Phytohormones on cell growth and taxol accumulation via cell culture of taxus wallichiana zucc

VIET NAM NATIONAL UNIVERSITY – HOCHIMINH CITY INTERNATIONAL UNIVERSITY STUDIES OF THE EFFECTS OF NUTRIENT MEDIA AND PHYTOHORMONES ON CELL GROWTH AND TAXOL ACCUMULATION VIA CELL CULTURE

VIET NAM NATIONAL UNIVERSITY – HOCHIMINH CITY

INTERNATIONAL UNIVERSITY

STUDIES OF THE EFFECTS OF NUTRIENT MEDIA AND PHYTOHORMONES ON CELL GROWTH AND TAXOL

ACCUMULATION VIA CELL CULTURE OF

TAXUS WALLICHIANA ZUCC

A thesis submitted to The School Biotechnology, International University

In partial fulfillment of the requirements for the degree of

M.Sc in Biotechnology

Student name: PHẠM CAO KHẢI – MBT02003 Supervisor: A/Prof Tran Van Minh

ACKNOWLEDGEMENTS

I wish to express my sincere gratitude to all those who gave me the possibility to complete this thesis

My profound appreciation goes to my advisor Associate Professor Tran Van Minh for his tremendous suggestions, encouragement and expertise I sincerely thank for his confidence and faith on me throughout my research project

I would like to thank Institute of Tropical Biology for giving me the permission to commence my research project in the first instance, to do the necessary research work and

to use the laboratory equipments I am bound to all officers from the institute for their stimulating support

I would like to send special thanks to my colleagues from Key Laboratory of Plant Cell Biotechnology for their consistent support I am immensely grateful to Mr Nguyen Trung Hau and Mr Nguyen Phi Vien Phuong for sharing their knowledge in tissue culture, technical advice and precious comments throughout this period I would like to give my special thank to Miss Mai Thi Phuong Hoa and Mr Do Tien Vinh for supporting me to identify taxol content of cultures using HPLC in this study I am also thankful to Dr Thanh

Do for looking closely at my thesis writing and offering outstanding suggestions for improvement I would like to acknowledge the International University through Science and Technology Development Program funding that supported my study

Finally, I wish to thank my family for their love and encouragement which enabled

me to achieve this goal

TABLE OF CONTENTS

ACKNOWLEDGEMENTS i

TABLE OF CONTENTS ii

ABBREVIATION v

LIST OF FIGURES vi

LIST OF TABLES vii

ABSTRACT viii

CHAPTER 1: INTRODUCTION 1

1.1 Problem Statement 2

1.2 Background Study 3

1.3 Objectives 5

1.4 Contents of the study 5

1.5 Long-term Goals 6

CHAPTER 2: LITERATURE REVIEW 7

2.1 Background information of the Taxus sp Genus 8

2.1.1 Scientific classification 8

2.1.2 Taxus sp in Vietnam 9

2.2 Biosynthesis of Secondary metabolites in plant 13

2.3 Background of Paclitaxel 15

2.3.1 Chemical Structure 15

2.3.2 Discovery History and Application 15

2.3.3 Activity Mechanism of Taxol 18

2.3.4 Taxol Production 18

2.4 Taxol Biosynthesis 21

2.4.1 First step: Geranylgeranyl pyrophosphate (GGPP) biosynthesis 21

2.4.2 Second step: The incorporation of GGPP in taxol biosynthesis 22

2.5 Plant cell culture for producing secondary metabolites 23

2.5.1 Optimization of cultural conditions 25

2.5.2 Selection of high-producing strains 25

2.5.3 Precursor feeding 26

2.5.4 Elicitation 27

2.6 Taxus cell culture for producing taxol 27

2.6.1 Background information of Taxus cell culture 27

2.6.2 Two stage culture 31

2.6.3 Effect of some physical and chemical factors on cell growth and taxol accumulation of Taxus cell suspension 32

2.7 Application of bioreactor techniques for producing of secondary metabolites 37

CHAPTER 3: MATERIALS AND METHODS 38

3.1 Materials 39

3.1.1 Plant Material 39

3.1.2 Media Components and Preparation 39

3.1.3 Laboratory Facilities 42

3.2 Methods 43

3.2.1 Experimental scheme 43

3.2.2 Callus culture 44

3.2.3 Cell suspension cultures 46

3.2.4 Cell Growth Measurement 50

3.2.5 Data Collection 51

3.2.6 Statistical Analysis 53

CHAPTER 4: RESULTS AND DISCUSSION 54

4.1 Callus Culture 55

4.1.1 Effect of mineral media and phytohormones on callus induction and growth 55

4.1.2 Proliferation of callus biomass 61

4.1.3 Determination of kinetic of callus growth 66

4.2 Cell suspension culture 67

4.2.1 Establishment of cell suspension culture 67

4.2.2 Effect of phytohormones on cell suspension proliferation 69

4.2.3 Effect of organic compounds on cell suspension proliferation 71

4.3 Elicitation 75

4.3.1 Effect of the phenylalanine and elicitors on taxol accumulation 75

4.3.2 Effect of adding time of elicitor on taxol accumulation 81

4.3.3 Effect of exposure time with elicitor on taxol accumulation 82

CHAPTER 5: SUMMARY AND CONCLUSION 84

REFERENCE 87

APPENDICES 99

IAA Indole-3-acetic acid

MS Murashige and Skoog

NAA Naphthalene acetic acid

WPM Loyd & McCown (1980) medium

B5 Gamborg (1968) medium

2,4-D 2,4-dichlorophenoxy acetic acid

ISO International Organization for Standardization

LIST OF FIGURES

Figure 2.1 Taxus wallichiana Zucc grown using cutting technique 9

Figure 2.2 Main pathway for leading to secondary metabolite in plant 14

Figure 2.3 Important compounds in Taxus sp 15

Figure 2.4 Schematic representation of taxol mechanism of action 18

Figure 2.5 Total taxol biosynthetic pathway in Taxus species 22

Figure 3.1 Taxus wallichiana Zucc 39

Figure 4.1 Callus induction on different mineral media 56

Figure 4.2 Callus induction and growth on B5 media with different auxins59 Figure 4.3 Callus proliferation on B5 media with different auxins 62

Figure 4.4 Difference in callus proliferation between treatments of sucrose supplementation 64

Figure 4.5 Callus proliferation on B5 media with various organics 65

Figure 4.6 Kinetics of callus growth of Taxus wallichiana Zucc 66

Figure 4.7 Initiation of cell suspension cultures 68

Figure 4.8 Morphology of single cells and cell clusters 70

Figure 4.9 Cell suspension proliferation on medium B5 with different organics 73

Figure 4.10 Kinetics of cell growth of Taxus wallichiana Zucc in liquid medium 74

Figure 4.11 Difference in cell growth between treatments of elicitor supplementation 80

LIST OF TABLES

Table 3.1 Media formation of callus proliferation 45

Table 3.2 Media Formation of cell suspension proliferation 47

Table 4.1 Influence of 2,4-D and mineral media on callus induction 57

Table 4.2 Influence of NAA and mineral media on callus induction 58

Table 4.3 Influence of 2,4-D and NAA on callus proliferation 61

Table 4.4 Influence of sucrose on callus proliferation 63

Table 4.5 Influence of Coconut water and Glycine on callus proliferation 65

Table 4.6 Influence of 2,4-D and NAA on cell proliferation 69

Table 4.7 Influence of organic compounds on cell proliferation 72

Table 4.8 Influence of the phenylalanine and elicitors on cell growth and taxol accumulation 76

