ISBN : 978-979-8969-06-5 International Conference on ... · Honorable Dean of Faculty of Biology...

PROCEEDINGISBN : 978-979-8969-06-5

International Conference on Biological Science Faculty of Biology Universitas Gadjah Mada 2011

(ICBS BIO-UGM 2011)

ISBN : 978-979-8969-06-5

International Conference on Biological Science Faculty of Biology Universitas Gadjah Mada 2011

(ICBS BIO-UGM 2011)

PROCEEDING ICBS BIO-UGM 2011

Published By:FACULTY OF BIOLOGY

UNIVERSITAS GADJAH MADAYOGYAKARTA

Jl. Teknika Selatan Sekip UtaraYogyakarta 55281

Phone: +62-274-580839; +62-274-6492350Fax : + 62-274-580839

E-mail : [email protected]

First Edition, October 2011

ISBN : 978-979-8969-06-5

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CONTENT iii

PREFACE iv

WELCOMING SPEECH v

OPENING REMARK vi

WELCOMING SPEECH viii

CONFERENCE COMMITTEE ix

ACKNOWLEDGEMENT x

PLENARY SESSIONSSession 1: Dr. Yam Tim Wing 1Session 2: Prof. Yasumasa Bessho 9Session 3: Prof. Christopher M. Austin 17Session 4: Drs. Langkah Sembiring, M.Sc., Ph.D 25Session 5: Hao Yu, Ph.D 36

THEMATIC ORAL PRESENTATION 39Topic 1. Molecular Biology, Genetic and Bioinformatics (O-MB) 39Topic 2. Ecology and Conservation (O-EC) 139Topic 3. Systematic and Evolution (O-SE) 209Topic 4. Physiology and Developmental Biology (O-PD) 293Topic 5. Biomedics (O-BM) 355

THEMATIC POSTER PRESENTATION 433Topic 1. Molecular Biology, Genetic and Bioinformatics (O-MB) 433Topic 2. Ecology and Conservation (O-EC) 465Topic 3. Systematic and Evolution (O-SE) 517Topic 4. Physiology and Developmental Biology (O-PD) 557Topic 5. Biomedics (O-BM) 605

LIST OF STUDENT COMMITTEE 643

LIST OF ORAL AND POSTER PARTICIPANTS 644

CONTENT

iii

PREFACE

Proceeding of the International Conference on Biological Science Faculty of Biology Universitas Gadjah Mada 2011 (ICBS BIO-UGM 2011), Advances in Biological Science: Education for Sustainable Development-based Tropical Biodiversity Management and Conservation for Supporting Human Prosperity, organized by and held at the Faculty of Biology, Universitas Gadjah Mada, Yogyakarta, Indonesia on September 23-24, 2011. The conference addressed a range of important research from various fields in biological science likely to play role tropical biodiversity management and conservation for supporting human prosperity. Three kinds of session were held at the conference: plenary session featuring keynote and invited papers, oral presentation session, and poster presentation session. This proceeding features a number of papers presented in these sessions, which represent 5 themes covered in the conference, i.e. genetics and molecular biology, ecology and conservation, systematics and evolution, physiology and developmental biology, and biomedics.

Many people have been involved in the production of these Proceedings, which is started in June 2011 with the launching of a call for abstracts. The abstracts were reviewed by both internal and external reviewers . Those selected abstracts were called for either oral or poster presentations and invited to submit full papers.

Lastly, on behalf of the organizing commite we would like to all participants for their kindness to be part of this conference. We would like to acknowledge each partnerships and sponsorship that involve during this event. I believe that this proceeding still has some weaknesses, therefore any constructive comments are welcome. We hope that the papers contain in this proceeding will prove helpful toward improving the scientific atmosphere. See you in the next two year ICBS 2013.

Yekti Asih Purwestri

Chair of the Organizing Commitee

iv

WELCOMING SPEECH FROM CHAIR PERSON OF THE ORGANIZING COMMITTEE

Distinguish guests• Executive Director of Indonesia-Managing Higher Education for Relevane and

Efficiency (I-MHERE) Project• Keynote speaker, invited speakers, participants, sponsorships, ladies and

gentlemenGood morning and May God shower us with His blessing.

