106

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

107

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.

108

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

109

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

113

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.

115

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.

116

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.