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ISBN : 978-97S.896946-5

International Conference on Biological ScienceFaculty of giology Universitas cadjah Mada 201l

(tcBs Bto"ucM 20ll)

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BIOLOGiCAL SCIEI{CE

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for Susrainable Developmcnt hasedTropical Biodiversry Managemenr

and Conserarion for Supporring

September 23'124'^ 2011Yogyakartd, INDONESIA

Education

Human P.orperiry

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Faculty ot Biology Universitas Gddjdh i\dda

ISBN : 978.979S96946-5

Intemational Conference on Biological ScienceFaculty of Biology Universitas cadjah l'rada 2011

(rcBs Bro-uGM 201r)

ADVANCES INBIOLOGICAL SCIENCE

Educlron tor Susrajnable Developmenr basedliopical Biodiversiry Management

and Cooservadon for SupportingHuman Prosperity

September 23'-24'" 2011

Yogyakarta, INDONESIA

PROCEEDING ICBS BIO UGM 2011

PublGhed aY:FACULTY OF BIOLOGY

UNIVERSITAS GANAH MADAYOGYIJ(ARTA

Jr.I€r6ik sdatan sekp ulaEYasyakatu 55231

Phone +62-274-5€o$3f]t 1s2 21444923fiFax : + 62-274-54m39

E mail I bioloql{gm@ugm.ao.id

All dqhts €sed€d (includho lho.€ of lEnslalion inlo olh€r languases)No .ad of lhis b€k mav b€ rcDrcdu@d in anv fom - bv phobpnnung,

micrcl5lm or anv oth€r means - nor tansmifi€d or 1ranslated into amaohine languag€ fihou{ witen pemission nom sle publishere.

Reoisl€Bd n.mes. tadenarks,6tc. used ln sis bmk, €v.n wh€n notsb*inelN na ed as su.h. aE not ro be @nsld6€d unpolected bv law

o-M801

Thecontinous Function of Kxift gene on secondary shoot Growth inItlicroprcpagation of Indonesian Black Orchld coerogvre patdurata

LindleyTrdnsgenic

Endano s€m dl Eqqis r. GtunF|

o , "o idE"d dn€ .€ ! - . ; d - . -odd E e N :ooaLnwd l ' r I6lbuni4ilyb94d}F|

' romalol hc\ beone i.recshg/ noonanlmB b_;DaM q aire6 and nrdyrq sene tuluororch'ds, which a; hlshly varued ;a;€ital planb flar.re con6.,anv b€ns eeneii€ltalte€d. ro imprcve lh€ quality or hdonesian black orchds we developed a @hveni6nlhelhod for lhe senetc modfddon of lhis orch d dsing ,Agrcbacre,,n l,nelac'e's Tho T_DNA or a dsamed Ti tlasnid dnlainins the coding res'on ot a neomvcnonosohohnsrc?se I qPne .r a selerrcblc mader @5 '-^mtullv 'rholdad lo :1bLi;rcE@mi or ir Bb;" ond rc@tcstne paNL@h I ron E'BREvtpFDtcLLLUS

la4r'

-

gene inro o.chid gen ome €su led in multislr ool lom ar on n P. .na bilis and c pahdLhta

6rt nat n V. trcotoL tn P. an.bilis. there was 31_90 shoors emersed l'.h one prolocom

(dev€loping odid €mbryo), and in C. pa'd,€|a rh€re was 4_7 sho

imnstomanfs prct\r@m. ftere is sfl a quesl on lo be addressedi ls iherc any slable omrid

tansfomant bal nalnlained Ihe tunclion ot inle€sl ssne in lneir semndarv grotr1h?

