Plant & Cell Physiol. 21(8): 1391-1404 (1980)
Studies on the production of Digitalis cardenolides by plant tissue culture I. Determ.ination of digitoxin and digoxin contents in first and second passage calli and organ redifferentiating calli of several Digitalis species by radioillllnunoassay
Central Research Institute, The japan Tobacco & Salt Public Corporation, 6-2. Umegaoka, Midoriku, Yokohama 227, japan (Received july 21, 1980)
With first and second passage calli induced from seedlings of Digitalis pur.fJurea, D. lanata, D. lutea, D. mertonensis, D. ambigua and D. ferruginea Gigantea and those induced from leaf discs of D. purpurea and D. lanata, the contents of digitoxin and digoxin equivalents were assayed and compared with the contents involved in the inocula. Although the total contents of digitoxin and digoxin equivalents in the first passage calli induced from seedlings varied between zero and nine times as high as in the original seedlings, those in the second passage calli were almost undetectable. The total contents of digitoxin equivalents in the first passage calli induced from leaf discs of D. purpurea were approximately equal to those in the original leaf discs, but those in the second passage calli were less than those in the inocula. In the first passage calli induced from leaf discs of D. lanata, the total contents of digitoxin equivalents decreased but those of digoxin equivalents slightly increased. However, in the second passage calli, the amounts of both cardenolides decreased. Root-forming calli accumulated no more digitoxin nor digoxin equivalents than completely dedifferentiated calli. However, shoot-forming calli accumulated considerable amounts of cardenolides, which were assayed as digitoxin and digoxin equivalents by radioimmunoassay. Key words: Cardenolides - Digitalis callus - Digitoxin - Digoxin - Radioimmunoassay - Redifferentiated organ.
Digitalis cardenolides, especially digoxin and digitoxin, are important in medicine. This study was carried out to produce these substances by plant tissue culture technique. The production of secondary products by cultured plant cells has been studied for many plant species by a number of investigators (8, 16). For example, shikonin formation by cultured cells of Lithospermum erythrorhizon (18) and carotenoid formation by those of Daucas carota (15) have been reported. However, in most cases, secondary products unique to the species are not produced or are produced by cultured cells in much lesser amount than by intact plants. Recently, some investigators succeeded in obtaining strains of cultured cells with high biosynthetic Abbreviations: BA, benzyladenine; 2,4-D, 2,4-dichlorophenoxyacetic acid; IAA, 3-indoleacetic acid; NAA, a-naphthaleneacetic acid. 1391
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Manabu Hagimori, Takashi Matsumoto and Takuro Kisaki
1392
M. Hagimori, T. Matsumoto and T. Kisaki
Materials and methods
Antisera preparation Digoxin antisera and 'digitoxin antisera were prepared as follows. Digitoxin and digoxin were coupled to human serum albumin by the method of Smith et al. (14), with 5.3 moles of digitoxin and 4.3 moles of digoxin coupled to 1 mole of albumin, as determined by spectroscopy. Four milligrams of the dialyzed and lyophilized conjugate was dissolved in 1 ml H 20 and emulsified with 1.5 ml complete Freund's adjuvant. Rabbits were immunized by one injection of 0.4 ml freshly prepared antigen emulsion into the toe pads followed by weekly intramuscular injections. Antisera were obtained 10 weeks after the first injection. Each 2 ml of antiserum was put into an ampule and lyophilized, then the ampule was sealed and stored at 4°C. For radioimmunoassay, the stock antiserum in the ampule was dissolved in 2 ml H 2 0 and further diluted with the buffer used in radioimmunoassay.
Radioimmunoassay procedure For radioimmunoassay 0.5 ml buffer (0.01 M KPi, 0.15 M NaCI, pH 7.3), 0.1 ml l % bovine serum albumin, 0.1 ml tracer (G-3H-digitoxin 7.4 Cijmmole or G-3Hdigoxin 10.6 Ci/mmole, The Radiochemical Centre, Amersham, 0.1 /-lCi/ml), 0.1 ml of digitoxin or digoxin standard or tissue extract or blank, and 0.1 ml antiserum solution were poured into a conical tube in an ice bath. After mixing, the mixture was incubated for 30 min at 24°C. The reaction was stopped by cooling in an ice bath and 0.5 ml of dextran-coated charocal suspension, prepared just before use by mixing 10 ml of the buffer containing 0.5 g of charcoal and 10 ml of the buffer containing 0.5 g of dextran, was added. After 10 min of contact time, the mixture was centrifuged at 2500 rpm for 10 min at O°C. A 0.5-mI portion of the supernatant
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activity by cell selection (17, 20, 21). We have also obtained strains of cultured cells of Nicotiana tabacum which accumulate ubiquinone-IO five times more than the parent strain by cell selection (9). The cardenolide productivity of cultured cells of the Digitalis species has been investigated by some workers and most of them reported that cultured cells did not produce cardenolides (1, 4, 6). However, Kartnig (7) reported that callus of D. purpurea accumulated purpureaglycoside A at 1 /-lg per g fresh weight at the tenth passage. If the early passage calli retain the activity to produce cardenolides to some extent, it might be possible to obtain cardenolide-accumulating strains from early passage calli by cell selection. Thus, we examined the ability of first and second passage calli of several Digitalis species to accumulate cardenolides. Then we determined the cardenolide contents in organ redifferentiating calli in order to find which state of cells of the Digitalis species is suitable for the biosynthesis of cardenolides. Radioimmunoassay using digoxin antisera has been reported to be suitable for the determination of digoxin levels in plant extracts (11,12,19). We also used this method for the determination of digoxin and digitoxin contents using digoxin antisera and digitoxin antisera, respectively.
Cardenolide contents in Digitalis callus
1393
containing antibody-bound haptene was put into a scintillation vial containing 3.5 ml scintillator (Instagel, Packard Instruments Company, Downers Grove) and counted. Standard and tissue extracts were run in duplicate.
Extraction
Induction andculture ofcallus Calli were induced from seedlings and leaf discs. Calli from seedlings: Calli were induced from 10-day-old seedlings of Digitalis purpurea, D. lanata, D. lutea, D. mertonensis, D. ambigua and D. ferruginea Gigantea. Murashige and Skoog's inorganic medium (10) containing 30 g/liter sucrose, 100 mgjliter myo-inositol, 2.0 mg/liter glycine, 1.0 mg/Iiter thiamin-HCl, 0.5 mgjliter nicotinic acid, 0.5 mgjliter pyridoxin·HCI and 8 g/liter agar was used as the basal medium. Eight different media which contained the constituents of the basal medium supplemented with 2 mg/liter 2,4-D (D), 2 mg/liter NAA (N), 2 mgjliter kinetin (K), 3 g/liter yeast extract (Y) and 3 g/liter malt extract (M) in eight combinations, DM, DKM, DY, DKY, NM, NKM, NY and NKY, were used. A seedling was placed on the solid medium in a test tube. After 50-70 days, all cultured material in the tube, which was designated the first passage callus, was harvested and part of the induced callus was transferred to the same fresh medium while the rest was subjected to radioimmunoassay. The second passage calli were harvested after 30-50 days of culture and treated by the same procedure used for the first passage calli. Calli from seedlings were cultured in the dark at 28°C. Calli from leaf discs: Pairs of leaf discs (4 mm in diameter) were obtained from symmetrical positions opposite each other across the central vein of leaves of D. purpurea and D. lanata. One of a pair was placed on the solid medium in a test tube for callus induction and the other was assayed for digoxin and digitoxin contents as a control. The calli from leaf discs were cultured in the dark or under a 12 hr daylight cycle (white fluorescent lamp, about 5,000 lux) at 28°C on four media, DM, DKM, NM and NKM. After 92 or 96 days, the first passage callus was harvested and the induced callus was transferred to the same fresh medium with the remainder subjected to radioimmunoassay. In preliminary experiments, the part of the leaf tissue and the part of the callus tissue of the first passage culture and the medium were assayed separately. The leaf tissue had become dark brown and contained relatively small amounts of cardenolides. Most of the cardenolides were detected in the callus tissue' and not in the medium. Therefore, in the regular experiments,
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From plant or callus tissue: About 1 g of fresh tissue was homogenized in 15 ml 80% ethanol. The homogenate was heated at 74°C for 4 hr and filtered. The filtrate was dried in vacuo and the residue was taken up in 1 ml 80% ethanol and diluted with 9 ml H20. When the concentration of digoxin or digitoxin equivalents was too high for radioimmunoassay, the extract was further diluted with H 20 to a desirable level. From agar medium: To about 15 ml of agar medium, on which callus had been cultured, about 85 ml of ethanol was added to give a final volume of 100 ml. After homogenization, the mixture was placed at room temperature overnight and filtered. Fifty milliliters of the filtrate was dried up in vacuo and the resideu was taken up in 1 m180% ethanol and diluted with 9 ml H 20.
1394
M. Hagimori, T. Matsumoto and T. Kisaki
all cultured material in the tube except the small part inoculated to the second passage culture was assayed together. The second passage calli were harvested after 37 or 38 days of culture and treated according to the same procedure used for the first passage calli. The cells inoculated to the next passage culture and the cells subjected to the assay were weighed. Four and five replicated experiments were done for calli from seedlings and leaf discs, respectively.
