Title Induction of Callus from Mistletoe and Interaction with its Host
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Title Induction of Callus from Mistletoe and Interaction with its Host
Title Author(s) Citation Issue Date URL Induction of Callus from Mistletoe and Interaction with its Host Cells Fukui, Mitsue; Azuma, Jun-Ichi; Okamura, Keizo 京都大学農学部演習林報告 = BULLETIN OF THE KYOTO UNIVERSITY FORESTS (1990), 62: 261-269 1990-12-20 http://hdl.handle.net/2433/191963 Right Type Textversion Departmental Bulletin Paper publisher Kyoto University 2 6 1 I n d u c t i o no fCaH 耶 f I 加E 路 s t l e t o e a n dI n t e r a c t i o nw i t hi t sH o s tC e l l s MitsueFUKUI,JUル i c h iA z UMA,and K eizo OKAMURA ヤドリギ@カルスの誘導と 宿主細胞との栴互作用 福井充枝・東 1 員 一岡村 Resume A 回l1u s was i n d u c e d from m i s t l e t o e (Viscum album L .v a rl u t e s c e n s M.), a nd e s t a b l i s h e d on a m o d i f i e d Murashig e Skoog (MS) medium h a v i n ga h a l fo ft h ei o n i c s t r e n g ho ft h eo r i g i n a l MS medium and s u p p l e m e n t e dw i t h1 .0 ppm o fk i n e t i n and 1 0 . 0ppmo f1 -n a p h t h a l e n e a c e t i ca c i da t2 8C u n d e r1 2・h rd aチ l e n g t ha ta b o u t 2, 5 0 0I I 工 X . 官官 c a l l u swasc r e a m i s hp a l ep i n kandm a i n l ys p h e r i c a lh a v i n ga v e r a g ed i a m e t e ro f7 . 5 μm. 百1 egrowtho ft h ec a l l u sr e a c h e damaximuma t 3w e e k si n c u b a t i o ni nPLAN 1E X t og i v ea b o u tt h r i c 氾 a mounto fd r yw e i g h t . Thec a l l u sc o n t a i n e d two 郡 l a c t o s eb i n d i n g 日 !c t i n sh a v i n gm o l e c u l a rw e i g h t so f7 7, 0 0 0and1 9, 4 0 0w h i c hw e r eo r i g i n a l l yo b s e r v e di n 1u so fm i s t l e t o ewas i nc o n t a c tw i t hi t sh o s t t h el e a v e so fm i s t l e t o e . When t h ec al b出 c hc al 1u s,b o t h凶 l l u s e sweref o u n dt ogroww i t h o u tc o n t a c ti n h i b i t i o n . 0 欄 要 ヤドリギ ( Viscumαlbum L .v a rl u t e s c e n s M.) から、カイネチン1.0ppm,ナフタレン酢費量 1 0 . 0 p p mを含む,イオン強度会半分にした M u r a s h i g e S k o o g(MS) 改変培地を用い, 2 80C、 1 2 時間の明暗府知,光度約 2 , 5 0 0 1 u xの条件下で、培謎し,カルスを誘導した。カルスは淡いピンク色 1 で、あった。また,プランテックスを用いて, 3週間措援した で,その細胞の平均間後は約7.5μm とき,その成長識が最大ζ l述 し , 絶 乾 畿 が 約 3 { 告になった カルス細胞には,分子・議 7 7, 0 0 0、 1 9, 4 0 0のガラクトース特異性レクチンが見出されたが,これらは,ヤドリギの諜にも問機に見 1 1 ¥ O された。一方、ヤドリギのカルスをブナのカルスに接触させたところ,拘カルスは接触 i 院議を起 さずに生長し統ける乙とがわかった。 1 .I NTRODUCTION P l a n tc 巴l l sa r e known t or e c o g n i z ee a c ho t h e rb ym u t u a lc o n t a c t and p r o t e c tt h e m s e l v e sa g a i n s te x t e r n a 1i n t r u d e r st h r o u g ht h ec o n t a c ta n df o l l o w i n g ‘c o n v e y a n c eo fi n f o r m a t i o n . Ak e yt oc l a r i f yt h i sr e c o g n i t i o ns y s t e mi se x p e c t e dt ob eo b t a i n e d from 2 6 2 a r a s i t ei n t e r a c t i o n s . 