amount of waste produced by human activities and the most attractive substrate for ‘biorefinery strategies’ to produce high-value products (example fuels, bioplastics or enzymes) through fermentation processes (Mazzoli et al., 2012). These cellulosic waste products can be used by fermentation for the production of useful products. Various alternatives are exercised to diminish this waste by elimination, purification and recycling (Ahmed et al., 2010; Athar et al., 2009). Cellulose is the most abundant organic polymer on this planet and is an important renewable energy source along with sugars and starches (Ahmed et al., 2009b). Cellulose degradation and its subsequent utilizations are important for global carbon sources. The value of cellulose as a renewable source of energy has made cellulose hydrolysis the subject of intense research and industrial interest (Ahmed et al., 2009a). Cellulases and hemicellulases are two important

*Corresponding author. E-mail: amerjamil@yahoo.com. Abbreviations: EXG, Exoglucanase; EG, endoglucanase; Ea, energy of activation.

classes of enzymes produced by microorganisms including filamentous fungi and secreted into the cultivation medium. Cellulase which can hydrolyze cellulose forming glucose and other commodity chemicals can be divided into three types: Endoglucanase (endo-1, 4-β-Dglucanase, EG, EC 3.2.1.4); exoglucanase (also called as cellobiohydrolase) (exo-1, 4-β-D-glucanase, CBH, EC 3.2.1.91) and β-glucosidase (1,4- β -D-glucosidase, BG, EC 3.2.1.21) (Ahmed et al., 2009 b). Cellulases are important enzymes in many proposed processes for producing fuels and chemicals from plant biomass (Wilson, 2012). They also play a key role in increasing the yield of the fruit juices, oil extraction and in improving the nutritive quality of bakery products and animal feed (Bhat, 2000). The application of cellulases to the hydrolysis of lignocellulosic materials (biomass) in order to further convert the released fermentable sugars into ethanol has increased because of their worldwide demand for renewable fuels (de Castro et al., 2010). Xylanases and cellulases together with pectinases account for 20% of the world enzyme market (Ahmed et al., 2007). Filamentous fungi have been used for more than 50 years for the production of industrial enzymes. Trichoderma, Humicola and Aspergillus species were

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shown to be interesting cellulase and xylanase producers (Ghori et al., 2011; Saadia et al., 2008; Irshad et al., 2008; Saleem et al., 2008). Aspergillus sp. is an important commercial source of cellulases for food, textile, and the pharmaceutical industries (Naika and Tiku, 2010). A. niger has been widely used for the production of cellulases, but limited information is available on the kinetics of EXG and EG from A. niger (Hanif et al., 2004). For fermentation processes, use of intensive raw material and process yield (product produced/substrate consumed), in addition to productivity are critical measures of performance and economic viability (Sattar et al., 2008; Ali et al. 2009). In a search for new and efficient cellulases to be used in industry, we optimized various fermentation conditions for the production of cellulases from A. niger NRRL 567 in our previous study (Ghori et al., 2011). As a continuation of our previous study, here we reported some properties and kinetics of the crude exoglucanase (EXG) and endoglucanase (EG) from A. niger NRRL 567.

measuring the enzyme activities in buffers at different pH (3, 3.5, 4, 4.5, 5). Optimum temperature of the crude EXG and EG activity was determined by measuring the enzyme activities at different temperatures (10 to 70°C, with 10°C interval). Effect of metal ions on enzyme activities The effect of several metal ions (Zn2+, Ca2+, Mn2+, Mg2+, Co2+, Cu2+, Fe2+ and Hg2+) on the crude activity of EXG and EG was studied by adding each ion at a concentration of 1 mM. The enzyme solution was pre-incubated with cations in sodium acetate buffer at 4°C for 30 min and the activity was measured under the standard conditions.

Determination of kinetic parameters of crude exoglucanase and endoglucanase EXG was assayed in 0.5M acetate buffer of pH 5.0 with variable concentrations of avicel as substrate. EG was assayed in 0.5M sodium acetate buffer, pH 5.0 with variable concentrations of carboxymethylycellulose (CMC). The data was plotted according to Lineweaver-Burk plot, Woolf-Augustinsson-Hofstee plot and HanesWoolf plot.

MATERIALS AND METHODS Microorganism A. niger NRRL 567 was taken out from our frozen glycerol stock stored at -80°C. A. niger was maintained on agar slants medium which consisted of (g/L); corn stover 20; CaCl2.2H2O 0.05; MgSO4, 0.05; KH2PO4, 1.5; Urea, 3; agar, 20 at 30°C (Ghori et al., 2011).

Activation energy (Ea), increase in reaction rate per 10°C rise in temperature (Q10) and heat of ionization (∆ ∆H) Activation energy of the EXG and EG was determined by using the data for optimum temperature as: Ea = Slope x R

Media and culture conditions

Where, R is the gas constant;

The inoculum for A. niger NRRL 567 consisted of (g/L); corn stover 20; CaCl2.2H2 O 0.05; MgSO4, 0.05; KH2PO4, 1.5; (NH4)2SO4, 2 at pH 3.5 and grown at 30°C on an orbital shaker working at 120 rpm. A 10 mL of liquid culture from the inoculum was transferred to 1000 mL Erlenmeyer flasks containing 250 mL fermentation medium under the same conditions as discussed above. The strain was grown with corn stover (4% w/v), 0.1 % molasses and 1% yeast at pH 3.5 at 30°C. Biomass was harvested by centrifugation at 10,000 × g for 10 min at 4°C. Resulting supernatant was tested for crude EXG and EG activity.

Slope = ∆H/R

Enzyme assay EXG activity was assayed in reaction mixture (1 mL) containing 1% (w/v) avicel, 0.05 M acetate buffer, pH 5.0 and appropriately diluted enzyme solution. After incubation at 60o C for 30 min, the reaction was stopped by adding 3 mL dinitrosalycilic acid reagent (DNS) (Shamala and Sereekanth, 1985). EG activity was assayed in reaction mixture (1 mL) containing 1% (w/v) carboxymethylcellulose (CMC), 1 mL of sodium acetate buffer, pH 5.0 and at 60°C. The reaction was stopped by adding 3 mL DNS reagent (Shamala and Sereekanth, 1985). One unit (IU) of enzyme activity was defined as the amount of enzyme required to liberate 1 µmole of glucose or pnitrophenol from the appropriate substrates under the standard conditions. Effect of pH and temperature on crude exoglucanase and endoglucanase Optimum pH of the crude EXG and EG activity was determined by

The value of activation energy was also used to calculate the increase in reaction rate for every 10°C rise in temperature with the help of following formula (Atkins, 1994):

Q10 =

Ea 1 1 ( − ) K T2 T1

Where, K is the velocity constant (proportional to the rate of reaction). Heat of ionization of cellulases was determined by checking enzyme activities at optimal pH at different temperatures (10, 15, 22, 28, 30, 35 and 40°C). ∆H was calculated as: ∆H = Slope x R x T.

RESULTS AND DISCUSSION Cellulase production by A. niger In our previous study, we found that maximum cellulase production from A. niger NRRL 567 was achieved with 4% corn stover, 0.1% molasses and 1% yeast sludge at optimal pH, temperature and incubation time of 3.5 and 30°C and 96 h, respectively (Ghori et al., 2011). We

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Figure 1. Optimum pH for crude cellulases from A. niger NRRL 567. ♦, EXG; ■, EG.

Figure 2. Optimum temperature for crude cellulases from A. niger NRRL 567. ♦, EXG; ■, EG.

used the same conditions in our current study for the kinetics studies of the crude EXG and EG from A. niger NRRL 567. Effect of pH and temperature on crude exoglucanase and endoglucanase Optimum pH for the crude EXG and EG from A. niger NRRL 567 was found to be 3.5 (Figure 1). Temperature optimum for crude EXG and EG was found to be 30°C (Figure 2). The optimal pH for fungal cellulases varies from species to species though in most cases the optimum pH

ranges from 3.0 to 6.0 (Niranjane et al., 2007; Ahmed et al., 2003, 2005). Our results are in agreement with the observation that stability of the fungal cellulases is commonly between pH 3.0 and pH 8.0 (Xu et al., 2006; Peng et al., 2009). Cellulases in general show optimum temperature between 30 to 55°C (Xu et al., 2006; Peng et al., 2009). Effect of metal ions on crude EXG and EG activities Several metal ions were assayed for their effects on crude EXG and EG activities. The results are shown in Table 1. Zn2+, Ca2+, Mn2+ and Co2+ enhanced EXG activity whereas Mg2+, Fe2+ and Hg2+ showed various

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Table 1. Effect of metal ions on activity of exoglucanase (EXG) and endoglucanase (EG).

Effector Control 2+ Zn 2+ Ca Mn2+ Co2+ 2+ Cu 2+ Mg 2+ Fe 2+ Hg a

Relative activity (%) of EXG 100 ± 2.1 121.2 ± 3.1 148.1 ± 2.3 155.6 ± 0.1 107.7 ± 1.7 121.2 ± 3.1 74.3 ± 2.0 67.5 ± 3.4 60.6 ± 2.3

Relative activity (%) of EG 100 ± 2.3 131.3 ± 3.1 154.6 ± 2.3 145.9 ± 0.1 123.7 ± 1.7 58.3 ± 3.1 70.9 ± 2.0 61.3 ± 3.4 49.6 ± 2.3

The final concentration of the various cations was 1mM. Data are given as mean ± SD.

Table 2. Kinetics parameters for crude EXG and EG from A. niger NRRL 567

Enzyme EXG EG

Lineweaver-Burk plot Km Vmax (mg/mL) (µmol/mL/min) 7.35 1.9 4.75 1.7

Hanes-Woolf plot Km Vmax (mg/mL) (µmol/mL/min) 7.48 1.91 4.13 1.58

degrees of inhibitory effects. Cu2+ enhanced EXG activity and inhibited EG activity. The presence of Mg2+, Fe2+ and Hg2+ is reported to inhibit the activity of cellulases as cited in other literature (Mawadza et al., 2000; Elshafei et al., 2009; Maheshwari et al., 2000; Murashima et al., 2002). It seems that heavy metals attack some certain groups at the active site of enzyme, for example the thiol groups, leading to the inactivation, whereas Mn2+ could enhance the substrate binding affinity of the enzyme and stabilize the confirmation of the catalytic site (Chauvaux et al., 1995; Tao et al., 2010). According to these results , Mg2+, Fe2+ and Hg2+ must be avoided in future cultivations for higher exoglucanase and endoglucanase production from A. niger NRRL 567. Effect of substrate concentration on crude EXG and EG Avicel was used as substrate for EXG whereas CMC was used as a substrate for EG. Kinetics parameters of EXG and EG from A. niger NRRL 567 are presented in Table 2. For crude EXG, by using the Lineweaver-Burk plot (1/V vs 1/S), the Km was 7.35 mg/mL and Vmax was 1.9 µmol/mL/min. By using Hanes-Woolf plot (S) vs. [S]/V), the Km was found to be 7.48 mg/mL and Vmax 1.91 µmol/mL/min. By using Woolf-Augustinsson-Hofstee plot (V/[S] vs. V), the Km was 4.53 mg/mL and Vmax was 1.46 µmol/mL/min (Table 2). For crude EG, by using the Lineweaver-Burk plot (1/V vs 1/S) the Km was 4.75 mg/mL and Vmax was 1.7

Woolf-Augustisson-Hofstee plot Km Vmax (mg/mL) (µmol/mL/min) 4.53 1.46 4.09 1.57

µmol/mL/min. By using Hanes-Woolf plot (S) V [S]/V), the Km was found to be 4.13 mg/mL and Vmax 1.58 µmol/mL/min. By using Woolf-Augustinsson-Hofstee plot (V/S vs. V), the Km was 4.09 mg/mL and Vmax was 1.57 µmol/mL/min (Table 2). Generally, fungal exoglucanases and endoglucanases show specificity toward various substrates. Literature suggests that the kinetic behavior of cellulases might be affected in the presence of other proteins (or substances) in the medium (Rastogi et al., 2010). Our results indicate small Km values for EXG and EG which demonstrates high affinity of EXG and EG with their respective substrate. It is, therefore, concluded that the EXG and EG produced during the present study were good catalytic agents for bioconversion of waste materials into useful products. Energy of activation (Ea) and enthalpy of activation energy (∆ ∆ H*) The energy of activation (Ea) for EXG and EG was 21.20 and 22.52 kJ mol-1; the slope was –2.551 and–2.7092, respectively. From these results, it was found that at 30°C, EXG and EG had maximum catalysis in the conversion of avicel into glucose by using activation energy (Ea) mentioned above. After this temperature, the enzyme starts becoming denatured and show less activity towards the conversion of substrate into product. The small amount of activation energies indicates a good relationship between the enzymes and their substrates. The enthalpy (∆H*) of activation of EXG and EG was 18.68 and 20.00 kJ mol-1. It was concluded that kinetically

Ghori et al.

EXG and EG from A. niger 567 were favorably good for the conversion of cellulose into glucose. Increase in reaction rate per 10°C (Q10) rise in temperature The Q10 values obtained for both EXG and EG were 1.38 and 1.4, respectively. These values show that there was an average of 1.37 times increase in the reaction rate of these enzymes when the temperature was increased from 20 to 30°C. Lower Q10 values demonstrate high catalysis as a distinctive feature of the enzymes catalysis as the Q10 of a catalyzed reaction is lower as compared to the same reaction uncatalyzed. Conclusion This study led us to conclude that this organism may serve as good source of exoglucanase and endoglucanase production, often deficient in many organisms. The optimum pH and temperature for the crude EXG and EG were found to be 3.5 and 30°C, respectively; hence these enzymes can be used in food industry where high temperature is not required. This study will help in the future production of cellulases at industrial scale from A. niger NRRL 567. REFERENCES Ahmed S, Qurrat-ul-Ain, Aslam N, Naeem S, Sajjad-ur-Rehman, Jamil A (2003). Induction of xylanase and cellulase genes from Trichoderma harzianum with different carbon sources. Pak. J. Biol. Sci. 6(22): 1912-1916. Ahmed S, Aslam N, Latif F, Rajoka MI, Jamil A (2005). Molecular cloning of cellulase genes from Trichoderma harzianum. (Eds.): Attaur-Rehman/ Choudhary/ Khan, Frontiers in Natural Product Chemistry. Bentham Science Publishers, The Netherlands. 1: 73-75. Ahmed S, Jabeen A, Jamil A (2007). Xylanase from Trichoderma harzianum: Enzyme characterization and gene isolation. J. Chem. Soc. Pak. 29(2): 176-182. Ahmed, S, Riaz S, Jamil A (2009a). Molecular cloning of fungal xylanases: an overview. Appl. Microbiol. Biotechnol. 84(1):19-35. Ahmed S, Bashir A, Saleem H, Saadia M, Jamil A (2009b). Production and purification of cellulose-degrading enzymes from a filamentous fungus Trichoderma harzianum. Pak. J. Bot. 41(3): 1411-1419. Ahmed S, Ahmad F, Hashmi SA (2010). Production of Microbial biomass protein by sequential culture fermentation of Arachniotus sp. and Candida utilis. Pak. J. Bot. 42(2): 1225-1234. Ali S, Ahmed S, Sheikh MA, Hashmi AS, Rajoka MI, Jamil A (2009). Lysine production by L-homoserine resistant mutant of Brevibacterium flavum. J. Chem. Soc. Pak. 31: 97-102. Athar M, Ahmed S, Hashmi AS (2009). Bioconversion of beet pulp to microbial biomass protein by Candida utilis. J. Chem. Soc. Pak. 31(1): 115-121. th Atkins P (1994). Physical chemistry, 5 ed., p. 1031. W.H. Freeman , Company, New York. Bhat MK (2000). Cellulases and related enzymes in biotechnology. Biotechnol. Adv. 18(5): 355-383. Chauvaux S, Souchon H, Alzari PM, Chariot P, Beguin P (1995). Structural and functional analysis of the metal-binding sites of Clostridium thermocellum endoglucanase CelD. J. Biol. Chem. 270(17): 9757-9762.

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de Castro AMDE, de Carvalho MLDA, Leite SGF, Pereira N. (2010). Cellulases from Penicillium funiculosum: production, properties and application to cellulose hydrolysis. J. Ind. Microb. Biotechnol. 37(2): 151-158. Elshafei AM, Hassan MM, Haroun BM, Abdel-Fatah OM, Atta HM, Othman AM. (2009). Purification and properties of an endoglucanase of Aspergillus terreus DSM826. J. Basic Microbiol. 49(5): 426-432. Ghori MI, Ahmed S, Malana MA, Jamil, A. (2011). Corn stoverenhanced cellulase production by Aspergillus niger NRRL 567. Afr. J. Biotechnol. 10(31): 5878-5886. Hanif A, Yasmeen A, Rajoka MI (2004). Induction, production, repression, de-repression of exoglucanase synthesis in Aspergillus niger. Bioresour. Technol. 94(3): 311-319. Irshad M, Ahmed S, Latif F, Rajoka MI (2008). Regulation of Endo- β-DXylanase and β- Xylosidase synthesis in Humicola lanuginosa. J. Chem. Soc. Pak. 30(6): 913-918. Maheshwari R, Bharadwaj G, Bhat M (2000). Thermophilic fungi: their physiology and enzymes. Microbiol. Mol. Biol. Rev. 64(3): 461-488. Mawadza R, Hatti-Kaul, Zvauya R, Mattiasson B (2000). Purification and characterization of cellulases produced by two Bacillus strains. J. Biotechnol. 83(3): 177-187. Mazzoli M, Lamberti C, Pessione E (2012). Engineering new metabolic capabilities in bacteria: lessons from recombinant cellulolytic strategies. Trends Biotechnol. 30(2): 111-119. Murashima K, Nishimura T, Nakamura Y, Kuga J, Moriya T, Simuda N. (2002). Purification, characterization of new endo-1, 4- βDglucanases from Rhisopus oryzae. Enzyme Microb. Biotechnol. 30(3): 319-326. Naika GS, Tiku PK. (2010). Characterization of functional intermediates of endoglucanase from Aspergillus aculeatus during urea guanidine hydrochloride unfolding. Carbohydr. Res. 345(11): 627-631. Niranjane AP, Madhou P, Stevenson TW (2007). The effect of carbohydrate carbon sources on the production of cellulase by Phlebia gigantean. Enzyme Microbial. Technol. 40(6): 1464-1468. Peng Y, Chi ZM, Wang XH, Li J (2009). Purification and molecular characterization of exo-beta-1,3-glucanases from the marine yeast Williopsis saturnus WC91-2. Appl. Microbiol. Biotechnol. 85(1): 8594. Rastogi G, Bhalla A, Adhikari A, Bischoff KM, Hughes SR, Christopher LP, Sani RK. (2010). Characterization of thermostable cellulases produced by Bacillus and Geobacillus strains. Bioresour. Technol. 101(22): 8798-806. Saadia M, Ahmed S, Jamil A (2008). Isolation and cloning of cre1 gene from a filamentous fungus Trichoderma harzianum. Pak. J. Bot. 40(1): 421-426. Saleem F, Ahmed S, Jamil A (2008). Isolation of a xylan degrading gene from genomic DNA library of a thermophilic fungus Chaetomium thermophile ATCC 28076. Pak. J. Bot. 40(3): 1225-1230 Sattar M, Ahmed S, Sheikh MA, Hashmi AS (2008). Fermentation of yeast sludge with Brevibacterium flavum to enhance lysine concentration. J. Chem. Soc. Pak. 30(4): 642-648. Shamala TR, Sreekantiah KR (1985). Production of cellulases and Dxylanase by some selected fungal isolates. Enzyme Microbial. Technol. 8(3): 178-182. Tao YM, Zhu XZ, Huang JZ, Ma SJ, Wu X, Long MN, Chen QX (2010). Purification and properties of endoglucanase from a sugar cane bagasse hydrolyzing strain, Aspergillus glaucus XC9. J. Agric. Food Chem. 58(10): 6126-6130. Wilson DB (2012). Processive and nonprocessive cellulases for biofuel _ production lessons from bacterial genomes and structural analysis. Appl. Microbiol. Biotechnol. 93(2): 497-502. Xu Z, Shih MC, Poulton JE (2006). An extracellular exo-beta-(1,3)glucanase from Pichia pastoris: purification, characterization, molecular cloning, and functional expression. Protein Expr. Purif. 47(1): 118-127.

African Journal of Biotechnology Vol. 11(28), pp. 7232-7237, 5 April, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.831 ISSN 1684â&#x20AC;&#x201C;5315 ÂŠ 2012 Academic Journals

Full Length Research Paper

Detoxification of cyanides in cassava flour by linamarase of Bacillus subtilis KM05 isolated from cassava peel Kasi Murugan1, 2*, Yashotha2, Kuppusamy Sekar 3 and Saleh Al- Sohaibani1 1

Department of Botany and Microbiology, College of Science, King Saud University, P.O.Box 2455, Riyadh-11451, Saudi Arabia. 2 P.G and Research Department of Microbiology, K.S.R. College of Arts and Science, Tiruchengodu-637209, Tamilnadu, India. 3 Research and Development Center, Bharathiyar University, Coimbatore-641046, Tamilnadu, India Accepted 27 October, 2011

Defensive cyanogenic glucoside linamarin accounts for 80% cyanide content of cassava and is known to cause severe diseases upon continual consumption. Detoxification of this cyanide would enhance the nutritive quality and hence market value of cassava flour. We isolated cyanogenic glucoside utilizing indigenous bacteria from cyanide rich cassava peel waste and exploited their potential for detoxification. Among the isolates, Bacillus subtilis KM05 utilized cyanogenic glycoside through assimilatory degradation with the release of hydrogen cyanide and ammonia. The partially purified linamarase (53 KDa) enzyme from this organism showed considerable activity (9.6 U/ml) and effected rapid cyanide reduction in cassava flour. The results indicate scope for enzymatic detoxification of cassava cyanide without compromising nutrients in sago industries. Key words: Cyanogenic glucoside, enzymatic treatment, linamarin, linamarase. INTRODUCTION Cassava, the vital food in the tropical areas of Africa, Asia and Latin America is the third most important source of calories in the tropics, after rice and corn. The enlarged root of the cassava plant contains a highly digestible starch of important nutritional value. Products derived from cassava are the principal food source of 500 million to 1 billion people in tropical countries (Sornyotha et al., 2010). Over 160 million tons of cassava is produced globally per annum, ranking it as the 4th crop in worldwide production after rice, wheat and maize (Ugwuanyi et al., 2007). This major stable food has two major deficiencies, that is, high content of the poisonous cyanogenic glucoside linamarin (and to a lesser extent,

*Corresponding author. Email: murutan@gmail.com Tel: +966 14675822.

lotaustralin), and low content of protein and free amino acid (Cooke and Coursey, 1981). The perishable tuber is normally stored as cassava flour, a substitute for wheat and rice. Cassava flour is produced primarily by the wet milling of fresh cassava roots which includes the following five main stages: preparation (peeling and washing), rasping/pulping/ grating, purification (starch washing), dewatering and drying, and finishing (milling and packaging). Upon tissue disruption, the cyanogenic glucosides are brought in contact with glycosidase and hydroxynitrile lyases and are degraded into cyanohydrins, hydrogen cyanide and ketones (Conn, 1980). When cassava products are used as a primary staple food, careful processing to remove these toxic constituents is required to avoid chronic cyanide intoxication (Onabolu et al., 2002). These cyanogens upon intake may cause cyanide poisoning with symptoms of vomiting, nausea, dizziness, stomach

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Figure 1. Enzyme catalyzed degradation of cyanogenic glycosides.

pains, weakness, headache and diarrhea and occasionally death (Akintonwa et al., 1994; Mlingi et al., 1995). While incomplete processing result in high cyanide exposure and associated severe diseases like tropical ataxic neuropathy, konzo, etc., the careful processing generally results in loss of proteins, vitamins and minerals. Hence, a well-organized method for detoxifying cassava without nutrient loss is desired. Though a number of processes aiming at the degradation and thereby reduction in cyanide content were developed, all of them either failed to yield the expected reduction in cyanide or resulted in the reduction of the nutrient composition. Silano et al. (1982) reported that about 50% of the nitrogen content of cassava is in the form of free amino acids and they may be lost during processing. Separation of the starch granules from the tuber in pure form is essential in the manufacture of cassava flour. The linamarin is a Î˛-glucoside of acetone cyanohydrin and ethyl-methyl-ketone-cyanohydrin whose Linamarin Î˛linkage can only be broken under high pressure, high temperature and use of mineral acids, while its enzymatic break occurs easily (Cereda and Mattos, 1996). Hence removal or conversion of these antinutrient cyanogenic glycosides would be possible through target specific enzymatic conversion if the methods of application are standardized. Linamarase or Î˛-D-glucosidase (EC 3.2.1.21), an enzyme found in many plants including cassava and in microorganisms converts the cyanide containing compounds into acetone cyanohydrins, which spontaneously decomposes to hydrogen cyanide (HCN)

(Rolle, 1998). The HCN then either dissolves readily in water or is released into the air (Figure 1). When the roots are completely disrupted, all linamarin will come out in contact with its hydrolytic enzyme (linamarase), resulting in hydrolysis and subsequent removal of the breakdown products during washing. Then, addition of exogenous linamarase directly into completely disrupted root is sufficient to hydrolyze all the free linamarin. Several investigators have explored linamarase to facilitate the process of cassava cyanide detoxification (Ikediobi and Onyike, 1982; Petruccioli et al., 1999; Yeoh and Sun, 2001). The objective of this study was to ameliorate cassava starch quality through bioprocesses, towards reduction of any remaining linamarin in the mash without any nutritional compromise and if possible simultaneously increase the protein content using linamarase isolated from indigenous bacteria degrading linamarin rich cassava peel wastes. MATERIALS AND METHODS Isolation of cyanide degrading bacteria Composite soil and partially degraded cassava peel were collected from waste dumping sites at the cassava processing unit, SPAC Tapioca products and Chellapa sago factory located in Poonachi, Erode district of Tamilnadu, India. The cyanide-utilizing microorganisms were isolated from the cassava peel soil waste by inoculating 1 g of sample into an isolation medium containing K2HPO.2H2O, 1.0 g; MgSO4.7H2O, 0.2 g; CaCl2, 0.01 g; NaCl2, 0.01 g; MnSO4 , 0.2 mg; CuSO4 .5H2O, 0.2 mg; ZnSO4, 0.2 mg; glucose, 2.0 g; tryptone, 1.0 g (Watanabe et al., 1998). The enriched culture

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was screened for linamarin utilization and linamarase production by spreading appropriately diluted growth on sterile nutrient agar plates having 2 ml of 1% p-nitrophenyl-β-D glucoside (PNPG). A control plate contained PNPG but not the organism. All plates were incubated at 30°C and examined after 24 h. Since linamarase is a β-glucosidase, PNPG brokedown into citron-yellow coloured nitrophenol which diffuse into the medium around the colony indicating linamarase activity. Positive isolates were graded in the same way as for linamarin assay, based on the strength and spread of the yellow coloration. Isolates that grew on the plate but without yellow coloration beyond the colony margin were considered negative (Ugwuanyi et al., 2007). The glycogenic cyanide utilization by the isolates was confirmed using a medium containing bacteriological peptone, 0.5 g; meat extract powder, 0.2 g; Tween 80, 0.1 ml; mineral solution (ZnSO4.7H2O, 1 g; CuSO4. 5H2 O, 0.5 g / 100 ml), 0.5 ml; agar, 1 g; sodium phosphate buffer, 0.1 M; and 0.01% linamarin [previously isolated from young leaves of cassava and partially purified as described by King and Bradbury (1995). Inoculated agar plates were then incubated at 37°C for 48 to 72 h. The breakdown of linamarin during growth was detected by means of placing aseptically a filter paper impregnated with alkaline picric acid into the headspace of agar slant. Discoloration of filter paper caused by HCN was taken as indication of linamarase activity (Okafor and Ejiofor, 1986). Controls were setup by incubating uninoculated medium. The cyanide degrading bacterial isolate obtained was characterized and identified on the basis of cell and colony morphology, Gram staining, physiological and biochemical reactions according to Bergey’s manual (Holt et al., 1994). Cyanide utilization pattern of the isolate Determination of hydrogen cyanide production (Lorck, 1948) A modified nutrient agar medium having peptone, 10 g; NaCl, 5 g with 4.4 g glycine/lit was prepared and sterilized. Cyanide degrading bacteria were streaked and Whatman filter paper No 1 soaked in sodium carbonate (2%) in 0.5% picric acid solution was placed on the top of the plate. Plates were sealed with parafilm and incubated at 37°C for four days. After incubation, colour change from yellow to brown was observed. Determination and quantification of ammonia release (Ahmad et al., 2002) Cyanide utilizing bacteria was inoculated into 10 ml of peptone water in each tube and incubated for 48 h at 37°C. After incubation, 0.5 ml of Nessler’s reagent was added into tubes and the colour change was observed (pale yellow to dark yellow). The released ammonia was assayed by mixing 0.5 ml samples with equal volume of a 1:3 dilution of commercially available Nessler’s reagent and measuring the absorbance at 420 nm using SmartSpec 3000 spectrophotometer (Bio-Rad Laboratories, Richmond, USA).

Enzyme extraction Cell-free extracts were prepared from cells grown in M9 minimal medium (without ammonium and citrate) adjusted to pH 9.5 on a rotatory shaker (Thermoscientific, USA) at 230 rpm and 30°C in the presence of 2 mM linamarin solution as nitrogen source and 50 mM acetate as carbon source (Luque-Almagro et al., 2005). Cells were harvested at the latest stage of the exponential growth and resuspended in 50 mM Tris/HCl buffer (pH 8.5). Cells were broken

by cavitations and three pulses of 5 S at 90 W. After centrifugation (Sigma Laborzentrifugen GmbH, Germany) at 19000 g, supernatants were collected and used as a source of enzymes. The cellfree extract was subjected to ammonium sulphate precipitation (30 to 60%) and the precipitate obtained after centrifugation, was dissolved in 20 mm Tris-HCl buffer (pH 8.0) and dialyzed against the same buffer. Protein concentrations were determined as described (Bradford, 1976) using bovine serum albumin as a standard. The extent of the purity of preparation and the molecular mass of the purified enzyme preparation were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) analysis. The test sample solution was prepared by dilution (1:1) in sample solubilizing buffer, which were placed for 10 min in a boiling (100°C) water bath. After cooling to room temperature, the samples were spinned for 1 min. The samples containing equal amount of proteins were loaded into the wells of polyacrylamide gels. The medium range molecular weight markers were used and electrophoresis was carried out at constant voltage of 75 V for 2 h. The gels were stained with 0.2% Coomassie brilliant blue solution. Enzyme assay The linamarase activity of the isolate was quantified as described (Ugwuanyi et al., 2007). Quantification of linamarase was based on the degradation of linamarin analogue PNPG and determination of the released p-nitrophenol (Ikediobi et al., 1980). The assay medium included 0.5 ml of enzyme extract, and 1.0 ml of 5 mM PNPG in 0.01 M phosphate buffer with pH 6.8. The mixture was incubated for 15 min at 65°C, and the reaction was terminated by the addition of 2 ml of 0.2 M borate buffer with pH 9.8. Colour of the released p-nitrophenol was measured at 425 nm in Genesys 20 Spectrophotometer against enzyme blank. Temperature inactivated enzyme was used as blank. One unit of linamarase activity was expressed as the amount that caused a change in absorbance of 0.01 units against an enzyme blank under the defined assay conditions (Ugwuanyi et al., 2007). Effect of enzymatic treatment on cassava flour An aliquot of 500 mg of cassava flour sample was put in a plastic vial, and a small filter paper impregnated with pH 6 buffer was placed in the vial. Linamarase, followed by 2.5 ml of water and a yellow picrate paper were added to the vial and the cap was immediately closed and left at 30°C overnight. The next day, the yellow–brown picrate paper was separated from the plastic backing strip and placed in 25 ml of water. The absorbance of the solution was measured at 510 nm and the total cyanide content in ppm was calculated by multiplying by 396 (Bradbury et al., 1999). To determine and compare the effect of enzyme treatment on cassava flour, two sets each containing 5 g of flour were taken in a beaker. One batch was well mixed with 6.25 ml water, the other with 5 ml water and 1.5 ml enzymatic extract. The beakers were placed in an incubator at 30°C for 5 h, after which the cyanide analysis was carried out as described (Cumbana et al., 2007). The changes in moisture (923.03), crude protein (Kjeldhal method, N×6.25), fat (Soxhlet method) and ash (923.10) were also determined as specified by the Association of Official Analytical Chemists (AOAC, 1995).

RESULTS 15 isolates with different colony morphology that showed

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Table 1. Taxonomic characteristics of B. subtilis KM05.

S/N 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Name of test Gram staining Endospore Acid fast staining Oxidase Catalase Vogus- Proskaur Methyl red Indole Citrate Esculin hydrolysis Casein hydrolysis Gelatin hydrolysis Growth at 6.5% NaCl Growth above 55.c

utilization of cyanide on screening medium were obtained and all the isolates were screened for their ability to utilize cyanogenic glycoside as their substrate. Positive isolates were graded based on the strength and spread of the yellow coloration in the PNPG medium. Out of 15 bacterial isolates, only one was selected based on the cyanogenic glycoside utilization pattern and discoloration of the picrate paper color from yellow to brown. Based on the cultural and biochemical tests, the cyanogenic glycoside utilizing bacteria were identified as Bacillus subtilis. The cultural and biochemical characteristics of the B. subtilis KM05 are presented in Table 1. They formed flat, round rough reticulate colonies having diameter of 5 to 7 mm on nutrient agar plates. It was observed that B. subtilis produced hydrogen cyanide (HCN) in addition to ammonia during cyanogenic glycoside utilization. Results obtained for the partial purification of B. subtilis KM05 cyanide degrading enzyme by conventional ammonium sulphate fractionation are shown in Figure 2. The protein precipitate formed within the saturation range of 30 to 60% was found to retain the enzyme activity. Electrophoretic analysis of crude extract and ammonium sulphate precipitate showed the presence of single band with a molecular mass of 53 KDa (Figure 2). The protein concentration in the enzyme preparation was found to be 0.23 mg/ml and the linamarase activity was found to be 9.6 U/ml. It was also inferred that the enzyme has the ability to degrade cyanogenic glycosides effectively. The results presented in Table 2 revealed that the untreated cassava flour had higher cyanide content and a significant reduction in both wet and enzyme treated flour. Before inoculation, the cyanide concentration of the flour was found to be 210 ppm/kg. The cell free extract on treatment of cassava flour extract over night led to rapid

Result Gram positive, straight, large, uniform multiseptate Positive Negative Negative Positive Negative Negative Negative Positive Positive Positive Positive Negative Negative

reduction in cyanide concentration of 8 ppm/kg, whereas in the untreated control, the cyanide concentration remained as 35 ppm/kg (Table 2). It was also observed that there was no significant change in the nutrient level except the small increase in protein. Results indicate that B. subtilis KM05 enzyme effectively detoxified the cassava cyanogenic glycosides. DISCUSSION The inadequate processing methods of cassava often result in linamarin and its hydrolytic product cyanohydrin, as a residue in the product. Hence, there is a need to improve the methods of processing cassava to foods that are popular in different communities in the tropics (Oluwole et al., 2002). The new processing method suggested to remove cyanogens from cassava flour involves mixing dry flour with water and leaving the wet flour in a thin layer in the shade for 5 h or for 2 h in the sun to allow the catalyzed breakdown of linamarin to hydrogen cyanide, facilitating a three to six fold reduction in total cyanide content of cassava flour. The wet flour obtained can be used for cooking on the same day (Bradbury and Denton, 2010). Even though a considerable level of cyanide content is reduced, many times, the residual cyanide level exceeds FAO limit of 10 ppm and also it depends exclusively on endogenous enzyme level which shows considerable variation. Several microorganisms including Bacillus sp. (AmoaAwua and Jakobsen, 1995), lactic acid bacteria (Cohen, 1994), Lactobacilli, Leuconostoc, Streptococci and yeasts (Obilie et al., 2004), Aspergillus, Fusarium, Penicillium and Trichoderma strains (Yeoh et al., 1995) are known for their detoxification activities on native cassava

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Figure 2. SDS-PAGE of B. subtilis KM05 linamarase showing lane 1 (medium range protein markers), lanes 2 and 3 (crude extracts) and lane 4 (ammonium sulphate precipitated sample).

Table 2. Changes in the nutrient and cyanide level in the treated and raw cassava flour.

