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 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 January, 2013 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. i 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 ii 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 iii 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 iv ABBREVIATIONS 2,4-D 2,4-Dichloropheonoxyacetic acid 2iP 2-isopentyladenine BA 6-Benzylaminopurine 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 Cs Colleagues Cw Coconut water YE Yeast Extract O-CHI Oligo Chitosan CH Casein hydrolysate MJ Methyl jamonate SA Salycilic acid PE Peptone ME Malt Extract DW Dry cell weight Ki Kinetin (6-Benzyl-aminopurine) LV Litvay (1985) PVP Polyvinylpyrrolidone v 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 vi 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 vii 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. viii ix Chapter 1: Introduction 1 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, 2 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 biological influences that affect secondary metabolites production in the nature are 3 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) 4 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 supplemented with different auxin (2,4-D or NAA) concentrations to determine 5 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. 1.5 Long-term Goals This study will provide a protocol to produce paclitaxel derivatives via Taxus wallichiana Zucc. cell culture in a shake-flash scale under laboratory conditions. Furthermore, this result is the bachground applied to produce paclitaxel on industrial scale for high productivity. Its success will ensure steady supply of paclitaxel compound to the market all year round as well as establish a proper protocol to prevent this precious medicinal plant genus from extinction by collection of its bark and whole plant for producing paclitaxel. 6 Chapter 2: Literature Review 7 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 P. N. H. et al., 1998). 2.1.1 Scientific classification Kingdom Plantae Subkingdom Tracheobionta Superdivision Class Spermatophyta Pinopsida Order Taxales Family Genus Taxaceae Taxus There are over 10 different species of Taxus distributed in the northern hemisphere regions, from Americas to Europe and Asia included: - Taxus brevifolia - Taxus baccata - Taxus cuspidate - Taxus chinensis - Taxus globosa - Taxus marei - Taxus wallichiana - Taxus sumatrana - Taxus floridana - Taxus canadensis Besides, there are also some different species and cultivars of the hybrid Taxus x media (T. baccata and T. cuspidate) (Seiki and Furasaki, 1996). 8 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 7-xylosyl10-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. V. et al., 1995). 2.1.2.2. Taxus wallichiana Zucc. Figure 2.1. Taxus wallichiana Zucc. grown using cutting technique 9  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 10 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 °C, and 80 - 90% in humidity. Soil: Taxus only grows best in well-drained acidic grey brown soil, yellow soil and yellow brown soil which have light mechanical components. In cultivation, Taxus spp. noted as requiring fertile soils, ample moisture and excellent drainage (Dirr, 1998). 11  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). 12 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. Furthermore, as cell culture systems are not affected by the environment such as 13 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). 14 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 sidechain 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 15 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 Xray 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 16 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 RhonePoulenc Rorer (currently Aventis) in the 1980s. It was shown to be twice as potent 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. 17 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 18 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, 10DAB 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 paclitaxelproducing 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 19 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. 20 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 21 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) 22 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 23 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 24 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 cells using cell aggregates of Coptis japonica (Yamada and Sato, 1981) which grew 25 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 5methyltryptophan, glyphosate and biotin have also been studied to select highyielding cell lines (Amrhein et al., 1985; Watanabe et al., 1982; Widholm, 1974). 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; 26 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 (FettNeto et al., 1993 and 1994). Feeding ferulic acid to cultures of Vanilla planifolia 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 from different explants of T. baccata including microspores. Subsequently, David 27 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, 2,4D 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 28 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 promoted taxane biosynthesis (Menhard et al., 1998; Yukimune et al., 1996, 2000; 29 Ketchum et al., 1999; Phisalaphong and Linden, 1999; Mirajilili and Linden, 1996). Jasmonic acid (JA) in 100 M concentration was added at the 7 th 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. 30 The variable amount of taxoids in callus lines of different Taxus species or the same Taxus species could be explained on the basis of the fact that secondary metabolite accumulation occurred due to dynamic equilibrium between the product formation, transport, storage, turnover and degradation of compounds. Taxol production can be enhanced by the use of self-immobilized cell aggregates, free and calcium alginate gel particle immobilized cells (Xu et al., 1998). In Viet Nam, there had been several studies of Taxus wallichiana ZUCC. cell and tissue culture (Taxus species belongs Lam Dong province) such as: Effect of sucrose, glucose, and fructose in proliferation cell suspension cultures (Tan Nhut D. et al., 2006); Primary studies on the callus induction and growth (Thanh Hien N. T. et al., 2004); Studies of parameters on growth of cell suspension cultures (Thuy Tien L. T. et al., 2006). However, there still exists lack of the studies of elicitors to increase Taxol content in cell cultures of Taxus wallichiana ZUCC. Besides, optimization of parameters in culture medium is very important to produce biomass in large scale for Taxus wallichiana ZUCC. cell lines such as pH, temperature, stirring speed, gas exchange which facilitate to growth and accumulate secondary metabolites still required at this time. 2.6.2 Two stage culture A two-stage suspension culture of T. media cells was carried out in a 5-l stirred bioreactor running for 36 days (Cusido, R. M. et al., 2002). The first stage, using a cell-growth medium, was continued until the cells entered their stationary growth phase on day 12. The second stage was then implemented using a production medium supplemented with the elicitor MJA (220 g/g fresh weight) and two putative precursors, mevalonate (0.38 mM) and N-benzoylglycine (0.2 mM). Under these conditions, 21.1 mg/l paclitaxel and 56.0 mg/l baccatin III were obtained after 8-d culture in the production medium. Choi et al. (2000) investigated the effects of temperature shift on cell growth and paclitaxel production to optimize a suspension culture of T. chinensis. Cell growth was optimum at 24°C while paclitaxel synthesis reached its maximum at 29°C. To minimize the inhibitory effect of the higher temperature on cell growth, the temperature was shifted to 29°C after maintaining the culture at 24°C for a certain time. Paclitaxel production increased dramatically, 31 reaching its maximum of 137.5 mg/l with an average productivity of 3.27 mg/l over 42 days. It was assumed that the metabolite flux for paclitaxel biosynthesis could be stimulated in the temperature range of 28-32°C. However, further enzymatic and metabolic evaluations are needed. In the Taxus chinensis cell line, paclitaxel has been found to exist mainly in the intracellular region, with most of the paclitaxel being deposited in the cell wall (Choi, H.K. et al., 2001). This finding can explain how cells that produce a high concentration of paclitaxel can survive under such a toxic condition without releasing paclitaxel. 2.6.3 Effects of some physical and chemical factors on cell growth and taxol accumulation of Taxus cell suspension 2.6.3.1 Light Light plays an important role in the growth, differentiation, tissue organization and production of certain primary and secondary metabolites. The spectral quality, intensity and period of light irradiation may affect plant cell cultures in one aspect or another (Zhong J.J. et al., 1991). Light was observed to have a marked influence in the cell growth and accumulation of secondary metabolites. Various research groups have demonstrated the stimulatory effect of light irradiation on the formation of compounds such as anthocyanins, vindoline, catharanthine, and caffeine in cell suspension cultures (Zhong J.J. et al., 1991; Kurata, H. et al., 1991). 2.6.3.2 Temperature Temperature significantly affects the growth of Taxus sp. cell suspension and taxol accumulation of cells. The rate of cell suspension growth of Taxus chinensis was the highest at 24 oC but was seriously reduced at higher temperature. Increase of temperature will cause a heat shock influence on cell activities (Choi et al., 2000). 2.6.3.3 Osmotic pressure Under osmotic stress, cell physiology changes drastically. Osmotic stress mimics drought stress, and can induce secondary metabolism in plant cells or foreign protein expression by genetically engineered cells (Terashima, M. et al., 1999 and Lee, J.H. et al., 2002). The effect of osmotic pressure on paclitaxel production was investigated in suspension cell cultures of T. chinensis (Kim, S.I. et 32 al., 1991). The highest paclitaxel production yield was achieved at an initial concentration of 60 g sucrose g/l (300 mOsm/kg). High osmotic pressure conditions generated by non-metabolic sugars such as mannitol or sorbitol and the addition of a non-sugar osmotic agent, polyethyl-eneglycol (PEG), also enhanced paclitaxel production. 2.6.3.4 Elicitors 2.6.3.4.1 Mechanism of Elicitation in Plant Cells Elicitors are substances from various sources that can induce physiological changes of the target living organism. In a broad sense, ‘elicitor’ that initiates or improves the biosynthesis of specific compounds and elicitation is a process of induced or enhanced plant biosynthesis of secondary metabolites associated with plant defense mechanisms (Radman R. et al., 2003). Such interactions usually result in an increase of the production or release of secondary metabolites (Zhao et al., 2005). Elicitors can be categorized into two major groups: biotic and abiotic. The biotic elicitors range from macromolecules such as oligosaccharides (e.g., chitin, chitosan) polysaccharides derived from plant cell wall (e.g. pectin or cellulose), micro-organisms, phospholipids and glycoproteins, to small molecules such as hydrogen peroxide, ethylene, methyl jasmonate, and salicylic acid. Abiotic elicitors usually refer to inorganic salts such as mercuric chloride (HgCl2), copper sulfate (CuSO4), calcium chloride (CaCl2), and vanadyl sulfate (VSO4) including mechanical stress agents such as ultraviolet radiation, wounding and chemicals that disturb membrane integrity. These elicitors interact with plant cells through different and complex mechanisms (Darvill and Albersheim, 1984; Benhamou, 1996). Elicitors have been used as an important means of enhancing the production of taxanes in cell cultures of Taxus species (Srinivasan V. et al., 1996; Yukimune Y. et al., 2001; Wu J. et al., 2001; Ciddi V. et al., 1995). The transduction of elicitor signals in plant cells may a useful mechanism in secondary metabolite production. The secondary messengers are generated and led to the activation of protein kinase cascades which may activate the biosynthetic ability for specific plant products. A mechanism for biotic elicitation in plants may 33 be summarized on the basis of elicitor-receptor interaction. When plant or plant cell culture is challenged by the elicitor a rapid array of biochemical responses occur in several steps described below (Radman et al., 2003). - Binding the elicitor to the plasma membrane receptor - Changes in ion flux across the membrane: Ca2- influx to the cytoplasm from the extracellular environment and intracellular Ca2- reservoirs; stimulation of K- and Clefflux - Rapid changes in protein phosphorylation patterns, protein kinase activation, mitogen activated protein kinase (MAPK) stimulation, G-protein activation - Synthesis of secondary messengers, Ins P3 and diacyl-glyceral (DAG), in which, mediating the intracellular Ca2- release and nitric oxide and octadecanoid signaling pathway - Cytoplasm acidification caused by H+ - ATPase inactivation, decrease in membrane polarization and extracellular pH increase - Activation of NADPH oxidase responseible for ROS and cytosol acidification - Cytoskeleton reorganization - Production of ROS such as the superoxide anion and H2O2 that might have a direct antimicrobial effect as well as contributing to the generation of bioactive fatty acid derivatives and being involved in the cross-linking of cell-wall-bound prolinerich proteins. H2O2 can act as a secondary messenger and it is involved in the transcriptional activation of defense genes - Accumulation of defense-related proteins or pathogenesis related proteins such as chitinases, glucanases and endopolygalacturonases contribute to the release of signaling pectic oligomers (endogenous elicitors), hydroxyproline-rich glycoproteins and protease inhibitors - Cell death at the infection site (hypersensitive response) - Structural change in cell wall (lignification of the cell wall, callus deposition - Transcriptional activation of the corresponding defence-response genes - Plant defense molecules such as tannins and phytoalexins are detected 2-4 hours after stimulation with the elicitor 34 - Synthesis of jusmonic and salicylic acids as secondary messengers - Systemic required resistance However, the study of the chronological order of these events and the interconnection and orchestration between them is complex and still under investigation. 