chapter 4 by using ethephon, ancymidol, precursors and organic...
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CHAPTER 4
PRODUCTION OF AJMALICINE, SERPENTINE AND CATHARANTHINE
BY USING ETHEPHON, ANCYMIDOL, PRECURSORS AND ORGANIC
COMPOUNDS IN VINCA ROSEA HAIRY ROOT CULTURES
4.1 INTRODUCTION
Ethylene, a gaseous plant hormone, plays an important role in plant secondary
metabolism. Exogenously applied ethylene is known to enhance the terpenoid
pathway during fruit ripening or leaf senescence.282 Addition of ethylene was found to
increase the accumulation of terpenoid phytoalexins in tobacco cultures.283,284
Significant improvements in the yield of taxanes were obtained by the addition of
ethephon to cell cultures of Taxus wallichiana.285 Similarly, exogenous ethylene
increased the accumulation of alkaloids in cell cultures of Vinca rosea.268
Growth retardants are synthetic substances, which reduce internodes’ length
in higher plants. The effect is the result of the inhibition of gibberellins
biosynthesis.286Among the sites of inhibition is the conversation of geranyl
diphosphate (GGPP) to ent-kaurene, which is catalyzed by the enzyme ent-kaurene
synthetase. This enzyme is inhibited by quaternary ammonium compound like
(2-chloroethyl)-trimethyl ammonium chloride compounds.287 Others sites of
inhibition of gibberellins biosynthesis are the oxidation step of ent-kaurence to ent-
kaureneoic acid catalyzed by cytochrome p450 dependent mono oxygenases. These
steps are inhibited by substituted pyrimidines such as ancymidol.286 Growth retardants
have been found to effect secondary metabolism in plant cell cultures. Growth
retardants were found to increase anthocyanin accumulation in cell cultures of carrot
by inhibiting the bio synthesis of gibberellins.288 Ancymidol was found to enhance the
production of forskolin significantly in cell cultures of coleus forskohlin.290 Similarly
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the accumulation of taxanes in cell cultures of T. wallichiana was increased by
treatment with ancymidol.285
As there are no reports of the effect of ethephon and ancymidol on hairy root
cultures of Vinca rosea, the present study was undertaken to see their influence on
alkaloid production.
4.2. MATERIALS AND METHODS
Stock Solutions
Ethephon (2-chloroerhyl phosphoric acid) and ancymidol were purchased
from Sigma chemical Co. Solutions of ethephon (1 mg/ml) and ancymidol (1 mg/1)
were prepared in double distilled water and ethanol, respectively. The solutions were
filter sterilized before addition to the cultures.
Culture Conditions
Hairy roots were cultured in 50 ml conical flasks containing 15 ml of ¼ MS
supplemented with sucrose (30 g/l). An inoculums size of about 30 mg fresh eight of
8-day-old hairy roots was used to initiate the cultures. The cultures ere incubated at
100 rpm at 25 c in the dark.
Addition of Ethephon and Ancymidol
Ethephon was tested at 10 and 50 uM while ancymidol was added at final
concentrated of 10 and 50 uM. The substances were added to cultures on day 21 and
the roots were harvested on day 25.
Extraction and Analysis of Alkaloids
Hairy roots were harvested, rinsed once with double distilled water and fresh
weights recorded. The roots were extracted and analyzed for alkaloids as described in
Chapter 2.
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Statistical Analysis
Analysis of variance (ANOVA) AND Turkey’s test was carried out using
Graph Pad Prism 5 (Graph Pad Software Inc., USA) for determining the significance
of treatment effects. A p value of <0.05 was considered significant.
4.3. RESULTS AND DISCUSSION
Ethephon and ancymidol were added to hairy root cultures of Vinca rosea in
the early stationary phase. The roots were harvested after 4 days of treatment.
Significant reduction in biomass fresh weight was observed with ancymidol treatment
(18% of control value). The effects of these substances on indole alkaloid production
are shown in table-21. However, there was no effect on the release profiles of the
release profiles of the alkaloids.
Treatment with ethephon increased significantly the accumulation of
ajmalicine at the highest concentration (50 µM) (fig. 27). Ajmalicine contents (1.59
mg/1) were enhanced by 1.2 times when compared to control (1.35mg/1). The
amounts of serpentine and catharanthine were not affected either as the lowest or
highest concentration of ethephon.
