Conference collection

4th International Conference on Mathematics and Natural Sciences (ICMNS 2012) Science for Health, Food and Sustainable Energy Bandung, Indonesia 8–9 November 2012

Editors Mitra Djamal Kiki Vierdayanti Intan Muchtadi Alamsyah Rino R. Mukti Widyaningrum Indrasari Muhammad Islahuddin Institut Teknologi Bandung, Bandung, Indonesia

All papers have been peer reviewed.

Sponsoring Organizations

Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung School of Life Sciences and Technology, Institut Teknologi Bandung School of Pharmacy, Institut Teknologi Bandung Calipha Land

Melville, New York, 2014 AIP Proceedings To learn more about AIP Proceedings visit http://proceedings.aip.org

Volume 1589

Editors Mitra Djamal

Rino R. Mukti

Mitra Djamal Institut Teknologi Bandung Department of Physics Jl. Ganesha 10 Bandung 40132 Indonesia

Institut Teknologi Bandung Department of Chemistry Jl. Ganesha 10 Bandung 40132 Indonesia

E-mail: [email protected]

Kiki Vierdayanti Institut Teknologi Bandung Department of Astronomy Jl. Ganesha 10 Bandung 40132 Indonesia

E-mail: [email protected]

Widyaningrum Indrasari Institut Teknologi Bandung Department of Physics Jl. Ganesha 10 Bandung 40132 Indonesia

E-mail: [email protected]

E-mail: [email protected]

Intan Muchtadi Alamsyah

Muhammad Islahuddin

Institut Teknologi Bandung Department of Mathematics Jl. Ganesha 10 Bandung 40132 Indonesia

Institut Teknologi Bandung Department of Mathematics Jl. Ganesha 10 Bandung 40132 Indonesia

E-mail: [email protected]

E-mail: [email protected]

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AIP Conference Proceedings, Volume 1589 4th International Conference on Mathematics and Natural Sciences (ICMNS 2012) Science for Health, Food and Sustainable Energy Table of Contents

Preface: 4th International Conference on Mathematics and Natural Sciences (ICMNS 2012) Mitra Djamal

1

Committees

2

EARTH AND SPACE SCIENCE On the effects of solar storms to the decaying orbital space debris Dhani Herdiwijaya and Abdul Rachman

3

Oscillation signature from multi-wavelength analysis on solar chromosphere Emanuel Sungging Mumpuni, Dhani Herdiwijaya, and Mitra Djamal

7

Deep coronal hole associated with quiescent filament Rasdewita Kesumaningrum and Dhani Herdiwidjaya

10

Orbit of the short-lived Sun-grazing comet C/1999 X3 B. Dermawan, H. R. T. Wulandari, P. Mahasena, R. W. Wibowo, Sulistiyowati, and C. S. Guritno

14

Behavior of long term interval of state a in GRS 1915+105 Febrie Ahmad Azizi, Kiki Vierdayanti, Putra Mahasena, and Chatief Kunjaya

18

Identifying solar wind structures related to Garuda 1 satellite anomaly by analyzing solar wind and IMF parameters Abdul Rachman and Dhani Herdiwijaya

22

The Bosscha Observatory's BIMA project Janette Suherli, Hakim L. Malasan, Mahasena Putra, Lidia Mayangsari, Dhimaz Gilang Ramadhan, and Gabriela Kezia Haans

26

Effect of initial inclination to the stability of triple star systems Y. Ghea, P. Mahasena, B. Dermawan, R. W. Wibowo, Sulistiyowati, C. S. Guritno, and H. R. T. Wulandari

29

Effects of dark matter on interior and evolution of main sequence stars Sulistiyowati, H. Wulandari, B. Dermawan, M. Ikbal Arifyanto, and I. Ibrahim

33

On the period determination of ASAS eclipsing binaries L. Mayangsari, R. Priyatikanto, and M. Putra

37

Analysis of blackdrop effect in the transit of Venus on June 6, 2012 A. Gunawan Admiranto, E. Sungging Mumpuni, Johan Muhamad, and Nana Suryana

42

Open cluster evolutions in binary system: How they dissolved R. Priyatikanto, M. I. Arifyanto, and H. R. T. Wulandari

45

Speckle observation of visual double stars at Bosscha observatory Muhammad Yusuf, Denny Mandey, I Putu Wira Hadiputrawan, Mahasena Putra, and Mochamad Irfan

49

Lunar occultation observation of μ Sgr: A progress report A. T. P. Jatmiko, G. P. Puannandra, R. D. Hapsari, R. A. Putri, Z. M. Arifin, G. K. Haans, and I. P. W. Hadiputrawan

53

Search for bright stars with infrared excess Moedji Raharto

57

The Hubble constant estimation using 18 gravitational lensing time delays Anton T. Jaelani and Premana W. Premadi

61

Volatiles in comets as probes to the early solar system Hitomi Kobayashi

65

PHYSICAL SCIENCES Pore shape characterization of Fontainebleau sandstone and its models using Fourier descriptor Fourier Dzar Eljabbar Latief and Umar Fauzi

