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Characterization of production of Paclitaxel and related Taxanes in Taxus Cuspidata Densiformis suspension cultures by LC,LC/MS, and LC/MS/MSCHAPTER THEREPLANT TISSUE CULTUREⅠ. Potential of Plant cell Culture for Taxane ProductionSeveral alternative sources of paclitaxel have been identified and are currently the subjects of considerable investigation worldwide. These include the total synthesis and biosynthesis of paclitaxel, the agriculture supply of taxoids from needles of Taxus species, hemisynthesis (the attachment of a side chain to biogenetic precursors of paclitaxel such as baccatin Ⅲ or 10-deacetylbaccatin Ⅲ), fungus production, and the production of taxoids by cell and tissue culture. This reciew will concentrate only on the latter possibility.Plant tissue culture is one approach under investigation to provide large amounts and a stable supply of this compound exhibiting antineoplastic activity. A process to produce paclitaxel or paclitaxel-like compounds in cell culture has already been parented. The development of fast growing cell lines capable of producing paclitaxel would not only solve the limitations in paclitaxel supplies presently needed for clinical use, but would also help conserve the large number of trees that need to be harvested in order to isolate it. Currently, scientists and researchers have been successful in initiating fast plant growth but with limited paclitaxel production or vice versa. Therefore, it is the objective of researchers to find a method that will promote fast plant growth and also produce a large amount of paclitaxel at the same time.Ⅱ. Factors Influencing Growth Paclitaxel ContentA.Choice of Media for GrowthGamborg's (B5) and Murashige & Skoog's (MS) media seem to be superior for callus growth compared to White's (WP) medium. The major difference between these two media is that the MS medium contains 40 mM nitrate and 20mM ammonium, compared to 25mM nitrate and 2mM ammonium. Many researchers have selected the B5 medium over the MS medium for all subsequent studies, although they achieve similar results.Gamborg's B5 media was used throughout our experiments for initiation of callus cultures and suspension cultures due to successful published results. It was supplemented with 2% sucrose, 2 g/L casein hydrolysate, 2.4 mg/L picloram, and 1.8 mg/L α-naphthalene acetic acid. Agar (8 g/L) was used for solid cultures.B. Initiation of Callus CulturesPrevious work indicated that bark explants seem to be the most useful for establishing callus. The age of the tree did not appear to affect the ability to initiate callus when comparing both young and old tree materials grown on Gamborg's B5 medium supplemented with 1-2 mg/L of 2,4-dichlorophenoxyacetic acid. Callus cultures initiated and maintained in total darkness were generally pale-yellow to light brown in color. This resulted in sufficient masses of friable callus necessary for subculture within 3-4 weeks. However, the growth rate can decline substantially following the initial subculture and result in very slow-growing, brown-colored clumps of callus. It has been presumed that these brown-colored exudates are phenolic in nature and can eventually lead to cell death. This common phenomenon is totally random and unpredictable. Once this phenomenon has been triggered, the cells could not be saved by placing them in fresh media. However, adding polyvinylpyrrolidone to the culture media can help keep the cells alive and growing. Our experience withcallus initiation was similar to those studies.Our studies have found that callus which initiated early (usually within 2 weeks ) frequently did not proliferate when subcultured and turned brown and necrotic. In contrast, calli which developed from 4 weeks to 4 months after explants were fist placed on initiation media were able to be continuously subcultured when transferred at 1-2 month intervals. The presence of the survival of callus after subsequent subculturing. The relationship between paclitaxel concentration and callus initiation, however, has not been clarified.C. Effect of SugarSucrose is the preferred carbon source for growth in plant cell cultures, although the presence of more rapidly metabolized sugar such as glucose favors fast growth. Other sugars such as lactose, galactose, glucose, and fructose also support cell growth to some extent. On the other hand, sugar alcohols such as mannitol and sorbital which are generally used to raise the sugars added play a major role in the production of paclitaxel. In general, raising the initial sugar levels lead to an increase of secondary metabolite production. High initial levels of sugar increase the osmotic potential, although the role of osmotic pressure on the synthesis of secondary metabolites is not cleat. Kim and colleagues have shown that the highest level of paclitaxel was obtained with fructosel. The optimum concentration of each sugar for paclitaxel production was found to be the same at 6% in all cases. Wickremesinhe and Arteca have provided additional support that fructose is the most effective for paclitaxel production. However, other combinations of sugars such as sucrose combined