S/N

Type of treatment

1 2 3

Raw flour Enzyme treated flour Wet treated flour

Moisture 14±0.4 13±0.5 13±0.6

Nutritional parameter Crude protein Fat 1.36±0.2 0.16±0.01 1.46±0.4 0.17±0.02 1.31±0.2 0.15±0.01

fermentation. To the authors’ knowledge, this study reports for the first time the potential of cassava peel waste degrading organisms in cassava detoxification meant for human usage. Species of Bacillus, Pseudomonas and Klebsiella oxytoca have been reported to biodegrade cyanide to non-toxic end-products using cyanide as the sole nitrogen source under aerobic and/or anaerobic environment (Kaewkannetra et al., 2009). Some previous studies reported Bacillus species involvement in detoxification associated with cassava fermentation. However, their role varies as determined by the type of fermentation (Amoa-Awua and Jakobsen, 1995; Oyewole and Odunfa, 1988). Oyewole (1992) noted that in the submerged fermentation process for ‘fufu’ production, Bacillus species which appear at the beginning of the fermentation became extinct towards the end of the process, while Amoa-Awua and Jakobsen (1995) reported the high occurrence and persistence of Bacillus species throughout the dough fermentation during the solid state fermentation (SSF) of cassava to ‘agbelima’ as done in Ghana. Hence instead of applying the whole cell

Ash 0.96±0.01 0.96±0.02 0.96±0.01

Cyanide 210±7 8±2 35±5

fermentation, the application of its enzyme would achieve the desired result. A cyanotrophic microorganism requires an assimilatory pathway that is able to convert cyanide into ammonium (Luque-Almagro et al., 2005). The formation of HCN and release of ammonia as an end product evidenced the assimilatory degradation of linamarin by B. subtilis KM05. It is also evident that this bacterium is able to grow, utilizing linamarin as the sole N source and acetate as the C source. Therefore, this strain offers new perspectives in the detoxification of cassava cyanide. The increase in the protein content of the cassava products could be attributed to the enzyme extract addition. It has been observed earlier that the SSF, employing various fungi improves the protein content and also reduces the antinutrients but separation of the fermentative organisms from the mass is practically difficult and limits their application. Hence, this study on nutrient enrichment and detoxification of cassava flour using enzyme from easily growing cheap, non-pathogenic bacteria B. subtilis KM05 would increase the productivity, efficiency and quality output in cassava based industrial processing operations

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in many developing countries. ACKNOWLEDGEMENT The authors acknowledge the Rector, KSU and the Deanship of Scientific Research, College of Science Research Center, King Saud University for their support of this project REFERENCES Ahmad A, Melih K, Metin G (2002). The removal of cyanides from water by catalytic air oxidation in a fixed bed reactor. J. Chem. Eng. Process. 41(6): 525-529. Akintonwa A, Tunwashe O, Onifade A (1994). Fatal and non-fatal acute poisoning attributed to cassava-based meal. In: Bokanga MA, Essers AJA, Poulter N, Rosling H, Tewe O (eds). International workshop on cassava safety. Acta Horticulturae -375, Leuven, ISHS. pp. 285-288. Amoa-Awua WKA, Jakobsen M (1995). The role of Bacillus species in the fermentation of cassava. J. Appl. Bacteriol. 79(3): 250-256. AOAC (1995). Official Methods of Analysis. Association of Official th Analytical Chemists. (15 Ed). Arlington. Association of Analytical Chemists. Bradbury MG, Egan SV, Bradbury JH (1999). Determination of all forms of cyanogens in cassava roots and cassava products using picrate paper kits. J. Sci. Food Agric. 79(4): 593-601. Bradbury JH, Denton IC (2010). Simple method to reduce the cyanogen content of gari made from cassava. Food Chem. 123(3): 840-845. Bradford MM (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72(1-2): 248-254. Cereda MP, Mattos MCY (1996). Linamarin - the toxic compound of cassava. J. Venom. Anim. Toxins. 2(1): 6-12. Cohen ME (1994). Detoxification of cassava by lactic acid bacteria. Process No. 1099, 39. Conn EE (1980). Cyanogenic compounds. Annu. Rev. Plant Physiol. 131(1): 433-451. Cooke RD, Coursey DG (1981). Cassava: A major cyanide containing food crop. In: Vennesland B, Conn EE, Knowles CJ, Wiseley J, Wissing F (eds). Cyanide in biology. Academic Press, New York, pp. 93-114. Cumbana A, Mirione E, Cliff J, Bradbury JH (2007). Reduction of cyanide content of cassava flour in Mozambique by the wetting method. Food Chem. 101(3): 894-897. Holt JG, Krieg NR, Sneath PHA, Staley JT, Williams ST (1994). Bergey’s Manual of Determinative Bacteriology. Williams & Wilkins, Baltimore. Ikediobi CO, Onyia GOC, Eluwah CE (1980). A rapid and inexpensive enzymatic assay for total cyanide in cassava (Manihot esculenta Crantz) and cassava products. Agric. Biol. Chem. 44(8): 2803-2809. Ikediobi CO, Onyike E (1982). The use of linamarase in gari production. Process Biochem. 17(4): 2-5. Kaewkannetra P, Imai T, Garcia-Garcia FJ, Chiu TY (2009). Cyanide removal from cassava mill wastewater using Azotobactor vinelandii TISTR 1094 with mixed microorganisms in activated sludge treatment system. J. Hazard. Mater. 172(1): 224-228. King NLR, Bradbury JH (1995) Bitterness of cassava: identification of a new apiosyl glucoside and other compounds that affect its bitter taste. J. Sci. Food Agric. 68(2): 223-230.

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Lorck H (1948). Production of hydrocyanic acid by bacteria. J. Plant Physiol. 1: 142 146. Luque-Almagro VM, Huertas MJ, Martínez-Luque M, Moreno-Vivián C, Roldán MD, García-Gil J, Castillo F, Blasco R (2005). Bacterial degradation of cyanide and its metal complexes under alkaline conditions. Appl. Environ. Microbiol. 71(2): 940-947. Mlingi NLV, Bainbridge ZA, Poulter NH, Rosling H (1995). Critical stages in cyanogen removal during cassava processing in southern Tanzania. Food Chem. 53(1): 29-33. Obilie EM, Tano-Debrah K, Amoa-Awua WK (2004). Souring and breakdown of cyanogenic glucosides during the processing of cassava into akyeke. Int. J. Food Microbiol. 93(1): 115-121. Okafor N, Ejiofor MAN (1986). The microbial breakdown of linamarin in fermenting pulp of cassava (Manihot esculenta Crantz). J. Microbiol. Biotechnol. 2: 327-338. Oluwole OSA, Onabolu AO, Sowunmi A (2002). Exposure to cyanide following a meal of cassava food. Toxicol. Lett. 135(1-2): 19-23. Onabolu AO, Oluwole OSA, Rosling H, Bokanga M (2002). Processing factors affecting the level of residual cyanohydrins in gari. J. Sci. Food Agric. 82: 966-969. Oyewole OB, Odunfa SA (1988). Microbiological studies on cassava fermentation for ‘lafun’ production. Food Microbiol. 5(3): 125-133. Oyewole OB (1992). Cassava processing in Africa. Application of biotechnology to traditional fermented foods. National Research Council, National Academy Press, WA, USA. pp. 89-92. Petruccioli M, Brimer L, Cicalini AR, Federici F (1999). The linamarase of Mucor circinelloides LU M40 and its detoxifying activity on cassava. J. Appl. Microbiol. 86(2): 302-310. Rolle RS (1998). Enzyme applications for agro-processing in developing countries: an inventory of current and potential applications. World J. Microbiol. Biotechnol. 14(5): 611-619. Silano V, Banzul HC, Bozzini A (1982). Improvement of nutritional quality of food crops-a state of the art report. In: FAO plant production and protection paper No. 34. FAO, Rome. Sornyotha S, Kyu KL, Ratanakhanokchai K (2010). An efficient treatment for detoxification process of cassava starch by plant cell wall-degrading enzymes. J. Biosci. Bioeng. 109(1): 9-14. Ugwuanyi JO, Harvey LM, McNeil B (2007). Linamarase activities in Bacillus spp. responsible for thermophilic aerobic digestion of agricultural wastes for animal nutrition. Waste Manage. 27(11): 15011508. Watanabe A, Yano K, Ikebukuro K, Karube I (1998). Cyanide hydrolysis in a cyanide-degrading bacterium, Pseudomonas stutzeri AK61, by cyanidase. Microbiology, 144(6):1677-1682. Yeoh HH, Tan TK, Loh CM (1995). Sources of fungal linamarases. World J. Microbiol. Biotechnol. 11(6): 678-680. Yeoh HH, Sun F (2001). Assessing cyanogen content in cassava-based food using the enzyme-dipstick method. Food Chem. Toxicol. 39(7): 649-653.

Full Length Research Paper

Effect of γ-irradiation on the physicochemical properties of mixed soy protein isolate/starch material Weihe Xu1, Boye Liu2, Hongshun Yang2, Kunlun Liu2, Suxian Jia2 and Fusheng Chen2 1

College of Chemistry and Chemical Engineering, Henan University of Technology, Zhengzhou 450052, China. 2 College of Food Science and Technology, Henan University of Technology, Zhengzhou 450052, China. Accepted 9 March, 2012

To elucidate the effect of γ-irradiation on the molecular structure and mechanical properties of biodegradable material composed by soybean protein isolated (SPI) and starch, SPI and starch were mixed and modified by succinic anhydride and irradiation. Different doses (0 to 50 kGy) γ-irradiation on the elongation, tensile strength, viscosity, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and color of the mixed material were investigated. Gamma-irradiation decreased the viscosity of the material due to cleavage of the polypeptide chains of SPI. Irradiation treatment also increased the yellowness index of the mixed material. This study reveals that γirradiation can be applied as a useful cross-linking measure to improve properties of mixed SPI and starch, which provide a valuable chance to replace conventional petroleum-derived plastics, adding more value to this vast agricultural resource. Key words: Soybean protein isolate, starch, biodegradable material, irradiation. INTRODUCTION Since the 20th century, research of synthesis of high polymer plastic progresses has advanced by leaps and bounds which brings the huge convenience for people's life (Derraik, 2002). However, with the growing production of plastic, it also brought lots of serious environmental pollution all over the world. For instance, groundwater and soil are seriously polluted; the safety of animals is also threatened (Lithner et al., 2011), landfill occupies land for a long time, and burning brings toxic gas which leads to further pollution (Lemieux et al., 2004). The annual generation of plastic wastes in China amounts to 4 million tons with only 10% recovered and recycled (dropped from about 20% in 1980), 20 to 30% incinerated or landfilled and 60 to 70% dumped, littered and washed away (Ren, 2003). As a result of increasing concerns of environmental pollution caused by nonbiodegradable petroleum-based plastics and exhausting of petroleum resources (Jan and Filip, 2009), researchers are interested in making natural degradable and environmentally-friendly plastics from renewable resources

*Corresponding author. E-mail: weihexu@haut.edu.cn. Tel: +86 371 67756837.

as alternatives to non-biodegradable petroleum-based plastics. Therefore, the search for new resources of sustainable development, exploration and research of environment-friendly materials become an important topic (Bordes et al., 2009). In order to solve these problems, people have continued to carry out new explorations; the research of soy protein isolate/starch biodegradable materials has become a hot issue recently. As inexhaustible natural polymer materials, soy protein isolate/starch can be easily modified by physicochemical methods (Park et al., 2000; Liu et al., 2008). At the same time, they are biodegradable, biocompatible and safe, etc. Unfortunately, biodegradable materials made from soybean proteins have poor mechanical and water resistant properties, which are limitations for plastic materials (Gonzalez et al., 2011). Therefore, it is necessary to add filling agents to improve plastics processing properties, and some suitable modified methods and small molecule plasticizers (Tian et al., 2009; Kumar et al., 2009; Mo et al., 2002; Kalichevsky et al., 1993) can be used to improve the plastic's function. Soybean protein isolated (SPI) contains a great deal of hydrophilic groups, such as hydrogen bond, carboxyl, hydroxy and amino, etc. Due to its structural characteristics, aldehyde group of starch

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can be integrated with SPI, which is helpful to increase the mixture molecular mass with the resulting materials having better water resistance properties. These biomaterials made by the pure SPI are rigid, brittle and expensive. As a filling agent, starch enhances the material’s process ability and reduces cost. In past studies, soybean protein-based materials were prepared by injection molding (Otaigbe et al., 1999), compression molding (Mo and Sun, 1999; Ogale et al., 2000) and lamination (Ghanbarzadeh and Oromiehi, 2009). As a new technology, irradiation technique has its own strong points, such as, been suitable for low temperature processing, has good maneuverability, high purity and good quality, less energy consumption and it meets the future trend of environmental protection. γ Irradiation affects soy proteins by causing conformational changes, oxidation of amino acids, rupture of covalent bonds and formation of protein free radicals (Cheftel et al., 1985). Chemical changes of the proteins that are caused by γ-irradiation are fragmentation, cross-linking, aggregation and oxidation caused by oxygen radicals which are generated by water radiolysis (Cho and Song, 2000). γ -Irradiation may generate free radicals on starch mole-cules and these free radicals are capable of hydrolyzing chemical bonds, thereby cleaving large molecules of starch into smaller fragments of dextrin (Grant and G’Appolonia, 1991). Through the irradiation technology, the SPI molecule combines with the starch molecule firmly in favor of increasing the material's capability and possessing a better exterior appearance. The objective of this study was to investigate the effects of starch (a filling agent), plasticizer (water and glycerol) and irradiation on the properties of soybean protein isolate based biodegradable materials used for plastics, including tensile strength and elongation at break, color, Fourier transform infrared spectroscopy (FTIR) and viscosity of the material plasticized by complex plasticizer. MATERIALS AND METHODS Sample preparation Powders of SPI (Shandong Wandefu Protein Co., Ltd.) and starch (Tianjin Kermel Co.,Ltd.) were mixed (94:6, 91:9, 88:12, 85:15, 82:18 w/w) and dispersed in epichlorohydrin (1:3, w/w), then 15% (w/w) succinic anhydride (Medicine group Chemical Co., Ltd.) was added and stirred for 0.5 h, respectively. The dispersion was vacuum filtrated and then air-dried. Plasticizers [1.5 g water, 1 g caprolactam (Tianjin Chemical Regent Co., Ltd. )] were added to the dried powders (10 g) in drops and mixed in a mixer. The mixture was equilibrated for 48 h to ensure an effective plasticization and even distribution of plasticizer. The mixture was then irradiated at 0, 10, 20, 30, 40 and 50 kGy at room temperature in air using a 60Co gamma irradiator (cobalt radiation source 120000 Ci, Tongweisuo Research Institute Co., Ltd, Henan Academy of Sciences, China). The plasticized soybean protein powder was placed into a molder and compression-molded in a heat press at 15 MPa with 120°C for15 min.

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Tensile testing and elongation at break The molded soybean protein/starch materials were equilibrated at 23°C and 50% relative humidity for 48 h before testing. The specimen was then cooled to room temperature at the same pressure and was cut into a dog-bone shape according to standard method ASTM D638-91 (ASTM Standard, 1993). The tensile strength (TS), elongation at break (E) and strain at break were calculated using an instron universal testing machine (CMT6503, Shenzhen SANS Test Machine Co., Ltd, China) according to the standard test method ASTM D638-91. The crosshead speed was 5 mm/min, and five replicates were tested for each sample.

Color measurements Colors were measured using a colorimeter (CR-400, Konica MinoltaCo., Ltd, Japan). Specimens (4 cm×4 cm) were placed on a white standard plate and the Hunter lab color was used to measure color: L = 0 (black) to L= 100 (white); a = _80 (greenness) to a = 100 (redness); and b = _80 (blueness) to b = 70 (yellowness). Total color difference (∆E) was calculated from five measurements which were taken at different locations on each specimen (Lee et al.,2005). ∆E =[(Lsample -Lstandard)2+(asample-astandard)2+(bsample-bstandard)2 ]0.5 Measurement of viscosity Viscosity of SPI solutions irradiated at various radiation doses was determined after 13 min mixing at 160~960 r/min at 25°C using a viscometer (RVA-4, New Port Co., Ltd, Australia). FTIR After samples were mixed with KBr, the mixture was then treated by the extrusion process (Souillac et al., 2002). FTIR spectra were collected with a QWF-510 FTIR spectrophotometer equipped with a standard DTGS detector. The spectra were recorded at the absorbance frequency from 4000 to 400 cm-1 mid infrared region at a resolution of 4 cm-1 with 128 co-added scans. At least triplicate spectra were recorded for each sample.

SEM SEM was used to characterize the microstructure of SPI/starch material. SPI/starch materials were made conductive by sputtercoating with a gold-palladium alloy coater (Hitachi Co., Ltd, e-1010 ions putter, Japan). The coated materials were dried in a desiccator. Samples were then examined using a SEM (FEI Co., Ltd, Quanta 200, USA) at an accelerating electron voltage of 20 kV. Micrographs for sample surface were obtained at 800×magnifications.

RESULTS AND DISCUSSION Effects of irradiation on properties of the mixed material The effect of radiation dose on E and TS of soy protein isolate/starch biodegradable materials are showed in Figure 1. The E increased and TS decreased with the

Tensible strength (MPa)

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Elongation (%)

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Irradi ation dose (kGy) Figure 1. Effect of radiation dose on E and TS of SPI/starch (88:12) biodegradable materials.

increase of radiation dose within the range of 0 to 50 kGy. The reason was that irradiation produced polymer radicals. Those radicals resulted in the lost in the main chain of two hydrogen atoms; alkyl radicals reacted with each other to form H-crosslinked or reacted with free radical of the end of molecular chain to form Tcorosslinked. After irradiation, water in samples produced excited state of oxidative free radicals that had oxygencontaining groups (carbonyl). It contributed to form amide link and improved the polymer surface polarity and the compatibility of them. At the same time, interface bonding strength was enhanced. The increase of the average molecular weight and partially increased polymer molecular weight could reduce the chance of cracking so that cross-linking increased the tensile strength (Kober and Gonzalez, 2007). Because the rate of forming free radicals was proportional to dose rate, increasing irradiation dose resulted in more cross-linking reaction. Therefore, with the increase in tensile strength, elongation at break was inversely deceased. Effect of radiation dose on water absorption capacity (WAC) of SPI/starch biodegradable materials are showed in Figure 2. It shows that WAC decreased with the increase of radiation dose from 10 to 50 kGy. Radiation effects were obvious in the range of 10 to 30 kGy. Starch contains many hydrophilic groups. When it comes to the range of 40 to 50 kGy, WAC of samples remained much stable. Introducing cross-links to polymer increased chemical stability. It seems that Îł-irradiation modified the conformation of proteins in the biomaterial mixture to a

certain extent that had more ordered and more stable structures, and then, the swelling of the biomaterial in solvent decreased. It further decreased the possibility of deformation owing to water absorption capacity. Besides, irradiation caused the formation of c-c bond which made hydrolysis more difficult. Therefore, the water resistance was improved and the solubility decreased (Lacroix et al., 2002). Effects of filling agent (starch) on properties of the mixed material Effects of starch content on E and TS of SPI/starch biodegradable materials at 30 kGy are showed in Figure 3. E increased and TS decreased with increasing starch content within the range of 6 to 18%. The compound between starch and protein was not completed by a single function but a comprehensive result which was produced by the covalent bond, electrostatic interaction, van der Waals forces, hydrogen bonding, hydrophobic interactions, ionic bonds, the role of volume exclusion and molecular entanglement (Kruif and Tuinier, 2011). These functions existed in different fragments and side chains of two kinds of macromolecules and maintained the structure of compounds. The Îľ-NH2 of protein molecules reacted with starch. They formed a proteinstarch compound which caused the protein subunit dissociation to occur and the polypeptide chain extended fully. Spatial structure changes increased the molecular

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WAC (%)

70 60 50 40 30 0

20 30 Irradiation dose (kGy)

Elongation (%)

Tensible strength (MPa)

Figure 2. Effect of radiation dose on WAC of SPI/starch (88:12) biodegradable materials.

Starch content (%) Figure 3. Effects of starch content on E and TS of SPI/starch biodegradable materials.

volume, improved flexibility and improved the material elongation. In addition, starch was cross-linked with protein amino acid residues that introduced exogenous moieties to protein molecules. It changed the distribution of protein molecular charge and resulted in the drop of surface charge of protein molecules. It also increased the

molecular volume, improved flexibility and improved the material elongation. Effects of starch content on WAC of soy protein isolate/ starch biodegradable materials at 30 kGy are shown in Figure 4. WAC slightly decreased with the increasing starch content within the range of 6 to 12%.

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WAC (%)

30 6

Starch content (%) Figure 4. Effects of starch content on water absorption capacity of SPI/starch materials.

biodegradable

Table 1. Effect of γ-irradiation on the color of SPI/starch biodegradable materials.

Irradiation dose(kGy) 0 10 20 30 40 50

L 81.72±0.54a 80.87±2.59a 82.06±1.24a 83.76±0.72a 84.44±0.87a 84.61±0.22a

a 4.36±0.11a 3.25±0.29b 2.31±0.12c 0.61±0.20d 0.06±0.05e -0.06±0.06e

b 14.20±0.08d 14.62±0.16cd 14.97±0.20c 15.10±0.13bc 15.49±0.14ab 15.96±0.24a

∆E 17.06±0.26a 16.73±0.20ab 16.42±0.07b 15.82±0.11c 15.47±0.09c 15.42±0.23c

Values are means of five replicates ± standard deviations. Values in the same column with different superscript letters indicate differences by multiple range test (P <0.05).

Increased starch content promoted cross-linking by irradiation of SPI/starch. It enhanced the effect of irradiation and decreased WAC (Salmoral et al., 2000). When the starch content was more than 12%, WAC increased with the increase of starch content. As the starch content increased, hydrophilic group was introduced, which added hydrophilic directly to SPI-starch compound, and the introduction of foreign groups also changed the conformation of proteins. Many hydrophilic groups were exposed, that easily formed hydrophilic network structure and improved hydrophilic ability of the materials. These results indicate that the optimum content of starch to biomaterials would result in forming a more compact structure and could help natural polymers to conquest the hydrophilic weakness (Kim et al., 2008). The analysis of hunter color values Table 1 shows the effect of γ-irradiation on the color of mixed materials. There were significant differences in

Hunter b value of irradiated materials. Increasing irradiation dose increased Hunter b value. Hunter b value at 50 kGy was 15.96, compared with 14.20 of the control sample. It was evident that γ-irradiation treatment increased in imparting yellow color to film. L value was also significantly increased and Hunter a value descended with increase of radiation dose. These results were in good agreement with the previous report that yellowness of materials were increased by UV radiation (Gennadios et al., 1998), addition of dialdehyde starch (Rhim et al.,1998), sodium doecyl sulfate (SDS) treatment (Rhim et al., 2002) and heat curing (Kim et al., 2002). Figure 5 shows effects of γ-irradiation on SPI/starch biodegradable material. It revealed that γ -irradiation treatment of SPI solutions also affected the viscosity of the mixed powder (Figure 5). When irradiation dose was under 30 kGy, non-covalent bonds and the disulfide bonds in SPI molecules were broken to cause subunit disaggregation and unfolded peptide chains, and even protein disaggregation and unfolding. Irradiation

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Figure 5. Effects of Îł-irradiation on viscosity of SPI/starch biodegradable material.

926 1045 Figure 6. (A) FTIR spectra of SPI/starch biodegradable material without irradiation treatment and (B) FTIR spectra of modified SPI/starch biodegradable material by irradiation (50 kGy).

significantly decreased viscosity due to the conformational change of protein molecules by oxygen radicals generated by radiolysis of water. It was widely accepted that the reduction in starch viscosity was caused by the theory that irradiation apparently caused fissures or splitting in starch granules (Abu et al., 2006). Under highpower gamma-irradiation, viscosity value for 50 kGy was 14 cP, compared with 53 cP of the 10 kGg. Therefore, the probability of the effective collision between protein and starch molecules was increased. Moreover, the activation energy of graft reaction could be reduced significantly, thus, increasing the reaction selectivity, the reaction rates were increased significantly (Guan et al., 2011). Figure 6 shows effects of y-irradiation on SPI/starch

biodegradable material on the FTIR spectra. When protein molecule binds with saccharide molecule covalently, hydroxyl group will be added. The result was that there was a wide peak in the wavelength range of 3700~3200 cm-1 and an absorption in the wavelength -1 range of 1260~1000 cm appeared on the infrared spectroscopy. Compared with the infrared spectroscopy of unirradiated biodegradable material, the infrared spectroscopy of irradiated SPI/starch biodegradable materials have the creation of new chemical functions in the wavelength range of 3700~3200 cm-1 and 1260~1000 -1 -1 cm . The absorption peak in the wavelength of 1045 cm was the effect of bond C-N, which proved the addition of -1 covalent bond. The absorption peak at 926 cm , which

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Table 2. Effect of γ-irradiation on secondary structure of protein.

Secondary structure α-Helix β-Pleated β-Turn Disordered structure

Unirradiated spi\starch material 15.22±0.12d 38.03±0.16a b 26.37±0.10 c 20.38±0.11

belongs to the wavelength range of 905~961 cm-1, was the bending vibration of the external plane of C-H double stranded. The infrared spectroscopy of irradiated SPI/ starch biodegradable materials have the creation of new chemical functions in the wavelength of 926 and 1045 cm 1 that proved the complex reaction of SPI and starch that were added. Acid amides I band (1600 to1700cm-1) is the most valuable band region for the investigation of the second structure of protein conformation (Byler and Susi, 1986). Infrared spectroscopy is a reliable method for the investigations that determines the second structure of protein according to the stretching vibration v(C-C) of C-C bond. Table 2 shows effects of y-irradiation on secondary atructure of protein. During the analysis of secondary structure of protein, as the increase of irradiation dose, amylase and amylo-pectin were broken and they had cross linking reaction with protein. α-Helix and β-pleated sheet structure were usually buried in the interior of a polypeptide chain. α-Helix was first formed during the establishment of the formation of globulin conformation. Compared with β-pleated sheet, α-helix was more unstable and was transformed into β-pleated sheet. So, α-helix greatly reduced under irradiation show polypeptide chains recombination in the interior of protein. The tensile strength and water resistance of biodegradable material closely related with the orderliness of molecular structure. Disordered structure greatly reduced after irradiation, which was consistent to the increase of tensile strength. This reduction also increased the degree of the smoothness of the material surface and reduced jut and gap. SEM Microstructure observed by scanning electron microscope showed that irradiated SPI/starch materials had glossier and smoother surface than the control material (Figure 7). Due to the good repeatability of the samples, the results were the same every time. The image displayed the consistency on the different observation point of the sample. Apart from tiny spots, the surface of SPI materials was unobvious rough and uneven. When unirradiated SPI/starch material with SPI/starch material irradiated was compared at 50 kGy, it was evident that

SPI/starch material irradiated at 50 kGy 1.02±0.13d 39.01±0.15b a 58.38±0.08 c 1.56±0.09

the former had some fissure and lump. It was possible that irradiation treatment enhanced cross-linking density of the material and reduced non-uniformity of raw materials, thereby causing the surface of the material to become more smooth which is significant for strengthening water resistance capability. The result is in good agreement with a previous report (Vachon et al., 2000) that the microstructure of protein films that were cast from irradiated film forming solutions was smoother than that of the control film. Conclusion This study clearly testifies that starch granules would be modified by γ -irradiation. γ -Irradiation treatment of the SPI caused the disruption of the ordered structure of the protein molecules as well as cleavage of the polypeptide chains of SPI, such as, reduced α-helix and enhanced βturn. Accompanied by the occurrence of the degradation, the cross-linking increased with the increasing dose. Therefore, the viscosity did not reduce much further above 30 kGy because of cross-linking of the proteins/ starch and aggregation of the polypeptide chains, resulting in change of water resistance capability and TS, and formation of more glossy and smoother surface. Starch is able to integrate with SPI, which is helpful to increase the mixture molecular mass with the resulting materials having better water resistance properties. These biomaterials made by the pure SPI are rigid, brittle and expensive. As a filling agent, starch enhances the material’s process ability and reduces cost. These effects may increase the applicability of such materials in biodegradable packaging or other industrial applications. As a result, γ-irradiation can be a useful tool (as a crosslinking agent) to improve mixed properties of SPI and starch; which offers a new method using the composite raw materials to replace conventional petroleum-derived plastics, adding new function field of the vast agricultural resource. ACKNOWLEDGEMENTS This research was funded in part by Innovation Scientists and Technicians Troop Construction Projects of Zhengzhou City. Project 31171790 and 20976037

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Figure 7. Microstructure of irradiated SPI films (800×magnification). A, unirradiated SPI material; B, unirradiated SPI/starch material; C, SPI/starch material irradiated at 50 kGy.

supported by National Natural Science Foundation of China contributed to this research also. The authors thank Zhengzhou University of Light Industry for their support. REFERENCES Abu JO, Duodu KG, Minnaar A (2006). Effect of γ-irradiation on some physicochemical and thermal properties of cowpea (Vigna unguiculata L. Walp) starch. Food Chem. 95: 386-393. ASTM Standard D 638-91 (1993). Standard Test Methods for Tensile Properties of Plastics. Am. Soc. Testing Mater. pp. 161-173. Bordes P, Pollet E (2009). Averous L. Nano-biocomposites: Biodegradable polyester/nanoclay systems. Prog. Polym. Sci. 34: 125-155. Byler DM, Susi H (1986). Examination of the secondary structure of proteins by deconvolved FTIR spectra. Biopolymers, 25: 469-487

Cheftel JC, Cuq JL, Lorient D (1985). Amino acids, peptides, and proteins. In: Fennema OR (Ed.), Food Chem. Marcel Dekker. pp. 279-343. Cho Y, Song K (2000). Effect of γ-irradiation on the molecular properties of BSA and β-lactoglobulin. J. Biochem. Mol. Biol. 33: 133-137. Derraik J (2002). The pollution of the marine environment by plastic debris: a review. Mar. Pollut. Bull. 44: 842-843. Gennadios A, Rhim JW, Handa A (1998). Ultraviolet radiation affects physical and molecular properties of soy protein films. J. Food Sci. 63: 225-228. Ghanbarzadeh B, Oromiehil AR (2009). Thermal and mechanical behavior of laminated protein films. J. Food Eng. 90(4): 517-524. Gonzalez A, Strumia MC, Igarzabal CIA (2011). Cross-linked soy protein as material for biodegradable films: Synthesis characterization and biodegradation. J. Food Eng. 106: 331-332. Grant LA, G’Appolonia BL (1991). Effect of low-level gamma-irradiation on water-soluable non-starchy polysaccharides isolated from hard red spring wheat flour and bran. Cereal Chem. 68: 651-660.

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Guan JJ, Zhang TB, Hui M (2011). Mechanism of microwaveaccelerated soy protein isolateâ&#x20AC;&#x201C;saccharide graft reactions. Food Res. Int. 44(5): 1-8. Jan K, Filip J (2009). Resources and future supply of oil. Energy Policy, 37: 441-464. Kalichevsky MT, Blanshard JMV, Tokarczuk PF (1993). Effect of water content and sugars on the glass transition of casein and sodium caseinite. Int. J. Food Sci. Tech. 28(1): 139-151. Kim JK, Jo C, Jin H (2008). Effect of gamma irradiation on the physicochemical properties of a starch-based film. Food Hydrocolloid, 22(2): 248-254. Kim Km,Weller CL, Hanna MA,Gennadios (2002). Heat curing of soy protein films at selected temperatures and pressures. Lwt-Food Sci. Technol. 35(2): 140-145. Kober E, Gonzalez ME (2007). Modification of water absorption capacity of a plastic based on bean protein using gamma irradiated starches as additives. Radiat. Phys. Chem. 76: 55-60. Kruif CG, Tuinier R (2011). Polysaccharide protein interactions. Food Hydrocolloid. 11(4): 555563.http://www.sciencedirect.com/science?_ob=PublicationURL&_hu bEid=1-s2.0S0268005X00X00203&_cid=271995&_pubType=JL&view=c&_auth= y&_acct=C000059901&_version=1&_urlVersion=0&_userid=3160871 &md5=3567e43db75a2aa3499954c3a66728dd Kumar R, Wang LX, Zhang LN (2009). Structure and mechanical properties of soy protein materials plasticized by thiodiglycol. J. Appl. Polym. Sci. 111(2): 970-977. Lacroix M, Le TC, Ouattara B (2002). Use of Îł-irradiation to produce films from whey, casein and soya proteins: structure and functionals characteristics. Radiat. Phys. Chem. 63: 827-832. Lee M, Lee S, Song KB (2005). Effect of g-irradiation on the physicochemical properties of soy protein isolate films. Radiat. Phys. Chem. 72: 35-40. Lemieux PM, Lutes CC, Santoianni DA (2004). Emissions of organic air toxics from open burning: a comprehensive review. Prog. Energ. Combust. 30: 1-32. Lithner D, Larsson A, Dave G (2011). Environmental and health hazard ranking and assessment of plastic polymers based on chemical composition. Sci. Total Environ. 409: 3309-3310. Liu WJ, Mohanty AK, Askeland P (2008). Modification of soy protein plastic with functional monomer with reactive extrusion. J. Polym. Environ. 16(3): 177-182. Mo XQ, Sun XS, Wang YQ (1999). Effects of molding temperature and pressure on properties of soy protein polymer. J. Appl. Polym. Sci. 73: 2595-2602.

Mo XQ, Sun XZ (2002). Plasticization of soy protein polymer by polyolbased plasticizers. J. Am. Oil Chem. Soc. 79(2): 197-202. Ogale AA, Cunningham PL, Dawson (2000). Viscoelastic, thermal, and microstructural characterization of soy protein isolate films. J. Food Sci. 65: 672-679. Otaigbe JU, Goel H, Babcock T (1999). Processability and properties of biodegradable plastics made from agricultural biopolymers. J. Elastom Plast. 31: 59-71. Park SK, Bae DH, Rhee KC (2000). Soy protein biopolymers crosslinked with glutaraldehyde. J. Am. Oil Chem. Soc. 77(8):879-884. Ren X (2003). Biodegradable plastics: a solution or a challenge. J. Clean Prod. 13: 27-28. Rhim JW, Gennadios A, Weller CL (2002). Sodium dodecyl sulfate treatment improves properties of cast films from soy protein isolate. Ind. Crop Prod. 15(3): 199-205. Rhim JW, Gennadios A, Weller CL (1998). Soy protein isolatedialdehyde starch films. Ind. Crop Prod. 8(3): 195-203. Salmoral EM, Gonzalez ME, Mariscal MP (2000). Comparison of chickpea and soy protein isolate and whole flour as biodegradable plastics. Ind. Crop Prod. 11: 227-236. Souillac PO, Middaugh CR, Rytting JH (2002). Investigation of protein/carbohydrate interactions in the dried state. 2. Diffuse reflectance FTIR studies. Int. J. Pharmaceut. 235: 207-218. Tian HF, Liu DG, Zhang LN (2009). structure and properties of soy protein films plasticized with hydroxylamine. J. Appl. Polym. Sci. 111(3): 1549-1556. Vachon C, Yu HL, Yefsah R (2000). Mechanical and structural properties of milk protein edible films cross-linked by heating and gamma irradiation. Agr. Food Chem. 48: 3202-3209.

Full Length Research Paper

Effects of different factors on the forward extraction of soy protein in reverse micelle systems Guanhao Bu, Haiyuan Liu, Fusheng Chen*, Kunlun Liu, Yingying Yang and Yanxiu Gao College of Food Science and Technology, Henan University of Technology, Zhengzhou 450001, China. Accepted 14 March, 2012

Reverse micelle extraction is a new technology for the extraction of protein. In this research, three kinds of reverse micelle systems, anionic surfactant sodium bis(2-ethylhexyl) sulfosuccinate (AOT) reverse micelle system, sodium dodecyl sulfate (SDS) reverse micelle system, and cationic surfactant cetyltrimethyl ammonium bromide (CTAB) reverse micelle system, were used to extract soy protein respectively. Effects of soy flour concentration, Wo ([H2O]/[AOT]), temperature, time, pH, ionic strength and ultrasonic power on forward extraction efficiency of soy protein were investigated. The effect of AOT reverse micelle diameter was studied as well. AOT reverse micelle system had higher extraction efficiency than SDS and CTAB systems. The main factors that affected the forward extraction were soy flour concentration, temperature and pH. The optimal conditions in AOT system were soy flour concentration being 0.007 g/ml, Wo 16, pH 6.5, temperature 34°C, time 20 min, KCl concentration of 0.1 mol/L and ultrasound power of 240 W. Under these conditions, the extraction efficiency of soy protein was 85.5%. The forward extraction efficiency of soy protein in AOT reverse micelle system increased with the increase of the reverse micelle diameter. Reverse micelle extraction is an effective way to extract soy protein. Key words: Soy protein, reverse micelle, forward extraction, sodium bis(2-ethylhexyl) sulfosuccinate (AOT), sodium dodecyl sulfate (SDS), cetyltrimethyl ammonium bromide (CTAB). INTRODUCTION Reverse micelle extraction is a novel technology for liquid-liquid extraction, which has recently received immense attention for the isolation and purification of proteins. The reverse micelles are nanometersized aggregates of surfactant molecules in non-polar solvents which are thermodynamically stable and optically transparent (Nandini and Rastogi, 2009). This technique offers several advantages such as low interfacial tension, ease of scale-up and continuous mode of operation. Reverse micellar extraction of lipase was carried out by many research groups using AOT or CTAB as surfactants (Naoe et al., 2007; Shen et al., 2005; Wu et al.,

*Corresponding author. E-mail: fushengc@yahoo.com.cn. Tel: +86-371-67756166. Fax: +86-371-67756667. Abbreviations: SDS, Sodium dodecyl sulfate; AOT, sodium bis(2-ethylhexyl) sulfosuccinate; CATB, cetyltrimethyl ammonium bromide.