2.6.3.4.2 Studies of elicitors in plant cell and tissue culture for taxol production Several researches have focused mainly on the biotic elicitors while the effects of abiotic elicitors on over production of secondary metabolites in plants are poorly understood. The elicitation is hypothesized to involve in the key messenger Ca2-, factors affecting cell membrane integrity, inhibition or activation of intracellular pathways and changes in osmotic pressure by acting stress agent (Radman et al., 2003). The primary reactions upon elicitation with a biotic elicitor are the recognition of the elicitor and its binding to a specific receptor protein on the plasma membrane and, the next step, inhibition of plasma membrane ATPase that reduces the proton electrochemical gradient across this membrane (Dörnenburg and Knorr, 1995). Chitin and chitosan are effective elicitors that are extensively used. Chitosan treatment greatly increased the membrane permeability of suspension cultured cells (Brodelius et al., 1989). Production of phytoalexins and the generation of hydrogen peroxide are also responses of plant elicited with chitosan. There is evidence on the signal transduction pathways involved in the elicitor actions. It has been reported that chitosan stimulates the accumulation of jasmonic acid, a signal molecule related to defense-gene regulation. Morever, treatment cultures of Rubia tinctorum enhanced the anthraquinone production (Vasconsuelo et al., 2004). The accumulation of paclitaxel and related taxanes in Taxus plants is thought to be a biological response to specific external stimuli (Yukimune Y. et al., 2001) and jasmonates have been reported to play an important role in a signal transduction process that regulates defense genes in plants (Farmer E.E. et al., 1990; Reymond P. et al., 2004; Zhao J. et al., 2005). It has been proposed that jasmonates are key signal transducers leading to the accumulation of secondary metabolites (Gundlach H. et al., 1992; Hu F.X. et al., 2008). Methyl jasmonate has been used to increase 35 paclitaxel production in cell cultures of T. canadensis (Phisalaphong M. et al., 1999; Linden J.C. et al., 2000; Senger R.S. et al., 2006) and T. cuspidata (Mirjalili N. et al., 1996). The biosynthesis and accumulation of paclitaxel and related taxanes in T. baccata are strongly promoted by jasmonic acid or its methyl ester (Bonfill M. et al., 2006; Moon W.J. et al., 1998; Yukimune Y. et al., 1996). The addition of methyl jasmonate to the culture medium has increased the production of paclitaxel (0.229%, 48.3 mg/l) and baccatin III (0.245%, 53.6 mg/l) in cell cultures at week 2 compared to the control, which yielded only 0.4 mg/l of both secondary compounds (Yukimune Y. et al., 1996). Furthermore, Miroslawa et al. (1997) reported that the taxol concentration under the elicitation of methyl jasmonate at 100 M was 38 times higher than that in the control culture without elicitor addition. Moon et al. (1998) reported that the time course of taxane production after methyl jasmonate addition differed from normal kinetics without elicitation. Baccatin III and 10deacetyl baccatin III were detected first, followed sequentially by paclitaxel, 10deacetyl taxol and cephalomine. Other abiotic elicitors such as vanadyl sulphate, silver nitrate, cobalt chloride, arachidonic acid, ammonium citrate, and salicylic acid have also been used to improve taxane production in T. baccata cell cultures. It was found that the addition of vanadium sulphate (VSO4) to the culture medium significantly stimulated callus growth as well as taxol and baccatin III content at the end of culture period (Cusidó R.M. et al., 1999). Cell suspension cultures grown from a selected callus line supplemented with 0.05 mM vanadium sulphate were shown to enhance the production of taxol and baccatin III by 2.5 times (5.2 - 13.1 g/g DW) and 3.6 times (4.4 - 16.0 g/g DW), respectively (Cusidó R.M. et al., 1999). A biotic elicitor from Rhyzopus stelonifera fungus (25 mg/L) used in combination with methyl jasmonate (10 mg/l) and salicylic acid (100 mg/l) was shown to improve taxol production 16fold when added at day 25 - 30 of culture to a growth medium (Khosroushahi A.Y. et al., 2006). 36 2.7 Application of bioreactor techniques for large scale of producing of secondary metabolites After optimizing the culture conditions and environmental and physical factors, the next step for large scale production of valuable secondary compounds is to scale up the culture. Bioreactors are needed to apply to large scale production because they allow a greater control of culture conditions. This is a critical step as various problems can arise when scaling-up from shake flasks to bioreactors. However, bioreactors operate as a biological factory for the production of high-quality products and provide many advantages listed as follows (Tautorus et al., 1991; Fulzele, 2000; Su, 2006). •Increased working volumes, •Homogeneous culture due to mechanical or pneumatic stirring mechanism, •Better control of cultural and physical environment, therefore easy optimization of growth parameters such as pH, nutrient media, temperature, etc. for achieving metabolite production, •Reproducible yields of end product under controlled growth conditions, •Enhanced nutrient uptake stimulating multiplication rates and yielding a higher concentration of yield of bioactive compounds, •Simpler and faster harvest of cells, •The opportunity to perform biosynthetic and/or biotransformation experiments related to metabolite production with enzyme availability. •Easier separation of target compounds because of lower complexity of extract, •Better control for scale-up. Although the use of bioreactors has been directed mainly for cell suspension cultures and secondary metabolite production, researches directed at improving bioreactors for somatic embryogenesis has been reported for several plant species as well. Bioreactors have also been used for the cultivation of hairy roots mainly as a system for secondary metabolite production (Ziv, 2000). Furthermore, innovative processes have been proposed for producing secondary metabolites selectively by enzymatic reactions (Takemoto, 2009; Abraham et al., 2005). 37 Chapter 3: Materials and Methods 38 3.1 Materials 3.1.1 Plant Material The 25-day-old stems and leaves were obtained from mature Taxus wallichiana Zucc. grown using cutting technique in experimental garden at Institute of Tropical Biology, Ho Chi Minh city, Viet Nam. Figure 3.1. Taxus wallichiana Zucc. 3.1.2 Media Components and Preparation Screening and optimizing of the culture medium are key factors for success in cell and tissue culture. In this study, stock solutions of the major components, such as inorganic salts, organic compounds, vitamins, and plant growth regulators were prepared and stored in refrigerator. 3.1.2.1 Inorganic Salts The Murashige and Skoog (1962), Gamborg (1968, B5), and Woody Plant Medium (WPM, 1981) inorganic salts were used as basal media for callus formation and maintenance; and then the best one for callus growth was used for all next experiments. The formation and composition of MS, B5, WP media were given in Appendix 1. Stock solutions of their macronutrients, micronutrients, vitamins, and amino acids were prepared and used the required amount during medium preparation. The NaFeEDTA stock was protected from light by storing in an amber glass bottle. 3.1.2.2 Plant Growth Regulators Auxin and cytokinins were the two major phytohormones used in different concentrations and combinations in various media for induction and multiplication of callus, cell suspension culture. 39 Auxins (2,4-D (2,4-Dichlorophenoxyacetic Acid), NAA (α-Naphthalene Acetic Acid)) and cytokinins (Ki (6-Furfurylaminopurine)) were prepared by dissolving in a minimal quantity of 1N NaOH and then making up the volume with sterilized distilled water. They were used or stored in freezer as stock for further use. The plant growth regulators used in the present study were synthetic compounds not to be denatured in high temperature. Therefore, they can be added to media prior to autoclaving. 3.1.2.3 Organic compounds - Sucrose was used as carbon source in preparation of culture media. It was completely dissolved in distilled water prior to application. - The coconut water which stimulates the plant cell growth is used quite popularly. It was thoroughly filtered to obtain a fine solution prior to using. - Glycine is one of the most commonly used amino acid which provides a source of reduced nitrogen for cell growth. It was completely dissolved in distill water prior to application. - Casein hydroxylase (CH) contains calcium, phosphate, micronutrients, vitamin and the mixture including 18 amino acids. Peptone (PE) originated from some low molecular-weight proteins, promoted the plant cell growth. Malt extracted (ME) from barley mainly provides the carbone source. They were completely dissolved in distill water prior to application. 3.1.2.4 Precursors Precursors have been used in various plant cell suspension cultures to improve the production of secondary metabolites. Some factors such as the concentration and the time of addition of the precursor were considered when applying the precursor to the cell culture medium. Phenylalanine which is a precursor in paclitaxel pathway provides both the phenylisoserine side chain and the benzoyl moiety at C-2 of paclitaxel. Therefore, we examined the effect of phenylalanine on Taxol biosynthesis of cell suspension of Taxus wallichiana Zucc. 3.1.2.5 Elicitors The elicitor is a substance that can initiate or improve the biosynthesis of specific compounds when it is introduced in low concentrations to a living cell 40 system (Radman R. et al., 2003). In this study, we tested the different concentrations of elicitors to determine the most proper concentration and type of elicitor for highest taxol accumulation. - Salicylic acid (SA), a phenolic hormone, found in plants with the functions in plant growth and development is involved in endogenous signaling, mediating in plant defense (Hayat, S. and Ahmad, A., 2007). - Methyl jasmonate (MeJA) is a volatile organic compound which can be used to signal the original plant’s defense systems. MeJA can induce the plant to produce multiple different types of defense chemicals such as photoalexins, nicotine or proteinase inhibitors. MeJA activates the proteinase inhibitor genes through a receptor-mediated signal transduction pathway (Farmer E. and Ryan C., 1990). Plants also produce jasmonic acid and methyl jasmonate in response to many biotic and abiotic stresses, which build up in the damaged parts of the plant. - Chitosan is a mostly acetylated β-1,4-linked D-glucosamine polymer, which acts as a structural component of the cell wall of several plant fungal pathogens such as Fusarium sp. Many literatures reported that chitosan and its derivatives have antimicrobial and plant-defense elicit function (Albersheim and Darvill, 1985). Zhang et al. (1991) reported that oligochitosans obtained by hydrolysis or degradation of chitosan was not only water-soluble but also more effective than chitosan. Chitosan-derived oligosaccharides were used in overproduction of taxol (six-fold increase) in cultures of Taxus canadensis (Linden J. and Phisalaphong M., 2000).  Preparation of Elicitors - Oligo-chitosan was dissolved in glacial acetic acid by adding drop wise at 60 oC for a period of 15 min and the final volume was made up with deionized water. The pH of the solution was adjusted at 5.7 with KOH prior to use as an elicitor (Komaraiah et al., 2003). - MJ and SA were dissolved in small amounts of methanol and the final volume was made up with deionized water. The pH of the solution was adjusted at 5.7 with KOH prior to use as an elicitor. 41 - Yeast extract was weight and dissolved in deionized water and autoclaved at 121 oC for 15 min. 3.1.2.6 Gelling agent Domestic agar (Ha Long, Viet Nam) was used as gelling agent with concentration at 0.8% and stored at cool and dry condition. While preparing the agar gel, we should keep their final volume constantly during boiling and remove all air bubble. 3.1.3 Laboratory Facilities This study was conducted mainly in Key Laboratory of Plant Cell Biotechnology, Institute of Tropical Biology. The plant tissue culture pilot was divided into three functional sections with different levels of aseptic condition: media preparation and culture evaluation section (Appendix. 2), aseptic transfer section (Appendix. 3), and environmentally controlled culture section (Appendix. 4). 42 3.2 Methods 3.2.1 Experimental scheme Sterilized explants Callus induction - Effect of Mineral medium - Effect of Growth regulators Selecting the type of proper callus Callus proliferation - Effect of Growth regulators - Effect of Organic compounds Examination of Dynamics of callus growth Establishment of suspension culture Proliferation of Cell suspension - Effect of Growth regulators - Effect of Organic compounds Examination of kinetics of cell growth and taxol content Elicitation of taxol biosynthesis Effect of Elicitors: Methyl jamonate, Salicylic acid, Oligochitosan. - Concentration of Elicitors - Adding time of Elicitors - Exposure time with elicitors  Effect of Precursor - Concentration of Phenylalanine  Determination of paclitaxel content 43 3.2.2 Callus culture 3.2.2.1 Callus induction Experiment 1: Examination of mineral media and phytohormones on induction and growth of callus tissue of Taxus wallichiana Zucc. - Goal: Determination of basal mineral media and phytohormones for callus induction and growth. - Plant materials: Young stems of Taxus wallichiana Zucc. - Culture medium: Experimental media including MS, B5, WP basal media supplemented with 20 g/l sucrose, 1.0 mg/l Kinetin (Thanh Hien N. T. et al., 2004), 2,4-D (1.0 - 5.0 mg/l) and NAA (1.0 - 5.0 mg/l) with different concentrations. - Procedure: The explants were washed with distilled water several times, immersed in bleach solution for 25 - 30 minutes, and rinsed under running tap-water for 1.5 - 2 hours. Then, their surface was disinfected with ethanol 70% for 30 seconds, followed by Javel solution 50% added with several drops of Tween-80 for 20 minutes and then they were rinsed with sterile distilled water. The sterilized young stems were cut into 1 cm-long segments under the laminar flow bench, and placed horizontally on the experimental media (5 explants per culture medium). The cultures were maintained in the dark at 25±2oC and 70±2% relative humidity. Weekly observations were carried out within 12 weeks to evaluate the formation and growth of callus. The explants were subcultured to the fresh medium at 4 week intervals. This experiment was designed completely randomized design with 5 replications. - Evaluated dimensions: Rate of callus-formed explants, fresh weight of callus, characteristics of callus, time of callus induction. 