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Morphogenetic and biosynthetic studies on tissue cultures of Atropa belladonna L.
Plant Science Letters, 13 (1978) 83--89 © Elsevier/North-HollandScientific Publishers Ltd.
83
MORPHOGENETIC AND BIOSYNTHETIC STUDIES ON TISSUE CULTURES OF A T R O P A B E L L A D O N N A L.
S U S A N EAPEN, T.S. R A N G A N , M.S. C H A D H A
and M.R. H E B L E
Plant Morphogenesis and Tissue Culture Section, Bio-Organic Division, Bhabha Atomic Research Centre, Bombay 400 085 (India.)
(Received December 1st,1977) (Accepted February 28th, 1978)
SUMMARY
The morphogenetic and biosynthetic ability of tissue cultures established from haploid and diploid plants of Atropa belladonna L. have been investigated. It was observed that the haploid tissue regenerated plantlets much more readily than did the diploids. The regenerants obtained in vitro were successfully transplanted to soil and grown to flowering stage. Haploid and diploid calli, regenerated young shoot buds and the in vitro obtained plants (at the stage of flowering) were subjected to chemical analysis. The total alkaloid contents in the calli and young shoot buds were very low (8--20 pg/g) as compared to the mature plant (1160 gg/g). Tropine, atropine, scopolamine and several unidentified Dragendorff-positive components were observed in the cultures. The alkaloid spectrum of the shoot buds was comparable to that of the unorganized calli. However, the relative proportion of atropine was more in the shoot buds as compared to the callus cultures. The regenerated flowering plants contained the principal belladonna alkaloids in quantities comparable to the plants raised from seeds.
INTRODUCTION
Haploid plants and tissues in vitro have been considered useful in somatic cell genetics and plant breeding [1,2]. Tissue cultures derived from haploid plants could be used to advantage in selection of mutants [3,4] since mutations in such cultures will be expressed without any masking effect by dominant alleles. Desirable mutants can be diplodized and homozygous inbred lines can be obtained. We have established tissue cultures from haploid and diploid plants of Abbreviations: BA, 6-benzyladenine; Kn, kinetin;MS, Murashige and Skoog; NAA, ~naphthaleneacetic acid;W and B, Wood and Braun.
84
Atropa belladonna L. with a view to study their morphogenetic behaviour and to obtain homozygous mutants with high alkaloid content through haploid cultures. The present report is concerned with the organ-forming ability of these cultures and their metabolic status in regard to the synthesis of tropane alkaloids. MATERIALS AND METHODS
Seeds o f Atropa belladonna L. (a m u t a n t variety developed by the Biology and Agriculture Division, Bhabha Atomic Research Centre, B o m b a y ) were used as the source material. While the diploid plant (2n = 72) was raised directly from the seeds, the haploid (n = 36) was obtained by anther culture. Tissue cultures were established from the leaves of one haploid and one diploid plant (about 0.6 m high and n o t bearing any flowers}. The leaves were surface-sterilized with 0.1% mercuric chloride and small leaf segments were cultured on W and B medium [5] supplemented with Lin and Staba vitamins [ 6 ] , NAA and Kn. For some experiments on organogenesis MS basal medium [7] was employed. The pH of the medium was adjusted to 5.8 and gelled with 0.9% agar. The cultures were maintained in diffuse light at 25 + 2°C. Haploid and diploid tissues from 5th, 6th and 7th passages growing on W & B + NAA + Kn medium were pooled and 50 g of the dry tissue was utilized for chemical analysis. Similarly tissues from 7th and 8th passages growing on MS + BA medium were pooled and 14 g of the dry tissue was analyzed. Visible shoot buds were removed from regenerating tissues and the callus and shoot buds were examined separately. The incubation period was generally 30 days, at the end o f which the tissues were harvested and dried. Plants that regenerated from callus cultures were grown in pots and harvested at the flowering stage f o r analysis.