On behalf of the Conference Organizing Committee, I extend a warm welcome to all participants to the second International Conference on Biological Science Faculty of Biology Universitas Gadjah Mada 2011 (ICBS BIO-UGM 2011), Advances on Biological Science : Education for Sustainable Development-based tropical biodiversity management and conservation for supporting human prosperity. Bio-conservation becomes a critical issue not only in Indonesia but also in global community. A good understanding on Education for Sustainable Development- based tropical biodiversity management is necessary to have the right policy regarding bio-conservation action.

For this year, the organizing committee has put together an interesting Scientific Program to accommodate the areas of Biology. The Program comprises of 6 plenary sessions of keynote and invited speakers. The parallel session of 82 oral presentations and more than 50 poster presentations. I realize that you are fully dedicated to the sessions but I do hope that you all will also take time to enjoy Yogyakarta, the multicultural city and may enjoy the special Merapi scenery, the most active volcano in the world.

I would like to take the opportunity to thank Prof Hubert Gijzen (Director of UNESCO-Jakarta ) as a keynote speakers and also to these following invited speakers, Hao Yu, Ph.D (National University of Singapore), Prof. Christ Austin (Charles Darwin University, Australia), Prof. Yasumasa Bessho, Ph.D (Nara Institute of Science and Technology, Japan), Dr. Yam Tim Wing (Senior Researcher Orchid Breeding and Conservation Singapore Botanic Gardens), Drs. Langkah sembiring, M.Sc. Ph.D (Faculty of Biology, Universitas Gadjah Mada) for delivering their valuable scientific information.

To make this program happen, I would like to gratefully acknowledge to Indonesia-Managing Higher Education for Relevane and Efficiency (I-MHERE) which support this conference. We also thank to the valuable contributions from personal and institutional sponsorship and funding including Ms. Sachiko Iida, PT Diastika Biotekindo, PT Roche, Prima Grafika Yogyakarta., and Drs. Agus Suryanto - Indogama Yogyakarta.

I also gratefully thank to the Dean and Vices Dean of Biology Faculty, Universitas Gadjah Mada for giving us opportunity and support to organize this conference. My deep appreciation to the Steering Committee, the Academic Reviewers (internal and external: Dr. Sentot Santoso from Institut fuer Klinische Immunologie und Transfusionsmedizin, Justus Liebig Universitaet Giessen, Germany and Prof. Yasumasa Bessho, Ph.D from Gene Expression Research, Biological Sciences, Nara Institute of Science and Technology, Japan), members of the Organizing Committee for their strong support, active participation, cooperation and hard works in preparing and organizing this event a success.

It is inevitable that there is a lack in organizing this conference and I profoundly apologize to all invited speakers, oral and poster presenters, attendants, donators and committee members.

I wish you a pleasant and rewarding two days of scientific discussion.

Thank you,

Yekti Asih PurwestriChair person of the Organizing Committee

v

OPENING REMARKS FROM THE DEAN of THE FACULTY OF BIOLOGY

Bismillahirrahmaanirrahiim.

Director of UNESCO Office Jakarta, Prof. Dr. Hubert Gijzen, Executive Direktor of Indonesian-Managing Higher education for relevance and Efficiency (I-MHERE) Project Honorable speakers and distinguished guest, dear participants,

Assalamu'alaikum wr.wb., may God give us healthy and happier life

Welcome to Yogyakarta, the city of youth, education, and culture. It's been an honour for me to be here in front of you to open the prestigious International Seminar with the special theme of "Advances in Biological Science: Education for Sustainable Development-based Tropical Biodiversity Management and Conservation for Supporting Human Prosperity", that invited our honorable speaker from the UNESCO as the keynote, Prof. Hubert Gijzen, Ph.D honorable invited speakers Dr. Yam Tim Wing From Singapore Botanic Garden, Singapore; Prof. Yasumasa Bessho, MD, Ph.D from NAIST, Japan; Prof. Christopher M. Austin, Ph.D from Charles Darwin University, Australia; Dr. Yu Hao from National University of Singapore, and Dr. Langkah Sembiring MSc, from the Faculty of Biology, Universitas Gadjah Mada, Indonesia.

My special gratitute to the speakers who have spent your time travelling to Indonesia in your such busy activity. This international seminar atrracts more than 400 scholars and students mostly come from Indonesia, and some participants come from abroad. This occassion is such a good opportunity for us to share our experiences in research and good practices of ESD based research and community service done, that could inspire students and other researchers, furthermore our keynote speaker today is the Director of UNESCO Jakarta Office, who will talk about Science, Technology and Innovation-an Engine for Sustainable Development.