2006; Yu et al., 2003). Isooctane, hexane and carbon tetrachloride were used as solvents, whereas, hexanol, isopropanol and butanol were used as co-surfactant/cosolvent. Shen et al. (2005) demonstrated that mixed reverse micelles consisting of CTAB for the extraction of industrial lipase resulted in maximum activity recovery of 70%. Reverse micelle can also preserve the properties of proteins and other bioactive molecules. The biotechnological relevance of these structures arises from their ability to solubilize water and hydrophilic molecules, such as proteins in their polar cores (Lye et al., 1994). Soy proteins are widely used in many kinds of foods as functional ingredients due to their high nutritional value, functional properties, and low cost. Soy proteins are used in a variety of foods such as salad dressings, soups, imitation meats, beverage powders, cheeses, non-dairy creamer, frozen desserts, whipped topping, infant formulas, breads, breakfast cereals, pastas, and pet foods. The Alkali dissolving acid sedimentation approach will reduce the activity of soy protein and as such new

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methods should be researched. Several research groups have engaged in the studying of the extraction of proteins by reverse micelles (Matzke et al., 1992; Zhao et al., 2011). Zhao et al. (2008a, b) investigated the extraction of soy 7S and 11S globulins from AOT reverse micelle. There are many factors that can affect the efficiency of extraction. The distribution of proteins between the micellar phase and the aqueous phase is largely determined by the environments of bulk aqueous phase, such as pH, ionic strength, and type of salts. Parameters related to the organic phase also affect the partition of protein, such as the concentration and type of surfactant, presence of co-surfactant, and type of solvent (Pires et al., 1996). Reverse micelle can be formed by anionic surfactant (such as AOT and SDS), cation surfactant (such as CTAB), and non-ionic surfactants (such as Spans and Tweens) in different systems where the factors that affect the extraction efficiency are different. The process of reverse micelle extraction, in general, consists of the forward extraction of protein from the feed aqueous phase to the reverse micelle organic phase and the backward extraction of protein from the reverse micelle organic phase to the recovery aqueous phase (Noritomi et al., 2006a). The objectives of this research were to investigate the forward extraction efficiency of soy protein in three reverse micelle systems, namely, anionic surfactant AOT and SDS systems, and cation surfactant CTAB system, and to study the effects of various factors such as soy flour concentration, Wo, temperature, time, pH, ionic strength, and ultrasonic power on forward extraction efficiency of soy protein, and to optimize the extraction conditions as well.

solution was added with various concentrations at (0.025, 0.05, 0.10, 0.15, 0.30, 0.40 mol/L) and pH at (5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5). Wo (6, 8, 10, 12, 14, 16) was the molar ratio of water to surfactant (Wo = [H2O]/[AOT]) and it was determined by the KarlFischer method. It was reverse micelle if the solution was transparent, and when otherwise, it was not reverse micelle. The second reverse micelle system was formed by sodium dodecyl sulfate (SDS)/isooctane/n-octyl alcohol and KCl solution. The third reverse micelle system was formed by cetyltrimethyl ammonium bromide (CTAB)/isooctane/n-octyl alcohol and KCl buffer solution. They were both prepared with the same procedure as that for the first reverse micelle system. Forward extraction of soy protein All extraction experiments were carried out in 250 ml Erlenmeyer flask with stoppers. Various concentrations of soy flour (0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04 g/ml) and reverse micellar systems were mixed together in Erlenmeyer flasks and extracted with sonication at 150, 180, 210, 240, 270, 300 W for 10, 20, 30, 40, 50, 60 min at different temperatures (25, 30, 35, 40, 45, 50, 55, 60°C), respectively. The undissolved residue was separated by centrifugation at 5000 rpm for 10 min and the volume of supernatant measured. Analysis of soy protein concentration Soy protein concentrations in organic of forward extractions were determined by spectrophotometer (UV-160A, Shimadzu, USA) at 280 nm (Zhao et al., 2010). Efficiency of forward extraction was estimated using the given equation: Forward extraction efficiency (%) =

total protein in the reverse micelle system ×100 total protein in soy flour

(1) Determination of reverse micelle diameter

MATERIALS AND METHODS Materials and chemicals Soy flour (100 mesh) was obtained from Anyang Mantianxue Food Manufacturing Co. Ltd (Anyang, China). It contained 377.5 g total protein, 223.7 g crude fat, and 68.5 g humidity per kilogram. Spectrophotometric-grade isooctane and Karl-Fischer titrant were obtained from Tianjin Kemiou Chemical Reagent Company (Tianjin, China). All other chemicals used in the experiment were of analytical grade. Chemical analysis Crude protein of soy flour was determined using the micro-Kjeldahl method (Concon and Soltess, 1973). Crude fat was measured by Soxhlet extraction (AOAC, 1984). The moisture content was determined by drying in an oven at 105°C until a constant weight was obtained. Preparation of three reverse micelle systems The first reverse micelle system was formed by sodium bis(2ethylhexyl) sulfosuccinate (AOT), isooctane and KCl solution. Various amounts of AOT were mixed with 50 ml isooctane and stirred at room temperature. When AOT dissolved completely, KCl

Diameter of reverse micelle (The Wo of reverse micelle was different) was determined directly by ZetaPlus instrument (Brookhaven Instrument Co., USA).

Experimental design and statistical analysis All analysis was carried out in triplicate. Efficiencies of extraction were expressed as means ± SD. On the basis of single factor experiments, further study was designed with Response Surface Analysis in the SAS 8.1 software (SAS Institute Inc., Cary, NC, USA). SAS statistical package was also used for regression analysis of the data and estimation of the coefficients of the regression equation. The statistical significance of the model was determined by the application of Fisher’s F-test. The significance of each coefficient was determined using the Fisher’s F-test and P value.

RESULTS AND DISCUSSION Effects of various factors on forward extraction efficiency of soy protein Figure 1 shows the forward extraction efficiencies of soy

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100

AOT

SDS

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80 60 40 20 0 0.010

0.015

0.020

0.025

0.030

0.035

0.040

Soy flour concentration (g/ml) Figure 1. Effects of soy flour concentrations on the forward extraction efficiency of soy protein using different reverse micelle systems. Extraction conditions were as follows: Wo 16, pH 6.5 for AOT and SDS, and pH 10 for CTAB, temperature 35째C, extraction time 20 min, KCl concentration 0.1 mol/L, ultrasound power 240 W for AOT, 210 W for SDS and 270 W for CTAB.

protein at various concentrations of soy flour using different reverse micelle systems (AOT, SDS and CTAB). Results indicate that when soy flour concentration varied from 0.01 to 0.04 g/ml, the forward extraction efficiency decreased gradually in these three systems. It was due to this concentration that the reverse micelle was constant, and soy protein which could enter the micelle was limited. These results are consistent with the conclusion of Sun et al. (2008). In addition, the extraction efficiency decreased more slowly in CTAB system than in AOT and SDS systems, this may be explained by the fact that CTAB held more water in the micelle, and the size of CTAB reverse micelle was bigger than AOT and SDS systems, therefore, it could take more soy protein (Ekwall et al., 1971; Fang and Yang, 1999; Li et al., 2006). As shown in Figure 2, the forward extraction efficiency increased with the increase of Wo. When Wo reached up to 16, the forward extraction efficiency reached a maximum in the three systems. The increase of Wo caused an increase of the reverse micelle size, meanwhile, the protein solubilization strongly depended on the reverse micelle size. The size of micelle relative to the size of a protein was critical to the ability of the micelle to solubilize protein (Sechler et al., 2010). The addition of protein to reverse micelles did not appreciably solubilize the protein until the diameter of the reverse micelle was similar to that of the protein (Matzke et al., 1992). With the increasing of Wo, some larger reverse micelles were formed and that were able to include plural protein molecules. This result is consistent with the report from

Leser et al. (2008). It could be observed in Figure 3 that the forward extraction efficiency increased slowly after 20 min. Zhao (2001) reported that the mass transfer rate of protein decreased with elongation of extraction time and the forward extraction efficiency slowly increased. Therefore, in order to save extraction time and obtain high extraction efficiency, the optimal extraction time was selected at 20 min. From Figure 4, it could be seen that the forward extraction efficiency reached the maximum when temperature was at 35째C in SDS and CTAB systems, and 40째C in AOT system. However, after that the forward extraction efficiency significantly decreased with temperature increased in the three systems. This may be explained by two reasons. Firstly, the water solubility in the reverse micelles reduced by decreased temperature. The decrease of the water solubility resulted in the decrease of solubilization of protein into the reverse micelle because the protein was entrapped into the reverse micelle by accompanying the water (Noritomi et al., 2006b). In addition, Kommareddi et al. (1993) found that the effect of increasing temperature on the microstructure of AOT reverse micelles included: faster tumbling of the reversed micelles, and increased lateral diffusion of the surfactant molecules due to the increase of thermal energy. This accounted for the forward extraction efficiency increased with the increasing temperature. However, higher temperature led to the expulsion of water from reverse micelles and the reduction of protein solubilization, thus the forward extraction efficiency

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80 70 60 50 40 30 20 6

Forward extraction efficiency of soy protein (%)

Figure 2. Effects of Wo on forward extraction efficiency of soy protein using different reverse micelle systems. Extraction conditions were as follows: soy flour concentration 0.01 g/ml, pH 6.5 for AOT and SDS, and pH 10 for CTAB, temperature 35째C, extraction time 20 min, KCl concentration 0.1 mol/L; ultrasound power 240 W for AOT, 210 W for SDS and 270 W for CTAB.

AOT

SDS

CTAB

80 75 70 65 60 10

30 40 Time (min)

Figure 3. Effects of extraction time on forward extraction efficiency of soy protein using different reverse micelle systems. Extraction conditions were as follows: soy flour concentration 0.01 g/ml, Wo 16, pH 6.5 for AOT and SDS, and pH 10 for CTAB, temperature 35째C, KCl concentration 0.1 mol/L; ultrasound power 240 W for AOT, 210 W for SDS and 270 W for CTAB.

reduced (Hilhorst et al., 1992). As shown in Figure 5, with the increase of the KCl concentration from 0.025 to 0.4 mol/L, the forward extraction

efficiency increased at the beginning, while it considerably decreased when KCl was higher than 0.1 mol/L in the three reverse micelle systems. KCl concentration

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90 85 80 75 70 AOT

SDS

CTAB

60 25

Temperature (째C) Figure 4. Effects of temperature on forward extraction efficiency of soy protein using different reverse micelle systems. Extraction conditions were as follows: soy flour concentration 0.01 g/ml, Wo 16, pH 6.5 for AOT and SDS, and pH 10 for CTAB, extraction time 20 min, KCl concentration 0.1 mol/L; ultrasound power 240 W for AOT, 210 W for SDS and 270 W for CTAB.

Forward extraction efficiency of soy protein (%)

100

AOT

SDS

CTAB

80 60 40 20 0

0.1

0.2

0.3

0.4

KCl concentration (mol/L) Figure 5. Effects of KCl concentration on forward extraction efficiency of soy protein using different reverse micelle systems. Extraction conditions were as follows: Soy flour concentration 0.01 g/ml, Wo 16, pH 6.5 for AOT and SDS, and pH 10 for CTAB, temperature 35째C, extraction time 20 min, ultrasound power 240 W for AOT, 210 W for SDS and 270 W for CTAB.

affected the transfer behavior of protein due to micelle size changes or screening of electrostatic interactions between the protein and the micelle wall (Goklen and Hatton, 1985). When concentration of salt ions was higher, the electrostatic repulsion of the surfactant polar

head could be reduced and the reverse micelles became smaller, thus, solubilization of water and biological molecules in reverse micelle decreased (Dekker et al., 1989). Figure 6 shows that the forward extraction efficiency

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80 60 40 20 5

pH Figure 6. Effects of pH on forward extraction efficiency of soy protein using different reverse micelle systems. Extraction conditions were as follows: soy flour concentration 0.01 g/ml, Wo 16, temperature 35°C; extraction time 20 min; KCl concentration 0.1 mol/L; ultrasound power 240 W for AOT; 210W for SDS and 270 W for CTAB

increased and reached the maximum at pH 6.5 in AOT and SDS systems, and then decreased. For CTAB system, the forward extraction efficiency increased and reached the maximum at pH 10 while it decreased from pH 10 to 12. The pH of aqueous phase plays a major role in controlling electrostatic interaction between enzyme and surfactant (Bansal-Mutalik and Gaikar, 2006). In the ionic reverse micellar systems, protein solubilization was regulated mainly by electrostatic interaction between the protein and the polar head of the surfactants (Zhao et al., 2010). AOT and SDS are anionic surfactant, so the reverse micelle polar heads of surfactant are negative electricity. The average isoelectric point of soy protein was about 4.5 and the protein was negatively charged above the isoelectric point. Therefore, it is deduced that electrostatic interaction cannot be a single driving force causing protein transfer at this time. Through the investigation, when the protein was negatively charged above the isoelectric point, it was inferred that the hydrophobic interaction between protein and surfactant led to protein solubilization (Paradkar and Dordick, 1994). However, the confirmatory mechanism of protein solubilization above the isoelectric point needed to be further researched. CTAB is a cationic surfactant with positive electricity polar head. Thus, pH should be above the average isoelectric point of soy protein. From the research, the extraction efficiency reached the maximum at pH 10. The conclusion could be drawn from the aforementioned research that reverse micelle formed by anionic

and cationic surfactants had different extraction pH and electrostatic interaction between the protein and the polar head of the surfactants was one driving force in extraction, while there may be other driving forces during the extraction. Figure 7 presents the effect of ultrasonic power on the forward extraction efficiency. In AOT and SDS reverse micelle systems, the extraction efficiency reached the maximum at 240 W, while in CTAB reverse micelle system, it was maximal at 270 W. Assessment of model and effects of independent variables on response AOT system had the highest efficiency based on the independent factor experiments. Therefore, further study was designed with Response Surface Analysis in the SAS software to gain the optimal conditions of forward extraction of soy protein in AOT reverse micelle system. The independent variables and their levels were shown in Table 1. The experimental conditions and the corresponding response value from the experimental design were presented in Table 2. The regression equation for the forward extraction efficiency of soy protein (Y) is thus, presented as: Y = 41.04649 − 223.7724 X 1 − 0.407296 X 2 + 16.91792 X 3 − 6337.437 X 1 X 1 +9.61332 X 1 X 2 − 2.952147 X 1 X 3 − 0.020037 X 2 X 2 + 0.256886 X 2 X 3 − 1.989303 X 3 X 3

(2) The effects of independent variables on response (Y) and

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90 AOT

SDS

180

210

CTAB

80 70 60 50 150

240

270

300

Ultrasond power (W) Figure 7. Effects of ultrasound power on forward extraction efficiency of soy protein using different reverse micelle systems. Extraction conditions were as follows: soy flour concentration 0.01 g/ml, Wo 16; pH 6.5 for AOT and SDS, and pH 10 for CTAB; temperature 35째C; extraction time 20 min; KCl concentration 0.1 mol/L.

Table 1. Independent variables and their levels employed in Response Surface Analysis experimental design.

Coded level -1.414 -1 0 1 1.414

Independent variables Soy flour concentration (g/ml) Temperature (째C) X1 X2 0.0037 33 0.010 35 0.025 40 0.040 45 0.046 47

analysis of variance (ANOVA) of regression model are presented in Table 3. For model fitted, the coefficient of determination (R2), which could check the goodness of a model was 0.9976. This implied that the sample variation of 99.76% for the forward extraction efficiency of soy protein was attributed to the independent variables and only 0.24% of total variation cannot be explained by the model. The data proved that the developed model was adequate to represent the actual relationship among the parameters chosen. It can be easily seen that the model was highly significant and the effect order of independent factor was soy flour concentration > pH > temperature (Table 3). The independent variable P-values suggested that X1 (soy flour concentration) and X3 (pH) significantly affected Y (forward extraction efficiency of soy protein) (P < 0.01). All of the quadratic terms also had significant effects on Y (P < 0.01). The response surface plot and

pH X3 4.9 5.5 7 8.5 9.1

their corresponding counter plot for the forward extraction efficiency of soy protein by the fitted second-order polynomial model are shown in Figures 8 to 10. Validation of the model For validation of the model, soy protein in the reverse micellar solution was extracted under the optimal conditions and the forward extraction efficiency was determined. The experimental value was compared with the predicted one in order to determine the validity of the model. The stationary point giving a maximum forward extraction efficiency of soy protein had the following critical values: soy flour concentration 0.007 g/ml, temperature 34째C, and pH 6.5. The predicted forward extraction efficiency for these conditions by SAS statistical package

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Table 2. Response Surface Analysis experimental design matrix with experimental values of the forward extraction efficiency of soy protein. a

Assay 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 a

Independent variables Soy flour concentration (g/ml) 0.010 (-1) 0.010 (-1) 0.040 (1) 0.040 (1) 0.0037 (-1.414) 0.046 (1.414) 0.025 (0) 0.025 (0) 0.025 (0) 0.025 (0) 0.025 (0) 0.025 (0) 0.025 (0) 0.025 (0) 0.025 (0)

Temperature (째C) 35 (-1) 45 (1) 35 (-1) 45 (1) 40 (0) 40 (0) 33 (-1.414) 47 (1.414) 40 (0) 40 (0) 40 (0) 40 (0) 40 (0) 40 (0) 40 (0)

Dependent variable pH Extraction efficiency (%) 8.5 (1) 79.2 5.5 (-1) 80.1 5.5 (-1) 78.5 8.5 (1) 77.9 7 (0) 85.8 7 (0) 78.3 7 (0) 83.7 7 (0) 84.1 4.9 (-1.414) 77.5 9.1 (1.414) 74.4 7 (0) 85.2 7 (0) 85.2 7 (0) 85.2 7 (0) 85.2 7 (0) 85.2

Values in parentheses are coded levels of independent variables.

Table 3. Significance of regression coefficients for response (Y) and analysis of variance (ANOVA) for model.

Source of variation X1 X2 X3 X1* X1 X1* X2 X1* X3 X2* X2 X2* X3 X3* X3 Model Error Total

Degrees of freedom 1 1 1 1 1 1 1 1 1 9 5 14

Sum of squares 28.125 0.080 4.805 15.685 1.040 0.009 1.936 7.424 154.547 203.310 0.490 203.8

Mean square 28.125 0.080 4.805 15.685 1.040 0.009 1.936 7.424 154.547 22.590 0.098

F value 287.087 0.817 49.046 160.107 10.613 0.090 19.759 75.780 1577.551 230.589

Pr > F 0.0001 0.408 0.0009 0.0001 0.023 0.776 0.007 0.0003 0.0001 0.0001

Significance ** ns ** ** * ns ** ** ** **

*Significant at the 0.05 level; ** Significant at the 0.01 level; ns, not significant at the 0.05 level.

was 86.2%, while the experimental value for these conditions was 85.5%. The results indicate that the experimental value (85.5%) was in agreement with the predicted one (86.2%). The effect of reverse micelle diameter on forward extraction efficiency The diameter of AOT reverse micelle was detected by ZetaPlus instrument and the extraction efficiency was studied at different diameter. Figure 11 shows the forward extraction efficiencies of soy protein at various diameters of AOT reverse micelle. The results indicate that with the

increased reverse micelle diameter, the forward extraction efficiency increased. Previous reports showed that large micelles afford more space to the enzyme and presumably permit greater conformational flexibility (Spreti et al., 1999; Zhao et al., 2004). Hieda et al. (2008) reported that the size of gold nanoparticles formed inside the water droplets was regulated by the size of reverse micelles. Conclusions The research demonstrated the feasibility of the forward extraction of soy protein from soy flour by three different

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Figure 8. Response surface and contour plots for the effects of soy flour concentration (X1) and temperature (X2) on forward extraction efficiency of soy protein (Y) at pH 7.

Figure 9. Response surface and contour plots for the effects of soy flour concentration (X1) and pH (X3) on forward extraction efficiency of soy protein (Y) at a temperature of 40째C.

reverse micelles. AOT reverse micelle system had higher extraction efficiency than SDS and CTAB systems. The main factors that affected the forward extraction were soy flour concentration, temperature and pH. The optimal conditions of forward extraction of soy protein in AOT

reverse micelle system were soy flour 0.007 g/ml, Wo 16, pH 6.5, 34째C, extraction time 20 min, KCl 0.1 mol/L, ultrasound power 240 W. Size of reverse micelle affected the forward extraction of soy protein. Electrostatic interaction between the protein and the polar head of the

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Forward extraction efficiency of soy protein (%)

Figure 10. Response surface and contour plots for the effects of temperature (X2) and pH (X3) on forward extraction efficiency of soy protein (Y) at soy flour concentration 0.025 g/ml.

80 70 60 50 40 30 20 2.0

2.5

3.0 3.5 4.0 Diameter (nm)

4.5

5.0

Figure 11. Effects of diameter of AOT reverse micelle on forward extraction efficiency of soy protein.

surfactants was one main driving force in extraction. The extraction mechanism of protein transferred from soy flour to reverse micelle should be also studied in the future.

ACKNOWLEDGEMENTS This study was supported by the National Natural Science Foundation of China (No. 20976037), the Doctor

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Foundation of Henan University of Technology (No. 2010BS014) and the Innovation Scientists and Technicians Troop Construction Projects of Zhengzhou City. REFERENCES AOAC (1984). Official Methods of Analysis, 14th ed. Association of Official Analytical Chemists, Washington, DC. Bansal-Mutalik R, Gaikar VG (2006). Reverse micellar solutions aided permeabilization of baker’s yeast. Process Biochem. 41(1): 133-141. Concon JM, Soltess D (1973). Rapid micro Kjeldahl digestion of cereal grains and other biological materials. Anal. Biochem. 53(1): 35-41. Dekker M, Hilhorst R, Laane C (1989). Isolating enzymes by reversed micelles. Ann. Biochem. 178(2): 217-226. Ekwall P, Mandell L, Solyom P (1971). The solution phase with reversed micelles in the cetyl trimethylammonium bromide-hexanolwater system. J. Colloid Interface Sci. 35(2): 266-272. Fang XL, Yang CF (1999). An Experimental Study on the Relationship between the Physical Properties of CTAB/Hexanol/Water Reverse Micelles and ZrO2–Y2O3 Nanoparticles Prepared. J. Colloid Interface Sci. 212(2): 242-251. Goklen KE, Hatton TA (1985). Protein extraction using reverse micelles. Biotechnol. Progr. 1(1): 69-74. Hieda J, Saito N, Takai O (2008). Size-regulated gold nanoparticles fabricated by a discharge in reverse micelle solutions. Surf. Coat. Tech. 202(22-23): 5343-5346. Hilhorst R, Fijneman P, Heering D, Wolbert RBG, Dekker M, Riet KV, Bijsterbosch BH (1992). Protein extraction using reversed micelles. Pure Appl. Chem. 64: 1765-1770. Kommareddi NS, John VT, Waguespack YY, McPherson GL (1993). Temperature and gas pressure induced microstructural changes in AOT water-in-oil microemulsions: characterization through electron paramagnetic resonance spectroscopy. J. Phys. Chem. 97(21): 57525761. Leser ME, Luisi PL, Palmieri S (1989). The use of reverse micelles for the simultaneous extraction of oil and proteins from vegetable meal. Biotechnol. Bioeng. 34: 1140-1146. Li JC, Zhang JL, Han BX, Gao YN, Shen D, Wu ZH (2006). Effect of ionic liquid on the polarity and size of the reverse micelles in supercritical CO2. Colloid Surface A. 279(1-3): 208-212. Lye GJ, Asenjo JA, Pyle DL (1994). Protein extraction using reverse micelles: kinetics of protein partitioning. Chem. Eng. Sci. 49(19): 3195-3204. Matzke SF, Creagh AL, Haynes CA, Prausnitz JM, Blanch HW (1992). Mechanisms of protein solubilization in reverse micelles. Biotechnol. Bioeng., 40(1): 91–102. Nandini KE, Rastogi NK (2009). Reverse micellar extraction for downstream processing of lipase: Effect of various parameters on extraction. Process Biochem. 44(10): 1172-1178. Naoe K, Takeuchi C, Kawagoe M, Nagayama K, Imai M (2007). Higher order structure of Mucor miehei lipase and micelle size in cetyltrimethyl ammonium bromide reverse micellar system. J. Chromatogr. B. 850(1-2): 277-284. Noritomi H, Ito S, Kojima N, Kato S, Nagahama K (2006a). Forward and backward extractions of cytochrome c using reverse micellar system of sucrose fatty acid ester. Colloid Polym. Sci. 284(10): 604-610.

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Noritomi H, Kojima N, Kato S, Nagahama K (2006b). How can temperature affect reverse micellar extraction using sucrose fatty acid ester. Colloid Polym. Sci. 284(6): 683-687. Paradkar VM, Dordick JS (1994). Mechanism of extraction of chymotrypsin into isooctane at very low concentrations of aerosol OT in the absence of reverse micelles. Biotechnol. Bioeng. 43(6): 529540. Pires MJ, Aires-Barros MR, Cabra JMS (1996). Liquid-liquid extraction of proteins with reversed micelles. Biotechnol. Progr. 12(3): 290-301. Sechler TD, DelSole EM, Deak JC (2010). Measuring properties of interfacial and bulk water regions in a reverse micelle with IR spectroscopy: a volumetric analysis of the inhomogeneously broadened OH band. J. Colloid Interf. Sci. 346(2): 391-397. Shen R, Liu JG, Xing JM, Liu HZ (2005). Extraction of industrial lipase with CTAB-based mixed reverse micelles. Chin. J. Process Eng. 5(3): 255-259. Spreti N, Alfani F, Cantarella M, D’Amico F, Germani R, Savelli G (1999). alpha-chymotrypsin superactivity in aqueous solutions of cationic surfactants. J. Mol. Catal. B: Enzyme, 6(1): 99-110. Sun XH, Zhu KX, Zhou HM (2008). Protein extraction from defatted wheat germ by reverse micelles: Optimization of the forward extraction. J. Cereal Sci. 48(3): 829-835. Wu JC, Chau PS, Chow Y, Li R, Carpenter K, Yu H (2006). Extraction of Candida rugosa lipase from aqueous solutions into organic solvents by forming an ion-paired complex with AOT. J. Chem. Technol. Biot. 81(6): 1003-1008. Yu YC, Chu Y, Ji JY (2003). Study of the factors affecting the forward and back extraction of yeast-lipase and its activity by reverse micelles. J. Colloid Interf. Sci. 267(1): 60-64. Zhao JT (2001). Study on the use of reverse micelle for the simultaneous extraction oil and protein. J. Zhengzhou Univ. Tech. 22: 54-56. Zhao L, Pal SK, Xia T, Zewail AH (2004). Dynamics of ordered water in interfacial enzyme recognition: bovine pancreatic phospholipase A2. Angew. Chem. Int. Edit. 43(1): 60-63. Zhao XH, Li YM, He XW, Zhong NJ, Xu ZB, Yang LS (2010). Study of the factors affecting the extraction of soybean protein by reverse micelles. Mol. Biol. Rep. 37(2): 669-675. Zhao XY, Chen FS, Chen JQ, Gai GS, Xue WT, Lee LT (2008a). Effects of AOT reverse micelle on properties of soy globulins. Food Chem. 111(3): 599-605. Zhao XY, Chen FS, Gai GS, Chen JQ, Xue WT, Lee LT (2008b). Effects of extraction temperature, ionic strength and contact time on efficiency of bis(2-ethylhexyl) sodium sulfosuccinate (AOT) reverse micellar backward extraction of soy protein and isoflavones from soy flour. J. Sci. Food Agric. 88(4): 590-596. Zhao XY, Chen J, Zhu QJ, Du FL, Ao Q, Liu J (2011). Surface characterization of 7S and 11S globulin powders from soy protein examined by X-ray photoelectron spectroscopy and scanning electron microscopy. Colloid Surface B. 86(2): 260-266.

African Journal of Biotechnology Vol. 11(28), pp. 7263-7269, 5 April, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.470 ISSN 1684â&#x20AC;&#x201C;5315 ÂŠ 2012 Academic Journals

Full Length Research Paper

The hymenopterous pollinators of Himalayan foot hills of Pakistan (distributional diversity) Hussain A, Khan M. Rahim, Ghffar A, Hayat Alia and Jamil A. Department of Entomology, Faculty of Agriculture, Azad Jammu and Kashmir University, Rawalakot, Azad Kashmir, Pakistan. Accepted 24 January, 2012

Studies were undertaken to explore the diversity of hymenopterans pollinators from a diverse agroecosystems of Himalayan foot hills comprising the orchards of pome and stone fruits at different altitudes from 2200 to 3000 m from sea level. Field experiments were conducted on seven commercial fruit orchards at five various localities. Out of the total 448 specimens, 60.94% were found in an antemeridian (A.M.) phase and 39.06% specimens were found in post-meridian (P.M.) indicating their activity both diurnal and crepuscular. Rank abundance values revealed that 9 species in 5 genera belonged to four families of order Hymenoptera comprising the diversity of Osmia cornifrons Panzer, Anthophora niveo-cincta (Smith), Anthophora himalayensis Rad., Anthophora crocea Bangham, Bombus tunicatus (Smith), Xylocopa dissimilis Lepel., Xylocopa rufescens Smith, Andrena harrietae Bangham and Andrena anonyma Cam. The calculated values of all diversity indices showed that the lowest diversity was found in a monoculture fruit habitat with well weeded orchards, whereas the diversity of pollinators was found greater in multiple cultures with partially weeded orchards particularly during the successional stage of full bloom in both pomes and stone fruits. A significant difference in the pollinatorsâ&#x20AC;&#x2122; population was seen in the orchards with undisturbed surroundings. The natural ecosystem offers more opportunities of refuges for the insect pollinators compare to those orchards with clean cultivation. Key words: Diversity, agro-ecosystem, fruit orchards, hymenopterans pollinators, monoculture. INTRODUCTION Pollination is one of the most important mechanisms in the maintenance and promotion of biodiversity and, in general, life on Earth. Many ecosystems, including agroecosystems, depend on pollinator diversity to maintain overall biological diversity. Pollination also benefits society by increasing food security and improving livelihoods (Khan and Khan, 2004). Pollinators are extremely diverse, with more than 16,000 pollinator bee species (Hymenoptera: Apidae) have been described worldwide (Michener, 2000; Kevan, 2003). The ecological relationship of the pollinators was recognized long before by Knutson et al. (1990) that cross pollination is the only means of maintaining the ecological diversity. Good pollination improves both fruit yield and size

*Corresponding author. E-mail: mrkhan40@yahoo.com.

(Gautier-Hion and Maisels, 1994; Free, 1993). It is estimated that bees accomplish more than eighty percent of the insect pollination. Yields of fruit, legumes and vegetable seeds often have been doubled or tripled by providing adequate number of bees for pollination (McGregor, 1976). The wild bees including bumble bees, leaf-cutting bees, alkali bees and carpenter bees are especially adopted for gathering pollens and nectars from flowers (Bohart, 1972). Pollinators other than honey bees are also extremely valuable although their value is difficult to estimate. Globally, the annual contribution of pollinators to the agricultural crop has been estimated at about US$54 billion (Buchmann and Nabhan 1996; Kenmore and Krell, 1998). In a recent survey from Pakistan, Jasra and Rafi (2008) concluded that 84% of the formers of northern area have no perception about the importance of pollination for their orchards and crops. The inadequacies have arisen from habitat fragmentation

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Table 1. Blooming months and availability of pollens on different flowering source of pome and stone fruits from High land of Pakistan.

Common name

Latin name

Blooming months

Pollen colour

Availability

Flowering quinc Walnut Crab apple Apple Plum Almond Wild cherry Peach Black cherry Pear Callery pear Blackberry Apricot

Chaenomeles japonica Juglans spp. Malus spp. Malus domestica Prunus spp. Prunus amygdalus Prunus avium Prunus persica Prunus serotina Pyrus communis

April to May April to May March to Jun April to May April to May February April to May April to May April to May April to May April to May May to Jun April to May

Yellow brown

Feral Cultivated Cultivated Cultivated Cultivated Cultivated Feral Cultivated Feral Cultivated Feral Feral Cultivated

Rubus spp. Prunus armeniaca

and destruction, pesticide use, and adverse effects of introduced species displacing natives (Ambrose and Kevan, 1990; Hingston and McQuillan, 1998; Kearns et al., 1998; Kevan, 1999, 2001; Kevan et al., 2002). The natural ecosystem in Northren part of Pakistan is still preserved but the high land insect fauna from a very rich horticulture zone of Pakistan is still unexplored. The present study was designed to prepare an inventory of hymenopterans pollinator bees from fruit orchards of Skardu District, which is unique part of land on the earth due to its geographical importance. MATERIALS AND METHODS Field survey Field surveys were undertaken in five locations of District Skardu, which is located in the north of Pakistan. The detailed survey of commercial fruit orchards in the five localities was conducted during early spring of 2009. These orchards and localities were City Park (37.5 acres), PCSIR Orchard (12.5 acres) and Agricultural Fruit Orchard Hamid Ghardh (7.5 acres) in Skardu City, Fruit Orchard of Shangrilla (Average of 35 acres) in Kachura, Agricultural fruit Orchard of Sermik (Average 10 acres) in Sermik, Agricultural Fruit Orchard Mehdiabad (Average10 acres) in Kharmang and Hashupi Fruit Orchard (11.5 acres) in Shighar. The clusters of orchard are located in a radius of 20 km radius. The occurrence and distribution of pollinators varies with the topographic change. The flowering period of major fruit plants of District Skardu is given in (Table 1). At each location roving survey was taken up twice in 15 days. In roving survey, the occurrence of pollinators was assessed by taking observations on five randomly selected plants. The hymenopterans pollinators in orchard ecosystem were sampled by using two methods; 1) by sweep net (30 × 60 × 45 cm) and 2) using malaise trap. In sweep net practice, twenty-five sweeps were made diagonally across each canopy and samples were placed in separate plastic sachets. A total of 12 samples were taken from each orchard at different time. The collection obtained from malaise trap was taken out after every 72 h from the previous collection. But the time of their activity in the field and their proportion in different localities was only possible to determine through sweep net collection in different times of the day. A maximum number of pollinator’s activity was also observed during the full bloom and fruit

Light olive Yellow white Grey Light brown yellow brown, light brown Reddish yellow Red yellow Yellow Light grey Yellow

Source for pollinator Good Fair Fair Good Good Fair Very good Good Minor Good Very good Minor Very good

setting. In order to study the proportion of each species within the local community, species diversity was computed based on ShannonWiener formula, also called the Shannon’s index or ShannonWiener index (Humphries et al., 1996), where H is the ShannonWiener (1963) biodiversity index; Pi is the proportion of each species in the sample (relative abundance); log e Pi is the natural log of Pi; and S is the number of species in the pollinators community. Species evenness (J) With a view to understanding the measure of how similar the abundance of different species, species evenness was calculated to estimate the equitability component of diversity (Pielou, 1969); H´= C {log10N - 1⁄N∑ (log10 nr log10)} Where, H is the Shannon-Wiener Richness index and S is the number of species in the community. Species richness (Ma) In order to assess how the diversity of the population is distributed or organized among the particular species, this index was calculated (Pielou, 1975) by; Ma = S-1 Log e N Where, S is the total number of species collected and N is the total number of individuals in all the species Simpson’s diversity index This accounts for both richness and proportion (per cent) of each species in the local community. The index has been defined in three different ways (Simpson, 1949); Simpson’s index (D): This denotes the probability that two randomly selected individuals in the community belong to the same species. The form of the Simpson’s index used was: S C = ∑ {ni (ni-1) ⁄ N (N-1)} i =1

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Table 2. Specific, generic and family wise detail of the specimens collected from different commercial fruit orchards of district Skardu.

Family Apidae

Genus Bombus

Species tunicatus

Anthophrodae

Anthophora

niveo-cincta himalayensis crocea

Anthophrodae

Xylocaopa

Dissimilis Dissimilis rufescens

Andrenidae

Andrina

harriete

Megachilidae

Osmia

anonyma cornifrons

Where, “ni” is the number of individuals in the” ith” species and “N” is the total number of individual in the sample. The form of the Nakamura’s index used was: S RI = ∑ Ri ⁄ S (M - I) r=i Where, “S” is the number of investigated species of insects; “M” is the number of rank of abundance (0, 1, 2, 3… M - I) and “R” is the rank value of “ith” species in the sample.

Rank abundance values In a biodiversity study, there are mainly four categories to be worked out; abundance, richness, evenness and dominance. For studying the species dominance of pollinators during the flowering period, rank abundance values were needed to be solved. This was done by taking the sum of individual species found throughout the crop period and ranks were given based on the dominance of hymenopterans species.