Ethephon undergoes degradation, releasing ethylene. Ethylene had been
shown to influence the alkaloid biosynthesis in Vinca rosea, but the results are
contradictory. Ajmalicine production as inhibited in cell cultures with limited gas
exchange, which contained lower concentrations of dissolved oxygen and higher
amounts of ethylene and carbon dioxide.111 In contrast, inhibition of ethylene
biosynthesis in shake flask and removal of carbon dioxide, ethylene or both from the
recirculation stream in a stirred and aerated bioreactor did not improve ajmalicine
production.289 But Yahia et al., (1998)268 reported that exogenous ethylene greatly
enhanced ajmalicine accumulation and concluded that there was no relation between
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the accumulation of alkaloid and the evolution of ethylene. Marginal stimulation of
ajmalicine production was seen in our study, which could due to up regulation of the
alkaloid pathway by ethylene.
Addition of ancymidol did not improve alkaloid accumulation in the present
study at the concentration tested (Fig.27). The alkaloid contents were lower than
control cultures with the highest concentration, which could be due to inhibition of the
growth of hairy roots. Ancymidol has been shown to increase the accumulation of
secondary metabolites in cell cultures of C. forskohlin and T. wallichiana.290,285 The
inhibition of gibberellins biosynthesis by ancymidol, which increases the availability
of GGPP, as suggested being responsible for the stimulation of forskolin or taxane
production. Similarly ancymidol was found to increase anthocyanin production in
carrot cell cultures.288 The failure of antymidol, a growth retardant, to increase
alkaloid production in the present study may be due to the fact that ajmalicine and
catharanthine are growth-associated secondary metabolites. Since serpentine, which is
non-growth-associated metabolite, is formed from ajmalicine there was also no
improvement in its production.
110
Table-21 Effect of Ethephon and Ancymidol on Alkaloid production in Hairy Root Cultures of Vinca rosea
Substance Concentration Alkaloid Contenta (mg/1) Ajmalicine Serpentine Catharanthine
Ethephon
10 µM
1.35±0.09
1.45±0.07
1.25±0.08
50 µM 1.59±0.05*
1.57±0.08
1.37+0.06
Ancymidol 10 µM 1.22±0.09
1.41±0.06
1.23±0.11
50 µM 1.14±0.11
1.33±0.07
1.19±0.07
Control Water 1.35±0.07 1.42±0.06 1.28±0.08
Control Ethanol 1.37±0.08 1.49±0.09 1.37±0.05
a n=3; values are mean ± standard deviation; *significantly different from
control (p<0.05).
111
Ethephon (10) Ethephon (50) Ancymidol (10) Ancymidol (50) Control (Water) Control (ethanol)0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0Ajmalicine
Serpentine
Catharanthine*
Concentration (micromoles)
Alk
alo
id c
on
ten
t (m
g/l)
Fig. 27 Effect of Ethephon and Ancymidol on Alkaloid Production in Vinca roseahairy root cultures. Data with * are significantly different from control (p<0.05)
112
4.4. EFFECT OF PRECURSORS AND ORGANIC COMPOUNDS ON ALKALOID PRODUCTION IN VINCA ROSEA HAIRY ROOT CULTURES
INTRODUCTION
Two convergent metabolic pathways supply the indole and iridoid precursors
for the biosynthesis of terpenoid indole alkaloids in Vinca rosea . The optimization of
culture conditions such as medium composition, including adding biosynthetic
precursor (s) to the medium, may enhance secondary metabolite production where the
productivity is limited by the lack of that particular precursor.291 Literature reveals
several reports about precursor feeding experiments in cell and tissue cultures of
Vinca rosea. Contradictory effects on TIA accumulation have been obtained for the
addition of tryptophan and tryptamine.96,298,131 It is generally accepted that most of
the Vinca rosea cell and tissue cultures have a limitation in the supply of terpenoid
precursors, which can be overcome by the addition of either loganin or
secologanin.130,131,299-,301
Tissue differentiation plays an important role in the production and
accumulation of secondary metabolites. Transformed root culture exhibit a higher
level of differentiation than cell suspension cultures and accumulate,in general, high
levels of metabolites. The indole and the terpenoid pathways were both found to be
not limiting during late exponential growth phase of hairy root cultures of Vinca
rosea.277 But the terpenoid pathway was found to be deficient during stationary phase
as the addition of geraniol, 10-hydroxygeraniol and loganin significantly enhanced the
accumulation of tabersonine.