Honorable and distinguished participants,

The seminar theme taken today is in line with vission of the Faculty of Biology UGM as the center of excellence for higher education that generates biologists who respect to our tropical biodiversity. Since 2010, Faculty of Biology UGM had obtained an ESD based research grant from the World Bank, through I-MHERE (Indonesian Management of Higher Education for Efficiency and Relevance) project. In this project has been conducted 3 activities, these are: improvement of publication and research quality, improvement of integrated collaboration research in tropical diversity with other Institutions, and community based activities that respect to biodiversity conservation. As stated in UNESCO HE information brief, the challenge for higher education in the context of ESD is to innovate the traditional learning environment and learning processes in such a way that they do not only support learning process in the formal education, but also in informal learning.

Our environment is now facing many dilemmas starting from global financial and economic crises highlights the risks of unsustainable economic development models and practices based on short-term goals. These aspects triger economic disparity between the poor and the rich countries, many complex societal contexts, and finally environmental degradation.

Education for Sustainable Development (EfSD) promotes quality education and its inclusive for all people. It is based on values, principles, and practices necessary to respond effectively to current and future challenges. UGM has shown commitment in Education for

vi

Sustainable Development and will continue to conduct ESD in the future. I hope that this Conference will continue to serve as a sustainable forum to provide opportunities for teachers, lecturers, researchers and professionals to share experience and present research activities and action programs. To everyone present here, I wish you have a productive and significant Conference that will benefit humankind, civilization as well as knowledge.

Lastly, I would like to extend my sincere appreciation and profound gratitude to the Director of UNESCO Jakarta and NAIST Japan for their supports. My special thanks should also go to the steering and organizing committee for their hard work in making this event a success. Thank you very much.

Yogyakarta, September 23rd, 2011

Sincerely yours,

Dr. Retno Peni Sancayaningsih, MSc.

vii

WELCOMING SPEECH FROM EXECUTIVE DIRECTORI-MHERE UGM

Honorable Dean of Faculty of Biology UGM, Dr. Retno Peni Sncayaningsih, M.Sc.Distinguish Keynote speaker Prof Hubert Gijzen (Director of Unesco in Indonesia)Distinguish Dr. Yam Tim Wing (Singapore), Prof. Yasumasa Bessho (Japan), Prof Christ Austin (Australia), Dr. Langkah Sembiring (UGM), Dr. Yu Hao (Singapore)Distinguish all of participants

Assalamu’alaikum wr.wb.

Welcome to Yogyakarta and participating in International Conference on Biological Science, by Faculty of Biology UGM.

This seminar was supported by IMHERE UGM (Indonesia Managing Higher Education for Relevancy and Efficiency). As we know, UGM get a competitive grant from World Bank trough Directorate General of Higher Education, from 2009 – 2012, and proposed program entitled “Education for Sustainable Development toward World Class Research University” by establishment of Center of Excellence (CoE) on 3 selected academic units, namely (i) “Tropical Biodiversity”, in Faculty of Biology (ii) “Medical Herbal and Supplements” in Faculty of Pharmacy and (iii) “Reduction Emission from Deforestation and Degradation (REDD)” in Faculty of Forestry.

Faculty of Biology has attempted for enhancement of the research quality on tropical biodiversity, development of the integrated research on utilizing biodiversity resources to enhance the EfSD and development of network capacity for national and international collaboration on research and community services through Regional Centre of Expertise (RCE) Yogyakarta.

This prestigious international seminar is one of our strategic activities to achieve better key performance indicator, especially in international publication and international research collaboration. As a new paradigm of competitive grant that developed by World Bank, called “Performance Based Contracts”, achievement of our key performance indicator in this year was 190% compare to targeted indicator for three years activities. We would like to continuing our “Research based Learning and Services for sustainable reputation as World Class Research University.

Please be enjoy to discuss and active participating in this seminar.

Wassalamu’alaikum wr.wb.

Sincerely yours,

Executive Director I-MHERE UGM

Dr. Cahyono Agus Dwikoranto, M.Agr.Sc.

viii

ix

CONFERENCE COMMITTEE ICBS 2011 FACULTY OF BIOLOGY UGM

1. Patron : Dean of Faculty of Biology2. Steering Committee : Dr. Retno Peni Sancayaningsih, M.Sc.

Drs. Langkah Sembiring, M.Sc., Ph.D. Dra. Mulyati, M.Si.