1 nt h ef i e l do f woody p l a n t, c h a r a c t e r i z a t i o no ft h ep l a n th o s tp m i s t l e t o eand i t sh o s ts 田 町1 t o be an a t t r a c t i v es t a r t i n gm a t e r i a l sf o rt h i sl i n eo f i n v e s t i g a t i o n . Althought h ep h y s i o l o g i c a l l ya c t i v ec o n s t i t u e n t so fm i s t l e t o e,i t sn u t r i e n t i t t l ei s r e l a t i o n s toward h o s t and anatomy o fm i s t l e t o eand h o s t were r e p o r t e dH 2), l knowna b o u tc e l l c e l li n t e r a c t i o nbetw配 nm i s t l e t o eandi t sh o st.官時間f o r e,i nt h i ss t u d y wei n t e n dt oi n v e s t i g a t et h ei n t e r a c t i o nb e t w e e nm i s t l e t o e(Viscumαlbum L . )andb e e c h (Fagus c r e 間 t a Bl . )i nv i t r o . F i r s t,we t r i e dt oi n d u c ea c a l l u s from m i s t l e t o e and c o n f i r m e dt h ep r e s e n s eo ft h esamek i n do fg a l a c t o s e b i n d i n gp r o t e i n si nb o t h1 悶 v e sand 凶lIu sc e l l so fm i s t l e t o eby p o l y a c r y l a m i d eg e l electrophoresis.τhen t h ep a r a s i t ec e l l s 蹴 ha s werec o n t a c t e dw i t hh o s tc e l l s by p l a c i n gt h ec a l l u so fm i s t l e t o eon t h a to fb E grown i nt h er e l a t i v es t a t e and a n a l y z e dw h e t h e ro rn o tt h e i rc o n t a c ti n h i b i t i o n o c c u r s . 附 2 .Experimental 2 .1 .P l a n t s V i sωm album L .v a rl u t e s c 側 sM .) growingona b e e c ha tt h eU n i v e r s i t y M i s t l e t o e( F o r 四 i nAshiu,Miyama-cho,Kyoto,was h a r v e s t e di n May,1 9 8 8 . S h o o t so fb e 邑c h (F α , gusc r e n a t aBL)growinga tt h eE x p e r i m e n t a lN u r s e r y,Sakyo ・・ku,Kyoto,wash a r v e s t e d une,1 9 8 8 . i nJ 2 . 2 .C e l lc u l t u r e s Smalls e c t i o n so fm i s t l e t o eweree x c i s e da s e p t i c a l l yfromana p i c a lmeristemandtwo l a t e s tl e a v e sands t e r i l i z e ds t e p w i s e l yf o r3 0s e cw i t h70% e t h a n o ,l 2 . 5 min with 0.2% b巴nzalkoniumc h l o r i d e,52 0minw i t h2%s o d i1 . lm h y p o c h l o r i t es o l1 . l t i o nandr i n s e ds e v e r a l h e y were t r a n s f e r r e dt o5 0 ml g l a s s t i m e s withs t e r i l ed i s t i l l e dw a t e r .S u b s e q u e n t l y,t 1 . l bec o n t a i n i n g2 5 ml o f am o d i f i e ds o l i d MS (Murashige and Skoog) 13 ) m e d i u m . I t t c o n t a i n e dah a l fo ft h ei o n i cs t r e n g t ho fm i n e r a ls a l t so ft h eo r i g i n a lMSmedium,1 0 0 . 0 mg/lm y o i n o s i t o l,0 . 5 mg/l n i c o t i n i ca c i d and 0.5mg/1 p y r i d o x i n e .HCl,0 . 