Precursor feeding studies in transgenic cell lines of Vinca rosea have
provided interesting insights into the biosynthesis of alkaloids. In cell line over
expressing the STR gene, combined feeding of loganin and tryptamine enhanced the
flux in the late growth phase.301 The optimal time for supplying the precursors was
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found to be at inoculation of the cells into the production medium and that multiple
feedings in such transgenic cell cultures could increase alkaloid levels
significantly.302, 303
On the basis of the knowledge of regulation of biosynthetic pathways, several
organic compounds have been added in order to improve the availability of precursors
for the production of alkaloids in cell cultures of Vinca rosea. Bioregulators like
Phenobarbital, trans-cinnamic acid, succinic acid and malic acid have been found to
enhance the flux into the alkaloid pathway, thereby improving the yields.190,304,246,247
Hence, it was felt interesting to study the effect of feeding loganin, alone or in
combination with tryptamine at different growth phases as well as the frequency of
feeding precursors on alkaloid production in hairy root cultures of Vinca rosea. Also,
the addition of various organic compounds was undertaken for testing the feasibility
of improving alkaloid yields.
4.5. MATERIALS AND METHODS
Stock Solutions
Loganin was kindly provided by Prof. Dr C.S.V.Rama Chandra Rao, Head, Dept. of
Biotechnology, MIC College of Technology, Kanchikacherla. Tryptamine
hydrochloride was purchased from Himedia Laboratories Pvt. Ltd., Mumbai.
Phenobarbital was obtained from Anglo-French Drug Co. (Eastern) Ltd., Bangalore.
Succinic and trans-cinnamic acids were purchased from commercial suppliers.
Solutions of loganin (1mg/ml), tryptamine hydrochloride (2mg/ml) and succinic acid
(60mg/ml) were prepared in double distilled water. Solutions of trans-cinnamic acid
(75mg/ml) and phenobarbital (2 mg/ml) were prepared in 50% ethanol. The solutions
were filter sterilized before addition to the cultures.
114
Culture Conditions
Hairy roots were cultured in 50ml conical flasks containing 15 ml of ¼ MS
supplemented with sucrose (30 g/l) for precursor feeding experiments. An inoculums
size of about 30 mg fresh weight of 8-day-old hairy roots was used to initiate the
cultures. For studies using the organic compounds, cultures were initiated in 100 ml
conical flasks for which an inoculums size of approximately 50 mg fresh weight was
transferred to 25 ml of media. The cultures were incubated at 100 rpm at 25ºC in the
dark.
Addition of Organic Compounds and Precursors
Required volumes of stock solutions of loganin and tryptamine hydrochloride
were added once or multiple times on day 13, 17 and 21, according to the design of
the experiment to get a final concentration of 52µM each while running suitable
controls.
The organic compounds were added to 21-day-old cultures. Phenobarbital was
tested at 50 and 250µM while trans-cinnamic acid, at 100 and 1000 µM
concentrations. Succinic acid was added at 5 and 10Mm concentrations while running
suitable controls.
Extraction and Analysis of alkaloids
Hairy roots were harvested at predetermined time intervals, rinsed once with
double distilled water and fresh weights were recorded. The roots were extracted and
analyzed for alkaloids as described in Chapter 2.
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Statistical Analysis
Analysis of variance (ANOVA) and Tukey’s test were carried out using Graph
Pad Prism5 (Graph Pad Software Inc., USA) for determining the significance of
treatment effects. A p value of <0.05 was considered significant.
4.6. RESULTS AND DISCUSSION
Effect of Feeding Biosynthetic Precursors
The effects of feeding loganin alone or in combination with tryptamine on
alkaloid accumulation in hairy root cultures of Vinca rosea was studied at different
growth phases. In this experiment, cultures were fed on day 13, 17 and 21 and
harvested 4 days after treatment. No significant effect on the growth of hairy roots
was observed with the addition of precursors when compared to control cultures.