Establishment ofshoot-Jorming calli
Results
Sensitivity andspecificity of radioimmunoassay The standard curves of digitoxin and digoxin radioimmunoassay are shown in Fig. 1. The ability of compounds other than digitoxin (or digoxin) to compete for the digitoxin antibody (or the digoxin antibody) is called cross reactivity. The percentage cross reactivity (OR %) is defined as OR ~ 0-
pico moles of digitoxin yielding BjBo=50% X 100 pico moles of compound under investigation yielding BjBo=50%
where Band Bo are the percentages of antibody-bound tracer in the presence and absence of unlabeled competetive substances, respectively. Table 1 shows the cross Table 1 Cross reactivities of several cardenolides, sterols andsaponin for digitoxin anddigoxin antisera
Substance Digitoxin Gitoxin Digoxin Gitaloxin Digitalin Strospeside Digitoxigenin Gitoxigenin Digoxigenin Digitonin Cholesterol Stigmasterol fJ-Sitosterol Campesterol a b
Digitoxin antiserum. Digoxin antiserum.
CR°;{, for 03 b 100
3.8 3.4 1.8 9.3
2.6 75 1.6 0.9
o o
o o o
9.8 1.0 100
0.2 2.4 0.3
8.2 0.1
136
o o o o o
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Shoot-redifferentiating calli were established by transferring third passage calli from seedlings to media which contained BA, lAA, kinetin or combinations of these substances in addition to the constituents of the basal medium and culturing them under a 12 hr day-light cycle at 28°0.
1395
Cardenolide contents in Digitalis callus
100-------------------,
A
80
o
CO 40 ........ 0)
0.1
0.4
1.0
4
oIGITOXI N
10
40
(ng)
100--------------------, B ,",80 o
0-
'-'60 o
0)
---. 40 CO
20 0.1
0.4
1.0
4
10
40
DIGOXIN ( ng )
Fig. 1. Digitoxin and digoxin standard curves obtained by plotting B /Bo % versus mass of unlabeled haptene on semilogarithmic scale. A, Digitoxin standard curve. B, Digoxin standard curve.
Table 2 Contents immunoassay
of digitoxin and digoxin equivalents in leaves 0./ several Digitalis species determined by radio-
Species
Dig. ftgfg f.w. a
D. purpurta D.lanata D. lutea
D. mertonensis
D. ftrruginea Gigantea D. ambigua a b
Digitoxin equivalents: pg per g fresh weight. Digoxin equivalents: pg per g fresh weight.
180 700 500 110
Digo. pgfg f.w.
8
400 28 8
260
22
330
9
b
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20
1396
M. Hagimori, T. Matsumoto and T. Kisaki
Table 3 Cell yield, morphology and contents of digitoxin and digoxin equiualents of the first passage calli D. purourea Medium a
NY NKY
M
O. 63±0. 37/
C C C C R R R R
1.01 ±O. 53 O. 53±0. 21 1.04±0. 35 2. 48± 1. 04 1. 98± 1. 10 1. 78±0. 62 1. 07 ±O. 25
Dig. ngjtube d 140± 90 370±480 220±200 260±240 230±310 330±270 390±300 420±140
70± 100± 130± 70± 50± 40± 70± 130±
40 60 80 10 30 40 30 70
Cultivation time (days) 70 70 70 70 57 57 57 57
30 h
150 6
Seedling
Digo. ngjtube 8
D.lutea
Medium DM DKM DY DKY NM NKM NY NKY
CY. g f.w.jtube
M
Dig. ngjtube
Digo. ngjtube
Cultivation time (days)
O. 34±0. 09 O.56±0.4 O. 12±O. 06 O. 11±0. 06 O. 90±0. 30 O. 58±0. 09 O. 67±0. 09 O. 29±0. 14
C C C C R R R R
20± 30 70±100 110± 70 90± 60 10± 20 10± 20 0 10± 20
110± 80 180± 60 240± 70 190± 140 80± 60 70± 70 110± 60 150± 60
70 70 70 70 55 55 55 55
Seedling
80
170 D. ambigua
Medium DM DKM DY DKY NM NKM
NY NKY Seedling a b C
d
cv. g f.w.jtube
M
Dig. ngjtube
O. 30±0. 17 O. 28±0. 18 O. 06±O. 02 O. 30±0. 39 1. 34±0. 61 1.61 ±O. 99 O. 23±O. 09 O. 09±O. 07
C C
110±110 460±570 20± 20 20± 20 20± 30 10± 20 160±240 360±350
C
C R R R R
90
Digo. ng/tube 50± 60± 20± 20± 10± 20± 40± 40±
60 80 20 20 30 20 80 80
Cultivation time (days) 70 70 70 70 60 60 60 60
20
The symbols of the media are explained in the text. Cell yield: g fresh weight per culture. Morphology of each culture: C, completely dedifferentiated callus; R, root-forming callus. Total content of digitoxin equivalents per culture.
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DM DKM DY DKY NM NKM
CY. g f.w.jtube b
C
1397
Cardenolide contents in Digitalis callus
qf several Digitalis species induced from seedlings
D. lanata CY. g f.w.jtube
Dig. ngjtube
Digo. ng/tube
C
10±20 10±20 30±40 20±30 40±40 50±60 10±20 20±30
110±220 110±200 70± 60 60± 60 160±120 190± 170 110± 140 90± 80
C C C
R R R R
130
Cultivation time (days)
70 70 70 70 56 56 56 56
130 D. mertonensis
CY. g f.w.jtube
M
Dig. ng/tube
O. 27±0. 04 O. 58±0. 48 O. 16±0. 10 1. 01 ±O. 48 2. 83± 1.58 1. 23±0. 96 2. 80±1. 37 2. 70±1. 65
C
0 40±80 20±20 50±40 20±40 40±60 10±20 0
C
C C
R R R R
Digo. ng/tube
10± 40± 70± 50± 50± 40± 20± 30±
140
20 10 40 20 70 50 40 20
Cultivation time (days)
70 70 70 70 50 50 50 50
30
D-.ferruginea Gigantea
e j
g h
CY. g f.w.jtube
M
Dig. ng/tube
0.07±0.10 O. 10±0. 09 O. 04±0. 02 O. 16±0. 12 O. 55±0. 24 O. 84±0. 29 0.37 ±O. 18 O. 48±0. 37
C
20±40 0 0 0 10±20 10±20 0 0
30± 50 20± 20 10± 20 10± 30 60± 60 90± 90 40± 30 70± 110
70
80
C C C
R R R R
Digo. ng/tube
Total content of digoxin equivalents per culture. Mean value of four experiments with standard deviation. Total content of digitoxin equivalents per seedling. Total content of digoxin equivalents per seedling.
Cultivation time (days)
70 70 70 70 70 70 70 70
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0.16±0.05 O. 34±0. 11 1. 05±0. 77 1. 22± 1.00 2. 26± 1. 06 1. 27±O. 45 O. 95±0. 75 O. 97±0. 49
M
1398
M. Hagimori, T. Matsumoto and T. Kisaki
Calli inducedfrom seedlings Table 3 shows the cell yields, morphology and contents of digitoxin and digoxin equivalents of the first passage calli from seedlings. The calli cultured on NAAcontaining media generally grew better than those on 2,4-D-containing media. Most of the former formed roots, while the latter were completely dedifferentiated. However, the effect of the difference in media on the contents of digitoxin and digoxin equivalents wes not clear. The contents of both substances varied between zero and nine times as high as in the original seedling. But the contents of digitoxin Table 4 Cellyields, morphology, contents and increasing rates of digitoxin equivalents of thefirst passage calli induced from leaf discs of Digitalis purpurea Medium a DM DKM
NM
Light condition
MO
Dig. ngjtube "
De
2. 17±0. 291 1.51±O. 44
C C
1, 240±370 1, 390±320
590± 1,000±
250 420
0.87 ±O. 18 O. 99±0. 22
L D
2. 33±0. 10 1. 07±O. 60
C C
1, 140±590 1, 760± 790
260 490± 2, 160± 1,280
0.84±0.44 1. 22±0. 42
L
2.11 ±O. 39 3. 36±0. 20
R R
1,060±490 1, 430±330
480± 430±
170 '110
O. 74±0. 26 1.02±0.11
2. 56±0. 37 2. 79±0. 35
R R
1, 390± 480 1,710±220
540± 630±
170 170
O. 99±0. 31 1. 25±0. 28
L
D
NKM
L D
Leaf disc
Increasing rate of digitoxin equivalents C
CY. g f.w.jtube "
d
Dig. ng/g f.w. b
1,400±250 g 210,000 ± 40, 000 h
See footnotes of Table 3. Concentration of ng digitoxin equivalents per g fresh weight of callus. C Ratio of the total content of digitoxin equivalents per culture to that per control leaf disc. d Cultured under a 12 hr-day light cycle. S Cultured in the dark. I Mean value of five experiments with standard deviation. g Mean value of total contents of digitoxin equivalents per control leaf disc with standard deviation. h Mean value of concentration of ng digitoxin equivalents per g fresh weight of control leaf disc with standard deviation. Cultivation time was 96 days. a
b
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reactivities of several compounds structurally analogous to digitoxin and digoxin. The data indicate that the digitoxin antiserum is specific to digitoxigenin and its glycoside, whereas the digoxin antiserum is specific to digoxigenin and its glycoside. Cardenolides other than digitoxigenin and its glycosides (or digoxigenin and its glycosides) showed CR% of less than 10% for digitoxin antiserum (or digoxin antiserum), and compounds other than cardenolides showed only negligible cross reactivities. Since digitoxin and digoxin were used as standards in the study, the experimental values are expressed as digitoxin equivalents or digoxin equivalents. Table 2 shows the concentrations of digitoxin and digoxin equivalents in the leaves of twomonth-old plants of Digitalis species measured by radioimmunoassay method using these antisera.