RESULTS AND DISCUSSION Occurrence of hymenopterous bee pollinators in orchard ecosystems Field surveys for collection of the bees were carried out during March 2010. The specimens were identified up to species level. A total of nine species belonging to five genera that fall in four families, were identified. The abundance, richness and evenness (equitability) of pollinators found in each sampled commercial fruit orchard and total abundance of each species and total abundance of species collected from all sampled commercial fruit orchards are in accordance with Jasara et al. (2000) and Jasara and Rafi (2008). Abundance, richness and evenness (equitability) of the hymenopterous pollinator bees was found at peak during

Number of species 1

the successional flowering time in the orchards of both pome and stone fruits in various localities. Previously, Verma and Pertap (1993) also highlighted the impact of mountain pollinators during spring from Himalayan region.

Ante-meridian (before noon) collection surveys During ante-meridian (A.M.) collection, a total of two hundred and seventy three (273) specimens were collected from all sampled commercial fruit orchards of district Skardu, which is the 60.94% of the total collected specimens in both ante-meridian (A.M.) and postmeridian (P.M.). It showed that hymenopterous pollinator bees prefer to visit flowers during ante-meridian (A.M.) phase as compared to post-meridian (P.M.) phase of the day time. There was no significant difference in abundance, richness and evenness (equitability) of the hymenopterous pollinator bees found in all commercial fruit orchards of district Skardu at post-meridian taxa collected at post-meridian. These results were in accordance with that of Louadi and Doumandji (1998). The diversity density of bee pollinators perhaps decreased in mono culture more rapidly.

Post-meridian (after noon) collection surveys During post-meridian (P.M.) collection, a total of one hundred and seventy three specimens (175) were collected from all sampled commercial fruit orchards; that is the 39.06% of the total specimens collected in both ante-meridian (A.M.) and post-meridian (P.M.). This indicated that hymenopterous pollinator bees less preferred to visit flowers during post-meridian (P.M.) phase as compared to ante-meridian (A.M.) phase of the day time. Abundance, richness and evenness

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45 40 35 30 25 20 15 10 5 0

40 26

16 4

Os mi a

An co tho rn ph ifr or on a s An ni ve tho oph ci nc or a ta hi ma la ye Bo ns mb is us tun Xy ic atu loc s op a di ssi An mi dr lis en ah ar r ie An ta dr e en aa no An ny th m op a ho ra cr Xy oc loc ea op a ru fes ce ns

No. of individuals

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Name of species

Figure 1. Diagrammatic representation of the specimens collected from all sampled commercial fruit orchards of district Skardu during post-meridian (P.M.).

(equitability) of the Hymenopterous pollinator bees found in all commercial fruit orchards of district Skardu at postmeridian (P.M.) are diagrammatically represented in Figure 1. Table 3 shows the collective rank list along with the list of taxa collected at post-meridian from different commercial fruit orchards of district Skardu. This ancient and co-evolved process arose at about, or before, the age of the dinosaurs and has allowed the marvelous radiation of flowering plants and pollinating insects that now comprise part of the green biofilm of the terrestrial surface, robed in the world’s largest membrane, the atmosphere (Thomas, 1993). These results are also in accordance with McGregor (1976) who estimated that bees accomplish more than 80% of the insect pollination. Yields of fruit, legumes and vegetable seeds often have been doubled or tripled by providing adequate number of bees for pollination. Shannon’s diversity index in perturbed situations are usually higher (Pileou, 1975). During the present study, four (4) diversity indices namely; Shannon-Wiener’s diversity index along with its equitability component, Margalef’s index, Simpson’s index and Nakamura and Toshima’s index were used for the calculation of abundance, richness and evenness (equitability). The calculated values and comparison of calculated values of four diversity indices for each sampled commercial fruit orchards of district Skardu are given in Table 4. Moreover, the pollinators’ guild (Table 5) indicated a significant difference of 14.38 and 8.64 from clean and bushy fields, reflecting the effect of cropping pattern on the availability of the pollinators. This pattern is followed by the each spp. of hymenopterous pollinators available during the full bloom. The calculated values of this index in different commercial fruit orchards of District Skardu ranged from

2.262 (Agricultural Fruit Orchard Mehdiabad) to 2.945 (PCSIR Fruit Orchard Skardu). The remaining commercial fruit orchards yielded values in between the above-mentioned figures (2.262 to 2.945). The calculated values showed that there is no big difference (0.921) and (0.988) in the richness and evenness of ShannonWiener’s diversity index, which meant that the hymenopterous pollinator bees are well distributed in all commercial fruit orchards of district Skardu. However, the maximum diversity value was calculated from the PCSIR Fruit Orchard Skardu (2.945) and minimum diversity value was calculated from the Agricultural Fruit Orchard Mehdiabad (2.262). Shannon’s equitability (J') Shannon’s equitability index measures the evenness (equitability) of the calculated species in the sample or sampling area (Shannon, 1963). The calculated values of the Shannon’s equitability index ‘J’ in sampled commercial fruit orchards of District Skardu ranged from 0.875 (Agricultural Fruit Orchard Mehdiabad) to 0.988 (Agricultural Fruit Orchard Hamid Ghardh). Out of the seven commercial orchards, the similarity and differences in calculated values were not significantly different in pollinators’ number as well in the fruit yields. This uniform distribution indicated the equitability of the hymenopterous pollinators (Table 2). The evenness and richness of the pollinators are coinciding in values from two diversity indexes (Shannon-Wiener’s diversity index) and (Shannon’s equitability index ‘J’) which support normal distribution of the pollinators’ species indicating less diverse pattern in the study area of monoculture.

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Table 3. The collective rank list along with the list of taxa collected from different commercial fruit orchards of district Skardu.

Rank

Name of Taxa

Abundance

City Park orchard

Hamid Gardh orchard

PCSIR Fruit Orchard

Shangrila orchard Kachura

Fruit Orchard Sermik

Agriculture Fruit Orchard Mehdiabad

Fruit Orchard Hushupi, Shigar 14

58 57 44 37 7 5 ∑N=448

8 9 8 9 7 0 N=98 8

9 15 8 8 0 0 N=75 7

10 9 7 5 0 5 N=66 8

9 5 6 6 0 0 N=57 7

5 6 5 4 0 0 N=49 7

8 7 6 3 0 0 N=47 6

9 6 4 2 0 0 N=56 7

Osmia cornifrons Anthophora niveo2 cincta Anthophora 3 himalayensis 4 Bombus tunicatus 5 Xylocopa dissimilis 6 Andrena harrietae 7 Andrena anonyma 8 Anthophora crocea 9 Xylocopa rufescens No of individuals No of species

Table 4. Calculated values of diversity Indices from different commercial fruit orchards of District Skardu, N.Areas.

S/N

Name Orchard

Shannon-Wiener’s index (H´)

Margalef’s index (D)

Simpson’s index (C)

Nakamura’s index (RI)

1 2 3

City park Agril. Fruit Orchard PCSIR Fruit Orchard

2.764 2.774 2.945

0.921 0.988 0.982

1.527 1.389 1.671

0.965 0.959 0.999

0.642 0.666 0.642

Orchard of Shangrila Kachura

2.945

0.982

1.671

0.999

0.642

Agril. Fruit Orchard Sermick

2.698

0.958

1.542

0.999

0.666

Agril. Fruit Orchard Mehdiabad

2.262

0.875

1.298

0.999

0.700

Agril. Fruit Orchard Hashupi, Shighar

2.617

2.932

1.490

0.999

0.666

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Table 5. Pollinators guild in a clean cultivated and a bush surrounding orchards.

S/N 01 02 03 04 05 06 07 08 09

Pollinators fauna Osmia cornifrons Anthophora niveo-cincta Anthophora himalayensis Bombus tunicatus Xylocopa dissimilis Andrena harrietae Andrena anonyma Anthophora crocea Xylocopa rufescens

Orchard with bushy surrounding 14.38 ± 0.14 9.34 ± 0.19 5.26 ± 0.06 4.37 ± 0.18 7.30 ± 0.15 3.34 ± 0.010 2.23 ± 0.16 4.12 ± 0.17 3.38 ± 0.26

Margalef’s index Margalef’s index is used to measure the richness of the species distributed in the sample or sampling area (Margalef, 1969). This index is used frequently in the biological data. The calculated values of the Margalef’s index in the seven commercial fruit of district Skardu ranged from 1.298 (Agricultural Fruit Orchard Mehdiabad) to 1.671 (PCSIR Fruit Orchard Skardu). The remaining sampled commercial fruit orchards yielded the values in between these two (Table 4). The yielded values of this index from all the sampled commercial fruit orchards indicated that there was no any big difference in the richness of hymenopterous pollinator bees in these orchards of district Skardu.

Orchard with clean cultivation 8.64 ± 0.08 7.20 ± 0.19 4.21 ± 0.14 3.84 ± 0.010 4.34 ± 0.14 3.24 ± 0.19 3.13 ± 0.18 2.24 ± 0.14 4.22± 0.25

indicated that the diversity of City Park Skardu and PCSIR Fruit Orchard Skardu was slightly higher then remaining all sampled commercial fruit orchard of district Skardu (Table 4). Conclusion The calculated values of all the indices from the entire sampled commercial fruit orchards showed that despite the big difference in the total number of individuals (abundance) there was no big difference in the richness and evenness of Hymenopterous pollinator bees in district Skardu. REFERENCES

Simpson’s index The calculated values of the Simpson’s index from sampled commercial fruit of district Skardu was calculated as: 0.965 (City park Skardu), 0.959 (Agricultural Fruit Orchard Hamid Ghardh Skardu) and the remaining sampled commercial fruit orchards as 0.999 (Table 4). The yielded values of this index indicated that the abundance of City Park Skardu and Agricultural Fruit Orchard Hamid Ghardh Skardu were slightly higher than remaining all sampled commercial fruit orchards of district Skardu.

Nakamura’s index (RI) Nakamura and Toshima’s index measures the richness of the species. The calculated value of Nakamura (RI) index ranged from 0 to 1. If the value tends to zero, the diversity will increase (Nakamura and Toshima, 1995). The calculated values of the Nakamura’s index (RI) from sampled commercial fruit orchards of district Skardu were calculated as: City park Skardu PCSIR Fruit Orchard Skardu (0.642) and the remaining sampled commercial fruit orchard as 0.666. The yielded values of this index

Ambrose JD, Kevan PG (1990). Reproductive biology of rare Carolinian plants with regard to conservation management. In: G.M. Allen, G.F.J. Eagles & S.D. Price (Eds). Conserving Carolinian Canada: Conservation Biology in the Deciduous Forest Region. University of Waterloo Press. pp. 281-290. Bohart G E (1972). Management of wild bees for the pollination of crops. Annual Review of Entomol. 17: 287-312. Buchmann SL, Nabhan GP (1996). The Forgotten Pollinators. Island Press, Washington, D.C., U.S.A. p. 292. nd Free, JB (1993). Insect pollination of crops. 2 edition, Academic press, London. Gautier-Hion A, Maisels, F (1994). mutualism between a leguminous tree and large African monkeys as pollinators. Behavioural Ecology Hingston AB, McQillan PB (1998). Does the recently introduced bumblebee, Bombus terrestris (Apidae) threaten Australian ecosystems. Australian J. Ecol. 23: 539-549. Jasara A W, Ashfaq S, Kasi AM (2000). Apple pollination in Balochistan, Pakistan. National Arid land Development and Research Institute, Ministry of Food, Agricultural and Livestock, Islamabad, p. 33. Jasara AW, Rafi MA (2008). Pollination management of apricot as a livelihood source in northern areas. Pak. J. Agric. Agricultural Engineering and Veterinary Science, 24: 34-40. Kearns CA, Innouye DW, Waser NM (1998). Endangered mutualisms; the conservation of plant-pollinator interactions. Annual Review Ecol. Syst. 29: 83-112. Kevan, PG (1999). Pollinators as bio indicators of the state of the environment: species, activity and diversity. Agriculture, Ecosystems & Environment, 74: 373-393. Kevan PG (2001). Pollination: Plinth, pedestal, and pillar for terrestrial productivity. The why, how, and where of pollination protection, conservation, and promotion. In: C.S. Stubbs and F.A. Drummond

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[eds] Bees and Crop Pollinationâ&#x20AC;&#x201C;Crisis, Crossroads, Conservation. Thomas Say Publication of the Entomological Society of America, Lanham, Maryland, U.S.A. pp. 7-68. Kevan, PG (2003). Pollination for the 21st century: integrating pollinator and plant inter dependence. In: K. Strickler and J.H. Cane [eds] For Nonnative Crops, Whence Pollinators of the Future. Thomas Say Publication of the Entomological Society of America, Lanham, Maryland, U.S.A. pp. 181-204. Kevan PG ,Clark EA and Thomas V (1990). Insect pollinators and sustainable agriculture. Am. J. Alternative Agric. 5: 13-22. Kenmore P, Krell R (1998). Global perspectives on pollination in agriculture and agro-ecosystem management. International workshop on the conservation and sustainable use of pollinators in Agriculture with emphasis on Bees October 7-9 Sao Paulo, Barazil. Khan M R, Khan M R (2004). The role of honey bees Apis mellifera L. (Hymenoptera: Apidae) in pollination of apple. Pak. J. Biol. Sci. 7: 359-362. Knutson R D, Taylor R G, Penson B J, Smith GE (1990). Economic impacts of reduced chemical use. Knutson and Associates, College Station, Texas pp 30- 31. Louadi K , Doumandji S (1998). Diversity and gathering activity of bees (Hymenoptera: Apoidea) in a therophyte lawn in Constantine (Algeria). Canadian Entomologist, 130: 691-702. Margalef SR (1969). Diversity and stability: A practical proposal: a model of instars- dependence. Brookhaven Symposium of Biology, 22: 25-37. McGregor S E (1976). Insect pollinators of cultivated crop plants. United State Department of Agriculture, Agriculture Hand book, p. 496. Michener C D (2000). The bees of the world. The Johns Hopkins University Press, pp 97- 98.

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Nakamura H, Toshima H (1995). Environmental evaluation by distribution of butterflies on the case in Kagawa prefecture using the RI index. J. Environ. Entomol. Zool. Japan, 6: 143-159. Shannon ER, W,Weiner W (1963). The mathematical theory of communication. University of Illinois Pres. Urbana, Illinois, p. 117. Simpson E H (1949). Measurement of diversity. Nature, 163: 688. Thomas VG, Kevan PG (1993). Ecological principles in sustainable agriculture. J. Agric. Environ. Ethics. 6: 1-9. Verma LR, Partap U (1993). The Asian Hive bee Apis cerana as a pollinator in Vegetable Seed Production. International Centre for Integrated Mountain Development Katmandu Nepal. p183.

African Journal of Biotechnology Vol. 11(28), pp. 7270-7285, 5 April, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.3341 ISSN 1684â&#x20AC;&#x201C;5315 ÂŠ 2012 Academic Journals

Full Length Research Paper

Ultrastructural study of spermatogenic stages in the protandrous sparid fish Diplodus cervinus cervinus (Lowe, 1838) from the South Eastern Mediterranean coast Nevine M. Abou Shabana Aquaculture Division, Spawning Laboratory, National Institute of Oceanography and Fisheries, Alexandria, Egypt. E-mail: anny666mb@yahoo.com. Tel: +20145579066. Fax: +2034801499 or +2034801174. Accepted 26 January, 2012

The current study was designed to study the ultrastructure of the spermatogenic stages of the protandrous hermaphrodite sparid Diplodus cervinus cervinus. Although, it is a useful tool to enhance understanding of germ cells differentiation in this economic species, none of the available references paid attention to the studied species. The testis of the studied specie is tubular in shape and the germ cells are arranged in cysts or clusters within the seminiferous lobules. Spermatogenesis occurs in several places along the length of each lobule and induced by the action of the somatic steroidogenic secretory cells which are known as Leydig cells. Such cells contained four main morphological structural characteristics a vesicular nucleus, ovoid and elongated mitochondria with tubular cristae, a number of smooth endoplasmic reticula, and a considerable amount lipid droplets in the cytoplasm. Spermatogenic cyst displays round shaped cells with large nuclei containing clumps of heterogenic dense chromatin and reduced cytoplasm known as primary spermatogonia. They undergo a series of mitotic divisions to reach the secondary spermatogonia stage; such cells irreversibly divide meiotically to form primary and secondary spermatocytes. Spermatids are seen in different stages of spermiogenesis, as indicated by the degree of chromatin condensation. They underwent a shape remodeling and a size reduction during spermiogenesis as the morphology of the spermatid nucleus gradually changed and several mitochondria and centrosomes moved to a position just behind the nucleus of the spermatid. Spermatozoan ultrastructure showed that it is anacrosomal primitive teleostean type (type I) with round head, the posterior part of the nucleus is indented a nuclear fossa which displays a bell-shaped in longitudinal section and circular in transverse section containing centeriolar complex, part of the basal body and cytoplasm. The chromatin is heterogeneously granular, high electron-dense containing tightly packed fibers. The mid piece is short containing mitochondrial ring which consists of spherical or ovoid mitochondria. The axoneme consists of nine double outer tubules and single microtubular constructions. In conclusion, the testis structure could be described as the unrestricted spermatogonial testicular type. More attention should be rewarded to the female of this species in order to give a clear view about the process of oogenesis. Key words: Spermatogenesis, Sparid, protandrous, hermaphrodite, anacrosomal, primitive teleostean spermatozoa. INTRODUCTION Diplodus cervinus cervinus is a member of family Sparidae commonly known as zebra sea bream. Sparids

(porgies or sea breams) is considered as one of the largest percoidei families, predominantly marine and dis-

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tributed in the Atlantic, Indian and Pacific Oceans (Nelson, 2006). The family includes 33 genera with about 115 species (Nelson, 2006) and 6 subfamilies: the Boopsinae, Denticinae, Diplodinae, Dagellinae, Pagrinae and Sparinae (Smith and Smith, 1986). Zebra sea bream occurs in the eastern Atlantic coast from the Bay of Biscay to the Cape Verde Island and from Angola to South Africa, as well as around Madeira and Canary Islands, and in the warmer zones of the Mediterranean Sea (Bauchot and Hureau, 1990). The species D. cervinus (Lowe, 1838) consists of three subspecies (Pajuelo et al., 2003), D. cervinus omanensis (Bauchot and Bianchi, 1984), D. cervinus cervinus (Lowe, 1838) and D. cervinus hottentotus (Smith, 1844), which are distributed in the Oman waters, eastern Atlantic and Mediterranean Sea, and Indian Ocean, respectively (De la Paz, 1975). This family is considered of high economic importance concerning extensive farming (Gow, 1994; Nelson, 1994; Kato et al., 2001; Taddei et al., 2001; GorshKov et al., 2002 and Gow et al., 2004). Sparids are characterized by a rudimentary type of hermaphroditism with a low level of protandry and a multiple spawning character (Pajuelo et al., 2008). Fine structural work on spermatogenesis and spermatozoa continues not only to enhance understanding of the germ cell differentiation but also to provide fresh insights into the relationships between various teleosts groups, especially at or above the family level (Cinquetti and Dramis, 2003; Medina et al., 2003; Gwo et al., 2003, 2004, 2005, 2006). Spermatogenesis process is associated by steroidogenic action of Leydig and Sertoli cells (Ee-Yung, 2008). Although Leydig and Sertoli cells have been identified in the testes of teleost fishes, the functions and activities of the Leydig cells and Sertoli cells with the germ cell developmental stages during spermatogenesis have not yet been clarified (Pudney, 1993, 1996). Sertoli cells show high phagocytic activity in the sea bream testis and it is likely that they are the only cell type involved in the phagocytosis of degenerating germ cells in this species (Elena et al., 2005). Spermatozoa ultrastructure has been studied in several groups of fishes (Jamieson, 1991, 2009) and the usefulness of this data in the identification of the phylogenetic relationships has been recognized. In particular these studies have shown that the organization of the spermatic organelles is very conservative in the member of the same family or subfamily (Gusmao-Pompiani et al., 2005; Maicchiolo et al., 2010); therefore, ultrastructural characters of spermatozoa can be well combined with usual morphologic characters, in phylogenetic analysis. Scarce information is present concerning D. cervinus cervinus in the north western Mediterranean region (Pajuelo et al., 2008) and eastern Mediterranean as well. None of the available references studied the spermatogenesis in D. cervinus cervinus in respect to

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steroid secreting cells using ultrastructure techniques, although it is a useful tool to enhance understanding of germ cells differentiation. The objective of the current study was to examine the ultrastructure of spermatogenic stages and spermatozoan of Zebra seabream belonging to family Sparidae and to compare it with those available on other fish groups to assess the phylogenetic relationship with other teleosts. MATERIALS AND METHODS Transmission electron microscope Mature male specimens of D. cervinus cervinus of total length 15 to 22 cm were obtained from fishermen, captured by hooks from deep seas of Kayet Bay shore in Alexandria from January to June 2007. Samples of testes and semen were collected after sacrifice of fish then fixed in 4% glutraldehyde in 0.1 M Phosphate Buffer pH 7.4 at 4°C for 2 to 3 h, post fixed in 1% Osmium tetroxide for one hour after washing in 5% Sucrose in 0.5 M Sodium cacodylate buffer (pH 7.5) for one hour, three changes .Tissues were dehydrated in ascending ethanol series and embedded in epoxy resin. Semi-thin sections (1 µm) were cut using a LKB ultramicrotome with a glass knife and stained with toluidine blue. When appropriate regions were found, ultrathin sections were subsequently made and stained with drops of 2% uranyl acetate followed by lead citrate for 30 min. Then, these sections were examined and photographed using Leica digital camera. Ultra thin sections were cut on Joel Jum 7 ultra microtome and were stained in 5% aqueous Uranyl acetate for half an hour followed by Lead citrate for 5 min (Reynolds, 1963) and examined in a JEM – X10 transmission electron microscope at 80 KV. Scanning electron microscope For the preparations of scanning microscopy, milt was collected by stripping males by gentle abdominal massage then immediately fixed 4% glutraldehyde buffered to pH 7.2 with sodium cacodylate then filtered on 0.22 micromole Millipore filter then they were dehydrated in a graded series of ethyl alcohol ,critical point dried using CO2 and gold coating.

RESULTS Light microscopy structure of testicular tissue The testis of D. cervinus cervinus is tubular in shape and the germ cells are arranged in cysts or clusters within the seminiferous lobules. Spermatogenesis occurs in several places along the length of each lobule and the testis structure could be described as the unrestricted spermatogonial testicular type (Figure 1). Spermatogonia are found near the periphery along the length of the lobule, while spermatocytes, spermatids and spermatozoa are found toward the interior. Sertoli cells are found surrounding the germ cells. In the testicular structure,

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Figure 1. A photo-micrograph of a semithin section in Diplodus cervinus cervinus testis showing the spermatogenic stages 1ry spermatogonia (Spg1),2ry spermatogonia(Spg2),1ry spermatocyte (Spc1), 2ry spermatocyte (Spg2), spermatid (Sd), spermatozoa (Sz) and sertoli (Ser) and leydig (Ly) cells (Toluidine blue)X1000.

Leydig and Sertoli cells which appeared near germ cells in germinal cysts were involved in spermatogenesis. Clusters of Leydig cells in the interlobular space were easily distinguishable from the connective tissue composing the walls of the seminiferous lobules. They are located in the interstitium, which is surrounded by seminiferous lobules. The ultrastructures and activity of Leydig cells, which varied with the different stages of germ cell development, were observed within the interstitium. In particular, well developed Leydig cells were found during the period of active meiotic division and before spermiation. Morphological changes and activities of Sertoli cells showed different characteristics with germ cell developmental stages. After spermiation Sertoli cells were involved in the formation of several phagosomes that originated from degenerating spermatids and whole sperm cells by phagocytosis. Electron microscopic observation of germ cell development during spermatogenesis Based on morphological characteristics and the development of germ cells in the seminiferous lobules of the testis, spermatogenic stages are classified into 4 successive stages: a) spermatogonia, b) spermatocytes,

c) spermatids and (d) spermatozoa stages. Spermatogonia Spermatogenesis occurred in cysts of the seminiferous lobules. spermatogonia were individually surrounded by Sertoli cell and displays a very low electron density and regular outlines. They are round cells containing thin layers of cytoplasm and large nuclei (N) (Figures 1, 2 and 3). Primary and secondary spermatogonia vary in size according to the stage of development. Early and late stages are characterized by large nuclei filled with small clumps of electron dense chromatin, some of these are placed on the nuclear membrane and the rest are scattered in the nucleoplasm. The primary spermatogonium apparently measures between 10 and 8.5 Âľm in diameter as it is the largest germ cell in the testis present throughout the year. They are slightly oval shaped or spherical and a single prominent nucleus (about 8 Âľm in diameter) contains a nucleolus. Chromatin material in the nucleus is frequently observed in different degrees of condensation according to mitotic stage. At this stage, in particular, some intermitochondrial cement and several mitochondria appear in the cytoplasm near the nuclear envelope of the primary spermatogonium and a clear

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Spg1

n N

Figure 2. An electron micrograph showing primary spermatogonia in Diplodus cervinus cervinus displaying a large heterogenic nucleus (N), accumulation of small amount of electron dense chromatin (Cr), small nucleolus (n), mitochondrion (M) and a perinuclear nuage (Pn).X10,000.

Figure 3. An electron micrograph showing developing secondary spermatogonia in Diplodus cervinus cervinus displaying a large heterogenic nucleus (N), more accumulation of chromatin (Cr), small nucleolus (n), mitochondrion (M) and a perinuclear nuage (Pn).X10,000.

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M Ly

Sd2 Spc1

Figure 4. An electron micrograph showing the primary spermatocytes (Spc1) in Diplodus cervinus cervinus with a large nucleus (N) surrounded by leydig cells(Ly), arrow lipid droplits and developing spermatids (Sd2) X 25,000.

perinucleolar nauge is also distinguishable (Figure 2).The primary spermatogonia divide mitotically to produce secondary spermatogonia which measure between 6.5 and 5.5 µm in diameter. A decrease in size and an increase in the electron density of chromatin clumps in late stages of spermatogonia is clearly detected (Figure 3). Also eccentric nucleoli are obvious; however, in late spermatogonia the nuclear material fills the entire space of the cell with a decrease in the late spermatogonial diameter which measures 5 µm. Spermatogonia cells are bordered by Sertoli cell processes (Figure 1). Secondary spermatogonia also contain intermitochondrial cement and several mitochondria in the cytoplasm. Spermatocytes Primary and secondary spermatocytes resulted from the mitotic division of the secondary spermatogonial cells. Primary spermatocytes are round to oval shaped as sometimes it is hard to distinguish the cellular limits. The

nuclei of the primary spermatocytes contain chromatin which is slightly denser than that of the secondary spermatogonia (Figure 4). During the period of meiotic division clusters of developing Leydig cells in the interlobular space of the interstitium appeared near the connective tissue composing the wall of the seminiferous lobule, and the cell were round to oval shaped. Each of the developing Leydig cells measure approximately 6 to 3.5 µm in diameter along its long and short axes showing three main morphological structural characteristics a vesicular nucleus, ovoid and elongated mitochondria with tubular cristae, lysosomes and lipid droplets in the cytoplasm (Figures 4 and 7). Secondary spermatocytes, which arise from the meiotic division of primary spermatocytes, are present in large numbers within the cysts. Compared to primary spermatocytes, secondary spermatocytes measure approximately between 4.5 and 3.5 µm in diameter showing a decrease in cellular and nuclear sizes (Figure 5). They possess spherical nuclei with condensed chromatin that is irregularly distributed in their cytoplasm which was reduced containing several

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Spc2 Sy

Figure 5. An electron micrograph of secondary spermatocytes (Spc2) in Diplodus cervinus cervinus showing a large electron dense heterogenic nucleus (N) displaying high level chromatin condensation and synaptonemal complex (Sy). X30,000.

mitochondria and lipid droplets. At this stage, the nucleoli are not visible, and chromosomic masses (referred to as synaptonemal complex) were frequently found. The synaptonemal complexes in the nucleus appear in the prophase during the 1st meiotic division (Figure 5). Spermatid Apparently, secondary spermatcytes give rise to spermatids through the 2nd meiotic division. Spermatids are seen in different stages of spermiogenesis, as indicated by the degree of chromatin condensation. They undergo a shape remodeling and a size reduction during spermiogenesis. In the early part of the spermatid stage (approximately 4 to 3 Âľm in diameter), nuclei are oval or round, and become smaller showing denser chromatin. At this stage, particularly, condensation of electrondense heterochromatin masses appeared in the nucleus (Figure

8). In an early stage of spermiogenesis, well-developed Sertoli cells, which possess ovoid nuclei, appeared near several spermatids, mitochondria , a number of glycogen particles and a few lipid droplets were present in the cytoplasm of Sertoli cells (Figures 6 and 14). At this time, the activity of Sertoli cells was very high, although no clear evidence of steroidogenesis was found. During the period of spermiogenesis, well-developed Leydig cells, which are located near the cysts containing secondary spermatocytes and spermatids, are ovoid or cuboidal, and each cell contains an ovoid vesicular nucleus possessing slightly condensed chromatin around the nuclear envelope (Figures 4 and 7) Spermiogenesis During spermiogenesis, the morphology of the spermatid nucleus gradually changed, and several mitochondria

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Spc2

Sd2

Ser

Sd1

M 5Âľm

Figure 6. An electron micrograph in Diplodus cervinus cervinus testis showing a sertoli cell (Ser) surrounding the secondary spermatocytes (Spc2) displaying round mitochondria (M) and developing spermatids (Sd1, Sd2) X 30,000.

and centrosomes moved to a position just behind the nucleus of the spermatid (Figures 8 and 10). The centriolar complex appears lateral to the nucleus and close to the plasma membrane to form the flagellum. Some mitochondria of spherical or ovoid shape are located near the centriole. At subsequent stage the centriole migrates to the basal pole of the nucleus (Figure 10). The process of spermiogenesis could be divided into the following stage based on formation of the flagellum, rotation and condensation of the nucleus and loss of the superfluous cytoplasm (exocytosis). Formation of the flagellum: The two centrioles, each with nine triplet microtubules, are arranged at right angles to each other and appear to be interconnected by osmiophilic filaments. Both centrioles lie close to the plasma membrane and the distal centriole (or its basal

body) starts to form the flagellum (Figure 10). The diplosome-flagellar axis is tangential to the nucleus and the mitochondria aggregate around the base of the flagellum. Golgi bodies appear as vesicular cisternae. The nucleus is electron dense with prominent patches of fine and coarse granular heterochromatin. Rotation and condensation of the nucleus: The cell in this stage displays a finely granular appearance because of the homogeneity of chromatin. The nucleus becomes indented and a nuclear fossa is formed. The proximal centriole is located within the nuclear fossa (Figure 10). A nuclear notch appears in the region between the proximal and distal centrioles. In addition, two fibrous bodies, each of which consists of osmiophilic disks alternating with lighter material, appear perpendicular to each other above the proximal centriole within the nuclear fossa;

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Figure 7. Sagittal section of Leydig cell in Diplodus cervinus cervinus showing a large ovoid nucleus (N) diplaying a contour layer of electron dense chromatin , mitochondrion(M), and the arrow pointing to variable size of lipid droplets (Ld) X 7,500.

Sd1 Sy

Figure 8. Sagittal section in Diplodus cervinus cervinus testis showing early developing spermatid (Sd1) displaying synaptoneamal complex (Sy) and mitochondria (M). X 30,000.

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F Ad Figure 9. An electron micrograph of scanning electron microscope showing the external morphology of Diplodus cervinus cervinus permatozoa, round head region (H) a muff-like middle piece (Mp) and a long tail or flagellum (F), the anterior depression (Ad), enlarged spermatozoan showing anterior depression and the middle piece of the spermatozoan (Mp). X15,000.

Dc C.C

5Âľm Figure 10. Sagittal section in Diplodus cervinus cervinus testis showing late spermatid (Sd2) and displaying the nuclear fossa (Nf), distal centriole(Dc) large mitochondria (M) and the vesicles of golgi bodies (Gb).X 50,000.

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Nf F Ad

1Âľm Figure 11. An electron micrograph in Diplodus cervinus cervinus developing spermatozoa displaying proximalcentriole (Pc) embedded in the nuclear fossa (Nf) and a large mitochondrion (M). X100,000.

similarly, the lower body connects with the proximal centriole. Moreover, a basal foot appears laterally in the basal part of the distal centriole and anchors it to the nucleus (Figure 11). The axoneme appears surrounded by a mitochondrion which is separated from the flagellum by the cytoplasmic canal (Figure 11). As development proceeds, the size of the spermatid decreases and the intercellular spaces enlarge to form a lumen in the cyst. Exocytosis: The nucleus becomes more compact with increasing dense and thick chromatin. The nuclear fossa is further expanded into the nucleus and completely surrounds the centriolar complex (Figure 10). As the nuclear fossa develops, the nucleus condenses and a small nuclear notch appears at the level of the proximal centriole, which gradually increases and becomes filled with electron dense material. Spermatozoon The mature spermatozoon is a relatively simple elonga-

ted cell composed of a head, a short mid piece and a relatively long tail or flagellum (Figures 9, 11 and 12). The head is spherical in shape, about 1.2 Âľm in diameter and has no acrosome. The nucleus is covered by a typical double layered undulating nuclear envelope. The nuclear envelope and the plasma membrane are applied tightly to the anterior part of the nucleus (Figure 12); however, the posterior part is indented by a nuclear fossa. The fossa is bell-shaped in longitudinal sections and circular in transverse sections and contains the centriolar complex, part of basal body and cytoplasm. The chromatin is heterogeneously granular, highly electron-dense containing tightly packed fibers and irregular small clear lacuna. The mid piece is short contains a mitochondrial ring which consists of spherical or ovoid mitochondria. The mitochondrion is completely separated from the mid piece by the cytoplasmic canal. The flagellum is surrounded by the flagellar plasma membrane. The axoneme consists of nine double outer tubules and two single central microtubular constructions. The central tubules are surrounded by a thin sheath and appear to be interconnected by a single strand. Each of the nine outer doublets consists of

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12 Ax M

Dc f

1µm Figure 12. An electron micrograph in Diplodus cervinus cervinus spermatozoa displaying the proximal centriole (Pc) and distal centriole (Dc) embedded in the nuclear fossa (Nf) and jointed together with fibrous tissue (f) where the axoneme rises (Ax) also showing the cytoplasmic collar of the sperm head (Co) and large mitochondrion (M). X75,000

sub-fibers A and B. Two arms arise from sub-tubule A of each doublet and extend toward the next tubule (Figure 13). DISCUSSION Spermatogenesis is a highly organized and coordinated process, in which diploid spermatogonia proliferate and differentiate to form mature spermatozoa. The duration of

this process is usually shorter in fish than in mammals and is also influenced by the water temperature (Nَõbrega et al., 2009). Spermatogenesis occurs during the annual reproductive cycle, so the testicular constituencies vary according to time and degree of maturity. It involves initial proliferation of spermatogonia through repeated mitotic division then a growth period to form primary spermatocytes. These then undergo reduction division (meiosis) to form secondary spermatocytes; leading to the spermatid phase, and following metamorphosis, into the

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Ad Dc Pc Co

F Ax 9+2 Figure 13. An electron micrograph of transmission electron microscope showing Diplodus cervinus cervinus mature spermatozoan displaying the deeply embedded centriolar complex into the nucleus ,the proximal and the distal centrioles (Pc, Ac), the anterior depression (Ad) ,the cytoplasmic collar( Co) and an enlarged region of axonem showing the 9+2 configuration of axonemal doublets.X25,000 and X50,000.

motile spermatozoa. The interstitium between lobules consists of interstitial cells, fibroblasts, blood and lymph vessels. The lobular component contains two cell types: germ cells and distinct somatic cells lining the periphery of the lobule. The terminology used to describe these somatic cells has been the source of much debate (Coward et al., 2002). In some species, the lobule boundary cells are considered more likely to be homologous to Sertoli cells as cells are found in close proximity to spermatids/developing sperm and possess various structures that indicate a phagocytic role (Billard, 1970; Billard et al., 1972; Grier, 1975, 1981) The present results show that Sertoli cells exist on the borders of cysts containing the primary spermatogonia. It is a spindle shape cell with large nucleus; the later has clumps of electron dense chromatin arranged on the nuclear membrane and some in the nuleoplasm. The

cytoplasm of Sertoli cells of D. cervinus cervinus contain a huge amount of lysosomes confirming the phagocytic function which has been described before. The present results conform to those prementioned concerning the function of Sertoli which said to play an important role in phagocytosis of degenerating and residual sperm cells (Grier, 1993; Loir et al., 1995; Cinquetti and Dramis, 2003). Moreover, Sertoli secrete fluid that generates the tubular lumen, and they phagocytise apoptotic germ cells, residual bodies discarded by spermatids during spermiogenesis, and residual sperm (Schulz et al., 2010). Also, Mattei and Mattei, (1982) mentioned another function of these somatic cells, that they are the storage site of steroids if they do not synthesize it. The second type of somatic interstitial cells which are known as Leydig cells are formed between seminiferous tubules of the investigated fish, it has large irregular

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Figure 14. An electron micrograph of Sertoli cell in Diplodus cervinus cervinus showing large nucleus (N), rough endoplasmic reticulum (REr), lysosomes(Ly) outside the sertoli cell membrane, large vacuole (V),and phagocytic cells (Ph). x 15,000.

nucleus with clumps of electron dense material, some of which are placed on the nuclear membrane. It has electron lucent cytoplasm in which smooth endoplasmic reticulum is scattered, mitochondria, free ribosomes and lipid droplets of different sizes. The current results were in agreement to those recorded by Cinquetti and Dramis (2003) on Leydig cells functions, these cells appear to be the main site of androgen synthesis throughout the reproductive cycle and undergo cycling morphological and functional changes correlated to the germinal epithelium. The Leydig cells of most teleost species undergo pronounced seasonal changes in lipid content. There is an accumulation of positive lipids at the end of the breeding season, presumably owing, to the build-up of substrate due to decreased androgen synthesis. The Leydig cells of Boleophthalmu pectinirostris have special morphological characteristics: a vesicular nucleus, mitochondria with tubulo-vesicular cristae, and a number of smooth endoplasmic reticulum (Ee-Yung, 2008). Some authors stated that lipid droplets are absent in healthy Leydig cells of other teleost species (Follenius and Porte, 1960; Follenius, 1968; Gresik et al., 1973). Some investigations confirmed the absence of lipid droplets in such cells in different species such as Thalassoma duperrey (Hourigan et al., 1989) and Esox lucius and E.

niger (Grier et al., 1989). Hormones secreted by interstitial Leydig cells into the extravascular space have ready access to the circulatory system or to target Sertoli cells, however, the interaction between Leydig and Sertoli cells in spermatogenesis is still unknown in fishes (Grier et al., 1989). The number of spermatogonial generations is genetically determined (A and B). Both in mammals (De Rooij and Russell, 2000) and fish, the type B spermatogonia are dividing more rapidly than the type A spermatogonia (Schulz et al. 2005). The different spermatogonial generations often pose problems as regards their identification and therefore receive particular attention. In general, stem cells are defined as the most undifferentiated cells of a particular lineage. They reside in a niche created by a supporting cell type, Sertoli cells, and the surrounding extracellular matrix. The niche is defined by its specific microenvironment that allows the stem cell to retain itâ&#x20AC;&#x2122;s stemness (Li and Xie, 2005; Hofmann, 2008). In species showing several spermatogonial generations, like guppy, zebrafish, or trout, a differentiation can be made based on cell/nuclear size and number of cells per cysts into early and late type spermatogonia (Schulz et al., 2010). The spermatogonia are large rounded cells with electron lucent cytoplasm and large round to oval nucleus.