1 mg/I ,I 3% S1 . lc r o s eand 1% a g a ra sl i s t e di nT a b l e1 . Auxin and c y t o k i n i n t h i a m i n e .HC ・ T a b l e1 . C o m p o s i t i o no ft h em o d i f i e dM u r a s h i g e S k o o gmedium Compound F i n a lc o n c . Compound (mg/l) 0 . 1 2 5 0 Na2MoOz・ 2HzO KI 0 . 4 1 5 0 C O C 1 2・6HzO 0 . 0 1 2 5 myoI n o s i t o l 1 0 0 . 0 N i c o t i n i ca c i d 0 . 5 P y r i d o x i n e .HCl 0 . 5 Thiamine・HCl 0 . 1 S u c r o s e 3 0 . 0 F i n a lc o n c . (mg/l) NH4N03 KH2P04 C a C 1 2・2H20 MgS04・7HzO F e S O q・7H20 Na2芯DTA MnS04・4H20 Z n S 0 4・7H20 8 2 5 . 0 8 5 . 0 2 2 0 . 0 1 8 5 . 0 1 3 . 9 0 1 8 . 6 5 1 1 . 1 5 4 . 3 0 2 6 3 epH o ft h emedium was a d j u s t e dt o5 . 7a n d w e r ea d d e di nv a r i o u sc o m b i n a t i o n s . 百1 0 0 o r2 0m i n . Thec u l t u r e s were k e p ta t2 8C t h emediumwast h e na u t o c l a v e da t1 2 0C f u n d e r1 2 h rd a y l e n g t ha ta b o u t 2, 5 0 0l u x . G r e e n and w h i t ec a l l u s e so fb e e c hw e r e i n d u c e dands u b c u l t u r e di nt h esamem o d i f i e d MSmedium s u p p l e m e n t e dw i t h1 .0ppm o f1 . 心 叩h t h a l e n e a c e t i ca c i d(NAA)( g r e e nc a l l u s )and0 . 5ppmo f2, 4 d i c h l o r o p h e n o x y a c e t i c a c i d( 2, 4D) ( w h i t ec a l l u s )t o g e t h e rw i t h0 . 5ppmo f6 ゐe n z y l a m i n o p u r i n e(BA) 2 . 3 .D e t e r m i n a t i o no fgrowthr a t eo fc a l l u s Onep i e c eo fc a l l u s,a v e r a g ew e i g h to f2mgi naw e ts t a t e,was p l a n t e d on a f i b e r mat i naPLANTEX CCP-102 (τDYOBO C o . , L t d . ),c o n t a i n i n g1 5 ml o ft h emedium d e s c r i b e da b o v es u p p l e m e n t e dw i t h1 .0ppmo fk i n e t i n(KIN) a sc y t o k i n i nand1 0 . 0ppm o fNAA a sa u x i n . 官1 ePLANTEXes w e r es 回 l e dw i t ha i r p e r m e a b l ep o l y v i n y l c h l o r i d e t a p 出 i no r d e rt op r e v e n tt h em i c r o o r g a n i s mc o n t a m i n a t i o n and l o s so fm o i s t u r ea n d i n c u b a t e da sd e s c r i b e da b o v e . At w e e k l yi n . t e r v a l sd u r i n g 4明 e e ki n c u b a t i o np e r i o d .c a l l u s e s were b l o t t e dw i t ha f i l t e rp a p e r (ADVAN1ECToyo N o . 2 )t o removem o i s t u r ea t t a c h e d on t h es u r f a c eo f c a l l u s . andt h e i rf r e s hw e i g h t s wered e t e r m i n e d . Th ec a l l u s e s weret h e nd r i e du n t i lt o r 回 c hc o n s t a n tw e i g h ti nano v e na t6 0C . and t h ea v e r a g ed r yw e i g h t was c a l c u l a t e d f o rt h r e es a m p l e s . 百1 eg r ・ owthr a t ewase s t i m a t e da si n c r e a s eo fd r yw e i g h to fc a l l u s c e l l sr e l a t i v et ot h eo r i g i n a ld r yw e i g h t . 2. 