PlantCell Reports
Plant Cell Reports (1996) 16:147-152
© Springer-Verlag 1996
Micropropagation of Croton sublyratus Kurza tropical tree of medicinal importance Wataru Shibata 1, Fukashi Murai 1, Toshiyuki Akiyama 2, Manoon Siriphol 3, Eisuke Matsunaga 3, and Hiroyuki Morimoto ~ i 2 3 4
Biomedical Research Laboratories, Sankyo Co., Ltd., 2-58 Hiromachi 1-chome, Shinagawa-ku, Tokyo, 114 Japan Exploratory Chemistry Research Laboratories, Sankyo Co., Ltd., 2-58 Hiromaehi 1-chome, Shinagawa-ku, Tokyo, 114 Japan Agricultural Technical and Research Laboratory, Thai Sankyo Co., Ltd., 607 Asoke-Dindaeng Road, Bangkok 10400, Thailand Plant Laboratory, Kirin Brewery Co., Ltd., 3377 Kitsuregawa-machi, Shioya-gun, Tochigi-ken, 329-14 Japan
Received 30 August 1995/Revised version received 13 June 1996 - Communicated by A. Komamine
ABSTRACT A shoot tip culture procedure has been developed for the rapid multiplication of Croton sublyratus Knrz, a tropical plant species cultivated in Thailand for the production of an anti-ulcer medicine, plaunotoi. Optimum conditions were established : (1) for the regeneration of shoots from shoot tips: (2) for axillary shoot formation and rooting and (3) for adaptation of regenerated plants to the open ground. The results demonstrate the feasibility of applying the shoot tip culture technique for enhancing production of plaunotol by cultivating uniform populations of C. sublyratus with higher plannotol levels. ABBREVIATIONS MS: Murashigeand Skoog B5: Gamborg'sB5 medium WPM: McCown'swoodyplant medium BA: benzyladenine (6-benzylaminopurine) IBA: indole-3-bu~ricacid KN: kinetin 2iP: N-(2-isopentenyl)-adenine 4PU: N-(2-chloro-4-pyridyl)-N'-phenylurea INTRODUCTION
Croton sublyratus Kurz (Euphorbiaceae: commonly known as "Plau-noi" in Thai) is a woody plant indigenous to Thailand, and it has been used in Thai herbal medicines as an anthelmintic and dermatological agent (Phaethanesura, 1967; Brun and Shumacher, 1987). In 1973, Ogiso et aL (1978 &1985) discovered an anti-peptic ulcer activity in the extract of Plau-noi, and this activity was attributed to an acyelie diterpene named plaunotol (Fig. 1). Plaunotol is now available commercially in both Japan and Thailand as a therapeutic medicine in the treatment of peptic ulcers (Kelnac, Sankyo Co., Ltd., Tokyo).
Correspondence to." F. Murai
~
CH20H
~ C I ~ O U Fig. 1. Chemical structure of plaunotol; (E,Z,E)-7hydroxymethyl-3,11,l 5-trimethyl-2,6,10,14-hcxadecateraen-I-ol Commercially produced plaunotol is obtained by extraction from leaves of Plau-noi cultivated in southern Thailand (Fig. 2) (Matsunaga and Domethong, 1990; Matsunaga et al., 1990a, 1990b). Since plaunotol content varies significantly in individual trees and a few selected trees containing a potent amount of plaunotol (so-called elite tree) were found, the need has arisen to establish a suitable method for the rapid propagation of these selected elite trees. Conventional techniques, such as stem and root cuttings, as well as grafting, despite being commonly adopted in cultivation practice (Matsunaga et al., 1990c), are not attractive approaches for producing a large number of elite trees within a short period. Tissue culture studies on Plau-noi have been restricted to the production of plaunotol (I) in callus and cell suspension cultures (Morimoto and Mural, 1989). The aim of this study was to establish a practical method for rapid and large-scale multiplication of Plaunoi by induction of shoot proliferation from apical and axillarymeristems. The technique outlined below coupled with the micro-grafting technique has been successfully applied to produce thousands of plants and they have grown well under field conditions in Thailand.
MATERIALS AND METHODS Plant M a t e r i a l Seeds of Croton sublyratus Kurz were collected from a
148 cultivated Thai source. Subsequently, they were germinated and cultivated in Vermiculite in a growth cabinet at 25 °C under a 16 hr photoperiod using Cool White fluorescent tubes (50 ttmol m -2 sec-1). Juvenile trees were produced by grafting fresh branches of C. sublyratus, developed by pruning onto the rootstock of C. oblongifolius Roxb., the latter being produced from seedlings. Detailed procedures and advantages of this technique have been reported elsewhere (Matsunaga et aL, 1990c). The plants were maintained in a greenhouse under a controlled temperature (30 °C) and relative humidity (80%). P r e p a r a t i o n o f shoot tips Terminal shoot tips (approximately 2 cm in length) were excised from two-month-old seedlings. They were sterilized using a 2.5% sodium hypochlorite solution containing 0.1% Tween 20 for 10 rain., and with 70% ethanol for 30 sec.; then washed 3 times with sterilized water, before being i_.nocu!ated onto a medinm~ MS medium (Murashige and Skoog, 1962) supplemented with 1.0% (w/v) sucrose and 0.2 mffl BA was used, and the initial pH was adjusted to 5.8, prior to the addition of 0.4% (w/v) Gelrite (Kelco Corp., San Diego, USA). The shoot cultures were raised in Magenta G7 vessels and were incubated in a growth cabinet under the above-mentioned conditions for 6 weeks. The shoot cultures established in vitro were maintained by subculturing 6 apical or nodal shoot segments in a box, approximately every six weeks. Cultures were also initiated from axillary buds excised from a juvenile tree grafted onto C. oblongifolius (vide infra) by the same procedures described above. P r o p a g a