3.2.2.2 Callus proliferation After 12 week of culture, appropriate callus masses selected from the callus induction experiment according to their histological and morphological observation were used for biomass proliferation. 44 Experiment 2: Examination of plant growth regulators on growth and proliferation of callus tissue of Taxus wallichiana Zucc. - Goal: Determination of appropriate concentration of phytohormone (NAA and 2,4-D) for proliferation of callus tissue. - Plant materials: The callus tissue was obtained from experiment 1 after 2 subcultures. - Culture medium: Experimental medium was the best mineral media (experiment 1) supplemented with sucrose (20 g/l), ascorbic acid (100 mg/l), 1.0 mg/l Kinetin (Thanh Hien N. T. et al., 2004), NAA (0, 2.0, 4.0 mg/l), and 2,4-D (0, 2.0, 4.0 mg/l), described in Table 3.1. This experiment was designed completely randomized design with 3 replications. Table 3.1 Media formation of callus proliferation Treatment Medium NAA (mg/l) 2,4D (mg/l) CP0 CP1 CP2 CP3 CP4 CP5 CP6 CP7 CP8 CIM CIM CIM CIM CIM CIM CIM CIM CIM 0 0 0 2 4 2 2 4 4 0 2 4 0 0 2 4 2 4 *CIM = the best mineral media (experiment 1) supplemented with 20 g/l sucrose, ascorbic acid 100 mg/l. - Procedure: 1g of callus with uniform clumps of friable reddish-brown callus were transferred to callus multiplication media (Table 3.1) and kept under dark condition at room temperature (25oC ± 2). The growth of callus was measured at the day 42. - Evaluated dimensions: fresh weight of callus and characteristics of callus. 45 Experiment 3: Examination of sucrose on growth of callus tissue of Taxus wallichiana Zucc. - Goal: Determination of the appropriate concentration of sucrose induced osmosis stress aimed to enhance growth of callus tissue. - Plant materials: The callus tissue was prepared in previous steps. - Culture medium: Experimental medium was the best medium determined in experiment 2 which were complemented with different concentrations of sucrose (20, 30, 40 g/l). - Procedure: 1g of callus were transferred to callus multiplication media and kept under dark condition at room temperature (25 oC ± 2). The cell growth was determined at the day 42. This experiment was designed completely randomized design with 3 replications. - Evaluated dimensions: fresh weight of callus and characteristics of callus. Experiment 4: Examination of organic ingredients on growth of callus tissue of Taxus wallichiana Zucc. - Goal: Determination of optimal medium for proliferation of callus tissue. - Plant materials: The callus tissue was prepared in previous steps. - Culture medium: Experimental medium was the best medium determined in experiment 3 which were complemented with different volumes of coconut water (50, 100, 150 ml/l) and Glycine (5, 10, 15mg/l). - Procedure: 1g of callus were transferred to callus multiplication media and kept under dark condition at room temperature (25 oC ± 2). The cell growth was determined at the day 42. This experiment was designed completely randomized design with 3 replications. - Evaluated dimensions: fresh weight of callus and characteristics of callus. 3.2.3 Cell suspension cultures In order to initiate cell suspension cultures, 2g fresh callus tissue of 35 day old callus was transferred into 50 ml of liquid medium containing the compositions similar to that of the best callus proliferation medium including ascorbic acid (100 mg/l), Ki (1.0 mg/l), sucrose, 2,4-D, NAA, glycine, Cw. Cell system was placed on a rotary shaker at 100 rpm in the dark. Temperature was maintained at 25 ± 2oC. 46 After 42 days of cultivation, all was filtered via a stainless steel mesh (600 - 800 µm) to collect fine suspension. Settled cell volume was measured after 15 minutes setting time (O’Looney et al., 2005). 3.2.3.1. Cell suspension proliferation Experiment 5: Examination of plant growth regulators (NAA; 2,4-D) on growth and proliferation of cell suspension of Taxus wallichiana Zucc. - Goal: Determination of the appropriate concentration of phytohormones for proliferation of cell suspension. - Plant materials: The fine cell suspension was prepared in previous steps. - Culture medium: Experimental medium included ascorbic acid (100 mg/l), Ki (0.5 mg/l) (Thuy Tien, 2006), sucrose, glycine, Cw according to the best result of experiment 4, NAA (0, 1.0, 3.0, 5.0 mg/l), and 2,4-D (0, 1.0, 2.0, 3.0, 4.0 mg/l), described in Table 3.2. Table 3.2. Media Formation of cell suspension proliferation NAA (mg/l) 2, 4 D (mg/l) 0 1 2 3 4 0 CSI1 CSI5 CSI9 CSI13 CSI17 1 CSI2 CSI6 CSI10 CSI14 CSI18 3 CSI3 CSI7 CSI11 CSI15 CSI19 5 CSI4 CSI8 CSI12 CSI16 CSI20 *CSI is an abbreviation of medium. Ingredients of CSI include sucrose (30 g/l), ascorbic acid 100 mg/l, glycine (10 mg/l), Cw (10 %). Numbers from 1 to 20 is a combination of NAA and 2,4-D in various concentrations. - Procedure: 1g of fresh cells was transferred to 25 ml of experimental media. This experiment was designed completely randomized design with 3 replications. - Culture conditions: The pH values of media were adjusted to 5.7±0.1 prior to autoclaving at 121 oC, 1 atm in 20 minutes. The cultures were maintained in the dark at 25 ± 2oC. Shaker speed was set at 100 rpm. Growth of cell suspension was measured after 42 days of cultivation. - Evaluated dimensions: cell density, fresh cell weight and dry cell weight. 47 Experiment 6: Examination of organic compounds on growth and proliferation of cell suspension of Taxus wallichiana Zucc. - Goal: Determination of optimal medium for proliferation of cell suspension. - Plant materials: The fine cell suspension was prepared in previous steps. - Culture medium: Experimental medium was the best media determined in experiment 5 which were complemented with PE, or ME, or CH at different concentrations (0, 0.1, 0.5, 1.0, 1.5, 2.0 mg/l). - Procedure: 1g of fresh cells was transferred to 25 ml of culture media. This experiment was designed completely randomized design with 3 replications. - Culture conditions: The pH values of media were adjusted to 5.7±0.1 prior to autoclaving at 121 oC, 1 atm in 20 minutes. The cultures were maintained in the dark at 25 ± 2oC. Shaker speed was set at 100 rpm. Growth of cell suspension was measured after 42 days of cultivation. - Evaluated dimensions: fresh cell weight and dry cell weight. Experiment 7: Examination of phenylalanine (PA) on taxol accumulation of cell suspension of Taxus wallichiana Zucc - Goal: Determination of the proper concentration of PA for enhancing taxol biosynthesis of cell suspension. - Plant materials: The fine cell suspension was prepared in previous steps. - Culture medium: Experimental medium was the best media determined in experiment 6 which were complemented with PA (0, 5, 10, 15, 20 mg/l) at the day 14 of cultivation. - Procedure: 1g of fresh cells was transferred to 25 ml of media eliciting taxol biosynthesis. This experiment was designed completely randomized design with 3 replications. - Culture conditions: The pH values of media were adjusted to 5.7±0.1 prior to autoclaving at 121 oC, 1 atm in 20 minutes. The cultures were maintained in the dark at 25 ± 2oC. Shaker speed was set at 100 rpm. Growth of cell suspension and paclitaxel content was measured after 42 days of cultivation. - Evaluated dimensions: dry cell weight, paclitaxel content. 48 3.2.3.2. Elicitation of paclitaxel biosynthesis in cell suspension cultures Experiment 8: Examination of some elicitors on taxol biosynthesis of cell suspension of Taxus wallichiana Zucc - Goal: Determination of the appropriate type and concentration of elicitor for enhancing taxol biosynthesis of cell suspension. - Plant materials: The fine cell suspension was prepared in previous steps. - Culture medium: Experimental medium was the best media collected in experiment 7 which were complemented with MJ (0, 10, 20, 30 mg/l), or SA (0, 50, 100, 150, 200 mg/l), or O-Chi (0, 5, 10, 15 mg/l) or YE (0, 500, 1000, 2000 mg/l) at the day 14 of cultivation. - Procedure: 1g of fresh cells was transferred to 25 ml of media eliciting taxol biosynthesis. This experiment was designed completely randomized design with 3 replications. - Culture conditions: The pH values of media were adjusted to 5.7±0.1 prior to autoclaving at 121 oC, 1 atm in 20 minutes. The cultures were maintained in the dark at 25 ± 2oC. Shaker speed was set at 100 rpm. Growth of cell suspension and paclitaxel content was measured after 42 days of cultivation. - Evaluated dimensions: dry cell weight and paclitaxel content. Experiment 9: Examination of elicitor addition time on taxol accumulation of cell suspension of Taxus wallichiana Zucc - Goal: Determination of the proper time for adding elicitor into the cell suspension cultures to enhance taxol biosynthesis of cells. - Plant materials: The fine cell suspension was prepared in previous steps. - Culture medium: Experimental medium was the best medium obtained in experiment 7 which were complemented with a proper elicitor at the appropriate concentration (experiment 8) at the different times (0, 7, 14, 21, 28 day). - Procedure: 1g of fresh cells was transferred to 25 ml of media eliciting taxol biosynthesis. This experiment was designed completely randomized design with 3 replications. 49 - Culture conditions: The pH values of media were adjusted to 5.7±0.1 prior to autoclaving at 121 oC, 1 atm in 20 minutes. The cultures were maintained in the dark at 25 ± 2oC. Shaker speed was set at 100 rpm. Growth of cell suspension and paclitaxel content was measured day 42. - Evaluated dimensions: dry cell weight and paclitaxel content. Experiment 10: Examination of exposure time with elicitor on taxol accumulation of cell suspension of Taxus wallichiana Zucc - Goal: Determination of the proper exposure time of cell suspension cultures with elicitor on taxol biosynthesis of cells. - Plant materials: The fine cell suspension was prepared in previous steps. - Culture medium: Experimental medium was the best media obtained in experiment 7 which were complemented with a proper elicitor (experiment 8) at the optimal concentration at the appropriate time (experiment 9). Growth of cell suspension and paclitaxel content was measured at different durations of elicitor exposure (0, 2, 4, 6, 8 days). - Procedure: 1g of fresh cells was transferred to 25 ml of media eliciting taxol biosynthesis. This experiment was designed completely randomized design with 3 replications. - Culture conditions: The pH values of media were adjusted to 5.7±0.1 prior to autoclaving at 121 oC, 1 atm in 20 minutes. The cultures were maintained in the dark at 25 ± 2oC. Shaker speed was set at 100 rpm. - Evaluated dimensions: dry cell weight and paclitaxel content. 3.2.4 Cell Growth Measurement In this study, measurement of growth of cultured plant cells was based on fresh weight, dry weight and cell density. - Fresh weights were determined by using a filter unit consisting of a stainless steel mesh placed in a Buchner funnel. Filter paper with defined dry weight was then placed on this mesh. 25 ml of evenly dispersed cell suspensions were then filtered. The cells were washed by distill water and weighed together with filter paper to determine final fresh weight. 50 - Dry weights were measured by drying the cells including filter paper (after determining their fresh weight) in an Oven (Universal Biochemical; India) at 40°C for 24 hours and took dry weight in a balance. - Cell numbers of cell suspensions were determined directly under microscope with assistance of a Neubauer counting chamber. To obtain a reliable value of the number of cells in suspension culture, cell clusters were first disaggregated by Chromium trioxide (CrO3). This was accomplished by incubating 1 ml of dispersed cell suspension with 2 ml of 8% CrO3. After disaggregation, mixtures were then filled to counting chamber for observation with 10X magnification. The average of the obtained number was the number of cells in 0.1 mm 3 and multiplied by 104 to get the cell number per milliliter of the mixture. The cell density was finally determined by multiplying it by 3 (mixture ratio is 2 volumes of 8% CrO3 over 1 volume of cell suspension). - Determination of kinetics of cell growth Callus growth curve: 1g of initial weight of callus was transferred onto the best media determined in experiment 4 and then measured callus fresh weight at various time intervals (7, 14, 21, 28, 35 and 42 days) to determine the callus growth rate at every 7 day time interval. Cell growth curve: 1g of fresh cells was transferred into 25 ml of liquid medium and then determined fresh cell weight and characteristics of cell suspension at different time intervals (7, 14, 21, 28, 35, 42 days) to determine the cell growth rate at every 7 day time interval. 3.2.5 Data Collection To callus initiation, frequency of callus induction was calculated by the number of responded explants dividing the number of initial explants and then multiplied by 100 and the mean of final weight (in gram) was also determined. Number of responded explants Callus formation frequency (%) = x 100 Number of initial explants To biomass proliferation, after 42 days of culture in each experiment, the mean of final weight (in gram) was determined and then transformed to growth index. It was calculated as the ratio of the accumulated and initial biomass. 