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Proceedings

2010+ 3F 118 ,

of Nagoya International OrchidCongress 2010

March 11, 2010

Nagoya Dome

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ccncricl.rdsrormrtion otlh. Indondl,n Bl'ckOrth;d

lcoelogn. p^daratuLi^t!:t !) rhtotlh lEtobrcteti|m tu efaciens tor NricroptuplSrrioD

Fnd ,ncsemD, i i ' qk t rgg ' \ umu ld r R 'nhR?{ i ' n i 'I r uNr iN l ' c t ' d r ' Ch i ro ro \4ach id '

r Faculry ol Biology, Univesird c.dj.h Md4 Jl Tcki ika sel.hn, S.kip ubri, Yogvrtrda 5 52 31 ,

rc du{e sctml, Cenler ror Reserch of BioiechnoloeJ, univesits Odj:n MdA Brek, Yogvlk@

rDivision olBiologicdl Sciencs, Cradsre SchooL of Science, Nagova Unrdsiry' Chikua ku, Nlcov4

<collesc or Biotecbnolog md Bioscicr.c' chubu Univesit, KsuBri, 'r3r 3501 Jrpm

ABSTRACIThe black orchid (Coslrg,e ,,iluzro Lindlev) is an endemic orchid fton E ast Kali m d t!'t is

chande.ted by ire hr; ie ol llosen. {ith sreenish sepals rd petals wiih a bltck hbellufr

Recendv. lhe DoDuldtion ol &is orchid *6 decre.scd md its extiD.tion ttuateied to be sxl|rci

b-"*; .f ol;;ftdi"s ".d/oi

hlbihr destuctiotr rhe h.$ !rcpagation of this oEhid is u€mdv

needed. Micspropagarion ol $e black orchid was caded out bv innoducins rhe AFbidopss 8en€xna4"dt tiL" 1a; at tr \o n-o t L\ t t t -'i. lEmbu-mon l ' { l o ad " . ; n . ' \ c . . , - d we I l v i \ ' mea ' r ! Le r u€J w : r ! l

. eb i c " i oN lnc e loeaD 'n r *e -ep ! : l ) Obr -n " rhe oo { s -Eoh b '

" - ,

" O ,. rn "- w*r. \,n\ i\'! Phr"e oo ' \Jl MuEd'ice drn S (ooC MS dnd \\UDqO\

. .K . r i ) . { - . rono rh .o ' p ld o r de r - oD in6 o ro n oa ro eme 'd ' k . ' i dcme i r

mn fmd ido lD ' . r ' d 15s -a \ . ! | dd pc ( . f r pv \edo ' oa '1d r u r -g ' 14 ! r ' iP l

i in LBAlao4 inio orchid protocod. Th. ; Lb show dEt rne best medium for t !,ro culbie of

the black orchid piotocom is a hnlf $tngh of MS (l/2 MS) ncdium lhd could incFase the grofrh

.rte of9.6% pobcod up to phde 6 an.r l2_week cuhivdion Bur after 12 weks. the bcst Eediun

for shoot ind;crion ro obbin p;nrleb n N? dedirq so lhat I I.5v" piotocoms bsone plrrtkts with

rhree leNes. Th. bed condirio. for gene ftnsfer is five month-old pmrocom. The tequdcv ofgenetic nasfom.tion using A bmefa;ieds is32 9% lor pcften vekor ed 4l'2% loip35S (NATI

t r ' r i o ( 6 p . o . - s(!y s(ds: Birciorchids, ci)etoere pnda.dh. bzs\. ncditn, prctocom seneti! hnsrolmdon,KNA T I, A sn baenuu turl ela. ie B

media m necd.d to obbin lhe mo* ruir$h

In othid rissuemediuN for s.ed geminadon sd shoo. inductonm Knudson c (Kc), va.in and wcni (\4v1, indMrashige 6d skmg (Mq wnh rddition of someoqmic cofrpl*es such d coconut tld (Ardifri,1993. Widi,eoety dan Sy!fti1, l99li DemN.bu ddl.. r99SjUnrri d a1,2006). hlafr er '1 0993)used xc, ,r,/ae,opr'r !\rr) nedim ror callusinduclion of Phabenopsis. sennani , al (2007)also uscd rhe N} Dedim ror srorvingPhalaenopsis orchid befoE rnd afrei genehcla$rolmrion of n\e orchid sing,4 rd4a.I.r'