72

Disaster risk management in prospect mining area Blitar district, East Java, using microtremor analysis and ANP (analytical network processing) approach Diyan Parwatiningtyas, Erlin Windia Ambarsari, Dwi Marlina, and Yogi Wiratomo

76

On the near-brane will-be-SimplestLittleHiggs scalar in Scherk-Schwarz breaking of 5-dimensional SU(6) GUT as a source of exotic multi-scalar Higgs A. Hartanto, F. P. Zen, and J. S. Kosasih

81

Fixed conditions for achieving the real-valued partition function of one-dimensional Gross-Pitaevskii equation coupled with time-dependent potential T. B. Prayitno

87

Model of a tunneling current in a p-n junction based on armchair graphene nanoribbons - an Airy function approach and a transfer matrix method Endi Suhendi, Rifki Syariati, Fatimah A. Noor, Neny Kurniasih, and Khairurrijal

91

Simulation of fluid flow in a U-shape self-siphon and its working space Nurhayati, W. Hidayat, Novitrian, S. Viridi, and F. P. Zen

95

Propagators and vertices of scalar-gravity interaction in the teleparallel theory of gravitation Kian Ming and Triyanta

99

A simple and inexpensive vertical deformation measurement system for soil compression tests Riska Ekawita, Hasbullah Nawir, Suprijadi, and Khairurrijal

104

Application analysis of Monte Carlo to estimate the capacity of geothermal resources in Lawu Mount Supriyadi, Wahyu Srigutomo, and Arif Munandar

108

Digital characterization and preliminary computer modeling of hydrocarbon bearing sandstone Fourier Dzar Eljabbar Latief and Tedy Muslim Haq

112

Characterization of an atrazine molecularly imprinted polymer prepared by a cooling method Idha Royani, Widayani, Mikrajuddin Abdullah, and Khairurrijal

116

Three-dimensional visualization and characterization of cracks in geothermal reservoir rock using image analysis of reconstructed μCT images: A preliminary study Fourier Dzar Eljabbar Latief and Selly Feranie

120

Development of a low cost potentiostat using ATXMEGA32 Abdul Muid, Mitra Djamal, and Rahadi Wirawan

124

Characterization of tsunamigenic earthquake in Java region based on seismic wave calculation Sugeng Pribadi, Afnimar, Nanang T. Puspito, and Gunawan Ibrahim

129

Geoelectrical dimensionality analyses in Sumatran Fault (Aceh segment) using magnetotelluric phase tensor Rudy Prihantoro, Nurhasan, Doddy Sutarno, Yasuo Ogawa, Has Priahadena, and Dini Fitriani

133

Multiple-state quantum Otto engine, 1D box system E. Latifah and A. Purwanto

137

COMPUTER SCIENCES AND COMPUTATIONAL Treatment model of dengue hemorrhagic fever infection in human body D. Handayani, N. Nuraini, N. Primasari, and K. P. Wijaya

141

Implementation of measure of relative tendency to phase image filtering spacial based Nurjannah Syakrani, Edy T. Baskoro, Tati L. R. Mengko, and A. B. Suksmono

145

The SWASH model for soliton splitting due to decreasing depth N. Subasita, H. Latief, and S. R. Pudjaprasetya

150

MATERIAL SCIENCES Solid polymer electrolyte from phosphorylated chitosan Iqbal Fauzi and I Made Arcana

154

Potentiometric measurement of polymer-membrane electrodes based on lanthanum Asep Saefurohman, Buchari, Indra Noviandri, and Syoni

159

Study of benzotriazole as corrosion inhibitors of carbon steel in chloride solution containing hydrogen sulfide using electrochemical impedance spectroscopy (EIS) Agus Solehudin and Isdiriayani Nurdin

164

The synthesis and characterization of 7-hydroxy-4-methylcoumarin and the investigation of the fluorescence properties of its 7-hydroxy-4-methylcoumarin-chitosan films Deana Wahyuningrum, Muhammad Zulqarnaen, and Veinardi Suendo

169

Effective atomic numbers of blue topaz at different gamma-rays energies obtained from Compton scattering technique S. Tuschareon, P. Limkitjaroenporn, and J. Kaewkhao

174

Ionic conductivity of Bi2NixV1-xO5.5−3x/2 (0.1 ≤ x ≤ 0.2) oxides prepared by a low temperature sol-gel route Rolan Rusli, Isaac Abrahams, Aep Patah, Bambang Prijamboedi, and Ismunandar

178

Isothermal oxidation behavior of ternary Zr-Nb-Y alloys at high temperature Djoko Hadi Prajitno, Syoni Soepriyanto, Eddy Agus Basuki, and Slameto Wiryolukito

182

High temperature oxidation behavior of austenitic stainless steel AISI 304 in steam of nanofluids contain nanoparticle ZrO2 Djoko Hadi Prajitno and Dani Gustaman Syarif

187

HF treatment effect for carbon deposition on silicon (111) by DC sputtering technique A. S. Aji and Y. Darma

191

Mechanical properties of paper sheets coated with chitosan nanoparticle Nurul Hidayati Fithriyah and Erdawati

195

Micromagnetic simulation dynamics of susceptibility spectrum in diamond-shaped ferromagnetics Ismail, D. Djuhana, and D.-H. Kim