t i o n of shoots For the purpose of effective shoot multiplication, two experiments were set up to investigate the effects of basal medium, sucrose, and gelling agents (Table 1), and also the effects of various concentrations of eytokinins, indicated in Table 2. The pH of the medium was adjusted to 5.8 before autoclaving. Six apical segments were cultured in each Magenta GA7 vessel sealed by Nesco film, and then incubated under controlled temperature (20 -24 °C), relative humidity (60 - 70%), and light (8 hr dark - 16 hr light). The number and length of shoots were recorded after 4 weeks. The number of leaves, if any, was also recorded. Elongation of shoots To promote growth, shoots were transferred to an MS medium supplemented with sucrose (1.0 %), solidified by Gelrite (0.2%). The pH of the medium was adjusted to 5.8 before autoclaving. Culture conditions during shoot elongation were the same as that during shoot multiplication. The shoots were cultured for 2 weeks to elongate fully. Root induction Shoots that attained a height of 3 cm were transferred to a Magenta GA7 vessel filled with agar, Gelrite, and Vermiculite moistened with various strengths of MS basal salts devoid of plant regulators. The experiments were
carried out under non-aseptic conditions in a growth cabinet. The number of shoots that rooted and the number' of roots per each rooted shoot were recorded 4 weeks after being transferred to the rooting medium (Table 3). Adaptation to the greenhouse and the open g r o u n d After 4 weeks the regenerated plants were transferred to a greenhouse equipped with a misting system. The plantlets were covered with polyethylene film, to maintain: humidity. They were watered once per week with onetenth- strength MS mineral salts. The film was then removed, and the cup was covered with cheese cloth. Initially for several days the plantlets were maintained under a high-humidity condition, and then they were acclimatized by decreasing the relative humidity (80 % to 60%) in the greenhouse. After a hardening period of 4 weeks, they were transferred to pots filled with soil and maintained in the greenhouse for at least 3 months. Four regenerated plants were then brought to southern Thailand and they were finally shifted to the field. C h e m i c a l analysis A quantitative analysis of plaunotol was carried out by the method described earlier (Morimoto and Murai, 1989), with slight modifications. Kitazawa et al. (1982) reported that the majority of plaunotol (I) occurred in the form of a fatty ester, and extraction with an alkaline methanol solution was undertaken to determine the total amount of' plaunotol in the leaves. Piaunotol levels of the 2nd, 3rd, and 5th leaves of the regenerated plantlets grown in the field for 10 to 15 weeks were analyzed (Table 4). The dried plant material (100 mg) was extracted with a mixture of methanol and 10% NaOH (9:1) under reflux for 1 hr. The extract was shaken with n-hexane, and the nh e x a n e layer was evaporated and dried under vacuum. The residue was dissolved in pyridine, followed by the addition of an internal standard (n-heptacosane). The mixture was treated with N,O-bis (trimethyi-silyl)-trifiuoroacetoamide, to prepare the corresponding trimethylsilyl derivatives. Quantitative analysis was undertaken with gas chromatography (Shimadzu GC-9A) using OV-17 (4%) on Chromosorb W (3 mm x 2.1 m) under the following conditions: Column Temperature, 250 °C; Carrier gas, He; detector, FID
RESULTS Shoot initiation Cultures of C. sublyratus were established from both seedlings and mature tree sources. Initially, an apical part of the seedlings was used for initiation of cultures. MS medium with 0.2 mg/i of BA and a phytohormone-free medium were tested. An initial stock of shoots was successfully established on a medium containing BA. All attempts to initiate an aseptic shoot culture from adult trees growing in the field were unsuccessful, due to serious problems of contamination and/or browning of the shoots. The problems were alleviated by taking the nodal explants from grafted trees grown in a greenhouse.
149 Sterilized shoots d e r i v e d f r o m a g r a f t e d t r e e w e r e e s t a b l i s h e d b y t h e s a m e p r o c e d u r e s d e s c r i b e d above. No distinction was o b s e r v e d b e t w e e n t h e r e s p o n s e o f t h e e x p l a n t s excised f r o m t h e a d u l t t r e e a n d t h e r e s p o n s e f r o m t h e seedlings. Shoot multiplication I n t h i s e x p e r i m e n t , n o d a l e x p l a n t s f r o m t h e in vitrog r o w n shoots w e r e used for assessing o p t i m a l r e q u i r e m e n t s for shoot development. Initially, n o d a l e x p l a n t s f r o m in vitro-grown shoots w e r e c u l t u r e d on v a r i o u s b a s a l m e d i a solidified b y a g a r o r b y G e i r i t e , u n d e r v a r i e d c o n c e n t r a t i o n s of s u g a r s u p p l e m e n t e d w i t h 0.2 mg/I of B A (Table 1~ Data_obtainedafter 6weeksrevealedthatMS m e d i u m c o n t a i n i n g 1.0% sucrose gelled w i t h G e i r i t e ( 0 . 4 % ) gave o p t i m a l r e s p o n s e s w i t h r e g a r d to t h e overall shoot development, as assessed b y total l e n g t h of t h e shoots a n d t h e n u m b e r of developed leaves.
developed a shoot c l u s t e r h a v i n g 2 to 4 shoots ( m o r e t h a n lq] m m in length), in a d d i t i o n to 6 to 12 apical a n d n o d a l s e g m e n t s (Fig. 3a).