4 .E x t r a c t i o no fm i s t l e t o ep r o t e i n s Th ef r 田 hl e a v e so fm i s t l e t o ewerec u ti n t os m a l lp i e c e sw i t hap a i ro fs c i s s o r sa f t e r washing w i t ht a pw a t e rf o l l o w e d by d i s t i l l e dw a t e r .F o r t y grams o fl e a v e sw e r e s u s p e n d e di n1 0 0ml o f0.25mM T r i sg l y c i n eb u f f e r (pH8 . 3 ),and h o m o g e n i z e df o r2 0 mina t 5C . Thed e e p g r ‘ e e n i s hs u s p e n s i o nwasc e n t r i f u g e df o r1 5mina t8 . 0 0 0xga t5O C t oremovei n s o l u b l em a t e r i a l s . Thee x t r a c t e ds o l u t i o nwasp o u r e di n t o1 5t i m e svolume ofωlda c e t o n e . Thep r e c i p i t a t e dm a t e r i a l sw e r er e c o v e r e dbyc e n t r i f u g a t i o na sd e s c r i b e d a b o v e, and washed w i t ha c e t o n et og i v ea c e t o n ep o w d e r . The a c e t o n e p o w d e r was ・ ( 印 p f 狂 {8 . 7 竹 )ぱ 0fw凶 h 1 せ i c hN a C α 1∞ c o民 n 1 χ c 印 e n t れ i s o l u b i l i z e di n0.05MT r i s H C lb u f f e r 2 . 5 . Aff i n i t ychromatography A c i d ω t r e a t e dS e p h a r o s e4Bwasp r e p a r e dbywashingt h eg e lw i t hc o l d0 . 2 NHClf o l l o w e d bys h a k i n gt h es u s p e n s i o no ft h ea c i dwashedg e l( g e l : 0 . 2 NHCl 1 : 2,v/v) a t5 0C f o r 1 4 3h r ) . 百1 eg e l was washed and e q u i l i b r a t e d 網 俳 0 幽 0 輔 0 町 口 2 6 4 0 0μ1 o fe a c hsamplewasa p p l i e dt ot h eg e l su s i n gM a l a c h i t eG r e e na sat r a c k i n g 1 0 00C,1 巴da t room t e m p e r a t u r ei n i t i a l l ya t 1mA/g e l,t h e n d y e . E l e c t r o p h o r e s i swasperform a t 2mA/g e la f t e rt h eb a n d sweremoved i n t ot h es e p a r a t i o ng e . l 1 l 1 ec a l i b r a t i o nk i t ( P h a r m a c i a )wasu s巴da st h emarkerp r o t e i n s .A f t e r ・e l巴c t r o p h o r e s i s,t h eg e lwass t a i n e df o r m a s s i eB r i l l i a n tB l u eR 2 5 0andd e s t a i n e dw i t h10%a c e t i ca c i d . p r o t e i n sw i t h0 . 2 5 % Coo 3 .Results and Discussion 3 . 1I n d u c t i o nandgrowtho fc a l l u sfrommis t 1e t o e Wheni n d u c t i o no fc a l l u sfromm i s t l e t o ewas t r i e du s i n gt h eMS medium c o n t a i n i n g v a r i o u s∞n c e n t r a t i o n sa n dt y p田 o fp l a n thormons,a l lgr田 ル c o l o r e dt r a n s p l a n t e dm i s t l e t o e t u r n e di n t o brown w i t h i no n ed a y . S i n c et h i ss e e m e dt ob ec a u s e d by t h eh i g hi o n i c s t r e n g t h i nt h eo r i g i n a l MS-medium, a sf r e q u e n t l yo b s e r v e di nt h e叩 s e so f woody 1 6 1 p l a n t s ,n e x twet r i e dt oi n d u c ec a l l u si nt h em o d i f i e d MS mediwn h a v i n ga h a l fo f i o n i cs t r e