1399
Cardenolide contents in Digitalis callus
and digoxin equivalents in the second passage cultures of these strains were all below the detection limit except for one strain and the contents in the third passage of this strain was also below the detection limit.
Calli inducedfrom leaf discs
Table 5 Growth rates, morphology, contents and increasing rates calli inducedfrom leaf discs ofDigitalis purpurea Medium a DM
DKM NM
NKM
Light condition a
Growth rate b
MO
of digitoxin equivalents of the second passage
Dig. ng/tube a
Dig. tiglg f.w. a
Increasing rate of digi toxin equivalents C
20± 30 100± 65
10±10 40±20
O. 2±0. 2 O. 3±0. 2
16.9±10. sa 10.0± 2.5
C
D L D
13.2± 2.1 14.1 ± 6.6
C C
60± 20 170± 50
30±20 70±20
0.7±0.5 O. 5±0. 1
L D
13.5± 4.5 6.5± 1. 2
C-R C-R
100± 100 140± 60
50±30 60±30
O. 7±0. 2 O. 8±0. 2
L
15.4± 5.8 13.4± 5.5
C-R
40± 50
C
20± 40
10±10 10±10
O. 2±0. 3 0.I±O.2
L
D
C
See footnotes of Tables 3 and 4. Ratio of the cell yield to the weight of the inoculated cells. C Ratio of the total content of digitoxin equivalents per culture to that per inoculated cells. The latter was calculated by multiplying the digitoxin equivalents concentration of the first passage culture of the strain by the weight of the inoculated cells of the second passage culture. Cultivation time was 37 days. a
b
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Tables 4 and 5 show the results obtained with first and second passage calli from leaf discs of D. purpurea, respectively. The total content of digitoxin equivalents in each culture offirst passage calli was approximately as high as that in the original leaf disc and was not substantially affected by the media or lgiht. On the other hand, the cell weight increased and therefore the concentration of digitoxin equivalents per g fresh weight dropped to between 1/50 and 1/1,000 that of the original leaf discs (210 pg/g fresh weight). The concentrations of digitoxin equivalents in the dark-grown calli on DM and DKM media were relatively high, but this was probably due to their relatively low growth rate because their digitoxin contents per tube were also as high as those in the original leaf discs (Table 4). During the second passage culture, the total content of digitoxin equivalents in each culture decreased from that in the inoculum, while the cells grew well. Thus the concentration of digitoxin equivalents per g fresh weight dropped to less than 1/2,000 that of leaf discs (Table 5). The calli cultured under light did not become green. Tables 6 and 7 show the results of experiments with first and second passage calli from leaf discs of D. lanata, respectively. During the first passage culture, though the total content of digitoxin equivalents in each culture tube decreased, that of digoxin equivalents slightly increased. Therefore the ratios of digoxin equivalents to digitoxin equivalents in the calli increased from about 1 in the original leaf discs to 2-4. However, the concentrations of digitoxin and digoxin equivalents per g fresh weight dropped to 1/60-1/1,500 and 1/15-1/600 of those in the original
R R
3. 88±0. 24 3. 28±0. 59
D
R
3. 17±0. 34
D
L
R
2. 66±0. 70
C
L
C
1. 27 ±O. 55
L
D
D
C
1. 27 ±O. 29 O. 97±0. 59
C
0.51±0.41 a
L
a
300
1,900± 1,800± 2,300±
6, OOO± 1,500 7, 100±2, 100 7, 400± 1,400
6, 400± 1, 400
3,100± 2, 100
7, 000±2, 400
600, 000±80, 000
300
600
6,300± 1,900
7, 300±2, 300
11,800± 6,800
9, OOO±4, 400
800
6,100±
d
b
20, 500± 11, 700
Digo. ng/g f.w.
7, 800±2, 000
8, 000±4, 800
D'IgO.ng / tu b e a
6
1. 22±0. 42
1. 10±0. 42
O. 92±0. 34
1. 17±0.50
6, 400± 1,
4,300±
3,300±
1,600±
1,800±
2,800±
100 J
600
900
400
700
900
3, 300± 1,800
1. 37±O. 57 1. 08±0. 28
2,500±
600
2, OOO± 1,000
1. 20±0. 45
1, 30±0. 75
Increasing rate of digoxin Dig. ngjtube " equivalents a
b
a
~ate Digo./ of digitoxin D' C equivalents a rg.
2.7±0.3 4. 5±2. 2 3. 9± 1. 4 2. 3±0. 9
1. 7±0.3
600 O. 45±0. 11 300 O. 27±O. 09 100 0.27 ±O. 08 200 O. 53±0. 09 400 O. 68±0. 12
500± 900± 1,300±
600,
OOO± 120,000 6
700±
2,300±
1. O±O. 2
2. 8±0. 9
3, 200 O. 54±0. 30
3,900±
3. 1±0. 3
3, 700 O. 32±0. 19 4. O±O. 7
a Increa;si!1g
300 O. 43±0. 17
2,000±
5,400±
,
DIg. ng/g f.w.
See footnotes of Tables 3 and 4. Concentration of ng digoxin equivalents per g fresh weight of callus. C Ratio of the total content of digoxin equivalents to that of digitoxin equivalents in the callus. d Mean value of total contents of ng digoxin equivalents per control leaf disc with standard deviation. 6 Mean value of concentration of ng digoxin equivalents per g fresh weight of control leaf disc with standard deviation, f Mean value of total contents of ng digitoxin equivalents per control leaf disc with standard deviation. g Mean value of concentration of ng digitoxin equivalents per g fresh weight of control leaf disc with standard deviation. Cultivation time was 92 days,
Leaf disc
NKM
NM
DKM
DM
Light a CY • g f .w. / tu b e a M Medium a condition
Table 6 Cell yields, morphology; contents and increasing rates of digoxin and digitoxin eqaivalents and ratios of digoxin equivalents to digitoxin equivalents of the first passage calli inducedfrom leaf discs of Digitalis lanata
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er:
~
Cf.l
~
~
=' 0.-
~
8"
S 0
~
ft
~
~
~
p.
S· 0
CIQ
~
t:t=
~
~ o
R R
6. 6±4. 6 8. 1± 1.4
L
D
R
10.8±2. 3
D
C
3.0±2.0
D
R
C
3.1 ± 1. 6
L
8.7±2.8
C
1.2±0. 5
D
L
C
2.7 ± 1. 9 0
L
o
See footnotes of Tables 3, 4, 5 and 6. Cultivation time was 38 days.
NKM
NM
DKM
DM
MO
Growth rate 0
Light Medium 0 condition 0
360± 140 140±
270±
280±
750±670 460±280
220±
1,110±
570±370 450± 170
590±
1,660±
70
190
270
160
880
500
960
1, 600± 1,600
340±480
340± 110
380±230
Digo. ngjtube " Digo, ngjg f.w. 0
O. 4±0. 1
O. 7±0. 3
O. 9±0. 9
O. 7±0. 3
0.4±0.2
O. 2±0. 3
O. 3±0. 2
O. 5±0. 5
Increasing rate of digoxin equivalents 0
120± 40
120± 50
120± 30
120± 40
270±210
140± 140
50± 40
60± 50
Dig. ngjtube 0
Increasing rate of digitoxin equivalents 0 0.4±0.4 O. 1±0. 07 O. 3±0. 3 O. 5±0. 3 0.8±0.4 O. 8±0. 4 O. 4±0. 2 O. 2±0. 09
Dig. ngjg f.w. 0
260±410 180± 60 310±370 520±460 50± 20 70± 60 60± 30 40± 20
Table 7 Growth rates, morphology, contents and increasing rates of digoxin and digitoxin equivalents of the second passage calli induced from leaf discs of Digi talis lanata
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~
~ Vol
e.
o
~
~.
~.
S· b
~
t:::l
an
0
(")
s.: n
g,.
0.. n t:::l
"1
~
0
1402
M. Hagimori, T. Matsumoto and T. Kisaki
Table 8
Concentrations of digitoxin and digoxin equivalents in shoot-forming calli of Digitalis species
Species D. purpurea
D. lanata
Medium a
Dig. ng/g f.w. b
Digo. ng/g f.w. C
BA 10 BA 10
18,200 12,000 9, 700 6,800 3,900 700
1,700 4,000 1,400 700 600 300
leaf discs (both were 600 /lg!g fresh weight), respectively. The effects of differences in media and light on the contents of both substances were not clear, whereas those on growth and morphology were similar to the case of D. purpurea. The relatively high concentrations of the cardenolides in the light-grown calli on DM and DKM media seem to be due to their relatively low growth rates (Table 6). During the second passage culture, the total contents of digoxin and digitoxin equivalents in each culture decreased from the amounts involved in the inoculum and their concentrations dropped to 1/200-1/9,000 and 1/600-1/20,000 of those in the original leaf discs, respectively (Table 7). The calli cultured under light did not become green.