Extraction and estimation of alkaloids The tissues, harvested after 30 days growth, were dried at 60°C for 24 h, powdered, macerated overnight with EtOH -- 28% NH4OH (9 : 1) mixture and refluxed with methanol. The methanol extract was evaporated to dryness and the residue thus obtained was dissolved in 0.1 N HC1 and extracted with CHC13. The CHC13 layer was washed with 0.1 N HC1. The acidic extract and the washings were pooled, made alkaline with N NaOH and extracted with CHC13, which gave crude basic fraction. This fraction was further dissolved in 0.05 N HCI, filtered and the basic components were regenerated and extracted with CHC13 as above. The CHCI3 extract was dried over Na2 SO4 and evaporated to dryness which gave basic fractions. Aliquots of this fraction were used for colorimetry and GLC analysis. Estimation of total alkaloids was done by the Vitali-Morin m e t h o d [ 8 ] , with slight modifications. The nitrated basic components were dissolved in 10 ml o f DMF, basified with 100 ~1 o f freshly prepared 3% KOH in methanol and the intensity of colour developed after 2 min was measured at 545 nm. The
85 total alkaloid contents were calculated using the atropine calibration curve. TLC was carried out using silica gel C (Acme Products, India) and two solvent systems, system 1:CHC13-EtOH -- 25% NH4OH (85 : 14 : 1) system 2: CHC13 saturated with 25% NH4OH. GLC analysis was carried out on 2 m, 65 cmm glass columns packed with 2% OV-17 or 3% SE-30 on Chromosorb W 80/100 mesh using N: as carrier gas and FID. The alkaloid mixtures were analyzed under following conditions; SE-30 : Isothermal 200°C, Temperature programme 150--210°C (8°/min); OV-17 : Isothermal 230°C, Temperature programme 160--250°C (4°/min). Each extract was analyzed under different conditions using authentic atropine, tropine and scopolamine standards. The identity of tropine, atropine, and scopolamine in the tissue extracts was confirmed through co-chromatography (GLC, 3% SE-30) with authentic samples. RESULTS A: Organogenesis and plantlet regeneration When the explants were cultured on W & B + NAA (2 mg/1) + Kn (0.5 mg/1), in about 3 weeks cell proliferation occurred from the periphery and an actively growing mass of tissue was formed. While the haploid (The terms haploid culture and diploid cultures are used to indicate the source) cultures showed a high degree (100%) of shoot bud formation the diploid cultures grew as an unorganised mass of tissue. Experiments were carried out to ascertain the organogenetic potential of these tissues under different nutritional and hormonal conditions. It was observed that the haploid as well as the diploid cultures exhibited 100% shoot bud regeneration on MS medium supplemented with BA (1 mg/1). The maximum number of shoot buds that developed per culture, however, was 6.7 + 0.91 in diploid cultures and 11.8 + 1.66 in haploid cultures. When the developing shoot buds were excised and cultured on modified Whites medium [9] supplemented with NAA (1 mg/1) root initiation occurred from the cut ends and by about 4--6 weeks complete plantlets developed. These plantlets were transferred to soil in pots and grown to maturity. Experiments to assess the regenerative ability of both haploid and diploid calli during serial subcultures revealed that while the haploid tissues retained the organogenetic potential, the diploid callus showed a decline in the organogenetic potential which was completely lost by the 8th serial passage (Table I). B: Alkaloid detection in regenerating and non-regenerating tissues on W & B medium The basic fraction isolated from regenerating haploid callus and unorganized diploid callus grown on W & B + NAA + Kn medium gave Vitali-Morin positive test. When aliquots of these fractions were subjected to TLC analysis, 8 Dragendorff-positive spots were observed of which 4 corresponded to the Rf values of atropine, scopolamine, tropine and scopoline. GLC analysis of the fractions showed that in addition to some minor peaks of which 3 correspond-
86
TABLE I COMPARATIVE ORGANOGENETIC BEHAVIOUR OF HAPLOID AND DIPLOID TISSUE CULTURES OF ATROPA BELLADONNA, DURING 5th-8th SERIAL PASSAGE ON MS + BA (1 rag/l). Each figure represents a mean of 24 cultures. Growth period: 30 days. The figure in parenthesis is the average No. of shoot buds/culture. Passage
Haploid %
Diploid
5 6 7 8
100 (11.8) 100 (7.7) 100 (7.5) 100 (4.8)
100 (6.7) 86.9 (2.7) 23.3 (0.7)
%
Nil
ed with the retention time of authentic tropine, atropine and scopolamine, several major peaks were also present. The relative proportion of tropine, atropine and scopolamine in the callus tissues were comparable unlike in the whole plant where atropine alone formed the major alkaloid (Figs. 1, 2 & 4). The overall pattern o f alkaloids in haploid and diploid callus was similar as observed by TLC and GLC (Figs. 1 and 2). Quantitative estimation of total alkaloids in the tissues showed a comparable alkaloid c o n t e n t in diploid (8 #g/g) and haploid callus (8 #g/g).
