PROCEEDING OF

INTERNATIONAL BIOLOGY CONFERENCE Science for Energy, Food and Environmental Sustainability

December 6th, 2012

Seminar Room, SCC (Student Community Center) Building lnstitut Teknologi Sepuluh Nopember

Sukolilo ~ Surabaya

IBOC2012 -- ......... Wli!iiiiH!'f!tilllll!4€4 - -~ ·. INTERNATIONAL BIOLOGY CONFERENCE

Organized by Department of Biology, lnstitut Teknologi Sepuluh Nopember (ITS}, Surabaya -Indonesia

Supported by lnstitut Teknologi Sepuluh Nopember (ITS), Surabaya- Indonesia

iTS lnstitut Teknologi Sepuluh Nopember

Proceeding of International Biology Conference (IBOC) 2012: Science for Energy, Food and

Environmental Sustainability

© Department of Biology, Faculty of Mathematic and Natural Sciences, lnsti tut Teknologi Sepu luh

Nopember- Surabaya, Indonesia

Text and layout editor

Front cove r

Farid K. Muzaki, S.Si, M .Si Roksun Nasihin, S.S i Talitha Rahma Nathan ia, S.Si Aplilia Fitriana, S.Si Dinda Zahidah, S.Si Andarini Ayu R, S.Si

lwenda Bella Subagio, S.Si

All rights reserve d. No part of this manuscript may be reproduced, stored in a retrieval system, or tran smitted by any other means without the prior perm ission of the copyright holder. Please direct

al l enquir ies to the publishers .

i ISBN 978-979-97316-1-6 I

Depa rtm ent of Biology Faculty of Mathematic and Natura l Sciences lnst itut Teknolog i Sepu luh Nopember

Kampus ITS Sukoli lo Jl. A. R. Haki m, Keputih- Su koli lo, Surabaya 60111

www.bio.it s.ac. id

PREFACE

We would like to thank to God for bless ing us until we are able to organize our first international annual event called The International Biology Conference (IBoC) 2012 at the end of this year. This conference is purposed to build a future and promising international networking between our department and other international parties which have a similar interest keeping environment balance for save the earth . Concerning to it, our first theme is "Science for Energy, Food and Environmental Sustai nability" . The first outcome we would gain is having a direct and personal contact, sharing and discussion with sc ientists around the world . Further on we would like to build a real scientific networking doing a real work keeping the earth balance and save. We are really grateful realizing that they are from tgypt, Thailand, Bangladesh, Malaysia and Indonesia . Thank you for your participations. We are also glad to inform that, this conference have been possible only because of the support from The Faculty of Mathematics and Natural Sciences and The lnstitut Teknologi Sepuluh Nopember (ITS), Surabaya-lndonesia. The Vice Rector, Prof. Darminto put several administrative and scientific advices. The big applause and thank is also going to our students from The Biology Department who has been managing the conference.

Thank you and best regards.

Surabaya, June 51h 2013

Head of Biology Department

Dr.rer.nat. Maya Shovitri

Preface

Contents

CONTENTS

Utilization of both cellulose and hemicellulose in agricultural solid waste for 1

production of biofuel- Arief WIDJAJA

Purification and characterization of autoinducer-2 inactivating enzyme in 3

periodontopathogenic bacterium Eikenella corrodens- Fariha Jasin MANSUR, Mohammad Minnatul KARIM, Ayako NAGAO and Hiroyuki AZAKAMI

Effect of Autoinducer-2 on biofilm formation of periodontopathogenic bacterium 5 Eikenella corrodens - Mohammad Minnatul KARIM, Tatsunori HISAMOTO, Yuichiro NOIRI, Shigeyuki EBISU, and Hiroyuki AZAKAMI

Initial study on fisheries resources in Lamong estuary, East Java 7 - Farid K. MUZAK!, Apri liana Mutia DEWI and D. Ali SAUWIBI

The effectivity of Micorhiza against Fusarium withered disease 17 in Tomato (Lycopersicon esculentum Mill.} Fortuna variety- YUNIS, Sri NURHATIKA and Krintansi lndah PURWANI