200

Fixation of chiral smectic liquid crystal (S)-(+)-4-(2-methyl-1-butyloyloxy)phenyl 4-[1-(propenoyloxy) butiloxy] benzoate using UV curing techniques Afrizal, Muhammad Hikam, Bambang Soegiyono, Asep Riswoko, Nurdelima, and Umeir

203

Composition, morphology and mechanical properties of sputtered TiAlN coating Esmar Budi, M. Mohd. Razali, and A. R. Md. Nizam

207

Effect of roll hot press temperature on crystallite size of PVDF film Ambran Hartono, Mitra Djamal, Suparno Satira, Herman Bahar, Ramli, and Edi Sanjaya

217

Simulation of quantum dots size and spacing effect for intermediate band solar cell application based on InAs quantum dots arrangement in GaAs I. B. Hendra P., F. Rahayu, and Y. Darma

221

Comparative study of radiation emission without and with target in a 2.2 kJ plasma focus device Muhammad Zubair Khan, Yap Seong Ling, and Wong Chiow San

225

Incorporation of network in synthesis of zircon-imprinted polymer and its effect on zircon ion extraction S. Aladin, M. B. Amran, B. Buchari, and I. M. Arcana

230

Cloning and construction of recombinant palI gene from Klebsiella oxytoca on pET-32b into E. coli BL21 (DE3) pLysS for production of isomaltulose, a new generation of sugar Maelita R. Moeis, Liska Berlian, Sony Suhandono, Alex Prima, Eli Komalawati, and Tati Kristianti

235

Hydrogen atom excitation in intense attosecond laser field: Gauge dependence of dipole approximation Ch. Aldarmaa, L. Khenmedekh, and O. Lkhagva

239

Surface hardening of St41 low carbon steel by using the hot-pressing powder-pack boriding method Sutrisno and Bambang Soegijono

241

Preparation of ruby red glasses from gold nanoparticles: Influence of stannic oxide Y. Ruangtaweep, P. A-nupan, and J. Kaewkhao

245

Structural investigation and microwave characteristics of (Ba0.2La0.8)Fe0.2Mn0.4Ti0.4O3 absorbing materials Azwar Manaf and Wisnu Ari Adi

249

The effect of spin-orbit coupling in band structure of few-layer graphene Muhammad Fauzi Sahdan and Yudi Darma

253

Synthesis of silver nanoparticles by chemical reduction at various fraction of MSA and their structure characterization Markus Diantoro, Rina Fitrianingsih, Nandang Mufti, and Abdulloh Fuad

257

Dynamic micromagnetic simulation of domain wall around the Walker breakdown in ferromagnetic nanowires driven magnetic pulse D. Djuhana, A. Manaf, A. Sumarta, Mardona, and D.-H. Kim

262

Biodegradation test of SPS-LS blends as polymer electrolyte membrane fuel cells Zufira Putri and I Made Arcana

266

Synthesis of thin film containing 4-amino-1,2,4-triazole iron(II) complexes Djulia Onggo

272

BIOSCIENCES AND BIOTECHNOLOGY Study of continuous-wave domain fluorescence diffuse optical tomography for quality control on agricultural produce Vebi Nadhira, Deddy Kurniadi, E. Juliastuti, and Adeline Sutiswan

276

Positive control study for the intact immature Swiss-Webster mouse uterotrophic assay Arlisa Alisjahbana and Ayda T. Yusuf

281

Environment effect on fruit ripening related gene to develop a new post harvest technology Fenny Dwivany, Rizkita Rahmi Esyanti, Jekson Robertlee, Indra Chandra Paramaputra, Rinda Kania Permatadewi, Dina Hermawaty Tambun, Resnanti Utami Handayani, Aksarani 'Sa Pratiwi, and Herafi Zaskia

285

Anti-oxidant properties and polyphenolic profile screening of Vitis vinifera stems and leaves crude extracts grown in Perlis, Malaysia Nursyahda Zakaria, Razauden Mohamed Zulkifli, Fazrena Nadia Md Akhir, and Norazah Basar

288

Silkworm (Bombyx mori) hemolymph unable to substitute fetal bovine serum in insect cell culture Irma H. Suparto, Chandra Nur Khalam, Willy Praira, and Dondin Sajuthi

292

Application of Markov chain to the pattern of mitochondrial deoxyribonucleic acid mutations Sandy Vantika and Udjianna S. Pasaribu

296

Does zero-water discharged technology enhance culture performance of pacific white shrimp (Litopenaeus vannamei Boone.)? Gede Suantika, Jayanty Anggraeni, Fahri Azhari Hasby, and Ni Putu Indah Yanuwiarti

300

In planta transformation method for T-DNA transfer in orchids Endang Semiarti, Aziz Purwantoro, Ixora S. Mercuriani, Anida M. Anggriasari, Seonghoe Jang, Sony Suhandono, Yasunori Machida, and Chiyoko Machida

303

The effect of leaf biopesticide (Mirabilis jalapa) and entomopathogenic fungi (Beauveria bassiana) combinations to some physiological characters and histology of Crocidolomia pavonana (Lepidoptera: Pyralidae) larvae Nur Tasmiah Sirajuddin and Tjandra Anggraeni

