PlantCell Reports

Plant Cell Reports (1986) 5:239-242

© Springer-Verlag 1986

Alkaloid production by hairy root cultures in Atropa belladonna Hiroshi Kamada 1, ,, Nobuyuki Okamura 2, Motoyoshi Satake 3, Hiroshi Harada 4, and Koichiro Shimomura 3 x Gene Experiment Center, University ofTsukuba, Sakura-mura, Ibaraki-ken, 305 Japan 2 Faculty of Pharmaceutical Sciences, Fukuyama University, 985 Higashimura-machi Aza Sanzo, Fukuyama-shi, Hiroshima-ken, 729-02 Japan Tsukuba Medicinal Plant Research Station, National Institute of Hygienic Sciences, 1 Hachirnandai Yatabe-machi, Tsukuba-gun, Ibaraki-ken, 305 Japan 4 Institute of Biological Sciences, University ofTsukuba, Sakura-mura, Ibaraki-ken, 305 Japan Received January 14, 1986 / Revised version received June 10, 1986 - Communicated by F. Constabel

Abstract

Hairy roots w e r e induced by i n o c u l a t i o n of stems of sterile plants of A t r o p a b e l l a d o n n a with A q r o b a c t e r i u m rhizogenes. The a x e n i c c u l t u r e of the h a i r y r o o t s i s o l a t e d from the stems p r o l i f e r a t e d 60 fold as based on the i n i t i a l f r e s h w e i g h t a f t e r o n e m o n t h of c u l t u r e . T h e p r e s e n c e of a t r o p i n e a n d s c o p o l a m i n e in h a i r y r o o t s w e r e e x a m i n e d by TLC and HPLC. Their a m o u n t s w e r e analyzed by GLC. The r e s u l t s s h o w t h a t the a m o u n t of the two alkaloids in the axenic cultures was the s a m e as or e v e n h i g h e r t h a n t h o s e of n o r m a l plants g r o w n in the field.

Introduction

The large-scale p r o d u c t i o n of secondary m e t a b o l i t e s such as p i g m e n t s and alkaloids by f i e l d g r o w n p l a n t s has b e e n l i m i t e d due to the d e p e n d e n c y of the m e t a b o l i s m on r a t e of propagation, cultivation of p l a n t s , and climate during the cultivation. Thus, n u m e r o u s i n v e s t i g a t o r s have tried to utilize plant tissue culture techniques to m i n i m i z e these disadvantages. In s p i t e of t h e s e e f f o r t s , the l a r g e - s c a l e p r o d u c t i o n of o n l y few substances such as shikonin has succeeded ( F u j i t a et al. 1981). Of several alkaloids w h i c h are t h o u g h t to be m a i n l y p r o d u c e d in roots of plants, Sz~ke et al. (1982) reported the p r o d u c t i o n of a t r o p i n e and s c o p o l a m i n e by callus cultures. The p r o d u c t i o n of t h e s e a l k a l o i d s by c a l l u s c u l t u r e , however, is g e n e r a l l y d i f f i c u l t ( B h a n d a r y et al. 1969, Hiraoka and Tabata, 1974, Hashimoto and Yamada, 1983). It seems that the b i o s y n t h e s i s of such a l k a l o i d s is c o r r e l a t e d w i t h the o r g a n i z a t i o n of cells as root. Recently, it has b e e n c l e a r l y d e m o n strated t h a t t h e Ri p l a s m i d present in A g r o b a c t e r i u m r h i z o g e n e s causes the transform a t i o n of plant cells by i n t r o d u c i n g T-DNA of

the Ri p l a s m i d i n t o g e n o m i c D N A of p l a n t c e l l s and t h a t on a h o r m o n e - f r e e m e d i u m the t r a n s f o r m e d plant cells give rise to m a s s i v e roots, so c a l l e d h a i r y r o o t s (White and Nester,1980, C h i l t o n et al. 1982, T e p f e r , 1984). The transformed plant cells can produce hairy roots even after A g r o b a c t e r i u m is eliminated. In t h i s r e p o r t , we demonstrate the i n d u c t i o n of h a i r y r o o t s , e s t a b l i s h m e n t of the c u l t u r e , and p r o d u c t i o n of a l k a l o i d s by the hairy root culture in Atropa belladonna. Materials