Table 2. Effect of eytokinins on multiplication of Plan-noi shoots grown for 6 weeks on MS medium (sucrose: 1.0 %) supported by Gelrite (0.4 %) Cytoldnin Mean * Mean shoot* Mean number a Verification cone. shoots length of rate (mg/l) /explant (mm) developed leaves (%) 0A 0.6 0.8 1.0 2.0
1.0 _+0.1 b 1.0 _+0.1 b 1.2_0.1 b 1.2 -+ 0.1 b 1.3 _+0.2 b
18.2 _+1.5 26.5 _+2.0 31.8 -+2.120.3 +--2.4 22.8 _+3.1
6A _+0A 6.4 _+0.5 8.2-+0.5 7.9 _+0.6 6.3 _+0.5
0.0 16.7 7~.10.0 33.3
0.4 0.6 2iP 0.8 t.u 2.0
1.0 _+0.1 s 1.1 _+0.1 b 1.0 _+0.1 b ,.l -~o.t 1.1 _+0.I b
24.7 _+1.6 27.7 _+3.1 31.7 _+2.5 32.5 -+3.4 16.0 _+0.8
7.2 _+0.3 7.6 _+0.4 7.8 _+0.4 7.6 -+ 0.5 6.1 _+0.5
0.0 7.6 0.0 0.0 23.1
0.4
1.5 -+ 0.1 b
•
Table 1. Effect of medium composition supplemented by IBA (0.2 rag/l) on the growth of shoots of Pla u-noi within 4 weeks Basal Sucrose Gelling agent Mean shoot a Mean number of a medium (%) (%) length (mm) developed leaves B5 b WPM c xl/2 MS MS MS MS MS
1.0 1.0 1.0 1.0 1.0 2.0 3.0
Gelrite Geirite Gelrite Gelrite agar Gelrite Gelrite
(0A) (0.4) (0.4) (0.4) (0.8) (0.4) (0.4)
22.2 _+2.4 d 44.1 -+4.3 d 49.0 -+ 4.2 d 72.9 _+8.0 e 20.6 _+1.2 ~ 38.2 _+2.2 e 20.6 _+0.8 ~
8.3 _+0.6 8.6_+0.5 9.4 _+0.8 9.8 _+0.9 5.4 _+0.8 6.5 _+0.5 0.2 _+0.5
a: The means _+the standard deviation are listed. b: (Gamborg et al., 1968) c: (Lloyd and MeCown 1980) d: 42 shoots were transferred into the medium. e: 30 shoots were transferred into the medium.
I n a second e x p e r i m e n t , t h e effects of cytokinins on t h e shoot g r o w t h a n d m u l t i p l i c a t i o n r a t e were e x a m i n e d u s i n g t h e m e d i u m m e n t i o n e d above. T h e n o d a l e x p l a n t s were c u l t u r e d f o r 6 weeks in m e d i a c o n t a i n i n g various c o n c e n t r a t i o n s of KN, 2iP, 4PU, a n d BA. As s h o w n in T a b l e 2, t h e c u l t u r e s r e s p o n d e d n e i t h e r to KIN n o r 2iP, a n d tiny c l u s t e r s of a x i l l a r y shoots were o b s e r v e d to o c c u r a t a h i g h c o n c e n t r a t i o n o f 4 P U (1.0 rag/I). The medium c o n t a i n i n g a low c o n c e n t r a t i o n of B A (0.4 mg/I) s u p p o r t e d shoot elongation, b u t e x h i b i t e d a low f r e q u e n c y of a x i l l a r y shoot formation. W i t h a n i n c r e a s e i n t h e level of B A (1.0 2.0 mg/i) in t h e m e d i u m , t h e n u m b e r of a d v e n t i t i o u s shoots also increased, while shoot elongation was n o t distinct. F u r t h e r m o r e , callus d e v e l o p m e n t o c c u r r e d in t h e b a s a l region o f those e x p l a n t s w h i c h w e r e in c o n t a c t w i t h t h e m e d i u m . U n d e r a m o d e r a t e level o f B A (0.6 -0.8 mg/l), t h e e x p l a n t developed n u m e r o u s a d v e n t i t i o u s shoots, besides showing m o d e g a ~ s h ~ elongatiem. N o f a ~ m b l g effects o c c u r r e d w i t h a d d i t i o n a l KN, 2iP o r 4 P U ( d a t a not shown). F r o m t h e above results, M S m e d i u m s u p p l e m e n t e d w i t h sucrose ( L 0 % ) , B A (0.6 rag/l), a n d G e l r i t e ( 0 . 4 % ) was used as a m u l t i p l i c a t i o n m e d i u m , i n w h i c h a single shoot
A
.
d
,
n
.
b
60.8 -+ 4.8
8.1 + 0.4
0.0
0.6 4PU 0.8 1.0 2.0
1.0 _+0.1 b 51.4 _+3.6 1.2 _+0.1 b 61.0 _+5.0 2.6 _+0.2 b 80.1 _+9.2 4.0 _+0.8 b 77.8 _+8.3
8.7 _+0.5 9.6 _+0.7 11.3 _+1.2 16.8 _+2.4
0.0 0.0 0.0 0.0
0.4 0.6 BA 0.8 1.0 2.0
2.8 _+0.7 c 3.9 + 1.0 c 4.1__.1.1 ~ 5.8 _+1.2 c 7.4 _+1.8 ¢
14.9 -+ 2.2 18.7 + 2.0 19.3_+1.8 27.7 _+2.5 31.7 -+4.0
7.7 2.4 22.2 24.4 70.8
72.1 + 6.9 99.0 __.8.6 91.6_+8.8 96.2 _+9.6 81.9 + 7.5
a: The means + the standard deviation are listed. b: Out of 12 initial explants (>3 mm in length) e: Out of 42 initial explants (>3 mm in length)
Rooting of shoots and plantlet development To p r o m o t e growth, t h e m u l t i p l i e d shoots w e r e t r a n s f e r r e d to M S m e d i u m devoid of p h y t o h o r m o n e s p r i o r to rooting. T h e shoots a t t a i n e d a h e i g h t g r e a t e r t h a n 10 m m w i t h i n 4 to 6 weeks. T h u s , M S m e d i u m gelled w i t h G e i r i t e ( 0 . 2 % ) p r o v e d to b e a p p r o p r i a t e f o r p r e - r o o t i n g elongation of shoots ( d a t a not shown).
