1

CHAPTER 1

INTRODUCTION

Higher plants are recognised as important sources of a wide range of

biochemicals, used as drugs, pesticides, flavorings and fragrances. Traditionally, these

substances have been extracted from naturally grown whole plants. On a commercial

basis, this approach involves large-scale crop cultivation (e.g. alkaloids from Vinca

rosea). Many plant products can now be produced by chemical synthesis, which can

be a more reliable, consistent and cost-effective method. Plant tissue culture provides

an alternative approach, which may be attractive under certain circumstances: if, for

example, the source plant is difficult to cultivate, has a long cultivation period or has a

low metabolite yield; if chemical synthesis has not been achieved or if it is technically

problematic. Metabolite yield by the tissue culture may significantly exceed that

observed in the parent plant. Thus, using this technology, the metabolite can be

produced under controlled and reproducible conditions, independent of geographical

and climatic factors.

Plants are known to produce a large array of natural products, also referred to

as secondary metabolites. Plant alkaloids have a rich chemical ecology that has been

exploited for medicinal purposes for thousands of years. Despite being highly

represented within today's pharmacopoeia, relatively little is known about the

biosynthesis, regulation and transport of these molecules. Understanding how nature

synthesizes plant alkaloids will enhance our ability to overproduce, i.e. to

metabolically engineer these medicinally useful compounds as well as new-to-nature

compounds (with potentially improved bioactivity) derived from these natural

scaffolds.

2

The Genus Vinca belongs to the family Apocynaceae and is closely related to

the genera Catharanthus. It is also known as Catharanthus roseus. The genus Vinca

comprises of the species Vinca difformis, Vinca erecta, Vinca herbacea, Vinca major,

Vinca minor, Vinca pubescens. Other English names occasionally used include Cape

Periwinkle, Rose Periwinkle, and Rosy Periwinkle. Periwinkle, also called Bara Massi

or Sada Bahar in Hindi, Kasi Kanigale in Kannada and Billaganneru in Telugu, is a

hardy plant growing wild in many places and now cultivated systematically for the

last few years. It has a pan tropical distribution, being naturalized in Africa, America,

Asia, Australia and Southern Europe and on some islands in the Pacific Ocean.

1.1. Allied drugs/ Substitutes:

Other Catharanthus species such as C. longifolius, C. trichophyllus and C.

lanceus are known to contain vindoline type alkaloids.There are three varieties in

Vinca rosea a) rose flowered (Fig.1) b) white flowered, and c) white flowered with

rose-purple spot in the center. Vinca rosea, as this species was originally named, was

published by Linnaeus in Syst. Nat., ed. 10, p. 944 (1759).

1.2. Taxonomy, Habit and Habitat of Vinca Rosea

Vinca rosea, the Madagascar periwinkle or rosy periwinkle, is an attractive

small sub shrub with graceful pink or white salver form flowers. Native to south-

eastern and eastern Madagascar, the plant is easily cultivated, and European colonists

exported it widely as an ornamental. It is now grown almost worldwide, and is found

naturalized in most tropical and subtropical regions following escapes from

cultivation. Madagascar periwinkle was used in Madagascar, and in many of the

countries to which it was later spread, as a folk treatment for diabetes

3

Traditionally, different parts of it are used in the treatments of various diseases

(viz. diabetes, menstrual regulators, hypertension, cancer and antigalactagogue etc.),

in numbers of countries (Australia, Brazil, China, Cook Island, Dominica, England,

Europe, France, French Guinea, India, Jamaica, Kenya, Mexico, Mozambique, North

Vietnam, Pakistan, Peru, Philippines, South Africa, South Vietnam, Taiwan,

Thailand, USA, Venda, Vietnam, West Indies etc.). Moreover, more than 130

alkaloids have been isolated from different parts; amongst which two important

alkaloids (Vinblastine and Vincristine are used in cancer treatment) present in very

low concentrations. Keeping these views; researcher continuously use different

approaches to enhance the level of important alkaloid to meet the required demand.

Vinca rosea commonly known as Madagascar periwinkleis a perennial,

evergreen herb, 30-100 cm height that was originally native to the island of

Madagascar. It has been widely cultivated for hundreds of year and can now be found

growing wild in most warm regions of the world. The leaves are glossy, dark green

(1-2 inch long), oblong – elliptic, acute, rounded apex; flowers fragrant, white to

pinkish purple in terminal or auxiliary cymose clusters; follicle hairy, many seeded, 2-

3 cm long; seeds oblong, minute, black. The plant is commonly grown in gardens for

beddings, borders and for mass effect. It blooms throughout the year and is

propagated by seeds or cuttings. The bloom of natural wild plants are pale pink with a

purple eye in the centre, but horticulturist has developed varieties (more than 100)

with colour ranging from white to pink to purple.

1.3. Traditional uses of Vinca rosea

The plant has historically been used to treat a wide assortment of diseases. It

was used as folk remedy for diabetes in Europe for centuries.1 In India, juice from the

leaves was used to treat wasp stings. In Hawaii, the plant was boiled to make a

4

poultice to stop bleeding. In china, it was used as an astringent, diuretic and

coughremedy.2

In central and south America, it was used as a homemade cold remedy to ease

lung congestion and inflammation. Throughout the Caribbean, an extract from the

flowers was used to make a solution to treat eye irritation and infections. It also had a

reputation as magic plant, European thought it could ward off evil spirits, and the

French referred to it as “violet of the sorcerers.” Western researchers finally noticed

the plant in 1950’s when they learn of a tea Jamaican were drinking to treat diabetes.

They discovered that the plant contains a mother lode of useful alkaloids (130 in all at

last count). Some, such as catharanthine, leurosine sulphate, lochnerine,

tetrahydroalstonine, vindoline and vindolinine lower blood sugar level, however,

others act as haemostatics (arrest bleeding) and two others, vincristine and vinblastine

have anticancerous properties. Periwinkle also contains the alkaloids reserpine and

serpentine, which are powerful tranquilizers.

.