Effect of Rhizobium and indigenous Mycorrhiza from Labang, Bangkalan- Madura 31

on the growth of peanut (Arachis hypogea)- Tutik NURHIDAYATI, Kristanti lndah PURWANI, Ni Ketut Dewi INDRAYANI and Triano Bagus SAPUTRO

Feed conversion of Silver Pompano (Trachinotus blochii) in culture media using 35

manipulated salinity- Nurlita ABDULGANI, Salim ARROKHMAN and Dewi HIDAYATI

Biosurfactant production by Pseudomonas putida T1 (8} using molasses as substrate 41

- Nur Hidayatul ALAMI, Lailatul MAGHFIROH, FATIMAH and NI'MATUZAHROH

Isolation and identification of Acetobacter from banana peels native Indonesia as a 49 potential bacteria for banana vinegar production - Dadang H.MUSTHOFA, Jonathan lvander KURNIAWAN Nurlaila SHOVIANITA, Ahmad ARIFIYANTO, Roksun NASIKHIN and Maya SHOVITRI

Hydrogen gas produced by isolated bacteria from septic tank- Maya SHOVITRI, 59 engah D. Kuswytasari, Ayuk RAHMAWATI and Elita S. AMBARNINGTYAS

Production of cellulase enzymes by Penici/lium sp in temperature, pH and different 67 agricultural waste- Irma ALFIAH and Nengah Dwianita KUSWYTASARI

Bi rd diversity in different types of wetland area in Keputih,

East Coast of Surabaya - Hubertus B. AJIE and Citra Fitrie RIANY

istribution study of birds conserved by Indonesian law in Wonorejo, Surabaya - s a DESMAWATI, AUNUROHIM and lndah TRISNAWATI

75

83

~~:.~~ --~ : --~,~~- t:~~;-·;.s:~~?~:~_:,-~ -~~ r- .- ~ ::.:~:, ,.•_ ;;~;' . . .

Study on habitat and bird community at western Mbeliling landscape, Flores - Nur Sita HAMZATI, Fer i IRAWAN , AUNUROHIM

Nuclear markers in biodiversity studies of some black corals species (Antipatharia, Hexacorallia) of North Sulawesi, Indonesia- Hapry F.N LAPIAN

Status of coral reefs before and after mass bleaching event 2010 in coastal water of PLTU Paiton - Farid K. MUZAKI and Dian SAPTARINI

95

103

111

Exploration of potential bacteria as biofertilizer from soil samples of Alas Purwo 119 National Park - Agus SUPRIYANTO, Tini SURTININGSIH, Tri NURHARIYATI, NI' MATUZAH ROH

Bioremediation of cooking oil contaminated soil using hydrocarbonoclactic 125 microbial consortium isolated from Wonocolo- Bojonegoro- NI' MATUZAHROH, lndana

ZULFA, Nur Hidayatul ALAMI, Tri NURHARIYATI

Grouping of epiphytes meiofauna on seagrass using canonical correspondence 133 analysis - lndah TRISNAWATI, M . MURYONO, M . Sjahid AKBAR, KURNIAWAN

Optimization of DNA extraction from seeds and fresh leaf tissues 141 of Soybean (Glycine max)- Oeke YUNITA, Benny SETIAWAN and EVELYN

Potential Arecaceae family for bioenergy, food, medicine 147

and environmental services in Purwodadi Botanic Garden- Rony IRAWANTO

Bacteria biodiversity in cow dung, capsule husk and seed cake 155 of Jatropha curcas Linn. anaerobic digesters - Roy HENDROKO, Ahmad WAHYUDI, Elizabeth

C. SITU MORANG, Nurita TORUAN and Satriyo K. WAHONO

Influence of incubation temperature and pH on activity of ligninolytic enzymes 161 in sugarcane baggase by Gliomastix sp. T3.7 - lma MufidatuiiLMI, Nengah Dwianita

KUSWYT ASA RI

The hypnotic effect of benzoylthioutea derivative {2.4 dichlorobenzoylthiourea) 167 in mice - Di ni KESUMA