and M e t h o d s

Induction of hairy roots S t e r i l e p l a n t s of A t r o p a b e l l a d o n n a L. were established by s h o o t tip c u l t u r e of f i e l d - g r o w n plants and m a i n t a i n e d by r e p e a t e d shoot culture on h o r m o n e - f r e e M u r a s h i g e and S k o o g ' s (MS) m e d i u m u n d e r 25oc, 18h l i g h t / 6 h dark, 4,000 lux. A g r o b a c t e r i u m rhizo~enes strain 15834 h a r b o r i n g Ri p l a s m i d (pRi 15834) g r o w n on Y E B a g a r m e d i u m ( V e r v l i e t et al. 1975) w a s i n o c u l a t e d by a n e e d l e o n t o s t e m s of the sterile plants. Culture of the hairy roots T w o to f i v e w e e k s a f t e r i n o c u l a t i o n , hairy roots a p p e a r e d on the inoculated sites. S e g m e n t s of the hairy roots w e r e cut off and c u l t u r e d on h o r m o n e - f r e e MS agar (I %) m e d i u m containing antibiotic (carbenicillin, I mg/ml). After several passages onto MS m e d i u m , s e g m e n t s of the g r o w i n g h a i r y r o o t s w e r e t r a n s f e r r e d and m a i n t a i n e d on h o r m o n e f r e e M S a g a r m e d i u m w i t h o u t a n t i b i o t i c . To e x a m i n e the p r o l i f e r a t i o n rate, s e g m e n t s of the h a i r y r o o t s g r o w n on the M S a g a r m e d i u m w e r e t r a n s f e r r e d into h o r m o n e - f r e e MS liquid m e d i u m a n d the f r e s h w e i g h t w a s m e a s u r e d before and after a f o u r - w e e k culture under 25 °C, continuous dark, 100 rpm. A n a l y s i s of a t r o p i n e and s c o p o l a m i n e E x t r a c t i o n : Hairy roots h a r v e s t e d after 2-4 w e e k s of c u l t u r e w e r e l y o p h i l i z e d a n d ground. The p o w d e r e d s a m p l e (40-460 mg) was

* Present address: Institute of Biological Sciences, University of Tsukuba, Sakura-mura, Ibaraki-ken, 305 Japan Offprint requests to: H. Kamada at his present address