C: Alkaloids in cultures grown on M S medium On MS + BA medium both the haploid and diploid callus exhibited organforming ability. The basic extracts o f these tissues were Vitali-Morin positive. TLC analysis showed t h a t their alkaloid pattern was similar and comparable to those observed with tissues grown on W & B medium. GLC analysis of the extract further confirmed the similarities. Quantitative estimation of total alkaloids in the tissues showed t h a t the diploid callus contained 11 ~zg/g and haploid callus contained 10 pg/g. The total alkaloid c o n t e n t in these tissues showed a marginal increase over those grown on W & B medium. D. Alkaloids in shoot buds and regenerated plant Basic extracts of shoot buds from haploid and diploid calli gave positive reaction in Vitali-Morin test. TLC analysis of these extracts showed the presence of 8 Dragendorff-positive spots as in the case of callus cultures. GLC analysis showed t h a t the pattern of alkaloids was comparable to t h a t of the callus cultures (Fig. 3). However, the relative proportion of atropine as compared to tropine and scopolamine was more than that observed in callus extracts (Figs. 2 & 3). A mature plant regenerated from callus cultures at the time of flowering was also analysed to examine its alkaloid pattern and to compare with t h a t o f callus cultures and y o u n g shoot buds. TLC analysis of the plant
87
I
0
5
2
,'o
1'0 MIN
MIN,
2
0
3
10 MIN
Mill
Figs. 1--4. Gas-liquid chromatography on 3% SE-30 of the tropane alkaloids isolated from, 1: diploid callus cultures grown on W & B medium; 2: haploid callus cultures grown on MS medium; 3: 30-day-old regenerated shoot buds from haploid callus grown on MS medium; 4 : regenerated plant at flowering stage. Peak 1, tropine; peak 2, atropine; peak 3, scopolamine.
e x t r a c t revealed the presence o f 7-Dragendorff-positive spots which included atropine, tropine, scopolamine and scopoline. Atropine was t he m ost a b u n d a n t alkaloid as c o m p a r e d t o others. A comparison o f t he alkaloid pat t ern in the whole plant and callus cultures showed t hat 6 c o m p o n e n t s present in t he whole plant were also present in cultured tissues and t w o additional c o m p o n e n t s observed in th e callus were absent in t he plant. GLC analysis o f the whole plant showed th at atropine was t he m os t a b u n d a n t alkaloid (Fig. 4). T h e alkaloid c o n t e n t in di f f er e nt callus cultures and s h o o t buds are shown in Table II.
88 TABLE II ALKALOID CONTENT IN CULTURED TISSUES AND REGENERATED MATURE PLANT OF ATROPA BELLADONNA Material
Growth medium
Passage No.
Morphogenetic response
Diploid Callus
W&B
5+6+7
Callus
MS
7 + 8*
Callus
MS
5
Regenerated shoot buds
MS
5
Unorganized growth Unorganized growth Shoot bud formation --
W&B
5+6+7
MS
7+8
MS
7+8
--
--
Haploid Callus Callus Regenerated shoot buds Regenerated flowering plant
Shoot bud formation Shoot bud formation ---
Total alkaloid ~g/g dry wt. 8 II
12 20
8 11 18 1160
* Cultures exhibited loss of organogenetic potential.