Table 3. Effect of strength of MS medium and support substrates on rooting of Plau-noi shoots within 4 weeks MS Support Rooted shoots medium / strength substrate Shoots tested 1 1/2 1/5 1/10 1/10 1/10
agar (0.8 %) agar (0.8 %) agar (0.8 %) agar (0.8 %) Gelrite(0.4%) Vermiculite
0/42 2/42 8/42 16/42 30/60 37•60
Roots" / shoot
Mean * root length (mm)
0.0 b 0.05 _+0.01 b 0.07 _+0.01 0.19 __.0.03 b 0.45 + 0.02 0.48 + 0.05 b 2.17 + 0.15 0.57 _ 0.05 c 9.95 _ 1.21 0.77 + 0.11 c 12.10 -+ 1.60
a: The means _+the standard deviation are listed. b: 42 shoots were tested e: 60 shoots were tested
150 Several experiments were carried out to determine a suitable basal medium (devoid of hormone and supplemented with sucrose (1.0 %)) which would be effective in supporting material for rooting under aseptic conditions. As shown in Table 3, a one-tenth MS medium supported by Vermiculite was found to be effective. In general, an increase in the percentage of rooted shoots was accompanied by an increase in the numbers of roots per rooted shoot. Addition of IBA did not cause a substantial enhancement in the high frequency of root induction. The most proliferative root formation was found in the shoots grown in one-tenth-strength MS medium supported by Vermiculite, as assessed by the numbers of roots per shoot and the total lengths of the roots within 4 weeks. More than 60% of the shoots were rooted, and in addition, they showed vigorous growth (Fig. 3b). The rooted plants (15 mm to 20 mm in height) were used in acclimatization studies without transplantation. With the method involving the use of a mist system in a greenhouse in the hardening stage, 80% of the plantlets survived for 4 weeks. The regenerated plants showed no morphological abnormalities. They were transferred into pots containing soil and were further maintained under greenhouse conditions. After 3 months, 100% of the planticts were successfully acclimatized (Fig. 3c) and four plants were transferred to the field, where they grew vigorously. Quantitative analysis o f plaunotol The plaunotol content of the micropropagated plantlets ( 6 months old) ranged from 0.7 to 0.8 % in the developed leaves and 1.3 % of the content was observed in juvenile leaf (Table 4), while an average of 1.0 % of plaunotol was detected in the leaves of the mother tree from which the initial shoot was taken. Table 4 Plaunotol content of mieropropagated plants Plaunotol content (% w/w) of respective leaves Plant number 2-nd 3-rd 5-th 1
2 3 4
0.8 0.7 _b b
0.8 0.7 0.7 0.8
1.3a 0.9 0.7 0.8
a: juvenile leaf b: leaf died
DISCUSSION The present study provides a method for micropropagation by induction of shoot proliferation from shoot tips of seedlings and nodal segments of adult trees of C. sublyratus Kurz, a woody tropical plant of medicinal importance. On average more than 3.5 shoots were sprouted from a single shoot tip during a six week period, and they were repeatedly snbcultured every six weeks. The propagated shoots can be rooted in the final six-weekculturing. The rooted shoots were transplanted to the
greenhouse and were finally shifted to the field. Further work is under way to improve the productivity of plaunotol. However, the micropropagated trees which have been successfully established so far have behaved uniformly in regard to leaf shape and plant form. Four plants have already grown well in the field and all plants produced plaunotol. Wide variations with respect to the yield of plaunotol in respective leaves and in populations have been disclosed by chemical analysis (Matsunaga et al, 1990b & 1990c). The present study revealed that the multiplied plantlets displayed characteristics, including the content of plaunotol in their leaves, that were identicaJ to their mother plant. The variation of the plaunotol content (0.7 - 0.8 in developed leaves) in the clonaily propagated plants was much smaller than that of normally grown plants (0.4 -1.1). Therefore, the procedures described here provide a future scope for rapid and ~ r scale multiplication of C. sublyratus, and offer a possible technique to raise the yield of plaunotoi using microproDa2ation initiated from a plant rich in plaunotol. Matsunaga et al., (1990e), reported that C. oblongifolius, a species closely related to C. sublyratus, showed various beneficial characteristics such as fast grow rate, high yield of the leaves and strong disease resistance. when compared with those of C. sublyratus, although C. oblongifolius is not capable of producing plaunotol in their leaves. It is of commercial value to combine the advantages of the two species with a grafting procedure. Trees produced by grafting (scion: C. sublyratus, rootstock: C. oblongifolius ) exhibited desirable characteristics induced by the rootstock and total plannotol production was higher for the grafted trees when compared with the self-rooted trees obtained from conventional cuttings. A combination procedure of rapid multiplication of selected elite trees in vitro with a mierografting technique resulted in the mass-production of elite trees with substantial improvement in productivity from grafting (Siriphol et aL, 1993). Using this procedure, thousands of' plants have been established under field conditions in Thailand. A detailed description of the methods and the results of mierografting will be presented in a subsequent paper.
151
Fig. 3 (a)
Fig. 2
Fig. 3 (b)
Fig. 3 (c)
Fig. 2. Mature tree of"Plau-noi" (Crotonsublyr~us Kurz) cuitiva~tedin the field. Fig. 3D Stages of plant regeneration from shoot tips of C. sul@ratu:~ (a) multiple shoot development (b) rooted plantlets (¢) regenarated plants
152 ACKNOWLEDGMENTS The authors would like to thank Dr. Akira Ogiso for his support and advice. Thanks are also extended to Mrs. Yuko Sugano and Miss Ternmi Takahashi, for their technical assistance.