240 w e i g h e d and s u b j e c t e d to the e x t r a c t i o n of alkaloids. An a p p r o p r i a t e v o l u m e of C H C I 3M eOH-NH4OH (I 5 : 5 : 1 )(I 0 m l e x t r a c t i o n s o l v e n t / ~ 0 0 m g d r y s a m p l e ) w a s a d d e d to the w e i g h e d s a m p l e , s o n i c a t e d for 10 min, t h e n k e p t at r o o m t e m p e r a t u r e for I h (Kagei et al. 1978). After filtration, the residue was w a s h e d t w i c e w i t h I ml of CHCI 3. The pooled f i l t r a t e w a s e v a p o r a t e d to d r y n e s s . To the r e s i d u e , 5 ml of C H C I 3 a n d 2 ml of IN H 2 S O 4 were added, then the solution was m i x e d well. The C H C I 3 p h a s e w a s r e m o v e d and the H 2 S O 4 p h a s e w a s a d j u s t e d to pH 10 w i t h 28% N H 4 O H in an i c e - b a t h . F r o m the s o l u t i o n , a l k a l o i d s were e x t r a c t e d once w i t h 2 ml and twice w i t h I ml of C H C I 3. The c o m b i n e d e x t r a c t s w e r e f i l t e r e d a f t e r a d d i n g a n h y d r o u s N a 2 S O 4 and t h e n the r e s i d u e w a s w a s h e d w i t h I ml of CHCI 3. The c o m b i n e d filtrates were e v a p o r a t e d to d r y n e s s at 40°C. T L C a n a l y s i s : The a l k a l o i d f r a c t i o n thus obtained was d i s s o l v e d in an a p p r o p r i a t e v o l u m e of M e O H , a p p l i e d on S i l i c a gel p l a t e (60 F254, Merk), a n d t h e n a n a l y z e d w i t h t w o d i f f e r e n t d e v e l o p i n g solvent systems, EtOAc2PrOH-10%NH4OH (9:7:3) a n d C H C 1 3 - a c e t o n e MeOH-28%NH4OH (75:10:15:2). The alkaloidal spots were detected with Dragendorff's reagent (Wagner et al. 1984). HPLC analysis : The alkaloidal fraction w a s a n a l y z e d in the T r i r o t o r V H P L C ( J a p a n S p e c t r o s c o p i c Co., Ltd. Tokyo) equipped w i t h a UV d e t e c t o r (model UVIDEC-100-V; Japan S p e c t r o s c o p i c Co., Ltd. T o k y o ) a n d all d a t a p r o c e s s i n g was done w i t h SIC C h r o m a t o c o r d e r 1 1 ( S y s t e m I n s t r u m e n t s Co., Ltd. Tokyo). An a p p r o p r i a t e v o l u m e of the s a m p l e d i s s o l v e d in M e O H w a s i n j e c t e d i n t o a T S K gel O D S - 1 2 0 T (Toyo S o d a M a n u f a c t u r i n g Co., Ltd. J a p a n ) column (5 ~um, 25cm x 4.6 m m internal diameter) and eluted w i t h a solution c o m p o s e d of 48 v o l u m e s of M e O H a n d 52 v o l u m e s of 10 m M sodium 1 - h e p t a n e s u l f o n a t e in HpO (pH 4.0 w i t h a c e t i c a c i d ) ( Y a m a d a et al. 19~4). The f l o w rate was 1.0 m l / m i n throughout the analysis. The e f f l u e n t w a s m o n i t o r e d by m e a s u r i n g the absorbance at 215 nm. G L C a n a l y s i s : To the d r i e d a l k a l o i d a l f r a c t i o n , 50 )/i of B S A ( W a k o P u r e C h e m i c a l Industries, Ltd. Japan) was added and heated in a d r y b l o c k h e a t e r at 4 2 ° C for 15 min. After a brief period at room temperature, the reaction mixture was diluted with an a p p r o p r i a t e v o l u m e of hexane and, just prior to analysis, cocaine was added as an internal standard (25)/i/ml). Gas l i q u i d c h r o m a t o graphy (GLC) was p e r f o r m e d on a S h i m a d z u GC9A ( S h i m a d z u Co., J a p a n ) e q u i p p e d w i t h a flame thermoionic d e t e c t o r (FTD), u s i n g a g l a s s c o l u m n (3 m m x 2 m) p a c k e d w i t h 5% 0V17 on S u p e l c o p o r t (100-200 mesh: Supelco, Inc.). The G L C c o n d i t i o n s w e r e as f o l l o w s ; i n j e c t i o n t e m p e r a t u r e , 230°C; d e t e c t o r t e m p e r a t u r e , 260°C; h e l i u m as c a r r i e r gas at a f l o w r a t e of 50 m l / m i n . GC-MS analysis : Identification of alkaloids was done by mass spectrometry. The G C - M S c o n d i t i o n s w e r e as f o l l o w s ; a 2 m x 3 mm glass tube packed with 3 % silicon OV-101, c o l u m n t e m p e r a t u r e of 1 8 0 - 2 4 0 ° C (temperature program, 5°C/min; isothermal at 240°C), h e l i u m as the c a r r i e r gas at a f l o w r a t e of 40 m l / m i n , T I M as t h e d e t e c t o r , and an ionizing energy of 20 eV. TMS-atropine; m/e (rel. int.) 361 [ M + ] (41), 140 (5), 1 2 4

(100), 95 (6), 94 (6), 83 (14), 82 (15), 73 (22). TMS-scopolamine; 375 [M+] (67), 193 (30), 154 (23), 138 (100), 128 (3), 108 (33), 103 (I01, 94 (63), 73 (46). D e t e c t i o n of opines Extraction and detection of o p i n e s ( a g r o p i n e and m a n n o p i n e ) w e r e p e r f o r m e d by the m e t h o d of P e t i t et al (1983). Hairy roots (ca. 50 mg fresh weight) w e r e ground in an E p p e n d o r f t u b e w i t h 50 ~ i of d i s t i l l e d w a t e r u s i n g a g l a s s rod. The extracts were c e n t r i f u g e d at 1 2 , 0 0 0 r p m for 2 min. T e n ~i of the supernatant were spotted on a W h a t m a n 3 M M p a p e r a n d t h e n the p a p e r w a s e l e c t r o phoresed at the constant voltage of 20 V/cm. The b u f f e r u s e d for the e l e c t r o p h o r e s i s w a s f o r m i c a c i d (99%), a c e t i c a c i d (99.5%) and distilled w a t e r (1:3:16, V / V / V ) ( O t t e n a n d Schilperoort, 1978). After drying the paper, o p i n e s w e r e v i s u a l i z e d by a l k a l i n e s i l v e r nitrate reagent (Trevelyan et al. 1950). Results

