35

CHAPTER 2

DEVELOPMENT AND ELICITATION OF VINCA ROSEA

HAIRY ROOT CULTURES

2.1 INTRODUCTION

Two anti cancer compounds, bisindole alkaloids, vinblastine and vincristine,

accumulated in low amounts by Vinca rosea plants, are of great interest to the

pharmaceutical industry. Vinblastine and vincristine at present, are produced by

extraction of the plant material or by semi-synthesis using the monomeric precursors,

Vindoline and catharanthine.208 Vindoline is accumulated at a relatively higher level

in the aerial parts, whereas catharanthine is present at a much lower level.209, 210Cell

and tissue cultures of Vinca rosea have been extensively studied as alternative sources

of anticancer and other alkaloids. Vinblastine and vincristine are not produced in

undifferentiated cell suspension cultures, the main reason being the absence of

vindoline production. Moreover, cell suspension cultures produce low levels of

alkaloids and are unstable.

Root cultures inculcated by Agrobacterium rhizogenes mediated

transformation have become of much interest in recent times as sources of useful

compounds due to their rapid growth and high productivity.211,212 Hairy root cultures

also possess a higher level of differentiation and an inherent genetic stability,

resulting in a stable production of secondary metabolites when compared to cell

suspension cultures. Potential of the hairy root cultures of Vinca rosea has been

investigated by some research groups with respect to the production of indole

alkaloids.210, 213-216 Transformed root cultures have been found to be productive by at

least one order of magnitude more than the cell suspension cultures.217 Parr et al., 218

reported an interesting result, the detection by immunoassay of a small level of

vinblastine in Vinca rosea hairy root cultures.The presence of bisindole alkaloids has

36

not been confirmed in any other hairy root lines produced since parr et al., (1988)218.

Another important result with transformed root cultures of Vinca rosea was that of

palazon et al., (1998)219 which demonstrated the ability of root lines to synthesize

vindoline. These findings in the literature promoted us to undertake studies on hairy

root cultures of Vinca rosea for exploring their potential as sources of therapeutically

interesting indole alkaloids.

2.2. MATERIALS AND METHODS

Table 5. Culture vessels, equipment and glassware

Round bottom flask (100 and 250ml)

Beakers (50,100,250,500 and 1000ml)

Pipettes (1,2,5 and 10ml)

Volumetric flasks (10,50,100 and 250ml)

Culture conical flasks (50 and 100 ml)

Screw cap tubes (5 and 10ml)

Conical flasks (250,500 and 1000ml)

Measuring cylinders (10,25,50,100,250,500 and 1000ml)

Separating funnels (125 and 250ml)

Culture tubes (50ml)

Petri dishes (90mm diameter)

Culture vessels and glassware were purchased from Borosil glass works limited,

Mumbai (Borosil) and Schott Duran.

37

The following are some of the major equipment used in thisstudy. ( Table 6)

Table 6. Major equipment used in the study

Bath sonicator (Loba 3.5 L100, Loba Chemie, Hyderabad)

Laminar flow clean air work station (Steri Vertical YSI 189, Yorko Sales Pvt. Ltd., New Delhi ) Vertical autoclave (Life Steriware, Yorko Sales Pvt. Ltd., New Delhi)

Electronic balance (Shimadzu ELB-300 And EIB 200G ,Shimadzu Philippines)

Ph meter (Elico LI 127, Elico Ltd .,Hyderabad)

Water purification system (Millipore Elix 3,Millipore India Ltd., Bangalore)

Refrigerated shaker incubator (Remi CIS 24BL / R15-24BL, Remi Electro Technik ltd, Vasai, India) Paper electrophoresis apparatus (Systronics Ltd.,Hyderabad)

Micro refrigerated centrifuge (MICRO 17TR, Hanil science Industrial Co., Korea)

Rotary evaporator (Super fit Instruments, Mumbai)

HPLC system (Shimadzu LC - 20AT Prominence chromatograph with binary pump/ detector PD M 20 A)

Plant Material

Seeds of Vinca rosea were kindly provided by Prof M.A Iyengar, Manipal

College of Pharmaceutical Sciences. The seeds were sown in the campus of

Siddhartha College of Pharmaceutical sciences, Vijayawada and grown to maturity.

Standards

The authentic samples of ajmalicine, serpentine and vinblastine were

purchased from Sigma chemical co. Catharanthine and Vindoline were kindly

provided by Prof Dr C.S.V.Rama Chandra Rao, Head, Dept.of Biotechnology, MIC

College of Technology, Kanchikacherla. Mannopine was gift sample from

38

Prof. Dr S Krishnan, Professor and Head, Dept. of Pharmaceutical Biotechnology,

College of Pharmacy, Sri Ramakrishna Institute of Paramedical Sciences,

Coimbatore. Rifampicin was kindly gifted by Prof.K.V.S.R.G.Prasad, Sri Padmavathi

Mahila Viswa Vidyalayam, Tirupati while Cefotaxime (Taxim) was purchased from

Alkem laboratories Ltd.

Bacterial Strains

A.rhizogenes strains A4, K599, 15834 and 9365, A.rhizogenes strain LBA

9402 was obtained from Himedia labs. A4 was grown on yeast mannitol broth (YMB)

agar medium while LBA 9402 was maintained on YMB+rifampicin (YMB+rif)

growth medium. K599, 15834 and 9365 were grown on luria beratani (LB) media.

Culture Media

Murashige and Skoog (MS) medium and indole 3-butyric acid were purchased

from HiMedia Laboratories Pvt.Ltd., Mumbai. Ingredients of bacterial media, other

chemicals and solvents were of either laboratory or analytical reagent grade and were

procured from HiMedia laboratories Pvt.Ltd. Mumbai; Loba chemie Pvt.Ltd.

Mumbai; Qualigens Fine chemicals, Mumbai; Merck Ltd., Mumbai; Ranbaxy Fine

chemicals Ltd., New Delhi; S.D.Fine-chem Ltd., Mumbai. Methanol and acetonitrile

of HPLC grade were obtained from Ranbaxy Fine chemicals Ltd., New Delhi.

STOCK SOLUTIONS OF MEDIA INGREDIENTS

All the stock solutions were stored in the refrigerator until used and fresh

solutions were prepared every two months.

Indole 3-Butyric acid (IBA) solution

50mg of IBA was accurately weighed, dissolved in 1ml of 0.1M sodium

hydroxide and the volume was made to 50ml with double distilled water.

39

Calcium chloride solution

15g of calcium chloride, dihydrate was accurately weighed, dissolved in

double distilled water in a volumetric flask and the volume was made to 100 ml.

Cleaning of glassware

All glassware contaminated or coming into contact with micro organisms was

decontaminated before washing .The surface debris, if any,was removed by brushing

the glassware in running tap water, soaked in detergent solution and washed with tap

water. Chromic acid mixture was then used to wash all the glass ware, followed by

second washing with detergent solution and running tap water. The glassware was

finally rinsed with distilled water and dried in hot air oven.