Organ-redijferentiating calli As shown in Tables 3 to 7, though almost all of the calli induced and cultured on the NAA-containing media formed roots, they accumulated no more cardenolides than the completely dedifferentiated calli.
Shoot-forming calli were established on
the basal medium supplemented with 10 mg/liter BA (BA 10) for D. purpurea and D. lanata, 1 mg/liter BA plus 1 mgjliter IAA (BAI) or 2 mgjliter kinetin (K2) for D. lutea and D. mertonensis, and BAI for D. ambigua and D.ferruginea Gigantea, under a 12 hr day-light cycle. The redifferentiated shoots became deep green simultaneously with redifferentiation, but their appearances were to some extent anomalous. In particular, the development of the leaves was imperfect. Table 8 shows the concentrations of digitoxin and digoxin equivalents in these shoot-forming calli. In every case, considerable accumulation of these substances was observed. Since the inoculum of these cultures contained no detectable amounts of the cardenolides, de novo synthesis of the cardenolides must have occurred in these redifferentiated shoots. The ratio of digoxin equivalents to digitoxin equivalents in shoot-forming calli of D. lanata was less than 1 in contrast with those in the dedifferentiated or root-forming calli. Discussion
Radioimmunoassay method combining high specificity with high sensmvity made analysis of very small amounts of digitoxin and digoxin equivalents in the calli of Digitalis species possible.
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D. lutea BAI D. mertonensis K2 BAI D. ambigua BAr D. ferruginea Gigantea a The symbols of the media are explained in the text. b Concentration of ng digitoxin equivalents per g fresh weight of callus. C Concentration of ng digoxin equivalents per g fresh weight of callus. Cultivation time was 40 days.
Cardenolide contents in Digitalis callus
1403
We are grateful to Dr. W. Ulrich in Institute fur Botanik der Technischen Hochschule, Darmstadt, Germany, and to Mr. T. Tanaka, director of the Medicinal Plant Garden of Tokyo Metropolitan Government, for the supply of Digitalis seeds and also to Dr. Y. Takanami, Dr. T. Fujimori and Dr. D. Yoshida in this institute for their valuable advice on the immunological experiments.
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The total contents of digitoxin and digoxin equivalents in each culture of the first passage calli from seedlings varied between zero and nine times as high as in the original seedlings. However, those in the second passage calli were almost undetectable in all the cultures examined. The increase of the cardenolide content during the first passage culture might be due to either the presence of a few cardenolideproducing cells in the population or their production by seedling cells which still remained in the undedifferentiated state during the early period of culture. Evans et al. reported that in seedlings of D. purpurea, synthesis of cardenolides kept pace with the growth ofthe seedling at 15 days after germination (2). The total contents of digitoxin equivalents in the first passage calli induced from leaf discs of D. purpurea were generally as high as those in the original leaf discs in spite of cell growth, and during the second passage culture, digitoxin equivalents decreased while the cells grew more rapidly. These results suggest that, in the case of Digitalis purpurea, even early passage callus such as the first or second passage callus lacks the ability to accumulate cardenolides. During the first passage culture of calli from D. lanata leaf discs, the total content of digitoxin equivalents decreased while that of digoxin equivalents slightly increased. The ability of D. lanata suspension culture cells to transform digitoxin into digoxin was reported by Heins et al. (5). Therefore, the increase of digoxin equivalents in the first passage calli might be due to the transformation of digitoxigenin glycosides into digoxigenin glycosides rather than to de novo synthesis. A decrease in the contents of both cardenolides during the second passage culture suggests that synthesis of cardenolides did not occur in the calli. The root-forming calli accumulated no more cardenolides than the completely dedifferentiated calli, but the shoot-forming calli accumulated them in considerably higher concentrations although these were lower than those in intact leaf. Though cardenolides are present in the entire Digitalis plant (3), these results are in accord with those by Hirotani et al. (6) that synthesis of cardenolides occurs in the leaf and not in the root. Rucker et al. reported that the roots newly differentiated from leaf tissue of D. purpurea contained cardenolides (13). However, they did not investigate whether the cardenolides in the roots were synthesized there de novo. The cardenolides might be transported from the leaf tissue to the roots. At least, the state of cells in the shoot seems to be more suitable for synthesis of cardenolides than that in the root. The cardenolide contents in shoot-forming calli of the six Digitalis species were quite variable. The variation seemed to be at least partially due to the variation in morphology, because a relatively high cardenolide-accumulating shoot-forming callus strain had relatively well developed leaves and vice versa. Expression and suppression of cardenolide-synthesizing activity in Digitalis cells seems to be closely linked to the state of the cell. Therefore, it seems necessary to cut the link in some way such as by inducing mutation to obtain a dedifferentiated culture strain with high cardenolide-producing capability.
1404
M. Hagimori, T. Matsumoto and T. Kisaki References
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( 1) Elze, H., H. Pilgrim and E. Teuscher: Die Biotransformation von Cholesterol-26- 14C durch Gewebekulturen von Evonymus europaea und Digitalis purpurea. Pharmazie 29: 727-728 (1974). ( 2) Evans, F. J. and P. S. Cowley: Variation in cardenolides and sapogenins in Digitalis purpurea during germination. Phytochemistry 11: 2729-2733 (1972). ( 3) Evans, F. J. and P. S. Cowley: Cardenolides and spirostanols in Digitalis purpurea at various stages of development. ibid. 11: 2971-2975 (1972). ( 4) Graves, J. M. H. and W. K. Smith: Transformation of pregnenolone and progesterone by cultured plant cells. Nature 214: 1248-1249 (1967). ( 5) Heins, M.,J. Wahl, H. Lerch, F. Kaiser and E. Reinhard: Preparation of fJ-methyldigoxin by hydroxylation of tJ-methyldigitoxin in fermenter cultures of Digitalis lanata. Planta Med. 33: 57-62 (1978). ( 6) Hirotani, M. and T. Furuya: Restoration of cardenolide-synthesis in redifferentiated shoots from callus cultures of Digitalispurpurea. Phytochemistry 16: 610-611 (1977). (7) Kartnig, T.: Cardiac glycosides in cell cultures of Digitalis. In Plant Tissue Culture and Its Biotechnological Application. Edited by VV. Barz, E. Reinhard and M. H. Zenk. p. 44-51. SpringerVerlag, Berlin, 1977. ( 8) Kurz, W. G. W. and F. ConstabeI: Plant cell cultures, a potential source of pharmaceuticals. Adv. Appl. Microbiol. 25: 209-240 (1979). ( 9) Matsumoto" T., T. Ikeda, N. Kanno, T. Kisaki and M. Noguchi: Selection of high ubiquinone-l0-producing strain of tobacco cultured cells by cell cloning technique. Agric. Biol. Chem, 44: 967-969 (1980). (10) Murashige, T. and F. Skoog: A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol. Plant. 15: 473-497 (1962). (11) Nickel, S. L. and E.]. Staba: Suitability of antidigoxin antiserum for digoxin in plant extracts. Lloydia 40: 230-235 (1977). (12) Nickel, S. L. and E. J. Staba: RIA-test of Digitalis plants and tissue cultures. In Plant Tissue Culture and Its Biotechnological Application. Edited by W. Barz, E. Reinhard and M. H. Zenk. p. 278-284. Springer-Verlag, Berlin, 1977. (13) Rucker, W., K. Jentzsch and M. Wicht!: Wurzeldifferenzierung und Glycosidbildung bei in vitro kultivierten Blattexplantaten von Digitalispurpurea L. Z. Pflanzenphysiol. 80: 323-335 (1976). (14) Smith, T. W., V. P. Butler, Jr. and E. Haber: Characterization of antibodies of high affinity and specificity for the Digitalis glycoside digoxin. Biochemistry 9: 331-337 (1970). (15) Sugano, N., S. Miya and A. Nishi: Carotenoid synthesis in a suspension culture of carrot cells. Plant & Cell Physiol. 12: 525-531 (1971). (16) Tabata, M.: Recent advances in the production of medicinal substances by plant cell cultures. In Plant Tissue Culture and Its Bio-technological Application. Edited by W. Barz, E. Reinhard and M. H. Zenk. p. 3-16. Springer-Verlag, Berlin, 1977. (17) Tabata, M. and N. Hiraoka: Variation of alkaloid production in Nicotiana rustica callus cultures. Physiol. Plant. 38: 19-23 (1976). (18) Tabata, M., H. Mizukami, N. Hiraoka and M. Konoshima: Pigment formation in callus cultures of Lithospermum erythrorhizon. Phytochemistry 13: 927-932 (1974). (19) Weiler, E. W. and M. H. Zenk: Radioimmunoassay for the determination of digoxin and related compounds in Digitalis lanata. ibid. 15: 1537-1545 (1976). (20) Yamada, Y. and F. Sato: The photoautotrophic culture of chlorophyllous cells. Plant & Cell Physiol. 19: 691-699 (1978). (21) Zenk, M. H., H. El Shagi, H. Arens,]. Stockigt, E. W. Weiler and B. Deus: Formation of the indole alkaloids serpentine and ajmalicine in cell suspension cultures of Catharanthus roseus. In Plant Tissue Culture and Its Bio-technological Application. Edited by W. Barz, E. Reinhard and M. H. Zenk. p. 27-43. Springer-Verlag, Berlin, 1977.
