• KSBB Journal
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• v.11 no.5
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• pp.600-605
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• 1996

Optimum inoculum concentration for the production of taxol was determined in Taxus brevifolia and Taxus cuspidata cell suspension cultures. By fresh weight, 2.5, 5, 7.5, 10 g/flask of cells were inoculated and cell growth as well as taxol production were examined. In both Taxus cell cultures, the higher the inoculum concentration, the shorter the length of the lag period. The optimum inoculum concentration for taxol production was found to be 5 g/flask. To produce taxol in large quantity, utilization of proper medium was thought to be important. In case of using a production medium with 6% sucrose, taxol production was noticed. Its level reached the maximum at the 9th day of culture and decreased afterwards. However, taxol was not detected from cell cultures in growth medium.

• Korean Journal of Plant Tissue Culture
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• v.27 no.5
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• pp.401-405
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• 2000

Influence of fructose addition to the cultivation medium on the production of taxanes and the growth of callus culture of Taxus baccata was studied. The cultures showed an ability to adjust to the substitution of some of the sucrose in the media by fructose and the fresh biomass accumulation was higher on the media containing different concentrations of fructose during the second cultivation period.

• Journal of Plant Biotechnology
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• v.2 no.1
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• pp.29-33
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• 2000

To develop an efficient propagation method for yew tree, zygotic embryos were cultured under various conditions. When dissected embryos were cultured on GA$_3$ containing media, the highest germination frequency was observed on WPM medium contaning 1.0 mg/L GA$_3$. For germination of the embryos, two different conditions were compared; culturing embryos with endosperm (Method I), and 2) culturing embryos only (Method II). Maximum germination was achieved in 0.5 mg/L GA$_3$ when embryos with endosperm were cultured on the media. Of the media tested, White and WPM medium were the most suitable on germination of embryos. The abnormality of yew embryos found was observed when it cultured on GA$_3$ or culture media. About 40% of the precociously germinated embryos could be developed into full seedlings. Seedlings contained taxol in high quantity (535 $\mu\textrm{g}$/g dry weight). In vitro techniques will be sewed as a useful tool for the development of transformed root cultures and biosynthesis studies.

• Korean Journal of Pharmacognosy
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• v.27 no.3
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• pp.262-266
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• 1996

Undifferentiated callus were induced from the young leaves of Taxus cuspidata by treatment with various combinations of plant growth hormones. The effects of light and culturing temperature on production of baccatin III, the precursor of taxol, were studied. The contents of baccatin III in the cultured callus were analysed by HPLC. The illumination of fluorescence and administration of isoleucine, one of the possible substrates in biosynthesis of terpenoids, to the culturing media increased the production of baccetin III.

• Biotechnology and Bioprocess Engineering:BBE
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• v.5 no.5
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• pp.350-354
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• 2000

The growth promotion of a Taxus brevifolia cell suspension culture was investigated using conditioning factors. The conditioning factors produced and secreted from cultured cells usually stimulate cell division and the production of secondary metabolites. Therefore, the effective incubation time for the optimal secretion of conditioning factors was firstly determined for the promotion of cell growth. Conditioned media obtained by cultivating for 2 and 5 days showed the promotion of initial cell growth during the early cell growth period. However, the positive effect of the conditioning factors on the initial cell growth did not continue because of the depletion of the medium nutrients. Accordingly, the addition of a carbon source to the conditioned medium prolonged the positive effect on the cell growth. The addition of sucrose to the conditioned medium resulted in the maximum cell density being reached 4 days earlier compared to the control group and an increased substrate yield.

• BMB Reports
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• v.38 no.6
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• pp.668-675
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• 2005

A full-length cDNA encoding taxadiene synthase (designated as TmTXS), which catalyzes the first committed step in the Taxol biosynthetic pathway, was isolated from young leaves of Taxus media by rapid amplification of cDNA ends (RACE). The full-length cDNA of TmTXS had a 2586 bp open reading frame (ORF) encoding a protein of 862 amino acid residues. The deduced protein had isoelectric point (pI) of 5.32 and a calculated molecular weight of about 98 kDa, similar to previously cloned diterpene cyclases from other Taxus species such as T. brevifolia and T. chinenisis. Sequence comparison analysis showed that TmTXS had high similarity with other members of terpene synthase family of plant origin. Tissue expression pattern analysis revealed that TmTXS expressed strongly in leaves, weak in stems and no expression could be detected in fruits. This is the first report on the mRNA expression profile of genes encoding key enzymes involved in Taxol biosynthetic pathway in different tissues of Taxus plants. Phylogenetic tree analysis showed that TmTXS had closest relationship with taxadiene synthase from T. baccata followed by those from T. chinenisis and T. brevifolia. Expression profiles revealed by RT-PCR under different chemical elicitor treatments such as methyl jasmonate (MJ), silver nitrate (SN) and ammonium ceric sulphate (ACS) were also compared for the first time, and the results revealed that expression of TmTXS was all induced by the tested three treatments and the induction effect by MJ was the strongest, implying that TmTXS was high elicitor responsive.