The ftsulb ol or PEvior exp{imed

INTRODUCTIONBlack ornid (Coclosa, ,'ai&ura findhy) is metrdenic oFhid or &e Provifte ofE$. klimmrm,Indonsix, th.t is L\rcaiened by .i.iidion ntover colledioq hlbiht dsdcdo4 md difiicuftisor cul'ivarlon dmlgn conveniional nedlods aEthe chief Eoblem of this oehid (Anliti, 1992)The uniquhss ofrhis orchid tu irs very shon (r-5days) blooninE period md dimcdt Poll'mahon(.Alditi, 1992) Mocov6, ft utro *edgemin.tion ol dJs orchid necds sPecisl conditionsu.h .s l-4 nonL\s incubdim in a ddk priorgemindiotr (wialusmdh, 2009, peMn.lcomunicdrlon) Fo. succsstul cultivriioq invr, seed geminaiion is rhe key se! (Aiditi &Ed$, 1993) In ordft ro obhin $e optimil

pror@om reslltd iD nulrishoob produd'on($od rl shoot liom oe pmhrcm) $ill beu*lul lor rbe mic.opFplgr.otr of bla.k orchidslvl'lrishoor o-nence in KN/\TI taEgenic pldhhas also becn rcponed by Chtr.k d al (1q96) iitmsgenic A bidoprs plani and Nnhmum d,l(1000) in Nicolihr h DendroblDm "Mrdamerho,s ln orchir, Ytr rl,t (2001) obrained thumulrnhoot froft.allus rhal dqivcd ftor cur offprorocoms rhd rmsfded by DoIl1 geE 6:I:,lall,omologous ii Den&obn,n!. F:ch sl'oot could bcrdependcdly gm{n'nro a phndd

llca we ftpod rhc gcndic tusfomlioi of.ri,v/rr cem ud.r th. .otrtol oa Cdl4o"r,L/'vr. ,rifl6 (CrMv)rj,badsitrn ,,q/,.84 sFiin I-BA,r40l nbpiorocoms orbiack orchids for nicioprcpagdroD

\IATERL{LS ^XD IIIETHODSPtbt nakn^ dhtt .,ttne .orditi,'\Irh'F s..ds atrd I modhntd orotocoms of bl,ckirchid\ w.a 6ed

n mkusumh (n\e ovnlr of Edra.d .nd I nsrahis Nmery, E.st Jas) seeds liom rrllv::4idg rflii (rilcDonrhs old in'it) vct soM in1 :rous cultuE h.da Knudson c 0.c). vacin &i . (vw), N.* Phahenopsis C,rP) (hlm .r .r.,::!r). $d MuAhice & Skooc (Ms) in h3lflid:-.:r.uisd, coneitrtioi or mrcmelemc'L\ w1d

.r:i rmsliftd i o rou kinds oro(hid nedim- \rs rP rd Kc merl!m !! rPPen,enlLl

j0 iq poh!, rjo , br l0

:r -rltus *ere in.ubrbd n 2i"c virh 1000-.. :rlhtrous lisbt

: : rd pl.nrl.b was exmred evlry week.

::::,: hn(omd,on of plarmid 35S ]KNAfl:i :!:!.i sector inro othid Ns crded otrrr r : : : : : : 3 rD$emdhodors r f r i d ia . l ( 200?) ,.::::1i rh. liquid DcJim d[i {s uscd ro-ji: ::: crorocom rrs hrlf ilength vw freditrmr: ::, nq I cerolt{m slM (rlio, /'&d-'

:: -- 0lj lrvl N,{{+ 5 $M2iF) supplunenred,r :10 msl Kmdisin for sclccritrgr:::::i: ftrs|o.nmi lrequen.r' orr:Ji:-ra.tr lf,s decided by fiu ruiio or d'e

Rf,J LLTS ,!\D DTSCUSSIONrt . i. t a!- aJ Btaet orc hid

'fhc blrck orhid (c ,",/az'a) is rn epiphrtrsynpodial orrhlil son p$udobnlbs eror pmrtrlift so leavss crch Fivc to sven uoies wereraangld n r rcldq izgnn.e, erch flo\vr tr7-12.d ir dinetr Seprls.nd Ferah G 3r0hand ihe ltrbellm (lit) is bldck seeds aromicrcscopj. Li size, inside the lnir (Fig 1)