Dr. Endang Semiarti, M.S., M.Sc.Prof. Dra. Endang S. Soetarto, M.Sc., Ph.D.Prof. Chris Austin (Charles Darwin University, Australia)Prof. Yasumasa Bessho, Ph.D (NAIST, Japan)

3. Academic Reviewer :Internal Reviewers : Prof. (ret). Dr. Jusup Subagja, M.Sc

Prof. (ret). Dr. Jesmant Situmorang, M.ScProf. (ret). Sukarti Moeljoprawiro, M.App.Sc., PhDProf. (ret). Dr. Nyoman Puniawati Soesilo, SU.Prof. (ret). Dr. IstiyatiProf. Dr. Endang Sutariningsih S., M.ScLangkah Sembiring, M.Sc. Ph.D.Dr. Suwarno HadisusantoDr. Endang Semiarti, M.S., M.Sc.Dra. Rarastoeti Pratiwi, M.Sc., Ph.DDr. Budi Setiadi Daryono, M.Agr.Sc.Dr. rer-nat. Ari Indrianto, SU.Dr. Niken Satuti Nur Handayani, M.Sc.Dr. Kumala Dewi, M.Sc.St.Dr. Rina Sri KasiamdariDr. L.Hartanto, M.Agr.Dr. Yekti Asih Purwestri, Dr. Woro Anindito, M.Sc.Dr.biol.hom. Nastiti Wijayanti

External Reviewers : Dr. Sentot Santoso. (Institut fuer Klinische Immunologie und TransfusionsmedizinJustus Liebig Universität Giessen, GermanyProf. Yasumasa Bessho, Ph.D. (Graduate School of Biological Science, Nara Institute of Science and Technology (NAIST), Japan)

4. Chief of Organizing : Dr. Yekti Asih Purwestri, M.Si.Committee

5. Vice of Chief of : Dr. L. Hartanto Nugroho, M.Agr.Organizing Committee

6. Secretary/Secretariate : Ardaning Nuriliani, S.Si., M.Kes.Dra. Ratna Susandarini, M.Sc.Widiastuti, S.Pd.Siti Nurhaida, S.EDimas Willy, SIPKukuh Madyaningrana

7. Treasurer : Dr. Diah RachmawatiYuni HartatiSamiyati, S.E, M.AccPardiso

8. Plenary and Scientific : Dr. Rina Sri KasiamdariSession Dr. biol.hom. Nastiti Wijayanti, M.Si.

Abdul Rahman Siregar, S.Si., M.Biotech.Dr. Woro Anindito Sri Tunjung, M.Sc Sari Darmasiwi, S.Si, M.BiotechAries Bagus Sasongko, S.Si, M.Biotech

9. Publication : Zuliyati Rohmah, S.Si., M.Si.Donan Satria Yudha, S.Si., M.Sc.Slamet Riyadi, S.SiAris SetiawanR. Nur Wigunadi

10. Funding and : Dr. Suwarno HadisusantoSponsorship Donan Satria Yudha, S.Si, M.Sc

11. Documentation : Drs. Abdul Rachman, M.Si.Sudarsono

12. Logistics : Drs. Sutikno, S.U.Drs. H. WiyonoYatin, S.PdHarjanaDodo PriyatnoNahrowiGiyartoBektiLarno

13. Refreshment : Dra. Siti Susanti, S.UKodrat WartiniRusna NurainiPrapti

14. Hospitality : Dr. Niken Satuti Nur HandayaniDr. Rarastoeti Pratiwi, M.ScDrs. Heri Sujadmiko, M.SiDra. Upiek Ngesti Wibawaning Astuti, M.KesDr. Maryani

15. Accommodation : Slamet Widiyanto, S.Si., M.Sc.Donan Satria Yudha, S.Si., M.Sc.HaryantoSuharjitoHarsono

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O-MB09

Embryonic Calli Induction, Proliferation and Regeneration of Rodent Tuber

Plant (Thyphonium flagelliforme Lodd.) by single node culture

Nesti F. Sianipar*1, Rustikawati2, Wilmar Maarisit1, Ariandana Wantho1,

Dessy Novitasari Romauli Sidabutar1

1,Department of Biology, Faculty of Science and Mathematics, Universitas Pelita Harapan, M.H. Thamrin Boulevard 1100 Lippo Village, Indonesia

2Department of Agronomy, Bengkulu University , Bengkulu *To Whom all correspondence should be addressed :

Dr. Nesti F. Sianipar Email : [email protected]