Table 4.9 Effect of adding time of elicitor on taxol accumulation 81

Table 4.10 Effect of exposure time with elicitor on taxol accumulation 82

ABSTRACT

Taxus wallichiana Zucc., an important medicinal plant, is the main source of

paclitaxel, a diterpene alkaloid of commercial interest for pharmacological properties

Toward the main objective of in vitro production of paclitaxel, the specific objectives of

this research were i) to investigate medium components for optimizing cell growth and paclitaxel accumulation via callus and cell suspension cultures ii) to enhance paclitaxel production using precursors and abiotic elicitors

For optimization of cell growth, high frequency of callus formation (100%) was obtained from young stem explants on WP solid medium, but B5 medium was the best one for callus growth supplemented with 2,4-D (4.0 mg/l) The friable and reddish, light brown callus masses collected for cell culture proliferated with high growth index (3.07) on medium B5 supplemented with 2,4-D (4.0 mg/l), NAA (2.0 mg/l), sucrose (30 g/l), glycine (10 mg/l) and Cw (10%) The growth rate of callus was highest between 14th and 28th day

of culture period, and the appropriate subculture time was day 35 favored optimal growth

of callus Subsequently, cell suspension cultures were established by transferring selected friable calluses to liquid medium for their further growth and enhancement of paclitaxel

biosynthesis Cell growth of Taxus wallichiana Zucc in liquid medium supplemented with

2,4-D (3 mg/l), NAA (3 mg/l), sucrose (30 g/l), Cw (10 %), Glycine (10 mg/l), Casein hydrolysate (1000 mg/l) increased significantly with high growth coeffection

For elicitation of paclitaxel biosynthesis, the suitable concentration of phenylalanine for paclitaxel production (0.518 mg/g DW) was 15 mg/l which was 1.254 times higher than the control The addition of O-chi in cell cultures strongly promoted the biosynthesis of paclitaxel whereas Yeast extract, Salycilic acid showed little effect O-chi was the most appropriate substrate for paclitaxel production and its optimal concentration was 5 mg/l for highest paclitaxel product (3.450 mg/g DW) which was 14.009 times higher than the control Subsequently, MJ for elicitation of paclitaxel production at the suitable concentration was 10 mg/l (1.658 mg/g DW) which increased paclitaxel content to 6.215 times higher than the control Moreover, time of elicitor addition on the culture medium determined was day 21 at stage of strong growth of cells and cell cultures were harvested

on day 27 Finally, paclitaxel content was highest (0.0413% of dcw) after 6 days of elicitor exposure

Chapter 1: Introduction

1.1 Statement of the problem

Taxus sp., a class of important medicinal plant, is the main raw source of

materials provided the bark and leaves for producing paclitaxel, a diterpene alkaloid that has a key role in cancer treatment The unique paclitaxel cytotoxicity mechanism promotes the assembly of tubulin and stabilizes the resulting microtubules Its activity against cancers of the ovary, breast, lung, esophagus, bladder, endometrium, and cervix, as well as Kaposi’s sarcoma and lymphoma, has been demonstrated in various clinical trials (Pezzutto, J., 1996) However, the main

limitations of Taxus sp are very slow growing and the low content of paclitaxel

(0.01% dry weight of bark) For example, the commercial isolation of 1 kg of Taxol

required about 6.7 tons of Taxus brevifolia bark, equivalent to 2,000 - 3,000 more

50-years-old trees (Hartzell, 1991; Croom, 1995; Suffness and Wall, 1995), and people need about more 1000 kg taxol per year to treat several kinds of cancer In addition, scientists recognized that harvesting trees would not provide a renewable source of this natural product because of death of whole trees This harvesting seriously affects ecosystem and biological diversity of Vietnam in recent years Devastation on floristic composition of primeval forest has pushed many valuable

plant species into their extinction Taxus wallichiana Zucc has been considered as a

rare species being in threatened risk and is gradually losing distribution areas Its quantity and quality is not guaranteed to develop and expand the species distribution (Nghia, 1999) On the other hand, it is due to its poor regeneration and requirement of severe conditions so the next generation is not virtually ensure continuity role This is a threat in the future (Tung et al., 1999)

In this situation, scientists have discovered efficiently alternative methods for producing taxol to meet the ever increasing demand of the market without

deforestation With the successful advances of in vitro technologies, plant cell and

tissue culture has become a powerful, promising stable and long-term alternative tool for the production of taxoids However, applications for enhancement of taxol biosynthesis are still the challenges for many scientists

The problems stem from the limited knowledge of paclitaxel biosynthesis pathway as well as the feasibility of proper cultivation methods In this case,

manipulation of inducing factors and bioprocessing strategies via cell culture of

Taxus wallichiana Zucc should be considered as appropriate solution to solve this

problem

1.2 Background Study

Taxus wallichiana Zucc narrowly distributes in some Asian countries such

as China, Myanmar, Nepal, Afghanistan, India, Philippines, and Vietnam (Chi V

V., 2004; Hop T., 2003) In Vietnam, Taxus wallichiana Zucc occurred in Khanh

Hoa province and some districts belong to Lam Dong province (Duc Trong, Don Duong, Lac Duong and Da Lat city) (Tien T V., 1997) distributed from 1300 to 1700m in height The habitat of this yew are in canyons with the dominant evergreen broadleaf trees, less coniferous trees, surrounded by three-leaf pines which tend to be toward the distribution zones of yew decreasing their population seriously

Environmental concerns associated with the harvest of yew trees, coupled with increasing demand, have prompted efforts to develop a more sustainable source of Taxol Consequently, alternative strategies to guarantee its supply have extensively been investigated, including (1) semi-synthesis from its natural precursor, (2) total synthesis, (3) production by fungi or bacteria, (4) gene cloning and (5) plant cell culture Although taxol has been prepared by total synthesis (Nicolaou K.C et al., 1994; Holton R.A et al., 1994), the process is not commercially viable Taxol can also be produced semi-synthetically from more abundant taxoids in Taxus needles However, extracting the semi-synthetic precursors is also very expensive and difficult

A more promising approach for the sustainable production of Taxol and

related taxanes is provided by plant cell and tissue cultures (Gibson et al., 1995)

The capacity of plant cell, tissue, and organ culture to produce and accumulate many of the same valuable chemical compounds as the parent plant in nature has

been almost recognized since the inception of in vitro technology The major

advantages of cell cultures include controlled environment for synthesis of bioactive secondary metabolites, independent from climatic and soil conditions; negative

eliminated (microorganisms and insects); it is possible to select cultivars with higher production of secondary metabolites; with automatization of cell growth control and metabolic processes regulation