DISCUSSION O u r results o n Atropa belladonna have s h o w n t h a t tissue c u l t u r e s o f h a p l o i d a n d d i p l o i d origin e x h i b i t m o r p h o g e n e t i c p o t e n t i a l . H o w e v e r , t h e d e g r e e o f s h o o t b u d f o r m a t i o n was m o r e in t h e h a p l o i d c u l t u r e s t h a n in t h e d i p l o i d cultures. While t h e d i p l o i d callus lost its o r g a n o g e n e t i c p o t e n t i a l b y 8 t h passage t h e h a p l o i d callus c o n t i n u e d t o d i f f e r e n t i a t e . This is c o n s i s t e n t w i t h t h e earlier w o r k b y Rashid a n d S t r e e t [ 1 0 ] w h e r e diploid c u l t u r e s o f Atropa lost t h e e m b r y o g e n i c p o t e n t i a l m u c h earlier t h a n t h e h a p l o i d cultures. H a p l o i d a n d d i p l o i d callus c u l t u r e s o f A tropa belladonna e x h i b i t e d d i s t i n c t d i f f e r e n c e s in t h e i r o r g a n o g e n e t i c ability o n W & B m e d i u m . While t h e h a p l o i d c u l t u r e s f o r m e d s h o o t b u d s t h e d i p l o i d callus r e m a i n e d u n o r g a n i z e d . H o w e v e r , t h e similarities in t h e i r alkaloid p a t t e r n a n d c o n c e n t r a t i o n i n d i c a t e d t h a t t h e origin o f c u l t u r e a n d t h e o r g a n o g e n e t i c ability d o n o t i n f l u e n c e t h e m e t a b o l i c events leading t o t h e s y n t h e s i s o f p r i n c i p a l alkaloids o f b e l l a d o n n a . T h e similarities in t h e alkaloid s p e c t r u m o f d i f f e r e n t i a t i n g h a p l o i d a n d d i p l o i d callus o n MS m e d i u m w i t h t h o s e g r o w n o n W & B m e d i u m f u r t h e r s u b s t a n t i a t e d t h e above observation.
89 The apparent similarities in the alkaloid spectrum of the unorganized callus and the differentiated shoot buds indicated that the extent of differentiation o f shoot buds has little contribution towards the formation of major alkaloids. However, the relative proportion of atropine as compared to other alkaloids was more in shoot buds than in callus. It is therefore evident that the shoot buds, despite possessing alkaloid pattern comparable to the callus, show a tendency towards acquiring the metabolic status of the whole plant where atropine forms the major alkaloid. The data suggested that the expression o f synthetic potentiality is possibly determined b y the development stages o f the plant rather than the process of differentiation. Thus, in tissue cultures of Scopolia parviflora the roots initiated from callus cultures showed alkaloid contents as low as those of the callus b u t the alkaloid content increased from 0.003--0.05% with the development of clusters of fibrous or somewhat thickened roots [ 1 1 ] . Variations in alkaloid pattern and concentration during different stages of growth and development of the plant Atropa belladonna have been demonstrated [ 12]. The presence of alkaloids in the callus and the regenerated shoot buds indicated that the alkaloid synthesis is n o t exclusively associated with the initiation and development of roots. Nevertheless, the immense differences in the alkaloid contents of the shoot buds (20 ug/g) and mature plant (1160 ~g/g)indicated that the process of r o o t formation and further development of the plant m a y have a strong influence on alkaloid biosynthesis. In tissue cultures of A. belladonna the dependence of alkaloid synthesis u p o n the organisation of roots has been well demonstrated [ 1 3 , 1 4 ] . REFERENCES 1 K.J. Kasha (ed.) Haploids in higher plants: Advances and potential,University of Guelph, Ontario, Canada, 1974. 2 I.K. Vasil and C. Nitsch, Z. Pflanzenphysiol.,76 (1975) 191. 3 P.S. Carlson, Science, 168 (1970) 487. 4 P. Maliga, L. Marton and A. Sz. Breznovits,Plant Sci, Lett., 1 (1973) 119. 5 H.N. W o o d and A.C. Braun, Proc. Natl. Acad. Sci. USA, 47 (1961) 1907. 6 M. Lin and E:J. Staba, Lloydia, 24 (1961) 139. 7 T. Murashige and F. Skoog, Physiol. Plant.,15 (1962) 473. 8 F.M. Freeman, Analyst, 80 (1955) 520. 9 N.S. Rangaswamy, Phytomorphology, 11 (1961) 109. 10 A. Rashid and H.E. Street,Plant Sci. Lett.,2 (1974) 89. 11 ~¢I.Tabata, Y. Yamamoto, N. Hiraoka and M. Konoshima, Phytochemistry, 11
(1972)940. 12 J.D. Phillipson and S.S. Handa, Phytochemistry, 15 (1976) 605. 13 S.B. Raj Bhandary, H.A. Collin, E. Thomas and H.E. Street, Ann. Bot., 33 (1969) 647. 14 E. Thomas and H.E. Street, Ann. Bot., 34 (1970) 657.