308

The effect of leaf biopesticide Mirabilis jalapa and fungi Metarhizium anisopliae to immune response and mortality of Spodoptera exigua instar IV A. Irma Suryani and Tjandra Anggraeni

312

The effect of the combination of two biological control agents, Mirabilis jalapa and Bacillus thuringiensis, to Spodoptera litura's immune response and their mortality Dina Maulina and Tjandra Anggraeni

316

The maturity of Nypa palm worm Namalycastis rhodochorde (Nereididae: Polychaeta) Junardi, Tjandra Anggraeni, Ahmad Ridwan, and Edy Yuwono

320

ENVIRONMENTAL SCIENCES Interaction of Sr-90 with site candidate soil for demonstration disposal facility at Serpong Budi Setiawan, Oktri Mila, and Safni

325

Accumulation of Pb and Cu heavy metals in sea water, sediment, and leaf and root tissue of Enhalus sp. in the seagrass bed of Banten Bay Faiza Fauziah and Devi N. Choesin

329

Effectiveness of a model constructed wetland system containing Cyperus papyrus in degrading diesel oil Danni Gathot Harbowo and Devi Nandita Choesin

334

Radioecologycal study of 239/240Pu in Bangka Island and Muria Peninsula: Determination of 239/240Pu in marine sediment and seawater as part of baseline data collecting for sitting of candidates of first Indonesia NPP Heny Suseno and Djarot S. Wisnubroto

338

Bioaccumulation factor of 137Cs in some marine biotas from West Bangka Indonesia Heny Suseno

342

137

Cs sorption into bentonite from Cidadap-Tasikmalaya as buffer material for disposal demonstration plant facility at Serpong B. Setiawan, H. Sriwahyuni, NE. Ekaningrum, and T. Sumantry

346

Isolation, identification and characterization of indigenous fungi for bioremediation of hexavalent chromium, nickel and cobalt Nelis Hernahadini, Sony Suhandono, Devi N. Choesin, Siti K. Chaerun, and Ade Kadarusman

350

Healthy latrine development model to achieve MDGs target Eddy S. Soedjono and Nurvita Arumsari

355

Geographically weighted lasso (GWL) study for modeling the diarrheic to achieve open defecation free (ODF) target Nurvita Arumsari, Brodjol Sutidjo S.U., and Eddy S. Soedjono

361

Numerical simulation of dissolved oxygen in Jakarta Bay Susanna Nurdjaman, Ivonne M. Radjawane, and Sripardi Jamelina

369

The relation between soil sulfate concentration and proanthocyanidin content of Selliguea feei Bory from around Ratu crater, Mount Tangkuban Perahu Vivi Novianti and Devi N. Choesin

373

Environment management conflict in Mount Tangkuban Perahu Nature Reserve and Nature Park, North Bandung, West Java Demak Ely Riana Damanik and Achmad Sjarmidi

378

Analysis of aerosol, cloud and precipitation interaction behavior using predator-prey model Rita Sulistyowati, L. Muhammad Musafar K., Wahyu Srigutomo, and Rizal Kurniadi

381

The impact of insecticides to local honey bee colony Apis cerana indica in laboratory condition Ramadhani E. Putra, Agus D. Permana, and Syayidah Nuriyah

385

Estimation of primary productivity in Banda Sea using the vertical distribution model Putri Kemili and Mutiara R. Putri

389

Synthesis and characterization of [BMIM]bromide using microwave-assisted organic synthesis method and its application for dissolution of palm empty fruit bunch Lucy Arianie, Deana Wahyuningrum, Zeily Nurrachman, and Dessy Natalia

394

FOOD SCIENCES Bioactive compounds and antioxidant activity analysis of Malaysian pineapple cultivars Chong Hang Chiet, Razauden Mohamed Zulkifli, Topik Hidayat, and Harisun Yaakob

398

Molecularly imprinted polymers-curcuminoids and its application for solid phase extraction Meyliana Wulandari, M. B. Amran, A. B. Descalzo Lopez, J. L. Urraca, and M. C. Moreno-Bondi

400

HEALTH AND MEDICAL SCIENCES Modeling of blood vessel constriction in 2-D case using molecular dynamics method M. Rendi A. S., Suprijadi, and S. Viridi

404

Molecular cloning and cold shock induced overexpression of the DNA encoding PhoR sensor domain from Mycobacterium tuberculosis as a target molecule for novel anti-tubercular drugs Gladys Emmanuella Putri Langi, Maelita R. Moeis, Ihsanawati, and Ernawati Arifin Giri-Rachman

407

A flavone derivative from Sesbania sesban leaves and its cytotoxicity against murine leukemia P-388 cells Hanhan Dianhar, Yana Maolana Syah, Didin Mujahidin, Euis Holisotan Hakim, and Lia Dewi Juliawaty

411

Color canals modification with canny edge detection and morphological reconstruction for cell nucleus segmentation and area measurement in normal Pap smear images Dwiza Riana, Dyah Ekashanti Octorina Dewi, Dwi H. Widyantoro, and Tati Latifah R. Mengko