Preparation of Media

The ingredients of bacterial media (Table-7) or MS (Table-8) were dissolved

in double distilled water. For making of MS medium, sucrose (30g/l) and calcium

chloride stock solution (2.94ml) were added. Required volume of IBA stock solution

was added in the preparation of modified MS media for the initiation and maintenance

of non-transformed root cultures. The volume of different media was made

approximately 95% of the final volume and the Ph was adjusted to the required value

by using 0.5M sodium hydroxide or 0.2 M hydrochloric acid. Once the medium was

made to final volume (required amount of agar was added for making solid media,

which was subsequently dissolved by heating), it was distributed in culture vessels

before autoclaving or the media and vessels were autoclaved separately and then

distributed aseptically later. The culture vessels were plugged with non-absorbent

cotton wool; the tops were covered with aluminium foil and autoclaved at 121oC (15

pounds/square inch) for 20 minutes. The thermo labile ingredients like Rifampicin

40

and Cefotaxime were added, filter sterilized (Milex 0.22µ PVDF 33mm, Millipore

corporation,MA,USA) after the autoclaved medium had cooled to at least 500C.

2.3 INDUCTION OF HAIRY ROOTS

Sterilization of plant material

Young leaf and stem pieces of Vinca rosea were collected and washed

thoroughly in running water. They were treated with Tween 20 (10 % v/v) for 2-3

minutes. The residuals of the surfactant solution were removed by thorough washing

in running water and finally with distilled water. Leaves were surface sterilized by

immersion in mercuric chloride (0.1% w/v) with occasional shaking for 2-3 minutes

while stem pieces were treated for about 10 minutes. Leaves and stem pieces were

then thoroughly rinsed with sterile water for 3 to 4 minutes .

Preparation of bacterial inoculum

1 to 2 loopfuls of bacterial colonies were aseptically removed from an

overnight culture of A.rhizogenes and transferred into 25 ml of YMB/YMB+RIF/LB

liquid medium in a 100 ml conical flask. The inoculated media were incubated on a

gyratory shaker at 100 rpm at 26°c in the dark for 48 hours to get a thick suspension

of bacteria.

41

Table -7 Composition of Bacteria Mediaa

aFor yeast mannitol broth+rifampicin(YMB+rif) growth medium,

Rifampicin was added at 100mg/l.Rifampicin was dissolved in small

amount of 0.1M hydrochloric acid and added after the autoclaved media

had cooled to about 500c. For the preparation of solid media ,agar was

added at 15g/l.

Ingredient

Quantity(g/l)

Yeast Mannitol Broth (YMB)

Luria-bertani(LB)

Di-potassium hydrogen Phosphate,trihydrate

0.655 -

Magnesium sulphate, heptahydrate

0.2

-

Sodium chloride

0.1

10

D-Mannitol

10

-

Yeast extract

0.4

5

Tryptone

-

10

Water

Up to 11

Up to 11

PH

7

7

42

Table-8 Composition of Murashige and Skoog (MS) Medium

Ingredient Quantity ( mg/l) Macronutrients

Potassium nitrate 1900

Ammonium nitrate

1650

Magnesium sulphate,heptahydrate 370

Dihydrogen potassium phosphate 170

Calcium chloride, dihydrate 440

Micronutrients

Boric acid

6.2

Magnesium sulphate,tetra hydrate 15.6

Zinc sulphate, heptahydrate 8.6

Sodium molybdate,dihydrate 0.25

Cupric sulphate, pentahydrate 0.025

Cobalt chloride,hexahydrate 0.025

Potassium iodide 0.83

Ferrous sulphate, heptahydrate DisodiumEDTA

27.8 37.3

Vitamins

Thiamine hydrochloride 0.5

Pyridoxine hydrochloride 0.5

Nicotinic acid 0.5

Myo-inositol 100

Sucrose 30000

PH 5.8 a Agar was added at 10g/l in the preparation of MS solid medium

43

Infection of explants with bacterial suspension

The leaf and stem pieces of Vinca rosea were trimmed to remove damaged

tissues and a number of small cuts and incisions were made on the petiole and major

veins of leaf explants and on stem pieces. The explants were infected by incubating

with bacterial suspension as described by Giri et al., (2001)220 for different periods of

time. The explants were then transferred to MS basal media containing sucrose (30g/l)

and agar (10g/l) and incubated at 26°C in the dark. After 48 hours of co-cultivation

the explants were washed with sterile doubled distilled water to remove any excessive

bacterial growth and incubated on MS basal media containing Cefotaxime (300mg/l).

Elimination of Bacteria and Subculture of Hairy Roots

When the emergent hairy roots were 20-30 mm long, leaf segments bearing

the roots were excised and transferred to 25ml of ¼ MS basal media containing

sucrose (30g/l) and Cefotaxime (300 mg/l) in a 100 ml conical flask . The hairy roots

were incubated on a gyratory shaker at 26°c at 100rpm in the dark. The hairy roots

were maintained by sub culturing in ¼ MS basal media containing sucrose (30g/l) at

the interval of 3 weeks and on ¼ MS basal agar media supplemented with sucrose

(30 g/l) at the interval of 4-5 weeks .

Non-transformed roots

Non-transformed roots were established from leaf explants on MS medium

containing sucrose (30g/l), IBA (0.5mg/l) and agar (10g/l). The explants were

incubated at 26°C in the dark.The roots were sub cultured on MS media of same

composition at the interval of 4 to 5 weeks.

44

Opine assay

100 mg fresh weights of transformed and non-transformed roots were ground

in an eppendroff tube with 100µl of 1% hydrochloric acid using a glass rod. The

extracts were centrifuged at 10,000 rpm for 5 minutes. The supernatants were streaked

on what man 3MM paper and then the paper was electrophoresed at a constant voltage

of 100 volts/cm for 15 minutes. The buffer used was formic acid: acetic acid: water

(30:60:910, v/v/v).314,315 after drying the paper, the opines were visualized by spraying

with alkaline silver nitrate reagent.315

Measurement of growth and frequency of hairy root induction

The frequency of hair root induction was calculated on the basis of percentage

of explants capable of being transformed. Inocula of 50-60 mg fresh weight,

consisting of five root tips of 40-50 mm long from 8-day-old culture, were transferred

to 100ml conical flasks containing 25 ml of ¼ MS basal medium supplemented with

sucrose (30g/l). The flasks were incubated on a gyratory shaker at 100 rpm in the dark

at 26°C. The growth index is defined as the harvest fresh weight at 28 days of culture

divided by the inoculum fresh weight.