n g t ho ft h eo r i g i n a lo n e . P r e l i m i n a r yf i n d i n g si n d i c a t et h a ta d d i t i o no f1 .0 ppm o f BA k e p tt h et r a n s p l a n t e dm i s t l e t o eg r e e nf o rt h r e e months w i t h o u t growth andi n d u c t i o no fc a l l u s . Ont h eo t h e rhanda d d i t i o no f1 .0ppm KIN wasf o u n dt ob e f a v o r a b l ef o1 't h egrowtho fm i s t l e t o ec a l l u s . B a s e d on t h e s er e s u l t s,t h ei n d u c t i o no f c a l l u sfromm i s t l e t o ewas c a r r i e do u tu n d e rv a r i o u sc o m b i n a t i o n so fh o r m o n s . A f t e r 4 w e e ki n c u b a t i o na t2 8C, o n l yo n ec a l l u sc o u l db ei n d u c e d from t h el e a fu n d e ra c o m b i n a t i o no f1 .0ppmo fKINand1 0 . 0ppmo fNAAa sshowni nF i g .1 . Th er e s i d u a lt i s s u e se i t h e r became d e a do rw e r ec o n t a m i n a t e d by m i c r o o r g a n i s m s d u r i n gt h ec u l t u r e . 官官 c a l l u sh a sb e e ne a s i l ys u b c u l t u r e di nt h e same medium f o r moret h a n2y e a r sa ti n t e r v a lo fa b o u t 3w e e k s . 官官印Ilu s出 a r ec r e a m i s hp a l ep i n k i nc o l o randm a i n l ys p h e r i c a li ns h a p eh a v i n ga v e r a g ed i a m e t e ro f7.5μma sshowni n F i g s .2 aand3 . Whene f f e c t so f2 , 4 幽D w erea n a l y z e dont h egrowth o ft h em i s t l e t o e叩Ilu sc e l l s,t h e c a l l u swasf o u n dt ogrowa tt h ec o n c e n t r a t i o nb e l o w1 0 . 0 ppm i nt h ep r e s e n c eo f1 .0 ppmo fK I N . However,t h ec o l o ro ft h ec a l l u sa t0 . 0 5ppmand0 . 1ppmo f2 , 4 D became d a r k e ront h e i rs u r f a c e,andc h a n g e di n t or e d d i s hp i n ka th i g h e rt h a n0 . 5ppm.S i m i l a r l y, t h em i s t l e t o ec a l l u sc o u l dgrowont h esamem o d i f i e dMSmedias u p p l e m e n t e dw i t h0 . 5 ppmo fBAand0 . 5ppmo f2, 4-D 01‘ 1 .0ppmo fNAAw h i c hwereu s e df o rb e e c hc al 1u s, a l t h o u g ht h egrowth r a t ei sn o ts of a s ta st h a tg i v e nu n d e rt h ec o m b i n a t i o no f KIN andNA A . Them i s t l e t o ec a l l u sgrownont h em巴diumc o n t a i n i n g0 . 5ppmo fBAand0 . 5 4D i sshowni nF i g .2 b . Thea d a p t a t i o no fm i s t l e t o ec al Iu st ot h egrowth ppmo f2, c o n d I t i o no fb e e c hc a l l u sw i l lb ed e s c r i b e dl a t e r . 1 nc o n c l u s i o n,t h ec a l l u s grew most a t1 0 . 0ppmo fNAAand1 .0ppmo fKINs u p p l e m e n t e di nt h eh a l fo fi o n i cs t r e n g t ho f MS medium. Th ec h a n g ei nr e l a t i v e growth r a t eo ft h ec a l l u sa f t e rs u b c u l t u r e on t h i s hormonal c o n d i t i o ni s shown i nF i g . 