Also, there was no effect on the release of alkaloids from hairy roots into the media.
The results of feeding precursors at different growth phases on alkaloid accumulation
are depicted in Table-22.
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Table-22 Effect of Feeding Precursors at Different Growth Phases
onalkaloid Production in Vinca rosea Hairy Root Cultures
Precursor Day of feeding
Alkaloid contenta (mg/I) Ajmalicine Serpentine Catharanthine
Loganin
13 0.82±0.06
0.51±0.05
0.63±0.06
17 1.47±0.1 1.08±0.1
1.03+0.1
21 3.27±0.1* 2.51±0.12* 1.25±0.11
Loganin + Tryptamine 13
0.78±0.07
0.48±0.05
0.59±0.06
17 1.21±0.07
1.14±0.06
0.98±0.06
21 1.49±0.08
3.27±0.13*
1.39±0.09
Control (water)
13 0.67±0.06
0.42±0.04
0.55±0.04
17 1.36±0.1 1.02±0.09 0.98±0.08
21 1.45±0.09 1.39±0.1 1.3±0.08
n=3; values are mean ± standard deviation; *significantly
different from control (p<0.05)
117
Feeding of loganin alone or in combination with tryptamine did not improve
significantly the alkaloid levels during early and late exponential growth phase
(Fig.28 and 29). However, the feeding of precursors at early stationary phase resulted
in significant improvements in the accumulation of ajmalicine and serpentine
(Fig. 30). With loganin feeding, ajmalicine production (3.27 mg/1) was improved 2.3
times and there was 1.8 timesincrease in the yield of serpentine (2.51 mg/1) when
compared to control cultures (1.42 and 1.39 mg/, respectively). Ajmalicine
accumulation was not affected with the combined feeding of loganin and tryptamine
but there was 2.4 times improvement in serpentine production (3.27mg/1) over the
control. Catharanthine levels were unaffected by either of these treatments.
The results indicate a limitation in the supply of terpenoid precursors during
stationary phase in the accumulation of ajmalicine and serpentine, the levels of which
can be improved by feeding loganin alone or in combination with tryptamine. Morgan
and Shanks (2000)277 also found a limitation in the supply of terpenoid precursors in
Vinca rosea hairy root cultures during the stationary phase. Moreover, highest levels
of alkaloids were obtained in transgenic cell lines after the combined addition of
loganin and tryptamine.302,303
118
Loganin Loganin + Tryptamine Control0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0 Ajmalicine
Serpentine
Catharanthine
Precursor
Alk
alo
id c
on
ten
t (m
g/l)
Fig. 28 Effect of feeding precursors on Alkaloid Production in Vinca rosea hairy root cultures At early exponential phase. Data with * are significantly different from control (p<0.05)
119
Loganin Loganin + Tryptamine Control0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6Ajmalicine
Serpentine
Catharanthine
Precursor
Alk
alo
id c
on
ten
t (m
g/l)
Fig. 29 Effect of feeding precursors on Alkaloid Production in Vinca rosea hairy root cultures at late exponential phase. Data with * are significantly different from control (p<0.05)
120
Loganin Loganin + Tryptamine Control0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
AjmalicineSerpentineCatharanthine
*
*
*
Precursor
Alk
alo
id c
on
ten
t (m
g/l)
Fig. 30 Effect of feeding precursors on Alkaloid Production in Vinca rosea hairy root cultures at early stationary phase. Data with * are significantly different from control (p<0.05)
121
In order to see the effect of multiple feeding of precursors on alkaloid
accumulation, cultures were fed once (day 13), twice (day 13and 17) and multiple
times (day 13, 17 and 21) and harvested on day 25. The results of these treatments are
shown in table-22. Alkaloid accumulation was not affected with single and double
feeding of loganin (Fig. 31). Similarly single and multiple feeding of loganin and
tryptamine combination did not increase the alkaloid levels (Fig.32). But significant
improvements in alkaloid levels were obtained with multiple feeding of loganin (Fig.