In tis Gpod. we anallzolho conlnous tuncton of KN,4r lEnsgen€ on 36@ndary

gDwlh of brack orchrd tmnstoma iijon lo undecEnd th€

slrbiliry of x 'nr as a forcign sene n omhid q6nome. I is Mdlr 10 elaboEte fof lhis orchid

due lo he E€ness or rhe b/adk orchid C padudra as an ldonesbn endemic orchid. Ihe

method could b6 imp €monl€d Jor o

MATERIAS AI{D I{ETHODS

Plant malef .ls.nd cultuE condlton

Four d€veloping independent shoots of 35s:(NAT1-bhck ordid lransfdmants lhal

are govins upon 10omg.Ir K.namycin{nlain ng N€wPhalaenopsis (NP)m€drum we€

usedasplanlnareda|sinthjserpeimeds'Theshet5assourceo'

hrb le.f dis6 and a sl€m to indu@ new shml romauon on .eseneEtron m6dium ii? v,ro.

on han siGnsrh of fret Pr.r.e,opsis (NP) nediun (3) wrh

addiron of 150 ml.lr coconut wal€r, and combinadon of plant gotlh .egulaloB 2_

ispenth$ytadqine l2iPl and Naphralene aoel,o a.d (NM) wiih Ero dr 1i 1 (015 and 3

pM). Ths .uhures weE incubaled at 25'C wilh 1000 lux mndnuous lisht Ths growlh ot

prclomm lke bodies (PLBS) and sh@b rr.n lhe explanls were examined every @ek For

conbl experiment, a similar s€t of expedment was also done uslnq

T.I)NA Consfuct fo. Generic lEn6f.ma0on and Detscdon of TEnsgene bv PcR

Genelic Fansfotratjon of plasmid 35S::KNAT1 and pcrcen vector inlo orchid was

cani6d out accordins lo th€ melhod or semiah 3, .r (l). The sf,clurc ol 35S::KNAT1

mnrain ng T DNA @n6[acl thal inseded ntoorchdgenomeisshoMinFg 1

Flgurc 1. Schemalic sltuctu€ ol35s::KNATlBordar, 35S CalW pomotel KNAT1 gen,":

enlaininq T-DNA. LB, L€il bodee RB, REhtHPT: Nygbmycin phosphobansneEsa; Tnos:

Genomc DNA of l€nsfomant planls and non lransfomanl reE solated €nd

eftdr€d using oIAGEN 9ONA extacdon kit (GmBH, Gemaiv) according lo the danoa

insrtudo^ lbm $e manul.ctuE. Purcd gDNA rbm $e me'sed

iEnstomanl 6xp.nls re€ deiecied by potmeEse chain reaclion {PcR) melhod for rh€

exislance of KNA|T gene lslno KNAIT sene specinc pdmer KNATIFI (5r

CTTCCTAAAGAAGGACGGCAG.3) ANd KNATIRJ (5' CCAGTGACGCTTTCTTTGGT-T'

3 ), ih.l ampl'lied 1.2 kb DNA fiagmenl.

RESULTS AND DISCUSSION

Moaholoqy orlhe shoor and !eav* ot lEnstomanl aE n.m3 as he same as non

vansidmant prant. rhe sbMh Ete of shoo(s) fom tansfomant and non_rransftrmanl

srem dpiani on halt sftngth NP m€dium silh !.rious omenlralion ot grcw$ GsulatoF

L€atnent shoed tlat sene€lly, inducton of shoot tomaion in t€

ihan lnat of non-r_ansfomsnt fable 1).'Itis dab indical€s ihot lhe smenii ot shools rrom

rsnsromant €&lanl may be hdu.6d by KNIII gena .ctvily rhal lnleg€ted in the othid

genoma, lhan lhal or inducl on by srclvlh boulalo6 endosenouslv or exogenousLv

Table 1. Th€ Grcuh of shoL lrom l,€nsromanl.nd non-lranslomant slem erplanis onhar slrcnqur NF Modium and vaious Con@ntElion of Grc h Requlalo6 Auxin and

rhsr6ilme shmL €mea nq dm.elanL (w€r)

Ths raslsr emerled sh@(s) iiom iransromanl srm tas htb reeks afrer explanl

inocularian on % NP medium wiuoul addirbnalsrcldh €guidloE and il NP+ o 15 uM NAA

and o.l5 [M 2iP,lhough n non l€nsfomant exp ant rhe shoots smerged at4 w66ks after

inodlation. Inieresrnqly. when lhe hLsher dnentElion ot srctlh rcgulaloB s€s added inlo

medium, lhe shmt tomation delar€d up lo 6 reeks in t€nslbmanl explaht and 3 reeks ror

