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©CAB International 2010. Medicinal Plant Biotechnology 176

(ed. Rajesh Arora)

Chapter 11

Genetic Transformation of Catharanthus roseus

(L.) G. Don. for Augmenting Secondary

Metabolite Production

Rajesh Arora, Archana Mathur, Ajay K. Mathur and C.M. Govil

Introduction

Catharanthus is a perennial short-lived plant that commonly grows in tropical countries

and is mainly cultivated as an ornamental plant in the tropical and subtropical world. The

plant grows in warm regions of the world and especially in the Southern United States. The

plant normally grows 8 to 18 inches in length and a 1-foot spread, although the trailing

types can spread up to 2 feet. It is highly branched and develops a woody base. The leaves

of the plant are petiolate, oblong in shape, thick and leathery in texture, while the

arrangement is opposite or alternate (Virmani et al., 1978). The plant is referred to by a

variety of names in various countries (Table 11.1.) and the various species are mildly

poisonous.

Flowers of the plant are single and never double and most modern varieties show

overlapping petals. The colour of the flowers ranges from pink, white or a mixture of both

to pale pink in colour with a dark violet dot in the centre. The chromosome number for all

Catharanthus species is 2n16. However, tetraploid plants grow faster and the flowers are

reported to be bigger in size (Virmani et al., 1978). The genus Catharanthus comprises

eight species (Fig. 11.1). Seven species of the genus are known from Madagascar; one is

restricted to India and Sri Lanka, while one species is cultivated in China. The plant is

believed to be a native of the West Indies. The plant is more commonly known as

Madagascar periwinkle.

Genetic Transformation of Catharanthus roseus 177

Fig. 11.1. The eight common species of Catharanthus.

Catharanthus: A Unique Medicinal Plant

The genus name, Catharanthus, refers to ‘pure flower’. Catharanthus is often considered

synonymous to Vinca, however, certain authors disagree with the proposition that

Catharanthus and Vinca are one and the same. As of now, Catharanthus and Vinca are

considered two different species.

Catharanthus roseus: A source of medicinally useful terpenoid indole alkaloids (TIAs)

The Catharanthus plant has been used in traditional medicine in several parts of the world

for a long time for treating a variety of ailments and the existence of local names in most

countries is evidence enough regarding the importance of this medicinal plant (Table 11.1).

Catharanthus roseus comes in various hues and shades, the most common being red and

white (Fig. 11.2).

However, in recent years Catharanthus roseus has gained popularity as a medicinal

plant mainly for its curative and palliative role in cancer therapy (Table 11.2 and Table

11.3). The plant is almost synonymous with cancer therapy and no wonder then that the

National Cancer Council of Malaysia (Majlis Kanser National, MAKNA) uses the

periwinkle logo as its symbol of hope for cancer patients (Loh, 2008).

The serendipitous discovery of the antineoplastic property of Catharanthus alkaloids in

the 1950s paved the path for the discovery of a number of plant-derived anticancer drugs.

The Catharanthus alkaloids are of wide importance in clinical medicine and vincristine

(VCR) and vinblastine (VLB) (Fig. 11.3) form an indispensable part of standard

chemotherapy regimens. VCR and VLB are integral components of virtually every curative

regimen for metastatic malignancy and are extensively used in adjuvant and neoadjuvant

drug regimens. Leads obtained from Catharanthus have proved that natural products

indeed are reliable sources of medicine and can solve several of the health problems being

faced by humans (Sharma and Arora, 2006; Arora et al., 2010).

178 Genetic Transformation of Catharanthus roseus

Table. 11.1. A few vernacular names of Catharanthus roseus.

Common name(s) Country

Ainskati, Billaganneru, Nayantara, Sadabahar, Ushamanjairi India

Atay-biya, Chichirica, Kantotan, Periwinkle, Tsitsirika Philippines

Boa-noite, Congorca Brazil

Brown man’s fancy, Consumption bush, Old maid, Periwinkle, Pink

flower, Ram goat rose, Red rose, Sailor’s flower, White tulip

West Indies

Caca poule Dominica

Chatilla Guatemala

Chavelita Peru

Dua can Vietnam

Liluvha Venda

Madagascar periwinkle Madagascar

Maua Kenya

Mini-mal, Patti-poo Sri Lanka

Nayantara Bangladesh

Nichinich-so, Nichinichi-so Japan

Ninfa Mexico

Periwinkle Jamaica, USA

Pervenchede de Madagascar French Guiana

Phaeng phoi farang, Phang-puai-fa-rang Thailand

Sadabahar Pakistan

Saponaire Rodrigues Islands

Tiare-tupapaku-kimo Cook Islands

Fig. 11.2. The most common red (top) and white varieties (bottom) of Catharanthus roseus.