414

Three-dimensional visualization and characterization of bone structure using reconstructed in-vitro μCT images: A pilot study for bone microarchitecture analysis Fourier Dzar Eljabbar Latief, Dyah Ekashanti Octorina Dewi, and Mohd Aliff Bin Mohd Shari

418

Simulation of hydrocephalus condition in infant head Erna Wijayanti and Idam Arif

422

PHARMACEUTICAL SCIENCES Determination of stability constants of aminoglycoside antibiotics with their metal complexes Vanny M.A. Tiwow

426

Isolation, identification, and antibacterial activity of chemical compounds from ethanolic extract of suji leaf (Pleomele angusifolia NE Brown) Faridah, Natalia, Maria Lina, and Hendig W

431

MATHEMATICS Reserve estimation Sutawanir, Agus Yodi Gunawan, Asep K. Permadi, and Nina Fitriyati

436

A survey on application of representation theory to molecular vibration Yohenry Prakasa and Intan Muchtadi-Alamsyah

440

A two-dimensional simulation of plasma leakage due to dengue infection N. Nuraini, Windarto, Swarna Jayanti, and Edy Soewono

444

Simulation for measuring the effect of flight retiming to the robustness of flight schedule K. Novianingsih, R. Hadianti, S. Uttunggadewa, and E. Soewono

448

Reflection wave on sloping beach N. Erwina and S. R. Pudjaprasetya

452

Study of behavior and determination of customer lifetime value(CLV) using Markov chain model Dony Permana, Sapto Wahyu Indratno, and Udjianna S. Pasaribu

456

Mathematical model of temephos resistance in Aedes aegypti mosquito population D. Aldila, N. Nuraini, E. Soewono, and A. K. Supriatna

460

Natural frequency of regular basins Sugih S. Tjandra and S. R. Pudjaprasetya

464

Spatial cluster for clustering the influence factor of birth and death child in Bogor Regency, West Java Rokhana Dwi Bekti and Ro'fah Rachmawati

468

On the analysis of parasite effect for Aedes aegypti and Aedes albopictus population Meta Kallista, Dipo Aldila, Nuning Nuraini, and Edy Soewono

472

Optimal control of diarrhea transmission in a flood evacuation zone N. Erwina, D. Aldila, and E. Soewono

476

Numerical modeling of 2D wave refraction and shoaling I. Magdalena and S. R. Pudjaprasetya

480

The stationarity of generalized STAR(2;λ1,λ2) process through the invers of autocovariance matrix Utriweni Mukhaiyar, Udjianna S. Pasaribu, Wono Setya Budhi, and Khreshna Syuhada

484

On the analysis of competitive displacement in dengue disease transmission Karunia P. Wijaya, Nuning Nuraini, Edy Soewono, and Dewi Handayani

488

Critical point analysis of phase envelope diagram Darmadi Soetikno, Rudy Kusdiantara, Dila Puspita, Kuntjoro A. Sidarto, Ucok W. R. Siagian, Edy Soewono, and Agus Y. Gunawan

492

Modeling mass drug treatment and resistant filaria disease transmission A. M. Fuady, N. Nuraini, E. Soewono, H. Tasman, and A. K. Supriatna

496

L(d,2,1)-labeling of sun graphs Diari Indriati, Titin S. Martini, and Novita Herlinawati

500

Stock and option portfolio using fuzzy logic approach Novriana Sumarti and Nanang Wahyudi

504

The comparison of isotropic and anisotropic semivariogram for Gauss model Rr. Kurnia Novita Sari and Udjianna S. Pasaribu

508

SUSTAINABLE ENERGY Heat loss model for flow assurance in a deep water riser Darmadi Soetikno, Isti Rodiah, Muhammad Islahuddin, Riska A. P. Kania, Agus Y. Gunawan, Pudjo Sukarno, Asep K. Permadi, and Edy Soewono

512

Analysis of factors that affect the potential of star fruit (Averhoa Bilimbi) and cactus (Gymnocalycium Hossei) extracts as alternative battery Sitti Rahmawati and Agnesstacia

516

A new principle for low-cost hydrogen sensors for fuel cell technology safety Martin Liess

522

In planta transformation method for T-DNA transfer in orchids Endang Semiarti, Aziz Purwantoro, Ixora S. Mercuriani, Anida M. Anggriasari, Seonghoe Jang, Sony Suhandono , Yasunori Machida, and Chiyoko Machida Citation: AIP Conference Proceedings 1589, 303 (2014); doi: 10.1063/1.4868806 View online: http://dx.doi.org/10.1063/1.4868806 View Table of Contents: http://scitation.aip.org/content/aip/proceeding/aipcp/1589?ver=pdfcov Published by the AIP Publishing

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In planta Transformation Method for T-DNA transfer in orchids Endang Semiarti1, 2*, Aziz Purwantoro3, Ixora S. Mercuriani2, Anida M. Anggriasari1, Seonghoe Jang4, Sony Suhandono5, Yasunori Machida6, Chiyoko Machida7 1

