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

  PROCEEDING

  ISBN : 978-979-8969-06-5 International Conference on Biological Science

Faculty of Biology Universitas Gadjah Mada 2011

(ICBS BIO-UGM 2011)

  CONTENT 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

iii

CONFERENCE COMMITTEE

  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 Prof. Dr. Endang Sutariningsih S., M.Sc

Langkah Sembiring, M.Sc. Ph.D.

Dr. Suwarno Hadisusanto

Dr. Endang Semiarti, M.S., M.Sc.

Dra. Rarastoeti Pratiwi, M.Sc., Ph.D Dr. 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 Kasiamdari Dr. 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 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 6. Secretary/Secretariate : Ardaning Nuriliani, S.Si., M.Kes.

  

Dra. Ratna Susandarini, M.Sc.

Widiastuti, S.Pd. Siti Nurhaida, S.E Dimas Willy, SIP Kukuh Madyaningrana

  7. Treasurer : Dr. Diah Rachmawati Yuni Hartati Samiyati, S.E, M.Acc Pardiso ix

  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 12. Logistics : Drs. Sutikno, S.U.

Drs. H. Wiyono

Yatin, S.Pd Harjana

Dodo Priyatno

Nahrowi Giyarto Bekti Larno

  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 x

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

  39

  46

  52

  59

  67

  68

  75

  76

  84

  93 101 102 110

  116 125 131

  O-MB01

The Continous Function of KNAT1 gene on Secondary Shoot Growth in

Micropropagation of Indonesian Black Orchid Coelogyne pandurata

_1*

Lindley Transgenic

_{1 1 2 3 1} Endang Semiarti , Eggie F. Ginanjar , Rizqie L.Nurwulan , Y. Machida and C. Machida ₂ Faculty of Biology, Universitas Gadjah Mada, Jl. Teknika Selatan, Sekip Utara, Yogyakarta 55281, Indonesia., ₃ Division of Biological Sciences, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya, Japan, ₄ College of Biotechnology and Bioscience, Chubu University, Kasugai, Japan,

• *Corresponding author:

  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 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

• 1

  are growing up on 100 mg. l 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

• 1

  addition of 150 ml.l 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.

  RB _{Pnos Tnos BAR Tnos NPTII p35S KNAT1} LB

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.

  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.

  The first time shoot emerging from explant (week) Growth regulators

NAA: 2-IP

  1

  2

  

3

  4

  5

  6

  7

  8 (0.00 : 0.00) NT T

  (0.15 : 0.15) NT T

  (3.00 : 3.00) NT T

  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 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.

  (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.

  Number of shoots No. Growth regulators (NAA: 2-IP) (µM) Transformant Non transformant

  1

  

11

  5 (0.00 : 0.00)

  2

  

36

  4 (0.15 : 0.15)

  3

  

13

  20 (3.00 : 3.00) Detection of KNAT1 gene 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.

  

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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