Plant Cell Reports (1984) 3:85-87
Plant Cell Reports © Springer-Verlag 1984
In vitro propagation of A tractylodes lancea N o b o r u H i r a o k a , N o b u y u k i Y a m a d a , T o m o k o K o d a m a , and Y u t a k a T o m i t a Niigata College of Pharmacy, 5829 Kamishinei-cho, Niigata 950-21, Japan Received February 15, 1984/Revised version received March 29, 1984 - Communicated by F. Constabel
ABSTRACT Shoot cultures of Atractylodes lancea DC. (Compositae) have been e s t a b l i s h e d by i n o c u l a t i n g the flower bud on L i n s m a i e r - S k o o g ' s m e d i u m s u p p l e m e n t e d with 10 -5 M n a p h t h a l e n e a c e t i c acid and l0 -5 M 6 - b e n z y l aminopurine. Shoots were m u l t i p l i e d on a m e d i u m containing 10 -6 M 6 - b e n z y l a m i n o p u r i n e . P r o p a g a t e d shoots rooted on a m e d i u m devoid of any p l a n t ...... v....... ~ re~alatots were t r a n s f e r r e d to p o t t i n g soil and finally to the field.
ABBREVIATIONS LS NAA BAP
Linsmaier and Skoog (1965) m e d i u m 1 - N a p h t h a l e n e a c e t i c acid 6-Benzylaminopurine
d i u m were a u t o c l a v e d at 121°C for 15 min. The cultures were kept in a p h o t o p e r i o d of 16h/day of f l u o r e s c e n t light, 3000-6000 ix, at 25 _+ 5°C. Each one of the m u l t i p l e shoots, w i t h the large leaves removed, was e x c i s e d and t r a n s f e r r e d to fresh medium. Subcultures were p e r f o r m e d at 4-week intervals. The rooted shoots were t r a n s f e r r e d to various k i n d s of p o t t i n g soil. The p o t t e d plants were covered with a vinyl sheet for the first 1-2 weeks to m a i n t a i n humidity° The cover was gradually r e m o v e d to a l l o w the plants to acclimatize to greenhouse conditions. After 1-2 months, the plants were t r a m s f e r r e d to the field. The chromosome numbers of the P r o p a g a t e d plants were d e t e r m i n e d by cytological o b s e r v a t i o n of the root-tip cells a c c o r d i n g to the Feulgen squash m e t h o d (Darlington and La Cour 1969).
RESULTS AND D I S C U S S I O N INTRODUCTION The rhizome of Atractylodes lancea DC. (Compositae) is one of the crude drugs p r e s c r i b e d clinically for diuretic, stomachic and sudorific p u r p o s e s in trad i t i o n a l Chinese medicine. Since the p l a n t bears no seeds in Japan, d i v i s i o n of rhizomes is a usual p r o cedure for its propagation. Only a few pieces of the rhizome r e a d y for t r a n s p l a n t a t i o n can be o b t a i n e d from a 3- to 4 - y e a r - o l d p l a n t by this method. Recently, Shoyama et al. (1983) r e p o r t e d the clonal m u l t i p l i c a tion of this p l a n t through m e r i s t e m culture. We have a t t e m p t e d i n d e p e n d e n t l y to e s t a b l i s h a procedure for r a p i d clonal propagation, starting from flower buds. This p a p e r d e s c r i b e s the results of these experiments.
M A T E R I A L S AND M E T H O D S Flower buds (i0 m m long, 3 m m in diameter) were c o l l e c t e d late in July o r e a r l y in A u g u s t from p l a n t s o f about one m o n t h before the first flowering. They were w a s h e d for half an hour in r u n n i n g water, sterilized for 7 min in an aqueous solution of 0.1% HgCI c o n t a i n i n g 0.1% Tween 80 in a 100-ml E r l e n m e y e r fl~sk on a shaker, and rinsed three times w i t h sterile water. Several o u t e r involucral scales were cut off from buds and the buds were cut v e r t i c a l l y into four pieces. Both the scales and the bud segments served as explants. The b a s a l m e d i u m was LS t h r o u g h o u t the experiments. Test tubes (30 x 130 mm) c o n t a i n i n g 20 ml of agar m e -
Offprint requests to: N. Hiraoka
Shoot Induction: Flower buds were used as explants instead of shoot tips because a limited number o f plants were available and each rhizome has only a few shoot tips. The 40 bud segments were p l a c e d on LS containing one of four combinations o f N A A and BAP at c o n c e n t r a t i o n s o f 10 -6 M and/or 10 -5 M. After one m o n t h of incubation, a single shoot was induced from the explants on the m e d i u m supplemented with 10 -5 M N A A and 10 -5 M BAP (Fig. l-A). Involucral scales formed only callus tissues regardless of the m e d i a c o m p o s i tions d e s c r i b e d above.