D.alrltm at ph^^ ol btoek t.hidTo s[lw. rhs sonh rb or t black orchid.-r,ry.,r*i.c *;d g"*i"di"., *. d*"di.d th.de! elopnetrral penod i o s ix phscs bsed on trgro*tl, phat*, nmelY PhNe l6: $seL/!.llowish embrr'o, phae 2/grcen edryo,Itrsel,tipolr embryo phase 4rii6t lclf rom.d embrvo.plus s^lcoid leaf lodcd enbryo sd phase6,third l€rl romed embryoembryo d*clofmmr obssntion shovcd d'a( ihecoLor ol die embryoyelloNjsh (phase l) inb sreen (!h$. 2) Lbree'eeks rner so*ing.enrbo. rormed a bi|old sructuis bl'ase 3), Nrsone side dtukd than dE oth{. lhc duker pol! oithe cmbryo changed inro leaf primordir (!ha* 4)d the fifri' weeki prorocom ti( so reN$ rIsven Nr.ks (!hme 5) dd prob.orm qih tbeLerv* at ekven wcck (phN 6) (Fis 2)

'rlelve weeks sher sowing, bsed on .hegrcwlh nte ofembryor, rhe d.ra r.v$led $! 1/2Ms nqlium r fi. be$ red'um to suppon anda.celene the groiih r.lc of black othd emrrrvos(rabc I). rhis ws indicded by 3162% ot the

Perc.nbge ofso*ing €nbryo d .ach phd{

Trbh r- Gtukb ofBlEk Orchid\ Embryo itr vmous Mcdim 3t T{dYt w'lc Alter sourg

illFis. 1. Ddelopnerhl pha*s ofTbe B

emerging ftom Lle shoot 1ip. Thde Esulb indicatedd halr-nrenstn.lemens .onraitrins mediun 6 meded lor black

When tre ddedprdNoms s plmi m€dtls, b!$d on $e d1e.!

shoos is NP mediud 1tuee oui or 26 protocomcached phse 5 {nhin eo nondrs of subctrltuieon NP mediu (Table Il). This is consistent w'ihrhe resll$ ol lrlam 4 !r ( 1993), rLo obhincd d\cb*l coddition lor c lus induc.ion on N} nediunIi becaus or n\e nitugctr md phosphde co'reit In

Fis. r. s[oos or rd$tomn G)

T3ble rr Gofrh ol blr.k orchid Pi

Nunbd ofprdmom Erh deEldptrdrl phrk

P M ! € , I P h s € : I P h * " 3 P M * a P h * . 5

of rh. black oEhid (c ,dirirl, Lindl9 NDg lv,tqdenrn

rr:::tr(v.doo

t \ \ " a . r ! t , 4 , t i . t n {an8 ia " t hk k -oPv - cv { r o io $ " $M de B r ' '

--:- ^k,A.-r.^k^ R 1ee or 'o \ '"'v b ftc g'0flo' g l' o' r . ; . i - ; ; "

; : i f o s ( ] \ " I na B3 . . o fhd . , i o ' r . r . d (Gm, . o e

r:---. *.. ,l-r *r li. t"q*-v or srn ot 4 nonlh-old prcb'om fld nlu$lc

;:-!io. i, *I"li'oryh'sh, "b"d

ar,5i" 05 d k*$ i'" " "

bldk orchid 'ulrue cchniques fta_ . : , .

. . ; . o ; - . , r , * : " " ,

s , . nd ! o \ . , Dpo to r . r u , d n , . - had ' " '' ' - . '

" " " * " o P o r D \ f r i N

; - - ' . . - ' ' " , , t s nd i i e ,PP ouo000 b 'v rd ' vFo \ ' I t u r o

- . l l i . * , r . . " " " , r , , , " . j , -d #o ' . c i . r . .po r . . ) . ' o .e rd

-...i*. r,'"-.*.. "tnarl

g-. '" I..b""l'gv (MExr) or rapm sd bv o

- .:=ir i'"ft ".vi--o

-isr,i impr;ve fte Acadenic Fmnti* Pmjdt fo' Fdv*e unwdn*.--?.r'

"i*" *nta

" t".i shoois d ha mabhing tud subrdv aoo MEXT 2005 ?009

a-:::: :1 morh' naturl orchid, Prar.e,opriJ': '..i i:ddi a ar, 200?) Th. ffulb til!iF:. -..i d: coNefldion sffort md othid AJdini J 1992 rudamenbh oforchid B'oLogv

; - '

om\ \ e j&qo ' Na \0 r . - -lo0l v 'oP oPdca'on or

,{crso$ LECDtrNlitr_T orchid Jobr.\vil'v &lmr' lnc -Nc*oYork i;,,

when Oecrxpresed in,,lra6id,prts P/r,r crrr

Crl]l.d, t., 19rz The Archid Baok. C^riindu.Unive^iry Pre$, Gred Bnhid.