83
MORPHOGENETIC AND BIOSYNTHETIC STUDIES ON TISSUE CULTURES OF A T R O P A B E L L A D O N N A L.
S U S A N EAPEN, T.S. R A N G A N , M.S. C H A D H A
and M.R. H E B L E
Plant Morphogenesis and Tissue Culture Section, Bio-Organic Division, Bhabha Atomic Research Centre, Bombay 400 085 (India.)
(Received December 1st,1977) (Accepted February 28th, 1978)
SUMMARY
The morphogenetic and biosynthetic ability of tissue cultures established from haploid and diploid plants of Atropa belladonna L. have been investigated. It was observed that the haploid tissue regenerated plantlets much more readily than did the diploids. The regenerants obtained in vitro were successfully transplanted to soil and grown to flowering stage. Haploid and diploid calli, regenerated young shoot buds and the in vitro obtained plants (at the stage of flowering) were subjected to chemical analysis. The total alkaloid contents in the calli and young shoot buds were very low (8--20 pg/g) as compared to the mature plant (1160 gg/g). Tropine, atropine, scopolamine and several unidentified Dragendorff-positive components were observed in the cultures. The alkaloid spectrum of the shoot buds was comparable to that of the unorganized calli. However, the relative proportion of atropine was more in the shoot buds as compared to the callus cultures. The regenerated flowering plants contained the principal belladonna alkaloids in quantities comparable to the plants raised from seeds.
INTRODUCTION
Haploid plants and tissues in vitro have been considered useful in somatic cell genetics and plant breeding [1,2]. Tissue cultures derived from haploid plants could be used to advantage in selection of mutants [3,4] since mutations in such cultures will be expressed without any masking effect by dominant alleles. Desirable mutants can be diplodized and homozygous inbred lines can be obtained. We have established tissue cultures from haploid and diploid plants of Abbreviations: BA, 6-benzyladenine; Kn, kinetin;MS, Murashige and Skoog; NAA, ~naphthaleneacetic acid;W and B, Wood and Braun.
84
Atropa belladonna L. with a view to study their morphogenetic behaviour and to obtain homozygous mutants with high alkaloid content through haploid cultures. The present report is concerned with the organ-forming ability of these cultures and their metabolic status in regard to the synthesis of tropane alkaloids. MATERIALS AND METHODS
Seeds o f Atropa belladonna L. (a m u t a n t variety developed by the Biology and Agriculture Division, Bhabha Atomic Research Centre, B o m b a y ) were used as the source material. While the diploid plant (2n = 72) was raised directly from the seeds, the haploid (n = 36) was obtained by anther culture. Tissue cultures were established from the leaves of one haploid and one diploid plant (about 0.6 m high and n o t bearing any flowers}. The leaves were surface-sterilized with 0.1% mercuric chloride and small leaf segments were cultured on W and B medium [5] supplemented with Lin and Staba vitamins [ 6 ] , NAA and Kn. For some experiments on organogenesis MS basal medium [7] was employed. The pH of the medium was adjusted to 5.8 and gelled with 0.9% agar. The cultures were maintained in diffuse light at 25 + 2°C. Haploid and diploid tissues from 5th, 6th and 7th passages growing on W & B + NAA + Kn medium were pooled and 50 g of the dry tissue was utilized for chemical analysis. Similarly tissues from 7th and 8th passages growing on MS + BA medium were pooled and 14 g of the dry tissue was analyzed. Visible shoot buds were removed from regenerating tissues and the callus and shoot buds were examined separately. The incubation period was generally 30 days, at the end o f which the tissues were harvested and dried. Plants that regenerated from callus cultures were grown in pots and harvested at the flowering stage f o r analysis.