5

Table 1. Traditional uses of the Vinca rosea world-wide

Country Use

Australia Hot water extract of dried leaves is taken orally for menorrhagia,

diabetes and extract of root bark is taken orally as febrifuge3,4

Brazil The hot water extract of dried entire plant is taken orally by

human for diabetes mellitus5,6

China Hot water extract of the aerial parts is taken orally as a menstrual

regulator.4,7

Cook Island Decoction of dried leaves used orally to treat diabetes, hyper

tension and Cancer8

Dominica Hot water extract of leaves is taken orally by pregnant woman to

combat primary inertia in child birth and the boiled leaves are

drink to treat diabetes9

England Hot water extract of dried entire plant is taken orally for the

curing of diabetes10

Europe Decoction of dried leaves is taken orally for diabetes mellitus3

France Hot water extract of entire plant is taken as an antigalactagogue 4

French Guinea Hot water extract of entire plant is taken orally as a cholagogue 11

India The hot water extract of dried entire plant is taken orally by

human for cancer. Hot water extract of dried leaves is taken

orally to Hodgkin’s disease. The root extract is taken orally for

menorrhagia 7, 12

Jamaica Hot water extract of dried leaves is taken orally for diabetes 13

Kenya Hot water extract of dried leaves is taken orally for diabetes13

Mexico Infusion of whole plant is taken orally for stomach problem 14

Mozambique Hot water extract of leaves is taken orally for diabetes and

rheumatism and the root extract is taken orally as hypotensive

and febrifuge 15

North Vietnam Hot water extract of the aerial parts is taken orally as a menstrual

regulator 16, 17

Pakistan Hot water extract of dried ovules is taken orally for diabetes18

6

Country Use

Peru Hot water extract of dried entire plant is taken orally by human

adults for cancers, heart disease and leishmaniasis19

Philippines Hot water extract of root is taken orally by pregnant women to

produce abortion7, 20, 21

South Africa Hot water extract of dried leaves is taken orally for menorrhagia

and diabetes 3

South Vietnam Hot water extract of the entire plant is taken orally by human

adults as an antigalactagogue 4,7

Taiwan Decoction of dried entire plant is used orally by human adults to

treat diabetes mellitus23 and liver disease22

Thailand Hot water extract of dried entire plant is taken orally for

diabetes23

USA Hot water extract of leaves are smoked as a euphoriant 24

Venda Water extract of dried root is taken orally for venereal disease 25

Vietnam Hot water extract of dried aerial parts is taken orally as drug in

Vietnamese traditional medicine, listed in Vietnamese

pharmacopoeia (1974 Edition) 26

West Indies Hot water extract of leafy stems is taken orally for diabetes 27

Antitumor Activity: The ethanol (70%) extract of Vinca rosea leaves was

administered intraperitoneally to female mice and proved to be highly active on CA-

Ehrlich ascites.28,29The chloroform extract of the leaves of Vinca rosea was active on

Leuk-P3885. The plant contains about 130 alkaloids (Table 2) of the indole group, out

of which 25 are dimeric in nature. Total alkaloids of the entire plant administered to

mice intraperitoneally at a dose of 10.0 mg/kg and orally at a 75.0 mg/kg were active

on Leuk-P1534.30-33

Further, the plant Vinca rosea was also reported to have a good number of

pharmacological activities. (Table 2 )

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Table 2. Pharmacological activities of Vinca rosea

S.No Source Pharmacological activity

Refere

nces

1 Hot water extract of dried leaves Animutagenic Effect 34

2

Methanol/water (1:1) extract of

dried leaf and stem Antifertility Activity 35

3 Total alkaloids of root Antihypertensive Activity 36

4 Acetone and water extracts of dried

aerial parts Antifungal Activity 37-40

5 Ethanol (70%) extract of leaves Antimitotic Activity 41

6 Ethanol extract (95%) of dried

leaves Anti-Inflammatory Activity 42

7 Hot water extract of dried leaves

Antihyperchcholesterollemic

Activity 43

8 Alkaloid fraction of the entire plant Antidiuretic Activity 44

9 Chloroform extract of root Antimalarial Activity 45

10 Hot water extract of dried aerial

parts Antihyperglycemic Activity

1, 46,

47

11 Benzene extract of dried flowers Antibacterial Activity

48,49,

36,44

12 Water extract of callus tissue Antiviral Activity 50

13 Ethanol (70%) extract of leaf and

stem Cardio tonic Activity 36

14 Total activity of root CNS Depressant Activity 36

15 Alkaloid fraction of dried leaves Cytotoxic Activity 51

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Table 3. Alkaloids isolated from different parts of Vinca rosea

Alkaloids

Extracted from

Β-Carboline Leaf Tryptamine,N,N-Dimethyl Cell suspension culture Tryptamine,Nb-Acetyl Cell suspension culture Apparicine Leaf, flower Ammocalline Plant extract, root Anthirine Plant extract, cell suspension culture Akuammicine Plant extract, leaf, root, callus culture, cell

suspension culture, shoots Iochrovicine Leaf Pericyclivine Plant extract, leaf Pleiocarpamine Cell suspension culture Cavincine Plant extract, leaf, root, callus culture, hair y root Iochnerine Cell suspension culture Tubotaiwine Callus culture, cell suspension culture Rosicine Leaf Catharanthine Plant extract, leaf, flower, seedlings, callus culture,

cell suspension culture, shoots Tabersonine Plant extract, leaf, seedlings, seed, callus culture,

cell suspension culture Venalstonine Root Akuammicine,12-Hydroxy Cell suspension culture Perivine Plant extract, leaf, flower, root, callus culture, cell

suspension culture Vinervine Cell suspension culture Coronaridine Flower Vincadifformine Leaf Cyclolochnerine,21-Hydroxy

Callus culture, cell suspension culture, shoots, hairy root

Iochneridine Leaf, callus culture, cell suspension culture, hairy root

Alstonine Root, callus culture Serpentine Leaf, root, seedlings callus culture, cell suspension

culture, shoots, ,hairy root Cathenamine Plant extract Vallesiachotamine Callus culture, cell suspension culture Isovallesiachotamine Callus culture, cell suspension culture Ajmalicine Callus culture, cell suspension culture Ajmalicine,19-Epi,3-Iso Plant extract, callus culture, cell suspension culture Ajmalicine, 3-Epi Plant extract, callus culture ,cell suspension culture Akuammigine Cell suspension culture Akuammiline, O-Deacetyl Leaf, callus culture Iochnericine Plant extract, leaf, cell suspension culture

9

Alkaloids

Extracted from

Minovincine Plant extract Preakuammicine Seedlings Rosamine Leaf Tabersonine,19-Hydroxy Cell suspension culture Tetrahydroalstonine Plant extract, flower, root, callus culture, cell

suspension culture, shoots, hairy root Vindolinine, Nb-Oxide Plant extract, cell suspension culture Vindolinine,19-Epi,N-Oxide

Cell suspension culture

Fluorocarpamine, N -Oxide Plant extract, leaf Perividine Plant extract Isositsirikine, 19,20-Cis-16 (R)-

