IankuYl'. E .. C(lm s, M " Li ~ Il . J .. l~ob!! \'a. 1'. :L il e! Tmllc().~() , A. (2001 • S<!(:( Só. & Tahm¡f. , 29. 77-482

In vifro p ropagaLion of Angelica pancicii Vaud ., an endungcrcd plant 'pccies in Bulgari a

E. TAN OVA'. M, e . NTOS I J, mAl" 1, P. ROBEV ~ AN D A. TRON<..XJSO ' e

I InSlinlto dc RCCIlr!\Il. NJlIUmlcs. ¡.\ gr IJJ()lugill u~ Se " ill¡¡ (IR, AS). CO)n~ j ¡IP 'fif)r d fll\'C: li~~ciL Ile: C¡~I Il (íic; (C le). p.O, Bo~ 1O~2 • •• 1 OXO, ~e" iJl IL, SJ1:lin (E· mml : lroncoso@irn¡ ·.c~i c,c~)

~ IllS ~ itu l ~ u [ [).u-tilll • BA 2~, }\C,I" , G, ROllctLcv :SI rcC[. 1 1 1 3 Sn li a. BlIlg lTÍo

(ACt~epted () .wfJl?r 20()())

lImDll;l '1"

1\lIgdicu ¡NU¡{,'ici l (V:md .. . ) i nn c lldcmi • c nd.ungcrcJ. pm lL'Cloo s~d~l> in Bul ~aria. hi~ ~inlit li o ll i, l11:¡jnly dlLe lo lh.c ..:xccss r hal've '.illg 11IIel l)) - eli fli c ll ll i sor secd ' crllli llalio ll s nd p i¡LIlI plq}¡l ~~ti n. ~OO ' from 1I1<Llure f ilS h:¡ro .. esle~l ill d irrC'rell l <=¡js u f BlIlgaria were U ed (lS plmu m;\lc ri lll , U L1CT ill .~c Y i I Jc (Spain) lh~ sCLxI ...... erc sulJj~cld

10 d illerellC germilmtion mcChnd~ mul lhc ~ccd l illgs l11ulti]:l liGl1<!d in l'¡,'TU . • C~u.!i dkl llol gcrmi Jl~I I~ IISi Elo g [muilio Ral ílCfl11inm;oll mClhod" evcn wbell s(!cd cnld .~wra1('~ (4 nmlllhs ~1 4°C)

m~d GJ\ • . applicatiolls wcre m ed. ombinit:lg sccd co ld 5.10rllgc (:l nlQITI h.- JLI . ~ e C) :and irl Ilitro c:u i lun: cm I i3 J\ot JIlc:d i una ""ill l 30 I ng, l" of G 1\ 1

o h i8h leve! uf g<!l1nlnlL~ ion ",'¡is t,en ' hed (63 ~ ), '/J ~i tm cul t llre (]f L ll l¡llcd c mbryo.!i was a Ihiro lll ..:ll lOd 01" j;:CfI11inmi n ilwcsligalcd. Tho ugh lne gc rl11ill il liulI

le>' 1 wHh ¡his Il1c lhod ¡lid nol n~ercome lhll l o f [lit: .'/7 v¡lrv ~ . ll1in¡u ' ,1 seed~. g m1in~ l i"n lim c W3!> c1c¡¡rly decre¡n~d ,md s d oold s1 q ge. was Im ncccs ¡Ir)".

Whole p l llll l l e.L~ (87%) were regeneratcd f m rhiwllUl $ '(Uing e pi mIs hy culluring ir¡ pirm. IIrsl on MS med.ium wilh 2 mg.!" 1 BAI' (rnult iplic . ion !mdlJlcn un lh~ S' 111": IJ l~dium witlL I m~ , l " KAA roo1illg),

Aflcr 60 (b ys IJf in '11ro {;uli ure. bOlll s 'd li l1g~ JlIlcl pl~l11 lcl , we re SlICCC. sfu ny lransplllll tc!.I ¡¡ud h¡mle neLlI m id e fld ili n~. tTol11 which wdl dC~'eI()J1ed ]:Ij¡lIl[ ~ Were obUti lled ,