2.4 ALKALOID PRODUCTION AND GROWTH RATE

Growth and production kinetics

Inocula of 50-60 mg fresh weight, consisting of five root tips of 40-50 mm

long from 8-day-old culture, were transferred to 100 ml conical flasks containing

25ml of ¼ MS basal medium supplemented with sucrose (30g/l). The flasks were

incubated on a gyrator shaker at 100 rpm at 26°c in the dark. The growth kinetics of

transformed root cultures was determined by checking the fresh weight (triplicate)

every fourth day up to 28 days. The contents of the flasks were harvested, blotted dry

45

and weighed. The roots were dried and extracted for alkaloids, which were

subsequently analyzed to determine the kinetics of production.

Alkaloids were extracted from hairy roots and media according to Jung et al.,

(1992)210 with modification. The roots were powdered and extracted three times with

10 ml methanol for 30 minutes in a bath sonicator at room temperature. After

evaporation of the methanol solution to dryness under vacuum, the residue was

dissolved in 20 ml of 1M hydrochloric acid and extracted with 20ml of ethyl acetate

at a low PH. The acid solution was adjusted to PH 10 with 10M sodium hydroxide and

extracted three times with an equal volume of ethyl acetate. The ethyl acetate solution

was evaporated to dryness under vacuum and the residue was dissolved in 1ml of

methanol (HPLC grade) for further analysis.

For the extraction of alkaloids from the media 20ml was acidified to PH 1.5 with

2M hydrochloric acid and extracted with ethyl acetate at low and high PH as in the

case of alkaloid extraction from hairy roots.

The hairy root extracts were subjected to TLC study. The solvent system

described by Asada and Shuler (1989)98 was used to separate the alkaloids which

consisted of chloroform: acetone: diethylamine (20:4:1, v/v/v).

Dragendorff reagent was used as spray reagent for visualization of the

separated compounds. The stock solution was prepared by mixing equal volumes of

solutions A and B (Wagner and Bladt, 1996).221 Solution A was prepared by

dissolving 0.85g basic bismuth nitrate in 10 ml glacial acetic acid (GAA) and 40 ml

water under heating. Solution B was prepared by dissolving 8 g of potassium iodide in

30 ml of water. The spray reagent was made by mixing 1 ml stock solution with 2 ml

GAA and 10 ml water.

46

Strips of filter paper were placed on the three walls of the TLC chamber and

allowed to soak in the solvent system. This was done 30 minutes prior to running the

chromatogram to ensure that the chamber is fully saturated with vapours of the

solvent system. Standard solutions of 100 µg/ml of ajmalicine, catharanthine and

serpentine were prepared in methanol. The standard solutions and alkaloid extracts

were applied 1.5 cm away from the bottom edge of the 5×10 cm TLC plates (Silica

gel 60 F254, 0.2mm, Merck). The solvent was allowed to evaporate after each

application to prevent spreading of the spots.

The TLC plates were placed into the chamber and the development was

continued till the solvent front had reached a distance of 9 cm. The plates were taken

out, solvent front marked and air-dried for visualization with Dragendorff reagent.

The alkaloid extracts were also analyzed by the HPLC.222 The HPLC system

consisted of a solvent delivery module LC-20AT (Shimadzu Corporation), a rheodyne

syringe loading sample injector with a 20 ml injection loop (Rheodyne, L.P.,), a

Shimadzu model SPD-20A detector with wavelength set at 254 nm and the spectra

recording from 200 to 360 nm, coupled to shimadzu LC20 computing integrator.

The analytical column used was Luna 18(2), 250 x 4.6 mm I.D., 5 µ particle

size (Phenomenax). The mobile phase consisted of a mixture of methanol:

acetonitrile:5mM diammonium hydrogen phosphate (Ph 7.3) (32:32:36, v/v/v)

degassed using bath sonicator. The chromatography was performed isocratically at a

flow rate of 1 ml/minute at room temperature.

Standard Graph of Alkaloids

Stock solutions of 100 µg/ml of ajmalicine, catharanthine and serpentine were

prepared in methanol (HPLC grade). Required volumes of the stock solution was

diluted with methanol to get working standard solutions of concentration of 1, 2, 3, 4,

47

5, 6 and 10 mg/ml for each alkaloid. The peak areas of each concentration were

recorded and the standard graph was constructed by plotting the concentration on X-

axis and the corresponding peak area on Y-axis.

2.5. RESULTS AND DISCUSSION

The leaf and the stem pieces of Vinca rosea, were utilized for establishing

hairy root cultures. The selection of highly productive cell lines has for long been a

well-known strategy for the production of secondary metabolites. Statistically, high

producing plants give rise to high producing cell lines.138 The total alkaloid yield was

from 0.85 to 1.07% w/w and contains high amounts of vincristine (0.002% w/w) and

vinblastine (0.0058% w/w).223 The transformed roots were easily induced on leaf

segments following inoculation with A. rhizogenes strain A4. Transformation of stem

pieces was not obtained with any of the strains tested. Bacterial strains like LBA9402,

K599 and 15834 have been successfully used to induce hairy roots in Vinca rosea by

other research groups.86,213,210,218,215 But these strains could not transform the leaf

explants or stem pieces under the conditions employed in the present study. Different

strains of A. rhizogenes vary in their transforming ability.224, 220,225 Protocols used for

establishing the hairy root cultures vary as well as the susceptibility of the plant

species to infection by Agro bacteria.226 The age and differentiation status of the plant

tissue can also affect the chances of successful transformation.227 The agropine strain

A4 is more virulent than other strains and has been known to transform a wide range

of plant species.

The young leaves were found to be more suitable for transformation than

mature ones. This may be due to the fact that young tissues and organs contain

actively growing cells and hence, are easily susceptible for infection with the bacteria.

Numerous roots appeared at the wound sites, 2-3 weeks after infection with the

48

bacterium (fig.2). Hairy roots developed mainly from the midrib and petiole portion

of the leaves. Leaf explants were incubated for different periods of time 10, 20 and 30

minutes. Maximal response was seen with explants treated for 20 minutes and about

50% of explants produced hairy roots.

In order to eliminate the bacteria, transgenic roots were excised along with

leaf segments and placed into ¼ MS basal liquid medium containing sucrose (30 g/l)

and Cefotaxime (300 mg/l). The roots were transferred to fresh media of the same

composition for two more passages at the interval of 12-15 days, which resulted in the

complete removal of the bacteria. The roots were white to light cream coloured, thin

and straight with regular branching and thin tips. They exhibited the characteristic fast

and plagiotropic growth (Fig. 3 and 4).

Transformed nature of Vinca rosea hairy roots was confirmed by opine assay.

Non-transformed roots established on MS agar medium supplemented with sucrose

(30mg/l) and IBA (0.5 mg/l) were used as control. A4 strain is an agropine strain and

produces agropine, mannopine and other related acids. Mannopine could be detected

in transformed roots while it was absent in non-transformed roots (Fig.5).