4,w h i c hi n d i c a t e 0 暢 2 6 5 F i g .2 F i g .1 C a l l u sgrowtho fm i s t l e t o e . C a l l u si n2weeksa f t e rs u b c u l t u r ei nPLANTEXesunder t h ehormonal c o n d i t i o n sd e s c r i b e di nt h ef i g u r e . l n d u c t i o no fc a l l u so fm i s t l e t o e . F i g .3 Microphotograph o fc a l l u sc el 1s I n d u c e dc a l l u si n 3 weeks a f t 巴r o fm i s t l e t o e . t r a n s p l a n t a t i o no fa s m a l lp o r t i o n o f m i s t l e t o ew i t hana p i c a lmeristem andtwo l e a v e sont h em o d i f i e d MS medium supplement 母dw i t h1 .0ppm o f KIN and 10.0 ppm o f NAA. Two c a l l u s e sa r ei n d u c e d from t h e 日 !a v e s . 2 6 6 6 . 0 " ' 5 . 0 0 L嗣 . I : : i4 . 0 0 ' - 0' 由 〉 3 . 0 ← 口 ! i2.0 1 . 0 O O 2 3 4 T i m e( w e e k s l F i g .4 r v e so fc a l 1 u sc e l l so fm i s t l e t o ea n db e e c hi nPLANTEX: m i s t l e t o e GrowthCl, c a l l u s(8) grown on t h em o d i f i e d MS m吋 iums u p p l e m e n t e dw i t h1 .0ppm o f KINa n d1 0 . 0ppmo fNAA,g r e e nb e e c hc a l l u sCA)o nt h em o d i f i e dMS medium s u p p l e m e n t e dw i t h0 . 5ppmo fBAa n d1 .0ppmo fNAA,w h i t eb e e c hc a l l u s(臨) o nt h em o d i f i e dMSmediums u p p l母m e n t e dw i t h0 . 5ppmo f2 , 4D. t op i c ku pt h e伺 l l u sc e l l s from t h ef i b e r mats i n s i d et h ePLANτE)ιτ' h em e r i t so f h e y showed t h a t t h ePLANTEX was t h o r o u g h l yr e p o r t e d by Yaiima and T a k i17l, and t t h egrowthr a t eo fsome阻 l l u sc e l l swas e n h a n c e d andmorea n t h o c y a n i n was p r o d u c e d fromab e e f s t e a kp l a n tc a l l u s . τ' h ep r e s e n ts t u d yp r o v i d e du st h enew u s a g eo f PLANτEX i nt h ef i e l do ft i s s u ec u l t u r e . t i o no fl e c t i n si nt h em i s t l e t o e白 l l u s 3 . 2D e t邑c 百1 ee x t r a c t so fm i s t l e t o ehaveb e e nu s凶 a部 i n s tv a r i o u sd i s e a s e ss i n c ea n c i e n tt i m e s . R e c e n t l ym i s t l e t o eh a sb e e n shown t oc o n t a i n D氾 a l a c t o s e b i n d i n gl e c t i n sw h i c ha r 邑 i m p o r t a n t components i nt h ee x t r a c t,and t h e i rc h e m i c a lp r o p e r t i e s and p h y s i o l o g i c a l a c t i v i t i e swerei n v e s t i g a t e d ト 1 1 ) B a s e dont h e s日 r e s u l t st h ep r e s e n s eo ft h esame l e c t i n si n t h em i s t l e t o ec a l l u sc e l l si se x p e c t e dt ob eu s e da s au s e f u li n d e xf o ri d e n t i f i c a t i o no f n a l抑 制 t h es o l u b l ep r o t e i n so fb o t hm i s t l佼 o e t h ec e l l s .1 no r d e rt oa s c e r t a i nt h i s,wea andi t sc a l l u s . At f i r s t,t h em i s t l e t o el e c t i nwasi s o l a t e dbyana f f i n i t ychromatography h a r o s e4B g e l s . At y p i c a lr e s u l ti s shown i nF i g . 