The motor coordination disorder effect of 2-chlorobenzoylthiourea in mice - Aguslina 175 KIRTISH ANTI

Community structure of benthic fauna in rocky shores of Balekambang - lwenda Bella 183 SUBAGIO, Di mas Ca hyo HARTRANTO and Erna ROFIDAH

The utilizat ion of potential Durian peel waste namely Biobriquettes as energy 189 resource - Wahidin NURIANA, Nurfa ANISA and MART ANA

Measured and modeled plant growth and productivity in relation to SRI (System of 191 Rice Intensification) method on local paddy cultivar under salinity stress - Mukhammad

MURYONO and lndah TRISNAWATI

Diversity and species distribution of mangrove in East Java - Fa ri d K. MUZAKI, Dian SAPTARIN I, Syaikhul MAHM UD, Muham mad ROMADH ON I, Lind a Novita SARI and Sofyan ARIS

Functional biodiversity of Arthropods fa una following seasonal variation in the

agricultural landscape - lndah TRI SNAWATI and Mukhammad MURYONO

Study of Potential Phytoplankton as a Cause of HABs (Harmful Algal Blooms) in

Northern Coast of East Java- Dian SAPTARIN I, Fit ri Wu landari EFFENDI, Wenny D. D. RA HMADIANI and Citra Fitri e RIANY

193

195

197

Toxicity test and chemical screening of Terminalia catappa leaf extract as a 199

biofungicide to control Phytopthora capsid growth on red pepper (Capsicum frutescens longa.) - Dini ERMAVITALIN I, Kristanti lndah PU RWANI, Tuti k NURHIDAYATI, Sri

URHATI KA

Optimisation of biosurfactant oroduction from Acinetobacter sp. P2(1) using glucose 201

substrate- FATIMAH, SUHARJONO, NI' MATUZAHROH, Tri ARDYATI, Afaf BAKTIR, Ahmad HONTOWI

Characterize lypolitic and proteolytic bacteria from domestic sewage - Nita CITRASARI, 203 I' MATUZAHROH and Tri NURHARIYANTI

Isolation of HS Avia n Influenza viruses from domestic poultry in Surabaya, East Java, 205

Indonesia, in 2010 - 2011 - Luh Ade Wilan KRISNA, Masaoki YAMAOKA, Aldise Mareta ASTRI, Emmanuel Djoko POETRANTO, Laksmi WULANDARI, Resti YUDHAWATI, Landia

SETIAWATI, Retno Asih SETYONINGRUM, Teridah Ermala GINTING, Akiko MAKINO, Kyoko _HI NYA and Yoshihiro KAWAOKA

Effectiveness of Moringa o/eifera seeds to reduce levels of Pb and Cr in Surabaya River

- AU NU ROHIM, Nengah Dwianita KUSWYTASARI and MUHARTO

Opisthobranchia of East Java- Fa rid Kama l MUZAK!, Dian SAPTARINI and Swiss WINNASIS

e composition of insects in farming land of ITS Campus- Surabaya- NURYATI, lndah ---:> JSNAWATI, Kristanti lndah PURWANI

Bird diversity and status in Lamong Bay Estuary, Surabaya- Ahmad YANUAR, Febri Eka

=~DANA

~pli cation indegenus Vessicula Arbuscular Mycorrhiza (VAM) from Socah village,

Bangkalan-Madura and Rhizobium of Peanut (Arachis hypogea)- Tutik NURHIDAYATI ::-o Triano Bagus SAPUTRO

207

209

211

213

. 215

;J eli minary study of Sidoarjo mud effluent effect to the water quality and fish survival 217

-;3t e - Dewi HIDAYATI, Norela SULAIMAN, M.Shuhaimi-OTHMAN and Ismail B.S

Estim ation of carbon stock on Mangrove in Pamurbaya - Nur jannatul FAIDAH, 219 bi hatuz ZAHROH, Sofyan ARIS, Putri LILIAN DAR!, Vita KARLINDA

Optimization of DNA extraction from seeds and fresh leaf tissues of Soybean (Glycine max)