51 Accumulated biomass - initial biomass Growth index = Initial biomass To determination of Paclitaxel content, dried cell suspension material was extracted thrice with MeOH (Methanol) in ultrasound pool at 40oC in 30 minutes, shaking at 10 minutes interval, and the methanolic fractions were filtered and evaporated to dryness. The residue was dissolved in 10 ml of MeOH, and then filtrated by filtered membrane (size 0.45µm) before analyzing paclitaxel content by high performance liquid chromatography (HPLC). Quantitative studies included standard addition experiments for the construction of calibration curves. HPLC analyses were performed in triplicate. Evaporation - The paclitaxel content in the extracted solution was analyzed by HPLC (SYKAM, Germany) with UV detection at 227 nm. The HPLC column was Symmestry C18 of 15 × 3.2 mm dimension (Brownlee Laboratory, Inc., Santa Clara, California). The mobile phase was methanol:water in a 70:30 volume ratio at a flow rate of 0.5 ml/min. - Standards: the stock solutions contain 1mg paclitaxel (Sigma; St. Louis, MO) dissolved in 10 ml HPLC grade methanol. The linearity of calibration curve was established with a series of solutions prepared by two-fold dilutions of the stock 52 solution with 100% methanol to the final concentrations which ranged from 31.25 ppm - 500 ppm. 3.2.6 Statistical Analysis Data for all characteristics were subjected to analysis of variance with the MSTATC statistical software package. 53 Chapter 4: Results and Discussion 54 Cell cultures require all the mineral nutrients as intact plants for their optimal growth and development. The ingredient of nutrient media is a vital factor to establish a successful cultivation. Each of tissue type has different demands on composition, concentration of compounds and depends on the studied targets. The current study was conducted to determine the optimal concentrations and combinations of plant growth regulators in the media as well as other factors for plant cell culture in Taxus wallichiana Zucc. via young stems callogenesis culture and single-cell proliferation in shake-flasks. 4.1 Callus Culture 4.1.1 Effects of mineral media and phytohormones on induction and growth of callus tissue of Taxus wallichiana Zucc. Callus induction is result of deep morphological modification, metabolism, and chemical signs in cell (Lindsey et al., 1993). Callus induction depends on genotype, explants, the composition of culture media, ratios of phytohormone especially the ratio of auxin to cytokinin, and environmental factors (Pierik, 1987). Optimization of the callus induction medium requires consideration of the mineral composition, the concentration of phytohormones and organic constituents. Up to date, modified versions of the B5 media have remained the most common nutrient mixture used for growing Taxus sp. cell culture, however others such as MS, SH, WPM and optimized media developed specifically for Taxus by Ketchum and Gibson have also been used (Zhong, 1995). Furthermore, both natural auxins (IAA) and synthetic auxins (2,4-D, NAA, picloram) used in combination with cytokinins (Ki, BA, 2iP, zeatin) have been used in growing Taxus sp. cell culture (Ketchum 1996). Therefore, callus induction of explants from Taxus wallichiana Zucc. was required reexamination of the basic mineral media and different concentrations of growth regulators to optimize the growth of explants. Three mineral media MS - B5 - WPM supplemented with Ki (1.0 mg/l), ascorbic acid (100 mg/l), 2,4-D (1.0 - 5.0 mg/l) or NAA (1.0 - 5.0 mg/l), sucrose (20 g/l) were used in this experiment to determine a proper medium for callus induction and growth in young stem segments. 55 Callus formation initiated along with the injured areas of young stem explants at different times with various ratios dependent on the type and concentrations of phytohormone and different mineral media. Rate of responding explants and callus growth was directly proportional to the increase of concentration of growth regulators. Explants turned into browning were observed together with the forming of callus. Callus did not induce from explants on the culture medium without plant growth regulators even after 45 days of culture. Figure 4.1. Callus inducted from young stem explants of Taxus wallichiana Zucc after 45 days on different mineral media. (A) Medium without phytohormone, (B) WP medium 2,4-D (4mg/l), (C) B5 medium with 2,4-D (4mg/l), (D) MS medium2,4-D (4mg/l). 56 Table 4.1 Influence of 2,4-D and mineral media on callus induction from stem segments Mineral 2,4-D (mg/l) Callusinducted time (days) MS MS MS MS MS B5 B5 B5 B5 B5 WPM WPM WPM WPM WPM 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 38 38 31 27 27 31 31 27 23 23 31 30 25 22 21 Rate of callus inducted explants* (%) Fresh weight of callus* (g) 52 d 68bc 84ab 100 a 100 a 64cd 76bc 96 a 100 a 100 a 72bc 84ab 100 a 100 a 100 a 1.18j 1.42h 1.68 fg 2.32b 2.11cd 1.23 ij 1.35 hi 1.74f 2.56a 2.20bc 1.07k 1.12 jk 1.57g 2.04de 1.91e * Different letters in one column indicate statistical significant differences at the level of 0.01%. Table 4.1 showed that the explants responded in the period of 27 - 38 days of culture with various ratios according to different concentrations of 2,4-D on the MS medium. The highest frequency of callus induction (100%) was recorded at treatment with 2,4-D (4.0 or 5.0 mg/l). The callus sprouting on MS medium with 2,4-D (4.0 mg/l) showed the significant increase (2.32g) after 12 weeks of culture. Similarly, the explants responded in the period of 23 - 31 days of culture with various ratios according to different concentrations of 2,4-D on the B5 medium. The highest frequency of callus induction (100%) was recorded at treatment with 2,4-D (4.0 or 5.0 mg/l). The callus growth on B5 medium with 2,4-D (4.0 mg/l) showed the highest increase of weight (2.56g) after 12 weeks of culture. Finally, the explants responded in the period of 21 - 31 days of culture with various ratios according to different concentrations of 2,4-D on the WPM medium. The highest frequency of callus induction (100%) was recorded at treatment with 57 2,4-D (3.0, 4.0, or 5.0 mg/l). The callus growth on WPM medium showed limited development even if they were maintained for long period in culture. Callus induction from young stems on WPM medium were better than those on other media (MS and B5), but B5 mineral medium was the best one for callus growth (Table 4.1). The friable, reddish, light brown callus masses induced on the media with 2,4-D showed the significant growth. Table 4.2 Influence of NAA and mineral media on callus induction from stem segments Mineral NAA (mg/l) Callusinducted time (days) Rate of callus inducted explants* (%) Fresh weight of callus* (g) MS MS MS MS MS B5 B5 B5 B5 B5 WPM WPM WPM WPM WPM 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 41 39 32 29 29 35 33 29 27 27 35 35 28 26 25 16 c 36 b 72 a 68 a 64 a 16 c 64 a 84 a 80 a 76 a 20bc 64 a 84 a 84 a 72 a 0.54g 0.79e 1.82ab 1.65 bc 1.52c 0.53g 0.71efg 1.94a 1.82ab 1.65 bc 0.57 fg 0.74ef 0.99d 1.15d 1.17d * Different letters in one column indicate statistical significant differences Table 4.2 showed that the explants responded in the period of 29 - 41 days of culture with various ratios according to different concentrations of NAA on the MS medium. The highest frequency of callus induction (72%) was recorded at treatment with NAA (3.0 mg/l). Similarly, the explants responded in the period of 27 - 35 days of culture with various ratios according to different concentrations of NAA on the B5 medium. The highest frequency of callus induction (84%) was recorded at treatment with NAA (3.0 mg/l). Finally, the explants responded in the period of 25 - 58 35 days of culture with various ratios according to different concentrations of NAA on the WPM medium. The highest frequency of callus induction (84%) was recorded at treatment with NAA (3.0 or 4.0 mg/l). Callus induction from young stems on WPM medium were better than those on other media (MS and B5), but B5 mineral medium was the best one for callus growth (Table 4.2). The callus mass on the media with NAA showed the limited growth and light yellow-compact nature and low ratio of responded explants. Possibly, concentration of NAA is not strong enough to break the hormone balance in explants. A B Figure 4.2. Callus inducted from young stem explants of Taxus wallichiana Zucc after 90 days on B5 media with different auxins. (A) NAA(4mg/l), (B) 2,4-D (4mg/l) 59 Callus induction from young stems on WPM medium were better than those on other media (MS and B5), but B5 mineral medium was the best one for callus growth (Table 4.1 and 4.2). Main reason can be concentration of NH4+ ion in the B5 medium lower than that in the MS medium and higher than that in the WP medium. According to Fett-Neto et al. (1993), the effect of mineral medium composition such as B5, SH (Schenk and Hildebrandt 1972) and modified SH medium on callus growth of T. cuspidate showed that all promoted callus growth whereas MS salts (Murashigeand Skoog 1962) suppressed growth. Growth of callus is influenced by many factors like concentration and rate of NO3- and NH4+ ions (Fett-Neto et al., 1993). Response of plant in vitro conditions to NO3- and NH4+ ions depends on differentiated levels of tissue, age, physiological status, genotype, and diversity of enzymes in cell. Activity of some enzymes can be stimulated or prevented depending on concentration of two ions above (Edwin, 1993). In addition, callus induction of T. brevifolia in different culture media (Gibson et al., 1993) showed that although several media formulations led to callus initiation, only a few allowed subsequent subculture and growth. These are similar to the results in our study. Bai et al. (2004) reported that the induction of a T. cuspidate callus line on B5 medium with NAA (0.5 mg/l) showed fast growth compared to other callus lines established in other media with higher NAA concentrations or other growth regulators, such as IAA. Gibson et al. (1993) also found that concentrations of 1.0 2.0 mg/l of 2,4-D allowed callus growth in T. brevifolia. In this study, the effect of 2,4-D was better in callus induction and growth from young stems than that of NAA. Rate of responding explants and callus growth was directly proportional to the increase of concentration of growth regulators. Explants placed in the media supplemented with various growth regulators showed different morphological changes. There were two kinds of callus formed: One was friable and reddish, light brown in color induced on the media with 2,4-D; the other was compact and light yellow in color on the media with NAA. Gibson et al. (1993) reported that the high variability in the cell growth observed was correlated with callus color. Light brown and friable calli showed higher in growth rate than dark and compact calli. Apparently, Callus tissues are friable to be suitable for suspension cultures with the 60 greatest degree of cell dispersion. As a result, Callus formed on medium with 2,4-D (4.0 mg/l) was used for next experiments and B5 medium was suitable for callus growth. 4.1.2 Proliferation of callus biomass 4.1.2.1 Effect of 2,4-D and NAA on callus proliferation Calli can be considered as homogeneous cell clusters after several subcultures on the same medium and their growth parameters were repeated during subsequent transfers onto fresh culture medium. In this experiment, the 3 rd generation callus masses reddish brown and fragile were used as a material source for biomass proliferation. Callus was transferred onto the medium supplemented with various compounds such as phytohormones, coconut water, amino acid, organic matters to optimize the cell growth. The medium for callus proliferation commonly have hormone concentration lower than that for callus formation. Hence, influence of growth regulators on proliferation of callus biomass were examined again in this case. The growth index and status of the callus were also considered along with fresh weight. B5 medium supplemented with sucrose (20 g/l), NAA and 2,4-D with different concentrations were tested. Table 4.3 Influence of 2,4-D and NAA on proliferation of callus biomass Treatment NAA 2,4-D (mg/l) (mg/l) Weight of fresh callus*(g) Growth index 3.2 0 0 0 2 1.52 d 2.40bc 0.52 1.40 3.3 0 4 2.53 b 1.53 0 2.36 bc 1.36 2.36 bc 1.36 2.54 b 1.54 3.04 a 2.04 bc 1.37 c 1.32 3.1 3.4 3.5 3.6 3.7 3.8 3.9 2 4 2 2 4 4 0 2 4 2 4 2.37 2.32 * Different letters in one column indicate statistical significant differences 61 Secondary callus grew faster than primary callus and turned into brownish yellow after 2 - 3 next subcultures. Addition of plant growth regulators with the different compositions and ratios in the culture media gained the different growth of callus. The callus growth from young stems on the B5 medium with 2,4-D was better than that on the B5 medium with NAA. Moreover, Callus was compact in nature, low in the growth index, and short in the longevity when NAA was alone complemented into the medium. Callus tissue grew slowly and changed gradually from reddish, brownish yellow to brown and died after 42 days of cultivation. A B Figure 4.3. Callus masses proliferated after 42 days on B5 media with different auxins. (A) NAA (4 mg/l), (B) 2,4-D (4.0 mg/l) + NAA (2.0 mg/l) 62 Fett-Neto et al. (1993) reported that the presence of auxin (1.0 - 8.0 mg/l) was important for the development of the callus. In this case, the use of 2,4-D (8.0 mg/l) in combination with GA3 (0.5 mg/l) had a positive effect on T. cuspidata callus growth. Moreover, Cusido et al. (1999) established a callus culture from stems of T. baccata using B5 medium and a combination of 2,4-D (4.0 mg/l), kinetin (1.0 mg/l) and GA3 (0.5 mg/l) which was optimal for callus development. In this experiment, these results obtained after 35 days of cultivation shown the treatment supplemented with 2,4-D (4.0 mg/l) and NAA (2.0 mg/l) resulted in effective growth of callus with the highest growth index 2.04 and lived longer than that on the others. Calli indicated the lowest growth index (1.32) on the medium without auxin. This demonstrates the high variability in the response of Taxus explants to plant growth regulators. The results described above show that the culture medium composition must be optimized for each Taxus species. Consequently, B5 medium supplemented with Ki (1.0 mg/l), ascorbic acid (100 mg/l), 2,4-D (4.0 mg/l), NAA (2.0 mg/l), Sucrose (20 g/l) was used in the next experiment for proliferation of callus biomass. 