with glucose also increased paclitaxel production.The presence of extracellular invertase activity and rapid extracellular sucrose hydrolysis has been observed in many cell cultures. These reports suggest that cells secrete or possess on their surface excess amounts of invertase, which result in the hydrolysis of sucrose at a much faster rate. The hydrolysis of sucrose coupled with the rapid utilization of fructose in the medium during the latter period of cell growth. This period of increased fructose availability coincided with the faster growth phase of the cells.D. Effect of Picloram and Methyl JasmonatePicloram (4-amino-3.5.6-trichloropicolinic acid) increases growth rate while methyl jasmonate has been reported to be an effective elicitor in the production of paclitaxel and other taxanes. However, little is known about the mechanisms or pathways that stimulate these secondary metabolites.Picloram had been used by Furmanowa and co-workers and Ketchum and Gibson but no details on the effect of picloram on growth rates were given. Furmanowa and hid colleagues observed growth of callus both in the presence and absence of light. The callus grew best in the dark showing a 9.3 fold increase, whereas there was only a 2-4 fold increase in the presence of light. Without picloram, callus growth was 0.9 fold. Unfortunately,this auxin had no effect on taxane production and the high callus growth rate was very unstable.Jasmonates exhibit various morphological and physiological activities when applied exogenously to plants. They induce transcriptional activation of genes involved in the formation of secondary metabolites. Methyl jasmonate was shown to stimulate paclitaxel and cephalomannine (taxane derivative) production in callus and suspension cultures. However, taxane production was best with White's medium compared to Gamborg's B5 medium. This may be due to the reduced concentration of potassium nitrate and a lack of ammonium sulfate with White's medium.E. Effect of Copper Sulfate and Mercuric ChlorideMetal ions have shown to play significant roles in altering the expression of secondary metabolic pathways in plant cell culture. Secondary metabolites,such as furano-terpenes, have been production by treatment of sweet potato root tissue with mercuric chloride. The results for copper sulfate, however, have not been reported. F. Growth Kinetics and Paclitaxel ProductionLow yields of paclitaxel may be attributed to the kinetics of taxane production that is not fully understood. Many reports stated inconclusive results on the kinetics of taxane production. More studies are needed in order to quantitate the taxane production. According to Nett-Fetto, the maximum instantaneous rate of paclitaxel production occurred at the third week upon further incubation. The paclitaxel level either declined or was not expected to increase upon further incubation. Paclitaxel production was very sensitive to slight variations in culture conditions. Due to this sensitivity, cell maintenance conditions, especially initial cell density, length of subculture interval, and temperature must be maintained as possible.Recently, Byun and co-workers have made a very detailed study on the kinetics of cell growth and taxane production. In their investigation, it was observed that the highest cell weight occurred at day 7 after inoculation. Similarly, the maximum concentration for 10-deacetyl baccatin Ⅲ and baccatin Ⅲ were detected at days 5 and 7, respectively. This result indicated that they are metabolic intermediates of paclitaxel. However, paclitaxel's maximum concentration was detected at day 22 but gradually declined. Byun and his colleagues suggested that paxlitaxel could be a metabolic intermediate like 10-deacetyl baccatin Ⅲ and baccatin Ⅲ or that pacliltaxel could be decomposed due to cellular morphological changes or DNA degradation characteristic of cell death.Pedtchanker's group also studied the kinetics of paclitaxel production by comparing the suspension cultures in shake flasks and Wilson-type reactors where bubbled air provided agitation and mixing. It was concluded that these cultures of Taxus cuspidata produced high levels of paclitaxel within three weeks (1.1 mg/L per day ). It was also determined that both cultures of the shake flask and Wilson-type reactor produced similar paclitaxel content. However, the Wilson-type reactor had a more rapid uptake of the nutrients (i.e. sugars, phosphate, calcium, and nitrate). This was probably due to the presence of the growth ring in the Wilson reactor. Therefor, the growth rate for the cultures from the Wilson reactor was only 135 mg./L while the shake flasks grew to 310 mg/L in three weeks.In retrospect, strictly controlled culture conditions are essential to consistent production and yield. Slight alterations in media formulations can have significant effects upon the physiology of cells, thereby affecting growth and product formation. All of the manipulations that affect growth and production of plant cells must be carefully integrated and controlled in order to maintain cell viability and stability.利用LC,LC/MS和LC/MS/MS悬浮培养生产紫杉醇及邓西佛米斯红豆杉中相关紫杉醇类的特征描述第三章植物组织培养Ⅰ.利用植物细胞培养生产紫杉的可能性紫杉醇的几个备选的来源已被确定，而且目前是全球大量调查的主题。