31). There were approximately 2 times improvements for the production of ajmalicine
(2.77 mg/1) and serpentine (3.04 mg/1) over the control cultures (1.42 and 1.42
mg/1). There was a significant increase in the content of catharanthine. Catharanthine
level (2.53 mg/1) was improved by 1.9 times over the control (1.31 mg/1). This is
quite different from that observed with single feeding of loganin at early stationary
phase in the previous experiment. Catharanthine accumulation may be inhibited by
catabolism to other products302,303 and multiple feedings might have resulted in the
maintenance of its levels.
Effect of Organic Compounds on Indole Alkaloid Production
Phenobarbital, trans-cinnamic acid and succinic acid were added to hairy root
cultures of Vinca rosea at early stationary phase to see their effect on alkaloid
accumulation. The culture were exposed to these organic compounds for 4 days and
the results are depicted in table-24. There was no significant effect on the release of
alkaloids by the addition of organic compounds.
122
Table-23 Effect of the Frequency of Feeding Precursors on Alkaloid
production in Hairy Root Cultures of Vinca rosea
Precursor Frequency
Of Feeding
Alkaloid contenta (mg/I)
Ajmalicine Serpentine Catharanthine
Loganin
Single 1.3±0.11 1.33±0.09
1.16±0.08
Double
1.37±0.09 1.44±0.09
1.2+0.06
Multiple
2.77±0.12* 3.04±0.13* 2.53±0.13*
Loganin + Tryptamine
Single 1.26±0.06
1.43±0.12
1.23+0.06
Double 1.25±0.09
1.37±0.07
1.42+0.12
Multiple 1.36±0.08 1.41±0.12 1.36±0.07
Control (water)
- 1.42±0.1
1.42±0.06
1.31+0.11
a n=3; values are mean ± standard deviation; *significantly different from control (p<0.05).
123
Single Double Multiple Control0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5 Ajmalicine
Serpentine
Catharanthine*
Frequency of Feeding
Alk
alo
id c
on
ten
t (m
g/l)
Fig. 31 Effect of the frequency of feeding loganin on Alkaloid Production in Vinca rosea hairy root cultures. Data with * are significantly different from control (p<0.05)
124
Single Double Multiple Control0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
Ajmalicine
Serpentine
Catharanthine
Frequency of Feeding
Alk
alo
id c
on
ten
t (m
g/l)
Fig. 32 Effect of the frequency of combined feeding of loganin and tryptamine on Alkaloid Production in Vinca rosea hairy root cultures. Data with * are significantly different from control (p<0.05)
125
Treatment with Phenobarbital increased the accumulation of ajmalicine and
serpentine but catharanthine levels were not affected much when compared to control
cultures (Fig. 33). Significant effects were seen with the lowest concentration (50
µm). Ajmalicine production (1.98mg/1) was improved by 1.4 times over the control
cultures (1.44 mg/1). There was 1.5 times increase in the accumulation of serpentine
(2.21 mg/1) when compared to control cultures (1.47 mg/1).
Cytochrome P450 enzyme is widely involved in the biosynthesis of indole
alkaloids in Vinca rosea.152,276 The cytochrome p450 monooxygenase, Geraniol 10-
hydroxylase (G10H), involved in an early step of the biosynthesis of secologanin, has
been suggested as a potential site for regulation. Simpson and Kelly (1989)304
employed cytochrome p450 inducers and inhibitors to study alkaloid production in
cell culture of Vinca rosea. Phenobarbital was shown to induce the activity of G10H,
thereby increasing the levels of ajmalicine.275 Similarly, Zhao et al., (2001 a)247 found
an improvement not only with the production of ajmalicine and serpentine in compact
callus clusters cultures but also with catharanthine. The failure of Phenobarbital
treatment to improve catharanthine accumulation in the present study could be due to
differences in the culture systems utilized, medium composition or growth conditions.
126
Table -24 Effect of Organic Compounds on Alkaloid Production in
Vinca rosea Hairy Root Cultures
Substance Alkaloid contenta (mg/I)
Ajmalicine Serpentine Catharanthine
Phenobarbital(50 µM)
1.98±0.16* 2.21±0.17* 1.36±0.08
(250 µM)
1.39±0.05 1.53±0.06 1.28+0.09
Trans-cinnamic acid (100 µM)
1.34±0.07 1.38±0.08 1.26±012
(1000 µM) 1.53±0.05 1.59±0.09
1.44±0.06
Succinic acid (5 mM) 1.43±0.09 1.53±0.12 1.44±0.1
(10 mM) 1.86±0.1
1.97±0.13
1.63±0.12
Control (Water) 1.31±0.11 1.45±0.1 1.36±0.09
Control (Ethanol) 1.44±0.12 1.47±0.09 1.25±0.07
a n=3; values are mean ± standard deviation; *significantly different from control (p<0.05).
