The Continous Function of KNAT1 gene on Secondary Shoot Growth in Micropropagation of Indonesia Black Orchid Coelogyne pandurata Lindley Transgenic - repository civitas UGM

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PROCEEDING

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 SESSIONS

Session 1: Dr. Yam Tim Wing

1 Session 2: Prof. Yasumasa Bessho

9 Session 3: Prof. Christopher M. Austin

17 Session 4: Drs. Langkah Sembiring, M.Sc., Ph.D

25 Session 5: Hao Yu, Ph.D

36 THEMATIC ORAL PRESENTATION

39 Topic 1. Molecular Biology, Genetic and Bioinformatics (O-MB)

39 Topic 2. Ecology and Conservation (O-EC)

139 Topic 3. Systematic and Evolution (O-SE)

209 Topic 4. Physiology and Developmental Biology (O-PD)

293 Topic 5. Biomedics (O-BM)

355 THEMATIC POSTER PRESENTATION

433 Topic 1. Molecular Biology, Genetic and Bioinformatics (O-MB)

433 Topic 2. Ecology and Conservation (O-EC)

465 Topic 3. Systematic and Evolution (O-SE)

517 Topic 4. Physiology and Developmental Biology (O-PD)

557 Topic 5. Biomedics (O-BM)

605 LIST OF STUDENT COMMITTEE

643 LIST OF ORAL AND POSTER PARTICIPANTS

644 CONTENT

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ix

CONFERENCE COMMITTEE

ICBS 2011 FACULTY OF BIOLOGY UGM

1. Patron : Dean of Faculty of Biology

2. 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.Sc

Prof. (ret). Sukarti Moeljoprawiro, M.App.Sc., PhD Prof. (ret). Dr. Nyoman Puniawati Soesilo, SU. Prof. (ret). Dr. Istiyati External Reviewers : Dr. Sentot Santoso.

(Institut fuer Klinische Immunologie und Transfusionsmedizin Justus Liebig Universität Giessen, Germany

Prof. 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

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8. Plenary and Scientific : Dr. Rina Sri Kasiamdari

Session 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.Biotech

Aries Bagus Sasongko, S.Si, M.Biotech 9. Publication : Zuliyati Rohmah, S.Si., M.Si.

Donan Satria Yudha, S.Si., M.Sc. Slamet Riyadi, S.Si

Aris Setiawan R. Nur Wigunadi

10. Funding and : Dr. Suwarno Hadisusanto Sponsorship Donan Satria Yudha, S.Si, M.Sc 11. Documentation : Drs. Abdul Rachman, M.Si.

Sudarsono

13. Refreshment : Dra. Siti Susanti, S.U Kodrat Wartini

Rusna Nuraini Prapti

14. Hospitality : Dr. Niken Satuti Nur Handayani Dr. Rarastoeti Pratiwi, M.Sc Drs. Heri Sujadmiko, M.Si

Dra. Upiek Ngesti Wibawaning Astuti, M.Kes Dr. Maryani

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

Suharjito Harsono

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LIST OF ORAL PRESENTER TOPIC 1: MOLECULAR BIOLOGY, GENETIC AND BIOINFORMATIC

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46

52

59

67

68

75

76

84

93

101

102

110

116

125

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O-MB01

The Continous Function of

KNAT1

gene on Secondary Shoot Growth in

Micropropagation of Indonesian Black Orchid

Coelogyne pandurata

Lindley Transgenic

Endang Semiarti1*, Eggie F. Ginanjar1, Rizqie L.Nurwulan1, Y. Machida2 and C. Machida3

1

Faculty of Biology, Universitas Gadjah Mada, Jl. Teknika Selatan, Sekip Utara, Yogyakarta 55281, Indonesia., 2 Division of Biological Sciences, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya, Japan, 3

College of Biotechnology and Bioscience, Chubu University, Kasugai, Japan, 4 *Corresponding author: endsemi@ugm.ac.id;

Abstract

Agrobacterium-mediated genetic transformation has become increasingly important tools for improving cultivars and studying gene function in plants. This is particularly true in orchids, which are highly valued ornamental plants that are continually being genetically altered. To improve the quality of Indonesian black orchids, we developed a convenient method for the genetic modification of this orchid using Agrobacterium tumefaciens. The T-DNA of a disarmed Ti plasmid containing the coding region of a neomycin phosphotransferase II gene as a selectable marker was successfully introduced into intact protocorms of the Black Orchid (Coelogyne pandurata L. Form East Kalimantan). The

BREVIPEDICELLUS (BP)/KNAT1 gene was under the control of the Cauliflower Mosaic Virus (CaMV) 35S promoter, and is a member of the family of class 1 KNOTTED-like homeobox (KNOX) genes in Arabidopsis thaliana that is required for the maintenance of indeterminate state of cells. the T-DNA containing BP/KNAT1 was transformed into the black orchid. The protocorms that were transformed with BP/KNAT1 produced multiple shoots, indicating that the BP/KNAT1 gene can be used to improve shoot formation for mass propagation of these orchids. In vitro culture using leaf discs of the 35S::KNAT1 transgenic Black orchid on hormon-free medium also resulted in multishoots production. These data indicate that the KNAT1 gene maintained its function in secondary shoot growth of transgenic black orchid. The method can be applied to the commercial production of orchids in Indonesia for both domestic and international trade.