Abstract

The rodent tuber plant (Thyphonium flagelliforme Lodd.) is a medicinal plant which shows detoxificying, antineoplastic or anti-cancer agent, antibacterial and antiviral activities. Contains bioactive compounds such as alkaloid, flavonoid, saponin, steroid and glycoside. However, the genetic variation in this plant is relatively low. The purpose of this study is to found optimal media for calli induction, proliferation of calli and shoots induction from embryogenic calli. Using tissue culture technique and to obtain optimal composition of plant regulators having the ability to regenerate plantlets from embryogenic calli. Single node of rodent tuber was sterilized and cultured on MS basal medium. Embryogenic calli were induced on MS basal medium and treated 1 mg/l NAA and 0.5 mg/l BAP. Proliferated calli were treated within various concentration of 2.4-D : 0.5 mg/l, 1 mg/l and Kinetin : 0.1 mg/l , 0.2 mg/l , 0.3 mg/l. The best embryogenic calli were produced on medium using 2.4 D 0.5 mg/l and Kinetin 0.1 mg/l. The embryogenic calli have genereted up to 14.38 plantlet per explant on MS basal medium and treated with 1 mg/l NAA dan 0.5 mg/l BAP. This study suggests the plantlet can be regenerated in significant amounts through the induction of embryogenic calli from single node.

Keywords : Thyphonium flagelliforme, single node, embryogenic calli, 2.4 D, NAA, Kinetin

INTRODUCTION

Rodent tuber (T. flagelliforme Lodd.) is a medicinal plant belonging the family

Araceae, native to Indonesia which are found in Java and grow well at an altitude of 100-300

m above sea level (Essai, 1986). It is an herbal plant that has a detoxifying agent. This plant

is found to have the potential to cure cancer. All parts of plant, namely roots, stems, leaves

and flowers contain bioactive compounds that function as anticancer agent. It can grow up

30 cm tall. This plant is also found in India and Sri Lanka (Nicolson and Sivadasan, 1981).

Rodent tuber is known to be useful in treating some diseases including cancers of

breast, colon, prostate gland, liver, leukemia and cervical cancer (Hoesen, 2007; Heyne,

1987). It contains antineoplastic or anticancer and antiviral cpds as well as (Teo and Ch'ng,

1996). Compounds which are efficacious in this plant are alkaloids, saponins, steroids and

International Conference on Biological Science Faculty of Biology Universitas Gadjah Mada 2011 (ICBS BIO-UGM 2011)

Faculty of Biology UGM - Yogyakarta, Indonesia, September 23rd-24th 201184

glycosides (Syahid, 2007). Medicinal plants contain bioactive compounds that can inhibit

pathogenic microorganisms such as bacteria, fungi and viruses (Lai et al., 2003). Choon et

al. (2008) has stated that the rodent tuber as an anti-cancer activity and induces apoptosis.

Rodent tuber plant is generally propagated vegetatively by tillers separation / hump

(Essai, 1986). Micropropagation of shoots can be induced by provision of optimal plant

growth regulators. The effectiveness of plant growth regulators auxin and exogenous

cytokinin depends on endogenous hormones in the plant tissues. Furthermore, cytokinins

(Benzyl Adenine) commonly used in the regeneration of in vitro culture of plant growth

regulators for this function in cell division and differentiation of adventitious buds (Bhojwani

and Razdan, 1981). In this study micropropagation in vitro methods through a single node

or shoot meristem can be induced shoot multiplication. The addition of plant growth

regulators BA and NAA on the media is expected to produce an optimal shoot multiplication.

The purpose of this study is to found optimal media for calli induction, proliferation

of calli and shoots induction from embryogenic calli.

MATERIALS AND METHODS

Plant Material

Rodent tubers were obtained from Balai Tanaman Obat Bogor. The material used was

single-node culture of node rodent tuber from Bogor. Node rodent tuber with buds were

aseptically used as explants.

Sterilization of explants.

The rhizomes of T. flagelliforme were washed thoroughly with detergent and rinsed in

running tap water to remove any soil particles. The single node tuber of T. flagelliforme were

used as explants resources. The buds were excised from the rhizomes and soaked in a a

solution fungicide and bactericide each for 2 hour. Thus, The explants of node tuber were

sterilized using 2.5 % and 1.5 % Clorox bleach with three drops of Tween-20 for 10

minutes respectively. Explants node tuber were sterilized again using Clorox bleach 1% for 5

minute and HgCl2 0.1 % for 5 minute respectively. Explants were rinsed again using sterile

water three times. Explant node tuber were grown in MS medium.