• n trodudion

Accnn.l ing to S lojallov cr a l. (1967 , the Angelic(J gcn ll .~ in Bulgaria is rq)rc . cnlcd b A,

J)J¡ ' e.~;tri.> L. A. pandcii Vauds, and /1. /l . hal1gdica L. Among t.hem, A. ,ytvesfris i tlle most

ex tended' ¡:\. al ¡lOn.~efi a is pracüc:J ll y eXlinct anJ A, pancicii i an endemic, endull"ere(i, protected spec ies, on ly growing ar prese lH in area ' at high aH itude in lhe lOp of lhe Bulgari an mOlllila.ins,

Tbi ... itualion can be n:lah;J lo twO factor: i) lh ex lve barve ti ng fo r use as m dic in:J.l

p lant. a nd ii lh dlfficuhie" of 'eed germinalion anu plalll propagulion. AJJitionall y, lhe

eX lended U e 0 1." oil f OI" a g:ricul ture and urhan izalion, .Ii re. an·d conlamination has redllced the gl'Owing are .. ~ fo r Lhe. spcc ics, cOlllribuling to Ihis regre. 8.101l ])l'Oce .

U ntil no w, [he main re. eaJ'clle relate d to AJi" lica pla tl ts ha ve hcc.n conncc lea w'¡ lh

produclion, extl'action and llS ofth i r chenl ical produclN ( KI::ITo la a nd Kallio , 1994: l3emard

and C la ir, 1997 .

... A UII!t1T fUT corruspuJlllcnL;e

477

E. IAN t;.OV/" r.l ¡,,;,¡ros. J. utQ.-\ : . 1'. R Il!!\',\ ,\ ::-''D ,\. TRON OSO

The objecliv S r lhis . ludy wcre k lcmify mcthoJs lO irnpwve th¡; s ~~J g~nn i llaLio n ano pl¡lnt mu lti p li c,11 ion 01' /\Hj?elica pancr Ji in nrdcr lO pr ~ Iye thi s ' ll dangeJ'e 1 spede .

l\¡latc l'inl Hnd mcthods

Seed. 1 mm maHlre f l1.l ilS harve. lcd in Ll iffcfCIlL arcas of Bul gar ia w~r~ uscu as phlll! ¡Hal~riaJ.

LaÍl.:r in SevLlIe (Spain). lwo grnups of ~ccd s wer form d, one ~H"II' d .. H 4°C and lhe otller llla.irHai ned • t rOom tcmpcrnlu re.

AJter four monLbs nI' ~wrage. 1800 ~eed~ of eacb g roup 000 per every trea~l1)elH) We l

sow n as fo ll ows : i) in germi na tion dish..:: .- wi lh p~rii tc-pca t ( 1: 1 y/v) s uh.~l rale m 23~C; i i) in Pctri dj she. w i lh fi lter paper sonkeJ rC. 1 e lively ~' i th \Vmer. KNO~ . O.2M . 01 LLtion or gibbe/'cllic <Id d (GA.l) solmioll al fom <'Ji f'feren t OIlC II tmtlon (2. 10, 20, 30 ~lg l ·I ). The Pet¡"i dishes we re inCllhlHed a l 23°C in Ihe Jark.

For the in ,..¡(ro e ! el'imelHs: i 240 seeds from ro fIl. st rage ( 1:: I el' IreaLmell L) muJ o Lhc r 240 arler onl y lWO month: of co l I ~tor.nge we l tl5;ed ;¡nd cuhured in 1_0 x 2. mm le. t tube wilh 1/ streug th MS mCtli urll (Mllmsltig~ mHj SktJ>l)g, 19(2) lUid adJ ili l)[IS 01'0, 5, 10, 20o!"30 mg.l ·1 of GA~. resl cC l i I¡' ly~ ii) Thc : ame Ilumbc rs f ísolalc I embi'yos ","cee cu ltll red I1l I'ilro 011 Ihe 'ame m dia a: abov . Culture ' w r ke pt in a growing hmnber with cool while nl,lorescenl ¡ubes aE 30 ~F.n"~" · 1 0 1" illumina l ion, 16 h li <rilllday :l[ld -3 ... 2" 001' lemp¡;:nlWr .