Several studies have been intensively conducted on various Taxus species (T baccata, T brevifolia, T cuspidate, T chinensis, etc.) producing paclitaxel via calli

and cell suspension cultures using different explants and media (Christen et al., 1991; Fett-Neto et al., 1992; Wickremeshinhe and Arteca, 1993) Suspension cell culture was also studied (Yoon and Park, 1994; Son et al., 2000) Besides, the

protoplast culture of T yunnanensis, selecting cell line that have highly yield, were

researched (Ketchum and Gibson, 1994; and Zhong et al., 1995) Nevertheless, there is little attention on bio-processing strategies and metabolic engineering

within cell suspension cultures of T wallichiana, a precious medicinal plant in Lam

Dong province, containing high quantity of paclitaxel and 10-deacetyl baccatin III

in bark and needles

Generally, one main problem in the application of plant cell culture technology to secondary metabolite production is a lack of basic knowledge concerning biosynthetic routes and the mechanisms regulating the metabolite accumulation Recently, there have been some reports addressing this important issue in plant cell cultures through elicitation, cell line modification by traditional and genetic engineering approaches, as well as biochemical study

Elicitation is effective in enhancing metabolite synthesis in some cases, such

as production of paclitaxel by Taxus cell suspension cultures (Yukimune Y et al.,

1996) and tropane alkaloid production by suspension cultures of Datura stramonium (Bellica R et al., 1993) Increasing the activity of metabolic pathways

by elicitation, in conjunction with end-product removal and accumulation in an extractive phase, has proven to be a very successful strategy for increasing metabolite productivity (Brodelius P et al., 1993)

Indeed, the tissues and cells typically accumulate large amounts of secondary compounds only under specific conditions That means maximization of the production and accumulation of secondary metabolites by plant tissues and cells required (i) manipulating the parameters of the environment and medium, (ii)

selecting high yielding cell clones, (iii) precursor feeding, and (iv) elicitation The elicitors can be polymeglucan, glycoprotein, organic acids, fungal cells, or disadvantage conditions such UV light, heavy metal salts; methyljamonate, chitosan (Linden and Phisalaphong, 2000), abscisic acid (Luo et al., 2001), salicylic acid (Yu

et al., 2001)

1.3 Objective

This research focuses on the development of an efficient technique of callus

and cell suspension cultures by using young stems obtained from mature T wallichiana Zucc using cutting technique in experimental garden at the Institute of

Tropical Biology (ITB), Vietnam Academy of Science and Technology

This study aims to establish an appropriate procedure to upgrade the effect of

producing paclitaxel via cell culture of Taxus wallichiana Zucc Specially, the

objectives of this study are:

 To develop an efficient procedure for inducing Taxus wallichiana Zucc

callus from young stem explants and collection of suitable high-producing callus tissue

 To determine appropriate modified-medium for growing callus tissue

 To establish a fine cell suspension to be used as the material for paclitaxel biosynthesis elicitation culture

 To determine appropriate modified-medium for growing cell suspension

 To determine appropriate modified-medium and culture technique for enhancing paclitaxel biosynthesis

1.4 Contents of the study

Our primary aim is to develop a cultivation protocol for producing paclitaxel

via cell suspension of Taxus wallichiana Zucc The successful production of paclitaxel using in vitro technology in this study will be achieved in three steps: (i)

callus induction, subculture, and selection of appropriate callus types; (ii) cell suspension establishment and subculture; and (iii) enhancing paclitaxel biosynthesis

of cells

Precisely, the young stem explants will be introduced to MS, or B5, or WPM

optimal medium for callus induction and growth For further growth of callus, we also examined some organic compounds such as sucrose, coconut water and glycine

to optimize callus proliferation Subsequently, suspension cultures will be initiated using callus which has previously been maintained in solid medium for several times of subculture, by that way the callus will grow at a consistent rate, indicating

a successful adaptation to the medium Initiation and subculture of cell suspension will be done using different concentrations and combinations of plant growth regulators such as 2,4-D, NAA as well as different organics like malt extract, peptone, casein hydrolysate Finally, this study focuses on a number of strategies aimed at yield-improvement in cell cultures for paclitaxel production: (i) manipulation of inducing factors by elicitors; (ii) bioprocessing strategies by combining of inducing techniques, two-stage cultivation, and feeding of precursors

Chapter 2: Literature Review

2.1 Background information of the Taxus sp genus

Taxus was discovered and used thousands of years ago Previously, Taxus was considered as a kind of tree having the most dangerous toxicity Most of the poisonous situations in cattle were due to eating needles of these trees One can die

if he approximately eats 150 needles of Taxus Nowadays, scientists determine the main toxicants of Taxus being alkaloid compounds belong to the taxoid group (Toan

There are over 10 different species of Taxus distributed in the northern

hemisphere regions, from Americas to Europe and Asia included:

Besides, there are also some different species and cultivars of the hybrid

Taxus x media (T baccata and T cuspidate) (Seiki and Furasaki, 1996)

2.1.2 Taxus sp in Vietnam

In Vietnam, there are two species of Taxus: Taxus chinensis and Taxus wallichiana Zucc

2.1.2.1 Taxus chinensis (Pilg.) Rebd

This is a group of evergreen trees tall from 15 to 20m Their needles are dark-green (about 1.5 to 2 cm in length, 2 to 3 mm in width), acute at the tip, arranged spirally on the stem, but the leaf bases twisted to range the leaves in two flat rows either side of the stem They distributed in Northern provinces such as Hoa Binh, Son La, Lai Chau, Lao Cai, Ha Giang, etc often grow on cliffs at 1000 m elevation (Khoi N.D et al., 1997)

Vietnamese Institute of Chemistry has published chemical ingredients of

Taxus chinensis From its bark collected in April, 1993 at Mai Chau district, the

researchers at this institute have isolated and determined the structure of 10-deacetyl baccatin III which can be a promising group of derivatives having activities equivalent to taxol 10-deacetyl baccatin III (4mg) extracted from 500g of its leaves is a vital intermediate in the semi-synthesis of taxol and toxotere (Tri M

7-xylosyl-V et al., 1995)

2.1.2.2 Taxus wallichiana Zucc

Figure 2.1 Taxus wallichiana Zucc grown using cutting technique

 Discovery

In 1931, French botanists have collected specimens of Taxus species

determined Taxus baccata var wallichiana (Zucc.) Hook at high mountains in Da

Lat city (1500 m)

In June 1994, people found several tens of big Taxus trees (about 30 to 40 m

in height) in the remote forest on the granite mountain at altitudes over 1500m belong to Don Duong district, Lam Dong province Recently, some Taxus trees were also discovered in Da Lat and Nha Trang city Based on assessment of their templates of leaves and fruits, Prof Dr Shemluck (Quinsigamond College,

Worcester, Massachusetts, America) determined that these are Taxus wallichiana

Zucc derived from the Himalaya range (Khoi N D et al., 1997; Trang D D., 1994)

 Economic and pharmaceutical values

Biomass (leaves, twig, bark and roots) of all Taxus wallichiana Zucc

contains a unique class of diterpenoid alkaloids that are the material source for producing a chemotherapeutic drug (Taxol) used to treat a range of human cancers

In Europe and Asia, the wood of the trees were prized for bows and also valued for fine musical instruments, cabinets, and utensils (Vance and Rudolf, 1974; Hartzell,

1991) The bark of T wallichiana (Himalayan yew) was used for preparing

beverages and medicines (Purohit et al., 2001)