Shoot M u l t i p l i c a t i o n : When the s h o o t was i n c u b a t e d on LS c o n t a i n i n g 10 -6 M BAP, w h i c h was analogous to the best culture conditions for in v i t r o shoot m u l t i p l i c a t i o n of p y r e t h r u m (Wambugu and Rangan 1981), it p r o d u c e d a new crop of m u l t i p l e shoots. By r e p e a t i n g this procedure, e n o u g h shoot m a t e r i a l s for the following e x p e r i m e n t s were obtained. Fig. 2 shows the effect of various kinds o f cytokinins on shoot m u l t i p l i c a t i o n d u r i n g four successive subcultures. Each one s h o o t e x p l a n t c o n s i s t e n t l y p r o d u c e d an additional shoot during the 4-week incubation p e r i od due to the a d d i t i o n of 10 -6 M BAP to the basal medium. The other cytokinins, kinetin, 6 - i s o p e n t e n y l a m i n o p u r i n e or zeatin~ were inferior to BAP in shoot multiplication. Fig. I-B shews typical features o f m u l t i p l e shoots thus obtained. ~ Fig. 3 shows the e f f e c t of auxins (in~ole-3-acetic
86
B
Fig. i.
A single shoot induced from a flower bud (A), a 4 - w e e k - o l d culture in the shoot m u l t i p l i c a t i o n p h a s e (B), a rooted shoot (C), p o t t e d p l a n t l e t s (D), and chromosomes in a r o o t - t i p cell of a p l a n t l e t (E).
acid, i n d o l e - 3 - b u t y r i c acid and NAA) on shoot multip l i c a t i o n d u r i n g four successive subcultures. All m e d i a contain 10 -6 M BAP and an auxin. The number of shoots per culture increased due to the addition o f
KINETIN
3
"
BAP
2
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10 -6 M i n d o l e - 3 - a c e t i c acid, 10-8-10 -5 M indole-3-
I
butyric acid or 10 -7 M NAA at the 14th passage. However, it d e c r e a s e d gradually with i n c r e a s i n g number of p a s s a g e s on the same medium. Eventually, w i t h d r a w a l of auxins r e s u l t e d in the m o s t vigorous s h o o t m u l t i p l i c a tion. Therefore, we chose LS + 10-6 M BAP as the optimal s h o o t - m u l t i p l i c a t i o n medium. By the 17th passage a consistent average of 2.0 shoots p e r culture was obtained. Shoyama e~ al. (1983) r e p o r t e d that the addition of 1 mg/l BAP and 0.5 mg/l NAA to Murashige and Skoog's m e d i u m s t i m u l a t e d m u l t i p l i c a t i o n of shoots at a rate of c a . i0 shoots p e r culture within 7 weeks. These differences in m u l t i p l i c a t i o n m a y arise from the origin of the p l a n t source, the type of explants or incubation period. Rooting of the Shoot: For initiation of roots, multiple shoots were t r a n s f e r r e d to LS m e d i a o f x l, x 2 and x 4 d i l u t i o n containing no growth regulators, -6 -5 i0 M i n d o l e a c e t i c acid or NAA and i0 M indoleacetic acid or NAA. The number of roots per culture and average root length d e c r e a s e d w i t h i n c r e a s i n g d i l u t i o n of the basal m e d i u m (Fig. 4). Considering these two factors and the p e r c e n t a g e of rooted-shoots, we chose LS of the full strength containing no growth regulators as a rooting medium. This finding is similar to that of Shoyama et al. w h i c h showed that Murashige and Skoog's m e d i u m with no growth regulators is optimal for r o o t i n g of shoots. Fig. I-C shows a rooted shoot. Transfer of Plantlets to Soil: The rooted cultures were t r a n s p l a n t e d in the following p o t t i n g m i x t u r e s : v e r m i c u l i t e only, a 6 : 2 : 2 v e r m i c u l i t e : s a n d : p e a t cont a i n i n g 1% slaked lime, 7:3 soil:sterile leaf m o l d or
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No.of p a s s a g e s after shoot i n d u c t i o n
Fig. 2.
Effects of BAP, kinetin, 6 - i s o p e n t e n y l a m i n o purine (IP) and zeatin at the c o n c e n t r a t i o n of 10 -6 M ( O ) and 10 -5 M ( e ) on the m u l t i p l i c a t i o n of shoots during subculture. Each p o i n t represents the mean w i t h s t a n d a r d error from five replicates.