REFERENCES Brnn V, Shumacher T (1987) Traditional Herbal Medicine in Northern Thailand. University. of California Press, Berkeley, p263 Gamhorg O L, Miller R A and Ojima K (1968) Exp. Cell Res., 50:151-158 Kitazawa E, Kurabayashi M, Kasuga S, Oda O, Ogiso A (1982) Ann. Rep. Sankyo Res. Lab. 34".39-41 Lloyd G and McCown B (1080) Comb. Proc. Int. Plant Propagator's Soc., 30:421-427 Matsunaga E, Domethong C (1990) Jap. J. Trop. Agr., 34:48-50 Matsunaga E, Horibe Y, Promdej C (1990a) Jap. J. Trop. Agr., 34:191-196 Matsunaga E, Domethong C, Boriboon M (1990b) Jap. J. Trop. Agr., 34:243-249 Matsunaga E, Domethong C, Boriboon M, Smitinand T (1990c) Jap. J. Trop.Agr~, "34:284-288 Morimoto H, Murai F (1989) Plant Cell Report 8:210-213 Murashige T and Skoog F (1962) Physiol. Plantarum 15:473-497 Ogiso A, Kitazawa E, Kurabayashi M, Sato A, Takahashi S, Noguchi H, Kuwano H (1978) Chem. Pharm. Bull., 26:3117-3123 Ogiso, A Kitazawa E, Kobayashi S, Komai T, Matsunuma N, Kataumi S (1985) Annu. Rep. Sankyo Res. Lab. 37:1-39 Phaet-hanesura P ed. (1967) Pramual Sapphakhun Ya Thai (Medicinal Uses of Thai Drugs). voi 2, The Association of School of Old-Style Medicine, Bangkok, Thailand, pp 265-266 Siriphol M, Matsunaga E, Shibata W and Akiyama T (1993) Thailand Patent No. 3526
Plant Cell Reports (1996) 16:147-152
© Springer-Verlag 1996
Micropropagation of Croton sublyratus Kurza tropical tree of medicinal importance Wataru Shibata 1, Fukashi Murai 1, Toshiyuki Akiyama 2, Manoon Siriphol 3, Eisuke Matsunaga 3, and Hiroyuki Morimoto ~ i 2 3 4
Biomedical Research Laboratories, Sankyo Co., Ltd., 2-58 Hiromachi 1-chome, Shinagawa-ku, Tokyo, 114 Japan Exploratory Chemistry Research Laboratories, Sankyo Co., Ltd., 2-58 Hiromaehi 1-chome, Shinagawa-ku, Tokyo, 114 Japan Agricultural Technical and Research Laboratory, Thai Sankyo Co., Ltd., 607 Asoke-Dindaeng Road, Bangkok 10400, Thailand Plant Laboratory, Kirin Brewery Co., Ltd., 3377 Kitsuregawa-machi, Shioya-gun, Tochigi-ken, 329-14 Japan
Received 30 August 1995/Revised version received 13 June 1996 - Communicated by A. Komamine
ABSTRACT A shoot tip culture procedure has been developed for the rapid multiplication of Croton sublyratus Knrz, a tropical plant species cultivated in Thailand for the production of an anti-ulcer medicine, plaunotoi. Optimum conditions were established : (1) for the regeneration of shoots from shoot tips: (2) for axillary shoot formation and rooting and (3) for adaptation of regenerated plants to the open ground. The results demonstrate the feasibility of applying the shoot tip culture technique for enhancing production of plaunotol by cultivating uniform populations of C. sublyratus with higher plannotol levels. ABBREVIATIONS MS: Murashigeand Skoog B5: Gamborg'sB5 medium WPM: McCown'swoodyplant medium BA: benzyladenine (6-benzylaminopurine) IBA: indole-3-bu~ricacid KN: kinetin 2iP: N-(2-isopentenyl)-adenine 4PU: N-(2-chloro-4-pyridyl)-N'-phenylurea INTRODUCTION
Croton sublyratus Kurz (Euphorbiaceae: commonly known as "Plau-noi" in Thai) is a woody plant indigenous to Thailand, and it has been used in Thai herbal medicines as an anthelmintic and dermatological agent (Phaethanesura, 1967; Brun and Shumacher, 1987). In 1973, Ogiso et aL (1978 &1985) discovered an anti-peptic ulcer activity in the extract of Plau-noi, and this activity was attributed to an acyelie diterpene named plaunotol (Fig. 1). Plaunotol is now available commercially in both Japan and Thailand as a therapeutic medicine in the treatment of peptic ulcers (Kelnac, Sankyo Co., Ltd., Tokyo).
Correspondence to." F. Murai
~
CH20H
~ C I ~ O U Fig. 1. Chemical structure of plaunotol; (E,Z,E)-7hydroxymethyl-3,11,l 5-trimethyl-2,6,10,14-hcxadecateraen-I-ol Commercially produced plaunotol is obtained by extraction from leaves of Plau-noi cultivated in southern Thailand (Fig. 2) (Matsunaga and Domethong, 1990; Matsunaga et al., 1990a, 1990b). Since plaunotol content varies significantly in individual trees and a few selected trees containing a potent amount of plaunotol (so-called elite tree) were found, the need has arisen to establish a suitable method for the rapid propagation of these selected elite trees. Conventional techniques, such as stem and root cuttings, as well as grafting, despite being commonly adopted in cultivation practice (Matsunaga et al., 1990c), are not attractive approaches for producing a large number of elite trees within a short period. Tissue culture studies on Plau-noi have been restricted to the production of plaunotol (I) in callus and cell suspension cultures (Morimoto and Mural, 1989). The aim of this study was to establish a practical method for rapid and large-scale multiplication of Plaunoi by induction of shoot proliferation from apical and axillarymeristems. The technique outlined below coupled with the micro-grafting technique has been successfully applied to produce thousands of plants and they have grown well under field conditions in Thailand.