Yew is the economic kind of tree Yew wood is reddish brown and very springy, traditionally used for timber and other appliances (Khoi N.D et al., 1997) Its leaves were anciently used to treat a range of diseases such as asthma, bronchitis, hiccup, etc (Chi V V., 2004) Taxus is the endemic species of Lam Dong province but our current indiscriminate deforestation significantly decreased a number of

Taxus trees and caused this species at high risk of extinction T wallichiana was

listed as an endangered species in the Viet Nam Plant Red Data Book – Rare and Endangered Plants (Nghia N H., 1999; Tien T V., 1999)

 Biology of Taxus wallichiana Zucc

Taxus wallichiana Zucc having reddish-brown bark, sepia core of stem,

smooth trunk, and wide spreading branches is a group of evergreen trees high up to

30m (Khoi N.D et al., 1997) generally long-lived, frequently 250 to 500 years old Their leaves are alternative and arranged in 2 rows along with their stem forming one 60-90o angle to the axis of the leaf-bearing branches Upside of leaves are

green, but green yellow in their underside Taxus wallichiana Zucc are dioecious

and have cross-pollination The male cone is composed of petiolate capitula with squamae on the base It bears 6 to 14 scutellate stamens, each of which has 4 to 9 anthers The female cone bears an acrogenous ovule, held by discal collar at the base and several bracts on the bottom The seeds of pyreniform are globular, borne inside the red fleshy cotyloid arils and ripen within the same year (Chi V V., 2004; Hop T., 2003; Trang D D., 1994)

Taxus wallichiana Zucc grows very slowly and like the light and good

moisture, but need shade condition for germination and development of seed in the early years The flowering time is from August to December and ripening from August to December of the next year Dispersal distance of seed is not far by 6-8m

in radius to the center of the tree Seed germination and development where there is high humidity and average light intensity is suitable The more plant grows, the higher light intensity is required for its growth The thick-coated seed in deep dormancy needs 2.5 years to germinate, so their ability of regeneration under natural conditions and in cultivation is poor Propagation is slow, generally required two years for natural germination of seed, required two to three months for artificial propagation of rooted cuttings Cultivation in plantations was difficult with individual plants slow to establish (Dirr, 1998; Chi V V., 2004; Hop T., 2003)

 Eco-biological condition

Climate: Taxus distributed in low and middle mountain-tropical climate where there are two distinct seasons in the year The rainy season lasts from April to October with the average rainfall by 1600 - 1800mm, an average temperature of 20

 Distribution

The five Asian Taxus spp appeared from lowland to montane zones in cool

climates with moderate to high, evenly distributed precipitation (Farjon, 2001)

Taxus wallichiana Zucc narrowly distributes in some Asian countries such as

China, Myanmar, Nepal, Afghanistan, India, Philippines, and Vietnam (Chi V V.,

2004; Hop T., 2003) In Vietnam, Taxus wallichiana Zucc occurred in Khanh Hoa

province, some districts belong to Lam Dong province (Duc Trong, Don Duong, Lac Duong and Da Lat city) (Tien T V., 1997) distributed from 1300 to 1700m in height The habitat are canyons with the dominant evergreen broadleaf trees, less coniferous trees, surrounded by three-leaf pines which tend to be toward the distribution zones decreasing Yew population seriously

 Status

Three Taxus species native to China (T chinensis, T cuspidata, and T fuana) reported was listed under the National First Category All native species of Taxus in China are listed Class I, which prohibits the collection of yew without the authorization of the Chinese Government T wallichiana is listed as endangered in

the China Plant Red Data Book - Rare and Endangered Plants (CITES, 2004)

In Viet Nam, Taxus wallichiana Zucc has considered as a rare species listed

in the Viet Nam Plant Red Data Book being in threatened risk and is gradually losing distribution areas Its quantity and quality is not guaranteed to develop and expand the species distribution (Nghia N H., 1999) On the other hand, it is due to its poor regeneration and requirement of severe conditions so the next generation is virtually very little not to make sure continuity role This is a threat in the future (Tung L X et al., 1999)

The biochemists belong to Institute of Highland Biology successfully studied

chemical components of Taxus wallichiana Zucc and content of 10-deacetyl baccatin III was extracted from its leaves (0.04% DW) higher than that of Taxus chinensis in Hoa Binh Province Levels of 10-deacetyl baccatin III in the leaves

greatly vary depending on each of different individuals and significantly reduce in the rainy season (Phan N H T., 1998)

2.2 Biosynthesis of Secondary metabolites in plant

Plants produce more than 30000 types of chemicals, including pharmaceuticals, pigments and other fine chemicals, which is four times more than those obtained from microbes These compounds belong to rather broad metabolic group, collectively referred to secondary products or metabolites The secondary metabolites do not perform vital physiological functions as primary compounds such as amino acids or nucleic acids, but these are produced to ward off potential predators, attract pollinator, or combat infectious diseases

The ability to synthesize secondary compounds has been selected throughout the course of evolution in different plant lineages when such compounds addressed specific needs The biosynthesis of secondary metabolites is often restricted to a particular tissue and occurs at a specific stage of development When there is a demand, the secondary compounds are then degraded and the stored carbon and nitrogen recycled back into the primary metabolism The balance between the activities of the primary and secondary metabolism is a dynamic one, which will be largely affected by growth, tissue differentiation and development of the plant Those factors determining the location and accumulation of secondary products in the intact plant are applied for the production of secondary products in plant cell cultures because machenism of secondary metabolites biosynthesis is similar

Studies on the production of plant metabolites by callus and cell suspension cultures have been carried out on an increasing scale sine the end of the 1950’s The prospect of using such culturing techniques for obtaining secondary metabolites such as active compounds for the pharmaceuticals and cosmetics, hormones, enzymes, proteins, food additives, and natural pesticides from the harvest of the cultured cells and tissues is very feasible The large scale cultivation of tobacco and

a variety of plant cells was examined from the late 1950’s to early 1960’s initiating more recent studies on the industrial application of plant cell culture techniques in many countries

Plant cell culture offers many advantages over field grown materials Climate

does not affect in vitro systems thus production is possible anywhere in the world

diseases and insects, they are potentially a much more reliable renewable source Optimization of nutrient or gas composition, and addition of elicitors produce higher yields enabling to meet increasing demand of people For example, the production of shikonin by cultured cells is about 23% shikonin per gram of dry weight compared to 1.5% gram per dry weight found in the plant’s roots (Lambie, 1990) Additionally, cell cultures produce more consistent product quality within a less complicated mixture, thus making the process of isolation and purification more economical

Figure 2.2 Main pathway for leading to secondary metabolite in plant

Abbreviation: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP),

glyceraldehyde-3-phosphate (GAP), non-protein amino acids (NPAAs), Acetyl coenzyme

A (Ac-CoA).