Keywords: Black orchids, secondary shoot induction, genetic transformation, Agrobacterium tumefaciens.

INTRODUCTION

Techniques on plant tissue culture for orchid micropropagation are useful for mass

production. Since the needs of orchids are always increase by the time for commercial

trades, the conservation efforts should also be elaborated. Mass propagation through in vitro

culture will become a good tool for these efforts. But, in orchids, there are many obstacles to

do tissue culture, due to the slow growth rate and the long life cycle of orchid. Recently, we

developed an efficient technique for orchid micropropagation through Agrobacterium–

mediated genetic transformation of Knotted1-like Arabidopsis thaliana (KNAT1) gene into

genomes of three genera of Indonesian orchids, i.e Phalaenopsis amabilis (L.) Blume,

Vanda tricolor Lindley and Coelogyne pandurata Lindley (1, 2).. The insersion of KNAT1

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

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gene into orchid genome resulted in multishoot formation in P. amabilis and C. pandurata,

but not in V. tricolor. In P. amabilis, there was 31-90 shoots emerged from one protocorm

(developing orchid embryo), and in C. pandurata there was 4-7 shoots emerged from one

transformant’s protocorm. There is still a question to be addressed: is there any stable orchid transformant that maintained the function of interest gene in their secondary growth?

In this report, we analyze the continous function of KNAT1 transgene on secondary

growth of black orchid transformant’s shoots in tissue culture condition, to understand the

stability of KNAT1 as a foreign gene in orchid genome. It is worth to elaborate for this orchid

due to the rareness of the black orchid C. pandurata as an Indonesian endemic orchid. The

method could be implemented for other Indonesian natural orchids.

MATERIALS AND METHODS

Plant materials and culture condition

Four developing independent shoots of 35S::KNAT1-black orchid transformants that

are growing up on 100 mg. l-1 Kanamycin-containing New Phalaenopsis (NP) medium were

used as plant materials in this experiments. The shoots as source of explant were cut into

two leaf discs and a stem to induce new shoot formation on regeneration medium in vitro.

Explants were cultivated on half strength of New Phalaenopsis (NP) medium (3), with

addition of 150 ml.l-1 coconut water, and combination of plant growth regulators

2-isopenthenyladenine (2iP) and Naphtalene acetic acid (NAA) with ratio of 1: 1 (0.15 and 3

μM). The cultures were incubated at 25°C with 1000 lux continuous light. The growth of protocorm like bodies (PLBs), and shoots from the explants were examined every week. For

control experiment, a similar set of experiment was also done using non-transformant black

orchid plant.

T-DNA Construct for Genetic Transformation and Detection of Transgene by PCR

Genetic transformation of plasmid 35S::KNAT1 and pGreen vector into orchid was

carried out according to the method of Semiarti et al. (1). The structure of 35S::KNAT1

containing T-DNA construct that inserted into orchid genome is shown in Fig. 1.

Figure 1. Schematic Structure of 35S::KNAT1 containing T-DNA. LB, Left border; RB, Right

Border, 35S: CaMV promoter; KNAT1 gene; HPT: Hygromycin phosphotransferase; Tnos: Nos terminal. Bar: 1.2 kb.

Pnos NPTII Tnos p35S KNAT1 BAR Tnos

RB LB

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

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Genomic DNA of transformant plants and non transformant were isolated and

extracted using QIAGEN gDNA extraction kit (GmBH, Germany) according to the manual

instruction from the manufacture. Pured gDNA from the emerged shoots from selected

transformant explants were detected by polymerase chain reaction (PCR) method for the

existance of KNAT1 gene using KNAT1 gene specific primer KNAT1F1 (5’

-CTTCCTAAAGAAGC-ACGGCAG-3’) and KNAT1R1 (5’-

CCAGTGACGCTTTCTTTGGT-T-3’), that amplified 1.2 kb DNA fragment.

RESULTS AND DISCUSSION

Phenotypic analyses

Morphology of the shoot and leaves of transformant are normal as the same as non

transformant plant. The growth rate of shoot(s) from transformant and non-transformant

stem explant on half strength NP medium with various concentration of growth regulators

treatment showed that generally, induction of shoot formation in transformant was faster

than that of non-transformant (Table 1).This data indicates that the growth of shoots from

transformant explant may be induced by KNAT1 gene activity that integrated in the orchid

genome, than that of induction by growth regulators endogenously or exogenously.

Table 1. The Growth of Shoots from transformant and non-transformant stem explants on

half strength NP Medium and Various Concentration of Growth Regulators Auxin and Cytokinin.