Fo r the Pl:1ri tl i.' hc, anJ in l'itro g:cn niliali }nS Ihe sectls were prcviulI sly di s inh:t: Lcd through ¡¡ 30 s ~r atlllent wilh 70% ethannl, fo ll ow J by g ntly .sl'irring in 40 ¡ solution off sodilll1l hypoeh loriLc (7% at: Li vc ehluri nc) plus ~Uln~ tlrops of l \\'ecn-20 for 20 l11 in at 3S"C. Fina lly, the ~ cds W ' I ~ lboroughly rinsed w ith SI ri I isüll ated wal r.

For mbryo xtnlc tion, ¡hc disinfee IC(.1 sct'ds were pl:lccd in Pcl ri d i~hes w ilh ~leril i7.ed

w~ltcr amI incuoatc d hll' 41-\ h in lhe dark, a l 2. ± ::!()<'C, calls ing the sccds 10 swcll .. Emhryo isolw imi wa_~ perfUrrilW u nJe r a bi.lIocular mieruscu¡ e in a frcc:-g~fm cnv ifOli rnCI1 [ by cuUi llg

off a latera l el¡on 01' th enao penn und fre ing ~he em )ryo fl'Om th r mai nin s ed ti sue wi I..h lhe sharp c no ni' a sea lpcl.

Arte .. 60 d a}'~ r ill ,'ifm culturc, p,irl f th 10m ti se ~d líllgs was lransplanted lo e -\l itro cO lld il iOl1s . From Lhe u lhe r part or Lhe scc.:Jl ings c.:xplan ís nr rmH , rh i/.omc.: , .~km . shoot, l e ~l f

hipocoty l amI co ty lcJ on w¡;r~ ase pti ca ll.y prcparcd and cu llLlr~d 111 vi/m in 120 x 25 mm lest

tube. irsl. u[} 1'.,.lS IllcJ iuln with 2 mg.l·1 01' BAP (1 ro pagalion) aud then on MS m 'Ll ium w'ith 1 mg.l·1 of NAA (foo ti ng). Th . nC l1ba~ íoCl ond i.li OlL we l'e Ih ~ "ame a indk,Hed foe lhe Íll ,'itro ge rm i na L i 011.

Thc ilJ l'itro devcJopcd sccdJ i ligs <Hitl plams W 'fe lJail~planl~J to oul:i Llc condi tioli s by harJ eni ng in 300 ce pOL.- wi th pcr l. i lC-p~al (1 : [ v/v ) .. u b~lratc. t:ovc.rccl wi Lh a tranS IXLn~nL

plas lic bago Evc ry ':'-3 dn)' 5. a picccof thccovering bag \Va. cul. un til [he plruíli s fin aJl}' in ru ll e ntac t with lhe gm ing chamb l' cnv ír nmc l 1.

Th sial isti al compari:on <llllong P' rc~rllages !l oe b n m<ld u¡:: ing Ih> I-Swdent te"! aecordi n g to La lTIoJl e, I 976_

7'

IN VITRO r~ rAGATlON 01' ,·l1.'G~"U ·,11', ,Ve/GI,'

R ulls and d íSClJs s íUfl

Afler fivc monlhs , [lO scodling~ develope<1 frolH (he 300 seeds, c.hil led m nOL, sown in [he gcnninalion di he wi~h p rlite-pL!:lt subslmLc. This result i m.l it:ated same form of eed donnancy a r I o lled for otlleT planl spL!c ic. (MiLmko and Diamallta!!Joll, 19 4; Jinb :Jnd C i 'ar 'S ,1 9Y7; ia amI KcrmoJc. 19(9) aJld 1hm faur monlhs ni" cnld s torage were i I1SIJ ITiciell1 lO break lhe dorman y.

S imilnc re ul were obla ined in lhe PL!lri-Ji sbc~ cxperimelllS, Only a f w 'old '-Ior 'd s d .. slat1ec! lO gcrminaL ' bUl lhe qllickly slOpp~d nrowill g alld d ice! , indicaling lhal cok) Lre<1 lmenllo" lhe r with Lhe pr SL!JlCC nI" GA; did 1101 pIUVUk.· seed gennination.