• Journal of Plant Biotechnology
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• v.6 no.4
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• pp.213-219
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• 2004

Zygotic embryos were excised from immature and mature seeds of the Himalayan yew, Taxus wallichiana. The embryos germinated precociously when kept in darkness for 5 weeks and developed into full seedlings within 10-12 weeks. The highest rate of embryo germination ($81\%$) was obtained in modified Lloyd & McCown' s woody plant medium containing macro and micronutrients at half strength supplemented with $1\%$ activated charcoal, which supported both the best embryonic growth ($43\%$) and seedling development ($32\%$). However, the supplementation of basal media with kinetin, thidiazuron, 6-benzyl aminopurine or $GA_3$ had no effect on the germination of the embryos. The embryos derived from immature seeds germinated but the frequency of embryonic growth was better in mature seeds. Stratification of seeds effected precocious germination of embryos. Seeds kept at $4^{\circ}C$ for 1 week germinated earlier and at a higher frequency irrespective of the stage of seed maturity, while the germination rate declined with prolonged cold treatment for 1 month at that same temperature. Analysis of taxanes in germinating seedlings revealed that root tissues contained high levels of taxol, 10-deacetyl-baccatin ill and baccatin ill as compared to shoots. Thus embryo culture technique appears to overcome the lengthy dormancy requirement of T. wallichiana seeds.

• Journal of Korean Society of Forest Science
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• v.70 no.1
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• pp.1-6
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• 1985

The purpose of this experiment lies in finding the most appropriate cutting condition of Taxus cuspidata Sieb. et Zucc. which has been considered valuable tree species in gardening. Statistical analysis was focused on the comparison of the average ratios of rooting between greenhouses and fields, based on the observation rooted autogenis. To conduct this survey, hardwood cutting of Taxus cuspidata Sieb. et Zucc. were performed at both places of experiment above on April 20, 1982 with rooting results calculated on October 31, 1982. After formation of cuttings, it was soaked in IBA 200 ppm for 12 hours and than put in a bed. A summary of the result is presented as follows; 1) Cutting at the greenhouse showed higher average rate of rooting than at fields. Same tendency was found regardless of the differences in treatment. 2) It was clear that IBA-treatment contributed remarkably to causing higher average rooting rate. As an evidence, when twenty centimeter cuttings were inserted sandy media, IBA-treated area resulted in eighty six percent of average rooting rate, as compared with only twenty three percent in nontreatment case. In case of field cutting, IBA-treatment brought about fifty three percent of rooting in comparison with eleven percent nontreatment. 3) When sandy soil, loam and brown soil were separately used as cutting media, the highest rooting rate was found in case of sandy soil, without any difference between the two experimental places above. 4) As a result of the analysis to seek the impact of the length of cuttings on rooting, the range of length form fifteen to twenty centimeter was apparently most appropriate. It was also found that the rate of rooting declined beyond twenty five centimeter. 5) Two kinds of rooting pattern were observed. One was the case that callus cell lump was created on the lower cut side of cuttings. Importantly, root radical were formed inside the lump to influence the germination of root system. The other relates to the case that adventitious root which look like lateral roots appeared at the stem region. In abstract, first, sandy soil was effectively recommended in case of hardwood cutting in April. Second, the most appropriate length of cuttings ranged between fifteen and twenty centimeters. Third, high density IBA treatment was clearly effective. Forth, for proper environmental management, both pre-disinfection of sail by sterilizer and maintenance of high relative humidity were essentially required.

• Proceedings of the Botanical Society of Korea Conference
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• 1995.06a
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• pp.40-56
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• 1995

During the past two decades, China has seen her great progress in plant biotechnology. Since the Chinese market of herb medicine is huge, while the plant resources are shrinking, particular emphasis has been placed in plant tissue and cell cultures of medicinal plants, this includes fast propagation, protoplast isolation and regeneration, cell suspension cultures and large scale fermentation. To optimize culture conditions for producing secondary compounds in vitro, various media, additives and elicitors have been tested. Successful examples of large scale culture for the secondary metabolite biosynthesis are quite limited : Lithospermum ery throrhizon and Arnebia euchroma for shikonin derivatives, Panax ginseng, P. notoginseng, P. quinquefolium for saponins, and a few other medicinal plants. Recent development of genetic transformation systems of plant cells offered a new approach to in vitro production of secondary compounds. Hairy root induction and cultures, by using Ri-plasmid, have been reported from a number of medicinal plant species, such as Artemisia annua that produces little artemisinin in normal cultured cells, and from Glycyrrhiza uralensis. In the coming five years, Chinese scientists will continue their work on large scale cell cultures of a few of selected plant species, including Taxus spp. and A. annua, for the production of secondary metabolites with medicinal interests, one or two groups of scientists will be engaged in molecular cloning of the key enzymes in plant secondary metabolism.

• Journal of the Korean Wood Science and Technology
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• v.27 no.3
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• pp.91-98
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• 1999

The content of taxol and related compounds in needle and stem tissues of exotic yews growing in the Chollipo Arboretum which located in the West Sea of Korea were determined by HPLC. A considerable variation in contents of taxol and the related compounds within species and among cultivars were observed. The content of taxol was much higher in the needles than those in the young stem tissues except T. X media cv. Vermuleun, T. baccata cv. Washingtonii, and T. X cuspidata cv. Nana, and the content of 10-deacetyl baccatin III(10-DAB III) was also slightly higher in the needle. The needle of T. cuspidata cv. Nigra collected from April was contained the high amount of taxol(1,027 ${\mu}g$ per g dry weight) and also contained high quantity in 10-DAB III(267 ${\mu}g$ per g dry weight). Concentration of taxol in needle was significantly greater than those of meristemic and ovary tissues, Exotic yews growing at the Chollipo Arboretum containing T. cuspidata cv. Nigra could be suitable materials as a source for the tissue and cell cultures for production of taxol.