Experiment 1. Calli induction in basic culture medium for optimum in vitro culture

growth of T. flagelliforme.

Callus was induced on MS basal medium treated with growth regulators 2,4 D : 0

mg/l, 0.1mg/l , 0.5 mg/l, 1 mg/l with BA 0.3 mg/l and NAA 1mg/l with BA 0.5 mg/l. The

sterilized explants cultured in five kinds of MS medium with addition of plant hormones.

Four explants of node tuber were used for each culture medium. The best medium was


Faculty of Biology UGM - Yogyakarta, Indonesia, September 23rd-24th 2011 85

induced embryogenic calli with supplemented NAA 1 mg/l and BA 0.5 mg/l. Parameter is

observed when the callus induction begins to form.

Experiment 2. Effect of 2,4 D and Kinetin on Proliferation embryogenic calli

The material used were the calli induced from single-node culture. Calli were

obtained from previous treatment media and then sub-cultured to a new media.

Embryogenic calli were proliferated on MS basal medium treated with growth regulators

2,4D and NAA. The design used was completely randomized design in factorial pattern with

3 replications per treatment. The first factor is 2.4 D :0.5 mg / l and 1 mg / l and the second

factor is the third level of kinetin: 0.1 mg / l, 0.2 mg / l and 0.3 mg / l. The observed

parameters is diameter of the calli, colour of calli and texture of calli.

Experiment 3. Effect of NAA and BA on Shoots Induction from calli embryogenic

Calli embryogenic were have proliferated and then its subcultured to media of

shoot induction. Calli embryogenic were regenerated to be shoots on MS media with

treatment NAA : 0.5 mg/l ; 1 mg/l dan 1,5 mg/l and BAP 0.5 mg/l. The parameters observed

were the number of shoots formed. These experiments the number of shoots formed from

each calli embryogenic after 8 weeks of culturing was recorded. The data were analyzed

using ANOVA and the means compared using Tukey's pairwise comparisons at P= 0.05.

The pH of the culture media for all the above experiments was adjusted to 5.6-5.8

before autoclaving at 121oC for 20 minute. The cultures were placed in a culture room with

the temperature regulated 22oC and 16 h fluorescent lighting with a light intensity 1000 lux.

RESULT AND DISCUSSION

1. Percentage of calli Induction

Callus induction in basic culture medium for optimum in vitro culture growth of

T. flagelliforme by single node culture. MS Media were supplemented with 2,4 D 0, 0.1 ,

0.5 or1 mg/l, or 0.3 mg/l BA couldn’t induce calli from single node culture of rodent tuber.

This could be due to the balance of the addition of auxin 2,4 D and cytokinin BA were not

optimal to calli induction.



Figure 1. Percentage of induced Calli in the media MS supplemented with 1 mg/ NAA with 0.5 mg/l BA

The MS media added with 1 mg/l NAA or 0.5 mg BA induced calli from single node

culture of rodent tuber up to 77.78 % (Fig.1.) The result showed that auxin NAA and

cytokinin BA can be induced calli of rodent tuber after 5 weeks. According to Bhojwani and

Radzan (1996), 2,4 D was a powerful normally used for callus induction. The same study

was done with the induction of callus from embryo using auxin 2.4 D in Wheat (Rahman et

al., 2008; Kamil, 2002).

Fig.2. Calli Induction from single node culture of rodent tuber from Bogor: (A ) Explants within three weeks of culture; (B) Calli induction within 5 weeks of culture; (C) Embryogenic calli winthin eight weeks of culture.



2. Proliferation Embryogenic Calli

The embryogenic calli were induced from single node culture could be proliferated in

optimal medium. The best medium to proliferated embryogenic calli was MS media

supplemented with 1 mg/l 2,4 D and 0.3 mg/l kinetin or 0.5 mg/l 2,4 D and 0.3 mg/l kinetin.

The resulted data obtained from various treatments showed that every treatment was

different (Table 1). The induced calli were friable, compact and globular structure. Produced

Calli were embryogenic indicated with a light green and yellowish green color (Table 1.)