Studies on the production of Digitalis cardenolides by plant tissue culture I. Determ.ination of digitoxin and digoxin contents in first and second passage calli and organ redifferentiating calli of several Digitalis species by radioillllnunoassay
Central Research Institute, The japan Tobacco & Salt Public Corporation, 6-2. Umegaoka, Midoriku, Yokohama 227, japan (Received july 21, 1980)
With first and second passage calli induced from seedlings of Digitalis pur.fJurea, D. lanata, D. lutea, D. mertonensis, D. ambigua and D. ferruginea Gigantea and those induced from leaf discs of D. purpurea and D. lanata, the contents of digitoxin and digoxin equivalents were assayed and compared with the contents involved in the inocula. Although the total contents of digitoxin and digoxin equivalents in the first passage calli induced from seedlings varied between zero and nine times as high as in the original seedlings, those in the second passage calli were almost undetectable. The total contents of digitoxin equivalents in the first passage calli induced from leaf discs of D. purpurea were approximately equal to those in the original leaf discs, but those in the second passage calli were less than those in the inocula. In the first passage calli induced from leaf discs of D. lanata, the total contents of digitoxin equivalents decreased but those of digoxin equivalents slightly increased. However, in the second passage calli, the amounts of both cardenolides decreased. Root-forming calli accumulated no more digitoxin nor digoxin equivalents than completely dedifferentiated calli. However, shoot-forming calli accumulated considerable amounts of cardenolides, which were assayed as digitoxin and digoxin equivalents by radioimmunoassay. Key words: Cardenolides - Digitalis callus - Digitoxin - Digoxin - Radioimmunoassay - Redifferentiated organ.
Digitalis cardenolides, especially digoxin and digitoxin, are important in medicine. This study was carried out to produce these substances by plant tissue culture technique. The production of secondary products by cultured plant cells has been studied for many plant species by a number of investigators (8, 16). For example, shikonin formation by cultured cells of Lithospermum erythrorhizon (18) and carotenoid formation by those of Daucas carota (15) have been reported. However, in most cases, secondary products unique to the species are not produced or are produced by cultured cells in much lesser amount than by intact plants. Recently, some investigators succeeded in obtaining strains of cultured cells with high biosynthetic Abbreviations: BA, benzyladenine; 2,4-D, 2,4-dichlorophenoxyacetic acid; IAA, 3-indoleacetic acid; NAA, a-naphthaleneacetic acid. 1391
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Manabu Hagimori, Takashi Matsumoto and Takuro Kisaki
1392
M. Hagimori, T. Matsumoto and T. Kisaki
Materials and methods
Antisera preparation Digoxin antisera and 'digitoxin antisera were prepared as follows. Digitoxin and digoxin were coupled to human serum albumin by the method of Smith et al. (14), with 5.3 moles of digitoxin and 4.3 moles of digoxin coupled to 1 mole of albumin, as determined by spectroscopy. Four milligrams of the dialyzed and lyophilized conjugate was dissolved in 1 ml H 20 and emulsified with 1.5 ml complete Freund's adjuvant. Rabbits were immunized by one injection of 0.4 ml freshly prepared antigen emulsion into the toe pads followed by weekly intramuscular injections. Antisera were obtained 10 weeks after the first injection. Each 2 ml of antiserum was put into an ampule and lyophilized, then the ampule was sealed and stored at 4°C. For radioimmunoassay, the stock antiserum in the ampule was dissolved in 2 ml H 2 0 and further diluted with the buffer used in radioimmunoassay.
Radioimmunoassay procedure For radioimmunoassay 0.5 ml buffer (0.01 M KPi, 0.15 M NaCI, pH 7.3), 0.1 ml l % bovine serum albumin, 0.1 ml tracer (G-3H-digitoxin 7.4 Cijmmole or G-3Hdigoxin 10.6 Ci/mmole, The Radiochemical Centre, Amersham, 0.1 /-lCi/ml), 0.1 ml of digitoxin or digoxin standard or tissue extract or blank, and 0.1 ml antiserum solution were poured into a conical tube in an ice bath. After mixing, the mixture was incubated for 30 min at 24°C. The reaction was stopped by cooling in an ice bath and 0.5 ml of dextran-coated charocal suspension, prepared just before use by mixing 10 ml of the buffer containing 0.5 g of charcoal and 10 ml of the buffer containing 0.5 g of dextran, was added. After 10 min of contact time, the mixture was centrifuged at 2500 rpm for 10 min at O°C. A 0.5-mI portion of the supernatant
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activity by cell selection (17, 20, 21). We have also obtained strains of cultured cells of Nicotiana tabacum which accumulate ubiquinone-IO five times more than the parent strain by cell selection (9). The cardenolide productivity of cultured cells of the Digitalis species has been investigated by some workers and most of them reported that cultured cells did not produce cardenolides (1, 4, 6). However, Kartnig (7) reported that callus of D. purpurea accumulated purpureaglycoside A at 1 /-lg per g fresh weight at the tenth passage. If the early passage calli retain the activity to produce cardenolides to some extent, it might be possible to obtain cardenolide-accumulating strains from early passage calli by cell selection. Thus, we examined the ability of first and second passage calli of several Digitalis species to accumulate cardenolides. Then we determined the cardenolide contents in organ redifferentiating calli in order to find which state of cells of the Digitalis species is suitable for the biosynthesis of cardenolides. Radioimmunoassay using digoxin antisera has been reported to be suitable for the determination of digoxin levels in plant extracts (11,12,19). We also used this method for the determination of digoxin and digitoxin contents using digoxin antisera and digitoxin antisera, respectively.
Cardenolide contents in Digitalis callus
1393
containing antibody-bound haptene was put into a scintillation vial containing 3.5 ml scintillator (Instagel, Packard Instruments Company, Downers Grove) and counted. Standard and tissue extracts were run in duplicate.
Extraction
Induction andculture ofcallus Calli were induced from seedlings and leaf discs. Calli from seedlings: Calli were induced from 10-day-old seedlings of Digitalis purpurea, D. lanata, D. lutea, D. mertonensis, D. ambigua and D. ferruginea Gigantea. Murashige and Skoog's inorganic medium (10) containing 30 g/liter sucrose, 100 mgjliter myo-inositol, 2.0 mg/liter glycine, 1.0 mg/Iiter thiamin-HCl, 0.5 mgjliter nicotinic acid, 0.5 mgjliter pyridoxin·HCI and 8 g/liter agar was used as the basal medium. Eight different media which contained the constituents of the basal medium supplemented with 2 mg/liter 2,4-D (D), 2 mg/liter NAA (N), 2 mgjliter kinetin (K), 3 g/liter yeast extract (Y) and 3 g/liter malt extract (M) in eight combinations, DM, DKM, DY, DKY, NM, NKM, NY and NKY, were used. A seedling was placed on the solid medium in a test tube. After 50-70 days, all cultured material in the tube, which was designated the first passage callus, was harvested and part of the induced callus was transferred to the same fresh medium while the rest was subjected to radioimmunoassay. The second passage calli were harvested after 30-50 days of culture and treated by the same procedure used for the first passage calli. Calli from seedlings were cultured in the dark at 28°C. Calli from leaf discs: Pairs of leaf discs (4 mm in diameter) were obtained from symmetrical positions opposite each other across the central vein of leaves of D. purpurea and D. lanata. One of a pair was placed on the solid medium in a test tube for callus induction and the other was assayed for digoxin and digitoxin contents as a control. The calli from leaf discs were cultured in the dark or under a 12 hr daylight cycle (white fluorescent lamp, about 5,000 lux) at 28°C on four media, DM, DKM, NM and NKM. After 92 or 96 days, the first passage callus was harvested and the induced callus was transferred to the same fresh medium with the remainder subjected to radioimmunoassay. In preliminary experiments, the part of the leaf tissue and the part of the callus tissue of the first passage culture and the medium were assayed separately. The leaf tissue had become dark brown and contained relatively small amounts of cardenolides. Most of the cardenolides were detected in the callus tissue' and not in the medium. Therefore, in the regular experiments,
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From plant or callus tissue: About 1 g of fresh tissue was homogenized in 15 ml 80% ethanol. The homogenate was heated at 74°C for 4 hr and filtered. The filtrate was dried in vacuo and the residue was taken up in 1 ml 80% ethanol and diluted with 9 ml H20. When the concentration of digoxin or digitoxin equivalents was too high for radioimmunoassay, the extract was further diluted with H 20 to a desirable level. From agar medium: To about 15 ml of agar medium, on which callus had been cultured, about 85 ml of ethanol was added to give a final volume of 100 ml. After homogenization, the mixture was placed at room temperature overnight and filtered. Fifty milliliters of the filtrate was dried up in vacuo and the resideu was taken up in 1 m180% ethanol and diluted with 9 ml H 20.
1394
M. Hagimori, T. Matsumoto and T. Kisaki
all cultured material in the tube except the small part inoculated to the second passage culture was assayed together. The second passage calli were harvested after 37 or 38 days of culture and treated according to the same procedure used for the first passage calli. The cells inoculated to the next passage culture and the cells subjected to the assay were weighed. Four and five replicated experiments were done for calli from seedlings and leaf discs, respectively.