Extraction and estimation of alkaloids The tissues, harvested after 30 days growth, were dried at 60°C for 24 h, powdered, macerated overnight with EtOH -- 28% NH4OH (9 : 1) mixture and refluxed with methanol. The methanol extract was evaporated to dryness and the residue thus obtained was dissolved in 0.1 N HC1 and extracted with CHC13. The CHC13 layer was washed with 0.1 N HC1. The acidic extract and the washings were pooled, made alkaline with N NaOH and extracted with CHC13, which gave crude basic fraction. This fraction was further dissolved in 0.05 N HCI, filtered and the basic components were regenerated and extracted with CHC13 as above. The CHCI3 extract was dried over Na2 SO4 and evaporated to dryness which gave basic fractions. Aliquots of this fraction were used for colorimetry and GLC analysis. Estimation of total alkaloids was done by the Vitali-Morin m e t h o d [ 8 ] , with slight modifications. The nitrated basic components were dissolved in 10 ml o f DMF, basified with 100 ~1 o f freshly prepared 3% KOH in methanol and the intensity of colour developed after 2 min was measured at 545 nm. The
85 total alkaloid contents were calculated using the atropine calibration curve. TLC was carried out using silica gel C (Acme Products, India) and two solvent systems, system 1:CHC13-EtOH -- 25% NH4OH (85 : 14 : 1) system 2: CHC13 saturated with 25% NH4OH. GLC analysis was carried out on 2 m, 65 cmm glass columns packed with 2% OV-17 or 3% SE-30 on Chromosorb W 80/100 mesh using N: as carrier gas and FID. The alkaloid mixtures were analyzed under following conditions; SE-30 : Isothermal 200°C, Temperature programme 150--210°C (8°/min); OV-17 : Isothermal 230°C, Temperature programme 160--250°C (4°/min). Each extract was analyzed under different conditions using authentic atropine, tropine and scopolamine standards. The identity of tropine, atropine, and scopolamine in the tissue extracts was confirmed through co-chromatography (GLC, 3% SE-30) with authentic samples. RESULTS A: Organogenesis and plantlet regeneration When the explants were cultured on W & B + NAA (2 mg/1) + Kn (0.5 mg/1), in about 3 weeks cell proliferation occurred from the periphery and an actively growing mass of tissue was formed. While the haploid (The terms haploid culture and diploid cultures are used to indicate the source) cultures showed a high degree (100%) of shoot bud formation the diploid cultures grew as an unorganised mass of tissue. Experiments were carried out to ascertain the organogenetic potential of these tissues under different nutritional and hormonal conditions. It was observed that the haploid as well as the diploid cultures exhibited 100% shoot bud regeneration on MS medium supplemented with BA (1 mg/1). The maximum number of shoot buds that developed per culture, however, was 6.7 + 0.91 in diploid cultures and 11.8 + 1.66 in haploid cultures. When the developing shoot buds were excised and cultured on modified Whites medium [9] supplemented with NAA (1 mg/1) root initiation occurred from the cut ends and by about 4--6 weeks complete plantlets developed. These plantlets were transferred to soil in pots and grown to maturity. Experiments to assess the regenerative ability of both haploid and diploid calli during serial subcultures revealed that while the haploid tissues retained the organogenetic potential, the diploid callus showed a decline in the organogenetic potential which was completely lost by the 8th serial passage (Table I). B: Alkaloid detection in regenerating and non-regenerating tissues on W & B medium The basic fraction isolated from regenerating haploid callus and unorganized diploid callus grown on W & B + NAA + Kn medium gave Vitali-Morin positive test. When aliquots of these fractions were subjected to TLC analysis, 8 Dragendorff-positive spots were observed of which 4 corresponded to the Rf values of atropine, scopolamine, tropine and scopoline. GLC analysis of the fractions showed that in addition to some minor peaks of which 3 correspond-
86
TABLE I COMPARATIVE ORGANOGENETIC BEHAVIOUR OF HAPLOID AND DIPLOID TISSUE CULTURES OF ATROPA BELLADONNA, DURING 5th-8th SERIAL PASSAGE ON MS + BA (1 rag/l). Each figure represents a mean of 24 cultures. Growth period: 30 days. The figure in parenthesis is the average No. of shoot buds/culture. Passage
Haploid %
Diploid
5 6 7 8
100 (11.8) 100 (7.7) 100 (7.5) 100 (4.8)
100 (6.7) 86.9 (2.7) 23.3 (0.7)
%
Nil
ed with the retention time of authentic tropine, atropine and scopolamine, several major peaks were also present. The relative proportion of tropine, atropine and scopolamine in the callus tissues were comparable unlike in the whole plant where atropine alone formed the major alkaloid (Figs. 1, 2 & 4). The overall pattern o f alkaloids in haploid and diploid callus was similar as observed by TLC and GLC (Figs. 1 and 2). Quantitative estimation of total alkaloids in the tissues showed a comparable alkaloid c o n t e n t in diploid (8 #g/g) and haploid callus (8 #g/g).
