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1、 Characterization of production of Paclitaxel and related Taxanes in Taxus Cuspidata Densiformis suspension cultures by LC, LC/MS, and LC/MS/MS CHAPTER THEREPLANT TISSUE CULTURE. Potential of Plant cell Culture for Taxane Production Several alternative sources of paclitaxel have been identified and
2、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
3、 or 10-deacetylbaccatin ), 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 antin
4、eoplastic 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 wo
5、uld 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
6、method that will promote fast plant growth and also produce a large amount of paclitaxel at the same time. Factors Influencing Growth Paclitaxel Content A. Choice of Media for Growth Gamborgs (B5) and Murashige & Skoogs (MS) media seem to be superior for callus growth compared to Whites (WP) medium.
7、 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. Gamborgs B5 medi
8、a 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 -naphthalene acetic acid. Agar (8 g/L) was used for solid cultures.B. In
9、itiation of Callus Cultures Previous 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 Gamborgs B5 medium supplemented with 1-2 mg
10、/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 foll
11、owing 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
12、 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 with callus initiation was similar to those studies. Our studies have found that callus which initiated early (
13、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. Th
14、e 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 Sugar Sucrose is the preferred carbon source for growth in plant cell cultures, although the presence of more rapi
15、dly 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 th
16、e 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