The re ·ll lts 01' Ihe ill vi/m A. {JllllCic ii eds gel'minalion ar shmv n in Lahlc l. ThL! chillcd 'e ds 'howed bL!llcr gcnnill¡j li Jl cate in every tr al111 111, ah hough Ibe Lime 01' culd . 1Orage was unly l \Vo mOJllhs. In b Ih gr tLpS o f sceds (e hillcd D" not) lhe ;:Itkl ition 01' A~ to 1he med iurn íncrea ed ed g nninahiliLy in cOTltra~L lO 1he Petri d ishe . . ' ¡pri lllen ts. Thus, in in 11;"'0 condit io n.s, cokl trcatlllen! plu lhe :lddition IJf GA} \VeTe d TcCl ivc in i l1'IPToving seed genninmioll .

Tahle l . !'>ClI.'tll ings nf 11. fJmrddi f l tu ir! ~'irf{l g~nllj~:\I'd llnd c ul11lmd «(}J I dlly.) L ... xl~ .

M ~<I iUl11

MS 1/.

MS 113 + 5 ni '.1"' (j ¡\ ,

M 1/3 + lO moJ 1 (lA ,

MS Ir. + 20 III'J ' "\J

MS In ~. 30 1Il ~.I ' GA ,

•• I rr'I1m N~ (Ir s~~Js

48

·18

~

S

~

Roulll s1(J rll~c S~~d rn lit % ~ f ~c d l ¡flg~ · u ()f SCL~h

( 1 :1'\ 48

l h .IH

JI RI'I .:1R

3(, ll B 4 ~

48

"kl ~1<' r;¡gc r.~. ~ 1 f ~~ct!d I i n;!!s

t ~ ill'l

_ o hU

SO hB

63 bB

,:! B

Di rfN~1l 1 I ~l[e ind ica.le signifi ncc :tl Ihe p fU)l le"cl; srrulil l e lle r ~ illdicme differcncc5 ~1ll01lg ~.oragc CI1llo ilillllS; Gll' iral l euc~ · i tl.dica[~ diffcrcn s among trca(ItI': n[ ..

In n e qllence, Lhe eornbinaLi on of ¡j lwo mon th<; cold lrcallll Ilt :lIl ) flt 1'irm cullurc 0 1"1

J/3 !VIS mcdium wi Lh 2t -30 mg. l ' of GA} r ~~u ll ' ¡j i Tl a higb levd uf A. fJlIIJcicii _' eed gcrm in al io n.

Th~ in l )illV lIhure 01' i olalea ' mbryo~ as a !1I011O iml rove and sil rt ' Jl lh i! rn inal ion proce ..• ha b n suceo::ss ru lly uscu ror spt:cies like )le Vo ... I man Ira, rose, nlivL! am) ju nipt.:rus ( hee, 1994; Pl urnrn~ r el l/r. , 19 5: L¡wzer ami Lab rg • 11.)<)6; Acebe uo (!! al .. 1 Y97: Canto el al .. 1 \,19 ). h r . lllt s of Ihe in I'itm gL~rlT1 i []aL i( ll1 lJ f i ~u l a¡ ' d e mbl'Y s of A. p(IIwirii are bown i n lablc 2. Nu ~ j gnij'jcnnt cli fferenc S w re found bCL\Vccll LhL! g~ rm i l1aL io ll r'Jl~~ flhc

embryo.s rrorn the two gr lIpS fr om or 'old slorcd} indic¡j! ing that the e Id tomge of Ihe SCCl.b bad /lu je inft I n ' on Ihe gL!rminabi l i Ly 01' lh~ isu lateJ embf'y s. Thi . j - in agree mcnl