MATERIALS AND METHODS Plant M a t e r i a l Seeds of Croton sublyratus Kurz were collected from a
148 cultivated Thai source. Subsequently, they were germinated and cultivated in Vermiculite in a growth cabinet at 25 °C under a 16 hr photoperiod using Cool White fluorescent tubes (50 ttmol m -2 sec-1). Juvenile trees were produced by grafting fresh branches of C. sublyratus, developed by pruning onto the rootstock of C. oblongifolius Roxb., the latter being produced from seedlings. Detailed procedures and advantages of this technique have been reported elsewhere (Matsunaga et aL, 1990c). The plants were maintained in a greenhouse under a controlled temperature (30 °C) and relative humidity (80%). P r e p a r a t i o n o f shoot tips Terminal shoot tips (approximately 2 cm in length) were excised from two-month-old seedlings. They were sterilized using a 2.5% sodium hypochlorite solution containing 0.1% Tween 20 for 10 rain., and with 70% ethanol for 30 sec.; then washed 3 times with sterilized water, before being i_.nocu!ated onto a medinm~ MS medium (Murashige and Skoog, 1962) supplemented with 1.0% (w/v) sucrose and 0.2 mffl BA was used, and the initial pH was adjusted to 5.8, prior to the addition of 0.4% (w/v) Gelrite (Kelco Corp., San Diego, USA). The shoot cultures were raised in Magenta G7 vessels and were incubated in a growth cabinet under the above-mentioned conditions for 6 weeks. The shoot cultures established in vitro were maintained by subculturing 6 apical or nodal shoot segments in a box, approximately every six weeks. Cultures were also initiated from axillary buds excised from a juvenile tree grafted onto C. oblongifolius (vide infra) by the same procedures described above. P r o p a g a t i o n of shoots For the purpose of effective shoot multiplication, two experiments were set up to investigate the effects of basal medium, sucrose, and gelling agents (Table 1), and also the effects of various concentrations of eytokinins, indicated in Table 2. The pH of the medium was adjusted to 5.8 before autoclaving. Six apical segments were cultured in each Magenta GA7 vessel sealed by Nesco film, and then incubated under controlled temperature (20 -24 °C), relative humidity (60 - 70%), and light (8 hr dark - 16 hr light). The number and length of shoots were recorded after 4 weeks. The number of leaves, if any, was also recorded. Elongation of shoots To promote growth, shoots were transferred to an MS medium supplemented with sucrose (1.0 %), solidified by Gelrite (0.2%). The pH of the medium was adjusted to 5.8 before autoclaving. Culture conditions during shoot elongation were the same as that during shoot multiplication. The shoots were cultured for 2 weeks to elongate fully. Root induction Shoots that attained a height of 3 cm were transferred to a Magenta GA7 vessel filled with agar, Gelrite, and Vermiculite moistened with various strengths of MS basal salts devoid of plant regulators. The experiments were
carried out under non-aseptic conditions in a growth cabinet. The number of shoots that rooted and the number' of roots per each rooted shoot were recorded 4 weeks after being transferred to the rooting medium (Table 3). Adaptation to the greenhouse and the open g r o u n d After 4 weeks the regenerated plants were transferred to a greenhouse equipped with a misting system. The plantlets were covered with polyethylene film, to maintain: humidity. They were watered once per week with onetenth- strength MS mineral salts. The film was then removed, and the cup was covered with cheese cloth. Initially for several days the plantlets were maintained under a high-humidity condition, and then they were acclimatized by decreasing the relative humidity (80 % to 60%) in the greenhouse. After a hardening period of 4 weeks, they were transferred to pots filled with soil and maintained in the greenhouse for at least 3 months. Four regenerated plants were then brought to southern Thailand and they were finally shifted to the field. C h e m i c a l analysis A quantitative analysis of plaunotol was carried out by the method described earlier (Morimoto and Murai, 1989), with slight modifications. Kitazawa et al. (1982) reported that the majority of plaunotol (I) occurred in the form of a fatty ester, and extraction with an alkaline methanol solution was undertaken to determine the total amount of' plaunotol in the leaves. Piaunotol levels of the 2nd, 3rd, and 5th leaves of the regenerated plantlets grown in the field for 10 to 15 weeks were analyzed (Table 4). The dried plant material (100 mg) was extracted with a mixture of methanol and 10% NaOH (9:1) under reflux for 1 hr. The extract was shaken with n-hexane, and the nh e x a n e layer was evaporated and dried under vacuum. The residue was dissolved in pyridine, followed by the addition of an internal standard (n-heptacosane). The mixture was treated with N,O-bis (trimethyi-silyl)-trifiuoroacetoamide, to prepare the corresponding trimethylsilyl derivatives. Quantitative analysis was undertaken with gas chromatography (Shimadzu GC-9A) using OV-17 (4%) on Chromosorb W (3 mm x 2.1 m) under the following conditions: Column Temperature, 250 °C; Carrier gas, He; detector, FID
RESULTS Shoot initiation Cultures of C. sublyratus were established from both seedlings and mature tree sources. Initially, an apical part of the seedlings was used for initiation of cultures. MS medium with 0.2 mg/i of BA and a phytohormone-free medium were tested. An initial stock of shoots was successfully established on a medium containing BA. All attempts to initiate an aseptic shoot culture from adult trees growing in the field were unsuccessful, due to serious problems of contamination and/or browning of the shoots. The problems were alleviated by taking the nodal explants from grafted trees grown in a greenhouse.
149 Sterilized shoots d e r i v e d f r o m a g r a f t e d t r e e w e r e e s t a b l i s h e d b y t h e s a m e p r o c e d u r e s d e s c r i b e d above. No distinction was o b s e r v e d b e t w e e n t h e r e s p o n s e o f t h e e x p l a n t s excised f r o m t h e a d u l t t r e e a n d t h e r e s p o n s e f r o m t h e seedlings. Shoot multiplication I n t h i s e x p e r i m e n t , n o d a l e x p l a n t s f r o m t h e in vitrog r o w n shoots w e r e used for assessing o p t i m a l r e q u i r e m e n t s for shoot development. Initially, n o d a l e x p l a n t s f r o m in vitro-grown shoots w e r e c u l t u r e d on v a r i o u s b a s a l m e d i a solidified b y a g a r o r b y G e i r i t e , u n d e r v a r i e d c o n c e n t r a t i o n s of s u g a r s u p p l e m e n t e d w i t h 0.2 mg/I of B A (Table 1~ Data_obtainedafter 6weeksrevealedthatMS m e d i u m c o n t a i n i n g 1.0% sucrose gelled w i t h G e i r i t e ( 0 . 4 % ) gave o p t i m a l r e s p o n s e s w i t h r e g a r d to t h e overall shoot development, as assessed b y total l e n g t h of t h e shoots a n d t h e n u m b e r of developed leaves.
developed a shoot c l u s t e r h a v i n g 2 to 4 shoots ( m o r e t h a n lq] m m in length), in a d d i t i o n to 6 to 12 apical a n d n o d a l s e g m e n t s (Fig. 3a).