and D i s c u s s i o n

The bacteria cultured on YEB agar m e d i u m for 3-7 days were inoculated on internodes of the sterile plants. A f t e r 2 to 4 w e e k s numerous hairy roots appeared on the inoculated site (Fig. IA) and grew extensively. The e l o n g a t i n g r o o t s w e r e cut off a n d put on h o r m o n e - f r e e MS a g a r m e d i u m c o n t a i n i n g c a r b e n i c i l l i n (I m g / m l ) . After several days of culture, e l o n g a t i n g root tips were cut off and transferred to h o r m o n e - f r e e MS agar m e d i u m w i t h o u t carbenicillin. This p r o c e d u r e was repeated several times until no colony of bacteria appeared. After transfer of the axenic hairy roots into the MS liquid m e d i u m (70 ml) in 100 ml E r l e n m e y e r flasks or o n t o s o l i d MS a g a r m e d i u m in p e t r i d i s h e s t h e y g r e w r a p i d l y and e x h i b i t e d e x t e n s i v e l a t e r a l b r a n c h i n g (Fig. IB a n d IC). As a control experiment, a d v e n t i t i o u s roots f o r m e d at t h e c u t e n d of s t e r i l e plants were c u l t u r e d on h o r m o n e - f r e e MS a g a r m e d i u m . However, t h e s e r o o t s g r e w s l o w l y and no lateral branching appeared. P i e c e s of the established hairy root cultures were subc u l t u r e d at 3-4 w e e k i n t e r v a l s on l i q u i d or agar-solidified MS medium without phytoh o r m o n e s for one year. The a v e r a g e w e i g h t i n c r e a s e of the h a i r y r o o t s in l i q u i d MS m e d i u m in five separate e x p e r i m e n t s was about 60 fold in o n e - m o n t h cultures. A g r o p i n e a n d m a n n o p i n e are w e l l k n o w n opines d e t e c t a b l e in hairy roots t r a n s f o r m e d w i t h Ri p l a s m i d (Petit et al. 1983). To e x a m i n e w h e t h e r t r a n s f o r m a t i o n w i t h the Ri p l a s m i d had o c c u r r e d , p r e s e n c e of a g r o p i n e and m a n n o p i n e in extracts of the hairy roots was examined by paper electrophoresis, f o l l o w e d by alkaline silver nitrate staining. Both agropine and m a n n o p i n e w e r e d e t e c t e d in the e x t r a c t s of the f r e s h l y i s o l a t e d h a i r y roots, but not in t h o s e of the h a i r y r o o t s a f t e r a p r o l o n g e d c u l t u r e (Fig. 2). This t e n d e n c y of d i s a p p e a r a n c e of a g r o p i n e a n d m a n n o p i n e d u r i n g l o n g t e r m c u l t u r e of h a i r y r o o t s has a l s o b e e n r e p o r t e d in t o b a c c o a n d carrot, even w h e n full length of the T-DNA of Ri p l a s m i d can be d e t e c t e d by S o u t h e r n - b l o t hybridization in g e n o m i c D N A of the h a i r y roots after p r o l o n g e d culture (Tepfer, 1984). Although agropine and mannopine w e r e not d e t e c t e d a f t e r p r o l o n g e d c u l t u r e of h a i r y

241

Fig. I. A : H a i r y r o o t s on s t e m s e g m e n t s . B:Hairy roots grown medium. C : H a i r y roots g r o w n in h o r m o n e - f r e e MS liquid medium. Table

I.

Alkaloid

on

hormone-free

content

alkaloid content atropine

Fig. 2. D e t e c t i o n of a g r o p i n e and m a n n o p i n e in the extracts of the hairy roots. A;agropine, M;mannopine, NS;neutral sugar. Lanes I and 5;Standard marker, Lane 2;Nont r a n s f o r m e d control roots, Lane 3;Freshly isolated hairy roots, Lane 4;Hairy roots after repeated s u b - c u l t u r e s (one year).

-S

0

-,A -.,NH i

I

~

~

I

2

3

-~T

Fig. 3. TLC pattern of the alkaloidal fraction. S;scopolamine, A;atropine, N H ; n o r h y o s c y a m i n e , T;tropine. Lanes I and 3;authentic compounds, Lane 2;extracts of the hairy roots.