Plant extract, cell suspension culture

Isositsirikine, 19,20-Trans-16 (R)-

Plant extract, cell suspension culture

Isositsirikine, 19,20-Trans-16 (S)-

Plant extract, leaf, cell suspension culture

Minovincinine Cell suspension culture Sitsirikine Plant extract, leaf, callus culture, cell suspension

culture, shoots Yohimbine Plant extract, leaf, root, callus culture, cell

suspension culture, hairy root Sitsirikine,Dihydro- Plant extract, leaf, root, callus culture, cell

suspension culture Perimivine Plant extract, root Tabersonine,11-Methoxy Plant extract, flower Almalicine, 7-Hydroxy -Indolenine

Callus culture

Ajmalicine Pseudo-Indoxyl Callus culture Akuammiline,10-Hydroxy- Deacetyl

Callus culture

Epimisiline,19(S) Hairy root Horhammericine Cell suspension culture, shoots Mitraphyllline Flower, callus culture Vincoline Plant extract, leaf Vindolinine Plant extract, leaf, cell suspension Vindolinine,19-Epi Plant extract, leaf, cell suspension culture Vincolidine Plant extract, leaf Akuammine Plant extract Lochnerinine Plant extract, leaf, cell suspension culture Lochrovidine Plant extract Tabersonine,19-Hydroxy-11- Methoxy

Plant extract

Iochrovine Plant extract

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Alkaloids

Extracted from

Vindolidine,O-Deacetyl- Leaf Akuammiline Plant extract, cell suspension culture Horhammericine,11-Methoxy

Cell suspension culture, shoots

Vincarodine Plant extract, leaf Vinosidine Root Vindoline,Deacetoxy- Cell suspension culture, leaf, seedlings Tabersonine,19-Acetoxy-11- Hydroxy-

Plant extract, leaf, cell suspension culture

Vindoline,Deacetyl- Plant extract, leaf Iochnerinine Leaf, root Tabersonine,19-Acetoxy-11- Methoxy

Cell suspension culture

Cathovaline Leaf Vindolidine Plant extract, flower Strictosidine Lactam Cell suspension culture, shoots, hairy root Vindoline Plant extract, leaf, flower, seedlings, shoots Akuammicine, Xylosyloxy- Cell suspension culture Strictosidine Plant extract, leaf. Root, seed, callus culture, cell

suspension culture Bannucine Plant extract, leaf Leurosivine Leaf Leurosine,17-Deacetoxy- Plant extract Vinblastine,4-Deacetoxy- Plant extract, leaf Vinblastine, Deacetyl- Plant extract Vinsedine Seed Leurosinine Plant extract Vinsedicine Seed Vinblastine,3’,4’-Anhydro- Leaf, shoots Vingramine Seed Vinblastine,4’-Deoxy- Plant extract, leaf Vinosidine Plant extract Vinblastine, N-Demethyl- Plant extract Vingrmine, Methyl- Seed Catharanthamine Plant extract, leaf Leurosine Plant extract, leaf, shoots Roseadine Plant extract, leaf Vincathicine Plant extract, leaf Roseamine Plant extract Vinblastine Plant extract, leaf, flower, seedlings, cell

suspension culture Vinblastine,20’-Epi- Plant extract, leaf Catharicine Plant extract, leaf, flower

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Alkaloids

Extracted from

Catharine Plant extract, leaf, shoot Leurosine, 5’-Oxo- Leaf Carosine Plant extract, leaf, flower Leurosine,N B’-Oxide Leaf Vinamidine Plant extract, leaf Vincristine Plant extract, leaf Leurosidine, N B-Oxide Plant extract Vinblastine,14’-Hydroxy- Plant extract Vinblastine, 15’hydroxy- Plant extract Neoleurocristine Plant extract, leaf Vindolidine Plant extract Leurosinone Leaf Neoleurosidine Plant extract, leaf Neoleurosidine,N B-Oxide Plant extract, leaf Vindolicine Plant extract, leaf Ammorosine Root Cathalanceine Root Cathindine Leaf, root, cell suspension culture Cavincidine Plant extract, leaf, root, callus culture, cell

suspension culture Lochneririne Leaf, root Maandrosine Plant extract, root Perosine Plant extract, leaf, root, callus culture Rovindine Plant extract, leaf Vinaphamine Plant extract, leaf Vinaspine Plant extract, leaf Vincamicine Plant extract, leaf

There is a continued commercial demand for a wide range of plant secondary

metabolites. Commercial production of secondary metabolites by de novo synthesis or

by biotransformation of externally fed substrates requires the successful cultivation of

plant organs on a large scale. Successful scaling up of synthesis of these compounds

by cultured cells/roots should reduce or eliminate the need to cultivate the source

plants under variable climatic conditions or, alternatively, the need to conduct

complex and expensive organic synthesis. The strategies used to optimize the product

yield include: (1) culture conditions, (2) selection of high yielding lines, (3)

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elicitation, (4) immobilization of cells, (5) hairy root culture, (6) biotransformation,

(7) permeabilization of cell, and (8) removal of secreted products.

The evolving commercial importance of secondary metabolites has in recent

years resulted in a great interest in secondary metabolism, particularly in the

possibility of altering the production of bioactive plant metabolites by means of tissue

culture technology. Plant cell and tissue culture technologies can be established

routinely under sterile conditions from explants, such as plant leaves, stems, roots,

and meristems for both the ways for multiplication and extraction of secondary

metabolites.

The capacity for plant cell, tissue, and organ cultures to produce and

accumulate many of the same valuable chemical compounds as the parent plant in

nature has-been recognized almost since the inception of in vitro technology. The

strong and growing demand in today’ smarketplace for natural, renewable products

has refocused attention on in vitro plant materials as potential factories for secondary

phytochemical products, and has paved the way for new research exploring secondary

product expression in vitro. However, it is not only commercial significance that

drives the research initiatives. The deliberate stimulation of defined chemical products

within carefully regulated in vitro cultures provides an excellent forum for in-depth

investigation of biochemical and metabolic pathways, under highly controlled

microenvironmental regimes.

Plant-produced secondary compounds have been incorporated into a wide

range of commercial and industrial applications, and fortuitously, in many cases,

rigorously controlled plant in vitro cultures can generate the same valuable natural

products have served as resources for flavors, aromas and fragrances, bio based fuels

and plastics, enzymes, preservatives, cosmetics (cosmeceuticals), natural pigments,

13

and bioactive compounds. There is a series of distinct advantages to producing a

valuable secondary product in plant cell culture, rather than in vivo in the whole crop

plant. These include: a) Production can be more reliable, simpler, and more

predictable, b) Isolation of the phytochemical can be rapid and efficient, as compared

to extraction from complex whole plants, c) Compounds produced in vitro can

directly parallel compounds in the whole plant, d) Interfering compounds that occur in

the field-grown plant can be avoided in cell cultures, e) Tissue and cell cultures can

yield a source of defined standard phytochemicals in large volumes, f) Tissue and cell

cultures are a potential model to testelicitation.