The growth of the established hairy root cultures of Vinca rosea was checked

in different strengths of MS basal media supplemented with sucrose (30 g/l). The

fastest growth and maximum biomass were obtained in ¼ MS medium, which was

therefore selected for further studies. Root fresh weight increased from 0.06 g to 1.72

g during a 28-day period. There was an increase of approximately 29 times from the

initial fresh weight and is higher than earlier reports by Parr et al.,(1988)218 and Islas

et al., (1994).227

For determining the growth kinetics of Vinca rosea hairy root cultures, cultures

were initiated from an 8-month-old-hairy root line, using a single flask to minimize

49

variation from flask to flask. Growth was checked every four days for 28-day culture.

For each stage, roots from three flasks were harvested for determining their fresh

weight and the results are presented in Table-9. As depicted in Fig.5, the hairy roots

exhibited an initial lag phase before entering an active growth phase around day 12

and reached stationary phase 20 days after inoculation. The results are in agreement

with those of Bhadra and Shanks (1997)228, Jung et al., (1994)229 and Toivonen et al.,

(1990).215

Hairy root cultures of Vinca rosea were found to contain a large number of

alkaloids as indicated by the presence of several peaks during HPLC analysis with

UV spectra resemblance to major alkaloids. Alkaloids were not detected in the

medium, which are generally accumulated within the roots.213,218,215 Ajmalicine,

serpentine and catharanthine were identified with TLC, HPLC (Fig.6) and UV spectra

(Fig.7) by comparison with authentic standards. Neither vindoline nor vinblastine

could be detected in our hairy root cultures.

50

Table-9 Kinetics of Growth and Alkaloid production in Hairy Root Cultures of Vinca rosea

Days of Incubation

Growth Indexa Alkaloid Contenta (mg/1)

Ajmalicine Serpentine Catharanthine

0 1±0.17 0.05±0.01 NDb 0.04±0.01

4 1.33±0.17 0.08±0.01

ND

0.06+0.01

8 2.17±0.28 0.15±0.01 0.03±0.01 0.13±0.01

12 5.33±0.33 0.32±0.04

0.11±0.01

0.34±0.04

16 10.83±0.5 0.65±0.05 0.27±0.03 0.59±0.03

20 19.17±0.83 1.22±0.08

0.85±0.06

1.12±0.05

24 25.67±1.17 1.39±0.02

1.42±0.08

1.36±0.06

28

28.67±1.78 1.49±0.05

1.81±0.09

1.52±0.04

a n=3, values are mean ±, standard deviation; -bnot detected

Fig.

51

Fig.5 Growth kinetics of Hairy root Cultures of Vinca rosea

52

Fig.6HPLC Chromatogram of (A) standard alkaloid mixture (B) hairy root extract

53

Fig.7 UV absorption spectra of Serpentine ( solid line), Catharanthine ( dashed line) and ajmalicine ( dotted line)

54

The solvent system consisting of chloroform: acetone: diethyl amine (20:4:1,

v/v/v) gave a good separation of the alkaloids. The Rf values of ajmalicine, serpentine

and catharanthine were found to be 0.77, 0.22 and 0.73 respectively. Base line

separation of the alkaloids was achieved by HPLC using the mobile phase consisting

of methanol: acetonitrile: 5Mm diammonium hydrogen phosphate (pH 7.3) (32:32:36,

v/v/v). The retention time of ajmalicine, serpentine and catharanthine were found to

be 17.77, 5.47 and 16.49 minutes respectively. Standard curves of the different

alkaloids were constructed using simple linear regression. The calibration curves were

found to be linear within 1-10 mg/ml range for ajmalicine and catharanthine while it

was 0.5-10 µg/ml for serpentine (Fig.8-10).

Characteristically, growth-associated products accumulate to maximum levels

during the exponential growth phase in concert with biomass.230 Non-growth-

associated products accumulate to maximum levels in the stationary phase, despite

slower growth and without a functional dependence on the trend of biomass

accumulation. The kinetics of alkaloid production in hairy root cultures of Vinca

rosea is shown in Table-9. The accumulation of ajmalicine started to increase from

day 12 onwards and reached to a maximum yield (26.5 µg/gram fresh weight, 1.22

mg/l) 20 days after inoculation (Fig. 11). The content of ajmalicine on day 24 and 28

was found to be 22.6 and 21.7 µg/g FW (1.39 and 1.49 mg/l,), respectively. The

variation of ajmalicine yield with biomass supports growth-associated accumulation,

increasing with the maximum yield approximately coincident with the start of

stationary growth phase and eventually decreasing at higher biomass levels.

55

Fig. 8 Standard Graph of Ajmalicine

56

Fig. 9 Standard Graph of Serpentine

57

Fig. 10 Standard Graph of Catharanthine

58

Fig. 11 Production kinetics of Hairy root cultures Of Vinca rosea

59

The synthesis of serpentine was found to be non-growth-associated with

minimum was found in late exponential phase and maximum in late stationary phase

of culture (Fig. 11). The yield of serpentine increased from a low of 10.1 µg/g FW

(0.27 mg/l) on day 16 to a high of 26.3 µg/g FW (1.81 mg/l) on day 28. The content

of serpentine on day 20 and 24 was found to be 18.5 and 23.1 µg/g FW (0.85 and 1.42

mg/l), respectively.

Catharanthine accumulation resembled that of ajmalicine (Fig. 11), with the

maximum yield of 24.3 µg/g FW (1.12 mg/l) occurring on day 20. The content of

catharanthine on day 24 and 28 was found to be 22.1 µg/g FW (1.36 and 1.52 mg/l,

respectively). Thus, accumulation of alkaloids in the hairy root lines followed the

same pattern as in earlier reports (Bhadra and Shanks, 1997228; Islas et al., 1994227;

Sim et al., 1994).231

2.6 ELICITATION OF HAIRY ROOT CULTURES OF VINCA ROSEA

In vitro plant cell cultures for the production of different secondary

metabolites have met with limited success due to their low yields.232 In order to

improve the yield of such products, various techniques have been used. An

extensively used technique for enhancing secondary metabolite yield is elicitation,

which is an integral part of any large scale process for secondary metabolite

production. An elicitor can be defined as a compound that not only induces

accumulation of antimicrobial phytoalexins in plants233 but also stimulates any type of

defense response.234 In addition to decreasing the time needed to reach a high product

concentration, elicitation as a process strategy can be easily integrated with other

yield enhancement methodologies like in situ product removal.230 Elicitation is also

useful in elucidation of metabolic pathways, knowledge that in turn can contribute

60

towards production of secondary metabolites by identifying the rate limiting steps

involved in biosynthesis.