.,]

llultlsh@t! pbducllon fon 3ss::KtlATi TEEr.m.nl ftplan!3

ln lhe cac€ ol numbpr o' qhooL p'@Lcto. lne lrdlslomanl elen oddred

munishooE film The hisher number of sh@ts *6 pFdued t'

rr.nslbma axolantslhan fEt in non lansfomant siem (Figr, Tsble 2) MunBhoot

pmduciion @€ aho Gpo'ted by Yu 6l .1. (4), \ten oondrcbium orcrrd HMeoh6\1 \DOH1)

inh_odu@d inlo otohid hyb d t€ndobirn 'Madame ftong In' lnl@duclon of KNAT 1 gen.

inlo some Dicol wss also induced mulllshool fomalion, ie Chuck et ar (O obseded

mulrishoor pbducdon in n6b,?ops6, and NishimuE ea 4,. (6) obLined muliishools rn

roba@ lEnslenic sxplanls. smlani el dl (7) repod€d lhat lhe ert6$ion ol KN,I1 g€ne

Ms lmDrded in a l€.r mlcd ot lEbrdopsit a$J,n€ttc ,eares2 nuianl, lhal podued

munishoors on mulad lea{ dbc in tlat we€ d[ured on homon€ free nedlom

non rranstomanl 6xpbln lt is lnlin. wih our previous d6ta in P.anabilb tlal sh@ts lomad

on lsrdis6 of35S::KNAI1 transfomantlbM on homon_ne€ NPmedium (1)

Figuc 2. Mullshoot romation ftlm stem expLnt of 35S::KrV,4r1 on 'l NP + 0 15 plv

NAA+ 0.15 pM). O I re€k O, sbninq rhoculal€d epant, 2i shool niliation (sm€Eing lear

pdmordia) 6m€ out fion explanl, reek 2i 7i w€6k_7, 9 i w8k_q I I ; Week_1 1, and 131

muhishoots emetgdd at w@k-13 €ar 1mm)

Muhlshools pmducion mig also bo Elaled to lhe amngemenr d endogeneous

phrlohomone blosynfrEsis pathway, such ss drlokinin and qibbeBlic acid (GA) uial

invoted in ell division, cell€lonsaton and shftl rom.tion As d€scribed bv GeoEe *6r

i3) lnar in planr tssue culr0€, during sdvehrif shoor fomalon lne _ncenrEtion ofcltokinin in ce incrcase, but lhe condenlErion of cA lil be decGased. tn tobaccorEnsqenc pranrs. de|Prcdudim ot KNATI proleii suppEsssd lhe aclrvily ot GI2o.r(Mc12) lhar bound io sme sequ€nc* in tho fi61 inton of fie cA2oox gone. Thisftmprgr Gdled L\e synth*s ot GA, in tum ir w]u acrivare ctokinin€ e4Dbssro' o. x\d?, ger€ w

enr I Js al\o ptv@np adt(€. o.rye,erD,esseo AAr, r, gele r he btd-r o,c1d strm

Table 2. Numberofshoot podudion fiom 35S:(NAT1 tEnstoma slem exptad aner 13see6 cutiivalion on % Np lvtedium supptemenred silh various soMh Egu atoE

D€recriohof 1014tt g€nein3ss:rt(NAr L:nsromanrpta,s genome

S6ven sh@rs .r lEnsromanrs and three nonr€.sromanr p ahs were ana yzed toT1 inro irs genomes The genomb DNA o, €ach ptanrs

@E ampmed ustno sp&ific otisonucteotde prtmeG lor &vA,, geres {KNAI1F1 andk\ATrR.i, rhdl Eubd n abdr,2 \Ddmdfeo DNAr€gnanl t orfte' tFe wa,e r.ga!v. ,F,g. r,. rFeee csL,6,nd,Lare

nar ^ryAr I ocno ( J .n,-qrd.eooainhinod irs aoriviry tor shool pbducrion in lh€.e omhids. ushs theso fou rin€s,micbprcpagarid of black orchid ( C. pandLakl en rn inprcued. .s rell as rhs us6 or fEhregenic lechnotogy ror othe. chaEcrer ihprdemenr or lhis ofch d i he use o, ceen

aeilr 961" w' ,aprcve ,re.T d qu€r? i boh r\oo, Tul,p,cab, a, a r,oure"*n(e

lr:nr Hoeefulx ir wi sive beaefir ro suppon borh conserysion ano cmmercar rrade of