Studies on plant secondary metabolites have been increasing over the last 50

years. These molecules are known to play a major role in the adaptation of plants to

their environment, but also represent an important source of active pharmaceuticals.

Plant tissue culture technologies were introduced at the end of the 1960s as a possible

tool for both studying and producing plant secondary metabolites. Different strategies,

using in vitro systems, have been extensively studied with the objective of improving

the production of secondary plant compounds. Undifferentiated cell cultures have

been mainly studied, but a large interest has also been shown in hairy roots and other

organ cultures. Specific processes have been designed to meet the requirements of

plant cell and organ cultures in bioreactors. Despite all of these efforts of the last 30

years, plant biotechnologies have led to very few commercial successes for the

production of valuable secondary compounds. Compared to other biotechnological

fields such as microorganisms or mammalian cell cultures, this can be explained by a

lack of basic knowledge about biosynthetic pathways, or insufficiently adapted

facilities. More recently, the emergence of recombinant DNA technology has opened

a new field with the possibility of directly modifying the expression of genes related

14

to biosyntheses. It is now possible to manipulate the pathways that lead to secondary

plant compounds. Many research projects are now currently being carried out and

should give a promising future for plant metabolic engineering.

Transgenic hairy root cultures have revolutionized the role of plant tissue

culture in secondary metabolite production. They are unique in their genetic and

biosynthetic stability, faster in growth, and more easily maintained. Plant hairy root

cultures are promising potential alternative sources for the production of high-value

secondary metabolites of industrial importance. Recent developments in transgenic

research have opened up the possibility of the metabolic engineering of biosynthetic

pathways to produce high-value secondary metabolites. The production of the

pungent food additive capsaicin, the natural colour anthocyanin and the natural

flavour vanillin are examples. Reliance of a plant on a specialized structure for

production of a secondary metabolite, in some cases, is a mechanism for keeping a

potentially toxic compound sequestered.

Extraction from the in vitro tissues is much simpler than extraction from

organized, complex tissues of a plant. Plant tissue culture techniques offer the rare

opportunity to tailor the chemical profile of a phytochemical product, by manipulation

of the chemical or physical micro environment, to produce a compound of potentially

more value for human use. While research to date has succeeded in producing awide

range of valuable secondary phytochemicals in unorganized callus or suspension

cultures, in other cases production requires more differentiated micro plant or organ

cultures. This situation often occurs when the metabolite of interest is only produced

in specialized plant tissues or glands in the parent plant. Genome manipulation is

resulting in relatively large amounts of desired compounds produced by plants

15

infected with an engineered virus, whereas transgenic plants can maintain constant

levels of production of proteins without additional intervention.

Plant cell culture systems represent a potential renewable source of valuable

medicinals, flavours, essences and colourants that cannot be produced by microbial

cells or chemical syntheses. However, only a few cultures produce these compounds

in commercially useful amounts. The low productivities are associated with our poor

understanding of the biochemistry of these systems. Recent advances in molecular

biology, enzymology, physiology and fermentation technology of plant cell cultures

suggest that these systems will become a viable source of important natural products.

The main problem is due to the lack of optimization of cultural conditions and

several strategies leading with increased accumulation of secondary metabolites. A

detail studies are required to know the proper enzyme functions at various levels,

product membrane permeability and adsorption for improvements towards achieving

a viable economic production methodology. In addition, over-expression of enzymes

and the genetic modification could be very useful via organogenesis or somatic

embryogenesis for the production of desired levels of secondary metabolite.

Periwinkle (Vinca rosea), is highly valued in the Pharmaceutical industry.

Different pharmacological studies and the traditional uses proved the high medicinal

properties of the Vinca rosea ; which continuously being used in the treatments of

numbers diseases. Study of periwinkle has increased because of its ability to produce

secondary metabolites such as terpenoid indole alkaloids.Today India is the third

largest manufacture of Vinblastine and Vincristine in the world and is exporting these

alkaloids to European countries.

Plant regeneration is a challenge for Vinca rosea, especially through

organogenesis using vegetative tissues. After testing numerous concentrations of BA

16

and NAA as well as different cultivars and explant types, it was found that variations

in regeneration capabilities were caused by many factors, especially genotype and

explant. In fact, Vinca rosea cultures have turned into a major model system in plant

cell biotechnology. In the last decades, a considerable amount of information has been

obtained on the growth and production of secondary metabolites by cell and tissue

cultures of Vinca rosea.

High demand and low yield of these alkaloids in the plant has led to research

for alternative means for their production. Various important alkaloids, mostly the

monomers were successfully identified in culture media with the enhanced yields;

however the commercial production is still far away. The low yield and high market

price of the pharmaceutically important alkaloids of Vinca rosea viz. vincristine,

vinblastine and ajmalicine have created interest in improved alternative routes for

their production such as using cell and tissue culture. The callus developed on

Murashige and Skoog (MS) media supplemented with different concentrations of

auxins and cytokinins was found to have variable alkaloid contents.

As a promising alternative to produce plant secondary metabolites, plant cell

culture technology has many advantages over traditional field cultivation and

chemical synthesis, particularly for many natural compounds that are either derived

from slow growing plants or difficult to synthesize with chemical methods.52

More than 130 alkaloids have been isolated from different parts but the

important alkaloids are present in very low concentrations. Keeping these views;

researcher continuously using different approaches to enhance the level of important

alkaloid to meet the required demand. Considerable progress has been accomplished

in tissue culture techniques for production of indole alkaloids from Vinca rosea.

Vinca rosea produces several commercially valuable secondary metabolites including

17

the anti-cancerous vinblastine, vincristine and anti-hypertensive alkaloids ajmalicine

and serpentine. Bioengineering efforts to synthesize these indole alkaloids in plant

tissue cultures of Vinca rosea have yielded varying responses.53,54,55 The low yield of

dimeric indole alkaloids from the plant (approximately 0.0005%) and their

consequent high price have stimulated numerous efforts to develop alternative

strategies for their production and thishas encouraged intense research for alternative

methods for the production of these alkaloids, such as synthesis or semisynthesis56 ,

cell and tissue culture 57.

Nevertheless, for an industrial large-scale production the alkaloid levels need

to be improved. In order to reach this goal a better understanding of the regulation of

secondary metabolism is needed. Various methods have been tested with the aim of

improving alkaloid production.