Elicitors of non-biological origin that induce phytoalexin synthesis are called

abiotic to distinguish them from elicitors of biological origin (biotic elicitors). Biotic

elicitors include enzymes (cellulose, pectinase,etc.,) that can liberate endogenous

elicitor from the plant cell walls,molecules that act as endogenous signals in the

defense mechanisms of plants (Jasmonic acid, salicylic acid, etc.,) and extracts of

diverse microorganisms.235,236,237 Examples of abiotic elicitors are ultraviolet

radiation, heavy metal salts and other chemical compounds with diverse mechanisms

of action.236 The mechanisms of actions of biotic and abiotic elicitors are considered

to be different. The modes of actions are complex and there are many hypotheses

regarding the mechanism of elicitation.236 The effect of the elicitors depends on many

factors, such as the concentration of elicitor, growth stage of the culture at the time of

elicitation, period of contact and time course of elicitation. Moreover, little is known

about the biosynthetic pathways of most secondary metabolites and hence, the effect

of an elicitor on a plant cell or tissue culture cannot easily be predicted.

Elicitation has been found to be effective in inducing the synthesis of

secondary metabolites in cell and organ cultures of different plants. For example,

taxol and other related taxanes in taxus species,238 tropane alkaloids in Solanaceous

species,239,241,242Azadirachtin in Azadirachta indica242 and Thiophenes in Tagetes

patula.232,243

A number of biotic and abiotic elicitors have been employed to stimulate

alkaloid biosynthesis in cell suspension cultures of Vinca rosea.442,453,35,70,246-249

Literature reveals a few reports on elicitation of hairy root cultures of Vinca rosea.

Fungal homogenates, hydrolytic enzymes and methyl jasmonate were employed by

61

Vazquez-Flota et al.,(1994)250 while Sim et al., (1994)231 succeeded in stimulating the

production and secretion of alkaloids by subjecting hairy roots to a synergic effect of

fungal elicitation and in situ adsorption. Jasmonic acid was found to be a unique

elicitor leading to an enhancement in flux to several branches in the alkaloid

pathway.251Inhibition of external Ca2+ flux across the plasma membrane by cadmium

chloride was found to enhance alkaloid production as well as induce their secretion

into the medium.252

Because of their stable and high productivity, hairy roots are a good culture

system for studying the effects of a wide range of elicitors. Hence, it was thought

worthwhile to study the effect of both abiotic and biotic elicitors on hairy root cultures

of Vinca rosea to explore more on their potential of as sources of alkaloids.

2.7. MATERIALS AND METHODS

Culture Conditions

Approximately an inoculum size of 50 mg from 8-day-old culture was

transferred to 25 ml of ¼ MS medium supplemented with sucrose (30g/l) contained in

100 ml conical flasks. The cultures were incubated in a gyratory shaker at 25º C at

100 rpm in the dark.

Stock Solutions

Arachidonic acid and water-soluble linoleic acid were obtained from Sigma

Chemical Co; other chemicals used in the present study were of highest purity grade

available and were purchased from commercial suppliers. Solutions of copper

sulphate (12.48 mg/ml), cadmiumacetate (13.32 mg/ml), aluminium chloride (600 mg

/ml), silvernitrate (8.5 mg/ml), sodium chloride (200 mg/l), mannitol (910 mg/ml) and

linoleic acid (350 µg/ml) were prepared by dissolving in double distilled water. Stock

62

solution of salicylic acid was prepared in double distilled water by using a few drops

of 0.1M sodium hydroxide. Arachidonic acid was dissolved in ethanol to get a stock

solution of 2.5 mg/l and the Ph of the solutions was adjusted to 5.5 wherever required

and filter sterilised.

Elicitation

Hairy root cultures were subjected to treatment with abiotic and biotic elicitors

on day 21. The abiotic elicitors were tested at three concentrations each. Copper

sulphate (100,500 and 1000µM), cadmium acetate (100,500 and 1000µM), aluminium

chloride (10, 50, and 100 mM) and silver nitrate (10,100, and 1000 µM) were tested

in one experiment. Sodium chloride (50,100, and 250Mm) were added in another

experiment. Salicylic acid (100 and 1000µM), arachidonic acid (1 and 5 mg/l) and

linoleic acid (50 and 250µM) were tested in a third separate experiment. The

incubation conditions were same as described above. The roots were harvested on day

23 in each study.

Alkaloid Analysis

After harvesting, hairy roots were washed once with double distilled water,

blotted and fresh weights recorded. Hairy roots were extracted and analyzed for

alkaloids by TLC and HPLC as described in Chapter 2.

Statistical Analysis

Analysis of variance (ANOVA) and Tukey’s test were carried out using prism

5 (GraphPad Software Inc., USA) for determining the significance of treatment

effects. A p value of <0.05 was considered significant.

63

2.8. RESULTS AND CONCLUSIONS

Hairy root cultures of Vinca rosea were treated with abiotic and biotic (signal

and signal transduction molecules) elicitors with an aim of increasing the production

of indole alkaloids. The accumulation of ajmalicine and catharanthine takes place

parallel to growth while the production of serpentine is non-growth phase were

exposed for elicitors and roots were harvested 48 hours after treatment for extraction

and analysis of alkaloids.

Elicitation with Metal Ions

Heavy metal ions especially at the highest concentration were generally found to be

detrimental to growth. Significant reduction of the root biomass occurred with copper

(340 mg, 23% of FW of control culture), cadmium (300mg, 21% of FW of control

culture) and silver (250mg, 17% of FW of control culture) treatments. This may be

due to the death of some cells because of stress condition as indicated by a change in

colour from cream to light brown to light black compared to the control roots where

the colour remained unchanged. The effects of elicitors on indole alkaloid production

are shown in table 10-12.

64

Table-10 Effect of Metal ions on Ajmalicine production in Hairy root cultures of Vinca rosea

Elicitor Concentration Ajmalicine Contenta(mg/1) CR CM CT Copper Sulphate

1000 µm 0.78±0.08 4.24±0.15 5.03±0.23*

500 µm 3.94±0.12 2.41±0.11 6.35±0.23*

100 µm 4.41±0.11 0.36±0.04 4.78±0.14*

Cadmium Acetate

1000 µm 0.65±0.04 0.45±0.05 1.09±0.09

500 µm

1.45±0.09

0.34±0.03

1.8±0.12

100 µm

3.20±0.16

ND

3.20±0.16*

Aluminium Chloride

100 mM

0.55±0.05 ND 0.55±0.05

50 mM

0.81±0.07

1.48±0.1

2.29±0.17*

10 mM 0.34±0.04 0.92±0.08 1.26±0.11

Silver nitrate

1000 µm 1.74±0.15 5.16±0.14 6.90±0.29*

100 µm

6.31±0.13

ND

6.31±0.13*

10 µm

5.98±0.17

ND

5.98±0.17*

Control

Water 1.73±0.14 ND 1.73±0.14

a n=3, values are mean ±, standard deviation; * significantly different from

control (p<0.05); AR- Ajmalicine in root ; AM- Ajmalicine in medium; AT -total

Ajmalicine; ND-not detected.