NR-field I

0.340 ± 0.031

NR 2 )

0.031 Z 0.004

HR 3 )

0.371

~ 0.013

of

MS

hairy

agar

roots

(% dry wt. ± SD) scopolamine

0.008

Z 0.005

trace 0.024 ± 0.010

I) N o r m a l roots from o n e - y e a r old plants g r o w n in the field (harvested in autumn). 2) N o r m a l roots from plantlets c u l t u r e d in vitro. 3) Hairy roots cultured for 4 weeks in liquid medium. Each value was o b t a i n e d from five independent experiments.

roots in t h i s r e p o r t , other phenotypic f e a t u r e s of the h a i r y r o o t s , s u c h as r a p i d g r o w t h and e x t e n s i v e lateral branching, w e r e m a i n t a i n e d . W h e n the h a i r y r o o t s of A t r o p a belladonna were cultured on M S m e d i u m containing phytohormones, plantlets regenerated and e x h i b i t e d characteristic features, such as w r i n k l e d leaves, shortened internodal length, and active adventitious root f o r m a t i o n (data not shown here). These phenotypic features observed in the regenerated plants have also been repOrted for t o b a c c o , c a r r o t and o t h e r p l a n t s p e c i e s (Tepfer, 1984, O o m s et al. 1985). From these facts, it s e e m s l i k e l y t h a t the Ri T - D N A is c o n s e r v e d during p r o l o n g e d culture of Atropa h a i r y roots, t h o u g h a g r o p i n e a n d m a n n o p i n e could not be detected. By u s i n g the e s t a b l i s h e d hairy root c u l t u r e s , p r o d u c t i o n of t r o p a n e a l k a l o i d s , such as a t r o p i n e and scopolamine, was examined. By TLC analysis, two spots corresponding to s c o p o l a m i n e and atropine w e r e d e t e c t e d on the TLC plate (Fig. 3). The r e s u l t s of T L C a n a l y s i s i n d i c a t e d t h a t the hairy roots produced alkaloids resembling t h o s e of n o r m a l p l a n t s g r o w n in the field. The a l k a l o i d s p r o d u c e d in the h a i r y r o o t s

242 were further a n a l y z e d by HPLC. The identification of these alkaloids as s c o p o l a m i n e and atropine was p e r f o r m e d by c o - c h r o m a t o g r a p h y with' standard compounds. T w o peaks separated by HPLC were isolated and confirmed as atropine and scopolamine by T L C a n a l y s i s . Atropine and s c o p o l a m i n e in hairy roots were also identified with GC-MS. Next, the c o n t e n t s of s c o p o l a m i n e a n d a t r o p i n e w e r e e x a m i n e d by GLC. As s h o w n in T a b l e I, G L C analysis of the root extract also d e m o n s t r a t e d the presence :of s c o p o l a m i n e and atropine. A l t h o u g h the r o o t s of the n o r m a l plantlets i_nnvitro c o n t a i n e d these alkaloids, the c o n c e n t r a t i o n s w e r e l o w e r t h a n t h o s e of the normal plants grown in a f i e l d . In contrast we found that the hairy roots contained scopolamine and atropine at concentrations of 0.024 % d W a n d 0.371 %dW, respectively. The c o n c e n t r a t i o n of atropine in the hairy roots was c o m p a r a b l e to that of the n o r m a l plants g r o w n in a field (Table I). T h e p r o d u c t i o n of tropane alkaloids, such as a t r o p i n e a n d s c o p o l a m i n e , has b e e n e x a m i n e d in callus cultures of various plant species, but large-scale production has been u n s u c c e s s f u l in a l m o s t all cases (Deus-Neuman a n d Zenk, 1984, H i r a o k a and T a b a t a , 1974, Hashimoto and Yamada, 1983). Recently, successfull p r o d u c t i o n was reported for cell l i n e s w h i c h o r i g i n a t e d f r o m r o o t s (Sz6ke et al. 1982) a n d for c e l l l i n e s r e s u l t i n g f r o m selection (Deus-Neuman and Zenk, 1984). However, these selected cell lines produced the alkaloids in high a m o u n t s only during the selection period, and the a m o u n t s b e c a m e lower after several sub-cultures under nonselective condition. As c o m p a r e d w i t h these e x p e r i m e n t s , the h a i r y r o o t c u l t u r e s y s t e m established in t h i s report constantly produced a t r o p i n e and s c o p o l a m i n e under nonselective conditions during a prolonged p e r i o d (at l e a s t one y e a r a n d p o s s i b l y for s e v e r a l y e a r s ) in t h e s a m e a m o u n t as or s l i g h t l y h i g h e r a m o u n t s t h a n t h o s e in r o o t s of p l a n t s grown in t h e f i e l d . It w a s reported t h a t in r o o t c u l t u r e s grown in medium containing a low concentration of auxin tropane alkaloids w e r e p r o d u c e d in c o m p a r a b l e a m o u n t s to t h o s e of the i n t a c t p l a n t s ( W e s t et al. 1957, B h a n d a r y et al. 1969, H a s h i m o t o et al. 1983, T a b a t a et al. 1972). These results indicate that the s y n t h e s i s of t r o p a n e a l k a l o i d s s e e m s to be closely correlated to m o r p h o l o g i c a l d i f f e r e n t i a t i o n as root. Thus, h a i r y r o o t c u l t u r e s m a y be a u s e f u l s y s t e m for l a r g e scale p r o d u c t i o n of these alkaloids. In our recent w o r k , t h i s s y s t e m has b e e n a p p l i e d s u c c e s s fully to o t h e r plant species, s u c h as S c o p o l i a , D a t u r a , a n d H y o s c y a m u s (data not shown). We a r e n o w e x a m i n i n g the o p t i m u m