Abstract

Oeke YUNITA, Benny SETIAWAN and EVELYN

Faculty of Pharmacy

University of Surabaya

Email: oeke .ynita@gmail.com

The effects of various components on extraction buffer such as SDS-NaCI, PVP, ~-mercaptoetha nol ,

extraction with Phenoi:Chloroform :lsoamyl acetate, and incubation time on the DNA extraction from

seeds and fresh leaves of Soybean (Glycine max) were stud ied. Based on results above, an optimized

method for DNA extraction from Soybean seeds and leaves were established. Extracting Soybean

seeds twice with Phenoi :Chloroform:lsoamyl acetate (25:24:1), incubating for 30 min and 1% SDS-2

M NaCI in extraction solution could promote the quantity and purity of DNA from seeds, respect ive ly.

The results also showed that high quality DNA from Soybean leaves could be extracted with

Phenoi:Chloroform:lsoamyl acetate (25:24:1) twice, incubating for 30 min and 0.5% SDS-2% PVP in

extraction solution, although the DNA quantity was less than DNA quantity obtained with

Nucleospin· Plant II method.

Keywords :Glycine max, DNA extraction, optimization

INTRODUCTION

Soybean (Glycine max) is an important commodity in Indonesian Protective Commitment in

International Trading Conference (WT0)(1) which is used in food and herbal supplement .(2)

Despite its important effect as herbal supplement, there were many reports that revealed its side

effects on skin, gastrointestinal, and respiratory reactions and in some cases anaphylaxisYl Before

further evaluation about its side effect, correct identification of the raw material must be performed

to ensure its safety. One of the most reliable methods for identification of herbal medicine materials

is by analyzing DNA. DNA markers are reliable for informative polymorphisms as the genetic

composition is unique for each species and is not affected by age, physiological conditions as well as

environmental factors. (4)

The application of genetic identification in some plant species has however been constrained by lack

of efficient DNA extraction techniques, because of the presence of polyphenols and metabolites that

interfere with further application of DNA, such as DNA fingerprinting. (S) Therefore this study had

performed optimization of DNA extraction from seeds and fresh leaves of soybean.

MATERIAL AND METHODS

Plant material

Soybean seeds were collected from local market at Surabaya on 2011. After being cultivated several

days in the soil, the leaves from the seeds were collected, washed free of dirt, mopped dry and

quickly stored at -80 2C until used.

Solutions

The extraction buffer consisted of 200 mM Tris-HCI pH 8.0, 250 mM NaCI, 25 mM EDTA, and 0.5 _

SDS was prepared. The following solutions were also prepared and stored at 4•c

Phenoi:Chloroform: lsoamylacetate (25:24:1), 70% Ethanol.

DNA extraction

Genom ic DNA extraction, modified from ICI Seeds Co.1996(6l. Seeds (Fresh leaves) 100.0 mg we re

ground and 400~-tl of extraction buffer (200 mM Tris-HCI pH 8.0, 250 mM NaCI, 25 mM EDTA, anc

0.5% SDS) were added . The mixture was added w ith 400~-tl phenol with occasional inversion and ther

centrifuged 12,000rpm 4•c for 10 min. Three hundreds micro littre of upper phase was mixed wi--

250~-tl isopropanol and incubated at room temperature for 10 min. After centrifugation 12,000 g c: :

42C for 10m in, the DNA pellet was washed with 600~-tl of 70% cold ethanol, centrifuged 12,000rpr

4•c for 10 min and DNA pe llet was dissolved in 300~-tl sterile water (for leaves) or 100~-tl (for see s

and kept at -20 2( until used.

Optimization of DNA extraction from Soybean seeds and leaves

Based on extraction procedure above, following modifications on DNA extraction method we~

done respectively: (1) with 2% PVP for leaves; (2) DNA extraction with phenol, Phe nc

Ch loroform:lsoamyl acetate (25 :24:1) once and twice; (3) set a different incubation time (0 min, 3: min, and 60 min); and (4) adjust the concentration of SDS in extraction solution to 0.5%, 1.0% a :

2.0%, respectively; (5) adjust the composition of SDS and NaCI (0.5% SDS- 250mM NaCI; 1% SDS-2'

NaCI) for seeds; (6) adjust the concentration of 6-mercaptoethano/ (1.0%, 5.0%, and 10.0%) fc·

leaves.