Establishment ofshoot-Jorming calli
Results
Sensitivity andspecificity of radioimmunoassay The standard curves of digitoxin and digoxin radioimmunoassay are shown in Fig. 1. The ability of compounds other than digitoxin (or digoxin) to compete for the digitoxin antibody (or the digoxin antibody) is called cross reactivity. The percentage cross reactivity (OR %) is defined as OR ~ 0-
pico moles of digitoxin yielding BjBo=50% X 100 pico moles of compound under investigation yielding BjBo=50%
where Band Bo are the percentages of antibody-bound tracer in the presence and absence of unlabeled competetive substances, respectively. Table 1 shows the cross Table 1 Cross reactivities of several cardenolides, sterols andsaponin for digitoxin anddigoxin antisera
Substance Digitoxin Gitoxin Digoxin Gitaloxin Digitalin Strospeside Digitoxigenin Gitoxigenin Digoxigenin Digitonin Cholesterol Stigmasterol fJ-Sitosterol Campesterol a b
Digitoxin antiserum. Digoxin antiserum.
CR°;{, for 03 b 100
3.8 3.4 1.8 9.3
2.6 75 1.6 0.9
o o
o o o
9.8 1.0 100
0.2 2.4 0.3
8.2 0.1
136
o o o o o
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Shoot-redifferentiating calli were established by transferring third passage calli from seedlings to media which contained BA, lAA, kinetin or combinations of these substances in addition to the constituents of the basal medium and culturing them under a 12 hr day-light cycle at 28°0.
1395
Cardenolide contents in Digitalis callus
100-------------------,
A
80
o
CO 40 ........ 0)
0.1
0.4
1.0
4
oIGITOXI N
10
40
(ng)
100--------------------, B ,",80 o
0-
'-'60 o
0)
---. 40 CO
20 0.1
0.4
1.0
4
10
40
DIGOXIN ( ng )
Fig. 1. Digitoxin and digoxin standard curves obtained by plotting B /Bo % versus mass of unlabeled haptene on semilogarithmic scale. A, Digitoxin standard curve. B, Digoxin standard curve.
Table 2 Contents immunoassay
of digitoxin and digoxin equivalents in leaves 0./ several Digitalis species determined by radio-
Species
Dig. ftgfg f.w. a
D. purpurta D.lanata D. lutea
D. mertonensis
D. ftrruginea Gigantea D. ambigua a b
Digitoxin equivalents: pg per g fresh weight. Digoxin equivalents: pg per g fresh weight.
180 700 500 110
Digo. pgfg f.w.
8
400 28 8
260
22
330
9
b
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20
1396
M. Hagimori, T. Matsumoto and T. Kisaki
Table 3 Cell yield, morphology and contents of digitoxin and digoxin equiualents of the first passage calli D. purourea Medium a
NY NKY
M
O. 63±0. 37/
C C C C R R R R
1.01 ±O. 53 O. 53±0. 21 1.04±0. 35 2. 48± 1. 04 1. 98± 1. 10 1. 78±0. 62 1. 07 ±O. 25
Dig. ngjtube d 140± 90 370±480 220±200 260±240 230±310 330±270 390±300 420±140
70± 100± 130± 70± 50± 40± 70± 130±
40 60 80 10 30 40 30 70
Cultivation time (days) 70 70 70 70 57 57 57 57
30 h
150 6
Seedling
Digo. ngjtube 8
D.lutea
Medium DM DKM DY DKY NM NKM NY NKY
CY. g f.w.jtube
M
Dig. ngjtube
Digo. ngjtube
Cultivation time (days)
O. 34±0. 09 O.56±0.4 O. 12±O. 06 O. 11±0. 06 O. 90±0. 30 O. 58±0. 09 O. 67±0. 09 O. 29±0. 14
C C C C R R R R
20± 30 70±100 110± 70 90± 60 10± 20 10± 20 0 10± 20
110± 80 180± 60 240± 70 190± 140 80± 60 70± 70 110± 60 150± 60
70 70 70 70 55 55 55 55
Seedling
80
170 D. ambigua
Medium DM DKM DY DKY NM NKM
NY NKY Seedling a b C
d
cv. g f.w.jtube
M
Dig. ngjtube
O. 30±0. 17 O. 28±0. 18 O. 06±O. 02 O. 30±0. 39 1. 34±0. 61 1.61 ±O. 99 O. 23±O. 09 O. 09±O. 07
C C
110±110 460±570 20± 20 20± 20 20± 30 10± 20 160±240 360±350
C
C R R R R
90
Digo. ng/tube 50± 60± 20± 20± 10± 20± 40± 40±
60 80 20 20 30 20 80 80
Cultivation time (days) 70 70 70 70 60 60 60 60
20
The symbols of the media are explained in the text. Cell yield: g fresh weight per culture. Morphology of each culture: C, completely dedifferentiated callus; R, root-forming callus. Total content of digitoxin equivalents per culture.
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DM DKM DY DKY NM NKM
CY. g f.w.jtube b
C
1397
Cardenolide contents in Digitalis callus
qf several Digitalis species induced from seedlings
D. lanata CY. g f.w.jtube
Dig. ngjtube
Digo. ng/tube
C
10±20 10±20 30±40 20±30 40±40 50±60 10±20 20±30
110±220 110±200 70± 60 60± 60 160±120 190± 170 110± 140 90± 80
C C C
R R R R
130
Cultivation time (days)
70 70 70 70 56 56 56 56
130 D. mertonensis
CY. g f.w.jtube
M
Dig. ng/tube
O. 27±0. 04 O. 58±0. 48 O. 16±0. 10 1. 01 ±O. 48 2. 83± 1.58 1. 23±0. 96 2. 80±1. 37 2. 70±1. 65
C
0 40±80 20±20 50±40 20±40 40±60 10±20 0
C
C C
R R R R
Digo. ng/tube
10± 40± 70± 50± 50± 40± 20± 30±
140
20 10 40 20 70 50 40 20
Cultivation time (days)
70 70 70 70 50 50 50 50
30
D-.ferruginea Gigantea
e j
g h
CY. g f.w.jtube
M
Dig. ng/tube
0.07±0.10 O. 10±0. 09 O. 04±0. 02 O. 16±0. 12 O. 55±0. 24 O. 84±0. 29 0.37 ±O. 18 O. 48±0. 37
C
20±40 0 0 0 10±20 10±20 0 0
30± 50 20± 20 10± 20 10± 30 60± 60 90± 90 40± 30 70± 110
70
80
C C C
R R R R
Digo. ng/tube
Total content of digoxin equivalents per culture. Mean value of four experiments with standard deviation. Total content of digitoxin equivalents per seedling. Total content of digoxin equivalents per seedling.
Cultivation time (days)
70 70 70 70 70 70 70 70
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0.16±0.05 O. 34±0. 11 1. 05±0. 77 1. 22± 1.00 2. 26± 1. 06 1. 27±O. 45 O. 95±0. 75 O. 97±0. 49
M
1398
M. Hagimori, T. Matsumoto and T. Kisaki
Calli inducedfrom seedlings Table 3 shows the cell yields, morphology and contents of digitoxin and digoxin equivalents of the first passage calli from seedlings. The calli cultured on NAAcontaining media generally grew better than those on 2,4-D-containing media. Most of the former formed roots, while the latter were completely dedifferentiated. However, the effect of the difference in media on the contents of digitoxin and digoxin equivalents wes not clear. The contents of both substances varied between zero and nine times as high as in the original seedling. But the contents of digitoxin Table 4 Cellyields, morphology, contents and increasing rates of digitoxin equivalents of thefirst passage calli induced from leaf discs of Digitalis purpurea Medium a DM DKM
NM
Light condition
MO
Dig. ngjtube "
De
2. 17±0. 291 1.51±O. 44
C C
1, 240±370 1, 390±320
590± 1,000±
250 420
0.87 ±O. 18 O. 99±0. 22
L D
2. 33±0. 10 1. 07±O. 60
C C
1, 140±590 1, 760± 790
260 490± 2, 160± 1,280
0.84±0.44 1. 22±0. 42
L
2.11 ±O. 39 3. 36±0. 20
R R
1,060±490 1, 430±330
480± 430±
170 '110
O. 74±0. 26 1.02±0.11
2. 56±0. 37 2. 79±0. 35
R R
1, 390± 480 1,710±220
540± 630±
170 170
O. 99±0. 31 1. 25±0. 28
L
D
NKM
L D
Leaf disc
Increasing rate of digitoxin equivalents C
CY. g f.w.jtube "
d
Dig. ng/g f.w. b
1,400±250 g 210,000 ± 40, 000 h
See footnotes of Table 3. Concentration of ng digitoxin equivalents per g fresh weight of callus. C Ratio of the total content of digitoxin equivalents per culture to that per control leaf disc. d Cultured under a 12 hr-day light cycle. S Cultured in the dark. I Mean value of five experiments with standard deviation. g Mean value of total contents of digitoxin equivalents per control leaf disc with standard deviation. h Mean value of concentration of ng digitoxin equivalents per g fresh weight of control leaf disc with standard deviation. Cultivation time was 96 days. a
b
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reactivities of several compounds structurally analogous to digitoxin and digoxin. The data indicate that the digitoxin antiserum is specific to digitoxigenin and its glycoside, whereas the digoxin antiserum is specific to digoxigenin and its glycoside. Cardenolides other than digitoxigenin and its glycosides (or digoxigenin and its glycosides) showed CR% of less than 10% for digitoxin antiserum (or digoxin antiserum), and compounds other than cardenolides showed only negligible cross reactivities. Since digitoxin and digoxin were used as standards in the study, the experimental values are expressed as digitoxin equivalents or digoxin equivalents. Table 2 shows the concentrations of digitoxin and digoxin equivalents in the leaves of twomonth-old plants of Digitalis species measured by radioimmunoassay method using these antisera.