, - . - - . : . . =

Figur€ 3, Dereclion of KNAtt q€ne in 35S::KNATI Blacl Orchid TBnstomanls Lanes (a_D)

show tEt 1.2 kb DNA lrasmenr duld be amplfred frcm Tou. iransto

ONA d sestad by Sly I en4me lhat used as DNA size maRer'

lmp@emenl of shool fomalbn ln

KNAu qene has stably nainrained

lransibmanl. Th€ mstDd en be

rndonasia lo' both domestc and

Research Compeinjon gEnt HB XVI

to Bunga Rinlee Orchid N!6ery,

Th€ Arabidopsis Kr"At7 gene can b€ used rof

dicEpropoqaton ot Black ordid (c. pard!6r.) The

iis tuncdon ln seondary shool so$1h ot bhck orchLd

applied ro *le dmmercial pbdudion or drchids in

Tho rcsearch was suppoded by lndmesian DGHE

2009-2010 No. LPPM'UGM/604/2009. We $.nk

Yogyakana lor the gifr of lruil ot dE Black orchtd

semiadi. E.. a. ndianlo, A. PlMntorc, s. lsminingsih N sus€no. T lshikaws Y

Ydhioka, Y Mach da, c, Mach da. 2007. AEobacFnum{edialed tansromal on

orflr6 ld orchrd sDecies Pt./a6napsis anabtts. Plant Bbtechnolw 2 265'272

s€miani, E , A. lndianro. E.a. suyanto, R L NuMlan. R R€siani Y Machida and c

Machida. 2010a. Genelic TEnslomar on of Indonesian Black Orchlds (coe'ogvre

p.tuluala Lind)ey) Jhrcush Ag@hactetun twefeciens tot Mimprcpagaran

Pnceodhss otNroc 2010. Naqoya Dme Japan

5.

F?avd6dqUDtrsbsGadrdU{a4

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1993. conlrct or cbwth and Devetopment otby cadon sftrces in phale*apsjs. Fknt

Yu, H , S.H. Yang and c.J. Goh. 2ooo. ooH1, a ch$ 1 roox cei, rs Requicd forMaiilenan.e of the Basic ptant Architecture and FtoEtTEnslion h orchid. p/antcetl R.pott. 12: 2114

chuck, G., c Lincoln, and s. Hake. 1s96. (NAt indued lobeEd @mensGm when derexprcssed in AEbid.p.is pLnt ceu. s: 1217-1j:ag

NishimuB, A.. M. ramaoki, T. S.kamolo, and M. Matsuoka.2uoo. oveF.kspresion ofbbacco riotud1-ttpe ctass t hm€obox gs,€s a[€6 vanbus teaf moahotosyPknt calt physialogy. 41.15): 5a3-5sa

semiadi, E., Y ueno,H Tsukaya, H. tGkada, c. Machida, andy.Mehid..2001.TheASYMETRtC LEAVES2 serc d/€bdopsb lharr6r6 €sutal€s romalon ol asymheric lanina, esizbtshmenl of venafon 6nd bpress on or nors@m,€bbdhomeobox genes in teav$. oe@/op n.nt.12s: 1171_17a3

GeoE6, E.F, M.A. Hall and GJ. de Rte . 2OOs. ptanr phpasan'n nssu. cdture 3dEdirion. sping€r. The N€henands .pp.1,2 175-1s7.2D5-216

Davles, P.J.2@4. pLANf troRMolv€s airsjzrthos,i sbr.r ransducuon, Adin!.KtlMra*denicplbtish.E. Londoh. pp5ss-ss4

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semiadi,8., A. rndf.nlo, A. Furenlob, N.A. Maiiwi, yM.L. FeronEsanli. F Nrdirah,r.s. Mercu ana, R. Dwiyani, H. rrekaE, y yoshioka, y Mrchida. c, Machda.2oi0b Hlgh-rrequoncy gen€tic l,?nsromarion of pralaerqpsls anrbi,,s orchidusins iomaro exu'ad4nriched medtum tor tle m€{uLruE of p@iocoh. Jouhar o,Haniu u@I scbhce & Biotechn'tagy asg):20r21o.