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  • 1
    ISSN: 1432-203X
    Keywords: Key words Orchid ; Particle bombardment ; PLB ; Transformation ; Key terminologyProtocorm A small storage organ formed from the germinating embryo ; possessing an apical meristem and a leaf primordium ; protocorm-like body (PLB) A somatic protocorm derived from in vitro culture of apical or axillary bud meristems ; primary PLBs PLBs induced by culturing apical meristem-tips aseptically ; secondary PLBs PLBs formed on the surface of a primary PLB in culture ; proliferate PLBs PLBs proliferating on the surface of either primary or secondary PLBs
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  A protocol is presented for genetically engineering Cymbidium orchid using particle bombardment. This protocol enabled the routine transformation of orchid plants that were previously difficult to transform. Liquid culture was used to generate a large number of protocorm-like bodies (PLBs) to be bombarded and to promote continued development of the bombarded meristematic tissue. Plasmid DNA (pKH200) carrying the GUS-INT and NPTII genes flanked by tobacco matrix attachment regions was introduced into the meristematic cells of PLBs by particle acceleration. The transformed PLBs were proliferated and selected for kanamycin resistance conferred by the introduced NPTII gene. Shoot regeneration was then induced from the kanamycin-resistant PLBs, and transgenic plantlets were produced. Both the kanamycin-resistant PLBs and regenerated shoots expressed the GUS-INT gene. The presence of the introduced gene in the transformed orchid plants was confirmed by PCR analysis, sequencing and Southern blot analysis of the PCR product. The recovered transgenic plants were established in soil and acclimatized in the greenhouse.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1432-203X
    Keywords: Key words Pilot-scale culture ; Taxane ; Taxol ; Fed-batch culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  A novel type of bioreactor was successfully developed for the production of taxol and its precursors by culturing cells of Taxus cuspidata (Japanese yew) on a pilot-scale. Rapidly growing cell lines were selected from callus cultures derived from immature embryos of yew. The cells were inoculated in 20-l capacity bioreactors of different types to test the growth performance. The models of small-scale bioreactors incorporated in this study included a balloon-type bubble bioreactor (BTBB), a bubble-column bioreactor (BCB), a BCB with a split-plate internal loop, a BCB with a concentric draught-tube internal loop, a BCB with a fluidized bed bioreactor, and two different models of stirred tank reactors. Among the reactors, BTBB appeared to be the most efficient in promoting cell growth. The doubling time of cell growth in BTBB was 12 days with a 30% inoculation cell density. The optimum time for medium replacement or feeding was 12–15 days after inoculation as determined by monitoring both the levels of sugars and medium conductivity. When yew tree cells were grown in different sizes (100–500-l) of BTBBs, more than 70% cell viability was recorded at the time of harvest. The growth pattern of the cells in the pilot-scale BTBB appeared to be the same as that of cells in the 20-l bioreactors. Approximately 3 mg/l of taxol and 74 mg/l total taxanes were obtained after 27 days of culture.
    Type of Medium: Electronic Resource
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