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Primary spermatogonia are larger in size than secondary spermatogonia. Distinctive changes are observed in D. cervinus cervinus spermatogonia to develop into spermatic cysts especially in nuclei that illustrate electron lucent nucleoplasm with few clumps of electron dense chromatin. An increase in the amount of this electron dense chromatin is noticed in the late stages than in the early ones filling the entire nucleus and gaining a heterogeneous appearance. Perinuclear nuage is always seen near to the nuclear pore and associated with the germ cells. The origin and function of the perinuclear nuage in the male germ cell is not clear (Huang et al., 2002). Some authors assumed that this dense material is composed of ribonucleoproteins and mRNA species showing a long half-life time, including vasa or piwi mRNA, specific products of the germ cell lineage (Knaut et al., 2000; Houwing et al., 2007). After final mitosis of spermatogonia (B), meiotic division started to take place to produce the primary spermatocyte subsequently followed by the secondary spermatocytes. Diplodus cervinus cervinus spermatocytes are characterized by large nuclei with higher electron density and the appearance of synaptonemal complex displaying diplosomal structure. Grier (1992) reported that Sertoli cells, primary spermatogonia, and the subsequent stages of germ cell development reside in spermatocysts. They are always sequestered from interstitial tissues by a basement membrane. Sertoli cells rest upon the basement membrane and their process form the borders of spermatocysts. The current results coincide with the findings on B. pectinirostris (Ee-Yung, 2008). Spermiogenesis consists of series of morphological changes that lead to the differentiation of spermatids into spermatozoa. The changes include nuclear condensation, elimination of organelles and cytoplasm, flagellum formation, and the rearrangement of cellular organelles along the spermatozoon cytoplasm (Jamieson, 1991). The spermatid of D. cervinus cervinus is non acrosomal with a flagellum originating perpendicular to the centriolar complex and deeply embedded into the nucleus. The current findings were in agreement to those recorded in family Scoloplacidae (Spadella et al, 2006). The nucleus has a condensed granulated chromatin, the condensation of chromatin in the nuclei of spermatids develops to maturity in a definite pattern, and it always starts adjacent to the developing flagellum. There are three types of spermiogenesis in fish, (type I, II, and III) have been described (Quagio-Grassiotto and Oliveira, 2008) based on the orientation of the flagellum to the nucleus, and on whether or not a nuclear rotation occurs. Type I is characterized by a perpendicular flagellum in relation to the nucleus with nuclear rotation; in type II, the flagellum develops parallel to the nucleus without nuclear rotation, and in type III, the flagellum is central without nuclear rotation (Mattei, 1970; Quagio-Grassiotto and Oliveira,

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2008). These patterns are reflected in the spermatozoa structure, and highly conserved within taxonomic units, and are, therefore, a powerful tool for phylogenetic analyses in fish (Jamieson, 1991; Quagio-Grassiotto and Oliveira, 2008). So the present result obeys type I spermatid, which possesses a perpendicular central deeply embedded flagellum with nuclear orientation. Stages of spermatogenesis in Diplodus cervinus c. were based on the modifications occurred in the nucleus and spermatid before spermatozoan formation. The distribution and organization of the cytoplasmic organelles and the implantation of the spermatid fossa of Diplodus cervinus c. happens according to the pattern described by Thiaw et al. (1988) and Zaki et al. (2005). A decrease in the volume is found in D. cervinus c. in stages of spermatocytes and spermatogonia. The centriole in D. cervinus c. is embedded in the nucleus; the proximal and distal centrioles are linked by electron dense filament to form the nuclear fossa, and the cytoplasmic collar attached to the proximal part of the flagellum. Sprando and Russell (1988) proposed three ways to reduce the cytoplasmic size in spermatid: 1- formation of residual bodies to be phagocytosed by Sertoli cells, 2- formation of tubular complexes followed by disintegration, 3dehydration then chromatin condensation. The elimination of the residual bodies in D. cervinus c. could be of the first type. Same believe was recorded by Zaki et al. (2005), while Huang et al. (2002) mentioned that the elimination in his study is of the second type although all these authors were examining Sparid fishes. Spermtozoan of D. cervinus c. has the characteristic morphology of teleosts. It is devoid of an acrosome as the rest of the Sparids and other bony fishes (Billard, 1970; Bacetti et al., 1984; Gow et al., 1993). Ginsburg (1968) reported that the absence of an acrosome could be attributed to the presence of micropyle in fish eggs. The head of sperm of D. cervinus c. is round shape as reported before in other species, in turbot (Suqnet et al., 1993), Oreochromis sp (Bern and Avtation, 1990), grey mullets (Brusle, 1982) and cyprinids (Bacetti et al.,1989), Sparidae (Gow et al., 2005). On the other hand, oval shaped head was reported in other species such as guppy (Billard, 1970), eel (Billard and Ginoburg, 1973), Stanoperca sp. (Matos et al., 2002), Mullidae and Siganidae (Gow et al., 2004) and Blue Spart, Clupeidae (Gow et al., 2006). The nucleus of the spermatozoan in D. cervinus c. has an anterior and a posterior nuclear depressions which was reported before in the Rainbow and becook trout , guppy ,Tilapia, turbout, Sparus aurata ,Diplodus vulgaris and Lithognathus mormyrus by Billard (1983a, b, 1970), Bern and Avtalion (1990), Suquet et al. (1993) and Boops boops by Zaki et al. (2005). On both sides of D. cervinus cervinus axial nuclear fossa, electron dense microfibrils exist connecting the

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distal centriole to the nucleus of the sperm head. Martinez-Soler et al. (2007) have reported that in Sepia officinalis the perinuclear microtubule system represents an element responsible for rigidity and shape of the sperm nucleus and has a role in nucleoplasmic transport. A short or reduced middle piece is found in D. cervinus cervinus spermatozoan, a feature which is common in teleosts with external fertilization (Nicander and SjoÈ den, 1971; Zaki et al., 2005), while in internally fertilized fish the spermatozoan has a longer middle piece (Matti, 1969). Depending on the orientation of the centriolar complex to the nuclear fossa, Matti (1991) had classified teleostean sperm into two types I and II. In type I the centriolar complex lies inside the nuclear fossa, while type II it lies outside the nuclear fossa. D. cervinus cervinus centriolar complex lies deeply inside the nuclear fossa (type I) as pre-mentioned in other sparid fishes (Gow et al., 2004; Zaki et al., 2005); turbout (Suquet et al., 1993) and Gymnotiforms (França et al., 2007). However, Gusmăo et al. (2005) reported that the sperms of the Sciaenidae family and other Percoidei are of type II nucleus. D. cervinus cervinus has one big mitochondrion lies beneath the head of the sperm as reported in other Sparids as a characteristic feature for this family (Gow et al., 2004; Zaki et al., 2005). The flagellum of D. cervinus cervinus has the common microtubular structure 9 + 2 which is observed in many teleosts, turbouts (Matti, 1969; Suquet et al., 1993; Gow et al., 2004; Zaki et al., 2005). From the present study and the previous ones on Sparids, the information provided on sperms morphology and the differences between species can be used to define the taxononomic position. Although the ultrastructural characterization of the sperm is not so useful in determining the phylogenetic relationships as in different groups of other animals (Jamieson, 1991; Matti, 1991). Conclusion and recommendation In order to learn more about this species, more attention should be rewarded to the study of oogenesis process. Also it is highly recommended to concern this economic fish species in aquaculture field and to establish an induced spawning protocol for fish farming as the majority of the sparids had been already farmed successfully. REFERENCES Baccetti B, Burrini AG, Collodel G (1989). Morphogenesis of decapitated and decaudated sperm defect in two brothers. Gamete Res. 23: 181-188. Bauchot ML, Bianchi G (1984).*Diplodus cervinus omanensis*, nouvelle sous-espèce de *Diplodus cervinus* (Lowe, 1841), capturée en mer d'Arabie (Pisces, Perciformes, Sparidae). Historie naturelle des

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Full Length Research Paper

Evaluation of the productive performance characteristics of red tilapia (Oreochromis sp.) injected with shark DNA into skeletal muscles and maintained diets containing different levels of probiotic and amino yeast Samy Yehya El-Zaeem1,3*, Talaat Nagy Amer2 and Nader Ezzat El-Tawil2 1

DNA Research Chair, Zoology Department, College of Sciences, P. O. Box 2455, King Saud University, Riyadh 11451, Saudi Arabia. 2 Experimental Station for Aquaculture Research (ESAR), Central Laboratory for Aquaculture Research, Agriculture Research Center, Alexandria, Egypt. 3 Animal and Fish Production Department, Faculty of Agriculture (Saba-Bacha), Alexandria University, Alexandria, Egypt. Accepted 14 March, 2012

This work aimed to study the effect of direct injection of shark (Squalus acanthias L.) DNA into skeletal muscles of red tilapia (Oreochromis sp.) fed at different additive levels (two probiotic levels; 0.3 and 0.5%, two amino yeast levels; 0.5 and 1.0% and a mixed of 0.3% probiotic and 0.5% amino yeast), on the productive performance. The results show that red tilapia injected with DNA had significant (P≤0.05) superiority of growth performance and feed utilization; besides the body composition was improved. In addition, the different levels of probiotic and amino yeast were more effective in stimulating most of the productive performance traits compared to the control group and the mixed of probiotic and amino yeast. The result indicates a possible easy and rapid way for improving red tilapia characteristics. Key words: red tilapia, shark DNA, direct injection, productive performance, probiotic, amino yeast. INTRODUCTION A quick method and rapid way for introducing foreign DNA injected directly into the muscle tissue was reported by Wolff et al. (1990) and Ono et al. (1990) in adult mice, by Thomson and Booth (1990) in rat and by Hansen et al. (1991), Rahman and Maclean (1992), Tan and Chan (1997), Xu et al. (1999), El-Zaeem (2004), Hemeida et al. (2004), El-Zaeem and Assem (2004), Assem and ElZaeem (2005), El-Zaeem (2012), and El-Zaeem et al. (2012) in fish. This procedure is useful because muscle injection is much easier than the others and very rapid

*Corresponding author. E-mail: selzaeem@yahoo.com, selzaeem@ksu.edu.sa or samy.elzaeem@alexagrsaba.edu.eg. Tel: +201003552398 or +966592299396.

results are obtained (Rahman and Maclean, 1992). The foreign DNA was presented extrachromosomally up to six months following injection (Wolff et al., 1990). Moreover, Sudha et al. (2001) reported that the expression of muscular injection of DNA was evident in several non muscle tissues, such as skin epithelia, pigment cells, blood vessel cells and neuron-like cells. A series studies have focused on the use of shark DNA to boost immune responses in fish (El-Zaeem and Assem, 2004; Assem and El-Zaeem, 2005). Sharks contain high levels of immunoglobulin (IgM) proteins, which act as antibodies and help initiate immune responses to bacterial invasions. Although IgM can be found at high levels in shark (up to 50% of the serum proteins), it has been reported to be present at much lower levels in fish such as Atlantic salmon, Halibut (Hippoglossus

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hippoglosus L.), Haddock (Melanogrammus aeglefinus L.) and Cod (Gadus morhua L.) (2, 8, 13 and 20% of the serum proteins, respectively) (Marchalonis et al., 1993; Magnadottir, 1998). When shark (Squalus acanthias L.) DNA was injected into the skeletal muscles of Nile tilapia (Oreochromis niloticus) and red belly tilapia (Tilapia zillii) fingerlings, fish showed significantly higher levels of total antibody activity, serum total protein, and globulin (ElZaeem and Assem, 2004; Assem and El-Zaeem, 2005). In addition, injected tilapia had significant growth enhancement and changes in proximate composition, with decreases in moisture and increases in both protein and lipid content. Injected fish showed high genetic polymorphism, indicating random integration of the shark genes into tilapia muscle DNA. Since the first use of probiotics in aquaculture, a growing number of studies have demonstrated their ability to control potential pathogens and to increase the growth rates and welfare of farmed aquatic animals (Gatesoupe, 1991, 1999; Wang et al., 2005; Wang and Xu, 2006; Wang, 2007). The beneficial effects of probiotics, such as improvement of feed utilization, modulation of intestinal microflora, enhancement of immune responses and antagonism to pathogens, have been demonstrated in a number of previous studies (Balcázar et al., 2006a,b; Irianto and Austin, 2002a,b; KesarcodiWatson et al., 2008; Merrifield et al., 2010a,b,c,d; Wang et al., 2008). Among the various benefits of probiotics, immunomodulatory activity is noteworthy in improving the overall health status of the host. However, there is limited research available for immunomodulatory activity of probiotics, especially for the long-term use of probiotics in fish diets. The most commonly used probiotics in aquaculture practices belong to lactic acid bacteria and Bacillus spp. (Wang et al., 2008). Brewer's yeast, Saccharomyces cerevisiae, can be used as a probiotic (Chiu et al., 2010; Lara-Flores et al., 2003) and also as growth promoter (Abdel-Tawwab et al., 2008; Lara-Flores et al., 2003; Li and Gatlin, 2003, 2005) in aquaculture. Brewer's yeast (S. cerevisiae) has been used as a feed supplement for various animals. It contains various immunostimulating compounds such as β-glucans, nucleic acids, mannan oligosaccharides and other cell wall components (Li and Gatlin, 2003, 2005; Oliva-Teles and Gonçalves, 2001). It has been observed that S. cerevisiae can positively influence the non-specific immune responses (Ortuno et al., 2002; Siwicki et al., 1994) as well as growth performance (Abdel-Tawwab et al., 2008; Li and Gatlin, 2003, 2005; Noh et al., 1994; Oliva-Teles and Gonçalves, 2001; Rumsey et al., 1991; Taoka et al., 2006) of some fish species. Therefore, the objective of this study was to evaluate the productive performance characteristics of red tilapia (Oreochromis sp.) injected with shark (Squalus acanthias L.) DNA (into skeletal muscles) and fed maintained diets containing different additive levels of probiotic and amino yeast.

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MATERIALS AND METHODS Fish origin The red tilapia, Oreochromis sp. fry used in this study was a hybrid, descended of an original cross of female Oreochromis mossambicus x male O. niloticus and obtained from Marine Fish Hatchery, 21 Km, Alexandria, Egypt. Red tilapias were transported to the laboratory of Breeding and Production of Fish, Animal and Fish Production Department, Faculty of Agriculture (Saba-Bacha), Alexandria University, Alexandria, Egypt. DNA extraction High molecular weight DNA was extracted according to Brem et al. (1988) method. Isolation of DNA was accomplished by reducing liver sample from shark (Squalus acanthias L.) to small pieces, which were then transferred to a microfuge tube and incubated overnight until the sample was digested in a buffer solution containing 50 mM Tris, 100 mM ethylenediaminetertracetic acid (EDTA, pH 8.0), 100 mM NaCl, 0.1% sodium dodecyl sulfate (SDS) and 0.5 mg/ml proteinase K. After incubation, samples were extracted twice for 15 to 20 min with one volume of phenol/ chloroform (1:1) and then again twice for 15 min with one volume of chloroform/isoamyle-alcohol (24:1). The aqueous phase was then precipitated with 2.5 volumes of 100% ethanol in the presence of 1/10 volume 3 M sodium acetate (pH 6.0). The pelleted DNA was washed with 70% ethanol and dissolved in 0.1x saline sodium citrate (SSC) buffer. The DNA concentrations were measured by UV spectrophotometry. The extracted DNA was restricted by Eco R1 restriction enzyme type II. It digested DNA between guanine and adenine according to Tsai et al. (1993). Experimental design Culture condition Red tilapias were acclimatized to laboratory conditions for two weeks. Fry with an initial body weight (2.9±0.11 g) were divided randomly to 12 groups and three replicates for each group, stocked at the rate of 10 fish per aquarium. The aquaria of dimensions 100 x 34 x 50 cm were supplemented with continuous aeration. Nearly half of the volume of water in the aquaria was changed daily by freshly stocked tap water and the aquaria were cleaned every day before feeding. Water temperature was maintained at 26°C by means of electric aquarium heaters. Fish were stocked at 1.0 fish/10 L and fed twice daily to satiation, six days a week. Fish were weighed at the beginning of experiment and then biweekly for eight weeks. Injection of foreign DNA in vivo The DNA concentration of 40 µg/0.1 ml/fish (El-Zaeem, 2004; ElZaeem and Assem, 2004; Hemeida et al., 2004; Assem and ElZaeem, 2005) were prepared using 0.1x SSC buffer and injected into red tilapia muscles using a hypodermic needle. The injection was applied on six groups of red tilapia fingerlings, while the other six groups were left without injection as a control. Diets formulation and preparation Six dietary treatments were tested in triplicate groups: the control diet with no dietary supplementation (C) and five other test diets which included; probiotic added at 0.3% (P1) and 0.5% (P2); amino

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Table 1. Composition and proximate analysis of red tilapia (Oreochromis sp.) diets used during the growth period.

Ingredient Wheat flour Wheat bran Soybean meal Yellow corn Fish meal Bone meal Vit. & Min. Mix* 1 Probiotic 2 Amino yeast Total Proximate analysis (%) Moisture Protein Fat Fiber Nitrogen free extract (NFE) Ash Metabolizable energy (kcal/100g)

C 31 18 23 14.2 11.5 2 0.3 0 0 100

P1 31 18 22.7 14.2 11.5 2 0.3 0.3 0 100

P2 31 18 22.5 14.2 11.5 2 0.3 0.5 0 100

A1 31 18 22.5 14.2 11.5 2 0.3 0 0.5 100

A2 31 18 22 14.2 11.5 2 0.3 0 1.0 100

P1+A1 31 18 22.2 14.2 11.5 2 0.3 0.3 0.5 100

10.63 23.84 7.31 4.24 44.07 9.91 244.05

10.73 23.82 7.30 4.20 44.07 9.88 244.23

10.71 23.84 7.29 4.21 44.04 9.91 243.85

10.83 23.70 7.29 4.13 44.18 9.87 244.14

10.96 23.69 7.21 4.02 44.30 9.82 243.51

10.78 23.83 7.24 4.19 44.10 9.86 243.87

* Content/kg of vitamin and minerals mixture (P- Fizer, Cairo, Egypt). Vitamin A, 4.8 MIU; Vitamin D, 0.8 MIU; Vitamin E, 4.0 g; vitamin K, 0.8 g; vitamin B1, 0.4 g; vitamin B2, 1.6 g; vitamin B6, 0.6 g; vitamin B7, 20.0 mg; vitamin B12, 4.0 g; folic acid, 0.4 g; nicotinic acid, 8.0 g; pantothenic acid, 4.0 g; colin chloride, 200 g; zinc, 22 g; cooper, 4.0 g; iodine, 0.4 g; iron, 12.0 g; manganese, 22.0 g; selenium, 0.04 g. 1 Probiotic; produced by Pura 2A. Company, Cairo, Egypt, containing: Lactobacillus sp. (L. plantarum, L. fermentum, L. delbrueckii), Bacillus subtilis, Saccharomyces cerevisiae, Rhodopseudomonas palustris and especially active additives. probiotic 1%, molasses 3%, water 96%. 2 Amino yeast; produced by New Vetro vit 10 th. of Ramadan city, Egypt containing: B. Glucan 44.50 g, Rhammose (1500.00 mg), Xylose (3000.00 mg), D L- Methionine (10110.00 mg), lysine (2600.00 mg), monnanollgo saccharides (200.00 g.)

yeast added at 0.5% (A1) and 1.0% (A2); and mixed of probiotic and amino yeast 0.3% + 0.5% (P1+A1). Total protein content for all dietary treatments was adjusted by the parallel decrease of soybean meal with increase level of dietary supplementation as described in Table 1. Metabolizable energy was calculated according to feedstuff values reported by NRC, (1993). Dry ingredients were passed through a sieve (0.6 mm diameter hole) before mixing into the diets. Mixtures were homogenized in a food grinder mixer. Water was then blended into the mixture at the ratio of 50% for pelleted. The diets were pelleted using meat grinder with a 1.5 mm diameter. Quantitative traits studied The following parameters were measured; body weight (BG), weight gain (WG), specific growth rate (SGR %/day), percent body weight increases (%BWI), feed intake, feed conversion ratio (FCR), protein efficiency ratio (PER), protein and energy retention percent (PR% and ER%). Initial and final whole body composition analyses were performed using the standard methods (AOAC, 1984) for moisture (oven drying), for protein (macro-kjeldahl method) and lipid (ether extract method).

Statistical analysis Data were analyzed using the following model (CoStat, 1986): Yijkl=µ+Ti+Aj+(TA)ij+ Bk+eijk

Where,Yijk is the observation of the ijkth parameter measured; µ is the overall mean; Ti is the effect of ith DNA; Aj is the effect of Jth additives; (TA)ij is the interaction DNA by additives; Bk is the effect of Kth block; and eijk is the random error. Significant differences (P≤0.05) among means were tested by the method of Duncan (1955).

RESULTS Data presented in Table 2 show that the final body weight (FBW), weight gain (WG), percent body weight increases (% BWI) and specific growth rate (SGR %/day) of red tilapia (Oreochromis sp.) injected with shark DNA were significantly higher (P≤0.05) than those of the noninjected fish. Moreover, the highest value of FBW was obtained by the red tilapia fed the highest level of probiotic, but did not differ (P≤0.05) significantly from those fish fed the lowest level of probiotic and the lowest and highest levels of amino yeast. The highest WG was achieved by the fish fed the highest level of probiotic, but did not differ (P≤0.05) significantly from those of fish fed the lowest level of probiotic and the highest level of amino yeast. Red tilapia fed the lowest level of probiotic show higher mean of % BWI, but did not differ (P≤0.05) significantly from that of fish fed the highest level. The

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Table 2. Effect of shark DNA injection and different additive levels on growth performance1 of red tilapia (Oreochromis sp.).

Treatment Type of DNA (T) DNA Non-DNA

IBW (g)

FBW (g)

WG (g)

2.92±0.10 2.83±0.11

14.87±1.76 b 12.81±0.81

Type of Additive (A) Control P1 P2 A1 A2 P1+A1

2.90±0.10 2.80±0.00 2.90±0.13 2.85±0.09 2.90±0.10 2.90±0.10

Interactions T x A DNA x C DNA x P1 DNA x P2 DNA x A1 DNA x A2 DNA X P1+A1 Non-DNA x C Non-DNA x P1 Non-DNA x P2 Non-DNA x A1 Non-DNA x A2 Non-DNA X P1+A1

2.90±0.10 2.80±0.00 3.00±0.00 2.90±0.10 3.00±0.00 2.90±0.00 2.90±0.00 2.90±0.00 2.80±0.00 2.80±0.20 2.80±0.00 2.90±0.00

%BWI

11.9±61.74 b 9.97±0.86

12.37±1.50 a 14.90±0.81 15.05±2.96a 13.63±0.99abc 13.93±1.20ab bc 13.16±0.77

9.48±0.16 a 12.10±0.81 12.15±3.32a 10.78±0.94b 11.03±1.10ab bc 10.26±0.81

12.45±0.15b 15.50±0.70ab 17.90±1.10a 14.44±0.22ab 15.10±0.30ab b 13.86±0.10 12.30±0.10b 14.30±0.30ab 12.20±0.20b 12.82±0.78b 12.75±0.15b 12.47±0.47b

9.55±0.05b 12.70±0.70ab 14.90±1.10a 11.54±0.12ab 12.10±0.30ab ab 10.96±0.20 b 9.40±0.20 11.50±0.30ab 9.40±0.40b 10.02±0.78b 9.95±0.15b 9.57±0.57b

SGR %/day a

2.90±0.20 b 2.70±0.16

327.21±16.98 a 432.14±33.25 417.56±101.12ab 378.06±33.51bc 379.34±29.21bc c 354.70±40.57

2.59±0.06 a 2.98±0.10 ab 2.91±0.31 2.84±0.09abc 2.77±0.12bcd cd 2.70±0.14

329.65±13.64 453.57±35.36 496.67±51.85 398.27±13.57 403.33±14.14 378.34±27.81 324.77±25.59 410.72±15.15 338.46±54.39 357.86±39.39 355.36±7.57 331.07±43.94

2.60±0.04 3.06 ±0.09 3.19±0.11 2.87±0.04 2.89±0.04 2.80±0.08 2.59±0.08 2.91±0.02 2.64±0.16 2.82±0.12 2.66±0.03 2.61±0.13

409.97±59.84 b 353.04±39.41

(1) Mortality rate was 0.0% for all fish injected and for the control. Means having different superscripts within column in a main effect are significantly different (P≤0.05).P1 and P2, 0.3 and 0.5% of probiotic, respectively; A1 and A2, 0.5 and 1.0 % of amino yeast, respectively. Initial and final body weight (IBW and FBW) = body weight at start and end of experiment. Weight gain (WG) = final weight - initial weight. Percent body weight increases (% BWI) = (final weight - initial weight) 100/ initial weight. Specific growth rate (SGR%/day) = (Ln final weight - Ln initial weight) 100 / number of days.

highest value of SGR %/ day was recorded by red tilapia fed the lowest level of probiotic, but did not differ (P≤0.05) significantly from those of fish fed the highest level of probiotic and the lowest level of amino yeast. Moreover, the highest record of FBW was obtained by red tilapia injected with shark DNA and fed the highest level of probiotic, but did not differ (P≤0.05) significantly from those of fish injected with DNA and fed the lowest level probiotic, the lowest and the highest levels of amino yeast and non-injected fish fed the lowest level of probiotic. Weight gain of red tilapia injected with shark DNA and fed with the highest level of probiotic showed a higher mean, but this mean did not differ (P≤0.05) significantly from that of all injected fish except for the control group and non-injected red tilapia fed the lowest level of probiotic. At the end of the experiment, crude protein and crude fat of red tilapia injected with shark DNA were signifi-

cantly (P≤0.05) higher than those of the non-injected group while, the moisture content decreased (P≤0.05) significantly by red tilapia injected with shark DNA. The highest records of moisture content, crude protein and crude fat were achieved by red tilapia fed the lowest and highest levels of probiotic and the lowest level of amino yeast, respectively. These records were significantly (P≤0.05) higher than those of the other groups. The highest body moisture was achieved by non-injected fish fed the lowest level of probiotic, and this moisture differed (P≤0.05) significantly from that of DNA injected fish fed the lowest level of amino yeast and their control group, and that of non-injected fish fed the highest level of amino yeast and their control group. Moreover, the highest body protein contents were obtained by red tilapia injected with shark DNA and fed the highest level of probiotic and differ (P≤0.05) significantly from those of non-injected fish fed with mixed probiotic, and those of

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Table 3. Effect of shark DNA injection and different additive levels on body composition of red tilapia (Oreochromis sp.).

Treatment At the start

Moisture 73.87

Crude protein 13.56

Crude fat 5.28

At the end Type of DNA (T) DNA Non-DNA

72.69±0.64 a 73.47±0.92

18.09± 0.72 b 17.24± 0.87

6.30±0.40 b 6.20±0.29

Type of Additive (A) Control P1 P2 A1 A2 P1+A1

72.02±0.78 a 74.02± 1.09 b 73.63±0.69 72.82±0.22d 72.76±0.33d 73.21±0.27c

17.23±1.02 c 17.51±0.66 a 18.72±0.60 18.17±0.32b 17.30±0.74cd 17.09±0.97d

6.17±0.06 b 6.31±0.26 b 6.26±0.72 6.48±0.32a 6.20±0.19bc 6.10±0.30c

Interactions T x A DNA x C DNA x P1 DNA x P2 DNA x A1 DNA x A2 DNA X P1+A1 Non-DNA x C Non-DNA x P1 Non-DNA x P2 Non-DNA x A1 Non-DNA x A2 Non-DNA X P1+A1

71.35±0.07c abc 73.08±0.11 73.03±0.04abc 72.64±0.06bc 73.04±0.05abc 72.98±0.03abc 72.70±0.09bc 74.95±0.21a 74.23±0.06ab 73.01±0.08abc 72.48±0.04bc 73.44±0.06abc

18.11±0.06ab ab 16.94±0.17 19.23±0.11a 18.43±0.11ab 17.93±0.20ab 17.92±0.23ab 16.34±0.08b 18.07±0.13ab 18.02±0.10ab 17.91±0.16ab 16.66±0.04ab 16.26±0.06b

6.17±0.08 6.09±0.03 6.88±0.04 6.75±0.14 6.07±0.18 5.84±0.08 6.17±0.06 6.53±0.04 5.64±0.10 6.21±0.06 6.32±0.08 6.35±0.07

amino yeast and their control (Table 3). Data presented in Table 4 show also that, feed intake, feed conversion ratio (FCR) and protein efficiency ratio (PER) had surpassed the red tilapia injected with shark DNA significantly (P≤0.05). The highest feed intake was achieved by red tilapia fed the highest level of amino yeast, but did not differ (P≤0.05) significantly from those of fish fed the lowest and the highest levels of probiotic and their control. The highest FCR was recorded by the control group, but did not differ (P≤0.05) significantly from that of fish fed the highest level of amino yeast. Red tilapia fed the lowest level of amino yeast show significant (P≤0.05) superiority of PER and PR%, but did not differ (P≤0.05) significantly from those of fish fed the lowest and the highest levels of probiotic. Moreover, the highest record of ER % was obtained by the red tilapia fed the lowest level of amino yeast, but did not differ (P≤0.05) significantly from that of fish fed the highest level of probiotic.

DISCUSSION Red tilapia injected with shark DNA had significant (P≤0.05) superiority of growth performance, body composition and feed utilization compared with non-injected group. The improvement of growth performance, body composition and feed utilization in the present work may be explained by Hemieda et al. (2004); they reported the genetic investigation of Nile tilapia injected directly with shark DNA (into skeletal muscles). The concentrations of such DNA up to 40 µg/0.1 ml/fish probably activated gradually cell proliferation in modified muscle tissues. Also, the measurements of DNA content in the muscles of modified fish indicated that shark DNA may be acting as a mutagen and it had no carcinogenic effect. This is mostly responsible for the enhancement of the productive performance shown in the modified fish injected with foreign DNA. Moreover, Martinez et al. (2000) and Lu et al. (2002) found that anabolic stimulation and average

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Table 4. Effect of shark DNA injection and different additive levels on feed utilization of red tilapia (Oreochromis sp.).

Treatment Type of DNA (T) DNA Non-DNA

Feed intake (g)

FCR

PER

PR%

ER%

19.18±2.47a b 17.63±1.76

1.62±0.16b a 1.78±0.21

2.60±0.25a b 2.37±0.27

49.28±6.21 48.04±5.87

27.89±3.45 26.99±3.92

Type of Additive (A) Control P1 P2 A1 A2 P1+A1

18.62±1.67ab 19.48±0.95a 19.27±3.77a 16.34±0.74c a 19.53±1.68 bc 17.20±1.01

1.97±0.21a 1.61±0.06b 1.60±0.08b 1.53±0.12b ab 1.78±0.13 b 1.69±0.19

2.14±0.25c 2.59±0.11ab 2.61± 0.15ab 2.75±0.24a bc 2.35±0.21 ab 2.50±0.32

41.21±4.57c 49.49±3.72ab 53.91±1.19a 55.45±4.21a bc 44.88±3.55 bc 47.04±2.98

23.37±1.74d 28.06±2.11bc 29.46±1.23ab 32.43±3.29a cd 25.11±2.00 bcd 26.21± 2.76

Interactions T x A DNA x C DNA x P1 DNA x P2 DNA x A1 DNA x A2 DNA X P1+A1 Non-DNA x C Non-DNA x P1 Non-DNA x P2 Non-DNA x A1 Non-DNA x A2 Non-DNA X P1+A1

17.95 ± 0.29 19.83 ± 0.86 22.91 ± 1.38 16.94 ± 0.61 20.88 ± 1.31 16.59 ± 0.98 19.29 ± 2.08 19.13 ± 0.91 15.61 ± 0.03 15.75 ± 0.14 18.68 ± 0.51 17.81 ± 0.56

1.88 ± 0.07 1.56 ± 0.02 1.54 ± 0.02 1.47 ± 0.07 1.73 ± 0.15 1.52 ± 0.07 2.01 ± 0.27 1.67 ± 0.00 1.67 ± 0.07 1.59 ± 0.14 1.83 ± 0.08 1.87 ± 0.06

2.22±0.11 2.67±0.05 2.71±0.05 2.84±0.18 2.43±0.30 2.76±0.16 2.06±0.37 2.51±0.08 2.51±0.15 2.65±0.33 2.28±0.14 2.34±0.09

40.37±2.56 52.61±0.54 53.45±0.62 56.17±3.83 44.09±5.32 49.00±3.15 42.04±7.30 46.38±1.53 54.37±1.74 54.73±6.05 45.68±2.67 45.07±1.15

22.73±1.23 29.87±0.40 28.59±0.47 32.43±0.21 25.34±3.10 28.40±1.73 24.02±2.43 26.26±0.51 30.34±1.12 32.43±5.69 24.89±1.46 24.02±0.82

Means having different superscripts within column in a main effect are significantly different (P≤0.05). P1 and P2, 0.3 and 0.5% of probiotic, respectively; A1 and A2, 0.5 and 1.0 % of amino yeast, respectively. Feed conversion ratio (FCR) = dry feed intake / gain. Protein efficiency ratio (PER) = gain / protein intake. Protein retention (PR%) = protein increment / protein intake (100). Energy retention (ER %) = energy increment / energy intake (100).

protein synthesis were higher in transgenic than that of non-transgenic fish. The results of the present study are consistent with these findings. The results obtained by El-Zaeem (2004), El-Zaeem and Assem (2004), Hemeida et al. (2004) and Assem and El-Zaeem (2005) showed that the dose of 40 µg/0.1 ml/fish of shark DNA was more effective in stimulating most growth performance, body composition and immunity traits of O. niloticus and T. zillii. These traits were significantly higher (P≤0.05) than those of the other injected doses of DNA and their control. El-Zaeem (2012) produced grey mullet, Mugil cephalus with accelerated growth through direct injection of foreign DNA isolated from the liver of shark (Squalus acanthias L.) or African catfish (Clarias gariepinus) into muscles of fingerlings fish at the dose of 40 µg/fish. The results showed a significant (P≤0.05) improvement in most of the growth performance and body composition parameters of grey mullet fingerlings injected with shark DNA compared to both grey mullet injected with catfish DNA and the control fish.