65

Table-11 Effect of Metal ions on Serpentine production in Hairy root cultures of Vinca rosea

Elicitor Concentration Serpentine Contenta(mg/1) S R SM ST Copper Sulphate

1000 µm 1.94±0.05 2.12±0.12 4.06±0.09*

500 µm 3.72±0.13 1.99±0.1 5.72±0.23*

100 µm 4.18±0.12 ND 4.18±0.12*

Cadmium Acetate

1000 µm 1.77±0.15 0.75±0.07 2.52±0.22*

500 µm 1.81±0.12 0.69±0.07 2.5±0.19*

100 µm 2.34±0.11 ND 2.34±0.11*

Aluminium Chloride

100 mM

3.02±0.12 ND 3.02±0.12*

50 mM

1.95±0.13

0.75±0.09

2.70±0.22*

10 mM 2.49±0.11 0.22±0.03 2.72±0.14*

Silver nitrate

1000 µm 1.40±0.09 ND 1.40±0.09

100 µm

3.34±0.13

2.58±0.09

5.93±0.22*

10 µm

3.53±0.13

ND

3.53±0.13*

Control

Water 1.42±0.17 ND 1.42±0.17

a n=3, values are mean ±, standard deviation; * significantly different from

control (p<0.05); SR- Serpentine in root ; SM- Serpentine in medium; ST-total

Serpentine; ND-not detected.

66

Table-12 Effect of Metal ions on Catharanthine production in Hairy root cultures of Vinca rosea

Elicitor Concentration Catharanthine Contenta(mg/1) CR CM CT Copper Sulphate

1000 µm 0.57±0.06 2.65±0.12 3.21±0.18

500 µm 1.98±0.1 1.45±0.07 3.43±0.17

100 µm 3.5±0.12 ND 3.50±0.12

Cadmium Acetate

1000 µm 0.53±0.06 ND 0.53±0.06

500 µm 1.1±0.11 ND 1.1±0.11

100 µm 2.3±0.13 ND 2.3±0.13*

Aluminium Chloride

100 mM

0.59±0.06 ND 0.59±0.06

50 mM

0.37±0.05

1.21±0.11

1.58±0.16

10 mM 0.29±0.04 2.06±0.08 2.34±0.12*

Silver nitrate

1000 µm 0.49±0.06 ND 0.49±0.06

100 µm

1.46±0.13 ND 1.46±0.13

10 µm

3.73±0.19 ND 3.73±0.19*

Control

Water 1.22±0.12 ND 1.22±0.12

a n=3, values are mean ±, standard deviation; * significantly different from

control (p<0.05); CR-Catharanthine in root ; CM-Catharanthine in medium;

CT-total Catharanthine; ND-not detected

67

Copper ions were found to stimulate alkaloid production as well as their

release into the medium (table 10-12). The highest concentration of cu²+ (1000 µM)

produced more release than the lower concentrations (500 and 100 µM). At 1000 µM

Cu, the amount of ajmalicine, serpentine and catharanthine found in the media were

4.24, 2.12 and 2.65 mg/l, respectively which were not detected in the media of control

cultures. Maximal production of alkaloid contents (root and media) was favoured with

lower concentration of the elicitor (Fig.12). Accumulation of ajmalicine (6.35 mg/l)

and serpentine (5.72mg/l) were improved with 500 µM Cu²+ by about 3.7 and 4 times,

respectively over the control cultures (1.73 and 1.42 mg/l, respectively). There was

nearly 3 times improvement in the production of catharanthine (3.5 mg/l) with the

lowest concentration of elicitor (100µM) when compared to control cultures

(1.22mg/l).

Elicitation with Cd²+ ions did not result in significant improvement in either

production or release of alkaloids, except for treatment with the lowest concentration

(100 µM). There were nearly 2 times improvements in the production of ajmalicine

(3.2mg/l), serpentine (2.52mg/l) and catharanthine (2.3mg/l) when compared to

control cultures (Fig. 13)

Treatment with aluminium chloride (100 mM) enhanced significantly the

production of serpentine (3.02 mg/l) by about 2 times while the accumulation of

ajmalicine and increases in the levels of TDC transcript and cellular tryptamine

(Zheng and Wu, 2004)249 Moreno-Valenzuela et al., (2003)252 found that cadmium

chloride which blocks ca2+ flux across the plasma membrane increased the total

alkaloid content of transformed root cultures of Vinca rosea by 25% and their release,

10 times. However, copper treatment decrease the total and individual valepotriate

contents of transformed roots of Valeriana locusta.255 Copper and cadmium were

found to affect the distribution of tropane alkaloids between hairy roots of

D.stramonium and the media, the effect being dependent on the concentration and

nature of the ion used for elicitation.255

68

1000 500 100 control0

1

2

3

4

5

6

7

AjmalicineSerpentineCatharanthine

*

*

*

**

*

**

*

Concentration (micromoles)

Alk

aloi

d co

nte

nt (

mg

/l)

Fig. 12 Effect of Copper Sulphate on Alkaloid Production in Vinca rosea hairy root cultures. Data with * are significantly different from control (p<0.05)

69

1000 500 100 control0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

AjmalicineSerpentineCatharanthine*

**

*

*

Concentration (micromoles)

Alk

alo

id C

on

ten

t (m

g/l)

Fig. 13 Effect of Cadmium Acetate on Alkaloid Production in Vinca rosea hairy root cultures. Data with * are significantly different from control (p<0.05)

70

100 50 10 control0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5Ajmalicine

Serpentine

Catharanthine

**

**

*

Concentration (milli moles)

Alk

alo

id C

on

ten

t (m

g/l)

Fig. 14 Effect of Aluminium chloride on Alkaloid Production in Vinca rosea hairy root cultures. Data with * are significantly different from control (p<0.05)

71

1000 100 10 control0

1

2

3

4

5

6

7

8Ajmalicine

Serpentine

Catharanthine*

**

*

* *

Concentration (micro moles)

Alk

aloi

d c

ont

ent

(mg

/l)

Fig. 15 Effect of Silver Nitrate on Alkaloid Production in Vinca rosea hairy root cultures. Data with * are significantly different from control (p<0.05)

72

Aluminim chloride was found to increase the accumulation of tropane

alkaloids (43-83%) in hairy root cultures of Brugmansia candiada with significant

release of scopolamine into the medium (Spollansky et al., 2000),240 Hamel et al.,