Plant growth regulators of 2,4-D is a strong auxin often used to induce callus

formation from various plant tissues (Bhojowani and Razdan, 1996). Plant growth regulators

of 2,4 –D is effective to initiate callus (Nagasawa and Finer, 1988). The use of auxin (2,4 D)

and cytokinins (Benzyl Adenine) will enhance the process of callus induction (Litz et al.,

1995). Cytokinins BA are commonly used in the process of regeneration in vitro culture

because this plant growth regulators are function in cell division and differentiation of

adventitious buds from callus (Bhojwani and Razdan, 1996).

Table 1. Effect 2.4 D and kinetin to calli proliferation of colour of calli and texture of calli after

10 weeks Treatment Colour of calli Texture of calli 1 mg/l 2.4-D + 0.3 mg/l Kinetin Light green friable, compact, globular

1 mg/l 2.4 -D + 0.2 mg/l Kinetin Greenish Yellow friable, compact

1 mg/l 2.4 -D + 0.1 mg/l Kinetin Light Yellow Translucent, slimy

0.5 mg/l 2.4-D + 0.1 mg/l kinetin Brownish yellow Translucent, slimy

0.5 mg/l 2.4-D+ 0.2 mg/l Kinetin Brownish yellow friable, compact

0.5 mg/l 2.4-D+ 0.3 mg/l Kinetin Yellowish green friable, compact, globular

The effect in addition of plant growth regulator 2.4-D and kinetin showed that each

treatment produced a significant different texture and colour of the embryogenic calli. The

combination of auxin and cytokinin concentrations determined optimal embryogenic callus

formation (George and Sherrington, 1984). Oluk and Kaskar (2005) stated that the addition

of kinetin and NAA can be induced embryogenic callus on Papaver somniferum plant.



Table 2. Effect of 2,4 D and Kinetin to calli proliferation of Rodent Tuber Diameter after 10 weeks

Source DF Seq SS Adj SS Seq MS

(mm)

F P

24D 1 30,6 30,6 30,6 0,26 0,614ns

Kin 2 5,6 5,6 2,8 0,02 0,976ns

24D*Kin 2 79,3 79,3 39,6 0,34 0,716ns

Error 18 2092,5 2092,5 116,3

Total 23 2208,0

Note : analyzed using ANOVA

The results showed that supplementation of 2.4 D and kinetin on MS medium

could propagated calli but not significant for calli diameter. Diameter of calli can be

achieved 39.6 mm per clump (Table 2). The development and proliferation of calli can be

produced a embryogenic calli (Fig. 3 and 4). Among the six treatment of MS media,

addition of 0.3 mg /l kinetin and 1mg/l 2,4-D or 0.5 mg/l 2.4 D tended to make the texture of

calli was friable, compact and globular stucture.

Fig 3. Development and proliferation of embryogenic Calli with supplemented 1 mg/l 2,4 D and 0.3 mg/ l Kinetin (A) Embryogenic calli within one week (B) Embryogenic Calli within four weeks (C) Embryogenic Calli within six weeks (D) Embryogenic Calli within ten weeks

Fig 4. Development and proliferation with supplemented 0.5 mg/l 2,4 D + 0.3 mg/ l Kinetin (A) Embryogenic calli within one week (B) Embryogenic Calli within four weeks (C) Embryogenic Calli within six weeks (D) Embryogenic Calli within ten weeks



3. Shoots induction from calli embryogenic

Embriogenic calli could be regenerated on various medium. Medium used for shoots

induction from embryogenic calli were MS medium with NAA and BA. Embriogenic

calli were regenerated to plantlet. All shoots produced from three combination of BA and

NAA with various concentrations (0.5 mg/l; 1 mg/l; 1.5 mg/l) added in MS medium

produced normal shoots (Fig.5). When the combination of concentration of 0.5 mg/l BA

and 1 mg/l NAA, the number of shoots induced from each clump of embryogenic calli

was significantly increased. The best medium which enabled embryogenic calli to

produced the highest shoot numbers (14.38 per clump) was the MS medium

supplemented with 1.0 mg/l NAA and 0.5 mg/l BA (Table 3).