127
50 250 control0.0
0.5
1.0
1.5
2.0
2.5Ajmalicine
Serpentine
Catharanthine
*
Concentration (micromoles)
Alk
aloi
d co
nten
t (m
g/l)
Fig. 33 Effect of Phenobarbital on Alkaloid Production in Vinca rosea hairy root cultures. Data with * are significantly different from control (p<0.05)
128
The addition of trans-cinnamic acid did not produce significant improvements
on alkaloid accumulation in hairy roots culture of Vinca rosea. Marginal increase in
alkaloid levels were observed with the highest concentration (Fig. 34). Trans-
cinnamic acid inhibits phenylalanine ammonia lyase and thus blocks the carbon flow
from shikimate in to phenyl propanoid pathway. Significant improvements in indole
alkaloid production were obtained in compact callus clusters after treatment with
trans-cinnamic acid, suggesting the considerable contribution of the indole branch in
alkaloid biosynthesis in that culture system.247 Godoy-Hernandez et al., (2000)245
found a significant improvement in alkaloid production in osmotically stressed cell
cultures of Vinca rosea. Accumulation and excretion of phenolic compounds from
plant cell cultures is generally seen when they are exposed to stress inducing
conditions like fungal homogenates and osmotic shock.53 In the present study, the
hairy root cultures were not subjected to any stress and did not show any browning.
Hence, trans-cinnamic acid could be ineffective in improving the alkaloid production.
129
100 1000 control0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0Ajmalicine
Serpentine
Catharanthine
Concentration (micromoles)
Alk
alo
id c
on
ten
t (m
g/l)
Fig. 34 Effect of trans-Cinnamic acid on Alkaloid Production in Vinca rosea hairy root cultures. Data with * are significantly different from control (p<0.05)
130
Significant effects of succinic acid treatment were seen on the accumulation of
ajmalicine (1.86 mg/1) and serpentine with the highest concentration (10mM).
Production of ajmalicine (1.86 mg/1) and serpentine (1.97 mg/1) was improved by 1.4
times each when compared to control cultures (1.31 and 1.45 mg/1, respectively) (Fig.
35). Catharanthine contents (1.63mg/1) were improved by 1.2 times over the control
culture (1.36mg/1). At the lowest concentration of succinic acid, however, alkaloid
production was not affected.
Alkaloid accumulation in cell cultures of Vinca rosea has significantly
improved by the addition of malic, succinic and citric acids.246,247All these acids
participate in the tricarboxylic acid cycle. Biosynthetic studies including ours
(Chapter 3) indicate that both acetate/mevalonate and triose phosphate/pyruvate
pathways (non-mevalonate) are important for alkaloid production in Vinca rosea.
Zhao et al., (2000a; 2001a)246,247 suggested that exogenous malate and succinate may
inhibit tricarboxylic acid cycle by a negative feedback control or they can be
transformed to pyruvate. Therefore, addition of malate or succinate may stimulate
indole alkaloid accumulation by increasing pyruvate pool or directing acetyl CoA and
pyruvate flux to alkaloid path way. A similar effect might have resulted in the
increased accumulation of alkaloids in the present study.
131
5 10 control0.0
0.5
1.0
1.5
2.0
2.5
Ajmalicine
Serpentine
Catharanthine*
*
Concentration (millimoles)
Alk
alo
id c
on
ten
t (m
g/l)
Fig. 35 Effect of Succinic acid on Alkaloid Production in Vinca roseahairy root cultures. Data with * are significantly different from control (p<0.05)
132
Alkaloid production in hairy root cultures is thus limited by the supply of
terpenoid precursors during stationary growth phase. Moreover, multiple feeding of
loganin increased the accumulation of alkaloids, giving further support to the
importance of the terpenoid pathway in alkaloid biosynthesis. Improvements in
productivity seem with Phenobarbital provide further evidences to the regulatory role
of G10H in alkaloid biosynthesis.