Faculty of Biology, Universitas Gadjah Mada,, Yogyakarta, Indonesia Study Center of Biotechnology, Universitas Gadjah Mada, Yogyakarta, Indonesia 3 Faculty of Agriculture, Universitas Gadjah Mada, Yogyakarta, Indonesia 4 Academia Sinica Biotechnology Center in Southern Taiwan (AS-BCST) of ABRC in Taipei, 5 School of Life Sciences and Technology, Institut Teknologi Bandung, Bandung, Indonesia 6 Division of Biological Sciences, Graduate School of Science, Nagoya University, Nagoya, Japan, 7 College of Biotechnology and Bioscience, Chubu University, Kasugai, Japan, 2

*E-mail: [email protected] Abstract. Transgenic plant technology is an efficient tool to study the function of gene(s) in plant. The most popular and widely used technique is Agrobacterium-mediated transformation in which cocultivation was done by immersing the plant tissues/organ in overnight bacterial cultured for about 30 minutes to one hour under in vitro condition. In this experiment, we developed more easier technique that omitted the in vitro step during cocultivation with Agrobacterium, namely in planta transformation method. Pollinaria (compact pollen mass of orchid) of Phalaenopsis amabilis and Spathoglottis plicata orchids were used as target explants that were immersed into bacterial culture for 30 minutes, then dried up the pollinaria, the transformed pollinaria was used to pollinate orchid flowers. The T-DNA used for this experiments were Ubipro::PaFT/A. tumefaciens GV3101 for P. amabilis and MeEF1α2 pro::GUS/ A. tumefaciens LBA 4404 for S.plicata. Seeds that were produced from pollinated flowers were grown onto 10 mg/l hygromicin containing NP (New Phalaenopsis) medium. The existance of transgene in putative transformant protocorm (developing orchid embryo) genome was confirmed using PCR with specific primers of either PaFT or GUS genes. Histochemical GUS assay was also performed to the putative transformants. The result showed that transformation frequencies were 2.1 % in P. amabilis, and 0,53% in S. plicata. These results indicates that in planta transformation method could be used for Agrobacterium-mediated genetic transformation, with advantage easier and more secure work from contaminants than that of the in vitro method. Key words: Genetic transformation, Agrobacterium, in planta, pollinaria, orchids PACS: 87.18.Cf

INTRODUCTION Agrobacterium-mediated genetic transformation in orchids is now widely used. Successful methods are depending on the success of Agrobacterium/Ti plasmid system integrate the T-DNA into plant cell’s genome, that has been facilitated by (1) the construction of modified Agrobacterium strains in which the genes responsible for pathogenicity have been deleted; (2) the design of intermediate vectors containing selectable markers for introducing foreign genes into the Ti plasmid and subsequently into plant cells; and (3) the development of efficient in vitro methods for transforming plant cells and tissues with engineered Agrobacterium strains [1]. The use of in vitro procedure in genetic transformation method often causes difficulties that resulted in failures of

producing transformed plants. Therefore, modification of genetic transformation without in vitro inoculation procedure is needed. Chang et al [2] has published a stable genetic transformation method of Arabidolpsis thallana by simple in plants inoculation of A. tumefaclens strain LBA4404 harboring a binary vector pBI121 severing of apical shoots at their bases, that called in planta transformation. On average, 5.5% of the newly formed shoots produced transformed progenies, that confirmed by Southern hybridization method and in situ histochemical GUS assay. This simple, efficient, and reproducible transformation system has been developed in Dendrobium orchid using some modifications (our unpublished data), but the

4th International Conference on Mathematics and Natural Sciences (ICMNS 2012) AIP Conf. Proc. 1589, 303-307 (2014); doi: 10.1063/1.4868806 © 2014 AIP Publishing LLC 978-0-7354-1221-7/$30.00

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efficiency of transformation was still very low. In our previous works [3, 4], we established a method of Agrobacterium-mediated transformation using intact protocorms as target materials, but it still could not avoid in vitro innoculation procedure. In this work, we modified the in planta transformation procedure using pollen (pollinia and pollinaria) of two orchid species as target materials, i.e a terrestrial sympodial orchid Spathoglottis plicata and epiphytic monopodial orchid Phalaenopsis amabilis. In planta transformation method was conducted by immersed the pollen in overnight cultured Agrobacterium, then the inoculated pollen were used to pollinate its flower (self pollination). The new generation from the silique were analyzed by both morphological and molecular approaches. The foreign genes are expected to be stably maintained and expressed in the resulting plants generated from the seeds and are inherited by progeny as typical Mendelian traits. The improvement technology for gene transfer in orchid plants will facilitate numerous studies on gene expression and regulation in orchids.

MATERIALS AND METHODS Plant Materials and Bacterial Strains Flowering plants of terestrial orchids Spathoglottis plicata and epyphitic orchids Phalaenopsis amabilis were used as plant materials.

FIGURE 1. Structure of the T-DNA of plasmids MeEF1α2pro::GUS::GFP for S. plicata and pGAS102 for P. amabilis Agrobacterium tumefaciens strain GV 3101 harboring the binary vector pCambia 1303 with MeEF1α2pro::GUS::GFP and 35Spro::GUS::GFP containing T-DNA (Fig. 1A) [5] were used for genetic transformation in S. plicata. A. tumefaciens strain LBA 4404 harboring pGA3426 (vector only) and pGAS102 that carry Ubi-pro::PaFT (P. amabilis Flowering Locus-T) for genetic transformation in P. amabilis (Fig. 1B).