Genetic Transformation of Catharanthus roseus 179

Table 11.2. Catharanthus alkaloids used in the treatment of different neoplasms.

Alkaloid Neoplasms

Vincristine

Vinblastine

Vindesine

Acute lymphocytic leukaemia

Lymphomas (lymphocytic, mixed cellular, histiocytic, non-differentiated,

nodular and diffuse types)

Hodgkin's disease

Lymphosarcoma

Carcinomas (breast, cervix, prostate, choriocarcinoma)

Primary brain tumours (Astrocytomas)

Neuroblastoma

Pediatric tumours

Embryonal rhabdomyosarcoma

Wilm's tumour

Monocytic leukaemia

Lymphomas

Hodgkin's disease

Lymphosarcoma

Mycosis fungoides

Reticulum cell sarcoma

Carcinomas

Methotrexate-resistant choriocarcinoma

Kaposi's sarcoma

Metastatic testicular cancer

Non-small cell lung cancer

Carcinomas of the breast

Fig. 11.3. Chemical structures of vincristine (left) and vinblastine (right), the clinically and

economically important terpenoid indole alkaloids of Catharanthus roseus.

180 Genetic Transformation of Catharanthus roseus

Table 11.3. Curative and palliative regimens containing bisindole Catharanthus alkaloids.

Acronym Drug Combinations Disease

ABOS

ABV

ABVD

EVAP

MOPP

Doxorubicin, bleomycin, vincristine, streptozocin

Doxorubicin, bleomycin, vinblastine

Daunomycin, Bleomycin, vinblastine,

dacarbasine

Etoposide,vinblastine, adriamycin, prednisone

Nitrogen mustard, vincristine, procarbazine,

prednisone

Hodgkin’s lymphoma

CHOP Cyclophosphamide, daunomycin, vincristine,

prednisone

Non-Hodgkin’s lymphoma

PVB Cisplatinum, vinblastine, bleomycin Testicular cancer

COP

MACOP-B

Cyclophosphamide, vincristine, prednisone

Methotrexate, daunomycin, cyclophosphamide,

vincristine, prednisone, bleomycin

Lymphoma

MVAC Methotrexate, vinblastine, daunomycin,

cyclophosphamide

Bladder cancer

POC Procarbazine, vincristine, cyclophosphamide Melanoma, Small-cell lung

cancer

VATH Vinblastine, daunomycin, Thio-TEPA, Halostin Breast cancer

VP Vinblastine/vindesine, cisplatinum Non-small cell lung cancer

- Doxorubicin, daunorubicin, bleomycin, vincristine

and vinblastine

AIDS-Kaposi’s sarcoma

Catharanthus roseus contains more than 120 different types of alkaloids, designated as

terpenoid indole alkaloids (TIAs). Among the TIAs, vincristine (VCR) and vinblastine

(VLB) find extensive use in the treatments of leukaemias, lymphomas, small cell lung

cancer and other malignancies (Noble, 1990; Pratt et al., 1994). Vincristine has been an

integral component of combination chemotherapy regimens for ALL (acute lymphoblastic

leukaemia) and plays an important role in the treatment of Hodgkin’s and non-Hodgkin’s

lymphomas. It has also been used in combination with other agents to treat Wilm’s tumour,

Ewing’s sarcoma, neuroblastoma, and rhabdomyosarcoma in children; and multiple

myeloma, breast cancer and small cell lung cancer in adults. Vinblastine is used for the

treatment of cell cancers of the testes and advanced Hodgkin’s disease, and is commonly

used in combination with other agents to treat carcinomas of the breast and bladder,

Kaposi’s sarcoma and other neoplasms (Rowinsky and Donehower, 1997).