1399
Cardenolide contents in Digitalis callus
and digoxin equivalents in the second passage cultures of these strains were all below the detection limit except for one strain and the contents in the third passage of this strain was also below the detection limit.
Calli inducedfrom leaf discs
Table 5 Growth rates, morphology, contents and increasing rates calli inducedfrom leaf discs ofDigitalis purpurea Medium a DM
DKM NM
NKM
Light condition a
Growth rate b
MO
of digitoxin equivalents of the second passage
Dig. ng/tube a
Dig. tiglg f.w. a
Increasing rate of digi toxin equivalents C
20± 30 100± 65
10±10 40±20
O. 2±0. 2 O. 3±0. 2
16.9±10. sa 10.0± 2.5
C
D L D
13.2± 2.1 14.1 ± 6.6
C C
60± 20 170± 50
30±20 70±20
0.7±0.5 O. 5±0. 1
L D
13.5± 4.5 6.5± 1. 2
C-R C-R
100± 100 140± 60
50±30 60±30
O. 7±0. 2 O. 8±0. 2
L
15.4± 5.8 13.4± 5.5
C-R
40± 50
C
20± 40
10±10 10±10
O. 2±0. 3 0.I±O.2
L
D
C
See footnotes of Tables 3 and 4. Ratio of the cell yield to the weight of the inoculated cells. C Ratio of the total content of digitoxin equivalents per culture to that per inoculated cells. The latter was calculated by multiplying the digitoxin equivalents concentration of the first passage culture of the strain by the weight of the inoculated cells of the second passage culture. Cultivation time was 37 days. a
b
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Tables 4 and 5 show the results obtained with first and second passage calli from leaf discs of D. purpurea, respectively. The total content of digitoxin equivalents in each culture offirst passage calli was approximately as high as that in the original leaf disc and was not substantially affected by the media or lgiht. On the other hand, the cell weight increased and therefore the concentration of digitoxin equivalents per g fresh weight dropped to between 1/50 and 1/1,000 that of the original leaf discs (210 pg/g fresh weight). The concentrations of digitoxin equivalents in the dark-grown calli on DM and DKM media were relatively high, but this was probably due to their relatively low growth rate because their digitoxin contents per tube were also as high as those in the original leaf discs (Table 4). During the second passage culture, the total content of digitoxin equivalents in each culture decreased from that in the inoculum, while the cells grew well. Thus the concentration of digitoxin equivalents per g fresh weight dropped to less than 1/2,000 that of leaf discs (Table 5). The calli cultured under light did not become green. Tables 6 and 7 show the results of experiments with first and second passage calli from leaf discs of D. lanata, respectively. During the first passage culture, though the total content of digitoxin equivalents in each culture tube decreased, that of digoxin equivalents slightly increased. Therefore the ratios of digoxin equivalents to digitoxin equivalents in the calli increased from about 1 in the original leaf discs to 2-4. However, the concentrations of digitoxin and digoxin equivalents per g fresh weight dropped to 1/60-1/1,500 and 1/15-1/600 of those in the original
R R
3. 88±0. 24 3. 28±0. 59
D
R
3. 17±0. 34
D
L
R
2. 66±0. 70
C
L
C
1. 27 ±O. 55
L
D
D
C
1. 27 ±O. 29 O. 97±0. 59
C
0.51±0.41 a
L
a
300
1,900± 1,800± 2,300±
6, OOO± 1,500 7, 100±2, 100 7, 400± 1,400
6, 400± 1, 400
3,100± 2, 100
7, 000±2, 400
600, 000±80, 000
300
600
6,300± 1,900
7, 300±2, 300
11,800± 6,800
9, OOO±4, 400
800
6,100±
d
b
20, 500± 11, 700
Digo. ng/g f.w.
7, 800±2, 000
8, 000±4, 800
D'IgO.ng / tu b e a
6
1. 22±0. 42
1. 10±0. 42
O. 92±0. 34
1. 17±0.50
6, 400± 1,
4,300±
3,300±
1,600±
1,800±
2,800±
100 J
600
900
400
700
900
3, 300± 1,800
1. 37±O. 57 1. 08±0. 28
2,500±
600
2, OOO± 1,000
1. 20±0. 45
1, 30±0. 75
Increasing rate of digoxin Dig. ngjtube " equivalents a
b
a
~ate Digo./ of digitoxin D' C equivalents a rg.
2.7±0.3 4. 5±2. 2 3. 9± 1. 4 2. 3±0. 9
1. 7±0.3
600 O. 45±0. 11 300 O. 27±O. 09 100 0.27 ±O. 08 200 O. 53±0. 09 400 O. 68±0. 12
500± 900± 1,300±
600,
OOO± 120,000 6
700±
2,300±
1. O±O. 2
2. 8±0. 9
3, 200 O. 54±0. 30
3,900±
3. 1±0. 3
3, 700 O. 32±0. 19 4. O±O. 7
a Increa;si!1g
300 O. 43±0. 17
2,000±
5,400±
,
DIg. ng/g f.w.
See footnotes of Tables 3 and 4. Concentration of ng digoxin equivalents per g fresh weight of callus. C Ratio of the total content of digoxin equivalents to that of digitoxin equivalents in the callus. d Mean value of total contents of ng digoxin equivalents per control leaf disc with standard deviation. 6 Mean value of concentration of ng digoxin equivalents per g fresh weight of control leaf disc with standard deviation, f Mean value of total contents of ng digitoxin equivalents per control leaf disc with standard deviation. g Mean value of concentration of ng digitoxin equivalents per g fresh weight of control leaf disc with standard deviation. Cultivation time was 92 days,
Leaf disc
NKM
NM
DKM
DM
Light a CY • g f .w. / tu b e a M Medium a condition
Table 6 Cell yields, morphology; contents and increasing rates of digoxin and digitoxin eqaivalents and ratios of digoxin equivalents to digitoxin equivalents of the first passage calli inducedfrom leaf discs of Digitalis lanata
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er:
~
Cf.l
~
~
=' 0.-
~
8"
S 0
~
ft
~
~
~
p.
S· 0
CIQ
~
t:t=
~
~ o
R R
6. 6±4. 6 8. 1± 1.4
L
D
R
10.8±2. 3
D
C
3.0±2.0
D
R
C
3.1 ± 1. 6
L
8.7±2.8
C
1.2±0. 5
D
L
C
2.7 ± 1. 9 0
L
o
See footnotes of Tables 3, 4, 5 and 6. Cultivation time was 38 days.
NKM
NM
DKM
DM
MO
Growth rate 0
Light Medium 0 condition 0
360± 140 140±
270±
280±
750±670 460±280
220±
1,110±
570±370 450± 170
590±
1,660±
70
190
270
160
880
500
960
1, 600± 1,600
340±480
340± 110
380±230
Digo. ngjtube " Digo, ngjg f.w. 0
O. 4±0. 1
O. 7±0. 3
O. 9±0. 9
O. 7±0. 3
0.4±0.2
O. 2±0. 3
O. 3±0. 2
O. 5±0. 5
Increasing rate of digoxin equivalents 0
120± 40
120± 50
120± 30
120± 40
270±210
140± 140
50± 40
60± 50
Dig. ngjtube 0
Increasing rate of digitoxin equivalents 0 0.4±0.4 O. 1±0. 07 O. 3±0. 3 O. 5±0. 3 0.8±0.4 O. 8±0. 4 O. 4±0. 2 O. 2±0. 09
Dig. ngjg f.w. 0
260±410 180± 60 310±370 520±460 50± 20 70± 60 60± 30 40± 20
Table 7 Growth rates, morphology, contents and increasing rates of digoxin and digitoxin equivalents of the second passage calli induced from leaf discs of Digi talis lanata
Downloaded from http://pcp.oxfordjournals.org/ at RMIT University Library on July 6, 2015
~
~ Vol
e.
o
~
~.
~.
S· b
~
t:::l
an
0
(")
s.: n
g,.
0.. n t:::l
"1
~
0
1402
M. Hagimori, T. Matsumoto and T. Kisaki
Table 8
Concentrations of digitoxin and digoxin equivalents in shoot-forming calli of Digitalis species
Species D. purpurea
D. lanata
Medium a
Dig. ng/g f.w. b
Digo. ng/g f.w. C
BA 10 BA 10
18,200 12,000 9, 700 6,800 3,900 700
1,700 4,000 1,400 700 600 300
leaf discs (both were 600 /lg!g fresh weight), respectively. The effects of differences in media and light on the contents of both substances were not clear, whereas those on growth and morphology were similar to the case of D. purpurea. The relatively high concentrations of the cardenolides in the light-grown calli on DM and DKM media seem to be due to their relatively low growth rates (Table 6). During the second passage culture, the total contents of digoxin and digitoxin equivalents in each culture decreased from the amounts involved in the inoculum and their concentrations dropped to 1/200-1/9,000 and 1/600-1/20,000 of those in the original leaf discs, respectively (Table 7). The calli cultured under light did not become green.
Organ-redijferentiating calli As shown in Tables 3 to 7, though almost all of the calli induced and cultured on the NAA-containing media formed roots, they accumulated no more cardenolides than the completely dedifferentiated calli.
