Large-scale growth and taxane production in cell cultures of Taxus cuspidata (Japanese yew) using a novel bioreactor.

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.

Expression of a human S-adenosylmethionine decarboxylase cDNA in transgenic tobacco and its effects on polyamine biosynthesis.

S-adenosylmethionine decarboxylase (SAMDC; EC 4.1.1.50) is a key regulatory enzyme in the polyamine biosynthetic pathway. Numerous studies have shown that the enzyme activity and polyamine levels are generally correlated with cellular growth in plants, animals and bacteria. In order to gain more insight into the role of polyamines in plants, human SAMDC cDNA under control of 35S promoter of cauliflower mosaic virus, along with a neomycin phosphotransferase gene, was transferred to tobacco (Nicotiana tabacum cv.Xanthi) via Agrobacterium tumefaciens. Transgenic plants showed the presence of SAMDC mRNA and a 2-4-fold increase in SAMDC activity. In the transformed tissues, putrescine levels were significantly reduced, while spermidine content was 2-3 times higher than the control tissues. Cellular spermine content was either increased or remained unchanged. Excised leaf segments from transformed plants frequently produced shoots even on callus inducing medium.

High efficiency shoot regeneration from callus of quaking aspen (Populus tremuloides Michx.).

Callus was induced from leaf segments of aspen (Populus tremuloides Michx.) on modified B5 (mB5) medium with 0.1 mg/1 benzyladenine (BA) and 0.5 mg/1 2,4-dichlorophenoxyacetic acid (2,4-D). The resulting callus was either subcultured to solidified Woody Plant Medium (WPM) with 0.5 mg/1 BA directly for shoot regeneration or sieved into liquid mB5 medium for suspension culture. After 3 weeks of suspension culture, when the callus clumps grew to 3-4 mm in diameter, they were transferred onto solidified WPM with 0.5 mg/1 BA for shoot regeneration. Almost 100% of the clumps formed shoots on WPM when subcultured directly from mB5 with an average number of 6 shoots per callus. When transferred from suspension culture in mB5 to WPM, an average of 6 shoots per callus were produced from 51% of calli. These shoots could be easily rooted on either mB5 or WPM with 0.2 mg/1 indole-3-butyric acid (IBA) and transferred to pots. Transplanted plants were kept under intermittent mist for 2-4 weeks before normal growth in the green house.