The indole alkaloids of Catharanthus represent a market of considerable economic

interest and there is a potential in-house demand as well as a world-wide international

market. The anti-cancer Catharanthus alkaloids have a market value as follows: vincristine

($3,5,00,000 per kg); vinblastine ($1,000,000/ kg) (Veltkamp et al., 1985). Their retail

value is more than $20,000/ g (Curtin, 1983). The high prices are mainly due to the costly

methods currently available for production of these compounds, and the low yields

obtained from the source plants, e.g., the yield of vincristine from C. roseus whole plants is

of the order of 0.0003–0.0005%, which happens to be the lowest yield of any medicinally

useful alkaloid produced on a commercial basis (Taylor and Farnsworth, 1975; Curtin,

1983). Nearly 500 kg of leaves are needed to produce just 1 g of purified vincristine,

meaning thereby that about 10,000–15,000 kg leaves are required to produce just 30 g of

Genetic Transformation of Catharanthus roseus 181

the drug. Up to 16 weeks may be required for processing. This is one of the reasons behind

the high cost of these alkaloids. In the 1970s, the annual world supply of these drugs was

of the order of 4.5 kg. The current annual demand for the dimeric/monomeric alkaloids is

as follows: VCR: 1 kg per year; VLB: 12 kg per year; ajmalicine: 5000 kg/ year.

India happens to be the third largest manufacturer of vinblastine and vincristine in the

world and these alkaloids are exported to Europe and America (Rama Rao and Gurjar,

1990). In 1985–86, Indian exports of Catharanthus roots alone were worth US$140,625,

and in 1987–88, US$171,875. In 1993–94 Vinca alkaloids alone worth > US$210,000 were

exported (Chaudhri, 1996). The demand of these alkaloids in the international market has

been steadily increasing.

Catharanthus roseus: Model medicinal plant system for biotechnological interventions

World-wide demand of TIAs, and the miniscule amount in which they are produced within

the plant, are the factors mainly responsible for the exorbitantly high cost of these

compounds. In view of this, tremendous efforts have been directed towards increasing the

content of VCR and VLB in the plants by modern biological techniques, including cell line

selection, fungal elicitation, biotransformation, and genetic transformation with

Agrobacterium rhizogenes (van der Heijden et al., 1989, 1998; Saito et al., 1992;

Verpoorte et al., 1993; Yeoman and Yeoman, 1997; Arora, 1998; Batra et al., 2004; Arora

et al., 2005; Sharma and Arora, 2006; Christie et al., 2009; Junaid et al., 2009; Natali et al.,

2009).

The establishment of 5-methyltryptophan (5-MT)-resistant callus lines has been

reported (Arora, 1998; Seth and Mathur, 2005). Seth and Mathur (2005) isolated four

variant callus lines showing differential tolerance to 5-MT, a tryptophan analogue. The

selected lines showed higher alkaloid content in the presence of 5-MT stress. The presence

of catharanthine and vindoline were demonstrated in two lines Nir L-60 and C6 L-120,

indicating a positive influence of tryptophan accumulation on diverting the metabolic flux

towards synthesis of the monomeric indole alkaloids. Shimoda et al., (2007) isolated four

disaccharides from the cell suspension of Catharanthus roseus after incubation with

capsaicin and 8-nordihydrocapsaicin in addition to glucosides and in undifferentiated

cultures. Sriram et al. (2007) studied flux quantification in central carbon metabolism of C.

roseus hairy roots by

C labelling and bondomer balancing. Echevarría-Machado et al.,

(2007) studied the response of transformed roots of C. roseus to very low concentrations of

salicylic acid. Pomahaová et al. (2009) recently reported improved accumulation of

improved accumulation of ajmalicine and tetrahydroalstonine in Catharanthus cells

expressing an ABC transporter. Jaleel et al. (2007) reported on the accumulation of

ajmalicine in Catharanthus roseus after treatment with giberellic acid.

The molecular events that regulate genes and the activators of terpenoid indole alkaloid

metabolism are far from being completely understood and much needs to be unraveled.