Shoot-forming calli were established on
the basal medium supplemented with 10 mg/liter BA (BA 10) for D. purpurea and D. lanata, 1 mg/liter BA plus 1 mgjliter IAA (BAI) or 2 mgjliter kinetin (K2) for D. lutea and D. mertonensis, and BAI for D. ambigua and D.ferruginea Gigantea, under a 12 hr day-light cycle. The redifferentiated shoots became deep green simultaneously with redifferentiation, but their appearances were to some extent anomalous. In particular, the development of the leaves was imperfect. Table 8 shows the concentrations of digitoxin and digoxin equivalents in these shoot-forming calli. In every case, considerable accumulation of these substances was observed. Since the inoculum of these cultures contained no detectable amounts of the cardenolides, de novo synthesis of the cardenolides must have occurred in these redifferentiated shoots. The ratio of digoxin equivalents to digitoxin equivalents in shoot-forming calli of D. lanata was less than 1 in contrast with those in the dedifferentiated or root-forming calli. Discussion
Radioimmunoassay method combining high specificity with high sensmvity made analysis of very small amounts of digitoxin and digoxin equivalents in the calli of Digitalis species possible.
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D. lutea BAI D. mertonensis K2 BAI D. ambigua BAr D. ferruginea Gigantea a The symbols of the media are explained in the text. b Concentration of ng digitoxin equivalents per g fresh weight of callus. C Concentration of ng digoxin equivalents per g fresh weight of callus. Cultivation time was 40 days.
Cardenolide contents in Digitalis callus
1403
We are grateful to Dr. W. Ulrich in Institute fur Botanik der Technischen Hochschule, Darmstadt, Germany, and to Mr. T. Tanaka, director of the Medicinal Plant Garden of Tokyo Metropolitan Government, for the supply of Digitalis seeds and also to Dr. Y. Takanami, Dr. T. Fujimori and Dr. D. Yoshida in this institute for their valuable advice on the immunological experiments.
Downloaded from http://pcp.oxfordjournals.org/ at RMIT University Library on July 6, 2015
The total contents of digitoxin and digoxin equivalents in each culture of the first passage calli from seedlings varied between zero and nine times as high as in the original seedlings. However, those in the second passage calli were almost undetectable in all the cultures examined. The increase of the cardenolide content during the first passage culture might be due to either the presence of a few cardenolideproducing cells in the population or their production by seedling cells which still remained in the undedifferentiated state during the early period of culture. Evans et al. reported that in seedlings of D. purpurea, synthesis of cardenolides kept pace with the growth ofthe seedling at 15 days after germination (2). The total contents of digitoxin equivalents in the first passage calli induced from leaf discs of D. purpurea were generally as high as those in the original leaf discs in spite of cell growth, and during the second passage culture, digitoxin equivalents decreased while the cells grew more rapidly. These results suggest that, in the case of Digitalis purpurea, even early passage callus such as the first or second passage callus lacks the ability to accumulate cardenolides. During the first passage culture of calli from D. lanata leaf discs, the total content of digitoxin equivalents decreased while that of digoxin equivalents slightly increased. The ability of D. lanata suspension culture cells to transform digitoxin into digoxin was reported by Heins et al. (5). Therefore, the increase of digoxin equivalents in the first passage calli might be due to the transformation of digitoxigenin glycosides into digoxigenin glycosides rather than to de novo synthesis. A decrease in the contents of both cardenolides during the second passage culture suggests that synthesis of cardenolides did not occur in the calli. The root-forming calli accumulated no more cardenolides than the completely dedifferentiated calli, but the shoot-forming calli accumulated them in considerably higher concentrations although these were lower than those in intact leaf. Though cardenolides are present in the entire Digitalis plant (3), these results are in accord with those by Hirotani et al. (6) that synthesis of cardenolides occurs in the leaf and not in the root. Rucker et al. reported that the roots newly differentiated from leaf tissue of D. purpurea contained cardenolides (13). However, they did not investigate whether the cardenolides in the roots were synthesized there de novo. The cardenolides might be transported from the leaf tissue to the roots. At least, the state of cells in the shoot seems to be more suitable for synthesis of cardenolides than that in the root. The cardenolide contents in shoot-forming calli of the six Digitalis species were quite variable. The variation seemed to be at least partially due to the variation in morphology, because a relatively high cardenolide-accumulating shoot-forming callus strain had relatively well developed leaves and vice versa. Expression and suppression of cardenolide-synthesizing activity in Digitalis cells seems to be closely linked to the state of the cell. Therefore, it seems necessary to cut the link in some way such as by inducing mutation to obtain a dedifferentiated culture strain with high cardenolide-producing capability.
1404
M. Hagimori, T. Matsumoto and T. Kisaki References
Downloaded from http://pcp.oxfordjournals.org/ at RMIT University Library on July 6, 2015
( 1) Elze, H., H. Pilgrim and E. Teuscher: Die Biotransformation von Cholesterol-26- 14C durch Gewebekulturen von Evonymus europaea und Digitalis purpurea. Pharmazie 29: 727-728 (1974). ( 2) Evans, F. J. and P. S. Cowley: Variation in cardenolides and sapogenins in Digitalis purpurea during germination. Phytochemistry 11: 2729-2733 (1972). ( 3) Evans, F. J. and P. S. Cowley: Cardenolides and spirostanols in Digitalis purpurea at various stages of development. ibid. 11: 2971-2975 (1972). ( 4) Graves, J. M. H. and W. K. Smith: Transformation of pregnenolone and progesterone by cultured plant cells. Nature 214: 1248-1249 (1967). ( 5) Heins, M.,J. Wahl, H. Lerch, F. Kaiser and E. Reinhard: Preparation of fJ-methyldigoxin by hydroxylation of tJ-methyldigitoxin in fermenter cultures of Digitalis lanata. Planta Med. 33: 57-62 (1978). ( 6) Hirotani, M. and T. Furuya: Restoration of cardenolide-synthesis in redifferentiated shoots from callus cultures of Digitalispurpurea. Phytochemistry 16: 610-611 (1977). (7) Kartnig, T.: Cardiac glycosides in cell cultures of Digitalis. In Plant Tissue Culture and Its Biotechnological Application. Edited by VV. Barz, E. Reinhard and M. H. Zenk. p. 44-51. SpringerVerlag, Berlin, 1977. ( 8) Kurz, W. G. W. and F. ConstabeI: Plant cell cultures, a potential source of pharmaceuticals. Adv. Appl. Microbiol. 25: 209-240 (1979). ( 9) Matsumoto" T., T. Ikeda, N. Kanno, T. Kisaki and M. Noguchi: Selection of high ubiquinone-l0-producing strain of tobacco cultured cells by cell cloning technique. Agric. Biol. Chem, 44: 967-969 (1980). (10) Murashige, T. and F. Skoog: A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol. Plant. 15: 473-497 (1962). (11) Nickel, S. L. and E.]. Staba: Suitability of antidigoxin antiserum for digoxin in plant extracts. Lloydia 40: 230-235 (1977). (12) Nickel, S. L. and E. J. Staba: RIA-test of Digitalis plants and tissue cultures. In Plant Tissue Culture and Its Biotechnological Application. Edited by W. Barz, E. Reinhard and M. H. Zenk. p. 278-284. Springer-Verlag, Berlin, 1977. (13) Rucker, W., K. Jentzsch and M. Wicht!: Wurzeldifferenzierung und Glycosidbildung bei in vitro kultivierten Blattexplantaten von Digitalispurpurea L. Z. Pflanzenphysiol. 80: 323-335 (1976). (14) Smith, T. W., V. P. Butler, Jr. and E. Haber: Characterization of antibodies of high affinity and specificity for the Digitalis glycoside digoxin. Biochemistry 9: 331-337 (1970). (15) Sugano, N., S. Miya and A. Nishi: Carotenoid synthesis in a suspension culture of carrot cells. Plant & Cell Physiol. 12: 525-531 (1971). (16) Tabata, M.: Recent advances in the production of medicinal substances by plant cell cultures. In Plant Tissue Culture and Its Bio-technological Application. Edited by W. Barz, E. Reinhard and M. H. Zenk. p. 3-16. Springer-Verlag, Berlin, 1977. (17) Tabata, M. and N. Hiraoka: Variation of alkaloid production in Nicotiana rustica callus cultures. Physiol. Plant. 38: 19-23 (1976). (18) Tabata, M., H. Mizukami, N. Hiraoka and M. Konoshima: Pigment formation in callus cultures of Lithospermum erythrorhizon. Phytochemistry 13: 927-932 (1974). (19) Weiler, E. W. and M. H. Zenk: Radioimmunoassay for the determination of digoxin and related compounds in Digitalis lanata. ibid. 15: 1537-1545 (1976). (20) Yamada, Y. and F. Sato: The photoautotrophic culture of chlorophyllous cells. Plant & Cell Physiol. 19: 691-699 (1978). (21) Zenk, M. H., H. El Shagi, H. Arens,]. Stockigt, E. W. Weiler and B. Deus: Formation of the indole alkaloids serpentine and ajmalicine in cell suspension cultures of Catharanthus roseus. In Plant Tissue Culture and Its Bio-technological Application. Edited by W. Barz, E. Reinhard and M. H. Zenk. p. 27-43. Springer-Verlag, Berlin, 1977.