4.1.2.2 Nutritional Factors The results in the previous experiments have determined the ingredients of mineral medium, concentration of growth regulators appropriate to proliferation of callus biomass. However, in in vitro culture, beside growth regulators, inorganic compounds, nitrogen source, agar, pH of medium, the plant cell growth also was affected by carbon source, organic compounds and other extractives (Minh T.V., 2006). Carbon source was found to be a significant factor in plant cell metabolism, manipulation of medium sucrose was demonstrated very effectively for improvement of culture productivity. Therefore, to enhance effective growth of callus, we studied the influence of different concentrations of sucrose on callus growth. Table 4.4 Influence of sucrose on proliferation of callus biomass Treatment Sucrose (g/l) 4.1 4.2 4.3 20 30 40 Weight of callus after 6 weeks (g) 2.63 b 3.20 a 2.44 b Growth index 1.63 2.20 1.44 63 The results in Table 4.4 recorded after 35 days of culture showed an important role of sucrose to callus growth. The optimal sucrose concentration was obtained at 30 g/l that yielded highest callus growth (3.20g Fresh weight). However, the higher concentrations of sucrose (40 g/l) led to inefficient callus proliferation. This phenomenon could be explained via disadvantages of high osmotic pressure in solid phase (Shibli et al., 1992). A B Figure 4.4. Callus masses proliferated after 42 days on B5 media with different concentrations of sucrose. (A) 30 (g/l), (B) 40 (g/l) According to Zhang YH et al. (1996), the final dry cell weight increased with an increase of initial sucrose concentration from 20 to 40 g/l in cell cultures of Panax notoginseng, but a higher sucrose concentration of 60 g/l seemed to repress the cell growth. A high sugar level was favorable to the synthesis of paclitaxel, which may be due to the high osmotic pressure and reduced nutrient uptake under the conditions. Protova (1974) also reported that addition of sucrose on medium caused the osmotic pressure increasingly: 1.7 bar (2%); 4 bar (4%); 8.2 bar (10%); and 12.7 bar (15%). Indeed, each plant species in the different stages of growth has a different demand on sucrose concentration and osmotic pressure. Coconut water (Cw) containing many kinds of sugar, amino acid, phytohormone and other metabolites which can stimulate cell growth is a common organic compound used in tissue culture. In addition, Chen et al. (2003) found that supplementation with amino acids (15 - 20 mg/l) greatly improved callus growth in T. yunnanensis callus cultures established on B5 medium. Besides, some previous studies also showed that callus induction and growth was stably maintained on 64 medium supplement with glycine or L-glutamic acid + L-aspartic acid, L-arginine or a mixture of 18 amino acids like Reinert and White medium (Minh T.V., 2006). A B Figure 4.5. Callus masses proliferated after 45 days on B5 media with Glycine and Coconut water. (A) Cw (100 ml/l) + Glycine (10 mg/l), (B) Cw (100 ml/l) Table 4.5 Influence of Coconut water and Glycine on proliferation of callus biomass Treatment 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 5.10 5.11 5.12 5.13 5.14 5.15 5.16 Cw (%) 0 0 0 0 5 10 15 5 10 15 5 10 15 5 10 15 Glycine (mg/l) 0 5 10 15 0 0 0 5 5 5 10 10 10 15 15 15 Fresh cell weight* (g) d 3.10 3.17 cd 3.53abcd 3.50abcd 3.13d 3.73abc 3.77ab 3.43bcd 3.83ab 3.87ab 3.77ab 4.07a 3.80ab 3.67abcd 3.57abcd 3.60abcd Growth index 2.10 2.17 2.53 2.50 2.13 2.73 2.77 2.43 2.83 2.87 2.77 3.07 2.80 2.67 2.57 2.60 * Different letters in one column indicate statistical significant differences In this experiment, Cw and glycine were separately used for the callus proliferation, stimulation of cell growth was not efficient (2.13g to 2.87g) while Cw in combination with glycine had a positive effect on cell growth (2.43g to 3.07g). 65 Based on the results of above experiments, the medium of callus proliferation was the B5 medium complemented with Ki (1.0 mg/l), ascorbic acid (100 mg/l), 2,4-D (4.0 mg/l), NAA (2.0 mg/l), glycine (10 mg/l), Cw (10 %), sucrose (30 g/l) induced the highest biomass of callus (F.W - 4.07g). This medium had used for the next experiments. 4.1.3 Determination of dynamic of callus growth In order to maintain the callus growth and select a kind of suitable callus for the aim of this research, these required regular subcultures at a proper time interval of the culture. Conventionally, Callus growth on solid medium contained five phases: Lag phase, exponential phase, linear phase, decelerating phase, and stationary phase. Therefore, examination of kinetics of callus growth to determine a proper time for subculture is necessary to maintain cell growth effectively. To establish the kinetics of cell growth, 1g of callus was cultured on the optimal medium (B5 + Ki (1.0 mg/l) + ascorbic acid (100 mg/l) + 2,4-D (4.0 mg/l) + NAA (2.0 mg/l) + glycine (10 mg/l) + Cw (10 %) + sucrose (30 g/l)) which had determined in the previous experiments and then measured fresh weight and characteristics of callus at every 7 day time interval (7 - 14 - 21 - 28 - 35 - 42). Figure 4.6. Kinetics of callus growth of Taxus wallichiana Zucc on optimal medium supplemented with 2,4-D (4.0 mg/l), NAA (2.0 mg/l), Ki (1.0 mg/l), ascorbic acid (100 mg/l), sucrose (30 g/l), Agar (8 g/l), Cw (10 %), Glycine (10 mg/l) Fig. 4.6 shown: During the lag phase in the first seven days, there was little callus growth until the day 7 of culture because the callus masses required a certain period of time to adapt with fresh medium. The exponential growth phase began on 66 the day 7 of culture, the calluses initially divided with stable growth and their weight and size increased slightly. In the stage of 7 - 28 day, the weight of callus proliferated excessively with the highest rate of growth (1.21g to 3.76g) and the callus growth reached stationary phase on day 35. In stage from 35 to 42 days, callus did not mostly grow both size and weight and color of callus changed from reddish brownish yellow to brown and turned into black brown on day 42. The above results showed that the most appropriate time for subculture was th on 35 day. At this time, the subculture of calluses on fresh medium was necessary for their further growth in the next generation. 4.2 Cell suspension culture 4.2.1 Establishment of cell suspension culture Compared to callus cultures, cell suspension cultures produce a large amount of cells from which metabolites can be more easily extracted. In addition, these cells can easily absorb the nutrient due to their surface completely contacted with the liquid medium. The best medium (CPM = B5 + Ki (1.0 mg/l) + ascorbic acid (100 mg/l) + 2,4-D (4.0 mg/l) + NAA (2.0 mg/l) + glycine (10 mg/l) + Cw (10 %) + sucrose (30 g/l)) for the callus proliferation was used for establishment of cell suspension. In order to initiate cell suspension cultures, 2g of fresh callus was transferred into 50 ml of liquid CPM medium. These cultures were placed on an orbital shaker at 100 rpm in the dark. Release of single cells and cell clusters from stem-derived callus masses into medium was clearly shown after 1 - 2 weeks of culture. The suspension cultures became fairly homogenous by centrifugal force (Fig. 4.7). After 42 days of cultivation, all was filtered via mesh (600 - 800 µm) to collect fine suspension. The establishment of homogenous cell suspension had brought certain advantages to the growth of cell mass due to fine cell suspensions which were achieved in other Taxus species. 67 A B C Figure 4.7. Initiation of cell suspension cultures. (A) Callus proliferation (B) Establishment of suspension culture (C) Orbital shaker system 68 4.2.2 Effect of phytohormones on cell suspension proliferation Generally, the media for callus cultures are also suitable for suspension cultures without the gelling agent. However, in some cases, the concentrations of auxins and cytokinins were required more exactly. Therefore, the established cell suspension cultures were transferred to fresh B5 medium supplemented with glycine (10 mg/l), Cw (10 %), sucrose (30 g/l), ascorbic acid (100 mg/l), Ki (0.5 mg/l), 2,4-D (1.0 - 4.0 mg/l) and NAA (1.0, 3.0, 5.0 mg/l) for proliferation of cell biomass. Table 4.6 Influence of combinations between 2,4-D and NAA on proliferation of cell biomass Treatment 6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9 6.10 6.11 6.12 6.13 6.14 6.15 6.16 6.17 6.18 6.19 6.20 Mean cell Fresh cell density weight (mg/l) (mg/l) (cells/ml x104) (g) NAA 0 1 3 5 0 1 3 5 0 1 3 5 0 1 3 5 0 1 3 5 2,4-D 0 0 0 0 1 1 1 1 2 2 2 2 3 3 3 3 4 4 4 4 2.3p 5.4k 5.7j 5.3k 2.9o 3.1n 6.5h 4.4l 3.6m 5.8j 6.9g 7.1f 3.2n 7.2f 7.6e 7.9d 10.5b 9.2c 11.7a 6.2i 2.11m 2.15m 2.90jk 3.28hij 2.46lm 2.93ijk 3.12hij 3.73fg 2.57kl 3.32hi 4.24e 4.61 d 3.50gh 3.90ef 6.20a 3.73fg 5.13bc 5.31 b 4.87cd 3.40gh Dry cell weight (mg) 198h 273 gh 281 gh 321 fg 214h 278 gh 305 fg 342efg 217h 320 fg 415cde 449bcd 356efg 385def 610a 348efg 489 bc 512b 451bcd 287 gh DCW/ FCW 0.094 0.127 0.097 0.098 0.087 0.095 0.098 0.092 0.084 0.096 0.098 0.097 0.102 0.099 0.098 0.093 0.095 0.096 0.093 0.084 * Different letters in one column indicate statistical significant differences 69 After 42 days of subculture, most of the treatments showed the positive results in cell suspension proliferation regarding to their weight and cell number determination. However, cell growth was slow with the control treatment (without phytohormones) or the treatments in low concentration of phytohormone. On the other hand, media supplemented with a combination of 2,4-D and NAA indicated the longer longevity and the considerable growth of cell clusters in suspension culture. Figure 4.8. Morphology of single cells and cell clusters on medium supplemented with 2,4 D (3 mg/l) + NAA (3 mg/l). (A) Cell division, (B, C, D) Cell aggregate In fact, the addition of NAA in the medium showed a low cell growth compared with the addition of strong auxin 2,4-D and cell growth on the medium with higher concentration of 2,4-D alone or in combination with low concentration of NAA was faster than that on the others. According to Table 4.6, the highest biomass yield of cell suspension culture (6.2g) was obtained in the medium containing 2,4-D (3.0 mg/l) and NAA (3.0 mg/l). The use of 2,4-D in combinations with NAA was more efficient compared with the use of 2,4-D or NAA alone in the 70 growth of cell suspension culture. Apparently, a hormonal balance would be vital for enhancing the growth of cells and low or high concentration of phytohormones inhibited cell growth. The effect of phytohormone on taxol production was also exemplified by Ketchum. In multiple-flask experiments of a single cell line, manipulations of auxin and cytokinin types produced taxol in concentrations between 1 mg/l and 14 mg/l. However, the results were not transposable between species, as it was also found that IAA/BA combination was superior for lines of Taxus sp. but NAA/Th was best for Taxus canadensis (Ketchum, 1996). Other researchers have noted similar results. For example, the recent patent application Phyton INC. reported the P/BA combination to be best for Taxus sp. but P/2iP for T. brevifolia, P/K for T.canadensis, N/BA for T.chinensis (Bringi, 1997). 4.2.3 Effect of organic compounds on cell suspension proliferation There are many factors to enhance biosynthesis of secondary metabolites in plant cell culture such as selection of suitable cell lines, composition of medium and environmental condition. Among a number of those, compositions of medium such as inorganic compounds and organic compounds had directly effects on cell growth and productivity of plant metabolites in cells. Although cell cultures are normally capable of synthesizing all of the required amino acids for cell growth and metabolite production from the nitrate and ammonium in the media, supplementation of amino acid compounds such as glutamine, casamino acid, phenyl alanine, serine, glycine, proline and mixtures of amino acids like casein hydrolysate has resulted in positive effects on enhancement of cell growth and taxol production (Wickremesinhe, 1993). The promotion of taxol production by these compounds is likely due to their role as precursors in the biosynthesis of the C13 side chain. However, concentration of amino acids added into the medium depends on each subject, nature of protein and desired products. 