Today India is the third largest manufacture of Vinblastine and Vincristine in

the world and is exporting these alkaloids to European countries. High demand and

low yield of these alkaloids in the plant has led to research for alternative means for

their production. Vinblastine is also modified structurally to yield deacetyl vinblastine

amide (Vindesine) introduced recently as Eldisine for use in the treatment of acute

lymphoid leukaemia in children. Biochemical coupling of alkaloids Catharanthine and

Vindoline to get dimeric compounds is also achieved.

Besides these, tissue culture techniques are developed for the development of

these dimeric alkaloids60 and the application of various biotechnological tools viz.

Optimization of Media Composition, Phytohormones, pH, Temperature, Light,

Aeration, Elicitors, Mutagenesis, High Cell Density Culture, Selection of Superior

cell lines, Bioreactors and Immobilization Methods, Hairy root culture, In Vitro

Somatic embryogenesis, Biosynthesis of alkaloids in Vinca rosea, Metabolic and

18

Genetic Engineering in alkaloids biosynthesis, Coupling method for Alkaloids

biosynthesis, Cellular Compartmentation for the enhancement of important secondary

metabolites present in different parts of Vinca rosea.

Vinca rosea is a renowned medicinal plant and is a rich source of alkaloids,

which are distributed in all parts of the plant. The alkaloid content of Vinca rosea

varies considerably in various parts; the maximum being in the root bark which

ranges from 0.15 to 1.34 % and even up to 1.79 in some strains.61 The plant contains

about 130 alkaloids of the indole group out of which 25 are dimeric in nature. Two of

the dimeric alkaloids vinblastine and vincristine mainly present in the aerial parts,

have found extensive application in the treatment of human neoplasma. Among the

monomeric alkaloids ajmalicine (raubacine) found in the roots has been confirmed to

have a broad application in the treatment of circulatory diseases, especially in the

relief of obstruction of normal cerebral blood flow. Vinblastine sulphate is used

particularly to treat Hodgkin’s disease besides lymphosarcoma, choriocarcinoma,

neuroblastoma, and carcinoma of breast, lungs and other organs in acute and chronic

leukaemia. Vincristine sulphate arrest mitosis in metaphase and is very effective for

treating acute leukaemia in children and lymphocytic leukaemia. It is also used

against Hodgkin’s disease, Wilkins’s tumour, neuroblastoma and reticulum cell

sarcoma.

Periwinkle organogenesis was first reported in the late 1970s by Dhruva et al

(1977)62 followed by Ramavat et al. (1978)63 and Abou-Mandouret al. (1979).64

However, the shoot regeneration rate was low. In 1989, Mollers and Sarkar65 induced

calluses from phytoplasma-infected stem tissues. These callus tissues differentiated

into plants on the MS medium with 0.25 mg•L−1 6-benzyladeine (BA), 1.0 mg•L−1

1-naphthalene acetic acid (NAA), and 10.0 mg•L−1 gentamicin. Further test

19

confirmed that phytoplasmas were eliminated. Recently, efficient plant generation of

periwinkle was accomplished mainly through somatic embryogenesis starting with

generative tissues such as anthers and immature zygotic embryos. Kim et al.

(1994)66induced somatic embryos from calluses derived from anthers in MS medium

supplemented with 1.0 mg•L−1 NAA and 0.1 mg•L−1 kinetin. Plants were also

regenerated from immature zygotic embryos in 2, 4-D containing MS medium

through a callusing phase (Kim et al., 2004)67. An efficient somatic embryogenesis

system has been established. Embryogenic calluses were developed from hypocotyls

and primary cotyledonary somatic embryos and somatic embryos were then

differentiated on medium supplemented with 1.0 mg•L−1 NAA and 1.5 mg•L−1 BA

(Junaid et al., 2007).68 However, the relatively high regeneration rate published was

achieved through somatic embryogenesis and only one cultivar was used.

20

1.4. Role of biotechnological approaches in Vinca rosea micropropagation and

enhancement of pharmaceutically active compounds being used in the treatment

of various diseases

Due to the pharmaceutical importance and the low content in the plant of

vinblastine and vincristine Vinca rosea became an important model system for

biotechnological studies on plant secondary metabolism. Researchers are focusing

their attention to enhance the alkaloids yield by various ways (chemically,

enzymatically, synthetically or by cell culture method).

The plant cell can be cultured at large scale69, but the yield of alkaloids

production is too low and limits commercial applications. In recent times, however,

two strategies have been commonly used for the enhancement of alkaloids.

a) In vitro cultivation of shoot via organogenesis and somatic embryogenesis, callus

or suspension by the optimization of media, phytohormones, temperature, pH, light,

aeration etc. In addition, high cell density culture, elicitor’s treatment, mutagenesis,

bioreactors and immobilization are also practiced to improve alkaloids yield.

b) Genetic engineering and over expression of biosynthetic rate limiting enzymes in

alkaloid biosynthesis pathways.

1.5. In-vitro Studies

In tissue culture, the response of culture has been influenced by a number of

factors which in turn regulate alkaloids yield. Some of them are discussed in brief

Media Composition: The yield of alkaloids in suspension culture is directly

influenced by the surrounding environmental conditions and genetic constitution of

the concerned plant material. Over the years efforts have been made in numbers for

optimization of culture media for better biomass and alkaloids production, some

patents have also been filed.70, 71, 72, 53 Carbon sources and inorganic compounds play a

21

significant role in indole alkaloid production. It was earlier reported that nitrogen and

phosphate both promoted growth but had an adverse effect on alkaloids yield73,74. The

inhibitory effect of nitrogen on alkaloid production has not always been observed.75

The effect of nitrogen on alkaloids production is dependent on carbon availability to

the cells which makes the carbon-to-nitrogen ratio (C/N ratio) an important factor to

be taken into account. By the determination of the cellular C/N ratio,76 three distinct

growth phases were identified: an active growth phase, an accumulation phase, and a

biomass decline phase (endogenous metabolism). They also noticed that phosphate

(0.56 Mm), nitrate (12.97 Mm) and low concentration of ammonia were beneficial for

maximum growth and increased alkaloids production. Similarly higher concentration

of sucrose only enhanced biomass, the optimized glucose (500Mm), ammonium and

phosphate (0- 12Mm) were previously used for higher alkaloids yield.77

Medium composition and day’s interval had direct effect on induction and

accumulation of indole alkaloids.78 A medium added with 6 % sucrose is favourable

for both biomass and alkaloids production in Vinca rosea.79 Liquid medium with 3-

6% maltose was also found to be highly effective for production of somatic

embryos.80 It has been reported81 that agitated liquid media added with BAP (1.0 mg/l)

was very productive for large-scale plant regeneration. Alteration in macro and

micronutrient of MS medium82 has also been used to promote growth and subsequent

alkaloid production.83

Surface methodology84, has been used for the rapid biomass growth and

increase in ajmalicine production in hairy root cultures. Similar results in cell

suspension culture have been noticed.77 Hairy root culture is a unique system, often

used for root specific indole alkaloids production.85 Recently, Batra et al86 have

observed an increase in growth and terpenoids indole alkaloids (ajmalicine and

22

serpentine) yield when left and right termini-linked Ri T-DNA gene integration were

made in hairy root cultures of Vinca rosea.