2.3 Taxol (Paclitaxel)

2.3.1 Chemical Structure

Biomass (leaves, twig, bark and roots) of all Taxus species contains many different compounds divided into main groups: isoprenoids, flavonoids, phenylpropanoids and phenol derivatives Isoprenoids is the most important group including terpenoids and diterpenoids Diterpenoids is the key and specific molecular component of compounds called taxoid of Taxus species (taxane diterpenoid) including taxol and related derivatives (Phan N.H.T et al., 1998; Altstadt et al., 2001)

(a) 10-deacetyl baccatin III (b) Taxol (paclitaxel)

Figure 2.3 Important compounds in Taxus sp

The empirical formula of paclitaxel is C47H51NO14 with a molecular weight

of 853.9 Structure of paclitaxel contains one taxane core, four oxetane rings at the C-4 and C-5 position of the taxane ring, and one N-benzoyl-3-phenylisoserine side-chain at C-13 position Paclitaxel is a white to off-white crystalline powder It is highly lipophilic, insoluble in water, soluble in some organic solvents such as ethylic, methanol, chloroform, and dimethyl sulfoxid, and melts at around 216 - 217

°C

2.3.2 Discovery History and Application

At the beginning of the 20th century, the pharmaceutical industry of medicines depended almost entirely on the extraction of several plants and improving the analytical chemical techniques to isolate and purify these medicinal compounds in large amounts

In the early 1960’s, Jonathan Hartwell at the United States National Cancer Institute organized collection of plants from the U.S for evaluation as potential

sources of anticancer drugs In this program, plant samples collected at random were supplied by U.S Department of Agriculture under an interagency agreement with the NCI In August 1962, USDA botanist Arthur Barclay and three college student field assistants collected 650 plant samples in California, Washington, and

Oregon, including bark, twigs, leaves, and fruit of Taxus brevifolia in Washington

states These different explants were extracted with solvents to obtain their medicinal agents tested as possible anticancer drugs on an animal test system such

as rabbit, mice and guinea pigs

Monroe Wall and Mansukh Wani (1966) reported the extract of the bark

Taxus brevifolia to be highly cytotoxic The isolation of the active ingredient of this

extract was completed by June 1967 In 1971, Monroe Wall and Mansukh Wani were identifying the pure and crystalline active substance in Pacific yew that is responsible for the anticancer activity and they named the substance “taxol”, referring to the botanical name of Taxus They solved the structure of taxol by X-ray crystallography Taxol as a novel compound showed a unique activity against breast cancer in the tested animals

When taxol was found to exhibit excellent activity against B16 melanoma, it was finally selected as a development candidate Thus, preclinical development started in 1977 The subsequent observation of significant in vivo activity against several human tumour xenograft systems, including the MX-1 mammary tumor, provided further evidence of its superior spectrum of activity In addition, Susan Horwitz and coworkers demonstrated the unique mechanism of action of taxol, namely, promotion of tubulin polymerization and stabilization of microtubules against depolymerisation, raising more interest in application of taxol

Formulation studies were completed in 1980, after which toxicology studies started Following the completion of these preclinical studies, approval was granted for the initiation of phase I clinical trials in 1983 The early phase I trials progressed slowly because of the scarcity and difficult large-scale isolation of taxol, its poor aqueous solubility, which hampered the development of a suitable formulation, and

an unacceptably high incidence of severe hypersensitivity reactions These could be traced back to Cremophor EL®, a polyethoxylated castor oil used as a co-solvent in

the pharmaceutical formulation of taxol, and were largely suppressed by the application of antiallergic premedication and a 24-hour infusion schedual This then provided a sound basis for moving to phase II trials, which started in 1985 The observation of responses in patients with ovarian cancer and breast cancer led to an increased demand for this agent, which severed the supply problem

In an effort to obtain adequate supplies of taxol, the NCI issued a Cooperative Research and development Award (CRADA), which was awarded to Bristol-Myers Squibb in 1991 The company moved rapidly to obtain FDA (Food and Drug Administration) approval In December 1992, the FDA approved the use

of taxol for second-line treatment of metastatic carcinoma of the ovary Taxol then became a registered trademark of Bristol-Myers Squibb, which forced scientists to use paclitaxel as a generic name

At the moment, Taxol® has also been FDA approved for the second-line treatment of breast cancer, the second-line treatment of AIDS-related Kaposi’s sarcoma, the first-line treatment of non-small cell lung cancer in combination with cisplatin, the adjuvant treatment of node-positive breast cancer administrated sequentially to standard doxorubicin-containing combination chemotherapy, and the first-line treatment of advanced carcinoma of the ovary Many clinical trials, mainly focused on combination chemotherapy, are ongoing Paclitaxel is one of the leading anticancer agents in the clinic and the largest-selling anticancer drug of all time In

2000, worldwide sales of Taxol® increased to $ 1,592 million In 2001, U.S sales sharply decreased to $ 545 million because of generic competition, while international sales increased to $ 625 million

A semisynthetic analogue of paclitaxel, docetaxel, was developed by Poulenc Rorer (currently Aventis) in the 1980s It was shown to be twice as potent

Rhone-as paclitaxel in in vitro studies Following clinical trials, which began in 1990,

Docetaxel was FDA approved for the treatment of advanced or metastatic breast cancer in 1996 Docetaxel has now also been approved for the second-line treatment

of non-small cell lung cancer

2.3.3 Activity Mechanism of Taxol

In 1977, taxol’s mechanism of action was identified by Susan Horowitz and coworkers at Albert Einstein College of Medicine in New York City Taxol interfered with cell division by binding to the protein tubulin, which is a key factor

in mitosis Unlike some other anticancer drugs which prevented tubulin from assembling into microtubules, taxol has unique anticancer activity Taxol bound to assembled microtubules and blocked them from disassembling, stopping the process

of cell division and growth Taxol is found to induce apoptosis, a process through which cells die in a controlled manner, and also has anti-angiogenic properties by preventing a blood supply to the site of the cancer (Crown et al., 2000) Taxol is administered in the form of intravenous infusion Taxol preparation should be diluted before infusion in dextrose or ringer’s solution to a final concentration of 0.3

to 1.2 mg/ml The solution is chemically and physically stable for 27 hours under room temperature (AMA Council Report, 1985)

Figure 2.4 Schematic representation of taxol mechanism of action (David G 2001) 2.3.4 Taxol Production

All of the parts of Taxus brevifolia contain a unique class of diterpenoid

alkaloids that are the material source for producing a Taxol used to treat a range of

human cancers (Vidensek et al., 1990) From 1967 to 1993, almost all paclitaxel produced was derived from bark from the Pacific yew However, the most difficult problem encountered in paclitaxel production is a limited supply of initial materials

arising from its very low content in the bark of T brevifolia (only about 0.01 -

0.05% of dry weight) and very slow growth of the yew

This resource problem could be partially solved by isolating paclitaxel and its precursors from fresh branches and leaves instead of bark (Witherup, K.M et al., 1990; Eric, L.M et al.; 2000; Zu, Y.G et al., 2006; Fu, Y.J et al., 2008; Li, S.M et al., 2009), because branches and leaves are renewable resources and their biomass is larger than that of bark In addition, several other taxane including 10-DAXP, 10-DAB III, and cephalomannine have been obtained from the needles which can be used for semi-synthetic production of paclitaxel (Madhusudanan, K.P et al., 2002; Ballero, M et al., 2003; Mroczek, T et al., 2001)

There are four alternative means of obtaining paclitaxel without affecting the forest: (1) semi-synthesis from its natural precursor, (2) total synthesis, (3) production by fungi or bacteria, and (4) plant cell or organ culture