5.τ' h es u b u n i t on a c i d t r e a t e d S巴p s t r u c t u r eo ft h el e c t i n sr e c o v e r e d from t h ef r a c t i o n se l u t e d with 0 . 1M l a c t o s e was a r 凶 タzedbysodium dodecyl sulfate g e le l e c t r o p h o r e s i s( i nt h ep r e s e n c eo f ふmer 伺 p t o nF i g . 6.τ' hr ・ e eminor and t h r e emajor p r o t e i n s e t h a n o l ) andt h er e s u l t sa r巴 showni d e s i g n a t e da sat ocanddt o fr e s p e c t i v e l yc o u l db ε d e t e c t e di nt h ef r a c t i o n bound t o r e a t e dS e p h a r o s e4Bg e l s .T h 告 c a l l u sc e l l swere f o u n dt oc o n t a i n two p r o t e i n s t h ea c i dt c o r r e s p o n d i n gt o a and fcomponents,wh 芭r e a stwo maior g a l a c t o s eb i n d i n gp r o t e i n s, dande ,w eren o td e t e c t e di nt h e凶 l l u sc e l l s . T h i sd i f f e r e n c e may b ed u et ot h ef a c t t h a tt h epathwayo ft h ep r o t e i ns y n t h e s i si sc h a n g e da f t e ri n d u c t i o no ft h ec a l l u s . 働 2 6 7 1 5 . 0 E C O 記1 0 . 0 0 . 0 5 相 口 0 . 0 4 出 u 巴 口 五3 . o ~ 5 . 0 〈 0 . 0 1 O G F i g .5 80 1 6 0 m l ) E l u t i o nv o l u m告 ( 400 480 母d A f f i n i t ychromatographyo fm i s t l e t o eg a l a c t o s eb i n d i n gl e c t i n sona c i d t r e a t S e p h a r o s e4 B . 邑t o n epowderf r o m4 0go fm i s t l e t o el e a v 巴 ,sw asa p p l i e donacolumn( 1. 7x Ac 田). T he l e c t i n s wer忠 告 l u t e dw i t h0 . 0 5M l a c t o s ei n0 . 0 5M T r i s網 目C lb u f f e r 1 8c (pH8 . 7 ) .Thearrow i n d i c a t e st h echang 邑 o ft h ee l u t i o nc o n d i t i o n s . 3 . 3I n t e r a c t i o nbetweenboth回 l l u s e so f m i s t l e t o eandb田 c h Regarding t h er e c o g n i t i o n mechanisms betweenp l a n tc e l l s,i t seems t o be q u i t ε i n t e r e s t i n gt oi n v e s t i g a t e what h a p p e n s whenh o s t andp a r a s i t ec e l l scomei nc o n t a c t with e a c ho t h e r . I no r d e r toωrry o u tt h i se x p告r i m e n ti nv i t r o,i ts h o u l dbe n e c 巴s s a r yt oc u l t u r et h eh o s t andp a r a s i t e 回 l l u si nt h e same medium.τ' h eg r e e n andw h i t eb申告c hc a l l u s e swere s u b c u l t u r e d i nt h esamem o d i f i e dMSmediumt h a twas u tu n f o r t u n a t e l y u s e df o rm i s t l e t o ec a l l u s,b t h e yw e r ・ egrownont h ed i f f e r e n thormonal c o n d i t i o n s . S i n c et r a n s p l a n t a t i o no ft h e b e e c hc a l l u si n t omediumo ft h em i s t l e t o e wasfoundt obed i f f i c u l tandt h em i s t l e t o e 回 l l u shadwidea d a p t a b i l i t yf o rd i f f e r e n t h em i s t l e t o ec a l l u s e s hormonalc o n d i t i o n s,t were i nt u r na d a p t巴d f o rt h ec o n d i t i o n s o ft h eb e e c h臼 l l u s cells.