\ iLh Istal1lb uli ancl cville ( l977 a utl Acebedo el al. ( J 997) r s~ l hs. Wllen 110 CoA , was a lelecl tn Ihe 113 MS I11cdiurn. [he i. olal d mbryos g nn inalcd oclLcr than tbe seed . which Illay bc J'cl nled to dorman y nssnci: llL!d w ilh thL! . eL!lI Li ~~lIes, N~ vc f'the l , , ¡he p re 11 :lge oF g rlll i naled emhryos i Tl thos' cundilio ll WfL J'mh r 10\1.', hlJl i Tl Tcas¡:t! w ilh Lile <ttlLliLi ( Il~ oC GA ; Lo Ihe med illJl1. fea hi ng si mi lar ruLes 01' gcnni nali il lo lhose btained fár lit . scc.ds

479

M' 1/3

MS 1 f3 + 111 m~ ,I' G/\ ,

M llJ 2 mgJ ·' CA ,

MS 1/3 + )0 nI!lJ ' GA ,

s~~u rruJIl ~ uf ~.eds

~

-IR

48

-18

4

J{ . 111 ,1 ra!;1J.

(,il ()f l"'".C.."'CdIJII~':p.

2 aA

~O ~ I l

5 all

52 ~B

r,o aB

:S~~d ¡'mm L,)ld ~Ior!lgc -u "f ""L'f.l, '" <1r scccll i Il!l~

48 ~5 nA

<l ~ ;;n ~B

4x M uB

· I~ :U aB

~I :-;: ... aB

Di ffcrc l1 t kLle", ind ica¡e ,igninc!IIJcc ", [he l'= fU ¡1 k; 1; .111:111 le l cr. ¡ndi _'le difr"re nees among 1U¡¡¡g· e ndi¡i n~~ 31)ilal kUcrs indi",,¡c difTcrent: ;~ anlO,lg 1I"~llJ)lCrl1.,.

chillcJ ror 2 mUlllh .. (LHb lc 1). This similar iJ1l'ifro germ inat ionl ve l 0 1' i\. pancieli ed and embJ'yos, i nOl lOlally in agree men l Wilh ll w rcsl{lL~ uf Ac¡; bcuo el a l. ( 1 ~ 1,J7) working wi.th ol ivc and Call1.OS t~{ c.'I_ ( 149H) wilh junip 'l1 ls. WhC1 illd i .Hc{1 a h lk:r re~p(ln!'e of llle is laled

embry , In vilm seed ami _ mbryo g rminat ion limes on 1/3 MS mediulll plu . 30 mg .J -1 fGA] wcrc

comparcd l'ig Llrc 1). tl2% uf emnryos fOHn el seedli n f!~ arter only 10 days fr m s w ing. N fLl tther genlJi nalic)]I was ubserwJ. Sccus starlt:u lo gcrrn inate a l. day J O rmm ~()\ving am i

I'egui l"ed 70 day o f c ulture [O J'cach a si ll1 ila r lcvd uf gc n ninalion lo Iha L of lh e c mh ryos . [sn llllion 01" clll bryo~ c lca rl)' s ho l11t:ned Ihe germina l iO¡l ti m ac o rd in g (:l (hat previ u. Jy r 1 l'led by Randolpb, 194 : Voyi<t[ i .... , 1965 ~ Hu. ¡ 975: Di amrllll -glou :lIId i\'1ilnllms. 1979; Cañaset al. , 1987; VuyialLis llnd PriEsa, 1994; AcchccJo Na!., 1997; Trnneos 1'10(. , 1997; · or

oLh r plant specics.

90

~ (: lnhr.l'\:'''' --o- ~I!d ...

·1

:11

.§f

.-< "'C ,' 11 .'" .'" .~

~

11)

Da_ ..

f igl'N 1, P fC~nLagc ;l lld ¡in.1 f Á ¡,-",ddi >eedl ing8 fOrll l;lqi [1 fmlll cmbryu '" ,~~tI, c lIltumu ;1.1 ','; Im 0 11 1/3 MS

l'Ileu i [nn plll. 30 IIlg, l·' u f Gl \"

The e p.lal t oot.lin d fmm lh rhizome or lhe Ü/ ~·itm scedling.· \Ver~ Lile u nl)' lis,>uc abk to fonn p l,lIl11 els \",'hc l1 cultun:d 111 '1¡ro, r~~lt: b i ng 11 '7% .. Thu . for Ihis expefíment n litlons lhcrc WaS a ve!)' diff r nt b~hav i rol" lh rhi7.0me e;.; p lanl in rd aLiol1 lO lhe uther un 's k~ Lc tl.