2.3.4.1 Fungal Resources

Stierle et al (1993) firstly reported a paclitaxel-producing endophytic

fungus, Taxomyces andreanae, which was isolated from yew trees Although the

yield of paclitaxel was low (24-50 ng/l), this finding stirred a great interest in biotechnologists Further, a few other reports on the isolation of paclitaxel-producing endophytic fungi have demonstrated that organisms other than Taxus species can also produce paclitaxel (Strobel, G et al., 1996; Wang, J.F et al., 2000)

So far, the greatest problem in fungal fermentation for paclitaxel production represents very poor and unstable yields The paclitaxel production by fungi does not exceed 70 μg/l of culture

2.3.4.2 Total Synthesis

Total synthesis of paclitaxel is a great challenge for organic chemists because

of four complicated rings (A, B, C rings and the oxetane ring) and 11 chiral centers

in the molecule Three total synthetic schemes have been completed: two by

Nicolaou K.C et al (1994) and Holton R.A et al (1994) and another one by Danishefsky S.J et al (1996)

Although the successful chemical total synthesis of paclitaxel is a great scientific achievement, it cannot be commercialized within a foreseeable future, because more than 20 steps are required, the chemical reagents are costly, the control of reaction conditions is difficult, and the production rate is very low (0.07% and 2.7% for the Holton and Nicolaou routes, respectively) The total synthesis is still too complex and expensive for industrial large scale production

2.3.4.3 Semi-Synthetical Production

An important biosynthetic precursor of paclitaxel, 10-deacetylbaccatin III,

was isolated in high yields from the needles of Taxus baccata in 1981 and from the bark of Taxus brevifolia in 1982 (ChauviBre, G et al., 1981; Kingston, D.G.I et al.,

1982) This compound serves as starting material for the semi-synthesis of paclitaxel through a coupling reaction with a protected side chain

The semisynthetic production of paclitaxel via the coupling of a phenylisoserine moiety with protected 10-DAB III has been extensively studied by many groups (Gueritte-Voegelein, F et al., 1986; Denis, J.N et al., 1988; Ojima, I

et al., 1992; Georg, G.I et al., 1993) The Ojima-Holton -lactam coupling method has been proven as the most efficient and versatile method among these coupling methods This semi-synthetic approach was a real break-through in the history of paclitaxel production (Kingston, D.G.I., 2001)

2.3.4.4 Plant cell culture

Considering the limitations of natural resources, feasible alternative methods for sustainable production of paclitaxel include plant tissue cultures (Hajnos, M.L

et al., 2001), cell suspension cultures (Michael, C.N and Susan, C.R., 2005; Yuan, Y.J et al., 2002; Huang, Y.F et al., 2005), hairy root cultures (Furmanowa, M and Syklowska-Baranek, K., 2000), and the induction of paclitaxel biosynthesis in cell culture systems (Wang, Y.D et al., 2004) Especially Taxus cell cultures have been considered as a promising means for paclitaxel production This technique can ultimately provide a continuous, reliable source of natural products

The major advantages of cell cultures include biosynthesis of secondary metabolites in controlled environment, independently from climatic and soil conditions, elimination of negative biological influences in the nature, selection of cultivars with higher production of secondary metabolites, and automatization of cell growth control and metabolic processes regulation, cost price can decrease and production increase

2.4 Taxol Biosynthesis

Taxol is a cyclic and polyoxygenated diterpenoid containing several functional groups and its biosynthesis begins with the formation of GGPP which is synthesized from three IPP molecules and the isomer dimethyl diphsophate (DMAPP) by the enzyme geranylgeranyl diphosphate synthase and putatively involves 19 additional steps However, several genes involved in the oxygenation of its core nucleus have not been identified The first committed step in taxane biosynthetic pathway is the conversion of geranygeranyl pyrophosphate (GGPP) to taxa 4(5), 11(12)-diene by taxadiene synthase (TASY) (Koepp et al., 1995; Ezekiel Nims et al., 2006)

2.4.1 First step: Geranylgeranyl pyrophosphate (GGPP) biosynthesis

The biosynthesis of GGPP are summarized into four main steps: (i) synthesis

of isoprene unit (IPP); (ii) repetitive fusion of isoprene in a sequence of elongation reactions to produce GGPP in the form of diphosphate; (iii) GGPP is then cyclized

by specific terpene synthases to yield different classes of terpenoid skeletons and that is considered as the first committed step in the formation of various terpenoid classes such as taxol; and (iv) followed by secondary enzymatic modifications steps (Buchanan B et al., 2000; Roberts S and Croteau R., 2001; Zhi-Hua L et al., 2006)

Terpene synthases (cyclases) operate at metabolic branch point involved in the regulation of terpenoid pathway flux and essentially catalyzes the first committed step leading to the various terpene classes (Gershenzon et al., 1993) Geranylgeranyl pyrophosphate (GGPP), the universal diterpene precursor, is cyclized into taxa 4(5), 11(12)-diene by taxadiene synthase (TASY), the first

committed enzymatic step in taxane biosynthetic pathway (Koepp et al., 1995 and Ezekiel N et al., 2006)

2.4.2 Second step: The incorporation of GGPP in taxol biosynthesis

After the biosynthesis of taxadiene from GGPP, taxadiene inter in a series of oxygenation, hydroxylation and combination with alkaloid-driven phenylalanine to form taxol as described in Figure 2

Figure 2.5 Total taxol biosynthetic pathway in Taxus species (Ezekiel N et al.,

2006)

The exact biosynthetic pathway to taxol and other taxanes may never be completely determined, however in the past few years much progress has been made

in discovering the building blocks are enzymes responsible for construction this complicated molecule While investigations were motivated initially by desire to understand biosynthesis as a means to increase yield in plant cell culture, a great deal of new knowledge has been collected due to the many ways in which the pathway to the taxanes has challenged accepted theories of plant biochemistry and

physiology

2.5 Plant cell culture for producing secondary metabolites

Plants produce more than 30,000 types of chemicals, including pharmaceuticals, pigments and other fine chemicals, which is four times more than those obtained from microbes However, natural habitats for medicinal plants are disappearing fast together with environmental and geopolitical instabilities; it is increasingly difficult to acquire plant-derived compounds The continued deforestation has caused a major threat to the plant species getting extinct over the years Clearly, the development of alternative and complimentary methods to whole plant extraction for secondary metabolites, especially medicinal value, is an issue of considerable socio-economic importance These factors have generated considerable interest in the use of plant cell culture technologies for the production

of pharmaceuticals (DiCosmo, F et al., 1989) The concept of plant cell culture includes the culture of plant organs, tissue, cells, protoplast, embryos, and plantlets The application of plant cell culture has three main aspects: the production of secondary metabolites, micropropagation, and the study of plant cell genetics, physiology, biochemistry and pathology

Plant cell culture is not affected by environmental, ecological or climatic conditions and cells can thus proliferate at higher growth rates than whole plants in cultivation The first step in plant cell culture is to develop a callus culture from the whole plant To maximize the formation of a particular compound, it is desirable to initiate the callus from the plant part that is known to be a high producer A suspension culture is then developed by transferring the relatively friable callus

masses into liquid medium and is maintained under suitable conditions of aeration, agitation, light, temperature and other physical parameters