(1998)256 suggested that aluminium may act as an elicitor as most of the genes up

regulated by it shared homologies with pathogenesis-related ones. Ag+ has been found

to be an effective inducer of secondary metabolite production in several studies. It

improved theaccumulation of tropane alkaloids by 5- to 8 times in transformed root

cultures of B.candida and increased significantly the release of scopolamine.239 The

addition of Ag+ resulted in more than 2 times improvement in the yield of tanshinones

in Salvia miltiorrhiza hairy root cultures.257

From the data it is clear that there is stimulation in the accumulation of

alkaloids in hairy root cultures of Vinca rosea in the case of copper and silver

treatment. However, alkaloid production was not affected substantially by treatment

with aluminium and cadmium, which may be due to toxic effects overwhelming any

possible elicitation. The toxic effects of elicitors may be mechanisms of action. The

cellular damage caused by copper and other metal ions especially to membranes could

release endogenous elicitors that increase the production of secondary metabolites as

a stress response to the damage.258 Moreover, the positive effects of silver nitrate on

indole alkaloid production may probably be associated with the stimulation of the

activity of enzymes involved in the formation of terpenoid moiety donor,

secologanine. Ag+ was shown to stimulate the activity of 3-hydroxy-3-methylglutaryl

CoA reductase (HMGR) and 1-deoxy-D-xylulose 5-phosphate synthase(DXS) in

hairy root cultures of S. miltiorrhiza, thereby enhancing the production of diterpenoid,

tanshinone.259 Thus, elicitation of hairy rootcultures of Vinca rosea with metal ions

appears to be an effective strategy for improving the production of indole alkaloids.

73

Treatment with Sodium Chloride and Mannitol

Salt stress by sodium chloride and osmotic shock by mannitol did not affect

adversely the growth of hairy roots of Vinca rosea. Sodium chloride addition

stimulated the production and release of alkaloids (Table-13). The maximum amount

of ajmalicine, serpentine and catharanthine found in the media were 1.39, 1.43 and

0.44 mg/l, respectively. The highest ajmalicine and serpentine yield (4.21 and 4.08

mg/l, respectively) were obtained with 250 mM concentration (Fig. 16).The yields

were about 2.5 times higher than the control values (1.83 and 1.52 mg/l for ajmalicine

and serpentine, respectively).However catharanthine yield (2.36 mg/l) was improved

by 1.8 times over the control cultures (1.25 mg/l).

Treatment with mannitol improved the production of catharanthine and

serpentine with significant amounts being retained within the root tissues (Table-

14).With regard to catharanthine production (4.54 mg/l), 3.6 times improvement was

seen with 200 mM of mannitol (Fig. 17).Serpentine production (4.16 mg/l) was

increased by 2.7 times while ajmalicine contents (3 mg/l) were improved by 1.6 times

over the control.

74

Table-13 Effect of Sodium Chloride on Alkaloid production in Hairy root cultures of Vinca rosea

Elicitor Concentration Alkaloid Contenta(mg/1) Of NaCl Root Medium Total

Ajmalicine 250 mM 2.83±0.13 1.39±0.09 4.21±0.22*

100 mM 2.25±0.13 0.59±0.09 2.84±0.2*

50 mM 1.54±0.14 ND 1.54±0.14

Control

1.83±0.12

ND

1.83±0.12

Serpentine 250 mM 2.65±0.14 1.43±0.11 4.08±0.25*

100 mM 1.27±0.12 ND 1.27±0.12

50 mM 1.1±0.09 ND 1.1±0.09

Control

1.52±0.07

ND

1.52±0.07

Catharanthine

250 mM 1.92±0.08 0.44±0.05

2.36±0.13*

100 mM 2.18±0.17 0.13±0.02

2.31±0.19*

50 mM 1.22±0.1 ND 1.22±0.1

Control

1.25±0.08

ND

1.25±0.08

a n=3, values are mean ±, standard deviation; * significantly different from

control (p<0.05); ND-not detected

75

250 100 50 control0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

AjmalicineSerpentineCatharanthine

*

*

*

*

Concentration (milli moles)

Alk

alo

id c

on

ten

t (m

g/l)

Fig. 16 Effect of Sodium chloride on Alkaloid Production in Vinca rosea hairy root cultures. Data with * are significantly different from control (p<0.05)

76

Table-14 Effect of Mannitol on Alkaloid production in Hairy root cultures of Vinca rosea

Elicitor Concentration Alkaloid Contenta (mg/1) of Mannitol Root Medium Total

Ajmalicine 400 mM 1.75±0.12 1.25±0.12 3.0±0.24*

200 mM 2.51±0.19 0.23±0.03 2.75±0.21*

100 mM 2.35±0.14 0.18±0.03 2.52±0.17

Control

1.83±0.12

ND

1.83±0.12

Serpentine 400 mM 3.26±0.08 ND 3.26±0.08*

200 mM 4.16±0.13 ND 4.16±0.13*

100 mM 2.91±0.09 ND 2.91±0.09*

Control

1.52±0.07

ND

1.52±0.07

Catharanthine 400 mM 4.13±0.14 ND

4.13±0.14*

200 mM 4.43±0.17 0.37±0.03

4.5±0.17*

100 mM 3.50±0.17 0.1±.02 4.54±0.19*

Control

1.25±0.08

ND

1.25±0.08

a n=3, values are mean ±, standard deviation; * significantly different from control (p<0.05); ND-not detected

77

400 200 100 control0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

Ajmalicine

Serpentine

Catharanthine* *

*

*

**

**

*

Concentration (milli moles)

Alk

aloi

d co

nten

t (m

g/l)

Fig. 17 Effect of Mannitol on Alkaloid Production in Vinca rosea hairy root cultures. Data with * are significantly different from control (p<0.05)

78

In Vinca rosea plants, drought has been found to promote alkaloid

accumulation.260,261,262 On the other hand salt stress and osmotic shock have been

used for increasing the alkaloid accumulation in cell suspensioncultures.90 Higher

levels of ajmalicine and catharanthine were found to be accumulated in a Vinca rosea

salt tolerant line.216 Zhao et al., (2000b)263 reported that osmotic shock was more

effective than salt stress in improving indole alkaloid yields. A combination treatment

of fungal elicitation and mannitol effectively improved alkaloid production in cell

cultures of Vinca rosea.264 The results of our study support the positive effects of salt

and osmotic stress in improving secondary metabolite production in plant cell

cultures.

Effects of signal and signal Transduction Molecules

Salicylic, arachidonic and linoleic acids were added to 21-day-old hairy root

cultures of Vinca rosea to study their effect on alkaloid production. Growth of the

cultures was not affected significantly with these treatments. But hairy roots turned

light brown with the addition of arachidonic acid. This is presumably a manifestation

of the hyper sensitive response and browning has also been observed with cell

cultures of Taxus species elicitation with arachidonic acid.238 The results pertaining

to the effect of these agents on alkaloid yields are presented in Table 15-17.

Treatment with salicylic acid did not improve the accumulation of ajmalicine

and serpentine (Fig. 18). However, the contents of catharanthine (2.04 mg/l) were

found to be increased by 1.6 times with the lowest concentration (100 µM). The lower

levels seen with salicylic acid treatment may be due to the inhibitory effect of the

elicitor on alkaloid biosynthesis especially at the highest concentration.