While the results of FCR and PER indicated that fish injected with shark DNA or catfish DNA had significant (P≤0.05) superiority compared to their control. El-Zaeem et al. (2012) stated that dietary protein can be spared down to 22% protein by direct injection of shark DNA into skeletal muscles of red tilapia. Thus, feed costs can be reduced by a further reduction in dietary protein. The results of the present work are consistent of these findings. In addition, the different levels of probiotic and amino yeast were more effective in stimulating most of the productive performance traits compared to the control group and the mixed of probiotic and amino yeast. These results are consistent with the findings reported by ElTawil et al. (2012) in Mugil cephalus; Amer and El-Tawil (2011) in red tilapia; Li and Gatlin (2004) in hybrid striped bass, Morone chrysops × M. saxatilis; Essa et al. (2010) in O. niloticus and Abdel-Tawwab et al. (2010) in Sarotherodon galilaeus. These stimulation may be due to improvement in intestinal microbial flora balance which in

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turn will lead to better nutrient digestibility, higher adsorption quality and increased enzyme activities (Waché et al., 2006; Suzer et al., 2008; Sáenz de Rodriganez et al., 2009). The result of the present study indicates a possible easy and rapid way for improving red tilapia (Oreochromis sp.) characteristics. ACKNOWLEDGMENT This project was supported by King Saud University, Deanship of Scientific Research, College of Science Research Center, Saudi Arabia. REFERENCES Abdel-Tawwab M, Mousa A, Mohammed M (2010). Use of live baker’s yeast Saccharomyces cerevisiae, in practical diet to enhance the growth performance of Galilee tilapia, Sarotherodon galilaeus (L.), and its resistance to environmental copper toxicity. J. World Aquacult. Soc. 41: 214-223. Abdel-Tawwab M, Abdel-Rahman AM, Ismael N (2008). Evaluation of commercial live baker's yeast, Saccharomyces cerevisiae as a growth and immunity promoter for fry Nile tilapia, Oreochromis niloticus (L.) challenged in situ with Aeromonas hydrophila. Aquaculture, 28: 185-189. Amer TN, El-Tawil NE (2011). Effect of green seaweed (Ulva sp.) and probiotic (Lactobacillus sp.) as dietary supplements on growth performance and feed utilization of Red tilapia (♀ O. mossambicus × ♂ O. niloticus). Abbassa Int. J. Aquacult. Vol. (4). No.1. AOAC, (Association of Official Analytical Chemists) (1984). Official th methods of analysis. 14 ed. Association of Official analytical Chemists, Arlington, Virginia. Assem SS, El-Zaeem SY (2005). Application of biotechnology in fifh breeding. II: Production of highly immune genetically modified redbelly tilapia, Tilapia zillii. Afr. J. Biotechnol. 5: 449-459. Balcázar JL, De Blas I, Ruiz-Zazuela I, Cunningham D, Vandrell D, Muzquiz JL, (2006a). The role of probiotics in aquaculture. Vet. Microbiol. 114: 173-186. Balcázar JL, Decamp O, Vendrell D, De Blas I, Ruiz-Zarzuela I (2006b). Health and nutritional properties of probiotics in fish and shellfish. Microb. Ecol. Health Dis. 18: 65-70. Brem G, Brenig B, Horstgen-Schwark G, Winnacker EL (1988). Gene transfer in tilapia (Oreochromis niloticus). Aquaculture, 68: 209-219. Chiu CH, Cheng CH, Gua WR, Guu YK, Cheng W (2010). Dietary administration of the probiotic, Saccharomyces cerevisiae P13, enhanced the growth, innate immune responses, and disease resistance of the grouper, Epinephelus coioides. Fish Shellfish Immunol. 29(6): 1053-1059. CoStat (1986). CoStat 3.03, Copyright, Co Hort Software. All rights reserved. P.O. Box 1149, Berkeley, CA 94701, USA. Duncan DB (1955). Multiple range and multiple F tests. Biometrics, 11: 1-42. El-Tawil NE, Amer TN, Ahmed MH, Seden ME (2012). Effect of different levels of probiotic (Lactobacillus sp.,) on fish performance, survival and feed efficiency of striped mullet Mugil cephalus fed on all plant diet. Egyptian J. Nutr. Feeds, In press. El-Zaeem SY, El-Tawil NE, Amer TN (2012). Effect of direct injection of Shark DNA into skeletal muscles on the productive performance characteristics of Red Tilapia (Oreochromis sp.) fed Different dietary regimes. Afr. J. Agric. Res. In Press. El-Zaeem SY, Assem SS (2004). Application of Biotechnology in fish breeding: production of highly immune genetically modified Nile Tilapia, Oreochromis niloticus with accelerated growth by direct injection of shark DNA into skeletal muscles. Egypt. J. Aquat. Biol. Fish. 8(3): 67-92.

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African Journal of Biotechnology Vol. 11(28), pp. 7294-7301, 5 April, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB12.037 ISSN 1684â&#x20AC;&#x201C;5315 ÂŠ 2012 Academic Journals

Full Length Research Paper

Effect of gene transfer of Chlorella vulgaris n-3 fatty acid desaturase on mouse breast cancer cells Meilan Xue1, Yinlin Ge1*, Jinyu Zhang1, Qing Wang2 and Yongchao Liu1 1

Department of Biochemistry and Molecular Biology, Medical College, Qingdao University, Dengzhou Roud 38#, Qingdao, Shandong, 266021, China. 2 Affiliated Hospital of Qingdao University, Qingdao 266003, China. Accepted 16 March, 2012

Chlorella vulgaris had the gene of n-3 fatty acid desaturase (CvFad3) which can synthesize the precursor of n-3 polyunsaturated fatty acids (PUFAs) or to convert n-6 to n-3 PUFAs. The objective of this study was to examine whether the CvFad3 gene from C. vulgaris can be functionally expressed in mammalian cells and whether its expression can exert a significant effect on cellular fatty acid composition. CvFad3 gene was inserted into plasmid pEGFP-C3 to construct eukaryotic expression vector pEGFP-C3-n-3 and expressed the n-3 Fad gene in mouse breast cancer cells (4T1 cells). Transfection of recombinant vector into 4T1 cells resulted in a high expression of n-3 fatty acid desturase. Lipid analysis indicated a remarkable increase in the level of n-3 PUFAs accompanied with a large decrease in the contents of n-6 PUFAs. Accordingly, CvFad3 gene significantly decreased the ratio of n-6/n-3 PUFAs of 4T1 cells membrane. The expression of CvFad3 gene decreased cellular proliferation and promoted cellular apoptosis. This study demonstrates that CvFad3 gene could dramatically balance the ratio of n-6/n-3 PUFAs. It would be an effective approach to modifying fatty acid composition of mammalian cells and also provided a basis for potential applications of this gene transfer in experimental and clinical settings. Key words: Chlorella vulgaris, CvFad3 gene, fatty acid desaturase, recombinant expression vector, fatty acid composition. INTRODUCTION Biological effects of polyunsaturated fatty acids (PUFAs) have been widely investigated around the world. In particular, their impacts on human health have attracted increasing public attention in recent years. PUFAs mainly include two groups: n-3 PUFAs and n-6 PUFAs. The n-3 PUFAs have been the subject of increasing investigation and have attracted considerable interest as pharmaceutical compounds and nutraceuticals (Connor, 2000; Salem et al., 1996). N-3 PUFAs are beneficial for humans and animals and have been verified to exert preventive and therapeutic effects on some diseases such as cardiovascular diseases, arthritis, cancer and neuropathic diseases (Kris-Etherton et al., 2004; Mozaffarian et al.,

*Corresponding author. E-mail: geyinlin@126.com. Tel: +86532-82991209.

2005). Clinical CancerResearchesindicate that breast, and colon cancer can be modified or inhibited their growth by supplying n-3 PUFAs in human diet (Bougnoux et al., 1999; Rao et al., 2001). In general, a balanced n6/n-3 ratio of the body lipids is essential for normal growth and development and plays an important role in the prevention and treatment of many clinical problems (Simopoulos, 2000).However, humans and mammals are incapable of synthesizing n-3 PUFAs in their bodies, so the levels of PUFAs in their bodies are, to a great extent, dependent on dietary intake (McLennan, 1998). Some plants, such as Chlorella vulgaris are able to synthesize the n-3 fatty acid. It is reported that a CvFad3 gene from C. vulgaris encoded the n-3 fatty acid desaturase (FAD), this enzyme, when expressed in Nicotiana tabacum can catalyze the conversion of n-6 PUFAs to n-3 PUFAs by introducing an n-3 double bond into their hydrocarbon chains (Suga et al., 2002). The objective of this study

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was to examine whether the Cv- Fad3 gene from C. vulgaris can be functionally expressed in mammalian cells in a high efficiency and whether its expression can exert a significant effect on cellular fatty acid composition. MATERIALS AND METHODS Construction of recombinant plasmid The plasmid pEGFP-C3 (BD Bioscience Clontech) was digested by XhoI and EcoRI and fractionated on 1% agarose gels, then the long objective bands were purified. The fragment of CvFad3 gene was amplified from mRNA of C. vulgaris by reverse transcriptasepolymerase chain reaction (RT-PCR) and then inserted into the plasmid of pEGFP-C3 (Figure 1A). The detailed steps were described as follows: The long segment pEGFP-C3 and the CvFad3 gene were ligated in 1:4(mol/mol) by T4 ligase. The construct was confirmed by enzymatic digestion and DNA sequencing. The eukaryotic recombinant expression vector was named pEGFP-C3-n-3. Cell culture and transfection with recombinant plasmid 4T1 cells were obtained from Shanghai Life Science of Chinese Academy of Sciences. 4T1 cells were routinely maintained in 1:1 (v/v) mixture of DMEM high glucose and 10% (vol/vol) fetal bovine serum (FBS), 37°C in a tissue culture incubator with 5% CO2 and 98% relative humidity. 4T1 cells were placed in 6-well plates and cultured as normal. The cells number and medium volume were the same in each well. After 24 h cultured, cells were transfected with recombinant plasmid pEGFP-C3-n-3 and pEGFP-C3-control for experiments when 75% of plate was covered by cells. Transfectants were carried out by adding X-fect polymer at the same culture medium with serum. After 5 h incubation, the transfection medium was replaced with normal culture medium. Forty-eight hours incubation cells were used for photographs, analysis of gene expression and fatty acid composition.

MTT assay The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Roche Diagnostics Corporation) assay was performed to determine cell growth and viability. 4T1 cells (1×104 plaque-forming units per milliliter) were grown in 96 well culture plate. Two groups for pEGFP-C3-n-3 and pEGFP-C3-control were set up, and each group had 10 unions. After 48 h of transfection, 20 µl MTT (5 mg/ml) labeling reagent was added to each well (100 µl medium). The solution was then incubated with 50 mL/L CO2, at 37°C for 4 h. The medium was discarded and 100 µl of the solubilization solution was then added into each well. Complete solubilization of the purple crystals was checked for and the spectrophotometric absorbency of the solution was measured at 490 nm. mRNA analysis Briefly, total RNA was extracted from cultured cells transfected after 48 h by using a total RNA isolation reagent (TRIzol, Invirogen), according to the manufacturer’s protocol. Amplifying the CvFad3 gene fragment, reverse transcription-polymerase chain reaction (RT-PCR) was done. The forward and reverse primers were 5’TTGCCGCTCCTGCCGAAGA-3’ and 5’-GGGTCACTGGGTCCGTGATGGT-3’, respectively. The condition for amplification was 94°C for 3 min, 94°C for 30 s, 61°C for 40 s, 72°C for 1 min, 72°C

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for 10 min. The amplification products were 793 bp. And the objective products were subjected to autoradiography. Gas chromatograph analysis After 48 h transfection, the 4T1 cells were collected from 6-well plates. The fatty acid composition of total cellular lipids was analyzed as described (Weylandt et al., 1996; Kang et al., 1992). Lipid was extracted with chloroform/methanol (2:1, vol/vol) containing 0.005% butylated hydroxytoluene (BHT, as antioxidant). Fatty acid methyl esters were prepared by using a 14% (wt/vol) BF3/methanol reagent. Fatty acid methyl esters were quantified with gas chromatography/mass spectrometry (GC/MS) by using an HP-5890 Series II gas chromatograph equipped with a Supelcowax SP-10 capillary column (Supelco, Bellefonte, PA) attached to an HP-5971 mass spectrometer. The injector and detector are maintained at 260°C and 280°C, respectively. The oven program is maintained initially at 150°C for 2 min, then ramped to 200°C at 10°C/min and held for 4 min, ramped again at 5°C/min to 240°C, held for 3 min, and finally ramped to 270°C at 10°C/min and maintained for 5 min. Carrier gas-flow rate is maintained at a constant 0.8 ml/min throughout. Total ion monitoring is performed encompassing mass ranges from 50-550 atomic mass units. Fatty acid mass is determined by comparing areas of various analyzed fatty acids to that of a fixed concentration of internal standard. Flow cytometric analysis After 48 h transfection, the 4T1 cells were collected from 6-well plates. Cell cycle and apoptosis were determined by Vybrant Apoptosis Assay Kit (Invirogen), according to the manufacturer's protocol, and measured by fluorescence activated cell sorting using a FACScan flow cytometer (Becton Dickinson). Data analysis Cell growth and viability data (MTT), fatty acid composition level was analysed by GraphPad Prism 5 software. The figures of cell cycle and apoptosis were made by WinMDI 2.9 software, each experiment was done 3 times. All data were expressed as the mean ± standard deviation (SD) and were analyzed using the Student's ttest. The level of significance was set at P<0.05.

RESULTS Enzymatic digestion of recombinant plasmid Recombinant plasmid pEGFP-C3-n-3 was digested by the fast-digest Xho I and EcoR I, the result corresponded to plasmid pEGFP-C3 and the objective CvFad3 gene (Figure 1B). Expression of CvFad3 gene in 4T1 cells The co-expression of EGFP allowed us to identify the cells that were transfected and expressed the transgene. The result is shown in Figure 3, 48 h after transfection about 40% of the cells exhibited bright fluorescence, indicating a high efficiency of gene transfer and a high expression level of the transgene. The cells expressed

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Figure 1. Construction and identification of recombinant plasmid pEGFP-C3-n-3. A: Construction of recombinant plasmid pEGFP-C3-n-3. The plasmid pEGFP-C3 was digested by XhoI and EcoRI, and the long objective bands were purified. The fragment of CvFad3 gene was amplified from mRNA of Chlorella vulgaris by RT-PCR and then inserted into the plasmid of pEGFP-C3. B: The identification of recombinant plasmid pEGFP-C3-n-3 and pEGFP-C3 by double enzyme Xho I/ EcoR I digestion. M 1kb DNA ladder; 1 2 3 Xho I/ EcoR I, digest pEGFP-C3-n-3; 4 Xho I/ EcoR I digest pEGFP-C3. Products of PCR were observed by 1.0% agarose electrophoresis.

CvFad3 gene mostly died, but few cells died in the cells only expressed EGFP gene, as shown in Figure 2. Furthermore, expression profile of the

transgene also was determined by RT-PCR. As shown in Figure 3, the mRNA of CvFad3 gene was not detected in cells transfected with pEGFP-

C3-control but was highly abundant in cells transfected with pEGFP-C3-n-3. The results indicate that CvFad3 gene has a very high

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Figure 2. Photomicrographs showing gene-transfer efficiency. 4T1 cells were transfected with pEGFPC3-control or pEGFP-C3-n-3. Forty-eight hours after tranfection, the group of pEGFP-C3-control was brighter than the group of pEGFP-C3-n-3. The cells expressed CvFad3 gene mostly died, but few cells died in the cells only expressed EGFP gene.

Effect of n-3 desaturase on fatty acid composition

Figure 3. Level of n-3 FAD transcript in 4T1 cells transfected with pEGFP-C3-control and pEGFP-C3n-3 after 48 h. Lane M 100 bp DNA ladder; lane 1 pEGFP-C3 -Control group; 2 lane pEGFP-C3-n-3group. Products of PCR observed by 2.0% agarose electrophoresis. This result indicates that: CvFad3 mRNA was not detected in 4T1 cells transfected with pEGFP-C3-Control, but was highly abundant in 4T1 cells transfected with pEGFP-C3-n-3.

Expression in 4T1 cells transfected with pEGFP-C3-n-3.

We tested whether the expression of CvFad3 gene in the 4T1 cells can lead to conversions of n-6 fatty acids to n-3 fatty acids and, thereby, a change in fatty acid composition. The fatty acid composition of total cellular lipids was analyzed by gas chromatograph. These results are summarized in Table 1. The fatty acid profiles are remarkably different between the control cells transfected just with the pEGFP-C3 and the cells transfected with the pEGFP-C3-n-3. In the cells expressing the CvFad3 gene, n-6 fatty acids were converted largely to the corresponding n-3 fatty acids, namely, 18:2n-6 to 18:3n3, and 20:4n-6 to 20:5n-3. As a result, the fatty acid composition of the cells expressing the n-3 fatty acid desaturase was changed significantly when compared with that of the control cells transfected with pEGFP-C3control. Importantly, the ratio of n-6/n-3 was reduced from 6:1 in the control cells to 1:1.2 in the cells expressing the n-3 fatty acid desaturase. Change of cellular proliferation To investigate the effect of expression of CvFad3 gene on 4T1 cells growth, we analyzed proliferation of cells. As shown in Figure 4, in the result of MTT kit (cell growth and viability assay), the absorbency of control cells was higher than that of experiment group. That is to say, the growth and viability of control cells was higher than that

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Table 1. PUFA composition of total cellular lipids from the control 4T1 cells and the transgenic cells expressing CvFad3 gene (area percentage, ±s, n = 3).

PUFAs composition n-6 PUFAs 18:2 n-6 20:4 n-6 22:5 n-6 Total n-3 PUFAs 18:3 n-3 20:5 n-3 20:6 n-3 Total n-6/n-3 ratio

Control group

Experiment group

7.27±0.134 4.73±0.064 1.62±0.041 13.62

2.13±0.049* 1.85±0.052* 0.86±0.039* 4.84

0.95±0.047 0.75±0.043 0.46±0.039 2.16 6.31

2.93±0.128* 2.04±0.051* 1.07±0.024* 6.04 0.80

Partial gas chromatograph determined fatty acid profiles of total cellular lipids extracted from the control cells transfected with pEGFP-C3-Control and the cells transfected with pEGFPC3-n-3. The fatty acid profiles are remarkably different between the control cells transfected just with the pEGFP-C3 and the cells transfected with the pEGFP-C3-n-3. In the cells expressing the CvFad3 gene, n-6 fatty acids were converted largely to the corresponding n-3 fatty acids, namely, 18:2n-6 to 18:3n-3, and 20:4n-6 to 20:5n-3. *P<0.01 compared to control group.

Figure 4. The proliferation of 4T1 cells (the control cells and the cells expressing n-3 fatty acid desaturase) were assessed by MTT. Note: 1: pEGFP-C3 –n-3 group 2: pEGFP-C3- Control group. *P<0.01 compared to control group. The absorbency of cells transfected with pEGFP-C3-n-3 was lower than that of the cells transfected with pEGFP-C3- Control.

of experiment cells with CvFad3 gene, because the more live cells or less of dead cells are, the higher the absorbency of cells is in 490 nm (P<0.01). This indicates that the expression of CvFad3 gene can cause 4T1 cells to die and/or inhibits the growth, leading to decrease of its cellular proliferation.

Change of cell cycle and apoptosis Compared with cells transfected with pEGFP-C3-control, in the 4T1 cells transfected pEGFP-C3-n-3, the percent of G0/G1 period was lower (P<0.01); while the percent of G2/M period was higher (P<0.01); the percent of S period

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Figure 5. Change of cell cycle. Note: A: pEGFP-C3 –Control group B: pEGFP-C3-n-3 group. Compared with cells transfected with pEGFP-C3-control, in the 4T1 cells transfected pEGFP-C3-n-3, the percent of G0/G1 period was lower (P<0.01); while the percent of G2/M period was higher (P<0.01); the percent of S period and APO period obviously increased (both P<0.01).

and APO period obviously increased (both P<0.01, Figure 5). These results indicate that CvFad3 gene could inhibit 4T1 cells proliferation, make cell cycle blocked at G2/M period, and cells could not cleavage subsequently. Apoptosis analysis showed that compared with cells transfected with pEGFP-C3-Control, in the 4T1 cells transfected with pEGFP-C3-n-3-control, the percent of early apoptosis and late apoptosis were higher (P<0.01, Figure 6). DISCUSSION In this study, we expressed the construct in 4T1 cells to estimate the product activity as well as expression of CvFad3 gene in mammals. This study clearly demonstrated that the CvFad3 gene from C. vulgaris can be expressed functionally in mouse breast cancer cells, and its expression could confer cells’ capability of converting n-6 PUFAs to corresponding n-3 PUFAs, leading to a balanced n-6/n-3 ratio, that could inhibit cell growth and induce apoptosis in breast cancer cells. We have known that n-6 PUFAs stimulate animal’s tumor and cancer growth and metastasis, whereas n-3 PUFAs exhibit inhibitory effects. A number of studies have shown n-3 fatty acid protection in rodent models of breast cancer. These include dietary supplementation in mouse transplantable tumors (Gabor et al., 1985) and human cell xenograft models (Rose et al., 1995) as well as chemically induced tumors in rats (Takahashi et al., 1994). Many signaling pathways that are relevant to carcinogenesis and tumor progression are differentially affected by n-3 and -6 PUFAs. For instance, n-6 PUFA products were reported to upregulate and activate cellular signaling mediators including protein kinase C, ras, ERK ½ and NF-κB

whereas n-3 PUFA products had the opposite effect. The ability of long-chain n-3 PUFAs to induce apoptosis in tumor cells has also been attributed to the increased susceptibility of these cells to lipid peroxidation (Stoll, 2002). Inhibition of tumor cell growth and invasion by n-3 PUFAs in a xenograft animal model was associated with decreased COX-2 and PGE2 levels (Kobayashi et al., 2006). Thus, n-3 PUFAs may act as a natural COX “inhibitor”. It is a significant method for controlling tumor development to balance the ratio of n-6/n-3 PUFAs (Simopoulos et al., 2002). Meanwhile, the n-3 and n-6 PUFAs are not interconvertible in the human body, because mammalian cells lack the n-3 fatty acid desaturase. With hectic schedule, poor dietary habits and low quality of food sources that people have today, experts recommend the consumption of nutritional dietary supplements, including fish oil supplements, which help fulfill the nutritional demand of all body organs (decrease the intake of n-6 fatty acids and increase the intake of n-3 fatty acids) (Simopoulos et al., 1999). Fish oil is oil derived from the tissues of deep-sea oily fish, but this oily fish does not actually produce docosahexaenoic acid/eicosapentaenoic acid (DHA/EPA) fatty acids, but instead accumulate them from consuming deep-sea micro-algae that produce these fatty acids. C. vulgaris is one kind of deep-sea micro-algaes, and it has the CvFad3 gene, encoding n-3 FAD which can catalyze the conversion of n-6 PUFAs to n-3 PUFAs. Therefore, an alternative approach that can quickly and effectively increase cellular n-3 PUFA contents and balance the n-6/n-3 ratio, without the need for a lengthy intake of fish oil supplements would be desirable. Because lack of an n-3 desaturase gene is the bottleneck for endogenous production of n-3 PUFAs in mammals, transfer of the n-3 desaturase gene from deep-sea micro-

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Figure 6. Change of cell apoptosis. Note: A: pEGFP-C3 –Control group B: pEGFP-C3-n-3 group. Compared with cells transfected with pEGFP-C3 –Control, in the 4T1 cells transfected pEGFP-C3-n-3–control, the percent of early apoptosis and late apoptosis were higher (P<0.01).

algaes into livestock is the key to overcoming this constraint, as these animals already possess most of these other desaturases and PUFA elongases (Wallis et al., 2002). This assumption has been shown to be feasible in transgenic mice generated with the n-3 desaturase gene from the roundworm C. elegans. A transgenic mouse model expressing the fat-1 gene increased the ratio of n3 and n-6 fatty acids in various tissues (Kang et al., 2004).In this study, we used eukaryotic recombinant expression vector to transfer the CvFad3 gene from C. vulgaris into mouse breast cancer cells, convert n-6 PUFA into n-3 PUFA to increase the level of n-3 PUFA and the ratio of n-3/n-6 PUFA. Our experimental results showed that the transgenic construct expressed the interest protein and the transgene product possessed its biological activity in the transfected mammal cells. Then, we demonstrated that the CvFad3 gene from C. vulgaris was functionally expressed and that its product has a significant effect on the fatty acid composition. The proportion of n-6 PUFAs decreased and n-3 PUFAs increased considerably, particularly for ALA, DHA and EPA, which correspondingly inhibited cell growth and induced apoptosis in breast cancer cells. All these results showed that the CvFad3 gene from C. vulgaris possesses great value for the production of n-3 PUFAs and cancer cell suppression in transgenic 4T1 cells. Our findings as presented here suggest that gene transfer of the CvFad3 gene from C. vulgaris could be such a desirable intervention that can quickly and effectively provide therapeutic and cancer-preventive effects of n-3 fatty acids. The molecular mechanisms

which account for these biological effects are not completely understood. Certainly, further study in this regard is warranted. ACKNOWLEDGEMENTS We thank Li Fangfang and Li Yanjun for their helpful comments and discussion on this study. REFERENCES Bougnoux P (1999). N-3 polyunsaturated fatty acids and cancer. Curr Opin Clin Nutr Metab Care. 2: 121-126. Connor WE (2000). Importance of n-3 fatty acids in health and disease. Am. J. Clin. Nutr. 71, 1 Suppl.: 171S-175S. Gabor H, Hillyard LA, Abraham S(1985). Effect of dietary fat on growth kinetics of transplantable mammary adenocarcinoma in BALB/c mice. J. Natl. Cancer Inst. 74:1299-1305. Kang JX, Man SF, Brown NE, Labrecque PA, Garg ML, Clandinin MT (1992). Essential fatty acid metabolism in cultured human airway epithelial cells. Biochim. Biophys Acta. 1128(2-3): 267-274. Kang JX, Wang J, Wu L, Kang ZB (2004). Transgenic mice: fat-1 mice convert n-6 to n-3 fatty acids. Nature. 427(6974): p. 504. Kris-Etherton PM, Hecker KD, Binkoski AE (2004). Polyunsaturated fatty acids and cardiovascular health. Nutr Rev. 62: 414-426. Kobayashi N, Barnard RJ, Henning SM, Elashoff D, Reddy ST, Cohen P, Leung P, Hong-Gonzalez J, Freedland SJ, Said J (2006). Effect of altering dietary omega-6/omega-3 fatty acid ratios on prostate cancer membrane composition, cyclooxygenase-2, and prostaglandin E2 . Clin. Cancer Res. 12: 4662-4670. McLennan PL, Abeywardena MY, Charnock JS (1998). Dietary fish oil prevents ventricular fibrillation following coronary artery occlusion and reperfusion. Am. Heart J. 116: 709-717. Mozaffarian D, Ascherio A, Hu FB, Stampfer MJ, Willett WC, Siscovick DS, Rimm EB (2005) Interplay between different polyunsaturated fatty acids and risk of coronary heart disease in men. Circulation,

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111: 157-164. Rao CV, Hirose Y, Indranie C, Reddy BS (2001). Modulation of experimental colon tumorigenesis by types and amounts of dietary fatty acids. Cancer Res. 61: 1927-19332. Rose DP, Connolly JM, Rayburn J, Coleman M (1995). Influence of diets containing eicosapentaenoic or docosahexaenoic acid on growth and metastasis of breast cancer cells in nude mice. J. Natl. Cancer Inst. 87: 587-592. Salem NJr, Reyzer M, Karanian J (1992). Losses of arachidonic acid in rat liver after alcohol inhalation. Lipids, 31Suppl: S153-S156. Simopoulos AP (2000). Human requirement for N-3 polyunsaturated fatty acids. Poultry Sci. 79: 961-970. Simopoulos AP, Leaf A, Salem Jr. N (1999). Workshop on the essentiality of and recommended dietary intakes for omega-6 and omega-3 fatty acids. J Am. Coll. Nutr. 18(5): 487-489. Simopoulos AP (2002). The importance of the ratio of omega-6/omega3 essential fatty acids. Biomed Pharmacother. 56:365-379. Stoll BA (2002). N-3 fatty acids and lipid peroxidation in breast cancer inhibition. Br. J. Nutr. 87: 193-198.

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Suga K, Honjoh K, Furuya N, Shimizu H, Nishi K, Shinohara F, Hirabaru Y, Maruyama I, Miyamoto T, Hatano S, Iio M (2002). Two lowtemperature-inducible Chlorella genes for delta12 and omega-3 fatty acid desaturase (FAD): isolation of delta12 and omega-3 fad cDNA clones, expression of delta12 fad in Saccharomyces cerevisiae, and expression of omega-3 fad in Nicotiana tabacum. Biosci Biotechnol. Biochem. 66(6): 1314-1327. Takahashi M, Minamoto T, Yamashita N, Kato T, Yazawa K, Esumi H (1994). Effect of docosahexaenoic acid on azoxymethane-induced colon carcinogenesis in rats. Cancer Lett. 83: 177-184. Wallis JG, Watts JL, Browse J (2002). Polyunsaturated fatty acid synthesis: what will they think of next? Trends Biochem. Sci. 27: 467473. Weylandt KH, Kang JX, Leaf A (1996). Polyunsaturated fatty acids exert antiarrhythmic actions as free acids rather than in phospholipids. Lipids, 31: 977-982.

Full Length Research Paper

Selection of ligand peptides with the ability to detect antibodies in enzootic bovine leukosis Elizangela Maira dos SANTOS1*, Rone CARDOSO2, Luiz Ricardo GOULART FILHO2, Marcos Bryan HEINEMANN1, Rômulo Cerqueira LEITE1 and Jenner Karlisson Pimenta dos REIS1 1

Departamento de Medicina Veterinária Preventiva, Escola de Veterinária – Universidade Federal de Minas Gerais (UFMG) 30 123-970, Belo Horizonte, MG, Brasil. 2 Instituto de Genética e Bioquímica, Laboratório de Nanobiotecnologia, Campus Umuarama – Universidade Federal de Uberlândia (UFU) 38 400-902, Uberlândia, MG, Brasil. Accepted 22 March, 2012

Peptides present in phages were selected using phage display technology and immunoassays to find out the antigenic mimetics of immunodominant epitopes of bovine leukosis virus (BLV). The use of antigenic mimetics may result in the enhancement of the sensitivity and specificity of the serologic diagnosis of enzootic bovine leukosis (EBL), contributing directly to disease control. The selections enabled the choice of clones which can be used as potential antigens in the diagnoses of diseases. The synthetic peptide produced from the selected sequences may be considered as an alternative for antigens in the serologic diagnosis of enzootic bovine leukosis. Key words: Diagnostic, antigens, enzootic bovine leukosis (EBL), phage display. INTRODUCTION The bovine leukosis virus (BLV) is a member of the genus Deltaretrovirus and an etiologic agent of enzootic bovine leukosis (EBL). The EBL shows a worldwide distribution and infects more frequently, the dairy cattle (Modena et al., 1984; Castro et al., 1992; Camargos et al., 2002). The economic damages caused by BLV infection include restrictions on the trading of the animal or its products such as semen and embryos between countries (Burny et al., 1980; Gutiérrez et al., 2009). Other economic damages include bovine deaths due to lymphosarcoma, carcass rejection at the slaughterhouse, and productive and reproductive decrease in infected animals (Camargos, 2005). BLV infection also decreases milk production and causes other losses to farmers (Da et al., 1993). As there are no protective vaccines against the virus, the control and eradication of EBL are carried out

*Corresponding author. E-mail: elmaira27@yahoo.com.br. Abbreviations: AGID, agar gel immunodiffusion; BLV, Bovine leukosis virus; EBL, enzootic bovine leucosis; ELISA, enzymelinked immunosorbent assay; PCR, polymerase chain reaction.

through diagnosis, segregation and slaughtering of the bovine carriers. For this reason, the sensitivity of the diagnostic technique utilized is regarded as a critical factor to prevent the spread of the disease in the flocks (Trono et al., 2001). Like other retroviruses, the BLV presents envelope glycoprotein gp51 (env) and core viral p24 (gag) as immunodominant proteins in vivo (Miller et al., 1981; Portetelle et al., 1989; Callebaut et al., 1993; Gatei et al., 1993; Willems et al., 1995; Doménech et al., 1997). The proteins gp51 and p24 are frequently used in diagnostic tests for the detection of specific BLV virus antibodies (Miller et al., 1981; Kittelberger et al., 1999). The most utilized tests for EBL diagnosis are agar gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA), but several factors may contribute to the inconsistency of results such that a need arises for the results to be confirmed by other more sensitive and specific techniques (Van Der Maaten, 1974; Portetelle et al., 1989; Reichel et al., 1998; Soutullo et al., 2001; Trono et al., 2001; De Giuseppe et al., 2004; Leroux et al., 2004; Paré and Simard, 2004; Alvarez et al., 2007; Lim et al., 2009). Techniques such as western blot and polymerase chain reaction (PCR) have been

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developed and used as complementary or confirmatory diagnostic tools for EBL and other retroviruses (Payne et al., 1989; Kim and Casey, 1992; Langemeier et al., 1996; Johnson et al., 1998; Reichel et al., 1998; Kittelberger et al., 1999; Llames et al., 2000; Trono et al., 2001; Choi et al., 2002; Alvarez et al., 2007; More et al., 2008). Even though many assays have been performed to improve antigen production, the traditional cell culture method of production which involves the use of strain FLK/BLV is still the mostly used method in antigen production for commercial diagnostic kits (Van der Maaten et al., 1974; Beier et al., 2004; Lim et al., 2009). The cell strain FLK used in BLV antigen production is also known for being infected with bovine diarrhea virus (Bolin et al., 1994; Dees et al., 1994; Beier et al., 2004; Lim et al., 2009) and this contamination may result in further problems with diagnoses related to the specificity of the reactions (Beier et al., 2004). Other problems common with the antigen productions techniques which utilize cell cultures is that the growth protocols usually includes the addition of bovine fetal serum. This serum contributes to the components that may cause unspecific reactions. Besides these factors, the presence of cell proteins that are not eliminated or that are co-purified during the obtention of BLV proteins may interfere with the viral proteins (Llames et al., 2000), thus, producing mistaken results in EBL diagnosis. Advances in vaccines and diagnostic tests development require a deeper understanding of pathogen antigenic structures and immunogenic proteins. The synthetic peptides have been utilized for mapping the antigenic sites in several viral systems (Neurath et al., 1990; Callebaut et al., 1991; Ball et al., 1992), presenting a wide potential when utilized as antigens in diagnostic techniques and as efficient vaccine components in the induction of the immunologic memory (Ball et al., 1992; Kabeya et al., 1996; Soutullo et al., 2001, 2005). Peptides selected by a phage display-technology such as antigenic mimetic of natural epitopes may be used to immunize animals. Some of them are capable of inducing new antibodies that present cross-reaction with the natural epitopes which are considered as mimetic immunogenic (Cardoso et al., 2009). In order to avoid the problems presented by use cell culture in the production of antigens from, antigenic mimetics and immunogenic capabilities are utilized as the foundations which help to find epitopes that are used in the development of vaccines and diagnostic techniques for many diseases (Folgori et al., 1994; Pasqualini et al., 1995; Sioud et al., 1996). In this study, the peptides present in phages were selected from phage display libraries and from immunoassays. These peptides were regarded as antigenic mimetics of the immunogenic epitopes of BLV proteins, having the potential for being used as antigens and in synthetic peptide production for the development of diagnostic techniques for EBL.