Several workers have attempted to elucidate the structure and function of key enzymes and

the enzymatic pathways and the molecular regulation with a view to augmenting secondary

metabolite production (Pasquali et al., 1992; van der Fits and Memelink, 2000; Siberil et

al., 2001; van der Heijden et al., 2004; Blondy et al., 2005; Courdavault et al., 2005; Dutta

et al., 2005, 2007; Barleben et al., 2007; Bernhardt et al., 2007; Suttipanta et al., 2007;

Kutchan et al., 2008; Stöckigt et al., 2008; Pereira et al., 2009). A simplified TIA

biogenetic pathway is represented in Fig. 11.4.

182 Genetic Transformation of Catharanthus roseus

Fig. 11.4. The Terpenoid Indole Alkaloid Biogenetic Pathway.

Genetic Transformation of Catharanthus roseus 183

Metabolic engineering approaches have been attempted by several other workers

(Rischer et al., 2006; Kutchan et al., 2008; Christie et al., 2009; Pereira et al., 2009).

The Promise of Hairy Roots

Hairy roots, which are produced as a result of transfer of T-DNA from A. rhizogenes- a

gram-negative bacterium, into the plant genome (Banerjee et al., 1995; Doran, 1997) have

been the subject of intense investigation due to their immense potential as an alternative

source of TIAs. However, Catharanthus has been a fairly recalcitrant crop as far as genetic

transformations are concerned, and in particular establishment of fast-growing clones has

been rather difficult. Several attempts have been made in recent years to obtain fast-

growing clones and increase the levels of TIA accumulation in in vitro plant organ systems

like hairy roots (Hughes et al., 2004; Arora et al., 2005). Hong et al., (2006) established

Catharanthus roseus hairy root cultures that were transgenic for the rol ABC genes from T-

DNA of the agropine-type Agrobacterium rhizogenes strain A

. The rol ABC hairy root

lines exhibited a wild-type hairy root syndrome in terms of growth and morphology on

solid medium. The transgenic hairy roots did not produce detectable levels of mannopine

and agropine which, in contrast, are often synthesized abundantly in wild-type hairy root

lines. The absence of these opines did not appear to cause the rol ABC lines to have higher

levels of terpenoid indole alkaloids than wild-type hairy root lines. Unlike wild-type lines,

rol ABC lines produced very similar levels of total alkaloids despite wide variations in

individual alkaloid contents. This work demonstrated that the three genes rol ABC are

sufficient to induce high-quality hairy roots in Catharanthus roseus.

Chung et al. (2009) have recently isolated four new flavonoid glucosides viz., 3,4-di-

O-methylquercetin-7-O-[(413)-2,6,10,14-tetramethylhexadec-13-ol-14-

enyl]--d-glucopyranoside (1), 4-O-methylkaempferol-3-O-[(413)-2,6,10,14-

tetramethylhexadecan-13-olyl]--d-glucopyranoside (2), 3,4-di-O-methylbutin-7-O-

[(61)-3,11-dimethyl-7-methylenedodeca-3,10-dienyl]--d-glucopyranoside

(3), and 4-O-methylbutin-7-O-[(61)-3,11-dimethyl-7-

hydroxymethylenedodecanyl]--d-glucopyranoside (4), along with the three known

compounds, from the methanolic extract of Catharanthus roseus hairy roots. The structures

of these compounds were elucidated spectroscopically. The new flavonoid glucosides

inhibited both MMP-9 activity and TNF- production in THP-1 cells treated with

lipopolysaccharide. These results do show that it is possible to transform Catharanthus and

produce a variety of chemical compounds. Magnotta et al. (2007) have recently reported

expression of deacetylvindoline-4-O-acetyltransferase in Catharanthus roseus hairy roots.

In this paper we report the successful induction of hairy root cultures in some elite

Indian genotypes of C. roseus using Agrobacterium rhizogenes strains and optimization of

culture conditions for some selected fast-growing clones.

Catharanthus roseus: The Medicinal Plant Model System

Five genotypes of Catharanthus roseus viz., Red-flowered (4n=32), White-flowered

(4n=32), Nirmal (2n=16), BRT-1 and BRT-2 were tested for their susceptibility towards

different A. rhizogenes strains. The explants (leaf, petiole, ovule, embryo, petal,