In plantaTransformation Bacterial colonies of Agrobacterium that carry desired T-DNA were inoculated in 5 ml of liquid LB medium containing 100 mg.L-1 Kanamycin, also Agrobacterium that carry pGA3426 and pGAS102 (Ubi-pro::PaFT) were inoculated in 5 ml of liquid LB medium with addition of 5 mg.L-1 Tetracycline antibiotics, then incubated at 28oC for 2 days. One ml of this cultures were subcultured in 9 ml of new medium containing 50 mg.L-1 Acetosyringone and incubated overnight. Ten ml of bacterial culture (OD600 = 0.8-1) were centrifuged at 5000 rpm for 10 minutes. Bacterial pellets then resuspensed with NP medium. Suspension of bacterial culture was then diluted with liquid NP medium at ratio 1: 4 (v / v) and used for innoculation. For S. plicata, separated pollen that called pollinia were immersed in bacterial suspension for 30 minutes-1 hour, then it was dried up prior to be used for pollination. For P. amabilis, a pair of pollen that called pollinaria was immersed in bacterial suspension for 30 minutes up to one hour. The pollinaria was then used for self pollination. Seeds of 3 months old pod that produced from S. plicata pollination, and 4 months old pod that was produced from the P. amabilis pollination were sown on NP Medium as described in [4]. Regenerated protocorm is then cut into 2 pieces. One piece is regenerated, while another piece is used for transgene integration analysis by PCR. Protocorms are then selected on NP medium that contain 10 mg.L-1 hygromycin. Four weeks old green protocorms were counted to determine the efficiency of transformation in orchids. GUS assay and Hygromycin resistancy analysis To detect β-glucuronidase activity, the plant tissue were incubated for 6-12 h in the GUS staining buffer containing 1 mM 5-bromo-4-chloro-3-indolyl β-D-glucuronide as a substrat as described by Jefferson et al. [6]. For hygromycin resistance test, 8 WAS developing protocorms were transfered onto NP medium + 10 mg.L-1 hygromycin for selection of transformants. The survived green protocorms were counted. Regenerated protocorms were then cut into 2 pieces. One piece was continuing to be regenerated, while another piece was used for molecular analyses by PCRFor detection of GUS gene in transforman. the plant DNA(s) were amplified using GUS specific primers GUS A F (5’-CAACGAACTGAAACTGGCAGA-3’), GUS A R (5’-GGCACAGCACATCAAAGAGA-3’) and detection of GFP gene using GFP specific primers GFP B F1 (5’-CTT-TTCACTGGAGTGGTCCC-3’), and GFPB R1 (5ATTTGAACTGGTGA-CACGAG-3’).Detection of

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Ubipro::PaFT insertion in P. amabilis transfor-mant genomes was performed using primer Ubi (forward: 5’-TTG-TCGATGCTCACCCTG-3’) and TNos (reverse: 5’-GATCTAGTAACATAGATGACACCGCG-3’). Specific primers (trnL-F C (5’CGA-AATCGG-TAGAC-GCTACG-3’), and trnL-F F (5’- GCCATGTGTAATCCTAGCAG-3’) Chloroplast DNA were used as internal control of PCR reaction.

RESULTS AND DISCUSSION Analysis of Hygromycin resistancy In planta transformation in orchids is an improvement from the common Agrobacteriummediated transformation method that omit in vitro procedure when innoculation of bacterial culture for T-DNA transfer into plant genome. In planta technique is very simple, in which pollinia /pollinaria (orchid pollens) just simply deepen in Agrobacterial culture, dried out the pollen, then used it for self pollination of the orchid flower. (Figure 2). Prior to in planta transformation, it is important to check and decide the concentration of antibiotic used for selection of transformants. In this work, we used Hygromycin resistant gene as selection marker. We analyzed wild type plants of both S. plicata and P. amabilis by sowing seeds on various concentration (0-15) mg.L-1 of Hyg selection medium. Both orchids shows that 10 mg.L-1 Hyg is the best concentration for selection, based on the data of LC 50 (Table 1) First we observed that after innoculation, the pollen became swollen, then used it for pollination. Twenty four infected pollinia were successfully pollinated with its original flower, some fruits produced. The next generation (seeds) were sown on 10 mg.L-1 Hygromycin containing medium. Three of them were intensively analyzed for morphological and molecular analysis. In this experiment, we used pollen/pollinia of two orchid species as target materials. Without any in vitro procedure, the technique become simpler, cheaper, and faster. Figure 2 shows the technique, that first pollen (pollinia/pollinaria) were taken from a flower by a clean toothpick, then immerse into a suspension of Agrobacterial culture in half strength of NP liquid medium (no LB medium at all) for 30 minutes-1 hour. After taken out and dried the pollen, it was put into gynoecium/column to pollinate the ovule in the original flower (self pollination). Three months after pollination, the fruit with matture seeds inside could be gathered. Seeds were sown on to NP medium for 3 weeks, then transferred the protocorms on to 10 mg.L-1 Hygromycin selection NP medium. The number of transferred protocorms was indicated