However, plant cells in suspension cultures often undergo spontaneous genetic variation in terms of accumulation of secondary metabolites, which leads to heterogeneous population of cells in a suspension culture This variation, known as somaclonal variation, has posed a commercial obstacle to the large scale production

of secondary metabolites Therefore, establishment of a high yielding genetically stable cell line would provide a suitable means for the large scale production of plant metabolites Besides, the regulatory mechanisms of secondary metabolism have also been poorly understood As a result, yields of metabolites will be improved with proper understanding of regulatory mechanisms, plant cell differentiation, intracellular organization, and cell physiological characteristics Increasing awareness in metabolic engineering of various metabolic routes would eventually lead to improvement of product accumulation by the cultured cells (Memelink, J et al., 2001)

These new technologies will extend and enhance the usefulness of plants as renewable resources of valuable chemicals There has been a considerable interest

in plant cell cultures as a potential alternative to traditional agriculture for the industrial production of secondary metabolites (Dicosmo and Misawa, 1995) The advantage of this method is that it can ultimately provide a continuous, reliable source of natural products The major advantages of cell cultures include (i) biosynthesis of secondary metabolites in controlled environment, independently from climatic and soil conditions; (ii) elimination of negative biological influences

in the nature (microorganisms and insects); (iii) selection of cultivars with higher production of secondary metabolites; and (iv) automatization of cell growth control and metabolic processes regulation, cost price can decrease and production increase

Culture productivity is a vital aspect in the practical application of plant cell culture technology to production of plant-specific bioactive metabolites Until now, various strategies have been developed to improve the production of secondary metabolites using plant cell cultures The cultured cells typically accumulate large amounts of secondary compounds only under specific conditions That means

maximization of the production and accumulation of secondary metabolites by plant tissue cultured cells requires (i) manipulating the parameters of the environment and medium, (ii) selecting high yielding cell clones, (iii) precursor feeding, and (iv) elicitation

2.5.1 Optimization of cultural conditions

A number of chemical and physical factors like media components, phytohormones, pH, temperature, aeration, agitation, light affecting production of secondary metabolites has been extensively studied (Lee and Shuler, 2000; Wang et al., 1999; Fett-Neto et al., 1995; Goleniowski and Trippi, 1999) Several products were found to be accumulated in cultured cells at a higher level than those in native plants through optimization of cultural conditions A wide variety of explant and media compositions have been used with success for callus induction viz MS, B5,

LS, Blaydes (Blaydes, 1996) etc Variations and modifications of these media are widely used by adding vitamins, inositol, sucrose and growth regulators, etc especially auxin for cell division For example, ginsenosides by Panax ginseng

(Choi et al., 1994; Furuya et al., 1984; Franklin and Dixon, 1994; Furuya, 1988),

rosmarinic acid by Coleus bluemei (Ulbrich et al., 1985), shikonin by Lithospermum erythrorhizon (Takahashi and Fujita, 1991), ubiquinone-10 by Nicotiana tabacum (Fontanel and Tabata, 1987), berberin by Coptis japonica (Matsubara et al., 1989),

accumulated in cultured cells were much higher in content than those in the intact plants

2.5.2 Selection of high-producing strains

Plant cell cultures represent a heterogeneous population in which physiological characteristics of individual plant cells are different Synthesis of several products in high amounts using selection and screening of plant cell cultures have been described by Berlin and Sasse (1985) Cell cloning methods provide a promising way of selecting cell lines yielding increased levels of product A strain

of Euphorbia milli accumulated about 7-fold the level of anthocyanins produced by

the parent culture after 24 selections (Yamamoto et al., 1982) Selection can be easily achieved if the interested product is a pigment (Fujita et al., 1984) Cloned

faster and produced a higher amount of berberin were cultivated the strain in a 14l bioreactor The growth of selected cell line increased about 6-fold in 3 weeks and the highest amount of alkaloid was produced 1.2 g/l of the medium and the strain was very stable, producing a high level of berberin even after 27 generations

Increased capsaicin and rosmarinic acid in PEP cell lines of Capsicum annuum were

reported (Salgado-Garciglia and Ochoa-Alejo, 1990) Selective agents such as methyltryptophan, glyphosate and biotin have also been studied to select high-yielding cell lines (Amrhein et al., 1985; Watanabe et al., 1982; Widholm, 1974)

5-Cell lines of T baccata growing under the same conditions show differing

capacities for producing paclitaxel in suspension cultures (Brunakova K et al., 2004) It has been observed that the production of paclitaxel is more affected by differences in biosynthetic activity among the cultured lines than by any other factor (Bonfill M et al., 2006) Paclitaxel and baccatin III production in cell lines obtained

by mixing low-, medial- and high-producing cell lines was higher than the mean productivity of individual lines before mixing (Bonfill M et al., 2006)

Brunakova et al (2004) observed great variability of growth and paclitaxel content among callus cultures originating from the same type of explants of different mother plants or from different parts of the same mother plant Out of the nine well-growing callus lines established after 18 months of cultivation, only one showed improved production (23.2 g/g DW) In another study by the same group,

a cell line VI/Ha was selected and cloned after 20 months of callus initiation, achieving a paclitaxel production of up to 0.0109 ± 0.0037% on an extracted dry weight basis (Brunakova K et al., 2005)

2.5.3 Precursor feeding

Exogenous supply of a biosynthetic precursor to culture medium may also increase the yield of the desired product This approach is useful when the precursors are inexpensive The concept is based on the theory that any compound,

an intermediate, in or at the beginning of a secondary metabolite biosynthetic route, stands a good chance of increasing the yield of the final product Attempts to induce

or increase the production of plant secondary metabolites, by supplying precursor or intermediate compounds, have been effective in many cases (Silvestrini et al., 2002;

Moreno et al., 1993; Whitmer et al., 1998) For example, amino acids have been added to cell suspension culture media for production of tropane alkaloids, indole

alkaloids etc Addition of phenylalanine to Salvia officinalis cell suspension cultures

stimulated the production of rosmarinic acid (Ellis and Towers, 1970) Addition of

the same precursor resulted stimulation of taxol production in Taxus cultures Neto et al., 1993 and 1994) Feeding ferulic acid to cultures of Vanilla planifolia

(Fett-resulted in increase of vanillin accumulation (Romagnoli and Knorr, 1988) Furthermore, addition of leucine led to enhancement of volatile monoterpenes in

cultures of Perilla frutiscens, whereas addition of geraniol to rose cell cultures led

to accumulation of nerol and citronellol (Mulder-Krieger et al., 1988)

2.5.4 Elicitation

Plants produce secondary metabolites in nature as a defense mechanism against attack by pathogens Elicitors are signals triggering the formation of secondary metabolites Use of elicitors of plant defense mechanisms, i.e elicitation, has been one of the most effective strategies for improving the productivity of bioactive secondary metabolites (Roberts and Shuler, 1997) Biotic and abiotic elicitors which are classified on their origin are used to stimulate secondary metabolite formation in plant cell cultures, thereby reducing the process time to attain high product concentrations (Barz et al., 1988; Eilert, 1987; DiCosmo and Tallevi, 1985) Production of many valuable secondary metabolites using various elicitors were reported (Wang and Zhong, 2002a, 2002b; Dong and Zhong, 2001;