Table 2. Effect of eytokinins on multiplication of Plan-noi shoots grown for 6 weeks on MS medium (sucrose: 1.0 %) supported by Gelrite (0.4 %) Cytoldnin Mean * Mean shoot* Mean number a Verification cone. shoots length of rate (mg/l) /explant (mm) developed leaves (%) 0A 0.6 0.8 1.0 2.0
1.0 _+0.1 b 1.0 _+0.1 b 1.2_0.1 b 1.2 -+ 0.1 b 1.3 _+0.2 b
18.2 _+1.5 26.5 _+2.0 31.8 -+2.120.3 +--2.4 22.8 _+3.1
6A _+0A 6.4 _+0.5 8.2-+0.5 7.9 _+0.6 6.3 _+0.5
0.0 16.7 7~.10.0 33.3
0.4 0.6 2iP 0.8 t.u 2.0
1.0 _+0.1 s 1.1 _+0.1 b 1.0 _+0.1 b ,.l -~o.t 1.1 _+0.I b
24.7 _+1.6 27.7 _+3.1 31.7 _+2.5 32.5 -+3.4 16.0 _+0.8
7.2 _+0.3 7.6 _+0.4 7.8 _+0.4 7.6 -+ 0.5 6.1 _+0.5
0.0 7.6 0.0 0.0 23.1
0.4
1.5 -+ 0.1 b
•
Table 1. Effect of medium composition supplemented by IBA (0.2 rag/l) on the growth of shoots of Pla u-noi within 4 weeks Basal Sucrose Gelling agent Mean shoot a Mean number of a medium (%) (%) length (mm) developed leaves B5 b WPM c xl/2 MS MS MS MS MS
1.0 1.0 1.0 1.0 1.0 2.0 3.0
Gelrite Geirite Gelrite Gelrite agar Gelrite Gelrite
(0A) (0.4) (0.4) (0.4) (0.8) (0.4) (0.4)
22.2 _+2.4 d 44.1 -+4.3 d 49.0 -+ 4.2 d 72.9 _+8.0 e 20.6 _+1.2 ~ 38.2 _+2.2 e 20.6 _+0.8 ~
8.3 _+0.6 8.6_+0.5 9.4 _+0.8 9.8 _+0.9 5.4 _+0.8 6.5 _+0.5 0.2 _+0.5
a: The means _+the standard deviation are listed. b: (Gamborg et al., 1968) c: (Lloyd and MeCown 1980) d: 42 shoots were transferred into the medium. e: 30 shoots were transferred into the medium.
I n a second e x p e r i m e n t , t h e effects of cytokinins on t h e shoot g r o w t h a n d m u l t i p l i c a t i o n r a t e were e x a m i n e d u s i n g t h e m e d i u m m e n t i o n e d above. T h e n o d a l e x p l a n t s were c u l t u r e d f o r 6 weeks in m e d i a c o n t a i n i n g various c o n c e n t r a t i o n s of KN, 2iP, 4PU, a n d BA. As s h o w n in T a b l e 2, t h e c u l t u r e s r e s p o n d e d n e i t h e r to KIN n o r 2iP, a n d tiny c l u s t e r s of a x i l l a r y shoots were o b s e r v e d to o c c u r a t a h i g h c o n c e n t r a t i o n o f 4 P U (1.0 rag/I). The medium c o n t a i n i n g a low c o n c e n t r a t i o n of B A (0.4 mg/I) s u p p o r t e d shoot elongation, b u t e x h i b i t e d a low f r e q u e n c y of a x i l l a r y shoot formation. W i t h a n i n c r e a s e i n t h e level of B A (1.0 2.0 mg/i) in t h e m e d i u m , t h e n u m b e r of a d v e n t i t i o u s shoots also increased, while shoot elongation was n o t distinct. F u r t h e r m o r e , callus d e v e l o p m e n t o c c u r r e d in t h e b a s a l region o f those e x p l a n t s w h i c h w e r e in c o n t a c t w i t h t h e m e d i u m . U n d e r a m o d e r a t e level o f B A (0.6 -0.8 mg/l), t h e e x p l a n t developed n u m e r o u s a d v e n t i t i o u s shoots, besides showing m o d e g a ~ s h ~ elongatiem. N o f a ~ m b l g effects o c c u r r e d w i t h a d d i t i o n a l KN, 2iP o r 4 P U ( d a t a not shown). F r o m t h e above results, M S m e d i u m s u p p l e m e n t e d w i t h sucrose ( L 0 % ) , B A (0.6 rag/l), a n d G e l r i t e ( 0 . 4 % ) was used as a m u l t i p l i c a t i o n m e d i u m , i n w h i c h a single shoot
A
.
d
,
n
.
b
60.8 -+ 4.8
8.1 + 0.4
0.0
0.6 4PU 0.8 1.0 2.0
1.0 _+0.1 b 51.4 _+3.6 1.2 _+0.1 b 61.0 _+5.0 2.6 _+0.2 b 80.1 _+9.2 4.0 _+0.8 b 77.8 _+8.3
8.7 _+0.5 9.6 _+0.7 11.3 _+1.2 16.8 _+2.4
0.0 0.0 0.0 0.0
0.4 0.6 BA 0.8 1.0 2.0
2.8 _+0.7 c 3.9 + 1.0 c 4.1__.1.1 ~ 5.8 _+1.2 c 7.4 _+1.8 ¢
14.9 -+ 2.2 18.7 + 2.0 19.3_+1.8 27.7 _+2.5 31.7 -+4.0
7.7 2.4 22.2 24.4 70.8
72.1 + 6.9 99.0 __.8.6 91.6_+8.8 96.2 _+9.6 81.9 + 7.5
a: The means + the standard deviation are listed. b: Out of 12 initial explants (>3 mm in length) e: Out of 42 initial explants (>3 mm in length)
Rooting of shoots and plantlet development To p r o m o t e growth, t h e m u l t i p l i e d shoots w e r e t r a n s f e r r e d to M S m e d i u m devoid of p h y t o h o r m o n e s p r i o r to rooting. T h e shoots a t t a i n e d a h e i g h t g r e a t e r t h a n 10 m m w i t h i n 4 to 6 weeks. T h u s , M S m e d i u m gelled w i t h G e i r i t e ( 0 . 2 % ) p r o v e d to b e a p p r o p r i a t e f o r p r e - r o o t i n g elongation of shoots ( d a t a not shown).