79

Significant improvements in ajmalicine and serpentine production were

observed with arachidonic and linoleic acids. Concentration of the elicitor seemed to

be important as positive effects were seen at higher doses (Fig. 19 and 20). Also, these

treatments induced the release of ajmalicine and serpentine but the site of

accumulation of catharanthine was not affected (Table 15-17). Arachidonic acid

(5 mg/l) enhanced the yield of ajmalicine (4.3 mg/l) by 2.5 times when compared to

the control (1.73 mg/l) (fig. 19). A greater improvement was obtained for serpentine

production (4.87 mg/l) which was 3.4 fold more than the control (1.45 mg/l).

However, there was no significant effect on catharanthine accumulation (1.61 mg/l)

when compared to control cultures (1.36 mg/l).

80

Table-15 Effect of signal and signal transduction molecules on Ajmalicine Production in Vinca rosea hairy root cultures

Substance Concentration Ajmalicine Contenta(mg/1) AR Am At

Salicylic acid 1000µM 1.36±0.12 ND 1.36±0.12

100 µM 1.74±0.16 ND 1.74±0.16

Arachidonic acid

5mg/1 3.5±0.16 0.8±0.04 4.3±0.19*

1mg/1 1.73±0.1 ND 1.73±0.1

Linoleic acid 250 µM 2.74±0.03 ND

2.74±0.13*

50 µM 1.54±0.12 ND

1.54±0.12

Control

Water Ethanol(50%)

1.65±0.16 1.73±0.06

ND ND

1.65±0.16 1.73±0.06

a n=3, values are mean ±, standard deviation; * significantly different from

control (p<0.05); Ar-ajmalicine in root; Am-ajmalicine in medium; At-total

Ajmalicine;ND-not detected

81

Table-16 Effect of signal and signal transduction molecules on Serpentine Production in Vinca rosea hairy root cultures

Substance Concentration Serpentine Contenta(mg/1) SR SM ST

Salicylic acid 1000µM 1.33±0.11 ND 1.33±0.11

100 µM 1.71±0.14 ND 1.71±0.14

Arachidonic acid

5mg/1 3.82±0.16 1.05±0.06 4.87±0.21*

1mg/1 1.42±0.12 ND 1.42±0.12

Linoleic acid 250 µM 3.54±0.14 0.11±0.02 3.65±0.16*

50 µM 2.42±0.15 ND

2.42±0.15*

Control

Water Ethanol(50%)

1.34±0.12 1.45±0.09

ND ND

1.34±0.12 1.45±0.09

a n=3, values are mean ±, standard deviation; * significantly different from control (p<0.05); SR-Serpentine in root;Sm- Serpentine in medium;St-total Serpentine ; ND-not detected

82

Table-17 Effect of signal and signal transduction molecules on Catharanthine Production in Vinca rosea hairy root cultures

Substance Concentration Catharanthine Contenta(mg/1) CR CM CT

Salicylic acid 1000µM 1.22±0.12 ND 1.22±0.12

100 µM 2.04±0.17 ND 2.04±0.17

Arachidonic acid

5mg/1 1.61±0.12 ND 1.61±0.12

1mg/1 1.2±0.07 ND 1.2±0.07

Linoleic acid 250 µM 1.35±0.13 ND 1.35±0.13

50 µM 1.38±0.1 ND

1.38±0.1

Control

Water Ethanol(50%)

1.24±0.1 1.36±0.09

ND ND

1.24±0.1 1.36±0.09

a n=3, values are mean ±, standard deviation; * significantly different from control (p<0.05); CR- Catharanthine in root;Cm- Catharanthine in medium; Ct-total Catharanthine; ND-not detected

83

1000 100 control0.0

0.5

1.0

1.5

2.0

2.5 Ajmalicine

Serpentine

Catharanthine

*

Concentration (micromoles)

Alk

alo

id C

on

ten

t (m

g/l)

Fig. 18 Effect of Salicylic acid on Alkaloid Production in Vinca rosea hairy root cultures. Data with * are significantly different from control (p<0.05)

84

5 1 control (ethanol)0

1

2

3

4

5

6AjmalicineSerpentineCatharanthine*

*

Concentration (mg/l)

Alk

alo

id c

on

ten

t (m

g/l)

Fig. 19 Effect of Arachidonic acid on Alkaloid Production in Vinca rosea hairy root cultures. Data with * are significantly different from control (p<0.05)

85

250 50 control0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5 Ajmalicine

Serpentine

Catharanthine

*

*

*

Concentration (micromoles)

Alk

aloi

d c

on

ten

t (m

g/l)

Fig. 20 Effect of Linoleic acid on Alkaloid Production in Vinca rosea hairy root cultures. Data with * are significantly different from control (p<0.05)

86

Treatment with linoleic acid increased significantly the accumulation of

serpentine (3.65 mg/l), which was 2.5 times more than the control (Fig. 20).

Ajmalicine yield (2.74 mg/l) was improved by 1.7 times but catharanthine production

was not affected.

Salicylic acid is a signaling molecule that plays an essential role in many

plant defense reactions and up regulates the expression of various defense related

genes.265 It also inhibits the biosynthesis of ethylene, a phytohormone that is also

active in plant defense mechanisms. Treatment with salicylic acid increased the

production and release of hyoscyamine and scopolamine in hairy roots of

B.candida.239 But salicylic acid did not increase the total yield of tropane alkaloids

but induced their release from transformed root cultures of A.belladona.266 Salicylic

acid has also been found to be ineffective in increasing the alkaloid contents in cell

cultures of Vinca rosea.244,267,246 Moreover, Yahia et al., (1998)268 demonstrated that

there was no relation between the accumulation of ajmalicine and the evolution of

ethylene in cell cultures of Vinca rosea.

Arachidonic acid is a fatty acid present in the lipids of plant pathogenic

Oomycete fungi and a potent elicitor of sesquiterpenoid phytoalexins and suppressor

of glycol alkaloid synthesis in Solanaceous Species.269 Arachidonic acid has been

used to promote the production of taxanes in cell cultures of Taxus species in several

studies.270,271,272 Moreover, arachidonic acid has been found to alter the expression of

HMGR isogenes in potato and tomato.269,273 Also, it has been reported that there

exists a cross talk between mevalonate and non-mevalonate pathways in the synthesis

of alkaloids during stationary growth phase of hairy root cultures of Vinca rosea.274

The positive effects of arachidonic acid in the present study may be due to

stimulation of the mevalonate pathway, which supplies terpenoid precursors towards

87

alkaloid biosynthesis. Moreover, the polyunsaturated fatty acids arachidonic and

linoleic acids were found to induce extremely rapid generation of hydrogen peroxide

in the cultured potato plant cells.275 Such a stimulation of free–radical generation may

also be associated with hairy roots of Vinca rosea upon treatment with arachidonic

and linoleic acids, which may result in the enhancement of alkaloid yields.