Tabel 3. Effect of various NAA and BA combination on production of T. flagelliforme Shoots from embryogenic calli after 8 weeks of culture

Treatment Shoots number from derived of calli

1.5 mg/l NAA + 0.5 mg/l BA 8,13b

1.0 mg/l NAA + 0.5 mg/l BA 14.38a

0.5 mg/l NAA+ 0.5 mg/l BA 4,25b

Means followed by the same letter are not significantly different (compared using Tukey's pairwise comparisons at P= 0.05 )

MS medium was the best basic medium for the in vitro production of multiple shoots

of T. flagelliforme (Sai et al., 2000). Tuber is commonly used a part of plant as explants in

micropropagation of rodent tuber. According Nobakht et al. (2009), MS medium contained 5

mg/l BAP and 1 mg/l NAA can produce the most number of shoots per explant. At a higher

concentration of NAA (0.1 – 1 mg/l) , roots were produced, but abnormally shortened and

thickened and a high NAA concentration (> 0.5 uM) inhibited shoot multiplication (Sai et al.,

2000)



Fig.5. Induction Shoots from embryogenic Calli (A) Shoots produced in MS in the presence 1 mg/l NAA + 0.5 mg/l BA, (B) Shoots produced in MS in the presence 0.5 mg/l NAA + 0.5 mg/l BA (C) Shoots produced in MS in the presence 1.5 mg/l NAA + BA 0.5 mg/l

CONCLUSION

Calli induction of rodent tuber from single node can be obtained in the treatment of

1 mg/l NAA and 0.5 mg/l BA within 5-8 weeks of culture. The best proliferation of

embryogenic calli on medium MS were added 1 mg/l 2,4-D and 0.3 mg/l kinetin; 0,5 mg/l

2,4-D and 0.3 mg/l kinetin. Texture of embryogenic calli were compact, friable and globular.

Calour of embryogenic calli were yellownish green and light green. The embryogenic calli

have been resulted up to 14.38 shoots per explant on MS basal medium and treated 1 mg/l

NAA and 0.5 mg/l BAP.

Acknowlegdement

This work was funded by DIKTI through Hibah Bersaing Project. The authors would like to

thank Directorate General of Hinger Education, Ministry of National Education, Indonesia.

REFERENCES

Bhojwani, SS dan Razdan MK. 1996. Plant Tissue Culture: Theory and Practice, a Revised Edition. Elsevier Science. Amsterdam: 767p.

Choon SL, Rosemal HMHM, Nair NK, Majid MIA, Mansor SM dan Navaratnam. 2008.Typhonium flagelliforme inhibits cancer cell growth in vitro and induces apoptosis: An evalution by the bioactivity guided approach. Journal of Ethnopharmacology 118 : 14-20

Essai. 1986. Medicinal herbs index in Indonesia. PT Essai indonesia. 357 hal

Heyne. 1987. Tumbuhan berguna Indonesia. Jilid I. Jakarta. 502 hal



Hoesen DSH. 2007. Pertumbuhan dan perkembangan tunas Typhonium secara in vitro. Berita Biologi. 8(5): 413-422.

George EF and Sherrington PD. 1984. Plant Propagation by Tissue Culture. Exgetics Ltd. Eversley, Basingstroke, Hans, RG 27, England.769 pp

Lai KC, Wan YK and Tengku-Muhammad TS. 2005. Comparison of cytotoxic between in

vitro and field plants of Thyphonium flagelliforme (Lodd.) Blume. Journal of plant biology. 48(1) : 25 -31.

Litz RE, Moon PA and Chavez VM. 1995. Somatic embryogenesis from leaf callus derived rom mature trees of the cycad ceratozamia hildae(Gymnospermae). Plant Cell, Tissue and Organ Culture 40 : 25 – 31.

Kamil H. 2002. Wheat Immature embryo culture for embryogenic callus induction. Journal of Biological Science 2 (8) : 520-521.

Oluk EA and Kaskar C. 2005. Somatic embryogenesis and shoot regeneration from callus cultures of Papaver somniferum L. Cv.Office-95*. Akadeniz Universitesi Ziraat Fakultesi Dergisi 18(2), 225-227.

Nagasawa A and Finer JJ. 1988. Induction of morphogenic callus of Garlic. Hort Science 23 (6) : 1068 – 1070.

Nobakht, G. M., Kadir, M. A., dan Stanslas, J. 2009. In Vitro Mass Propagation of Typhonium flagelliforme as Affected by Plant Growth Regulators. African Journal of Biotechnology 8: 6840—6843.

Rahman MM, Shamsuddin AKM and Asad U. 2008. In Vitro Regeneration from Mature Embryos in Spring Wheat . Int. J. Sustain Crop prod. 3(2): 76-80

Sai ST, Keng CL, Pargini N and Teo KH. 2000. In Vitro propagation of Thyphonium

flagelliforme (Lodd) Blume. In vitro cell. Dev.Biol-Plant 36 : 402-406.



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