Table 3. Effect of strength of MS medium and support substrates on rooting of Plau-noi shoots within 4 weeks MS Support Rooted shoots medium / strength substrate Shoots tested 1 1/2 1/5 1/10 1/10 1/10
agar (0.8 %) agar (0.8 %) agar (0.8 %) agar (0.8 %) Gelrite(0.4%) Vermiculite
0/42 2/42 8/42 16/42 30/60 37•60
Roots" / shoot
Mean * root length (mm)
0.0 b 0.05 _+0.01 b 0.07 _+0.01 0.19 __.0.03 b 0.45 + 0.02 0.48 + 0.05 b 2.17 + 0.15 0.57 _ 0.05 c 9.95 _ 1.21 0.77 + 0.11 c 12.10 -+ 1.60
a: The means _+the standard deviation are listed. b: 42 shoots were tested e: 60 shoots were tested
150 Several experiments were carried out to determine a suitable basal medium (devoid of hormone and supplemented with sucrose (1.0 %)) which would be effective in supporting material for rooting under aseptic conditions. As shown in Table 3, a one-tenth MS medium supported by Vermiculite was found to be effective. In general, an increase in the percentage of rooted shoots was accompanied by an increase in the numbers of roots per rooted shoot. Addition of IBA did not cause a substantial enhancement in the high frequency of root induction. The most proliferative root formation was found in the shoots grown in one-tenth-strength MS medium supported by Vermiculite, as assessed by the numbers of roots per shoot and the total lengths of the roots within 4 weeks. More than 60% of the shoots were rooted, and in addition, they showed vigorous growth (Fig. 3b). The rooted plants (15 mm to 20 mm in height) were used in acclimatization studies without transplantation. With the method involving the use of a mist system in a greenhouse in the hardening stage, 80% of the plantlets survived for 4 weeks. The regenerated plants showed no morphological abnormalities. They were transferred into pots containing soil and were further maintained under greenhouse conditions. After 3 months, 100% of the planticts were successfully acclimatized (Fig. 3c) and four plants were transferred to the field, where they grew vigorously. Quantitative analysis o f plaunotol The plaunotol content of the micropropagated plantlets ( 6 months old) ranged from 0.7 to 0.8 % in the developed leaves and 1.3 % of the content was observed in juvenile leaf (Table 4), while an average of 1.0 % of plaunotol was detected in the leaves of the mother tree from which the initial shoot was taken. Table 4 Plaunotol content of mieropropagated plants Plaunotol content (% w/w) of respective leaves Plant number 2-nd 3-rd 5-th 1
2 3 4
0.8 0.7 _b b
0.8 0.7 0.7 0.8
1.3a 0.9 0.7 0.8
a: juvenile leaf b: leaf died
DISCUSSION The present study provides a method for micropropagation by induction of shoot proliferation from shoot tips of seedlings and nodal segments of adult trees of C. sublyratus Kurz, a woody tropical plant of medicinal importance. On average more than 3.5 shoots were sprouted from a single shoot tip during a six week period, and they were repeatedly snbcultured every six weeks. The propagated shoots can be rooted in the final six-weekculturing. The rooted shoots were transplanted to the
greenhouse and were finally shifted to the field. Further work is under way to improve the productivity of plaunotol. However, the micropropagated trees which have been successfully established so far have behaved uniformly in regard to leaf shape and plant form. Four plants have already grown well in the field and all plants produced plaunotol. Wide variations with respect to the yield of plaunotol in respective leaves and in populations have been disclosed by chemical analysis (Matsunaga et al, 1990b & 1990c). The present study revealed that the multiplied plantlets displayed characteristics, including the content of plaunotol in their leaves, that were identicaJ to their mother plant. The variation of the plaunotol content (0.7 - 0.8 in developed leaves) in the clonaily propagated plants was much smaller than that of normally grown plants (0.4 -1.1). Therefore, the procedures described here provide a future scope for rapid and ~ r scale multiplication of C. sublyratus, and offer a possible technique to raise the yield of plaunotoi using microproDa2ation initiated from a plant rich in plaunotol. Matsunaga et al., (1990e), reported that C. oblongifolius, a species closely related to C. sublyratus, showed various beneficial characteristics such as fast grow rate, high yield of the leaves and strong disease resistance. when compared with those of C. sublyratus, although C. oblongifolius is not capable of producing plaunotol in their leaves. It is of commercial value to combine the advantages of the two species with a grafting procedure. Trees produced by grafting (scion: C. sublyratus, rootstock: C. oblongifolius ) exhibited desirable characteristics induced by the rootstock and total plannotol production was higher for the grafted trees when compared with the self-rooted trees obtained from conventional cuttings. A combination procedure of rapid multiplication of selected elite trees in vitro with a mierografting technique resulted in the mass-production of elite trees with substantial improvement in productivity from grafting (Siriphol et aL, 1993). Using this procedure, thousands of' plants have been established under field conditions in Thailand. A detailed description of the methods and the results of mierografting will be presented in a subsequent paper.
151
Fig. 3 (a)
Fig. 2
Fig. 3 (b)
Fig. 3 (c)
Fig. 2. Mature tree of"Plau-noi" (Crotonsublyr~us Kurz) cuitiva~tedin the field. Fig. 3D Stages of plant regeneration from shoot tips of C. sul@ratu:~ (a) multiple shoot development (b) rooted plantlets (¢) regenarated plants
152 ACKNOWLEDGMENTS The authors would like to thank Dr. Akira Ogiso for his support and advice. Thanks are also extended to Mrs. Yuko Sugano and Miss Ternmi Takahashi, for their technical assistance.
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