Hu et al., 2001; Lee and Shuler, 2000)

2.6 Taxus cell culture for producing taxol

2.6.1 Background information of Taxus cell culture

Researches towards the development of Taxus sp cell cultures began prior to the discovery of taxol Cultures of Taxus sp gameophyte and pollen were

established in 1953 by Larue and 1959 by Tuleke These early studies investigating

the organogenetic potential of Taxus sp microspores were followed up by Lepage and Degivry who published several papers related to the germination of Taxus sp

embryos in 1970 Rohr (1973) was the first scientist who developed callus cultures

and Plastira (1976) standardized mineral and phytohormone composition of the culture medium required for efficient proliferation of the calli generated from

mature stem explants in T baccata L Basal media such as Gamborg’s B5

(Gamborg et al., 1968; Wickremesinhe and Arteca, 1994), MS (Murshige and Skoog, 1962) and Woody Plant Medium (WPM) (Loyd and McCown, 1981) have been used for the initiation, proliferation and maintenance of the callus and cell suspension cultures in Taxus species It has been first reported the production of

taxol by Taxus sp callus culture in 1989 (Christen, 1991) Between 1991 and 1996 approximately 75 papers including 18 patents were published on Taxus sp cell and

tissue culture (Jaziri, 1996) Variability in growth response as well as taxol production in callus cultures derived from different genotypes has demonstrated (Brunakova et al., 2004)

Frequently, supplementation of various phytohormones as well as organic substances such as casein hydrolysate, polyvinyl pyrolidone (PVP), ascorbic acid and other essential amino acids such as glutamine, aspartic acid, proline, phenylalanine along with vitamins in the medium enhanced cell growth and proliferation Most of the research groups have observed that auxins (IBA, NA, 2,4-

D and picloram) at the varying level of 1.0 - 10.0 mg/l used in combination with cytokinins (BAP or Kinetin) at the level of 0.1 - 0.5 mg/l were effective for the callus initiation and growth Thuy Tien L T et al (2006) reported that the growth

of Taxus wallichiana cell suspension was best in culture media complemented

sucrose (30 g/l) and 2,4-D (5.0 mg/l), Kinetine (0.5 mg/l) Further, the callus

induction and growth of Taxus wallichiana Zucc was best on B5 medium

containing 4.0 mg/l 2,4-D and 1.0 mg/l Kinetin (Thanh Hien N T et al., 2004).The influence of plant growth factors on the rate of cell proliferation is complicated and depends on both the basal medium composition and plant genotype Inorganic compounds like VOSO4 have also been reported to be instrumental in enhancement

of the cell growth and multiplication in different Taxus species

A major problem associated with Taxus cultures is secrete phenolics, hampering the growth of calli and blackening or browning the medium and explants after a certain period that results in death of the cells (Gibson et al., 1993) Use of

anti-oxidants like PVP, citric acid, ascorbic acid, activated charcoal and frequent subcultures of cell cultures have been prevented ill effects of phenolics leach-outs The inoculum size, period of subculture, light intensity and photoperiod are also the key factors for the multiplication, phenotypic appearance and proliferation of Taxus cells (Wickremsinhe and Arteca, 1993; Navia-Osorio et al., 2002).

Several protocols have been reported for the production of some important taxoids and up-scaling of these cell cultures (Navia-Osorio et al., 2002) Almost every Taxus species has been studied in terms of the optimization of the growth media to yield maximum biomass (Mirajalili and Linden, 1996) Ketchum et al

(1995) formulated a new TMS growth medium for the callus cultures of T brevifolia Similarly, Fett-Neto et al (1992) studied the effect of various nutritional components on paclitaxel production in T cuspidata cell cultures

The kinetics of taxol production in plant cell culture has been extensively studied by numerous groups with varying results Srinivasan et al (1995) studied

the kinetics of biomass accumulation and paclitaxel production in T baccata cell

suspension cultures This study showed that there is a strong correlation between biomass production and taxol accumulation in callus cultures Paclitaxel has been found to accumulate in high amounts (1.5 mg/l of culture) in the second phase of

the growth curve A similar level of paclitaxel has also been observed in T brevifolia suspension cultures (Kim et al., 1995) Seki et al (1997) and Morita et al (2005) studied on plant cell cultures of T cuspidata for the continuous production

of taxoids In this case, addition of carbohydrates (sucrose and fructose) in the midway of the growth cycle increased the rate of the cell growth and paclitaxel accumulation Generally, the growth curve of cells grown without elicitors is biphasic in shape with taxane production reaching a maximum at the end of the growth cycle (Shuler, 1995; Gibsom, 1995)

Supplementation of biotic and abiotic elicitors in the cell suspension cultures

of Taxus has been affected the growth of cell biomass as well as paclitaxel production by pathway stimulation Several reports have shown that addition of methyl jasmonate in the second growth phase of suspension cultures, strongly

Ketchum et al., 1999; Phisalaphong and Linden, 1999; Mirajilili and Linden, 1996) Jasmonic acid (JA) in 100 M concentration was added at the 7th day after subculture also increased taxane content in the culture medium (Baebler et al., 2002) In addition, chitosan derived oligosaccharides were also used to stimulate the effect of methyl jasmonate in over-production (six-fold increase) of taxol in cultures

of Taxus canadensis (Furmanowa et al., 1997; Linden and Phisalaphong, 2000)

Paclitaxel and baccatin III production in suspension cultures of T media can

be improved by a two-stage culture method with adding methyl jasmonate (220 g/l FW) together with mevalonate (0.38 mM) and N-benzoylglycine (0.2 mM) Under these conditions, 21.12 mg/l of paclitaxel and 56.03 mg/l of baccatin III were obtained after 8 days of the culture in the production medium (Cusido et al., 2002)

Aspergillus niger, an endophytic fungus, isolated from the inner bark of T chinensis, added as an elicitor (40 mg/l) in the late exponential-growth phase,

resulted in more two-folds increase in the yield of the taxol and about a six-folds increase in the yield of the total taxoids (Wang et al., 2001) Addition of a trivalent ion of a rare earth element, lanthanum (1.15 to 23.0 M) also promoted taxol production in suspension cultures of Taxus species (Wu et al., 2001)

Ketchum et al (2003) reported that addition of carbohydrate during the growth cycle increased the production rate of paclitaxel, which accumulated in the culture medium (14.78 mg/l) It is observed that higher initial sucrose concentration

in culture medium repressed cell growth leading to a longer lag phase This could be overcome by a low initial sucrose concentration (20 g/l) and subsequent sucrose feeding (fed-batch culture) resulting in a high taxane yield of 274.4 mg/l (Wang et al., 2000) The studies of addition of different amino acids to the culture medium promote to increase the taxoid production in these cultures, and phenylalanine was

found to assist in maximum taxol production in T cuspidata cultures (Long and

Caroteau, 2005) It has also been shown that initial addition of 1.0 - 2.0 mM phenylalanine into the medium, followed by addition of 73.0 mM sucrose and 173.3

mM mannitol at the 28th day of culture, strongly promoted cell growth and taxoid production