Temperature: For in-vitro study temperature range from 200C – 300C has been

considered best for better biomass and growth of cultures, but contradictory

information have been reported about the alkaloids yield. Temperature in low range

had inhibitory99, stimulatory100, and no effect on alkaloid yield. In the tested cell lines

under different temperature range (20°C, 25°C, 30°C), highest serpentine production

was101 recorded at 25°C and, no effect was recorded at temperature39,45 and 32°C

while in hair y root culture low temperature enhanced alkaloid yield.102

PH of Culture Medium: In-vitro biomass and alkaloid production are directly

influenced by the pH values of the medium; values with a range of 5.5-6.5 did not

have much effect on alkaloids yield. The value 5.5 was found to be optimum for

serpentine production.96 It has been reported97 that alkaloids produced by suspension

culture were stored in vacuole and simultaneously storage capacity changed as the

changes of pH in the medium and vacuole take place. Low and higher values of pH

were used to release intracellular alkaloids into the culture medium.98 It is quite

known that the optimized value (5.5-5.8) occasionally fluctuates during culture time

and influences in-vitro responses including alkaloid yield.

Light: Light is an important factor for both ex vitro and in-vitro morphogenetic study.

Its presence, absence, time and intensity directly influence anabolic and catabolic

processes, particularly secondary metabolism.99, 103 Most of the study of the effect of

light was made on serpentine and ajmalicine where serpentine content was directly

related to the intensity of light in Vinca rosea,104 same was true for vindoline105 and

however, another alkaloid catharanthine was decreased in the absence of light. But it

has also been reported75, that light did not affect yield but it affect the accumulation

23

site. However, 15h per day exposure instead of 24 improved serpentine accumulation.

Although, dark-grown culture was much better in comparison to light grown, where

serpentine and ajmalicine content were decreased (serpentine from 79%-14% and

ajmalicine 78%-18%). Gradual transfer of dark grown culture of Vinca rosea towards

the light increased serpentine content, however, continuous exposure of light

decreased serpentine level.101 It has been optimized that 12h light period91, for better

callus growth and alkaloid production, however, dark period more than 12h decreased

alkaloid contents. It has been found106, that an increased chloroplast number and

enhanced chlorophyll accumulation in response to light influenced serpentine

production. Besides, exposure of monochromic light such as blue (450 nm) or red (670

nm) did not affect growth and alkaloid accumulation, showed constant ajmalicine and

serpentine synthesis which decreased further under white light.91, 106

Phyto hormones: The role of plant growth regulators in alkaloids production of Vinca

rosea has been extensively studied, but the response varies with genetic makeup of

the used explant, type and quantity of phytohormones.71,87 The cytokinin applied

exogenously either alone or in combination with auxins to suspension cultured cells

enhanced alkaloids accumulation in tumorous and non-tumorous cell lines.88,89

Enzyme peroxidase play a significant role in alkaloids biosynthesis, addition of 2,4-D

to the culture medium however, reduced the peroxidase activity.90

An increase, 91 in vindoline and catharanthine concentration by using 0.1 mg/l

BAP and 0.1 mg/l NAA added MS medium had been reported. Exogenously supplied

cytokinin increased ajmalicine and serpentine content in untransformed callus from

cotyledons92. At the protein level it was shown that endogenously produced cytokinin

did not mimic the effect of exogenously applied cytokinin in Vinca rosea,93 and they

also noticed that the protein pattern of Ipt transgenic callus lines were insensitive to

24

exogenously used cytokinin. A28 KD polypeptide and simultaneous ajmalicine

accumulation was noted on omission of 2, 4-D in medium and by the use of NaCl

treatments.94, 95

Aeration: Different types of gases, mainly CO2 and ethylene, are usually evolved

within the culture. In many cases these gases reduce O2 level in close vessels, inhibit

plant culture growth and secondary metabolism. High dissolved oxygen and improved

gaseous permeability at aerated condition stimulated secondary metabolism as

observed by 107, when ajmalicine production was increased with high oxygen level.

Improved oxidative metabolism at rich O2 level is believed to be the reason for better

product conversion. Aeration has been provided in culture to influence the alkaloids

synthesis and to make it more efficient modern stirring devices have been employed

along with traditional shake flask.101,108,109,110,111 Different types of fermenters have

also been used; shikonin and ginseng, the two important secondary metabolites have

been commercially produced by the use of fermenters. Several researchers112, 113 have

suggested the use of bioreactors in secondary metabolites production in plant cell

culture of Vinca rosea. An impeller with a speed of 100 rpm was most appropriate for

the accumulation of alkaloids; however, higher impeller speed increased

callus/suspension growth. The rate of ajmalicine production was studied114 by using

different vessels including shake flask and bioreactors. He found that biomass was not

affected by different culture vessels; however, ajmalicine production was decreased

with over feeding of biomass in shake flask and fermenter.

Elicitors: New groups of triggering factors which are better known as elicitors have

been reported to stimulate the secondary metabolites.115 The substance used as

elicitors may be of biotic and abiotic in origin. Biotic elicitors include microbial

filtrates (Yeast, Pythium and other fungal filtrate), while abiotic elicitors comprise of

25

simple inorganic and organic molecules (vanadyl sulphate, oxalate, UV irradiation

etc.).

It has been reported116 that addition of Phytium aphanidermatum filtrate

increased the accumulation of phenolic compounds instead of alkaloids production.

Effect of different concentrations of Pythium vexans extract was studied by117, who

had noticed that low elicitor concentration increased serpentine production but no

effect was on catharanthine yield. Addition of nicotinamide (8.2 mm) in Vinca rosea

cell lines was used to enhance the anthocyanin accumulation.118 The extract of

Pythium aphanidermatum in a hormone free cell lines responded well and induced

enzymes {(TDC and anthranilate synthase (AS)} which catalyse the biosynthesis of

several intermediates and subsequently accumulated tryptamine.53 Several inorganic

compounds (sodium chloride, potassium chloride and sorbitol) had also a positive

effect on catharanthine accumulation.87

The addition of vanadyl sulphate119 to cell suspension culture increased

catharanthine, serpentine and tryptamine production but was concentration dependent.