conditions necessary for r a p i d g r o w t h of hairy roots and high production of t h e alkaloids. N o t e a d d e d in p r o o f : We l e a r n e d t h a t s o m e c o m p a r a b l e results in H y o s c y a m u s m u t i c u s are b e i n g o b t a i n e d by Drs. H. E. F l o r e s and P. F i l n e r at L o u i s i a n a S t a t e U n i v e r s i t y a f t e r the p r e p a r a t i o n of this manuscript.

Acknowledgment W e t h a n k Mr. T. T a n a k a f o r c r i t i c a l r e a d i n g of t h i s m a n u s c r i p t . U t i l i z a t i o n of cocaine during GLC analysis was done under control of Dr. H. Shiomi of F u k u y a m a Univeristy.

References B h a n d a r y SBR, Collin HA, T h o m a s E, Street HE (1969) Ann Bot 33:647-656 Chilton M-D, Tepfer DA, Petit A, David C, C a s s e - D e l b a r t F, T e m p ~ J (1982) Nature 295:432-434 D e u s - N e u m a n B, Z e n k M H (1984) P l a n t a M e d i c a 50:427-431 Fujita Y, Hara Y, Suga C, M o r i m o t o T (1981) Plant cell Reports 1:61-63 H a s h i m o t o T, Y a m a d a Y (1983) Planta Medica 47:195-199 Hiraoka N, Tabata M (1974) P h y t o c h e m 13:16711675 H u f f m a n GA, W h i t e FF, Gordon MP, Nester EW (I 984) J Bacteriol 157:269-276 Kagei K, H e m m i S, Shirai H, H a s e g a w a S, T o y o s h i m a S (1978) S h o y a k u g a k u Zasshi 32:222227 O o m s G, Karp A, Burrell MM, Twell D, Roberts J (1985) Theoret Appl Genet 70:440-446 Otten LABM, S c h i l p e r o o r t RA (1978) B i o c h i m Biophys Acta 527:497-500 P e t i t A, D a v i d C, D a h l GA, E l l i s JG, G u y o n P, C a s s e - D e l b a r t F, T e m p ~ J (1983) Mol Gen Genet 190:204-214 ¢ Sz~ke E, Dung NN, V e r z a r - P e t r l G, Potoczki A (1982) Acta Bot Acad Sci Hung 28:403-410 Tabata M, Y a m a m o t o H, Hiraoka N, K o n o s h i m a M (i972) P h y t o c h e m 11:949-955 Tepfer DA (1984) Cell 37:959-967 T r e v e l y a n WE, Procter DP, H a r r i s o n JP (1950) Nature 166:444-445 V e r v l i e t G, Holsters M, Teuchy H, M o n t a g u M van, Schell J (1975) J gen Virol 26:33-48 W a g n e r H, Bladt S, Zgainski EM (1984) "Plant Drug Analysis. A Thin Layer C h r o m a t o g r a p h y Atlas" (Translated by A. Scott) SpringerVerlag, Berlin, pp 300-301 West FRJr, M i k a ES (1957) Bot Gaz 119:50-54 W h i t e FF, Nester EW (I 980) J B a c t e r i o l 141:1134-1141 Y a m a d a S, Noda N, H a y a k a w a J, Uno K (1984) Yakugaku Zasshi 104:199-203