Detection of quality and quantity of DNA from the seeds and leaves of Soybean

DNA purity (A260/A280 ratio) and its concentration were measu red with NanoDrc:

spectrophotometer.

RESULTS and DISCUSSION

This study had used seeds and leaves of soybean fo r extracting the DNA. Soybean seeds were useo -

this study because seeds frequently being sold at the local market and they were usually consulT'<::

or developed into the another nutritive form, such as tofu, milk. The seeds were also quite stab le -

long term storage. The leaves were chosen for DNA extraction due to their continued availa bil :­

whole year round.

Figure 1. shows DNA yield from seeds and leaves which were incubated on different incubation t irr::

Increasing the incubation time of leaves and seeds in extraction buffer until 60 minute could increc:E::

the DNA yield until 430.47 ng/~-tl from seeds and 937.10 ng/~-tl from leaves, but the DNA qua lit :=:

decreased if compared with the DNA quality from seeds and leaves wh ich incubated 30 minute -

extraction buffer. Puchooa and Venkatasamy had also used incubation time 35 minute to extract p · ::

DNA from Trochetia boutoniana.(7)

142- IBOC 2012

<l: z 0 ·v; :::::: ro :::1. ..... ...._ ..... tlO c c aJ-"' c 0

:::.:::

• ~tEDS

Figure 1. Effect of different incubation time on DNA yield from seeds and

fresh leaves of soybean

Prob lems encountered in the isolat ion and purification of high molecular weight DNA from certain

plant species include co-iso lation of highly viscous polysaccharides and inh ibitor compounds like

polypheno ls and other secondary metabolites which directly or indirectly interfere with subsequent

application process, as reported by several resea rchers .!8•9

'

Figure 2. shows DNA yield from soybean seeds and leaves wh ich were extracted on extract ion buffer

contain ing Sodium Dodecyl Sulphate (SDS) at several concentrations . Extraction buffer conta ining

0.5% SDS could increase the DNA yield until 248 . 97ng/~l from seeds and 2% SDS increased t he DNA

yield until 451.194ng/~l from leaves. SDS extraction was usually used to increase the efficiency of

removing proteins from the ext racted DNA and several researchers had used 0.5%-2% SDS to extract

genomic DNA.!10'11

' Because of the lower purity of DNA extracted with >1%SDS from leaves, this study

only used the 0.5% SDS in extract ion buffer.

<( z 0 ·v; =­ro :::1. ..... ...._ ..... tlO c c Q)-

"' c 0

:::.:::

• seeds

leaves

SDS concentration

Figure 2. Effect of different incubation time on DNA yield from seeds and

fresh leaves of soybean

I

} ~I i

I !

The major differences in extraction method for seeds or leaves on table 1. mainly concern t ::

ingredients of the extraction buffer. Each plant organ (seeds or leaves) may require its releva :

protocol depending on the demand of the level of DNA purity. Reducing agents such as

mercaptoethanol (0.5-3%) was also usually included in inhibiting oxidation process, which eithe·

directly or indirectly caused damage to DNA. PVP (2.5-3%) could also improve the colour of the D :..

obtained. Addition of high concentration of NaCI (2.5-3 M) increased the solubility of polysaccharid e~

in ethanol, effectively decreasing co-precipitation of the polysaccharides and DNA.17'8'12

l

Table 1. The Yield of Soybean DNA from Seeds and Leaves after Optimization of DNA Extractior

with Different Parameters

Soybean Organs Methods and Parameters DNA concentration (ng/J.I.I)

Seeds Modified Incubation 30 min 289,07 method time from ICI SDS 0.5% 248.97 Seeds co .16l concentration