τ' h er e s u l t s c 1e a r l yi n d i c a t et h a tt h em i s t l日t o e叩l1u s e s c o u l da c t u al 1y grow on t h e same media 造 量 f 1 F i g .6 SDS♂ o l y a c r y l a m i d eg e le l e c t r o p hか r e s i s o f g a l a c t o s e b i n d i n g l e c t i n s f r o mm i s t l e t o e(1)and c a l l u sc e l l s (1) . E l e c t r o p h o r e s i swas p e r f o r m e di n 10% p o l y a c r y l a m i d eg 告I s and g e l s wer巴 s t a i n e dw i t hCoomassieB r i l l i a n tB l u e R-250 a sd e s c r i b e di nt h e t e x t . 2 6 8 c a l F i g .7 訪れち告患 Growtho fm i s t l e t o ec a l l u sc e l l song r e e n(a)andw h i t 母 ( b )b e e c hc a l lu s . h em i s t l君t o ec a l l u s e s w邑r ep u ton A f t e ra d a p t a t i o no ft h egrowthc o n d i t i o n s,t t h ec a l l u sc e l l so f b邑e c h and c u l t u r e df o r 2 weeks a sd e s c r i b e di nt h et e xt . M i s t l e t o ec a l l u s告sa r ei n d i c a t e dby t h ea r r o w s . nt h ePLAN 1E X. F i g u r e2 bshowst h ea d a p t a t i o no f i nwhicht h eb e e c hc a l l u s巴sgrewi hcallus.τnerefore,a f t e rs u b c u l t u r e df o r m i s t l e t o et ot h ec o n d i t i o no ft h e white b田 c 1etoe回 l l u swas t r a n s p l a n t e d on t o po ft h eb e e c h 3 weeks,a s m a l lp i e c eo ft h e mist l l u s,andi n c u b a t巴da t2 50C . Asshowni nF i g . 7,no c o n t a c ti n h i b i t i o no c c u r r e di n both c a s e s,and t h em i s t l e t o e c a l l u s e s grew t o g e t h e r with thεbeech o n e s . However,t h em i s t l e t o ec a l l u sd i dn o t l u s,andt h ec o n t a c tbetweentwoc a l l u s e swasn o ts os t r o n ga s i n t r u d ei n t ot h eb e e c h回 l e yc o u l de a si 1y be separated by using t w e e z e r s . Although a d e t a i l e d we e x p ε c t e d . Th 号c o g n i t i o nb母tweent h e h o s t and t h ep a r a s i t ec a l l u si sd i f f i c u l tt o d i s c u s s i o nont h er t i g a t e,t h i ss t u d yi st h ef i r s t attempt t oc o n t a c tt h eh o s t and p a r a s i t告 m i s t l e t o e i n v巴s 叩 nv i t r o .F u r t h e ri n v e s t i g a t i o n sf o r f u n c t i o n s o f g a l a c t o s eb i n d i n g l e c t i n si n c a l l u s e si m i s t l e t o eandi n t e r a c t i o n sbetweent h em i s t l e t o eandb e e c hc e l l sa r enowi np r o g r e s s by l Iu s e s . u s i n gs u s p e n s i o n c u l t u r e d田 幽 4 .References 1 ) K U I J T,J .andTOTH, R . : Ult r a s t r u c t u r eo fangiosperm haustoria-a r e v i e w . Ann. Bot. 4 0 .1 1 2 1 1 1 3 0,1 9 7 6 . . :Haustoria o fphanerogamic p a r a s i t e s . Ann. Rev. 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