71 Table 4.7 Influence of organic compounds on proliferation of cell biomass Treatment 7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8 7.9 7.10 7.11 7.12 7.13 7.14 7.15 7.16 ME PE CH (mg/l) (mg/l) (mg/l) 0 0 0 0.1 0.5 1.0 1.5 2.0 0.1 0.5 1.0 1.5 2.0 0.1 0.5 1.0 1.5 2.0 Fresh cell weight (g) Dry cell weight (g) 5.93 de 6.17cde 6.23cde 6.53 cd 6.33cde 5.87 de 6.07cde 6.17cde 6.97 bc 6.67 cd 5.43e 6.40cde 6.77 cd 8.13a 7.70ab 6.03cde 0.609bcd 0.612bcd 0.600 cde 0.602bcd 0.620bcd 0.553de 0.591 cde 0.553de 0.633 bcd 0.643bc 0.523 e 0.637bc 0.664 abc 0.736 a 0.684ab 0.604 bcd * Different letters in one column indicate statistical significant differences Peptone originated from some low molecular-weight proteins and malt extracted from barley, they can promote the plant cell growth. The results in table 4.7 showed that effect of malt extract and peptone on cell growth was little different in some treatments from 7.2 to 7.11 and slightly better than the control treatment. Concentration of these compounds at 1.0 g/l showed the best results with 6.53g and 6.97g in fresh weight respectively and the cell growth decreased at higher contents. Although they are not used popularly any more, they have some important functions in culture of Citrus family, promoting embryogenic process in the first stage. Some recent studies showed the key role of malt with somatic embryo culture of Citrus sinensis and Citrus spp. (Jumin, 1995). Casein hydroxylase containing calcium, phosphate, micronutrients, vitamin and the mixture including 18 amino acids improved rate of cell growth in the study of Cardamine pratensis culture with lack of phosphate and it also recovered the insufficient addition of glutamine in culture medium (Bister - Miel et al., 1985). In this study, addition of Casein hydrolysate at various contents all enhanced the 72 growth of cell suspension. The growth of cell suspension gradually increased with the increase of concentration of casein hydrolysate in culture medium and the maximum growth of cell suspension obtained at 1000 mg/l of casein hydrolysate with 8.13g in fresh weight. This result indicated that casein hydrolysate (1000 g/l) is the most appropriate compound for cell growth of Taxus wallichiana Zucc. (Table 4.7). However, it decreased the growth of cell suspension at 1500 mg/l. Agrawal (2000) and Jha et al. (1997) also conducted addition of casein hydrolysate at 250 and 500 mg/l into culture medium of Taxus wallichiana Zucc. Further, in Taxus brevifolia, Taxus baccata, Taxus cuspidate, Taxus media and Taxus chinensis, casein hydrolysate was supplemented into the medium of callus and cell suspension culture with 1000 (mg/l) in concentration as well (Agrawal, 2000; Choi et al., 2001; Kim et al., 2001; Wickremesinhe et al., 1993). Figure 4.9. Cell suspension inducted from young stem explants of Taxus wallichiana Zucc was proliferated on medium supplemented with (A) Casein hydrolysate (1000 mg/l) (B) Malt extract (1000 mg/l) (C) Peptone (1000 mg/l) (D) Control 73 4.2.4. Examination of kinetic of cell growth in optimal liquid medium Figure 4.10. Kinetics of cell growth of Taxus wallichiana Zucc in liquid medium supplemented with 2,4-D (3 mg/l), NAA (3 mg/l), ascorbic acid (100 mg/l), Ki (0.5), sucrose (30 g/l), Cw (10 %), Glycine (10 mg/l), Casein hydrolysate (1000 mg/l) A growing cell suspension culture of Taxus wallichiana Zucc. on a fresh weight basis is shown in Fig. 4.10. Cell growth typically exhibited a lag period in the initial 0-7 days, a rapid growth period between day 7 and day 28, and a stationary phase thereafter. An initial lag phase in culture growth was observed up to 7 th day and the fresh weight of cells was increased by 57.3% after 7 days of culture and subsequently escaped its exponential growth phase after the day of 28. The growth of cell suspension was slowed down after 28 days of culture implying the decelerating phase of the suspension culture. The maximum growth index was about 6.453 and the highest average growth rate (0.230 g/day) after 28 days and maintained for several days that indicated the beginning of a stationary phase. The cell biomass increased slightly during the stationary phase between 28 and 42 d. On the basis of growth of cell suspension cultures, we investigated the effects of MJ, SA, and O-Chi on cell growth and paclitaxel accumulation. 74 4.3. Elicitation 4.3.1. Effect of the phenylalanine and elicitors on accumulation of taxol production in cell suspension of Taxus wallichiana Zucc The biosynthesis and accumulation of a number of secondary metabolites take place in specialized cells during specific developmental stages such as differentiation and pigmentation in the organs and/or whole plants (Roja and Heble, 1996). In terms of cell growth kinetics, cell growth usually incorporates an exponential phase, but most secondary metabolites are produced during the stationary phase. In this phase, there have also been many new enzymes frequently appeared in stationary phase, absently during the lag or log phases. Some recent studies showed that addition of organic compounds into culture medium had good impacts on content of secondary metabolites. Besides, complementation of organic compounds also promoted biosynthetic pathway for producing new compounds which plant cells do not possess. Scientists have conducted to enhance the productivity of Taxus cell cultures by including a biogenetic precursor (Fett-Neto et al., 1994; Muranaka et al., 2004), Nutrient feeding (Choi et al., 2000), elicitor treatment with chitosan (Zhang et al., 2000), methyl jasmonate (Yukumune et al., 1996; Mirjalili and Linden, 1996; Ketchum et al., 1999) and jasmonic acid (Baebler et al., 2002). Fleming et al. (1994) also reported that phenylisoserine was incorporated into a side chain of paclitaxel during its biosynthesis. Furthermore, Addition of yeast extract into the culture medium enhanced diosgenin content in Dioscorea deltoidea or influence of chitosan on stimulation of some genes related to biosynthetic pathway of the artemisinin compound in cell of Artemisia annua L., substrate of valencene on synthesis of nothatone, substrate of codeinone on synthesis of codeine, etc were also conducted (Minh T.V., 2006). From these findings, we speculated that production of paclitaxel could be enhanced by addition of these compounds as elicitors or biogenetic precursors in cell suspension cultures of T. wallichiana Zucc. 75 Table 4.8 Influence of the phenylalanine and elicitors on cell growth and accumulation of taxol production in cell suspension Treatment PA YE MJ SA (mg/l) (mg/l) (mg/l) (mg/l) 8.1 0 8.2 5 8.3 10 8.4 15 8.5 20 8.6 500 8.7 1000 8.8 2000 8.9 10 8.10 20 8.11 30 8.12 10 8.13 50 8.14 100 8.15 150 8.16 200 8.17 8.18 8.19 O-Chi (mg/l) 5 10 15 Dry cell weight (DCW) Taxol content (% DCW) 0.927cd 1.148a 1.018b 0.820ef 0.643hij 0.923cd 0.980bc 0.832 e 0.652hi 0.551kl 0.599ijk 0.910cd 0.861de 0.688gh 0.755fg 0.573jkl 0.617hljk 0.476m 0.512lm 0.02299 0.02018 0.04306 0.05181 0.04092 0.02551 0.05368 0.04394 0.16583 0.12872 0.08985 0.02891 0.05053 0.07537 0.04863 0.05527 0.34501 0.28449 0.22622 * Different letters in one column indicate statistical significant differences Phenylalanine supplementation has been reported to enhance secondary metabolite production in plant cell cultures (Shinde et al., 2009). Data in table 4.8 showed that the supplementation of phenylalanine at the range of 5 to 20 mg/l caused slight variation on the cell biomass. Phenylalanine (5 mg/l) produced maximum biomass (1.148g DW/culture) followed by 10, 15, 20 mg/l and control. The lowest growth was observed in 20 mg/l phenylalanine (0.64g DW/culture). Phenylalanine enhanced the biomass accumulation by 23.84% with supplementation of 5 mg/l. However, the most suitable concentration of phenylalanine for the highest paclitaxel production was 15 mg/l (0.518 mg/g DW) which was 1.254 times higher than the control. Higher concentration of phenylalanine seemed to be unsuitable for paclitaxel production and the minimum paclitaxel accumulation was obtained in 5 mg/l phenylalanine (0.02018 % DCW). 76 The optimized concentration of phenylalanine for paclitaxel production from Taxus chinensis was 15 mg/l (Luo and He, 2004) and this concentration is 5 times higher than optimum concentration of that for flavonoid production. In addition, Fett-Neto A.G. and DiCosmo F. (1996) found that paclitaxel yields in the cell culture of Taxus cuspidata were improved up to six times by feeding phenylalanine and other potential paclitaxel side-chain precursors. Furthermore, Khosroushahi et al. (2006) reported that, in Taxus baccata, addition of phenylalanine increased the Taxol amount. In Psoralea corylifolia hairy root culture, the effect of phenylalanine on the production of isoflavones showed that phenylalanine at 2 mM concentration increased the production of daidzein and genistein by 1.3 fold compared with the control. Daidzein and genistein levels were greatly inhibited when concentration of phenylalanine was increased to 10 mM (Shinde et al., 2009). In addition, Phenylalanine at low concentration (< 33 mg/l) showed a negative effect on the cell growth and a significant positive effect on phenylethanoid glycosides biosynthesis. After 20 days culture, the cell biomass and phenylethanoid glycosides content decreased with the increase of phenylalanine concentration over 33 mg/l (Ouyang et al., 2005). Considering this phenylalanine supplementation which is expected to increase the metabolic flux through phenylpropanoid biosynthetic pathway and elevate the level of targeted compound. Yeast extract (YE) used as an ingredient of plant media nowadays is a source of amino acids and vitamins, especially inositol and thiamine (Robbin and Bartley 1937). In a medium consisting only of macro- and micro-nutrients, addition of YE showed that it was essential for tissue growth (White, 1934; Robbins and Bartley, 1937). Yeast elicitor is one of the most commonly used elicitors for enhancement the secondary metabolites. Yeast extract was known to effectively bind to receptors on the plant cells. It induced the synthesis of phenylalanine ammonium lyase activity and enhanced secondary metabolites accumulation in plant cell cultures of Cistanche deserticola (Cheng et al., 2005). However, not all plant species response to this elicitor. In some case, yeast elicitor reduced the secondary metabolite such as camptothecin production in cell suspension cultures of Ophiorrhriza pumila (Saito et al., 2001). Yeast extract used as elicitor in Taxus wallichiana Zucc. cells 77 unsignificantly improved the paclitaxel production. In this experiment, direct addition of Yeast Extract into the medium at the beginning significantly improved taxol content in cell suspension, taxol content gradually increased along with increase of Yeast Extract concentration from 1000 to 2000 mg/l. Growth of the Taxus wallichiana Zucc. cell suspension cultures was slightly affected by addition of yeast extract. The highest paclitaxel content (0.537 mg/g DCW) was obtained from the cultures elicited by 1000 mg/l yeast extract. The MJ treatment led to a clear repression of cell growth in cell suspension cultures of Taxus wallichiana Zucc. (Table 4.8) and induced cell browning as well. When the cultures were treated with 10 mg/l MJ, cell growth was decreased by 29.89% relative to the control cultures. Treatments with 20 and 30 mg/l MJ showed reduction of 40.75% and 35.59% in cell growth, respectively. However, addition of MJ in different concentrations all increased level of paclitaxel in cell cultures and the most suitable concentration of MJ for the highest paclitaxel production was 10 mg/l (1.658 mg/g DW) which was 6.215 times higher than the control. Higher concentration of MJ seemed to be unsuitable for paclitaxel production. MJ has been studied for its inhibitory effects on cell growth and many other metabolic activities in plants (Cosio et al., 1990). In particular, Lois et al. (1989) reported that MJ concentrations above 0.01 mM inhibited root growth in some species. Suspension cell cultures are generally more sensitive to stress than cultured roots, and in our study, the growth of suspended cells was respectively decreased after treatment with 10, 20, and 30 mg/l MJ. In view of the inverse relationship between the production of biomass and the accumulation of secondary metabolites, the cell growth inhibition caused by MJ treatment may favor the synthesis of secondary metabolites. In fact, although MJ inhibited the growth of cells, it resulted in a higher production of paclitaxel. Similarly, indole alkaloid is produced by Catharanthus roseus (Sim et al., 1994) and Arnebia euchroma (Fu and Lu, 1999), and taxoids are produced by Taxus cuspidate (Ketchum et al., 1999). MJ has proved to be an effective signaling molecule that can strongly stimulate taxane biosynthesis in cultured Taxus cells (Wang et al., 2001). MJ has also been shown to induce 78 accumulation of camptothecin in Camptotheca acuminata (Song and Byun, 1998) and tropane alkaloids in Scopolia parviflora (Kang et al., 2004). Exogenously supplied SA has been shown to affect a variety of processes in plants, including stomatal closure, seed germination, fruit yield, and glycolysis (Klessing and Malamy, 1994). SA and its chemical derivative (acetylsalicylic acid, ASA) can enhance the productivity of some secondary metabolites in plant cell cultures. In our experiment, SA had a negative effect on growth, similar to that of MJ. All concentrations of SA slightly decreased cell growth, but did not stimulate cell browning. When the cultures were treated with 100 mg/l SA, cell growth decreased to 26.02% of the control culture growth while level of paclitaxel in cell culture increased to 2.279 times of the control (0.754 mg/g DW). Higher concentration of SA seemed to be unsuitable for paclitaxel production. Manthe et al. (1992) reported a 25% reduction in the growth of Vicia faba L. after 7 d of treatment with 5 mM SA. Besides, in a suspension culture of Hyoscyamus muticus treated with 40 mM SA, the production of lubimin increased by 50%, while in a transformed root culture of the same species, solavetivone production increased by 48% following the addition of 4 mM SA (Mehmetoglu and Curtis, 1997). Chitosan and derivatives have been proven recently to significantly improve accumulation and stimulation biosynthesis of secondary metabolites in plant cell suspension cultures (Cheng et al., 2006). The cells treated with O-chi changed the color from dark-orange to gray. The cell growth was decreased when increased concentrations of O-chi (Figure 4.11). When the cultures were treated with 5 mg/l O-chi, cell growth was decreased by 33.66% relative to the control cultures. Treatments with 5 and 10 mg/l O-chi showed reduction of 48.82% and 44.95% in cell growth, respectively. However, addition of O-chi in different concentrations all increased level of paclitaxel in cell cultures and the most suitable concentration of O-chi for the highest paclitaxel production was 5 mg/l (3.450 mg/g DW) which was 14.009 times higher than the control. Higher concentration of O-chi seemed to be unsuitable for paclitaxel production. Kim et al. (1997) reported that indirubin production from suspension culture of Polygonium tinctorum increased with complementation of chitosan and it also has been found to be the most effective in 79 increasing of plumbagin accumulation in suspension cultures of Plumbago rosea (Komaraiah et al., 2003). Paclitaxel content in cell suspension in table 4.8 showed that most treatments were higher in taxol content than the control. The most suitable concentration of phenylalanine for paclitaxel production was 15 mg/l (0.518 mg/g DW) which was 1.254 times higher than the control. In the elicited treatments, 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. Next, SA (100 mg/l) increased level of paclitaxel in cell culture to 2.279 times of the control (0.754 mg/g DW). And finally, the paclitaxel content (0.537 mg/g DCW) was obtained from the cultures elicited by 1000 mg/l yeast extract. Fig. 4.11. Cell suspension inducted from young stem explants of Taxus wallichiana Zucc was elicitated with A. Phenylalanine (15 mg/l) B. Oligo-chitosan (5 mg/l) C. Salysilic acid (100 mg/l) D. Methyl jamonate (10 mg/l) 80 However, these results may be different owing to factors such as the concentration or kind of elicitors. Therefore, to achieve maximum stimulation of paclitaxel biosynthesis, both the adding times and the exposure times of the elicitor need to be optimized. 