SDS-NaCI ratio SDS 1%; NaCI 227.94 2M

Steps amount Phenoi:Chloro 237.29 on extraction form:

lsoa mylacetat e (25 :24:1)-two steps

Nucleospin Plant II method 244.80 Leaves Modified Incubation 30 menit 770.87

method time from ICI SDS 0.5% 351.36 Seeds concentration co. (G) PVP 2% 184.06

Steps amount Phenoi:Chloro 609.00 on extraction form :

lsoa mylacetat e (25 :24:1)-two steps

6- 1% 770.87 mercaptoetha

no/

Nucleospin Plant II method 328.29

Note:

A260/A280 ratio in all experiment were >1.8, which indicated that DNA was quite pure and amenable

for another application

The yield of soybean DNA ranged from 227.94 to 289,07 ng per 100.0 mg of seeds and from 184.0E

to 770.87 ng per 100.0 mg of leaves. Some experiments showed that DNA concentrations frorr

soybean seeds and leaves with the modified ICI Seeds co. method we re higher than DN;.

concentrations wh ich were obtained with Nuc/eospin • Plant II method, but DNA concentration frorr

the seeds were lower than DNA concentration from the leaves, it might be caused the abunda nce

144- IBOC2.0.12

protein and polysaccharides in the seeds which interfered the extraction process. A260/A280 ratio

more than 1.9 of DNA extracted from the seeds could indicate polysaccharide contam ination, but the

NA from the seeds should be amenable for subsequent applications such as amplification by

Polymerase Chain Reaction (PCR) method because Abd-Eisalam had reported that DNA ranged from

SO to 100 ng per 100 mg of starting material, enough to conduct PCR.'11l

CONCLUSION

Optimal condition for DNA extraction from Soybean seeds were 1%SDS-2M NaCI in t he extracti on

buffer which incubated 30 minute and extraction by two steps of Phenol: Chloroform : Isoamyl

acetate. Optimal condition for Soybean leaves were 0.5%SDS, 2%PVP, 1%.

REFERENCES

1. Arifin B. New Challenge on Food Economy. Economic Review 2009, No. 216: 1-8

2. BalkE et al. Effects of Soy on Health Outcomes. AHRQ Publication 2005, 05-E024-2

3. Sicherer SH and Sampson HA. Food Allergy. J. Allergy Clin lmmunol 2006, 117 (2supp

primer):S470-S475

4. Yip et al. DNA methods for Identification of Chinese Medicinal Materials. Chinese Medicine

2:9.

5. Hanania U et al. An Improved Method for Isolating High-Quality DNA from Vitis vin ifero c'e·

Plant Molecular Biology Reporter 2004, 22: 173-177

6. Pardal SJ . Transformasi Kedelai dengan Gen Proteinase Inhibitor II melalui Agrobacterium oc

Penembakan Partikel. Bogar: Sekolah Pascasarjana lnstitut Pertanian Bogar, 2004

7. Puchooa D. and Venkatasamy K. A Protocol for the Isolation of DNA from Trochetia boutonior:J

International Journal of Agriculture & Biology, 2005, 07(1):82-85.

8. Chaudhary et al. Protocol for Isolation of Genomic DNA from Dry and Fresh Leaves of ig,...:;

species Suitable for RAPD and Restriction Digestion, Advances in Biological Research 2008, 2(5-6

83-89.

9. Chen Q et al. Optimization of DNA Extraction from Seeds of Sorghum sudanense (Piper) Sta p .

Not. Bot. Hart. Agroboc Cluj, 2009, 37(1): 256-260.

10. Abdulova Get al. Isolation and Purification of Nuclear DNA from Excised Cotyledons of Cucuro·:::

pepo L. (zucchini), Bulg. J. Plant Physiol., 2002, 28(3 -4}, 3-11.

11. Abd-Eisalam KA et al. Isolation of High-Quality DNA from Cotton and Its Fungal Pathogens,

Journal of Plant Diseases and Protection, 2007, 114(3):113-116.

12. Ranjan S. et al. Standardization of Extraction of Genomic DNA and PCR-RFLP conditions of Air ... ~

stracheyi: A high Altitude Plant, Academia Arena, 2010, 2(7):11-14.