At 25 ppm, catharanthine and ajmalicine were primarily accumulated, and at 50-75

ppm tryptamine accumulation was only noticed. Moreover, the effect of heavy metal

was studied120 where addition of copper (200µm) increased total indole alkaloid

accumulation which was correlated with decreased tryptamine concentration.

Several stress factors (fungal elicitor, vanadyl sulphate and potassium

chloride) were used and it was found that the alkaloids accumulation was

concentration dependent121 The optimal concentration of fungal elicitor, vanadyl

sulphate and potassium chloride into medium increased alkaloids accumulation,

however, higher concentration had toxic effects and resulted in the loss of cell

26

viability. Two fold increase in alkaloids yield was noticed added tryptophan, fungal

elicitor and vanadyl sulphate to the culture production medium.122

Exposure of 2, 2-azobis dehydrochloride (AAPH, an oxidative stress agent)

and UVB irradiation to Vinca rosea increased nicotinamide and trigolline content.123

Simultaneously phenylalanine ammonia lysate (PAL) activity was also increased. The

increase in PAL activity caused by 2µm AAPH was prevented by 0.1 mm 3-amino

benzomide, which is an inhibitor of poly (ADP-ribose) polymerase. This suggests that

nicotinamide and its metabolites function as signal transmitter in response to the

oxidative stress, since poly-polymerase has defensive metabolic functions. The level

of vinblastine and leurosine increased in response to irradiation with near (370 nm)

ultraviolet light124, 125 in shoot culture of Vinca rosea; however, catharanthine and

vindoline content were decreased. Leaves were more sensitive to dimeric alkaloid

accumulation in comparison to shoot, however, 126 near ultraviolet’s irradiation in

whole plant of Vinca rosea, accumulation of dimeric alkaloids was increased.

Yeast extract induces transcription of the biosynthetic gene encoding

strictosidine (STR) in cultured Vinca rosea cells and alkalinization of the culture

medium. The active principle from yeast extract was partially purified and found to be

of a proteinacious in nature.127 Age of culture is very important factor for the elicitor’s

to be effective;128 addition of elicitors is preferred after a few days of inoculation of

the culture when the cells are rapidly dividing.

Mutagenesis: Mutagenesis plays a potent role in the alteration of the genetic

constitution which leads to produce new varieties. Penicillium is the most classic

example, with many other successful cases. Process of mutagenesis in diploid plants is

very complex. Mutagenesis enhance alkaloids yield but the route of biosynthesis and

the necessary regulation procedure are not elucidated yet clearly. Therefore, mutation

27

at target site in duplicate is really difficult. In spite of several limitations in this

process, scientists in numbers have used mutagens.129 Some p-flurophynylalanine

resistant cell lines of Nicotiana tabacum and N. glauca accumulated higher level of

phenolics.129 In case of Vinca rosea, it was noticed that a tryptophan analog resistant

mutant accumulated catharanthine in both growth and production medium. Similarly

several research groups used x-rays where more serpentine was produced. Beside

these examples, some successful reports are available in other group of crops where

mutagenesis improved metabolic accumulation.

High Cell Density Culture: In order to increase secondary metabolites production,

high cell density culture feeding has been attempted with or without much success.

Study on Vinca rosea in relation to high cell density was found to be unsuccessful.

Ajmalicine production was very low when inoculam potential was increased to 2:8

from 1:9 mg/g.75 Moreover, 130 low-density cultures increased alkaloids yields. It has

also been remarked107 that low oxygen level and inadequate nutrient uptake are

among the possible causes for low metabolic accumulation during high cell density

culture.

Selection of Superior Cell Lines: Isolation and selection of superior lines from the

heterogeneous cell populations help to improve the yield of alkaloids. These cells

show genetic variability which was further diversified by the use of various mutagenic

agents. Ajmalicine and serpentine level were increased in Vinca rosea by the selection

of superior cell lines.131

Bioreactor and Immobilization: In tissue culture, for alkaloids production research

has been mainly focused on suspension culture which requires a rotatory shaker. For

large-scale production, however, large size culture vessel fermenter/bioreactor is most

important. In both types of systems a stirring device is provided for improved

28

aeration.75, 132,133There are several important vessels fitted with compressors which

provide filtered air. For plant culture growth and productivity, it is recommended that

bioreactors with low shear stress are much more suitable than those of high shear

stress. Bioreactors with improved mechanical designs are regularly introduced in

bioreactors industry with innovated impeller which helps to regulate shear

agitation.134

In Vinca rosea,immobilization of plants cells has been suggested for better

accumulation of terpenoids. 98,135,136,137 Immobilization not only maintains the cells

viable for a longer period of time but also helps in extracellular alkaloids

accumulation. Alginate mediated immobilized cells enhanced the accumulation of

tryptamide, ajmalicine and serpentine.131, 138 The use of agar and agarose are found to

be effective for long-term maintenance of cells. In the last few years surface

immobilization has been proposed using different types of matrices for large- scale

production of alkaloids.139,140 In some other cases, negative influence of

immobilization on cell was noticed;137 gel or matrices entrapment on polysaccharide

sheet is employed in many plant systems and in Vinca rosea it is fairly successful.

Hairy Root Culture: Root contains a variety of secondary metabolites which produce

alkaloids. High rooting can be induced by genetic transformation using

Agrobacterium rhizogenes. Induced roots grew with a faster rate in hormone free

medium with high accumulation of secondary metabolites in Vinca rosea.64 In

transgenic Vinca rosea root, a significant increase in ajmalicine and catharanthine was

noticed.26,141 Other groups used various types of bioreactors/fermenters to improve the

growth of hairy roots and then for better production of secondary metabolites.142, 76

29

In-vitro Somatic Embryogenesis: Although somatic embryogenesis (SE) has been

reported in a wide variety of plant genera17, 143 in Vinca rosea it has been reported for

the first time.80 Earlier, a preliminarily study on plant regeneration from immature

zygotic embryo was reported in Vinca rosea.65 The advantage of SE is that the initial

cell populations can be used as a single cellular system and their genetic manipulation

are easy and are similar to microorganisms.