4.3.2 Effect of adding time of elicitor on taxol accumulation of cells of Taxus wallichiana Zucc. Table 4.9. Effect of adding time of elicitor on taxol accumulation of cells of Taxus wallichiana Zucc. Treatment Time (day) DCW (g) % Taxol 6.1 0 0.192d 0.01454 6.2 7 0.381c 0.09475 6.3 14 0.461 bc 0.30466 6.4 21 0.532b 0.36935 6.5 28 a 0.09443 0.684 * Different letters in one column indicate statistical significant differences Culture medium = B5 + 2,4-D (3 mg/l) + NAA (3 mg/l) + Ki (0.5 mg/l) + ascorbic acid (100 mg/l) + sucrose (30 g/l) + Cw (10 %) + Glycine (10 mg/l) + Casein hydrolysate (1000 mg/l) + O-chi (5 mg/l) + PA (15 mg/l) In this scenario, determination of the most appropriate time of the elicitor complementation on the culture medium aimed to enhance paclitaxel accumulation of cells. Results in table 4.9 showed that elicitor addition time affected cell biomass proliferation and paclitaxel accumulation. In addition of 5 mg/l O-chi to the culture medium on day 0, dry weight of cells and paclitaxel content gained 0.192g and 0.0145% of dcw, respectively. This yield was lower than that gained on day 7 (0.381g and 0.0948 of DCW, respectively) and paclitaxel content increased 5.52 times compared with treatment 6.1 (addition of elicitor on day 0). However, the paclitaxel content in the treatment 6.3 and 6.4 supplemented with O-chi on day 14 and 21 when the growth of cells was strongest was highest (2.216 times and 2.898 times of paclitaxel content in treatment 6.2, respectively), and cell biomass gained 0.461g and 0.532g, respectively. In treatment 6.4 on day 28, cell biomass and paclitaxel accumulation of cells gained 0.684g and 0.944 mg/g DCW, 81 respectively. Although weight of cell biomass was higher than the others, paclitaxel content was lower than that in treatment 6.2, 6.3 and 6.4. Therefore, to enhance paclitaxel accumulation in cells, elicitor addition time on the culture medium determined was day 21 at stage of strong growth of cells. 4.3.3. Examination of exposure time with elicitor on taxol accumulation of cell suspension of Taxus wallichiana Zucc The growth of cell suspension was stable after 21 days of culture, and the cell cultures were supplemented with 5.0 mg/l of O-chi and paclitaxel accumulation of cells was measured at different times of exposure to O-chi (0 - 2 - 4 - 6 - 8 days). Culture medium = B5 + 2,4-D (3 mg/l) + NAA (3 mg/l) + Ki (0.5 mg/l) + ascorbic acid (100 mg/l) + sucrose (30 g/l) + Cw (10 %) + Glycine (10 mg/l) + Casein hydrolysate (1000 mg/l) + O-chi (5 mg/l) + PA (15 mg/l) Table 4.10. Effect of exposure time with elicitor on taxol accumulation of cell suspension of Taxus wallichiana Zucc Treatment Time (day) DCW % Paclitaxel 10.1 0 0.643a 0.10045 10.2 2 0.619b 0.30126 10.3 4 0.571c 0.34412 10.4 6 0.465e 0.40127 10.5 8 0.482d 0.29139 * Different letters in one column indicate statistical significant differences During the culture period, we introduced O-chi on day 21 (exposure time 0 day) and observed the effects up to 29 day (exposure time 8 day). Time course of cell growth and paclitaxel accumulation of Taxus wallichiana Zucc. cell cultures treated by 5.0 mg/l O-chi were shown in Table 4.10. Growth of cell suspension of Taxus wallichiana Zucc. markedly decreased according to increase of elicitor exposure time. Levels of paclitaxel increased 1.99- and 2.99-fold over treatment 10.1 by 2 days - 6 days and declined 8 days. In treatments 12.1 - 12.4, the elicitor exposure time from 0 - 8 days decreaesd growth of cell biomass from 0.643 - 0.482 g, but paclitaxel content of cells increased from 1.005 - 4.013 mg/g DCW. In treatment 12.4, paclitaxel content was highest (0.0413% of dcw) after 6 days of 82 elicitor exposure. Weight of cell biomass (0.482 g) decreased in treatment 12.5 (on day 8 of elicitor exposure), and paclitaxel content (0.2914% of dcw) also declined. These could be explained as following: the highest paclitaxel accumulation of cells impacted cell growth and caused cell death. 83 Chapter 5: Summary and Conclusions 84 Studies of the effects of nutrient media and phytohormones on cell growth and taxol accumulation via cell culture of Taxus wallichiana Zucc. were carried out at Key Laboratory of Plant Cell Biotechnology - Institute of Tropical Biology. The results of the investigation are summerised here under. 5.1. Cell cultures - High frequency of callus formation (100%) was obtained from young explants on WP solid medium supplemented with 2,4-D (4.0 mg/l), but B5 medium was the best one for callus growth with 2,4-D (4.0 mg/l). - The friable and reddish, light brown callus 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%). - 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. 5.2. 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 in cells 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. - In addition, elicitor addition time on the culture medium determined was day 21 at stage of strong growth of cells and cell cultures were harvested on day 27. 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Composition of the MS (Murashige and Skoog, 1962), B5 (Gamborg, 1968,), and WPM (Woody Plant Medium, 1981) Media Component Macronutrients (g/l) Micronutrients (mg/l) Vitamine & aminoacid (mg/l) NaFeEDTA (g/l) MS WPM B5 CaCl2.2H2O - 0.0725 0.15 CaCl2. 0.334 - - Ca( NO3)2.4H2O - 0.556 - KNO3 1.9 - 2.5 K2SO4 - 0.99 - (NH4)2SO4 - - 0.134 KH2PO4 0.17 0.17 - NaH2PO4.2H2O - - 0.15 MgSO4.7H2O 0.37 0.37 0.25 NH4NO3 1.65 0.4 - CoCl2.6H2O 0.025 0.025 0.025 CuSO4.5H2O 0.025 0.025 0.025 H3BO3 6.20 6.20 3 KI 0.83 0.83 0.75 MnSO4.H2O 22.3 22.3 10 Na2MoO4.H2O 0.25 0.25 0.25 ZnSO4.H2O 8.60 8.60 2 Glycine 2.00 2.00 0.00 Myo-Inositol 100 100 100 Nicotinic acid 0.50 0.50 1.00 Pyridoxine HCl 0.50 0.50 1.00 Thiamine-HCl 0.10 0.10 10.00 NaEDTA 0.0373 0.0373 0.0373 FeSO4.7H2O 0.0278 0.0278 0.0278 99 Appendix 2. Facilities in Media Preparation and Culture Evaluation Section Name Brand and Model Place of origin Analytical balances Sartorius – TE313S Japan pH meter VWR Scientific – 8005 USA Hot plate and magnetic stirrer IKA – 3581201 USA Refrigerator Electrolux – ER3198D WH Japan Autoclave Hirayama – HV 85 Japan Fume hood Cole Palmer - EW-33730 USA Microwave Panasonic – NNC2003S Japan Water purification system Aquatron A8000 England Inverted light microscope Nikon – Eclipse 90i Japan Dissecting microscope Nikon – SMZ 1B Japan Appendix 3. Facilities in culture transferring area Name Brand and Model Place of origin Laminar air flow hood Sanyo – MCV B131S (T) Japan Alcohol burner Schoot Germany Forceps, spatulas, scalpel, and Vietnam disposable blades Parafilm roll Sigma – P7793 Malaysia Appendix 4. Facilities in environmentally controlled culture Name Brand and Model Culture shelves Place of origin Vietnam Orbital shakers Stuart USA Incubators Sanyo – MCO 18 Japan 100 Appendix 5. Statistic analysis Experiment 1: Examination of mineral media and phytohormones on induction and growth of callus tissue of Taxus wallichiana Zucc. Data File:Influence of 2,4-D and mineral media on callus induction Error Mean Square = 78.64 Error Degrees of Freedom = 59 LSD value = 14.93 at alpha = 0.010 A N A L Y S I S O F V A R I A N C E T A B L E Degrees of Sum of Mean Freedom Squares Square F-value Prob. ----------------------------------------------------------------------Between 14 18500.541 1321.467 16.803 0.0000 Within 59 4640.000 78.644 ----------------------------------------------------------------------Total 73 23140.541 Coefficient of Variation = 10.29% Var. V A R I A B L E No. 2 1 Number Sum Average SD SE -----------------------------------------------------------------1 5.00 360.000 72.000 10.95 3.97 2 5.00 420.000 84.000 8.94 3.97 3 5.00 500.000 100.000 0.00 3.97 4 5.00 500.000 100.000 0.00 3.97 5 5.00 500.000 100.000 0.00 3.97 6 5.00 320.000 64.000 16.73 3.97 7 5.00 380.000 76.000 16.73 3.97 8 5.00 480.000 96.000 8.94 3.97 9 5.00 500.000 100.000 0.00 3.97 10 5.00 500.000 100.000 0.00 3.97 11 5.00 260.000 52.000 10.95 3.97 12 5.00 340.000 68.000 10.95 3.97 13 5.00 420.000 84.000 8.94 3.97 14 5.00 500.000 100.000 0.00 3.97 15 4.00 400.000 100.000 0.00 4.43 -----------------------------------------------------------------Total 74.00 6380.000 86.216 17.80 2.07 Original Order Ranked Order Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 = = = = = = = = = = = = = = = 72.00 84.00 100.0 100.0 100.0 64.00 76.00 96.00 100.0 100.0 52.00 68.00 84.00 100.0 100.0 BC AB A A A CD BC A A A D BC AB A A Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean 15 9 3 4 5 14 10 8 13 2 7 1 12 6 11 = = = = = = = = = = = = = = = 100.0 100.0 100.0 100.0 100.0 100.0 100.0 96.00 84.00 84.00 76.00 72.00 68.00 64.00 52.00 A A A A A A A A AB AB BC BC BC CD D Data File:Influence of 2,4-D and mineral media on callus growth Error Mean Square = 0.006000 Error Degrees of Freedom = 60 LSD value = 0.1303 at alpha = 0.010 101 A N A L Y S I S O F V A R I A N C E T A B L E Degrees of Sum of Mean Freedom Squares Square F-value Prob. ----------------------------------------------------------------------Between 14 15.807 1.129 190.635 0.0000 Within 60 0.355 0.006 ----------------------------------------------------------------------Total 74 16.162 Coefficient of Variation = 4.53% Var. V A R I A B L E No. 2 1 Number Sum Average SD SE -----------------------------------------------------------------1 5.00 5.880 1.176 0.07 0.03 2 5.00 7.080 1.416 0.07 0.03 3 5.00 8.410 1.682 0.10 0.03 4 5.00 11.580 2.316 0.11 0.03 5 5.00 10.550 2.110 0.08 0.03 6 5.00 6.130 1.226 0.05 0.03 7 5.00 6.770 1.354 0.06 0.03 8 5.00 8.720 1.744 0.05 0.03 9 5.00 12.800 2.560 0.07 0.03 10 5.00 11.010 2.202 0.08 0.03 11 5.00 5.340 1.068 0.08 0.03 12 5.00 5.580 1.116 0.06 0.03 13 5.00 7.830 1.566 0.08 0.03 14 5.00 10.210 2.042 0.08 0.03 15 5.00 9.560 1.912 0.09 0.03 -----------------------------------------------------------------Total 75.00 127.450 1.699 0.47 0.05 Original Order Ranked Order Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 = = = = = = = = = = = = = = = 1.176 1.416 1.682 2.316 2.110 1.226 1.354 1.744 2.560 2.202 1.068 1.116 1.566 2.042 1.912 JK H FG B CD IJ HI F A BC K JK G DE E Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean 9 4 10 5 14 15 8 3 13 2 7 6 1 12 11 = = = = = = = = = = = = = = = 2.560 2.316 2.202 2.110 2.042 1.912 1.744 1.682 1.566 1.416 1.354 1.226 1.176 1.116 1.068 A B BC CD DE E F FG G H HI IJ JK JK K Data File:Influence of NAA and mineral media on callus induction Error Mean Square = 117.3 Error Degrees of Freedom = 60 LSD value = 18.23 at alpha = 0.010 A N A L Y S I S O F V A R I A N C E T A B L E Degrees of Sum of Mean Freedom Squares Square F-value Prob. ----------------------------------------------------------------------Between 14 44160.000 3154.286 26.883 0.0000 Within 60 7040.000 117.333 ----------------------------------------------------------------------Total 74 51200.000 102 Coefficient of Variation = 18.05% Var. V A R I A B L E No. 2 1 Number Sum Average SD SE -----------------------------------------------------------------1 5.00 100.000 20.000 14.14 4.84 2 5.00 320.000 64.000 8.94 4.84 3 5.00 420.000 84.000 8.94 4.84 4 5.00 420.000 84.000 8.94 4.84 5 5.00 360.000 72.000 10.95 4.84 6 5.00 80.000 16.000 8.94 4.84 7 5.00 320.000 64.000 8.94 4.84 8 5.00 420.000 84.000 8.94 4.84 9 5.00 400.000 80.000 14.14 4.84 10 5.00 380.000 76.000 8.94 4.84 11 5.00 80.000 16.000 8.94 4.84 12 5.00 180.000 36.000 8.94 4.84 13 5.00 360.000 72.000 10.95 4.84 14 5.00 340.000 68.000 10.95 4.84 15 5.00 320.000 64.000 16.73 4.84 -----------------------------------------------------------------Total 75.00 4500.000 60.000 26.30 3.04 Original Order Ranked Order Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 = = = = = = = = = = = = = = = 20.00 64.00 84.00 84.00 72.00 16.00 64.00 84.00 80.00 76.00 16.00 36.00 72.00 68.00 64.00 BC A A A A C A A A A C B A A A Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean 8 3 4 9 10 5 13 14 15 2 7 12 1 6 11 = = = = = = = = = = = = = = = 84.00 84.00 84.00 80.00 76.00 72.00 72.00 68.00 64.00 64.00 64.00 36.00 20.00 16.00 16.00 A A A A A A A A A A A B BC C C Data File:Influence of NAA and mineral media on callus growth Error Mean Square = 0.006000 Error Degrees of Freedom = 30 LSD value = 0.1739 at alpha = 0.010 A N A L Y S I S O F V A R I A N C E T A B L E Degrees of Sum of Mean Freedom Squares Square F-value Prob. ----------------------------------------------------------------------Between 14 11.224 0.802 131.909 0.0000 Within 30 0.182 0.006 ----------------------------------------------------------------------Total 44 11.406 Coefficient of Variation = 6.64% 103 Var. V A R I A B L E No. 2 1 Number Sum Average SD SE -----------------------------------------------------------------1 3.00 1.620 0.540 0.09 0.05 2 3.00 2.380 0.793 0.10 0.05 3 3.00 5.450 1.817 0.10 0.05 4 3.00 4.940 1.647 0.10 0.05 5 3.00 4.570 1.523 0.07 0.05 6 3.00 1.600 0.533 0.03 0.05 7 3.00 2.140 0.713 0.08 0.05 8 3.00 5.820 1.940 0.10 0.05 9 3.00 5.460 1.820 0.06 0.05 10 3.00 4.960 1.653 0.09 0.05 11 3.00 1.720 0.573 0.04 0.05 12 3.00 2.230 0.743 0.04 0.05 13 3.00 2.980 0.993 0.08 0.05 14 3.00 3.450 1.150 0.05 0.05 15 3.00 3.520 1.173 0.07 0.05 -----------------------------------------------------------------Total 45.00 52.840 1.174 0.51 0.08 Original Order Ranked Order Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 = = = = = = = = = = = = = = = 0.5400 0.7933 1.817 1.647 1.523 0.5333 0.7133 1.940 1.820 1.653 0.5733 0.7433 0.9933 1.150 1.173 G E AB BC C G EFG A AB BC FG EF D D D Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean 8 9 3 10 4 5 15 14 13 2 12 7 11 1 6 = = = = = = = = = = = = = = = 1.940 1.820 1.817 1.653 1.647 1.523 1.173 1.150 0.9933 0.7933 0.7433 0.7133 0.5733 0.5400 0.5333 A AB AB BC BC C D D D E EF EFG FG G G Experiment 2: Examination of plant growth regulators on growth and proliferation of callus tissue of Taxus wallichiana Zucc. Data File:Influence of 2,4-D and NAA on callus growth Error Mean Square = 0.006000 Error Degrees of Freedom = 18 LSD value = 0.1820 at alpha = 0.010 A N A L Y S I