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Plant Cell Reports © Springer-Verlag 1984
In vitro propagation of A tractylodes lancea N o b o r u H i r a o k a , N o b u y u k i Y a m a d a , T o m o k o K o d a m a , and Y u t a k a T o m i t a Niigata College of Pharmacy, 5829 Kamishinei-cho, Niigata 950-21, Japan Received February 15, 1984/Revised version received March 29, 1984 - Communicated by F. Constabel
ABSTRACT Shoot cultures of Atractylodes lancea DC. (Compositae) have been e s t a b l i s h e d by i n o c u l a t i n g the flower bud on L i n s m a i e r - S k o o g ' s m e d i u m s u p p l e m e n t e d with 10 -5 M n a p h t h a l e n e a c e t i c acid and l0 -5 M 6 - b e n z y l aminopurine. Shoots were m u l t i p l i e d on a m e d i u m containing 10 -6 M 6 - b e n z y l a m i n o p u r i n e . P r o p a g a t e d shoots rooted on a m e d i u m devoid of any p l a n t ...... v....... ~ re~alatots were t r a n s f e r r e d to p o t t i n g soil and finally to the field.
ABBREVIATIONS LS NAA BAP
Linsmaier and Skoog (1965) m e d i u m 1 - N a p h t h a l e n e a c e t i c acid 6-Benzylaminopurine
d i u m were a u t o c l a v e d at 121°C for 15 min. The cultures were kept in a p h o t o p e r i o d of 16h/day of f l u o r e s c e n t light, 3000-6000 ix, at 25 _+ 5°C. Each one of the m u l t i p l e shoots, w i t h the large leaves removed, was e x c i s e d and t r a n s f e r r e d to fresh medium. Subcultures were p e r f o r m e d at 4-week intervals. The rooted shoots were t r a n s f e r r e d to various k i n d s of p o t t i n g soil. The p o t t e d plants were covered with a vinyl sheet for the first 1-2 weeks to m a i n t a i n humidity° The cover was gradually r e m o v e d to a l l o w the plants to acclimatize to greenhouse conditions. After 1-2 months, the plants were t r a m s f e r r e d to the field. The chromosome numbers of the P r o p a g a t e d plants were d e t e r m i n e d by cytological o b s e r v a t i o n of the root-tip cells a c c o r d i n g to the Feulgen squash m e t h o d (Darlington and La Cour 1969).
RESULTS AND D I S C U S S I O N INTRODUCTION The rhizome of Atractylodes lancea DC. (Compositae) is one of the crude drugs p r e s c r i b e d clinically for diuretic, stomachic and sudorific p u r p o s e s in trad i t i o n a l Chinese medicine. Since the p l a n t bears no seeds in Japan, d i v i s i o n of rhizomes is a usual p r o cedure for its propagation. Only a few pieces of the rhizome r e a d y for t r a n s p l a n t a t i o n can be o b t a i n e d from a 3- to 4 - y e a r - o l d p l a n t by this method. Recently, Shoyama et al. (1983) r e p o r t e d the clonal m u l t i p l i c a tion of this p l a n t through m e r i s t e m culture. We have a t t e m p t e d i n d e p e n d e n t l y to e s t a b l i s h a procedure for r a p i d clonal propagation, starting from flower buds. This p a p e r d e s c r i b e s the results of these experiments.
M A T E R I A L S AND M E T H O D S Flower buds (i0 m m long, 3 m m in diameter) were c o l l e c t e d late in July o r e a r l y in A u g u s t from p l a n t s o f about one m o n t h before the first flowering. They were w a s h e d for half an hour in r u n n i n g water, sterilized for 7 min in an aqueous solution of 0.1% HgCI c o n t a i n i n g 0.1% Tween 80 in a 100-ml E r l e n m e y e r fl~sk on a shaker, and rinsed three times w i t h sterile water. Several o u t e r involucral scales were cut off from buds and the buds were cut v e r t i c a l l y into four pieces. Both the scales and the bud segments served as explants. The b a s a l m e d i u m was LS t h r o u g h o u t the experiments. Test tubes (30 x 130 mm) c o n t a i n i n g 20 ml of agar m e -
Offprint requests to: N. Hiraoka
Shoot Induction: Flower buds were used as explants instead of shoot tips because a limited number o f plants were available and each rhizome has only a few shoot tips. The 40 bud segments were p l a c e d on LS containing one of four combinations o f N A A and BAP at c o n c e n t r a t i o n s o f 10 -6 M and/or 10 -5 M. After one m o n t h of incubation, a single shoot was induced from the explants on the m e d i u m supplemented with 10 -5 M N A A and 10 -5 M BAP (Fig. l-A). Involucral scales formed only callus tissues regardless of the m e d i a c o m p o s i tions d e s c r i b e d above.