C: Alkaloids in cultures grown on M S medium On MS + BA medium both the haploid and diploid callus exhibited organforming ability. The basic extracts o f these tissues were Vitali-Morin positive. TLC analysis showed t h a t their alkaloid pattern was similar and comparable to those observed with tissues grown on W & B medium. GLC analysis of the extract further confirmed the similarities. Quantitative estimation of total alkaloids in the tissues showed t h a t the diploid callus contained 11 ~zg/g and haploid callus contained 10 pg/g. The total alkaloid c o n t e n t in these tissues showed a marginal increase over those grown on W & B medium. D. Alkaloids in shoot buds and regenerated plant Basic extracts of shoot buds from haploid and diploid calli gave positive reaction in Vitali-Morin test. TLC analysis of these extracts showed the presence of 8 Dragendorff-positive spots as in the case of callus cultures. GLC analysis showed t h a t the pattern of alkaloids was comparable to t h a t of the callus cultures (Fig. 3). However, the relative proportion of atropine as compared to tropine and scopolamine was more than that observed in callus extracts (Figs. 2 & 3). A mature plant regenerated from callus cultures at the time of flowering was also analysed to examine its alkaloid pattern and to compare with t h a t o f callus cultures and y o u n g shoot buds. TLC analysis of the plant
87
I
0
5
2
,'o
1'0 MIN
MIN,
2
0
3
10 MIN
Mill
Figs. 1--4. Gas-liquid chromatography on 3% SE-30 of the tropane alkaloids isolated from, 1: diploid callus cultures grown on W & B medium; 2: haploid callus cultures grown on MS medium; 3: 30-day-old regenerated shoot buds from haploid callus grown on MS medium; 4 : regenerated plant at flowering stage. Peak 1, tropine; peak 2, atropine; peak 3, scopolamine.
e x t r a c t revealed the presence o f 7-Dragendorff-positive spots which included atropine, tropine, scopolamine and scopoline. Atropine was t he m ost a b u n d a n t alkaloid as c o m p a r e d t o others. A comparison o f t he alkaloid pat t ern in the whole plant and callus cultures showed t hat 6 c o m p o n e n t s present in t he whole plant were also present in cultured tissues and t w o additional c o m p o n e n t s observed in th e callus were absent in t he plant. GLC analysis o f the whole plant showed th at atropine was t he m os t a b u n d a n t alkaloid (Fig. 4). T h e alkaloid c o n t e n t in di f f er e nt callus cultures and s h o o t buds are shown in Table II.
88 TABLE II ALKALOID CONTENT IN CULTURED TISSUES AND REGENERATED MATURE PLANT OF ATROPA BELLADONNA Material
Growth medium
Passage No.
Morphogenetic response
Diploid Callus
W&B
5+6+7
Callus
MS
7 + 8*
Callus
MS
5
Regenerated shoot buds
MS
5
Unorganized growth Unorganized growth Shoot bud formation --
W&B
5+6+7
MS
7+8
MS
7+8
--
--
Haploid Callus Callus Regenerated shoot buds Regenerated flowering plant
Shoot bud formation Shoot bud formation ---
Total alkaloid ~g/g dry wt. 8 II
12 20
8 11 18 1160
* Cultures exhibited loss of organogenetic potential.