Growth regulators

NAA: 2-IP

The first time shoot emerging from explant (week)

1 2 3 4 5 6 7 8

(0.00 : 0.00) _T _NT

(0.15 : 0.15) _T _NT

(3.00 : 3.00) _T _NT

T= transformant; NT = Non-transformant

The fastest emerged shoot(s) from transformant stem was two weeks after explant

inoculation on ½ NP medium without additional growth regulators and ½ NP+ 0.15 µM NAA

and 0.15 µM 2iP, though in non transformant explant the shoots emerged at 4 weeks after

inoculation. Interestingly, when the higher concentration of growth regulators was added into

medium, the shoot formation delayed up to 6 weeks in transformant explant and 8 weeks for

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non transformant explant. It is inline with our previous data in P.amabilis, that shoots formed

on leaf discs of 35S::KNAT1 transformant grown on hormon-free NP medium (1).

Multishoots production from 35S::KNAT1 Transformant Explants

In the case of number of shoot production, the transformant stem produced

multishoots from one stem explant. The higher number of shoots were produced in

transformant explants,than that in non transformant stem (Fig.2, Table 2). Multishoot

production were also reported by Yu et al. (4), when Dendrobium Orchid Homeobox1 (DOH1)

introduced into orchid hybrid Dendrobium “Madame Thong In”. Introduction of KNAT 1 gene

into some Dicot was also induced multishoot formation, i.e Chuck et al. (5) observed

multishoot production in Arabidopsis, and Nishimura et al. (6) obtained multishoots in

tobacco transgenic explants. Semiarti et al. (7) reported that the expression of KNAT1 gene

was improved in a leaf mutant of Arabidopsis, assymetric leaves2 mutant, that produced

multishoots on mutant leaf disc in that were cultured on hormone free medium.

Figure 2. Multishoot formation from stem explant of 35S::KNAT1 on ½ NP + 0.15 µM

NAA+ 0.15 µM). 0 ; week 0, starting inoculated explant, 2; Shoot initiation (emerging leaf

primordia) come out from explant, week-2; 7; Week-7, 9 ; Week-9, 11; Week-11, and 13;

multishoots emerged at week-13 (Bar: 1mm)

Multishoots production might also be related to the arrangement of endogeneous

phytohormone biosynthesis pathway, such as cytokinin and gibberelic acid (GA) that

involved in cell division, cell elongation and shoot formation. As described by George et al.

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(8), that in plant tissue culture, during adventif shoot formation the concentration of

cytokinin in cell increase, but the concentration of GA will be decreased. In tobacco

transgenic plants, overproduction of KNAT1 protein suppressed the activity of GA20ox

(Ntc12) that bound to some sequences in the first intron of the GA20ox gene. This

complex reduced the synthesis of GA, in turn it will activate cytokinin synthase gene,that

caused multishoot production.. Overexpression of KNAT1 gene will also eliminate apical

domination in the shoot tip, so that the determinated cells will switch into undeterminated

cells (9, 10). It is reasonable that multishoot production in this experiment has also proved

the activities of overexpressed KNAT1 gene in the black orchid stem.

Table 2. Number of Shoot Production from 35S::KNAT1 transformant stem explant after 13

weeks cultivation on ½ NP Medium supplemented with various growth regulators.

Detection of KNAT1gene in 35S::KNAT1 transformant plant’s genome

Seven shoots of transformants and three non-transformant plants were analyzed to

prove the prescence of 35S::KNAT1 into its genomes. The genomic DNA of each plants

were amplified using specific oligonucleotide primers for KNAT1 genes (KNAT1F1 and

KNAT1R1), that resulted in about 1.2 kb amplified DNA fragment. Four out of seven shoots

showed positive results, but the other three were negative (Fig. 3). These results indicate

that KNAT1 gene still integrated into the genomes of four orchid transformant lines and

maintained its activity for shoot production in these orchids. Using these four lines,

micropropagation of black orchid (C. pandurata) can be improved, as well as the use of the

transgenic technology for other character improvement of this orchid. The use of Green

Flourescent Protein (GFP) as a reporter gene as described previously (11), combine with

KNAT1 gene will improve the orchid quality in both shoot multiplication and flourescence

plant. Hopefully, it will give benefit to support both conservation and commercial trade of

Indonesian natural orchids.

No. Growth regulators (NAA: 2-IP) (µM)

Number of shoots

Transformant Non transformant

1 _{(0.00 : 0.00)} 11 5

2 _{(0.15 : 0.15)} 36 4

3 _{(3.00 : 3.00)} 13 20

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Figure 3. Detection of KNAT1 gene in 35S::KNAT1 Black Orchid Transformants. Lanes (A-D)

show that 1.2 kb DNA fragment could be amplified from four transformants. λ indicates λ

DNA digested by Sty I enzyme that used as DNA size marker.

CONCLUSION

The Arabidopsis KNAT1 gene can be used for improvement of shoot formation in

micropropagation of Black Orchid (C. pandurata). The KNAT1 gene has stably maintained

its function in secondary shoot growth of black orchid transformant. The method can be

applied to the commercial production of orchids in Indonesia for both domestic and

international trade.

Acknowledgement

The research was supported by Indonesian DGHE Research Competition grant HB XVII

2009-2010 No. LPPM-UGM/604/2009. We thank to Bunga Rintee Orchid Nursery,

Yogyakarta for the gift of fruit of the Black Orchid .

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