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METHODOLOGY Purification of IgG from bovine sera Bovine sera samples were tested for BLV infection in the Retroviruses Laboratory of Veterinary School, Universidade Federal de Minas Gerais (UFMG), using agar gel immunodiffusion (AGID) test as recommended by Miller and Van der Maaten (1977) with the antigen gp51 produced in FLK cells. In order to obtain total IgG from a positive pool sera for EBL from 20 animals and total IgG from a negative pool sera from 20 different animals, a HP column Hitrap Protein G in ÄKTA system was used following the manufacturer’s instructions (GE Healthcare®). The total IgG obtained from 3 ml of each pool were neutralized after the elution of the column with buffer 1 M Tris-HCl of pH 8.0, lyophilized, frozen and diluted in deionized\sterilized water. The concentrations of purified IgG and gp51 protein were estimated by spectrophotometry at 280 nm and by using the Bradford method (Bradford, 1976). The samples were submitted to electrophoresis in 16% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie-blue R250. The molecular weight marker Bench Mark Protein Ladder (Invitrogen) was applied to the gel for comparison. Biological selection (biopanning) The Ph.D. -12TM library - New England BioLabs® was used for the selection of peptide sequences bound to purified IgG from BLVpositive animal sera. This library contains random and linear peptides which consist of 12 amino acids and presents about 2.7 × 109 possible sequences. The positive purified IgG were utilized as target for peptide selection. The wells of microtitration plate (MaxiSorpTM - Nunc) were adsorbed using 150 µl of IgG positive for BLV at the concentration of 100 µg/ml in 0.1 M NaHCO3, pH 8.6. Phages at the concentration of 4.0 × 1010 of the original library were diluted in 90 µl of Tris-buffered saline Tween-20 (TBST) solution and added to the wells adsorbed with the targets (total positive IgG). Three selection cycles were performed for the enrichment of the phages containing the linking peptides. From the 2nd cycle, the washing buffer stringency increased from 0.1 to 0.5% with Tween-20 in all the washings. In the 3rd cycle, 15 washings were carried out, and after this step, the phages containing the linking peptides were withdrawn. The procedures for biopanning, amplification, precipitation and titration of the phages were performed according to the instructions given by the manufacturer (New England BioLabs®). Sequencing For the sequencing reaction, 500 ng of template DNA (DNA of each phage), 5 pmol of -96 gIII primer (5’-OH CCC TCATAG TTA GCG TAA CG-3’ - Biolabs) and Premix (DYEnamic ET Dye Terminator Cycle Kit – Amersham Biosciences) were used. The reaction was carried out in a plate thermocycler (MasterCycler – Eppendorf). The reading of the sequencing was carried out in a MegaBace 1000 (Amersham Biosciences) automatic sequencer. The DNA sequences obtained from the automatic sequencer were processed by the appropriate software equipment (Sequence Analyzer, Base Caller, Cimarron 3.12, Phred 15). Translation and analysis of sequences From the DNA sequences, analysis of deduction in silico were performed for the peptide sequences using the software DNA2PRO

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(available at https://relic.bio.anl.gov/programs.aspx). The software AAFREQ (https://relic.bio.anl.gov/aafreqs.aspx) was used for calculating the frequency and diversity of amino acids inside the population of selected peptides. The search for similarities between the peptides selected was accomplished using the software Clustal W (version 18.1 available at www.ebi.ac.uk/clustalW/), through multiple alignments (data not shown). Wild phage The phage M13KE (New England BioLabs®) was used in the immunoassays as negative control for the reactions. This phage does not present peptides fused to viral capsid. According to the manufacturer’s information, this clone may be useful as control or in titration of phage stocks. The procedures of amplification, precipitation and titration of the wild phage were the same as those used for the phages selected from the libraries. Phage-ELISA (IgG) High affinity plates (MaxiSorpTM - Nunc) were adsorbed with positive and negative purified IgG. 44 ELISA plate wells were adsorbed with 1 µg of positive IgG diluted in a bicarbonate buffer (0.1 M NaHCO3, pH 8.6) and the other 44 wells of the same plate were adsorbed with 1 µg of negative IgG. In each half of the microplate, four wells were used as controls without IgG adsorption. The plate was incubated for 18 h at 4°C under agitation, washed twice in 0.05% PBST and blocked with PBS solution with 5% skimmed powdered milk for 1 h at room temperature. After that, the plates were washed three times. The phage samples were precipitated with polyethylene glycol (PEG)/NaCl and with concentrations estimated by UV absorbance spectrophotometry at the wave lengths of 269 and 320 nm, they were diluted to the concentration of 1x1011 phages in the PBS solution with 1% skimmed powdered milk. Each phage sample was added in duplicate to the wells with positive and negative IgG adsorbed and incubated for at least 1 h at 37°C. The wild phage (negative control) was added to the adsorbed and non-adsorbed wells with the IgG according to the layout of plates. The plates were washed six times with 0.05% phosphate buffered saline with Tween 20 (PBST) and incubated for 1 h at 37°C with anti-M13-peroxidase-conjugated (1:5000, Sigma) in PBS solution with 1% skimmed powdered milk. By the end of the incubation, the plates were washed six times with 0.1% PBST solution and the reaction was initiated by the addition of 0.03% H2O2 and o-phenylendiamin (OPD) 1 mg/mL in 0.1 M citratephosphate with pH 5.0. The reaction was interrupted by adding sulphuric acid (2 N) and the optical density (OD) was determined by plate spectrophotometry (Titertek Multiskan Plus, Flow Laboratories, USA) at 490 nm. The cut-off value was calculated based on the OD obtained for the wild phage (cut-off = wild phage average value + 2X the standard deviation value for wild phage). The ELISA index was calculated by taking the OD average of the duplicates divided by the cut-off value. Values greater than one were considered positive. In order to verify the difference between the ELISA indexes obtained from the reactivity of positive and negative IgG clones, the paired t-test was performed with the software STATA 10.0.

Dot blot (IgG) The Dot blot was performed to verify the reactivity of the phages with positive and negative IgG. Nitrocellulose membranes (0.2 µm, Amersham Pharmacia) were sensitized with each phage in the concentration of 1011 pfu. The wild phage and 1 µg of gp51 were

also sensitized. After the sensitization, the membranes were blocked with PBS with 5% skimmed powdered milk for 1 h under agitation at room temperature and washed once in 0.05% Trisbuffered saline Tween-20 (TBST) . Positive and negative IgG were used as primary antibodies in the concentration of 400 µg diluted in block solution and incubated within the membranes for 1 h under agitation and at room temperature. After five washings with Tris-buffered saline Tween-20 (TBST) 0.05%, the reaction was initiated by adding 3,3'-diaminobenzidine (DAB) (Sigma Chemical) for visual reading and interrupted with deionized H2O. Western blot (IgG and sera) Western blot (WB) was performed to confirm if the peptides fused to protein III of the selected clones were immunoreactive with positive IgG and antibodies from positive sera for BLV, and capable of differentiating the positive and negative samples. For the WB assays, an electrophoresis in 16% SDS-PAGE was performed with each selected phage (1x1012 pfu), wild phage (negative control, 1x1012 pfu), and gp51 (10 µg). The samples were electrotransferred for 2 h in 280 mA at 4°C to nitrocellulose membranes (0.2 µm, Amersham Pharmacia) which were blocked by TBS solution with 5% skimmed powdered milk for 1 h under agitation at room temperature. After a wash with 0.05% Trisbuffered saline Tween-20 (TBST) solution (50 mM Tris-HCl pH 7.5, 150 mM NaCl and 0.05% Tween 20), positive and negative IgG (400 µg), and positive and negative sera samples for BLV (1:500) were diluted in blocking solution and incubated in the membranes for 1 h under agitation at room temperature. The membranes were washed three times in 0.05% Tris-buffered saline Tween-20 (TBST) solution. Secondary antibody anti-IgG bovine conjugated to peroxidase (1:5000, Sigma) which was diluted in blocking solution was added to each membrane. They were incubated for 1 h under agitation at room temperature, and afterwards, washed three times in 0.05% Tris-buffered saline Tween-20 (TBST) solution. The reaction was initiated with DAB (Sigma) for visual reading and interrupted with deionized H2O. ELISA (sera) Only clones non-reactive with negative IgG and clones reactive with the positive IgG were selected. The immunoreactivity of the phages selected using positive and negative sera samples for BLV infection was tested by ELISA. The bovine sera samples had previously been tested by AGID according to the protocol of Miller and Van der Maaten (1977). The samples of the selected phages and of the wild phage were diluted to the concentration of 1×1011 in 0.1 M NaHCO3 with a pH of 8.6 for the adsorption in the wells of ELISA plates (MaxiSorpTM Nunc). After adsorption (18 h at 4°C), the plates were washed twice in 0.05% PBST and blocked with PBS with 5% skimmed powdered milk for 1 h. After three washings, 20 positive sera samples and 20 negative sera samples were diluted to 1:100 in PBS solution with 1% skimmed powdered milk and added in duplicates to the wells. The incubation was for 1 h at room temperature. Wells that did not receive sera samples were considered as blank reaction. Six washings were carried out. The secondary antibodies anti-IgG bovine conjugated to peroxidase (1:5000, Sigma) which were diluted in PBS solution with 1% skimmed powdered milk were incubated in the wells for 1 h at room temperature. The plate was washed six times and the reaction was initiated as described for Phage-ELISA with IgG. The cut-off value was calculated based on the average values of OD from the wild phage with positive and negative sera and the values of all the phages tested with all the negative sera + 2X the

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Figure 1. Electrophoresis SDS-PAGE (16%) of the positive and negative samples. M, Molecular weight marker; + pool, positive pool sera for bovine enzootic leukosis (EBL) from 20 animals; - pool, negative pool sera for EBL from 20 animals; + IgG, positive IgG purified from positive pool sera; - IgG, negative IgG purified from negative pool sera; - sera, sera from negative animal for EBL. SDS-PAGE, Sodium dodecyl sulphate polyacrylamide gel electrophoresis.

standard deviation of the same values. From the cut-off, the positive and negative ELISA indexes were calculated thus for each phage sample tested: (i) positive ELISA index for each phage = OD average value obtained with all positive sera/cut-off; (ii) negative ELISA index for each phage = OD average value obtained with all the negative sera/cut-off. The ratio of positive and negative ELISA indexes (EI+:EI-) for each sample was calculated. Phages that presented the highest EI+:EI- were selected.

the stabilization of peptide structure. The peptide was synthesized by Genscript (www.genscript.com). The VLB-Ph presents 60 amino acid residues with molecular weight 6117.1, isoelectric point (IP) 12.4 and a positive charge in pH 7.0 of + 4.4. The peptide was diluted in sterilized/deionized water and its concentration was determined by spectrophotometry at 280 nm via the method of Bradford (1976). In order to verify the reactivity of the peptide VLB-Ph with the bovine sera samples, ELISA tests were performed.

Dot blot (sera) Dot blot was performed to verify the immunoreactivity of the phages selected with positive and negative sera for BLV infection. Twenty positive sera samples and 20 negative sera samples were tested at 1:1000 dilution in blocking solution as previously described.

Bioinformatics analysis The peptides selected by immunoassays were analyzed using bioinformatics tools. The search for the similarities between the sequences was performed via the software Clustal W version 18.1 (www.ebi.ac.uk/clustalW/). The sequences of the selected peptides were analyzed to determine their homology with the sequences of BLV proteins stored in the “GENEBANK” through the software Basic Local Alignment Search Tool (BLAST) (http://www.ncbi. nlm.nih.gov/BLAST/). To find the maximum similarity among all the selected peptides in the immunoassay and the BLV proteins of interest as revealed by Blast, the software MATCH available in the site http://relicbio.anl.gov/match.aspx/ was used.

ELISA VLB-Ph The 96-wells’ high affinity plates (MaxiSorpTM - Nunc) were adsorbed with 1 µg of the diluted peptide in bicarbonate buffer (0.1 M NaHCO3, pH 8.6) for 18 h at 4°C. After the adsorption period, the plates were washed and blocked as previously described. 30 positive sera samples for BLV infection and 30 negative sera samples previously tested by AGID according to the protocol of Miller and Van der Maaten (1977) were diluted 1:100 in PBS solution with 1% skimmed powdered milk and added in duplicates to the wells. After 1 h incubation at room temperature and six washings, secondary antibody anti- IgG bovine conjugated to peroxidase (1:5000, Sigma) which was diluted in PBS solution with 1% skimmed powdered milk was incubated in the wells for 1 hour at room temperature. The plate was washed six times and the reaction was initiated as previously described. The analysis of the results was carried out using the t-test in the software Prisma 5.0 (GraphPad).

RESULTS Synthesis of the peptide containing mimetic epitopes of the BLV

Immunoglobulins purification

The system of multiple mimetic peptides (“multiple antigens peptides”) from the selected sequences was utilized. The synthetic peptide VLB-Ph was planned with repetitions in tandem, presenting the same spacer (GGGS) and had C-terminal amide for

The IgG purification process from a pool of bovine sera presented efficiency and had an approximate yield of 7 µg/µl. Figure 1 presents the electrophoretical patterns of the purified IgG with respect to the sera pool and

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Figure 2. Phage-ELISA: Reactivity index of phages for positive and negative IgG. ELISA, Enzyme-linked immunosorbent assay.

molecular weight marker (M). The presence of a band of about 190 KDa in the sera pool samples as well as on the purified IgG corresponding to the approximated molecular weight of the bovine IgG was verified. Biopanning All the eluates containing amplified and non-amplified phages obtained by biopanning were titrated to estimate the quantity of phages selected in the three cycles using the Ph.D.-12TM library. The titers of entrance of phage in the selection cycles were greater than the output titers which increased gradually from the 1st to the 3rd selection cycles. According to the reading of the sequencing reaction, 44 sequences were obtained from the selection done through Ph.D.-12TM library and were later translated by the software DNA2PRO. All the sequences were different in comparison to each other. According to the results obtained by the software AAFREQ, the most frequent amino acids were alanine, histidine, proline, threonine and tryptophan, and the search for similarities between the sequences performed using the software Clustal W version 18.1 revealed some common motifs between the selected peptides which include AHKW, AhxW, HKW, HxW, FAPT and WAPT. The initial selection of the phages by immunoassays using purified IgG Phage ELISA The initial selection of the phages by immunoassays was

carried out using ELISA. The aim was to select the phages reactive with positive IgG and those which are non-reactive with negative IgG. From the 44 phages obtained after sequence analysis, just 23 were reactive with positive IgG and non-reactive to negative IgG. The only phages which were reactive with positive IgG included A1, A3, A4, A9, C5, C7, D4, D5, D10, E6, F1, F5, F8, F10, G5, G6, G10, H1, H6, H7, H8, H10 and H11. The reactivity index of positive IgG varied a lot among the selected phages (Figure 2). In order to verify the difference between the ELISA indexes obtained, the paired t-test was performed by the software STATA 10.0. The value p< 0.001 shows a significant reactivity difference of the phages with positive and negative IgG. Western blot The 23 selected phages, wild phage and gp51 protein were electro-transferred to nitrocellulose membranes after SDS-PAGE 16%. No reactivity of the phage samples and gp51 with the negative IgG was observed. In addition, only the phage samples G10 and H1, and wild phage (negative control) did not react with the positive IgG, other phage samples and gp51 reacted with the positive IgG (data not shown). Dot blot According to the results of Dot blot (data not shown), the phages A9, C7, G5, D4, F5, H11, D5, H6, D10 and F10 presented the best results in respect of the difference of

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3.5

Positive EI/Negative EI

3 2.5 2 1.5 1 0.5 0

Antigens Figure 3. Phages selected by ELISA from the analysis of the reactivity of 20 positive sera and 20 negative sera for EBL. ELISA, Enzyme-linked immunosorbent assay; EBL, enzootic bovine leukosis.

reactivity with the positive and negative IgG. The wild phage did not react in the assay, and the gp51 did not differentiate the positive and negative IgG, but reacted with both.

(antigens) with greater ability to differentiate the positive sera and the negative sera. The phages that presented the best results were selected and they include A3, A4, A9, D4, D10, F5, F10, G5 and H11 (Figure 4).

Reactivity of protein gp51 immunoglobulins and sera samples

Dot blot

with

purified

The gp51 did not present reactivity in the ELISA experiments performed using positive and negative IgG or the sera samples (Figure 3). Through an electrophoresis SDS-PAGE, it was possible to evaluate the quality of the sample of the protein gp51 utilized in the tests (data not shown). The protein presents mass with an approximate value of 60 KDa (Llames et al., 2000).

According to the results obtained by Dot blot, the phages that differentiated sharply the positive (coloured dots) and negative sera samples (uncoloured dots) were A3, A4, A9, D4, D10, F5, F10, G5 and H11 (Figure 4), thus, confirming the ELISA results. The wild phage did not react with the sera, and gp51 did not diferentiate the samples (data not shown). Western blot

The analysis of the reactivity of the phages with sera samples The 23 phages selected with purified IgG were also evaluated in the immunoassays with positive and negative sera for BLV. Phage ELISA The selected phage samples, wild phage and gp51 were adsorbed in the ELISA plate and incubated with sera samples. Positive ELISA indexes (EI +) and negative ELISA indexes (EI -) were calculated and the greater values were used to determined the phage samples

All the nine phage samples presented the reactivity of pIII protein (~ 42 KDa) with positive sera (visible coloured bands) and non-reactivity with negative sera (no visible coloured bands) (Figure 5). The wild phage did not present any reaction to the sera, and gp51 did not diferentiate between positive and negative sera (data not shown). According to the results obtained from the immunoassays with purified IgG and with field sera samples, the phages with sequences that presented better antigenic features which differentiated the positive and negative sera were: (i) A3 (ahkwdiplstsg), (ii) A4 (ahkweavqppmt), (iii) A9 (ipastidllppl), (iv) D4 (hkpppqtrlmha), (v) D10 (ghkwspivqpsp), (vi) F5 (lprsaidwlapv), (vii) F10 (wyppmhifapts), (viii) G5

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Positive sera samples

Negative sera samples

Figure 4. Dot blot test of the reactivity of selected phages with the positive and negative sera samples for EBL. The superior part of the black arrow indicates membranes incubated with positive sera samples, while the inferior part of the black arrow indicates membranes incubated with negative sera samples; W, wild phage. EBL, Enzootic bovine leukosis.

PIII Positive sera sample

Negative sera sample

Figure 5. Western blot assay for the reactivity test using phages as antigens in positive and negative sera samples for EBL. M, molecular weight marker; superior membrane, positive sera incubation; inferior membrane, negative sera incubation.

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Figure 6. Reactivity analysis of 30 positive sera samples and 30 negative sera samples for BLV infection with the synthetic peptide VLB-Ph in ELISA via the software Prisma 5.0. BLV, Bovine leukosis virus; ELISA, enzyme-linked immunosorbent assay.

(nmlqiappfpas) and (ix) H11 (tvtfaptydsss). Bioinformatics analysis The similarity between the sequences were analyzed by multiple alignments with the software Clustal W version 18.1 which identified the common motifs AHKW, HKW or HKx, FAPT and PPM. The selected peptide sequences were also analyzed for homology with BLV proteins sequences deposited in the â&#x20AC;&#x153;GENEBANKâ&#x20AC;? through the software BLAST. According to these results, the sequences of the peptides show similarity with the BLV sequences G4 (ABE28439.1), tax (ACR15161.1), gp51 (CAR64532.1), p34 (AAC82590.1), gp30 (AAP32011.1), gp60 (NP777385.2), p24 (AAA42784.1), protease (AAB50411.1), integrase (ACR15158.1), reverse transcriptase (ACR15158.1), rex (ABE28498.1), env (ACR15160.1) and pol (AAA42785.1). The software MATCH was used to calculate the maximum point of similarity between some of the selected peptides and the BLV proteins of interest from Blast search. The peptides from phages D10, G5 and A9 showed maximum similarities with p24, the main BLV capsid protein encoded by the gene gag, while the peptide from phage D4 showed maximum similarity with glycoprotein gp51 of the viral envelope encoded by gene env. Synthesis of the peptide containing epitopes mimetics of the bovine leukosis virus (BLV) According to the results obtained from the immunoassays and bioinformatics analysis, the peptide sequences

displayed by phages D4 and G5 were selected for the development of a synthetic peptide. The synthetic peptide was designed with two repetitions of each sequence intercalating between the linker GGGS- and C-terminal amidation for the stabilization of the peptide structure. The peptide called VLB-Ph was produced, presenting the sequences hkpppqtrlmha and nmlqiappfpas which correspond to the sequences of phages D4 and G5, respectively in the following manner: VLB-Ph HKPPPQTRLMHAGGGSNMLQIAPPFPASGGGSHKPP PQTRLMHAGGGSNMLQIAPPFPAS- NH3 The reactivity of the synthetic peptide VLB-Ph in ELISA with sera samples The statistics which invovled the use of the software Prisma 5.0 for t-test with the values of the ODs indicated a difference among the results for positive and negative sera samples with the peptide VLB-Ph in ELISA. The p value was 0.0114, showing a significant difference between the results (Figure 6). DISCUSSION Phage display techniques which were utilized in the discovery of epitopes are important for the development of diagnostic platforms and vaccines (Smith and Petrenko, 1997; Ziegler et al., 1998; Casey et al., 2009). This methodology enables the identification of new antigens with no previous information on the properties of

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the antibodies, avoiding the preparation and characterization of the individual monoclonal antibodies (Parmley and Smith, 1988, 1989). In this study, selected peptides were regarded as epitopes mimicking the proteins of bovine leukosis virus (BLV). The peptides were obtained by immunologic selection from library Ph.D.-12TM presented in phages against purified immunoglobulins of a pool of sera from animals naturally infected by BLV. The isolation of peptides that present similarities with the BLV proteins was carried out through repeated selection cycles of the peptides by immunologic affinity to the biologic target (positive IgG). So, the selection cycles and amplification tend to generate specific peptides recognized by the antibodies showing higher titers, producing specific subpopulations of clones with affinity for IgG (Parmley and Smith, 1988, 1989). After biologic selection (biopanning), the phages clones were characterized and validated through the usage of specific criteria. Phage titration in the beginning and at the end of the selection cycles were carried out to obtain the enrichment of the eluates during the selective process. Presumably, the phages with higher affinity to the positive IgG remained bound and phages with low affinity were removed in the washing processes. The output titers gradually increased from the 1st to the 3rd round, indicating the enrichment of positive clones and that the selections were directed towards the target (Ziegler et al., 1998; Kirsch et al., 2008; Leong et al., 2008). Based on the results of the selection done through the library Ph.D.-12TM, 44 different sequences were obtained with high frequencies of the amino acids alanine, histidine, proline and tryptophan, and common motifs between the selected peptides AHKW, AhxW, HKW, HxW, FAPT and WAPT, suggesting that these are the main participants in the interactions with target for biological selection. The initial selection of the phages through immunoassays was performed with the aim of identifying phages that react only with positive IgG and not with negative IgG. From the 44 peptide sequences obtained, only 23 were selected based on the results of ELISA, Dot blot and Western blot with purified IgG. Based on the large ratios of positive and negative indexes (EI+:EI-) in both ELISA and Dot blot serum assays, nine phages were selected because of their potential to differentiate between positive and negative sera samples. The results of the assays using the wild phage (negative control) and the results obtained from the Western blot proved that only peptides fused to protein III were specifically recognized by purified IgG and antibodies from sera samples. Based on the similarity analysis, these peptide sequences were found to be similar to protein p24 and glycoproteins gp51 and gp30 which show immunedominant epitopes as well as important proteins such as Tax, Rex and G4 which are related to the regulation of viral expression and tumor growth during the

development of the infection (Burny et al., 1980; Miller et al., 1981; Walker et al., 1987; Willems et al., 1993, 1995). The peptides from phages D10, G5 and A9 presented great similarities with the protein p24 which is considered as the main protein of the BLV capsid (Miller et al., 1981), while the peptide from phage D4 presented a great similarity with the glycoprotein gp51 of the viral envelope. These two proteins are considered the most immunegenic during BLV infection since many researchers have shown that the infected bovines developed a primary response to specific antibodies directed to the envelope glycoprotein gp51 and the core viral p24 protein (Deshayes et al., 1980; Portetelle et al., 1980; Walker et al., 1987). Using bioinformatics analysis, common motifs AHKW, HKW or HKx, FAPT and PPM were found in the nine selected peptides, indicating the importance of certain amino acids in distinghuishing between peptides and IgG (Cortese et al., 1995) as they potentially mimic the BLV protein epitopes. The sequences of the nine peptides selected showed similarities with various BLV proteins and played important regulatory, structural and immunodominant roles during the infection. Also, based on the results of the study, the phage display technique may be considered as a major tool to obtain a database of epitopes selected by immunologic interaction with antibodies that recognize the BLV proteins, but this technique needs to be verfied by different techniques. The immunoreactivity of the antigenic protein gp51 which is produced in FLK cells was evaluated in the assays with purified IgG and with sera samples. In the ELISA tests, the protein gp51 did not show reactivity with the positive or negative IgG, but presented an almost insignificant reactivity with the sera samples. Due to this, it was not possible to use gp51 as a control or a competitor in phage ELISA assays. In the Western blot and Dot blot, the gp51 showed reactivity, but it did not differentiate positive and negative samples. The lack of reactivity of gp51 may be related to the absence of standardization of this kind of antigenic material to the techniques utilized since this antigen is prepared for AGID. Considering the low differentiation ability of gp51 in the immunoassays, these mimetic peptides are potentially much better antigens for EBL diagnostics. With the immunoassay results and similarities found between the peptide sequences obtained from the selection with the library Ph.D.-12TM and the BLV immunodominant proteins, one can consider the selected nine peptides as mimetics of immunodominant epitopes of the virus. The mimetic antigens may work as specific probes for antibodies in the diagnoses of diseases in the same way that viral proteins are used in tests for illnesses caused by retroviruses. Moreover, they enable the emphasis on a specific recognition, excluding signals not related to the disease diagnosis and avoiding crossreactions (Smith and Petrenko, 1997). The results of the immunoassays and bioinformatics

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were utilized as criteria for selecting peptide sequences that constitute the synthetic peptide VLB-Ph, and ELISA tests performed with the VLB-Ph showed significant differences between positive and negative sera samples, although a great number of the samples need to be evaluated before the standardization of the use of this peptide in serologic tests. This study demonstrated that mimetic antigen selection of the BLV proteins from the phage display libraries may represent an important tool to define epitopes that are recognized by antibodies during viral infections, which avoids the expensive and complex preparation and characterization of monoclonal antibodies. The use of mimetic peptides which are obtained from the phage display libraries selection as antigens in diagnostics has been suggested as an alternative for the use of gross viral antigens. Using such peptides may eliminate the high ratio of unsuitable epitopes present in the preparations of gross antigens, producing a more efficient and specific diagnosis (Casey et al., 2009). The use of mimetic peptides may also work as a positive control precisely defined and suitable for diagnostic tests, and as an alternative whenever the original antigen is hard to obtain, unstable, or in the case of a virus with erratic distribution (Ziegler et al., 1998). The mimetic peptides selected in this work can be potentially utilized as antigens in EBL diagnosis, development of vaccines, and as a basis for the production of synthetic peptides. According to the results obtained, the synthetic peptide VLB-Ph has the potential for use as antigen in ELISA for the diagnosis of EBL. This study provided alternatives for the development of new methodologies for EBL diagnosis via the production of new antigens that may be used in routine and large scale procedures, contributing directly to the control and prevention of the disease studied. REFERENCES Alvarez I, Gutierrez G, Vissani A, Rodriguez S, Barrandeguy M, Trono K (2007). Standardization and validation of an agar gel immunodiffusion test for the diagnosis of equine infectious anemia using a recombinant p26 antigen. Vet. Microbiol. 121: 344-351. Ball JM, Rushlow KE, Issel CJ, Montelaro RC (1992). Detailed Mapping of the Surface Unit Glycoprotein of Equine Infectious Anemia Virus by Using Synthetic Peptide Strategies. J. Virol. 66(2): 732-742. Beier D, Riebe R, Blankenstein P, Starick E, Bondzio A, Marquardt O (2004). Establishment of a new bovine leukosis virus producing cell line. J. Virol. Methods, 121(20): 239-246. Bolin SR, Ridpath JF, Black J, Macy M, Roblin R (1994). Survey of cell lines in the American Type Culture Collection for Bovine Viral Diarrhea Virus. J. Virol. Methods, 48(2): 211-221. Bradford MM (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72(7): 248-254. Burny A, Bruck C, Chantrenne H, Cleuter Y, Dekegel D, Ghysdael J, Kettmann R, Leclerq M, Leunen J, Mammerickx M, Portetelle D (1980). Bovine leukemia virus: mol. boil. epidemiol. In: Klein, G. (Ed.), Viral Oncol. New York, Raven Press. pp. 231-289. Callebaut I, Burny A, Krchnák V, Gras-Masse H, Wathelet B, Portetelle D (1991). Use of Synthetic Peptides to Map Sequential Epitopes Recognized by Monoclonal Antibodies on the Bovine Leukemia Virus

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External Glycoprotein. Virol. 185(1): 48-55. Callebaut I, Vonèche V, Mager A, Fumière O, Krchnak V, Merza M, Zavada J, Mammerickx M, Burny A, Portetelle D (1993). Mapping of B-neutralizing and T-helper cell epitopes on the bovine leukemia virus external glycoprotein gp51. J. Virol. 67(9): 5321-5327. Camargos MF, Melo CB, Leite RC (2002). Frequência de soropositividade para a Leucose Enzoótica Bovina em rebanhos de Minas Gerais. Cienc.Vet. Trop. 5(1): 20-26. Camargos MF (2005). Vírus da Leucemia Bovina: Epidemiologia Molecular e Diagnóstico. 95f. Tese (Doutorado em Ciência Animal) Escola de Veterinária, Universidade Federal de Minas Gerais, Belo Horizonte. Cardoso R, Homsi-Brandeburgo MI, Rodrigues VM, Santos WB, Souza GRL, Prudencio CR, Siquieroli ACS, Goulart LR (2009). Peptide mimicking antigenic and immunogenic epitope of neuwiedase from Bothrops neuwiedi snake venom. Toxicon, 53(2): 254-261. Casey JL, Coley AM, Parisi K, Foley M (2009). Peptide mimics selected from immune sera using phage display technology can replace native antigens in the diagnosis of Epstein-Barr virus infection. Protein Eng. Des. Sel. 22(2): 85-91. Castro RS, Leite RC, Abreu JJ (1992). Prevalence of antibodies to selected viruses in bovine embryo donors and recipients from Brazil, and its implications in international embryo trade. Trop. Anim. Health Prod. 24(3): 173-176. Choi KY, Liu RB, Buehring GC (2002). Relative sensitivity and specificity of agar gel immunodiffusion, enzyme immunosorbent assay, and immunoblotting for detection of anti-bovine leukemia virus antibodies in cattle. J. Virol. Methods, 104(1): 33-39. Cortese R, Monaci P, Nicosia A, Luzzago A, Felici F, Galfré G, Pessi A, Tramontano A, Sollazzo M (1995). Identification of biologically active peptides using random libraries displayed on phage. Curr. Opin. Biotechnol. 6(1): 73-80. Da Y, Shanks RD, Stewart JA, Lewin HA (1993). Milk and fat yields decline in bovine leukemia virus-infected Holstein cattle with persistent lymphocytosis. Proc. Natl. Acad. Sci. USA. 90(14): 65386541. Dees C, Godfrey VL, Foster JS, Schultz RD, Travis CC (1994). Stabilization of the p53 gene product in two bovine leukemia virus infected cell lines. Cancer Lett. 86(1): 33-40. De Giuseppe A, Feliziani F, Rutili D, De Mia GM. (2004). Expression of the bovine leukemia virus envelope glycoprotein (gp51) by recombinant baculovirus and its use in an enzyme-linked immunosorbent assay. Clin. Diagn. Lab. Immunol. 11(1): 147-151. Deshayes L, Levy D, Parodi AL, Levy JP (1980). Spontaneous immune response of bovine leukemia virus infected cattle against five different viral proteins. Int. J. Cancer, 25(4): 503-508. Doménech A, Llames L, Goyache J, Suárez G, Gómez-Lucía E (1997). Macrophages infected with bovine leukaemia virus (VLB) induce humoral response in rabbits. Vet. Immunol. Immunopathol. 58(3): 309-320. Folgori A, Tafi R, Meola A, Felici F, Galfre G, Cortese R, Monaci P, Nicosia A (1994). A general strategy to identify mimotopes of pathological antigens using only random peptide libraries and human sera. EMBO J. 13(9): 2236-2243. Gatei MH, Good MF, Daniel RC, Lavin MF (1993). T-cell responses to highly conserved CD4 and CD8 epitopes on the outer membrane protein of bovine leukemia virus: Relevance to vaccine development. J. Virol. 67(4): 1796-1802. Gutiérrez G, Alvarez I, Fondevila N, Politzki R, Lomónaco M, Rodríguez S, Dus Santos MJ, Trono K (2009). Detection of bovine leukemia virus specific antibodies using recombinant p24-ELISA. Vet. Microbiol. 137(3): 224-234. Johnson M, Rommel F, Moné J (1998). Development of a syncytia inhibition assay for the detection of antibodies to bovine leukemia virus in naturally infected cattle; comparison with Western blot and agar gel immunodiffusion. J. Virol. Methods 70(2): 177-182. Kabeya H, Ohashi K, Ohishi K, Sugimoto C, Amanuma H, Onuma M (1996). An effective peptide vaccine to eliminate bovine leukaemia virus (VLB) infected cells in carrier sheep. Vaccine, 14(12): 11181122. Kim CH, Casey JW (1992). Genomic variation and segregation of equine infectious anemia virus during acute infection. J. Virol. 66(6):

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Payne SL, Rushlow KE, Dhurva BD, Issel CJ, Montelaro RC (1989). Localization of conserved and variable antigenic domains of equine infectious anemia virus envelope glycoproteins using recombinant env-encoded protein fragments produced in Escherichia coli. Virology, 172(1): 609-615. Portetelle D, Bruck C, Mammerickx M, Burny A (1980). In animals infected by bovine leukemia virus (VLB) antibodies to envelope glycoprotein gp51 are directed against the carbohydrate moiety. Virology, 105(1): 223-233. Portetelle D, Dandoy C, Burny A, Zavada J, Siakkou H, Gras-Masse H, Drobecq H, Tartar A (1989). Synthetic peptides approach to identification of epitopes on bovine leukemia virus envelope glycoprotein gp51. Virology, 169(1): 34-41. Reichel MP, Tham KM, Barnes S, Kittelberger R (1998). Evaluation of alternative methods for the detection of bovine leukaemia virus in cattle. N. Z. Vet. J. 46(4): 140-146. Sioud M, Forre O, Dybwad A (1996). Selection of ligants for polyclonal antibodies from random peptide libraries: potential identification of (auto) antigens that may trigger B and T cell responses in autoimmune diseases. Clin. Immunol. Immunopathol. 79(2): 105-114. Smith GP, Petrenko VA (1997). Phage Display. Chem. Rev. 97(2): 391410. Soutullo A, Verwimp V, Riveros M, Pauli R, Tonarelli G (2001). Desing and validation of an ELISA for equine infectious anemia (EIA) diagnosis using synthetic peptides. Vet. Microbiol. 79(2): 111-121. Soutullo A, García MI, Bailat A, Racca A, Tonarelli G, Borel IM (2005). Antibodies and PBMC from EIAV infected carrier horses recognize gp45 and p26 synthetic peptides. Vet. Immunol. Immunopathol. 108(3): 335-343. Trono KG, Perez-Filgueira DM, Duffy S, Borca MV, Carrillo C (2001). Seroprevalence of bovine leukemia virus in dairy cattle in Argentina: comparison of sensitivity and specificity of different detection methods. Vet. Microbiol. 83(3): 235-248. Van der Maaten MJ, Miller JM, Boothe AD (1974). Replicating C-type virus particles in monolayer cell cultures of tissues from cattle with lymphosarcoma. J. Natl. Cancer Inst. 52(2): 491-497. Walker PJ, Molloy JB, Rodwell BJ (1987). A protein immunoblot test for the detection of bovine leukemia virus p24 antibody in cattle and experimentally infected sheep. J. Virol. Method, 15(3): 201-211. Willems L, Kettmann R, Dequiedt F, Portetelle D, Voneche V, Cornil I, Kerkhofs P, Burny A, Mammerickx M (1993). In vivo infection of sheep by bovine leukemia virus mutants. J. Virol. 67(7): 4078-4085. Willems L, Gatot JS, Mammerickx M, Portetelle D, Burny A, Kerkhofs P, Kettmann R (1995). The YXXL signaling motifs of the bovine leukemia virus transmembrane protein are required for in vivo infection and maintenance of high viral loads. J. Virol. 69(7): 41374141. Ziegler A, Mayo MA, Torrance L (1998). Synthetic antigen from a peptide library can be an effective positive control in immunoassays for the detection and identification of two Geminiviruses. Virology, 88(12): 1302-1305.