fI r 60 days nf hr ~ ri!m cu lture, both sCl:dl iug.s :mJ p l a nlJd~ were :-ulli ~jent l y lI eve l p~ 1 to be lran ~pl anled lo ollls i d~ co nditi n, he in "';'¡ro 1 [,mt material r , i ~ l · d P rfecl ly [he lmnsplam ing and hardcníng pro swith 25 le loss ol" plallts. In cO llsequerlcc . combi ning se~J e Id stora" 2 mnnlhs al 4° ) ,me.! ilr Fin c uh n: u n l/3 MS m~dll11 n wieh JO III~. I · I 01' G . ,1 hi gh level nI' g.erm inat iü rl wm; reached (óY,iJ). h J vifruculll1 of i ~f) l a l d mh r}'n~ was anuthcr im resting m lh el r g .. liDa ü n, ¡\ lIhough (he ge rm ination leve! wi!h th is rnclhod did noL ovcrcmTI~ tlmt t r lhe in l'i//lJ g mi l1lled s ~d s, lhe ge rminal ion li me wns c lenrl y dccl'~a. ed and . eed cold st rag wa' unn ~S'l ry,

\Vh )Ie 11 mi ts (87%) we r regeneraled fmm rhil':nma ~l:~J l i n g cxpl<lnts by 1:11ltLlJ'ing ÚJ

'i l ro, l1rsl nn rvlS I11cdium \vit h 2 lug .J"' BAI ( IIm l ( i p l~nl l i( n) ana lhell (1 11 I ~ sallP med iu m \>vith I mg .l· l NAA ti' uti ll g).

A I'te l' 10 lay~ f ¡lo Llirm c lll ture, h )Lh scedli ngs am.! p lanlk ls W~l'c ~l I CCe1>S tln }' tl'.u1S­

r~ ant -.d and ha rd~n~d Lu ou lsiJ . e ndji j , n~, ohl.li ni I g w ,11 ti v lope l A. flonddi planls. Th • combillill l Il 0 1" pr c~(hlres is prnposcu as :m dTc chvc IlIc(hod r .. Lh e rwpagal it n of A. p mdcii p l'1 111S.

nel'enmces

A el _dL , M . 1., Lavee, S., Llluin , J. anJ TWIKlt.W . /\. ~ Ii) 7) . IIj n/m gtrTn i.~:\ l iiln nr clll ll r~' . ~ IOr pccclill" up sccd! illg d~ ~'c l pm nI in nli\"l! ¡'reedLmg pnli!rmllm~~~ .. SdcwUI l/IJfEJudrw(R', {jI;' ZU7- 215,

Berniml. C. iIllU . 1 ~I.i f, ,( 19L)'1 , E~~Cll l. i~1 nih ~r 1 hrl!l! A rJg.'/i,'tI L. ,pc-d~s ~rtJwin~ i.n rnull·t •. 1. Roul ·ik J-es:>em­il- ',. C~m l Slrc Ull, HI.: Allul\~J ] 11IIi ' hi ll~ e '11' J~ I! i ' 11. M~I~nu n" 1.1 , '~8 ;2(M.

<l nIOh, 'l. . ~e ,'as, J . . 7Jir;il('. R, ami 'fronc()sn, ,o!¡ . l' i'JL)i'i ). ~ Ill >r~'H I"(! "~l"l l ~ <Jml dc ',' ~ lulimetl l ,)f }¡mr¡rt·nl.l" On"'Pi/nl.\" ~llb~[I. ax.\· ednt, anJ rmrcmnrr{>(l. St' t'd Su'('I¡n' ~'lffll¡'('/IIl(/I.Jg.v, U" 19:1-1'J1l.