S O F V A R I A N C E T A B L E Degrees of Sum of Mean Freedom Squares Square F-value Prob. ----------------------------------------------------------------------Between 8 3.677 0.460 82.951 0.0000 Within 18 0.100 0.006 ----------------------------------------------------------------------Total 26 3.777 Coefficient of Variation = 3.12% 104 Var. V A R I A B L E No. 2 1 Number Sum Average SD SE -----------------------------------------------------------------1 3.00 4.570 1.523 0.08 0.04 2 3.00 7.200 2.400 0.07 0.04 3 3.00 7.580 2.527 0.06 0.04 4 3.00 7.090 2.363 0.07 0.04 5 3.00 7.080 2.360 0.10 0.04 6 3.00 7.620 2.540 0.09 0.04 7 3.00 9.130 3.043 0.09 0.04 8 3.00 7.120 2.373 0.04 0.04 9 3.00 6.960 2.320 0.06 0.04 -----------------------------------------------------------------Total 27.00 64.350 2.383 0.38 0.07 Original Order Mean Mean Mean Mean Mean Mean Mean Mean Mean 1 2 3 4 5 6 7 8 9 = = = = = = = = = 1.523 2.400 2.527 2.363 2.360 2.540 3.043 2.373 2.320 Ranked Order D BC B BC BC B A BC C Mean Mean Mean Mean Mean Mean Mean Mean Mean 7 6 3 2 8 4 5 9 1 = = = = = = = = = 3.043 2.540 2.527 2.400 2.373 2.363 2.360 2.320 1.523 A B B BC BC BC BC C D Experiment 2: Examination of organics on proliferation of callus tissue of Taxus wallichiana Zucc. Data File:Influence of sucrose on callus growth Error Mean Square = 0.008000 Error Degrees of Freedom = 6 LSD value = 0.2708 at alpha = 0.010 A N A L Y S I S O F V A R I A N C E T A B L E Degrees of Sum of Mean Freedom Squares Square F-value Prob. ------------------------------------------------------------------------Between 2 0.941 0.471 62.651 0.0001 Within 6 0.045 0.008 ------------------------------------------------------------------------Total 8 0.986 Coefficient of Variation = 3.15% Var. V A R I A B L E No. 2 1 Number Sum Average SD SE -----------------------------------------------------------------1 3.00 7.880 2.627 0.06 0.05 2 3.00 9.600 3.200 0.08 0.05 3 3.00 7.320 2.440 0.11 0.05 -----------------------------------------------------------------Total 9.00 24.800 2.756 0.35 0.12 Original Order Mean Mean Mean 1 = 2 = 3 = 2.627 3.200 2.440 Ranked Order B A B Mean Mean Mean 2 = 1 = 3 = 3.200 2.627 2.440 A B B 105 Data File:Influence of Coconut water and Glycine on callus growth Error Mean Square = 0.09100 Error Degrees of Freedom = 32 LSD value = 0.5017 at alpha = 0.050 A N A L Y S I S O F V A R I A N C E T A B L E Degrees of Sum of Mean Freedom Squares Square F-value Prob. ------------------------------------------------------------------------Between 15 3.479 0.232 2.559 0.0126 Within 32 2.900 0.091 ------------------------------------------------------------------------Total 47 6.379 Coefficient of Variation = 8.37% Var. V A R I A B L E No. 2 1 Number Sum Average SD SE -----------------------------------------------------------------1 3.00 9.300 3.100 0.40 0.17 2 3.00 9.500 3.167 0.35 0.17 3 3.00 10.600 3.533 0.35 0.17 4 3.00 10.500 3.500 0.10 0.17 5 3.00 9.400 3.133 0.25 0.17 6 3.00 11.200 3.733 0.31 0.17 7 3.00 11.300 3.767 0.31 0.17 8 3.00 10.300 3.433 0.35 0.17 9 3.00 11.500 3.833 0.35 0.17 10 3.00 11.600 3.867 0.21 0.17 11 3.00 11.300 3.767 0.21 0.17 12 3.00 12.200 4.067 0.25 0.17 13 3.00 11.400 3.800 0.17 0.17 14 3.00 11.000 3.667 0.15 0.17 15 3.00 10.700 3.567 0.45 0.17 16 3.00 10.800 3.600 0.36 0.17 -----------------------------------------------------------------Total 48.00 172.600 3.596 0.37 0.05 Original Order Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 = = = = = = = = = = = = = = = = 3.100 3.167 3.533 3.500 3.133 3.733 3.767 3.433 3.833 3.867 3.767 4.067 3.800 3.667 3.567 3.600 Ranked Order D CD ABCD ABCD D ABC AB BCD AB AB AB A AB ABCD ABCD ABCD Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean 12 10 9 13 11 7 6 14 16 15 3 4 8 2 5 1 = = = = = = = = = = = = = = = = 4.067 3.867 3.833 3.800 3.767 3.767 3.733 3.667 3.600 3.567 3.533 3.500 3.433 3.167 3.133 3.100 A AB AB AB AB AB ABC ABCD ABCD ABCD ABCD ABCD BCD CD D D 106 Experiment 5: Examination of plant growth regulators (NAA; 2,4-D) on growth and proliferation of cell suspension of Taxus wallichiana Zucc. Data File:Effect of NAA and 2,4-D on cell suspension proliferation Error Mean Square = 0.02800 Error Degrees of Freedom = 40 LSD value = 0.3695 at alpha = 0.010 Original Order Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 = = = = = = = = = = = = = = = = = = = = 2.117 2.150 2.895 3.281 2.462 2.933 3.119 3.725 2.571 3.315 4.236 4.611 3.499 3.904 6.197 3.728 5.125 5.314 4.873 3.398 Ranked Order M M JK HIJ LM IJK HIJ FG KL HI E D GH EF A FG BC B CD GH Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean 15 18 17 19 12 11 14 16 8 13 20 10 4 7 6 3 9 5 2 1 = = = = = = = = = = = = = = = = = = = = 6.197 A 5.314 B 5.125 BC 4.873 CD 4.611 D 4.236 E 3.904 EF 3.728 FG 3.725 FG 3.499 GH 3.398 GH 3.315 HI 3.281 HIJ 3.119 HIJ 2.933 IJK 2.895 JK 2.571 KL 2.462 LM 2.150 M 2.117 M Error Mean Square = 1284. Error Degrees of Freedom = 40 LSD value = 79.12 at alpha = 0.010 Original Order Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 = = = = = = = = = = = = = = = = = = = = 198.3 272.7 281.3 321.3 214.0 277.7 305.3 341.7 217.0 320.3 415.0 449.3 355.7 385.3 610.3 347.7 488.7 511.7 451.3 276.7 Ranked Order H GH GH FG H GH FG EFG H FG CDE BCD EFG DEF A EFG BC B BCD GH Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean 15 18 17 19 12 11 14 13 16 8 4 10 7 3 6 20 2 9 5 1 = = = = = = = = = = = = = = = = = = = = 610.3 511.7 488.7 451.3 449.3 415.0 385.3 355.7 347.7 341.7 321.3 320.3 305.3 281.3 277.7 276.7 272.7 217.0 214.0 198.3 A B BC BCD BCD CDE DEF EFG EFG EFG FG FG FG GH GH GH GH H H H 107 Experiment 5: Examination of organics on growth and proliferation of cell suspension of Taxus wallichiana Zucc. Error Mean Square = 0.1490 Error Degrees of Freedom = 32 LSD value = 0.8631 Variable 2 Fresh cell weight A N A L Y S I S O F V A R I A N C E T A B L E Degrees of Sum of Mean Freedom Squares Square F-value Prob. ------------------------------------------------------------------------Between 15 20.999 1.400 9.385 0.0000 Within 32 4.773 0.149 ------------------------------------------------------------------------Total 47 25.773 Coefficient of Variation = 5.98% Var. V A R I A B L E No. 2 1 Number Sum Average SD SE -----------------------------------------------------------------1 3.00 17.800 5.933 0.25 0.22 2 3.00 18.500 6.167 0.51 0.22 3 3.00 18.700 6.233 0.15 0.22 4 3.00 19.600 6.533 0.25 0.22 5 3.00 19.000 6.333 0.67 0.22 6 3.00 17.600 5.867 0.21 0.22 7 3.00 18.200 6.067 0.71 0.22 8 3.00 18.500 6.167 0.21 0.22 9 3.00 20.900 6.967 0.31 0.22 10 3.00 20.000 6.667 0.38 0.22 11 3.00 16.300 5.433 0.25 0.22 12 3.00 19.200 6.400 0.20 0.22 13 3.00 20.300 6.767 0.21 0.22 14 3.00 24.400 8.133 0.35 0.22 15 3.00 23.100 7.700 0.56 0.22 16 3.00 18.100 6.033 0.35 0.22 -----------------------------------------------------------------Total 48.00 310.200 6.462 0.74 0.11 Original Order Ranked Order Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 = = = = = = = = = = = = = = = = 5.933 6.167 6.233 6.533 6.333 5.867 6.067 6.167 6.967 6.667 5.433 6.400 6.767 8.133 7.700 6.033 DE CDE CDE CD CDE DE CDE CDE BC CD E CDE CD A AB CDE Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean 14 15 9 13 10 4 12 5 3 2 8 7 16 1 6 11 = = = = = = = = = = = = = = = = 8.133 7.700 6.967 6.767 6.667 6.533 6.400 6.333 6.233 6.167 6.167 6.067 6.033 5.933 5.867 5.433 A AB BC CD CD CD CDE CDE CDE CDE CDE CDE CDE DE DE E Error Mean Square = 0.001000 Error Degrees of Freedom = 32 LSD value = 0.07071 Variable 3 Dry Cell Weight 108 A N A L Y S I S O F V A R I A N C E T A B L E Degrees of Sum of Mean Freedom Squares Square F-value Prob. ------------------------------------------------------------------------Between 15 0.122 0.008 5.894 0.0000 Within 32 0.044 0.001 ------------------------------------------------------------------------Total 47 0.166 Coefficient of Variation = 6.03% Var. V A R I A B L E No. 3 1 Number Sum Average SD SE -----------------------------------------------------------------1 3.00 1.828 0.609 0.03 0.02 2 3.00 1.836 0.612 0.04 0.02 3 3.00 1.799 0.600 0.04 0.02 4 3.00 1.808 0.603 0.02 0.02 5 3.00 1.861 0.620 0.01 0.02 6 3.00 1.660 0.553 0.03 0.02 7 3.00 1.774 0.591 0.05 0.02 8 3.00 1.659 0.553 0.05 0.02 9 3.00 1.899 0.633 0.03 0.02 10 3.00 1.930 0.643 0.02 0.02 11 3.00 1.569 0.523 0.04 0.02 12 3.00 1.911 0.637 0.04 0.02 13 3.00 1.991 0.664 0.04 0.02 14 3.00 2.209 0.736 0.04 0.02 15 3.00 2.052 0.684 0.04 0.02 16 3.00 1.811 0.604 0.04 0.02 -----------------------------------------------------------------Total 48.00 29.597 0.617 0.06 0.01 Original Order Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 = = = = = = = = = = = = = = = = 0.6093 0.6120 0.5997 0.6027 0.6203 0.5533 0.5913 0.5530 0.6330 0.6433 0.5230 0.6370 0.6637 0.7363 0.6840 0.6037 Ranked Order BCD BCD CDE BCD BCD DE CDE DE BCD BC E BC ABC A AB BCD Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean 14 15 13 10 12 9 5 2 1 16 4 3 7 6 8 11 = = = = = = = = = = = = = = = = 0.7363 0.6840 0.6637 0.6433 0.6370 0.6330 0.6203 0.6120 0.6093 0.6037 0.6027 0.5997 0.5913 0.5533 0.5530 0.5230 A AB ABC BC BC BCD BCD BCD BCD BCD BCD CDE CDE DE DE E 109 Experiment 8: Examination of some elicitors on taxol biosynthesis of cell suspension of Taxus wallichiana Zucc Error Mean Square = 0.001000 Error Degrees of Freedom = 38 LSD value = 0.07001 at alpha = 0.010 A N A L Y S I S O F V A R I A N C E T A B L E Degrees of Sum of Mean Freedom Squares Square F-value Prob. ------------------------------------------------------------------------Between 18 1.977 0.110 143.152 0.0000 Within 38 0.029 0.001 ------------------------------------------------------------------------Total 56 2.006 Coefficient of Variation = 3.63% Var. V A R I A B L E No. 2 1 Number Sum Average SD SE -----------------------------------------------------------------1 3.00 2.781 0.927 0.03 0.02 2 3.00 3.443 1.148 0.05 0.02 3 3.00 3.055 1.018 0.05 0.02 4 3.00 2.460 0.820 0.03 0.02 5 3.00 1.928 0.643 0.03 0.02 6 3.00 2.769 0.923 0.04 0.02 7 3.00 2.940 0.980 0.01 0.02 8 3.00 2.496 0.832 0.01 0.02 9 3.00 1.956 0.652 0.02 0.02 10 3.00 1.654 0.551 0.02 0.02 11 3.00 1.796 0.599 0.01 0.02 12 3.00 2.730 0.910 0.03 0.02 13 3.00 2.583 0.861 0.02 0.02 14 3.00 2.064 0.688 0.02 0.02 15 3.00 2.265 0.755 0.02 0.02 16 3.00 1.720 0.573 0.01 0.02 17 3.00 1.851 0.617 0.01 0.02 18 3.00 1.428 0.476 0.02 0.02 19 3.00 1.536 0.512 0.03 0.02 -----------------------------------------------------------------Total 57.00 43.455 0.762 0.19 0.03 Original Order Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 = = = = = = = = = = = = = = = = = = = 0.9270 1.148 1.018 0.8200 0.6427 0.9230 0.9800 0.8320 0.6520 0.5513 0.5987 0.9100 0.8610 0.6880 0.7550 0.5733 0.6170 0.4760 0.5120 Ranked Order CD A B EF HIJ CD BC E HI KL IJK CD DE GH FG JKL HIJK M LM Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean 2 3 7 1 6 12 13 8 4 15 14 9 5 17 11 16 10 19 18 = = = = = = = = = = = = = = = = = = = 1.148 A 1.018 B 0.9800 BC 0.9270 CD 0.9230 CD 0.9100 CD 0.8610 DE 0.8320 E 0.8200 EF 0.7550 FG 0.6880 GH 0.6520 HI 0.6427 HIJ 0.6170 HIJK 0.5987 IJK 0.5733 JKL 0.5513 KL 0.5120 LM 0.4760 M 110 Experiment 9: Examination of elicitor addition time on taxol accumulation of cell suspension of Taxus wallichiana Zucc Error Mean Square = 0.001000 Error Degrees of Freedom = 10 LSD value = 0.08183 at alpha = 0.010 A N A L Y S I S O F V A R I A N C E T A B L E Degrees of Sum of Mean Freedom Squares Square F-value Prob. ------------------------------------------------------------------------Between 4 0.399 0.100 156.988 0.0000 Within 10 0.006 0.001 ------------------------------------------------------------------------Total 14 0.405 Coefficient of Variation = 5.60% Var. V A R I A B L E No. 2 1 Number Sum Average SD SE -----------------------------------------------------------------1 3.00 0.577 0.192 0.03 0.01 2 3.00 1.143 0.381 0.01 0.01 3 3.00 1.384 0.461 0.02 0.01 4 3.00 1.597 0.532 0.02 0.01 5 3.00 2.053 0.684 0.04 0.01 -----------------------------------------------------------------Total 15.00 6.754 0.450 0.17 0.04 Original Order Mean Mean Mean Mean Mean 1 2 3 4 5 = = = = = 0.1923 0.3810 0.4613 0.5323 0.6843 Ranked Order D C BC B A Mean Mean Mean Mean Mean 5 4 3 2 1 = = = = = 0.6843 0.5323 0.4613 0.3810 0.1923 A B BC C D Experiment 10: Examination of exposure time with elicitor on taxol accumulation of cell suspension of Taxus wallichiana Zucc Error Mean Square = 1.000e-006 Error Degrees of Freedom = 10 LSD value = 0.002588 at alpha = 0.010 A N A L Y S I S O F V A R I A N C E T A B L E Degrees of Sum of Mean Freedom Squares Square F-value Prob. ------------------------------------------------------------------------Between 4 0.077 0.019 42.358 0.0000 Within 10 0.005 0.000 ------------------------------------------------------------------------Total 14 0.081 Coefficient of Variation = 3.82% Var. V A R I A B L E No. 2 1 Number Sum Average SD SE -----------------------------------------------------------------1 3.00 1.929 0.643 0.02 0.01 2 3.00 1.858 0.619 0.02 0.01 3 3.00 1.713 0.571 0.03 0.01 4 3.00 1.396 0.465 0.01 0.01 5 3.00 1.446 0.482 0.03 0.01 -----------------------------------------------------------------Total 15.00 8.342 0.556 0.08 0.02 111 Original Order Mean Mean Mean Mean Mean 1 2 3 4 5 = = = = = 0.6430 0.6193 0.5710 0.4653 0.4820 Ranked Order A B C E D Mean Mean Mean Mean Mean 1 2 3 5 4 = = = = = 0.6430 0.6193 0.5710 0.4820 0.4653 A B C D E 112 [...]... 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... 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... 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) 4 selecting high yielding cell clones, (iii) precursor feeding, and (iv) elicitation The elicitors... 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... 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 24 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... production This technique can ultimately provide a continuous, reliable source of natural products 20 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... 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... 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. .. 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 ... 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 ... 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 ... 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