DISCUSSION O u r results o n Atropa belladonna have s h o w n t h a t tissue c u l t u r e s o f h a p l o i d a n d d i p l o i d origin e x h i b i t m o r p h o g e n e t i c p o t e n t i a l . H o w e v e r , t h e d e g r e e o f s h o o t b u d f o r m a t i o n was m o r e in t h e h a p l o i d c u l t u r e s t h a n in t h e d i p l o i d cultures. While t h e d i p l o i d callus lost its o r g a n o g e n e t i c p o t e n t i a l b y 8 t h passage t h e h a p l o i d callus c o n t i n u e d t o d i f f e r e n t i a t e . This is c o n s i s t e n t w i t h t h e earlier w o r k b y Rashid a n d S t r e e t [ 1 0 ] w h e r e diploid c u l t u r e s o f Atropa lost t h e e m b r y o g e n i c p o t e n t i a l m u c h earlier t h a n t h e h a p l o i d cultures. H a p l o i d a n d d i p l o i d callus c u l t u r e s o f A tropa belladonna e x h i b i t e d d i s t i n c t d i f f e r e n c e s in t h e i r o r g a n o g e n e t i c ability o n W & B m e d i u m . While t h e h a p l o i d c u l t u r e s f o r m e d s h o o t b u d s t h e d i p l o i d callus r e m a i n e d u n o r g a n i z e d . H o w e v e r , t h e similarities in t h e i r alkaloid p a t t e r n a n d c o n c e n t r a t i o n i n d i c a t e d t h a t t h e origin o f c u l t u r e a n d t h e o r g a n o g e n e t i c ability d o n o t i n f l u e n c e t h e m e t a b o l i c events leading t o t h e s y n t h e s i s o f p r i n c i p a l alkaloids o f b e l l a d o n n a . T h e similarities in t h e alkaloid s p e c t r u m o f d i f f e r e n t i a t i n g h a p l o i d a n d d i p l o i d callus o n MS m e d i u m w i t h t h o s e g r o w n o n W & B m e d i u m f u r t h e r s u b s t a n t i a t e d t h e above observation.
89 The apparent similarities in the alkaloid spectrum of the unorganized callus and the differentiated shoot buds indicated that the extent of differentiation o f shoot buds has little contribution towards the formation of major alkaloids. However, the relative proportion of atropine as compared to other alkaloids was more in shoot buds than in callus. It is therefore evident that the shoot buds, despite possessing alkaloid pattern comparable to the callus, show a tendency towards acquiring the metabolic status of the whole plant where atropine forms the major alkaloid. The data suggested that the expression o f synthetic potentiality is possibly determined b y the development stages o f the plant rather than the process of differentiation. Thus, in tissue cultures of Scopolia parviflora the roots initiated from callus cultures showed alkaloid contents as low as those of the callus b u t the alkaloid content increased from 0.003--0.05% with the development of clusters of fibrous or somewhat thickened roots [ 1 1 ] . Variations in alkaloid pattern and concentration during different stages of growth and development of the plant Atropa belladonna have been demonstrated [ 12]. The presence of alkaloids in the callus and the regenerated shoot buds indicated that the alkaloid synthesis is n o t exclusively associated with the initiation and development of roots. Nevertheless, the immense differences in the alkaloid contents of the shoot buds (20 ug/g) and mature plant (1160 ~g/g)indicated that the process of r o o t formation and further development of the plant m a y have a strong influence on alkaloid biosynthesis. In tissue cultures of A. belladonna the dependence of alkaloid synthesis u p o n the organisation of roots has been well demonstrated [ 1 3 , 1 4 ] . REFERENCES 1 K.J. Kasha (ed.) Haploids in higher plants: Advances and potential,University of Guelph, Ontario, Canada, 1974. 2 I.K. Vasil and C. Nitsch, Z. Pflanzenphysiol.,76 (1975) 191. 3 P.S. Carlson, Science, 168 (1970) 487. 4 P. Maliga, L. Marton and A. Sz. Breznovits,Plant Sci, Lett., 1 (1973) 119. 5 H.N. W o o d and A.C. Braun, Proc. Natl. Acad. Sci. USA, 47 (1961) 1907. 6 M. Lin and E:J. Staba, Lloydia, 24 (1961) 139. 7 T. Murashige and F. Skoog, Physiol. Plant.,15 (1962) 473. 8 F.M. Freeman, Analyst, 80 (1955) 520. 9 N.S. Rangaswamy, Phytomorphology, 11 (1961) 109. 10 A. Rashid and H.E. Street,Plant Sci. Lett.,2 (1974) 89. 11 ~¢I.Tabata, Y. Yamamoto, N. Hiraoka and M. Konoshima, Phytochemistry, 11
(1972)940. 12 J.D. Phillipson and S.S. Handa, Phytochemistry, 15 (1976) 605. 13 S.B. Raj Bhandary, H.A. Collin, E. Thomas and H.E. Street, Ann. Bot., 33 (1969) 647. 14 E. Thomas and H.E. Street, Ann. Bot., 34 (1970) 657.