Cu ,,~ , L,A" Clr l>lmn li llCl, L. :LI1,1 Vie~Il I ~ . v. ( 1 ') IJ7~ . V~gd:lli,'~ I'rup~~g¡Ll i"n t lf uJi \·~ 11 ~ , f l llll l ¡" ,·ir"'· ,' lIl ur"d

~ ll.Ibrl s. J'hmJ ' ·wlr' ..... 5.:1, (,~ .7. Chee, 1-' ..... (199-H./1l n'm! clllw r.' <J f ly~QljC ' 111 ry ~ fl f "I",)X 11. spL'<.' ic •. l·h)rIS ' i ~n~~. ' 1 ';o;. ' ndri:l . ll.: T/wAm",-,'cm:

S" h'fy .I(¡r IJnnimllllnd SCÍ<'lrL·(·. JUtlC 29. 695 7, D i¡UI1:IPr0/J-loLl . .'>. , MitrOlb' , K. ( 191'11 . • u r 1" l'U lUI"(! fr¡ l ·ir/·/! (! rC lll hIy 11 J' -l]jv i ' r j ().!",1 ,' ,'tr(¡j'I"I,'(J l. .

l'llI:oIal.,·U.'rJ, c'R. 1\ ~I d, ci l';¡ris, .s.'r, 1). l.NII, 15~7-L el. 1-1 u, '. y. ( l' 7. ). !i: L'irm L·u llLln.: uf rud i II I~JIl ¡¡¡ry ~III br)' . ~ l'f el~\' " n 11" 1 "'P ' ·i 's • .r. 1\ 111. SOl'. H"r.'¡' ·. 'id., I.~J. __ '1-

225, 1 st. mboul i, A . iIllll Nc,'i II ~, P .• 1977 . Elude o.Ie la "'d:'lffil,11l ' e" ti l::\i ~ rnL:n 'e~ '" (,I jo" ieT i {),'m , 'w·"p • .,. L. 1. Mi~~ ~n

~\';dl! lle~ (j' llnC dmlllmIL'~ ~mh~)'mIl Lirc. C. R. Al·~d . P¡l.Ii . ' I'i .. D. 2114 , l~flJ ~:;(l( ,

J illk.s. R,L, uwl i :!re, , L. f 1 91 J. Emx:t~ n1' s (' iLkill~ , w"shlllg ~mJ W;1rI 1I prcln!" lrtl~ l l( l,n [ht: gel'mjJI~Li n 1:>[

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R:Uluolph . L.F. (1945), Embr)'o culWre or iñs see<l , N . Ga't/élt, 1, 24 1- 246. Sloj~no • N .. SLefano" , R. and Georgiev, SL (1967), Fiom rr(l Btdgtm'a, 852 j)

Tronc()~() . A., Z:lmtc. R .• 1Ile! CanLll . • M. (1997). C'>n,~ ..... mLilln "iJl i" )·jlm PrllP~8~liollof cncbnge~ ... l 'pedl!~ fTom GmzaJemil N"luml Park (eady resull l, Ú¡g(U'C{(UtI, 19. ?03- 71O,

Vuyiutz;'" D.G. ( 1965). DUrn'lilllC)' ~"cl ll"rm;lIil1 i" n "f oli"c "lIlb'Y0!i ¡" affccLeJ by L""'I'"mlurD . PhisY(J/' p/(j lrrf!rr'¡'I. 9!i, 444-44·8.

Voyiatús. D,G. anu J>ri tsa T, (l994), Theo'USel and dis~ppe. ranccorflllmive dOHllancy of oli\'e embryo ~s aITecleu by ase. Acta lIonin,J/llme, 35'-6, 14,8-15 L

Xia, J.H. "nu Kern'.udc. A.R. ( 1999). AIl.lysc. Iu dClcrmi"., Lhc role of cmbryo i"'UL"turiLy in úorn'l"ncy Illllinlo,".nce {lf )'cUU'" cedO( (Clrm7laeC}'¡Nlri.\' "",,/k{j["'.<Í.') seeds: Sy nllmsi, 8",:1 acculIlulaL;oll uf 'lumsc lirúLcills ,,,KI proLe;'i' implicated. ili d ·si.",,¡li·o¡llolerJnce.. J -~XIl· bOL, O!l.for·d: Oxforo .,i\'crsily Press. Jan. 50. 107-[ Ilt