Howe11. S. H. 1993 Malnlar Getetj1 afPlaitDevelapne C^nbndae univeBity Pn$

klm, M o, S lchihashi and s MaEui. 19q3.Contol of srortn and dcvelopnent otptutoomlike bndy liom callus by ca$onew.s i Phalaercpss. Pkht Biatechnotast,

!in.oln, c, J. Long, .I Ymacncl'i, K serikaq4^nd s. Hxke \994. A Khanedl Lik2 hnneabareene ii AtobiCapttvegdative nensen md drmalically alieBleav. frorphology rhen ovecxprcssd ntsBenic p1db, Plant ce Re?atus 6:

Nrhnur. A., M Tmaoki, T salanoio, and MMnbuokr 2000. ovq-expr.$ion or robaccoKtuuedt ttpe cLL$ t hamoba\ sena zt\qsnnous realnoTholosy Pk,t cett Phi'iotag

S.oirfti. E., A. hdrianto, A Pwmroko, s.

7 /F'1rit t ^:lE+*L*dE)\l!1 t ti"78 cutosn.Prdarba<'l t e/n'\) l - t"EaW*

' l r o \ ' su ]oDo tuzqh L ingg ' \ t r f l u l u r .R ' r i b R ' \ r i an lEIE#'I" 3l'E+I\+'

rFacu|tyofBiology,UnivesilBGadjanMada,n'Teknikaselah,sek]pUt

:Graduxrc s.hool, cenb for R*cmh ofBiotecbnologv, UniveBibs cdj

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16Fa-ttt-+tF (coeroglhcpaid@raLindley) t:i:'l ,' l: it7A, t <t2 t60)a'/!b)

t . 1E):^Z < J, , F Llzj+tt&.8<q+ ti$At 6 a. F:li. : o t vri. j6*!l*qi I &,1!L1l!d)rirlir. t , <fEdo,E&r.i9f L<t\6. :,)t vo,(r€ialf*eae+w1:h7r" teItr.r1tt Knated|kEAtubita?s^ Lhdliandl.r-NAI|)lB,E+X7 lJF)\t7 \ a !,Vt tI+^r-1..1't,t,-+t \:t frtt\-(, u-trAi:1?;i4/l. id+tjitfl! L<. vscii md wci' (vw) 4iltra l: r t rE*LLTE l . :*r^rd,Hr. i . " : /=- ln &o+t+&'nx 3 dE.r\<1i- l : . F t . .5t€-r:-ilt (wf. Ne{ PhaLaenopsc CNP). Mmhis. skoog (Ms) . Knudsonc (Kc) ) t)&;'1. fr:r:. i6r;+g,\|:&i6olE l''-r.o4:6RBd)&;1. H=t.' A hthelaciet:t:E^aaa4 ER. ' -7a ta L \a l t 7- | 35s-KNAr l t pcBcn ^2 r -o)4) ' t l i .L"J , t , n '4, id)7o l' : - 4Jil* oFjErSlg lr l/2Ms art-L. 12jaru')Bj!t96%0)/d l':_rnr., =4 16 9),Lt.rr-t . lh'L.\2:67AUe')'r MlroEHl- l: NP Erl!,rftEta 9.1L'5 o/o

.7 = | =- L/ti 3 *Zn'/Ft Lt. fiE+€7.l.Rida7E l': - )'t:! 5 , Br}&t'r-tro'a&,'-, ,EE?+^r+tr pcreen ^2 4-E.L r(tr 32 9%' p35s KNArl t"!)trr432%r:'r'

kmirirgsih, N. Susho, T Lhikdd, Y Yoshioka,Y lvlrchidi, c, Machid. 2007.Asrcbacbriufr-ncdisted ftnsfomliomr thewltd atch\d sp..ica Phalaenaps h anob itis Pknl

unhr| R {d D w Puspiianinstys.2006Peogdh Brhan organik dan Nd{ rerhrd.pPedmbuhli AngsFk Hitm (coelo$aeprdunlr Lindi) drlam kulM in tu.,,bdr6tr6 vol.7. No.ri 314-343

Dem*bu, Sofia, B. Dodo, do D Kojoh 1993Penss@sn lmban Air Kclapa dd BdhoSub$ilusi Aw padr kultu jadngan Phss,kimn, dm agerekN5iondt sai[ dat LtnataRi I, haggdt 17-tBNov.mber 2003. UniveBita Lampung.

rvididtEty, D. dln slaFil. 1994 Pctrgarh AirKdapa erb!&p pembenbkan prctocom l*cbodies (!lb) dri angstk vad& dalm nediun.dn. Junat Holtilaltutu 4.2:7 | t3.

Ytr H. s. H. Yrg, and c J. Coh 200l,{sf ,ro.i.f i(,medirbdDedNrir orchid th rhe da$ I trur g?,eDO t Pknt Cell Repart 20:3ar'345