Shoot M u l t i p l i c a t i o n : When the s h o o t was i n c u b a t e d on LS c o n t a i n i n g 10 -6 M BAP, w h i c h was analogous to the best culture conditions for in v i t r o shoot m u l t i p l i c a t i o n of p y r e t h r u m (Wambugu and Rangan 1981), it p r o d u c e d a new crop of m u l t i p l e shoots. By r e p e a t i n g this procedure, e n o u g h shoot m a t e r i a l s for the following e x p e r i m e n t s were obtained. Fig. 2 shows the effect of various kinds o f cytokinins on shoot m u l t i p l i c a t i o n d u r i n g four successive subcultures. Each one s h o o t e x p l a n t c o n s i s t e n t l y p r o d u c e d an additional shoot during the 4-week incubation p e r i od due to the a d d i t i o n of 10 -6 M BAP to the basal medium. The other cytokinins, kinetin, 6 - i s o p e n t e n y l a m i n o p u r i n e or zeatin~ were inferior to BAP in shoot multiplication. Fig. I-B shews typical features o f m u l t i p l e shoots thus obtained. ~ Fig. 3 shows the e f f e c t of auxins (in~ole-3-acetic
86
B
Fig. i.
A single shoot induced from a flower bud (A), a 4 - w e e k - o l d culture in the shoot m u l t i p l i c a t i o n p h a s e (B), a rooted shoot (C), p o t t e d p l a n t l e t s (D), and chromosomes in a r o o t - t i p cell of a p l a n t l e t (E).
acid, i n d o l e - 3 - b u t y r i c acid and NAA) on shoot multip l i c a t i o n d u r i n g four successive subcultures. All m e d i a contain 10 -6 M BAP and an auxin. The number of shoots per culture increased due to the addition o f
KINETIN
3
"
BAP
2
I I -?I j?~l ?-?-o
10 -6 M i n d o l e - 3 - a c e t i c acid, 10-8-10 -5 M indole-3-
I
butyric acid or 10 -7 M NAA at the 14th passage. However, it d e c r e a s e d gradually with i n c r e a s i n g number of p a s s a g e s on the same medium. Eventually, w i t h d r a w a l of auxins r e s u l t e d in the m o s t vigorous s h o o t m u l t i p l i c a tion. Therefore, we chose LS + 10-6 M BAP as the optimal s h o o t - m u l t i p l i c a t i o n medium. By the 17th passage a consistent average of 2.0 shoots p e r culture was obtained. Shoyama e~ al. (1983) r e p o r t e d that the addition of 1 mg/l BAP and 0.5 mg/l NAA to Murashige and Skoog's m e d i u m s t i m u l a t e d m u l t i p l i c a t i o n of shoots at a rate of c a . i0 shoots p e r culture within 7 weeks. These differences in m u l t i p l i c a t i o n m a y arise from the origin of the p l a n t source, the type of explants or incubation period. Rooting of the Shoot: For initiation of roots, multiple shoots were t r a n s f e r r e d to LS m e d i a o f x l, x 2 and x 4 d i l u t i o n containing no growth regulators, -6 -5 i0 M i n d o l e a c e t i c acid or NAA and i0 M indoleacetic acid or NAA. The number of roots per culture and average root length d e c r e a s e d w i t h i n c r e a s i n g d i l u t i o n of the basal m e d i u m (Fig. 4). Considering these two factors and the p e r c e n t a g e of rooted-shoots, we chose LS of the full strength containing no growth regulators as a rooting medium. This finding is similar to that of Shoyama et al. w h i c h showed that Murashige and Skoog's m e d i u m with no growth regulators is optimal for r o o t i n g of shoots. Fig. I-C shows a rooted shoot. Transfer of Plantlets to Soil: The rooted cultures were t r a n s p l a n t e d in the following p o t t i n g m i x t u r e s : v e r m i c u l i t e only, a 6 : 2 : 2 v e r m i c u l i t e : s a n d : p e a t cont a i n i n g 1% slaked lime, 7:3 soil:sterile leaf m o l d or
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No.of p a s s a g e s after shoot i n d u c t i o n
Fig. 2.
Effects of BAP, kinetin, 6 - i s o p e n t e n y l a m i n o purine (IP) and zeatin at the c o n c e n t r a t i o n of 10 -6 M ( O ) and 10 -5 M ( e ) on the m u l t i p l i c a t i o n of shoots during subculture. Each p o i n t represents the mean w i t h s t a n d a r d error from five replicates.
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