as Total protocorm examined, the survival protocorms in this selection medium is termed as transformant candidates. The transfor-mant candidates were checked in DNA level by PCR using Hygromycin resistance gene’s primers, GUS and GFP reporter genes primers. The result shows that half of transformant candidates do not contain the TDNA. A pair of trnL-F primers of intergenic region between tRNA Leucine and tRNA Phenylalanine of cp DNA as internal control that could amplified 1.2 kb DNA fragmen. (Figure3). GUS assay reported that T-DNA has integrated into orchid genome, inwhich blue color can be detected in the midvein of orchid leaves (Figure 3). pMeEF1a2 promoter originally isolated from seeds of Manihot esculenta [5], we did a transient experiment using a young leaves of M. esculenta, and got the same result as the same as in orchids. TABLE 1. Hygromycin resistancy of S. plicata Protocorms Concentr ation of Hygromi cin (mg. L-1) 0

Numb er of proto corms

Survi ve proto corm

Death protoco rm

Percentage of death protocorm (%)

163

163

0

0

2,5

396

352

44

11,11

5

288

248

40

13,89

7,5

372

236

136

36,56

10

252

124

128

50,79*

12,5

320

132

188

58,75

15

368

140

228

61,96

*LC50 of S.plicata to Hygromycin was reached at the

Inconcentration planta transformation technique of 10 mg.L-1 Hyg

A

D

B

C

E

F

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FIGURE 2. In planta Transformation in Orchids. (A-C) S. plicata. A. Pollinia was taken out from flower, (B) Pollinia before innoculation, (C) After innoculated with Agrobacterial culture. (D-F) P. amabilis. (D) Protocorm of non transformant (NT) as control, (E) Protocorms of pGA transfered, and (F) Protocorms transferred by pUbi::PaFT.

biological relation of soil bacteria Agrobacterium and dicot plants, in which dicot plant cells produced chemostatic signal molecules acetosyringone as wounding response.In monocots, usually we added lycopene, vit C or fruit extract as triggers for wounding response to Agrobacterium [4, 7]. TABLE 2. Efficiency of In Planta Transformation in Orchids Plant

Genotype

S. NT plicata 35S::GUS::GFP pMeEF1α2::GUS::G FP

P. amabilis

B

C

Total Hygr Protocorm examined (+)

Eff. PCR Transf (+) orm. (%)

1011

186

0

0

1307

279

2

0.72

1508

379

2

0,53

0

0

NT

2091

pUbi:: HPT pUbi::PaFT

2203 2246

0 279 49

17 49

0,80 2.10

CONCLUSION

FIGURE 3. Transfoormants of S. plicata and detection of transgenes. (A) Growth of shoot from protocorms, (B) PCR products of transformant using 3 sets of primers: GUS, GFP, and trnL-F intergenic of chloroplast DNA as internal control. T-DNA were integrated into genome of half of survival transpormants. (C) GUS analysis of leaves of NT, and two transformants. The blue color is detected at the midvein in the leaves of transformants but not in NT leaf. Bar = 1 cm

Efficiency of transformation Transformants of both S. plicata and P. amabilis orchids, that grew on Hygromycin selection medium were confirmed by PCR and GUS assay. It is likely that the efficiency of in planta transfor-mation in P. amabilis is higher than that in S. plicata (Tabel 2), although it is not extreemely different, i.e (0.530.72)% in S. plicata and (0.8-2.1)% in P. amabilis. It might be related to the structure of pollinia/pollinaria, inwhich polli-naria of P. amabilis consists of two compact pollinia that connected by a structure of discus viscidium, that make it easy to be handled. Intact protocorm as target plant materials were used for infection of Agrobacterium with efficiency of transformation in a range of (0.1-1.7) % [7].. Chang et al [2] got high efficiency of transforma-tion (5.5%) in Arabidopsis, by using inflorescence for in vivo transformation. It might be due to the natural

Agrobacterium-mediated transformation in orchid can be approached by In planta Transfor-mation using pollen/pollinia to omit tissue culture step during T-DNA transfer. Efficiency of in planta transformation in S.plicata orchid is (0.53-0.72)% and in P. amabilis orchid is (0.8-2.1)%.

ACKNOWLEDGMENTS This work is supported by a Research Grant of STRANAS 2012 from DGHE, Ministry of Education RI contract No. 001/SP2H/PL/Dit.litabmas/III/2012

REFERENCES 1.

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5. S.Suhandono, J. Hughes., K. Brown, and M.A. Hughes, Expression and Structure of An Elongation Factor-1a Gene (MeEF1) from Cassava (Manihot esculenta Crantz). Euphytica 120:49-58 (2001) 6. Jefferson, R.A. 1997. Cambia , online accessed www.cambia.org.au 7. E. Semiarti, A. Indrianto, A. Purwantoro,Y. Machida and C. Machida, “Agrobacterium-Mediated Transformation of Indonesian Orchids for Micropropagation”. In: Genetic Transformation, edited by M.Alvarez, InTech-Open Publisher, http://www.intechweb.org/ ISBN 978-953-307-364-4, (2011),.

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