Full Length Research Paper

Prediction of metabolisable energy of poultry feeds by estimating in vitro organic matter digestibility Dragan Palić1*, Djordje Okanović1, Djordje Psodorov1, Natalija Džinić2, Slobodan Lilić3, Vladislav Zekić4 and Dragan Milić4 1

Institute of Food Technology, University of Novi Sad, Bulevar cara Lazara 1, 21000 Novi Sad, Serbia. 2 Faculty of Technology, University of Novi Sad, Bulevar cara Lazara 1, 21000 Novi Sad, Serbia. 3 Institute of Meat Hygiene and Technology, Kacanskog 13, 11000 Belgrade, Serbia. 4 Faculty of Agriculture, University of Novi Sad, Trg D. Obradovića 3, 21000 Novi Sad, Serbia. Accepted 16 March, 2012

The aim of this study was to develop equations to predict the in vivo apparent metabolisable energy (AME) of poultry feeds using an in vitro method for estimation of organic matter digestibility. In this study, a total of 57 samples of feedstuffs and 23 samples complete diets for poultry were used. Dry matter (DM), crude protein (CP), crude fibre (CF), crude fat (CFat) and crude ash (CA) of the diets were determined. A modified method for estimating the enzymatic digestibility of organic matter (EDOM) was used. For the determination of in vivo ME, the rooster digestibility assay was followed. Obtained laboratory results, that is in vitro and proximate analysis values were regressed against the in vivo ME values and equations for predicting the in vivo ME of feeds for poultry have been derived. Using CA, CF, CFat and in vitro EDOM as predictors, the following equation for predicting the in vivo ME in poultry feeds was derived: ME (MJ/kg DM) = 5.46 – 0.2166 x CA – 0.0946 x CF + 0.2219 x CFat + 0.1054 x EDOM (R2 = 0.844, RSD = 1.10). Using only EDOM as predictor generated the equation: ME (MJ/kg DM) = -0.41 + 0.1769 x EDOM (R2 = 0.689; RSD = 1.63). Results show that using only EDOM as a predictor was not as accurate as when the other variables were included. Key words: Metabolisable energy, prediction, poultry, feeds, organic matter digestibility. INTRODUCTION Quality control of animal feeds is commonly based on chemical analysis for determining the composition of the nutrients, example gross energy, protein, etc. It is questionable to what extent the results of chemical analysis of feed reflect its real quality, as these are only slightly influenced by physical and chemical treatments of feed, such as milling, heat treatment, enzyme treatment, etc., which all greatly influence the digestibility and thus availability of nutrients to the animal (Boisen, 2000; Palic et al., 2009). One of the most important parameters of

*Corresponding author. E-mail: dragan.palic@fins.uns.ac.rs. Tel: +381 21 485 3793. Fax: +381 21 450 725. Abbreviations: AME, Apparent metabolisable energy; DM, dry matter; CP, crude protein; CF, crude fibre; CFat, crude fat; EDOM, enzymatic digestibility of organic matter; CA, crude ash.

feed quality is its energy, since it is needed for execution of metabolitic processes and animal activity. Not all energy of the feed (gross energy) will be utilized by the animal, but only a bio-available portion called metabolisable energy (ME). This parameter serves as an accurate indicator of feed quality, can be reliably used for feed quality control and is crucial for diet formulation (Farrel, 1999). Metabolisable energy is directly proportional to digestibility of nutrients, as it directly affects their availability and absorption (Čolović et al., 2011).The accepted method for direct determination of ME of feeds is by in vivo trials. These are often expensive and time-consuming. In vitro methods used for predicting ME are attractive because of rapidity and low cost (Farrel, 1999) and can be estimated directly from parameters accessible in the feeds (Noblet and Perez, 1993). Therefore, there has always been a need for reliable laboratory methods and related equations for

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prediction of the in vivo ME values of feeds, in order to implement an adequate system of quality control (Alvarenga et al., 2011). A number of methods have been developed for predicting the AME of feeds for monogastric animals, including method by Valdes and Leeson (1992) using a two step in vitro technique with pepsin, pancreatin, bile acids and enterokinase. The repeatebility of the method was similar to in vivo trials but the residual standard deviation of the prediction was high for some of the studied diets. Boisen and Fernadiz (1997) modified the in vitro method by a three step enzymatic incubation of total tract energy digestibility in pigs. The authors investigated the relationship between the in vitro enzyme digestibility of organic matter (EDOM) and in vivo total tract digestibility of energy for 90 samples of 31 different feedstuffs. Their results showed a close relationship between predicted and determined digestibility of energy. In vitro metabolisable and digestible energy contents could also be predicted from chemical composition, employing either non-nutrients (ash and dietary fibre) or nutrients (starch, crude protein and crude fat) (Lijwgrex at al., 1992). The objective of this study was to develop equations to predict the in vivo ME of poultry feedstuffs from the digestibility of organic matter as determined by an in vitro method not used nowadays for poultry feeds, in order to mimic what actually happens in the gastro-intestinal tract of the animal. MATERIALS AND METHODS A total of 57 feedstuffs and 23 commercial complete diets for poultry was used in this study. Laboratory analysis Proximate analysis Dry matter (DM), crude protein (CP), crude fibre (CF), crude fat (CF) and ash were determined according to AOAC official methods (2000). In vitro determination of enzyme digestible organic matter (EDOM) The three-step procedure of Boisen and Fernadez (1997) was modified to 2-step thus using incubation of feed sample with pepsin for 75 min, followed by incubation with pancreatin for 18 h. Solubilised protein was precipitated with sulphosalicylic acid. Insolubilised and precipitated materials were collected after filtration and then dried and finally ashed. Based on the results from determined dry matter and ash in the sample and residue, respectively, EDOM was calculated. In vivo determination of true metabolisable energy (TMEn) The method used is a procedure for determining digestibility of

nutrients (McNab and Fisher, 1982, 1984; Fisher and McNab, 1987). This is a rapid bioassay technique in which 50 g of the test feed is introduced into the crop of an adult rooster by means of a stainless steel funnel and tube. Each test feed is replicated among six roosters. Excreta are collected during 48-h period. These are dried, weighed and analysed. Endogenous energy or amino acid losses are determined in roosters kept under the same conditions, but glucose is fed in place of the test ingredient. These endogenous losses are used to calculate the true digestibility of the test nutrient.

Statistical analysis Using statistical package STATISTICA (Data Analysis Software System), v.8.0. (2008), obtained EDOM and proximate analysis values were regressed against the in vivo ME results.

RESULTS The results of the proximate analysis, in vivo TMEn and in vitro EDOM are shown in Tables 1 and 2. Obtained laboratory results, that is, in vitro and proximate analysis values were regressed against the in vivo ME values and equations for predicting the in vivo ME of feeds for poultry, was derived as follows: Using both EDOM and proximate analysis results for regression analysis led to the following regresion parameters (Table 3). Using crude ash (CA), crude fibre (CF), crude fat (CFat) and in vitro EDOM as predictors, the following equation for predicting the in vivo ME in poultry feeds was derived: ME (MJ/kg DM) = 5.46 â&#x20AC;&#x201C; 0.2166 x CA â&#x20AC;&#x201C; 0.0946 x CF + 0.2219 x CF + 0.1054 x EDOM R2 = 0.844, RSD = 1.10 On the other hand, using only EDOM values for regression analysis led to the following regression parameters (Table 4). Regression of the in vitro EDOM values against in vivo ME results generated the following equation for predicting the in vivo ME in poultry feeds: ME (MJ/kg DM) = - 0.41 + 0.1769 x EDOM R2 = 0.689; RSD = 1.63 Results of the regression analysis showed that using only EDOM as a predictor is not as accurate as when the other variables were included. DISCUSSION Poultry, like other livestock species, eat to meet energy requirement. Therefore, food intake can be predicted accurately if the energy concentration of a diet is known precisely and no essential nutrients are limiting. This information is crucial for diet formulations (Farrel, 1999). Energy value of diets is of great importance for animal feed manufacturers and end users. The amount of

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Table 1. Results of proximate analysis, in vivo true metabolisable energy (TMEn) and in vitro enzyme digestible organic matter (EDOM) determination in complete diets of poultry.

CP (%) 19.97 26.77

CF (%) 1.90 4.24

CFat (%)

Ash (%)

Min Max

DM (%) 87.99 88.77

2.79 8.82

Min Max

85.30 89.84

17.19 25.99

2.06 7.25

Min Max

88.90 89.85

16.63 27.37

3.68 8.87

Diet

Starter

Grower

Finisher

TMEn (MJ/Kg DM)

In vitro EDOM (%)

5.21 6.25

OM (%) 93.75 94.79

14.71 16.51

77.85 82.68

4.32 9.74

4.28 11.39

88.61 96.00

12.60 17.18

71.50 86.76

7.50 11.06

6.43 8.29

91.71 93.57

14.63 16.92

83.31 84.94

No = Number of samples; DM = Dry matter; CP = Crude protein; CF = Crude fibre; CFat = Crude fat; OM = Organic matter; min = Lowest value; max = Highest value.

Table 2. Results of proximate analysis, in vivo true metabolisable energy (TMEn) and in vitro enzyme digestible organic matter (EDOM) determination in feedstuffs for poultry.

CF (%)

CFat (%)

Ash (%)

OM (%)

TMEn (MJ/Kg DM)

In vitro EDOM (%)

88.70 91.22

CP (%) 69.01 75.45

0.19 0.75

7.10 15.11

13.32 15.86

84.14 86.68

13.53 17.58

87.27 95.49

Min Max

90.61 93.82

38.20 41.49

5.50 9.98

15.86 20.96

4.85 5.59

94.41 95.15

17.31 18.17

70.76 82.73

Min Max

88.09 90.55

17.45 20.05

33.16 34.70

1.04 3.54

7.08 10.99

89.01 92.92

4.68 6.13

28.42 36.82

Soya oilcake

Min Max

88.44 90.21

52.25 53.85

2.66 5.78

0.78 4.45

6.81 7.48

92.52 93.19

12.36 14.69

73.36 79.12

Sunflower oilcake

Min Max

88.87 92.00

37.74 41.51

15.68 23.69

0.92 6.79

5.57 8.83

91.17 94.23

7.14 11.97

53.00 60.72

Wheat bran

Min Max

87.16 89.68

16.45 17.85

8.85 10.21

3.34 4.23

4.38 5.61

94.39 95.62

9.58 11.23

51.72 60.21

White maize

Min Max

87.20 87.28

8.62 8.68

1.46 4.13

3.82 4.22

1.58 1.59

98.41 98.42

15.82 17.92

74.52 79.72

Yellow maize

Min Max

87.15 88.53

8.07 9.95

2.46 3.22

2.69 6.79

0.99 1.89

98.11 99.01

15.51 17.55

82.36 85.57

DM (%)

Feedstuff

Fishmeal

Min Max

Full fat soya

Alfalfa hay

No = Number of samples; DM = Dry matter; CP = Crude protein; CF = Crude fibre; CFat = Crude fat; OM = Organic matter; min = Lowest value; max = Highest value.

available energy in feeds is described either by its metabolizable energy (ME) or by organic matter digestibility (OMD) (PojiÄ&#x2021; et al., 2008). Metabolisable energy is the most widely accepted value when expressing feed energy for poultry (Nwokolo, 1986;

Farrell et al., 1991), however, its capability to estimate feed energy contents must be validated with in vivo determined values (Losanda et al., 2009, 2010). In vitro methods used for predicting ME are rapid and not expensive (Farrel, 1999) as compared to in vivo

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Table 3. Regression parameters using EDOM and proximate analysis results.

Source Regression Residual Total

d.f. 4 80 84

s.s. 553.36 96.43 649.79

m.s. 138.341 1.205 7.736

v.r. 114.77

F pr. <0.001

Response variate: TMEn; fitted terms: constant, ash, crude fibre, crude fat and EDOM. Df, Degree of freedom; ss, sum of square; ms, mean of square; Fpr, final probability; vr, variance.

Table 4. Regression parameters using EDOM values.

Source Regression Residual Total

d.f. 1 72 73

s.s. 432.0 191.4 623.5

m.s. 432.028 2.659 8.540

v.r. 162.50

F pr. <0.001

Response variate: TMEn; fitted terms: constant, EDOM.

determination of digestibility which is time-consuming and costly. Therefore, there has been a need for quick and reliable in vitro methods for determining nutrient digestibility in single feedstuffs for use in feed formulations and for control of complete diets (Boisen and Fernandez, 1997). Estimation of ME values can be done by directly collecting the excreta of the animals but this method is relatively complex, expensive and needs several weeks for obtaining the results and requires separating urine from faeces which can be difficult (Noblet and van Milgen, 2007). There are several reports that metabolizable energy values vary according to the type of poultry used in their determination. Slinger et al. (1964) reported that chicks obtained more ME from high energy diets and less ME from low energy diets than did turkeys. Several different equations to predict ME have been derived based on physical characteristics such as bulk density of grains, dry matter or organic matter digestibility, and chemical characteristics of a foodstuff or diet using proximate or other analyses (Farrel, 1999; Campbell et al., 1986). But in some cases, prediction equations may be less effective, particularly for processed ingredients. Nehring and Haenlein (1973) produced regression equations to predict ME based on the digestibility of each of the chemical components as determined in the Weende system of analysis. Furthermore, Farrel (1999) reported that general prediction equations, no matter how precisely they are derived, do not usually have universal application, particularly when used to predict ME of some individual ingredients as opposed to formulated diets. Knowledge of the metabolisable energy of feedstuffs and complete diets for poultry is important in making decisions in poultry production, as feed cost has a major impact on economic parameters of poultry production (Tica et al., 2009)

Conclusion Based on the results of this study, it can be concluded that the ME of feeds for poultry can be successfully predicted using the enzymatic procedure for determining the organic matter digestibility. Results of the statistical analysis showed that using only EDOM as a predictor is not as accurate as when the other variables from proximate analysis are included. ACKNOWLEDGEMENT The authors are grateful to the Ministry of Science and Technological Development of Republic of Serbia for supporting this study through projects III-46009 and III46012.

REFERENCES Alvarenga RR, Rodrigues PB, Zangeronimo MG, Freitas RTF, Lima RR, Bertechini AG, Fassani EJ (2011). Energetic values of feedstuffs for broilers determined with in vivo assays and prediction equations. Anim. Feed Sci. Technol. 168: 257-266. AOAC Official Methods, (2000). Official Methods of Analysis of AOAC International, 17th Edition AOAC International, Gaithersburg, MD 20877-2417, USA. Boisen S, Fernandez JA (1997). Prediction of the total tract digestibility of energy in feedstuffs and feed diets by in vitro analyses. Anim. Feed Sci. Technol. 68: 277-286. Boisen S (2000). In vitro digestibility methods: History and specific approaches. In: Moughan PJ, Verstegen MWA, Visser Reineveld M. (eds). Feed evaluation. Principles and practice. Wageningen Pers. pp. 153-168. Campbell GL, Salmon RE, Classen HL (1986). Prediction of metabolizable energy of broiler diets from chemical analysis. Poult. Sci. 65(11): 2126-2134. Čolović R, Palić D, Modika YK, Barnes P (2011). Nonlinear models for predicting metabolizable energy of poultry diets. Food Feed Res. 38(1): 33-37.

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Farrell DJ,Thomson E, Du Preez JJ, Hayes JP (1991). The estimation of endogenous excreta and the measurement of metabolisable energy in poultry feedstuffs using four feeding systems, four assay methods and for diets. Br. Poult . Sci. 32: 483-489. Farrell DJ (1999). In vivo and in vitro techniques for the assessment of the energy content of feed grains for poultry: a review. Aust. J. Agric. Res. 50: 881-888. Fisher C, McNab JM (1987). Techniques for determining the ME content of poultry feeds. In: Haresign W, Cole DJA (eds). Recent Advances in Animal Nutrition Butterworths, London, pp. 3-17. Lijwgrex W, Graham PH, Aman B, Raj S, Kotarbinskab M (1992). Predicting the energy value of pig feeds using in vitro or analytical methods. Anim. Feed Sci. Technol. 39: 31-39. Losada B, García-Rebollar P, Cachaldora P, Álvarez C, Méndez J, De Blas JC (2009). Comparison of the prediction of apparent metabolizable energy content of starchy grains and cereal byproducts for poultry from its chemical components, in vitro analysis or near-infrared reflectance spectroscopy, Spanish J. Agric. Res. 7(4): 813-823. Losada B, García-Rebolla P, Álvarez C, Cachaldora P, Ibanez MA, Méndez J, De Blas JC (2010). The prediction of apparatent metabolisable energy content of oil seeds and oil seed by-products for poultry from its chemical components, in vitro analysis or nearinfrared reflectance spectroscopy. Anim. Feed Sci. Technol. 160: 6272. McNab JM, Fisher C (1982). The choice between apparent and true metabolisable energy systems –recent evidence. Proceedings 3rd European Symposium on Poultry Nutrition. pp. 45-55. McNab JM, Fisher C (1984). An assay for true and apparent metabolisable energy. Proceedings XVII World's Poultry Congress. pp. 374-376. Nehring K, Haenlein GFW (1973). Feed evaluation and ration calculation based on net energy. J. Anim. Sci. 36: 949-964.

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Noblet J, Perez JM (1993). Prediction of digestibility of nutrients and energy values of pig diets from chemical analysis. J. Anim. Sci. 71: 3389-3398. Noblet J, Jacquelin-Peyraud Y (2007). Prediction of organic matter and energy in the growing pig from an in vitro method. Anim. Feed Sci Technol. 134: 211-222. Nwokolo E (1986). A comparison of metabolizable energy content of eight common feed ingredients determined with young guinea fowls (keets) and pullet chicks. Anim. Feed Sci. Technol. 15: 1-6. Palic D, Siebrits FK, Coetzee SE (2009). Determining the optimum temperature for dry extrusion of full fat soybeans. South Afr. J. Anim. Sci. 39 Supplement 1: 69-74. Pojić M, Palić D, Mastilović J, Janić-Hajnal E (2008). The introduction of a me-thod for determination of organic matter digestibility in feeds into routine laboratory practice. Food process. quality, Safety, 35(3): 151-154. Statistica (Data Analysis Software System), v.8.0. (2008). StatSoft. Inc. Tulsa. OK. USA. (http://www.statsoft.com/ Slinger SJ, Sibbald IR, Pepper WF. (1964). The relative abilities of two breeds of chicken and two varieties of turkeys to metabolise dietary energy and dietary nitrogen. Poult. Sci. 43: 329-333. Tica N,Okanović Dj, Zekić V, Filipović S (2009). The effect of extruded corn on economical results of broiler production. Food Processings Qual. Saf. 36(3-4): 59-64. Valdes EV, Leeson S (1992). Measurement of metabolizable energy in poultry feeds by an in vitro system. Poult. Sci. 71: 1493-1503.

African Journal of Biotechnology Vol. 11(28), pp. 7318-7322, 5 April, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.852 ISSN 1684â&#x20AC;&#x201C;5315 ÂŠ 2012 Academic Journals

Full Length Research Paper

Identification and characterization of variants in the 5' flanking region of bovine growth hormone gene GENG Rong-qing1,2#, WANG Lan-ping1# and CHANG Hong2* 1

College of life science and technology, Yancheng teachers university, Yancheng Jiangsu Province, P.R China, 224002. 2 Animal science and technology college, Yangzhou university, Yangzhou Jiangsu Province, P.R China, 225009. Accepted 14 March, 2012

Sequence variations within the 5' flanking region of bovine growth hormone gene were identified from six bovine species raised in China. Cloned and sequenced amplified fragments revealed difference in length because of the insertion and deletion mutation. A total of thirty one variation sites were identified in this region within species and among species. Several new single nucleotide polymorphisms (SNPs) within bovine species were detected in the 5' flanking region with exception in swamp buffalo. Some important regulatory elements such as TATA box, CRE, NRE3, dPit1 and pPit1 were identified in the 5' flanking in six bovine species. The conservation of regulatory elements may be consistent with functional constraint during the course of evolution. Key words: Bovine species, growth hormone gene, variation, regulatory element. INTRODUCTION Growth hormone (GH), the product of the GH gene, is a multifunctional hormone, produced in the vertebrate pituitary gland, which promotes the postnatal growth of skeletal and soft tissues. The bovine growth hormone (bGH) gene, mapped to chromosome 19 contains about 2856 nucleotides, mainly distributed in five exons and four introns (Gordon et al., 1983; Fries et al., 1993). During the last decade, extensive researches have shown a close relationship between polymorphism of bGH gene and production traits (Grochowska et al., 2001; Ge et al., 2003; Wu et al., 2005; Zhou et al., 2005; China et al., 2007; Pawar et al., 2007; Dario et al., 2008; Tatsuda et al., 2008; Ishida et al., 2010; Mullen et al., 2010; Matsuhashi et al., 2011). Therefore, it has been considered a candidate gene for production traits, such as milk production and carcass traits. The 5' flanking region of bGH gene contains repetitive regions and protein binding sites, such as the transcription initiation sites and transcription factor binding sites, which are associated with gene expression control. Recent developments in molecular biotechnologies

*Corresponding author. E-mail: bio501@126.com.

#These authors contributed equally to this work.

have opened the possibility of identifying and using genomic markers and multiple genes for the genetic improvement of livestock (Montaldo and Meza-Herrera, 1998; Dekkers, 2004; Margawati, 2012). This has provided opportunities to enhance response to selection, in particular for traits that are difficult to improve by conventional selection. The use of genetic markers or more effective of marker-assisted selection (MAS) for desired important traits would be more valuable and useful and even more efficient in important trait selection of superior livestock. Application of molecular biotechnology approaches will enable improvement in productivity, reduction in costs, enrichment of milk compositions and extension of shelf life products. Bovine species are one of the most economically important domestic animals in China (Chang, 2009). It plays an important role in the economies of livestock sector. It provides food, or more specifically animal protein in human diets, income, employment and possibly foreign exchange. Our objective was to identify and characterize variations in the 5' flanking region of GH gene in different bovine species. MATERIALS AND METHODS Applying simple random sampling in typical colony method in the central area of habitat, a total of 103 blood samples were obtained

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Figure 1. Electrophoretic patterns of the PCR products of the GH1 fragment (DL2000 DNA marker was used as contrast band).

Figure 2. Electrophoretic patterns of the PCR products of the GH2 fragment (DL2000 DNA marker was used as contrast band).

from six bovine species, 18 samples of Leiqiong cattle (Bos indicus) from Guangzhou province, 19 samples each of Mangolian cattle (Bos taurus) and Bazhou yak (Bos grunniens) from Xinjiang autonomous region, 13 Gayal (Bos frontalis) from Yuunnam province, 18 Haizi buffalo (Bubalus bubalis) from Jiangsu province and 16 Nili-Ravi buffalo (Bubalus bubalis) from Guangxi province. These flocks were raised in the region of semi-agricultural and semi-pastoral or agricultural areas by natural grazing. The sampling was carried out in spring in the same year. Genomic DNA was isolated from the blood samples by a standard phenol/chloroform protocol. Two pair of primers for PCR amplification and sequencing was designed according to published nucleotide sequence information of the bGH gene (Gordon et al., 1983). One amplified fragment was named as GH1 (the PCR primer pair were P1 5'GGTGGGTTGCCTTTCTCTTCT-3' and P2 5'-TGTCATCATCCCGTCTCCACT-3'), including partial 5' flanking region. The other amplified fragment was named as GH2 (the PCR primer pair were P1 5'-TCTCAAGCTGAGACCCTGTGT-3' and P2 5'GGCCAAATGTCTGGGTGTAGA-3'), including a region from 5' flanking region to part of the first intron. PCR was performed using an initial denaturation at 94°C for 5 min, followed by 32 cycles of denaturation at 94°C for 50s,

annealing at 60°C for 50s and extension at 72°C for 60s, ending with a final extension at 72°C for 8 min, in a GeneAmp PCR system 9700 (Applied Biosysems, Foster, CA, USA). Amplified DNA fragments were subjected to electrophoresis on a 1% agarose gel and target fragments were cut from gel for purify with spin columns (Watson Biotechnologies, Shanghai, China). Purified fragments were cloned into PMD 18-T Vector (Takara, Dalian, China) before sequencing. Sequencing was performed in both directions using an ABI PRISM 3730 BigDye Terminator Cycle Sequencing Kit (Applied Biosysems, Foster, CA, USA). Sequences were manually trimmed to remove vector sequences and sequences were assembled with the DNASTAR software. Sequences were aligned by Clustalx1.83 package and the polymorphism results were exported using MEGA 4.0 (Tamura et al., 2007).

RESULTS AND DISCUSSION Two kind of fragments GH1 (Figure 1) and GH2 (Figure 2) were amplified in all samples from six bovine species. In the GH1 fragment, three different types in length (465,

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Table 1. Distribution of genotype frequencies of GH2 fragment of different bovine species.

Bovine species

GH1-A

GH1-B

GH1-C

GH1-D

GH1-E

GH1-F

GH1-G

GH1-H

Bos taurus

1.00

0.00

Bos indicus

0.77

0.06

0.17

0.00

Bos grunniens

0.95

0.00

0.05

0.00

Bos frontalis Bubalus bubalis (Swamp buffalo) Bubalus bubalis (River buffalo)

0.00 0.00 0.00

1.00 0.00 0.00

0.00 1.00 0.00

0.00 0.00 0.87

0.00 0.00 0.13

Table 2. Distribution of genotype frequencies of GH2 fragment of different bovine species.

Bovine species Bos taurus

GH2-A

GH2-B

GH2-C

GH2-D

GH2-E

GH2-F

1.00

0.00

Bos indicus

0.00

0.06

0.94

0.00

Bos grunniens

1.00

0.00

Bos frontalis Bubalus bubalis (Swamp buffalo) Bubalus bubalis (River buffalo)

0.00 0.00 0.00

0.08 0.00 0.00

0.92 0.00 0.00

0.00 1.00 1.00

467 and 473 bp) were identified according to sequencing results. The difference in length was due to a TG deletion sequence raised in Leiqiong cattle, while an ACTGCT insertion was raised in Haizi buffalo and Nili-Ravi buffalo. Twenty-six polymorphism sites were identified and denominated as GH1-A, GH1-B, GH1-C, GH1-D, GH1-E, GH1-F, GH1-G and GH1-H genotypes (Table 1). Bos taurus, Bos indicus and Bos grunniens shared genotype GH1-A and possessed the most frequency within population, while Bos frontalis and Bubalus bubalis possessed unique genotypes respectively. In the GH2 fragment, two different types in length (450 and 453 bp) were identified according to sequencing results. The difference in length was due to an AGA deletion sequence raised in Leiqiong cattle, Haizi buffalo and Nili -Ravi buffalo. Only seven polymorphism sites were identified and denominated as GH2-A, GH2-B, GH2-C, GH2-D, GH2-E and GH2-F genotypes (Table 2). Except B. taurus and B. grunniens shared genotype GH2A and possessed 100% frequency within population, B. indicus, Bos frontalis and Bubalus bubalis all possessed unique genotypes. Through assembling fragment GH1 and GH2, we got the sequences of 5' flanking region of bGH gene, which cover almost the entire length. There was difference in length of 5' flanking region among different bovine species because of the insertion and deletion. Except the mutation of insertion and deletion, total thirty one variation sites were identified in this region. The predominant substitution model of nucleotide was transition and transition was higher than transversion with the ratio of 1.6.

Multiple alignment results are shown in Figure 3. Regarding the single nucleotide polymorphisms (SNPs), no variation was observed within Haizi buffalo. Compared with published bGH gene M57764, one, four, three, one and one new SNPs were identified within Mangolian cattle, Leiqiong cattle, Bazhou yak, Gayal and Nili-Ravi buffalo, respectively. Analysis of sequences upstream of the ATG start site of the human and other animal GH genes has revealed various regulatory sequences (Wallis et al., 2001). Such regulatory elements of TATA box, CRE, NRE3, dPit1 and pPit1 were all identified in the six bovine species. The TATA box was named for its conserved DNA sequence, which is most commonly TATAAA, and many eukaryotic genes had a conserved TATA box. The TATA box element within bGH gene promoter was strongly conserved. A cyclic AMP response element (CRE) was found between the Pit-1 sites in the human GH gene promoter (Eberhardt et al., 1996) and appeared in the corresponding region in six bovine species. NRE3 probably represented a binding site for transcription factor YY1 (Park and Roe, 1996) and was conservative in all bovine species. Two putative binding sites for the Pit1 transcription factor (Theill and Karin, 1993) of other mammalian GH genes (Krawczak et al., 1999) were observed in the corresponding positions in bGH, the distalone overlapping the NRE3. It is notable that the distal and proximal sites in bovine species were very close to each other, but no changes in recognized regulatory elements in the promoter region, suggesting that regulation of bGH gene may be considerably conservative during the course of evolution. It was

Geng et al.

1 180 M57764 GTACTGGGGT GGGTTGCCTT TCTCTTCTCC AGGGGATTTA TCTGACCCAG GGATTGAACC GCATTTGCAG CTAGATTCTT TACGGCTGAG CCACCTGGGA AGCCCATTCG CTTCT----GCTGCTGCTA AGTTGCTTCA GTCGTGTCCG ACCTG-TGCG H1 -------... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .....----.......... .......... .......... ..TCTG.... H2 -------... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .....----.......... .......... .......... ..TCTG.... H3 -------... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .....----.......... .......... .......... ..TC--.... H4 -------... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .....----.......... .......... .......... ..TCTG.... H5 -------... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .....----.......... .......... .......... ..TCTG.... H6 -------... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .....----.......... .......... .......... ..TCTG.... H7 -------... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .....----.......... .......... .......... ..TCTG.... H8 -------... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .....----.......... .......... .......... ..TCTG.... H9 -------... .......... .......... .......... .......... .......... .......... .......... ....T..... .......... .......... .....----.......... .......... .......... ..TCTG.... H10 -------... .......... .......... .......... .......... .......... .......... .......... ....T..... .......... .......... .....----.......... .......... .......... ..TCTG.... H11 -------... .......... .......... ....C..C.T .......... .......... .......... ..G....... .......... .......... ........T. .....ACTGC .......... .......... ........A. ..TCTG...A H12 -------... .......... .......... ....C..C.T .......... .....TG... .......... ..G....... .......... .......... ........T. .....ACTGC .......... .......... ........A. ..TCTG...A H13 -------... .......... .......... ....C..C.T .......... .....TG... .......... ..G....... .......... .......... ........T. .....ACTGC .......... .......... ........A. ..TCTG.... 181 TRE 360 M57764 ACGCCATAGA CAGCAGCCCA CCAGGT--CC CCGTCCCTGG GATTCTCCAG GCAAGAACAT TGCCATTTCC TCCTCCAATG CATGAAAGTG AAAAGTGAAA GTGAAGTCAC TCAGTTGTGT GCGACCCCAT GGACTGCAGC CTTCCAGAAT GGGGTGCCAT H1 .......... .......... .....CTC.. .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... H2 .......... .......... .....CTC.. .......... .......... .......... .......... .T........ .......... .......... .......... .......... .......... .......... .......... .......... H3 .......... .......... .....CTC.. .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... H4 .......... .......... .....CTC.. .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... H5 .T........ .......... .....CTC.. ..A....... .......... ..........

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TGAGTCTCCT -GCTGCTGCT .......... -......... .......... -......... .......... -......... .......... -......... .......... -......... .......... -......... .......... -......... .......... -......... .......... -......... .......... -......... .......... T......... .......... T......... .......... T.........

PEA3 TGGAGTGGGT CCGACCCTCA .......... .......... .......... .......... .......... .......... .......... .......... ..........

Figure 3. Comparison of the nucleotide sequences of bovine GH gene 5' flanking region (compared with GenBank accession No. M57764). Positions of regulatory elements are shown as discussed in the text. “.” Represents identity to bovine GH sequence, “-” represents a deletion. H1 and H2, sequences from Bos taurus; H3 to H6, sequences from Bos indicus; H7 and H8, sequences from Bos grunniens; H9 and H10 represent sequences from Bos frontalis; H11, sequence from swamp buffalo (Bubalus bubalis); H12 to H13 represent sequences from river buffalo (Bubalus bubalis).

notable that the number of substitutions outside the defined elements also showed very strict conservatively

within species and among species. In addition, some SNPs were located near binding site

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of transcription factor PEA3 (Roth et al., 1990) and transcription factor TRE (Theill and Karin., 1993). However, these transcription factor binding sites were scattered within a SINE/BovA2 repeatitive region. Previous studies (Ge et al., 2003; Ferraz et al., 2006) may explain the high variability in this region, and this region may not really be the functional promoter for the bGH gene. A palindrome was also identified between pPit1 and TATA box, which maybe a regulatory sequence. Conclusion Great differences appear in the distribution of genotypes of bGH gene 5' flanking region among Chinese bovine species. It seems related to species differences and living environment to a certain extent. It also may be very important to adapt the living conditions and play a vital role for growth and development. The 5' flanking region of bGH gene has a considerable number of mutations and although they are not located in known binding sites, they should be tested in transient expression assays using a gene reporter, in order to analyse their potential transcriptional activity. Given the mutations of the bGH gene is likely to influence growth and development, it may therefore be an indicator that could be used for the genetic improvement of bovine quantitative traits. Combining bGH gene polymorphism data with breeding value information could conceivably improve genetic selection in beef cattle production and increase the potential economic benefit to the beef cattle industry. ACKNOWLEDGEMENTS This research was supported by the Project of the Basic Natural Science Foundation for Colleges and Universities in Jiangsu Province (Grant No.08KJB230002 & 09KJD230001), the Qing Lan Project for Colleges and Universities in Jiangsu Province and the National Natural Science Foundation of P.R.China (Grant No.30571323). REFERENCES Chang H (2009). Animal Genetic Resources. Sci. press, Beijing, China. China S, Supamit M, Voravit S, Panwadee S, Skorn K, Sornthep T (2007). Effect of genetic polymorphism of bovine growth hormone gene on preweaning growth traits in a Thai multibreed beef population. Nat. Sci. 41: 484-492. Dario C, Carnicella D, Ciotola F, Peretti V, Bufano G (2008). Polymorphism of growth hormone GH1-AluI in Jersey cows and its effect on milk yield and, composition. Asian. Australasian Anim. Sci. 21(1): 1-5. Dekkers JCM (2004). Commercial application of marker- and geneassisted selection in livestock:strategies and lessons. J. Anim. Sci. 82: E313-E328. Eberhardt NJ, Jiang SW, Shepard AR, Arnold AM, Trujillo MA (1996). Hormonal and cell-specific regulation of the human growth hormone and chorionic somatoma mmotropin genes. Progress in Nucleic Acid Res.Mol. Biol. 54: 127-163.

Ferraz AL, Bortolossi JC, Curi RA, Ferro M, Ferro J A, Furlan LR (2006). Identification and characterization of polymorphisms within the 5â&#x20AC;&#x2122;flanking region, first exon and part of first intron of bovine GH gene. J. Anim. Breed. Genet. 123: 208-212. Fries R, Eggen A, Womack J E (1993). The bovine genome map. Mamm. Genome, 4(8): 405-428. Ge W, Davis ME, Hines HC, Irvin KM, Simmen CM (2003). Association of single nucleotide polymorphisms in the growth hormone and growth hormone receptor genes with blood serum insulin-like growth factor I concentration and growth traits in Angus cattle. J. Anim. Sci. 81: 641-648. Gordon DF, Quick DP, Erwin CR, Donelson JE, Maurer RA (1983). Nucleotide sequence of the bovine growth hormone chromosomal gene. Mol. Cell Endocrinol. 33: 81-95. Grochowska R, Sorensen P, Zwierzchowski L, Snochowski M, Lovendahl P (2001). Genetic variation in stimulated GH release and in IGF-I of young dairy cattle and their associations with the leucine/valine polymorphism in the GH gene. J. Anim. Sci. 79: 470476. Ishida T, Umebayashi A, Tsuruta S, Akashi R, Harada H (2010). Polymorphisms in growth hormone gene and their associations with calf weight in Japanese black cattle. Anim. Sci. J. 81(6): 623-629. Krawczak M, Chuzhanova N A, Cooper DN (1999). Evolution of the proximal promoter region of the mammalian growth hormone gene. Gene, 237:143-151. Margawati ET (2012). A global strategy of using molecular genetic information to improve genetics in livestock. Reprod. Dom. Anim. 47(Suppl. 1): 7-9. Matsuhashi T, Maruyama S, Uemoto Y, Kobayashi N, Mannen H, Abe T, Sakaguchi S, Kobayashi E (2011). Effects of bovine fatty acid synthase, stearoyl-coenzyme A desaturase, sterol regulatory element-binding protein 1, and growth hormone gene polymorphisms on fatty acid composition and carcass traits in Japanese Black cattle. J. Anim. Sci. 89(1): 12-22. Montaldo HH, Meza-Herrera CA (1998). Use of molecular markers and major genes in the genetic improvement of livestock. Electronic J. Biotechnol. 1(2):83-89. Mullen MP, Berry DP, Howard DJ, Diskin MG, Lynch CO, Berkowicz EW, Magee DA, MacHugh DE, Waters SM (2010). Associations between novel single nucleotide polymorphisms in the Bos taurus growth hormone gene and performance traits in Holstein-Friesian dairy cattle. J. Dairy Sci. 93(12): 5959-5969. Park KY, Roe JH (1996). Identification of a negative regulatory site in the upstream region of bovine growth hormone gene. Biochemical and Biophysical Res. Commun. 219: 354-358. Pawar RS, Tajane KR, Joshi CG, Rank DN, Bramkshtri B P (2007). Growth hormone gene polymorphism and its association with lactation yield in dairy cattle, Indian J. Anim. Sci. 77(9): 884-888. Tamura K, Dudley J, Nei M, Kumar S (2007). MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24: 1596-1599. Tatsuda K, Oka A, Iwamoto E, Kuroda Y, Takeshita H, Kataoka H, Kouno S (2008). Relationship of the bovine growth hormone gene to carcass traits in Japanese black cattle. J. Anim. Breed. Genet. 125: 45-49. Theill LE, Karin M (1993). Transcriptional control of GH expression and anterior pituitary development. Endocrinol. Rev. 14: 670-689. Wallis OC, Zhang YP, Wallis M (2001). Molecular evolution of GH in primates: characterisation of the GH genes from slow loris and marmoset defines an episode of rapid evolutionary change. J. Mol. Endocrinol. 26: 249-258. Wu X L, Macneil MD, De S, Xiao QJ, Michal JJ, Gaskins CT, Reeves JJ, Busboom JR, Wright RW, Jiang Z (2005). Evaluation of candidate gene effects for beef backfat via Bayesian model selection. Genetica, 125:103-113. Zhou GL, Jin HG, Liu C, Guo SL, Zhu Q, Wu YH (2005). Association of genetic polymorphism in GH gene with milk production traits in Beijing Holstein cows. J. Biosci. 30: 595-598.

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