1.6. ALKALOID BIOSYNTHESIS IN VINCA ROSEA

Beside alkaloids, many other secondary metabolites have been isolated from

Vinca rosea, which include monoterpenoids, glucosides (loganin, secologanin,

deoxyloganin, dehydrologanin) steroids (catasteron, brassinolides), phenolics,

flavaonoids and anthocyanins. Metabolites are in fact the end products of a complex

process comprising the involvement of several enzymes, genes, regulatory genes and

(transport through) intra-and inter-cellular compartments. The TIA (terpenoids indole

alkaloids) are condensation products of two biosynthetic routes which require

coordination of the amount of the intermediates supplied by both pathways. The

biosynthesis of vinblastine requires the participation of at least 35 intermediates, 30

enzymes, 30 biosynthetic agents, 2 regulatory genes and 7 intra and intercellular

compartments.

Study on the biosynthesis of alkaloids was performed at the end of the 1950s

for Vinca rosea. Plants were grown in an atmosphere containing1 CO2 and after the

extraction of alkaloids; many labeled alkaloids have been detected by using column

and paper chromatography. Among the isolated alkaloids vinblastine and vincristine

were found only in a very low quantity. Thereafter, to increase the level of vincristine

and vinblastine, cell cultures of Vinca rosea were used. Biosynthesis of alkaloids by

in vitro cell culture has the advantages to manipulate the physiological (rapid growth,

30

ease of precursor feeding, etc) and genetical process. During the biosynthesis of

alkaloids of Vinca rosea various types of proteinacious compounds have been

reported in different biosynthetic pathways.144-158,127,180

In alkaloids biosynthesis, the role of several enzymes have been discussed in

Vinca rosea, a few of them have been purified, identified, characterized and their

encoding genes were also cloned.159-184,,116 Feeding of terpenoids precursors to Vinca

rosea cell suspension cultures increased alkaloids production.130, 194,195 Addition of

tryptophan (0.5 Mm) to Vinca rosea cells resulted in high intracellular levels of

tryptamine but did not influence ajmalicine accumulation much.146 As in other

feedback inhibitions, product accumulation depend upon the product degradation and

this phenomenon has been reported in cell suspension culture of Vinca rosea.185-205

1.7 Economic aspects: The therapeutic and economic importance of the dimeric

alkaloids vinblastine and vincristine have stimulated the research on biotechnology of

Vinca rosea (see Table 4).

Table 4. Economic aspects of Vinca rosea alkaloids

Alkaloid Production by

cell cultures

(g1-1)

Market

price

(USS-kg-1)

Market Volume

(kg-year)-1 )

Ajmalicine 0.2 (SC) 2.000 5000

Vinblastine traces (ShC) 1,000.000 12

Vincristine traces (ShC) 3,500.000 1

*Data obtained from Verpoorte et al. 199158 and Verpoorte et al. 1993b130

Abbreviations: SC: cell suspension cultures: ShC: shoot cultures.

31

At present the production of these alkaloids by suspension cultures has not

been achieved. However, the monomeric alkaloid ajmalicine is also of pharmaceutical

interest (Table 6) and it can be produced by suspension cultured cells. The market for

ajmalicine has an annual demand of approximately 3600 kg with a market price of

US$ 2000 kg. The production costs of ajmalicine from natural source (Vinca rosea

roots) is estimated at US$ 619.kg -l (Verpoorte et al. 1991)69. Drapeau et al. (1987b)

59 calculated the production costs for 1 kg ajmalicine by means of large-scale

cultivation of Vinca rosea cells. Considering a yield of 0.6% ajmalicine on dry weight

basis, and of a production volume of 800 kg ajmalicine per year, the production costs

were calculated at US$ 3215 for an amount of biomass containing 1 kg ajmalicine.

The high costs of the biotechnological process were considered to be a result of the

low product formation rate (0.26 mg g-l). Van Gulik et al. (1988)207 also studied the

economical feasibility of ajmalicine production by cell suspension cultures. Their

calculation was based on a yearly production of 3000 kg ajmalicine with a yield of

0.9% on dry weight basis. The estimated costs were US$ 1500 for an amount of

biomass containing 1 kg of ajmalicine. Considering a specific growth rate twice as

high and ajmalicine yields 10 times higher the estimated cost was US$ 430 per

amount of biomass containing 1 kg ajmalicine. This cost is still considered high for

competing with the traditional method. The low productivity of the biomass was

considered the major factor hampering commercial production by large-scale

cultivation of Vinca rosea cells.

Different pharmacological studies and the traditional used proved the high

medicinal properties of Vinca rosea; which continuously being used in the treatments

of number of diseases. Various important alkaloid, mostly the monomers were

successfully identified in culture media with the enhanced yields; however the

32

commercial production is still far away. The main problem is due to the lack of

optimization of cultural conditions and several strategies leading with increased

accumulation of secondary metabolites. Detailed studies are required to know the

proper enzyme functions at various levels, product membrane permeability and

adsorption for improvements towards achieving a viable economic production

methodology. In addition, over-expression of enzymes and the genetic modification

could be very useful via organogenesis or somatic embryogenesis for the production

of desired levels of secondary metabolite.

33

Proposed Research Study

Higher plants continue to be important sources of biologically active substances

in spiteof developments in the field of synthetic chemistry. But production from

plants is always not satisfactory and therefore, there has been much interest in the

methods of biotechnological production. The technique of plant tissue culture is an

important biotechnological tool for study of wide ranging problems in the physiology

and biochemistry of higher plants and it offers an avenue to further evaluate and

exploit the metabolic potentialities of higher plants for the bioproduction of useful

plant metabolites.

Cell and tissue cultures of Vinca rosea have been studied for many years as an

important and alternative source of therapeutically potential alkaloids. A review of

literature indicates studies on improving the productivity of hairy root cultures. But

there is a need for extended studies on the biosynthesis of alkaloids. Hence it is

proposed to undertake a systematic study to explore the biosynthetic potentialities of

hairy root cultures of Vinca rosea with elicitors, permeabilizing agents, metabolic

inhibitors, and precursors.

34

The proposed plan of work was phased out on following lines:

1. Development / Propagation of hairy root cultures of Vinca rosea, extraction

procedures and TLC and HPLC profiles of various alkaloids of hairy root

cultures.

2. Estimation of growth and production kinetics of hairy root cultures.

3. Studies on the biosynthesis of IPP with various metabolic inhibitors like

lovastatin, chlorocholine chloride, diphenylamine, fosmidomycin, DL-

glyceraldehyde and sodium pyrophosphate.

4. Evaluation of the effect of ethylene and ancymidol determination of the effect

of the precursor’s loganin and tryptamine and organic compounds, on alkaloid

production.

5. Improving the Production of Ajmalicine, Serpentine and Catharanthine by

different elicitors and study the influence of permeabilization of hairy root

cultures for improving the production and release of alkaloids using Tween 20,

DMSO and Chitosan.